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ROLE OF WNT SIGNALING PATHWAY IN COLITIS-TO-CANCER TRANSITION
By
ANITHA SHENOY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2012
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© 2012 Anitha Shenoy
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To Lord ‘SAI’ who made this dissertation possible
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ACKNOWLEDGMENTS
Though only my name appears on the cover, several people have contributed
during the process and completion of this dissertation. It is my pleasure to thank all
those people who have made this dissertation possible and because of whom my
graduate experience has been one that I will cherish forever. I owe my deepest
gratitude to my mentor Dr. Edward Scott who gave me the freedom to explore on my
own, and at the same time the guidance to recover when I stumbled. He taught me how
to question thoughts and express ideas. His patience and support helped me overcome
many crisis situations and finish this dissertation. I hope that someday I would be as
good an advisor to my students as he has been to me. I am also deeply grateful to my
co-advisor Dr. Emina Huang with whom I had an opportunity to work closely in the lab.
Although a surgeon, her passion, love and dedication for basic science research have
been tremendous and always inspired me. Her constructive criticisms on my scientific
views and ideas helped me evolve as a better scientist and taught me innumerable
lessons and insights on the workings of academic research in general. I am indebted to
my committee members Drs. Edward Chan, and Maurice Swanson for their continuous
encouragement and guidance. The passion and effort to science that I felt from them I
will remember. My sincere thanks to Dr. Lung-Ji Chang who was involved in the
packaging of lentiviral particles. Also my heart felt thanks to Drs. Henry Appelman and
Myron Chang for performing the histological and statistical analyses respectively. In
addition, I must thank all my lab mates especially Drs. Liya Pi, Robert Fisher, Koji
Hosaka, Greg Marshall, Mark Krebs and Amanda Loguidice for their help and advices.
My special thanks to Li Lin, Elizabeth Butterworth, Tata Goluguri, Lu Corrie, Gary Brown
and Dustin Hart without whom my journey through PhD would have been incomplete. I
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will cherish memories I had with my fellow graduate students Niclas Bengston,
Seungbum Kim, Huiming Xia and David Lopez. I would also like to thank the staffs of
the core facilities at UF, Neal Benson, Marda Jorgensen, Mike Rule and Doug Smith for
their support and expertise. I cannot thank enough my friends and family. My parents,
Ramesh Shenoy and Asha Shenoy have always believed in me and have supported in
every step of my life. I am blessed to have parents like them. Savitha Shenoy and
Akshatha Shenoy are more of friends than sisters to me. They have seen me through
thick and thin and have always been there for me. My parents-in-law, Anandraya
Shenoy and Late. Veena Shenoy have always encouraged me to pursue whatever I am
keen on and interested in. For all their time and devotion I can only send them smiles in
return. All my love to my sons Sai and Krishna whose smiling faces revive me back to
life every evening after all the hard work of the day. Lastly, I could never have survived
the last few years without my husband Vinayak Shenoy to maintain my sanity and keep
me on track. It is his unconditional love that has kept me going and without him nothing I
have accomplished to date would have been possible.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 9
LIST OF ABBREVIATIONS ........................................................................................... 10
ABSTRACT ................................................................................................................... 15
CHAPTER
1 BACKGROUND AND SIGNIFICANCE ................................................................... 17
The Colon ............................................................................................................... 17 Structure and Function of the Colon ................................................................. 17
Microarchitecture of Colon ................................................................................ 18 Colonic Crypts .................................................................................................. 18
Goblet cells ................................................................................................ 19
Enteroendocrine cells ................................................................................ 19 Colonocytes ............................................................................................... 19
Pathology of Colon ................................................................................................. 19 Colon Cancer ................................................................................................... 20
Etiology of colon cancer ............................................................................. 20
Symptoms of colon cancer ......................................................................... 21 Diagnosis and stage determination of colon cancer ................................... 21
Treatment for colon cancer ........................................................................ 22 Colitis................................................................................................................ 22
Inflammatory Bowel Disease ............................................................................ 23 Ulcerative colitis ......................................................................................... 23 Colitis-associated colon cancer (CAC) ....................................................... 23
Stem Cells .............................................................................................................. 24 Embryonic Stem Cells ...................................................................................... 25 Adult Stem Cells ............................................................................................... 26 Cancer Stem Cells ........................................................................................... 27
Colon cancer stem cells ............................................................................. 28
Precursor-colon cancer stem cells ............................................................. 29 Stem Cell Assays ............................................................................................. 29
Non-adherent sphere assay ....................................................................... 29 Colony forming assay ................................................................................ 29
Limiting dilution assay (LDA) ...................................................................... 30 Xenograft transplantation ........................................................................... 30
Wnt Signaling Pathway ........................................................................................... 30 Wnt/ β-Catenin Signaling in the Colon .............................................................. 32
Wnt/ β-catenin signaling in colon development .......................................... 32
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Wnt/ β-catenin signaling in adult colon ....................................................... 33
Wnt/ β-catenin Signaling in Colon Diseases ..................................................... 33 Wnt/ β-catenin signaling in CRC ................................................................ 33
Wnt/ β-catenin signaling in CAC ................................................................ 34
2 MATERIALS AND METHODS ................................................................................ 42
Human Subjects and Animals ................................................................................. 42 Cell Culture ............................................................................................................. 42 Dual-Fusion Wnt Reporter and Lentiviral Transduction .......................................... 42
Xenograft Dissociation and FACS .......................................................................... 43 In vitro Limiting Dilution Assay (Clonogenic Potential) ............................................ 44 In vivo Limiting Dilution Assay (Tumorigenic Potential) and Serial Passages ......... 44
RNA Extraction and Real Time PCR....................................................................... 45 β-Catenin Knockdown ............................................................................................. 46
In vivo Indomethacin Treatment and -Catenin Knockdown Tumors ...................... 47
Immunostaining and Quantification ......................................................................... 47 Immunoblotting ....................................................................................................... 48
Statistical Analysis .................................................................................................. 48
3 EARLY ACTIVATION OF WNT/ -CATENIN SIGNALING IN INFLAMATION-DYSPLASIA-CARCINOMA SEQUENCE IS ASSOCIATED WITH COLITIS-TO-CANCER TRANSITION .......................................................................................... 56
Early Activation of Wnt/ β-Catenin Signaling in Non-Dysplastic Colitic Colon ......... 56
Overlap of Active Wnt/ β-Catenin Signaling with pCCSC Marker ALDH in Non Dysplastic Colitic Colon ....................................................................................... 57
Dual Fusion Wnt Reporter ...................................................................................... 58 Validation of the Wnt Reporter Constructs .............................................................. 59
Wnt Reporter Constructs in pCCSCs and CCSCs .................................................. 59
4 HIGH WNT ACTIVITY CONFERS SUSTAINED TUMOR INITIATING POTENTIAL ON PRECURSSOR COLON CANCER STEM CELL ......................... 77
Wnthigh pCCSCs Exhibit CCSC Properties While Wntlow pCCSCs Correspond to Transit-Amplifying Cell Population ....................................................................... 77
High Wnt Activity Confers More Efficient CCSC Activity to ALDHhigh Cells ............. 79
5 INHIBITION OF SUSTAINED WNT ACTIVITY IN WNTHIGH PCCSC REDUCES TUMOR GROWTH RATE ....................................................................................... 94
β-Catenin Knockdown Decreases the Tumor Growth Rate .................................... 94
Pharmacological Inhibition of -Catenin Delays the Rate of Tumor Growth ........... 95
6 DISCUSSION ....................................................................................................... 100
LIST OF REFERENCES ............................................................................................. 106
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BIOGRAPHICAL SKETCH .......................................................................................... 116
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LIST OF TABLES
Table page 2-1 Antibodies utilized for Immunohistochemistry ..................................................... 50
2-2 Antibodies utilized in Western blotting ................................................................ 50
4-1 Tumorigenic and self-renewal potential of Wnthigh vs Wntlow cells derived from CT-1 pCCSCs. .................................................................................................... 92
4-2 Tumorigenic and self-renewal potential of Wnthigh vs Wntlow cells derived from CT-2 pCCSCs. .................................................................................................... 92
4-3 Tumorigenic and self-renewal potential of Wnthigh vs Wntlow cells derived from CA-1 pCCSCs. ................................................................................................... 93
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LIST OF FIGURES
Figure page 1-1 Colon in digestive system ................................................................................... 35
1-2 Microarchitechture of colon ................................................................................ 36
1-3 Crypt of Colon ..................................................................................................... 37
1-4 Eitiology of colon cancer ..................................................................................... 38
1-5 Colonoscopic images of normal and ulcerative colon ......................................... 38
1-6 Models of heterogeneity in solid tumors ............................................................. 39
1-7 Wnt signaling pathway ........................................................................................ 40
1-8 Mutations that occur in adenoma to carcinoma sequence in CRC and dysplasia to carcinoma sequence in CAC .......................................................... 41
2-1 Lentiviral constructs used in the study ................................................................ 51
2-2 A representative image of the FACS histogram................................................. 54
2-3 Overview of experimental design. ....................................................................... 55
3-1 Wnt/ β -catenin signaling in normal, colitis and CRC colon ................................ 62
3-2 Active Wnt signaling pathway as a percentage of crypt epithelial cells. ............. 63
3-3 Wnt/β-catenin signaling in ALDH+ cells of normal, colitis and CRC colon .......... 64
3-4 ALDH+ and nuclear/cytoplasmic β-catenin staining as a percentage of crypt epithelial cells ..................................................................................................... 65
3-5 Active β-catenin staining on SW480 and HEK293 cells ...................................... 66
3-6 FACS analyses of TTLG and TLG transduced HEK293 and SW480 cells. ........ 67
3-7 Wnt activity in SW480 and HEK293 cells to validate the dual fusion Wnt reporter. .............................................................................................................. 68
3-8 The eGFP expression (Wnt activity) in TTLG and TLG transduced colitis sphere cells (pCCSC) ......................................................................................... 70
3-9 The eGFP expression (Wnt activity) in TTLG and TLG transduced colon cancer sphere cells (CCSC) ............................................................................... 71
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3-10 Bioluminescence imaging of tumors. .................................................................. 72
3-11 Real-time PCR of Wnt target genes in Wnthigh and Wntlow fractions of CT-2 pCCSCs ............................................................................................................. 73
3-12 Real-time PCR of Wnt target genes in Wnthigh and Wntlow fractions of CT-1 pCCSCs ............................................................................................................. 74
3-13 Real-time PCR of Wnt target genes in Wnthigh and Wntlow fractions of CCSCs ... 75
3-14 TTLG-eGFP fractions (2% highest and lowest) of the indicated sphere isolates stained for activated β-catenin. .............................................................. 76
4-1 No difference in clonogenic potential of CT-2 ALDHhigh-Wnthigh and ALDHhigh-Wntlow cells. ........................................................................................................ 80
4-2 No difference in clonogenic potential of CA-1 ALDHhigh-Wnthigh and ALDHhigh-Wntlow cells. ........................................................................................................ 81
4-3 Clonogenic potential of CT-1 Wnthigh and Wntlow cells. ....................................... 82
4-4 Wnt activity in Wnthigh and Wntlow colitic primary tumors .................................... 83
4-5 Histology of ALDHhigh-Wntlow and ALDHhigh-Wnthigh derived primary tumors ....... 84
4-6 Clonogenic potential of Wnthigh and Wntlow cells derived from CT-2 primary and secondary tumors ........................................................................................ 85
4-7 Clonogenic potential of Wnthigh and Wntlow cells derived from CA-1 primary and secondary tumors ........................................................................................ 86
4-8 At lower dilutions, Wnthigh colitic secondary tumors grew faster than Wnthigh CT-2 primary tumors ........................................................................................... 87
4-9 Primary colitic Wntlow tumor creates a phenocopy of a Wnthigh tumorors ............ 88
4-10 Tumor from single Wnthigh cell derived from primary ALDHhigh-Wnthigh xenograft. ........................................................................................................... 89
4-11 Histology of single Wnthigh cell derived tumor (left) and 500 cells derived ALDHhigh primary tumor (right) ............................................................................ 90
4-12 β-catenin and Muc 2 staining in single cell tumor. .............................................. 91
5-1 FACS analysis indicating the -catenin knockdown by shRNAs......................... 96
5-2 Western data indicating the level of β-catenin in CT-2 TTLG eGFP cells transduced with shRNA Sc, #1 and #2. ............................................................. 97
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5-3 ShRNA against β-catenin decreases the tumor growth rate ............................... 98
5-4 Inhibition of tumor growth rate by indomethacin ................................................. 99
6-1 A schematic diagram that suggests the CSC hierarchy in CAC ....................... 105
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LIST OF ABBREVIATIONS
ALDH Aldehyde dehydrogenase
APC Adenomatous polyposis coli
ASC Adult stem cells
CAC Colitis-associated cancer
CBC Crypt base columnar cells
CCSC Colon cancer stem cells
CMV Cytomegalovirus
CRC Colorectal cancer
CSC Cancer stem cells
DCAMKL1 Doublecortin-like & Ca/Calmodulin-dependent protein kinase-like 1
eGFP Enhanced green Fluorescent protein
ESC Embryonic stem cells
FACS Fluorescence activated cell sorting
FAP Familial adenomatous polyposis
FDA Food and drug administration
FOBT Fecal occult blood test
HEK Human embryonic kidney cells
HNPCC Hereditary nonpolyposis colorectal cancer
IBD Inflammatory bowel disease
IPSC Induced Pluripotent stem cells
mRFP Monomeric red Fluorescent protein
pCCSC precursor-Colon cancer stem cells
RNAi RNA interference
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SC Stem cells
sFRP secreted Frizzled-related protein
ShRNA Short hairpin RNA
SiRNA Small interfering RNA
TC Transduction control
TCF T-cell factor
TLG minimal Thymidine kinase promoter-Firefly Luciferase-eGFP
TTLG TCF/LEF binding site- minimal Thymidine kinase promoter-Firefly Luciferase-eGFP
UC Ulcerative colitis
WIF Wnt inhibitory factor
Wnt Wingless
YFP Yellow Fluorescent protein
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
ROLE OF WNT SIGNALING PATHWAY IN COLITIS-TO-CANCER TRANSITION
By
Anitha Shenoy
May 2012
Chair: Edward William Scott Major: Medical Sciences – Molecular Cell Biology
One of the severe complications of ulcerative colitis (UC) is colorectal cancer
(CRC). However, very little is known about the transition from colitis-to-cancer. The
transition involves a poorly understood inflammation-dysplasia-carcinoma sequence,
while sporadic CRC is associated with well-characterized adenoma-carcinoma
sequence. One of the early events in this sequence of sporadic CRC is the activating
mutation in genes involved in Wnt/β-catenin signaling. Similar to sporadic CRC, in the
current study we have demonstrated an early activation of Wnt/β-catenin signaling in
the colitis-to-cancer transition. We observed intermediate level of Wnt activity in the
cells of UC when compared to normal colon and CRC by performing β-catenin
immunostaining on patient derived colon tissues. These Wnt-pathway-active cells
constitute a major subpopulation (52%+7.21) of ALDH+ cells that in UC are referred to
as precursor-colon cancer stem cells (pCCSC). By in vitro clonogenicity assays and
serial xenograft transplantations we established the ability of Wnthigh pCCSCs to exhibit
cancer stem cell (CSC) properties like self-renewal and tumor initiation. Moreover, a
single Wnthigh pCCSC was sufficient to initiate the tumor, suggesting the association of
Wnt/β-catenin signaling with the transformation of pCCSCs to CCSCs and thus the
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colitis-to-cancer transition. This was confirmed by shRNA-mediated down-regulation of
β-catenin in Wnthigh pCCSCs in vivo. Furthermore, pharmacological inhibition of Wnt/β-
catenin signaling was achieved by using FDA approved drug, indomethacin that
resulted in reduced tumor growth rate. Thus, high levels of Wnt/β-catenin signaling not
only further demarcate (with ALDH positivity) the tumor-initiating cell compartment of the
non-dysplastic epithelium of UC patients, but also represent a plausible diagnostic
marker and a therapeutic target for early intervention in the colitis-to-cancer transition.
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CHAPTER 1 BACKGROUND AND SIGNIFICANCE
The Colon
The colon, otherwise called the large intestine constitutes the lower
gastrointestinal tract. It connects small intestine to the rectum. The colon occupies all
four quadrants of the abdomen. The segmented appearance of the colon is due to
‘haustra’ of the colon. ‘Haustra’ are the small pouches that cause sacculations in the
colon.
Structure and Function of the Colon
The colon starts at the last part of the small intestine, known as the ileum. The
ileum is connected to the caecum, which is the first part of the colon in the lower right
quadrant of the abdomen. The rest of the colon is divided into four parts (Figure 1-1):
1. The ascending colon travels up the right side of the abdomen. 2. The transverse colon runs across the upper abdomen. 3. The descending colon travels down the left abdomen. 4. The sigmoid colon is a short curving of the colon, just proximal to the rectum.
Embryologically, the ascending colon to proximal transverse colon is developed
from the mid-gut and the distal transverse colon to sigmoid colon from the hindgut. The
colon is involved in the final stages of digestive process. It maintains the fluid balance of
the body. It absorbs certain vitamins, minerals and stores waste before it is eliminated.
The bacterial flora on the lining of the colon processes indigestible material (such as
fiber). The rest of the undigested matter along with mucus forms the feces. As the feces
make its way through the colon, the lining absorbs most of the water and the feces enter
the rectum.
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Microarchitecture of Colon
The wall of the colon is made up of four distinct layers: the mucosa, the
submucosa, the muscularis propria and the serosa (Figure 1-2).
1. Mucosa: It is the innermost layer of the colon. Three components of mucosa are- a single layer of epithelial cells that form the inner most layer of the mucosa, lamina propria or basement membrane lies outside the epithelium that is made up of connective tissues like blood and lymphatic vessels and the third is muscularis mucosae, which is a thin smooth muscle layer that forms the outer layer of mucosa. The mucosa comprises of crypts, which are straight, tubular structures that are separated by lamina propria (Figure 1-2).
2. Submucosa: As the name indicates it lies beneath the mucosa and contains connective tissue in which blood vessels, lymphatics and nerve vessels are embedded.
3. Muscularis propria: It is made up of circular and longitudinal muscle layer. Circular muscle layer is made up of smooth muscle cells that help move waste materials along the colon on contraction. Longitudinal muscle layers run length wise along the colon and in conjunction with circular muscles help in the propelling movement called peristalsis.
4. Serosa: Serosa forms a single layer on outside of colon and is called the visceral peritoneum.
Colonic Crypts
Crypts form important part of the mucosal layer of colon. It extends vertically down
from the flat surface of the mucosa and penetrates deep into the submucosa (Figure 1-
2). These crypts are lined by single layer of epithelial cells that consists of several
differentiated cell types and is in turn lined with mesenchymal cells (Figure 1-3) (1). The
bottom of the crypt contains stem cells (SCs) that are actively cycling (2). The
remainder of the crypt is largely occupied by transit-amplifying cells, which are
estimated to divide twice a day and are key to the rapid renewal of the epithelium (3). At
the top of the crypt, proliferation halts, and cells differentiate into either secretory cells
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like goblet cells that produce mucin and enteroendocrine cells that produce certain
digestive enzymes or absorptive colonocytes (4)(Figure 1-3).
Goblet cells
Goblet cells are one of the types of secretory cells of colon. These cells secrete
protective mucins and trefoil proteins that are required for the movement and effective
expulsion of gut contents. Goblet cells also provide protection against chemical damage
and shear stress. Most abundantly secreted gastrointestinal mucin is Muc2, which is a
widely used goblet cell marker (2). Inhibition of Notch pathway in the intestinal
epithelium results in a vast conversion of epithelial cells into goblet cells (5, 6).
Enteroendocrine cells
Enteroendocrine are alternatively termed as neuroendocrine cells. These cells
secrete specific peptide hormones. Based on morphology and expression of specific
intestinal hormones or marker gene expression, enteroendocrine cells are of 15
different subtypes. Enteroendocrine cells are present throughout the mucosa,
representing approximately 1% of the cells lining the intestinal lumen (7).
Colonocytes
Colonocytes are absorptive cells and are also called as columnar cells. These
cells are highly polarized cells that carry an apical brush border, which is responsible for
absorbing and transporting nutrients across the epithelium. Colonocytes constitute more
than 80% of all intestinal epithelial cells (2).
Pathology of Colon
Many disorders affect the colons ability to function properly. The most common
disorders of colon are colon cancer, colitis, diverticulitis, colon polyps and irritable bowel
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syndrome. Common symptoms of these diseases are diarrhea or constipation
depending on the disease.
Colon Cancer
Colon cancer along with rectal cancer is termed as colorectal cancer (CRC).
Colorectal cancer (CRC) is currently the third leading cancer diagnosed and is also the
third leading cause of cancer-related deaths in the United States as reported by the
American Cancer Society in ‘Colorectal cancer Facts and Figures 2011-2013 (8). The
development of colorectal cancer is characterized by a sequence of events, which
gradually transforms cells from normal colonic epithelium to adenoma and eventually to
adenocarcinoma (Figure 1-7).
Etiology of colon cancer
There are several causes of colon cancer (Figure 1-4). Among them, sporadic
colon cancer contributes to 75% of the cases. Sporadic CRC occurs in people without
any identifiable predisposing etiology. In other words these individuals have no or very
little family history of the disease. Familial cases, such as familial adenomatous
polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), and the
hamartomatous polyposis syndromes usually have a family history of CRC.
Approximately 15% of CRC is attributed to familial CRC (9). Close to 10 % of CRCs are
due to genetic reasons, where the genes that function as tumor suppressors or, less
frequently, oncogenes are mutated at germline level. Inflammatory bowel disease (IBD)
associated CRC accounts for 1% of all the cases of CRC. The risk for CRC increases
with both the duration and the length of distribution of IBD within the colon. Those
patients with IBD are at 6 times higher risk to develop CRC compared to general
population (10, 11).
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Symptoms of colon cancer
Many cases of CRC may present with no symptoms. However, common
symptoms like bloody stools, abdominal distension, unexplained weight loss, persistent
nausea, narrow ribbon-like stools and increased frequency of bowel movement might
indicate colon cancer (12).
Diagnosis and stage determination of colon cancer
Colon cancer if detected at an early stage colon cancer is completely curable.
However very few screening /diagnostics tools are currently available to detect colon
cancer. Colonoscopy and fecal occult blood test (FOBT) are most commonly used.
Colonoscopy is a procedure, in which a colonoscope is used that allows the
visualization of entire length of the colon. During this procedure a small piece of colon
tissue could be isolated for biopsy. Simultaneously, polyps, largely the precursors of
sporadic CRC could be removed. The second test called FOBT helps in the detection of
small amounts of blood in the pathology including colon cancer, thus it a less sensitive
test. However, FOBT along with colonoscopy helps in affirmation of colon cancer (13).
Other newly available screening tests are CT colonography and DNA tests of stool. CT
colonography also known as virtual colonoscopy is as sensitive as colonoscopy and is a
non-invasive test. However, this test includes risk of exposure to radiation and requires
colonoscopy to confirm and remove the detected lesions (13). On confirmation of colon
cancer, several other tests like CT or MRI scans of the abdomen, pelvic area, chest, or
brain may be performed to stage the cancer i.e, to determine the severity of the disease
(14). Staging of colon cancer indicates the extent to which the cancer has invaded or
spread. There are five different stages of cancer:
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Stage 0: innermost layer of colon is involved
Stage I: inner layers of the colon are cancerous
Stage II: Cancer has invaded through the muscle wall of the colon
Stage III: Cancer has spread to the lymph nodes
Stage IV: Cancer has spread to other organs like liver, lungs etc
These stages are further characterized based on TMN (tumor, node, metastases)
system (13).
Treatment for colon cancer
Three main therapies used in the treatment of CRC are surgery, chemotherapy
and in the case of rectal cancer, radiation therapy. The first line of treatment for CRC is
surgery, where the cancerous colon is cut out by a procedure that is referred to as
colectomy. Treatment that is offered after the surgical procedure is referred to as
adjuvant therapy. Stage III cancer patients receive chemotherapy as adjuvant therapy.
The most commonly used chemotherapeutic agents for CRC are Irinotecan, oxaliplatin,
capecitabine, and 5-fluorouracil. Targeted adjuvant therapy includes monoclonal
antibodies like cetuximab (Erbitux) and panitumumab (Vectibix) against epidermal
growth factor receptor, and bevacizumab (Avastin) against Vascular endothelial growth
factor. Radiation therapy is commonly used in rectal cancer when compared to colon
cancer.
Colitis
Colitis is inflammation of the inner lining of the colon and is associated with
diarrhea, pain, and blood in the stool. Different types of colitis are IBD, infectious,
ischemic and microscopic colitis. Infectious colitis is caused by pathogens like virus and
bacteria. Ischemic colitis is the inflammatory condition of colon caused by temporary
loss or lack of blood supply. Microscopic colitis is the collective term often used for
collagenous and lymphocytic colitis. It is called so as it is confirmed by examining a
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sample of colon tissue using a microscope. IBD is chronic inflammation of entire or
certain parts of digestive tracts. It includes ulcerative colitis (UC) and Crohn’s disease.
Inflammatory Bowel Disease
Ulcerative colitis (UC) and Crohn’s disease are two chronic inflammatory
conditions, which constitute IBD. UC is always restricted to the colon and rectum and
affects the innermost lining. However, Crohn’s disease commonly affects the last part of
the small intestine (called the terminal ileum) and parts of the colon, but can also attack
any part of the digestive tract from mouth to anus. Also, unlike UC, Crohn’s disease
often spreads deep into the wall of affected part of the digestive tract.
Ulcerative colitis
Ulcerative colitis is an idiopathic, chronic inflammatory condition that tends to
fluctuate between periods of remission (inactivity) and relapse (activity). It usually
begins at the rectum and spreads proximally into the colon in a symmetrical,
circumferential and uninterrupted fashion. Figure 1-5 shows the colonoscopic image of
normal and UC colon. The incidence of UC is 1.2 to 20.3 cases per 100,000 persons
per year, and its prevalence is 7.6 to 246.0 cases per 100,000 per year (15). Common
symptoms of UC include diarrhea with bloody stools, rectal bleeding, abdominal pain
and cramping, anemia and weight loss. Arthritis, mouth sores, skin rashes, and eye
inflammation are accompanying symptoms in some individuals (16). Similar to CRC,
colonoscopy and FOBT are commonly used diagnostic tests that are conducted in UC.
Colitis-associated colon cancer (CAC)
CAC is a type of CRC, which is one of the most severe complications of colitis.
Based on meta-analysis on 116 studies, the overall incidence of colorectal cancer in
any patient with UC was reported to be 3.7% (17). Also this study found an increased
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risk for CRC with increased duration of UC (17). Unlike the conventional adenoma to
adenocarcinoma progression in CRC, CAC advances through inflammation-to-dysplasia
and dyplasia-to-adenocarcinoma sequence (18). So far, no direct evidence of genetic
cause has been found for the increased risk of CRC in patients with UC. However,
several molecular responses such as generation of reactive oxygen species,
microsatellite instability, telomere shortening and chromosomal instability have been
attributed to inflammation driven genomic stress that leads to CRC (19). Although these
studies have shed light on understanding of inflammation associated carcinogenesis,
the markers based on these studies lack sensitivity or specificity to be used as reliable
biomarkers to assess the risk of colorectal cancer in patients with UC (19). On the other
hand screening for senescence markers, α-methylacyl-CoA-racemase and mutations in
p53 have been suggested to be potential pre-neoplastic markers in UC (20-23).
Stem Cells
Stem cells can be maintained indefinitely via their property of self-renewal. Further
they have the ability to differentiate into multiple cell lineages. Thus self-renewal and
ability to differentiate are the characteristic feature of stem cells and combination of
these properties are often referred to as ‘stemness’ (24, 25). Based on the potential to
differentiate there are 5 types of stem cells. They are
1. Totipotent stem cells: It is a single cell that has the ability to differentiate into all kinds of cells that makes an organism including extra embryonic tissue like placenta. Example: spores and zygote (25, 26)
2. Pluripotent stem cells: These cells have the ability to differentiate into any of the three germ layers i.e., endoderm, mesoderm and ectoderm
3. Multipotent stem cells: These cells have the ability to differentiate into multiple cell types but to limited number of lineages. Example: hematopoietic stem cells.
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4. Oligopotent stem cells: These cells differentiate into very few cell types. Progenitor cells belong to this category Example: lymphoid and myeloid progenitor cells (27)
5. Unipotent stem cells: These cells can differentiate into only one cell type. The ability of the liver to regenerate is attributed to unipotent property of hepatocytes.
Stem cells are found in embryo as well as in adult somatic tissue. Stem cells
derived from the inner cell mass of blastocyst stage of embryo are referred to as
embryonic stem cells and those found in adult tissues are referred to as adult stem
cells.
Embryonic Stem Cells
Embryonic stem cells (ESCs) are pluripotent, self-renewing cells that are derived
from blastocyst inner cell mass and are required for development of embryo proper (28).
ESCs obtained from mouse and human embryos can be grown in culture indefinitely
retaining their self-renewal property (29-31). Though ESC is very promising in
regenerative and tissue replacement therapy, apart from ethical controversies, the
availability and the perfect matching donor ESCs for the patient are the major issues.
These drawbacks could be overcome by using induced pluripotent stem cells (IPSC),
which are reprogrammed somatic cells that share several features with ESCs, including
a similar morphology in culture, the re-expression of pluripotency markers and the ability
to differentiate into distinct cell lineages (28). Though IPSCs can be successfully
generated from mouse and human cells (32-36), its application in regenerative and
tissue replacement therapy needs further research and perfection in terms of
differentiating the IPSCs into specific lineages. However it is advancing the potential for
personalized medicine.
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Adult Stem Cells
Stem cells that reside in the organs of adult organisms are known as Adult stem
cells (ASC) or somatic stem cells. ASC are organ specific and are multipotent,
quiescent, self-renewing cells that have the ability to differentiate into various cell
lineages that constitute that organ. Adult stem cells are found in all the body tissues and
function in tissue homeostasis and repair. They can be isolated from bone marrow,
intestine, liver, brain, dental pulp, hair follicles, skin, skeletal muscle, adipose tissue and
blood (37).
Colon Stem cells: Colon stem cells are a type of adult intestinal stem cells that are
specific to colon. Colon stem cells are usually found at the base of the colon crypt and
are part of the epithelial cell compartment. The estimated number of colon stem cells is
between four and six per crypt (38). Colon stem cells can divide either in symmetric or
asymmetric fashion in order to maintain the balance between self-renewal and
production of daughter cells (39-42). Symmetric division results in two intrinsically
similar daughter cells where the stem cell can either divide into two self-renewing stem
cells or two daughter cells, which have the potential to differentiate. This kind of division
occurs when there is a need for expansion of stem cell pool, as in during embryonic
development, disease and cancer or after tissue injury (43) . Alternatively, asymmetric
division results in daughter cells with different fates, where one daughter cell maintains
stemness and the other differentiates (44). During homeostasis, asymmetric division not
only maintains the number of stem cells but also contributes to the constant production
of transit-amplifying daughter cells (41).
Two models namely classic and stem cell zone model, define the exact identity of
colon stem cells. As per the classic model, cells at position + 4 from bottom of crypt
27
represent the crypt stem cell population, which were originally identified based on DNA
label retaining ability (45). The +4 cells cycle actively and they retain the label from
asymmetric segregation of old and new DNA strands (46, 47). However, the weakness
of this model is that there are no studies that link the +4 cells to their cellular progeny
(2). On the other hand, in more recent model, crypt base columnar cells (CBC)
spanning cell positions + 1 to + 4 termed the ‘stem cell zone’ could be clonally tracked
and thus represent the crypt stem cell population (38, 48-50). In order to identify, track
or isolate these stem cells, a definite stem cell marker is required. In mice, based on in
vivo lineage tracing studies, LGR5, Bmi1, CD133/ prominin 1, mTert and Sox9 were
shown to be reliable but not perfect intestinal markers. Microarray profiling of LGR5+
cells revealed that Ascl2 and Olfm4 are restricted to CBC cells. Based on positional
information Dcamkl1 and Musashi-1 were also indicated as stem cell markers (51). In
human colon crypts CD29, LGR5, MsiI, DCAMKL1 and ALDH1 are the often-used stem
cell markers (52-56).
Cancer Stem Cells
CSC are those cancer cells which have the capacity to self-renew, continually
sustain tumorigenesis and differentiate into various lineages of cells that constitute the
tumor (57). Two models have been proposed for tumor heterogeneity- 1) the Stochastic
or Clonal evolution model and 2) the Hierarchical or CSC model (Figure 2-6). As per the
stochastic model, every cancer cell has equal potential to proliferate, however, only few
cells clonally expand. On the other hand, according to hierarchical model, only limited
number of cells in a cancer cell pool has the ability to proliferate extensively and clonally
expand. Thus CSC model follows hierarchical organization with CSC being at the top of
the hierarchy (58). CSCs were first reported in acute myeloid leukemia as the rare
28
subset of cancer cells that had the ability to induce leukemia when transplanted into
immunocompromised mice (59, 60). Apart from leukemia, based on specific markers,
CSCs have been isolated and propagated from several solid tumors such as cancers of
breast, stomach, intestine, pancreas, brain, prostate, melanoma, lung, bone and head &
neck (61). CSCs because of their quiescent nature are highly resistant to chemotherapy
and targeted therapies (62). Hence, it becomes all the more essential to identify these
CSCs and study the mechanisms that evade these cells from death. There are both ex
vivo and in vivo model systems to validate the existence and to study the properties of
CSCs. Non-adherent sphere assay and colony forming assay that are described in
detail in later sections are ex vivo systems and in vivo systems include the xenograft
transplantation in the flanks and gold standard being orthotopic transplantation in
immunocompromised mice. In order to determine the stem cell frequency, in vitro and in
vivo limiting dilution assays (LDA) are frequently performed in CSC research.
Colon cancer stem cells
Cancer stem cells in the cancerous colon are referred to as CCSCs. Several
markers for CCSCs have been identified. CCSCs were first independently described
and identified by two groups in 2007 (63, 64). These groups showed that CD133 could
be used as marker for CCSCs. Dalerba et al. demonstrated that ESA+/CD44 and
CD166 were subpopulation of CD133 and could be used as a marker for CCSCs (65).
Other normal stem cell markers like MsiI, CD29, and LGR5 have been shown to be
expanded in colon cancer (66, 67) and thus could be used as a CSC marker in colon
cancer. Also increased nuclear β-catenin could be used as a marker for colon cancer
stem cells (68). We have shown that ALDH is a better marker of CCSC than either
CD133 or CD44 alone (56).
29
Precursor-colon cancer stem cells
Similar to CCSCs, recently, we have identified ALDHhigh population in the normal-
appearing, non-dysplastic colonic epithelium of UC patients. This suggested for the first
time that CAC might have a CSC origin. Because of their capacity to initiate the colitis-
dysplasia-cancer transition, we refer to these cells as precursor-CCSCs (pCCSCs). Our
findings to date indicate that pCCSCs are a valuable model to characterize the colitis-to-
cancer transition (69). Using this model could pave a path for development of methods
for early disease diagnosis and targeted drug therapy, which together might prevent the
progression from colitis to cancer.
Stem Cell Assays
Non-adherent sphere assay
In this assay, cells are grown as non-adherent cultures in serum-free defined
media to form spheres. This assay is more commonly used to determine the stem cell
activity in putative CSC. Nonetheless, while interpreting these results one has to be
cautious as both progenitor cells as well as stem cells could be propagated as spheres
(62). One of the important properties of CSC is self-renewal. Sphere cultures that could
be passaged more than five times demonstrate the self-renewal property and provide
evidence of CSCs. However, this assay is not useful for quantifying stem cell frequency.
Colony forming assay
Clonality is another important characteristic of stem cells, where the cells clonally
expand. This assay is well studied in neural stem cell system. This property is tested by
growing the cells on collagen matrix or agar gel, where the cells are plated thinly to form
multilineage colonies. This assay provides a read out for differential proliferative
potential of SC (70). This assay therefore could be used for CSCs too.
30
Limiting dilution assay (LDA)
LDA in tumor biology is used to quantify the frequency of CSC. Thus it also helps
in determining the effectiveness of the marker that is used to isolate and purify CSCs.
LDA was first used in tumor studies by Hewitt in 1958 (71). This assay could be carried
out both in vivo and in vitro to determine the CSC properties like tumorigenic and
clonogenic potential respectively.
Xenograft transplantation
The gold standard to determine the CSC activity is to transplant the cells either in
the flanks or orthotopically in immunocompromised mice. Several different kinds of
immunocompromised mice like nude, NOD-SCID, NOD-SCID ILR -/- are used for this
purpose. However, a melanoma CSC study by Quintana et al., demonstrated that NOD-
SCID ILR -/- mice are more permissive to tumor formation when compared to NOD-
SCID mice (72).
Wnt Signaling Pathway
Wnt signaling pathway is involved in regulation of events ranging from
development to diseases. Wnt (wingless) genes, first discovered in Drosophila encode a
large family of secreted cysteine rich glycoproteins (73). Wnt protein sequences are
highly conserved across species, where mammals have 19 Wnt genes, which can be
classified into twelve distinct subfamilies based on their amino acid sequences (74). The
biological activity of these Wnt proteins depends on palmitoylation of the conserved
cysteine residues (75). Though there is no direct evidence of mechanism of
palmitoylation, it is speculated that porcupine/MOM1 mediates this process (76). In the
extracellular matrix, Wnt proteins interact with other secreted proteins such as sFRPs
(soluble Frizzled receptor protein) and WIF (Wnt inhibitory factor) that inhibit the Wnt
31
activity. Based on the functional activity of the Wnts, it is distributed into two groups: 1.
that activates canonical Wnt/ β-catenin signaling pathway and 2. non-canonical Wnt/
Ca2+ signaling pathway (73, 77). Wnts activate the signaling pathway in the cells by
binding to family of seven transmembrane spanning receptors named Frizzled (Fz) and
single transmembrane spanning protein LRP (lipoprotein related protein). Wnts can
either bind simultaneously to Fz and LRP or just Fz alone. Functional complex formed
by binding of Wnt to Fz and LRP leads to the formation of nuclear Tcf/β-catenin
complexes. This constitutes canonical Wnt / β-catenin signaling pathway (76). On the
other hand, decreased LRP expression or its down-regulated through secreted factors
such as Dickkopfs, channelizes the binding of Wnt to Fz and activates Tcf/β-catenin
independent cellular processes like increased Ca flux through Wnt/ Ca2+ signaling
pathway, repression of Tcf-mediated transcription, and cytoskeletal rearrangements.
These processes together constitute non-canonical Wnt signaling pathway(78). β-
catenin and transcription factor Tcf (T-cell factor) are the main players of Wnt/ β-catenin
signaling pathway. In the absence of Wnt, β-catenin is targeted to proteosomal
degradation by β-catenin destruction complex. This destruction complex is made up of
scaffold proteins- Axin and APC and β-Catenin phosphorylating proteins- CKI (Casein
Kinase I) (at ser 45 of β-catenin) and GSK3β (Glycogen synthase kinase 3 β) (at serine
33, 37 and threonine 41 residues of β-catenin after the priming phosphorylation by CKI).
The β-catenin phosphorylated by destruction complex is recognized by F-box-containing
protein β-TrCP, which mediates ubiquitination and proteosomal degradation of β-
catenin. This results in low levels of cytoplasmic β-catenin and repression of TCF
induced transcription (Figure 1-7). However in the presence of Wnt, the cytoplasmic tail
32
of LRP in Wnt-Fz/LRP complex gets phosphorylated, which allows docking of Axin to
LRP (79). Recruitment of Axin to the membrane disrupts the destruction complex, thus
resulting in high levels of cytoplasmic β-catenin. Also on binding of Wnt to Fz and LRP,
DSH (Dishivelled) associates with Fz and GSK3β binding protein, Frat. Thus DSH is
thought to be involved in the process of stabilizing the β-catenin in the cytoplasm (76).
Free β-catenin then translocates into nucleus and relieves the repression of TCF thus
inducing the transcription of Wnt target genes (Figure 1-7). Some of the important Wnt
target genes are Axin2, LGR5, cyclinD1, c-myc, CD44 etc. Most of these target genes
are involved in cell survival and proliferation.
Wnt/ β-Catenin Signaling in the Colon
Wnt signaling pathway plays an important role in maintaining the homeostasis of
normal colon function. In addition, it is involved in various processes of gut
development, and in various colon disorders.
Wnt/ β-catenin signaling in colon development
Wnt signaling plays a vital role in the gut epithelial development. Gut originates
from endoderm and in ascidan embryos β-catenin was found to be essential in
endoderm formation (76, 80). TCF4 was also demonstrated to be involved in gut
development where TCF knockout mice lost intestinal stem cell and progenitor
population and the mice died before crypt formation (81). In addition, Wnt genes were
shown to be involved in gut patterning during mouse and chick gut development (82).
One of the target genes of Wnt, cdx1 is expressed in the developing intestinal
endoderm (83). Thus Wnt signaling is quintessential in endoderm formation, patterning
and cytodifferentiation of the gut tube during development.
33
Wnt/ β-catenin signaling in adult colon
Wnt/ β-catenin /Tcf4 pathway is critical in maintaining the proliferative
compartment of the adult gut epithelium. In the colon, β-catenin is present in all
epithelial cell membranes along the crypt. However, nuclear accumulation of β-catenin
is specifically found in the epithelial cells located at the bottom of colonic crypt, that
marks the colon stem cells (82). The Wnt target gene, cdx1 is expressed in the
proliferative crypt compartment during differentiation (84). So far, several studies have
indicated strong link between Wnt signaling and maintenance of transit-amplifying cells
(76). Though LGR5, another Wnt target, as well as wnt pathway activator marks the
stem cells of adult intestine, its role in them is not well elucidated (53, 85).
Wnt/ β-catenin Signaling in Colon Diseases
Most of the colon diseases are associated with mutation in one or the other Wnt
signaling components. FAP is an autosomal dominant inherited disorder that is caused
by germline mutation in the APC gene (86). This syndrome is characterized by the early
onset of hundreds to thousands of nonmalignant polyps throughout the colon. If left
untreated, this syndrome develops into colon cancer by age of 35-40 years. Other colon
diseases, which involve aberrant Wnt signaling, are CRC and colitis-associated
cancers.
Wnt/ β-catenin signaling in CRC
The gene often mutated in sporadic CRC is APC (87). APC is mutated in 80% of
the sporadic CRCs (88). It is the first gene to be mutated in the adenoma to carcinoma
sequence of the sporadic CRC (Figure 1-8) (89). Other components of the Wnt pathway
like β-catenin and axins are also found to be mutated in sporadic CRCs (88).
34
Wnt/ β-catenin signaling in CAC
Mutational analysis of Wnt pathway genes in the progression from dysplasia to
carcinoma in CAC has ranked these mutations towards the end of the dysplasia-
carcinoma sequence (Figure 1-8) (90). However recent immunohistochemical analysis
have indicated early activation of Wnt signaling in colitis-to-cancer transition (91). Lee et
al. have also demonstrated activation of β-catenin via PI3K/Akt pathway in mouse
model of intestinal inflammation (92). However, the role of Wnt signaling in the transition
of colitis to cancer is not well studied.
35
Figure 1-1. Colon in digestive system. (www.kolorektum.cz/index-en.php?pg=for-the-public--colorectal-cancer)
36
Figure 1-2. Microarchitechture of colon. (Redrawn from www.afritz.org/casebook /samplepage _anatomy.pdf)
37
Figure 1-3. Crypt of Colon. (Medema JP and Vermuelen L, 2011 Nature)
38
Figure 1-4. Eitiology of colon cancer (Modified from www.genomedical.com/you_and_your_family/colon_cancer_risk.cfm)
Figure 1-5. Colonoscopic images of normal and ulcerative colon. (Virtual Medical centre
and Mohammad F El-Baba, Medscape )
39
Figure 1-6. Models of heterogeneity in solid tumors. a. Cancer cells of different phenotypes have the potential to proliferate extensively, but only few cells exhibit clonogenecity or tumorigenecity in vitro b. Only a subset of cancer cells consistently proliferates in clonogenic assays and can form new tumors on transplantation, whereas most cancer cells have limited proliferative potential. (Reya T et al. 2011 Nature)
40
Figure 1-7. Wnt signaling pathway. Left: In the absence of the Wnt ligand Right: In the presence of the Wnt ligand.
41
Figure 1-8. Mutations that occur in adenoma to carcinoma sequence in CRC and dysplasia to carcinoma sequence in CAC. (Jianlin Xie et al. 2008 World J Gastroenterol)
42
CHAPTER 2 MATERIALS AND METHODS
The optimized materials and methods described in this chapter have been
developed and adapted over a number of years from multiple previous reports and own
observations.
Human Subjects and Animals
Colitic and colon cancer patient tissues were retrieved under pathologic
supervision with Institutional Review Board approval at the University of Florida and
University of Michigan. Normal colon tissues were obtained from a local organ
procurement organization (Life Quest). Inbred NOD-SCID mice (5-6 weeks old) were
used. Mice were maintained under pathogen-free conditions and experiments were
approved by the University of Florida Institutional Animal Care Committee.
Cell Culture
ALDHhigh sphere-cell isolates were obtained from Ulcerative colitis (pCCSC) and
CRC patients (CCSC) and cultured in serum–free media as previously described by
Carpentino et al (69). Serum–free media are referred to as defined media.
Dual-Fusion Wnt Reporter and Lentiviral Transduction
Dual-fusion genes in the below-mentioned constructs were obtained from triple-
fusion constructs (93) (a generous gift from Sanjiv Gambhir). A dual fusion Wnt reporter,
TTLG (6XTCF/LEF binding site array, minimal Thymidine kinase (TK) promoter, Firefly
Luciferase and eGFP) was constructed using the 6X TCF/LEF binding sites and a
minimal TK promoter from Top-flash (Millipore). The dual-fusion gene consisting of
Firefly Luciferase and eGFP linked by a spacer was inserted downstream of the minimal
TK promoter in the lentiviral vector backbone pTYFcHS4WPRE (Figure 2-1A). The
43
negative control, TLG (minimal TK promoter, Firefly Luciferase and eGFP) was
constructed in a similar manner as TTLG, but lacked the 6X TCF/LEF binding site array
(Figure 2-1B). The transduction control, hRL-mRFP was constructed with constitutively
active promoter EF1 and dual-fusion genes made up of humanized Renilla Luciferase
and mRFP inserted into the lentiviral vector pTYF-EF (Figure 2-1C). These three
constructs as described in Figure 2-1 were packaged into a lentivirus as previously
described (94, 95). The spheroidal cultures of ALDHhigh pCCSC and CCSC were
lentivirally transduced with TTLG at 2.5 MOI. Flow cytometry was used to sort the
spheroidal cultures transduced with TTLG for the brightest 2% of the population, to
ensure that every cell in the culture was TTLG transduced. These TTLG-eGFPhigh
(Wnthigh) cells were expanded in vitro so that cultured cells mimicked the TTLG
transduced parental ALDHhigh population with heterogeneous levels of eGFP
expression. It is this expanded TTLG-eGFPhigh cells that are used throughout the study
to further isolate Wnthigh or Wntlow population.
Xenograft Dissociation and FACS
Xenograft tissues were minced thoroughly, digested with collagenase and
centrifuged. Resulting cell pellets were washed with HBSS/2%FBS. To isolate epithelial
cells from xenograft derived cells, anti-epithelial specific antigen (ESA)-FITC (Biomeda,
Foster City, CA) were used at a dilution of 1:40. Nonviable cells were eliminated by
using a viability dye, DAPI, just prior to submission for flow cytometry. Murine cells were
eliminated by staining for mouse major histocompatibility complex, H2Kd (1:40)
(Southern Biotech, Birmingham, Alabama). To detect H2Kd, PE-Cy5 secondary
antibody was used at 1:200 dilutions. Flow cytometry was performed on a FACS Aria
44
(BD Immunocytometry Systems, Franklin Lakes, NJ). Side and forward scatter profiles
were used to eliminate cell doublets.
In vitro Limiting Dilution Assay (Clonogenic Potential)
Cells with high and low eGFP intensities (corresponding to Wnthigh and Wntlow
cells) (Figure 2-2) were deposited at 1, 2, 3, 4, 6, 8, 10, 12, 16, 18, 20 and 24 cells per
well of 96-well, ultra-low adhesion plates (Corning,Corning, NY) containing defined
media. The above-indicated number of cells was added into 8 wells for each number.
Clonal frequency and statistical significance were evaluated with the Extreme Limiting
Dilution Analysis (ELDA) 'limdil' function
(http://bioinf.wehi.edu.au/software/elda/index.html). This assay was carried out with
ALDHhigh-Wnthigh spheroidal cultures as well as with the ESA+/H2Kd- cells obtained
from dissociated primary and secondary xenografts (Figure 2-3) .
In vivo Limiting Dilution Assay (Tumorigenic Potential) and Serial Passages
For primary tumors, 10, 100 and 1000 ALDHhigh-Wnthigh colitis and colon cancer
sphere cells with the 2% lowest and the 2% highest eGFP intensities, and
corresponding to ALDHhigh-Wnthigh and ALDHhigh-Wntlow cells (Figure 2-2), were
deposited by FACS, onto a 96-well plate containing defined medium admixed with
Matrigel at a 1:1 ratio such that the total volume was 100ul. Cells were injected
subcutaneously into the hind flanks of NOD-SCID mice. For secondary tumors, the
ALDHhigh-Wnthigh and ALDHhigh-Wntlow primary tumors were dissociated and the 10, 100,
and 1,000 ESA+/H2Kd- cells with the 10% highest and 10% lowest eGFP intensities –
corresponding to Wnthigh and Wntlow – were injected into NOD-SCID mice as described
above (Figure 2-3). Single cell injections were also carried out as secondary tumors with
both Wnthigh and Wntlow cells derived from primary tumors of indicated sphere isolates.
45
Also single cell secondary tumors of corresponding isolate were established with
ALDHhigh cells derived from primary ALDHhigh tumor. Tertiary tumors were generated
with 10, 100 and 1000 ESA+/H2Kd- cells with the 10% highest and lowest eGFP
intensities from Wnthigh secondary tumors (Figure 2-3).
RNA Extraction and Real Time PCR
Total RNA was extracted from the 2% highest and lowest TTLG-eGFP spheroidal
cultures using RNAeasy mini kits (Qiagen, Valencia, CA) in accordance with the
manufacturer's protocol. cDNA synthesis was performed using superscript III Reverse
transcriptase (Invitrogen, Carlsbad, CA). Real time PCR was carried out using SYBR
green detection reagents on a BioRad CFX machine with the following parameters:
95oC, 30 sec for one cycle followed by 40 cycles of 95oC, 5 sec for denaturation, and
60oC, 10 sec for annealing and extension. The melt curve was at 65-95oC for 5 sec/
cycle. The primers used for different Wnt target genes were 1. CyclinD1: (sense) 5’-
CCG TCC ATG CGG AAG ATC-3’and (antisense) 5’-ATG GCC AGC GGG AAG AC-3’,
2. c-myc : (sense) 5’-TCA AGA GGC GAA CAC ACA AC-3’and (antisense) 5’-GGC
CTT TTC ATT GTT TTC CA-3’ , 3. LGR5 : (sense) 5’-CTT CCA ACC TCA GCG TCT
TC-3’and (antisense) 5’-TTT CCC GCA AGA CGT AAC TC-3’, 4. Axin2 : (sense) 5'-TTA
TGC TTT GCA CTA CGT CCC TCC A- 3' and (antisense) 5'-CGC AAC ATG GTC AAC
CCT CAG AC-3' . As a control, β-actin primers were used (sense) 5’-GCT CAC CAT
GGA TGA TGA TAT CGC-3’ and (antisense) 5’- GAC CTG GCC GTC AGG CAG CTC
G-3’.
46
β-Catenin Knockdown
ShRNA vectors - construct 1, TRCN0000003843 (#1); and construct 2,
TRCN0000003844 (#2) against -catenin in the pLKO.1 vector backbone (The RNAi
Consortium) and pLKO.1 - scramble shRNA (Plasmid 1864 from Addgene) were
packaged into lentiviral particles using the 2 plasmid system. The two plasmids are
pMD2.G (Addgene #12259), the envelope plasmid and psPAX2 (Addgene #12260), the
packaging plasmid. Lentiviral preparation is a 4 days procedure.
Day 1: Transfection: Single well of 6 well plate with 80% confluent HEK293T cells is used for transfection. For transfection, 0.7ug each of PMD2.G, psPAX2 and pLKO.1-shRNA was added into 40ul of plain DMEM. To another 40ul plain DMEM, 6ul of Transit reagent 293 (Mirus 2700) was added. Both tubes were incubated for 20 minutes at room temperature (RT). Following that the plasmids containing plain DMEM is slowly mixed with transit reagent containing plain DMEM and the mixture is again incubated at RT for 20 minutes. This mixture is then added drop wise into 2ml of complete media containing 80% confluent HEK293T cells. The plate is incubated overnight at 37oC.
Day 2: The media was changed with fresh 2ml of defined media after a wash with 1X PBS
Day 3: 2 ml of virus containing media was collected and replaced with fresh 2ml of defined media again. The virus containing media was spun at high speed to get rid of the dead cells and the supernatant was saved on ice in cold room.
Day 4: Second collection of virus containing media was carried out similar to that on day 3. The day 2 and day 3 supernatants were mixed and alliquoted to be stored at -80oC.
The ALDHhigh/Wnthigh colitis and cancer sphere cells with the 2% highest eGFP
intensities were transduced in presence of 8 g/ml of polybrene, with each of all three
shRNA lentivirus containing supernatant and were selected using puromycin (0.25
g/ml).
47
In vivo Indomethacin Treatment and -Catenin Knockdown Tumors
One hundred ALDHhigh-Wnthigh sphere cells with the 2% lowest and highest eGFP
intensities (corresponding to ALDHhigh-Wnthigh and ALDHhigh-Wntlow cells), admixed with
Matrigel at 1:1 ratio such that the total volume was 100 ul, were injected subcutaneously
into the hind flanks of NOD-SCID mice. At day 4 post-injection, the mice were injected
with 2.5-mg/kg indomethacin (Calbiochem, Billerica, MA) or DMSO i.p. every 12 hours
for 6 weeks. For β-catenin knockdown tumors 100 each of control (scrambled) shRNA
and #2 shRNA treated Wnthigh pCCSCs and CCSCs were prepared for injections as
mentioned above and were injected subcutaneously into the hind flanks of NOD-SCID
mice. Tumor size was measured twice a week using manual calipers for 6 weeks.
Immunostaining and Quantification
Paraffin embedded 5um colon sections from normal, colitis and colon cancer were
used. -catenin antibody (BD Transduction Laboratories, San Jose, CA) was used at
1:800 dilutions with citrate retrieval. For dual staining of -catenin and ALDH, Dako
retrieval was used for -catenin (1:600 dilution) followed by mouse on mouse (M.O.M)
kit (Vector labs, Burlingame, CA) used for ALDH (BD Transduction Laboratories, San
Jose, CA) (1:100) staining. M.O.M kit was also used for staining the xenograft tissue
sections, where -catenin antibody was used at 1: 400 dilutions. Active--catenin (ABC)
antibody (Millipore, Long beach, CA) (recognizes active form of β-catenin,
dephosphorylated on Ser37 or Thr41) was used at a 1:100 dilution on cytospun TTLG-
eGFPhigh and TTLG-eGFPlow colitic pCCSC and CCSCs. Cytospun cells were fixed
using 1:1 methanol and acetone for 30 min at -20oC. All the secondary antibodies were
used at 1:500 dilutions. Visualization was carried out under the same microscopy
48
settings at room temperature using laser spinning confocal microscopy (Leica TCS SP2,
Wetzlar, German) with the accompanying software (Slidebook, Irving, Tx). Detailed
descriptions of antibodies are in Table 2-1. Quantification was carried out by counting
the cells in 4 different fields of each tissue section at 40X magnification. The number of
epithelial cells counted ranged from 4000 to 6000 epithelial cells per disease condition.
Immunoblotting
For total protein isolation from shRNA transduced cells, the cells were first spun
down and washed with 1X PBS. Total proteins were then extracted in Radio-
immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) with protease inhibitors (Roche) and
PMSF (phenylmethylsulfonyl fluoride) at a final concentration of 1mM. The proteins
were separated using 10% SDS-polyacrylamide gel electrophoresis and the total -
catenin was detected using anti-human -catenin raised in mouse (BD Transduction
Laboratories, San Jose, CA) at 1: 2000 dilutions. Anti-mouse HRP conjugated
secondary antibody (Amersham Piscataway, NJ) was used at 1:10,000 dilutions. Later
the membrane was stripped to detect the housekeeping protein -Actin using anti-
human -Actin raised in mouse (Sigma) at 1:5000 dil. Detailed descriptions of
antibodies are in Table 2-2.
Statistical Analysis
Data are presented as means + standard error as indicated in the figure legends.
Statistical significance was defined as p < 0.05 after an unpaired Student’s t-test or a
one-way analysis of variance. To compare tumor growth rates, a mixed linear model
was used with tumor volume as the response variable and with time and group as
49
explanatory variables. We included subject (mouse) as a random effect, and we
assumed a compound symmetric covariance structure.
50
Table 2-1. Antibodies utilized for Immunohistochemistry
Protein Host Concentration Retreival Company Cat#
-catenin Mouse 1) 1:800 2) M.O.M Kit (Dual stain)- 1: 600 3) M.O.M Kit (Xenografts)- 1:400
Citrate BD Biosciences
610153
ALDH Mouse M.O.M Kit (Dual stain)- 1:100
Citrate BD Transduction Laboratories
611194
Muc2 Mouse 1:100 Citrate Vector Labs VP-M656
Active--catenin (ABC)
Mouse 1:100 Acetone: Methanol (1: 1)
Millipore 05-665
Table 2-2. Antibodies utilized in Western blotting
Protein Host Concentration Mol Wt (KDa)
Company Cat#
-catenin Mouse 1:2000 92 BD Biosciences
610153
-Actin Mouse 1:5000 42 Sigma A1978
51
Figure 2-1. Lentiviral constructs used in the study. Constructs include A. TTLG-Wnt reporter, B. TLG-negative control and C. The transduction control (TC)
52
Figure 2-1. Continued
53
Figure 2-1. Continued
54
Figure 2-2. A representative image of the FACS histogram. Unfilled histogram is the autoFluorescent peak of the cells. The gates on either side of the filled histogram which is an eGFP peak represents the Wntlow and Wnthigh population that were sorted
55
Figure 2-3. Overview of experimental design. This figure describes the in vitro clonogenicity and in vivo tumorigenicity assay conducted at different generations using indicated cell fractions.
56
CHAPTER 3
EARLY ACTIVATION OF WNT/ -CATENIN SIGNALING IN INFLAMATION-DYSPLASIA-CARCINOMA SEQUENCE IS ASSOCIATED WITH COLITIS-TO-
CANCER TRANSITION
In patients with ulcerative colitis (UC), the development of colorectal cancer (CRC)
is a common and serious complication (17, 96). Moreover, few tools are available for
detecting the colitis-to-cancer transition and making early diagnoses at more treatable
stages. Furthermore, the pathogenesis of colitis-associated cancer (CAC) is unclear,
although it likely includes an accumulation of mutations and influences of the
microenvironment. A gene frequently found mutated in the adenoma-to-carcinoma
sequence in CRC is Adenomatous Polyposis Coli (APC), an important component of the
Wnt (wingless) /β-catenin signaling pathway (89, 97). Also Wnt/β-catenin signaling is
involved in the maintenance of adult intestinal homeostasis and crypt structure, and the
proliferation of intestinal epithelial progenitor cells (98, 99). However, in colitis, the role
of Wnt/β-catenin signaling is poorly understood. Initial mutational studies examining the
pathogenesis of the colitis-to-cancer transition suggested that over-activation of the
Wnt/β-catenin pathway is much less frequent in the pathogenesis of CAC than in
sporadic CRC and occurs later in the pathogenic cascade (90). However, in this study
we re-examined these results by performing immunohistochemistry for total β-catenin
on human tissue samples derived from normal colon, colitis and CRC patients. Also we
created a lentiviral dual fusion Wnt reporter (reporter genes being Firefly luciferase and
eGFP) to track the Wnt activity in cells both in vitro and in vivo.
Early Activation of Wnt/ β-Catenin Signaling in Non-Dysplastic Colitic Colon
To determine the importance of Wnt/β-catenin signaling in the colitis-to-cancer
transition, we performed immunohistochemistry for β-catenin in colon samples from
57
healthy controls, colitis patients with non-dysplastic colon, and sporadic CRC patients.
Active Wnt/β-catenin cells are characterized by nuclear and/or cytoplasmic β-catenin
staining (100). In an unstimulated cell most of the endogenous β-catenin is found on the
cell membrane, where it interacts with α-catenin and E-cadherin to help arbitrate cell
adhesion (100, 101). Based on the localization of β-catenin in the cells we scored the
Wnt active cells (Wnt/ β-catenin pathway active cells will be hence forth referred as Wnt
active cells) as percentage of crypt epithelial cells. We observed a 2.5 fold increase in
the number of Wnt active cells in colitis when compared to normal colon (Figures 3-1, 3-
2). In CRC an increase of 4.5 fold over normal colon was observed (Figures 3-1, 3-2).
These results indicate an intermediate number of Wnt active cells in colitic crypt when
compared to normal colon and CRC. This suggests the outlines of a disease
mechanism that parallels the transition of colitis to CAC. Most important, our
immunostaining data suggest an early role for Wnt/β-catenin signaling in pCCSCs
during the colitis-to-cancer transition.
Overlap of Active Wnt/ β-Catenin Signaling with pCCSC Marker ALDH in Non Dysplastic Colitic Colon
ALDH is shown to be a reliable marker for pCCSCs in colitis (69). Similar to Wnt
active cells in colitis, our lab has shown earlier that the percentage of ALDH+ cells
(pCCSC) in colitis is also intermediate compared to normal colon and colon cancer (69)
(Figures 3-3, 3-4). This prompted us to perform the co-immunostaining of β-catenin and
ALDH on colon samples obtained from healthy controls, colitis patients with non-
dysplastic colon, and sporadic CRC patients. Indeed, about 52% of the ALDH+ cells in
colitis were Wnt-active, indicating that Wnt-active cells represent a major subpopulation
58
of pCCSCs (Figure 3-3, 3-4). In order to further confirm this finding we created a dual
fusion Wnt reporter, which could track the Wnt activity in vitro and in vivo.
Dual Fusion Wnt Reporter
A reporter construct consists of an enhancer specific to a gene or pathway of
interest, a minimal promoter sensitive to the enhancer used and a reporter gene
regulated by the pair of the minimal promoter and enhancer that is part of the construct.
Reporter genes are those genes whose expression could be followed by relatively easy
assay. Commonly used reporter genes are those that express luminiscent protein (firefly
luciferase, renilla luciferase), Fluorescent protein (eGFP, mRFP,YFP etc) and Lacz
protein. In this study we created a canonical Wnt pathway specific lentiviral reporter,
TTLG comprising of Wnt pathway specific enhancer with TCF/ LEF binding sites,
minimal TK promoter and dual fusion reporter gene. The dual fusion reporter gene is
made up two genes – firefly luciferase (luminescent protein) and eGFP (Fluorescent
protein) that are fused to one another by a spacer. A detailed method of generation of
this construct is described in Chapter 2. Transduction of TTLG into cells reports the
active Wnt pathway. The luminescent reporter, firefly luciferase helps track the Wnt
active cells in vivo and the Fluorescent reporter eGFP facilitates the isolation of Wnt
active cells by Fluorescence activated cell sorting (FACS) assay. Apart from TTLG two
other lentiviral vectors, TLG and transduction control (TC) were generated. Unlike
TTLG, TLG lacks the TCF/ LEF binding sites. The construct reports the background
luminescent and fluorescent signals that are generated by minimal TK promoter in the
absence of the TCF/ LEF binding sites. TC is made up of distinct luminescent and
Fluorescent proteins when compared to TTLG and TLG. Renilla luciferase and mRFP
are the respective luminescent and Fluorescent proteins that were employed. Also
59
instead of the TCF/ LEF binding sites and minimal TK promoter, it has a ubiquitous EF1
enhancer. These transduction controls are always transduced along with reporters. This
serves two purposes while comparing the pathway or gene of interest that the reporter
is reporting in more than two cell types- 1) It helps us to determine the transduction
efficiency of the virus in a particular cell type and 2) it helps in the normalization of the
luminescent or Fluorescent signals that are measured from the reporters.
Validation of the Wnt Reporter Constructs
In order to validate the dual fusion Wnt reporter TTLG, we used SW480, a colon
cancer cell line with truncated (at codon 1338) APC protein (102, 103) as positive
control and HEK293 cells as negative control. SW480 has constitutively active Wnt
pathway due to the truncation in the APC gene (104). This was confirmed by performing
immunocytochemistry on SW480 and HEK293 cells using anti ABC antibody (Figure 3-
5). Following that, TTLG & TC were also cotransduced into SW480 and HEK293 cells.
TLG & the control construct TC were cotransduced too. FACS analysis for eGFP
expression revealed increased Wnt signaling activity (henceforth referred as Wnt
activity) in SW480 when compared to HEK293 cells (Figure 3-6). Dual luciferase assay
using luminometer confirmed high Wnt activity in SW480, which was 17 fold greater
than that of HEK293 cells (Figure 3-7A). It is also noteworthy that the eGFP and firefly
luciferase expression in TLG transduced cells (both in SW480 and HEK) determined by
FACS analysis as well as luminescence visualization assay respectively was very
minimal (Figure 3-6, 3-7B, 3-7C).
Wnt Reporter Constructs in pCCSCs and CCSCs
To confirm Wnt activity in pCCSCs, we transduced TTLG into pCCSCs and
CCSCs. CCSCs, were operationally defined as ALDH+ cells derived from cancerous
60
colon that have demonstrated the ability to undergo serial passaging through
immunocompromised mice with limited cell numbers while retaining the ability to
recapitulate the primary tumor (56). pCCSCs were operationally defined as ALDH+ cells
derived from colitic colon that (i) were isolated from nondysplastic colitic colon, (ii) could
be serially passaged through immunocompromised mice, and (iii) which developed,
over the passages, first an anaplastic phenotype, then a poorly differentiated
adenocarcinoma histological phenotype (69). Successful transduction of pCCSCs and
CCSCs are shown in Figures 3-8 and 3-9. Based on flow data, where eGFP expression
was normalized to mRFP, we found that ~28% of pCCSCs and ~50% of CCSCs had an
activated Wnt pathway. Firefly luciferase assays on TTLG transduced pCCSCs and
CCSCs confirmed the in vivo propagation of Wnt active cells during tumor formation
(Figure 3-10). TTLG specificity was verified by comparing Wnt target genes (Figure 3-
11, 3-12, 3-13) in TTLG-eGFPhigh and TTLG-eGFPlow populations of pCCSCs and
CCSCs. These correspond, respectively, to Wnthigh and Wntlow populations of cells.
TTLG specificity was confirmed by nuclear and/or cytoplasmic β-catenin staining (using
anti-ABC antibody), which indicated active Wnt/β-catenin signaling (Figure 3-14). Using
quantitative PCR, Wnthigh pCCSCs were distinguishable from CCSCs based on
differences in Wnt target gene expression profiles such as that for c-myc (Figures 3-11
and 3-12). The fold differences in c-myc expression in CCSCs are consistent with the
results of Vermeulen et al (68). We speculate that decrease in expression of some Wnt
target genes in Wnthigh population (Figure 3-12, 3-13) could be a result of CpG island
methylation as reported by de Sousa E Melo et al (105). Collectively, our findings not
61
only confirm that TTLG is a valid Wnt reporter, but also validates the findings of
Immunohistochemistry that active Wnt signaling forms major subpopulation of pCCSCs.
62
Figure 3-1. Wnt/ β -catenin signaling in normal, colitis and CRC colon. Nuclear/ cytoplasmic β-catenin (β-cat) (white arrows) staining suggesting Wnt signaling. β-catenin limited to membranes indicates low/no Wnt activity. Scale bar: 10μm
63
Figure 3-2. Active Wnt signaling pathway as a percentage of crypt epithelial cells. Bars indicate mean±SEM (normal colon-n=3; colitis-n=5; CRC-n=4, 4000- 6000 epithelial cells were counted per condition), *p<0.05, **p<0.001, ***p<0.0001.
64
Figure 3-3. Wnt/β-catenin signaling in ALDH+ cells of normal, colitis and CRC colon.
Immunohistochemistry shows co-localization (white arrows) of ALDH+ cells (green), nucleus (blue) and nuclear/cytoplasmic β–catenin (red). C. β–catenin staining as a percentage of crypt epithelial cells. Scale bar: 10μm.
65
Figure 3-4. ALDH+ and nuclear/cytoplasmic β-catenin staining as a percentage of crypt
epithelial cells. Bars indicate mean±SEM (normal colon-n=3; colitis-n=5; CRC-n=4, 4000- 6000 epithelial cells were counted per condition), *p<0.05
66
Figure 3-5. Active β-catenin staining on SW480 and HEK293 cells
67
Figure 3-6.FACS analyses of TTLG and TLG transduced HEK293 and SW480 cells.
Red peak: TLG, Blue peak: TTLG, and Gray peak: Autofluorescence of the cells.
68
A Figure 3-7. Wnt activity in SW480 and HEK293 cells to validate the dual fusion Wnt
reporter. A. Dual luciferase assay was carried out using luminometer to measure the luminescence, which corresponds to Wnt activity. Wnt activity was normalized to HEK 293 cells. Activity of Wnt in SW480 was found ~ 17 times greater than that in HEK293T cells B. Expression of Renilla Luciferase in SW480 cells cotransduced with TTLG and TC (First wells) and TLG and TC (Second wells). C. Expression of Firefly Luciferase in SW480 cotransduced with TTLG and TC (First wells) and TLG and TC (Second wells). Last wells in A and B are controls that are not transduced.
69
Figure 3-7. Continued:
70
Figure 3-8. The eGFP expression (Wnt activity) in TTLG and TLG transduced colitis
sphere cells (pCCSC) showing successful transduction. Scale bar: 50m
71
Figure 3-9. The eGFP expression (Wnt activity) in TTLG and TLG transduced colon
cancer sphere cells (CCSC) showing successful transduction. Scale bar:
50m
72
Figure 3-10. Bioluminescence imaging of tumors. Tumors were generated from TTLG
(Rt) and TLG (Lt) transduced eGFP+ cancer and colitis sphere cells. Imaging was carried out after 2 weeks of injection.
73
Figure 3-11. Real-time PCR of Wnt target genes in Wnthigh and Wntlow fractions of CT-2
pCCSCs. Real-time PCR of Wnt target genes was performed on the 2% highest and lowest TTLG-eGFP fractions of CT-2 pCCSCs. Bar heights indicate the log2 of the fold change in expression of 4 genes in TTLG-eGFPhigh (Wnthigh) and the TTLG-eGFPlow (Wntlow) fractions. Error bars denote mean+SEM.
74
Figure 3-12. Real-time PCR of Wnt target genes in Wnthigh and Wntlow fractions of CT-1
pCCSCs. Real-time PCR of Wnt target genes was performed on the 2% highest and lowest TTLG-eGFP fractions of CT-1 pCCSCs. Bar heights indicate the log2 of the fold change in expression of 4 genes in TTLG-eGFPhigh (Wnthigh) and the TTLG-eGFPlow (Wntlow) fractions. Error bars denote mean+SEM.
75
Figure 3-13. Real-time PCR of Wnt target genes in Wnthigh and Wntlow fractions of
CCSCs. Real-time PCR of Wnt target genes was performed on the 2% highest and lowest TTLG-eGFP fractions of CCSCs. Bar heights indicate the log2 of the fold change in expression of 4 genes in TTLG-eGFPhigh (Wnthigh) and the TTLG-eGFPlow (Wntlow) fractions. Error bars denote mean+SEM.
76
Figure 3-14. TTLG-eGFP fractions (2% highest and lowest) of the indicated sphere isolates stained for activated β-catenin (ABC) localized to nucleus and/or cytoplasm. Scale bars: 25μm. ABC: red; Nucleus: blue; Merged stains: pink.
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CHAPTER 4 HIGH WNT ACTIVITY CONFERS SUSTAINED TUMOR INITIATING POTENTIAL ON
PRECURSSOR COLON CANCER STEM CELL
As described in Chapter 3, similar to CCSCs we found early activation of Wnt
signaling pathway in pCCSC. We have previously reported that pCCSCs transform into
CCSCs and have a capacity to initiate the colitis-dysplasia-cancer transition (69).
However, mechanisms underlying this transition are unknown. Because initiation of
sporadic CRC has been associated with activating mutations in Wnt/β-catenin signaling
(106, 107), pCCSCs (Figures 3-3, 3-4) and CCSCs exhibit high Wnt/β-catenin signaling
(68), we hypothesized that Wnt/β-catenin signaling is associated with clonogenic and
tumor initiating potential of pCCSCs. In order to test this hypothesis we performed in
vitro clonogenic and in vivo tumorigenic assays on pCCSCs derived from two colitis
patients CT-1 and CT-2, where CT-2 was mainly focused on for detailed study and
CCSCs served, throughout the study, as a control for pCCSCs.
Wnthigh pCCSCs Exhibit CCSC Properties While Wntlow pCCSCs Correspond to Transit-Amplifying Cell Population
To determine the functional significance of Wnt/β-catenin signaling in pCCSCs and
CCSCs, we subjected ALDHhigh-Wnthigh and ALDHhigh-Wntlow cells (Figure 2-2) to in vitro
clonogenic assays (limiting dilution assays [LDA]) and in vivo tumorigenic assays under
limiting dilution conditions (Figure 2-3). Tumors so obtained are designated as primary
tumors. However, there were no significant differences in the overall frequency of
clonogenicity or of tumor formation (Figures 4-1, 4-2, 4-3A, Tables 4-1, 4-2 and 4-3 -
Primary tumors). These results may be attributed to a starting cell population of
ALDHhigh pCCSCs and CCSCs that are already enriched for transit amplifying and stem
cells (13). As our study involved enrichment, the integrity of the cell populations may not
78
be absolute, and therefore, some finite level of heterogeneity in the Wnthigh and Wntlow
populations may be present. This heterogeneity was confirmed by eGFP expression
studies: FACS analysis revealed heterogeneity of eGFP expression in ALDHhigh-Wntlow
and ALDHhigh-Wnthigh tumors (Figure 4-8A) where some cells in the ALDHhigh-Wntlow
tumor had increased eGFP expression corresponding to high Wnt activity. However,
total β-catenin staining in these tumors revealed a significantly greater percentage of
Wnt-active cells in ALDHhigh-Wnthigh tumors than in ALDHhigh-Wntlow tumors (Figure 4-4).
Also, the resulting ALDHhigh-Wntlow tumors phenocopied the histological appearance of
ALDHhigh-Wnthigh tumors (Figure 4-5). In order to obtain a higher percentage of
enrichment with increased Wnt activity and to be able to extricate CSC and transit-
amplifying cell populations, we serially passaged these primary tumors based on two
extreme levels of Wnt activity (Figure 2-3).
Tumor initiation is a property of both transit-amplifying cells and stem cells (SC).
However, like SCs, CCSCs retain the ability to both initiate tumors on serial passaging
and to undergo self-renewal (108). Wnt/β-catenin signaling has been implicated in the
self-renewal of adult colon SCs and CCSCs (76, 109). To demonstrate this
phenomenon and also to enrich for stem-like cells in pCCSCs, secondary and tertiary
tumors were generated from the brightest (top 10%) and dimmest (bottom 10%)
fluorescent cells derived from primary ALDHhigh-Wnthigh xenografts. Simultaneously, an
in vitro LDA was performed to determine clonogenicity (Figure 2-3). Clonal frequency
correlated well with tumor-forming potential wherein Wnthigh cells formed tumors at a
greater frequency than Wntlow cells (Figures 4-3B, 4-3C, 4-6, Tables 1 and 2 Secondary
and Tertiary tumors). Wntlow cells largely failed to grow with subsequent passages
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(Tables 4-1,4-2 and 4-3 Secondary and Tertiary tumors). Also, tumors derived from
Wnthigh cells in subsequent passage grew at a faster rate following injections of 10 cells
(Figure 4-8). Similar results were obtained with secondary tumors derived from primary
ALDHhigh-Wntlow tumors of CT-2 (Figure 4-9). This indicates that pCCSCs generate self-
renewing Wnthigh cells. Similar results were obtained with control CCSCs (Figure 4-7,
Table 4-3). Thus, altogether our data demonstrates that high Wnt-activity is associated
with sustained tumor-initiation and self-renewal.
High Wnt Activity Confers More Efficient CCSC Activity to ALDHhigh Cells
To test whether high Wnt activity convenes an additional level of enrichment to
already existing pCCSC marker ALDHhigh, we performed single cell injections of
ALDHhigh cells derived from ALDHhigh primary tumor and Wnthigh and Wntlow cells derived
from ALDHhigh-Wnthigh primary tumor. We had 25% (5 of 20 injected) success rate with
tumor formation from single Wnthigh cell, whereas none of the ALDHhigh or the Wntlow
single cells developed into a palpable mass (Figure 4-10 and Table 4-2). Resulting
Wnthigh tumor displayed histological characteristics of well-differentiated
adenocarcinoma in contrast to the poorly differentiated adenocarcinoma phenotype of
primary ALDHhigh tumor and the Wnthigh tumors at the primary stage (Figure 4-11 and 4-
5). Furthermore, besides the differential β-catenin expression, the tumor revealed
immunocytochemical positivity for the goblet cell marker Muc2 thus confirming the
tumor heterogeneity (Figure 4-12). These results confirm the greater level of CCSC
enrichment bestowed by high Wnt activity on ALDHhigh cells.
80
Figure 4-1. No difference in clonogenic potential of CT-2 ALDHhigh-Wnthigh and ALDHhigh-
Wntlow cells. A. Wnthigh and Wntlow cell fractions were plated for limiting dilution assays. The y-axis indicates clonogenic potentials mathematically defined as the minimum number of cells required to form a single sphere plotted with 95% Confidence Intervals (error bars).
81
Figure 4-2. No difference in clonogenic potential of CA-1 ALDHhigh-Wnthigh and ALDHhigh-
Wntlow cells. Wnthigh and Wntlow cell fractions were plated for limiting dilution assays. A. The y-axis indicates clonogenic potentials mathematically defined as the minimum number of cells required to form a single sphere plotted with 95% Confidence Intervals (error bars).
82
Figure 4-3. Clonogenic potential of CT-1 Wnthigh and Wntlow cells. Wnthigh and Wntlow cell
fractions were plated for limiting dilution assays. The y-axis indicates clonogenic potentials mathematically defined as the minimum number of cells required to form a single sphere plotted with 95% Confidence Intervals (error bars). A. I: cell subsets derived from CT-1 ALDH+ sphere cells; B. Io: cell subsets enriched from CT-1 primary tumors; C. 2o: cell subsets enriched from CT-1 secondary tumors.
83
Figure 4-4. Wnt activity in Wnthigh and Wntlow colitic primary tumors. Upper Panel: Immunohistochemistry. Lower Panel: Quantification of nuclear and/or cytoplasmic β-catenin staining in Wnthigh and Wntlow primary xenografts (CT-2). Scale bar: 25um. Data are mean+SEM
84
Figure 4-5. Histology of ALDHhigh-Wntlow and ALDHhigh-Wnthigh derived primary tumors.
Histology shows poorly differentiated adenocarcinoma phenotype with occasional lumens, which are holes in the column of cells. Scale bar: 50um. I: cell subsets derived from ALDH+ dissociated sphere cells.
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Figure 4-6. Clonogenic potential of Wnthigh and Wntlow cells derived from CT-2 primary
and secondary tumors. Wnthigh and Wntlow cell fractions were plated for limiting dilution assays. The y-axis indicates clonogenic potentials mathematically defined as the minimum number of cells required to form a single sphere plotted with 95% Confidence Intervals (error bars). A. Io: cell subsets enriched from CT-2 primary tumors; B. 2o: cell subsets derived from CT-2 secondary tumors
86
Figure 4-7. Clonogenic potential of Wnthigh and Wntlow cells derived from CA-1 primary and secondary tumors. Wnthigh and Wntlow cell fractions were plated for limiting dilution assays. The y-axis indicates clonogenic potentials mathematically defined as the minimum number of cells required to form a single sphere plotted with 95% Confidence Intervals (error bars). A. Io: cell subsets enriched from CA-1 primary tumors; B. 2o: cell subsets derived from CA-1 secondary tumors
87
Figure 4-8. At lower dilutions, Wnthigh colitic secondary tumors grew faster than Wnthigh CT-2 primary tumors. Tumor growth curves of Wnthigh primary tumors (CT-2) and Wnthigh secondary tumors (CT-2) all generated from injections of 10 cells/recipient mouse
88
Figure 4-9. Primary colitic Wntlow tumor creates a phenocopy of a Wnthigh tumor. A. Overlay of FACS profiles based on the intensity of eGFP expression from H2Kd-/ESA+ cells derived from CT-2 primary Wnthigh vs Wntlow tumors. B. Left: Schematic of the experimental design. Right-top: Clonogenic potential of Wnthigh vs Wntlow cells derived from CT-2 primary Wntlow tumors. Right-bottom: Tumorigenic potential of Wnthigh vs Wntlow cells derived from CT-2 primary Wntlow tumors induced by injection of 100 cells. Bar graphs show mean+SEM, ***p<0.0001.
89
Figure 4-10. Tumor from single Wnthigh cell derived from primary ALDHhigh-Wnthigh xenograft. First two panels show the ALDHhigh-Wnthigh single cell with green fluorescence indicative of high Wnt activity. Panel 3 shows the tumor that is generated from ALDHhigh-Wnthigh single cell.
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Figure 4-11. Histology of single Wnthigh cell derived tumor (left) and 500 cells derived ALDHhigh primary tumor (right). Right- the tumor is poorly differentiated with only occasional lumens, which are the holes in the columns of cells. Scale bar: 50um.
91
Figure 4-12. β-catenin and Muc 2 staining in single cell tumor. Differential expression of β-catenin (left) and Muc 2 (right) in single Wnthigh cell derived tumor. Scale bar: 25um. Single cell tumor study was performed with CT-2 pCCSCs.
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Table 4-1. Tumorigenic and self-renewal potential of Wnthigh vs Wntlow cells derived from CT-1 pCCSCs.
Xenograft Passage Subset Cells Injected
1000 100 10
Primary Tumor (Io) Wnt high (I) 6/6 2/7 1/7 Wnt low (I) 4/6 7/7 3/7
Secondary Tumor (2o) Wnt high (Io) 3/3 6/6 4/6 Wnt low (Io) 0/3 0/6 0/6
Tertiary Tumor (3o) Wnt high (2o) 6/6 5/6 3/6 Wnt low (2o) 1/6 0/6 0/6
Enriched cell subsets obtained from pCCSCs were injected into the flanks of NOD-SCID mice as indicated. Ratios show the number of tumors after twelve weeks at the given number of cells injected (numerator) and number of mice (denominator). p-values are for differences between Wnthigh and Wntlow. In column 2: "I" indicates cell subsets derived from CT-1 ALDH+ sphere cells; "Io" indicates cell subsets enriched from CT-1 primary tumors; "2o" indicates cell subsets derived from the CT-1 secondary tumors. Table 4-2. Tumorigenic and self-renewal potential of Wnthigh vs Wntlow cells derived from
CT-2 pCCSCs.
Xenograft Passage Subset Cells Injected
1000 100 10 1
Primary Tumor (Io) Wnt high (I) 3/3 5/7 3/6 ND Wnt low (I) 3/3 5/7 3/6 ND
Secondary Tumor (2o) Wnt high (Io) 5/6 6/6 2/6 4/20 Wnt low (Io) 5/6 2/6 0/6 0/20 ALDHhigh 10) ND ND ND 0/20
Tertiary Tumor (3o) Wnt high 2o) 5/6 4/6 2/6 ND Wnt low (2o) 1/6 0/6 0/6 ND
Enriched cell subsets obtained from pCCSCs were injected into the flanks of NOD-SCID mice as indicated. Ratios show the number of tumors after twelve weeks at the given number of cells injected (numerator) and number of mice (denominator). p-values are for differences between Wnthigh and Wntlow. In column 2: "I" indicates cell subsets derived from CT-2 ALDH+ sphere cells; "Io" indicates cell subsets enriched from CT-2 primary tumors; "2o" indicates cell subsets derived from the CT-2 secondary tumors.
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Table 4-3. Tumorigenic and self-renewal potential of Wnthigh vs Wntlow cells derived from CA-1 pCCSCs.
Xenograft Passage
Subset Cells Injected
1000 100 10
Primary Tumor (Io)
Wnt high (I) 5/6 2/6 2/6 Wnt low (I) 5/6 4/6 1/6
Secondary Tumor (2o)
Wnt high (Io) 5/5 7/8 3/9 Wnt low (Io) 3/5 1/8 0/9
Tertiary Tumor (3o)
Wnt high (2o) 6/6 5/6 1/6 Wnt low (2o) 0/6 0/6 0/6
Enriched cell subsets obtained from pCCSCs were injected into the flanks of NOD-SCID mice as indicated. Ratios show the number of tumors after twelve weeks at the given number of cells injected (numerator) and number of mice (denominator). p-values are for differences between Wnthigh and Wntlow. In column 2: "I" indicates cell subsets derived from CA-1 ALDH+ sphere cells; "Io" indicates cell subsets enriched from CA-1 primary tumors; "2o" indicates cell subsets derived from the CA-1 secondary tumors.
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CHAPTER 5 INHIBITION OF SUSTAINED WNT ACTIVITY IN WNTHIGH PCCSC REDUCES TUMOR
GROWTH RATE
One of the important strategies to study the function of a gene or pathway is
termed loss-of-function. This forms the basis of reverse genetics, which involves gene
silencing to determine the role of a particular gene. In order to affirm the role of high
Wnt activity in pCCSC and CCSCs, we took the reverse genetics approach. The gene
can be silenced either at transcriptional level or post-transcriptional level. Gene
targeting by homologous recombination ‘knocks out’ the gene at transcriptional level.
However, oligodeoxyribonucleic acids, ribozymes and small interfering RNA (siRNA) /
short hairpin RNA (shRNA) ‘knocks down’ the genes at post-transcriptional level (110).
Inhibition of β-catenin reduces the canonical Wnt signaling activity (111). Here we set
out to inhibit β-catenin to decrease the Wnt activity. The Wnt signaling pathway plays a
crucial role in proliferation and survival, both in development and disease (112). Thus
‘knocking out’ β-catenin at transcriptional level was not a suitable option and hence we
took the second approach of ‘knocking down’ β-catenin at post-transcriptional level
using shRNA. The process of silencing the gene using siRNA/ shRNA is termed as RNA
interference (RNAi). Further, to elevate the relevance and significance of this study in
clinical context we used a pharmacological β-catenin inhibitor (113, 114), indomethacin
(indo), which is an FDA approved non-steroidal anti-inflammatory drug (NSAID).
β-Catenin Knockdown Decreases the Tumor Growth Rate
So far we have shown high Wnt signaling pathway associates with increased
tumorigenicity and self-renewal, thus correlating to CSC property of pCCSCs. To test
the idea that high level of sustained Wnt activity is necessary for tumor-initiation and
growth in colitis we used RNA interference approach, where we inhibited β-catenin
95
using two shRNAs, #1 and #2 (115). Scambled shRNA was used as control. The
efficacy of the knockdown was confirmed by FACS analysis (eGFP expression as the
measure of knockdown) (Figure 5-1), which was further confirmed by western analysis
for β-catenin (Figures 5-2). Blockade by ShRNA #1 was so effective that cells did not
survive more than a week in culture. Hence, in vivo studies were carried out with shRNA
#2 transduced cell population.Tumors derived from shRNA #2 treated Wnthigh pCCSCs
and CCSCs grew at significantly slower rate compared to Sc treated Wnthigh pCCSC
and CCSC tumors (Figures 5-3A and 5-3B).
Pharmacological Inhibition of -Catenin Delays the Rate of Tumor Growth
We then used the FDA approved non steroidal anti inflammatory drug (NSAID),
indomethacin, to attenuate Wnt/β-catenin signaling in pCCSCs by suppressing β-
catenin expression (113, 114). Wnthigh tumors in vehicle-treated mice grew significantly
faster (p< 0.005) than the Indomethacin treated animals (Figure 5-4). The decreased
Wnt activity in indomethacin tumors was confirmed by immunostaining for β-catenin
(Figure 5-4). This suggests a definitive role for high-level Wnt signaling in determining
the rate at which colitis progresses towards CAC. Thus, therapeutic targeting of Wnt/β-
catenin signaling may serve to delay or mitigate the progression of colitis to cancer.
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Figure 5-1. FACS analysis indicating the -catenin knockdown by shRNAs. FACS analysis of CT-2 TTLG eGFP cells transduced with shRNA #1, #2 and Sc for egfp expression
97
Figure 5-2. Western data indicating the level of β-catenin in CT-2 TTLG eGFP cells transduced with shRNA Sc, #1 and #2.
98
Figure 5-3. ShRNA against β-catenin decreases the tumor growth rate. Tumor growth
curves of shRNA Sc and #2 treated ALDHhigh-Wnthigh cell generated primary tumors. A. CT-2 colitic tumor. B. CA-1 tumor. Tumors were generated by injection of 100-cells/recipient mouse. Bars indicate mean±SEM.
99
Figure 5-4. Inhibition of tumor growth rate by indomethacin. A. Tumor growth curves of
indomethacin- and DMSO-treated ALDHhigh-Wnthigh primary tumors (CT-2) generated by injection of 100-cells/recipient mouse. Bars indicate mean±SEM. B. Decreased Wnt active cells were detected in Indomethacin treated tumors when compared to DMSO treated ones by staining for β-catenin. Scale bar: 25um
100
CHAPTER 6 DISCUSSION
In the present study, we have demonstrated early activation of the Wnt/β-catenin
signaling in the CCT based on immunohistochemical analysis, as well as in vitro and in
vivo functional assays. We confirmed the association of high levels of Wnt signaling with
sustained tumor-initiation, tumor heterogeneity and self-renewal, the three properties
that are unique to CSC. Moreover, we show the importance of high Wnt activity as a
method to further enrich pCCSCs within the ALDHhigh population that promotes the
progression from colitis to cancer.
To prove the importance of specific signaling pathway in a disease, the criteria of
Koch’s postulate should be met. Koch’s postulates were originally posited as applicable
to bacterial agents that are responsible for major disease outbreaks (116). Koch’s
postulates state that: 1) the agent should be present abundantly in every case of the
disease; 2) the agent must be isolated from the diseased host and grown in vitro
culture; 3) the agent must reproduce the disease when it is delivered to a susceptible
host; and 4) the agent must be recovered back from the secondary animals in which the
agent was introduced. However, the logic of this postulates are applicable beyond
infectious diseases (117). In this study, in addition to satisfying the criteria for cancer
stem cells we worked towards fulfilling the Koch’s postulates in establishing the role of
high Wnt signaling in the colitis-to-cancer transition. Corresponding to each element of
the Koch’s postulates we have shown that 1) high Wnt activity is prevalent in pCCSCs
2) Wnthigh cells could be isolated and cultured in vitro 3) Wnthigh cells could be
introduced as secondary and tertiary tumors 4) Wnthigh cells could be recovered from
101
these tumors each time. Apart from satisfying these postulates, we have also shown
that inhibition of β-catenin in Wnthigh cells reduced the tumor growth rate.
The contribution of Wnt/β-catenin pathway activation to the colitis-to-cancer
transition has been controversial. While initial reports suggested that activation of
Wnt/β-catenin signaling occurs late in the pathogenesis (118-120), more recent studies
have reported early activation (91, 121) similar to colorectal cancer (89). However, the
conclusions of the initial reports were mainly based on mutational analysis of the Wnt
signaling pathway components, while recent findings (including that of our own)
suggesting an early activation of this pathway are based on immunohistochemical data.
Furthermore, we found no mutations in Wnt target genes including APC and β-catenin
in the colitis and cancer sphere isolates that we used in this study. In murine models of
colitis, Lee et al., reported that the PI3K/PTEN cascade mediated activation of the
Wnt/β-catenin signaling in the development of dysplasia (92). Moreover, activation of
Wnt/β-catenin signaling results through cross talk with other pathways such as TGF-
β/BMP, Hedgehog (Hh), Notch, and mitogen-activated protein kinase (MAPK) during
development, adult homeostasis, stem cell maintenance and in diseases (122-126).
Based on these previous findings we speculate that early activation of Wnt/β-catenin in
CCT could be the result of pathway cross talk rather than simply mutations in the
pathway components.
In this study we have used two colitis sphere isolates (pCCSC) derived from two
different colitis patients to determine the importance of high Wnt activity in colitis-to-
cancer transition. The small sample number is due to the low success rate in generating
spheres from ALDHhigh cells derived from non-dysplastic colitic colon, which is
102
approximately 5-13% (69). The frequency of sphere formation from colitis derived
ALDHhigh cells is similar to the incidence of colitis-associated cancer in our regional
population, which is rare. This unique tool required multiple methodologies and duration
of three years to cultivate as sphere isolates, for the studies included within this
dissertation. Sporadic CRC derived sphere isolate (CCSC) was used as a control
throughout the study, since CCSCs with high Wnt/β-catenin signaling have been
demonstrated to display cancer stem cell properties (68). Thus, it was appropriate to
compare CCSCs as control vs pCCSCs.
Stemness is defined by sustained tumorigenicity with self-renewal and
recapitulation of tumor heterogeneity – the ability of a cell population to serially
propagate tumors with maintenance of tumor phenotype from a very low number of
cells. In this study, we demonstrated that Wnthigh pCCSCs exhibit CSC properties
(Figure 6-1). In primary tumors, we observed that Wntlow pCCSCs were as clonogenic
and tumorigenic as Wnthigh pCCSCs. Similar results were also obtained with control
CCSCs that corroborated with the results of David et al. (127). However, in contrast to
our outcome, they reported limited tumorigenic potential with differential Wnt activity.
This discrepancy could possibly be due to differences in the starting population.
Moreover, unlike our report, their report was based on single xenograft passage. We
attribute the equal clonogenic and tumorigenic potential of Wnthigh and Wntlow cells to
stem/transit-amplifying cells from an enriched ALDHhigh starting population, which was
confirmed by serial passaging of Wnthigh as well as Wntlow tumors. Failure of Wntlow cells
to consistently generate tumors with serial in vivo passages suggested that this cell
population includes a transit-amplifying subpopulation. Serial passaging further aided in
103
verifying the contribution of Wnt/ β-catenin activity to the transformation of pCCSCs into
CCSCs and thus their ability to undergo transformation into frank colon cancer. Our
results with subsequent passages of Wnthigh pCCSCs are comparable to those we
obtained from CCSCs derived from sporadic CRC, which were used as a control in this
study. The ability of Wnthigh CCSCs to propagate with serial in vivo passages are in
agreement with the results of Vermeulen et al., for sporadic CRCs where they reported
the ability of Wnthigh colon cancer cells to self-renew (68).
In this study, we compared the tumorigenic potential of colitis derived (CT-2)
ALDHhigh and ALDHhigh-Wnthigh cells at single cell level and demonstrated that a single
ALDHhigh-Wnthigh cell was sufficient to initiate the tumor formation and satisfy all the
three criteria of CSC. Despite the presence of Wnthigh cells in ALDHhigh cell population,
the ALDHhigh single cell failed to generate a tumor, as we have previously shown that
the minimum number of colitis derived ALDHhigh cells that will lead to xenograft tumor
formation in mice is 50 (69). Therefore, having an additional level of high Wnt activity
enrichment to ALDHhigh cells confers more efficient CCSC activity to ALDHhigh cells.
Thereafter, we tested whether high Wnt activity could be used as a therapeutic
target to mitigate CCT. In order to do so, we knocked down β-catenin using RNAi
strategy to reduce the Wnt activity. We observed reduction in tumorigenicity of Wnthigh
pCCSCs, on β-catenin knockdown, which was further confirmed by pharmacological
inhibition of β-catenin using NSAID, indomethacin. Previously, indomethacin was used
as a pharmacotherapy for the treatment of UC. However, one particular case reports
described relapse of IBD in 22 patients treated with indomethacin (128-130). But later
on it was found that, out of the 22 patients, only 2 patients showed a significant
104
association of drug treatment to relapse, thus weakening the theory of relapse with
indomethacin treatment in IBD (130). Our study indicates the potential benefits of
indomethacin in attenuating CCT. Accordingly, perhaps other modalities which mitigate
WNT signaling might be effective in preventing the CCT.
Collectively, we have demonstrated the importance of the Wnt/β-catenin
signaling activity in mediating colitis-to-cancer employing CSC based approach. While
ALDH may be a more inclusive marker for pCCSCs and CCSCs, the use of ALDHhigh/
Wnthigh as a marker may provide a more specific method of screening for pCCSCs in
patients with colitis. This combination of markers – ALDHhigh+ Wnthigh – thus proves to
be a marker panel that is indicative of an increased risk of malignant transformation in
UC patients. Those chronic UC patients bearing an epithelial phenotype exhibiting high
Wnt activation might be best served by a prophylactic colectomy. Additional clinical
studies are warranted to rigorously demonstrate such a correlation.
105
Figure 6-1. A schematic diagram that suggests the CSC hierarchy in CAC. In CSC model, ALDHhigh-Wnthigh cell is at the top of the hierarchy, which has the ability to self-renew and maintain sustained tumorigenicity.
106
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BIOGRAPHICAL SKETCH
Anitha Shenoy was born in March of 1979, in Sagar, Karnataka, India. She
completed her Bachelors in Physics, Chemistry and Mathematics with first rank from
Bangalore University. She then went on to complete her Master of Science in medical
biochemistry from Manipal University. She then served as lecturer in Department of
Biochemistry at Sri Devraj Urs Medical College, Kolar, India for 2 years. Following that,
she worked for a year as a research scientist in Technology Information, Forecasting &
Assessment Council Centres of Relevance & Excellence (TIFAC-CORE), Manipal Life
Science Centre. Later, Anitha enrolled into the Interdisciplinary Program in Biomedical
Sciences at the University of Florida in August of 2007. She joined the laboratory of Dr.
Edward Scott and studied about colon cancer stem cells in colitis and colon cancer. She
received her Ph.D. in May 2012. As her scientific achievements, she is the co-author in
scientific journals such as Blood and IVOS. She also has one first author and one
second author articles currently under submission. She presented her works in
international meetings such as the 2010, 2011 and 2012 American Association of
Cancer Research conferences.
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