Download IGPR-1 is a novel adhesion molecule involved in

Boston University
OpenBU
http://open.bu.edu
Theses & Dissertations
Boston University Theses & Dissertations
2015
IGPR-1 is a novel adhesion
molecule involved in colorectal
tumor growth
Woolf, Nicholas Taylor
http://hdl.handle.net/2144/16006
Boston University
BOSTON UNIVERSITY
SCHOOL OF MEDICINE
Thesis
IGPR-1 IS A NOVEL ADHESION MOLECULE INVOLVED IN COLORECTAL
TUMOR GROWTH
by
NICHOLAS TAYLOR WOOLF
B.A., Connecticut College, 2011
Submitted in partial fulfillment of the
requirements for the degree of
Master of Arts
2015
© 2015 by
NICHOLAS TAYLOR WOOLF
All rights reserved
Approved by
First Reader
Nader Rahimi, Ph.D.
Associate Professor of Pathology and Laboratory Medicine
Second Reader
Barbara Slack, Ph.D.
Associate Professor of Pathology and Laboratory Medicine
DEDICATION
This thesis is dedicated in loving memory of Irving and Ethel Woolf, Janice Hazen and
Mary Louise MacMullan. Although they have passed away, memories of their love and
compassion remain and continue to inspire me.
iv
ACKNOWLEDGMENTS
First and foremost, I would like to thank Dr. Nader Rahimi for all of his support
and guidance. The conversations he’s had with me since I joined his lab have been of
immeasurable value to me. I thank Drs. Barbara Slack, Vipul Chitalia and Rosana Meyer
for their comments and advice on several aspects of my project. Dr. Manisha Mehta is
recognized and thanked for her histology and immunohistochemistry work that is
included in this thesis. I also want to thank my friends and fellow lab members Emad
Arafa and Jordan Shafran for their constant encouragement and for engaging in many
spirited conversations with me about current events in the world of sports.
I thank the Department of Pathology, including Dr. Daniel Remick and Dr. Chris
Andry for accepting me into the Master’s program and for their full support and
guidance. I especially want to thank Debbie Kiley, administrative assistant of the
Department, for her help, support and positive attitude that makes the Department run
smoothly.
I would be remiss if I did not thank my good friends Terry Hsieh, Nisma Mujahid,
Anjali Jacob, Omar Mohtar, Tom Shin and Jake Kantrowitz; they are all extremely
kindhearted people and have always been willing to commiserate over our shared plights
of uncooperative experiments and looming deadlines. I value you all among my closest
friends and colleagues! I also thank my close friends from LCA and Connecticut College
and my extended family members for their constant love, support and camaraderie.
And last but never least, I’d like to thank Mom, Dad and my sister Kristina. You
have all taught me some of life’s most important lessons. Thank you for your unwavering
v
encouragement and advice throughout all the phases of my life. I love the three of you so
much!
vi
IGPR-1 IS A NOVEL ADHESION MOLECULE INVOLVED IN COLORECTAL
TUMOR GROWTH
NICHOLAS TAYLOR WOOLF
ABSTRACT
Colorectal cancer (CRC) is among the most prevalent and lethal cancers in the
United States. The mechanisms by which tumor cells sense their microenvironment have
profound importance in driving the progression of malignancy and evasion from
treatment. Specialized microenvironment-sensing cell surface receptors such as cell
adhesion molecules allow tumor cells to survey and respond to their microenvironment.
We have recently identified a novel cell adhesion molecule named immunoglobulincontaining and proline-rich receptor 1 (IGPR-1) that is normally expressed in both
endothelial and epithelial human cell types; however, its potential role in human
malignancy remains unknown. To investigate the role IGPR-1 plays in CRC tumor
growth, we overexpressed IGPR-1 in human HT29 and HCT116 colon adenocarcinoma
cells and examined the effect of IGPR-1 on tumor growth and the mechanisms involved
in a cell culture system. The data demonstrate that overexpression of IGPR-1 enhances
CRC cell proliferation and survival in vitro. Furthermore, we demonstrate that the
extracellular domain of IGPR-1 is required for its ability to support tumor growth. While
deletion of the extracellular domain of IGPR-1 impaired its ability to promote tumor cell
survival, stimulation of the chimeric IGPR-1 consisting of the extracellular domain of
human colony stimulating factor-1 receptor (CSF-1R) fused to the transmembrane and
cytoplasmic domains of IGPR-1 promoted tumor cell survival. Additionally, the presence
vii
of serine 186 and 220 in the cytoplasmic domain is important for IGPR-1 activity in
tumor cells. This work identifies IGPR-1 as an important protein in the regulation of
CRC cell growth and survival, and this makes it a possible therapeutic target in the
clinical management of CRC.
viii
TABLE OF CONTENTS
TITLE……………………………………………………………………………………...i
COPYRIGHT PAGE……………………………………………………………………...ii
READER APPROVAL PAGE…………………………………………………………..iii
DEDICATION ................................................................................................................... iv
ACKNOWLEDGMENTS .................................................................................................. v
ABSTRACT ...................................................................................................................... vii
TABLE OF CONTENTS ................................................................................................... ix
LIST OF FIGURES ........................................................................................................... xi
LIST OF ABBREVIATIONS ........................................................................................... xii
INTRODUCTION .............................................................................................................. 1
Colorectal Cancer Epidemiology ............................................................................. 1
Molecular Pathogenesis of Colorectal Cancer ....................................................... 1
Colorectal Cancer Metastasis .................................................................................. 8
IGPR-1: A Novel Cell Adhesion Molecule .............................................................. 9
METHODS ....................................................................................................................... 12
RESULTS ......................................................................................................................... 15
DISCUSSION ................................................................................................................... 25
ix
LIST OF JOURNAL ABBREVIATIONS........................................................................ 28
REFERENCES ................................................................................................................. 30
CURRICULUM VITAE ................................................................................................... 36
x
LIST OF FIGURES
Figure
Title
Page
1
IGPR-1 is expressed in colorectal tissue
11
2
Overexpression of IGPR-1 promotes tumor cell survival
16
3
cIGPR-1 is an inducible molecule that promotes CRC
18
survival only in the presence of CSF-1
4
IGPR-1 mediates its pro-survival effect via stimulation of
20
its extracellular domain
5
Serine phosphorylation mediates IGPR-1’s downstream
effects in HT29 cells
xi
23
LIST OF ABBREVIATIONS
APC ......................................................................................... Adenomatous Polyposis Coli
cIGPR-1 .................................................................................................... Chimeric IGPR-1
CIMP ............................................................................. CpG Island Methylation Phenotype
CIN .................................................................................................Chromosomal Instability
CK1 ..............................................................................................................Casein Kinase 1
CRC...........................................................................................................Colorectal Cancer
CSF-1 ...................................................................................... Colony-Stimulating Factor-1
DCC ............................................................................................... Deleted in Colon Cancer
DMEM ...........................................................Dulbecco’s Modification of Eagle’s Medium
Dvl........................................................................................................................Disheveled
EMT .............................................................................. Epithelial-Mesenchymal Transition
EpCAM .......................................................................... Epithelial Cell Adhesion Molecule
FAP .................................................................................. Familial Adenomatous Polyposis
GSK3β .................................................................................. Glycogen Synthase Kinase 3β
HEK ........................................................................................... Human Embryonic Kidney
HNPCC ........................................................... Hereditary Nonpolyposis Colorectal Cancer
IGPR-1 ........................................ Immunoglobulin-containing and Proline-rich Receptor 1
LOH ................................................................................................. Loss of Heterozygosity
LRP 5/6 ........................................... Low-density Lipoprotein Receptor-related Protein 5/6
MAPK ............................................................................ Mitogen-Activated Protein Kinase
MSI ................................................................................................ Microsatellite Instability
xii
MSS...................................................................................................... Microsatellite-Stable
PAE ............................................................................................. Porcine Aortic Endothelial
RFLP ............................................................... Restriction Fragment Length Polymorphism
sIGPR-1........................................................................................................ Soluble IGPR-1
TCF .................................................................................................................. T Cell Factor
xiii
INTRODUCTION
Colorectal Cancer Epidemiology
Colorectal cancer (CRC) is third highest among all cancers in terms of incidence
and mortality in both men and women in the United States. CRC incidence and mortality
rates have both dropped by 3% in the US during the past decade, especially in the adult
population aged 65 and older (Siegel, et al. 2014). Unfortunately, this encouraging trend
is not reflected worldwide, as CRC incidence rates have increased for both men and
women in areas such as Eastern Europe, Asia and South America (Center, et al. 2009).
This dichotomy in regional CRC incidence is partly due to recent economic and dietary
changes, which have led to increased food intake and westernized food choices and
portions in nations such as the Czech Republic, Slovakia and Japan (Center, et al. 2009).
The high-calorie western diet and weight gain are both known risk factors for CRC
(Hartz, et al. 2012 and Kasdagly, et al. 2014). Although more economically developed
nations like the United States have similar dietary habits, they have also implemented and
promoted colon cancer prevention measures for a longer period of time. These measures
include regular fecal occult blood tests and colonoscopies in adult patients after age 50,
and they have helped to save lives by identifying colon polyps at earlier, premalignant
stages. Nevertheless, if CRC incidence and mortality rates are to be further reduced
worldwide, more effective screening and treatment modalities are needed.
Molecular Pathogenesis of Colorectal Cancer
CRC Stem Cells
1
It is now widely recognized that many tumors, including colorectal adenomas,
begin with genetic alterations in cells with stem-like characteristics. The changes in
oncogenes or tumor-suppressor genes are then passed on as these cells divide and
differentiate. In CRC, tumor-initiating changes affect multipotent stem cells that reside in
the colonic crypts of Lieberkühn (van der Flier and Clevers 2009). These crypt stem cells
are responsible for rapidly replenishing the epithelial cell layer that lines the colonic
lumen. The cells can divide either symmetrically or asymmetrically; symmetrical division
produces two differentiated daughter cells or two stem cells, while asymmetrical division
produces one daughter cell and one stem cell. In this manner, a single stem cell can
produce progeny that come to dominate the cell population of a specific crypt in a
process called monoclonal conversion. This process and the eventual crypt fission that
follows have been proposed as the mechanism by which a single mutation in a crypt stem
cell can spread and produce an adenoma (Zeki, et al. 2011). These stem cells are also
thought to play a crucial role in metastasis, since they would serve as the tumor-initiating
cells after implantation in the secondary organ site (Brabletz, et al. 2005). Given CRC
stem cells’ crucial importance in tumor progression, researchers and clinicians have
sought to target them as part of the next generation of CRC detection and treatment
strategies.
Getting a reliable molecular phenotype for CRC stem cells has proven to be a
difficult task, with various flow cytometry experiments returning multiple potential cell
surface markers for CRC-initiating stem cells. In two separate studies, the authors found
that human CRC-derived CD133+ cells (comprising just 2.5% of the tumor cell mass)
2
were the only cells able to reproduce the original tumor in immunodeficient mice, and
they grew in an undifferentiated state for more than a year in vitro (O’Brien, et al. 2007
and Ricci-Vitiani, et al. 2007). However, shortly after these findings were reported,
another group found that a population of human CRC cells positive for epithelial cell
adhesion molecule (EpCAM), CD44 and CD166 were the only cells capable of forming
tumors when injected subcutaneously into NOD/SCID mice (Dalerba, et al. 2007). This
study also found that CD133 expression was heterogeneous across multiple human CRC
samples, which raises doubts about the previous authors’ findings. Still, it has also been
reported that when tumors contain cells expressing CD133 and CD44 heterogeneously,
only cells that express both of these molecules are able to form new tumors in mice
(Haraguchi, et al. 2008 and Chen, et al. 2011). Further complicating matters, another
recent study has suggested that a population of CD24+ stem cells should be targeted in
CRC treatment strategies, since these cells are more likely to undergo epithelialmesenchymal transition (EMT) and metastasize (Okano, et al. 2014). Taken together,
these studies paint an unclear picture regarding a universal molecular phenotype for the
CRC stem cell, but several promising molecules have been identified that could be
targeted by future CRC treatments.
The CRC Tumorigenic Progression Pathway
The first of two major tumorigenic pathways in CRC involves initial dysfunction
in the Wnt signaling pathway. Specifically, alterations in the tumor-suppressing molecule
adenomatous polyposis coli (APC) are the first step in what is known as the classic
pathway. APC is part of a complex that also includes glycogen synthase kinase 3β
3
(GSK3β), casein kinase 1 (CK1) and Axin. When the Wnt receptor is inactive, this
complex sequesters a molecule called β-catenin and phosphorylates it, which targets it for
proteasomal degradation. However, when Wnt ligands bind the Frizzled receptor, its coreceptor low-density lipoprotein receptor-related protein 5/6 (LRP5/6) binds Axin and
another molecule called disheveled (Dvl) binds GSK3β. These actions sequester key
components of the destruction complex and render it inactive, allowing β-catenin to
translocate to the nucleus and serve as a transcriptional co-activator with T cell factor
(TCF) transcription factors. This leads to increased expression of several target genes,
many of which promote increased cell growth and proliferation (van der Flier and
Clevers 2009).
The mutations in APC were originally discovered in patients with familial
adenomatous polyposis (FAP), an autosomal dominant syndrome that leads to the
formation of hundreds to thousands of individual colonic polyps and requires total
colectomy early in life (Fearon 2011). While germline APC point or missense mutations
account for a small percentage of colorectal tumors, around 80% of sporadic colorectal
tumors originate with somatic APC mutations (Fearon 2011). Most of these mutations
occur in the β-catenin-binding domain of APC and prevent proper phosphorylation and
destruction of β-catenin. The resulting β-catenin accumulation in the cytoplasm
eventually leads to an increase in nuclear β-catenin, which induces constitutive
transcription of several oncogenes and results in unchecked cellular proliferation (Fearon
2011). The high frequency of APC mutations in sporadic colorectal adenomas has
resulted in APC being dubbed the “gatekeeper” molecule in the classic pathway of
4
colorectal tumorigenesis (Kwong and Dove 2009). APC dysfunction is an early step in
this pathway because, while it does lead to the formation of tubular adenomas, other
mutations are required to complete the classic colorectal adenoma-carcinoma sequence.
The subsequent gene mutations in the classic pathway increase the dysplastic
nature of the colorectal adenoma until it becomes a carcinoma. Generally regarded as the
second major mutated protein in the sequence, KRAS is a well-known upstream
enzymatic effector of the pro-survival and pro-growth mitogen-activated protein kinase
(MAPK) pathway. KRAS was one of the earliest genes identified as mutated in CRC via
the restriction fragment length polymorphism (RFLP) technique (Vogelstein, et al. 1988).
It is now known that this gene is subject to missense mutations at several codons, all of
which promote constitutive KRAS activation by inhibiting its intrinsic GTPase function
(Zoratto, et al. 2014). This allows the KRAS enzyme to stay bound to GTP and activate
the MAPK pathway continuously, which leads to a marked increase in tumor size.
Another important genetic alteration identified early on by RFLP is the loss of
heterozygosity (LOH) at chromosome 18 (Vogelstein, et al. 1988). LOH results in
chromosomal instability (CIN) in the q arm of chromosome 18 and deletion of key genes
within this area of the chromosome, including deleted in colon cancer (DCC) (Zoratto, et
al. 2014). Loss of DCC and other genes located on 18q (including tumor suppressors
SMAD2 and SMAD4) speeds tumor progression by further promoting a state of genetic
chaos in CRC cells (Grady and Markowitz 2002). CIN is the major epigenetic disruption
of the classic CRC pathway, and it affects many chromosomal sites in addition to 18q.
5
One of the chromosomal sites that can be affected by CIN is 17p, which is home
to the crucial gene TP53 (Fearon 2011). Mutation or loss of TP53 is the final step in the
classic pathway of colorectal tumorigenesis. This gene produces the transcription factor
p53, widely known as the “guardian of the genome” because it is responsible for ensuring
the integrity of every cell’s DNA. Normally, when a cell’s DNA becomes irreversibly
damaged, p53 expression increases and it activates transcription of pro-apoptotic genes
(e.g. BAX) that promote cell death before any genomic errors are propagated via mitosis
(Grady and Markowitz 2002). However, when TP53 is the victim of inactivating
mutations, the corresponding p53 protein can no longer bind to its designated promoter
regions and serve as an essential genomic watchdog. This enables a cascade of mutations
and other genetic aberrations that lead to the onset of colorectal adenocarcinoma and
eventual metastasis.
The Alternative (Serrated) Tumorigenic Pathway
While the classic pathway gives rise to tubular adenomas, the alternative pathway
produces serrated adenomas, which are so-named due to their jagged, tooth-like
appearance. Serrated colorectal adenomas are the hallmark of an inherited syndrome
called hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. It arises due to
germline mutations of DNA mismatch repair genes such as hMSH2 and hMLH1, which
causes widespread microsatellite instability (MSI) and CpG island methylation (Grady
and Markowitz 2002). Microsatellites are DNA repeats that occur frequently throughout
the genome and are usually made up of no more than six nucleotides. These areas of
DNA are especially susceptible to DNA polymerase errors that result in base pair
6
mismatches (Zoratto, et al. 2014). When several mismatch repair genes are mutated, these
errors go uncorrected and promote other gene mutations (Jass 2002). Sporadic tumors
with MSI also tend to have a CpG island methylation phenotype (CIMP), where the
transcription of tumor suppressor genes is negatively regulated via hypermethylation of
CpG islands in their promoter regions (Jass 2002 and Fearon 2011). Tumor suppressor
genes such as APC and TP53 are less commonly mutated in the serrated pathway;
nevertheless, they are included in a broad number of genes that can be affected by MSI
and CIMP.
The serrated pathway is responsible for approximately 15% of sporadic CRC, and
most of these cases involve both MSI and CIMP and have gain-of-function mutations of
the BRAF oncogene (Grady and Markowitz 2002 and Fearon 2011). BRAF is an antiapoptotic serine/threonine kinase, and several activating mutations (including the wellknown V600E mutation) can result in its constitutive activity early in the alternative
pathway’s sequence; it plays a significant role in the initial serration of the epithelial
crypts (Zoratto, et al. 2014). Although the serrated pathway is not as common, its unique
genetic disruptions should be kept in mind when devising CRC screening and treatment
strategies.
The classic and alternative tumorigenic pathways have well-established molecular
mechanisms, but their key genes and patterns of mutation have yet to be optimally
leveraged for the benefit of CRC screening efforts in the United States. While
colonoscopies have proven effective at identifying polyps, there is no cheap, effective
screening test that looks at the status of any of the genes discussed above. With this in
7
mind, some clinicians have surmised that traits such as CpG methylation or TP53
mutational status could be tested in future strategies (Davies, et al. 2005). While there are
certainly technologies available to screen for these characteristics, clinical utility and
financial concerns continue to prevent their adoption as a commonplace clinical test for
CRC.
Colorectal Cancer Metastasis
A cancer patient’s prognosis is worsened considerably once cells from the
primary tumor metastasize and form secondary tumors at other locations in the body.
Colorectal tumors, like all other solid tumors, gain the ability to invade and metastasize
via an extraordinary phenotypic change known as the epithelial-mesenchymal transition
(EMT) (Hanahan and Weinberg 2011). This process is marked by transcriptional
repression of several epithelial cell markers including E-cadherin, a cell adhesion
molecule that is essential for the formation of adherens junctions between neighboring
cells (Onder, et al. 2008 and Polyak and Weinberg 2009). Loss of E-cadherin is
significant not only because it allows cells to break free from the mass of other tumor
cells, but also because it is associated with increased cell invasion and anchorageindependent cell survival (Onder, et al. 2008 and Prindull and Zipori 2004). Expression
of E-cadherin is inhibited by a transcription factor called Snail, which also acts to
promote transcription of mesenchymal proteins such as vimentin. Overexpressing Snail
in human CRC cells leads to increased motility and invasiveness, and it also increases
expression of previously-mentioned cancer stem cell markers CD133 and CD44 (Fan, et
al. 2012). This suggests that CRC cells undergoing EMT also gain a cancer stem cell
8
phenotype that allows them to survive in the circulation and become more
chemoresistant. These cells must mimic a mesenchymal cell’s survival strategy in order
to survive the arduous journey from the primary tumor, through the bloodstream and to a
distant, secondary tumor site.
Normally, cells that inappropriately break free from neighboring cells and the
extracellular matrix (ECM) undergo a specific type of apoptosis called anoikis (Gilmore
2005). Epithelial and endothelial cells are susceptible to anoikis because they are
supposed to remain in close contact with other cells and the ECM; conversely,
mesenchymal and hematopoietic cells are resistant to anoikis because they must be able
to detach and migrate easily within their respective tissues (Gilmore 2005 and Taddei, et
al. 2012). Loss of E-cadherin, among other changes that occur during the EMT, activates
several pro-survival signaling pathways, including the PI3K/Akt and Ras/MAPK
pathways (Guadamillas, et al. 2011 and Kim, et al. 2012). Specifically, CRC cells have
demonstrated elevated expression of PI3K, especially in HT29 and SW480 cell lines
(Wang, et al. 2003 and Wang, et al. 2013). An increase in these signaling molecules
allows metastatic tumor cells to complete their mesenchymal mimicry, thus avoiding
anoikis and surviving in the circulation.
IGPR-1: A Novel Cell Adhesion Molecule
Our laboratory has previously identified a molecule named immunoglobulincontaining and proline-rich receptor 1 (IGPR-1) as a novel cell adhesion molecule that
regulates endothelial cell-cell interaction, cell migration and angiogenesis. IGPR-1 is
expressed in higher mammalian species, including horses, cats, dogs, chimpanzees and
9
humans, but it is not expressed in rats or mice. IGPR-1 is a 31 kDa protein and contains a
single extracellular immunoglobulin domain, a transmembrane domain and a cytoplasmic
region that is rich in proline residues (Rahimi, et al. 2012). When expressed in vitro, the
observed molecular weight of IGPR-1 increases to 55 kDa, which is most likely due to
glycosylation at multiple extracellular sites (Rahimi, et al. 2013). Of note, IGPR-1 is only
expressed in endothelial and epithelial cell types.
To determine the role IGPR-1 plays in angiogenesis, IGPR-1 was overexpressed
in porcine aortic endothelial (PAE) cells. Overexpression resulted in an increase in
capillary tube formation by these cells, which indicates that IGPR-1 promotes
angiogenesis. IGPR-1 overexpression also led to a change in PAE cell morphology, an
increase in actin filament formation, an increase in focal adhesion complexes and a
decrease in cell motility (Rahimi, et al. 2012). These findings all support IGPR-1’s role
as a pro-angiogenic cell-adhesion molecule in endothelial cells; however, the role it plays
in diseases involving endothelial or epithelial cell pathology has not yet been elucidated.
A recent screen of cancer cell types in our laboratory demonstrated that IGPR-1 is
expressed in various cancer types, including breast, lung and colorectal (unpublished
data). Recently, we have also found that IGPR-1 is expressed heterogeneously in more
than 100 colorectal tumor tissue samples taken from patients at Boston Medical Center
(Figure 1). The overarching goal of this project was to determine the role of IGPR-1 in
CRC growth and survival in an in vitro system and the mechanisms involved. Our
findings demonstrate that IGPR-1 promotes colorectal tumor growth and survival in vitro
only upon activation of its extracellular domain.
10
Figure 1: IGPR-1 is expressed in colorectal tissue. Immunohistochemical staining
demonstrates expression of IGPR-1 in both normal colonic crypts (arrow) and
adenomatous colonic tissue (arrowhead). Magnification= 40X.
11
METHODS
Cell Lines and Growth Conditions
HEK293, HT29, HCT116 and B16F cell lines were purchased from ATCC
(Manassas, VA). Cells were grown in 10 cm culture dishes (Corning, Corning, NY).
Culture medium was Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5 g/L
glucose, L-glutamine and sodium pyruvate supplemented with 10% FBS and
penicillin/streptomycin (Corning). To maintain expression of viral vectors in transduced
cells, their media was supplemented with 1 µg/mL puromycin.
Plasmids and Antibodies
IGPR-1, ΔN-IGPR-1, A220 IGPR-1 and A186 IGPR-1 cDNA constructs were
cloned into retroviral vector pMSCV.puro as previously described (Rahimi, et al. 2012).
cIGPR-1 plasmids were produced by cloning the extracellular domain of the CSF-1
receptor and the transmembrane and cytoplasmic domains of IGPR-1 into retroviral
vector pMSCV.puro in a procedure modeled after previous creation of chimeric VEGFR
molecules (Rahimi, et al. 2000).
Polyclonal rabbit IGPR-1 antibody was produced as previously described
(Rahimi, et al. 2012). Polyclonal rabbit PLCγ1 (sc-81) and monoclonal mouse Hsp70 (sc24) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Virus Production and Transduction
The pMSCV.puro vectors containing IGPR-1 constructs or empty vectors were
transfected into 293-GPG cells, and viral supernatants were collected for 3 days as
previously described (Rahimi, et al. 2000). Concentrated virus was transduced into HT29,
12
HCT116 and B16F cells by adding 3 mL of virus-containing DMEM and 10 µg of
polybrene to cells in 6 cm culture dishes (Corning). After 16 hours, this media was
removed and puromycin-supplemented DMEM was added to ensure survival of
transduced cells only.
Western Blotting
Whole-cell lysates were used for Western blot analysis as described previously
(Meyer, et al. 2011). Briefly, whole-cell lysates normalized for protein content were
subjected to SDS-PAGE and gel contents were transferred to PVDF membranes.
Membranes were blocked in 2% milk solution and exposed to IGPR-1, PLCγ1 or Hsp70
antibody as appropriate. Membranes were then incubated in either goat anti-rabbit (sc2004) or goat anti-mouse (sc-2055) horseradish peroxidase-conjugated secondary
antibodies purchased from Santa Cruz Biotechnology. Protein bands were detected by an
ECL system (EMD Millipore, Billerica, MA).
MTT Cell Proliferation Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell
proliferation assay was performed as described previously (Mosmann 1983). 5x104 cells
were plated in 400 µL of DMEM + 1% FBS in each well of a 24-well low attachment
plate (Corning). Cells were distributed such that n=4 wells were used per cell
type/treatment group (0, 10 and 50 ng CSF-1) per time point (0, 2 and 4 days). At each
time point, 5 µL of MTT dye (Promega, Madison, WI) was added to each well, and 100
µL of solubilization solution (Promega) was added after two hours of incubation at 37°C
and 5% CO2. After an additional hour of incubation, 190 µL of solution from each well
13
was transferred to a 96-well plate (Corning) and read at an absorbance of 570 nm in a
microplate reader (VERSA max, Molecular Devices, Sunnyvale, CA). The resulting data
were used to generate percent survival curves, with day 0 values serving as 100%
survival baselines.
Statistical Analyses
The Student’s two-tailed t-test (assuming equal variances) was used to analyze
cell survival data in experiments comparing two cell lines. For experiments that
compared three or more cell lines, the one-way ANOVA with Tukey’s post-hoc test was
used to analyze the results. An alpha value of P < 0.05 denoted a significant difference
between two groups in all cell survival comparisons. T tests were performed using
Microsoft Excel and ANOVA tests were performed online at http://www.vassarstats.net.
14
RESULTS
IGPR-1 Activity Promotes Cancer Cell Survival
Cell adhesion molecules play a central role in tumor growth, cell survival and
activation of pro-survival signaling pathways, including the PI3K and MAPK cascades
(Okegawa, et al. 2004 and Rodriguez, et al. 2012). We sought to determine the effect
IGPR-1 has on survival and growth in microsatellite-stable (MSS) and microsatellite
instability (MSI) CRC cell lines. We used HT29 cells as the MSS line; these cells have
mutations in the APC and P53 genes (Comes, et al. 2007). HCT116 cells were chosen as
the MSI cell line, and in addition to loss of expression of DNA mismatch repair genes,
they contain mutations in β-catenin, KRAS and TGF-βIIR genes (Duldulao, et al. 2012
and Comes, et al. 2007). A mouse B16F melanoma cell line was also used as a non-CRC
control. All three of these cell lines express extremely low levels of IGPR-1
endogenously, but overexpression of the molecule via retroviral vector transduction
proved successful (Figure 2, A-C).
IGPR-1 overexpression had a significant pro-survival effect on all three of these
cell lines in a nutrient-poor, 3D culture system (Figure 2, A-C). Specifically, IGPR-1
expression in HT29 cells was associated with an overwhelmingly positive trend in cell
survival and growth, rather than the blunted, negative trend seen in HCT116 cells (Figure
2, A-B). This suggests that IGPR-1 not only promotes increased HT29 cell survival, but
also increased cell proliferation in a culture system that mimics anchorage-independence.
15
Figure 2: Overexpression of IGPR-1 promotes tumor cell survival. (A) HT29 cells
expressing empty vector vs. IGPR-1 were subjected to a cell survival assay every 48
hours as described in Materials and Methods. Cells expressing IGPR-1 showed a
16
significantly higher survival percentage after 4 days (*P<0.01). (B) HCT116 cells
expressing empty vector vs. IGPR-1 were subjected to the same cell survival assay
protocol. Cells expressing IGPR-1 died at a slower rate than cells expressing empty
vector (*P<0.05; ^P<0.01). (C) Mouse B16F melanoma cells expressing empty vector or
IGPR-1 were subjected to a cell survival assay that began on day 2 of incubation as
described in Materials and Methods. Contrary to cells expressing empty vector, cells
expressing IGPR-1 did not experience a drop in survival percentage after 6 days
(*P<0.05). All graphs show means of a four-well experiment that was conducted in
triplicate. All error bars denote one standard deviation from the mean of four wells.
Chimeric IGPR-1 (cIGPR-1) Indicates that Activation of IGPR-1 Stimulates Tumor
Growth
Prior work with IGPR-1 in PAE cells has demonstrated that stimulation of the
molecule’s extracellular immunoglobulin domain via trans dimerization is required for it
to regulate endothelial cell morphology and adhesion (Rahimi, et al. 2012). To determine
whether IGPR-1 could be stimulated in a controlled manner in the absence of its wildtype extracellular domain, we generated a chimeric IGPR-1 receptor containing the
extracellular portion of the colony-stimulating factor-1 (CSF-1) receptor and the
transmembrane and cytoplasmic domains of IGPR-1 (Figure 3A). This “chimeric IGPR1,” hereafter called cIGPR-1, is an inducible version of the molecule, the activation of
which could theoretically be controlled via treatment with the CSF-1 ligand.
17
After inducing cIGPR-1 expression in HT29 cells via a retroviral vector system,
we treated cells in the 3D culture system with 0 ng, 10 ng or 50 ng of CSF-1. Subsequent
assays performed at two and four days post-culturing demonstrated that IGPR-1
promoted survival only in cells treated with either 10 ng or 50 ng of CSF-1 (Figure 3B).
This result suggests that cIGPR-1 can only be activated if its extracellular domain is
stimulated with the appropriate ligand; the intracellular portion of IGPR-1 is incapable of
auto-stimulation.
18
Figure 3: cIGPR-1 is an inducible molecule that promotes CRC survival only in the
presence of CSF-1. (A) The cartoon depicts an inducible version of IGPR-1 that was
created by combining the extracellular domain of the CSF-1 receptor with the
transmembrane and cytoplasmic domains of IGPR-1. This molecule was successfully
cloned into a viral vector and expressed in HT29 cells. (B) HT29 cells expressing cIGPR1 promoted survival in a 3D culture system only after treatment with media containing 10
1
2
ng or 50 ng of CSF-1 ( P<0.01; P<0.01). The graph shows means of a four-well
experiment that was conducted in triplicate. All error bars denote one standard deviation
from the mean of four wells.
The Extracellular Immunoglobulin Domain is Required for IGPR-1 Activation
To further explore the role that IGPR-1’s extracellular domain plays in the
molecule’s activity, we cloned a soluble, myc-tagged version of IGPR-1’s extracellular
domain, expressed it in human embryonic kidney (HEK-293) cells and collected it once it
had been secreted in the cells’ media (Figure 4A). HT29 cells expressing empty vector
vs. IGPR-1 were treated with either untreated serum-poor media or soluble IGPR-1
(sIGPR-1)-enriched media. Cell survival assay findings showed that HT29 cells treated
with sIGPR-1 proliferated dramatically and had significantly higher survival percentages
than cells treated with control media (Figure 4B). This suggests that sIGPR-1 is capable
of activating membrane-bound IGPR-1 via trans dimerization. This activation, when
added to baseline IGPR-1 activity, has an additive effect on HT29 cell survival and
proliferation.
19
20
Figure 4: IGPR-1 mediates its pro-survival effect via stimulation of its extracellular
domain. (A) The cartoon depicts the structure of sIGPR-1 as compared to the original
IGPR-1 molecule. sIGPR-1 production in HEK-293 cells’ media was confirmed via
Western blot. (B) IGPR-1-expressing HT29 cells treated with media containing soluble
IGPR-1 had a significantly higher growth rate than cells treated with normally
conditioned media in a 3D culture system. HT29 cells expressing an empty vector also
demonstrated a significant increase in growth upon treatment with soluble IGPR-1 (all
numerals denote P<0.01 for the corresponding pairs of data points). (C) HT29 cells
expressing ΔN-IGPR-1 were similar to cells expressing empty vector in terms of survival
percentage (^P<0.05, †P<0.05, *P<0.01; data representative of two independent
experiments). The graphs in (B) and (C) show means of four-well experiments that were
conducted in duplicate. All error bars denote one standard deviation from the mean of
four wells.
We also wanted to investigate the effect that deletion of the immunoglobulin
domain would have on IGPR-1’s activity in HT29 cells. We transduced HT29 cells with
a viral vector containing an N-terminus-truncated version of IGPR-1 (ΔN-IGPR-1) that
has 133 fewer amino acids than the native molecule (Rahimi, et al. 2012). Cells
expressing ΔN-IGPR-1 had a stagnant survival pattern similar to what is seen in cells
expressing an empty vector, while cells expressing IGPR-1 continued to survive and
proliferate at a significantly higher rate (Figure 4C). Taken together with the cIGPR-1
and sIGPR-1 data, this seems to suggest that stimulation of an extracellular domain,
21
regardless of whether or not it is the wild-type immunoglobulin domain, is essential for
IGPR-1’s activation and subsequent pro-survival and pro-growth effects in HT29 CRC
cells.
Serine Residue 220 is Required for IGPR-1 Activity
Prior mass spectrometry analysis of IGPR-1 revealed several putative serine
phosphorylation sites in the molecule’s cytoplasmic domain (Figure 5A). Since many
membrane-bound receptors and adhesion molecules initiate their intracellular effects via
protein phosphorylation, we investigated the effect that mutating two of these serine
residues would have on IGPR-1’s functionality in HT29 CRC cells. Site-directed
mutagenesis was used to create two mutant IGPR-1 molecules: one with a Ser-Ala point
mutation at residue 220 and the other with the same point mutation at residue 186. These
mutant IGPR-1 molecules were overexpressed in HT29 cells, and their growth and
survival in the 3D culture system was assessed (Figure 5A). The cell survival assay
showed that the point mutation at residue 220 caused a more dramatic drop in cell
proliferation compared to the point mutation at reside 186, although it was still not as
effective as the wild-type molecule at promoting cell growth (Figure 5B). This data
suggests that phosphorylation of serine 220 is the cornerstone of IGPR-1’s intracellular
signaling mechanism.
22
Figure 5: Serine phosphorylation mediates IGPR-1’s downstream effects in HT29
cells. (A) Prior tandem mass spectrometry identified several putative serine
phosphorylation sites in IGPR-1’s cytoplasmic domain. Subsequent site-directed
mutagenesis created Ser-Ala point mutations at residues 220 and 186, and these mutated
IGPR-1 molecules were successfully overexpressed in HT29 cells. (B) Cells expressing
A220 mutant IGPR-1 experience stagnant growth, while cells expressing A186 mutant
IGPR-1 do proliferate, albeit at a significantly lower rate than cells expressing wild-type
IGPR-1 (*P<0.01, ^P<0.01, †P<0.05). The graph shows means of a four-well experiment
23
that was conducted in duplicate. All error bars denote one standard deviation from the
mean of four wells.
24
DISCUSSION
This study demonstrated that IGPR-1, a novel cell adhesion molecule, promotes
CRC cell growth and survival. This effect is dependent on both the activation of IGPR-1
via its extracellular domain and the presence of specific serine residues in its cytoplasmic
domain. Although many cell adhesion molecules have typically been associated with
prevention of cancer progression, some have been identified as promoters of invasion and
metastasis. Epithelial cell adhesion molecule (EpCAM) was recently associated with
prostate cancer cell proliferation, invasion and metastasis via activation of the prosurvival PI3K/Akt pathway. EpCAM knockdown was also associated with increased
sensitivity to chemotherapeutic agents (Ni, et al. 2013). E-cadherin, though it is often
regarded as the epithelial, anti-metastatic adhesion molecule associated with the EMT,
has been implicated in the growth and survival of inflammatory breast carcinoma and
glioblastoma. It has also been associated with pro-survival PI3K/Akt and MAPK
signaling in a large number of ovarian cancers, which paradoxically express higher levels
of E-cadherin than what is observed in normal ovarian tissue (Rodriguez, et al. 2012).
IGPR-1 functions similarly in CRC by supporting cell growth and survival in an in vitro
model that mimics anchorage-independent conditions.
Because cell adhesion molecules play an important role in cancer progression,
they have garnered considerable interest as potential cancer biomarkers/therapeutic
targets. EpCAM’s importance as a cancer stem cell marker and a promoter of metastasis
has made it a popular biomarker candidate for breast and ovarian cancers. Studies have
shown that serum levels of EpCAM were significantly elevated in patients with either
25
breast or ovarian cancer, but the molecule did not have any prognostic effect in both
patient populations (Karabulut, et al. 2014 and Tas, et al. 2014a). EpCAM has also shown
promise as an oncogenic marker that can be exploited to target epithelial cancers for drug
delivery (Simon, et al. 2013). For example, a recent study showed that curcumin-loaded
nanoparticles functionalized with EpCAM aptamers were able to bind HT29 CRC cells
much more effectively than a control nanoparticle, and this resulted in significantly
higher cytotoxicity (Li, et al. 2014). IGPR-1 could also be exploited in a similar manner
to target proliferating CRC cells.
Vascular cell adhesion molecule-1 (VCAM-1) has also garnered interest for its
role as a potential marker of tumor invasion. While VCAM-1 had no diagnostic or
prognostic value in breast cancer patients, it was found at higher levels in ovarian cancer
patients with metastatic disease (Karabulut, et al. 2014 and Tas, et al. 2014a). VCAM-1
levels were also elevated in lung cancer patients’ serum, and higher VCAM-1 content
was associated with platinum-based chemotherapy unresponsiveness and lower survival
rates (Tas, et al. 2014b). This suggests both a diagnostic and prognostic role for VCAM-1
in lung cancer patients treated with a specific therapeutic regimen. Lastly, VCAM-1 was
found to be significantly elevated in CRC patients’ serum, and it was associated with
poor prognosis in these patients (Okugawa, et al. 2009). Although the question of
whether IGPR-1 can be detected at appreciable levels in the serum of cancer patients
remains unanswered, its role in CRC growth and survival is an encouraging sign for its
potential as a diagnostic or prognostic marker.
26
While the current study demonstrated a significant pro-growth and pro-survival
effect in CRC cells expressing IGPR-1, it was limited by a lack of confirmation of these
findings with another method such as flow cytometry. We also did not examine the effect
of knocking down IGPR-1 in a CRC cell line that expresses it endogenously, but we have
identified Colo205 cells as a candidate cell line for this purpose (data not shown).
In conclusion, we found that IGPR-1 is a cell adhesion molecule that promotes
growth and survival when expressed in colorectal cancer cells. We also demonstrated that
this effect is dependent upon both the activation of IGPR-1 via its extracellular
immunoglobulin domain and the presence of the specific serine residue 220 in its
cytoplasmic region. Future studies should investigate both the signaling mechanism by
which IGPR-1 mediates its pro-growth and pro-survival effects in CRC cells and the
effect IGPR-1 expression has on CRC tumor growth and metastasis in an in vivo mouse
model.
27
LIST OF JOURNAL ABBREVIATIONS
Acta Biochim Pol
Acta Biochimica Polonica
Adv Exp Med Biol
Advances in Experimental Medicine and Biology
Ann Surg Oncol
Annals of Surgical Oncology
Annu Rev Genomics Hum Genet
Annual Review of Genomics and Human Genetics
Annu Rev Pathol
Annual Review of Pathology
Annu Rev Physiol
Annual Review of Physiology
Biochim Biophys Acta
Biochimica et Biophysica Acta
CA Cancer J Clin
CA: A Cancer Journal for Clinicians
Cancer Causes Control
Cancer Causes & Control: CCC
Cancer Epidemiol Biomarkers Prev Cancer Epidemiology, Biomarkers & Prevention
Cancer Med
Cancer Medicine
Cancer Res
Cancer Research
Cell Death Differ
Cell Death and Differentiation
Clin Exp Metastasis
Clinical & Experimental Metastasis
Dis Colon Rectum
Diseases of the Colon and Rectum
Expert Opin Drug Deliv
Expert Opinion on Drug Delivery
Int J Biochem Cell Biol
The International Journal of Biochemistry & Cell
Biology
Int J Cell Biol
International Journal of Cell Biology
Int J Nanomedicine
International Journal of Nanomedicine
Int J Oncol
International Journal of Oncology
28
J Biol Chem
The Journal of Biological Chemistry
J Cell Sci
Journal of Cell Science
J Immunol Methods
Journal of Immunological Methods
J Pathol
The Journal of Pathology
J Surg Res
The Journal of Surgical Research
Mol Biol Cell
Molecular Biology of the Cell
Mol Cell Biol
Molecular and Cellular Biology
N Eng J Med
The New England Journal of Medicine
Nat Rev Cancer
Nature Reviews. Cancer
Nat Rev Gastroenterol Hepatol
Nature Reviews. Gastroenterology & Hepatology
PLoS One
Public Library of Science One
Proc Natl Acad Sci USA
Proceedings of the National Academy of Sciences
of the United States of America
Surg Clin North Am
The Surgical Clinics of North America
Tumour Biol
Tumour Biology: The Journal of the International
Society for Oncodevelopmental Biology and
Medicine
World J Gastroenterol
World Journal of Gastroenterology: WJG
29
REFERENCES
Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T. 2005. Opinion: Migrating cancer
stem cells - an integrated concept of malignant tumour progression. Nat Rev Cancer
5(9):744-9.
Center MM, Jemal A, Ward E. 2009. International trends in colorectal cancer incidence
rates. Cancer Epidemiol Biomarkers Prev 18(6):1688-94.
Chen KL, Pan F, Jiang H, Chen JF, Pei L, Xie FW, Liang HJ. 2011. Highly enriched
CD133(+)CD44(+) stem-like cells with CD133(+)CD44(high) metastatic subset in
HCT116 colon cancer cells. Clin Exp Metastasis 28(8):751-63.
Comes F, Matrone A, Lastella P, Nico B, Susca FC, Bagnulo R, Ingravallo G, Modica S,
Lo Sasso G, Moschetta A, Guanti G, Simone C. 2007. A novel cell type-specific role
of p38α in the control of autophagy and cell death in colorectal cancer cells. Cell
Death Differ 14(4):693-702.
Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T, Gurney A, Huang EH,
Simeone DM, et al. 2007. Phenotypic characterization of human colorectal cancer
stem cells. Proc Natl Acad Sci USA 104(24):10158-63.
Davies RJ, Miller R, Coleman N. 2005. Colorectal cancer screening: Prospects for
molecular stool analysis. Nat Rev Cancer 5(3):199-209.
Duldulao MP, Lee W, Le M, Chen Z, Li W, Wang J, Gao H, Li H, Kim J, Garcia-Aguilar
J. 2012. Gene expression variations in microsatellite stable and unstable colon
cancer cells. J Surg Res 174(1):1-6.
30
Fan F, Samuel S, Evans KW, Lu J, Xia L, Zhou Y, Sceusi E, Tozzi F, Ye XC, Mani SA,
et al. 2012. Overexpression of snail induces epithelial-mesenchymal transition and a
cancer stem cell-like phenotype in human colorectal cancer cells. Cancer Med
1(1):5-16.
Fearon ER. 2011. Molecular genetics of colorectal cancer. Annu Rev Pathol 6:479-507.
Gilmore AP. 2005. Anoikis. Cell Death Differ 12 Suppl 2:1473-7.
Grady WM and Markowitz SD. 2002. Genetic and epigenetic alterations in colon cancer.
Annu Rev Genomics Hum Genet 3:101-28.
Guadamillas MC, Cerezo A, Del Pozo MA. 2011. Overcoming anoikis--pathways to
anchorage-independent growth in cancer. J Cell Sci 124(Pt 19):3189-97.
Hanahan D and Weinberg RA. 2011. Hallmarks of cancer: The next generation. Cell
144(5):646-74.
Haraguchi N, Ohkuma M, Sakashita H, Matsuzaki S, Tanaka F, Mimori K, Kamohara Y,
Inoue H, Mori M. 2008. CD133+CD44+ population efficiently enriches colon cancer
initiating cells. Ann Surg Oncol 15(10):2927-33.
Hartz A, He T, Ross JJ. 2012. Risk factors for colon cancer in 150,912 postmenopausal
women. Cancer Causes Control 23(10):1599-605.
Jass JR. 2002. Pathogenesis of colorectal cancer. Surg Clin North Am 82(5):891-904.
31
Karabulut S, Tas F, Tastekin D, Karabulut M, Yasasever CT, Ciftci R, Guveli M, Fayda
M, Vatansever S, Serilmez M, Disci R, Aydiner A. 2014. The diagnostic, predictive,
and prognostic role of serum epithelial cell adhesion molecule (EpCAM) and
vascular cell adhesion molecule-1 (VCAM-1) levels in breast cancer. Tumour Biol
35(9): 8849-60.
Kasdagly M, Radhakrishnan S, Reddivari L, Veeramachaneni DN, Vanamala J. 2014.
Colon carcinogenesis: Influence of western diet-induced obesity and targeting stem
cells using dietary bioactive compounds. Nutrition 30(11-12):1242-56.
Kim YN, Koo KH, Sung JY, Yun UJ, Kim H. 2012. Anoikis resistance: An essential
prerequisite for tumor metastasis. Int J Cell Biol 2012:306879.
Kwong LN and Dove WF. 2009. APC and its modifiers in colon cancer. Adv Exp Med
Biol 656:85-106.
Li L, Xiang DX, Shigdar S, Yang WR, Li Q, Lin J, Liu KX, Duan W. 2014. Epithelial
cell adhesion molecule aptamer functionalized PLGA-lecithin-curcumin-PEG
nanoparticles for targeted drug delivery to human colorectal adenocarcinoma cells.
Int J Nanomedicine 9: 1083-96.
Meyer RD, Srinivasan S, Singh AJ, Mahoney JE, Gharahassanlou KR, Rahimi N. 2011.
PEST motif serine and tyrosine phosphorylation controls vascular endothelial
growth factor receptor 2 stability and downregulation. Mol Cell Biol 31(10): 201025.
Mossman T. 1983. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J Immunol Methods 65(1-2): 55-63.
32
Ni J, Cozzi P, Hao J, Beretov J, Chang L, Duan W, Shigdar S, Delprado W, Graham P,
Bucci J, Kearsley J, Li Y. 2013. Epithelial cell adhesion molecule (EpCAM) is
associated with prostate cancer metastasis and chemo/radioresistance via the
PI3K/Akt/mTOR signaling pathway. Int J Biochem Cell Biol 45(12): 2736-48.
O'Brien CA, Pollett A, Gallinger S, Dick JE. 2007. A human colon cancer cell capable of
initiating tumour growth in immunodeficient mice. Nature 445(7123):106-10.
Okano M, Konno M, Kano Y, Kim H, Kawamoto K, Ohkuma M, Haraguchi N, Yokobori
T, Mimori K, Yamamoto H, et al. 2014. Human colorectal CD24+ cancer stem cells
are susceptible to epithelial-mesenchymal transition. Int J Oncol 45(2):575-80.
Okegawa T, Pong RC, Li Y, Hsieh JT. 2004. The role of cell adhesion molecule in cancer
progression and its application in cancer therapy. Acta Biochim Pol 51(2): 445-57.
Okugawa Y, Miki C, Toiyama Y, Koike Y, Inoue Y, Kusunoki M. 2009. Serum level of
soluble vascular cell adhesion molecule 1 is a valuable prognostic marker in
colorectal adenocarcinoma. Dis Colon Rectum 52(7): 1330-6.
Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. 2008. Loss of Ecadherin promotes metastasis via multiple downstream transcriptional pathways.
Cancer Res 68(10):3645-54.
Polyak K and Weinberg RA. 2009. Transitions between epithelial and mesenchymal
states: Acquisition of malignant and stem cell traits. Nat Rev Cancer 9(4):265-73.
Prindull G and Zipori D. 2004. Environmental guidance of normal and tumor cell
plasticity: Epithelial mesenchymal transitions as a paradigm. Blood 103(8):2892-9.
33
Rahimi N, Dayanir V, Lashkari K. 2000. Receptor chimeras indicate that the vascular
endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of
VEGFR-2 in endothelial cells. J Biol Chem 275(22): 16986-92.
Rahimi N, Rezazadeh K, Mahoney JE, Hartsough E, Meyer RD. 2012. Identification of
IGPR-1 as a novel adhesion molecule involved in angiogenesis. Mol Biol Cell
23(9):1646-56.
Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R.
2007. Identification and expansion of human colon-cancer-initiating cells. Nature
445(7123):111-5.
Rodriguez FJ, Lewis-Tuffin LJ, Anastasiadis PZ. 2012. E-cadherin’s dark side: Possible
role in tumor progression. Biochim Biophys Acta 1826(1):23-31.
Siegel R, Desantis C, Jemal A. 2014. Colorectal cancer statistics, 2014. CA Cancer J Clin
64(2):104-17.
Simon M, Stefan N, Pluckthun A, Zangemeister-Wittke U. 2013. Epithelial cell adhesion
molecule-targeted drug delivery for cancer therapy. Expert Opin Drug Deliv 10(4):
451-68.
Taddei ML, Giannoni E, Fiaschi T, Chiarugi P. 2012. Anoikis: An emerging hallmark in
health and diseases. J Pathol 226(2):380-93.
Tas F, Karabulut S, Serilmez M, Ciftci R, Duranyildiz D. 2014a. Clinical significance of
serum epithelial cell adhesion molecule (EPCAM) and vascular cell adhesion
molecule-1 (VCAM-1) levels in patients with epithelial ovarian cancer. Tumour Biol
35(4): 3095-102.
34
Tas F, Karabulut S, Bilgin E. 2014b. Serum levels of vascular cell adhesion molecule-1
(VCAM-1) may have diagnostic, predictive, and prognostic roles in patients with
lung cancer treated with platinum-based chemotherapy. Tumour Biol 35(8): 7871-5.
van der Flier LG and Clevers H. 2009. Stem cells, self-renewal, and differentiation in the
intestinal epithelium. Annu Rev Physiol 71:241-60.
Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura
Y, White R, Smits AM, Bos JL. 1988. Genetic alterations during colorectal-tumor
development. N Engl J Med 319(9):525-32.
Wang G, Wang F, Ding W, Wang J, Jing R, Li H, Wang X, Wang Y, Ju S, Wang H.
2013. APRIL induces tumorigenesis and metastasis of colorectal cancer cells via
activation of the PI3K/Akt pathway. PLoS One 8(1):e55298.
Wang M, Vogel I, Kalthoff H. 2003. Correlation between metastatic potential and
variants from colorectal tumor cell line HT-29. World J Gastroenterol 9(11):262731.
Zeki SS, Graham TA, Wright NA. 2011. Stem cells and their implications for colorectal
cancer. Nat Rev Gastroenterol Hepatol 8(2):90-100.
Zoratto F, Rossi L, Verrico M, Papa A, Basso E, Zullo A, Tomao L, Romiti A, Lo Russo
G, Tomao S. 2014. Focus on genetic and epigenetic events of colorectal cancer
pathogenesis: Implications for molecular diagnosis. Tumour Biol 35(7):6195-206.
35
CURRICULUM VITAE
Nicholas Taylor Woolf
676 Massachusetts Ave, Apt 4
Boston, MA 02118
[email protected]
617-640-2616
Year of Birth: 1988
Education
2017 (expected)
MD at Boston University School of Medicine, Boston, MA
2015
MA in Pathology and Laboratory Medicine at Boston University
School of Medicine, Boston, MA
2011
BA in Biological Sciences (Cell/Molecular Concentration) with
Honors at Connecticut College, New London, CT
2007
H.S. Diploma at Lexington Christian Academy, Lexington, MA
Publications and Presentations
1. Woolf AD, Woolf NT. 2005. Childhood lead poisoning in two families associated with
spices used in food preparation. Pediatrics 116: e314-e318.
2. Woolf NT, Lui JCK, Finkielstain G, Barnes K, Baron J. Changes in Gene Expression
Associated with Somatic Growth Deceleration in Mammals. Presented at the 2007 NIH
Summer Internship Program Poster Session. Bethesda, MD, August 3, 2007.
3. Woolf NT, Ohja J, Cashikar AG. Protein Aggregation Dynamics in Mammalian Cells.
Presented at the 2008 STAR Program Poster Session. Augusta, GA, July 17, 2008.
4. Woolf AD, Law T, Yu HYE, Woolf NT, Kellogg M. 2010. Lead poisoning from use of
bronze drinking vessels during the late Chinese Shang Dynasty: an in vitro experiment.
Clin Toxicol 48: 757-761.
5. Woolf NT, Moniz RJ. IL-10 and Its Role in Chronic Viral Infections. Presented at
Dana-Farber Cancer Institute in a Marasco laboratory meeting. Boston, MA, July 29,
2010.
36
6. Woolf NT. A Cold-hardening Strategy for the Marsh Amphipod Orchestia grillus.
Presented at the Connecticut College Biology/Botany Seminar Series. New London, CT,
May 13, 2010 and May 5, 2011.
7. Woolf, NT. Mechanism of cold acclimation in the salt marsh amphipod Orchestia
grillus. Connecticut College Honors Thesis in Biology. Submitted May 5, 2011.
8. Landesman-Bollag E, Shah A, Woolf NT, Gower A, Lee M-J, Fried S, Seldin D.
Reduced Dosage of CK2 Catalytic Subunit Genes Affects Adipogenesis in vitro.
Presented at the Evans Memorial Department of Medicine 100th Anniversary Celebration
Poster Session, Boston, MA, October 6, 2012.
9. Woolf N, Shafran J, Arafa E, Mehta M, Meyer R, Rahimi N. Immunoglobulin and
proline-rich receptor-1 (IGPR-1) is expressed in colon carcinoma cells and promotes
tumor growth. (in preparation)
Research Experience
2013-2015
MA Student, Rahimi Lab, Department of Pathology and Laboratory
Medicine, BUSM, Boston, MA
Thesis project: IGPR-1 is a novel adhesion molecule involved in colorectal
tumor growth
• Applied for and successfully received stipend funding from the CrossDisciplinary Training Program in Nanotechnology and Cancer (XTNC)
fellowship at Boston University for the 2013-2014 academic year
• Reviewed several concepts of cancer cell growth and metastasis in the current
literature, including the epithelial-mesenchymal transition and anoikis resistance
• Conducted MA thesis research on the effect of IGPR-1, a novel cell adhesion
molecule, on colorectal cancer cell growth and survival
• Maintained several HT29, HCT116, SW480, SW620 and Colo205 cell lines for
Western blots, MTT assays, and immunoprecipitation
• Took courses in basic and applied pathology, statistics, the business of science,
nanomedicine and post-translational modification
2012
Student Researcher, Seldin Lab, Evans Biomedical Research Center,
BUSM, Boston, MA
• Learned about the role CK2 plays in development, cancer and possible
connections to adipogenesis
• Worked on two projects that examined the effect that knocking down expression
of CK2 in mice had on adipogenesis
• Removed muscle, white adipose, brown adipose, liver, kidney, spleen and
pancreas from mice with varying levels of CK2 expression
• Ground up muscle and adipose tissue, then performed BCA quantification and
Western blot analysis on each sample (probing for UCP1 and UCP3 expression)
37
• Isolated mouse embryonic fibroblasts (MEFs) from embryos on Day 13 of
development, then grew and split these cells in culture
• Differentiated MEFs into adipocytes via a stringent protocol, then stained cells
with Oil Red O to quantify levels of differentiation
2010
Student Researcher, Marasco Lab, Dana-Farber Cancer Institute,
Boston, MA
• Acquired new knowledge concerning the details of innate and cell-mediated
immune responses
• Worked on a project focused on isolating fully-human monoclonal antibodies to
Interleukin-10
• Investigated the role IL-10 plays during both normal and chronic disease states
in the human immune system
• Performed molecular cloning and phage display techniques in an effort to isolate
promising antibody candidates
• Attended lectures given by either new postdoctoral candidates seeking to join
the lab or by experts in the field of immunology
2009-2011
Student Researcher, Loomis Lab, Biology Department, Connecticut
College, New London, CT
Thesis Project: Mechanism of cold acclimation in the salt marsh amphipod
Orchestia grillus
• Investigated the nature of the cold acclimation strategy of the amphipod
Orchestia grillus
• Determined LT50 values for animals collected in the spring and summer of 2009
• Monitored and recorded hysteresis activity in the hemolymph of 5 animals from
each collection
• Compiled supercooling points from all animals that were exposed to low
temperatures and produced an average SCP for each seasonal collection group
2008
Student Researcher, Cashikar Lab, Medical College of Georgia,
Augusta, GA
• Worked on a study that aimed to discover which molecular chaperone molecules
play prominent roles in helping mammalian cells recover from protein
aggregation
• Purified DNA from colonies of E. coli bacteria
• Cultured mammalian HEK-293T cells
• Transfected HEK-293T cells with a GFP-tagged plasmid vector
• Performed Hematoxylin and Eosin (H & E) staining of mouse brain tissue
• Performed immunohistochemistry to visualize protein oligomers in mouse brain
tissue
2007
Student Researcher, Baron Lab, National Institutes of Health,
Bethesda, MD
38
• Investigated the role certain genes play in mammalian somatic growth
deceleration
• Performed real-time RT-PCR on rat organ samples to quantify levels of these
genes
• Performed gel electrophoresis to confirm purity of cDNA used
• Assisted with a pilot study on rats by measuring tail lengths and weights of rat
pups
Skills
Computer: Adobe Photoshop, Microsoft Word, PowerPoint and Excel
Laboratory: Cell differentiation, molecular cloning of DNA, PCR, GST pulldown, SDSPAGE, Western blotting, immunoprecipitation, mammalian cell culture, viral culture,
collection and transduction of mammalian cells, hematoxylin and eosin (H&E) staining,
Gram staining
Awards
2013-2014
Fellow in the Boston University Cross-Disciplinary Training in
Nanotechnology for Cancer (XTNC) Program
2011
Recipient of the E. Frances Botsford Prize for excellence in biology and
service to the department at Connecticut College, New London, CT
Professional Affiliations
Member, American Association for the Advancement of Science
Member, American Medical Association
Member, American College of Physicians
Member, Massachusetts Medical Society
39