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FRONTIERS IN TUMOUR PROGRESSION
October 24-27, 2010
Madrid, Spain
Frontiers in Tumour
Progression
October 24-27, 2010
Madrid, Spain
Frontiers in Tumour Progression
October 24-27, 2010
Madrid, Spain
Index
Scientific Organisers:
Zena Werb, UCSF, USA
Douglas Hanahan, ISREC, EPFL, Switzerland
Erwin Wagner, CNIO, Spain
Co-organisers from Nature Publishing Group:
Barbara Marte, Nature, UK
Nicola McCarthy, Nature Reviews Cancer, UK
Sownya Swaminathan, Nature Cell Biology, UK
Administrative Organisers:
Mercedes Moro, CNIO, Spain
Carolina G. Sabaté, CNIO, Spain
Ralf Dahm, CNIO, Spain
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Programme
5
Keynote Lectures
13
Session 1
Session 2
Session 3
Session 4
Session 5
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27
37
45
51
Speakers’ biosketches
61
Poster session A
Poster session B
87
159
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Programme
Nature CNIO Cancer Symposium “Frontiers in Tumour Progression”
09:30 - 09:45
24th–27th October 2010
Palacete de los Duques de Pastrana, Madrid, Spain
SUNDAY, OCTOBER 24th
Welcome Address and Keynote Lectures:
18:00 - 19:00
19:00 - 20:00 20:30 - 22:30 Joan Massagué, Memorial Sloan-Kettering Cancer Center, New York, USA
Mediators and targets of metastasis
Michael Karin, University of California San Diego (UCSD), USA
Lymphocyte-derived cytokines control tumor development and metastatic progression
09:45 - 10:15 Michael Stratton, The Wellcome Trust Sanger Institute, Hinxton, UK
Evolution of the cancer genome
10:15 - 11:00 Coffee Break
11:00 - 11:30 Jacqueline F. Bromberg, Memorial Sloan-Kettering Cancer Center, New York, USA
Targeting the IL-6/Jak pathway in malignancies
11:30 - 11:45 Juan Guinea-Viniegra, CNIO, Madrid, Spain
Short talk: Skin cancer: A c-Fos-p53 axis controls keratinocyte differentiation
11:45 - 12:00 Benjamin Beck, Université Libre de Bruxelles, Brussels, Belgium
Short talk: Defining the impact of the vascular niche in regulating cancer stem cells of skin
tumors
Welcome Reception at the Pio XII Hotel
MONDAY, OCTOBER 25th
SESSION 1: Genetics, Mouse
Models and Mechanisms
Chair: Mariano Barbacid, CNIO, Madrid, Spain
Rafael Blasco-Patiño, CNIO, Madrid, Spain
Short talk: C-Raf, the key member of the ERK-MAPK pathway for K-RasG12V-induced NSCLC
initiation
12:00 - 12:30 Kent W. Hunter, Centre for Cancer Research, National Cancer Institute (NCI), Bethesda, USA
Systems genetics of breast cancer metastasis susceptibility
12:30 - 14:00 Lunch
09:00 - 09:30 Tyler Jacks, Massachusetts Institute of Technology (MIT), Cambridge, USA
Studying tumor evolution in mouse models of cancer
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Programme
SESSION 2: The
Metastatic Cancer Cell
Chair: Zena Werb, UCSF, San Francisco, USA
TUESDAY, OCTOBER 26th
SESSION 3: Inflammatory
Modulators
Chair: Maria S. Soengas, CNIO, Madrid, Spain
14:00 - 14:30
14:30 - 14:45
Hartmut Beug, Research Institute of Molecular Pathology (IMP), Vienna, Austria
Annexin A1 strongly suppresses EMT and metastasis by strengthening epithelial polarity
09:00 - 09:30 Neta Barashi, Hadassah Hebrew University Hospital, Jerusalem, Israel
Short talk: Inflammation induced hepatocellular carcinoma is dependent on CCR5
Lisa Coussens, University of California San Francisco (UCSF), USA
Inflammation and cancer: Reprogramming immune response as an anti-cancer therapeutic strategy
09:30 - 09:45 Catherine Muller, Institute of Pharmacology and Structural Biology, Toulouse, France
Short talk: Bidirectional crosstalk between mature adipocytes and breast cancer cells
stimulates tumor invasion
09:45 - 10:15 Mark Taketo, Kyoto University Graduate School of Medicine, Kyoto, Japan
Suppression of colon cancer metastasis by Aes through inhibition of Notch signalling
10:15 - 11:00 Coffee Break
11:00 - 11:30
Johanna Joyce, Memorial Sloan Kettering Cancer Center, New York, USA
Systems biology analysis of tumor and stromal genes in different metastatic microenvironments
11:30 - 11:45 Julie Wallace, The Ohio State University, Columbus, USA
Short talk: The role of stromal Pten signaling in the breast tumor microenvironment
11:45 - 12:15 Shahin Rafii, Howard Hughes Medical Institute, New York, USA
Angiocrine factors-derived from Akt-activated vascular niche initiate and maintain tumor growth
Lunch
14:45 - 15:00
Tobias Pusterla, German Cancer Research Center, Heidelberg, Germany
Short talk: RAGE signaling in mouse models of inflammation-associated carcinogenesis
15:00 - 15:30
Stefano Piccolo, University of Padova, Italy
A microRNA targeting Dicer for metastasis control
15:30 - 16:15
Coffee Break
16:15 - 16:45 Erik Sahai, London Research Institute, Cancer Research UK, London, UK
Imaging the metastatic process
16:45 - 17:00
17:00 - 17:30 17:30 - 20:00 8
Bojana Gligorijevic, Albert Einstein College of Medicine, New York, USA
Short talk: Invadopodia: Do they exist in vivo and are they the first step of metastasis?
Zena Werb, University of California San Francisco (UCSF), USA
Role of the tumor microenvironment in breast cancer metastasis and response to therapy
12:15 - 14:00 Poster Session with wine and cheese
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Programme
SESSION 4:
Mechanism-based Anti-metastatic Therapies
Chair: Manuel Hidalgo, CNIO, Madrid, Spain
WEDNESDAY, OCTOBER 27th
SESSION 5: Angiogenesis
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14:00 - 14:30 Theresa Guise, Indiana University School of Medicine, Indianapolis, USA
Molecular mechanisms of bone metastases: Implications for therapy
14:30 - 15:00 Lee Ellis, MD Anderson Cancer Center, University of Texas, Houston, USA
Targeting the tumor vasculature: Do we need to move beyond the obvious?
15:00 - 15:30 Joerg Huelsken, ISREC, Swiss Federal Institute of Technology Lausanne (EPFL), Switzerland
Stem cell niche interactions in metastasis
15:30 - 16:00 Fred de Sauvage, Genentech Inc., South San Francisco, USA
Targeting the hedgehog pathway in cancer
16:00 - 16:45 Coffee Break
16:45 - 18:00 Manuel Hidalgo, CNIO, Madrid, Spain
Round Table Discussion on “Questions from the viewpoint of a clinician” chaired by Manuel Hidalgo
18:00 - 20:00 Poster Session with wine and cheese
and Anti-angiogenic Therapy
Chair: Douglas Hanahan, Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland
09:00 - 09:30 Kari Alitalo, University of Helsinki, Finland
Targeting (lymph)angiogenesis and lymphatic metastasis
09:30 - 09:45 Sebastien Tabruyn, University of Liege, Belgium
Short talk: MiR-146a: A new angiostatic miRNA with tumor-suppressive properties
09:45 - 10:00 Amparo Acker-Palmer, University of Frankfurt, Germany
Short talk: EphrinB2 repression mediates anti-angiogenesis-induced glioma invasion
10:00 - 10:30 Peter Carmeliet, Katholieke Universiteit Leuven (KUL), Belgium
Tumor vessel normalization: Genetic insight and therapeutic potential
10:30 - 11:15 Coffee Break
11:15 - 11:45 Donald McDonald, University of California San Francisco (UCSF), USA
Synergistic effects of inhibiting VEGF/VEGFR and HGF/c-Met signaling in tumors
11:45 - 12:15 Robert S. Kerbel, University of Toronto, Canada
Preclinical modelling of adjuvant and metastatic antiangiogenic therapies
12:15 - 12:45 Douglas Hanahan, ISREC, Swiss Federal Institute of Technology Lausanne (EPFL), Switzerland
Determinants of malignant progression
12:45 – 13:00 Closing remarks
13:00 - 14:30 Lunch and Departure
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SUNDAY, OCTOBER 24th
Keynote
Lectures
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Keynote Lectures. Sunday, October 24th
Mediators and targets of metastasis
Joan Massagué
Memorial Sloan-Kettering Cancer Center, Howard Hughes Medical Institute, New York, USA
To form metastases circulating cancer cells (CTCs) that egress into a target organ must be able to survive in the newly
invaded microenvironment. Identifying these survival mechanisms may provide therapeutic strategies for the eradication
of disseminated cancer cells after primary tumor removal. In breast cancer, where relapse develops in an organ-specific
manner, we identified various genes that prime CTCs for initial infiltration of specific organs (lung, brain, bone marrow)
as well as genes for tumor self-seeding, the re-infiltration of a primary tumor by CTCs for amplification of aggressive
clones. We recently found that the survival of newly disseminated cells depends on a distinct set of mediators. SRC
primes breast cancer cells for survival in the bone marrow because it potentiates AKT activation by CXCL12 and IGF1,
cytokines that are abundant in the marrow microenvironment. In the lung microenvironment the survival of newly
seeded breast cancer cells depends on other factors, including the stem cell niche ECM protein tenascin-C. Inhibition of
SRC and tenascin-C suppressed the ability of disseminated breast cancer cells to survive and form aggressive lesions.
Lymphocyte-derived cytokines control tumor
development and metastatic progression
Michael Karin
Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology and Pathology, UCSD
School of Medicine, La Jolla, USA
Inflammation and immunity can intersect with tumor development in more than one way. While chronic inflammation
promotes tumor development, many tumors that do not arise in the context of underlying inflammation still exhibit an
inflammatory microenvironment. Furthermore, in certain cases, inflammation may act to suppress anti-tumor immunity,
but it can also be used to enhance the efficacy of cancer immunotherapy. Undoubtedly, we need to learn much more
about how inflammation and immunity affect tumor development. To study the pathogenic roles of tumor-elicited
inflammation, we have used mouse models of prostate and breast cancers, two of the most common malignancies in
men and women, respectively, which usually do not evolve in the context of underlying inflammation or infection. Yet,
in both cases, we found that tumor-elicited inflammation plays a key role in promoting metastatic spread and in the
case of prostate cancer, it contributes to the failure of androgen ablation therapy. Interestingly, in both types of cancer,
metastatogenesis depends on the accumulation of activated IκB kinase α (IKKα) in the nuclei of primary cancer cells,
where it acts both as an activator of chromatin modifiers that control cell cycle progression and as a repressor of an
anti-metastatic gene, called maspin. In both cases, IKKα, whose activation has also been observed in advanced human
tumors, may be activated upon production within the tumor microenvironment of two members of the TNF family of
cytokines: lymphotoxin (LT)α:β and RANK ligand (RANKL). While the cells responsible for production of these cytokines
during metastatic progression of prostate cancer remain to be identified, B cells were found to be a major source of
LTα:β during development of castration resistant cancer. In breast cancer, however, the major culprits in metastatic
progression are RANKL-producing regulatory T cells (Treg). Both in prostate and breast cancers, the recruitment of
lymphocytes into the primary tumor is likely to depend on activation of myofibroblasts which produce a number of tumor
promoting chemokines. LTα:β, RANKL, IKKα and the mechanisms responsible for myofibroblast activation, as well as
the chemokines they produce provide several new opportunities for therapeutic intervention.
Keynote
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Genetics, Mouse
Models and
Mechanisms
Chair: Mariano Barbacid
CNIO, Madrid, Spain
MONDAY, OCTOBER 25th
Session
1
Session 1
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Session 1. Monday, October 25th
Studying tumor evolution in mouse models of cancer
Tyler Jacks
Koch Institute for Integrative Cancer Research, Department of Biology, Howard Hughes Medical Institute,
Massachusetts Institute of Technology, Cambridge, USA
Short
Talk
C-Raf, the key member of the ERK-MAPK pathway for
K-RasG12V-induced NSCLC initiation
Rafael B. Blasco1#, Sarah Francoz
, Marta Cañamero2, Pierre Dubus3, Manuela Baccarini4 and Mariano
1#
Barbacid1*
Molecular Oncology Program and 2Biotechnology Program, Centro Nacional de Investigaciones Oncológicas
(CNIO), Madrid, Spain; 3EA2406 University of Bordeaux 2, Bordeaux, France; 4Center for Molecular Biology,
University of Vienna, Max F. Perutz Laboratories, Vienna, Austria
1
Like other solid tumors, lung adenocarcinoma progresses through multiple stages including tumor initiation, expansion
in the lung, and metastasis of the primary tumor to distant organs. Dissection of this process requires quantitative
and tractable models that recapitulate the human disease at the genetic and histological level. We have employed
autochthonous mouse models of lung adenocarcinoma that recapitulate tumor initiation, progression and metastasis in
the human disease both genetically, and pathologically. We have established oncogenic Kras as the initiating event in
lung adenoma and amplification of the oncogenic signaling is associated with progression to adenocarcinoma. The p53
tumor suppressor limits tumor progression in this model. Restoration of the Trp53 locus has pinpointed the adenomaadenocarcinoma transition as the stage in which p53 function is critical for tumor suppression. Expression of oncogenic
Kras in the absence of p53 leads to the development of distant metastases after long latency. Identification of the
lentiviral integration site in each tumor allows the clonal relationship between metastases and the primary tumors
to be unequivocally established. Gene expression and DNA copy number analyses of metastasis-derived cell lines
and primary lung tumors have uncovered a metastasis associated gene expression signature and recurrent genomic
alterations. Integration of these datasets, other genome-wide analyses and human tumor data are providing insights into
the molecular mechanism and biology of lung cancer progression and metastasis.
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The K-Ras oncogene is mutated in about 25% of non-small cell lung carcinomas (NSCLC), one of the most common
types of human cancer. To date, there are no approved therapies to treat NSCLC based on K-Ras inhibition. Although
Ras proteins are not druggable targets, they are known to signal through a cascade of kinases that, in principle, could
be targeted with small molecule inhibitors. To determine which kinases are critical in mediating K-Ras oncogenic
signaling in NSCLC, we have crossed our K-Ras+/LSLG12Vgeo strain, known to develop lung adenocarcinomas upon
Cre-mediated recombination of the K-RasLSLG12Vgeo allele, with mice carrying either null or conditional mutations for
each of the members of the Raf, Mek and Erk families of kinases. Our results indicate that ablation of individual Mek
or Erk kinases did not affect K-RasV12-induced lung tumorigenesis. However, combined elimination of either Mek1
and Mek2 or Erk1 and Erk2 in lung tissue, effectively impaired development of both adenomas and adenocarcinomas.
These observations provide genetic evidence that the tumorigenic properties of K-Ras oncogenes are solely mediated
by the Mek/Erk kinase pathway, at least in lung tissue. Unfortunately, widespread depletion of Mek (Mek1 and Mek2)
or Erk (Erk1 and Erk2) kinases in adult mice induced multiple defects that resulted in the death of the animals in less
than 3 weeks. Ablation of B-Raf had neither effect on tumor development nor Erk phosphorylation, suggesting that in
the absence of B-Raf, c-Raf (and/or A-Raf) effectively mediated K-Ras oncogenic signaling. Surprisingly, K-RasV12
induced few adenomas or adenocarcinomas in the absence of c-Raf. Indeed, these tumors expressed c-Raf, suggesting
inefficient recombination of the c-Raflox alleles. These findings indicate that K-Ras oncogenic signaling is selectively
mediated by c-Raf and that the absence of this kinase cannot be compensated by either B-Raf or A-Raf. Importantly,
widespread depletion of c-Raf did not affect the overall health of the mice, suggesting that pharmacological inhibition of
this kinase will be well tolerated. These observations indicate that c-Raf could be a relevant therapeutic target to treat
NSCLC induced by K-Ras oncogene.
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Session 1. Monday, October 25th
Evolution of the cancer genome
Targeting the IL-6/Jak pathway in malignancies
Michael Stratton
Jacqueline Bromberg1, Paul Gao , Qing Chang , Pasquale Sansone , Min Zhang , Rosario Andre ,
Wellcome Trust Sanger Institute, Hinxton, UK
1
1
1
1
2
Hector Peinado2, Cong Yan3, William Pao4 and David Lyden2
Memorial Sloan-Kettering Cancer Center, New York, USA; 2Weill Medical College of Cornell University,
New York, USA; 3Indiana University, Indianapolis, USA; 4Vanderbilt University, Nashville, USA
1
All cancers carry somatically acquired changes in their genomes. Some, termed “driver” mutations, are causally
implicated in cancer development. The remainder are “passengers”, and bear the imprints of mutational processes
operative during cancer development. Following the advent of second generation sequencing technologies the provision
of whole cancer genome sequences has become a reality. These sequences generate comprehensive catalogues of
somatic mutations, including point mutations, rearrangements and copy number changes and provide insights into
the evolutionary processes underlying the development of individual human cancers including the factors generating
variation and the forces of selection. These insights will form the foundation of our understanding of cancer causation,
prevention and treatment in the future.
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Persistent activation of the IL-6/Jak/Stat3 pathway is a common feature of many solid tumors. The highest levels
of IL-6 and activated Stat3 are found on the edge of tumors primarily in fibroblasts, immune cells, endothelial and
lymphatic vessels. Furthermore, paracrine IL-6 promotes autocrine IL-6 expression and signaling. We have examined
the consequences of targeting this signaling pathway in transgenic and xenograft models of breast and lung cancer. We
have determined that mice deficient in IL-6 develop mammary tumors but these do not metastasize to the lung which
correlates with a decrease in the recruitment of myeloid cells including cd11b/gr1+ cells. Conversely, constitutive
activation of Stat3 in the bone marrow promotes expansion of cd11b/gr1+ cells. These data suggest a role in the IL-6/
Stat3 pathway in recruiting myeloid cells to metastatic sites of disease. Similarly, in transgenic models of lung cancer
we observe a reduction in the rate of tumor progression in mice lacking IL-6. Further analyses revealed that tumors
from mice deficient for IL-6 were less vascularized and more differentiated. We have also examined the role of Jak
inhibition in xenograft models of breast and lung cancer and observe a marked reduction in primary tumor growth as
well as metastatic progression. This was associated primarily with a reduction in angiogenesis and in the recruitment
of myeloid cells to the primary tumor and to metastatic sites. In sum, these data suggest a therapeutic role in targeting
the IL-6/Jak/Stat3 pathway in the treatment of solid tumors.
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Session 1. Monday, October 25th
Short
Short
Talk
Talk
Skin cancer: a c-Fos-p53 axis controls keratinocyte
differentiation
Juan Guinea-Viniegra1, Rainer Zenz , Latifa Bakiri
2
1
and Erwin F. Wagner1
Defining the impact of the vascular niche in regulating
cancer stem cells of skin tumors
Benjamin Beck, K.K. Youssef, G. Driessens, S. Goossens, A. Caauwe, C. Paulissen, P. Carmeliet, J. Haigh,
BBVA-Foundation Cancer Cell Biology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO),
Madrid, Spain; 2Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria
and C. Blanpain
The proto-oncogene c-fos is a major nuclear target for signal transduction pathways controlling several cellular
processes. To investigate the function of c-Fos in epidermal homeostasis and skin tumorigenesis, mice with specific,
conditional deletion of c-fos in the epidermis (c-fosΔep) were generated. Mice lacking c-fos in keratinocytes show no
obvious skin phenotype, while in vitro treatment of c-fosΔep keratinocytes with Ca2+ induced premature and irreversible
differentiation. Molecularly, this is a consequence of increased p53 expression, which correlates with increased TACE/
ADAM17 (TNF-α Converting Enzyme) protein expression and subsequent Notch1 activation. In the context of oncogenic
RasV12 signaling, c-fosΔep keratinocytes also exhibit premature differentiation in vitro. Importantly, tumor-prone K5SOS-F transgenic mice lacking epidermal c-fos show dramatically reduced papilloma formation. Detailed molecular
analyses revealed that the small tumors consist of highly differentiated cells that overexpress p53, TACE and active
Notch1. Moreover, epidermis-specific inducible deletion of c-fos in K5-SOS-F transgenic mice also inhibited tumor
growth likely through induction of differentiation. This novel pathway seems to operate also in human cells as c-fos
knock-down or inhibition in several Squamous Cell Carcinoma (SCC) cell lines induced their differentiation in a p53dependent manner. Furthermore, highly differentiated human skin carcinomas express low levels of c-Fos and increased
levels of p53 and TACE, correlating with the findings in the mouse model. In summary, stress-induced induction of p53,
and consequently TACE/ADAM17 - a putative novel p53 target - in c-fos deficient keratinocytes leads to increased
expression and activation of the Notch1 pathway, which induces premature keratinocyte differentiation and prevents
papilloma development. We propose that c-Fos inhibition could be applied in differentiation-inducing therapeutic
strategies in skin cancer.
Cancer stem cells (CSCs) are characterized by high clonogenic potential and the ability to reform the parental tumors
upon transplantation. CSC have been described in many types of human cancers including squamous tumors of the
skin. Angiogenesis and neovascularization are key events during tumor initiation, progression and metastasis. In this
study, we investigated the impact of the vascular niche and VEGF signaling in controlling CSC functions from squamous
skin tumors. We first show that in papilloma, CSC are localized in close contact to the vascular niche. Inhibition of
VEGFR2, which is only expressed by endothelial cells, by the administration of blocking monoclonal antibodies inhibits
angiogenesis and induces tumor shrinkage that correlates with a decrease in the pool and the renewal potential of CSC.
Conversely, increasing VEGF secretion by tumor epithelial cells increases the pool of CSC by promoting their renewal
potential. Importantly, conditional deletion of VEGF in specifically in epithelial tumor cells induced loss of CSC and tumor
shrinkage, indicating that the secretion of VEGF by is required for the maintenance of CSC through a paracrine and/or an
autocrine mechanisms. Taken together, our results indicate that the vascular niche and the secretion of VEGF by tumor
cells controlled the maintenance, renewal and differentiation potential of CSC.
1
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Short
Talk
Université Libre de Bruxelles, Belgium
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Tumour Progression
Session 1. Monday, October 25th
Systems genetics of breast cancer metastasis
susceptibility
Kent W. Hunter
Centre for Cancer Research, National Cancer Institute (NCI), Bethesda, USA
Metastatic disease is the primary cause of breast cancer mortality. Despite decades of research however, the etiology
of the terminal stages of cancer progression is poorly understood. Recent evidence has suggested that many factors
other than somatic mutation play an important role in the development of metastatic disease. One of these previously
unrealized factors is inherited metastatic susceptibility. Using a systems biology approach based on a highly metastatic
mouse mammary tumor model we demonstrated that the genetic background upon which the tumor arose has a
significant impact on the metastatic efficiency. Subsequent work identified Sipa1 as the first candidate metastasis
efficiency locus. Further efforts have identified a number of additional metastasis efficiency genes that interact either
physically or transcriptionally with Sipa1. The polymorphic metastasis efficiency genes have been found not only to
impact the physiologic manifestation of metastasis, but also to induce prognostic gene expression signatures that can
discriminate human breast cancer patient outcome. Association studies have demonstrated that these polymorphisms
are significantly associated with distant metastasis-free survival in human populations as well as mouse inbred
strains. Current investigations are relying on a variety of genomic and genetic tools to not only identify additional
metastasis susceptibility genes but also to investigate the cellular mechanisms underlying metastatic predisposition.
Unexpectedly a large number of the candidate metastasis susceptibility genes appear to be functioning within a common
pathway associated with basal transcriptional control. Further investigations into the role of this pathway in metastatic
susceptibility may provide greater insights into the complex biology underlying predisposition for disseminated disease
and potentially offer new insights for prevention, control and/or treatment for the terminal stages of cancer.
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The Metastatic
Cancer Cell
Chair: Zena Werb
UCSF, San Francisco, USA
MONDAY, OCTOBER 25th
Session
2
Session 2
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Tumour Progression
Session 2. Monday, October 25th
Short
Talk
Annexin A1 strongly suppresses EMT and metastasis by
strengthening epithelial polarity
Inflammation induced hepatocellular carcinoma is
dependent on CCR5
Hartmut Beug1, Sabine Maschler , Christoph A. Gebeshuber , Eva-Maria Wiedemann , Memetcan
Neta Barashi1, Ido D. Weiss , Ori Wald , Hanna Wald , Katia Beider
1
2
1
Alacakaptan , Martin Schreiber and Ivana Custic
1
3
1
IMP – Research Institute of Molecular Pathology, Vienna, Austria; 2IMBA – Institute of Molecular
Biotechnology, Vienna, Austria; 3Medical University of Vienna, Dept. of Obstetrics and Gynecology,
Vienna, Austria
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1
1
1
1
Michal Abraham1, Shiri Klein1, Daniel
Goldenberg1, Jonathan Axelrod1, Eli Pikarsky2, Rinat Abramovitch1,3, Evelyne Zeira1, Eithan Galun1 and Amnon Peled1
Goldyne Savad Institute of Gene Therapy, 2Department of Pathology and 3MRI Lab HBRC, Hadassah Hebrew
University Hospital, Jerusalem, Israel
1
1
Metastasis is the major cause of carcinoma-induced death, but mechanisms involved are still rather poorly understood.
Metastasis crucially involves epithelial-to-mesenchymal transition (EMT), which is based to a large extent on events
deregulating epithelial polarity. Here we identify Annexin A1 (AnxA1), a protein with important functions in intracellular
vesicle trafficking, as an efficient suppressor of EMT and metastasis in breast cancer. AnxA1 levels were strongly reduced
in EMT of mammary epithelial cells, in metastatic murine and human cell lines, in metastatic mouse breast cancer
and, most importantly, in tissue samples from metastatic, lethal human breast carcinoma, present in a large human
breast cancer tissue array. RNAi-mediated AnxA1 knockdown required cooperation with oncogenic Ras to induce TGFβindependent EMT and metastasis in non-metastatic cells and stimulated the expression of E-cadherin repressors like
snail and delta EF-1. Strikingly, forced AnxA1 expression in metastatic human mammary carcinoma cells with an EMT
phenotype reversed this EMT, down-regulated E-cadherin repressors and abolished metastasis, supporting a function
of AnxA1 as a gatekeeper of epithelial polarity. AnxA1 knockdown stimulated multiple signalling pathways, but only
Tyk2/Stat3 (and to a lesser extent Erk1/2 signalling) were essential for EMT, as shown using pharmacological inhibitors.
EMT caused by AnxA1 knockdown plus Ras may require the synthesis of complex glycolipids, since a pharmacological
inhibitor of respective synthases reversed EMT in both AnxA1-RNAi- and TGFβ plus hyperactive MAPK signallinginduced EMT. In contrast, a specific potassium ionophore (Salinomycin) – claimed to specifically target breast cancer
stem cells – inhibited only Ras plus TGFβ induced EMT, suggesting that AnxA1-RNAi-induced EMT employs different
molecular mechanisms.
Clinical and epidemiologic studies have suggested an association between chronic inflammation and cancer, and it is
estimated that 25% of malignancies are initiated by infections and chronic inflammation. However, the mechanisms
linking chronic inflammation to tumorigenesis remain largely unresolved. We proposed that chemokine and chemokine
receptors play an essential role in the chronic inflammation stage and can induce tumor formation. To test this hypothesis,
we studied the Mdr2 knockout mouse strain, which spontaneously develops chronic liver inflammation followed by
hepatocellular carcinoma (HCC), a model for inflammation induced liver cancer. In a previous gene expression array
study Rantes, a pro-inflammatory chemokine, was shown to be up-regulated in the inflamed liver of Mdr2 Ko mice.
We therefore set to investigate the importance of the chemokine receptors for Rantes, CCR5 and CCR1 in this model.
We examined whether depletion of CCR5 or CCR1 can attenuate liver inflammation and damage and alter tumor
development. We found that in mice lacking CCR5 but not CCR1 receptor, inflammation was significantly reduced with
a significant reduction of macrophage recruitment to the liver parenchyma. The diminished macrophage accumulation
in the liver of Mdr2:CCR5 double KO mice was associated with reduced periductal proliferation and abrogation of
fibrosis. Furthermore, the pre-neoplastic lesions that are represented by dysplastic changes in the liver of Mdr2 KO mice
are absent in the Mdr2:CCR5 double KO mice. Indeed Mdr2:CCR5 double KO mice 60% decrease in tumor incidence.
Interestingly, although knocking out CCR1 did not attenuate liver inflammation and tumor initiation, tumor progression
was reduced both in Mdr2:CCR1 and Mdr2:CCR5 double KO mice. Our results indicate that CCR5 has a critical and
unique role in the development of liver cancer that is induced by chronic liver inflammation, and can serve as a potential
target for cancer prevention and treatment.
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Frontiers in
Tumour Progression
Session 2. Monday, October 25th
Short
Talk
RAGE signaling in mouse models of inflammationassociated carcinogenesis
Tobias Pusterla ,
1
Stefano Piccolo1, Graziano Martello , Antonio Rosato , Francesco Ferrari , Andrea Manfrin , Michelangelo
2
Astrid Riehl , Lars Wiechert , Julia Németh , Ilan Stein , Silke Marhenke , Thomas
Longerich4, Christoffer Gebhardt5, Peter Schirmacher4, Arndt Vogel6, Angelika Bierhaus3, Eli Pikarsky2, Peter Angel1
and Jochen Hess1,7
1
1
1
2
6
German Cancer Research Center (DKFZ), Division of Signal Transduction and Growth Control, Heidelberg,
Germany; 2Department of Pathology and the Lautenberg Center for Immunology, Hebrew UniversityHadassah Medical School, Jerusalem, Israel; 3Department of Internal Medicine, 4Institute of Pathology,
5
Department of Dermatology, University Hospital Heidelberg, Germany; 6Hannover Medical School, Hannover,
Germany; 7Department of Otolaryngology, Head and Neck Surgery, University of Heidelberg, Germany
1
The Receptor for Advanced Glycation-End products (Rage) is a multiligand receptor and member of the immunoglobulin
super-family of surface receptors mainly involved in chronic inflammatory disorders. Rage engagement by extracellular
inflammatory mediators (S100 proteins and HMGB1) sustains and prolongs the inflammatory response mainly
via activation of NF-kB signalling. Rage and its ligands were also shown to be overexpressed in tumors, enhancing
tumor progression and metastasis by still unknown mechanisms. In order to shed light on the role played by Rage
in inflammation-associated tumor development, Rage-/- mice were studied in two different models: the DMBA-TPA
model in the skin and the Mdr2-/- model in the liver. In the DMBA-TPA skin cancer model, Rage-/- mice were protected
from tumor development. Gene expression profiling applied on wt and Rage-/- samples of TPA-treated mouse back
skin revealed that Rage signaling is only required for sustained alterations in transcript levels. Moreover we found a
significant enrichment for several transcription factor binding sites (Sp1, Tcfap2, E2f, Myc, Egr). Accordingly, we could
confirm aberrant expression and regulation of members of the E2f protein family in Rage-/- keratinocytes. In the liver, we
analyzed the role played by Rage by crossing Rage-/- with Mdr2-/- mice. Rage-/- Mrd2-/- (dKO) mice developed smaller
and fewer hepatocellular carcinomas than Mrd2-/- mice. Surprisingly lack of Rage did not affect onset of hepatitis but
rescued liver from damage: at early time points, dKO showed significant lower ALT serum levels and less liver fibrosis
than in Mdr2-/- mice. These results show that Rage-/- mice are protected in two different models of inflammation-driven
tumorigenesis. We show that Rage plays a pivotal role in the pre-malignant chronic inflammatory phase, sustaining
inflammation and tissue damage. As a consequence, the lack of Rage may create a less suitable environment for tumor
development and progression.
30
A microRNA targeting Dicer for metastasis control
3
4
1
Cordenonsi1, Sirio Dupont1, Elena Enzo1, Vincenza Guzzardo5, Maria Rondina2, Thomas Spruce6, Anna R. Parenti5, Maria
Grazia Daidone7 and Silvio Bicciato4
Department of Histology, Microbiology and Medical Biotechnologies, University of Padua School of
Medicine, Italy; 2Department of Oncology and Surgical Sciences, University of Padua, Italy; 3Istituto
Oncologico Veneto, Padua, Italy; 4Center for Genome Research, Department of Biomedical Sciences,
University of Modena and Reggio Emilia, Modena, Italy; 5Department of Medical Diagnostic Science and
Special Therapies, Section of Pathology, University of Padua, Italy; 6MRC Clinical Sciences Centre, Imperial
College London, UK; 7Department of Experimental Oncology, National Cancer Institute, Milan, Italy
1
While specific microRNAs (miRNAs) can be upregulated in cancer, global miRNA downregulation is a common trait of
human malignancies. The mechanisms of this phenomenon and the advantages it affords remain poorly understood.
Here we identify a microRNA family, miR-103/107, that attenuates miRNA biosynthesis by targeting Dicer, a key
component of the miRNA processing machinery. In human breast cancer, high levels of miR-103/107 are associated with
metastasis and poor-outcome. Functionally, miR-103/107 confer migratory capacities in vitro and empower metastatic
dissemination of otherwise-non-aggressive cells in vivo. Inhibition of miR-103/107 opposes migration and metastasis
of malignant cells. At the cellular level, a key event fostered by miR-103/107 is induction of epithelial-to-mesenchymal
transition (EMT), attained by downregulating miR-200 levels. These findings suggest a new pathway by which Dicer
inhibition drifts epithelial cancer toward a less-differentiated, mesenchymal fate to foster metastasis.
31
Frontiers in
Tumour Progression
Session 2. Monday, October 25th
Imaging the metastatic process
Erik Sahai
London Research Institute, Cancer Research UK, London, UK
The acquisition of invasive behaviour enables the tumour cells to move into either the surrounding tissue or the
vasculature and thereby spread to other parts of the body. The focus of our research is investigating why cancer cells
become invasive and how they move. Until recently research into cancer cell movement has used highly artificial
environments that do not accurately mimic the tumour environment leading to many contradictory results that could
not be confirmed in vivo. The tumour environment is highly complex with numerous non-tumour cell types, variable
blood flow and a diverse range of extracellular matrix components. To study cell motility in this environment we perform
intravital multi-photon confocal imaging of tumours in anaesthetised mice. To complement intravital imaging, we have
established a range of three-dimensional ‘organotypic’ cultures that allow us to model many aspects of the tumour
environment in vitro. By using these systems it has become clear that cancer cells in transit between primary and
secondary sites have distinct characteristics. We will discuss the changes in cell signalling and differentiation status
that occur in disseminating cancer cells.
32
Short
Talk
Invadopodia: do they exist in vivo and are they the first
step of metastasis?
Bojana Gligorijevic, Marco Magalhaes, Jeff Wyckoff and John Condeelis
Albert Einstein College of Medicine and Gruss-Lipper Biophotonic Center, New York, USA
Invadopodia are actin-rich protrusions present in metastatic cancer cells. They have been studied extensively from the
perspective of cell biology, mostly in vitro in monolayers of cells plated on collagen-based matrices. In these models,
invadopodia are defined as axial cell protrusions which degrade the surrounding matrix. Based on in vitro studies of
invadopodium size, structural components and proteolytic activity under different conditions, several invadopodium
assembly models have been proposed. Invadopodia were further assumed to be essential for invasion and metastasis.
However, the existence of invadopodia in vivo has never been observed nor tested. It is unclear, therefore, if any of
these models represent the biological reality in primary tumor sites, or if these structures are an artifact of specific
in vitro experimental conditions. Testing the presence of invadopodia in vivo, deciphering conditions for assembly
and characteristics of their structure could unify the invadopodia model. Further, in vivo studies would determine if
invadopodium formation is an essential early step in metastasis. If so, the density of invadopodia might be used as
a marker for predicting metastasis. We have used a combination of structural markers (cortactin and actin/TKS5),
morphology and detection of proteolytic activity to test if invadopodia appear in vivo. Our current data suggests the
presence of invadopodia-like structures in the primary tumors. By comparing information extracted from intravital
multiphoton microscopy and ex vivo immunohistofluorescence, we were able to demonstrate the presence of, and
quantified density of invadopodia in different microenvironments of breast carcinoma in mice. We further show that
silencing N-WASP, protein essential for the assembly of the invadopodium core, results in reduced formation and matrix
degradation of invadopodia in cell culture and in their in vivo homologs. These treatments in turn result in a significant
reduction in average cell motility in the tumor which affected the invasion, intravasation, circulating tumor cell density
and lung colonization. These findings suggest that indeed invadopodia exist in vivo and that they catalyze essential steps
in metastasis.
33
Frontiers in
Tumour Progression
Session 2. Monday, October 25th
Role of the tumor microenvironment in breast cancer
metastasis and response to therapy
Zena Werb1, Jonathan Chou , Sylvain Provot , Marja Lohela
1
1
1
and Mikala Egeblad1,2
Department of Anatomy, University of California, San Francisco, USA and 2Cold Spring Harbor Laboratory,
Cold Spring Harbor, USA
1
Both extrinsic and intrinsic mechanisms regulate the development of the aberrant tumor organ and regulate its
progression. We have used genetic and in vivo imaging techniques to study the interaction between epithelial cancer
cells and stromal cells present in the tumor microenvironment during tumor progression. Transcriptional regulation
of epithelial differentiation, cell-cell interaction and motility is a key intrinsic event in tumor progression. We have
investigated GATA3, a master regulator for luminal differentiation, which is lost upon malignant conversion, and found
that it allows the malignant tumor cells to disseminate throughout the body. The progressing tumor cells develop in an
extrinsic microenvironment rich in inflammatory cells, growth factors, angiogenesis and activated stroma that promotes
neoplastic risk. All of these components communicate with each other to contribute to the aberrant tumor organ.
The inflammatory myeloid cells in the microenvironment increase dramatically upon the malignant conversion of the
tumor cells. These myeloid cell contribute many factors to the microenvironment including MMPs. The inflammatory
cells modulate the tumor ecology, altering vascular permeability and responses to chemotherapy. The dynamic interplay
of tumor cells and host cells responding to the tumor contribute to tumor evolution and evasion of therapeutic responses.
34
35
Inflammatory
Modulators
Chair: Maria S. Soengas
CNIO, Madrid, Spain
TUESDAY, OCTOBER 26th
Session
3
Session 3
36
37
Frontiers in
Tumour Progression
Session 3. Tuesday, October 26th
Inflammation and cancer: reprogramming immune
response as an anti-cancer therapeutic strategy
Bidirectional crosstalk between mature adipocytes and
breast cancer cells stimulates tumor invasion
Lisa M. Coussens
Beatrice Dirat1,2,3, Camille Attané1,3, Yuan Yuan Wang1,2,3, Marta Dabek1,2, Stéphanie Dauvillier1,2, Ludivine Bochet1,2,3,
1,2
Philippe Valet1,3 and
Department of Pathology, Helen Diller Family Comprehensive Cancer Center, University of California, San
Francisco, USA
The concept that leukocytes are components of malignant tumors is not new; however, their functional involvement
as promoting forces for tumor progression has only recently been appreciated. We are interested in understanding
the molecular mechanisms that regulate leukocyte recruitment into neoplastic tissue and subsequent regulation those
leukocytes exert on evolving cancer cells. By studying transgenic mouse models of skin, lung and breast cancer
development, we have recently appreciated that adaptive leukocytes differentially regulate myeloid cell recruitment,
activation, and behavior, by organ-dependent mechanisms. Thus, whereas premalignant progression, including
chronic inflammation, activation of angiogenic programming, tissue remodeling and malignant conversion during skin
carcinogenesis is B cell, Ig and FcγR–dependent, during mammary carcinogenesis by contrast, TH2-polarized CD4+ T
cells play a dominant role in regulating pro-tumor and pro-metastatic properties of M2-polarized macrophages and
dendritic cells, that together regulate metastasis of malignant mammary epithelial cells to lung. To be presented will
be recent insights into organ and tissue-specific regulation of epithelial cancer development by adaptive and innate
immune cells, and thoughts on how these properties can be harnessed for effective anticancer therapeutics.
LMC acknowledges generous support from the NIH and NCI, and Department of Defense Era of Hope Scholar Award
(W81XWH-06-1-0416) and Investigator-Initiated Research Award in Mesothelioma (PR080717).
38
Short
Talk
Catherine Muller
University of Toulouse, UPS, IPBS, Toulouse, France; 2Cancer Biology Department, Institute of Pharmacology
and Structural Biology CNRS UMR 5089, Toulouse, France; 3Metabolism and Obesity Department, Institut
National de la Santé et de la Recherche médicale, INSERM U858, Toulouse, France
1
In breast cancer, early local tumour invasion results in immediate proximity of cancer cells to adipose tissue. We
demonstrated that breast tumour cells cocultivated with mature adipocytes exhibit increased invasive capacities in vitro
and in vivo. In turns, adipocytes exhibit delipidation and decreased adipocyte markers associated to the occurrence of
an activated phenotype marked by the overexpression of proteases and pro-inflammatory cytokines (IL-6, IL-1β), IL-6
playing a key role in the stimulation of tumour invasion. We named these cells, Cancer- Associated Adipocytes (CAAs).
The presence of CAAs was confirmed in human breast tumours. In addition to the contribution in the settlement of an
inflammatory state favouring tumour progression, we showed that the crosstalk between CAAs and cancer cells also
affect tumour metabolism. Free fatty acids (FFAs), released during tumour cells-induced lipolysis can be taken up by
cancer cells themselves. These FFAs are used by tumour cells to perform FA β-oxidation, an activity that we found
present at high levels in tumoral but not in normal mammary epithelial cells. Blocking FA β-oxidation, by etomoxir
specifically decreased the invasion of tumour cells that have been co-cultivated with adipocytes. Collectively, our data
brought in vitro and in vivo evidences that: i) invasive cancer cells dramatically impact surrounding adipocytes ; ii) peritumoral adipocytes (CAAs) exhibit modified phenotype and specific biological features ; iii) CAAs modify the cancer cell
characteristics/phenotype leading to their more aggressive behaviour by secreting pro-inflammatory molecules and
providing FFAs to support tumour metabolism. Our results support the innovative concept that adipocytes participate in
a highly complex vicious cycle orchestrated by cancer cells to promote tumour progression that might be amplified in
obese patients, a condition that is associated to poor prognosis and increase local and distant invasion.
39
Frontiers in
Tumour Progression
Session 3. Tuesday, October 26th
Suppression of colon cancer metastasis by Aes through
inhibition of Notch signaling
Systems biology analysis of tumor and stromal genes
in different metastatic microenvironments
Mark Taketo
Johanna Joyce1, Steve Mason , Franck Rapaport , Lisa Sevenich , Joan Massague
Kyoto University Graduate School of Medicine (Pharmacology), Japan
1
Metastasis is responsible for most cancer deaths. Here we show that Aes (or Grg5) gene functions as an endogenous
metastasis suppressor. Expression of Aes was decreased in liver metastases compared with primary colon tumors in
both mice and humans. Aes inhibited Notch signaling by converting active Rbpj transcription complexes into repression
complexes on insoluble nuclear matrix. In tumor cells, Notch signaling was triggered by ligands on adjoining blood
vessels, and stimulated transendothelial migration. Genetic depletion of Aes in ApcΔ716 intestinal polyposis mice caused
marked tumor invasion and intravasation that were suppressed by Notch signaling inhibition. These results suggest
that inhibition of Notch signaling can be a promising strategy for prevention and treatment of colon cancer metastasis.
40
1
2
1
1
and Christina Leslie2
Cancer Biology and Genetics Program and Computational Biology Program, Sloan-Kettering Institute, New
York, USA
2
Metastasis is a multistage process that requires cancer cells to escape from the primary tumor, survive in the circulation,
seed at distant sites and colonize these foreign tissue environments. Each of these processes involves rate-limiting
steps that are influenced by stromal cells of the tumor microenvironment.
Our goal is to dissect and model the complex and reciprocal interplay between the tumor and stromal cells of the
microenvironment. To this end, we devised an experimental and computational strategy to enable the simultaneous
analysis of tumor and stromal genes in lesions from three distinct metastatic microenvironments. We made use of
a recently developed system where organ-specific metastatic variants were selected in vivo from a parental breast
cancer cell line and home to the bone, lung and brain in xenografted animals. We captured the stromal contribution
by removing intact whole tumors and subjecting them to microarray analysis. An important technological advance that
has allowed us to simultaneously query tumor and stromal gene expression is the HuMu ProtIn (Protease/ Inhibitor)
array. This microarray was designed to distinguish between human (Hu) and mouse (Mu) gene expression from whole
xenograft tumors, so we can determine whether differentially expressed proteases and endogenous inhibitors are of
tumor (human) or stromal (mouse) origin.
After profiling intact bone, lung and brain metastases at different stages of development, we developed a computational
strategy for inferring tumor-stromal interactions in the metastatic microenvironment. Specifically, we used a partial least
squares (PLS) regression approach to predict stromal gene expression from tumor expression profiles and vice versa.
We have experimentally validated predicted tumor and stromal genes to confirm their causal role in the metastatic
tumor microenvironment. We then used a mutual information computation to identify the tumor genes most impacted
by our key stromal genes in the model, and vice versa. By developing statistical models that can predict stromal/tumor
interplay and its impact on tumor progression and metastasis, we have gained new insight into how cancers develop and
ultimately how we might treat cancer with combination therapies that target the tumor along with its microenvironment.
41
Frontiers in
Tumour Progression
Session 3. Tuesday, October 26th
Short
Talk
The role of stromal Pten signaling in the breast tumor
microenvironment
Angiocrine factors-derived from Akt-activated vascular
niche initiate and maintain tumor growth
Julie A. Wallace1, Anthony J. Trimboli , Anand Merchant , Lianbo Yu , Parul Gulati , Sean Cory , Gustavo
Shahin Raffi
1
1
2
2
3
Leone1 and Michael C. Ostrowski1
Tumor Microenvironment Program, Comprehensive Cancer Center, The Ohio State University, Columbus,
Ohio, USA; 2Center for Biostatistics, Office of Health Sciences, The Ohio State University, Columbus, Ohio,
USA; 3McGill Center for Bioinformatics, McGill University, Québec, Canada
1
Pten is a tumor suppressor that has been shown to impact several signaling pathways involved in cancer. Previous
work in our laboratory using conditional mouse knockouts demonstrated that specific deletion of Pten in fibroblasts
caused higher tumor incidence and tumor burden in the well characterized ErbB2 breast cancer model. Gene expression
analysis of Pten null fibroblasts revealed increased expression of many genes reportedly involved in inflammation,
and an increase in angiogenesis in tumors lacking fibroblast Pten was observed which was at least in part due to
increased Ets2 signaling. Most significantly, our mouse Pten gene signature was able to completely separate normal
human stroma from tumor stroma, implicating the relevance of our studies to human disease. As a next step, we
aimed to determine the effect of Pten signaling in fibroblasts on gene expression in other proximal cell types in the
mammary gland, namely epithelial cells, endothelial cells and macrophages, and how these changes may also be
contributing to tumor progression. Pure populations of these cells were isolated from mammary glands with or without
Pten in fibroblasts both in the absence or presence of the ErbB2 oncogene, and were subjected to microarray analysis.
Examination of genes found to be differentially regulated in macrophages revealed several cytokines implicated in
the M2 macrophage phenotype, which may implicate a role for Pten signaling in macrophage polarization in a cell
nonautonomous fashion. Most interestingly, these individual gene signatures from the different cell compartments were
able to almost perfectly separate normal human stroma from tumor stroma on their own, thus further supporting the
relevance of stromal Pten signaling in human breast cancer. Implications from these studies have the potential to shape
the future of cancer therapeutics, particularly with developing personalized treatment strategies based on both tumor
and stromal characteristics.
42
Howard Hughes Medical Institute, New York, USA
Interaction of tumor cells with their niche cells could be essential for initiation and progression of tumor growth. We have
discovered that endothelial cells (ECs) within the tumor microenvironment are not just passive conduits to deliver oxygen
and nutrients, but also establish an instructive vascular niche, which by elaboration of paracrine trophogens, known as
angiocrine factors, directly promote tumor growth (1). Activation of Akt-mTOR pathway in the ECs stimulate expression of
angiocrine factors, including Notch-ligands, IGFBPs, FGFs and TGF-modulators, that induce expansion of hematopoietic
and hepatic stem and progenitor cells (2,3). Specifically, angiocrine expression of Notch ligands and IGFBP2 promoted
expansion of authentic long-term repopulating hematopoietic stem cells (2,3), while angiocrine expression of Wnt2 and
hepatocyte growth factor (HGF) induce liver regeneration (4). Similarly, Akt-activated ECs supported long-term expansion
of hematopoietic and epithelial tumors in serum- and cytokine-free conditions. To determine the contribution of activated
ECs to tumor growth in vivo, we employed a model in which Akt1 could selectively and conditionally be activated in the
ECs. Notably, overexpression of myristoylated-Akt1 in ECs in the adult mice was sufficient to initiate and maintain tumor
growth through release of angiocrine factors. Targeting specific angiocrine factors in tumor ECs will not only provide for
an effective means to block tumor growth, but also prevent rebound hypoxia-induced angiogenesis associated with the
use of anti-angiogenic factors that induce EC-apoptosis and vascular collapse.
43
Mechanism-based
Anti-metastatic
Therapies
Chair: Manuel Hidalgo
CNIO, Madrid, Spain
TUESDAY, OCTOBER 26th
Session
4
Session 4
44
45
Frontiers in
Tumour Progression
Session 4. Tuesday, October 26th
46
Molecular mechanisms of bone metastases:
implications for therapy
Targeting the tumor vasculature: do we need to move
beyond the obvious?
Theresa Guise
Lee M. Ellis
Indiana University, Indianapolis, USA
MD Anderson Cancer Center, University of Texas, Houston, USA
Bone metastases cause significant morbidity and once housed in bone, the tumors are incurable. Tumors produce
factors which stimulate osteoclasts and osteoblasts to dysregulate normal bone remodeling. The bone microenvironment
alters the behavior of metastatic tumor cells, driving a feed-forward cycle that makes skeletal metastases refractory
to treatment and cure. Transforming growth factor beta (TGFβ) is a central factor in this vicious cycle. It is deposited
into mineralized bone matrix by osteoblasts, released and activated by osteoclastic bone resorption, and changes the
phenotype of tumor cells.
In mouse models, TGFβ blockade inhibits osteolytic bone metastases due to breast cancer prostate cancer and
melanomas by blocking tumor-produced osteolytic and prometastatic factors (PTHrP, IL-11, CTGF). It also increases
bone mass, independent of effects on cancer cells, by increasing osteoblast activity and reducing osteoclast activity.
These effects are potentiated with the use of a bisphosphonate, zoledronic acid.
Since the bone microenvironment is hypoxic, we tested the interaction between TGFβ and hypoxia signaling. We found
that bone metastases are hypoxic and 1% O2 increases hypoxia-inducible factor (HIF)1á in MDA-MB-231 breast cancer
cells. Combined treatment with 1% O2 and TGFβ additively increased mRNA expression and promoter activity of
prometastatic factors VEGF and CXCR4, suggesting that HIF1á promotes bone metastasis via crosstalk with TGFβ. Our
results show that hypoxia/HIF1á signaling promotes bone metastasis, which were inhibited when preventively HIF1á
through genetic or pharmacologic approaches. Bone metastases development was further inhibited when targeting
both HIF1α and TGFβ signaling. In contrast to beneficial findings of TGFβ blockade in osteolytic bone metastases,
TGFβ signaling blockade increases growth of the osteoblastic prostate xenograft LuCAP23.1. These effects are likely
due to the direct effect of TGFβ blockade to alter the host response by increasing osteoblast differentiation. Taken
together, these data suggest that inhibition of TGFβ signaling may be effective in osteolytic disease, but may accelerate
osteoblastic bone metastases due to its effects to stimulate osteoblast activity.
Other tumor mediators of osteoblastic metastases have been implicated, such as endothelin-1 (ET-1), and are targets
for therapy. Tumor produced-ET-1 stimulates osteoblast activity via the endothelin A receptor (ETAR). ETAR blockade
abrogates osteoblastic disease in mouse models and improves survival in men with prostate cancer metastases to
bone. ET-1 suppresses osteoblast production of the Wnt pathway inhibitor DKK1 to result in the dysregulated new bone
formation associated with osteoblastic disease. Furthermore, blockade of ETAR is associated with reduced bone mass
in bone unaffected by tumor.
In summary, tumor bone microenvironment is rich in factors that cause cancer cells to thrive. Blockade of these factors
have important implications for the skeletal health of cancer patients. The result can improve bone metastases, but may
have differential effects which depend on the osteolytic or osteoblastic metastatic phenotype. Such therapy may alter
bone remodeling at sites unaffected by tumor.
Despite a great deal of publicity regarding anti-angiogenic (more specifically, anti-VEGF) therapies, results in the clinic
vary a great deal. For instance, single agent VEGF targeted therapies are highly effective in patients with metastatic renal
cell carcinoma. However, the use of VEGF targeted agents in tumors such as melanoma or pancreatic cancer does not
show any efficacy. It is essential to understand the mechanisms of action (MOA) of these agents in order to improve our
therapeutic outcomes.
With varied results with VEGF targeted therapies, we must recognize that there are likely multiple MOAs. Potential
MOAs of VEGF targeted therapies include 1) anti-angiogenic mechanisms, 2) normalization of the vasculature, 3)
vascular constriction, 4) direct effect on tumor cells, 5) offsetting the effects of stress/chemotherapy, 6) reversal of
immunosuppression due to VEGF, and 7) disruption of the cancer stem cell niche. The relative role of each of these
mechanisms of action will be discussed.
Although the vasculature of tumors delivers oxygen and nutrients, it has also been hypothesized that endothelial cells
contribute to the growth of tumor cells in a paracrine fashion. Gilbertson and colleagues have shown that glioma stem
cells reside in the perivascular niche (Cancer Cell 2007). We studied the potential role of endothelial cell (EC) derived
paracrine factors on promoting the CSC phenotype in human colorectal cancer (CRC) cells. Co-culturing of CRC cells with
ECs markedly increased the ALDH-positive population, sphere forming ability, and increased CD133 and CD44 protein
levels. Similarly, treatment of CRC cells with conditioned medium from ECs significantly increased the ALDH-positive
population, sphere forming ability, and the expression of CD133 and CD44. Conditioned medium from ECs decreased
spontaneous apoptosis in CRC and CRC cells exposed to EC conditioned medium also displayed decreased sensitivity
to chemotherapy.
47
Frontiers in
Tumour Progression
Session 4. Tuesday, October 26th
48
Stem cell niche interactions in metastasis
Targeting the Hedgehog pathway in cancer
Joerg Huelsken
Frederic de Sauvage
ISREC, Swiss Federal Institute of Technology Lausanne (EPFL), Switzerland
Genentech Inc., South San Francisco, USA
Mortality from cancer arises primarily from metastatic spread to distant organs, however currently there is no curative
treatment which specifically targets this process. Recent evidence suggests that breast cancer and other solid tumors
possess a rare population of cells capable of extensive self-renewal that are central for tumor initiation and maintenance.
We now identify two major mechanisms which govern the formation of metastasis in a murine breast cancer model.
We characterize a small sub-population of cancer cells to be essential for the early step of metastatic colonization,
i.e. the initial expansion of cancer cell colonies in the lung. We in addition find stem cell niche interactions to be
essential for this process. We identify an extracellular matrix molecule expressed in the normal mammary gland and
in the primary tumor, to be induced by tumor cells infiltrating the lung. This ECM protein is required to allow stem cell
maintenance via regulation of Wnt signaling and blockade of its function prevents lung metastasis. This suggests a novel
treatment strategy which targets the education of host stroma cells by disseminated tumor cells to prevent de-novo
niche formation and to inhibit metastatic disease.
The Hedgehog (Hh) pathway is an ancient signaling cascade that directs patterning in most animals and is crucial
for proper development. While Hh signaling is very active during embryogenesis, it remains relatively quiet in adult
life. However, aberrant reactivation of the pathway in adult tissue can lead to the development of cancer. Hh pathway
activation in tumors such as basal cell carcinoma (BCC) and medulloblastoma is the result of inactivating mutations
in PATCHED (PTCH) or activating SMOOTHENED (SMO) mutations. Targeting the Hh pathway with small molecule
antagonists therefore provides a new therapeutic opportunity for the treatment of these tumor types. GDC-0449, a
systemic Hedgehog (Hh) pathway inhibitor, was tested in a first-in-human, first-in-class, phase I study BCC patients.
Strong anti-tumor activity was observed in patients with locally advanced, multifocal or metastatic BCC, thereby
confirming the potential benefit of inhibiting aberrant Hh signaling in tumors where the pathway is mutated. In addition
to its role in BCC and medulloblastoma, Hh pathway activation in other solid tumor types, such as pancreatic, ovarian
or colon cancer is the result of upregulation of Hh ligand expression in epithelial tumor cells, which acts in a paracrine
manner to activate the pathway in surrounding stromal fibroblasts. These may in turn provide growth factor(s) and/or a
microenvironement that promotes tumor growth directly or by affecting other compartments such as the vasculature.
Inhibition of the Hh pathway may also provide benefit in tumors where Hh ligands are overexpressed and is currently
being explored in the clinic with GDC-0449.
49
Angiogenesis and
Anti-angiogenic
Therapy
Chair: Douglas Hanahan,
ISREC, EPFL, Laussanne,
Switzerland
WEDNESDAY, OCTOBER 27th
Session
5
Session 5
50
51
Frontiers in
Tumour Progression
Session 5. Wednesday, October 27th
Targeting (lymph)angiogenesis & lymphatic metastasis
Kari Alitalo and collaborators
Haartman Institute and Finnish Institute for Molecular Medicine, Biomedicum Helsinki, University of Helsinki,
Finland
Short
Talk
MiR-146a: a new angiostatic miRNA with tumorsuppressive properties
Julie Halkein1, Karolien Castermans1, Ludovic Malvaux1, Vincent Lambert2, Agnes Noel2, Joseph A. Martial1,
1*
and Ingrid Struman1*
Sebastien P. Tabruyn
Unit of Molecular Biology and Genetic Engineering, GIGA-Research, University of Liege, Belgium; 2Laboratory
of Tumor and Development Biology, GIGA-Research, University of Liege, Belgium
1
Vascular endothelial growth factor (VEGF) stimulates angiogenesis and permeability of blood vessels via its two
receptors VEGFR-1 and VEGFR-2, but it has only little lymphangiogenic activity. The third receptor, VEGFR-3, does not
bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. Homozygous
VEGFR-3 targeted mice die around midgestation due to failure of cardiovascular development, whereas transgenic mice
expressing the VEGFR-3 ligand VEGF-C or VEGF-D show evidence of lymphangiogenesis and VEGF-C knockout mice
have defective lymphatic vessels. VEGF-C provides effective treatment of lymphedema, even in a genetic mouse model
where VEGFR-3 is mutant (Milroy’s disease). The proteolytically processed form of VEGF-C binds also to VEGFR-2, as
we have recently shown in crystal structure analysis, and is angiogenic and in vivo. Thus VEGF-C and VEGF-D appear
to provide both angiogenic and lymphangiogenic activity. VEGF-C overexpression induces capillary lymphangiogenesis
and growth of the draining lymphatic vessels, intralymphatic tumor growth and lymph node metastasis. Furthermore,
soluble VEGFR-3 and antibodies blocking VEGFR-3 inhibit embryonic and tumor lymphangiogenesis and lymphatic
metastasis. These results have indicated that paracrine signal transduction between tumor cells and the lymphatic
endothelium is involved in lymphatic metastasis. - We have recently found a role for VEGFR-3 signaling also in settings
of physiological and pathological angiogenesis. VEGF-C and VEGFR-3 blocking antibodies provided significant inhibition
of tumor angiogenesis and growth and they improved tumor growth inhibition by anti-VEGF therapy. Enhanced VEGFR-3
expression was often detected in endothelial sprouts, and blocking of VEGFR-3 signaling inhibited angiogenic sprouting.
Blocking of the Notch signaling pathway lead to widespread endothelial VEGFR-3 expression and excessive angiogenesis,
which was inhibited by blocking VEGFR-3. The Notch pathway and the PDZ domain protein CLP24 that interacts with the
VEGFR-2 and VEGFR-3 pathways were also shown to regulate lymphatic vessel growth and development.
Our studies indicate that VEGF-C and VEGFR-3 provide new targets to complement current anti-angiogenic therapies.
Furthermore, our studies indicate that antibody combinations may be used for increased efficacy of inhibition of
angiogenic signal transduction pathways.
52
MiRNAs have emerged as important players in tumor progression and angiogenesis. In the present study, using the 16
kDa N-terminal fragment of human prolactin (16K hPRL) as a model of endogenous angiostatic factor, we indentified
miR-146a as a new mediator of angiostatic response. In vitro, 16K hPRL induced up-regulation of miR-146a via a NFκB- dependent pathway. Functional assays revealed that miR-146a overexpression reduced proliferation and induced
apoptosis of endothelial cells without affecting migration and tubulogenesis. Transcriptomic analysis revealed that
expression of 389 genes mainly involved in regulation of immune response, proliferation and apoptosis was significantly
affected in endothelial cells overexpressing miR-146a. NRAS was further identified as a new target that could contribute
to miR-146a anti-proliferative action. In an ex vivo aortic ring assay miR-146a overexpression reduced sprout formation.
Furthermore, in vivo in a mouse model of age-related macular degeneration, we showed that overexpression of miR-146a
decreased pathological neovascularization. Since, miRNAs have been shown to be present in blood inside exosomes,
we then explored whether miR-146a can be used as circulating biomarker to monitor 16K hPRL angiostatic response.
Blood from B16F10 melanoma-bearing mice treated with 16K PRL displayed a significant higher level of circulating
miR-146a than the control animals. In vitro, endothelial cells treated with 16K hPRL released exosomes loaded with
a higher content of miR-146a. In addition, exosomes from endothelial cells overexpressing miR-146a interfered with
B16-F10 melanoma cell proliferation. These results suggest that, in response to angiostatic factors, endothelial cells
release miRNA-containing exosomes that affects tumor cell properties. In conclusion, our data present miR-146a as a
new angiostatic miRNA produced by endothelial cells with intrinsic effect on the endothelium and acting at distance as
a tumor-suppressor.
53
Frontiers in
Tumour Progression
Session 5. Wednesday, October 27th
Short
Talk
EphrinB2 repression mediates anti-angiogenesisinduced glioma invasion
Helge zum Buttel1*, Conny Depner2*, Fabian Finkelmeier2, Guido Reifenberger3,
1*&
and Till Acker2*&
Amparo Acker-
Palmer
Frankfurt Institute for Molecular Life Sciences (FMLS) and Institute of Cell Biology and Neuroscience,
Goethe University Frankfurt, Germany; 2Institute of Neuropathology, Giessen University, Germany;
3
Department of Neuropathology, Heinrich-Heine-University Duesseldorf, Germany; *equal contribution;
&
Corresponding author
Tumor vessel normalization: genetic insight and
therapeutic potential
Peter Carmeliet
Katholieke Universiteit Leuven (KUL), Belgium
1
Research over the last decades has focused primarily on promoting or blocking blood vessel branching. More recent
insights show that vessels in pathological disorders, especially in cancer, are often abnormal in structure and function.
Normalizing these vessels offers unprecedented opportunities for improved treatment of pro- and anti-angiogenic
disorders. We will discuss principles, players and examples of anti-angiogenic normalization.
Diffuse invasion of the surrounding brain parenchyma is one of the major obstacles in neurosurgical removal and
treatment of gliomas. Recent evidence suggests that gliomas evade anti-angiogenic therapies by induction of an
invasive phentoype. Hypoxia is a key regulator of tumor invasion however the molecular machinery responsible for
this highly invasive phenotype is currently unknown. Eph/ephrin guidance controls colorectal cancer progression by
compartmentalizing the expansion of tumor cells. Here we show that the ephrinB2 gene is progressively deleted in human
glioma strongly correlating with malignancy. Importantly, loss of ephrinB2 in murine astrocytomas increases invasion
in an orthotopic xenograft model and accelerates tumor growth. Moreover, the genetic inactivation is complemented by
a hypoxic downregulation of ephrinB2 expression. Mechanistically, we demonstrate that HIF-1alpha induces the EMT
repressor SIP1 which in turn binds to the promoter of ephrinB2 to silence its expression. In vivo, downregulation of SIP1
results in a striking reduction of tumor growth. Most importantly, downregulation of SIP1 counteracts the highly invasive
behavior of tumors induced after treatment with Avastin. Our results identify the genetic and microenvironmental
downregulation of ephrinB2 as crucial initial step that promotes tumor invasion and growth by abrogation of repulsive
signals. This novel pathway may represent an attractive therapy target to inhibit tumor invasion and counteract tumor
intrinsic mechanisms to evade current antiangiogenic treatment strategies.
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Session 5. Wednesday, October 27th
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Synergistic effects of inhibiting VEGF/VEGFR and
HGF/c-Met signaling in tumors
Preclinical modelling of adjuvant and metastatic
antiangiogenic therapies
Donald M. McDonald
Robert S. Kerbel
University of California San Francisco (UCSF), USA
Sunnybrook Health Sciences Centre and Dept. of Medical Biophysics, University of Toronto, Canada
Inhibitors that target vascular endothelial growth factor (VEGF) in tumors stop sprouting angiogenesis, prune VEGFdependent tumor vessels, and transform tumor vessels that survive are more normal. These changes in the vasculature
have complex effects on tumor cells. Pruning of tumor vessels can have multiple downstream effects including increased
intratumoral hypoxia, which in turn can activate pathways that promote invasiveness and metastasis. The mechanisms
underlying evasive resistance after inhibition of VEGF are not fully understood, but hypoxia-induced activation of HIF1α and pro-invasive pathways involving hepatocyte growth factor (HGF) and its receptor c-Met are likely factors.
Tumors in RIP-Tag2 transgenic mice treated with function-blocking anti-VEGF antibody or the multi-targeted receptor
tyrosine kinase (RTK) inhibitor sunitinib, which inhibits VEGF receptors but not c-Met, are smaller but more invasive and
metastatic. The change in tumor phenotype is accompanied by increased expression of HIF-1α and c-Met and evidence
of epithelial-mesenchymal transition (EMT) in tumor cells. Increased invasiveness and metastasis associated with antiVEGF therapy can be reduced or prevented by concurrent inhibition of c-Met. Similarly, treatment with RTK inhibitors
that blocks both VEGFR and c-Met can reduce invasiveness and reduces metastases. Together, our findings indicate that
evasive resistance that follows inhibition of VEGF signaling is accompanied by greater intratumoral hypoxia, activation
of HIF-1α, c-Met, and EMT, and can be reduced or blocked through the synergistic effects of inhibiting c-Met and VEGF
signaling together.
A long standing problem in cancer research is the frequent discordance between the often very encouraging therapeutic
results obtained in preclinical mouse models compared to using identical or similar therapeutic protocols in subsequent
phase II or phase III clinical trials. One possibility accounting for this discrepancy is that most clinical trials involve patients
with extensive visceral and advanced metastases. Though more difficult to treat, this circumstance has rarely been
modeled in preclinical mouse investigations. Likewise studies using models of neoadjuvant therapy or postoperative
adjuvant therapy of early stage microscopic disease are rare. Thus, we have developed models of both advanced and
early stage metastatic disease using human tumor xenografts for therapy studies, mainly evaluating outcomes using
various antiangiogenic drugs and/or metronomic chemotherapy.
Several findings using such models appear promising in terms of their (increased) clinical relevance, and some
unexpected discoveries have been made as well, e.g. i) in contrast to transplanted primary tumors which often respond
extremely well to single agent targeted biologic drugs such as bevacizumab (or trastuzumab), little or no effect is
observed when treating mice with advanced metastatic disease; ii) in situations where a therapy is found to be effective
when treating advanced metastatic melanoma or breast cancer, a frequent finding is the eventual emergence of brain
metastases, likely the result of prolonged survival of the mice, thus allowing microscopic brain metastases sufficient
time to grow into macroscopic lesions. In addition, metronomic chemotherapy protocols having unusually robust activity
when treating advanced metastatic disease but showing unimpressive activity when treating the same line as an
established primary orthotopic tumor transplant have been noted. A version of these protocols moved forward to phase II
clinical trial evaluation and has shown highly encouraging results, and is now being evaluated in a phase III trial. Finally,
with respect to adjuvant disease models, results have been obtained showing antiangiogenic TKIs such as sunitinib
or sorafenib can accelerate the growth of micrometastases (but not established tumors), an effect not observed with
antiangiogenic monoclonal antibodies.
Taken together, the results of this research program (which has now been extended to developing models for advanced
ovarian cancer, renal cell carcinoma, colorectal cancer, and hepatocellular carcinoma) have not only led to what may
turn out to be potentially promising clinical treatments in the clinic, but also models relevant to studying aspects of the
molecular and cell biology of metastasis.
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Session 5. Wednesday, October 27th
Determinants of malignant progression
Douglas Hanahan
ISREC, Swiss Federal Institute of Technology Lausanne (EPFL), Switzerland
The Hanahan group and a number of alumni have long investigated a first generation mouse model of cancer, the RIPTag transgenic mice that develop of multi-focal pancreatic neuroendocrine tumors via an ostensibly linear pathway of
multi-stage tumorigenesis. The stepwise lesional progression occurs in a relatively synchronous fashion, culminating
in solid (non-invasive) tumors and invasive cancers; metastasis is rare, and the model was not considered particularly
amenable to studying metastasis. The frequent but incomplete penetrance of solid tumors progressing to invasive
carcinomas has enabled insightful studies by multiple laboratories on the invasive phenotype, collectively revealing
multiple determinants of and contributors to the invasive growth capability. These include upregulation of IGF2 and its
signaling via IGF1R and IR, down-regulation of E-Cadherin and loss of cell-cell adherens function, increased activity of
cysteine cathepsin proteases and heparanase supplied by inflammatory cells, and impaired tumor angiogenesis. More
recently, we have added to this growing list the loss of desmosomal adhesion function (Chun & Hanahan, PLOS-Genetics
2010a). Additionally, we have identified a polymprphic invasion suppressor locus that can dominantly suppress invasive
growth; ALK, a receptor tyrosine kinase, is implicated as a component of the modifier locus, one that is comparatively
highly expressed by the invasion-permissive allele, whose pharmacological inhibition impairs invasive growth (Chun et
al, PNAS, 2010b). Finally, we have implicated a subtype of mouse and human PNET that appears to spawn the infrequent
liver metastases from highly invasive primary tumors, as revealed by miR, mRNA, and CGH profiling. (Olson et al 2009,
and unpublished); the met-like primary tumors may arise form an early branched pathway, suggesting that metastatic
potential can be endowed early, and is not necessarily conveyed by rare mutants arising within a primary tumor, as
envisioned by the classical linear progression model. These latter new developments will be discussed.
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Speakers’
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Short scientific biographies of speakers in
accordance with the order of the programme
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Joan Massagué, Ph.D. delineated the TGFβ pathway from membrane receptors and Smad transcription
factors to CDK inhibitors that mediate tumor suppressor responses. This pathway is now known to be
crucial in embryo development and tissue homeostasis. Its disruption causes congenital disorders and
cancer. His lab is currently identifying mechanisms that mediate metastasis by breast and lung cancers
to bones, lungs, and brain. Joan Massagué is a member of MSKCC and HHMI, and adjunct director of IRB
Barcelona. He is an elected member of the US National Academy of Sciences, the Institute of Medicine,
and EMBO.
Joan Massagué
Memorial Sloan-Kettering
Cancer Center, New York, USA
Michael Karin has dedicated his career to elucidating signaling pathways that control the genomic
response to environmental stress and inflammation and the mechanisms by which these pathways
link inflammation and microbial infections to increased cancer risk, accelerated tumor progression
and metastatic spread. His major discoveries include identification of the AP-1 (Jun:Fos) transcription
factor as a target for tumor promoters and the establishment of the Jun kinase (JNK) and IκB kinase
(IKK) signaling pathways and their role in inflammation, tumorigenesis and metastatogenesis. While
identifying basic mechanisms that link infection and inflammation to tumor development and metastatic
progression, Karin has also provided several key druggable targets that can be used for the therapy and
prevention of inflammation-driven cancers and metastatic disease.
Michael Karin
University of California San
Diego (UCSD), USA
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Dr. Tyler Jacks is the Director of the Koch Institute for Integrative Cancer Research at the MIT and an
HHMI Investigator. He has pioneered the use of gene targeting technology in the mouse to study cancerassociated genes and to construct mouse models of many human cancer types, including cancers of the
lung, brain and ovary. He is currently Past President of the American Association of Cancer Research. In
addition to other awards, Dr. Jacks was elected to the National Academy of Sciences and the Institute of
Medicine of the National Academies in 2009.
Tyler Jacks
Massachusetts Institute
of Technology (MIT),
Cambridge, USA
Michael Stratton is Director of the Wellcome Trust Sanger Institute, where he is Joint Head of the Cancer
Genome Project, and is Professor of Cancer Genetics at the Institute of Cancer Research. He qualified
in medicine at Oxford University and Guy’s Hospital, trained as a histopathologist at the Hammersmith
and Maudsley Hospitals and obtained a PhD in the molecular biology of cancer at the Institute of Cancer
Research, London.
His primary research interests have been in the genetics of cancer. He mapped to chromosome 13 and
identified the high risk breast cancer susceptibility gene BRCA2, has subsequently identified moderate
risk breast cancer susceptibility genes including CHEK2, ATM, BRIP1 and PALB2 and characterised the
histopathological features of breast cancers in individuals carrying susceptibility alleles. He identified the
gene for hereditary cylindromatosis, a highly disfiguring predisposition to adnexal skin tumours, as well
as other susceptibility genes for testis, colorectal, thyroid, and childhood cancers.
In 2000 he initiated the Cancer Genome Project at the Wellcome Trust Sanger Institute which conducts
high throughput, systematic genome-wide searches for somatic mutations in human cancer. The primary
aims of this work are to identify new cancer genes, to understand processes of mutagenesis and to
reveal the role of genome structure in determining abnormalities of cancer genomes. Through these
studies he discovered mutations in the BRAF gene in malignant melanoma, mutations of the ERBB2
gene in lung cancer, and mutations of histone methylases and demethylases in renal and other cancers.
He has described the basic patterns of somatic mutation in cancer genomes and used next generation
sequencing to generate catalogues of somatic mutations in cancer genomes.
Michael Stratton
The Wellcome Trust Sanger
Institute, Hinxton, UK
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I am a physician-scientist with a long-standing interest in the IL-6/JAK/Stat3 signaling pathway. My
laboratory focuses on the mechanisms by which IL-6/Jak/Stat3 signaling mediates tumorigenesis (in
models of breast, lung, melanoma and thyroid cancer). My group began studying the mechanisms of
Stat3 activation in epithelial tumors and determined that aberrant regulation of IL-6 was the principal
cause of persistent Stat3 phosphorylation. Importantly, disruption of this pathway in models of lung
and breast cancer using a number of approaches (biochemical and genetic) has led to inhibition of
tumorigenesis. We are studying the molecular mechanisms by which abrogation of this pathway
modulates cancer development and growth and growth. These studies include the cross-talk between
cancer cells and the host cells or microenvironment and the mechanisms by which the PI3K, Ras-RafMEK-ERK and Jak/Stat3 pathway are dependent upon each other. We are also determining the most
effective dosing schedule of targeted inhibitors to these critical pathways.
Jacqueline F.
Bromberg
Dr. Hunter received a B.S. in biochemistry with Highest Honors from the Pennsylvania State University in
1985 and a Ph.D. in Biology with a focus on retrovirology from the Massachusetts Institute of Technology
in 1991. He did his postdoctoral fellowship in mouse genetic and genomics, also at MIT, before joining
the faculty of the Fox Chase Cancer Center in 1996. In 1999, he joined the Laboratory of Population
Genetics at the National Cancer Institute as an Investigator and became a Senior Investigator in 2007. He
currently is a member of the Laboratory of Cancer Biology and Genetics at the NCI.
Kent W. Hunter
Centre for Cancer
Research, National
Cancer Institute (NCI),
Bethesda, USA
Memorial Sloan-Kettering
Cancer Center, New York, USA
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Prof. Dr. Hartmut Beug (HB), born March 10th 1945, studied biology at Freiburg University (-1964).
Diploma thesis and Ph-D work: Dictyostelium cell adhesion. Since 1973, he worked as a postdoc and
group leader at DKFZ and EMBL, pursuing cooperative work describing the identification, characterization
and cellular readouts of three leukaemia oncogenes (erbA, erbB, myb-ets). In 1988, he took up a senior
scientist position at the IMP Vienna, where he identified mechanisms in self renewal of normal and
leukemic erythroid progenitors. At IMP. he also worked on epithelial to mesenchymal transition (EMT) and
metastasis in breast cancer, characterizing loss of epithelial polarity and signalling pathways involved.
Hartmut Beug
Research Institute of
Molecular Pathology (IMP),
Vienna, Austria
Stefano Piccolo is Professor of Molecular Biology at the University of Padua School of Medicine. He
worked with Eddy De Robertis at HHMI-UCLA, where he unveiled how extracellular antagonism to
growth factors is a key mechanism by which neural tissues is induced. Dr. Piccolo’s is interested in
how cells integrate and transduce distinct extracellular signals to regulate cell fate in a quantitative and
combinatorial fashion. His group contributed to the fields of embryonic development, cancer growth and
metastasis.
Stefano Piccolo
University of Padova, Italy
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Erik Sahai currently runs the Tumour Cell Biology laboratory at the Cancer Research UK London Research
Institute. His group aims to understand the process of cancer metastasis. In particular, his research
focuses on why cancer cells become invasive and the mechanics of how they move around. The tumour
environment is highly complex with numerous non-tumour cell types, variable blood flow and a diverse
range of extracellular matrix components. To study cell motility in this environment his group perform
intravital multi-photon confocal imaging of tumours in anaesthetised mice. To complement intravital
imaging, a range of three-dimensional ‘organotypic’ cultures are used. These allow many aspects of
the tumour environment to be modelled in vitro. These approaches have led his group to focus on
heterogeneity in cell signalling within tumours, the role of stromal fibroblasts, and the mechanism of both
single and collective cancer cell motility.
Before setting up his group in 2004 he worked with John Condeelis in New York. During this time he
learnt how to image cell motility in living. This followed on from his post-doctoral work studying cancer
cell behaviour in 3D environments with Chris Marshall. His PhD thesis work was carried in the lab of
Richard Treisman.
Erik Sahai
London Research Institute,
Cancer Research UK,
London, UK
Dr. Zena Werb received her B.Sc. in Biochemistry from the University of Toronto, and her Ph.D. in Cell
Biology from Rockefeller University. After postdoctoral studies at the Strangeways Research Laboratory
in Cambridge England, she was recruited to the faculty of the University of California, San Francisco,
where she is currently Professor and Vice-Chair of Anatomy. Dr. Werb is a member of the UCSF Diller
Family Comprehensive Cancer Center and the The Eli and Edythe Broad Center of Regeneration Medicine
and Stem Cell Research at the University of California, San Francisco.
She is recognized internationally for her fundamental discoveries about the molecular and cellular bases
of extracellular matrix proteolysis and their roles in the normal functioning and pathogenesis of tissues.
Her studies have led to new paradigms about the role of the cellular microenvironment and intercellular
communication in breast development and cancer. Her honors include a Guggenheim Fellowship, the
FASEB Excellence in Science Award, the Charlotte Friend Award of the American Association for Cancer
Research and the E.B. Wilson Medal from the American Society of Cell Biology. Dr. Werb is an elected
member of the National Academy of Sciences and of the Institute of Medicine, and a fellow of the
American Academy of Arts and Sciences. She has the honorary degree of Doctor in Medicine from the
University of Copenhagen. She has been an elected officer of the American Society for Cell Biology and
the American Association for Cancer Research. She has published more than 400 papers. She serves
or has served on the editorial boards of Science, Cell, Cancer Cell, Developmental Cell and Genes and
Development.
Zena Werb
University of California
San Francisco (UCSF), USA
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Dr. Coussens is a Professor in the Pathology Department and Helen Diller Family Comprehensive Cancer
Center at the University of California, San Francisco. The Coussens lab focuses on the role of immune
cells and leukocyte proteases as critical regulators of solid tumor development, including skin, lung (non
small cell and mesothelioma) and breast cancer development. She is currently Co-Director of the Mouse
Pathology Core and the Cancer, Immunity and Microenvironment Program at UCSF.
Educated in Kyoto University (MD, PhD, 1978). After postdoctoral training and faculty positions in USA, he
returned to Japan (Banyu-Merck) in 1992. In 1996, he accepted a professorship at University of Tokyo,
and moved to Kyoto University in 2000.
These ~15 years, he has been working on mouse models of colon cancer. He constructed Apc knockout
mice (FAP model), established the role of COX2 in adenoma expansion, constructed invasive colon cancer
model (Apc/Smad4 compound mutant), found the critical role of BM derived cells in invasion, and showed
the possibility of preventing liver metastasis by blocking BM cell recruitment.
Lisa Coussens
University of California San
Francisco (UCSF), USA
Mark Taketo
Kyoto University Graduate
School of Medicine, Japan
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Johanna Joyce is an Assistant Member in the Cancer Biology Program at Memorial Sloan Kettering
Cancer Center (MSKCC), New York, USA and an Assistant Professor in Cornell University Graduate School
of Medical Sciences. She received her Ph.D. in Biology from the University of Cambridge, UK, and did her
postdoctoral training with Doug Hanahan at UCSF, USA. She joined the MSKCC in December 2004, and
was named to a Geoffrey Beene Junior Faculty Chair in 2007. Her research interests are to understand
the mechanisms by which stromal cells in the tumor microenvironment regulate cancer development,
metastasis, and response to therapy, with a particular focus on the roles of bone-marrow derived cells
and proteasest.
Johanna Joyce
Memorial Sloan-Kettering
Cancer Center, New York, USA
Shahin Rafii, MD has been studying the role of vascular niche in the regulation homeostasis of organspecific normal and malignant stem and progenitors. He has identified specific endothelial-derived
paracrine trophogens, known as angiocrine factors, that regulate stem cell self-renewal and tumor
initiation. He has developed an innovative technology to generate abundant stable and functional
endothelial cells from human embryonic stem cells that could be therapeutically transplanted for organ
or employed for tumor targeting. He is Arthur B. Belfer Professor of Genetic Medicine, HHMI investigator
and Director of Ansary Stem Cell Institute at Weill-Cornell Medical College in New York.
Shahin Rafii
Howard Hughes Medical
Institute, New York, USA
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Theresa Guise
Indiana University School
of Medicine, Indianapolis,
USA
Theresa A. Guise, M.D., is Professor of Medicine and Jerry W. and Peg S. Throgmartin Professor of
Oncology in the Department of Medicine, Division of Endocrinology at Indiana University. She graduated
from the University of Pittsburgh School of Medicine in 1985 and completed an internal medicine
residency in 1988. A fellowship in endocrinology and metabolism followed at University of Texas from
1989 to 1992. She served on the Endocrinology faculty of the University of Texas Health Science Center
at San Antonio until-2002, where she also held the Zachry Chair for Translational Research at the
Institute for Drug Development of the Cancer Therapy and Research Center. She joined the University of
Virginia in 2002 as the Gerald Aurbach Professor in the Division of Endocrinology and Mellon Investigator
of the University of Virginia Cancer Center, where she directed a research in skeletal complications of
malignancy and the Metabolic Bone Disease Clinic until July 2009 at which time she moved to Indiana
University. She is an Associate Editor for The Journal of Bone and Mineral Research and serves on
the editorial boards of Bone, BoneKEy and Clinical Translational Research as well as on the Board of
Directors for the International Bone and Mineral Society, the Paget Foundation for Bone Diseases, Bone
and Cancer Foundation and Cancer and Bone Society. She received the Fuller Albright award of the
American Society for Bone and Mineral Research in 1999, was elected to the American Society for
Clinical Investigation (ASCI) in 2004 and the Association of American Physicians in 2008. Dr. Guise
served as Secretary-Treasurer and Councilor of the ASCI (2006-2010) and permanent member of NIH
study section for Skeletal Biology and Skeletal Regeneration (2004-2009; Chair 2008, 2009). She is
currently Vice President of the Cancer and Bone Society and has been appointed to the Susan Komen
Scientific Advisory Council. Her research interests lie in the area of skeletal complications of malignancy
with specific interest in the molecular mechanisms of bone metastases.
Lee Ellis graduated from the University of Virginia School of Medicine, completed his surgical residency
at the University of Florida, and completed a surgical oncology fellowship at the M. D. Anderson Cancer
Center. He is a member of the NCI GI Cancers Steering Committee. Dr. Ellis serves on 8 editorial boards,
including JCO, Cancer Research and Clinical Cancer Research (Senior Editor). Dr. Ellis has authored over
300 publications, 3 books, and 30 book chapters. He has authored editorials in Nature, NEJM, Nature,
Cell, Cancer Cell, Nature Medicine, JCO, and Lancet Oncology. Dr. Ellis plays a leadership role in ASCO,
AACR, and Keystone Scientific Symposia.
Lee Ellis
MD Anderson Cancer
Center, University of
Texas, Houston, USA
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Prof. Joerg Huelsken received his PhD in 1998 at the Humboldt University and did postdoctoral research
in the laboratory of Walter Birchmeier at the Max-Delbrueck Center for Molecular Medicine, Berlin. He
joined ISREC as an associate scientist and an NCCR project leader in January 2003 and, in 2005, was
nominated Assistant Professor at the EPFL School of Life Sciences.
Frederic de Sauvage obtained his PhD summa cum laude from the Catholic University of Louvain in
Belgium. He joined the laboratory of David Goeddel at Genentech as a postdoctoral fellow in 1990 and
was hired as a Scientist in 1992. In 1994, Dr. de Sauvage’s team at Genentech discovered Thrombopoietin
(TPO), the long sought regulator of platelet production. His laboratory made many discoveries in the field
of hematopoeisis before switching his focus to the Hedgehog pathway in the late 1990s. His work led
to the development of GDC-0449, a Hedgehog Pathway Inhibitor currently tested in clinical trials for the
treatment of various cancers.
Joerg Huelsken
ISREC, Swiss Federal
Institute of Technology
Lausanne (EPFL),
Switzerland
Frederic
de Sauvage
Genentech Inc.,
South San Francisco, USA
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Dr. Kari Alitalo is a tenured Research Professor of the Academy of Finland and the Scientific Director
of the Molecular/Cancer Biology Program and Centre of Excellence in Cancer Biology at University of
Helsinki. Significant achievements by Dr. Alitalo include the discovery and characterization of the first
lymphangiogenic growth factor VEGF-C, and the isolation of lymphatic endothelial cells. He has also
been central in the characterization of the receptors for VEGF-B, VEGF-C and VEGF-D. He has invented
molecular therapies for lymphedema that are now entering clinical trials. He has elucidated the role of
the VEGF-C receptor VEGFR-3 in tumor angiogenesis. Furthermore, his studies demonstrated the central
role of VEGF-C/VEGFR-3 signaling in tumor lymphangiogenesis and tumor metastasis.
Kari Alitalo
Prof. Peter Carmeliet has been studying the molecular basis of neurovascular biology and disease, with
a focus on angiogenesis and neurodegenerative disorders.
Since 1991, P. Carmeliet has focused his interest on blood vessels. In doing so, he has made contributions
to the understanding of how blood vessels form (angiogenesis), how they function in normal health
and how their abnormal growth or malfunctioning contributes to diseases such as cancer, ischemic
heart disease and neurodegeneration. His research efforts utilize a variety of approaches to identify
and characterize new factors involved in angiogenesis, and validate therapeutic strategies in relevant
in vivo models of human disease. In particular, he studied the role of VEGF, PlGF, hypoxia-signaling
and oxygen sensing in angiogenic disorders and vessel growth and investigate how vessel pattering
is regulated by guidance molecules. Novel gene candidates, involved in (lymph)angiogenesis and the
link between angio/neurogenesis, are also studied in (small) animal models, like zebrafish, Xenopus
tadpoles and mouse models. The latest research line studies the role of oxygen sensors at the crossroad
of metabolism and angiogenesis. Promising results can be translated to preclinical studies identifying
new therapeutic approaches in pathological conditions characterized by vessel growth disregulation.
Dr. Carmeliet was promoted to full Professor at the University of Leuven and is currently Director of the
Vesalius Research Center, VIB-KU Leuven. He trained more than 60 pre- and postdoctoral students. and
published over 350 research articles and reviews in peer reviewed journals, with more than 33.000
citations.
Peter Carmeliet
Katholieke Universiteit
Leuven (KUL), Belgium
University of Helsinki,
Finland
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Professor McDonald is a Member of the Comprehensive Cancer Center, Senior Staff Member of the
Cardiovascular Research Institute, and Professor of Anatomy at the University of California, San Francisco.
His research focuses on cellular mechanisms of angiogenesis, with the goal of understanding how blood
vessels can be used as targets for treatment of cancer and how angiogenesis inhibitors can be used to
reduce tumor growth, invasion, and metastasis.
Professor McDonald’s laboratory revealed that inhibitors of VEGF can reduce the formation of new
tumor vessels, prune existing tumor vessels, and induce changes in other tumor vessels that favor more
efficient delivery of anti-cancer drugs. His research team discovered that blood vessels in tumors rapidly
regrow after VEGF inhibitors are stopped, thereby justifying sustained use of the drugs or implementation
of alternate strategies to stop the regrowth. He is currently studying the next generation of angiogenesis
inhibitors that hold even greater promise for helping patients with cancer by reducing tumor invasiveness
and metastasis by blocking pathways that promote epithelial-mesenchymal transition in addition to
slowing tumor growth by stopping angiogenesis.
Donald
McDonald
Professor McDonald has published more than 180 research articles, teaches medical students, graduate
students, postgraduate scientists, and practicing clinicians, and has given hundreds of lectures at major
medical centers, hospitals, and research and medical conferences throughout the world on mechanisms
of angiogenesis and the complex actions of angiogenesis inhibitors on tumors.
Dr. Robert Kerbel is a Professor and Canada Research Chair in Tumor Biology, Angiogenesis and
Antiangiogenic Therapy at the University of Toronto. He is recognized for his many contributions to the
fields of metastasis and tumor angiogenesis research. Among the most notable of these are multiple
discoveries concerning metronomic chemotherapy, especially with respect to its mechanistic basis and
its application for the treatment of advanced metastatic disease. He also established a link between
the fields of oncogenes and tumor angiogenesis, uncovered new mechanisms of acquired resistance to
antiangiogenic drugs and how they may enhance chemotherapy efficacy, and alter tumor progression.
Robert S. Kerbel
University of Toronto,
Canada
University of California San
Francisco (UCSF), USA
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Douglas Hanahan, PhD, is currently Director of the Swiss Institute of Experimental Cancer Research
(ISREC), and Professor of Molecular Oncology in the School of Life Sciences at EPFL in Lausanne.
Hanahan received a bachelor’s degree in Physics from MIT, and a Ph.D. in Biophysics from Harvard,
where he was a Harvard Junior Fellow. He worked at Cold Spring Harbor Laboratory first as a graduate
student and then as a faculty member, before moving in 1988 to UCSF where he was until earlier
this year a Professor in the Dept. of Biochemistry & Biophysics and a member of the Comprehensive
Cancer Center and the Diabetes Center. His accomplishments have been recognized by appointments
as an American Cancer Society Research Professor (2000), a Fellow in the American Academy of Arts
& Sciences (2007), a member of the Institute of Medicine of the US National Academies (2008), and a
member of the US National Academy of Science (2009).
Douglas
Hanahan
ISREC, Swiss Federal
Institute of Technology
Lausanne (EPFL),
Switzerland
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Session A
Poster
Session A
Poster
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Poster Session A
Increased anti-tumor efficacy and prolonged
survival with Aflibercept (VEGF Trap) plus docetaxel
combination treatment is associated with enhanced
changes to tumor vasculature
Cristina Abrahams, Baosheng Li, Asma Parveen and Gavin Thurston
1
Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, Spain; 2Oncología Médica, Institut
d’Investigacions Biomèdiques Agustí Pi y Sunyer (IDIBAPS), Institut Clínic de Malalties Hemato-Oncològiques
(ICMHO), Hospital Clínic, Barcelona, Spain; 3Cirugía Torácica, Hospital Clinic, Barcelona, Spain; 4CIBER de
Enfermedades Respiratorias, Bunyola, Spain
Numerous preclinical and clinical studies have cited that VEGF blockade in combination with chemotherapy increases
anti-tumor efficacy. However, the mechanistic basis underlying improved effects resulting from combination therapy
is not well understood. This study focused on characterizing the mechanisms for enhanced effects of aflibercept plus
docetaxel on HT1080, a human fibrosarcoma line that is resistant to anti-VEGF treatment. Immunodeficient SCID
mice bearing subcutaneous HT1080 tumors (size ~100 mm3) were treated every 3-4 days with either control protein
(hFc) plus docetaxel vehicle, aflibercept, docetaxel, or the combination. Following three treatments, tumors were left
to grow until the average size of the treatment group reached ~500mm3. We observed that combination treatment
with aflibercept plus docetaxel was significantly more efficacious than either single agent. Although treatments with
docetaxel alone and in combination with aflibercept both initially regressed tumor size, the combination was significantly
more effective at delaying long-term tumor regrowth. Moreover, two treatment cycles of combination treatment with
aflibercept plus docetaxel further delayed tumor growth and prolonged survival compared to tumors that were given one
treatment cycle. Analysis of tumors harvested 24 hrs after the end of the first treatment cycle revealed that combination
treatment produced a dramatic increase in pyknotic nuclei and decrease in the number of proliferating cells. Strikingly,
Aflibercept or docetaxel alone had only minimal effects on the tumor vessels, whereas the combination treatment
resulted in pruned vessels with less branching, an increase in TUNEL-positive cells that are associated with the tumor
vasculature, and a marked decrease in tumor pericytes. These results suggest that improved efficacy observed in
tumors that are impervious to VEGF blockade may be due to taxanes (docetaxel) sensitizing the vasculature to anti- VEGF
treatment (aflibercept), thus resulting in enhanced effects on the tumor vessels. Studies like this aimed at understanding
mechanisms underlying the increased effectiveness of combination therapy may facilitate the improvement of current
treatment regimens to treat certain patients with anti-angiogenic and chemotherapy agents.
1
2
Caveolin-1-independent fibroblast activation in lung
squamous cell carcinoma
Jordi Alcaraz1,4, Marta Puig
, Alícia Giménez1, Abel Gómez3, Pere Gascón2 and Noemi Reguart2
1,2
Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, Spain; 2Oncología Médica, Institut
d’Investigacions Biomèdiques Agustí Pi y Sunyer (IDIBAPS), Institut Clínic de Malalties Hemato-Oncològiques
(ICMHO), Hospital Clínic, Barcelona, Spain; 3Cirugía Torácica, Hospital Clinic, Barcelona, Spain; 4CIBER de
Enfermedades Respiratorias, Bunyola, Spain
1
Lung squamous cell carcinoma (SCC) is the most frequent form of non-small cell lung cancer, which is the leading
cause of cancer death in western countries. Because tumor associated fibroblasts (CAFs) have been involved in tumor
initiation, growth, invasion, angiogenesis and metastasis in a variety of solid tumors including lung cancer, there is
growing interest in developing specific therapies against CAFs-dependent tumor-progression effects. While our
understanding of the mechanisms underlying these tumor-progression effects is still scarce, recent studies in breast
cancer support the hypothesis that loss of caveolin-1 (cav1) expression is necessary to transform normal fibroblasts
into tumor-promoting CAFs. However, this hypothesis has not been examined in lung SCC. To address this question, we
examined the expression of cav1 and alpha-smooth muscle actin (a-sma), a common marker for activated fibroblasts
or myofibroblasts, in primary human lung fibroblasts isolated from either tumor-free regions or SCC tissue. Cultured
CAFs from SCC tissue exhibited increased spreading, reduced proliferation and increased a-sma expression (all markers
of fibroblast activation) compared to non-cancer associated fibroblasts (NCAFs). In contrast, cav1 expression and
localization was similar in both NCAFs and CAFs. Our findings reveal that the tumor microenvironment of lung SCC is
enriched in activated fibroblasts, and suggest that, unlike breast cancer, loss of cav1 is not required for these fibroblast
alterations.
Poster
88
89
3
Frontiers in
Tumour Progression
Poster Session A
4
Distinctive clinicopathological associations of
amplification of the cortactin gene at 11q13 in head
and neck squamous cell carcinomas
New old friend: MTA inhibits melanoma cell
proliferation and halts melanoma tumor progression
Gustavo Jesús Alvarez-Alija , Sofia T. Menéndez , Dario García-Carracedo , Eva
Matias Avila and Juan A. Recio
1
1
1
Allonca1, Gonzalo Mancebo2, Carlos Suárez2, Juan Pablo Rodrigo2 and Juana Maria García-Pedrero1
Instituto Universitario de Oncología del Principado de Asturias, Hospital Universitario Central de Asturias,
Universidad de Oviedo, Spain; 2Servicio de Otorrinolaringología, Hospital Universitario Central de
Asturias, Universidad de Oviedo, Spain
Pedro Andreu Pérez, Javier Hernandez-Losa, Rosa Gil, Judit Grueso, Ana Pujol, Javi Cortés,
Institut de Recerca del Hospital Vall d´Hebron, Barcelona, Spain
1
Amplification of the 11q13 region is a prevalent genetic alteration in head and neck squamous cell carcinoma (HNSCC).
We investigated the clinical significance of cortactin (CTTN) and cyclin D1 (CCND1) amplification in HNSCC tumour
progression. CTTN and CCND1 amplification was analysed by differential and realtime PCR in a prospective series
of 202 laryngeal/pharyngeal carcinomas. CTTN mRNA and protein expression were respectively determined by realtime RT-PCR and immunohistochemistry, and correlated with gene status. Molecular alterations were associated with
clinicopathological parameters and disease outcome. CTTN and CCND1 amplifications were respectively found in 75
(37%) and 90 (45%) tumours. Both correlated with advanced disease; however, only CTTN amplification was associated
with recurrence and reduced disease-specific survival (p = 0.0022). Strikingly, CTTN amplification differentially
influenced survival depending on tumour site (p = 0.0001 larynx versus p = 0.68 pharynx) and was an independent
predictor of reduced survival in the larynx (p = 0.04). Furthermore, CTTN overexpression correlated significantly with
reduced disease-specific survival (p = 0.018). Taken together, these data indicate that CTTN may serve as a valuable
biomarker to identify patients with laryngeal tumours at high risk of recurrence and poor outcome.
Melanoma is the most deadly form of skin cancer without effective treatment, it has very poor prognosis and its
incidence rates are increasing every year. Several genetic studies have shown that the genomic region 9p21-22 is
homozygously deleted in melanoma, removing important genes as p14arf, MTAP and CDKN2A (a tumor suppressor
gene) whose deletion is directly related with the development of melanoma. Recently it has been reported that lost
of MTAP can be by itself a risk factor in melanoma. MTAP is a key enzyme in the methionine salvage pathway and
controls the levels of Methylthioadenosine (MTA) a nucleoside with differential effects on normal and transformed cells.
MTA has been widely demonstrated to promote anti-proliferative and pro-apoptotic responses in different cell types.
In our study we have assessed the therapeutic potential of MTA in melanoma treatment. To investigate the therapeutic
potential of MTA we performed in vitro proliferation assays in different mouse and human melanoma cell lines wild type
for RAS and BRAF or harboring different mutations in RAS pathway. We also have tested its therapeutic capabilities in
vivo in a xenograft mouse melanoma model and using a wide range of molecular techniques and tissue culture we
investigated its anti-proliferative and pro-apoptotic properties. In vitro experiments showed that MTA treatment inhibited
melanoma cell proliferation and viability in a dose dependent manner, where BRAF mutant melanoma cell lines appear
to be more sensitive. Importantly, MTA was effective inhibiting in vivo tumoral growth. The molecular analysis of tumor
samples and in vitro experiments indicated that MTA induces cytostatic rather than pro-apoptotic effects inhibiting the
phosphorylation of Akt and S6 ribosomal protein and inducing the down-regulation of cyclin D1. Our study shows how
MTA inhibits melanoma cell proliferation and in vivo tumor growth, revealing a new potential of this naturally occurring
drug for melanoma treatment.
Poster
90
91
Frontiers in
Tumour Progression
Poster Session A
Molecular subtypes of pancreatic adenocarcinoma with
different biological characteristics and therapeutical
responses
Sadanandam Anguraj,
Eric A. Collisson, Peter Olson, William J. Gibb, Morgan Truitt, Shenda Gu,
Janine Cooc, Jennifer Weinkle, Grace E. Kim, Lakshmi Jakkula, Heidi S. Feiler, Andrew H. Ko, Kathleen L. Danenberg,
Margaret A. Tempero, Paul T. Spellman, Douglas Hanahan and Joe W. Gray
Swiss Institute of Experimental Cancer Research (ISREC), EPFL, Lausanne, Switzerland
Pancreatic ductal adenocarcinoma (PDA) is a lethal disease. Presentation is late, so surgical cure is attempted in only
a minority, and resected patients usually recur quickly with incurable, metastatic disease. Metastatic PDA patients
are typically treated with gemcitabine, alone or in combination with other chemotherapeutic agents, with only modest
clinical benefit. Recent studies in other cancers demonstrate the utility of improving therapy by developing cancer
subtype specific treatments. Development of subtype specific therapies for PDA is urgently needed. We describe here a
broadly integrative approach to PDA subtype discovery and analysis that identifies gene expression based PDA subtypes
with different prognosis in primary patient samples and couples them with biological information and subtype specific
drug responses observed in human and mouse PDA cell line panels cultured and treated in vitro.
5
6
A novel function for Cyclin A2: control of cell invasion
and metastasis via RhoA signaling
Nikola Arsic,
Nawal Bendris, Bénédicte Lemmers, Marion Peter, Christina Begon, Cosette Rebouissou, Gilles
Gadéa, Frédéric Bibeau and Jean Marie Blanchard
Institut de Génétique Moléculaire de Montpellier CNRS, France Université Montpellier 2, France Université
Montpellier 1, France; 2Centre de Recherche en Biochimie Macromoléculaire CNRS, Montpellier, France;
3
CRLC Val d’Aurelle-Paul Lamarque Montpellier, France
1
Cyclin A2 plays a key role at two critical points in the cell cycle: during the S phase, where it activates CDK2, and
during the G2 to M transition where it activates CDK1. Even though Cyclin A2 over-expression has been detected in
some tumours, its precise role in adult physiopathology is still elusive due to the fact that its depletion resulted in very
early embryonic mortality. We were recently able to demonstrate a novel function for Cyclin A2 in the regulation of actin
cytoskeleton dynamics and cell motility. Using an shRNAmediated knock down approach two major phenotypes were
observed. The first one, in accordance with previous data consisted in a cell accumulation at the G2/M boundary of the
cell cycle. In addition, depletion of Cyclin A2 had no significant effect on proliferation of several adult cell lines. The second
phenotype was completely unexpected. Namely, Cyclin A2-depleted cells displayed significant morphological changes
correlating with a rearrangement of actin cytoskeleton and a redistribution of adhesion plaques. These morphological
changes were observed in all types of cells tested: primary cell lines, established or transformed cells from both human
and mouse origin. All these changes resulted in increased motility and invasion abilities of the cells together with a
reduced activity of RhoA GTPase, a key regulator of cell migration. Conversely, forced expression of Cyclin A2 had a
completely opposite effect on all these properties: migration, 3D matrix invasion and RhoA activity. Even more interesting
was the fact that this novel role of Cyclin A2 was independent of its kinase activating ability and rather mediated by
its direct binding to RhoA. Finally, Cyclin A2 was found differentially expressed in matched human primary tumors and
their corresponding metastases. We propose that Cyclin A2 is a multifunctional protein involved in cell cycle control,
cytoskeletal rearrangements, cell migration and cancer metastasis spread.
Poster
92
93
Frontiers in
Tumour Progression
Poster Session A
7
8
Investigating c-Fos functions in liver cancer using tetswitchable mouse models
ERp29 induces phenotypic change of basal-like breast
cancer cells
Latifa Bakiri, Holly A. Gefroh and Rainer Hamacher and Erwin F. Wagner
I Fon Bambang1, Yuan Kun Lee
Genes, Development and Disease Group, BBVA Foundation-Cancer Cell Biology Programme, Spanish
National Cancer Research Centre (CNIO), Madrid, Spain
Manipulation of the mouse genome to generate gain- or loss-of function mice has provided powerful tools for
understanding the molecular processes governing development and pathogenesis. We have previously demonstrated
that the protooncogene c-Jun, a component of the dimeric AP-1 transcription factor, is required for chemically-induced
mouse liver tumorigenesis. The in vivo function of the c-Jun partner c-Fos, in liver cancer remains to be defined. We
have utilized a recombinase-mediated single-copy transgene integration strategy in Embryonic Stem (ES) cells to rapidly
and efficiently generate mice carrying a tetracycline (tet)-switchable allele of c-Fos as well as Jun~c-Fos forced dimers.
These mice allow temporal, dose-dependent, reversible, but also tissuespecific expression of c-Fos monomers and
forced dimers in vivo. Our initial results indicate that broad ectopic expression of c-Fos using a tetracycline activator
expressed from the Rosa26 locus is lethal in adult mice with signs of liver dysfunction. When c-Fos dimerization
was restricted to a single partner c-Jun, the resulting Jun~c-Fos expressing mice survived and exhibited enlarged
fibrotic livers with multiple cancer nodules. Histological analyses indicated that broad ectopic expression of Jun~cFos triggers spontaneous liver inflammation, fibrosis, hepatocytes and bile duct hyperproliferation, loss of normal liver
architecture and carcinogenesis. Interestingly, liver function parameters were reverted to normal values and tumor
nodules largely decreased, when Jun~c-Fos expression was turned off, indicating a requirement for sustained Jun~cFos expression for the maintenance of the liver cancer phenotype. We plan to exploit this novel mouse model to address
the interaction between c-Fos/AP-1 and other genes functionally implicated in liver tumor development such as p53.
We are also investigating the possible cell-autonomous involvement of hepatocytes in tumor development using liver
and hepatocyte-spec.
2
and Daohai Zhang1
Department of Pathology, National University of Singapore; 2Department of Microbiology, National University
of Singapore
1
Endoplasmic reticulum protein-29 (ERp29) is a novel ER resident playing a critical role in protein unfolding and secretion.
However, its function in cancer progression has not been fully investigated. While differential expressions of ERp29
between tumor and non-tumor tissues, as well as tumor tissues within different stages have been reported, its correlations
with tumor progression in various cancers have been inconsistent. We have previously shown that ERp29 is a potential
tumor suppressor in breast cancer. Importantly, overexpression of ERp29 in MDA-MB231 cells resulted in Mesenchymal
to Epithelial Transition (MET), characterized by morphological change from lamellipodium-type of extensions to a more
cohesive and epithelioid phenotype. This phenotypic change was further verified by cytoskeletal rearrangement, loss
of mesenchymal markers and gain of epithelial markers. Further study demonstrated that ERp29 could upregulate
transcription repressor of E-cadherin (e.g. Slug, Twist, and Ets-1). Moreover, overexpression of ERp29 in MDA-MB-231
cells upregulated expression of β- catenin and translocated it to membrane where it forms complexes with Ecadherin.
This suggests the involvement of Wnt/β-catenin signaling in the ERp29- regulated MET. Similar phenotypic change
was also observed in another ERp29- transfected basal-like breast cancer cell line, BT549, with a rearrangement of
cytoskeleton. However, there were no significant changes in the expression of epithelial and mesenchymal markers, as
well as the E-cadherin regulators. These findings may indicate the mechanism of ERp29-induced morphological change
occurs in a cell-context dependent manner. Other mechanisms (e.g. cell polarity) underlying the ERp29-regulated
phenotypic change are currently under investigation.
Poster
94
95
Frontiers in
Tumour Progression
Poster Session A
9
10
The Src kinase Blk impairs melanoma cell invasiveness
through inhibition of RhoA activation
Dual role for bone marrow FAK in primary tumour
growth and metastasis
Rubén A. Bartolomé, Jan Kleinovink, Ana Serrano, Nohemi Arellano and Joaquin Teixidó
Silvia Batista1, L. Reynolds , B. Tavora , V. Kostourou , M. Frame
Centro de Investigaciones Biológicas (CSIC), Madrid, Spain
We previously showed that activation of G13 in melanoma results in tyrosine phosphorylation of p190RhoGAP that
leads to inhibition of RhoA activation associated with defective cell invasion. p190RhoGAP phosphorylation is blocked
by inhibitors of Src kinases, and in a preliminary comparative screening with cells that show defective p190RhoGAP
phosphorylation, we identified the Src kinase Blk as a candidate to trigger the phosphorylation of this GAP protein. We now
show that Blk and p190RhoGAP associate in melanoma cells, and that Blk is required for p190RhoGAP phosphorylation.
Using siRNA for Blk or overexpression of wild type or constitutively-activated mutant forms of this kinase, we found
that Blk regulates RhoA activation and invasion of melanoma cells. Finally, short-term in vivo melanoma cell metastasis
assays indicated that Blk activity impairs the homing of these cells to lungs. Together, these data indicate that activation
of the G13- Blk-p190RhoGAP pathway inhibits RhoA activation and the invasion of melanoma cells.
1
1
1
2
and K. Hodivala-Dilke1
Institute of Cancer, Bart’s & The London, Queen Mary’s School of Medicine & Dentistry, John Vane Science
Centre, Charterhouse Square, London, UK; 2The Beatson Institute for Cancer Research, Cancer Research UK
Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow, UK
1
Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, regulates integrinmediated adhesion and cell migration and
contributes to cell proliferation and survival. Constitutive deletion of FAK results in a phenotype where the embryos die
at embryonic stage E8.5 due to severe defects in late gastrulation (1). Therefore, to examine the effect of the stromaldeletion of FAK in mice FAK floxed (FAKfl/fl) mice were crossed with RERTnERT/ERTCre mice where Cre-ERT2 is under
the control of the promoter of the large subunit of RNA polymerase II in order to induce FAK deletion the majority of the
cells after tamoxifen treatment (2). RERTnERT/ERTCre; FAKfl/fl, tamoxifen-treated, mice showed significantly decreased
B16F0 tumour size and tumour blood vessel density when compared with controls. These results suggest that stromaldeletion of FAK in adult mice is sufficient to inhibit tumour growth. Furthermore, WT mice transplanted with bone
marrow (BM) from RERTnERT/ERTCre; FAKfl/fl and treated with tamoxifen to induce FAK deletion in the BM compartment,
showed a tendency for decreased B16F0 tumour growth with no significant difference in blood vessel density. These
data suggest that the lack of FAK in the BM may affect some aspects of primary tumour growth that are not dependent
on angiogenesis. However BM-FAK deleted mice showed a significant increase in the total number of B16F0 metastasis
when compared with controls indicating that FAK plays a different role in primary tumour growth and metastasis.
Poster
96
97
Frontiers in
Tumour Progression
Poster Session A
11
12
A new connection between HIF-1alpha and mitochondria
dysfunction in head and neck paraganglioma through
deregulation of microRNA-210 expression
MUC1 cytoplasmic dimers are required for Src
recruitment and ICAM-1 binding induced cell invasion
Sandra Bernaldo de Quirós, Iriana Zambrano, Milagros Balbín, Aurora Astudillo,Carlos Suarez and
Department of Laboratory Medicine and Pathology, Heritage Medical Research Centre, University of Alberta,
Edmonton, Canada; 2Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton,
Canada
Ashlyn J. Bernier1, Jing Zhang , Andrew R.E. Shaw
1
María-Dolores Chiara
Servicio de Otorrinolaringología y Diagnóstico Molecular, Hospital Universitario Central de Asturias and
Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Spain
Paragangliomas of the head and neck (HNPGL) are rare, mostly benign tumors of the parasympathetic nervous system.
They occur most commonly in the carotid body, the main peripheral sensor of physiological hypoxia. They are highly
vascular and usually characterized by an indolent, non-invasive growth pattern. Most cases are sporadic, but a significant
number (10–50%) have been shown to be familial. Mutations in genes encoding subunits of succinate dehydrogenase
(SDH, complex II in the mitochondrial respiratory chain) have been implicated in the familial forms of the disease. These
genes encode subunits (SDHB, SDHC, SDHD, and SDHA) or associated proteins (SDHFA2) of the SDH complex, which plays
a central role in energy metabolism as both an enzyme of tricarboxylic acid cycle and as complex II of the mitochondrial
respiratory chain. The mutation of subunits of SDH has been shown to result in the accumulation of succinate which
inhibits HIF prolyl hydroxylases (PHDs), leading to the stabilization of HIF-1, a key factor in the hypoxia response. The
activation of HIF-1 by inhibition of PHDs has been termed ‘pseudo-hypoxia’. These mechanistic insights suggest that
low oxygen levels have a central role in the pathogenesis of HNPGLs. In this study, we have characterized the hypoxic/
pseudo-hypoxic molecular signature of HNPGLs by analysis of HIF-1 protein expression, and hypoxicrelated genes and
miRNAs. HIF-1 protein accumulation was found in 9 out of 28 tumors and this significantly correlated with the levels of
expression of hypoxicrelated genes. Among the hypoxia related miRNAs, miR-210 was found overexpressed in the HNPGL
tumors that showed the highest hypoxic/pseudo-hypoxic response. Recently, miR-210 has emerged as a new player that
regulates mitochondrial metabolism under hypoxia by repressing the expression of the ironsulfur (Fe-S) cluster scaffold
protein ISCU1/2. Accordingly, we found that the RNA levels of miR-210 and ISCU were inversely correlated. in HNPGL
tumor samples. Furthermore, HNPGL-derived cells exposed to hypoxia increased miR-210 and decreased ISCU RNA
levels, respectively. These data suggest that a HIF-1-miR- 210-ISCU signaling pathway may be causing mitochondrial
dysfunction in human HNPGLs. However, this pathway is not a common to all HNPGLs and does not correlate with the
presence or absence of germinal mutations of SDH genes in patients. Collectively, the data suggest the existence of a
novel mechanism involved in the pathogenesis of HNPGLs that is independent of SDH inactivation but dependent of
HIF-1. The convergence of both pathways in the mitochondria suggests that different molecular defects that modify the
homeostasis of this metabolic organelle are responsible for both familial and non-familial paragangliomas.
2
and Judith C. Hugh1
1
The mucin MUC1, a type I transmembrane glycoprotein, is overexpressed in breast cancer and has been correlated with
increased metastasis. We were the first to report binding between MUC1 and Intercellular adhesion molecule-1 (ICAM1), which is expressed on stromal and endothelial cells throughout the migratory tract of a metastasizing breast cancer
cell. Subsequently, we found that MUC1/ICAM-1 binding results in the initiation of pro-migratory calcium oscillations,
cytoskeletal reorganization, and simulated transendothelial migration in breast cancer cells. These effects were found to
be dependent on the activity of Src kinase, a non-receptor tyrosine kinase also implicated in breast cancer initiation and
progression. Here, we further investigate the mechanism of MUC1/ICAM-1 signalling, focusing on Src recruitment and
cell motility. Our aim is to determine key steps in transmission of the MUC1/ICAM-1 signal, which will provide potential
targets for anti-metastatic therapies. We report that MUC1 forms cytoplasmic domain dimers which are necessary for
constitutive Src recruitment, ICAM-1 induced calcium oscillations and simulated transendothelial migration. Investigation
of the nature of these dimers revealed they are not covalently linked constitutively or following ICAM-1 binding, but do
become tyrosine phosphorylated following ICAM-1 binding. In contrast to previously published reports, we found that
membrane proximal cysteine residues were not required for dimerization or ICAM-1 induced signalling, and believe this
may suggest the existence of parallel pathways with differing environmental triggers. Our data extends previous studies
on MUC1 dimerization in cell signalling into the field of cell migration and cancer metastasis. Further research is required
to elucidate the role of MUC1 dimerization in the diverse pathways it has been implicated in and investigate the potential
of synergistic inhibition of multiple pathway components in breast cancer therapeutics.
Poster
98
99
Frontiers in
Tumour Progression
Poster Session A
13
14
Exploring the interplay between MET and Tumor
Necrosis Factor alpha
Identification of cooperating Ras effector pathways in
lung cancer
Viola Bigatto, Francesca De Bacco, Paolo M. Comoglio and Carla Boccaccio
Leander Blaas1, Monica Musteanu , Rainer Zenz , Thomas Hoffmann , Daniel Schramek , Beatrice
1
1
1
2
Institute for Cancer Research and Treatment (IRCC), University of Turin, Italy
Grabner1, Hans-Peter Kantner1, Mathias Mueller3, Thomas Kolbe3, Thomas Rülicke3, Josef Penninger2, Richard Moriggl1,
Lukas Kenner1,4, Dagmar Stoiber1, Helmut Popper5, Emilio Casanova1 and Robert Eferl1
Tumor necrosis factor alpha is a pro-inflammatory cytokine secreted either by tumor cells or activated tumor-associated
macrophages. Binding of TNF alpha to its receptor (TNFR) triggers a complex biological response, including modulation
of apoptosis and induction of cell invasion, which may lead to tumor progression. TNF alpha is a powerful inducer of
transcription factor NF-kB, known to be required for protection from apoptosis. Recently, we have shown that NF-kB
mediates upregulation of the MET oncogene (the tyrosine kinase receptor for HGF) in response to ionizing radiation.
The overexpressed Met receptor, on its turn, promotes survival and invasion of irradiated cells. We thus hypothesised
that MET could be a critical effector of the biological response induced by TNF alpha. Indeed we showed that TNF alpha
induces MET mRNA transcription and protein expression in a time-dependent manner. This response requires the NFkB functional subunit p65 RelA. We also found that TNF alpha induces cell scattering, wound healing and invasiveness,
mimicking the effects of Met activation by HGF. These activities are fully impaired in the presence of small-molecule Met
tyrosine kinase inhibitors or in cells were Met has been knocked-down. Moreover, we found that, in cells lacking MET
expression or function, the TNF alpha effect turns from anti- to pro-apoptotic. Our data show that MET, which is often
overexpressed in human cancers, is a transcriptional target of TNF alpha, and that it plays a critical role in mediating the
TNF alpha pro-survial and pro-invasive activities on tumor cells.
Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria; 2Institute of Molecular Biotechnology,
Vienna, Austria; 3Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna,
Austria; 4Clinical Institute of Pathology, Medical University of Vienna, Austria; 5Institute of Pathology, Medical
University of Graz, Austria
1
Oncogenic mutations in Ras proteins are commonly found in human tumors. Oncogenic Ras activates several
downstream effector pathways. Among them, the MAPK, Pi3K and Ral pathways are considered to be important for Rasinduced tumor formation. However, it is still unknown which individual pathway(s) support tumorigenesis most potently.
Moreover, recent data have suggested that the requirement for Ras effector pathways in oncogenic transformation is
cell-typespecific. Here we describe the development of a Ras effector (RasE) Multi-Hit mouse model that was used to
identify the importance and cooperation of Rasinduced MAPK, Pi3K and Ral pathways in lung cancer. We demonstrate
that all three pathways collaborate in lung cancer formation and invasiveness. Among individual pathways, activation of
Pi3K or combinatorial activation of MAPK/Ral and MAPK/Pi3K induced lung tumors most potently. Interestingly, activation
of Ral was incompatible with Pi3K-induced lung tumor formation in the absence of MAPK activation. Our results reveal
important cooperativities of Ras downstream effector pathways in lung cancer formation. We suggest that the differential
activation pattern of Ras effector pathways in lung tumors identified by the Multi - Hit approach can be used to predict
the efficacy of combinatorial drug treatment regiments.
Poster
100
101
15
Frontiers in
Tumour Progression
Poster Session A
16
A novel CDK-independent function for Cyclin A2: control
of cell invasion and metastasis via RhoA signaling
Dissecting the molecular mechanisms of basal cell
carcinoma initiation and malignant progression
Jean Marie Blanchard1, Nikola Arsic , Nawal Bendris , Bénédicte Lemmers , Marion Peter , Christina
Cédric Blanpain1, Khalil Kass Youssef , Gaelle Lapouge , Sandrine Rorive , Cindy Michaux , Benjamin
Institut de Génétique Moléculaire de Montpellier CNRS, France Université Montpellier 2, France Université
Montpellier 1, France; 2Centre de Recherche en Biochimie Macromoléculaire CNRS, Montpellier, France;
3
CRLC Val d’Aurelle-Paul Lamarque Montpellier, France
1
1
1
1
1
Begon , Cosette Rebouissou Gilles Gadéa and Frédéric Bibeau
1
1,
2
3
1
Cyclin A2 plays a key role at two critical points in the cell cycle: during the S phase, where it activates CDK2, and during
the G2 to M transition where it activates CDK1. Even though Cyclin A2 over-expression has been detected in some
human and mouse tumours, its precise role in adult physiopathology is still elusive due to the fact that its depletion
resulted in very early (5.5 day post-coitum) embryonic mortality. We were recently able to demonstrate a novel function
for Cyclin A2 in the regulation of actin cytoskeleton dynamics and cell motility. Using an shRNAmediated knock down
approach two major phenotypes were observed. The first one, in accordance with data from the literature, consisted in
a cell accumulation at the G2/M boundary of the cell cycle. In addition depletion of Cyclin A2 had no significant effect on
proliferation of several adult cell lines. The second phenotype was completely unexpected. Namely, Cyclin A2-depleted
cells displayed significant morphological changes (increased size and diameter) correlating with a rearrangement of
actin cytoskeleton and a redistribution of adhesion plaques. These morphological changes were observed in all types of
cells tested: primary cell lines, established or transformed cells from both human and mouse origin. All these changes
resulted in increased motility and invasion abilities of the cells together with a reduced activity of RhoA GTPase, a key
regulator of cell migration. Conversely, forced expression of Cyclin A2 had a completely opposite effect on all these
properties: migration, 3D matrix invasion and RhoA activity. Even more interesting was the fact that this novel role of
Cyclin A2 was independent of its kinase activating ability and rather mediated by its direct binding to RhoA. Finally,
Cyclin A2 was found differentially expressed in matched human primary tumors and their corresponding metastases.
We propose that Cyclin A2 is a multifunctional protein involved in cell cycle control, cytoskeletal rearrangements, cell
migration and cancer metastasis spread.
1
1
2
1
beck1, Isabelle Salmon2 and Véronique Del Marmol3
IRIBHM, Université Libre de Bruxelles, Belgium; 2Department of Pathology, Hôpital Erasme, Université Libre
de Bruxelles, Belgium; 3Department of Dermatology, Hôpital Erasme, Université Libre de Bruxelles, Belgium
Basal cell carcinoma (BCC) is one of the most frequent types of cancer in human and results from the constitutive
activation of hedgehog signaling pathway. Using mice conditionally expressing constitutively active Smoothened mutant
(SmoM2) in different cellular compartments of the skin epidermis, we have recently found that BCC arise most frequently
from long-term resident progenitor cells of the interfollicular epidermis (Kass Youssef et al. Nature Cell Biology 2010).
Here, we develop a novel strategy to isolate by flow cytometry oncogene targeted cells at different time following
SmoM2 expression and determine the molecular changes associated with tumor progression. Transcriptional profiling
of oncogene expressing cells at different time points following tumor initiation indicate that upon SmoM2 expression
interfollicular progenitors undergo a progressive and profound reprogramming into embryonic hair follicle progenitors
and suggest that canonical Wnt/beta-catenin signaling represent an early step in this reprogramming event. Deletion of
beta-catenin in SmoM2 expressing cells blocks the reprogramming of oncogene targeted cells into embryonic HF fate
and tumor progression. Analysis of human tumors samples also show evidence of Wnt activity and embryonic hair follicle
reprogramming at the leading edge of human nodular BCC. Our data indicate that reprogramming of adult interfollicular
cells into embryonic hair follicle progenitor is a critical event in BCC initiation and tumor progression and wnt/betacatenin signaling regulates this process in a cellular autonomous manner. Our study provides novel and important
insights into the molecular mechanisms underlying BCC initiation and malignant progression.
Poster
102
103
Frontiers in
Tumour Progression
Poster Session A
17
18
Src kinase and caveolin-1 promote metastasis through
a common pathway
The miR-200 family regulates cell migration and
metastasis in lung adenocarcinoma
Heiko Blaser1, Enrico Arpaia
Irene C. Blat, Monte M. Winslow and Tyler E. Jacks
1,2
, Miguel Quintela-Fandino1,3, Amber Ablack4, Hon Sing Leong4, Shruti
Nambiar4, Evan F. Lind1, Jennifer Silvester1, Cherina K. Fleming1, Vincent Nadeem1, James J. Pan1, Pamela S. Ohashi1,
John D. Lewis4 and Tak W. Mak1,2
The Campbell Family Institute for Cancer Research, University Health Network, Toronto, Canada;
Departments of Medical Biophysics and Immunology, University of Toronto, Canada; 3MD Anderson Spain/
Spanish National Cancer Centre (CNIO), Clinical Investigation Unit, Breast Cancer Group C, Madrid, Spain;
4
Translational Prostate Cancer Research Group, London Regional Cancer Program, London, Canada
1
The Koch Institute for Integrative Cancer Research, Department of Biology at MIT, Howard Hughes Medical
Institute, Cambridge, USA
2
The participation of caveolins in cell movement has sparked interest in studying their potential roles in metastasis.
However, the functions of caveolins in cancer are not clear because depending on the cell-type, caveolin-1 (Cav-1) can
act either as a tumor suppressor or an oncogene. Previously we showed that the small GTPase RhoC is crucial for tumor
cell migration/invasion and metastasis in mice. RhoC regulates p130Cas and Rac1 activation, and has the ability to bind
Cav-1 through its ‘caveolin-binding domain’ (CBD). We exogenously expressed the CBD in B16F10 melanoma cells to
disrupt the interaction of CBD-containing proteins with Cav-1. Surprisingly, CBD expression profoundly decreased cav1 mRNA levels and also reduced p130Cas and Rac1 activation. Besides, the lack of RhoC did reduce the activation of
p130Cas and Rac1 however only RhoA appeared to play a role in cav-1 transcription. Rho GTPases are implicated in Srcmediated migration of cancer cells. Thus we investigated the role of Src in this context. Interestingly, the expression of
a dominant negative form of Src (DN-Src) reduced cav-1 transcripts. Notably, the common effect resulting from CBD or
DN-Src expression was the reduction of Cav-1. Besides, B16F10 cells lacking Cav-1 showed reduced Src and p130Cas
activation. Importantly, B16F10 cells expressing CBD or DN-Src showed reduced ability to extravasate and survive in
secondary sites, in both recipient mice and chicken embryos. Reconstitution of Cav-1 in CBD- or DN-Src-expressing
tumor cells caused them to regain their aggressive nature and to metastasize similar to the parental cells. Our data
support the existence of a positive feedback loop involving Src and Cav-1 in which Src controls cav-1 transcription, and
Cav-1 controls Src activation. Activated Src then drives Rac1 activation and thus metastasis. We suggest that Cav-1 may
serve as a scaffold crucial for linking the functions of small GTPases and Src in metastasis.
miRNAs are post-transcriptional regulators of genes involved in such canonical signaling pathways as the TGF-β and
EGF pathways, both of which are frequently altered in tumor cells. miRNA downregulation results in upregulation of
its target genes and ultimately aberrant signaling of pathways involved in invasion and migration, for example. To
better understand how alterations in miRNA expression could lead to metastasis, we used a lung adenocarcinoma
mouse model in which a lentivirus expressing the Cre recombinase activates oncogenic K-Ras and deletes p53. Cell
lines derived from the primary lung tumors (T) in these mice give rise to distant metastases (M) that can be related by
their lentiviral integration site. We expression profiled 30 paired T and M cell lines and identified three members of the
miR-200 family – miR-200b, miR-200c, miR-429 – to be significantly downregulated in the M lines compared to their
corresponding T line. To explore the functional contribution of the miR-200s to metastasis, we designed a miRNA sponge
to stably inhibit endogenous miR-200 levels in T lines. We found miR-200 depletion promoted cell migration towards
either serum or EGF. Transplantation of the sponge-expressing lines via tail vein led to in vivo formation of more invasive,
metastatic tumors compared to controls. Previously, the miR-200 family has been shown to inhibit the TGF-β target gene
and the EMT mediator, ZEB1. We have identified another miR-200 target, Epidermal Growth Factor Receptor Substrate8
(Eps8), which mediates actin based cell motility. In miR-200 low M lines, Eps8 is expressed three-fold higher than in the
corresponding T lines. To deplete the miR-200s in vivo in an appropriate tumor microenvironment, we have designed a
bipartite lentivirus expressing the miR-200 sponge along with Cre in the lung. Our findings implicate the miR-200s as
a class of metastasis regulators that modulate expression of genes functionally relevant in tumor cell dissemination.
Poster
104
105
Frontiers in
Tumour Progression
Poster Session A
19
20
Sumoylation of HDAC2 regulates cancer relevant gene
expression patterns
The p38α MAPK phosphorylated forms
André Brandl, Tobias Wagner, Katharina Uhlig, Thorsten Heinzel and Oliver H. Krämer
Institute of Molecular and Cell Biology, A-STAR, Singapore
Friedrich Schiller University Jena, Center for Molecular Biomedicine (CMB), Institute of Biochemistry and
Biophysics, Department of Biochemistry, Jena, Germany
Histone Deacetylases (HDACs) are crucial regulators of gene expression. HDAC2 has unique functions in vivo and is
often overexpressed in primary tumors. Various posttranslational modifications govern the functions of HDAC2. Here,
we demonstrate covalent modification of HDAC2 with the small ubiquitin-related modifier SUMO1 in vitro and in vivo.
The SUMO-specific protease SENP1 counteracts HDAC2 sumoylation. In contrast to observations made for other HDACs,
sumoylation of HDAC2 is not required for nuclear localization and catalytic activity in HDAC2-positive and -negative
cells. However, sumoylated HDAC2 is tied into the signaling of the cancer-related transcription factor NF-B. Our data
for the first time demonstrate that SUMO modification of an HDAC itself contributes to the epigenetic regulation of gene
expression patterns. We reveal unanticipated novel functions for HDAC2 sumoylation, which might be applicable to other
enzymes and modulators.
Anna Brichkina and Dmitry V. Bulavin
p38α is a widely expressed MAP kinase family member that is known for its important role in the regulation of
numerous processes including inflammation, environmental stress response and cellular proliferation. Stress-induced
phosphorylation of p38α results in two forms of phospho-p38α. While one is dually phosphorylated on Thr180 and
Tyr182, the major form is phosphorylated on Tyr182 alone by multiple kinases including MKK6 and Src-family kinases.
In addition, Tyr182 single phosphorylated p38α has broad substrate specificity. These data support the existence of noncanonical pathway in the activation of p38α MAPK. To investigate the mechanism by which the phospho-Tyr182 form
is generated, we performed mass spectrometry analysis of p38-associated proteins and found several new proteins
bound to p38α. We show that some of these proteins can specifically modulate Tyr-182 phosphorylation of p38α.
The mechanism by which these newly identified proteins selectively regulate p38α phosphorylation is currently under
investigation.
Poster
106
107
21
Frontiers in
Tumour Progression
Poster Session A
22
The role of the Wnt-signaling antagonist Sclerostin
in the development of osteolytic lesions in multiple
myeloma
Osteoblasts orchestrate collagen remodelling via
tumour cell-dependent regulation of Endo180 in
metastatic bone disease
Giacomina Brunetti1, S. Colucci , A. Oranger , G. Mori , M. Centonze , R. Rizzi , V. Liso
Matthew Caley1, G. Kogianni , A. Adamarek , V. Fonseca , J. Gronau , J. Rhim , J. Cobb , J. Waxman
1
1
2
1
3
3
and M. Grano1
Department of Human Anatomy and Histology University of Bari, Italy; Department of Biomedical Science
University of Foggia, Italy; 3Hematology Section, Department of Pathology, University of Bari, Italy
1
2
Multiple myeloma (MM) is characterized by osteolytic bone lesions, adiacent to tumor foci within bone, that arise as
a consequence of osteoblast inactivation and osteoclast activation. Canonical Wnt signaling is central to normal bone
homeostasis, and secretion of Wnt signaling inhibitors by MM cells contributes to MM-related bone destruction and
disease progression. In this study, we investigated the effects of MM cells on osteoblast differentiation and the potential
role of Sclerostin, a Wnt signaling antagonist, in the inhibition of osteoblastogenesis in MM. In our study, we first showed
that MM cells expressed Sclerostin at mRNA and protein levels. Moreover, using a co-culture system between human
bone marrow stromal cells (BMSCs) and MM cells we demonstrated that MM cells induce an inhibitory effect on alkaline
phosphatase, collagen I and AP-1 transcription factor expression in BMSCs. This inhibition was reverted by the addition
of the anti-Sclerostin neutralizing monoclonal antibody. In the co-culture system, this antibody also partially rescued
the inhibitory effect on BMSC mineralization. Moreover, knowing that the Wnt signaling activation affect the RANKL/
OPG axis, we studied in our cocolture system the effect of the anti- Sclerostin monoclonal antibody on the expression of
these two cytokines. In particular, we found that human MM cells up-regulated RANKL expression, but strongly downregulated OPG production by BMSCs at mRNA and protein levels. The presence in the co-culture of the neutralizing
antibody reduced RANKL and increased OPG expression. Thus, our data indicate that Sclerostin could be one of the
factors inducing the uncoupling between formation and resorption in MM patients by the direct inhibition of osteoblast
differentiation and the indirect stimulation of osteoclast activity.
1
1
1
1
3
2
1
and
J. Sturge1
Molecular Cell Biology Section, Division of Cancer, Department of Surgery & Cancer, Faculty of Medicine,
Imperial College London, Hammersmith Hospital, London, UK; 2Center for Prostate Disease Research,
Uniformed Services University of the Health Sciences, Bethesda, USA; 3Biosurgery & Surgical Technology,
Division of Surgery, Department of Surgery & Cancer, Faculty of Medicine, Imperial College London, Charing
Cross Hospital, London, UK
1
A major clinical complication in patients with advanced prostate cancer is metastasis to bone where osteoblastic and/
or osteoclastic activity results in a highly destructive tissue remodelling process. Recent in vivo studies have identified
important functions for the collagen-binding receptor, Endo180, in both bone development and tumour progression. We
have confirmed the role of Endo180 as a collagen uptake receptor in epithelial cancer cells and identified a new role
for Endo180 in stromal cell collagen secretion. We have established a co-culture model of human bone metastases
using primary bone osteoblasts (hOBs) and prostate cells (PCs) derived from different stages of disease progression.
Using IF, FACS and RNAi we have quantified changes in Endo180 levels in both PCs and hOBs in our model. We have
also reproduced these changes in single cell systems to determine their effect on collagen uptake and production.
Direct co-culture induced an increase in Endo180 levels in PCs and a decrease in Endo180 levels in hOBs. The changes
in Endo180 levels in these co-cultures were coordinated with alterations in mineralisation and alkaline phosphatase
activity. Reproducing these changes using RNAi or protein overexpression confirmed the role of Endo180 in PC collagen
uptake but identified a new role in stromal cells. Depletion of Endo180 in hOBs did not lead to a decrease in the ability
of hOBs to take up labelled collagen it did however lead to a correlating decrease in collagen matrix production by hOBs.
We propose a novel role for Endo180 in stromal cell collagen secretion as well as confirming it’s previously identified role
in epithelial tumour cells. We have demonstrated a new model for investigating bone metastases and using this model
have demonstrated a new model for investigating bone metastases and using this model have demonstrated a role for
Endo180 in osteolytic tumours.
Poster
108
109
23
Frontiers in
Tumour Progression
Poster Session A
24
SADB kinase prevents genomic instability by regulating
correct centrosome duplication
mTOR mediates Wnt-induced epidermal stem cell
exhaustion and aging
Ana Clara Carrera1, María Alvarado-Kristensson
Rogerio M. Castilho1,2, Cristiane H. Squarize
, María Josefa Rodríguez3, Virginia Sílio1 and
1, 2
José M. Valpuesta
3
Department of Immunology and Oncology, 3Department of Macromolecular Structures, Centro Nacional
de Biotecnología/CSIC, Madrid, Spain; 2Center for Molecular Pathology, Dept. Laboratory Medicine, Lund
University, Malmoe University Hospital, Sweden
1
Symmetrical cell division requires cell duplication of DNA and protein content to generate two daughter cells.
Centrosomes also duplicate during cell division, but the mechanism controlling this process is incompletely understood.
The presence of supernumerary centrosomes often correlates with DNA instability. Centrosomes are composed of two
barrel-shaped mother and daughter centrioles surrounded by a pericentriolar matrix. At G1/S transition, a procentriole
forms adjacent to each parental centriole, which will continue to grow during S phase. At G2/M, the two centrosomes
separate; the daughter centriole matures and instructs mitotic spindle formation. Since errors in centrosome duplication
lead to aneuploidy, to prevent genomic instability, centrosome duplication is linked to DNA replication. We describe an
alternative splice form of SadB kinase encoding a short SADB Ser/Thr kinase whose activity fluctuates during the cell
cycle, localizes to centrosomes, and controls centrosome duplication. Reduction of endogenous SADB levels diminished
centrosome numbers, whereas enhanced SADB expression induced centrosome amplification. SADB exerted this action
through phosphorylation of g-tubulin on S131, since expression of a phosphomimetic Ser131-to-Asp g-tubulin mutant
alone increased centrosome numbers, whereas non-phosphorylable Ala131-g-tubulin impaired centrosome duplication.
We propose that SADB kinase activity controls centrosome homeostasis and ensures genomic stability by regulating
phosphorylation of g-tubulin.
, Lewis A. Chodosh3, Bart O. Williams4
1,2
and J. Silvio Gutkind1
Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National
Institutes of Health, Bethesda, USA; 2Department of Periodontics and Oral Medicine, University of Michigan,
Ann Arbor, USA; 3Department of Cancer Biology and Abramson Family Cancer Research Institute, University
of Pennsylvania School of Medicine, Philadelphia, USA; 4Laboratory of Cell Signaling and Carcinogenesis,
Van Andel Research Institute, Grand Rapids, USA
1
The Wnt family consists of 19 secreted cysteine-rich glycoproteins that initiate signaling by interacting with the
N-terminal extracellular cysteine-rich region of the Frizzled family of seven-span transmembrane receptors, and with
either LRP5 or LRP6, two members of the low-density-lipoprotein receptor-related (LDL-R) protein family. Wnt can
stimulate several major signaling cascades, and the canonical Wnt/β-catenin signaling is an important event in the
origin of several neoplasias. Although expression levels of Wnt-1 is associated to tumor development in some organs
including mammary gland, other tumors display reduced levels of Wnt1 (Oncomine Microarray database). Here, we
investigate the long-term consequences of ectopic Wnt-induced signaling by overexpressing Wnt- 1. Surprisingly, we
found that Wnt1 initially causes a rapid growth followed by the disappearance of the epidermal stem cell compartment
and progressive depletion of skin appendances. While Wnt1 expression induced the activation of both β- catenin and
mTOR in the epithelial compartment, stem cell exhaustion and epidermal phenotype were uniquely associated with
mTOR activation. The ability of the Wnt1 gene to signaling through mTOR and induce cellular senescence rationalize its
low expression levels found in other tumors providing important evidence that modulation of Wnt1 levels is a suitable
strategy for cancer treatment. These findings demonstrated that epidermal stem cells are endowed with a protective
mechanism resulting in cell senescence upon the persistent stimulation of Wnt1 that activate mTOR, thereby helping to
maintain the genetic integrity of the stem cell population, which may ultimately contribute to suppress tumor formation.
Nonetheless, the Wnt1/mTOR ability to induce senescence in stem cells represents strong evidence that this approach
may be a powerful tool in the combat of cancer cells presenting stem cell-like properties.
Poster
110
111
25
Frontiers in
Tumour Progression
Poster Session A
Gene amplification of the transcription factor DP1 and
the the p120ctn in human lung cancer
Sandra D. Castillo1, Barbara Angulo , Ana Suarez-Gauthier , Lorenzo Melchor , Pedro P. Medina , Lydia
2
2
3
26
Overexpression of Skp2 increases the expression of
MMP-2 and MMP-9 and invasion of A549 human lung
cancer cells
2
Sanchez-Verde4, Juan Torres-Lanzas5, Guillermo Pita6, Javier Benitez3 and Montse Sanchez-Cespedes1,2
Genes and Cancer Group, Programa de Epigenetica y Biologia del Cancer, PEBCIDIBELL, L’Hospitalet de
Llobregat, Barcelona, Spain; 2Lung Cancer Group, Spanish National Cancer Centre (CNIO), Madrid, Spain;
3
Human Genetics Group, Spanish National Cancer Centre (CNIO), Madrid, Spain; 4ImmunohistochemistryHistology Unit, Spanish National Cancer Centre (CNIO), Madrid, Spain; 5Thoracic Surgical Department,
Hospital Virgen de la Arrixaca, Murcia, Spain; 6Unidad de Genotipacion-CEGEN, CNIO. *Present address:
Hospital Universitario Madrid Norte Sanchinarro, Madrid, Spain
1
The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In
the present work we report the identification of novel amplified genes in lung cancer by means of global gene expression
analysis. To screen for amplicons we aligned the gene expression data according to the position of transcripts in the
human genome and searched for clusters of overexpressed genes. We found several clusters with gene overexpression,
suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters,
at chromosomes 11q12 and 13q34, confirmed the presence of amplification, spanning about 0.4-Mb and 1- Mb, for
11q12 and 13q34, respectively. Amplification at these regions occurred each at a frequency of three percent. Moreover,
quantitative RT-PCR of each individual transcript within the amplicons allowed us to verify the increased in gene
expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1,
at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumors with gene amplification.
We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To determine further the oncogenic
properties of DP1 we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by
small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein overexpression reduced
cell viability by 50 percent. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12,
occurring each at a frequency of three percent of non-small cell lung cancers. TFDP1, which encodes the E2F-associated
transcription factor DP1 is a candidate oncogene at 13q34.
Hui-Chiu Chang
Laboratory Science and Biotechnology, College of Health Sciences; Graduate Institute of Medicine, College
of Medicine, Kaohsiung Medical University; National Sun Yat-Sen University-Kaohsiung Medical University
Joint Research Center, Taiwan
Skp2 is one of the components of the E3 ubiquitin ligase which is required for the degradation of tumor suppressor p27.
Overexpression of this oncogene is frequently found in human cancers and has been shown to be associated with poor
prognosis. In addition to induce p27 degradation and enhance cellular proliferation, Skp2 also plays a role in promoting
tumor metastasis. However, the underlying mechanism is unclear. In this study, we established Skp2-overexpressing
stable transfectants from A549 human lung cancer cells. We found that these stable transfectants exhibited increased
migratory and invasive abilities. In addition, expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 was upregulated. Increase of MMP-2 and MMP-9 activity and neutralization of these two MMPs by antibodies reduced cell
invasion were confirmed by enzymatic assay and gelatin zymography. Our results also revealed that Sp1 was involved
in the induction of MMP-2 and MMP-9 by Skp2 because treatment of mithramycin or knockdown of Sp1 by siRNA
attenuated their expressions. Collectively, we provide the first evidence that upregulation of MMP-2 and MMP-9 is one
of the mechanisms by which Skp2 promotes cell invasion.
Poster
112
113
Frontiers in
Tumour Progression
Poster Session A
27
28
Deregulation of lipid metabolism in head and neck
paragangliomas: a novel insight into the pathogenesis
of the disease
Unbiased high-throughput genome-wide analysis of
translocation formation reveals non-random patterns
María-Dolores Chiara, Anna Merlo, Sandra Bernaldo de Quirós, Iriana Zambran, Milagros Balbín and
Howard Hughes Medical Institute, The Immune Disease Institute, and the Program in Cellular and Molecular
Medicine of Children’s Hospital, Department of Genetics, Harvard Medical School, Boston Massachusetts
Dept of Biomedical Sciences and Human Oncology and CERMS, University of Turin, Italy
Carlos Suarez
Servicio de Otorrinolaringología y Diagnóstico Molecular, Hospital Universitario Central de Asturias and
Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Asturias, Spain
Head and neck paragangliomas of the (HNPGL) are rare, mostly benign tumors of the parasympathetic nervous system.
They occur most commonly in the carotid body, the main peripheral sensor of physiological hypoxia. They are usually
characterized by an indolent, non-invasive growth pattern. Most cases are sporadic, but a significant number (10–50%)
have been shown to be familial, associated with mutations in genes encoding subunits of succinate dehydrogenase
(SDH) mitochondrial complex II (SDHB, SDHC, SDHD, and SDHA) or associated proteins (SDHFA2), which plays a central
role in energy metabolism. In this study, we compared the transcriptional profile of HNPGLs and normal carotid bodies.
Among the differentially expressed genes, a significant number, associated with PPAR/adipocytokine signaling pathways
and lipid metabolism, were found down-regulated in tumors. We also found a significant overlap between de HNPGL
deregulated pathways and genes associated with obesity, atherosclerosis, type II diabetes, and hypercholesterolemia.
Similar results linking cancer with metabolic diseases has been recently appreciated suggesting that a variety
of phenotypically diverse disease states are linked through a common transcriptional program involving metabolic
pathways. Interestingly, suppression of ADIPOQ, one of the most dramatically repressed genes in HNPGLs, is known to
decrease mitochondrial content and enzymes, causing mitochondrial dysfunction and insulin resistance. Defects in the
mitochondria, the centers of cellular metabolic activity, is known to precede much of the hallmark pathology of familial
paraganglioma associated with SDH mutations. The transcriptional profile identified here is common to both familial and
non-familial HNPGLs. Therefore, our data show that one putative pathological mechanism that plays a prominent role in
the pathogenesis of non-familial HNPGLs is lipid and mitochondrial metabolic dysfunction.
Roberto Chiarle, Monica Gostissa, Darienne Meyers, Susanna Lewis and Frederick W. Alt
Recurrent chromosomal translocations are characteristic features of many types of cancers, and are involved in
cancer initiation and progression. There are several basic mechanistic factors that are required for the generation of
most translocations. First, DNA Double Strand Breaks (DSBs) must be present simultaneously at the two participating
loci. Second, the two broken loci must either be in close proximity or be moved into close proximity to be joined.
Finally, cellular DNA repair pathways must be available to join the two broken loci to complete the translocation. These
mechanistic factors can vary in different normal and mutant cells and, as a result, substantially influence the frequency
at which particular translocations are generated in a given cell type. Recurrent translocations in tumors are often
considered to represent very low frequency events that are strongly selected at the cellular level. To gain further insights
into the mechanisms promoting translocations regardless the oncogenic selection, we have developed in vitro and in
vivo systems to identify translocation partners from a fixed DNA DSB via a high-throughput genome-wide sequencing
analysis. These systems employ cycling primary B cells where I-SceI recognition sequence was targeted either in
the Switch 1 region or in the c-myc locus, and DSBs were generated upon I-SceI expression. Our data show that
translocations from defined DSBs are mostly AID dependent and, more frequently, are generated within loci in the same
chromosome rather than different chromosomes. Similar patterns are also found in ATM and H2AX deficient cells. Thus,
unbiased genomewide analysis of translocation formation reveals non-random translocation patterns that highlight the
importance of mechanistic factors, such as DSB frequency and location, and the organization of chromosomes within the
nucleus. These results have great implications on the interpretation of data obtained from cancer genome sequencing
project.
Poster
114
115
29
Frontiers in
Tumour Progression
Poster Session A
30
Anti-metastatic mechanism of Tian-Xian Liquid (TXL)
and its bioactive fractions in human colorectal cancer
cells and xenograft models
Trans-differentiation of breast cancer by GATA3 reduces
primary growth and metastasis
Elli SM. Chu ,
Laboratory of Cancer Biology and Genetics, National Cancer Institute, Maryland, USA
1
S.C.W. Sze , H.P. Cheung , W.K. Liu , T.B. Ng and Y. Tong
1
1
2
2
1
School of Chinese Medicine, LKS Faculty of Medicine, The University of Hong Kong; 2School of Biomedical
Sciences, Faculty of Medicine, The Chinese University of Hong Kong
Isabel Chu, Aleksandra Michalowski and Jeffrey Green
1
Colorectal carcinoma is the second most prevalent cancer with an up-rising trend in Hong Kong (Hong Kong Cancer
Registry). Traditional Chinese medicine acts as a complementary alternative for tumour therapy with minimal sideeffects and traumatic injuries. Tian-Xian Liquid (TXL), one of the well-known natural medicinal herbal formulations, has
been commercially used as an anticancer dietary supplement for a decade without known adverse effects. This study
aimed to investigate the anti-metastatic property of TXL and its bioactive fractions [butanol fraction (BU), ethyl-acetate
fraction (EA) and aqueous fraction (WA)] at molecular level on human colorectal cancer in vitro (HT-29 cancer cells) and
in vivo (nude mice xenografts). For the cell model, TXL and its bioactive fractions have similar anti-proliferative effects by
MTT assay. At 4-hour-incubation, IC50 values were obtained at 1% (V/V) TXL, 1.25% (V/V) BU, 5% (V/V) EA and 0.3125%
(V/V) WA. At IC50, TXL and its bioactive fractions significantly reduced the MMP2 and MMP7 expressions at mRNA level
by real-time PCR. At protein level, TXL, BU and EA correspondingly down-regulated MMP2 (active form) and MMP7
protein from 24 to 48 hours; TXL and BU also down-regulated VEGF protein expression; however, no such effect was
found in WA-treated cells. Further, only TXL, EA and WA effectively inhibited the cell migration at 48 hours incubation by
woundhealing assay. For the xenografts models, MMP2 and MMP7 mRNA expressions were reduced by TXL-, BU- and
EA-treated xenografts; however no effects on MMP2 protein expression in all drug-treated xenografts. The VEGF protein
expression was significantly down-regulated in TXL- and WA-treated xenografts. Further, TXL, BU and WA effectively
inhibited the tumor growth without altering the body weight of the xenografts. In summary, the Chinese medicinal
formulation, TXL, demonstrated the most effective anti-metastatic ability on human colorectal cancer in vitro and in vivo.
Low-grade breast cancer is associated with increased differentiation and reduced metastases, suggesting that
reprogramming tumor cells to a more differentiated state could improve outcome. Previous differentiation strategies
for breast cancer have failed. Utilizing a novel differentiation therapy approach, we have reprogrammed aggressive,
basal, triple-negative Breast Cancer (BrCa) (generally with poor prognosis) towards a less aggressive phenotype by
manipulating expression of the key mammary luminal differentiation transcription factor, GATA3. GATA3 is essential
for programming undifferentiated mammary cells into a luminal subtype while myoepithelial/basal cells fail to express
GATA3. Significantly, GATA3 expression is highly correlated with the luminal, more differentiated BrCa phenotype. We
hypothesized that ectopic expression of GATA3 in metastatic, basal BrCa cell lines will reprogram them to a more
differentiated, less metastatic phenotype. Over-expressing GATA3 in human basaltype MDA-MB-231 (231-GATA3) BrCa
cells induced significant morphological changes in 2- and 3-D cultures compared to control cells (231-Emty). 231-Empty
cells maintained a spindle, elongated morphology, while 231-GATA3 cells became rounded and larger. In 3-D Cultrex,
231-GATA3 cells appeared smaller, more organized, and rounded compared to 231-Empty cells. Microarray profiling
of 231- GATA3 vs. 231-Empty cells revealed gene expression changes associated with increased adhesion, reduced
extracellular matrix remodeling factors and reduced metastasis. Western blot confirmed re-expression of E-cadherin
and reduction of fibronectin in 231-GATA3 cells, indicative of a more luminal phenotype. The gene most downregulated
by GATA3 was Lysyl Oxidase (LOX) and confirmed by qPCR. LOX has been shown to be critical for metastatic progression
through matrix remodeling. Knock down of GATA3 in the GATA3 positive BT474 cell line increased LOX expression.
Retrospective analysis of microarray profiles of BrCa cells revealed an association of LOX with the basal subtype and a
statistical inverse association between LOX and GATA3. SCID mice injected with 231-GATA3 cells by tail vein showed
significantly fewer metastatic lesions in the lung compared to control cells (p<0.05). Importantly, restoration of LOX
in 231-GATA3 cells by lentiviral transduction reversed the reduced metastatic phenotype. This demonstrates that
expression of GATA3 reprogrammed a poorly differentiated basal cell towards a less metastatic phenotype and the
GATA3 dependent LOX down-regulation is responsible for the reduced observed metastasis. These findings suggest that
transcription factor-induced differentiation pathways may be potentially novel therapeutic molecular targets to inhibit
metastatic disease progression in combination with standard therapeutic treatments.
Poster
116
117
31
Frontiers in
Tumour Progression
Poster Session A
Deubiquiting enzyme, OTUB1 modulates active RhoA
and p53 levels in prostate cancer that have a functional
relevance in regulating cell invasion
Yin-Choy Chuan1, Benedikt Kessler , Deigo Iglesias-Gato
2
3
32
Fibroblast gene expression profiles reflect the stage of
tumor progression in oral squamous cell carcinoma:
selection of key markers by transcriptome-interactome
analysis
and Amilcar Flores-Morales1
NNF Center for Protein Research, University of Copenhagen, Denmark; 2Department of Clinical Medicine,
University of Oxford, UK; 3Department of Molecular medicine and Surgery, Karolinska Institute, Sweden
1
Nicola Cirillo,
Kue Peng Lim, Yazan Hassona, Wenbin Wei, Sok Ching Cheong, Gayani Pitiyage, E. Ken
Parkinson and Stephen S Prime
School of Oral and Dental Sciences, University of Bristol, UK; 2Institute of Cancer Studies, University
of Birmingham, UK; 3Cancer Research Initiatives Foundation, Sime Darby, Subang Jaya Medical Centre,
Selangor, Malaysia; 4Department of Clinical and Diagnostic Oral Sciences, Queen Mary’s School of Medicine
and Dentistry, Institute of Cell and Molecular Sciences, London, UK
1
Metastasis of tumors in the course of cancer progression remains one of the most frequent causes of deaths in humans
and there is a clear lack of very efficient treatment strategies that could help to interfere with or reverse this process.
With much attention placed on the critical of ubiquitin-proteosome pathway mediated events in cancer progression, the
role of deubiquiting enzymes (DUBs) in cancers is only beginning to unravelled. In our laboratory, we are interested the
role of a particular subset of the deubiquiting enzymes family contains an ovarian tumor domain (OUT) in prostate cancer
progression, specifically on cancer cells invasion and metastasis. We used siRNA based knockdown of OUT family
members in prostate cancer cell lines and study the effect on cancer cells invasion through matrigel. We discovered that
one of the members, otubain 1 (OTUB1) siRNA knockdown inhibited androgen induced prostate cancer cells invasion.
This effect is mediated through the reduced activity of small GTPase RhoA. The same effect was observed in PCa
cells overexpressing dominant negative formed of OTUB1 (C91S mutant). By screening the phosphorylation profiles of
kinases, we found increased p53 protein level in OTUB1 knockdown PCa cells. Knockdown of p53 also leads to increase
PCa cells invasiveness but the molecular details remain to be determined. This studies described here provide a novel
insights into mechanisms underlying prostate cancer cells invasion and metastasis, which involves the interplays of
OTUB1, RhoA and p53. If this novel pathway turns out to be highly relevant for the regulation of migration and invasion,
it will uncover new molecular entry points for pharmaceutical interventions in the future for prostate cancer treatment.
Background: In view of the importance of the tumour stroma in the promotion of epithelial cancer development and
spread, this study examined gene expression in fibroblast strains from normal oral mucosa, dysplastic epithelial tissue,
and genetically stable and unstable oral squamous cell carcinoma (OSCC) usingeffective anti-metastatic ability on human
colorectal cancer in vitro and in vivo t Affymetrix gene expression microarrays. Methods: Fibroblasts were isolated from
early passage cultures of normal oral mucosa, dysplastic tissues and genetically stable (wild type p53 and p16INK4A)
and unstable (inactivation of p53 and p16INK4A) oral squamous cell carcinomas (OSCC). Genome wide transcriptional
profiles were generated using the GeneChipR cDNA whole transcript microarray platform. Invasion assays and network
analysis were performed according to standard procedures. Results: Fibroblasts were distinguished according to the
stage of tumour development. Tumour progression was associated with down-regulation of cell cycle- and cytokinesisrelated genes and up-regulation of genes encoding transmembrane proteins including cell adhesion molecules, changes
that paralleled with alteration in growth rates and migration. Fibroblasts from genetically unstable OSCC displayed
a senescent phenotype and stimulated the invasion of a non-tumorigenic keratinocyte cell line into fibroblast-rich
collagen gels. Transcriptome-interactome analysis revealed that the differentially transctipted genes (DTG) in fibroblasts
from unstable OSCC, especially those up-regulated, were highly connected in terms or protein-protein interactions. In
particular, expression of ACTA2, ITGA6 and MMP1 was associated with more aggressive behavior and poor prognosis.
Conclusions: The results show that fibroblasts have unique gene expression profiles according to the stage of epithelial
tumour progression and in the later stages of this process, the fibroblasts have a greater capacity to stimulate epithelial
invasion in vitro. Highly connected DTG were predictive of invasive behavior.
Poster
118
119
Frontiers in
Tumour Progression
Poster Session A
Human CD271-positive melanoma cells associated with
metastatic potential are able to establish the cellular
heterogeneity of the original patient tumor and to
sustain long-term tumor growth
33
34
Characterization of human colorectal cancer stem cells
resistance to chemotherapy
Victoria da Silva-Diz1, Diana Riba-Artés , Mireia Gausachs , Maria Urpí , Ramón Salazar , Gabriel
1
2
1
3
Capellà2, Alberto Villanueva2 and Purificación Muñoz1
Cancer Epigenetics and Biology Program (PEBC), Biomedical Research Institute of Bellvitge (IDIBELL);
Laboratory of Translational Research, Catalan Institute of Oncology (ICO/IDIBELL); 3Medical Oncology
Service (ICO/IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain
1
Gianluca Civenni, Anne Walter, Nikita Kobert, Daniela Mihic-Probst, Benedetta Belloni, Burkhardt Seifert,
Holger Moch, Reinhard Dummer, Maries van den Broek and Lukas Sommer
Institute of Anatomy, University of Zurich; Oncology, University Hospital Zurich; Department of Dermatology,
University Hospital Zurich; Department of Pathology, Institute of Surgical Pathology, University Hospital
Zurich; Biostatistics Unit ISPM, University Zurich, Switzerland
Human melanoma is composed of distinct cell types reminiscent of neural crest derivatives and particularly, contains
multipotent cells that express the neural crest stem cell markers CD271(p75NTR) and Sox10. Upon transplantation
into Nude or NOD/SCID mice, CD271-positive but not CD271-negative cells formed tumors that fully mirrored
the heterogeneity of the parental melanoma and could be passaged more than 5 times. In contrast, in even more
immunocompromized NOD/SCID/IL2rγnull mice, or in NK cell-depleted Nude or NOD/SCID mice, both CD271-positive
and CD271-negative tumor cell fractions established tumors. However, tumors resulting from either fraction did not
phenocopy the parental tumors, and tumors derived from the CD271-negative cell fraction could not be passaged
multiple times. This observation argues against the use of NOD/SCID/IL2rγnull mice as recipients of xenografts in the
context of cancer stem cell research. Together, our findings identify CD271-positive cells as melanoma stem cells. Our
observation that a relatively high frequency of CD271/Sox10-positive cells correlates with higher metastatic potential
and worse prognosis further supports that CD271-positive cells within human melanoma represent genuine cancer
stem cells.
2
Current chemotherapy for colorectal cancer is based in combined 5-fluorouracil (5-FU) and oxaliplatin (FUOX)
administration with or without other new drugs. However, colorectal cancer is one of the major cause of death in
developmental countries and an important percentage of patients show resistance to this treatment. Tumor initiating
cells or cancer stem cells (CSC) are able to reconstitute a tumor of similar characteristics to the primary tumor after
transplantation into immunodeficient mice. CSCs show the ability to self-renew and give rise to non-CSC progeny,
supporting the tumor growth. Recent studies suggest that CSCs may be intrinsically resistant to therapy, leading to
tumor relapse after chemotherapy. To gain insight into the dynamics of CSCs during tumor progression and in response
to chemotherapy, we have orthotopically implanted and perpetuated in nude mice 14 human colorectal carcinomas
(CRC) that produce local and distal dissemination. We have characterized the chemotherapy response in 7 of these
perpetuated xenografts. Five of these tumors showed a significant reduction of tumor growth after FUOX treatment
compared to the correspondent non-treated control tumors. To evaluate the role of CSCs in chemotherapy response, we
analyzed survival of non-CSCs and CSCs -identified by CD133/CD44/EpCAM cell surface markers - after treatment. Our
results show that in 2 out of 5 chemotherapy responsive tumors, CSCs were more resistant to FUOX than non-CSCs.
Nevertheless, in 3 out of 5 tumors, CSCs were as sensitive as non-CSCs or even more sensitive to treatment. This
differential pattern of CSC response to chemotherapy observed in vivo was reproduced in vitro assays. Indeed, spheres
isolated from different orthotopic CRC exhibit variable sensitivity to in vitro FUOX treatment, following a response pattern
similar to that observed in CSC after in vivo treatment. These results indicate that the resistance to chemotherapy is
not an intrinsic characteristic of CSCs and offer the possibility to characterize sensitive and resistant CSCs to FUOX to
identify genes and pathways directly involved in chemoresistance.
Poster
120
121
Frontiers in
Tumour Progression
Poster Session A
35
36
Snail1-Twist cross-talk regulates Zeb1 expression
during epithelial-to-mesenchymal transition
JunD is essential for c-Fos-induced osteosarcoma
progression
Natalia Dave, S. Guaita-Esteruelas, S. Gutarra, A. Frias, M. Beltran and A. García de Herreros
Jean-Pierre David1,2, Vice Mandic , Julia Luther , Arnt F. Schilling
Institut Municipal Investigació Medica (Parc de Recerca Biomèdica de Barcelona), Spain
1
2
Joachim Grün5, Andreas Grützkau5, Georg Schett2 and Michael Amling3
, Christina Böhm2, Nico Andreas1,
3,4
Bone Cell Differentiation Group, Deutsches Rheuma-Forschungszentrum (DRFZ), Berlin, Germany;
Department of Internal Medicine 3, Rheumatology and Immunology; University of Erlangen-Nuremberg,
Germany; 3Center for Biomechanics & Skeletal Biology, University Medical Center Hamburg Eppendorf,
Germany; 4Biomechanics Section, TUHH Hamburg University of Technology, Germany; 5Regine von Ramin
Laboratory, Deutsches Rheuma-Forschungszentrum (DRFZ), Berlin, Germany
1
Snail1 and Zeb1 are E-cadherin-transcriptional repressors induced during epithelialmesenchymal transition (EMT). In
this work we have analysed the factors required for Zeb1 expression during EMT. In NMuMG cells treated with TGF-,
Snail1 RNA and protein are induced one hour after addition of the cytokine whereas Zeb1 up-regulation requires six
to eight hours. Zeb1 gene expression is markedly dependent on Snail1 since depletion of this protein prevents Zeb1
protein and RNA up-regulation. Moreover, depletion of Twist transcriptional factor retards Zeb1 stimulation by TGF- or
decreases Zeb1 expression in other cellular models indicating that this factor is also required for Zeb1 expression.
Accordingly, Snail1 and Twist, cooperate in the induction of Zeb1: co-transfection of both cDNAs is required for the
maximal expression of Zeb1 mRNA. Unexpectedly, the expression of Snail1 and Twist shows a mutual dependence
although to a different extent; whereas Twist depletion retards Snail1 up-regulation by TGF-, Snail1 is necessary for the
rapid increase of Twist protein and later up-regulation in Twist mRNA induced by the cytokine. Therefore, these results
indicate that Snail1 and Twist are mutually controlled and are involved in the regulation of Zeb1 expression.
2
c-Fos overexpression causes osteosarcoma due chondo-osteoblast transformation. Binding to DNA requires c-Fos
dimerisation with one of the three Jun members. Expression studies in osteosarcomas developing in c-fos transgenic
mice (c-fostg) and in cells isolated from tumors suggested a role for JunD in c-Fos-induced tumor. We generated c-fostg
mice lacking JunD to test this hypothesis. Although tumor incidence was unaffected, a drastic decreased tumor size was
observed in absence of JunD. Thus, JunD is required for the progression but not the initiation of osteosarcoma. Despite
an increased expression of the cyclin-dependent kinase inhibitor p27, no change in proliferation or apoptosis could
be detected and, in agreement, no clear defect in cell division and apoptosis was detected in comparative Affymetrix
gene chips analysis of the tumors. However, a reduced mineralization of the tumors was demonstrated by von Kossa
staining. Surprisingly, the expressions of typical bone and cartilage markers were all unaffected. In contrast, we found
an increased osteoclast numbers in JunD deficient tumors that we confirmed by makers analysis. The phenotype was
not cell autonomous as shown by the normal osteoclastogenesis of monocytes isolated from c-fostg mice lacking
JunD and not caused by increased levels of the essential osteoclastogenic cytokines M-CSF or RANKL. Interestingly,
Sfrp1, a Wnt inhibitor also known to inhibit osteoclast differentiation, was strongly decreased in tumors lacking JunD.
Importantly, Sfrp1 was also downregulated in the bone of JunD deficient mice that develop a bone phenotype resembling
Sfrp1 deficiency. Thus, our work reveals an unexpected role for JunD, a non-oncogenic Jun member, in c-Fos–induced
tumor progression. It indicates that c-Fos-induced tumorigenesis may depend on its association with JunD. These
data also suggest that locally increasing bone resorbtion by osteoclast may represent a therapeutic option to control
osteosarcomas progression.
Poster
122
123
37
Frontiers in
Tumour Progression
Poster Session A
Screen for inhibitors of cell migration in cancer
metastasis using adenoviral knock-down
Multisite phosphorylation of Erk5 in mitosis
Remko de Pril1, Annemarie Lekkerkerker , Desire van Steenhoven , Ilhem Maghrani , Ester Frische , Tim
1
1
1
38
1
Perera2, Janine Arts2, Martin Page2, David Fischer1 and Richard Janssen1
Galapagos, Leiden, The Netherlands; 2Ortho Biotech Oncology R&D, Beerse, Belgium
1
Enhanced cell migration is a hallmark of metastatic cancer cells. The propensity of cancer cells to close an open wound
in a cell monolayer is thought to predict this ability. Using our adenoviral shRNA knock-down library we have established
a high-throughput wound healing assay to identify novel genes involved in cell migration. Therefore, a 96-pin scratch
tool was designed to apply a constant mechanical scratch-wound in the cellular monolayer. Cells were fixed at a
preset time period after scratching and the plates were imaged on an InCell Analyzer1000. We used transmitted light
imaging for segmentation and quantification of the scratch wound that remained open. Accordingly, we developed an
algorithm to measure the open space, which comprises the area of the wound as well as open spaces between cells.
Genes whose knock-down inhibit cell migration can be identified by their effect on the open wound. In addition, genes
that affect either proliferation or viability can thereby be counter screened as these disrupt the monolayer. Using this
approach we demonstrated that two knock-down constructs targeting a known player in motility, CXCR4, inhibit wound
healing, validating our set-up. Using this wound healing assay we have identified a number of novel genes associated
with cancer cell motility. These targets are currently validated for their effect in 3D invasion using Boyden chambers. We
use confocal imaging on a Pathway 435, of both the seeded cells in the top compartment and the invaded cells on the
bottom of the filter. In this way, we have identified a number of control genes which inhibit invasion through matrigel.
Using this assay we will validate the hits that inhibit migration, for their ability to additionally inhibit 3D invasion. As our
adenoviral knock-down libraries focus on drugable targets, these validated targets can quickly be employed to generate
small molecule compounds or antibody therapeutics targeting cancer metastasis.
Elena Díaz-Rodríguez and Atanasio Pandiella
Instituto de Biología Molecular y Celular del Cáncer. CSIC-Universidad de Salamanca, Spain
The MAPKs routes are highly conserved signalling pathways implicated in multiple cellular responses, including the
control of cell proliferation, differentiation and survival. In the last few years one of them, the MAPK Erk5, has been
indentified and involved in the regulation of cellular proliferation in response to proliferative signals, cytokines and
cellular stresses. Besides, its hyperactivation has been associated with highly aggressive forms of breast and prostate
cancers. The classical pathway of Erk5 activation involves dual phosphorylation at its TEY microdomain by its upstream
kinase, MEK5. Nevertheless we have found a second pathway involving kinase activities distinct from MEK5 that
controls Erk5 phosphorylation in mitosis. In fact, Erk5 is phosphorylated at the beginning this phase of the cell cycle and
dephosphorylated at its end. Studies aimed at identifying the kinase involved suggested that CDK1 activity is required to
sustain Erk5 phosphorylation in mitosis, as treatment with RO3306, a CDK1 inhibitor, reversed mitotic phosphorylation
of Erk5. The mitotic phosphorylation of Erk5 occurs at several residues located at its unique C-terminal region, within
a subdomain implicated in the control of its subcellular location. Furthermore, molecular studies using Erk5 phosphomutants indicated that phosphorylation at these sites may participate in the control of the transit of Erk5 between the
cytosol and the nucleus, besides regulating its transcriptional activity. Together, our results demonstrate the existence
of a second Erk5 phosphorylation pathway that is activated in mitosis and may participate in the regulation of Erk5
functions. Given the link between Erk5 levels and patient prognosis in breast and prostate cancers, it will be interesting
to evaluate the degree of activation of this pathway in patients in terms of phosphorylation at the new sites identified
and correlate them with clinical data such as overall survival or disease progression.
Poster
124
125
Frontiers in
Tumour Progression
Poster Session A
DARPins: a new generation of protein therapeutics for
oncology
Ignacio Dolado, Douglas Phillips, Talitha R. Bakker and Michael T. Stumpp
Molecular Partners AG, Schlieren-Zürich, Switzerland
Cancer therapeutics is currently dominated by two contrasting artilleries: small molecules and therapeutic antibodies.
Small molecules (~ 1 kDa) have constituted the standard of care for the last 40 years with remarkable success in some
cases (e.g. Imatinib). Unfortunately, their use has usually been limited by specificity and toxicity issues. Conversely,
antibodies (~ 150 kDa) have emerged in recent years as a therapeutic option able to overcome the limitations of small
molecules, but with significant room for improvement on the efficacy, biodistribution and developability sides. In between
both worlds lie DARPins (designed ankyrin repeat proteins), a novel class of binding molecules that capitalize on the
small size and potency of small molecules, combined with the specificity and low toxicity of antibodies. DARPins are
small (14 kDa), single domain proteins which can be easily selected to bind any given target protein with high affinity
and specificity. Their favorable molecular properties, including high stability, specificity and affinity, as well as small size
and adjustable pharmacokinetics, make them ideal candidates for oncology applications. For instance, DARPins have
been successfully used for the inhibition of kinases, proteases and drug-exporting membrane proteins of relevance
in oncology. In vivo, DARPins generated against angiogenic factors or designed to serve as payload-delivery vehicles
have shown outstanding tumor accumulation, low toxicity, and high therapeutic indexes in preclinical tumor models.
Furthermore, DARPins can be engineered to carry various effector functions, combine multiple binding specificities, or
restrict their therapeutic activity to an anatomic area of interest, thus enabling completely new drug formats. Taken
together, DARPins are a new generation of protein therapeutics with the potential to surpass existing therapeutic options.
39
40
Expression of kinin system components and functional
activity of the bradykinin b1 receptor in breast cancer
cells
Pamela Ehrenfeld ,
1
Alejandro Lizama1, Daniela P. Nualart1, María F. Pavicic1, Carola E. Matus1, Carlos B.
Gonzalez1, Rafael Burgos1, Andrew F. G. Quest2 and Carlos D. Figueroa1
1
Institutes of Anatomy, Histology & Pathology, Physiology and Pharmacology Universidad Austral de Chile,
Valdivia, Chile; 2Centro de Estudios Moleculares de la Celula (CEMC), Facultad de Medicina, Universidad de
Chile, Santiago, Chile
We and others have shown that the activation of bradykinin B1 (B1R) and B2 receptors in breast cancer cells stimulates
proliferation in a manner dependent on epidermal growth factor receptor (EGFR) activity and subsequent signaling via
the ERK1/2 mitogen-activated protein kinase pathway. However, other components of the kinin system involved remain
to be identified. Here, we characterized in more detail the kinin system in estrogen-sensitive MCF-7 and estrogeninsensitive MDA-MB-231 breast cancer cells. Using RT-PCR, immunocytochemistry and Western blotting, we identified
tissue kallikrein hK1, the enzyme responsible for kinin generation, high and low molecular weight kininogens, the
kinin-releasing substrates, and B1R. We also evaluated the ability of B1R to regulate the release of extracellular matrix
metalloproteases (MMPs) on breast cancer cells stimulated with Lys-des[Arg9]-bradykinin (LDBK), the natural B1R
agonist. Western blotting and gelatin zymography were used to evaluate presence of MMP-2 and MMP-9 in the incubation
media. Stimulation of B1R with 1 nM LDBK induces the accumulation of MMPs within 5 to 30 min of stimulation.
Coincidently, immunocytochemistry showed that cancer cells have reduced levels of MMPs after stimulation. This
effect was blocked by pre-treating the cells with a B1R antagonist and a B1R siRNA. Activation of the ERK1/2 pathway
and EGFR transactivation were required for MMPs release because both the MEK1 inhibitor, PD98059, and AG1478,
an inhibitor of the EGFR tyrosine kinase activity, blocked this event. The importance of EGFR-dependent signaling for
MMPs release was confirmed by transfecting cells with the dominant negative EGFR mutant HERCD533. Additionally,
LDBK induced an increase in MMPs mRNA levels when compared to unstimulated control cells. Our results establish
the existence of a kallikrein-kinin system in breast cancer cells and suggest that B1R activation may increase the
invasive activity of cancer cells by releasing MMPs that degrade the extracellular matrix and thereby favor metastasis. A
better understanding of the role of this system in breast cancer should lead to the identification of potentially attractive
alternative therapeutic targets for treatment of this neoplasia.
Poster
126
127
Frontiers in
Tumour Progression
Poster Session A
Platelet adhesion as determinant of hematogenous
metastasis
Georg Enders, Benedikt Kellermann and Costanza Chiapponi
Institute for Surgical Research at the WBex, LMU, Munich, Germany
41
42
CCL2 down-regulation form macrophages by stressinduced norepinephrine is protective in a human model
of malignant melanoma
Tim D. Eubank1, Michael T. Bailey
, Amy C. Gross3, Laura A. Sumner1, Clay B. Marsh1,3, Ronald Glaser3, 4, 5
2,3
Many types of tumors release cancer cells that migrate to distant sites in the body. Here the tumor cells lodge but are
exposed to shear forces and the impact of immune cells from the blood. So it is not surprising that at least in model
systems only a small fraction of tumor cells survive. Unfortunately the remaining tumor cells dictate the patient’s fate.
Hence a better understanding of the initial events in the homing and survival of tumor cells should offer therapeutic
options. The intravenous injection of mouse B16 melanoma cells, which mimics several steps in hematogenous
metastasis, was used to gain insight into the early steps. The tumor cells tend to have no specific receptor for interaction
with the endothelial cells. Their physical dimension as well as the activation of the coagulation system by the expressed
tissue factor (F3) favouring aggregate formation allowed the tumor cells to get stuck in the microcirculation of the lung.
This was followed by platelet adhesion forming a kind of thrombus which offers not only shielding but also growth
factors by platelet degranulation. The inhibition of platelet adhesion or activation could consequentially reduce the local
formation of metastases. The inhibition of platelet adhesion by anti-gpIIbIIIa and its consequences was investigated
further. Gene expression analysis and plasma mediators were determined to get more information on the initial
response. The results show a clear reduction of the number of metastases after blocking platelet gpIIbIIIa. The plasma
concentration for Vegfa and Tgf-beta was lower than in control animals, presumably reflecting the lower degranulation
of platelets. Given that the inhibition of platelet adhesion should interfere with the early events of tumor cell homing we
measured the expression of a tumor specific marker in the lung. Interestingly enough this gave a higher signal for the
treated animals and indicates a higher initial number of tumor cells present in the lung. These must have a lower survival
rate as indicated by the reduced number of metastases at the end of the experiment. Local expression of markers for
cytotoxic lymphocytes (CD8, perforin1, Gzma) was higher in gpIIbIIIa blocked animals and points to the fact of higher
local cytotoxicity. This may be supported by a better accessibility of the tumor target cells resulting in a lower number
of metastases at the end. Interference with the early settlement can be achieved by blocking platelet adhesion. This not
only reduces the chances for local survival but seems to make the tumor cells more accessible to the immune cells and
offers especially in combination with other protocols a good therapeutic opportunity.
and Eric V. Yang1, 3, 4
1
Division of Pulmonary Medicine, Allergy, Critical Care and Sleep Medicine, Department of Internal Medicine,
The Ohio State University, Columbus, USA; 2Section of Oral Biology, The Ohio State University, Columbus,
USA; 3Institute of Behavioral Medicine Research, The Ohio State University, Columbus, USA; 4Department of
Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus,
USA; 5Comprehensive Cancer Center, The Ohio State University Medical Center, Columbus, USA
Stress is implicated in the progression of various tumor types. We subjected female C57Bl/6 mice to six hour periods of
restraint stress (RST) or home cage control (HCC) for seven days. After, we subcutaneously injected B16F10 malignant
melanoma tumor cells onto one side of these mice. The RST/HCC paradigm was continued for 14 additional days. We
observed that tumors in RST mice grew significantly slower than those tumors in the mice of the HCC group (doublingtime: HCC=3.7 days; RST=6.5 days). Cytokine and immunohistochemical analysis of the tumors revealed that RST
contributed to a reduction in both CCL2 (HCC=887±115 vs. RST=560±90 pg/ml/100mg tissue) and F4/80+ tumor
macrophage presence (HCC=1.30±0.55 vs. RST=0.75±0.30% positively-stained cells per HPF), while HPLC analysis
of the livers illustrated an increase in norepinephrine output (HCC=0.78±0.02 vs. RST=1.22±0.23ng/ml/mg tissue).
In vitro analysis of B16F10 tumor cells suggested the slower tumor growth rates in the RST mice were not due to
proliferative or cytotoxic effects from CCL2 or norepinephrine. Further, we illustrate that norepinephrine down-regulates
CCL2 production from both mouse bone marrow-derived macrophages and human monocyte-derived macrophages
and that pre-treatment with the pan-β-adrenergic receptor inhibitor, Nadolol, rescues this activity. The role of CCL2 in
the stressrelated suppression of tumor growth was highlighted by finding that melanoma tumors injected into CCL2deficient mice were not affected by RST. In conclusion, this study suggests that stress-associated norepinephrine release
was protective in a mouse model of malignant melanoma by regulating CCL2 production, macrophage infiltration and
tumor growth.
Poster
128
129
Frontiers in
Tumour Progression
Poster Session A
KSHV induces transcriptional reprogramming of
lymphatic endothelial cells into an invasive cell type
43
44
Fang Cheng ,
Loss of hepatic Stat5a/b increases HCC incidence and
aggressiveness but abrogates negative side effects
of GH overexpression in a murine inflammatory liver
cancer model
University of Helsinki, Genome-Scale Biology Program, Helsinki, Finland; 2University of Helsinki, Molecular
Cancer Biology Program, Helsinki, Finland; 3Karolinska Institute, Department of Oncology-Pathology,
Stockholm, Sweden; 4UCL, Cancer Institute, London, UK
Katrin Friedbichler1, Madeleine Themanns , Kristina Mueller , Jan-Wilhelm Kornfeld , Luigi
1
Simonas Laurinavicius1, Nami Sugiyama1, Peter Biberfeld3, Stephen Henderson4, Mervi
Aavikko1, Lauri A. Aaltonen1, Chris Boshoff4, Kari Alitalo2, Kaisa Lehti1 and Päivi M. Ojala1
1
Kaposi’s sarcoma (KS) tumors consist of spindle-shaped endothelial cells (ECs) that express markers of lymphatic
endothelium, smooth muscle cells, macrophages and dendritic cells. Upon infection with KSHV, lymphatic ECs (LECs)
develop spindle morphology, the hallmark of KS tumor cells, but do not show other properties of transformed cells.
To better mimic the microenvironment of infected cells in vivo, we have developed a 3-dimensional (3D) organotypic
spheroid outgrowth assay for KSHV-infected LECs. Interestingly, our results show that while control LEC spheroids sprout
very little if at all, the KSHV-LEC spheroids show extensive sprouting. We have also analyzed the cellular differentiation
status of KSHV-LEC and control LEC spheroids by confocal microscopy and qRT-PCR using a variety of markers for cell
differentiation. Our data strongly suggests that KSHV induces transcriptional reprogramming of LECs into a mesenchymal
cell type via a process called endothelial to mesenchymal transition (EndMT). We are currently deciphering which cellular
pathways are activated and involved in the KSHV-induced EndMT by performing a global gene expression analysis
of LEC and KSHV-LEC spheroids. EndMT has recently been recognized as a potential source for cancer associated
fibroblasts (CAFs). To assess if the KSHV-induced EndMT could act as a source for KS-associated CAFs we have looked
for the presence of KSHV-positive mesenchymal cells in a panel of KS biopsies. Intriguingly, cells positive for both KSHV
LANA and mesenchymal markers such as aSMA or vimentin can be readily detected in the majority of the KS biopsies
analyzed. EndMT endows cells with migratory and invasive properties as a result of the loss of tight-junctions and by
expression of N-cadherin. We have analysed the invasive potential of the KSHV-LECs, and the results suggest that KSHVLECs can invade the collagen matrix. Inhibition and siRNA knock-down experiments indicate that invasion and spheroid
sprouting are dependent on specific matrix metalloproteinases. We are currently investigating the role of viral genes in
the transcriptional reprogramming and increased invasion.
1
1
2
Terracciano3, Andrey Kozlov4, Susanne Haindl4, Tricia Behling4, Lukas Kenner5, Thomas Kolbe6, Mathias Müller7, Kenneth
Snibson8, Markus Heim9 and Richard Moriggl1
Ludwig Boltzmann Inst. for Cancer Research, Vienna, Austria; 2Inst. for Genetics, Dept. of Mouse Genetics
and Metabolism, Univ. of Cologne, Germany; 3Inst. of Pathology, Univ. Hospital Basel, Switzerland; 4LBI
for Traumatology, Vienna, Austria; 5Inst. of Clinical Pathology, Medical Univ. of Vienna, Austria; 6Inst. of
Laboratory Animal Science & Biomodels Austria, Vetmeduni Vienna, Austria; 7Inst. for Animal Breeding and
Genetics, Vetmeduni Vienna, Austria; 8Centre for Animal Biotechnology of the Univ. of Melbourne, Victoria,
Australia; 9Dept of Biomedicine, Univ. Hospital Basel, Switzerland
1
The Janus kinase (Jak)/Signal transducer and activator of transcription (Stat) signaling pathway regulates fundamental
cellular functions, such as proliferation, survival, and differentiation as well as developmental processes. Importantly, it
is one of the core pathways which are frequently deregulated in primary tumors. Stat5 transcription factors have a key
role in hematopoiesis, lipid metabolism, reproduction, and the immune response. Hepatic Stat5 transcription factors
regulate postnatal body growth, sexual maturation, RNA biogenesis as well as metabolism and they have a protective
effect on the liver. Important growth factors and cytokines such as growth hormone (GH), prolactin, and insulin are
able to activate hepatic Stat5 proteins. In clinical studies and mouse models, both hyperactivation and deletion of
Stat5b in hepatocytes have been shown to be involved in the development of hepatocellular carcinoma (HCC). Using
growth hormone transgenic (GHtg) mice and conditional knockout mice lacking hepatic Stat5a/b (Stat5Δhep, AlfpCre),
we analyzed the role of Stat5 in the development of inflammatory liver cancer caused by hyperactivated GH signaling.
Interestingly, gigantism and premature mortality in GHtg mice was found to be dependent on Stat5. Moreover, the
polymorphonuclear shape and increased size of hepatocytes in GHtg mice were only found in the presence of Stat5. By
contrast, the liver tumor phenotype seen in these mice was aggravated upon deletion of Stat5 and was accompanied by
increased hepatic fat deposition (hepatosteatosis). Our results suggest that hepatic Stat5 proteins prevent the formation
of aggressive HCC and fatty liver disease via their hepatoprotective functions. At the same time, they are key factors in
controlling organ and body size as well as systemic inflammation secondary to hyperactivated GH signaling.
Poster
130
131
45
Frontiers in
Tumour Progression
Poster Session A
3-Phosphoinositide–Dependent Kinase 1 controls
breast tumor growth in AKT-independent manner
Inhibition of IL-6/Jak/Stat3 signaling: a novel and
potent targeting therapy for lung cancer
Paolo Armando Gagliardi1, Laura di Blasio , Francesca Orso , Giorgio Seano , Roberto Sessa ,
1
2
1
46
1
Daniela Taverna2, Federico Bussolino1 and Luca Primo3
Dept. of Oncological Science, Institute for Cancer Research and Treatment, Univ. of Turin, Candiolo,
Italy; 2Dept. of Oncological Science, Molecular Biotechnology Center, Univ. of Turin, Torino, Italy; 3Dept. of
Biological and Clinical Science, Institute for Cancer Research and Treatment, Univ. of Turin, Italy
1
Aberrant phosphatidylinositol 3-kinase (PI3K) signalling occurs commonly in several carcinomas. Although
3-Phosphoinositide-dependent protein kinase 1 (PDK1) is the pivotal element of PI3K pathway, its role in tumor
progression is not understood. The Ser/Thr kinase PDK1 binds the phosphatidyl inositol trisphosphate (PIP3) produced
by PI3K and activates Akt/PKB and other kinases of AGC family. Recent evidences indicated that PDK1 is overexpressed
in many breast carcinoma and potentiates the oncogenic effects of upstream lesions on the PI3K pathway. We show that
breast cancer cells with PI3KCA mutations (T-47D) or with different genetic alterations (MDA-MB-231) are dependent
by PDK1 for the growth in absence of adhesion and tumor formation in mice. Actually, the reduction of PDK1 activity by
RNAi or chemical molecules strongly inhibits the growth in soft-agar of these cell lines and increases their sensitivity to
anoikis. Nevertheless, PDK1 down-regulation does not alter the proliferation rate of these cells when grown in adhesion.
In xenografts model, the PDK1-silenced cells exhibit reduced tumor growth by increasing the apoptosis inside the
tumor. Re-expression of wild-type PDK1 in silenced cells rescues the phenotype while PDK1 kinase-dead does not. The
requirement of the kinase activity is in contrast with the unaltered phosphorylation of a PDK1 substrate. In fact, Akt1
is normally phosphorylated in PDK1-silenced cells. The involvement of Akt1, as possible PDK1 downstream effector in
breast tumorigenicity, has been excluded by the introduction in PDK1-silenced cells of a constitutively active form of
Akt1, which is unable to rescue the impaired phenotype. These results suggest a widespread role of PDK1 in breast
cancer that is not exclusively related to tumors harboring oncogenic mutation in the PI3K pathway. On the other hand,
we proved that the PDK1 role in breast cancer can be independent from Akt that is still considered the main PI3K
downstream effector.
Sizhi P. Gao1, Rosario Andre , Hector P. Selgas , Lucia Regales , Juliann Chmielecki , Volodia D. Gueorguiev ,
2
2
1
3
1
Juan Jiu1, Blake Hefter1, Erez Schori1, William Pao3, David Lyden2 and Jackie F. Bromberg1
Memorial Sloan-Kettering Cancer Center, New York, USA; 2Cornell University Weill Medical College, New
York, USA; 3Vanderbilt University, Nashville, USA
1
Non-small cell lung cancer (NSCLC) is the leading cause of death in the United States. A fraction of these tumors
harbor somatic activating mutations in the epidermal growth factor receptor (EGFR) leading to constitutive activation
of the kinase. Targeting this pathway using a class of tyrosine kinase inhibitors (TKI) such as gefitinib or erlotinib
induces significant initial responses in patients with NSCLC expressing activating EGFR mutations, however, disease
progression invariably occurs in part due to the outgrowth of cancers with EGFR TKI resistant secondary mutations.
There are many ongoing attempts to overcome this problem, but the current efforts are mostly focused on EGFR itself
and their effectiveness remains inconclusive. The interleukin-6 (IL-6) /Jak/Stat3 signaling pathway is critical not only
for inflammation and immunity, but also for oncogenesis. Stat3 is persistently activated in many human cancers,
promoting tumorigenesis through the regulation of genes important for proliferation, apoptosis, invasion, angiogenesis
and suppression of anti-tumor immunity. We previously determined that Stat3 is activated in mutant EGFR expressing
NSCLC cell lines and primary tumors in part through autocrine upregulation of the IL-6 gene that activates the gp130/
Jak signaling. In our current report, we studied the in vivo effect of inhibiting the IL-6/Jak/Stat3 signaling in EGFR
TKI-resistant models of NSCLC. Our data demonstrated that Jak inhibition by a novel orally available Jak inhibitor
markedly repressed in vivo growth of 3 different TKI resistant NSCLC cell lines as well as a TKI sensitive cell line.
Immunohistochemical analyses of these samples revealed reduced proliferation and angiogenesis in Jak inhibitor
treated tumors. In addition, administration of an IL-6R blocking antibody to xenograft tumors of a TKI resistant NSCLC
cell line also achieved significant reduction in tumor growth compared to controls. We further tested the potential role of
IL-6 in a transgenic murine model of TKI-resistant, mutant EGFR mediated lung cancer. Our results demonstrated mice
lacking IL-6 still developed lung tumors but at a much slower rate of tumor progression compared to IL-6 expressing
littermates. Additional examinations showed that mice lacking IL-6 developed tumors with papillary features compared
to IL-6 expressing littermates whereas most of the tumors were of the more advanced, solid/pleiomorphic subtype. IL-6
deficient lung tumors had decreased proliferation and angiogenesis compared to tumors from wildtype littlermates. In
summary, our findings lead us to hypothesize that the IL-6/Jak pathway should be considered as a novel therapeutic
target for this disease, especially TKI resistant NSCLC.
Poster
132
133
47
Frontiers in
Tumour Progression
Poster Session A
Expression and clinical significance of the Kv3.4
potassium channel subunit in the development
and progression of head and neck squamous cell
carcinomas
Activation of LKB1-Akt pathway independent of
Phosphoinositide 3-Kinase plays a critical role in
the proliferation of hepatocellular carcinoma from
nonalcoholic steatohepatitis
Juana María García-Pedrero1, S. T. Menéndez , J. P. Rodrigo , D. García-Carracedo , E. Allonca ,
1
1
1
1
G. Álvarez- Alija , S. Casado-Zapico , M. F. Fresno , C. Rodríguez and C. Suárez
1
2
3
2
48
1
Servicio de Otorrinolaringología, Hospital Universitario Central de Asturias and Instituto Universitario
de Oncología del Principado de Asturias, Oviedo, Spain; 2Departamento de Morfología y Biología Celular,
Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Spain; 3Servicio de
Anatomía Patológica, Hospital Universitario Central de Asturias, Oviedo, Spain
1
The concept of ion channels as membrane therapeutic targets and diagnostic/prognostic biomarkers is attracting growing
interest. We therefore investigated the expression pattern and clinical significance of the Kv3.4 potassium channel
subunit in the development and progression of HNSCC. Kv3.4 mRNA levels were determined by real-time RT-PCR in
both HNSCC tissue specimens and derived cell lines. Kv3.4 protein expression was evaluated by immunohistochemistry
in paraffin-embedded tissue specimens from 84 patients with laryngeal/pharyngeal squamous cell carcinomas and 67
patients with laryngeal dysplasias. Increased Kv3.4 mRNA levels were found in 15 (54%) of 28 tumours, compared to
the corresponding normal epithelia and varied mRNA levels were detected in 12 HNSCC-derived cell lines. Increased
Kv3.4 protein expression was observed in 34 (40%) of 84 carcinomas and also at early stages of HNSCC tumourigenesis.
Thus, 35 (52%) of 67 laryngeal lesions displayed Kv3.4-positive staining in the dysplastic areas, whereas both stromal
cells and normal adjacent epithelia exhibited negligible expression. No significant correlations were found between
Kv3.4-positive expression in HNSCC and clinical data; however, Kv3.4 expression tended to diminish in advanced-stage
tumours. Interestingly, patients carrying Kv3.4-positive dysplasias experienced a significantly higher laryngeal cancer
incidence than did those with negative lesions (p = 0.0209). In addition, functional studies using HNSCC cells revealed
that Kv3.4 blockade by siRNA leads to the inhibition of cell proliferation via selective G2/M cell cycle arrest. These
data demonstrate for the first time that Kv3.4 expression is frequently increased during HNSCC tumourigenesis and
correlated significantly with a higher cancer risk. Our findings support a role for Kv3.4 in malignant transformation and
provide original evidence for the potential clinical utility of Kv3.4 expression as a biomarker for cancer risk assessment.
Juan L. García-Rodríguez,
Nuria Martínez-López, Marta Varela-Rey, David Fernández-Ramos,
Mercedes Vázquez-Chantada, Nieves Embade, Ashwin Woodhoo, Luis Espinosa-Hevia, Francisco J. Bustamante,
Luis A. Parada, Manuel S Rodriguez, Shelly C. Lu, José M. Mato and Maria L. Martínez-Chantar
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas
(Ciberehd), Technology Park of Bizkaia, Spain
LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration.
Mice lacking the methionine adenosyltransferasesubtype. IL-6 deficient lung tumors had decreased proliferation and
angiogenesis compared to tumors from wildtype littlermates. In summary, our findings lead us to hypothesize that the
IL-6/Jak pathway should be considered as a novel therapeutic target for this disease, especially TKI resistant NSCLC.
(MAT) gene MAT1A exhibit a chronic reduction in hepatic S-adenosylmethionine (SAMe) levels, basal activation of LKB1,
and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These
results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a
marked reduction in hepatic MAT1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe-deficient
[SAMe-D]) from MAT1A knockout (MAT1A-KO) mouse HCC to examine the role of LKB1 in the development of liver tumors
derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe-D cells. LKB1 regulates
Akt-mediated survival independent of phosphoinositide 3-kinase, adenosine monophosphate protein–activated kinase
(AMPK), and mTORC2. In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in
the cytoplasm and the regulation of herpesvirus-associated ubiquitin-specific protease (HAUSP) and Hu antigen R (HuR)
nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53
and p-LKB1(Ser428) in a NASH-HCC animal model (from MAT1A-KO mice) and in liver biopsies obtained from human
HCC derived from both alcoholic steatohepatitis and NASH. Conclusion: The SAMe-D cell line is a relevant model of HCC
derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a
practical tool for uncovering new therapeutic strategies.
Poster
134
135
Frontiers in
Tumour Progression
Poster Session A
Recruitment of monocytes/macrophages by tumour cell
clot formation is essential for metastatic cell survival
Ana Maria Gil-Bernabe, Spela Ferjancic, Monika Tlalka, Lei Zhao, Thomas Tapmeier, Danny Allen,
Karla Watson, Sally Hill and Ruth Muschel
Gray Institute for Radiation Oncology and Biology, University of Oxford, UK
Coagulation is required for both spontaneous and experimental metastases. Many tumour cells express Tissue Factor
(TF), a key molecule in the coagulation cascade. Here, we identify the recruitment of monocytes/macrophages as
an essential component of the mechanism by which coagulation enhances metastasis. Methods Clot formation and
monocyte/macrophage recruitment were determined by microscopy. These processes were manipulated by changes in
TF and TFPI expression, pharmacological inhibition of coagulation and the use of mice with genetically altered monocytes/
macrophages. Results TF expression resulted in clot formation and cell survival in the lung. The cytoplasmic domain of
TF, involved in cell signalling, didn’t impair clot formation or cell survival. Monocytes/macrophages were recruited to the
tumour cells in a coagulation dependent interaction. This recruitment peaked at 8h after the injection of tumour cells.
The population of monocytes/macrophages recruited to the tumour cells through the clots on their surface is CX3CR1+,
Cd11b+, CD45+, Cd11c-, CD3- and Gr1-, with variable expression levels of F4/80 and CD68. Moreover, impairment
of monocyte/macrophage function, in Cd11b-DTR and in Mac1 KO models, decreases tumour cell survival in the lung
despite clot formation. Finally, we show that coagulation does not duplicate the processes involved in the formation of a
premetastatic niche. Conclusions The formation of clots on the surface of tumour cells, mediated by their expression of
TF, causes the recruitment of a population of monocytes/macrophages that enhances the survival of the tumour cells in
the lung. Clot formation per se is not sufficient for this survival, but requires functional monocytes/macrophages.
49
50
Semaphorin 3A blocks tumor invasiveness and prevent
resistance to anti-angiogenic therapies by overriding
cancer hypoxia
Federica Maione1,2, Stefania Capano1,2, Claudia Meda1,2, Lorena Zentilin3, Mauro Giacca3, Federico Bussolino1,2, Guido
1,2
Serini2,4 and
Enrico Giraudo
Laboratory of 1Transgenic Mouse Models and 4Cell Adhesion Dynamics, Institute for Cancer Research and
Treatment (IRCC), Candiolo, Italy; 2Department of Oncological Sciences, University of Torino, School of
Medicine, Candiolo, Italy; 3Molecular Medicine Laboratory, International Centre for Genetic Engineering and
Biotechnology (ICGEB), Trieste, Italy
Class 3 Semaphorins (Sema3), are implicated in the regulation of both physiological and tumor angiogenesis. We
have recently shown that endothelial Sema3A is an endogenous inhibitor that is lost during tumor progression and
its reintroduction into a mouse model of pancreatic islet b-cell carcinogenesis(RipTag2), resulted in reduced vascular
density, blood vessel normalization, restoration of tumor normoxia, and inhibition of tumor growth. Here, we show
that the treatment of tumor-bearing RipTag2 with exogenous Sema3A mice induced a dramatic reduction of tumor
invasiveness, the reappearance of E-cadherin and a down-modulation of vimentin, two known targets of cancer hypoxia
that are also regulated during epithelial-mesenchymal transition (EMT). Then, we sought to investigate if the preventive
administration of exogenous Sema3A was able to overcome the evasive resistance observed in RipTag2 upon treatment
with Sunitinib, an anti-angiogenic agent that inhibits several tyrosine kinase receptors. Notably, we observed a dramatic
reduction of tumor volume, cancer invasiveness, liver and peripancreatic lymph node metastases in RipTag2 treated for 2
weeks with Sema3A followed by 2 weeks of Sunitinib treatment, compared to Sunitinib-treated controls. Moreover, while
Sunitinib-treated tumors were highly hypoxic and displayed few pericyte-covered vessels, the combinatorial regimen of
Sema3A and Sunitinib normalized the vasculature and restored tumor normoxia. Finally, Real-Time RT-PCR and confocal
microscopy analysis revealed a strong increase of E-cadherin expression and a complete inhibition of vimentin in tumors
treated with Sema3A and Sunitinib as compared to Sunitinib alone. Therefore, re-expression of Sema3A in tumors
may safely harness the therapeutic potential of anti-angiogenic drugs, by normalizing the vasculature, inhibiting tumor
hypoxia, and modulating the expression of EMT markers and other hypoxic-induced genes activated by anti-angiogenic
treatments.
Poster
136
137
51
Frontiers in
Tumour Progression
Poster Session A
AICAR induces apoptosis independently of AMPK and
p53 through upregulation of the BH3-only proteins BIM
and NOXA in chronic lymphocytic leukemia cells
Diana M. González-Gironès , Antonio F. Santidrián , Daniel Iglesias-Serret , Llorenç Coll-Mulet ,
1
1
1
1
Ana M. Cosialls1, Mercè de Frias1, Clara Campàs1, Eva González-Barca2, Esther Alonso3, Verena Labi4, Benoit Viollet5, 6,
Adalberto Benito7, Gabriel Pons1, Andreas Villunger4 and Joan Gil1
1
Departament de Ciències Fisiològiques II, IDIBELL–Universitat de Barcelona, L’Hospitalet de Llobregat,
Spain; 2Departament d’Hematologia Clínica, IDIBELL–Institut Català d’Oncologia, L’Hospitalet de Llobregat,
Barcelona, Spain; 3Servei d’Hematologia, IDIBELL–Hospital de Bellvitge, L’Hospitalet de Llobregat, Barcelona,
Spain; 4Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria;
5
Institut Cochin, Université Paris Descartes, Paris, France; 6Inserm U567, Paris, France; 7Servicio de
Nefrología, Hospital Marqués de Valdecilla-IFIMAV, Santander, Spain
AICAR (5-aminoimidazole-4-carboxamide riboside or acadesine) induces apoptosis in chronic lymphocytic leukemia
(CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the
mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well known molecular target,
adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662
failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from AMPKalfa1-/mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective
of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway.
Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3only subgroup including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from NOXA-/- or BIM-/- mice were
partially protected from the cytotoxic effects of AICAR. Consistently, B cells from NOXA-/-/BIM-/- mice resisted induction
of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL- 2. These findings support the notion
that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to
conventional chemotherapy.
52
Prognostic significance of lymphangiogenesis in
pharyngolaryngeal carcinoma patients
María Victoria González1, D. Garcia-Carracedo , J.P. Rodrigo
1
, A. Astudillo1,3 and C. Suarez Nieto1,2
1,2
Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Asturias,
Spain; 2Servicio de Otorrinolaringología, 3Servicio Anatomía Patológica, Hospital Universitario Central de
Asturias (HUCA), Oviedo, Asturias, Spain
1
Lymphatic vessel spread is considered a major route for head and neck squamous cell carcinoma metastasis. Formation
of new lymphatic vessels could facilitate the process, raising the malignant potential of these tumours. Recent
identification of lymphatic markers allows the study of the lymphangiogenesis phenomenon. We searched for molecular
events involved in the lymphangiogenic process that could have prognostic value in laryngeal/pharyngeal carcinoma
patients. Methods 104 paraffin-embedded pharyngeal/laryngeal samples were studied. Immunohistochemical analysis
of podoplanin and double immunofluorescent analysis of Ki-67 and D2-40 were performed. Lymph vessel density (inside
the tumour mass, at its periphery or considered as a whole) and the presence of tumour emboli inside lymphatics were
recorded. The proliferative state of endothelial lymphatic cells was evaluated. Results Lymphatic vessels were detected
inside the tumour mass (75%) and in the surrounding tissue (80%); some of them in a proliferative state. Tumour emboli
were detected in a high proportion of the cases (45%). Lymphatic vessel density was higher in the pharyngeal cases
(p=0.0029), in greater size (p=0.039), more advanced stage primary tumours (p=0.006) and in carcinomas of patients
with affected nodes (p=0.019). The presence of tumour emboli and a high global vessel density were indicators of poor
prognosis (recorded as death from tumour) in the laryngeal group (p=0.015 and p=0.027, respectively), but notably not
in the pharyngeal one. Interestingly, high global vessel density showed a negative prognostic value among pathologically
staged N0 laryngeal carcinomas (p=0.03). Conclusions The lymphangiogenic process correlated with aggressive tumour
features (pN category, tumour size, tumour stage), but might play different roles in tumours arising from different
anatomic sites. Our results suggest that detection of tumour emboli and assessment of global vessel density using the
D2-40 antibody, may be useful in the clinical practice, as predictors of reduced survival among pN0 laryngeal carcinoma
patients.
Poster
138
139
Frontiers in
Tumour Progression
Poster Session A
Study of the molecular mechanism of LIF induction by
TGF-beta
Alba Gonzalez Juncà
Vall d’Hebron University Hospital, Barcelona, Spain
53
54
Phosphatidylinositol 3-kinase gamma inhibition
ameliorates inflammation and tumor growth in a model
of colitis-associated cancer
Ana González-García1, Jesús Sánchez-Ruiz , Juana M. Flores
1
Glioblastoma (GBM) is the most common tumor of the adult brain, and it is one of the deadliest tumors, with a median
survival of 15 months, despite of the therapies. Because of that, it is of outmost importance to underlie the molecular
mechanisms that drive the glioma progression, aggressivity and recurrence, in order to find new treatments. Recently,
our group has demonstrated the importance of the cytokine TGF-beta in glioma progression, showing that those patients
with an increased TGF-beta pathway activity have worse prognosis. We are focused in the study of the molecular
mechanisms that drive this oncogenic effect of TGF-beta. We want to underlie which are the mediators of this oncogenic
effect, and one of the important mediators is the cytokine LIF (Leukemia Inhibitory Factor). We have demonstrated that
the induction of LIF by TGF-beta is crucial for the Glioma Initiating Cells (GICs) self – renewal, enhancing the tumor
formation and recurrence. We are especially interested in studying the molecular mechanisms of LIF induction by TGFbeta, as not all the tumors induce LIF in response to TGFbeta. After a study of the LIF promoter region, we found that
Runx1 transcription factor is important for the LIF induction by TGF-beta. We are studying the role of Runx1 in the
induction of LIF by TGF-beta and, furthermore, its implication in glioma progression. Our hope is that, the knowledge
about the molecular mechanisms that are involved in the gliomagenesis, will lead us to develop further therapies against
this outmost incurable disease.
2
and Ana C. Carrera1
Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Campus de Cantoblanco,
Madrid, Spain; 2Animal Surgery and Medicine Department, Facultad de Veterinaria, Universidad Complutense
de Madrid, Spain
1
A large body of evidence supports a correlation between inflammation and cancer, although the molecular mechanisms
that govern this process are incompletely understood. Phosphatidylinositol 3-kinase is an enzyme that regulates the
immune response and contributes to cell transformation in several tumor types. Here we addressed the role of the PI3Kγ
isoform in inflammatory bowel disease (IBD) and in the development of colitis-associated cancer. Methods: PI3Kγ-/- and
control mice were repeatedly treated with dextran sulfate sodium (DSS) to induce chronic colitis and colitis-associated
cancer. Colorectal tumor burden and colon inflammation were evaluated in these mice. Leukocyte populations in colon
were characterized by flow cytometry analysis. Results: PI3Kγ-deficient mice had a lower incidence of colitis-associated
tumors, as well as reduced tumor multiplicity and smaller tumor size compared to controls. Reduced tumor development
paralleled less colon inflammation in PI3Kγ-deficient mice. Analysis of leukocyte populations in the colon of PI3Kγdeficient mice showed defective activation and infiltration of myeloid cells and defective recruitment of T cells to the
colon compared to controls. Conclusions: PI3Kγ regulates the innate immune response in a murine model of ulcerative
colitis, thereby controlling colon inflammation and tumor formation.
Poster
140
141
55
Frontiers in
Tumour Progression
Poster Session A
Enterocyte restricted deletion of p53 in enterocytes
facilitates invasive colon cancer – a new mouse model
for translational studies
56
FGF signalling in osteosarcoma disease progression –
from initiation to metastasis
Agi Grigoriadis1, H. Unsworth , D. Weekes , T. Kashima , A. Patiño-Garcia , C. Zandueta
1
1
1
Sarah Schwitalla, Valentin Becker, Irina Kerle, Andreas Meining and
Florian R. Greten
2nd Dept. of Medicine, Klinikum rechts der Isar, Technical University Munich, Germany
Several mouse models of intestinal tumorigenesis have been developed, but only few models exist that closely mimic
human disease because tumors primarily develop in the small intestine or do not become invasive. According to the
well established multi-step carcinogenesis of colon cancer established by B. Vogelstein about 20 years ago, loss of p53
is associated with invasion of colon cancer. To functionally test the relevance of p53 in intestinal tumor progression we
examined mice with an intestinal epithelial cell (IEC)-restricted deletion of p53 (Tp53ΔIECmice). Unchallenged mice
do not display any overt phenotype. However, when these mice are exposed to the carcinogen azoxymethane, which
induces mutations in Ctnnb and therefore leads to the activation of the Wnt pathway, Tp53ΔIEC mice rapidly develop
invasive cancer in the distal colon while wildtype mice only develop non-invasive adenomas. Tp53ΔIEC mice show
pronounced NF-κB activation and comparative gene expression analysis confirmed upregulation of several NF-κB target
genes that can also specifically be found at the invasion front of human colon cancers. Moreover, because of the distal
location tumor growth can easily be monitored by mini-endoscopy. Using endoscopically guided confocal laser scanning
micoroscopy (CLSM) we determined blood vessel density in tumors of Tp53ΔIEC mice and could confirm the increased
density of vessels that correlated with advanced tumor stages suggesting the occurence of an angiogenic switch.
Collectively, Tp53ΔIEC mice comprise a novel model that will be helpful for further analysis of the molecular changes
responsible for colonic tumor progression and that can be used for pre-clinical evaluation of novel therapies.
2
2
and F. Lecanda2
Dept. Craniofacial Development, Guy’s Hospital, King’s College London, UK; Center for Applied Medical
Research (CIMA), University of Navarra, Pamplona, Spain
1
2
Osteosarcoma (OS) is a highly malignant bone-forming tumour that occurs mostly in children and adolescents. Adjuvant
chemotherapy with surgery has improved OS survival, but this rate still remains at ~60%. Drug resistance and frequent
pulmonary metastases, present in ~80% of patients at diagnosis, are the main barriers to improving OS survival. The
molecular mechanisms determining the progression of primary bone tumour to secondary lung tumour are not known
and hold the key to improved OS survival. Our analysis of a transgenic c-Fos-overexpressing mouse model of OS
demonstrated that induction of c-Fos expression in osteoblasts resulted in upregulation of FGFR1, suggesting that
FGFR1 is a novel c-Fos/AP-1 target gene for osteoblast transformation. Further studies showed high FGFR1 levels in
human OS tumours as well as at early sites of tumour formation, implicating deregulated FGFR1 signalling in tumour
initiation. In murine osteoblasts c-Fos and bFGF cooperated in stimulating anchorage-independent growth, and the FGFR
inhibitor SU5402 abolished both murine and human OS colony growth. We performed orthotopic intratibial injection of
cells expressing dominant-negative (dn) FGFR1 constructs into nude mice. Aggressive lesions developed rapidly with
no apparent differences in the area of the primary osteolytic areas formed by dnFGFR1 and control clones. However,
examination of the lungs revealed a significant 2 to 5-fold increase in the number and area of spontaneous lung
metastases in dnFGFR1- injected mice and this was confirmed using in vitro invasion and migration assays. These data
have identified FGF/FGFR signalling as a potential therapeutic target for OS development and metastasis.
Poster
142
143
57
Frontiers in
Tumour Progression
Poster Session A
58
Fine mapping of genomic aberrations in triple negative
breast cancers using Affymetrix SNP 6.0 arrays
A new model for prostate cancer early disease
progression
Anita Grigoriadis1,*, Emanuele de Rinaldis
Julian H. Gronau1, Giolanta Kogianni , Matthew P. Caley
, Mamunur Rashid1, Patrycja Gazinska1, Ireneusz Shinomiya2,
Cheryl Gillett , Pierfrancesco Marra , Sarah Pinder and Andrew Tutt1
2
1
1,*
2
Breakthrough Breast Cancer Research Unit, Guy’s Hospital, King’s Health Partners AHSC, London, UK;
Department of Research Oncology, King’s College London, Guy’s Hospital, London, UK
1
2
Triple-negative breast cancers (TNBC) are defined by very low or absent immunohistochemical (IHC) expression of
estrogen (ER) and progesterone receptor (PR) and low expression or lack of amplification of human epidermal growth
factor receptor-2 (HER2). They represent a heterogeneous group of breast cancers that usually have an aggressive
phenotype, a relatively poor prognosis with early relapse, with targeted therapies not currently available. TNBC are
genetically very unstable and display complex patterns of copy number alterations (CNA). Our aim was to define a
comprehensive, high-resolution map of CNA occurring in TNBC, trying to dissect their patterns and to infer the impact
on the underlying biological pathways. Using high-resolution SNP6.0 Affymetrix arrays we performed genome-wide DNA
copy number profiling on a group of 83 TNBCs and 33 non-TNBCs (19 ER+/ HER2- and 14 ER-/HER2+). All samples were
fine-needle microdissected to ensure high content of neoplastic cells (> 70%). Circular binary segmentation (cbs) was
used to determine significant CNA, and a pool of 18 germline DNA formed the genomic baseline. Our analysis revealed
a high level of genetic instability, whereby 196 and 229 cytobands with a minimum size of 2kb were identified for being
recurrently amplified or deleted, respectively in more than 30% of TNBCs. In agreement with previous studies, gains on
3q22.3–q26.33; 6p25.3–11.1; 7q32.2–q36.3; 8p11.1– q24.3; 10p15.3–p12.1; 12p13.32–p13.2; and losses on 1p36;
5q11.1-q35.3; 8p23.3–p12; 7p13.3–q21.3 were observed. Comparative analysis of TNBC and non-TNBC identified
specific TNBC amplicons (e.g 1p36.33; 1p34.2) and deletions (e.g 9q22; 19p13.3). Interestingly, the latter included a
number of miRNAs (e.g hsa-let7a-1 on 9q22; hsa-mir-7-3 on 19p13), known to exert a role as tumour suppressors in
cancer. Oncogenes such as FGFR2, EGFR and IGF1R were found in regions of focal high level amplification (FCA, <10Mb).
These regions were mainly observed with low average frequency (~6% TNBC), with IGF1R being the most recurrently
amplified (~12% TNBC). To infer the impact of genetic alterations on biological pathways, genes frequently amplified
or deleted (>30%) in TNBC and non-TNBC were analyzed using a comparative gene set enrichment approach. Among
others, the “immunological”, “inflammatory” and “genetic disorders” pathways emerged as being more frequently
affected by events of amplification in TNBC (p-value < 0.001). Finally, to investigate whether the patterns of CNAs might
be involved in tumour development and progression, we performed co-occurrence analysis of frequent CNAs. A number
of chromosomal regions were frequently co-amplified, including regions on chromosome 6q11 with 7p12 and 19q13,
19q and 5p; and co-deleted, such as 17p13 and 5q11, 6q26 and 17p12. This work corroborates and extends existing
knowledge of TNBC, providing in-depth insights into the genetic events underpinning this group of breast cancers.
1
1
and Justin Sturge1
Prostate Cancer Metastasis Team, Department of Oncology, Division of SORA, Imperial College London,
Hammersmith Hospital Campus, London, UK
1
Prostate cancer is a common disease of men greater than 50 years of age. It usually originates from the luminal
epithelial cells residing in the glandular part of the tissue. In most cases prostate cancer is asymptomatic during the
early stages of the disease and the symptoms become apparent once the disease has metastasised to other parts of
the body. For these reasons alternative biomarkers to help indicate progression of the disease and new therapies to
delay onset of metastasis are needed. Metastasis is a complex multi-step process, a common hallmark of which is the
increased expression of proteins involved in remodelling of the extracellular matrix and promotion of cell migration. It
is often hypothesised that cancer cells of epithelial origin mimic developmental epithelial to mesenchymal transition
(EMT) in order to acquire an invasive phenotype. Expression of Endo180 by breast cancer cell lines positively correlates
with a more migratory and invasive phenotype. In vivo evidence supporting this comes from recent tissue microarray
based studies that report the expression of Endo180 on highly invasive basal-like breast and prostate tumour cells.
We designed a cell-line based model of prostate cancer disease progression in an attempt to best fit the epithelialto-mesenchymal transition (EMT) hypothesis. The model included cells derived from normal and/or benign prostate
hyperplasia (BPH) (RWPE1, RC165N), low (RC92a) and intermediate (RC58T) grade primary prostate cancer and three
commonly used prostate cell lines derived from metastatic lesions in the lymph nodes (LNCaP), bone (PC3) and brain
(DU145). For the first time we compare these cell lines in respect of their cytoskeletal features and expression of
epithelial and mesenchymal markers: E-cadherin/alphacatenin, vimentin, cytokeratin and CD44. We addressed their
migratory potential and their ability to invade into organotypic matrixes. Additionally, we have examined the expression
of components of the Endo180/uPA/uPAR-axis.
Poster
144
145
Frontiers in
Tumour Progression
Poster Session A
Embryogenesis meets tumorigenesis – nodal signaling
determines tumorigenicity and invasiveness of
pancreatic cancer stem cells
59
60
Folic acid inhibits vascular endothelial cell migration
Tien-Chi Hou1* and Wen-Sen Lee
2*
Department of Medicine and Graduate Institute of Medical Sciences, Taipei Medical University, Taiwan;
These authors contributed equally to this work.
1
2
Christopher Heeschen, Enza Lonardo, Stephan Huber, Patrick C. Hermann, Maria-Theresa Mueller,
*
Clinical Research Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; Department
of Surgery, Campus Grosshadern, Ludwig-Maximilian-University of Munich, Germany
Folic acid (a water soluble vitamin) has been known as a co-factor for de novo nucleotides synthesis. Recent studies
on folic acid have focused on its involvement on neural crest folding and its ability to cure Crohn’s disease. Here, we
report that folic acid exerts an anti-migration activity in human vascular endothelial cells. In the angiogenic process,
migration and proliferation of endothelial cells are two critical steps. Folic acid (10 uM) inhibited the migration of human
umbilical vein endothelial cells (HUVEC). Western blot analysis demonstrated that treatment of HUVEC with folic acid at
a concentration of 10 uM inhibited the membrane translocation of RhoA, but did not affect the levels of total protein of
RhoA, suggesting that folic acid might reduce the RhoA activity. Over-expression of constitutively active RhoA construct
(RhoA V14) abolished the folic acid-inhibited migration in HUVEC. However, pretreatment of HUVEC with a ROCK (a kinase
associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the RhoA V14-induced prevention
effect on the folic acid-induced inhibition of HUVEC migration. These results suggest that inhibition of Rho GTPases might
account for the folic acid-induced migration inhibition of HUVEC. Moreover, pretreatment of HUVEC with a Src inhibitor,
PP2, which blocked the RhoA activity, inhibited the folic acid-induced migration inhibition in HUVEC. Taken together,
our results suggest that folic acid might inhibit endothelial cell migration through a Src/RhoA-mediated pathway. The
findings from the present study suggest the potential applications of folic acid in the treatment of angiogenesis-related
disease conditions.
Irene Miranda and Iker Rodriguez
Nodal belongs to the TGF-β superfamily and is an important regulator of embryonic stem cell fate. Recent reports also
suggest that Nodal expression determines a more metastatic phenotype in melanoma. As we have shown that migrating
cancer stem cells (CSC) drive metastasis in pancreatic cancer, we investigated whether nodal/activin regulate selfrenewal and invasiveness of therapy-resistant pancreatic CSC. Here, we show that pancreatic CSC can be enriched
by sphere formation assays or expression of the stem cell marker CD133 as evidenced by expression of pluripotency
markers and markedly enhanced tumorigenicity. While differentiated pancreatic cancer cells express only low levels of
nodal, either functional enrichment for cancer stem cells or prospective isolation of CD133+ cells resulted in a drastic
increase in nodal expression. Inhibition of nodal signaling significantly, but reversibly reduced self-renewal capacity
of CSC in vitro and in vivo. Nodal inhibition also resulted in a nearly complete elimination of their migratory activity.
Intriguingly, gemcitabine, which was not able to eliminate untreated CD133 positive cells, virtually depleted cancer
stem cells following blockade of nodal signaling. Consistently, this combination treatment was capable of abrogating
the tumorigenic properties of orthotopically engrafted pancreatic cancer cells resulting in long-term survival of a large
number of animals. Taken together, our data demonstrate that self-renewal capacity of pancreatic cancer stem cells
is regulated by nodal. Modulation of this pathway provides a novel therapeutic strategy for targeted elimination of
tumorigenic cancer stem cells in order to overcome their resistance towards standard chemotherapy.
Poster
146
147
Frontiers in
Tumour Progression
Poster Session A
Progesterone receptor activation of extra-nuclear
signaling pathways in regulating p53 expression in
vascular endothelial cells during angiogenesis
Sung-Po Hsu and Wen-Sen Lee
Graduate Institute of Medical Sciences, Taipei Medical University, Taiwan
61
62
Genetic modulation of heparanase levels reveals
critical roles in promoting lymphangiogenesis and
tumor invasion during pancreatic neuroendocrine
tumorigenesis
Karen Hunter1,2, Carmela Palermo , Jemila Kester , Karoline Dubin , Laura Tang , Israel Vlodavsky
1
Previously, we showed that progesterone exerted an anti-angiogenic activity and inhibited the proliferation of human
umbilical vein endothelial cells (HUVEC) through a p53-dependent pathway (Hsu et al., Cellular and Molecular Life Sciences.
2008, 65:3839-3850). Now we investigate further the molecular mechanism underlying the hormone activity. In cultured
HUVEC, progesterone increased the formation of progesterone receptor (PR)-Src complex and Src activity. These effects
were abolished by pretreatment with RU486, suggesting the involvement of PR in these progesterone-induced activities.
Luciferase reporting assay demonstrated that the progesterone-increased p53 transactivity was prevented by PP2 (a
Src inhibitor), Src Kinase Inhibitor I (SKI-I), Ras inhibitory peptide, or a farnesyltransferase inhibitor (FTI). Pretreatment
with PP2 or SKI-I prevented the progesterone-induced increase of p53 protein level and membrane translocation of Kras.
Transfection of HUVEC with dominant-negative Erk2 prevented the progesterone-induced increases of the p53, p21 and
p27 protein levels and decrease of thymidine incorporation. Moreover, progesterone-induced phosphorylation of Raf-1
and Erk1/2 was abolished by pretreatment with Ras inhibitory peptide (Ras InP) or sulindac sulfide (a Raf-1 inhibitor).
Pretreatment of HUVEC with Src antisense oligonucleotide, U0126 (an Erk2 inhibitor) or Bay11-7082 (a NF-kB inhibitor)
blocked progesterone-induced increase of p53 protein level. Furthermore, the p53-induced increase of MDM2 protein
level after progesterone treatment was inhibited by administration of U0126 or Bay11-7082. The progesterone-induced
up-regulation of the promoter activity of p53 was prevented by pre-transfection of HUVEC with dominant-negative Erk2
or pretreatment with Bay11-7082 or CAPE (a NF-kB inhibitor). In addition, progesterone-induced increase of NF-kB
nuclear translocation and NF-kB binding onto the p53 promoter were abolished by pretreatment of HUVEC with U0126.
1
1
Johanna Joyce1
3
4
and
Cancer Biology and Genetics Program; 2Gerstner Sloan-Kettering Graduate School of Biomedical Sciences;
Department of Pathology, Memorial Sloan- Kettering Cancer Center, New York, USA; 4The Ruth and Bruce
Rappaport Faculty of Medicine, Technion, Haifa, Israel
1
3
Pancreatic neuroendocrine tumors are a relatively rare, but clinically challenging tumor type. Currently, little is known
about the cell of origin and prognostic indicators of these tumors and insights into these mechanisms are critical for
improving patient treatment. Using tumor tissue microarrays we have found that expression of an endoglycosidase,
heparanase, is correlated with increasing malignancy and metastasis in patients with neuroendocrine tumors. Heparanase
is the sole enzyme capable of cleaving heparan sulfate side chains within heparan sulfate proteoglycans(HSPGs),
an abundant component of the extracellular matrix. To elucidate the mechanisms by which heparanase promotes
pancreatic neuroendocrine tumors we utilized the RIP1-Tag2(RT2) transgenic mouse model. Previous work has shown
that heparanase is upregulated during RT2 tumor progression, further implicating this enzyme in tumor promotion. We
have crossed transgenic mice that constitutively overexpress human heparanase(hpa-tg) to RT2 mice. We additionally
have generated heparanase knockout RT2 mice to examine the effects of genetically modulating heparanase levels in
vivo. Analysis of hpa-tg RT2 mice revealed increased tumor invasion, while heparanase knockout mice exhibit decreased
tumor invasion, consistent with an important role for heparanase in remodeling the extracellular matrix through
processing of HSPGs. Interestingly, hpa-tg RT2 mice also develop a novel class of highly invasive tumors that have lost
markers of islet cell differentiation, termed poorly differentiated invasive carcinomas. Characterization of these tumors
by lineagespecific markers is being complemented by cell culture experiments to elucidate the underlying mechanisms.
Additionally, we have found that hpa-tg RT2 mice exhibit increased peritumoral lymphangiogenesis. These data indicate
that heparanase influences multiple facets of tumorigenesis and can have effects on both the tumor microenvironment
and cell differentiation.
Poster
148
149
Frontiers in
Tumour Progression
Poster Session A
63
64
Netrins-1 and -4 promote glioblastoma cell
proliferation, migration and invasion
Network analysis of cell-specific bidirectional Eph/
ephrin signalling in co-culture
Marko Hyytiäinen, Irene Ylivinkka, Yizhou Hu and Jorma Keski-Oja
Claus Jørgensen1, A. Sherman , G.I. Chen , A. Pasculescu , A. Poliakov , M. Hsiung , B. Larsen ,
University of Helsinki, The Haartman Institute, Molecular Cancer Biology Research Program, Helsinki, Finland
2
2
2
3
2
2
D. Wilkinson3, R. Linding4 and T. Pawson2
Cell Communication Team, Section of Cell & Molecular Biology, The Institute of Cancer Research, London,
UK; 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 3Division of
Developmental Neurobiology, National Institute for Medical Research, London UK; 4Cellular & Molecular
Logic Team, Section of Cell & Molecular Biology, The Institute for Cancer Research, London, UK
1
Netrins were originally identified to have roles in axonal guidance. More recently, they have been implicated in
angiogenesis, and cancer cell survival. Netrin-1 is expressed more in grade IV gliomas than in normal brain and netrin-4
is expressed more in invading glioma cells than in tumor stroma, suggesting that netrins have a promoting role for
some aspects of tumor growth/invasion. We have studied the effects of ntns -1 and -4 on grade IV glioblastoma cell
behavior, by using overexpression and knockdown models in U251MG cells. Overexpression of netrin-1 increased the
invasiveness of human glioblastoma cells whereas partial knock-down of netrin-1 by shRNAs reduced the invasiveness.
Similarly, netrin-1 overexpression increased and partial knockdown decreased migration of human glioblastoma cells.
Suppression of netrin-4 expression in U251MG cells inhibited cell motility. Overexpression of netrin-4 did not alter cell
migration, but addition of exogenous recombinant netrin-4 led to concentration dependent promotion of cell migration.
U251MG cells express endogenous ntn-1 and -4. Overexpression of ntn-1 or -4 did not affect cell proliferation.
However, knockdown of the expression of ntn-1 and -4 decreased proliferation rates. The reduced growth rate of ntn-1
knockdown cells was not compensated by overexpression of ntn-4, and ntn-1 overexpression did not compensate for
reduced growth rate in ntn-4 knockdown cells. Moreover, suppression of netrin-1 expression was still able to decrease
cell proliferation in netrin-4 silenced U251MG cells. This suggests that netrin-1 and -4 have separate signaling routes
in mediating cell proliferation. To reveal possible signaling routes and receptors netrins may have, we have carried out
tandem affinity purification and mass-spectrometry identification of binding proteins. We have identified numerous
signaling receptors binding directly or indirectly to netrins. The effects and functions of these receptors will be discussed.
Contact-initiated signalling between cells is an important mechanism for the development and maintenance of tissue
structure. During metastasis cells escape the normal restrictive regulatory mechanisms. As such, de-regulation of
receptors involved in maintaining tissue boundaries, such as the Eph receptors and ephrins, has recently been linked
to metastatic behaviour. Signalling between the transmembrane Eph receptors and ephrins results in a bi-directional
signal, which regulates cell migration and adhesion to control cell sorting. Until now it has been problematic to
systematically study cell-specific signalling networks in distinct populations of interacting cells, primarily because the
unique properties of each cell type are lost once co-cultured cells are processed for biochemical analysis. Here, we
present an analysis of bidirectional Eph/ephrin signalling in the context of direct cell-cell interaction. We have developed
a novel proteomic strategy, quantitative analysis of Bidirectional Signaling (qBidS), based on lineage-specific labelling
with stable isotopes of arginine and lysine, combined with mass spectrometric identification and relative quantification
of tyrosine-phosphorylated peptides. We identified and quantified tyrosine phosphorylation in both EphB2 and ephrin-B1
expressing cells. In addition, we performed a siRNA screen for signalling molecules involved in EphB2/ephrin-B1
controlled cell sorting, which identified several nodes that also were modulated by phosphorylation. Through integrative
and data-driven network modelling we identified cell specific utilisation of signalling networks and tyrosine kinases,
revealing asymmetric signalling. In summary, we have identified novel and cell specific regulatory mechanisms essential
for cell sorting and boundary maintenance.
Poster
150
151
65
Frontiers in
Tumour Progression
Poster Session A
66
Podoplanin is regulated by AP-1, and promotes platelet
aggregation and cell migration in osteosarcoma
Engineering melanoma progression in a humanized
environment in vivo
Takeshi G. Kashima1,2, Akiko Kunita , Agamemnon E. Grigoriadis
Gregor Kiowski1, Thomas Biedermann , Daniel Widmer , Gianluca Civenni , Charlotte Burger , Reinhard
1
2
and Masashi Fukayama1
Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Department
of Craniofacial Development, King’s College London, Guy’s Hospital, London, UK
1
Podoplanin is a type-I surface transmembrane sialomucine-like protein expressed by a wide range of cell types
including lymphatic endothelial cells as well as osteocytes in bone. We previously showed that podoplanin is a platelet
aggregation-inducing factor and promotes tumour lung metastasis in vivo. Here we investigated the function, regulation
and expression of podoplanin in osteosarcoma (OS). Podoplanin expression was observed in three human OS cell lines
(MG-63, HOS and U-2 OS) with platelet aggregation-inducing ability which was blocked by either gene silencing using
siRNA or by a neutralising anti-podoplanin antibody. Overexpression of podoplanin in non-metastatic OS cell line, Dunn,
promoted cell migration, which was also suppressed by the neutralising anti-podoplanin antibody without attenuating
cell proliferation. Both podoplanin and TGF- 1 were upregulated by exogenous c-Fos induction in the non-transformed
MC3T3- E1-derived osteoblastic cell AT9.2, and were highly expressed in OS that develop in c-Fos overexpressing
transgenic mice. Clonal cell lines derived from c-Fos transgenic OS showed high podoplanin expression at cell surface.
Furthermore, immunohistochemical analysis of human OS tissue microarrays showed staining of tumour cells embedded
in an excess of irregular neoplastic bone matrix in 100% (n=65) of tumours. Podoplanin immunoreactivity was also
observed in OS subtypes, with 65% of 80 osteoblastic OS, 100% of 19 chondroblastic OS and 79% of 34 fibroblastic OS
showing cell surface staining at various levels. Podoplanin protein expression in human OS was also confirmed using
Western blot analysis. The mRNA level of podoplanin was significantly higher in metastatic tumours than in primary
tumours. Our data suggests that podoplanin is aberrantly expressed in OS, is under AP-1 transcriptional control and is a
candidate molecule for therapeutic targeting and suppression of OS pulmonary metastasis.
2
3
1
1
Dummer3, Lukas Sommer1 and Ernst Reichmann2
Cell and Developmental Biology, Institute of Anatomy, University of Zurich, Switzerland; 2Department of
Surgery, University Children’s Hospital Zurich, Switzerland; 3Department of Dermatology, University Hospital
Zurich, Switzerland
1
To overcome the lack of effective therapeutics for aggressive melanoma, new research models closely resembling the
human disease need to be established. Thus, we developed a fully orthotopic, humanized in vivo model for melanoma
faithfully recapitulating human disease initiation and progression. To this end, human melanoma cells were seeded
into engineered human skin substitutes constructed from freshly isolated human keratinocytes and dermal fibroblasts.
Transplantation onto the back of immunocompromised rats consistently resulted in development of melanoma displaying
the hallmarks of their parental tumors. Importantly, all initial steps of disease progression were recapitulated, including
the incorporation of the tumor cells into their physiological microenvironment, transition of radial to vertical growth, and
establishment of highly vascularized, aggressive tumors with dermal involvement. Because all cellular components can
be individually manipulated using this approach, it not only provides a means to resolve the controversies surrounding
melanoma-initiating cells in a physiological environment, but also to dissect tumor cell autonomous and non-autonomous
pathways regulating human disease progression.
Poster
152
153
Frontiers in
Tumour Progression
Poster Session A
Tumor suppression in a mouse prone to inflammation
Rama Khokha, Carlo Hojilla, Hartland Jackson, Virginie Defamie, Alison Aiken and Purna Joshi
67
68
c-Myb-induced migration and invasiveness of breast
and colon cancer cells are associated with upregulation of cathepsin D and MMP9
Ontario Cancer Institute/University Health Network, University of Toronto, Canada
Mice lacking the Timp3 gene exhibit heightened inflammatory response to a number of extrinsic stimuli. Given the
active role of inflammation in tumorigenesis we tested whether Timp3 modulates breast or liver cancer development
and progression. Mice lacking Timp3 were bred into three independent genetic models of breast cancer, as well as
carcinogen-induced hepatocellular carcinoma development. In all three models of breast cancer, MMTV-PyMT, MMTVNeu, and MMTV-Wnt1, Timp3 deficiency consistently resulted in mammary tumor suppression, accompanied by
decreased tumor incidence, tumor burden and metastases. Similarly, liver tumorigenesis was also significantly reduced
in a Timp3 null background. The function of adaptive and innate immunity and of TNF signaling is being elucidated as
potential contributors to TIMP3-mediated tumor suppression.
Lucia Knopfova, Lucie Pekarcikova, Petr Benes and Jan Smarda
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
The c-Myb transcription factor is essential regulator of stem-progenitor cells in bone marrow, colon epithelia and
neurogenic niches in brain. Malfunction of c-Myb results in malignancies of several types including breast and colon
cancer. c-Myb promotes proliferation and inhibits differentiation/apoptosis of various cancer cells. However, its role
in control of invasion and metastasis of tumor cells remains unclear. This study shows that overexpession of c-Myb
enhances ability of human breast cancer MDA-MB-231 cells and mouse colon cancer CT26 cells to migrate and
invade Matrigel in vitro. Real-time analysis of cell migration and invasion using the xCELLigence RTCA system revealed
dynamics of c-Mybinduced effects with rapid onset of migration and invasion during first 6 and 12 hours, respectively.
Real-time PCR screening revealed up-regulation of cathepsin D and matrix metalloproteinase 9 (MMP9) mRNAs coding
for proteases that promote invasive behavior in c-Myb-overexpressing cells. Further experiments confirmed that
increased production of cathepsin D and matrix metalloproteinase 9 correlate with c-Myb-induced invasive phenotype
in MDA-MB-231 cells. Cathepsin D was identified as Myb-responsive gene by transactivation assays. Our results show
that c-Myb promotes human breast and mouse colon cancer cells invasiveness by enhancing cathepsin D and MMP9
production. Therefore, c-Myb could be one of the key regulators of tumor invasion and useful molecular target for
cancer prognosis and treatment. This work was supported by grants 301/09/1115 and 204/08/H054 of Czech Science
Foundation, IAA501630801 of GAAV CR, MUNI/C/0099/2009 of Masaryk University and MSM0021622415 of Ministry of
Education CR.
Poster
154
155
Frontiers in
Tumour Progression
Poster Session A
69
70
V-SRC-transformed chicken cell lines as a model
system for identification of metastasis-associated
genes
A novel mechanism of Zoledronate mediated tumor
inhibition governed by modulated TGFb levels and
polarized neutrophils
Denisa Kovářová, Jan Kosla, Kateřina Trejbalová, Jiří Plachý and Jiří Hejnar
Institute of Molecular Cancer Research, University of Zurich, Switzerland
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Metastatic spread to distant tissues and organs represents a critical step in the tumor progression. Genes involved
in this process have not yet been fully characterized. To identify them, we use our original model system of chicken
vsrc- transformed tumor cell line PR9692. Rare subclones of this cell line have lost their ability to induce metastases
after inoculation into syngeneic chickens although they display a similar level of v-src overexpression as parental line
PR9692. We compared the expression profiles of metastatic and non-metastatic cells using oligonucleotide microarrays,
assuming that metastasis-associated genes are among those with differential expresssion. To confirm the metastatic
capacity of these candidate genes we employ the knockdown induced by plasmid-delivered shRNAs. We present here
the efficiency of our RNAi system in the PR9692 cell line using the GFP reporter. Expression of shRNAs was optimized
and multiple vectors carrying Pol II or Pol III promoters were tested. Pol III promoter-directed shRNA decreased the GFP
expression more than 10 times in almost 22 % of cells. Using the same Pol III promoter-directed system we revealed the
importance of v-src overexpression for the maintenance of cell transformation and metastatic potential. We knockeddown the expression of v-src in PR9692 cells and resulting stable subclones were inoculated into chickens to analyse
their metastatic capacity. We show that in most v-src knock-downs of PR9692 cells, the incidence of metastases
decreased and the growth of primary tumors declined in all cases. This indicates that v-src plays an essential role
in maintaining the malignant phenotype. Other candidate genes will be assessed for their involvement in metastasis
induction.
Sushil Kumar, Sibel Mete and Reto Schwendener
Zoledronate is a potent inhibitor of osteoclast activity and used as a drug for the treatment of osteoporosis and skeletal
related events associated with bone metastases (1, 2). Recent reports suggest that zoledronate can also repress soft
tissue tumor growth and bone metastasis in mice and humans (3, 4). The antitumor activity of zoledronate has been
assigned to its ability to inhibit tumor angiogenesis largely by inhibiting MMP9 expression in macrophages (3, 5). Here,
we show a novel mechanism that is indispensable for anti-tumorigenic properties of zoledronate. Using mouse tumor
models, namely Lewis lung (LLC) and 4T1 breast carcinoma we could confirm the antitumorigenic action of zoledronate.
To our surprise, tumor growth inhibition in these models coincided with hyperinfiltration of neutrophils suggesting a
role of neutrophils in zoledronate’s therapeutic effects in cancer. Upon molecular characterization, we found these
neutrophils polarized to the M1 type in zoledronate treated tumors. Interestingly, we noticed a decrease in TGFb levels
in serum as well as in myeloid cells of the tumors treated with zoledronate. Furthermore, the neutrophil recruiting
chemokine CXCL5 mRNA was significantly upregulated in zoledronate treated tumors corroborating the increase in
neutrophil recruitment. Although zoledronate treated tumors displayed unaltered proliferation rate widespread necrosis
was observed in that could be the major cause of tumor growth suppression. Finally, we tested functional relevance of
M1 neutrophil recruitment in tumor growth inhibition by depleting neutrophils in zoledronate treated mice. The efficacy
of Zoledronate in tumor growth inhibition was blunted by neutrophil depletion with growth of untreated tumors equals to
that of zoledronate treated tumors depleted of neutrophils. Since the immune system is important for controlling tumor
progression as well as in chemotherapeutic treatment, we analyzed the CD8 T cell population and observed an increase
in zoledronate treated tumors when compared to control. Furthermore, this zoledronate mediated increase in CD8 T
cells was reduced to control levels by neutrophil depletion suggesting a role of M1 neutrophils in this phenomenon.
To understand the molecular basis of this immunogenic action of M1 neutrophils, we measured mRNA levels of key
molecules namely IL-2, IL12, arginase, and IFN-gamma that modulate immune system. We noticed an increase in IL2,
IL12, and IFN-gamma levels in neutrophils isolated from zoledronate treated tumors whereas arginase levels were
reduced. We are further strengthening our study by testing the functional relevance of TGFb in these cellular events as
well as further resolving the molecular phenotype of M1 neutrophils by genomic approaches. Nonetheless, our results
highlight a novel property of zoledronate to modulate TGFb levels and enhanced recruitment of M1 neutrophils that is
indispensable for its therapeutic efficacy in cancer.
Poster
156
157
71
Frontiers in
Tumour Progression
Poster Session A
72
Altered expression of p120-catenin during epithelialmesenchymal transition (EMT): act as tumor
suppressor or metastasis promoter?
The adhesion inducing and antiproliferative effects of
the novel GnRH-III conjugates, as potential anticancer
drug delivery substances
Akiko Kunita1, Andreas Wicki , Albert B. Reynolds
1
1
2
and Gerhard Christofori1
Department of Biomedicine, University of Basel, Switzerland; 2Department of Cancer Biology, Vanderbilt
University, Nashville, USA
1
Loss of E-cadherin-mediated cell-cell junctions has been correlated with cancer cell invasion and poor patient survival.
In many cancer cells, the loss of E-cadherin coincides with a gain of N-cadherin expression. The cadherin switch is
a hallmark of epithelial-mesenchymal transition (EMT). Notably, the loss of E-cadherin is sufficient to induce EMT in
vitro and malignant tumor progression in mouse models in vivo. However, the mere loss of cell adhesion may not be
sufficient and additional signals are required to cause tumor cells to invade surrounding tissue. In this study, we found
that the long isoform (isoform 1) of p120-catenin (p120), an important constituent of the E-cadherin adhesion complex,
was upregulated upon loss of E-caderin in MCF7 breast cancer cells and normal murine mammary gland (NMuMG)
cells. p120 has emerged as a key player in promoting E-cadherin stability and adherens junction integrity at the plasma
membrane, but also has additional roles in the cytoplasm and nucleus, and has been proposed as a potential invasion.
Furthermore, p120 has various isoforms, and the isoform shift from the short isoform to the long isoform was found to be
associated with invasive or metastasizing phenotype. To investigate the role of the long isoform of p120 during EMT, we
have employed siRNA-mediated ablation of p120 expression during TGF-beta-induced EMT in NMuMG cells. As a result,
p120 dissociated from the membrane, accumulated in the cytoplasm. Both E-cadherin and N-cadherin levels were
down-regulated in p120-deficient cells both in the presence and absence of TGF-beta. Beta-catenin levels were not
affected by treatment of TGF-beta in control cells, whereas TGF-beta treatment reduced the expression of beta-catenin
in p120-depleted cells. These results suggest that depletion of p120 destabilizes the E-cadherin cell adhesion complex
followed by a loss of beta-catenin. Taken together, the long isoform of p120 may behave as tumor promoter during EMT.
Eszter Lajkó,
2
U. Leurs, 2M. Manea, 3I. Szabó, 3G. Mező and 1L. Kőhidai
Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary; 2Department
of Analytical Chemistry, University of Konstanz, Germany; 3Research Group of Peptide Chemistry, Eötvös
Loránd University of Sciences, Hungarian Academy of Sciences, Budapest, Hungary
1
One of the most promising strategies of the targeted cancer therapy is based on findings that the peptide hormone
receptors express primarily on tumorous cells. The binding sites for gonadotropin-releasing hormone (GnRH) were found
on several human tumors. The GnRH-III isoform has not found in human yet, but it can specifically bind to the human
GnRH receptors, and it lacks of endocrine side effects. Thus, the GnRH-III can be used as a carrier to deliver cytotoxic
agents directly to tumorous cells. The controlled modification of adhesion of the tumor cells has high therapeutic
significance in respect of the tumorigenesis and formation of metastasis. Thus, in our present work (i) the effects on
adhesion, and (ii) antiproliferative / cytotoxic properties of the GnRH-III conjugates were evaluated. The tested ligands
were GnRH-III based molecules containing one or two cytotoxic agents (Daunomicin - Dau, Methotrexate – Mtx). The
adhesion of the monocytic model-cell (Mono Mac 6 cell line) was detected by the electric impedance based technique of
xCELLigence® (Roche). To analyse the antiproliferative effects of the conjugates the cell count, aggregation and viability
were measured by the Casy TT® (Roche) after 24, 48, 72 hrs. The concentration optimum of the positive effects on the
adhesion of the Dau containing conjugates ((GnRH-III–(Dau)8, GnRH-III–(Dau4-Dau8)) was 10-10 – 10-8M. All of the
analysed conjugates could significantly decrease the number of the monocytes. The strongest inhibition of growth was
detected in the case of GnRH-III–(Dau4-Dau8) and GnRH-III–(Mtx-Dau)8 (71% - 63%) at 10-6M. Both conjugates could
already elicit their antiproliferative effects after 24h with similar acivity to the free drugs. In conclusion, the GnRH-III
conjugates can favourably change the adhesive ability of metastasing tumor cells, and their notably antiproliferative
effects underline the prospective significance of GnRH-III conjugates as drug delivery systems in treatment of tumors.
Poster
158
159
Frontiers
in Tumour
Progression
Session B
Poster
Session B
Poster
161
Frontiers in
Tumour Progression
Poster Session B
Stem cell-specific activation of ancestral myc
protooncogenes in the early metazoan Hydra
Markus Hartl, Stella Glasauer, Taras Valovka, Bert Hobmayer and Klaus Bister
Institute of Biochemistry, Institute of Zoology and Center for Molecular Biosciences (CMBI), University
of Innsbruck, Austria
1
2
Heat Shock Protein (HSPs) and Multidrug Resistance
(MDR) gene upregulation in different adenocarcinomas
– characteristics in patients with peritoneal carcinosis
assigned to HIPEC therapy
Maria Lazariotou1, Malte Vetterlein , Markus Frank , Natasha Frank , Christoph Thomas Germer , Jörg
2
The myc oncogene was originally identified in the genome of highly oncogenic retroviruses (v-myc). The cellular c-myc
proto-oncogene encodes the transcription factor Myc controlling fundamental cellular processes. Deregulation of myc
leads to tumorigenesis and is a hallmark of many human cancers. We have identified two ancestral forms of myc from
the early diploblastic cnidarian Hydra [1]. Hydra myc1 and myc2 are specifically activated in all rapidly proliferating
cell types of the interstitial stem cell system. The ancestral Hydra Myc1 and Myc2 proteins display the principal design
of their vertebrate derivatives, with the highest degree of sequence identities confined to the bHLH-Zip domains. The
promoter of the myc2 gene contains the consensus sequence 5’-CACGTG-3’ (E-box) to which recombinant Hydra
Myc proteins bind with high affinity. The myc2 gene is located immediately adjacent to the Hydra CAD gene encoding
carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase required for pyrimidine nucleotide
biosynthesis. The human CAD gene represents a bona fide transcriptional Myc target. The 2213-amino acid Hydra
CAD protein displays 62% sequence identity with the 2225-amino acid human ortholog. Similar to the human CAD
regulatory region, the Hydra CAD promoter contains two Myc binding sites in close proximity to the transcription start
site, suggesting that CAD is a very early and essential transcriptional Myc target. Also, the oncogenic potential of Myc has
evolved very early since hybrid proteins composed of segments from the retroviral v Myc oncoprotein and of Hydra Myc1
or Myc2 display cell transforming activity. Our results suggest that the principal functions of the Myc master regulator
arose very early in metazoan evolution, allowing their dissection in a simple model organism showing regenerative
ability but no senescence. [1] Hartl, Mitterstiller, Valovka, Breuker, Hobmayer, Bister (2010). Proc. Natl. Acad. Sci. USA
107:4051-4056.
3
3
4
Pelz4, Martin Gasser4 and Ana Maria Waaga-Gasser2
Department of Cardiothoracic Surgery, University of Wuerzburg, Germany; 2Department of Surgery I,
Molecular Oncology and Immunology, University of Wuerzburg, Germany; 3Children’s and Brigham and
Women’s Hospital, Harvard Medical School, Boston, USA; 4Department of Surgery I, University of Wuerzburg,
Germany
1
Peritoneal carcinosis (PC) is diagnosed in 10-20% of gastric, colorectal and ovarian cancer. Hyperthermic intraperitoneal
chemotherapy (HIPEC) is increasingly integrated into multimodal treatment strategies. We analysed relevant HSPs that
confer resistance to physical stresses (elevated temperatures) as well as profiles of the specific MDR gene ABCB5 in
patients with PC. Patients with different adenocarcinomas and additional PC that underwent HIPEC therapy between
10/08 and 03/10 in our department (gastric, colorectal and ovarian cancer, n=9) were included in the study. HIPEC
therapy was performed under specific conditions (1 hr permanent chemotherapeutical flux via external pump into the
abdominal cavity after resection of relevant tumour masses with elevated temperature up to 43°C). Tumours before and
after HIPEC therapy were analysed for HSPs, MDR1, ABCB5 and additional CD133 expression, characteristic for putative
tumour initiating cells in PC, using immunohistology, Western Blot and RT-qPCR. Hsp70/72 expression was upregulated
in all investigated tumours both before and after HIPEC therapy. This could be demonstrated immunohistologically, by
Western Blot and RT-qPCR analysis. Upregulated protein and gene expression was also shown for ABCB5 and CD133
pre- and post HIPEC therapy although expression analysis on the protein level immediately after hyperthermia therapy
(1 hr) of 43°C suggests hyperthermia related protein degradation to some degree. However, immunohistological protein
expression and Western blot results strongly correlated with each other in individualised studied patient tumours.
Therapeutic approaches like HIPEC based therapies to achieve antiproliferative and apoptosis inducing effects in
patients with PC of different adenocarcinomas seem to be significantly influenced by highly conserved HSP mechanisms
as well as multidrug resistance genes. Studying HSP and MDR expression profiles in the tumours seem to be a valuable
tool for prognostic evaluation of individual patients with PC before HIPEC therapy and guiding therapy in regard to usage
of different chemotherapeutical agents.
Poster
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163
Frontiers in
Tumour Progression
Poster Session B
ROR1 is a pseudo-kinase involved in MET-driven
tumorigenesis
3
4
Wip1–dependent control of obesity and atherosclerosis
Xavier Le Guezennec1, Elena Kostromina, Nicolette Marshall, Calvina Kek, Weiping Han and
Luca Lazzari, Alessandra Gentile, Silvia Benvenuti, Livio Trusolino and Paolo M. Comoglio
Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School,
Candiolo, Italy
The human kinome includes ROR1, a yet poorly characterized orphan receptor tyrosine kinase, featuring an extracellular
HGF-like ‘kringle’ motif and a cytoplasmic putative tyrosine kinase domain. To investigate ROR1 contribution to human
cancer, we undertook an integrated screening in a panel of 43 human cancer cell lines. Analysis of growth response
to RNAi-mediated knockdown of ROR1, paralleled by analysis of basal ROR1 expression and tyrosine phosphorylation,
revealed that the extent of receptor phosphorylation (but not receptor expression) predicts sensitivity to ROR1 silencing:
only two cell lines, HS746T (gastric carcinoma) and NCI-H1993 (non-small cell lung carcinoma), exhibited high levels of
ROR1 tyrosine phosphorylation and experienced growth impairment when ROR1 was genetically inactivated. Similarly
to in silico analysis of its tyrosine kinase domain sequence, highlighting significant aminoacid variations within region
important for enzymatic activity, in vitro biochemical assays demonstrated that ROR1 is a pseudo-kinase, devoid of
autocatalytic activity. These data also suggested that detected ROR1 phosphorylation could have reference to passive
trans-phosphorylation by other tyrosine kinases. Intriguingly, in silico and biochemical analysis revealed that the two
phospho-ROR1-positive cell lines are both characterized by genomic amplification and constitutive activation of the MET
tyrosine kinase receptor. Pharmacologic inhibition of MET abrogated ROR1 phosphorylation, whereas overexpression
of other constitutive active tyrosine kinase receptors (EGFR and ErbB2) failed to induce ROR1 phosphorylation. ROR1
silencing in HS746T and NCI-H1993 cells impaired cellular proliferation and anchorage independent growth in vitro and
resulted in dramatic inhibition of tumorigenesis in vivo. These data enlightened an unexpected collaboration of pseudokinase ROR1 in the malignant phenotype sustained by the MET oncogene.
Dmitry Bulavin1
1
Dept of Cell Cycle and Tumorigenesis, Institute of Molecular and Cellular Biology, Proteos, Singapore; 2Lab
of Metabolic Medicine, Singapore Bioimaging Consortium, Biomedical Sciences Institutes, Helios, Singapore
Our lab showed that Wip1(wild type p53induced phosphatase 1) plays an important role in the inhibition of ataxiatelangiectasia mutated (ATM) serine/threonine protein kinase, a key protein in the cellular response to ionizing radiation.
Published evidence highlights a relationship between ATM deficiency and metabolic syndrome in several parameters
such as insulin resistance, glucose intolerance, adiposity, blood pressure and atherosclerosis using apoE -/- mice
models. The involvement of Wip1 in metabolism remains so far uncharacterized but the relation of Wip1 with ATM from
these recent progress underlines a potential for Wip1 to extend its function beyond DNA damage, oncogenesis and
exert effects in metabolic disorders. Wip1 null mice models when fed a high fat diet show resistance to weight gain and
display a reduced adiposity. Analysis of Wip1 null mice in apoE -/- background upon western diet feeding for 8 weeks
reveals a significant reduction in blood pressure as well as a decrease in aortic plaques when compared with apoE
-/- littermates. ATM deletion fully revert apoE-/- Wip1-/- phenotype and appear as a functional determinant in limiting
fat accumulation and atherosclerosis in apoE-/- Wip1 -/- mice. In Vitro Bone marrow derived macrophages from apoE-/Wip1-/- mice subjected to oxLDL treatment are characterised by a resistance to foam cell formation conversion which is
relieved upon ATM inhibition or deletion. We conclude that Wip1 through ATM exerts new functions in metabolic disorders
and might be of therapeutic interest for treating obesity and atherosclerotic disorders.
Poster
164
165
5
Frontiers in
Tumour Progression
Poster Session B
6
RhoB modulates metastasis progression and resistance
in lung adenocarcinoma
Crucial role of TLX signaling pathway in adult
neurogenesis and gliomagenesis
Fernando Lecanda*, Diego Luis-Ravelo , Iker Antón, Carolina Zandueta, Pablo Salazar-Colocho, María
Hai-Kun Liu1, Ying Wang , Thorsten Belz , Dagmar Bock , Andrea Takacs , Bernhard Radlwimmer , Sebastian
*
1
1
1
1
2
José Pajares, Jackeline Agorreta, Ignacio Wistuba, Luis M. Montuenga and Javier De Las Rivas
Barbus2, Guido Reifenberger3, Peter Lichter2 and Günther Schütz1
Division of Oncology, Adhesion and Metastasis Laboratory; 2Biomarkers Laboratory, Center for Applied
Medical Research (CIMA), University of Navarra, Pamplona, Spain; 3Department of Pathology, The University
of Texas - M. D. Anderson Cancer Center, Houston, Texas, USA; 4Bioinformatics and Functional Genomics
Research Group, Cancer Research Center, University of Salamanca (CSIC/USAL), Spain; *These authors
contributed equally to this work.
Division of Molecular Biology of the Cell I, German Cancer Research Center (DKFZ), Heidelberg, Germany;
Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany; 3Department
of Neuropathology, Heinrich-Heine-University, Düsseldorf, Germany
1
Lung adenocarcinoma frequently disseminates to a variety of organs including the skeleton, a process associated with
poor prognosis. By means of comparative transcriptomic analysis, extensive in vitro and in vivo functional and clinical
validation, we identified RhoB, a small GTPase involved in cytoskeletal organization and endosome trafficking as a key
component in lung adenocarcinoma cancer progression and treatment resistance. Clinical and in silico analysis revealed
a marked association between high RhoB levels and poor survival, especially in patients who received adjuvant chemoand radiotherapy. Consistent with this, RhoB conferred significant taxane-chemoresistance and survival advantage to
- irradiation in vitro and in vivo. In an orthotopic tumor model, overexpression of RhoB in lung cancer cells favored
an increased metastatic spread. More importantly, in a model of systemic inoculation RhoB overexpression promoted
high osseous prometastatic activity associated with high tumor burden and aggressive osteolysis leading to decreased
survival. Reciprocally, in vivo genetic inhibition of RhoB levels or pharmacological blockade of RhoB activity correlated
with decreased metastatic bone colonization and increased life span. These effects were associated with impaired
invasion and metalloproteolytic activities in vitro. Thus, RhoB promotes early dissemination, robust osseous colonization
and confers treatment resistance. We postulate that RhoB belongs to a novel class of “genes of recurrence” conferring
dual roles in metastasis progression and refractoriness. Our data suggest that RhoB could be a predictive marker for
treatment resistance in human lung adenocarcinomas and a potential therapeutic target.
1
2
Malignant gliomas are the most common primary brain tumors and associated with frequent resistance to therapy as
well as poor prognosis. Here we demonstrate that the nuclear receptor tailless (Tlx), which in the adult is expressed
exclusively in astrocyte-like B-cells of the subventricular zone, acts as a key regulator of neural stem cell (NSC)
expansion and brain tumor initiation from NSCs. Overexpression of Tlx antagonizes age-dependent exhaustion of NSCs
in mice and leads to migration of stem/progenitor cells from their natural niche. The increase of NSCs persists with
age and leads to efficient production of newborn neurons in aged brain tissues. Tlx overexpressing NSCs initiate the
development of glioma-like lesions and gliomas. We also demonstrate that Tlx transcripts are overexpressed in human
primary glioblastomas, and Tlx gene is amplified in some of the human brain tumors. Our unpublished work showed
that Tlx inhibits the expression of its target genes PTEN and p21 in NSCs, inactivation of those two genes specifically
in adult NSCs leads to glioma formation, which mimics the Tlx overexpressing phenotype. PTEN-deficient neural stem/
progenitor cells are more sensitive to rapamycin. Furthermore, we provide evidence that an increase of p53-dependent
cellular senescence contributes to the loss of PTEN-deficient neural stem/progenitor cells. Genetic ablation of p53
together with PTEN loss leads to a continuous increase in stem cell proliferation and brain tumor formation. Our study
clearly demonstrates how NSCs contribute to brain tumorigenesis driven by a stem cell specific transcription factor, thus
identifies a crucial pathway for NSC maintenance and brain tumor initiation.
Poster
166
167
Frontiers in
Tumour Progression
Poster Session B
7
8
Cancer initiating cells in K-RasG12V induced lung
adenocarcinoma
Dicer1 is a synthetic lethal partner of tumour
suppressor p53 in retinoblastoma
Sara Mainardi, Chiara Ambrogio and Mariano Barbacid
Irina Lambertz, David Nittner, Alexander Schramm, Frederic Clermont, Pieter Mestdagh, Søren Jensby Nielsen, Frank
Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
Despite the high incidence and mortality rate of lung adenocarcinoma, we still know very little regarding the cell type
that gives origin to this tumor type. In an effort to identify the cancer initiating cells (CICs) responsible for the onset of
lung adenocarcinoma, we have used the K-Ras+/LSLG12Vgeo strain, a mouse tumor model that closely recapitulates this
human disease. These mice carry an endogenous K-RasG12V oncogene whose expression is controlled by a knocked-in
4OHT-inducible CreERT2 recombinase. The targeted K-Ras allele also expresses a beta-galactosidase color marker
(β-Gal) in a bicistronic fashion to allow identification of K-RasG12V expressing cells at the single cell level. Activation of
the resident CreERT2 recombinase by a single injection of 4OHT leads to the expression of the endogenous K-RasG12V
oncoprotein in a limited number of lung cells. About one third of these cells (identified by β-Gal expression) initiate
unscheduled proliferation 1-2 weeks after turning on oncogene expression, leading to the generation of areas containing
≥10 β-Gal-positive cells within one month that eventually progress to larger areas (≥100 β-Gal-positive cells) during
the following month. We first determined whether these cells were preferentially located in any of the well defined
lung structures including the bronchiolar and alveolar epithelium, as well as bronchiolaveolar duct junctions which
are supposed to house the putative bronchiolaveolar stem cells (BASCs). Cell counts in sections obtained from mice
sacrificed 1 month after 4OHT injection revealed random activation of the oncogene in all these regions with a preference
for proliferation in the alveolar epithelium. Evaluation of the composition of these groups of β-Gal positive cells indicated
that the vast majority expressed SPC, a marker for alveolar type II cells, while only few small groups contained
CC10-positive Clara cells or double SPC-CC10-positive BASCs. None of the cells contained the main lung cell type,
alveolar type I cells. Thus, lung CICs are primarily derived from alveolar type II cells, a cell type that represents 3-5%
of all lung cells. Moreover, using a laser capture microdissection technique, we managed to selectively isolate the cells
that proliferated forming small groups (100-200 cells) one month after induction of the K-Ras+/LSLG12Vgeo expression.
Good quality RNA was extracted from the dissected cells and gene expression was evaluated by Agilent microarray and
validated by qRT-PCR. Current efforts are aimed at analyzing the gene expression data, as well as to isolate live CICs,
in order to determine whether they represent a unique subpopulation of alveolar type II pneumocytes, with the ultimate
goal of understanding what makes these cells permissive for the oncogenic properties of K-Ras oncogenes.
Speleman, Jo Vandesompele, Michael A. Dyer, Johannes Schulte and
Jean-Christophe Marine
Laboratory for Molecular Cancer Biology, VIB-KULeuven, Belgium; Department of Pediatric Hematology and
Oncology, University Children’s Hospital Essen, Germany; Center for Medical Genetics, Ghent University
Hospital, Belgium; Exiqon A/S, Vedbaek, Denmark; Department of Developmental Neurobiology, St Jude
Children’s Research Hospital, Memphis, USA
p53 tumour suppression function is lost in the vast majority of human cancers. Therefore, identification of synthetic lethal
interactors of p53 should lead to conceptually simple and attractive approaches to selective targeting of cancer cells.
DICER, an RNAse III endonuclease playing a critical role in miRNA biogenesis, exhibits reduced stability and/or activity in
human cancers. Consistently, we and others have shown that Dicer1 functions as a haploinsufficient tumour suppressor
in mice. Surprisingly, however, homozygous deletions or loss-of-function mutations in DICER1 have never been reported
in human tumours and homozygous loss of Dicer1 appears to be strongly selected against in a K-Ras-induced mouse
model of lung cancer. These data raised the possibility that Dicer1 is required for tumour formation. We now demonstrate
that targeted homozygous loss of Dicer1 completely prevents the formation of retinoblastoma in mice in which the Rb and
p53 tumour suppressor pathways are inactivated. Strikingly, we find that Dicer1 deficiency selectively kills Rb-deficient
tumour initiating cells in which p53 is inactivated while sparing cells that retain functional p53. miRNA profiling of mouse
and human primary retinoblastomas showed dramatic overexpression of the pro-oncogenic miR17-92 cluster in all
samples analyzed. High-resolution array-CGH indicates that in ~20% of human retinoblastoma patients overexpression
of miR-17-92 results from copy number alterations. Crucially, functional inactivation of the miRNAs encoded by the miR17-92 cluster is sufficient to decrease the viability of human retinoblastoma cells. Our data provide genetic evidence of
a synthetic lethal interaction between Dicer and p53 and important insights into the molecular mechanism underlying
selective pressure against Dicer1 loss during the process of tumorigenesis. Our data also designate members of the
miR-17-92 cluster as a highly selective therapeutic target for the treatment of retinoblastoma.
Poster
168
169
9
Frontiers in
Tumour Progression
Poster Session B
Oncogene regulation by tumour microenvironment:
Epstein Barr Virus-encoded microRNAs repress BCL6 in
diffuse large B cell lymphomas
Implication of the protease ADAMTS1 in basement
membrane deposition in physiological-and tumorassociated vasculature
Daniel Martín-Pérez1, Pierfrancesco Vargiu , Santiago Montes-Moreno , David González , Socorro M.
Estefania Martino-Echarri, C. Casal, M.C. Plaza-Calonge and J.C. Rodríguez Manzaneque
1
1
2
Rodríguez1, Rufo Rodríguez3, Manuela Mollejo3, Josep Castellvi4, Margarita Sánchez-Beato1 and Miguel A. Piris1
Lymphoma Group, Molecular Pathology Programme; 2Bioinformatics Core Unit, Structural Biology and
Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 3Department
of Pathology, Hospital Virgen de la Salud, Toledo, Spain; 4Department of Pathology, Vall d’Hebron University
Hospital, Barcelona, Spain
1
Epstein Barr Virus (EBV) is a lymphotropic virus that has been associated with the development of some cancers including
several hematologic malignancies such as Burkitt lymphoma (BL), Hodgkin lymphoma and diffuse large B cell lymphoma
(DLBCL). The virus interferes with the normal development of B cells and some viral proteins mimic extrinsic survival signals
for B cells in cancer which lead to the activation of the NF-κB pathway. BCL6 is a transcriptional repressor that plays a
key role during B cell development and is necessary to form germinal centres. It is the most commonly involved oncogene
in DLBCL and has been shown to repress NF-kB in this tumour type. In some B cell lymphomas the expression of BCL6
is opposite to the EBV infection, however the mechanism of this phenomenon and its biological meaning remain elusive.
In this study, we have analysed the microRNA (miRNA) expression profile of 36 cases of DLBCL using Agilent’s Human
miRNA microarray and we have found a cluster of 22 mature EBV-encoded microRNAs that are upregulated in EBV-positive
DLBCL cases. Ten of these miRNAs were predicted to potentially target BCL6 using the miRanda algorithm. Additionally,
we performed immunohistochemical studies and in situ hybridisation on a cohort of 149 DLBCL cases and we found an
inverse correlation between the expression of BCL6 and the presence of EBV. In fact, more than 75% of EBV negative
cases expressed BCL6 whereas only 3% of EBV positive cases expressed BCL6. This phenomenon was not observed in
BL and DLBCL cell lines where EBV presence seemed to be independent of the BCL6 expression levels. However we still
could correlate the expression of BCL6 with the expression of several viral miRNAs. To explore the possibility that some
viral miRNAs could help to repress BCL6, we selected ebv-miR-BART3, ebv-miRBART7, ebv-miR-BART9 and ebv-miRBART17-5p for further functional validation based on the calculated score by miRanda. Then we cloned the 3’ UTR of BCL6
in a luciferase reporter system and co-transfected this vector with synthetic miRNAs in HEK293T cells. At least three of
these miRNAs were able to induce a decrease in the luciferase signal when the 3’UTR of BCL6 was present. Moreover, we
investigated the effect of these miRNAs on endogenous BCL6 in BL and DLBCL cell lines. Transfection of synthetic miRNAs
induced a decrease of BCL6 expression in most cases, although the response was variable between cell lines. Transfection
of anti-miRNA molecules in two EBV-positive DLBCL cell lines led to increased levels of endogenous BCL6 protein only in
the cell line with lower levels of viral miRNAs. These results highlight the importance of BCL6 downregulation in the context
of EBV-mediated transformation probably allowing the activation of NF-κB by the virus.
170
10
GENYO (Pfizer-University of Granada-Andalusian Government Centre for Genomics and Oncological
Research), Granada, Spain
Solid tumors are considered as a group of highly dynamic pathologies due to their rapid growth, requiring the formation
of new blood supply that involves an intense remodeling of the extracellular space, driven by the action of specific
proteases. Our main interest includes ADAMTS1, a member of the family ADAMTS (a disintegrin and metalloproteinase
with thrombospondin motifs) of extracellular proteases. ADAMTS1 has been previously described as anti-angiogenic, but
also as pro-tumorigenic and pro-metastatic. Although these properties seem to be contradictory, we recently reported
a dual action of ADAMTS1 by its contribution to alternative tumor vascularization phenomena and the acquisition of an
endothelial-like phenotype by tumor cells. Also, its anti-angiogenic action has been proposed in the Tc1 mouse model of
Down’s Syndrome. Still, the specific proteolytic actions of this protease are unknown for different biological contexts. By
proteomic approaches we have been able to identify relevant molecules in the process of neo-vascularization, including
the basement membrane components Nidogen 1 and Nidogen 2. Importantly, various reports show that these proteins
appear proteolyticaly cleaved under basal conditions. Our more recent findings showed the unbalance of this proteolysis
process in breast tumor samples compared with control tissue, so we are investigating the action of ADAMTS1 in this
context, in conjunction with further studies that include the close characterization of vascularization events in distinct
tissues of wild-type and ADAMTS1-deficient mice. With a combination of in vivo and in vitro assays we are able to
elucidate the role of this protease for a correct deposition of basement membrane components, a hallmark to distinguish
among a physiological- or a pathological-associated vasculature.
Poster
171
Frontiers in
Tumour Progression
Poster Session B
The role of c-Raf in liver cancer development
11
12
Synergistic inhibition of ErB and IGF-IR in pancreatic
cancer cells
Gabriele Maurer and Manuela Baccarini
Max F. Perutz Laboratories - Faculty of Life Science Department of Microbiology and Immunobiology Vienna,
Austria
Raf kinases are serine/threonine specific kinases which function as a link between active RAS and the MEK/ERK
module that can regulate growth, differentiation and apoptosis downstream of a variety of extracellular signals. Genetic
studies in mice have shown that the Raf proteins (A-Raf, B-Raf and C-Raf) carry out non-redundant functions in mouse
development. c-raf deficient embryos die around midgestation and show increased apoptosis in the fetal liver. The
Ras-Raf- MEK-ERK/MAPK pathway can be activated at high frequency in human tumors. C-Raf is essential for the
development and maintenance of Ras-driven epidermal tumors. Increased activation of C-Raf protein was found in
hepatocellular carcinoma (HCC) biopsies. To study the function of C-Raf in liver cancer development in vivo, we generated
two conditional c-raf knock-out mouse lines, the Albumin-cre (hepatocyte and bile duct specific) and the Mx-cre
(hepatocyte and bone marrow specific) transgenic line. Liver tumors are induced by a model of chemical carcinogenesis
based on the tumor initiator diethylnitrosamine (DEN) and the tumor promoter phenobarbital. 7.5 months after DEN
injection, the results were surprising but clear-cut: liver tumor mass was dramatically increased in c-raf Δ/ΔAlbumin
(knockout) mice compared to c-raf F/F (wildtype) mice. Ablation of c-raf before tumor initiation in hepatocytes and bone
marrow derived cells rescued this phenotype, indicating that C-Raf is essential for the support of tumorigenesis by bone
marrow derived cells. Our results also suggest that C-Raf is required in the initiation stage of liver carcinogenesis but not
during progression. We propose that C-Raf has two critical functions in chemical liver carcinogenesis: a cell-autonomous
tumor suppressor function in parenchymal cells and a tumorpromoting function in non-parenchymal cells.
Adela Mazo, Anna Vidal-Pla, Susanna Pomés, Nerea Urtasun and Neus Carbó
University of Barcelona, Institute of Biomedicine, Barcelona, Spain
Pancreatic cancer is a particularly challenging malignacy given its usually advanced stage at diagnosis and its rather
limited response to treatments. Although gemcitabine is the backbone of routine therapy in advanced disease, novel
drugs are urgently needed for improving the treament of this cancer. Overexpression and/or activation of TyrK receptors
are common features in pancreatic cancer. The aim of this study was to evaluate if combined inhibition of EGFR, Her-2
and IGF-IR may overcome resistances observed in monotherapy strategies. Additionally, molecular changes involved in
all treatments have been analyzed. Several human pancreatic cancer cell lines (NP9, NP18, NP29, and CP15) expressing
variable levels of the above mentioned receptors were treated with different concentrations of the dual inhibitor of EGFR/
Her2, lapatinib, and the IGFIR inhibitor, NVP-AEW541, and cell viabilities were determined by MTT assay. Treatments
with combinations of these inhibitors resulted in dramatic decreases of viability in all the cell lines tested. The potent
synergy was evidenced by the corresponding coefficients of drug interaction (CDI), which ranged from 0.01 and 0.55.
Flow cytometry analysis demonstrated that the combination enhanced cell cycle arrest at G1 and apoptotic cell death.
The analysis by Western Blot of proteins involved in ErbB and IGF-IR signalling pathways demonstrated that, although
each drug was able to reduce slightly Akt, Erk or IRS-1 phosphorylation, only the combined treatment caused a marked
inhibition of the activated status of all three effectors. In conclusion, the combined inhibition of ErB and IGF-IR may solve
the resistance due to bidirectional transactivation of these receptors, representing a very promising therapy against a
significant percentage of human pancreatic tumors.
Poster
172
173
13
Frontiers in
Tumour Progression
Poster Session B
The molecular characterization of the antiangiogenic
compound aeroplysinin-1 reveals it as a selective
inducer of apoptosis in endothelial cells
14
FGF receptor-4 regulates tumor invasion by coupling
FGF-signaling to extracellular matrix degradation
Pipsa Meller1, Nami Sugiyama , Markku Varjosalo , Jouko Lohi , Marko Hyytiäinen , Sami Kilpinen , Olli
1
Miguel-Ángel Medina1,3, Beatriz Martínez-Poveda , Salvador Rodríguez-Nieto , Boris Zhivotovsky ,
1
2
2
María Victoria Ruiz-Pérez1, Melissa García-Caballero1 and Ana R. Quesada1,3
Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Spain;
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden; 3Unit
742, CIBER de Enfermedades Raras (CIBERER), Málaga, Spain
1
2
Aeroplysinin-1 (AE1) is a brominated metabolite extracted from the sponge Aplysina aerophoba and characterized by
our group as a potent antiangiogenic compound. Since AE1 had been previously described as an inhibitor of the receptor
tyrosine kinase EGFR, we wanted to test whether the antiangiogenic action of this compound was due to inhibition of
another tyrosine kinase, VEGFR2, involved in activation of the angiogenic program. This was not the case, since AE1 was
unable to inhibit VEGFR2 activity. This observation led us to suspect an action of AE1 on some key proteins downstream
in the VEGFR2 signaling pathway. By using docking modeling, we inferred potential direct interactions of AE1 with both
ERK and AKT. These predictions were experimentaly confirmed by showing that AE1 repressed both the Erk and Akt
pathways in endothelial cells (ECs) but not in tumor or normal cells, indicating that components of these pathways are
major targets in the molecular mechanism of AE1 in ECs. The effect on the Akt survival pathway could suggest a proapoptogenic effect of AE1. In this work we also provide evidences of a selective induction of apoptosis by AE1 in ECs. In
fact, AE1 induced chromatin condensation and nuclear fragmentation and increased the percentage of cells with subdiploid DNA content in ECs, but not in tumor cells. In ECs, AE1 induced activation of caspases, as well as the cleavage
of apoptotic substrates such as poly (ADP-ribose) polymerase and lamin-A in a caspasedependent mechanism. Our
data indicated a relevant role of the mitochondria in the apoptogenic activity of this compound. [Supported by grants
TRACE PT2008-0145 and PS09/02216 (Spanish Ministry of Science and Innovation), Fundación Ramón Areces, and
P08-CTS-03759 and funds from group BIO-267 (Andalusian Government). The “CIBER de Enfermedades Raras” is an
initiative from the ISCIII (Spain)].
2
1
1
3
Kallioniemi , Signe Ingvarsen , Lars H. Engelholm , Jussi Taipale , Kari Alitalo , Jorma Keski-Oja and Kaisa Lehti1
3
4
4
2,5
1
1
Molecular Cancer Biology Research Program, and 2Genome-Scale Biology Research Program, Biomedicum
Helsinki, University of Helsinki, Finland; 3Institute for Molecular Medicine Finland (FIMM), University
of Helsinki, Finland; 4The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark; 5Department of
Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden
1
Aberrant expression and polymorphism of fibroblast growth factor receptor-4 (FGFR4) has been linked to tumor
progression and anti-cancer drug resistance. We describe here a novel mechanism of tumor progression by matrix
degradation involving epithelial-to-mesenchymal transition in response to membrane-type-1 matrix metalloproteinase
(MT1-MMP, MMP-14) induction at the edge of tumors expressing the FGFR4-R388 risk variant. Both FGFR4 and
MT1-MMP were upregulated in tissue biopsies from several human cancer types including breast adenocarcinomas,
where they were partially co-expressed at the tumor/stroma border and tumor invasion front. The strongest overall
co-expression was found in prostate carcinoma. Studies with cultured prostate carcinoma cell lines demonstrated that
the FGFR4-R388 variant, which has previously been associated with poor cancer prognosis, increased MT1-MMPdependent collagen invasion. In this experimental model, knock-down of FGFR4-R388 or MT1-MMP by RNAi blocked
tumor cell invasion and growth in collagen. This was coupled with impaired phosphorylation of FGFR substrate-2 and Src,
up-regulation of Ecadherin, and suppression of cadherin-11 and N-cadherin. These in vitro results were substantiated
by reduced MT1-MMP content and in vivo growth of prostate carcinoma cells after the FGFR4-R388 gene silencing. In
contrast, knock-down of the alternative FGFR4-G388 allele enhanced MT1-MMP and invasive tumor cell growth in vivo
and within three-dimensional collagen. These results will help to explain the reported association of the FGFR4-R388
variant with the progression and poor prognosis of certain types of tumors.
Poster
174
175
Frontiers in
Tumour Progression
Poster Session B
Deregulation of lipid metabolism in head and neck
paragangliomas: a novel insight into the pathogenesis
of the disease
15
16
Appearance of high level of cell-malignancy traits in
breast cancer primary culture: a case report
Luigi Minafra1, Rossana Norata , Carmelo Lupo , Gianluca Di Cara , Cristina Messa
1
Anna Merlo, Sandra Bernaldo de Quirós, Iriana Zambran, Milagros Balbín, Carlos Suarez and María-Dolores
Chiara
Servicio de Otorrinolaringología y Diagnóstico Molecular, Hospital Universitario Central de Asturias and
Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Spain
Head and neck paragangliomas of the (HNPGL) are rare, mostly benign tumors of the parasympathetic nervous system.
They occur most commonly in the carotid body, the main peripheral sensor of physiological hypoxia. They are usually
characterized by an indolent, non-invasive growth pattern. Most cases are sporadic, but a significant number (10–50%)
have been shown to be familial, associated with mutations in genes encoding subunits of succinate dehydrogenase
(SDH) mitochondrial complex II (SDHB, SDHC, SDHD, and SDHA) or associated proteins (SDHFA2), which plays a central
role in energy metabolism. In this study, we compared the transcriptional profile of HNPGLs and normal carotid bodies.
Among the differentially expressed genes, a significant number, associated with PPAR/adipocytokine signaling pathways
and lipid metabolism, were found down-regulated in tumors. We also found a significant overlap between de HNPGL
deregulated pathways and genes associated with obesity, atherosclerosis, type II diabetes, and hypercholesterolemia.
Similar results linking cancer with metabolic diseases has been recently appreciated suggesting that a variety
of phenotypically diverse disease states are linked through a common transcriptional program involving metabolic
pathways. Interestingly, suppression of ADIPOQ, one of the most dramatically repressed genes in HNPGLs, is known to
decrease mitochondrial content and enzymes, causing mitochondrial dysfunction and insulin resistance. Defects in the
mitochondria, the centers of cellular metabolic activity, is known to precede much of the hallmark pathology of familial
paraganglioma associated with SDH mutations. The transcriptional profile identified here is common to both familial and
non-familial HNPGLs. Therefore, our data show that one putative pathological mechanism that plays a prominent role in
the pathogenesis of non-familial HNPGLs is lipid and mitochondrial metabolic dysfunction.
and Cecilia Gelfi
2
3
4,5
4,6
1
LATO-HSR Giglio, Cefalù (Palermo), Italy; 2La Maddalena Hospital, Palermo, Italy; 3Department of
Experimental Oncology and Clinical Application, University of Palermo, Italy; 4Institute of Molecular
Bioimaging and Physiology, CNR, Segrate (Milan), Italy; 5University of Milan Bicocca, Italy; 6Department of
Sciences and Biomedical Technologies, University of Milan, Italy
Cultivation of primary cells from individual breast cancer tissues is a fundamental approach for the in vitro study of a wide
variety of neoplastic cell properties. In addition, primary cells mimic more closely the in vivo behaviour, as compared to
the immortalized breast cancer cell lines. We report the isolation and characterization of a neoplastic cell population from
a ductal infiltrating breast cancer. Cells were isolated from the tumor immediately after surgery, by enzymatic digestion
and differential cell centrifugation methods. Preliminary tests for BRCA1 and BRCA2 germline mutations suggested the
sporadic origin of this tumor. Following enzymatic treatment, cells started to move away from the tissue boundaries,
acquiring a stellate morphology and a high proliferative capacity, resulting into a number of multilayered foci, typical
of highly malignant cells lacking contact inhibition. Immunological characterization has shown a complex expression
pattern of biomarkers. The cells were: negative for ER, PgR, HER2, and c-Kit; weakly positive for CK8/18, CK5/6, and for
p63 and p53; positive for Ki-67, smooth muscle actin (SMA), vimentin and EGFR. To determine whether the observed
biomarkers status corresponded with mRNA expression, we conducted RT-PCR analyses extended to the stem cell
markers CD24, CD44, OCT4. mRNA expression of all selected markers was observed, suggesting a strong transcriptional
activity. Present results allow to focus on following aspects: the co-expression of epithelial and mesenchymal markers
may suggest that cells undergo to an epithelial-mesenchymal transition or that they originate from a common breast
progenitor with bilinear (glandular and myoepithelial) differentiation potential. Moreover, present data are a further
strong indication that primary cell cultures represent a powerful system for the screening of a number of markers to
feed-back to the patient individualized treatment.
Poster
176
177
17
Frontiers in
Tumour Progression
Poster Session B
NO modified antiviral drug Saquinavir® reduces
metastatic potential of coloncancer cells
Effect of bioactive natural extracts in tumor
progression
Marija Mojic1, Danijela Maksimovic-Ivanic , Sanja Mijatovic , Djordje Miljkovic , Yousef Al-Abed , Stanislava
1
1
1
18
3
Stosic-Grujicic1 and Ferdinando Nicoletti2
Institute for Biological Research “Sinisa Stankovic”, Belgrade, Serbia; 2Department of Biomedical Sciences,
University of Catania, Italy; 3Department of Medicinal Chemistry, The Feinstein Institute for Medical
Research, Manhasset, New York, USA
1
Saquinavir® is an antiviral drug commonly used for treatment of HIV infection, which also exhibits direct antitumoral
action that cannot be assigned solely to immune system reconstitution. Due to various pharmacological limitations
of Saquinavir® it is necessary to raise its dose over the time, thus manifesting serious adverse effects of the drug. To
overcome this problem, a nitric oxide (NO) - donating group was covalently attached to Saquinavir®. As a result, the new
compound Saq-NO has diminished side effects, sustained antiviral activity while its antitumoral action is intensified,
as confirmed in vitro and in vivo on various cancer cell lines. In this study, the anticancer effect of Saq-NO and the
contribution of attached NO was examined on CT26CL25 mouse colon cancer line. As metastasis remains the main cause
of death in colon cancer patients, the antimetastatic potential of Saq-NO was explored for the first time. Both in vitro
and in vivo, Saq-NO reduced cell viability more efficiently than parental drug by permanent inhibition of cell proliferation,
independent from the continual drug presence. In contrast to other NO-modified compounds, Saq-NO liberated hardly
detectable quantities of NO but cell viability reduction was significantly higher in comparison to conventional NO donors.
Treatment with nitric oxide/peroxynitrite scavengers did not prevent drug promoted decrease in cell viability confirming
the irrelevance of NO in drug effectiveness. Unlike the same dose of Saquinavir, Saq-NO pretreatment decreased colon
cancer cells’ ability to attach to the compounds of extracellular matrix for approximately 25%, while cell migration and
invasiveness were reduced for more than 50%. Although molecular basis of this phenomenon remains to be investigated,
results suggest that Saq-NO might affect metastatic phenotype on several levels. Taken together, modified drug could be
qualified as a new chemical entity with more potent antitumor properties than original drug.
Susana Molina1*, Margarita González-Vallinas , Mónica R. García-Risco , Tiziana Fornari , Susana
1*
2
2
Santoyo , Guillermo Reglero and Ana Ramírez de Molina
2
1,2
1
Institute IMDEA Food (Institute for Advanced Studies of Madrid), Madrid, Spain; 2Food Science Department,
Universidad Autónoma de Madrid, Spain. * These authors contributed equally to this work.
1
In recent years much effort is being dedicated to the development of herbal therapeutics in cancer. It has been shown that
extracts from common herbs possess a variety of bioactivities including antioxidant and anti-inflammatory properties
pointing at a beneficial effect as chemopreventive agents. Furthermore, natural supplements with antioxidant activity
have been reported to reduce the incidence or mortality of gastrointestinal tumors. However, different antioxidants display
different effects and clinical trials are controversial, maybe due to the lack of knowledge of the specific mechanism of
action of these bioactive compounds. Supercritical fluid extracted herbs (HE) with known antioxidant activity was used
to analyze their effect on colon and mammary cancer cells and determine whether this activity is mediated through a
specific mechanism of action. Antiproliferative effect was analyzed by MTT assay in SW620 and HT-29 human colon
cancer cells and in MDA-MB-231 and MCF-7 mammary cancer cells, determining growth inhibition (GI50), citostacity
(TGI50) and citotoxicity (LC50). HE exhibited dosedependent viability suppression and induced cell death in tumoral
cells. Furthermore, HE treatment inhibited the transforming properties of cancer cells as determined by anchorageindependent growth assays. Results obtained varied among the cell systems, suggesting a different behavior depending
on the type of cancer. To study the mechanism of action of HE in cancer cells, gene expression analysis after treatment
was developed under different conditions according to the observed biological effects, using Agilent G4112F Whole
Human Genome Microarray. Gene ontology analysis was performed using Ingenuity Pathways Analysis. Gene expression
analysis revealed that the most significant altered network was involved in cell death and protein degradation. Lipid and
carbohydrate metabolismrelated genes were also found altered with a greater score than those of genes related to oxi.
Poster
178
179
19
Frontiers in
Tumour Progression
Poster Session B
The role of DUSP1 in angiogenesis and lung cancer
metastases
20
Invasion mechanisms in mesenchymal tumor cells
Chiara Mondello1, Cristina Belgiovine , Roberta Frapolli , Katiuscia Bonezzi , Ilaria Chiodi , Giulio Chiesa ,
1
Verónica Moncho-Amor ,
1
Inmaculada Ibañez-de-Cáceres2, Eva Bandres3; Beatriz Martínez-Poveda4,
Jose-Luis Orgaz4, Isabel Sánchez-Pérez1, Sandra Zazo5, Ana Rovira6, Joan Albanell6, Benilde Jiménez4, Federico Rojo5,6,
Cristobal Belda- Iniesta2 , Jesús García-Foncillas3 and Rosario Perona1
1
Instituto de Investigaciones Biomédicas C.S.I.C./U.A.M., Translational Oncology Unit C.S.I.C./U.A.M; CIBER de
Enfermedades Raras (CIBERER) Valencia, Spain; 2Oncology Service, Research Unit H. La Paz, Madrid, Spain;
3
Dept. of Medical Oncology, CIMA, Pamplona, Spain; 4Instituto de Investigaciones Biomédicas C.S.I.C./U.A.M.
Dept. Molecular & Cellular Biology of Cancer, Madrid, Spain; 5Medical Oncology Department, Hospital del
Mar-IMAS, and Cancer Research Program, IMIM-Hospital del Mar, Free University of Barcelona, Spain;
6
Anatomía Patologica, Fundación Jimenez Díaz, Madrid, Spain
MAPK constitute a family of proteins that control cell proliferation, cell migration and apoptosis. Their activation requires
phosphorylation of the threonine and tyrosine residues, while dephosphorylation is mediated by a dual specific family of
protein phosphatases (DUSPs). The first member identified in this family was DUSP1 and its expression is stimulated by
inflammation, oxidative stress or growth factors and is expressed in early stages in NSCLC and other types of tumours
such as breast cancer. The aim of this study is to gain insight into the cellular pathways involving DUSP1 actions using
a double strategy that combines siRNA and microarray technologies. Specifically, gene expression profiles of H460v
(empty vector) and H460cri (DUSP1 siRNA) cells were compared using an array platform. Results showed that some of
statistically significant genes were involved in angiogenesis and tumor progression. Angiogenesis studies revealed in
H460cri conditioned media a decrease in the migration of HMVEC cells and in vivo assays with matrigel plugs, showed
lower vessel density. This was also observed in solid tumours derived from H460cri cells, which correlated with the
decrease observed on VEGFC expression in tumours. On the other hand, we observed less migration and invasive
capacity of H460cri compared to H460v cells. When cells were inoculated in nu-/nu- mice, H460cri cells induced
fewer tumours and metastasis. Metastasis induced by H460cri cells were only found in lungs. However, the animals
inoculated with H460v cells developed tumours in several tissues and lower median survival time was observed. Finally,
in human NSCLC specimens we found a significant correlation between DUSP1 and VEGFC expression. All together, our
results suggest that DUSP1 targeting in NSCLC, would be a good strategy to inhibit tumor angiogenesis and progression
and therefore DUSP1 might represent a good biomarker for combined treatment with chemotherapeutic drugs and
angiogenic inhibitors.
2
3
1
1
Maurizia Mello-Grand4, Francesco Favero4, Angelo P. Dei Tos5, Giulia Taraboletti3 and Maurizio D’Incalci2
Istituto di Genetica Molecolare, CNR, Pavia, Italy; Istituto di Ricerche Farmacologiche “Mario Negri”, 2Milano
and 3Bergano, Italy; 4Fondo Edo Tempia, Biella, Italy; 5Ospedale Generale di Treviso, Italy
1
A high degree of motility and invasiveness characterizes cells of advanced and metastatic tumors. Cancer cells use two
major types of movements: the mesenchymal movement, which is typical of cells of mesenchymal origin and depends
on matrix protease activity, and the amoeboid one, which is characteristic of cells with a rounded shape and relies on
the activity of the RhoA-dependent kinase ROCK. We investigated motility and invasiveness in telomerase immortalized
fibroblasts (cen3tel), which became progressively able to induce sarcomas and metastases in nude mice. In vitro assays
showed increased motility and invasiveness in tumorigenic cells compared to the non tumorigenic ones, paralleled by
a change in morphology, from fibroblastic to polygonal. When grown in 3D matrices, tumorigenic cen3tel cells showed
a rounded morphology with actin cortical rings. This observation, together with the decrease in several matrix protease
gene expression, suggested that tumorigenic cells had switched from a mesenchymal to an amoeboid movement. Indeed,
cell invasion decreased upon treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor RO
28-2653. The increased invasiveness of tumorigenic cen3tel cells was associated with a reduced expression of RhoE,
a cellular inhibitor of ROCKI, and Snail, a main player in the epithelial-mesenchymal transition. In cen3tel cells, ectopic
RhoE expression decreased their invasive and metastatic capacity, while ectopic Snail expression led to a swap to an
MMP-dependent mechanism of invasion. These results indicate that the increased motility and invasiveness associated
with transformation of human fibroblasts can be linked to a switch towards the amoeboid movement. Moreover, they
point to RhoE and Snail as regulators of the type of invasion mechanism adopted by mesenchymal tumor cells.
Poster
180
181
Frontiers in
Tumour Progression
Poster Session B
21
22
Hepatocyte-specific deficiency of Stat5 and GR
signaling causes non-alcoholic fatty liver disease and
promotes hepatic dysplasia
Does constitutively active jak2 perturb mammary gland
development and lead to cancer?
Kristina M. Mueller ,
1
Maria Muñoz Caffarel1, Rosa Zaragoza , Anthony R. Green
2
1
Jan-Wilhelm Kornfeld2, Katrin Friedbichler1, J. Andrew Pospisilik3, Harald
Esterbauer4, Susanne Haindl5, Andrej Kozlov5, Kay-Uwe Wagner6, Luigi Terracciano7, Markus Heim8 and Richard
Moriggl1
Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria; 2Institute for Genetics, University of
Cologne, Germany; 3Max Planck Institute of Immunobiology, Freiburg, Germany; 4Department of Laboratory
Medicine, Medical University Vienna, Austria; 5Ludwig Boltzmann Institute for Traumatology, Vienna, Austria;
6
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha,
USA; 7Institute of Pathology, University Hospital Basel, Switzerland; 8Department of Biomedicine, University
Hospital Basel, Switzerland
1
Hepatic steatosis is one hallmark of non-alcoholic fatty liver disease (NAFLD), which is the most common chronic liver
disease in the western world. Growth hormone (GH)-activated signal transducer and activator of transcription 5 (Stat5)
and the glucocorticoid receptor (GR) are critical signal integrators in liver during metabolic or physiological stress.
Here, we address the function of hepatic Stat5 and GR proteins in lipid homeostasis. We used the Cre-loxP system to
generate hepatocyte-specific deletion of Stat5 and/or the GR in mice. Control and GR knockout mice displayed normal
liver morphology, whereas steatosis and hepatomegaly were observed in Stat5 and Stat5/GR mutants. While steatosis
in the Stat5 mutants remained mild, compound deletion of Stat5 and GR induced severe, progressive steatosis and
steatohepatitis. Genes involved in lipogenesis were induced in both Stat5-deficient lines, possibly as a result of elevated
levels of the key lipogenic transcription factors PPARg and SREBP1c. Loss of hepatic Stat5 caused growth hormone
resistance, while deletion of GR resulted in prominent hypercortisolism. The combination of both conditions in Stat5/GR
mutants led to massive peripheral lipodystrophy and subsequent hepatic fat deposition. Additionally, increased oxidative
phosphorylation and reactive oxygen species (ROS) production by liver mitochondria accompanied by deregulation of
hepatic antioxidant enzymes were observed in Stat5 and Stat5/GR mutant mice. Interestingly, the compound loss of
Stat5 and GR signaling in liver led to the development of dysplastic tumors. The onset of tumors was detected at 9 month
and the incidence of tumor formation increased with age. This model suggests a critical role of hepatic Stat5 and GR in
lipid and hepatocyte homeostasis.
3
and Christine J. Watson1
Department of Pathology, University of Cambridge, UK; Department of Biochemistry and Molecular Biology,
University of Valencia, Spain; 3Department of Haematology, Cambridge Institute for Medical Research, UK
2
Signalling through the JAK/STAT pathway is required for mammary gland development and is frequently deregulated in
cancer. STAT5 is essential for lobuloalveolar development during pregnancy and deletion of STAT5 results in lactation
failure. In contrast, STAT3 is a critical initiator of post-lactational cell death and tissue re-modelling. Paradoxically, both
STAT3 and STAT5 have been shown to be oncogenes in the breast and other tissues such as prostate. Conditional
deletion of JAK2 in the mouse results in failed lactation. Constitutive activation of JAK2 would be predicted to result
in hyperactivation of STATs 3 and 5 with concomitant breast tumourigenesis. However, it is difficult to predict the
outcome of hyperactivation of these STATs as they play opposing roles in normal mammary gland development. We have
analysed the effect of constitutive activation of JAK2 on mammary cell fate by transducing mammary epithelial cells with
retrovirus harbouring either normal JAK2 or the constitutively active JAK2 mutant JAK2V617F. We have observed that
constitutive activation of JAK2 perturbs cell signalling leading to the hyperactivation of STAT5, but not STAT3. Moreover,
JAK2V617F expression increases mammary epithelial cell proliferation, alters functional differentiation and induces a
switch to a more basal phenotype. We have also analysed the phenotype of JAK2V617F mice that express the JAK2
mutant selectively in the mammary gland and we have observed that they show increased pregnancyinduced branching,
accelerated differentiation and increased mammary epithelial cell proliferation. All these effects seem to be mediated by
STAT5 overactivation. Taken together, these results show that the constitutive activation of JAK2 in mammary epithelial
cells perturbs cell signalling and that this deregulated JAK2 affects mammary epithelial cell fate. Our work provides
insights into signalling downstream of constitutively active JAK2/STAT5 and could be important for understanding the
molecular mechanisms of breast tumourigenesis.
Poster
182
183
Frontiers in
Tumour Progression
Poster Session B
Loss of function of p53 and Rb induces alterations in
the homeostasis of bulge and hair germ stem cells of
hair follicle linked to skin tumors development
Purificación Muñoz ,
1
Sònia Solé-Sánchez*1, Victoria da Silva-Diz*1, Diana Riba-Artés1, Maria Urpí1,
Rosa Penin2, Joaquim Marcoval2, Herbert Auer3 and Eduard Batlle4
1
Cancer Epigenetics and Biology Program (PEBC), Biomedical Research Institute of Bellvitge (IDIBELL),
Barcelona, Spain; 2Pathology Service, Hospital Universitario de Bellvitge/IDIBELL, Barcelona, Spain;
3
Functional Genomics Core Facility and 4Oncology Programme, Institute for Research in Biomedicine (IRB),
Barcelona, Spain
Multipotent stem cells populations are responsible of hair follicle regeneration during homeostasis. Bulge stem cells
(bulge SC), identified by 6-integrin/CD34 and keratin 15 (K15) expression are infrequently dividing stem cells localized
in the upper permanent region of hair follicle. In contrast, their progeny, hair germ stem cells (HG SC), are a more
proliferative population characterized by the expression of K15, Lgr5 and P-cadherin. Several studies have implicated
epidermal stem cells in squamous (SCC) and basal cell carcinoma (BCC) development as well as in skin appendage
tumors. Due to the fact that mutations in p53 and Rb have been related with tumorigenesis, we have studied the effect
of loss of function of p53 and Rb in the homeostasis of bulge SC and HG SC and its impact in early steps of skin cancer.
We have used the multi-step skin cancer mouse model K14-HPV16, that over-expresses the viral oncoproteins E6 and
E7 in stem cells and basal keratinoctyes and develops spontaneous SCC in the skin. These mice were crossed with K15EGFP mice which express EGFP in the bulge and HG SC of hair follicle. Compared to wild-type skin (K14-HPV16 +/+/
K15-EGFP Tg/+), the preneoplasic skin of E6/E7 transgenic mice showed a significant increase in the number of hair
follicles, together with a selective expansion of the HG SCs compartment in the absence of significant changes in the
whole population of bulge SCs. However, both stem cells populations presented an increased ratio of proliferating cells
compared to wt mice. Furthermore, we have observed that the percentage of cells with nuclear staining of b-catenin
is increased per hair follicle in transgenic mice. Altogether, these results indicate that the loss of p53 and Rb function
leads to activation of bulge and HG SCs with a resulting expansion of only the HG SC population, indicating an important
disequilibrium between both hair follicle stem cell populations. The analysis SCC developed in E6/E7 transgenic mice
showed the presence of CD34+ cells, previously described as cancer stem cell in mouse SCC, but also of K15+ cells in
these neoplasic lesions. Interestingly K15+-tumor cells were more abundant in SCC in situ and become clearly reduced
in SCC poorly differentiated. These results suggest that the abnormal activation and expansion of hair follicle stem cells
induced by loss of function of p53 and Rb can be relevant during the initial steps of tumorigenesis.
23
24
Impact of UFT, CTX and UFT/CTX metronomic chemotherapy
on tumor markers of local invasion and distant metastasis
in an orthotopic breast cancer xenograft model
Raquel Muñoz1, Shan Man , Ping Xu , Geoffrey A.Wood , JohnWong , Christopher Jedeszko , Alicia Viloria-Petit
2
2
3
4
2
5
and Robert S. Kerbel2
1
Department of Biochemistry, Physiology and Molecular Biology, Faculty of Sciences, University of Valladolid,
Valladolid, Spain; 2Cellular and Molecular Biology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada;
3
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Ontario, Canada; 4Department of
Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada; 5Department of Biomedical
Sciences, Ontario Veterinary College, University of Guelph, Ontario, Canada
Metronomic chemotherapy has been previously shown to target tumor angiogenesis and cause potent anti-tumor effects in
a number of preclinical models, including some involving treatment of advanced visceral metastatic disease, with minimal
associated host toxicity. We have previously reported that metronomic chemotherapy with UFT (an oral 5-FU prodrug) and
cyclophosphamide (CTX), given alone or as a double metronomic combination, was remarkably efficient at inhibiting local
primary tumor invasion and distant microscopic tumor cell metastasis in an orthotopic tumor xenograft model, namely using the
231/LM2-4 cell line, a highly metastatic variant of MDA-MB231 human breast cancer cells (Munoz et al. Cancer Research 66
(7): 3386-91, 2006). These results constituted part of the basis for the initiation of a phase II clinical trial with very encouraging
results (Dellapasqua et al. J Clin Oncol 26: 4899-905, 2008). In order to investigate the mechanisms by which metronomic UFT
and UFT/CTX suppress local primary tumor invasion and distant micrometastasis of 231/LM2-4 orthotopic tumor xenografts,
we evaluated the effect of these drugs on the expression of a number of molecular tumor markers at the invasive margin
compared with the central tumor area, using immunohistochemistry. Markers included enzymes involved in the metabolism
of UFT (dihydropyrimidine dehydrogenase, thymidine phosphorylase, thymidylate synthase); of cell proliferation, apoptosis
and angiogenesis (Ki67, TUNEL, CD31, VEGFR2, VEGF and TSP-1); and growth factor receptor systems known to promote
epithelialmesenchymal- transition (EMT) and breast cancer invasion, including: c-Met, phosphrylated c-Met (pTyr1003), TGFβ1
and phosphorylated-Smad2 (a marker of TGFβ canonical signalling). No significant effects were observed on tumor levels of
enzymes associated with UFT response and metabolism, VEGF, TSP-1, apoptosis, TGFβ1, activated/phosphorylated Smad2
and c-Met among the different treatment groups. In agreement with previous reports, we observed a significant suppressive
effect of metronomic CTX treatment on vascular density. We also observed a consistent effect of metronomic UFT/CTX in the
level of phosphorylated c-Met in 231/LM2-4 xenografts, suggesting this receptor as a potential mediator of the antimetastatic
properties of metronomic UFT/CTX. We also observed a direct correlation between phosphorylated c-Met and cell proliferation,
which was limited to the tumor tissue itself and absent in areas of tumor stroma. We present additional evidence of the antiinvasive properties of metronomic UFT in threedimensional (3D) organotypic structures formed by 231/LM2-4 cells cultured
on reconstituted basement membranes. Taken together, our results reveal a novel target of metronomic UFT/CTX therapy,
and the anti-invasive/metastatic properties of this therapy which, as detected by our in vitro analysis of 3D cultures, appears
independent of its anti-angiogenic properties.
Poster
184
185
25
Frontiers in
Tumour Progression
Poster Session B
Identification and analysis of synthetic lethal
interactions with c-Myc
26
Circulating sphingosine-1-phosphate and erythrocyte
sphingosine kinase-1 activity as novel biomarkers for
early prostate cancer detection
Daniel J. Murphy1, Lidan Liu , Jannes Ulbrich , Torsten Wustfeld , Michael Krause , Theresia Kress , Lars
1
1
2
3
1
Zender2 and Martin Eilers1
Biocenter, University of Würzburg, Germany; 2Helmholtz Centre for Infection Research, Braunschweig,
Germany; 3Institute of Molecular Biology and Tumor Research, Universtity of Marburg, Germany
1
Myc family oncogenes contribute to the genesis of multiple forms of cancer and enhanced expression of c-Myc is
a hallmark of many human tumors. Overexpression of Myc is known to sensitize cells to apoptosis, suggesting that
additional signal transduction events protect tumor cells from Myc-induced death. In order to identify components of
such protective signaling, we performed a synthetic lethal siRNA screen of the human kinome in U2OS-MycERTAM
cells, using immuno-fluorescence for cleaved-PARP as a measure of apoptosis. U2OSMycERTAM cells transfected with
779 siRNA pools were treated with 4- hydroxytamoxifen (4-OHT), to activate MycER, or with solvent control. Differential
cleaved-PARP fluorescence between 4-OHT treated and untreated populations indentified multiple kinases required
for viability of Myc overexpressing cells, including two members of the same family, AMPK and ARK5. While AMPK
is a well-established regulator of cellular metabolism, much less is known about ARK5. Previous reports have linked
ARK5 to cellular survival under nutrient deprivation, as well as to cell migration, metastasis and genomic instability.
ARK5 function requires phosphorylation by LKB1 and is additionally regulated by AKT. Here we present evidence that
ARK5 is required for viability of multiple cell types overexpressing Myc. This protective function requires the catalytic
lysine of ARK5 as well as the LKB phosphorylation site. Depletion of ARK5 prevents tumor formation by hepatocytes
overexpressing Myc and AKT in a murine model of Hepatocellular Carcinoma (HCC) and immunohistochemical staining
of human HCC samples reveals clear overexpression of ARK5. Thus, ARK5 represents a credible target for therapeutic
intervention in human HCC with Myc overexpression.
Joao Nunes1, Lysann Sauer , Justin Sturge
1
1
1
and Dmitry Pshezhetskiy1
Department of Surgery & Cancer, Hammersmith Campus, Faculty of Medicine, Imperial College London, UK
There are only limited markers available for screening normal populations and for monitoring prostate cancer treatment
and these markers lack sensitivity and selectivity. Sphingosine-1-phosphate (S1P) is a secreted lipid second messenger
fundamental to cell growth and migration, the immune response, angiogenesis and malignant transformation. We have
previously shown that sphingolipid metabolism is implicated in the control of prostate cancer growth and that the
regulation of S1P in prostate cancer sensitizes cells to the effects of chemotherapy. In this present study we have
measured plasma S1P levels in patients with prostate cancer (PCa) with localised, locally advanced or metastatic
disease (n=88) and compared levels with age matched controls with benign prostate hyperplasia (BPH) (n=110) or
young healthy males with the very small chance of having PCa foci (n=20). Levels of circulating S1P were significantly
higher in healthy subjects (10.36+/-0.69 pmol/mg prot, p<0.0001) and patients with BPH (9.39±0.75, p=0.0013) than
in patients with PCa (6.89±0.58), (Anova p=0.0019) and were an early marker of PCa progression. Furthermore, S1P
levels were correlated with PCa progression to hormonal unresponsiveness and with the development of lymph nodes
metastases. There was no correlation between S1P levels and the presence of distant metastases or with Gleason score.
Plasma S1P significantly correlated with PSA levels. During the course of the study 9 patients have died of prostate
cancer. Importantly, their circulating S1P levels were 5.11±0.75 vs 7.02±0.22 pmol/mg protein in the surviving patients
(n=79, p=0.0439), allowing to propose circulating S1P as a significant prognostic marker predicating for prostate
cancer mortality. Our data suggest that the decrease in circulating S1P during PCa progression may stem from a highly
significant down-regulation of erythrocyte sphingosine kinase-1 (SphK1) activity (2.14+/-0.17 pmol/mg prot/min in PCa
patients vs 4.7+/-0.42 in healthy individuals, p<0.0001). This current study has provided a potential mechanism for
cancer-related anaemia and compelling evidence that plasma S1P and erythrocyte SphK1 activity are markers with huge
potential for the diagnosis and monitoring of PCa.
Poster
186
187
27
Frontiers in
Tumour Progression
Poster Session B
Classical and alternative NF-κB activation in
lymphomas; the involvement of NIK in the regulation of
both pathways
Lina Odqvist1, Margarita Sánchez-Beato , Santiago Montes-Moreno , Ken H. Young , José A. Martinez1
1
2
Climent , Lydia Sánchez and Miguel A. Piris
3
5
1
Molecular Pathology Programme & 5Biotechnology Programme, Spanish National Cancer Research Centre
(CNIO), Madrid, Spain; 2Department of Pathology & Laboratory Medicine, University of Wisconsin, USA;
3
Oncology Area, Molecular Oncology Laboratory, Centre for Applied Medical Research (CIMA), Pamplona,
Spain
1
The NF-κB signaling pathway regulates the transcription of many genes involved in both cancer initiation and progression
and several lymphomas rely on NF-κB activity for proliferation and survival. The NF-κB-Inducing Kinase (NIK) is essential
for the activation of the alternative NF-κB pathway by inducing the phosphorylation of the NF-κB member p100, which
leads to its processing to p52 and its subsequent nuclear translocation. A role for NIK in the classical NF-κB pathway
has been shown as well. Given its importance in NF-κB activation, NIK is an attractive therapeutic target which is still
to be evaluated in lymphomas. Here, we study the frequency and extent of alternative and classical NF-κB activation in
different types of lymphomas and the implication of NIK in both pathways. We observed a frequent alternative NF-κB
activation in lymphoma cell lines, since high protein levels of p52 were detected in the majority of mantle cell lymphoma,
Hodgkin lymphoma (HL), chronic lymphocytic leukemia, diffuse large B cell lymphoma (DLBCL) and T-cell lymphoma
(TCL) cell lines. The expression of p52 was associated with NIK expression, Epstein-Barr virus (EBV) infection or with
truncated forms of p100. Knockdown of NIK resulted in a decreased processing of both p100 and p105, indicating an
implication of NIK in both the classical and alternative pathway. Analysis of the nuclear expression of p52 (alternative
NF-κB) and p50 (classical NF-κB) by immunohistochemistry in lymphoma samples revealed a frequent activation of
both pathways in TCL, HL and DLBCL. We further evaluated a panel of 301 DLBCL samples and observed that 34 and
28 % of the cases expressed nuclear p52 and p50, respectively. Interestingly, a significant positive correlation between
EBV and p52 expression was established (p<0.001), where 93% of the LMP1 positive cases also expressed nuclear
p52. Additionally, over 50% of the p52-positive cases also expressed nuclear p50, suggesting that both pathways
are frequently activated in the same tumor. Taken together, our results show that the alternative and classical NF-κB
pathways are activated in a subset of human lymphomas and suggest a frequent overlap between the two pathways.
Furthermore, an implication of EBV in the activation of the alternative pathway was proposed and NIK was identified as
a regulator of both pathways.
28
Metastasis in Trp53-deficient squamous cell carcinoma
associated with increased mTOR and TGF activity and
deregulated miRNA expression
Jesús M. Paramio,
Ana Belén Martínez-Cruz*, Olga Bornachea*, Mirentxu Santos, Ramón GarcíaEscudero, Marta Dueñas, Carmen Segrelles, Corina Lorz, Agueda Buitrago, Clotilde Costa, Xabier Aguirre1, Teresa
Grande2, Antonio Maraver3, José M Ariza, Felipe Prosper1, Montse Sánchez-Céspedes4 and Manuel Serrano3
Molecular Oncology Unit, Division of Biomedicine, CIEMAT, Madrid, Spain; 1Center for Applied Medical
Research, University of Navarra, Pamplona, Spain; 2Unit of Medical Applications, CIEMAT, Madrid, Spain;
3
Tumour Suppression Group, Spanish National Cancer Research Centre, Madrid, Spain; 4Genes and Cancer
Group, Programa Epigenetica i Biologia del Cancer PEBC–IDIBELL Hospital Duran i Reynals, Barcelona, Spain
Missense mutations in Tp53 gene are associated with increased metastatic potential. However, little is known about
the complete loss of function of p53 in tumor metastasis. Importantly such truncating mutations of p53 occur in several
human tumors, such as breast cancer bearing BRCA1 mutation. Here we show that squamous cell carcinoma generated
by the specific ablation of Trp53 gene in mouse epidermis are highly metastasis prone. Metastases are associated with
activated mTOR- and TGF -signal transduction leading to early repression of Np63 expression and induction of epithelialmesenchymal transition. During this process we also observed deregulated expression of specific miRNAs in primary
tumors. In particular, the upregulation of miR-21, which correlated with repression of some of its targets, conferred,
in cooperation with TGF , increased invasive properties to non-transformed human keratinocytes. Moreover increased
miR21 was also found in human metastatic tumors harboring mutations in TP53 gene. Collectively these data identify
novel mechanisms, originated by p53 loss in epithelia, leading to metastasis in vivo, and connect mTOR, TGF repression
of Np63 and altered miRNA expression as major mechanisms of EMT and metastasis in squamous malignancies. These
findings may open new avenues for preclinical testing of specific inhibitors in human tumors bearing complete loss of
p53.
Poster
188
189
29
Frontiers in
Tumour Progression
Poster Session B
Melanoma cells produce multiple laminin isoforms and
strongly migrate on α5-laminin(s) via several integrin
receptors
30
Snail cooperates with KrasG12D in a gene dose
dependent fashion to accelerate pancreatic ductal
adenocarcinoma progression in genetically engineered
mice
Manuel Patarroyo1, Yuko Oikawa , Johan Hansson , Takako Sasaki , Patricia Rousselle , Anna
1
2
3
4
Domogatskaya5, Sergey Rodin5, and Karl Tryggvason5
Department of Dental Medicine, Karolinska Institutet, Stockholm, Sweden; 2Department of OncologyPathology, Karolinska Institutet, Stockholm, Sweden; 3Shriners Hospital for Children, Portland, Oregon,
USA; 4Institut de Biologie e Chimie de Protéines, Université Lyon I, Lyon, France; 5Department of Medical
Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
1
Melanoma cells express and interact with laminins (LMs) and other basement membrane (BM) components during
invasion and metastasis. In the present study we have investigated the production and migration-promoting activity of
laminin isoforms in cutaneous melanoma. Immunohistochemistry of five melanoma specimens and immunoprecipitation/
Western blotting of five melanoma cell lines indicated expression of laminin-111/121, laminin-411/421 and
laminin-511/521 and, to a lower extent, laminin-211. Laminin-332 was not detected. In functional assays, laminin-111,
laminin-332 and laminin-511, but not laminin-211 and laminin-411, strongly promoted haptotactic migration of the
cell lines either constitutively or following stimulation with insulin-like growth factors I and II. Placenta (fragmented)
and recombinant (intact) laminin-511 were similarly active, and isolated recombinant IVa domain of LMα5 chain also
promoted cell migration. Integrin screening of the cell lines demonstrated predominant expression of αVβ3 integrin
and considerable levels of laminin-binding integrins α3β1 and α6β1, but not of α7β1 and α6β4. Function blocking
antibodies in cell migration assays identified α6β1 integrin as the major receptor for laminin-111, and both α3β1
and α6β1 integrins for laminin-332 and laminin-511. In contrast, isolated LMα5 IVa domain-promoted melanoma cell
migration was largely mediated via αVβ3 integrin and inhibited by RGD peptides. Thus, melanoma cells produce multiple
laminin isoforms and exhibit a laminin isoform-specific migratory response. Considering the ubiquitous expression of
α5-laminins in melanoma cells and in tissues/anatomical structures targeted by melanomas as well as the strong
migration-promoting activity of these laminin isoforms, α5-laminins emerge as putative primary ECM mediators of
melanoma invasion and metastasis via α3β1 and other integrin receptors.
Mariel C. Paul1, Barbara Seidler , Angelika Schnieke , Roland M. Schmid , Guenter Schneider
1
2
Dieter Saur
1
1
and
1
Technische Universität Munich, Klinikum rechts der Isar, II. Medizinische Klinik, Munich, Germany,
Technische Universität Munich, Livestock Biotechnology, Munich, Germany
1
2
Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with 5 year survival rates below 3 percent. The transcription
factor Snail plays a crucial role in epithelial-mesenchymal transition (EMT) and is overexpressed in PDAC. We investigated
the role of Snail for pancreatic carcinogenesis and EMT in a genetically engineered mouse model of PDAC. A conditional
Snail expression model was generated by targeting the Rosa26 locus with a lox-Stop-lox (LSL) silenced Snail cassette
(LSL-R26Snail/+). Expression of Snail in the pancreas can be activated in this model using a pancreas specific Cre
driver line (Ptf1aCre/+). Mice with Snail expression from one Rosa26 allele (Ptf1aCre/+;LSL-R26Snail/+) exhibited
normal development. Increasing gene dose by expressing Snail from both Rosa26 alleles resulted in dramatic growth
retardation most likely due to impaired exocrine pancreatic function. In the context of concomitant KrasG12D expression,
R26Snail/+ mice developed aggressive locally invasive PDAC. The median survival of these animals was shortened to 6
months compared to 16 months in Ptf1aCre/+;LSL-KrasG12D/+ mice. All PDAC from Ptf1aCre/+;LSLKrasG12D/+; LSLR26Snail/+ mice showed a well differentiated morphology without signs of EMT or increased metastasis. Investigation
of precursor lesions revealed acceleration of PanIN formation and progression accompanied by a markedly increased
PanIN proliferation index. Increasing Snail expression levels by using homozygous LSL-R26Snail/Snail mice lead to
a dramatic acceleration of PDAC formation and a reduced median survival of 2 months. Therefore, Snail expression
accelerates pancreatic tumor progression in a gene dose dependent fashion; however, it is not sufficient to induce overt
EMT and metastasis of PDAC. Our findings provide new insights into Snail gene function as a tumor promoter in vivo.
Investigation of the underlying mechanisms will open potential novel therapeutic strategies for this dismal disease.
Poster
190
191
31
Frontiers in
Tumour Progression
Poster Session B
c-Myc triggers macrophage alternative activation and
controls macrophage activity and survival in tumors
32
Oscar M. Pello1, Maria De Pizzol , Massimiliano Mirolo , Laura Soucek , Angelo Amabile , Luca Zammataro ,
Analysis of oxidative DNA binding at sequence level
along the codons 5 and 7 of the tumour suppressor
gene p53 in a group of smokers and non smokers by
ligation mediated PCR
Department of Immunology and Inflammation, Istituto Clinico Humanitas, Rozzano, Italy; 2Department of
Pathology and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco,
USA
1
1
1
2
1
1
Andrea Doni1, Alberto Mantovani1 and Massimo Locati1
1
Microenvironment switches on different signallings that polarize macrophages towards an inflammatory (M1) or an
alternative (M2) status, which are phenotypically different and linked to different cell functions. Tumour-associated
macrophages (TAMs), also display an alternative-like activation phenotype. Though several stimuli sustaining macrophage
polarization have been described, the key transcription factors involved in this process have not been identified. Here
we show that the transcripicion factor c-Myc is specifically expressed on IL-4-activated M2 macrophages in a PI3Kdependent manner. Microarray analysis of macrophages where c-Myc activity was blocked by using c-Myc activation
inhibitors or c-Myc expression was inhibited by using lentiviral shc-Myc, showed a prevention in the acquisition of
alternative phenotype markers. The regulation of M2 markers is, in some cases, directly mediated by the interaction
of c-Myc with the gene target promoter, including SCARB-1, CTSC or ALOX-15; in other cases, like MRC-1 and CD209,
c-Myc indirectly controls their expression by regulating other IL-4 signalling mediators such as STAT6 and PPARγ.
We validate these results by using macrophages from a MycER KI mouse model where endogenous c-Myc has been
replaced by a c-MycER fusion, which is functional only by providing tamoxifen. Remarkabely, we confirmed that upon
IL-4 stimulation, ALOX-15 and MRC-1 expression was inhibited in c-Myc knock out macrophages. In addition, we found
that human TAMs, in vitro and in vivo, express c-Myc, which is involved in the acquisition of TAMs protumoral properties
by regulating the expression of TGF-β, VEGF, MMP-9 and HIF-1α. Moreover, we demonstrate that c-Myc expression in
TAMs is needed for macrophage survival in tumors. These results suggest c-Myc as a key regulator of macrophage
polarization and biology and is therefore a good candidate as a putative target for therapy in M2 macrophage-associated
pathologies, including tumours.
Marco Peluso1, Munnia Armelle , Petcharin Srivatanakul , Marcello Ceppi
1
2
3
and Paolo Boffetta4
ISPO-Cancer Prevention and Research Institute, Florence, Italy; National Cancer Institute, Bangkok,
Thailand; 3National Cancer Institute, Genoa, Italy; 4Mount Sinai School of Medicine, New York, USA
2
Smoke contains a plethora of short lived reactive oxygen species (ROS) resulting in persistent oxidative stress, a known
cancer risk factors. The most damaging ROS species is the hydroxyl radical, that is responsible for a number of oxidative
base modifications. If not repaired, DNA damage can be fixed in mutations and induce permanent modifications of
DNA, that can lead to cancer. To characterize the preferential binding sites due to ROS along the p53 exons 5 and 7, a
gene involved in lung cancer, we have treated in vitro A549 cells with a system of xantine/xantine oxidase at different
concentrations (0-5 mU/ml) for 30 min. and 2 hrs. We analyzed the formation of mutational hot spots binding using the
Ligation mediated PCR (LMPCR), a five step procedure used to analyze the levels of mutational hot spot binding (MHSB).
We have obtained a standard and a calibration curve (p<0.001). In addition, when the MHSB were analysed, a statistical
significance was osbserved (p< 0.001). LMPCR is based on the convertion of modified bases to single strand breaks,
hybridization and extention with a gene specific oligomer by asymmetric PCR, ligation of a double strand linker to bluntended DNA fragments, linear and exponential PCR amplification of DNA fragments, size fractionation and detection of
the oxidative MHSB on a polyacrylamide gel with IR detection. After enhancing the sensitivity of LMPCR, we measured
MHSB along the exons 5 and 7 of the p53 in smokers and non-smokers. Our aim was to provide MHSB analysis in points
associated to lung cancer development. We detected specific and reproducible MHSB patterns at base levels in treated
cells and human samples. MHSB was express in pixelx106 for each base, transfering the image obtained from the LiCor
to Image-Quant. A correspondence of MHSB with the distribution in the p53 mutational spectra of smokers was found.
Furthermore, the levels of MHSB were significantly related to smoking habits (p<0.005).
Poster
192
193
33
Frontiers in
Tumour Progression
Poster Session B
STAT3 synergizes with the Neu oncogene in mammary
tumorigenesis
Sara Pensa1,4, Isaia Barbieri , Tania Pannellini , Christine J. Watson , Paolo Provero , Piero Musiani
1
2
3
1
2
Valeria Poli1
34
Analysis of genetic epidemiology of alpha1-antitrypsindeficiency in patients suffering colorectal cancer
(preliminary results)
and
Molecular Biotechnology Center, University of Turin, Italy; 2Ce.S.I., Anatomia Patologica, University
G. D’annunzio, Chieti, Italy; 3Department of Pathology, University of Cambridge, UK; current address:
Department of Pathology, University of Cambridge, UK
1
The transcriptional activator STAT3 is constitutively activated in nearly 70% of both solid and haematological tumours
that often become addicted to its activity, and is therefore referred to as an oncogene. Although STAT3 is known to
contribute to several aspects of tumorigenesis, including survival, proliferation, angiogenesis, tissue invasion and
immune evasion, the molecular mechanisms involved are not fully understood. To investigate the role of STAT3 in
mammary tumorigenesis, we intercrossed MMTV-Her2Neu transgenic mice (NeuT), which develop multifocal mammary
adenocarcinomas at high multiplicity, with knock/in mice expressing physiological levels of a constitutively active form
of STAT3 (STAT3C). We observed that NeuT mice carrying a STAT3C allele develop faster growing, more invasive, less
differentiated tumours with dramatically decreased levels of apoptotic cells. Interestingly, cells derived from STAT3Cexpressing tumors (NeuT/STAT3C cells) show strongly increased migratory and invasive activities. NeuT/STAT3C cells
form more dynamic cell-cell contacts, displace actin from the membrane while forming abundant actin stress fibres and
are more tumorigenic in vivo. By comparing the gene expression profiles of NeuT/STAT3C and NeuT/STAT3WT cells, we
identified a few genes known to be involved in the cross-talk between cells and extracellular environment which are
upregulated in both STAT3C-expressing cells and tumors. We focused our attention on Cten, a peculiar member of the
Tensins family known to be required for EGF-induced cell migration. Cten silencing in NeuT;STAT3C cells significantly
inhibits their migration potential and partially reverts their cell-cell junctions defects. Thus, constitutively active STAT3
can synergize with the Neu oncogene to promote cell survival and trigger cytoskeleton and cell junctions reorganization,
leading to enhanced motility and invasion/metastases, and Cten may represent a key target for these activities.
Sergio Pérez-Holanda Fernández*,
1
Ignacio Blanco Blanco and 3Manuel Menéndez
2
Department of Surgery, Hospital Valle Nalón, Langreo, Asturias, Spain; 2Coordinator of the Alpha1Antitrypsin Deficiency Spanish Registry, the Alpha One International Registry (AIR); 3Alpha-1 Antitripsin
Reference Laboratory, Instituto Nacional de Silicosis, HUCA, Oviedo, Asturias, Spain
1
Recent studies have shown Alpha1-antitrypsin may inhibit the carcinogenic process in colorectal cancer (CRC).
Therefore, AT deficiency (ATD) could predispose to the development of the aforementioned colonic carcinogenic process.
OBJECTIVES: (1) To obtain the AT gene frequency in subjects suffering from colorectal carcinoma hospitalized at Health
Care Area VIII in Asturias, in order to calculate ATD prevalence rate. (2) To compare the ATD prevalences of that cohort
of patients which those found in the general population in our province (Blanco I et al. Med Clin 1999, 113:366-70).
Methods: Informed consent in writing shall be obtained from each patient suffering CRC. Blood samples will be taken
from each subject. Phenotypic characterisation shall be carried out by means of isoelectric focusing (IEF method), and
the AT serum concentrations will be measured following an enzymatic method, at the AT Reference Laboratory of the
Instituto Nacional de Silicosis (Oviedo, Asturias). The prevalence of every phenotype will be calculated by applying HardyWeinberg statistics (Sofaer JA. In: Emery AEH, Rimoin DL, editores. Principles and practice of medical genetics (2.a ed.).
Cap. 9. Edimburgo: Churchill Livingstone, 1990), and presented as mean values expressed per thousand inhabitants,
with a 95%CI.
Poster
194
195
35
Frontiers in
Tumour Progression
Poster Session B
Role of p120 catenin during skin wound healing and
cancer
Mirna Perez-Moreno1, Carolina Epifano
1
36
Role of Skp2 in senescence and neuroendocrine
transdifferentiation of prostate cancer cells induced by
androgen depletion
and Diego Megias2
Epithelial Cell Biology Group, BBVA Foundation-Cancer Cell Biology Programme and 2Confocal Microscopy
Core Unit, Spanish National Cancer Research Center (CNIO), Madrid, Spain
1
The skin is a self-renewing tissue composed of epidermis, dermis, hair follicle and subcutaneous fat. Normal epidermis
maintains homeostasis through the equilibrium between cellular proliferation and differentiation. Epidermal progenitor
cells contribute to the replenishment of the epidermis during the lifetime of the organism, and they are crucial during
tissue repair. Alterations in this process may lead to skin inflammatory diseases including cancer. Wound healing is a
physiological process that involves epidermal cell proliferation, cell migration, and the inflammatory response in order
to repair the tissue. It consists of three phases: inflammatory phase, proliferative phase, and remodeling phase. Upon
injury, an acute response from epidermal progenitor cells is required to repair the tissue and reestablish normal skin
homeostasis. Among the crucial events that are required to repair the epidermis, cellular adhesion in epithelial cells
plays a major role. In this way, cells can migrate to heal the wound, interact later with each other, acquire their proper
cell polarity, and differentiate to form a functional epidermal barrier. We are interested in the role of the adherens junction
protein p120 catenin (p120), which binds to and stabilize cadherins and regulate Rho GTPase activity. Using mouse
genetics, it has been shown that the absence of p120 in epidermal progenitors of skin provokes a chronic inflammatory
skin disease due an increase in RhoA activity and an activation of the NFkB pathway (Perez-Moreno et al., Cell 124, 63144 (2006); Perez-Moreno et al., PNAS 105(40):15399-404 (2008). We are now studying the role of p120 in epidermal
progenitor cells during wound healing, in particular its role in cell migration and the crosstalk with inflammatory cells
that allows the resolution of inflammation. Using primary keratinocytes in vitro, we have already observed that absence
of p120 leads to alterations in cell proliferation, changes in cell attachment to extracellular matrices due to changes in
focal adhesion stability, and overall alterations in polarized cell migration. These studies will help elucidate the role of
adherens junctions molecules, in particular p120 catenin, in the wound healing response of epidermal progenitors to
elicit proper tissue regeneration.
Zuzana Pernicova1,2, Eva Slabakova
, Eva Slavickova1,2, Alois Kozubik1,2 and Karel Soucek1,2
1,2
Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno,
Czech Republic; 2Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech
Republic
1
Prostate cancer is one of the most frequently diagnosed cancers in men. Patients usually benefit form the conventional
anti-androgen therapy, but the disease recurs in more severe and aggressive manner and cancer cells become
independent on androgens. Neuroendocrine transdifferentiation (NED) of cancer cells probably contributes to acquisition
of this androgen independence. Cancer cells express neuroendocrine phenotype, which include expression of markers of
NED, arrest in cell cycle progression and changes in the cellular morphology. We showed that androgen depletion leads
not only to NED and development of neuroendocrine-like phenotype, but also to development of senescence in prostate
cancer epithelial cells. Induction of senescence is generally considered as a potent barrier to tumorigenesis. However, the
ability of metabolically active senescent cells to secrete broad spectrum of biological factors and boost tumorigenicity of
malignant cells represents a dark side of senescence. Using detection of selected factor of this senescence secretome
(IGFBP3), we assessed that NED can be associated also with induction of this senescence secretome. Recently, the role
of Skp2 was described. As Skp2 expression is under the control of androgen receptor, we employed RNA interference
approach to elucidate its role in induction of NED and senescence. Our results demonstrate that downregulation of
Skp2 in LNCaP cells increases number of senescent cells and this was confirmed in another prostate cancer cell lines
LAPC4. In summary our results show that androgen depletion can induce both NED and senescence in prostate epithelial
cancer cells and suggest the role of Skp2 in modulation of these processes. This work was supported by grant No.
310/07/0961 of the Czech Science Foundation, grants Nos. NS9600-4 and 9956-4/2008 of the Ministry of Health of the
Czech Republic, and grants Nos. AV0Z50040507 and AV0Z50040702 of the Grant Agency of Academy of Sciences of
the Czech Republic.
Poster
196
197
Frontiers in
Tumour Progression
Poster Session B
37
38
LYL1 dysfunction in adult mice affects tumor
angiogenesis and lymphangiogenesis
Glucose starvation induces melanogenesis in B16F10
mouse melanoma cells through oxidative stress
Valérie Pinet1,2,3,#, Nelly Pirot
Bruno Piva and Bruno L. Diaz
1,2,3
, Virginie Deleuze1,2,3, Rawan El-Hajj1,2,3, Christiane Dohet1,2,3, Fred
Sablitzky4, Philippe Couttet5 and Danièle Mathieu1,2,3,#
Institut de Génétique Moléculaire de Montpellier UMR 5535 CNRS, France; 2Université Montpellier 2, France;
3
Université Montpellier 1, France; 4Institute of Genetics, The University of Nottingham, UK. 5Novartis Institute
for Biomedical Research (NIBR), Basel, Switzerland. #DM and VP share senior authorship.
1
The two related basic helix loop helix genes, LYL1 and TAL-1 are active in both hematopoietic and endothelial lineages.
While Tal-1 is essential for both hematopoietic and vascular development, the role of Lyl1 appears to be distinct as
deficient mice are viable and display only modest hematopoietic defects, such as reduced long term hematopoietic
stem cells (LTHSC) and peripheral B cells. Up to now, nothing was known concerning the endothelial function of LYL1.
We took advantage of the mild phenotype of Lyl1-deficient mice to investigate its role in adult endothelial lineage. In
this study, we reveal a role for Lyl1 as a regulator of the formation of both new blood and lymphatic vessels in adults
(angio- and lymphangiogenesis). Compared to wild-type littermates, tumors implanted into Lyl1-deficient mice grew
faster and display highly angiogenic vessels, as evidenced by enlarged lumen, reduced pericyte coverage and increased
permeability. Noteworthy, Lyl1-deficient tumor vessels exhibited up-regulation of Tal-1 and its target gene VE-Cadherin
as well as Angiopoietin-2, three major actors in angiogenesis. Hematopoietic reconstitutions demonstrated that this
sustained tumor angiogenesis did have an endothelial origin. Importantly this angiogenic phenotype was not restricted to
tumors since microvessels forming in Matrigel or originating from aortic explants of Lyl1-deficient mice were also more
numerous and larger than their wild-type counterparts. LYL1 depletion in human endothelial cells revealed that LYL1
controls the expression of molecules involved in the stabilization of vascular structures. Our data suggest that LYL1 may
serve as a stabilization factor contributing to the maturation of newly formed blood vessels in adults. Surprisingly, tumor
lymphangiogenesis appeared to be differently affected by the absence of Lyl1 function. Indeed, a strong reduction in
lymphatic tumor infiltration, as revealed by the lymphatic marker LYVE-1, was observed in Lyl1-deficient mice compared
to their wild-type littermates. Our current works aim to determine the origin of this lymphatic phenotype and to evaluate
the consequences of Lyl1 deficiency on metastatic spreading.
Federal University of Rio de Janeiro, Brasil
Tumor cells have defects in their regulatory circuitry that govern cell proliferation and homeostasis. In a tumor mass,
vasculature is disorganized leading to poor distribution of oxygen and nutrients that induces high stress levels in cells.
Such stress is known to make cells more resistant to chemotherapy. Melanoma resistance to chemotherapy may be
related to its origin in transformed melanocytes and its inherent ability to synthesize melanin in response to stress such
as UV radiation that may provide protective capacity for the melanoma cell. The objective of this study was to analyze the
regulation of melanogenesis induced by glucose deprivation using as model the B16F10 cells. B16F10 cells cultured in
DMEM without glucose for 24 hours, showed induction of tyrosinase activity followed by melanogenesis. Cells cultured
without glucose for 24 hours also showed reduction in proliferation rate and reorganization of actin filaments, but with no
effect on cell viability. Supplementation of culture medium with pyruvate prevented melanogenesis in glucose-deprived
cells. Nutrient stress-induced melanogenesis was also inhibited when anti-oxidant capacity of cells was increased by
N-acetylcysteine or reduced glutathione treatment. Cells treated with SB202190, a p38 inhibitor, exhibited an increase
of melanin synthesis when cells were cultured under glucose deprivation. In conclusion, glucose deprivation is a potent
stress stimulus for B16F10 leading to melanogenesis, proliferation arrest and morphological changes due to oxidative
stress. Concomitant p38 activation seems to counteract the effects of nutrient stress in B16F10 cells.
Poster
198
199
39
Frontiers in
Tumour Progression
Poster Session B
Chromosomal, epigenetic and microRNA-mediated
inactivation of lrp1b and modulation of the tumor
microenvironment
40
Strategic alliance of E2F1 and EGFR in cancer
metastasis
Brigitte Pützer1, Vijay Alla , David Engelmann , Annett Niemetz , Jens Pahnke , Anke Schmidt , Manfred
1
Hugo Prazeres1,2,3, Joana Torres , Fernando Rodrigues , Marta Teixeira , Maria Pastoriza , DanielaGomes ,
1
4
1
5
6
J. Cameselle-Teijeiro7, Anxo Vidal5, Teresa C. Martins3, M. Sobrinho-Simões1,2 and Paula Soares1,2
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Portugal; 2Medical
Faculty, University of Porto, Portugal; 3Molecular Pathology, 4Endocrinology and 6Anatomic Pathology Service
of the Portuguese Institute of Oncology of Coimbra, EPE, Coimbra, Portugal; 5Department of Physiology,
School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain; 7Department
of Anatomic Pathology, Clinical University Hospital (CHUS), SERGAS, University of Santiago de Compostela,
Santiago de Compostela, Spain
1
The Low density lipoprotein receptor Related Protein (LRP1B), encoding an endocytic LDLfamily receptor, is amongst
the 10 most significantly deleted genes across 3,312 human cancer specimens. We undertook the present study to
elucidate the mechanisms of LRP1B inactivation in cancer cells and to investigate the key roles of this lipoprotein
receptor in the carcinogenesis. Here we show that LRP1B inactivation (by chromosomal, epigenetic and microRNAmediated mechanisms) modulates the tumor microenvironment to confer cancer cells an increased invasive capacity.
LRP1B displays frequent DNA copy number loss and CpG-island methylation resulting in mRNA under-expression. By
using CpG-island reporters methylated in vitro, we found that DNA methylation disrupts a functional binding site for the
histone-acetyltransferase p300 located at intron 1. We identified and validated a microRNA targeting LRP1B (miR-548a5p) which is overexpressed in cancer cell lines as a result of 8q22 DNA gains. Restoration of LRP1B impaired in vitro and
in vivo tumor growth, inhibited cell invasion. LRP1B re-expression led to overall changes in the extracellular medium,
among which are growth factors, cytokines and metalloproteinases. These findings link DNA copynumber changes,
an epigenetic transcription factor and a specific microRNA with the modulation of the extracellular microenvironment
through LRP1B (de) regulation.
1
1
2
1
Kunz , Stephan Emmrich , Marc Steder and Dirk Koczan
3
1
1
4
Department of Vectorology and Experimental Gene Therapy, Biomedical Research Center (BMFZ);
Department of Neurology; 3Department of Dermatology and Venerology, and Institute for Immunology,
University of Rostock Medical School, Germany
1
2
The E2F1 transcription factor is considered to be the ‘final frontier’ of the G1 to S phase boundary that promotes
cell cycle progression. Abnormalities in E2F1 gene expression have been described in various tumor types including
malignant melanoma. Specifically dysregulation of E2F1 is common in advanced tumors that are associated with poor
patient survival, suggesting that its oncogenic properties extend beyond the ability to stimulate aberrant growth of
neoplastic cells. Tumor metastasis continues to be the most significant problem in the field of cancer, but the molecular
alterations leading to tumor progression are unclear. To investigate the association of enhanced E2F1 activity with
aggressive phenotype, we performed a gene-specific silencing approach in a metastatic melanoma model. Knockdown
of endogenous E2F1 via E2F1 small hairpin RNA (shRNA) expression increased Ecadherin expression of metastatic
melanoma cells and reduced their invasive potential but not their proliferative activity. Although growth rates of xenograft
tumors from metastatic melanoma cells expressing E2F1 shRNA were unaltered, mice implanted with E2F1 knockdown
cells showed a significantly smaller area of metastases per lung than control mice. We identified epidermal growth
factor receptor (EGFR) as a direct target of E2F1 and demonstrated that inhibition of receptor signaling abrogates E2F1induced invasiveness. Our data emphasize for the first time the importance of the E2F1 – EGFR interaction as a driving
force in melanoma progression that may serve as a paradigm for E2F1-induced metastasis in other human cancers (J
Nat Cancer Inst. 2010;102(2:127-133).
Poster
200
201
41
Frontiers in
Tumour Progression
Poster Session B
42
A novel and selective PDK1 inhibitor reduces breast
cancer cell invasion and tumour growth
Synergy between HIF1a and LOX is critical for tumor
progression
Claudio Raimondi1, Tania Maffucci
Caroline Reynaud1, Floriane Pez , Frédéric Dayan , Jérôme Durivault , Bastien Kaniewski , Géraldine
1
and Marco Falasca1
Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Institute
of Cell and Molecular Science, Centre for Diabetes, Inositide Signalling Group, London, UK
1
Impairment of metastasis development is a critical target for cancer therapy. We recently reported that phospholipase
Cγ1 (PLCγ1) is involved in regulation of motility and invasion of cancer cells and is required for metastasis development
and progression. Experimental metastasis assays in nude mice revealed that inducible knockdown of PLCγ1 strongly
inhibits development of MDA-MB-231- derived lung metastasis and reverts metastasis formation. In an effort to develop
anti-metastatic drugs, different inositol phosphates compounds were tested to identify potential PLCγ1 inhibitors. We
found that a synthetic derivative of inositol pentakisphosphate, Ins(1,3,4,5,6)P5, inhibits cell migration and 3D invasion
in MDAMB- 231 and MDA-MB-435 human breast cancer cell lines and in TSA murine mammary adenocarcinoma cells
and reduces calcium release upon EGF stimulation indicating a potential inhibition on PLCγ1 activity. Kinase profile
assay, performed in vitro to test the potential inhibitory effect of the Ins(1,3,4,5,6)P5 synthetic derivative on different
kinases showed a specific inhibition of the 3- phosphoinositide-dependent-protein kinase 1 (PDK1) with an IC50 of 26
nM. Knockdown of PDK1 using the small interfering RNA technology in breast cancer cell line MDA-MB-231 showed an
impairment in cell migration and invasion and inhibition of EGF-induced calcium mobilisation. In addition, it has been
recently shown that PDK1 is a critical determinant for resistance to tamoxifen anti-cancer drug. Our experiments show
that combined treatment of the Ins(1,3,4,5,6)P5 synthetic derivative with tamoxifen, paclitaxel, and curcumin in MCF7
and MDAMB- 468 cells results in additive or more than additive effects, and therefore suggest that this novel PDK1
inhibitor can be potentially used in combination with other drugs to increase their anti-cancer activity.
1
2
2
1
Aimond , Philippe Clezardin , Pascal Sommer and Jacques Pouysségur
1
3
1
2
Institute of Biology and Chemistry of Proteins, Lyon, France; 2nstitute of Signalling, Developmental Biology
and Cancer Research, Nice, France; 3INSERM, Lyon, France
1
The microenvironment of solid tumors is exposed to hypoxic conditions which lead to the activation of Hypoxia-Inducible
Factor 1 (HIF1), a key transcription factor involved in cellular adaptation to changes in oxygen level. HIF1 plays a critical
role in various cellular and physiological events, inducing the expression of several transcriptional targets such as Lysyl
Oxidase (LOX). LOX is an amine oxidase that catalyzes crosslinking of fibrillar collagens and elastin in the extracellular
matrix. Furthermore, LOX expression in tumor cells lines correlates with tumor progression and metastatic potential.
Using three different human colorectal carcinoma cell lines, LOX was stably overexpressed or knocked down by lentiviral
transduction. In these models, we pointed out that besides HIF1-dependant regulation of LOX, LOX can also act on
the HIF1 pathway under hypoxic conditions. Indeed, LOX enzymatic activity upregulates HIF1a protein synthesis, and
this action is mediated by the PI3K/AKT pathway. Thus, these results emphasize the existence of a mutual regulation
between two main actors of tumoral progression: HIF-1a and LOX. To further determine the implication of both proteins
in tumor progression, we generated human colorectal carcinoma cell lines modulating LOX and/or HIF1a expression.
Our results show that LOX enzymatic activity increase cell proliferation and clonogenic potential in vitro and this role
is partly dependant of HIF1a. Using Balb/c nude mice, we have also shown that LOX and HIF-1a act in synergy to favor
tumor formation and progression. Taken together, our results demonstrate a direct crosstalk between HIF1a and LOX in
the tumor microenvironment and underline a critical role of this mutal regulation in tumor progression in vitro and in vivo.
Poster
202
203
Frontiers in
Tumour Progression
Poster Session B
PI3K facilitates the autocrine induction of TGFβ2 by
TGFβ in glioblastoma
43
44
Tumour progression involves a functional switch
of Endo180/CD280 from cell-cell adhesion to cell
migration
Laura Rodón and Joan Seoane
Vall d’Hebron Institute of Oncology, Barcelona, Spain
Human glioblastoma (GBM) is one of the most aggressive and recalcitrant human tumors and is virtually not curable.
GBM presents a high TGFβ-Smad activity that confers poor prognosis. In many cases, the increased TGFβ-Smad activity
is due to high expression of TGFβ2 ligand. In this work, we observed that TGFβ induces its own expression generating
a malignant autocrine loop. This phenomenon explained the increased secretion of TGFβ2 and hence the hiperactivity of
the TGFβ pathway present in high grade gliomas. We identified a new crosstalk between the PI3K and TGFβ signaling
pathway at the level of TGFβ2 secretion. We demonstrated that hiperactivation of PI3K signaling increased TGFβ
mediated expression of TGFβ2. These results were confirmed in human GBM specimens. This work provides new
molecular targets to restore normal TGFβ function as well as new therapeutic strategies against this disease.
Mercedes Rodriguez-Teja, Ai Minamidate, Jonathan Waxman and Justin Sturge
Prostate Cancer Metastasis Team, Prostate Cancer Group, Department of Oncology, Faculty of Medicine,
Imperial College London, UK
Epithelial integrity in glandular tissue is maintained by the intact epithelial cells adhesion as well as the homeostasis of
the mechanical forces between the cells and the surrounding matrix. The prostate is comprised of glandular tissue that
contains multiple ductal structures called acini, formed by polarized epithelial cells that represent the origin of luminal
breast and prostate cancer. We have shown that the collagen binding receptor Endo180 (CDC280; MCR2; uPARAP)
localizes to the apical and lateral membranes of the normal glandular epithelium in prostate gland sections. Using an in
vitro 3D culture model of prostate gland acini we have shown that depletion of Endo180 from prostate epithelial cells
affects the glandular acini architecture by promoting cell scattering into the surrounding matrix following the disruption
of cell-cell adhesion. This disruption of acini structure can be rescued by ectopic expression of Endo180. In particular,
we have demonstrated that blocking the function of the CTLD4 domain of Endo180 by an antibody that binds to this
ectodomain of the receptor disrupts acini formation, thereby mimicking the genetic depletion of Endo180 from the
prostate epithelial cells. In contrast, treatment of acini with an alternative mouse monoclonal antibody that recognises
the CTLD2 domain of Endo180 induces an increase of 40% in acini size. No significant difference was observed in the
migratory abilities of tumour cells treated with either antibody, however depletion of Endo180 from the tumours cells
reduced their migratory ability. These results have led us to propose a model in which Endo180 works as cell-cell
adhesion molecule in prostate epithelium via a mechanism that involves the CTLD4 ectodomain of the receptor; and that
during malignant transformation a functional switch takes place from this role in cell-cell adhesion to a pro-metastatic
role where the receptor works to enhance cell migration.
Poster
204
205
45
Frontiers in
Tumour Progression
Poster Session B
Oncogenic ras induced senescence in pre-malignant
lung tumors requires Thrombospondin-1
Kwan-Hyuck Baek, Alexander Zaslavsky, Dongha Bhang and
46
A SILAC quantitative proteomics analysis for the
identification of bladder cancer metastasis-associated
candidates
Sandra Ryeom
Department of Cancer Biology, University of Pennsylvania School of Medicine, Abramson Family Cancer
Research Institute, Philadelphia, USA
Marta Sánchez-Carbayo1, Laura Grau , Jose L. Luque-Garcia , Pilar González-Peramato , Dan
1
2
3
Theodorescu4, Marta Gil5, Joan Palou6 and Joaquin Bellmunt7
Tumor Markers Group, Spanish National Cancer Research Center, Madrid, Spain; 2Department of Analytical
Chemistry, University Complutense of Madrid, Spain; 3Pathology Department, Hospital Universitario La Paz,
Madrid, Spain; 4Mellon Urologic Cancer Institute, University of Virginia, US; 5Oncology Department, Institut
Catalá d’Oncologia, Barcelona, Spain; 6Urology Department, Fundació Puigvert, Barcelona, Spain; 7Oncology
Department, Hospital del Mar, Barcelona, Spain
1
Progression of pre-malignant lesions is restrained, in part, by oncogenic activation of cellular senescence. Many studies
have demonstrated that precocious signalling of the Ras network in vivo induces senescence, confirming its physiological
relevance as a tumor suppressive mechanism. While Ras-induced senescence is mediated by the key p16INK4a/Rb and/
or p19ARF/p53 tumor suppressor pathways, as well as a number of cell extrinsic signals, there has not yet been any
comprehensive delineation of senescence effector pathways downstream of p53. Oncogenic Ras triggers senescence
in many cell types include lung epithelium. The lung exhibits high levels of the angiogenesis inhibitor thrombospondin-1
(TSP-1). Unexpectedly, we found that TSP-1 plays a critical but previously unidentified role in suppressing K-ras-induced
lung tumorigenesis independent of its anti-angiogenic function. Oncogenic ras-induced senescence is abrogated by
Tsp-1 deficiency, leading to a marked decrease in overall survival in a mouse lung tumor model driven by sporadic
activation of oncogenic K-ras. Similarly, Tsp-1-/- lung epithelial cells and fibroblasts are refractory to oncogenic Rasinduced senescence, which is restored upon TSP-1 re-expression or enforced p53 expression in vitro. Moreover, our
data indicate that K-ras induces TSP-1 upregulation in a p53-dependent manner leading to a positive feedback loop
promoting oncogenic Ras signaling via MAP kinase effector pathways. Collectively, these data indicate that TSP-1 is a
critical and cell-autonomous mediator of oncogenic Ras-induced senescence in the lung functioning downstream of
p53 activation.
Bladder cancer is most clinically aggressive when metastases develop, associated with the shortest patient survival.
To identify molecular pathways associated with the metastatic process in bladder cancer, we carried out a proteomic
analysis of an invasive T24 and a metastatic derived T24T bladder cancer cell line using a SILAC (Stable Isotope
Labelling by Amino acids in Cell culture) approach. Protein extracts were fractionated, separated by SDS-PAGE and
analyzed by nanoflow LC-ESI-LTQ mass spectrometry (MS). Comparative analysis of the invasive and metastatic bladder
cancer cells identified 831 proteins with high confidence (>99%). Protein expression data from SILAC were compared
with mRNA expression data obtained from oligonucleotide microarrays. Relatively weak correlations were observed,
consistent with the poor associations between RNA and protein expression previously reported. The involvement of
the proteins identified in biological networks served to propose how selected candidates may participate in molecular
pathways associated with bladder cancer metastasis. Verification analyses included Western Blot using independent
protein extracts. Immunohistochemistry analyses of proteins differentially expressed, such as Cul3, a protein involved
in the ubiquitination and subsequent proteasomal degradation of target proteins, were performed on bladder tumours
spotted onto tissue microarrays (n=284). Cul3 was associated with tumor staging, lymph node metastasis and diseasespecific survival. Further functional analyses revealed that Cul3 silencing diminished the proliferative, migration and
invasive rates of T24T cells, and restored the expression of other metastasis related cytoskeleton proteins identified
by SILAC such as ezrin, moesin, filamin A or caveolin. Hence, this approach not only identified proteins and molecular
mechanisms involved in bladder metastasis but also revealed a role for Cul3 in bladder cancer progression, metastasis
and clinical outcome.
Poster
206
207
47
Frontiers in
Tumour Progression
Poster Session B
Identification of the mechanism of sphingosine kinase
1–mediated prosaposin regulation in prostate cancer
cells
48
C-JUN promotes BCR-ABL induced lymphoid leukemia
by preventing down-regulation of CDK6
Ruth Scheicher, Karoline Kollmann and Veronika Sexl
Lysann Sauer1, Joao Nunes , Carlos R. Morales , Stuart Pitson , Jonathan Waxman
1
Dmitry Pshezhetskiy1
2
3
1
and
Tumour Microenvironment and Chemotherapy Group, Hammersmith Campus, Faculty of Medicine, Imperial
College London, UK; 2Department of Anatomy and Cell Biology, McGill University, Montreal, Canada; 3Centre
for Cancer Biology, SA Pathology, Adelaide, Australia
1
Prosaposin is a neurotrophic secreted protein involved in cancer progression, metastasis and chemoresistance. Recent
reports suggest a prosaposin-mediated activation of sphingosine kinase 1 (SK1), an oncogenic lipid kinase associated
with the promotion of cancer cell growth, motility and metastasis. Here we investigate the mechanism of the crosstalk
between prosaposin and SK1 signalling pathways. Prosaposin knockout mice exhibited an involution of the prostate
gland which correlated with a significant reduction of prostate SK1 activity. In prostate cancer cell lines SK1 activity
correlated with the amount of secreted, but not intracellular prosaposin, indicating a role for the prosaposin/GPCR
signalling. The specific knockdown of prosaposin in prostate cancer cells induced a significant decrease in SK1 activity
and expression on mRNA and protein level. Additionally, prosaptide TX14A, derived from the trophic sequence of
saposin C, enhanced SK1 activity and up-regulated SK1 mRNA expression. SK1 activation was essential for prosaposin
secretion and expression during serum deprivation. The effect was further amplified upon overexpression of SK1 and
was drastically abrogated by specific knockdown or SK1 inhibitor. Exogenous sphingosine-1-phosphate (lipid product
of SK1) triggered the secretion of prosaposin in a dose-dependent manner. Overall, prosaposin plays a crucial role in
SK1 regulation in advanced prostate cancer cells. Our data indicate a reciprocal regulation of prosaposin expression and
secretion by Sk1 activation.
Department of Pharmacology, Medical University of Vienna, Austria
The down-regulation or loss of the AP-1 transcription factor JUNB occurs in myeloid and B lymphoid leukemia and is
associated with a highly malignant phenotype. In contrast, the AP-1 member c-JUN is considered to be a protooncogene
and is frequently up-regulated in human hematopoietic malignancies. The transcription factor c-JUN and its upstream
kinase JNK1 have been shown to promote BCR-ABL induced B lymphoid leukemogenesis. The mechanism how JNK1
accelerates leukemogenesis has been elucidated; JNK1 is required to allow the continuous expression of the antiapoptotic protein BCL2. Using gene targeted mice we show that c-JUN promotes tumor formation independently of
JNK1. The lack of c-JUN did not impair the viability or apoptotic behavior of the tumor cells, but significantly reduce
proliferation. The decreased proliferation of leukemic c-JunΔ/Δ cells is associated with the loss of cyclin dependent
kinase 6 (CDK6). Interestingly, the lack of c-JUN did not interfere with CDK6 expression in non transformed cells. Only
upon transformation by the BCR-ABL oncogene c-JUN expression is critical for the maintenance of CDK6 expression.
Moreover, CDK6 exerts a non-redundant role for tumor cell proliferation. This was verified by re-expression of CDK6 in
BCR-ABL transformed c-JunΔ/Δ cells which reconstitutes tumor cell growth. In summary our study describes that c-JUN
regulates CDK6 expression in BCR-ABL transformed leukemic cells and thus drives tumor growth. Moreover, our data
reveal a critical and non-redundant role for CDK6 in BCR-ABL driven leukemia.
Poster
208
209
49
Frontiers in
Tumour Progression
Poster Session B
Therapeutic effect of γ-secretase inhibition in a mouse
model of lung adenocarcinoma
50
Jun proteins inhibit autophagy and induce cell death
Eitan Shaulian and Orli Yogev
Antonio Maraver and
Manuel Serrano
Tumour Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
The γ-secretase protease complex regulates a variety of cell processes 1, notably including the Notch pathway, which is
critical for the function of stem cells and can be oncogenic when constitutively active 2-5. A large proportion of human and
murine lung adenocarcinomas have the Notch pathway hyperactive, and we hypothesized that γ-secretases would play a
role in tumour initiation and cancer maintenance in the lung. Interestingly, genetic disruption of the γ-secretase complex
by combined deletion of its key components presenilin 1 and 2, results in complete protection from KrasG12Vdriven lung
adenocarcinomas. Even more, genetic elimination of RBP-Jκ also hampers the development of KrasG12V-driven lung
adenocarcinomas, demonstrating that the canonical Notch pathway is involved in the protection mentioned above. We
also show that the activity of the Notch pathway increases during tumour progression suggesting an oncogenic addiction
for the Notch pathway in this type of tumours. To test this hypothesis, mice with endogenous lung adenocarcinomas
were treated with a small molecule γ-secretase inhibitor 6 and this resulted in complete arrest of cancer growth, often
accompanied by cancer regression, as measured by positron electron tomography (PET). Immunohistochemical analyses
of treated cancers indicated loss of Hes1 and active Erk, accompanied by decreased proliferation and higher apoptosis.
Together, these results support a critical role for γ-secretase activity in lung cancer initiation and maintenance, and
provide proof for the therapeutic potential of γ -secretase inhibitors against lung adenocarcinoma.
Department of Biochemistry and Molecular Biology, IMRIC, Hebrew University-Hadassah Medical School,
Jerusalem, Israel
The growing number of biological functions affected by autophagy ascribes a special significance to identification
of factors regulating it. The AP-1 transcription factors are involved in most aspects of cellular proliferation, death or
survival, yet no information regarding their involvement in autophagy was available until recently. We have demonstrated
that the AP-1 proteins JunB and c-Jun but not JunD, c-Fos or Fra-1 inhibit autophagy. JunB inhibits autophagy induced
by starvation, overexpression of a short form of ARF, smARF, a potent inducer of autophagy, or even after rapamycin
treatment. In agreement, acute repression of JunB expression, by JunB knockdown potently induces autophagy. As
expected from autophagy-inhibiting proteins, Jun B and c-Jun expression is reduced by starvation. Decrease in JunB
mRNA expression as well as post transcriptional events down-regulate JunB protein expression after starvation. The
transcriptional activities of c-Jun and JunB, are required for autophagy inhibition and JunB incapable of heterodimerizing
is a less effective inhibitor of autophagy. Genomic screen reveled a potential target regulating the inhibition of autophagy.
In addition and most importantly, inhibition of autophagy in starved HeLa cells by JunB enhances apoptotic cell death.
The ability of AP-1 proteins to regulate autophagy thereby to modulate cell death after exposure to environmental
stresses will be discussed.
Poster
210
211
51
Frontiers in
Tumour Progression
Poster Session B
Loss of MMP9 inhibits onset of angiogenesis and
promotes tumor invasion in two independent models of
carcinogenesis
52
RelB reduces cell-cell adhesion and promotes
invasiveness of mesenchymal glioma cells
Raquel Sitcheran1, M. Narcisa Predescu , Dhivya Ramakrishnan , Hyeong-Il Kwak , Ian Parney
1
Ksenya Shchors , Hiroaki Nozawa , Jin Xu , Fanya Rostker , Lamorna Swigart- Brown , Gerard Evan
1,3
2
1
1
1
1,4
and Douglas Hanahan2,3
Department of Pathology and 2Department of Biochemistry and Biophysics, University of California San
Francisco (UCSF), USA; 3Swiss Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de
Lausanne (EPFL), Switzerland; 4Department of Biochemistry, University of Cambridge, UK
1
Due to the role of metalloproteinases (MMP) in extracellular matrix (ECM) re-arrangement and the promotion of
neoangiogenesis, it was anticipated that inhibition of their activity in vivo would have an anti-tumorigenic effect.
However, the use of MMP inhibitors in clinical trials provided no survival benefits for patients. In order to understand
the reasons behind the apparent inefficacy of MMP inhibition, we here investigate the long-term effects of inhibition
of MMP9 activity in two different mouse models of pancreatic neuroendocrine carcinogenesis in vivo, namely Rip2Tag
and Myc;Bclxl. While the contribution of MMP9 activity to mobilization of VEGF and the initiation of tumor angiogenesis
in the Rip2Tag model is well established (Bergers G et al., 2000; Nozawa H et al., 2006), we now demonstrate a similar
role of MMP9 in Myc-driven tumor angiogenesis in vivo. Interestingly, MMP9 is rapidly induced following Myc-activation
in the Myc;BclXl model, in an Il-1-beta-dependent manner. The pharmacological inhibition of MMP9 activity in vitro
interfered with Myc-mediated VEGF A release. Moreover, tumors initiated in MMP9-defecient animals exhibited retention
of ECM-bound VEGF A. However, inhibition of angiogenesis was not sufficient to interfere with tumor progression, and the
emerging tumors exhibit a profoundly invasive phenotype in both models. Further analysis has revealed that neoplastic
lesions formed in MMP9-deficient animals had higher levels of tissue hypoxia and a more mature vasculature than
the tumors which developed in the control group. Taken together our data suggests that MMP9-deficient tumors are
indifferent to the lack of VEGF-mediated angiogenesis, shifting instead to a more invasive mode, in contrast to their
wild-type counterparts. Thus, MMP9-deficient tumor models mimic an evasive resistance observed in mouse models
and patients subjected to anti-angiogenic therapies. Such MMP9-deficient models could be employed as a genetic tool
to understand the sequence of events behind the emergence of tumors resistant to anti-angiogenic agents, ultimately
leading to the development of new strategies to increase efficacy angiogenesis inhibitors.
Bayless
1
1
2
and Kayla
1
1
Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas,
USA; 2Department of Neurological Surgery, Mayo Clinic, Minnesota, USA
High-grade gliomas have one of worst prognoses among all types of human tumors due to enhanced cell survival
and migration, increased invasiveness into normal brain tissue, and robust vascularization. In spite of advances in
treatment, these tumors almost always recur and new paradigms for therapy are desperately needed. Based on gene
expression profiling, gliomas have been classified into distinct tumor subtypes (Phillips et al., 2006; Verhaak et al.,
2010). However, the biological significance of particular tumor subtypes is unknown. Using primary glioblastoma tumor
explants and U87-MG glioma cells, we have found that the NFkappaB transcription factor, RelB, is highly expressed in
gliomas of a mesenchymal subtype and that elevated RelB expression and increased copy number are both associated
with diminished patient survival. Our data reveal that in mesenchymal glioblastoma tumor cells RelB, but not RelA, drives
an atypical epithelial-tomesenchymal transition (EMT); shRNA-mediated suppression of RelB in mesenchymal glioma
tumor cells induces increased expression of both N- and EApplications cadherin, as well as loss of vimentin. Moreover,
mesenchymal gliomas in which RelB expression has been silenced exhibit a significant increase in cell-cell adhesion,
which is consistent with increased expression of desmosome and tight junction proteins. Finally, we show that loss of
RelB in mesenchymal glioma cells completely abrogates tumor invasion in 3D-collagen matrices that mimic the tumor
microenvironment, and also potently inhibits tumor formation in xenograft mouse models. Together our findings reveal
a novel role for RelB in regulating the loss of cell-cell adhesion that is associated with an atypical EMT in mesenchymal
glioma cells. Thus, our data suggest that RelB, rather than RelA/p65, may be a more efficacious therapeutic target for
these devastating tumors.
Poster
212
213
53
Frontiers in
Tumour Progression
Poster Session B
Docosahexaenoic acid enhances colon cancer cell
sensitivity to TRAIL-induced apoptosis
Belma Skender1, 2, Alena Vaculová , Alois Kozubík
1
1, 2
54
TGF-beta1 induced EMT of benign and transformed
prostate epithelial cells-analysis of expression of
transcription factors and miRNAs
and Jiřina Hofmanová1, 2
Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno,
Czech Republic; 2Department of Animal Physiology and Immunology, Faculty of Science, Masaryk University,
Brno, Czech Republic
1
Docosahexaenoic acid (DHA), n-3 polyunsaturated fatty acid, is an important component of cellular membranes, and
an effective modulator of proliferation, differentiation or apoptosis in various cancer cell types. In addition to its direct
effects on the cytokinetic processes, DHA can also indirectly modulate the cytotoxic action of some endogenous/
exogenous agents. We investigated the sensitizing effect of DHA on apoptosis triggered by TRAIL (TNF-related apoptosis
inducing ligand) in SW620 human epithelial cell line derived from colon cancer metastasis. TRAIL is an interesting
member of tumor necrosis factor (TNF) family known for its ability to selectively induce apoptosis in cancer cells, but
not in most normal cells. However, many cancer cells including colon are still resistant to cytotoxic effects of TRAIL. We
showed that pretreatment with physiologically relevant concentrations of DHA resulted in increased apoptosis of colon
cancer cells subsequently treated with TRAIL. We focused on investigation of the molecular mechanisms responsible for
the effects observed, particularly the involvement of caspases and their endogenous inhibitors, pro- and anti-apoptotic
Bcl-2 family proteins, and mitochondria. Our results support the role of DHA as a modulator of epithelial cancer cell
sensitivity to apoptosis, and suggest the potential molecules to be targeted in future therapeutic interventions. This
work was supported by grants No. 524/07/1178, 301/07/1557 and 303/09/H048 GA CR and No. AVOZ5004050 AS CR.
Eva Slabakova, Zuzana Pernicova, Eva Slavickova, Alois Kozubik and Karel Soucek
Department of Cytokinetics, Institute of Biophysics AS CR, Brno, Czech Republic; Department of
Experimental Biology, Masaryk University, Faculty of Science, Brno, Czech Republic
Most prostate cancer–related deaths are associated with advanced disease and metastasis. Epithelial-mesenchymal
transition (EMT) is viewed as an essential early step facilitating dissemination of tumour cells. Transcription factors of
the ZEB family are thought to be involved in tumour progression, thus having potential clinical interest. Members of the
miR-200 family, which regulate expression of ZEB transcription factors, have been found downregulated during EMT,
suggesting an important role in inhibition of EMT. The aim of this study is to analyze the expression and function of
selected transcription factors and miRNAs in the EMT of benign and tumorigenic prostate cells in order to identify key
molecules responsible for tumorigenicity and invasivity of prostate cancer. In our experimental system, we used parental
and tumorigenic clones derived from non-tumorigenic prostate epithelial cell line BPH-1. All of them can undergo EMT
after TGF-beta1 treatment. To investigate the molecular mechanisms driving this process, we analyzed expression of
selected transcription factors and miRNAs. Among the genes analyzed, SNAI2/Slug is rapidly and strongly upregulated in
parental BPH 1 cells. Changes in expression levels of ZEB1 and ZEB2 mRNA and miRNA of miR-200 family are observed
after extended periods of TGF-beta1 treatment and correlate with progression of EMT but may not be responsible
for rapid upregulation of EMT-driving transcription factors. Tumorigenic clones of BPH-1 - CAFTD have substantially
increased expression of vimentin and SNAI2/Slug compared to the parental cell line. In conclusion, our results suggest
that in prostate epithelial cells, endogenous SNAI2/Slug is an important regulator of EMT and tumorigenicity. This
work was supported by grant No. 310/07/0961 and 303/09/H048 of the Czech Science Foundation, IGA MZD 96004/2008, IGA MZD 9956-4/2008, and by the Academy of Sciences of the Czech Republic, grants no. AV0Z50040507 and
AV0Z50040702.
Poster
214
215
55
Frontiers in
Tumour Progression
Poster Session B
Metastatic outgrowth is associated with overexpression of collagen-I, fibronectin, and periostin
genes and organization of the encoded proteins to
networks regulating cell adhesion, migration, and
growth
Johanna Soikkeli , Piotr Podlasz
1
Loss of function of p53 and Rb induces alterations in
the homeostasis of bulge and hair germ stem cells of
hair follicle linked to skin tumors development
Sònia Solé-Sánchez ,
*1
Victoria da Silva-Diz*1, Diana Riba-Artés1, Maria Urpí1, Rosa Penin2, Joaquim
Marcoval2, Herbert Auer3, Eduard Batlle4 and Purificación Muñoz1
, Miao Yin , Pirjo Nummela , Tiina Jahkola , Susanna Virolainen ,
Leena Krogerus1, Päivi Heikkilä1, Karl von Smitten4, Olli Saksela5 and Erkki Hölttä1
1,2
1
1
3
56
1
Department of Pathology, Haartman Institute, University of Helsinki, Finland; 2Division of Animal Anatomy,
Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury,
Olsztyn, Poland; 3Department of Plastic Surgery, Helsinki University Central Hospital, Finland; 4Breast
Surgery Unit, Helsinki University Central Hospital, Finland; 5Department of Dermatology, Helsinki University
Central Hospital, Finland
1
The outgrowth of micrometastases to macrometastases and further spreading of the metastases are the main
cause of death from solid cancers. However, the molecular mechanisms involved, especially in real human cancers,
remain poorly understood. To identify factors important for the outgrowth of metastases, we first compared the gene
expression profiles of melanoma lymph node micro- and macrometastases and found a common activation of the
transforming growth factor beta (TGF-beta) signaling pathway, which was confirmed by immunohistochemical staining
of phosphorylated SMAD2. Moreover, we found a concerted up-regulation of the TGF-beta-inducible genes periostin
(POSTN), fibronectin (FN1), collagen-I (COL-I), and versican (VCAN) to be consistently associated with the metastatic
outgrowth. By immunohistochemical staining and confocal microscopy, we found the respective four extracellular matrix
proteins to form together intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases
from lymph node, liver, and lung. Our in vitro functional analyses suggested that the fibrillar networks regulate adhesion
and migration of tumor cells, fibroblasts, and endothelial cells, by POSTN acting as an anti-adhesive molecule, which
counteracts the adhesive functions of FN1 and COL-I. Further, we found cellular FN and POSTN to be specifically upregulated in the newly forming/formed tumor blood vessels, providing cues and constituents for vessel formation. In
addition, we found knockdown of FN1 expression by short hairpin RNA expression to dramatically inhibit tumor and
stromal cell growth. Targeting TGF-beta receptors and TGF-beta-regulated matrix proteins, especially FN1 and POSTN,
may thus open up new possibilities to treat metastatic disease, because of interfering with multiple key processes of
metastasis: tumor/stromal cell migration and growth, and tumor angiogenesis.
1
Cancer Epigenetics and Biology Program (PEBC), Biomedical Research Institute of Bellvitge (IDIBELL),
Barcelona, Spain; 2Pathology Service, Hospital Universitario de Bellvitge/IDIBELL, Barcelona, Spain;
3
Functional Genomics Core Facility and 4Oncology Programme, Institute for Research in Biomedicine (IRB),
Barcelona, Spain
Multipotent stem cells populations are responsible of hair follicle regeneration during homeostasis. Bulge stem cells
(bulge SC), identified by 6-integrin/CD34 and keratin 15 (K15) expression are infrequently dividing stem cells localized
in the upper permanent region of hair follicle. In contrast, their progeny, hair germ stem cells (HG SC), are a more
proliferative population characterized by the expression of K15, Lgr5 and P-cadherin. Several studies have implicated
epidermal stem cells in squamous (SCC) and basal cell carcinoma (BCC) development as well as in skin appendage
tumors. Due to the fact that mutations in p53 and Rb have been related with tumorigenesis, we have studied the effect
of loss of function of p53 and Rb in the homeostasis of bulge SC and HG SC and its impact in early steps of skin cancer.
We have used the multi-step skin cancer mouse model K14-HPV16, that over-expresses the viral oncoproteins E6 and
E7 in stem cells and basal keratinoctyes and develops spontaneous SCC in the skin. These mice were crossed with K15EGFP mice which express EGFP in the bulge and HG SC of hair follicle. Compared to wild-type skin (K14-HPV16 +/+/
K15-EGFP Tg/+), the preneoplasic skin of E6/E7 transgenic mice showed a significant increase in the number of hair
follicles, together with a selective expansion of the HG SCs compartment in the absence of significant changes in the
whole population of bulge SCs. However, both stem cells populations presented an increased ratio of proliferating cells
compared to wt mice. Furthermore, we have observed that the percentage of cells with nuclear staining of b-catenin
is increased per hair follicle in transgenic mice. Altogether, these results indicate that the loss of p53 and Rb function
leads to activation of bulge and HG SCs with a resulting expansion of only the HG SC population, indicating an important
disequilibrium between both hair follicle stem cell populations. The analysis SCC developed in E6/E7 transgenic mice
showed the presence of CD34+ cells, previously described as cancer stem cell in mouse SCC, but also of K15+ cells in
these neoplasic lesions. Interestingly K15+-tumor cells were more abundant in SCC in situ and become clearly reduced
in SCC poorly differentiated. These results suggest that the abnormal activation and expansion of hair follicle stem cells
induced by loss of function of p53 and Rb can be relevant during the initial steps of tumorigenesis.
Poster
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217
57
Frontiers in
Tumour Progression
Poster Session B
58
Chemoprevention and treatment of tumors by mTOR
inhibitors upon epithelial-specific deletion of PTEN
The p65 subunit of NF-kB and PARP1 assist Snail1 in
activating Fibronectin transcription
Cristiane H. Squarize1,2, Rogerio M. Castilho
Jelena Stanisavljevic1,3, Montserrat Porta-de-la-Riva
1,2
and J. Silvio Gutkind2
Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, USA;
2
Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda,
USA
1
The tumor suppressor PTEN is the major negative regulator of PI3K/mTOR pathway, which has emerged as one of the
most frequently targeted pathways in human cancers. Genomic aberrations are present in a variety of PI3K signaling
components resulting in the deregulation of cellular growth and survival, which contributes to a competitive growth
advantage, metastatic potential, and resistance to therapy. Pten heterozygosity and tissue-specific deletion in mice
leads to dysplasic changes in the prostate, colon, and skin, and to spontaneous tumor development, confirming the
notion that PTEN plays an important role in cancer predisposition, establishment, and progression, as well as in human
hamartoma syndromes. Indeed, inactivating mutations of PTEN have been linked to several autosomal dominant
hamartoma syndromes including Cowden’s disease, Bannayan- Riley-Ruvalcaba syndrome, and Lhermitte-Duclos
syndrome, which are all characterized by the presence of lesions and tumors in multiple organs, increased tumor
susceptibility, and development of variety of malignant tumors. We observed that epithelial-specific deletion of Pten in
mice led to the development of multiple dermal lesions, and breast and thyroid tumors as age progressed, strikingly
resembling Cowden’s disease. This animal model provided an opportunity to explore novel therapeutic approaches in
epithelial originated tumors that present loss of PTEN. We show here that Rapamycin administration, which inhibits a key
downstream target of Akt, mammalian target of rapamycin (mTOR), promotes the rapid regression of advanced lesions.
Furthermore, we found that chronic administration of rapamycin, previously to disease manifestation, was sufficient to
halt tumor development, and to prolong animal survival. These findings suggest that mTOR inhibition with rapamycin
represent a suitable therapeutic option for the chemoprevention and treatment of epithelial tumors involving defective
PTEN function.
, Raquel Batlle1, Antonio García de Herreros1,2
1,3,4
and Josep Baulida
1
Programa de Recerca en Càncer, IMIM-Hospital del Mar, Barcelona, Spain; 2Departament de Ciències
Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain; 3These authors made an equale
contribution; 4Current address: Cancer and Human Molecular Genetics, Catalan Institute of Oncology (ICO) Bellvitge Institute for Biomedical Research (IDIBELL), L’Hospitalet, Barcelona, Spain
1
Epithelial-mesenchymal transition is a morphogenetic process accompanied by epithelial gene repression and
mesenchymal gene activation. Snail1 acts as a transcription repressor of epithelial genes, such as E-cadherin (CDH1), by
binding to consensus sequences (E-boxes) in their proximal promoters and recruiting cofactors. Although expression of
Snail1 affects transcription of mesenchymal genes, underlying molecular mechanisms are poorly understood. Growing
evidence indicates that NF-kB plays a central role in EMT and metastasis by affecting the expression of mesenchymal
genes. During EMT, a pool of the NFkB subunit translocates from the cell membrane to the nucleus where it binds to
the promoter of the mesenchymal gene Fibronectin (FN1). Here we describe a molecular cooperation between p65NFkB and Snail1 in the activation of FN1: while PARP1 may allow formation and/or stabilisation of the complex and
mediate the regulation of the chromatin architecture required for the activation, p65NF-kB may trigger the complex to
the promoter. Interaction between Snail1 and NF-kB allows a switch from Snail1’s classical epithelial targets to those
requiring their cooperation, such as FN1. In addition to FN1, other mesenchymal genes are sensitive to NF-kB; therefore,
by collaborating with p65NF-kB and PARP1, Snail1 may regulate a wide range of mesenchymal genes activated in
epithelial cells undergoing EMTas well as in the invasion programs of mesenchymal cells.
Poster
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Frontiers in
Tumour Progression
Poster Session B
Role of embryonic EMT-inducers at early phases of
tumour progression
59
60
FGF receptor-4 polymorphism acts as an activity switch
of a membrane-type-1 matrix metalloproteinase-FGFR4
complex
Stéphane Ansieau1,3, Clémence Thomas
1,2,3
, Anne-Pierre Morel1,2, George Hinkal1,2, Frédérique Fauvet1,2,
Stéphanie Courtois-Cox1, Julie Pourchet1,2 and Alain Puisieux1,2,3,4
Nami Sugiyama1, Markku Varjosalo , Pipsa Meller , Jouko Lohi , Kui Ming Chan , Zhongjun Zhou , Kari
Inserm, U590, Lyon, France; 2Centre Léon Bérard, Lyon, France; 3Université Lyon I, Lyon, France; 4Institut des
Sciences Pharmaceutiques et Biologiques, Lyon, France
Alitalo1, Jussi Taipale2,4, Jorma Keski-Oja1 and Kaisa Lehti1
Senescence and apoptosis are major oncosuppressive barriers, induced in premalignant lesions and able to constrain
the promitotic effects of oncoproteins. Placed at the crux of signalling pathways, p53 turned out being the key regulator
of these two failsafe programs. Loss of p53 function that accounts for malignant conversion, results from indirect
inactivation mechanisms, mutations of p53 likely occurring at later stages. Among the identified p53 inactivation
mechanisms is the reactivation of the embryonic TWIST genes, as frequently observed in a variety of carcinomas as
well as sarcomas, melanomas, gliomas and neuroblastomas. TWIST1 was originally identified as a prometastatic factor
through its ability to foster an epithelial-mesenchymal transition (EMT), a transdifferentiation process associated with
the acquisition of cell motility. Further examination of TWIST1/2 properties led us to demonstrate that the two proteins
additionally override H-RasG12V-or N-Myc-induced failsafe programs and cooperate with the mitogenic proteins in
promoting murine cell transformation, providing them with a tumorigenic phenotype when xenografted in nude mice. By
performing the oncogenic cooperation assays in human primary epithelial cells, failsafe program escape was next found
to be concomitant with an EMT, leading us to propose that TWIST proteins might favour early cancer cell dissemination.
By demonstrating that EMT additionally provides cells with stem-like properties, including self-renewal capabilities, our
results suggest that the reactivation of the TWIST embryonic genes might actually provide cells with several proliferative
and survival advantages at the primary site of tumour development. By combining various in vitro transformation
models, we now demonstrate that EMT directly takes part in the transformation process, strengthening our conclusions
that embryonic transcription factors and the associated EMT are determinant in initiating primary tumour formation.
Tumor cells use membrane-type-1 matrix metalloproteinase (MT1-MMP) for invasion and metastasis. However, the
signaling mechanisms that underlie MT1- MMP regulation in cancer have remained unclear. Using a systematic gainoffunction kinome screen for MT1-MMP activity, we have here identified kinases that significantly enhance MT1-MMP
activity in tumor cells. In particular, we discovered a MT1-MMP/fibroblast growth factor receptor-4 (FGFR4) membrane
complex that either stimulates or suppresses MT1-MMP and FGFR4 activities, depending on a tumor progressionassociated polymorphism in FGFR4. The FGFR4-R388 allele, linked to poor cancer prognosis, increased collagen invasion
by decreasing lysosomal MT1-MMP degradation. FGFR4-R388 induced MT1-MMP phosphorylation and endosomal
stabilization, while, surprisingly, the increased MT1-MMP in return enhanced FGFR4-R388 autophosphorylation. A
phosphorylation-defective MT1-MMP was stabilized on the cell surface, where it induced simultaneous FGFR4-R388
internalization and dissociation of cell-cell junctions. In contrast, the alternative FGFR4-G388 variant down-regulated
MT1- MMP, and the over-expression of MT1-MMP and particularly its phosphorylationdefective mutant vice versa
induced FGFR4-G388 degradation. These results provide a mechanistic basis for the FGFR4-R388 function in cancer
invasion.
1
2
1
1
3
3
Molecular Cancer Biology Research Program, and 2Genome-Scale Biology Research Program, Biomedicum
Helsinki, University of Helsinki, Finland; 3Department of Biochemistry, Center of Development, Reproduction
and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong; 4Department of Biosciences and
Nutrition, Karolinska Institutet, Sweden
1
Poster
220
221
Frontiers in
Tumour Progression
Poster Session B
HGF does not influence the expression of HO1 in
esophageal cancer cells
Agata Szade, Agnieszka Węgrzyn, Halina Waś, Józef Dulak and Alicja Józkowicz
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Krakow, Poland
Esophageal cancer is one of the most frequent and dangerous tumors. Currently many different studies on new drugs
for cancer treatment are concentrated on inhibiting various growth factors. One of the least explored in this field
is hepatocyte growth factor (HGF). HGF was shown to induce production of proangiogenic factors and migration of
esophageal cancer cells. It was also demonstrated that HGF induces the expression of heme oxygenase-1 (HO-1) in the
mouse model of severe acute renal failure. Moreover HO-1 was proven to mediate the anti-apoptotic effect of HGF in
mesangial cells. HO-1 catalyses the enzymatic breakdown of heme into the ferrous ion, carbon monoxide and biliverdin,
which is subsequently transformed into bilirubin. It works as a cytoprotective, anti-apoptotic, anti-inflammatory and
proangiogenic factor. We have previously reported that HO-1 overexpression can increase viability, proliferation,
and angiogenic potential of melanoma cells as well as facilitate tumor growth and metastasis. However, its role in
esophageal cancer development remains unclear. Here we investigated whether stimulation with HGF can influence the
HO-1 pathway in esophageal cancer cells. Experiments were performed in human esophageal adenocarcinoma cell line
(OE33) treated with HGF (10-200 ng/ml, 6 and 24 hours) and HO-1 inducers: hemin (10-100 μM, 6 and 24 hours) and
cobalt protoporphyrin (CoPPIX, 10-100 μM, 6 and 24 hours). In such cells we measured expression of genes involved in
HO-1 pathway: HO-1, biliverdin reductase (BVR), and ferritin (FTH - heavy chain and FTL - light chain) and HGF pathway:
HGF, MET, matriptase (ST14), urokinase (PLAU) and Kunitz-type protease inhibitor-1 and-2 (SPINT1 and SPINT2). We did
not find any significant effect of HGF on expression of HO-1-associated genes, regardless of time of treatment, although
we found a strong and dose-dependent induction of HO-1 mRNA in response to hemin and CoPPIX, with maximal fold
of induction after 6 and 24 hours, respectively. Incubation with HO-1 inducers did not modulate expression of BVR,
but slightly changed the level of FTH and FTL. Activation of HO-1 did not significantly influence the expression of HGFassociated genes: MET, matriptase and HAI-1. Expression of HGF was increased in dose-dependent manner after 6
hours of incubation with hemin, reaching 4 fold induction. CoPPIX induced HGF expression 2 times at the doses of 10
μM and 30 μM after 6 hours of incubation. Doesdependent tendency to increase SPINT2 expression by 24 h incubation
with CoPPIX was observed, reaching 2-fold induction at the dose of 100 μM. In conclusion, quantitative analysis of mRNA
suggests that expressions of genes of HO-1 pathway are not regulated by HGF in esophageal adenocarcinoma cell line
OE33. From the genes of HGF pathway only HGF, and SPINT2 are slightly affected by HO-1 inducers.
61
62
Metastasis-regulation by microRNAs: is there a role in
tumor progression?
Marcell Szasz,
Z. Baranyak, L. Lukacs, M. Szendroi, G. Arato, J. Fillinger, I. Soltesz, Z. Hanzely, K. Balint,
A. Richardson, M. Micsinai and J. Kulka
Semmelweis University, 2nd Department of Pathology and Department of Orthopaedics, Korányi National
Institute for Tuberculosis and Pulmonology, National Institute of Neurosurgery, Harvard Medical School,
Brigham and Women’s Hospital, New York University, Sackler Institute, USA
Despite the achievements, understanding of genetic programs and molecular mechanisms required for cancer
metastasis are still incomplete. Genes that specifically regulate the process of metastasis are useful tools to elucidate
molecular mechanisms and may become markers for anti-metastatic therapy. Recently, several non-coding regulatory
RNA-genes were identified, which play roles in various steps of metastasis. MicroRNAs are ideally suited to regulate
tumor metastasis due to their capacity to coordinately repress numerous target genes, thereby potentially enabling their
intervention at multiple steps of the invasion-metastasis cascade. In breast cancer, pro- (mir-10b, -21, -373, -518d,
- 520c) and anti-metastatic (mir-31, -34, -126, -200b, -205, -206, -335) microRNAs were identified. We previously
studied miR-31, whose expression correlates inversely with metastatic recurrence in human breast carcinomas. In
this study we aimed to analyze the expression of these molecules: we compared their expression in primary breast
carcinomas (without relapse: 23 cases, and with recurrence: 23 patients) and their corresponding distant metastatic
sites and 17 additional metastasic samples (applicable: 40 samples). As published studies investigating this topic usually
lack comparative data, because of sample unavailabilty, in our collected material we are taking the identified microRNAs
under further scrutiny to elucidate their role in tumor progression.
Poster
222
223
Frontiers in
Tumour Progression
Poster Session B
Ets2-mediated global genetic and epigenetic alterations
to the myeloid genome throughout the course of tumor
progression
63
64
E2F1 regulates cellular growth by mTOR signaling
Albert Tauler, Sebastian Real, Lilia Espada and Nathalie Meo-Evoli
Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia. Universitat de Barcelona, Spain
David A. Taffany, Haritha Mathsyaraja, Sudarshana M. Sharma, Tahera Zabuawala and
Michael C. Ostrowski
Department of Molecular and Cellular Biochemistry, Tumor Microenvironment Programme, Comprehensive
Cancer Center, The Ohio State University, Columbus, USA
Cancer metastasis remains a deadly aspect of invasive breast cancer, yet the underlying genetic impetus driving
metastasis remains poorly understood. Discoveries in recent years have placed increasing emphasis on the cells of
the surrounding tumor microenvironment in promoting metastasis, such as the abilities of macrophages and other
myeloid cells to promote angiogenesis and potentially prime the premetastatic niche. Previous work in our lab suggests
the transcription factor Ets2 plays an important macrophage-specific role in breast cancer progression and metastasis
by suppressing anti-angiogenic genes such as Thbs1, Thbs2, Timp1 and Timp3 through both direct repression and
recruitment of histone deacetylase. Currently, experiments are underway to, through microarray analysis and chromatin
immunoprecipitation combined with massively parallel sequencing (ChIP sequencing), unearth the global genetic and
epigenetic mechanisms through which Ets2 may regulate gene expression within the cells of the myeloid lineage and to
determine how this regulation may promote the spread and proliferation of metastatic tumor cells. Myeloid samples from
Ets2+ and Ets2- mice have been harvested from bone marrow and lungs at two days, seven days, and fourteen days
following tail-vein injection of metastatic breast tumor cells, and ChIP sequencing will be performed for transcription
factors Ets2 and PU.1 as well as a variety of histone marks. As the samples and resulting data are further analyzed, we
hope to gain a comprehensive picture of Ets2-mediated gene expression changes within the myeloid compartment that
affect tumor metastasis along with the mechanisms through which they are activated or repressed.
During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of
inducing growth by regulating mTOR activity. The activation of mTOR by E2F1 is dependent on both E2F1’s ability to
bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in
this process. In contrast to E2F1, E2F3 is unable to activate mTOR, suggesting that growth activity could be restricted
to individual E2F members. The effect of E2F1 on the activation of mTOR does not depend on Akt. Furthermore,
over-expression of TSC2 does not interfere with the effect of E2F1, suggesting that the TSC1/TSC2 complex does
not participate in E2F1 growth signaling. Immunolocalization studies demonstrate that E2F1 activates mTOR activity
by potenciating its association to the late endosome vesicles, a mechanism similar to that of amino acid mediating
mTOR activation signaling. From these studies, E2F1 emerges as a key protein that integrates cell division and growth,
essential for cell proliferation.
Poster
224
225
65
Frontiers in
Tumour Progression
Poster Session B
Integrated proteomic and siRNA screen analysis to
study the migratory phenotype of breast cancer cell
lines
Platelet Derived Growth Factor-B and Transforming
Growth Factor-beta cooperate in the induction of
epithelial to mesenchymal transition in lung cancer
cells
Safia Thaminy1,2, Yong Zhou , Ching-Yun Chang , Berend Snjider , Carey Sheu , Shuang He , Olga Vitek
2
3
1
2
3
66
3
and Ruedi Aebersold1,2,4,5
Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, Zurich, Switzerland; 2Institute
for Systems Biology, Seattle, Washington, USA; 3Departments of Statistics and Computer Science, Purdue
University, West Lafayette, Indiana, USA; 4Competence Center for Systems Physiology and Metabolic
Diseases, Zurich, Switzerland; 5Faculty of Science, University of Zurich, Switzerland
1
The mechanisms controlling cancer cells migration and invasion are not well understood. Beside genetic alteration, it
has been shown that the tumor microenvironment and changes in protein glycosylation of cancer cells play an important
role in the metastasis process (1,2). To elucidate the molecular mechanisms leading to cell invasiveness, we selected
as model the low (MCF7) and high (Hs578T) invasive breast cell lines and treated them in hypoxia to reconstitute the
tumor microenvironment. We then used a mass spectrometry –based approach to identify and quantified N-glycoprotein
expressed in these cell lines (3), and combined it with a wound healing screen to identify N-glycoproteins involved
in cell migration. Using a label-free quantification method (4), we determined changes in protein expression of ~450
N-glycoproteins and demonstrated the validity of our quantification by western blot analysis. Our analysis revealed
that N-glycoprotein expression profiles could be used to classify the cell lines similarly to the established luminal/
basal gene signature. We found that MCF7 and Hs578T cell lines showed a time-dependent response to the hypoxia
treatment and regulated different set of N-glycoproteins. To assess if N-glycoprotein regulation could be associated
with the invasiveness potential of the cell lines, we performed a high throughput wound healing screen. Using a custom
designed siRNA library, we identified in a primary screen 18 N-glycoproteins that affect the migration of the Hs578T cell
line. Interestingly, the migratory phenotype was enriched for hypoxia-regulated proteins. Further experiments are under
progress to confirm the importance of these regulated proteins in cell migration. These results suggest that proteomic
technology, coupled to a wound healing screen, could capture protein expression that supports migratory phenotype.
This new approach could lead to the discovery of therapeutic targets.
Francisco Miguel Torres, Isabel Cuartas, Ludmila Prudkin and Joan Seoane
Vall d’Hebron Institute of Oncology (VHIO), Hospital Universitari Vall d’Hebron, Barcelona, Spain
TGF-beta is a cytokine with a well known role in the control of epithelial tissue homeostasis. However, TGF-beta, promote
tumor progression in certain cancers. One of the effectors of the TGF-beta protumorogenic role is PDGF-B; a cytokine
involved in angiogenesis and cell proliferation. Epithelial to Mesenchymal Transition (EMT) is a process where epithelial
cells are transformed into a mesenchymal phenotype, related with the acquisition of protumourogenic traits. Here, we
study the role of the induction of PDGF-B by TGF-beta in EMT, in lung cancer cells. We observed that not only TGF-beta
induces PDGF-B expression, but the presence of PDGF-B cooperates with TGF-beta to induce its own expression in an
autocrine loop. In addition, we found that PDGF-B and TGF-beta cooperate in the induction of EMT through Snail and
Slug, two well known inducers of EMT. The blockade of PDGF-B signal inhibit EMT progression. Therefore, in lung cancer
cells, TGF-beta induces PDGF-B and PDGF-B itself facilitates its own expression generating an autocrine loop. PDGF-B
promotes TGF-beta induced EMT, being a crucial factor in the TGF-beta oncogenic effect.
Poster
226
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Frontiers in
Tumour Progression
Poster Session B
67
68
The hemopexin domain of matrix metalloproteinase-9
regulates cell survival and migration in B-cell chronic
lymphocytic leukemia
Toll-like receptor (TLR) 7 and 8 expression in CD133+
cells in colorectal cancer points to a specific role for
TLRs in tumourigenesis and tumour progression
Estefanía Ugarte-Berzal, Javier Redondo-Muñoz, Elvira Bailón, MercedesHernández del Cerro, José
Ana Maria Waaga-Gasser2, Martin Gasser , Martin Grimm , Maria Lazariotou , Mia Kim , Andreas
A. García-Marco and Angeles García-Pardo
Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of B-cells in peripheral
blood and the subsequent infiltration of lymphoid organs. Localization in these niches likely contributes to B-CLL cell
survival and thus, characterization of the molecules/mechanisms involved in these processes is important. We previously
reported that matrix metalloproteinase-9 (MMP-9) is present at the B-CLL cell surface via binding to alpha4beta1
integrin and the CD44v isoform, and this results in inhibition of cell migration and increased survival. While the first
function requires MMP-9 catalytic activity, the second one results from cell signaling induced by MMP-9/alpha4beta1
integrin interaction. To further characterize the role of MMP-9 in B-CLL pathology, we have studied the region of MMP-9
responsible for these functions. Using recombinant MMP-9 containing individual domain deletions, we found that the
carboxy-terminal region (PEX9) is necessary for MMP-9 binding to the cell surface and for induction of cell survival.
Accordingly, the recombinant protein GST-PEX9 bound to B-CLL cells both in soluble and immobilized forms, while GST
alone did not. Cell binding was inhibited by blocking alpha4beta1 with antibodies or peptides, indicating that this integrin
was the receptor for PEX9. Indeed K562 cells, which lack alpha4beta1 expression did not attach to GST-PEX9 while
K562-alpha4beta1 transfected cells did. Binding of GST-PEX9 to alpha4beta1 increased B-CLL cell survival and this
involved Lyn kinase activation and Mcl-1 upregulation. GST-PEX9 also blocked B-CLL cell migration through HUVEC or
Matrigel, indicating that the PEX domain is responsible for the MMP-9 functions that contribute to B-CLL pathogenesis.
Targeting this domain may therefore constitute an efficient therapy for this malignancy. Suppported by grant SAF200907035 from the Ministerio de Ciencia e Innovación and by the Fundación de Investigación Médica Mutua Madrileña.
1
2
4
1
Rosenwald3 and Christoph Thomas Germer1
Department of Surgery I, University of Wuerzburg, Germany; 2Department of Surgery I, Molecular Oncology
and Immunology, University of Wuerzburg, Germany; 3Institute of Pathology, University of Wuerzburg,
Germany; 4Department of Cardiothoracic Surgery, University of Wuerzburg, Germany
1
Nuclear factor-kappaB (NF-κB) was identified as a key modulator in driving inflammation to cancer. Toll-like receptor
(TLR) stimulation results in activation of NF-κB and mitogen-activated protein kinases (MAPKs) that have been shown to
recruit mitotic and cyclooxygenase-2 (COX-2) induced pathways in carcinogenesis. Here we asked whether different TLR,
COX-2 and stem cell marker expression profiles in colorectal cancer (CRC) provide further evidence for this hypothesis
from a clinical perspective. We analysed gene and protein expression of TLR7, 8, 9, 10, COX-2 and CD133 in CRC patients
(n=65). FACS and immunofluorescence staining of TLR7 and 8 were specifically used to detect expression on CD133+
colon cancer initiating cells. In addition, cells derived from patients normal colon tissue and CRC were analysed on a
flow cytometer (FACS-Analysis). Statistical analysis was performed using Kaplan-Meier method for the overall survival
time in association with TLR7 and 8 expression and univariate analysis of significance for TLR7 and 8 expression
differences in survival curves. Multivariate analysis by using the Cox Proportional Hazards Model was performed on
all parameters that were found to be significant on univariate basis. Survival time was determined as the time from
tumour resection to tumour specific death and as the time from tumour resection to time of obvious recurrence. Gene
analysis demonstrated significantly upregulated TLR7, 8, 9, 10, and COX-2 expression in CRC tumor tissues. Moreover,
immunofluorescence double staining and FACS analysis of isolated tumour cells from primary tumours and from HT
29 tumour cell line showed co-expression of TLR7 and 8 with CD133 and gave evidence for a subpopulation of colon
cancer-initiating cells. In addition, in multivariate analysis TLR8-expression was found to be an independent prognostic
factor. We found significantly increased TLR7 and 8 expression in CD133+ colon cancer cells. We conclude, persistent
TLR-specific activation of NF-κB in CRC and particularly in tumour initiating cells may thus sustain further tumour
growth and progression through perpetuated signalling known from inflammatory and tissue repair mechanisms with
consecutive self-renewal in pluripotent tumour cells. Furthermore, activation through self-ligands or viral RNA fragments
may putatively maintain this inflammatory process, suggesting a key role in cancer progression.
Poster
228
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Frontiers in
Tumour Progression
Poster Session B
69
70
EMT and metastases in the AY-27/F344 rat model of
bladder cancer
Functional interplay between tetraspanins and
proteases
Kate Williamson, Alvin Lee, Vincent Koo, Perry Maxwell, Anthony J. Glackin, Neil Anderson, Osama Sharaf
María Yáñez-Mó1, Dolores Gutiérrez-López , Alvaro Gilsanz , Pilar Gonzalo , Alicia G Arroyo , Francisco
Eldin, Sam B. Gray, Daniel Calder, Patricia Carberry, Lynn McCallum, Gareth McClean and Jenny Watson
2
2
3
3
Sánchez-Madrid1,3 and Carlos Cabañas2
Queen’s University Belfast, Belfast Health & Social Care Trust, Craigavon Area Hospital and University
College Dublin, Ireland
Servicio de Inmunología, Hospital de la Princesa, Instituto de Investigación Sanitaria Princesa, Madrid,
Spain; 2Centro de Biología Molecular Severo Ochoa, Madrid, Spain; 3Departamento de Biología Vascular e
Inflamación, CNIC, Madrid, Spain
The F344/AY-27 rat model of bladder cancer is orthotopic, immunocompetent and mimics human disease. We inoculated
38 female F344 rats intravesically with AY- 27 cells and after 16 days 29/32 (91%) had bladder tumors, of these
18/29 (62%) were muscle invasive. After 35 days the remaining six animals all had muscle invasive disease. Following
serial sectioning we found no evidence of metastases in lung, liver, brain or kidney organ resections. This absence of
metastases occurred in the presence of immunophenotypic characteristics associated with Epithelial to Mesenchymal
transition (EMT). There was a reduction and redistribution from membranous to cytoplasmic E-cadherin immunoreactivity
in 14/23 (64%) tumors. Vimentin was expressed in tumor areas in 22/23 (96%) bladders whilst histologically “normal”
urothelium was vimentin negative. Cytokeratin expression was reduced in 19/22 (86%) tumors when compared with
normal urothelium. In 14/20 (70%) bladders S100A4 expression in urothelium was lower than in tumor areas. Notably
S100A4 staining was absent in basal cells. Because S100A4 is associated with metastases, we explored the effects
of forced expression of S100A4 in AY-27 cells and subsequently in the F344/AY-27 model. Transfection of rat S100A4
into AY-27 cells induced four-fold expression in the resultant “S100A4” cells. In vitro, forced S100A4 induced a spindle
phenotype and enhanced growth, whilst TGF-β1 induced polarised vimentin immunoreactivity and reduced cell size.
S100A4 and TGF-β1 acted synergistically to induce numerous pseudopodia and to significantly enhance would closure.
Three of nine rats developed metastases following inoculation with S100A4 cells, two had concomitant invasive bladder
tumors. We conclude that the F344/AY-27 model is poised on the threshold of a metastatic phenotype which is inducible
through four fold increased expression of S100A4. Our data suggest that TGF-β1 cooperates with S100A4 to promote
metastases.
Tetraspanin-enriched microdomains (TEMs) are specialized platforms in the plasma membrane that include adhesion
receptors and enzymes. Our group has observed that tetraspanin CD9 associates with ADAM17 on the surface of
leukocytes and endothelial cells. The occurrence of CD9-ADAM17 association was established by different
complementary approaches: co-localization, in situ Proximity Ligation Assays, co-immunoprecipitation, chemical crosslinking and pull-down experiments employing recombinant proteins. We demonstrated, through overexpression and
silencing, that CD9 exerts negative regulatory effects on ADAM17-mediated shedding of TNF-α and ICAM-1 in different
cell types. In addition, we demonstrated that membrane-anchored metalloproteinase MT1- MMP/MMP14 is included
in TEMs in endothelial cells. MT1-MMP regulates pericellular matrix proteolysis in cancer invasion and angiogenesis
by initiating a metalloproteinase cascade via the enzymatic maturation of pro-MMP2. In endothelial cells, MT1-MMP
was found to be directly associated with CD151, as demonstrated by coimmunoprecipitation and FRET experiments.
Knockdown of this tetraspanin, but not of CD9, resulted in an increase in MMP2 maturation. However, endothelial cells
depleted of CD151 or derived from CD151 deficient mouse presented an aberrant collagenolysis. Insertion of MT1MMP into TEMs was shown to be crucial for its association and functional coordination with integrins, thus, a ternary
CD151-MT1-MMP-alpha3beta1 integrin complex spatiotemporally directs pericellular proteolysis during endothelial cell
migration and angiogenesis. Together, all these emerging evidences point to Tetraspanin-Enriched Microdomains (TEMs)
as potential regulators of membrane protein shedding and matrix degradation and rearrangement.
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We would like to thank our sponsors:
Centro Nacional de Investigaciones Oncológicas (CNIO)
Melchor Fernández Almagro, 3
28029 Madrid, Spain
www.cnio.es
Coordination and edition: Mercedes Moro, Carolina G. Sabaté and Ralf Dahm, CNIO
Direction of art and production: Versus Gráficas S.A.
Photographic archive: CNIO
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© Fundación CNIO Carlos III, 2010
Printed in Spain.