Download cell biology of cancer

cell biology of cancer
december 3 • san francisco, ca
Moscone Center | Room 104
San Francisco, CA | Saturday, December 3, 2016
8:00 am – 5:30 pm
The 2016 ASCB Doorstep Meeting l www.ascb.org/doorstep
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Thanks to Our Supporters
We thank ASCB President Peter Walter for conceiving this inaugural Doorstep Meeting on the Cell Biology
of Cancer, and for appointing its two organizers: Ira Mellman, Genentech, and Alan Ashworth, UCSF Helen
Diller Family Comprehensive Cancer Center. Both are experts in this field and we appreciate
their input.
Thanks to everyone for making this meeting happen!
Peter Walter
Ira Mellman
Alan Ashworth
University of California
Genentech
University of California
San Francisco School of
Helen Diller Family
Medicine/HHMI
Comprehensive Cancer Center
A Special Thank You to the Following Supporters:
The 2016 ASCB Doorstep Meeting l www.ascb.org/doorstep
Contents
Meeting Schedule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Poster Session Abstracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Cell Biology of Cancer Doorstep Meeting
SCHEDULE
7:45 am
Registration Opens
Registration is located outside Room 104.
Do not go to the Registration Area for the ASCB Annual Meeting.
8:00 am
Breakfast and Opening Remarks
8:30 am
The Cell Biology of Cancer Immunotherapy Opening Remarks by ASCB President Peter Walter
Ira Mellman, Genentech
It is now clear that manipulating the immune system can elicit dramatic and durable responses
in cancer patients, indicating that the interaction between tumors and the immune system
is a fundamental feature of cancer biology. As this is a recent appreciation, the cell biological
mechanisms underlying this interaction remain largely unexplored, thus creating a remarkable
opportunity to investigate problems guided by hypotheses generated by human clinical studies.
9:05 am
DNA Repair and Cancer Pathogenesis and Therapy
Alan Ashworth, UCSF Helen Diller Family Comprehensive Cancer Center
DNA repair pathways are commonly defective in cancer leading to pathogenic mutations and cancer
progression. I will describe how a better understanding of these repair pathways is giving insights
into how cancers develop and change over time as well as providing new therapeutic opportunities.
9:40 am
Forcing Form and Function
Valerie Weaver, University of California, San Francisco School of Medicine
I will discuss tissue mechanics—stem cells and mammary development—and relevance to
breast cancer.
10:15 am
Coffee Break
10:30 am
Stromal-Immune Interactions in Cancer Shannon J. Turley, Genentech
I will discuss our recent and ongoing work on the interactions between stroma and immune cells
in healthy lymphoid tissues and in cancer. Particular emphasis will be placed on the regulation of
dendritic cell and T cell motility and function by tissue-resident mesenchymal stroma.
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11:05 am
Macrophages Are a Cellular Toolbox That Promotes Tumor Progression and
Metastasis
Jeffrey W. Pollard, University of Edinburgh
One of Metchnikoff’s insights into macrophage function was to define two states: “Physiological
Inflammation” and “Pathological Inflammation.” The former was the role of macrophages in
maintaining homeostasis and the latter was their function in response to challenges from outside.
Our research on macrophages has provided insights into both roles and confirmed Metchnikoff’s
original ideas. We have shown macrophages regulate many aspects of development and participate
in tissue repair as well as immune responses. Our recent work has indicated that tumors redirect
many of these macrophage roles in development and repair to enhance their survival and
progression to malignancy. These roles include the enhancement of angiogenesis, promotion
of epithelial cell invasion and survival as well as suppression of immune responses. My talk will
emphasize these commonalities with a particular emphasis of the roles of macrophage synthesized
WNTs.
11:40 am
SWI/SNF (BAF) Complex Mutations in Cancer Charles W.M. Roberts, St. Jude Children’s Research Hospital
Data emerging over the last several years implicate the SWI/SNF (BAF) chromatin remodeling
complex as a major tumor suppressor as inactivating mutations in at least nine SWI/SNF subunits
have collectively been identified in over twenty percent of all cancers. Insights into the normal
function of SWI/SNF complexes, the mechanisms by which mutation of the complexes drive cancer
formation, and potential therapeutic vulnerabilities created by mutation of the complex will be
presented.
12:15 pm
Lunch Break with Roundtable Discussions & Poster Session
• 12:15 pm - Lunch Buffet Opens
• 12:30-1:30 pm – Roundtable Discussions with a speaker(s) or other attendees over lunch
[table sign-up available at Registration]
• 1:30-2:20 pm – Poster Session
2:20 pm
Cancer Therapy Resistance: A Cell Biologist’s Perspective Joan Brugge, Harvard Medical School
Despite dramatic advances in the treatment of cancer, therapy resistance remains the most
significant hurdle in improving the outcome of cancer patients. The tumor ‘ecosystem’ is highly
robust and able to adapt to the cancer therapies designed to induce tumor cell death. Genetic
and phenotypic heterogeneity within individual tumors represents the major source of therapy
resistance. This presentation will describe the challenges associated with the complexity of cancer
resistance and potential approaches to address this important problem.
2:55 pm
Lymphatic Vessels in Inflammation and Cancer: New Insights into Immune
Regulation
Melody A. Swartz, Institute of Molecular Engineering
Lymphatic vessels, which become activated and expand in chronic inflammation and cancer,
contribute to innate and adaptive immunity in multiple ways. In addition to carrying cells, antigens,
and signals from the periphery to the lymph node, they are lined with endothelial cells that
directly interact with immune cells to alter their activation status and skew responses. This talk
will highlight recent advances in our understanding of how lymphangiogenesis contributes to
inflammation and promotes cancer metastasis, and suggest new directions for therapeutic targeting
for immunomodulation.
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3:30 pm
The Autophagy Adaptor p62/SQSTM1 Is a Key Mediator of Liver Cancer
Development
Michael Karin, University of California San Diego
Liver cancer (hepatocellular carcinoma/HCC), one of the most common malignancies worldwide,
is the end stage of many chronic liver diseases. A common pathological feature of such diseases
and HCC are aggresomes containing the autophagy adaptor p62/SQSTM1. We found that p62
accumulation is a key driver of liver tumorigenesis due to its ability to protect HCC initiating cells
from oxidant-induced death through activation of the NRF2 dependent antioxidant response.
4:05 pm
Afternoon Break
4:20 pm
The Regulation of Metastasis
Sean J. Morrison, University of Texas Southwestern Medical Center
My laboratory studies the biology of cellular self-replication, in normal stem cells and in cancer
cells. While cancers commonly arise from mutations that inappropriately activate stem cell selfrenewal pathways, we are also interested in identifying ways in which the self-replication of cancer
cells differs from the self-renewal of normal stem cells. We hypothesize that cancer cell metastasis
requires mechanisms, including metabolic adaptations, that are not normally employed by stem
cells under physiological conditions.
4:55 pm
Towards a Tumor Cell Atlas: Dissecting the Tumor Ecosystem with Single Cell
Genomics
Aviv Regev, MIT, Broad Institute, HHMI
Tumors consist of diverse cells—both malignant and non-malignant—and it is increasingly
recognized that the diversity of cell types, states, and interactions all play a critical role in
tumorigenesis and metastasis, as well as present opportunities for clinical intervention. To explore
the distinct genotypic and phenotypic states of tumors, we use single cell genomics, to obtain,
process, and analyze single-cell profiles of any tumor cells (malignant and non-malignant) from any
clinical cancer sample and animal models. We study malignant, immune, stromal, and endothelial
cells in the tumor, to understand heterogeneity in proliferation, inflammation, spatial context,
differentiation programs and drug resistance in malignant cells, and to understand the functional
state of immune and stromal cells, and infer the molecular basis of interactions between different
cell types. Working with animal models, we then dissect the mechanistic basis of these states and
their impact on the tumor. Working across diverse tumor types, this would lead to a new human
tumor cell atlas and an integrative understanding of the cellular ecosystem in tumors.
5:30 pm
Closing Remarks
6:00 pm
Keynote with Public Service, Kaluza, Gibco, and Fellows Award Presentations
Keynote: Genes, Genomes, and the Future of Medicine
Richard P. Lifton, The Rockefeller University
All Doorstep Meeting attendees are invited to attend the ASCB Keynote Address at 6:00 pm in Hall
E of the Moscone Center, as well as the Opening Reception that follows the Keynote. If you are
registered for the Annual Meeting and already have your Annual Meeting badge, you may wear
that badge to gain entry. If you are not registered for the Annual Meeting or have not picked up
your badge, please wear your Doorstep Meeting badge to gain entry.
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Save the Date
cell biology of
degeneration and repair
in the nervous system
2017 ascb doorstep meeting
december 2 • philadelphia, pa
Look for more information this Spring!
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Poster Session Abstracts
1:30 – 2:20 pm
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Centrosome Amplification Drives Spontaneous Tumor Development In Mammals
Michelle Levine, Bjorn Bakker, Bram Boeckx, Diether Lambrechts, Floris Foijer, and Andrew Holland
Centrosome number is normally tightly controlled in cycling cells, so that upon entering mitosis a cell contains two
centrosomes to organize the bipolar spindle and ensure equal chromosome segregation. However, extra centrosomes
are frequently observed in both solid and non-solid cancers and are correlated with high tumor grade and poor
prognosis. Despite the strong correlation between supernumerary centrosomes and tumor development, it remains
unclear whether and how extra centrosomes contribute to tumor development. To address this question, we developed
a mouse model in which the levels of Polo-like kinase 4 (Plk4), the master regulator of centrosome copy number, can be
inducibly elevated to promote the creation of extra centrosomes in the absence of additional genetic defects.
We show that a modest increase in Plk4 levels drives robust and sustained centrosome amplification across multiple
tissues in vivo. Centrosome amplification increased the frequency of chromosome misseggregation and aneuploidy. To
address the role of extra centrosomes in tumorigenesis, we drove centrosome amplification in the APCmin intestinal
neoplasia model. Supernumerary centrosomes promoted an increase in tumor initiation events in the intestine, but
did not influence tumor progression. Additionally, we tested the effect of centrosome amplification in the absence
of additional genetic insults. Importantly, we provide the first evidence to show that centrosome amplification is
sufficient to promote spontaneous tumorigenesis. Tumors in Plk4 overexpressing animals exhibited robust centrosome
amplification and exhibited striking aneuploidy and ongoing chromosome segregation errors. This recapitulates the
karyotypic alterations observed in genetically unstable human tumors. Together, our findings demonstrate that extra
centrosomes can be a driver of tumorigenesis in mammals.
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Crosstalk Between CLCb/Dyn1-Mediated Adaptive CME And EGFR Signaling Increases Metastasis
Ping-Hung Chen, Nawal Bendris, Yi-Jing Hsiao, Carlos R. Reis, Marcel Mettlen, Hsuan-Yu Chen, Sung-Liang Yu and Sandra
L. Schmid*
Signaling receptors are internalized and regulated by clathrin-mediated endocytosis (CME). Two clathrin light
chain isoforms, CLCa and CLCb are integral components of the endocytic machinery whose differential functions
remain unknown. We report that CLCb is specifically up-regulated in non-small cell lung cancer (NSCLC) cells and
associated with poor patient prognosis. Engineered single CLCb-expressing NSCLC cells, as well as ‘switched’ cells that
predominantly express CLCb, exhibit increased rates of CME and altered clathrin-coated pit dynamics. This ‘adaptive
CME’ resulted from up-regulation of dynamin 1 (Dyn1) and its activation through a positive feedback loop involving
enhanced EGF-dependent Akt/GSK3β phosphorylation. CLCb/Dyn1-dependent adaptive CME selectively altered EGFR
trafficking, enhanced cell migration in vitro, and increased the metastatic efficiency of NSCLC cells in vivo. We define
molecular mechanisms for adaptive CME in cancer cells and a role for the reciprocal crosstalk between signaling and
CME in cancer progression.
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Epithelial Membrane Protein 2 Regulates APC-Mediated 3D Morphogenesis And Apical-Basal Polarity
Alyssa C. Lesko1,2, Carolyn Ahlers1,2, Jenifer R. Prosperi1,2,3; 1Department of Biological Sciences, Harper Cancer Research
Institute, University of Notre Dame, Notre Dame, IN; 2College of Science, University of Notre Dame; 3Department of
Biochemistry and Molecular Biology, Indiana University School of Medicine
Adenomatous Polyposis Coli (APC) is well known as a negative regulator of the Wnt signaling pathway; however, it also
regulates polarity proteins, such as Dlg and Scribble (Scrib) through its C-terminus binding domains. Loss of apicalbasal polarity disrupts several cellular processes including epithelial structure and intracellular signaling, and is an early
marker for tumor development. We demonstrated that APC knockdown (APCKD) in Madin-Darby Canine Kidney (MDCK)
cells altered epithelial morphogenesis and inverted polarity in 3D culture. Microarray analysis identified several targets
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with changed expression upon APC loss, including epithelial membrane protein 2 (EMP2). Interestingly, reintroduction of
the C-terminal fragment of APC or knockdown of EMP2 in APCKD cells decreased cyst size and restored apical polarity.
These data demonstrate a novel role for EMP2 in mediating polarity and cyst size, and suggest that the APC C-terminus
may be involved in EMP2 regulation and signaling. In this study, we seek to investigate both direct and indirect
mechanisms of the regulation of apical polarity by the APC C-terminus. First, we evaluated Scrib as a potential direct
regulator of APC-mediated polarity and found APC loss decreases Scrib expression. To assess the indirect regulation
of apical polarity by the APC C-terminus, we investigated transcriptional activation of EMP2 using bioinformatics and
transcription factor DNA/protein arrays. EMP2 promoter screens (ConTra v2 webserver) identified 22 transcription
factor binding sites including signal transducer and activator of transcription (STAT3), specificity protein 1 (Sp-1), and
core binding factor (CBF). Interestingly, APCKD MDCK cells exhibit increased expression of STAT3, Sp-1, and CBF in DNA/
protein arrays compared to control MDCK cells. These studies have identified Scrib as a possible mechanism by which
APC directly controls apical polarity and STAT3, SP-1, and CPF as possible APC-mediated transcriptional regulators of
EMP2. Future studies in the laboratory will determine whether Scrib is the central signaling modality between EMP2 and
APC in the regulation of polarity, and investigate the MAPK/ERK Scrib-mediated pathway as a downstream mechanism
of EMP2 signaling. Additionally, we will continue to dissect the transcriptional mechanism(s) of EMP2 regulation through
ChIP assays and reporter assays using mutant EMP2 promoter constructs. Understanding the interaction of APC and
EMP2 and the influence on apical-basal polarity will identify key players in the role of APC in disease progression.
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Targeting Endoplasmic Reticulum-Resident Proteins for the Treatment Of B Cell Cancer
Chih-Hang Anthony Tang1, Juan R. Del Valle2 and Chih-Chi Andrew Hu1; 1The Wistar Institute, Philadelphia, PA,
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Department of Chemistry, The University of South Florida, Tampa, FL
The IRE-1/XBP-1 pathway is the most conserved endoplasmic reticulum (ER) stress response. We discovered that genetic
deletion of XBP-1 significantly decelerates the progression of chronic lymphocytic leukemia (CLL) in Eµ-TCL1 mice.
We set up a high throughput in vitro screening platform to look for effective inhibitors that can block the IRE-1/XBP-1
pathway. We discovered a specific inhibitor, B-I09, which blocks the IRE-1/XBP-1 pathway with high potency and efficacy.
B-I09 clearly suppresses activation of the IRE-1/XBP-1 pathway as evidenced by the decreased mRNA and protein levels
of XBP-1 in intact cells. B-I09 specifically targets mouse CLL cells in vivo by inducing apoptosis. We also discovered that
genetic deficiency of XBP-1 compromises the BCR signaling which is crucial for the survival of CLL cells. To test whether
pharmacological inhibition of XBP-1 could enhance the effect of inhibitors to the BCR signaling, we combined B-I09
with Bruton’s Tyrosine Kinase (BTK) inhibitor, ibrutinib, to treat human CLL cells. A true and strong synergistic effect in
pharmacology was observed using the Chou-Talalay combination index method. Such results suggested that B-I09 can
decelerate the growth of CLL either as a single agent or in combination with ibrutinib. Our finding in synergism may
be important because B-I09 may help ibrutinib to achieve higher cytotoxicity in CLL cells at a lower dose, addressing
ibrutinib’s toxicity issue. Our studies also led us to discover that IRE-1 interacts with another ER-resident protein called
STING. STING is critical for cytoplasmic DNA sensing and interferon production. The agonists of STING are recently
shown to activate anti-tumor functions of T and NK cells, resulting in efficient control of cancer in mouse models. We
showed that the IRE-1/XBP-1 pathway is required for the interferon-producing function of STING, and that the agonists
of STING selectively trigger mitochondria-mediated apoptosis in normal and malignant B cells. Upon stimulation, STING
was degraded less efficiently in B cells, implying that prolonged activation of STING can lead to apoptosis. Transient
activation of the IRE-1/XBP-1 pathway partially protected agonist-stimulated malignant B cells from undergoing
apoptosis. In CLL-bearing mice, injection of the STING agonist, 3’3’-cGAMP, induced apoptosis and regression of
leukemia. Similarly efficacious effects were elicited by 3’3’-cGAMP injection in syngeneic or immunodeficient NSG mice
grafted with malignant B cells. These data suggested that, in addition to boosting anti-tumor immune responses, STING
agonists can directly eradicate CLL as well as other types of B cell malignancies in vivo. IRE-1 and STING are thus useful
ER-resident protein targets for the treatment of B cell cancer.
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Mechanisms to Maintain Centromere Stability in Human Cells
Simona Giunta, Hironori Funabiki; The Rockefeller University
Cell division is the fundamental process of faithfully transmitting genetic information into each daughter cell. The
region of human chromosomes that enables cell division is the centromere, a highly specialized genomic locus made
of repetitive DNA sequences. Extensive sequence homology of the centromeric tandem arrays suggests that the
repeat structure must be tightly regulated to avoid genome instability. Here, we employed chromosome-orientation
fluorescence in situ hybridization at the centromere (which we named c-CO-FISH) to assess centromere dynamics.
This technique facilitated an unprecedented exploration into the behavior of core centromere regions and provided
evidence of ongoing mutational events occurring during a single cell cycle in human cells. We found that, while human
chromosomes undergo infrequent recombination events at the centromere, C-SCE increase during carcinogenesis and
cellular senescence, indicating that primary cells may have mechanisms to suppress satellite changes. To investigate this,
we removed the centromere-specific histone H3 variant CENP-A and observed an increase in centromere alterations
by c-CO-FISH. CENP-A is the most basal protein in the constitutive centromere-associated network (CCAN). Downregulation of other CCAN components such as CENP-C and CENP-T also led to repeats instability. Interestingly, induction
of chromosomes missegregation by different methods did not enhance centromere rearrangements, suggesting
that CENP-A, CENP-C and CENP-T may actively maintain alpha-satellite DNA repeat integrity besides their role during
chromosome segregation. We also present the first analysis of centromere repeats organization in human metaphase
chromosomes combining c-CO-FISH and super-resolution 3D structural illumination microscopy (SIM) to visualize
centromere organization within a single chromatid, while preserving DNA morphology in absence of a denaturation
step. 3D-SIM revealed that human alpha-satellite DNA exhibits a ring-like configuration at mitotically arrested
centromeres in a CENP-A-dependent manner. Together, our work points toward a novel vulnerability in genome stability
specific to centromeres.
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The Oncogene MYC Alters Mitotic Function via TPX2
Julia Rohrberg, Alexandra Corella, Sanjeev Balakrishnan and Andrei Goga; Department of Medicine, Cell and Tissue
Biology, University of California, San Francisco
The MYC oncogene is amplified or over-expressed in some of the most prevalent and difficult to treat human
malignancies including breast and liver cancers and lymphomas. MYC expression is correlating with chromosomal
instability and aneuploidy, a hallmark of aggressive cancer. To investigate if MYC directly causes mitotic abnormalities we
compared mitosis in RPE1 cells (RPE NEO) and RPE1 cells engineered to overexpress MYC (RPE MYC). We found that RPE
MYC cells are aneuploid and have more mitotic abnormalities such as multipolar spindles. Furthermore, RPE MYC cells
spend longer time in mitosis and have impaired microtubule nucleation. We observed the same MYC depended defects
in human mammary epithelial cells (HMECs).
To understand how MYC induces mitotic defects we combined bioinformatics analysis of gene expression data and in
vitro screening approaches in RPE NEO versus RPE MYC cells. We found that RPE MYC cells are highly sensitive to the
loss of the microtubule binding protein TPX2. After loss of TPX2 RPE MYC cells cannot form the mitotic spindle and
proceed normally through mitosis and eventually die. We validated this novel synthetic lethal interaction in breast
cancer cell lines that were previously characterized to have high and low MYC level. Interestingly, TPX2 expression can
be induced by MYC and the expression of TPX2 and MYC correlate in vitro and in vivo.
We found that the microtubule binding protein TPX2 is a novel synthetic-lethal interaction partner of MYC and
hypothesize that MYC can induce mitotic abnormalities via TPX2. Mechanistically understanding the mechanism of this
vulnerability will help understanding cancer development and can potentially provide novel therapies against MYCdriven cancer.
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Carcinoma-Associated Fibroblasts Lead Cancer Cell Invasion Through Fibronectin Assembly
Youmna Atieh, Sophie Richon, Andrew Clark, Carina Grass, Paolo Maiuri, Vasily Gurchenkov, Danijela Matić Vignjević
During the progression of carcinoma, cancer cells migrate through the stroma towards the blood vessels in order to
reach the circulation and metastasize. Carcinoma-associated fibroblasts (CAFs) accumulate in invasive tumors. However,
how cancer cells and CAFs cooperate during invasion remains unclear. Do CAFs assist cancer cell invasion by secreting
pro-invasion diffusible molecules or by remodeling the extracellular matrix (ECM)?
Using cancer cell spheroids embedded in collagen I containing CAFs or their conditioned media, we found that CAFs,
and not normal fibroblasts (NAFs), induce cancer cell invasion mainly by remodeling the matrix. Addition of matrix
metalloproteases (MMP) inhibitors did not block invasion of cancer cells in the presence of CAFs. On the contrary,
inhibition of actomyosin contractility completely abolished cancer cell invasion. Contraction of CAFs resulted in
fibronectin deposition. Depletion of fibronectin in CAFs using siRNA, reduced cancer cell invasion indicating that
CAF-mediated cancer cell invasion was primarily dependent on CAFs’ ability to deposit fibronectin. We are currently
investigating the role of integrins in CAF-mediated fibronectin assembly and cancer cell invasion. We also noticed that
cancer cells migrate towards CAFs suggesting a possible chemotactic response. Using Dunn’s chemotaxis chamber,
we found that cancer cells migrate along a gradient of CAF-conditioned media and a gradient of fibronectin. Finally,
orthotopic injections of cancer cells and CAFs in the colon wall of mice revealed that CAFs stimulate metastasis of cancer
cells to the liver.
In conclusion, our data show that CAFs promote cancer cell invasion by depositing fibronectin that can guide cancer cells
favoring metastasis formation.
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Go or Grow: Loss of Nonmuscle Myosin IIA in Glioblastoma Blocks Tumor Invasion and Enhances Nonmuscle Myosindependent Proliferation
Hannah Picariello1,2, James Crish2, Athanassios Dovas4, Jan Lammerding3, Peter Canoll4, Steven Rosenfeld1,2; 1Department
of Molecular Medicine, Lerner College of Medicine of Case Western Reserve University, 2 Department of Cancer Biology,
Lerner Research Institute, Cleveland Clinic Foundation, 3Meinig School of Biomedical Engineering and Weill Institute for
Cell and Molecular Biology, Cornell University, Ithaca, 4Departments of Pathology and Cell Biology, Columbia University
Medical Center
The propensity of glioblastoma (GBM) to disperse widely through brain severely limits all therapies for this uniformly
fatal disease. We have previously shown that GBM cells use nonmuscle myosin II (NMMII) isoforms to crawl through
the tight confines that characterize brain extracellular space. Of the three NMMII isoforms (IIA, IIB, and IIC), NMMIIA is
significantly up-regulated in GBM and its expression correlates inversely with patient survival. This has motivated us to
test the hypothesis that specifically targeting NMMIIA will reduce GBM dispersion and enhance survival in a rodent preclinical model of this disease. We tested this hypothesis by injecting a bicistronic retrovirus, encoding for PDGF and the
cre recombinase, into the cerebral white matter of mice carrying floxed alleles for NMMIIA and the tumor suppressor
PTEN. The resulting GBMs, deleted for both NMMIIA and PTEN, are considerably less invasive in vivo and have
severely impaired migration as assessed by several in vitro invasion assays. However, we also find that these tumors
are considerably more lethal than those intact for NMMIIA, because they are more proliferative and grow to a much
larger size. We have found by RNAseq analysis that end-stage tumors with intact NMMIIA have similar gene expression
signatures to similarly sized end-stage NMMIIA-deleted tumors, suggesting that NMMIIA deletion produces an initial
growth advantage that becomes less significant as the NMMIIA-deleted and NMMIIA intact tumors approach similar
end-stage sizes. This in turn suggests that NMMIIA deletion alters the tumor microenvironment in such a way as to
increase proliferation signaling. This leads us to our underlying hypothesis—that anything which inhibits dispersion of
tumor cells that secrete growth factors, such as PDGF, will accelerate proliferation by increasing the local concentration
of these growth factors.
We predict that effective therapy for GBM will require simultaneously targeting both invasion and proliferation. We
have attempted to block both by injecting our bicistronic retrovirus into mice with floxed alleles for PTEN, NMMIIA,
and NMMIIB. These two NMMII isoforms account for >90% of the NMMII expressed in our murine GBM models. We
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find that the injected mice have significantly increased survival with most never forming lethal tumors in vivo, and that
shRNA-mediated suppression of both NMMIIA and NMMIIB in GBM cells in vitro leads to cytokinesis defects that inhibit
tumor formation and slow proliferation. We therefore conclude that targeting both predominant NMMII isoforms in
glioma may represent an effective method to halt both glioma migration and proliferation.
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Mesenchymal Stem Cells Respond to Matrix Stiffness to Promote Mammary Carcinoma Proliferation via Prosaposin
Secretion
Seiichiro Ishihara, David R. Inman, Wan-Ju Li, Suzanne M. Ponik, Patricia J. Keely
The tumor microenvironment contains cancer cells, non-cancerous cells, and extracellular components such as
extracellular matrix (ECM). Previous studies found that interactions between the cells and the microenvironment
contribute to cancer progression via chemical and mechanical stimuli. Recently, it has been reported that mesenchymal
stem cells (MSCs) differentiate into cancer associated fibroblasts (CAFs) in response to chemical stimuli from cancer
cells and thereby promote cancer progression. However, the contribution of mechanical stimuli, such as stiffness of
the ECM, to MSCs in cancer is poorly understood. In this study, we revealed that MSCs showed CAF phenotypes and
promote mammary cancer progression in response to mechanical stimuli. On a stiff substrate, MSCs treated with
conditioned media from cancer cell culture expressed increased levels of alpha smooth muscle actin (alpha-SMA), a
marker of CAFs, compared to the MSCs cultured on a soft substrate. MSCs grown on a stiff substrate displayed higher
expression and activity of YAP and increased phosphorylation of myosin light chain (MLC) compared to a MSCs grown
on a soft substrate. In addition, knockdown of YAP by shRNA decreased expression of alpha-SMA and phosphorylation
of MLC in MSCs on a stiff substrate. Pharmacological inhibition of MLC phosphorylation by H1152 treatment also
reduced expression of alpha-SMA and activity of YAP in MSCs. Cell-cell communication between MSCs and carcinoma
cells was bi-directional, as conditioned medium from MSCs cultured on a stiff substrate, but not a soft substrate,
increased proliferation of mammary carcinoma cells. The soluble factor prosaposin was highly secreted by the MSCs
on a stiff substrate, and the addition of recombinant prosaposin increased proliferation of mammary carcinoma cells.
Furthermore, secretion of prosaposin was promoted in YAP-overexpressed MSCs on a soft substrate. These results
suggest that increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSCs to CAFs via
YAP and actomyosin contractility, which may trigger progression of mammary cancer via secretion of prosaposin.
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Tissue Mechanics Regulate Heterochromatin Formation in Breast Epithelial Cells
Josette Northcott, Russell Bainer, Valerie Weaver
Alterations to breast tissue architecture during normal development and cancer progression are known to be
accompanied by a plethora of transcriptional changes and a modified nuclear organization. We showed that the
extracellular matrix (ECM) stiffens progressively and in concert with disrupted tissue organization in breast cancer,
and that a stiffened ECM compromises breast tissue architecture. Whether ECM rigidity disrupts tissue architecture
to induce gene expression changes and whether this occurs at the level of chromatin organization remains largely
unknown. Using the normal human breast epithelial cell lines HMT-3522 S1 and MCF10A, we observed that the
formation of polarized 3D multicellular structures is associated with an increase in heterochromatin (marked by
H3K9me2 and H3K27me3) and a reduction in total RNA content, as compared to cells grown in 2D. In contrast, increased
ECM stiffness alone was sufficient to cause a loss of heterochromatin in MCF10A cells. Analysis of the spatial coherence
of gene expression changes across chromosomes demonstrated excess coincidence of differentially expressed genes
and lamina associated domains (LADs), where genes localized inside LADs were more likely to have altered levels of
expression in response to changes in ECM stiffness or multi-cellularity. Furthermore, ChIP-seq experiments revealed
a negative association between gene expression levels and enrichment for the LAD-associated H3K9me2 mark. Taken
together, our data indicate that cellular architecture and tissue stiffness have a direct effect on epigenetic regulation
of gene expression by regulating heterochromatin formation. Ongoing studies are focused on determining whether
chromatin remodeling occurs in response to mechanosignalling through signal transduction pathways and/or direct
forces via the linker of the nucleoskeleton and cytoskeleton (LINC) complex. This work supported by: NIH/NCI
R01CA192914, 1U01CA202241-01 and DOD W81XWH-13-1-0216.
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Using Quantitative Super-Resolution Microscopy to Optimize Multivalent Her2-Targeting Ligands
O. Golfetto, R. Jorand, S. Biswas, D. L. Wakefield, S. J. Tobin, K. N. Avery, K. Meyer, J. C. Williams, T. Jovanovic-Talisman
Monoclonal antibodies (mAbs) are critical components of many current and next generation cancer therapeutics. In a
number of clinical studies, combination of mAbs targeting the same antigen but different epitopes (e.g., trastuzumab
and pertuzumab) extends progression-free survival in breast cancer patients(1). According to our hypothesis, the
mechanism underlying such improved clinical outcomes involves antibody-mediated clustering of the surface-expressed
antigens: this reorganization ultimately leads to more comprehensive signaling inhibition, down-regulation of the
targeted receptor, and/or improved antibody‐dependent cell‐mediated cytotoxicity. Herein, we examined the ability
of therapeutic mAbs to affect clustering of human epidermal growth factor receptor 2 (Her2). Her2 clustering was
quantitatively interrogated with pointillistic super-resolution microscopy techniques(2) – which can measure the
distribution of proteins with nanometer scale precision and single molecule sensitivity. We established the organization
of Her2 tagged with photoactivatable GFP (paGFP) overexpressed in breast cancer MDA-MB-468 cells and determined
that Her2-paGFP largely behaves like endogenous protein. Importantly, treating these cells with therapeutic mAbs
profoundly affected Her2 organization in a valency dependent manner: while Her2 clustering was affected minimally
by the monovalent Fab domain of trastuzumab, divalent trastuzumab or pertuzumab had a distinct and pronounced
clustering effect. Critically, a combination of divalent trastuzumab and divalent pertuzumab produced the highest
level of clustering. To demonstrate that multivalent ligands can effectively replace a second antibody, we leveraged
recently discovered meditope technology(3). Meditopes are cyclic peptides that can bind tightly to a broad array of
re-engineered, meditope-enabled mAbs (memAbs) without affecting antigen binding; thus they are a rapid and sitespecific means to manipulate and/or add functionality to mAbs. Compared to the clustering induced by the combination
of trastuzumab and pertuzumab, a tetravalent meditope Fc analog combined with memAb trastuzumab not only
produced far greater clustering but was also more effective at inhibiting EGF-dependent activation of Her2, EGFR and
Akt. Thus, the multivalent meditope ligands and memAbs efficiently change geography and activation of Her2 receptor;
and they may represent a highly effective approach for cancer therapy.
1 Swain, S. M., Baselga, J. et al. The New England journal of medicine 372, 724, (2015).
2 Sengupta, P., Jovanovic-Talisman, T. et al. Nat. Methods 8, 969, (2011).
3 Donaldson, J. M., Zer, C. et al. Proc Natl Acad Sci U S A 110, 17456, (2013).
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Multivariate Analysis of Single Cells Reveals New Links Between Cell Adhesion, Small Gtpases, and the Regulation of
YAP/TAZ
Julia E. Sero and Chris Bakal; Dynamical Cell Systems Team, Division of Cancer Biology Institute of Cancer Research
Genetic and chemical perturbations often affect cell morphology and context, such as density, and may thus affect
signalling pathways indirectly. Multivariate analysis of single cells using high content imaging allows us to normalize
for changes in cell shape and identify direct effects of gene knockdown and drug treatment. In these studies, we
performed RNAi screens targeting all human Rho GTPases, GEFs, and GAPs to uncover new pathways that regulate the
mechanosensitive oncogenic transcriptional regulators YAP and TAZ in breast epithelial cells. YAP/TAZ are frequently
upregulated or hyperactivated in breast cancer, and are regulated by a number of chemical and physical stimuli. We
identified the Rac1/Cdc42 GEF beta-PIX, as well as Rac1, Cdc42, and their effector kinase PAK2 as key mediators of YAP
activation in response to cell spreading and ECM adhesion.
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13
Screening for Centriole Abnormalities in Cancer Identifies Novel Causes of Centriole Amplification
Gaëlle Marteil, Adan Guerrero, André F. Vieira, Pedro Machado, Susana Mendonça, Marta Mesquita, Beth Villarreal,
Irina Fonseca, Maria Eugenia Francia, Katharina Dores, Erin Tranfield, Joana Paredes, David Pellman, Susana A. Godinho,
Mónica Bettencourt-Dias
Centrosomes are the major microtubule organising centres of animal cells. Deregulation in their number occurs in
cancer and was recently shown to accelerate tumorigenesis. However, incidence, consequence and origins of this
abnormality are poorly understood. Here, we screened the NCI-60 panel of cancer cell lines and a set of patient samples
to systematically analyse centriole structure and number. Our screen shows that centriole amplification is widespread,
more prevalent in aggressive carcinomas and requires loss of p53 function. Moreover, we identified a recurrent feature
of cancer cells: centriole size deregulation. Further experiments demonstrated that centriole over-elongation promotes
amplification through both centriole fragmentation and ectopic procentriole formation. Furthermore, we showed that
overly long centrioles form over-active centrosomes that nucleate more microtubules, a known cause of invasiveness,
and perturb chromosome segregation. Our screen established centriole amplification and over-elongation as common
features of cancer cells and identified novel causes and consequences of those abnormalities.
14
Extracellular Matrix Malleability Regulates Breast Cancer Cell Invasion
K.M. Wisdom1, R.M. Desai2, D.J. Mooney2, O. Chaudhuri1; 1Mechanical Engineering, Stanford University, Stanford, CA,
2
School of Engineering and Applied Sciences, Harvard University, Cambridge, MA
Ductal Carcinoma, the most common form of breast cancer, progresses from noninvasive to invasive when the
mammary epithelium invades through the nanoporous basement membrane (BM) into the collagen-rich stromal tissue.
Recent studies have demonstrated that tumor cells are capable of both protease-dependent invasion, mediated by
enzymatic degradation of the extracellular matrix (ECM), and protease-independent invasion, involving cells squeezing
through pores. It is thought that proteases are required for BM invasion due to the nanoporosity of the BM. Most of
the studies investigating how pore size affects invasion have utilized rigid or elastic pores. However, many physiological
tissues and extracellular matrices exhibit inelastic behaviors. They can be viscous, with matrix flowing in response to
forces, and this flow can be irreversible, making the tissue or matrix malleable. We hypothesized that in physiological
tissues that are sufficiently malleable, cell-generated forces could physically dilate pores and facilitate invasion without
requiring proteases. Here, we investigated how ECM malleability regulates protease-independent invasion by breast
cancer cells. We developed a system of hydrogels, consisting of interpenetrating networks (IPNs) of alginate and
reconstituted basement membrane (rBM), in which malleability can be modulated independent of stiffness and ligand
density. These IPNs, which present BM ligands and are nanoporous, were then used for 3D culture of a highly invasive
breast cancer cell line (MDA-MB-231). Cells encapsulated in IPNs with low malleability exhibited rounded morphologies
and amoeboid blebbing, and did not invade into the matrix. Cells in IPNs with high malleability extended oscillatory,
actin-rich protrusions into the matrix in a protease-independent manner and were more migratory. Inhibition of Rac1
and Arp 2/3 complexes diminished protrusive activity, while inhibition of ROCK promoted protrusive activity. Protrusions
were preceded by actin-rich plaques in which actin co-localized with cortactin or β1 integrin but excluded paxillin, as in
invadopodial precursors, suggesting the protrusions to be invadopodia. In combination, these findings reveal that cells
can physically invade through nanoporous tissues in a protease-independent manner if the tissues are malleable.
15
Cellular Neighborhood Influences the Equilibria of Phenotypic States in a Heterogeneous Population
Amanda Paulson, B.S.1,2; Chithra Krishnamurthy, Ph.D.1; Zev Gartner, Ph.D.1; 1Department of Pharmaceutical Chemistry
University of California, San Francisco, 2Biomedical Sciences Graduate Program University of California, San Francisco
Tumor heterogeneity poses serious challenges in the diagnosis and treatment of cancer, and derives from both genetic
and non-genetic sources. Recent evidence suggests that steady state distributions heterogeneous cell states in tumors
and tumor cell lines can arise from stochastic state transitions within a plastic tumor cell population. However, the
role of the local microenvironment in determining the probability of state transitions, and thus the overall distribution
of cell types, has not been explored. To investigate the role of the microenvironment on phenotype distributions,
we utilize the transformed epithelial cell line MCF10AT. MCF10AT cells have a stable distribution of epithelial and
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mesenchymal phenotypes, which can be distinguished by their differential expression of cell surface markers CD49f and
CD24. Enriched populations of these cells relax to the same steady state population-level distribution as the parental
cell line over time. Surprisingly however, single cell clones established from the parental cell line are homogeneous
in their morphology, span a range of EMT phenotypes, and do not return to the parental steady state phenotype
distribution. Transcriptional analysis of the stable epithelial and mesenchymal clones reveal differential expression
levels of ECM molecules, secreted factors, and cell-adhesion molecules, suggesting a role for local cell-cell and cell-ECM
communication in reinforcing cellular phenotypes. Consistent with this notion, we found laminin and fibronectin biased
the parental population towards the epithelial and mesenchymal phenotypes, respectively.
Additionally, when populations of epithelial- or mesenchymal-like cells purified by CD49f and CD24 were co-cultured
with stable clones, the distribution cell states was altered. Thus, we hypothesize that local feedback from the
extracellular matrix, neighboring cells or secreted factors can counterintuitively both maintain a heterogeneous
population or stabilize homogeneous cell states in a differentiation hierarchy. To better understand the interactions
between heterogeneous cell states within a population, we generate a cellular Potts model to allow us to explore the
impact of differential growth rates and probabilistic cell-fate changes, together with cell-cell interactions that locally
modify these probabilities. Moreover, we investigate how perturbing local microenvironmental cues can influence
cellular phenotypic transitions. These results may have significant implications for our understanding of tumor
heterogeneity and its relationship with the local microenvironment.
16
Pten Deletion in a Mouse Model of Synovial Sarcoma Results in a Pro-Inflammatory Microenvironment that
Generates Lung Metastases
Jared J. Barrott1,2,3, Alexander J. Lazar4, Kevin B. Jones1,2,3; 1Department of Orthopaedics, University of Utah,2Department
of Oncological Sciences, University of Utah, 3Huntsman Cancer Institute, University of Utah, 4Departments of Pathology
and Translational Molecular Pathology, M.D. Anderson Cancer Center
Solid tumor metastasis is a complex biology, impinged upon by a variety of dysregulated signaling pathways. PI3’-lipid
signaling has been associated with metastasis and inflammation in many cancers, but the relationship between tumor
cell intrinsic PI3’-lipid signaling and inflammatory cell recruitment has remained enigmatic. Elevated PI3’-lipid signaling
associates with progression of synovial sarcoma, a deadly soft-tissue malignancy initiated by a t(X;18) chromosomal
translocation that generates an SS18-SSX fusion oncoprotein. Here, we show in genetically engineered mouse models
(GEMMs) of locally induced expression of SS18-SSX1 or SS18-SSX2 that Pten silencing dramatically accelerated and
enhanced sarcomagenesis without compromising synovial sarcoma characteristics. PTEN deficiency increased tumor
angiogenesis, promoted inflammatory gene expression and enabled highly penetrant spontaneous pulmonary
metastasis. PTEN deficient sarcomas revealed infiltrating myeloid-derived hematopoietic cells, particularly macrophages
and neutrophils, recruited via PI3’-lipid-induced CSF1 expression in tumor cells. Moreover, in a large panel of human
synovial sarcomas, enhanced PI3’-lipid signaling also correlated with increased inflammatory cell recruitment and
CSF1R signal transduction in both macrophages and endothelial cells. Thus, both in the mouse model and in human
synovial sarcomas, PI3’-lipid signaling drives CSF1 expression and associates with increased infiltration of the monocyte/
macrophage lineage as well as neutrophils.
17
CaV1 Calcium Channels Regulate Carcinoma Cell Collective Invasion
Grasset E.1, Bonan S.1, Bozec A.2, Maiel M.1, Duranton C.3, Meneguzzi G.1, and Gaggioli C.1; 1INSERM, U1081, CNRS,
UMR7284, Institute for Research on Cancer and Aging, Nice (IRCAN), University of Nice Sophia Antipolis, Medical School,
28 Avenue Valombrose, F-06107 Nice, France, 2 Face and Neck University Institute, 31 Avenue de Valombrose, 06103,
Nice, France, 3 LP2M CNRS-UMR7370, University of Nice-Sophia Antipolis, Medical School, 28 Avenue Valombrose,
F-06107 Nice, France.
Metastatic spreading of carcinoma cells is a complex process involving cross talk between tumor cells and stroma.
To disseminate the surrounding extracellular matrix (ECM), carcinoma cells follow a mode of invasion defined as
“collective” because of the migration pattern of cohesive cancer cells. The cellular mechanisms sustaining such cell
migration pattern have been recently described; however, the underlying signaling pathways remain poorly understood.
Thus, identification of small molecule inhibitors able to block collective carcinoma cell invasion would potentially help to
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identify the molecular mechanisms governing this mode of invasion and also pave the way to therapeutic strategies for
patients suffering from aggressive carcinoma.
To this aim we screened small molecule libraries to identify agents able to inhibit epidermoïd carcinoma SCC12 cells
invasion in vitro using ‘three-dimensional” organotypic invasion assays that mimic mesenchymal and epidermal tumor
cell interactions in culture. This model enables SCC12 cells to invade the ECM in response to a carcinoma associated
fibroblast (CAF) activity that remodels the ECM by creating tracks subsequently used by SCC12 cells to collectively
penetrate the matrix.
Screening of 378 compounds identified 52 chemicals that efficiently block SCC12 cells invasion in vitro. Despite the
pleotropic panel of inhibitors, CaV calcium channel inhibitors were specifically selected for further investigations.
Indeed, 9 out of 11 CaV blockers inhibited SCC12 cells invasion without any detectable toxic effect on SCC12 and CAF
cells. Furthermore, knockdown of CaV1.1 in SCC12 cells was sufficient to inhibit their collective invasion capacity.
Mechanistically, we demonstrated that CAF-dependent matrix stiffening induces CaV1.1 expression in SCC12 cells
and consequently calcium entry. Thereafter, calcium signaling regulates different signaling pathways. In particular
calcium regulates: the activity of the small GTPase Cdc42, the induction of partial epithelial-to-mesenchymal transition
(conservation of E-cadherin expression) and cell migration, therefore leading to carcinoma cell invasion. Interestingly,
in biopsies of human head and neck carcinoma, CaV1.1 expression was found to significantly correlate with carcinoma
invasion and matrix remodeling. Moreover, patient-derived carcinoma spheroid assays confirmed the inhibitory effect of
CaV1 blockers on carcinoma invasion.
In light of these results, we propose that CaV1 calcium channels are key regulators of carcinoma collective invasion.
Thus, CaV1 blockers such as Verapamil or Diltiazem (FDA approved drugs), could be used in combination with
therapeutics for patient with aggressive carcinoma.
18
Corruption of Signaling Filters in Cancer Cells Can Drive Cellular Misperception and Hyperproliferation
L.J. Bugaj1, A. Sabnis2,3, A. Mitchell4, J. Garbarino1, T. Bivona2,5, J.E. Toettcher6, W. Lim1,7,8;
1
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 2Helen
Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Franciso, CA, 3Department
of Pediatrics, University of California, San Francisco, San Francsicso, CA, 4Program in Systems Biology, University of
Massachusetts Medical School, Worcester, MA, 5Division of Hematology and Oncology, University of California, San
Francisco, San Francisco, CA, 6Department of Molecular Biology, Princeton University, Princeton, NJ, 7Center for Systems
and Synthetic Biology, University of California, San Francisco, San Francisco, CA, 8Howard Hughes Medical Institute, San
Francisco, CA
Introduction
Within cancer, it is believed that oncogenes drive disease through constitutively high levels of pathway signaling. An
alternative and untested hypothesis is that cancer mutations might ‘rewire’ a cell’s perception of external signals,
so that a cell might errantly interpret non-proliferative signals as proliferative. Building upon recent work using
optogenetics to quantitatively characterize signal filtering properties within living cells, we tested the hypotheses that
input/output responses within the Ras/Erk pathway might be altered in cancer cells, and that these alterations could
contribute to disease phenotypes like hyperproliferation.
Methods
Optogenetic activation of Ras signaling was accomplished using inducible membrane recruitment of a Ras activator
(optoSOS). Optogenetic experiments were conducted both through 1) live cell fluorescent microscopy and 2) newly
developed custom devices for fully controllable illumination of 96- and 384- well plates followed by single cell
immunofluorescence.
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Results
We measured how a panel of Ras/Erk mutant non-small cell lung cancer lines responded to dynamic Ras inputs, and
we found a BRAF mutant with significantly extended kinetics of Erk activation (~10-fold) and inactivation (~20 fold)
compared to non-transformed cell types. We showed that these changes were a function of the mutant BRAF, and a
further search uncovered other BRAF-mutant cell lines that showed similar behavior. We found that cancer-associated
paradox-activating BRAF inhibitors also extend Erk kinetics within non-transformed cells, and we used these as a model
to study whether changes in Erk kinetics and signal filtering can result in cellular misperception. We found that while
wild-type and rewired cells responded similarly to constant signals, rewired cells were unable to discriminate dynamic
signals from constant ones, leading to improper accumulation of downstream immediate early genes and CyclinD1. By
measuring proliferative response to a systematic scan of dynamic Ras input signals, we further demonstrated that, while
wild-type and rewired cells both proliferated under high amounts of Ras, rewired cells also proliferated across a range of
weaker Ras inputs that the wild-type cells ignored.
Conclusions
This study represents a new paradigm for understanding cancer through a functional lens and highlights the unique
ability of optogenetics to quantitatively probe the disease state of signaling networks in living cells. Rather than
cataloguing the mutations within a cancer cell, we found that the cell’s internal computation of external inputs can be
fundamentally changed in a manner contributing to disease.
19
Tributyltin Alters Interleukin 1 beta and Interleukin 6 Production and mRNA Expression from Human Immune Cells
Shyretha Brown1, Mariam Boules1, Nafisa Hamza2, and Margaret M. Whalen2 ;1Department of Biological Sciences,
Tennessee State University, 2Department of Chemistry, Tennessee State University
Tributyltin (TBT) is an organotin compound that has been used as a biocide in a variety of industrial applications and
contaminates the environment. Mammals exposed to TBT have shown increased incidences of tumors. Interleukin 1
beta (IL-1β) is a pro-inflammatory cytokine that is produced by monocytes and macrophages. Interleukin 6 (IL-6) is an
anti-inflammatory agent, as well as a pro-inflammatory mediator, that is produced by T lymphocytes and monocytes.
Both IL-1β and IL-6 are responsible for immune response regulation and cellular growth. The current study examines
whether TBT modifies production (intracellular levels/secretion) and mRNA expression of IL-1β and IL-6 from human
lymphocytes (monocyte-depleted (MD) peripheral blood mononuclear cells (PBMCs)). MD-PBMCs were exposed to TBT
concentrations of 2.5 to 200 nM for 10 min, 30 min, 6 h, and 24 h. IL-1β and IL-6 secretion was monitored using enzymelinked immunosorbent assay (ELISA), intracellular protein levels were analyzed by western blot, and mRNA expression
was examined using RT-PCR. The results indicated that 24 h exposures to TBT elevated IL-1β secretion compared to
control cells at all concentrations of TBT and intracellular levels were increased at TBT concentrations of 2.5 to 100nM.
24 h exposures to 200 nM TBT generally decreased IL-6 secretion compared to control cells while intracellular levels
were increased at all TBT concentrations. RT-PCR indicated that increases in gene expression were seen at the higher
concentrations of TBT for both cytokines. These data indicate that secretion and/or intracellular levels of IL-1β and IL-6
are increased at every TBT exposure indicating that TBT stimulates IL-1β and IL-6 production in MD-PBMCs which may
potentially affect immune competence and cancer invasiveness.
20
Macrophage-Dependent Cytoplasmic Transfer Drives Melanoma Metastasis in Vivo
Minna Roh-Johnson1, Arish N. Shah1, Julie di Martino2, Raj Kumud Poudel1, Rafael E. Hernandez3, Susumu Antoku4,
Lalita Ramakrishnan5, Jose Javier Bravo-Cordero2, and Cecilia B. Moens1; 1 Fred Hutchinson Cancer Research Center,
Basic Sciences Division, Seattle, WA, USA, 2 Icahn School of Medicine at Mount Sinai, Division of Hematology and
Oncology, Department of Medicine, New York, NY, USA, 3Seattle Children’s Hospital, Infectious Disease, Seattle, WA,
USA, 4Columbia University, Department of Pathology and Cell Biology, New York, NY, USA, 5University of Cambridge,
Department of Medicine, Cambridge, England
During cancer progression, interactions between tumor cells and immune cells play critical roles in the initiation
of metastasis. In malignant melanoma, it is unclear how a melanocyte transitions from a premalignant nevus to an
invasive tumor cell, and which components in the tumor microenvironment regulate this switch. A major limitation to
understanding this switch in vivo is the lack of genetically tractable model systems that are amenable to high-resolution
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imaging. We have overcome this obstacle by directly visualizing tumor cells and their interactions with stromal cells
in zebrafish. We found that injecting either human or zebrafish melanoma cells results in dissemination from the site
of injection. Live imaging revealed that when in contact with tumor cells, macrophages form long, thin, and dynamic
protrusions. We hypothesized that these protrusions may be a mechanism for cell-cell communication between tumor
cells and tumor-associated macrophages that may influence metastatic potential. Consistent with this hypothesis,
we varied macrophage recruitment to the primary tumor by altering gene function of myd88, a key regulator in the
inflammatory response pathway, and found that depleting zebrafish larvae of myd88 reduced macrophage recruitment
to the primary tumor and subsequent tumor cell contact, whereas specific expression of constitutively active myd88
in macrophages increased macrophage recruitment and tumor cell contact. These genetic backgrounds resulted in
decreased and increased melanoma dissemination, respectively. These results suggest that first, expression of Myd88 in
macrophages is sufficient to drive melanoma cell metastasis; and second, that contact between macrophages and tumor
cells promotes tumorigenesis in vivo. Recently it has been shown that tumor progression is accelerated by exchange
of active molecules between tumor cells (Zomer et al., 2015). Thus, we tested whether macrophages were capable of
transferring cytoplasm to tumor cells to promote metastasis. We found that, indeed, macrophages transfer cytoplasm
to tumor cells. Importantly, we discovered that macrophage cytoplasmic transfer was correlated with melanoma cell
dissemination. These results suggest that recruitment of macrophages to the primary tumor and cytoplasmic transfer
of molecules potentially through cell contact-mediated protrusions may be a mechanism by which macrophages drive
tumor cell dissemination.
21
Down-Regulation of MLCK Promotes Cell Migration and Invasive Behavior of Breast Epithelial Cells via Increased
Expression of EGFR and ERK/JNK Signaling
Dayoung Kim and David M. Helfman
Alterations in the actin cytoskeleton and changes in the expression of various cytoskeletal proteins are a feature of
transformed cells and tumors. Myosin light chain kinase (MLCK) expression is down-regulated in various cancers,
including invasive and metastatic breast tumors. However, little is known about how loss of MLCK expression
contributes to tumor progression. Here, we provide evidence for the role of MLCK in breast cancer following inhibition
of its function using siRNAs or pharmacological inhibitors in untransformed MCF10A breast epithelial cells. Genetic
depletion of MLCK disrupted cell-cell adhesions, increased single-cell or collective cell migration and invasion, and
enhanced formation of linear actin filaments and focal adhesions at the leading edge of migratory cells. Adhesion to
fibronectin was increased with development of lamellipodia. These changes were not associated with induction of
genes associated with epithelial-to-mesenchymal transition (EMT), but rather with the increased expression of EGFR
and HER2 and activation of their downstream MEK-ERK and JNK-Paxillin signaling pathways. Inactivation of EGFR, ERK or
JNK prevented the loss of cell-cell adhesions and increased migration in MLCK down-regulated cells. In addition, downregulation of MLCK cooperated with HER2 in MCF10A cells to promote cell migration and invasion and decreased levels
of MLCK is associated with a poor prognosis in HER2 positive breast cancer patients. Thus, increased expression of EGFR
and HER2 following down-regulation of MLCK promotes cell migratory behaviors. Finally, inactivation of the catalytic
function of MLCK using pharmacological agents inhibited cell migration and invasion, and did not affects epithelial
adhesions. Full-length, kinase-dead or N-terminal fragment of MLCK expression recovered actin organization and
impaired adhesions in MLCK down-regulated cells. These results reveal that catalytic and non-catalytic function of MLCK
have distinct roles in the regulation of epithelial migration and invasion. Thus, loss of MLCK contributes to the migratory
properties of epithelial cells resulting from changes in cell-cell and cell-matrix adhesions, and increased EGFR and HER2
signaling. These findings suggest that decreased expression of MLCK might play a critical role during tumor progression
by facilitating the metastatic potential of tumor cells.
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22
The Dynamic Control of GSK3β Localization and Function via PI3K-Akt-mTOR Signaling and Endomembrane Traffic
Stephen Bautista, Ivan Boras, and Costin N. Antonescu
Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that regulates numerous cellular processes such as
apoptosis, self-renewal, growth, and metabolism. GSK3 activity is controlled through different regulatory residues
that may inhibit or enhance kinase activity. For example, many signals trigger GSK3 S9 phosphorylation, including
phosphatidylinositol-3-kinase (PI3K)-Akt, which negatively regulates GSK3 kinase activity towards some substrates.
Importantly, GSK3 exhibits distinct functions depending on its localization to the nucleus or cytosol; however, the
signals that control GSK3 localization are poorly understood. Previous studies suggested that PI3K may control GSK3
nucleocytoplasmic shuttling, To resolve the signals that control GSK3 function by controlling its nucleocytoplasmic
shuttling, we employed various chemical and molecular perturbations of PI3K-dependent signaling pathways, and
examined the effects on GSK3 cellular localization and dynamics. We found that PI3K-Akt-mTOR signaling controls
GSK3 nuclear localization. As PI3K-Akt-mTOR signaling exhibits reciprocal regulation with endomembrane traffic, we
also examined the effect of perturbations of clathrin-mediated endocytosis and endosomal traffic on GSK3 localization
and function. Our results indicate that GSK3 nucleocytoplasic shuttling is regulated by PI3K-Akt-mTOR, and further
suggest that organization of these signals on specific endomembrane compartments is critical for spatial control of
GSK3 and this GSK3 function. Given the role of GSK3 in diseases such as cancer, obtaining a better understanding of
how endomembrane traffic processes organize different mitogenic and metabolic signaling pathways to regulate GSK3
subcellular localization and thus GSK3 function may give insight to the development of novel cancer biomarkers or
better anti-cancer drugs.
23
Wnt5a Signaling Through Organelle Dynamics Controls Cell Polarity and Drives Directional Persistence in Migrating
Melanoma Cells
Mary Katherine Connacher, Natalie Ahn
Wnt5a plays an emerging role in cancer malignancy and strongly enhances cell invasion in melanoma. We discovered
a new intracellular complex, named the “Wnt5a-receptor-actomyosin polarity (WRAMP) structure”, which provides a
novel mechanism to link Wnt5a, a signaling ligand that controls cell polarity, to rear-directed signaling and directional
cell movement. This structure involves the formation of late endosomes which interact dynamically with F-actin at the
cell posterior to create the WRAMP structure. Our objective is to determine how this structure functions as well as the
mechanisms for its formation and rear-polarization.
We demonstrate that various melanoma cell lines, and some other cell lines, form WRAMP structures. Treatment
with exogenous Wnt5a leads to the formation of more structures. In migrating cells, the WRAMP structure is found
at the rear and is associated with tail-end retraction. The presence of the WRAMP structure is correlated with an
increase in the speed as well as the directional persistence of cell migration. A key marker for the WRAMP structure is
melanoma cell adhesion molecule (MCAM), which undergoes internalization into endosomes in response to Wnt5a. We
show that both MCAM and the Wnt5a receptor, Fzd3, co-localize in endosomes at WRAMP structures, suggesting cointernalization of signaling effectors. We used a mass spectrometry strategy, “Dynamic Organelle Trafficking by Signaling
(DOTS-MS)”, to identify other proteins that polarize at WRAMP structures. The ERM proteins, ezrin and moesin, localize
with PI4,5P2 at the WRAMP structure in response to Wnt5a. We demonstrate that both ezrin and moesin are required
for WRAMP structures to form. Live-cell imaging provided temporal information about the recruitment of components
to WRAMP structures. The polarization of ezrin to the site of the WRAMP structure precedes MCAM, which precedes
myosin IIb, suggesting that PI4,5P2 might be responsible for directing the WRAMP structure to the cell rear.
We previously reported that the formation of the WRAMP structure is coupled to a rear-polarized increase in
cytoplasmic Ca2+, followed by actomyosin contractility. We now have identified the presence of the ER-associated Ca2+
sensor, STIM1, and the plasma membrane Ca2+ channel, ORAI1, at the WRAMP structure, suggesting that the rearpolarized Ca2+ elevation involves STIM-mediated opening of ORAI channels. Thus, the WRAMP structure explains how
cells can integrate cell polarity with the mobilization of Ca2+ and subsequent membrane contractility, key steps needed
for cell movement and invasion. We propose that the WRAMP structure provides a mechanism by which cells that are
initially unpolarized acquire directional persistence in response to Wnt5a signaling and become invasive.
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24
Targeting Endoplasmic Reticulum-Resident Proteins for the Treatment of B Cell Cancer
Chih-Hang Anthony Tang1, Juan R. Del Valle2 and Chih-Chi Andrew Hu1; 1The Wistar Institute, Philadelphia, PA,
2
Department of Chemistry, The University of South Florida, Tampa, FL
The IRE-1/XBP-1 pathway is the most conserved endoplasmic reticulum (ER) stress response. We discovered that genetic
deletion of XBP-1 significantly decelerates the progression of chronic lymphocytic leukemia (CLL) in Emu-TCL1 mice.
We set up a high throughput in vitro screening platform to look for effective inhibitors that can block the IRE-1/XBP-1
pathway. We discovered a specific inhibitor, B-I09, which blocks the IRE-1/XBP-1 pathway with high potency and efficacy.
B-I09 clearly suppresses activation of the IRE-1/XBP-1 pathway as evidenced by the decreased mRNA and protein levels
of XBP-1 in intact cells. B-I09 specifically targets mouse CLL cells in vivo by inducing apoptosis. We also discovered that
genetic deficiency of XBP-1 compromises the BCR signaling which is crucial for the survival of CLL cells. To test whether
pharmacological inhibition of XBP-1 could enhance the effect of inhibitors to the BCR signaling, we combined B-I09
with Bruton’s Tyrosine Kinase (BTK) inhibitor, ibrutinib, to treat human CLL cells. A true and strong synergistic effect in
pharmacology was observed using the Chou-Talalay combination index method. Such results suggested that B-I09 can
decelerate the growth of CLL either as a single agent or in combination with ibrutinib. Our finding in synergism may
be important because B-I09 may help ibrutinib to achieve higher cytotoxicity in CLL cells at a lower dose, addressing
ibrutinib’s toxicity issue. Our studies also led us to discover that IRE-1 interacts with another ER-resident protein called
STING. STING is critical for cytoplasmic DNA sensing and interferon production. The agonists of STING are recently
shown to activate anti-tumor functions of T and NK cells, resulting in efficient control of cancer in mouse models. We
showed that the IRE-1/XBP-1 pathway is required for the interferon-producing function of STING, and that the agonists
of STING selectively trigger mitochondria-mediated apoptosis in normal and malignant B cells. Upon stimulation, STING
was degraded less efficiently in B cells, implying that prolonged activation of STING can lead to apoptosis. Transient
activation of the IRE-1/XBP-1 pathway partially protected agonist-stimulated malignant B cells from undergoing
apoptosis. In CLL-bearing mice, injection of the STING agonist, 3’3’-cGAMP, induced apoptosis and regression of
leukemia. Similarly efficacious effects were elicited by 3’3’-cGAMP injection in syngeneic or immunodeficient NSG mice
grafted with malignant B cells. These data suggested that, in addition to boosting anti-tumor immune responses, STING
agonists can directly eradicate CLL as well as other types of B cell malignancies in vivo. IRE-1 and STING are thus useful
ER-resident protein targets for the treatment of B cell cancer.
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Establishing the Involvement of Exosomal NM23 in Pro-Angiogenic Communication Between Triple Negative Breast
Cancer Cells and Their Vascular Targets
Suzann Duan, Senny Nordmeier, Iain L.O. Buxton; Department of Pharmacology, University of Nevada, Reno School of
Medicine
Decades of research has nurtured the idea that breast cancer cells of the primary tumor prime the metastatic
microenvironment prior to establishing metastases at distant sites in the body. While this process is thought to facilitate
neocolonization of dormant circulating tumor cells, the mechanisms by which breast cancer cells communicate to their
remote targets remain largely unknown. Of special interest are exosomes, cell-derived nanoscale carriers of biological
information that act as novel mediators of cell-to-cell communication and have been implicated in a variety of cancers.
Our lab has previously shown that triple negative human breast cancer (MDA-MB-231) cells release exosomes carrying
NM23, a nucleoside diphosphate kinase (eNDPK) that is suggested to act as an upstream mediator of vasodilation and
pro-angiogenic activity through its action of elevating extracellular ADP/ATP levels. To further elucidate the role of
eNDPK in breast cancer signaling, we first establish that MDA-MB-231 cells elaborate exosomes that target the lung
prior to the development of metastases. Next, we show that these exosomes induce a pro-angiogenic phenotype in
murine pulmonary endothelial cells and stimulate proliferative effects on pulmonary vasculature in vivo. Lastly, we
demonstrate that treatment of pulmonary endothelial cells with the NDPK-specific inhibitor ellagic acid (EA) and an
antagonist to the ADP/ATP-activated P2Y1 receptor results in amelioration of pro-angiogenic characteristics.
The 2016 ASCB Doorstep Meeting l www.ascb.org/doorstep
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The studies reported here suggest the involvement of exosomes and eNDPK in cell-cell communication between
primary tumor cells and their vascular targets. The significance of uncovering the mechanism(s) by which cancer cells
metastasize is emphasized by the fact that recurrence of cancer at distant sites is associated with the most negative
outcome in women diagnosed with breast cancer. Implication of eNDPK-mediated signaling in metastasis may challenge
current and invasive treatment methods, such as immediate resection of the primary tumor, in favor of developing
novel small molecule inhibitors of eNDPK signaling. Finally, eNDPK can be further explored as a biomarker for detecting
the early stages of primary tumor development and metastasis.
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Hemodynamic Profiles Tune the Arrest and Extravasation of Circulating Tumor Cells
Follain Gautier1, Osmani Naël1, Allio Guillaume1, Fekonja Nina1, Harlepp Sebastien2, Goetz Jacky1; 1INSERM U1109 MN3T - Tumor Biomechanics, 67200 Strasbourg; 2IPCMS - UMR 7504 - DON, 67200 Strasbourg
Cancer progression is a complex multi-step process in which a growing and invasive primary tumor spreads to distant
organs to form life-threatening metastases. While multiple evidences suggest that metastatic seeding is driven by
tumor cell-intrinsic as well as local environmental cues, contribution of mechanical cues such as vascular architecture or
hemodynamic factors has been poorly studied. In our laboratory, we develop intravital correlative microscopy, a pioneer
imaging tools, allowing us to track single metastatic cells at high-resolution (Karreman et al. 2016). Using the latter, we
aim to elucidate the impact of blood flow on the arrest, the adhesion and the extravasation of circulating tumor cells in
vivo by combining intravital and high-speed imaging as well as biophysical analysis of blood flow parameters. Because
arrest of circulating tumor cells (CTCs) in vivo occurs mostly in hemodynamically favorable vessels, we questioned
whether tuning blood flow would alter both their arrest and the extravasation efficacies. For doing so, we exploit the
advantages of 2-days post-fertilization ZF embryos where hemodynamic profiles can be tuned using pharmacological
approaches. We have successfully used several drugs that either increase or decrease flow velocities in the ZF embryo,
without impact key traits of tumor cells in vitro (adhesion, migration). Interestingly, blood flow tuning affects multiple
steps of tumor cell metastasis in the living ZF embryo. First, using live imaging of CTCs upon injection, we observed
that low flow forces favored the initiation of stable tumor cell arrest while high flow forces tend to favor circulation of
the CTCs, impeding its arrest. Using optical tweezers applied both in vivo and in vitro, we identified a threshold a flow
and adhesion forces favoring successful arrest of CTCs, independently of the mechanical constrinats imposed by vessel
architecture. We then developed a quantitative mapping of the arrest of CTCs at 3h post-injection (hpi) over several
embryos and observed that tuning flow forces significantly altered the favored location of arrest of CTCs. Finally, using
quantitative mapping of extravasation at 24 hpi, we observed that while low flow forces drastically impaired tumor
cell extravasation, high flow forces stimulated the extravasation efficacies suggesting that flow forces can stimulate the
transmigration of CTCs. Using our intravital CLEM approach, we are currently testing a potential contribution of the
endothelial in this process. Altogether, these results demonstrate that hemodynamic profiles at metastatic sites can
regulate important steps of the metastasis cascade.
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Mechanisms of Transcriptional Regulation Under Oxidative Stress, a Job for the
Nuclear Structural Protein NuMA
Shirisha Chittiboyina1, Swaathi Jayaraman1, Patricia Abad1, Kurt B. Hodges1, Jacob Galan2, W. Andy Tao2, Sophie A.
Lelièvre1; 1Department of Basic Medical Sciences, 2Department of Biochemistry, Purdue University, West Lafayette, IN
Changes in higher order chromatin organization and local epigenetic modifications are driving cancer onset and
progression by influencing gene transcription. Yet, how nuclear architecture cooperates with epigenetic pathways to
control phenotypes remains to be understood. We have been studying the structural protein, nuclear mitotic apparatus
(NuMA) for its function at the boundary of nuclear architecture and chromatin organization with an impact on major
aspects of cellular homeostasis, such as phenotypically normal differentiation, nucleolus-based stress response and
DNA repair. Through stringent mass spectrometry analysis that involved antibodies spanning three different regions of
NuMA, we identified the transcriptional coactivator lens epithelium derived growth factor (LEDGF) as an interacting
partner. LEDGF has a protective role by up-regulating antioxidant proteins in case of oxidative stress (OS), a major
promoter of breast cancer that influences nuclear architecture and epigenetic pathways such as DNA methylation and
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The 2016 ASCB Doorstep Meeting l www.ascb.org/doorstep
histone modifications, notably H3K9me2. Our preliminary investigation indicated that LEDGF expression was decreased
in invasive triple negative breast cancer (TNBC) compared to the preinvasive stage. Interestingly, increased H3K9me2
expression represses LEDGF and correlates with increased expression of chromatin remodeling factor, remodeling and
spacing factor (RSF-1) protein that we have found to be another binding partner of NuMA. RSF-1 acts as a transcriptional
repressor when overexpressed in case of OS. Our central hypothesis is that NuMA-LEDGF-RSF interaction controls
cancer progression under stress by influencing transcriptional regulation. Western blot analysis reveals that LEDGF
expression is highest in TNBC MDA-MB 231 and BT-20 cells compared to luminal A, MCF7 cells and luminal B, BT-474
cells. Moreover, tissue microarray analysis of 80 invasive breast tumor samples suggests that LEDGF is expressed only in
TNBC cases. To test our hypothesis we are using the HMT-3522 model of TNBC progression that in 3D cell culture mimics
the phenotypically normal luminal epithelium (non-neoplastic S1 cells), ductal carcinoma in-situ (DCIS; S1-derived S2
cells) and invasive ductal carcinoma (IDC; S2-derived T4-2 cells). Induction of OS with 250 μM H202 in S1 cells decreases
NuMA expression, yet NuMA interaction with LEDGF is strengthened compared to control. NuMA and RSF-1 follow a
similar expression pattern as LEDGF with lower expression in T4-2 cells compared to S2 cells, and increased expression
under OS in S2 cells. These results support the idea that microenvironmental stress influences nuclear mechanisms
of transcription control, and calls for further investigation of the pathways regulated by NuMA-LEDGF and/or RSF-1
interaction.
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Can We Use Cell Shape as Biomarker of Cancer Cell Invasiveness?
Elaheh Alizadeh1, Samanthe M. Lyons2, Katherine Schaumberg2, Joshua Mannheimer2, Jordan Castle3,
Zachary R Bodmer1,2, Ashok Prasad1,2; 1Department of Chemical and Biological Engineering, 2School of Biomedical
Engineering, 3Department of Biology, Colorado State University
Introduction: This work is based on the hypothesis that shape of the cell on substrates is determined by the active
mechanical properties of the cytoskeleton. Since invasive cancer cells are believed to have altered mechanical
properties compared with non-invasive cells, it follows that changes in shape of cancer cells may correlate with the
acquisition of invasive capacity. The central aim of this study is trying to understand whether this correlation exists and
is reproducible across cancers.
Materials and Methods: We characterize shape changes for four osteosarcoma paired cell lines, two pairs of human and
two pairs of murine cell lines. Each pair consists of a cell line that has high invasive capacity, and which is derived from
its paired low invasive line after selection for metastasis. We cultured cells on a substrate, then they were fixed and
stained to capture boundary of nuclei and cell’s image. Images were processed to eliminate background noise. In order
to quantitatively describe cell’s shape, we chose a set of 29 geometric shape descriptors. As a more general method we
also used Zernike Moments, which are calculated from a set of orthogonal basis functions. Since we are dealing with
high dimensional data we use principal component analysis (PCA) to examine changes in cell shape. We also used a
machine learning neural network classifier to distinguish between different cell populations.
Results and Discussion: By analyzing geometric parameters, we discovered two types of shape changes in the cancer
cell lines under study. In type 1, high invasive lines were smaller, more elongated and more irregular with respect to
their paired low invasive lines. Three of the paired lines belonged to this class. However, type 2 shape changes showed
an opposite trend with high invasive line being larger, less elongated and less irregular in comparison with its low
invasive pair[1]. For Zernike moments we developed a novel method that looks at shape changes in the space of the
first two principal components. This method allows us to classify multiple classes and quantify the distance between
them in multidimensional space. Using this method we observed the same two types of shape changes with descriptive
parameters. Our machine learning algorithm was able to distinguish between high invasive lines from their low invasive
lines with good accuracy[1].
Conclusions: With these methods, we can understand and identify shape changes of aggressive cancer cells. If these
findings hold across a wide variety of cancers, they may be able to provide crucial information for assessing the
invasiveness of cancer cells.
Reference : [1] Lyons, S. M., Alizadeh, E., Mannheimer, J., Schuamberg, K., et al., Biology Open 2016, 5, 289-299.
The 2016 ASCB Doorstep Meeting l www.ascb.org/doorstep
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YAP-Independent Mechanotransduction of Mammary Epithelial Cells in 3D Culture Model of Cancer Invasion
Joanna Y. Lee1, Jessica Chang2, Sungmin Nam1, and Ovijit Chaudhuri1; 1Department of Mechanical Engineering, Stanford
University, 2Department of Genetics, Stanford School of Medicine
One of the critical steps in breast cancer is cell invasion, when a cancer cell breaks from the primary tumor and ‘invades’
through the basement membrane (BM) into the collagen-1 (col-1) rich stroma, enabling travel to distant tissues.
Interestingly, increased stiffness of the extracellular matrix (ECM) correlates with invasion and 3D culture models of
normal mammary epithelium show that enhanced stiffness induces an invasive phenotype. Studies have converged
upon the finding that YAP, a transcriptional regulator that is deregulated in diverse cancers, is the transducer of ECM
stiffness. However, these studies were primarily performed in 2D culture and involved col-1, a ligand that activates
distinct signaling pathways and is not normally found in the BM. Here, we examined the role of culture dimensionality,
matrix stiffness, and col-1 density, in the activation of YAP during stiffness-induced invasion. For 3D culture, we
generated interpenetrating networks (IPNs) of reconstituted basement membrane (rBM) and alginate, which allow
stiffness to be tuned in the absence of col-1 and independently of ECM protein concentration and matrix pore size.
Traditionally used hydrogels composed of rBM and col-1, with stiffness tuned by increasing concentrations of col-1,
were also produced for 3D culture. As a control, polyacralymide (PAM) gels with the same range of stiffness were used
in 2D culture studies. Our results show that enhanced 3D stiffness increases MCF10A cell invasion and proliferation.
However, in contrast to results from 2D culture, invasive phenotypes in 3D cultured cells did not correlate with nuclear
localization of YAP, indicating lack of YAP activity. The dispensible nature of YAP in 3D stiffness sensing was supported
by RNA-seq analysis, which showed a lack of gene expression of YAP downstream targets. RNA-seq identified 389
differentially expressed genes in response to enhanced 3D culture stiffness, indicating the likelihood of a novel set of
transcription factors involved in 3D mechanosensing. Interestingly, 310 other genes were differentially expressed in
response to increased col-1 density at higher stiffness, suggesting that post-invasion exposure to col-1 in the stroma
induces significant changes in cell phenotype. Furthermore, YAP colocalized with F-actin in YAP-activating 2D culture
conditions, but not in YAP-inactivating culture conditions. As actin network architecture is substantially altered by matrix
dimensionality, association of YAP with actin may explain YAP’s diminished role in 3D mechanosensing. Since stiffness,
but not YAP activation, promotes invasion and proliferation in 3D culture, these results indicate that YAP does not
mediate stiffness sensing during BM invasion in breast cancer.
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Real-Time Visualization of Early Metastasis Events in Danio Rerio
Colin D. Paul1, Alexus Devine1, Kevin Bishop2, Raman Sood2, and Kandice Tanner1; 1Laboratory of Cell Biology, Center
for Cancer Research, National Cancer Institute, National Institutes of Health, 2Translational and Functional Genomics
Branch, National Human Genome Research Institute, National Institutes of Health
Metastasis, the process by which cancer cells travel from a primary tumor to establish lesions in distant organs, is the
cause of most cancer-related deaths. One critical process during metastasis is the transit of cells from a primary tumor
and through the vasculature or lymphatic systems to a distant site. However, visualization of cellular behavior in the
vasculature is difficult in most model systems, where final cell destination is not known beforehand. Here, we used
subclones of breast adenocarcinoma cells injected into the circulation of embryonic zebrafish as a model xenograft
system of metastasis. The zebrafish vasculature contains vessels on the scale of human capillaries. Real-time intravital
imaging revealed metastatic spread to be an inefficient process, with less than 20% of cells passing through a given
organ remaining there following 14 h of imaging. Additionally, there was no significant difference in the organ-specific
residence time or migration speed of cells of each clone in the organ vasculature. Instead, cell capture and migration
rate were dependent on vessel topography, and groups of cells had both longer residence times and greater interactions
with host macrophages and neutrophils. To elucidate the role of topography in the absence of host cells, we engineered
a microfluidic device containing microchannels with cross-sectional area on the scale of the vasculature. Again, cell
migration speed was not dependent on ECM but was instead dictated by topography. Our findings indicate that local
topography and not ECM composition drives early metastatic spread.
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The 2016 ASCB Doorstep Meeting l www.ascb.org/doorstep