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Role of Wnt Signaling Pathway in Colitis-to-Cancer Transition

Permanent Link: http://ufdc.ufl.edu/UFE0044003/00001

Material Information

Title: Role of Wnt Signaling Pathway in Colitis-to-Cancer Transition
Physical Description: 1 online resource (116 p.)
Language: english
Creator: Shenoy, Anitha Kota
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: ccsc -- colitis -- colon
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 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/Beta-catenin signaling. Similar to sporadic CRC, in the current study we have demonstrated an early activation of Wnt/Beta-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 Beta-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/Beta-catenin signaling with the transformation of pCCSCs to CCSCs and thus the colitis-to-cancer transition. This was confirmed by shRNA-mediated down-regulation of Beta-catenin in Wnthigh pCCSCs in vivo. Furthermore, pharmacological inhibition of Wnt/Beta-catenin signaling was achieved by using FDA approved drug, indomethacin that resulted in reduced tumor growth rate. Thus, high levels of Wnt/Beta-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.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Anitha Kota Shenoy.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Scott, Edward W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044003:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044003/00001

Material Information

Title: Role of Wnt Signaling Pathway in Colitis-to-Cancer Transition
Physical Description: 1 online resource (116 p.)
Language: english
Creator: Shenoy, Anitha Kota
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: ccsc -- colitis -- colon
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 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/Beta-catenin signaling. Similar to sporadic CRC, in the current study we have demonstrated an early activation of Wnt/Beta-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 Beta-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/Beta-catenin signaling with the transformation of pCCSCs to CCSCs and thus the colitis-to-cancer transition. This was confirmed by shRNA-mediated down-regulation of Beta-catenin in Wnthigh pCCSCs in vivo. Furthermore, pharmacological inhibition of Wnt/Beta-catenin signaling was achieved by using FDA approved drug, indomethacin that resulted in reduced tumor growth rate. Thus, high levels of Wnt/Beta-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.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Anitha Kota Shenoy.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Scott, Edward W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044003:00001


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1 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 DOC TOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 20 12

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2 20 12 Anitha Shenoy

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3 made this dissertation possible

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4 ACKNOWLEDGMENTS Though only my name appears on the cover, several people have contribu ted 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 situ ations 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 sur geon, 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 insight s 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 sinc ere 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 w hom my journey through PhD would have been incomplete. I

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5 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 staff s of th e core facilities at UF, Neal Benso n, 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 hav e 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. L astly, 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 poss ible.

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6 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 Etiolog y 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/ Catenin Signaling in the Colon ................................ .............................. 32 Wnt/ catenin signaling in colon development ................................ .......... 32

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7 Wnt/ catenin signaling in adult colon ................................ ....................... 33 Wnt/ catenin Signaling in Colon Diseases ................................ ..................... 33 Wnt/ catenin signaling in CRC ................................ ................................ 33 Wnt/ 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 D issociation 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 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 S EQUENCE IS ASSOCIATED WITH COLITIS TO CANCER TRANSITION ................................ ................................ .......................... 56 Catenin Signaling in Non Dysplastic Colitic Colon ......... 56 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 CONF ERS SUSTAINED TUMOR INITIATING POTENTIAL ON PRECURSSOR COLON CANCER STEM CELL ......................... 77 Wnt high pCCSCs Exhibit CCSC Properties While Wnt low pCCSCs Correspond to Transit Amplifying Cell Population ................................ ................................ ....... 77 High Wnt Activity Confers More Efficient CCSC Activity to ALDH high Cells ............. 79 5 I NHIBITION OF SUSTAINED WNT ACTIVITY IN WNT HIGH PC CSC REDUCES TUMOR GROWTH RATE ................................ ................................ ....................... 94 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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8 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 116

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9 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 Wnt high vs Wnt low cells derived from CT 1 pCCSCs. ................................ ................................ ................................ .... 92 4 2 Tumorigenic and self renewal potential of Wnt hig h vs Wnt low cells derived from CT 2 pCCSCs. ................................ ................................ ................................ .... 92 4 3 Tumorigenic and self renewal potential of Wnt high vs Wnt low cells derived from CA 1 pCCSCs. ................................ ................................ ................................ ... 93

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10 LIST OF FIG URES 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 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 catenin signaling in ALDH+ cells of normal, colitis and CRC colon .......... 64 3 4 catenin staining as a percentage of crypt epithelial cells ................................ ................................ ................................ ..... 65 3 5 catenin staining on SW480 and HEK293 cells ................................ ...... 66 3 6 FACS analyses of TTLG and TLG transduced HEK293 and SW480 cell s. ........ 67 3 7 Wnt activity in SW480 and HEK293 cells to validate the dual fusion Wnt reporter. ................................ ................................ ................................ .............. 68 3 8 The eGFP expression (Wnt act ivity) 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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11 3 10 Bioluminescence imaging of tumors. ................................ ................................ .. 72 3 11 Real time PCR of Wnt target genes in Wnt high and Wnt low fractions of CT 2 pCCSCs ................................ ................................ ................................ ............. 73 3 12 Real time PCR of Wnt target genes in Wnt high and Wnt low fractions of CT 1 pCCSCs ................................ ................................ ................................ ............. 74 3 13 Real time PCR of Wnt target genes in Wnt high and Wnt low fractions of CCSCs ... 75 3 14 TTLG eGFP fractions (2% highest and lowest) of the indicated sphere catenin. ................................ .............................. 76 4 1 No difference in clonogenic potential of CT 2 ALDH high Wnt high and ALDH high Wnt low cells. ................................ ................................ ................................ ........ 80 4 2 No difference in clonogenic potential of CA 1 ALDH high Wnt high and AL DH high Wnt low cells. ................................ ................................ ................................ ........ 81 4 3 Clonogenic potential of CT 1 Wnt high and Wnt low cells. ................................ ....... 82 4 4 Wnt activity in Wnt high and Wnt low c olitic primary tumors ................................ .... 83 4 5 Histology of ALDH high Wnt low and ALDH high Wnt high derived primary tumors ....... 84 4 6 Clonogenic poten tial of Wnt high and Wnt low cells derived from CT 2 primary and secondary tumors ................................ ................................ ........................ 85 4 7 Clonogenic potential of Wnt high and Wnt low cells derived from CA 1 primary and secondary tumors ................................ ................................ ........................ 86 4 8 At lower dilutions, Wnt high colitic secondary tumors grew faster than Wnt high CT 2 primary tumors ................................ ................................ ........................... 87 4 9 Primary colitic Wnt low tumor creates a phenocopy of a Wnt high tumorors ............ 88 4 10 Tumor from single Wnt high cell derived from primary ALDH high Wnt high xenograft. ................................ ................................ ................................ ........... 89 4 11 Histology of single Wnt high cell derived tumor (left) and 500 cells derived ALDH high primary tumor (right) ................................ ................................ ............ 90 4 12 catenin and Muc 2 staining in single cell tumor. ................................ .............. 91 5 1 FACS analysis indicating the catenin knockdown by shRNAs ......................... 96 5 2 catenin in CT 2 TTLG eGFP cells transduced with shRNA Sc, #1 and #2. ................................ ............................. 97

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12 5 3 catenin decreases the tumor growth rate ............................... 98 5 4 Inhibi tion of tumor growth rate by indomethacin ................................ ................. 99 6 1 A schematic diagram that suggests the CSC hierarchy in CAC ....................... 105

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13 LIST OF ABBREVIATION S ALDH Aldehyde dehydro genase 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 de pendent 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 kid ney 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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14 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 Lucife rase eGFP UC Ulcerative colitis WIF Wnt inhibitory factor Wnt Wingless YFP Yellow Fluorescent protein

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15 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 20 12 Chair: Edward William Scott Major: Medical Sciences Molecular Cell Biology One of the severe complications of ulcerative coli tis (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/ catenin signaling. Similar to sporadic CRC, in the current study we have demonstrated an early activation of Wnt/ ca tenin 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 catenin immunostaining on patient derived colon tissues These Wnt pathway active c ells 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 Wnt high pCCSCs to exhibit cancer stem cell (CSC) properties like self renewal and tumor initiation. Moreover, a single Wnt high pCCSC was sufficient to initiate the tumor, suggesting the association of Wnt/ catenin signaling with the transformation of pCCSCs to CCSCs and thus the

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16 colitis to cancer transition. This was confirmed by shRNA mediated down regulation of catenin in Wnt high pCCSCs in vivo Furthermore, pharmacological inhibition of Wnt/ catenin signaling was achieved by using FDA approved drug, indomethacin that resulted in reduced tumor growth rate. Thus, high levels of Wnt/ catenin signaling not only further demarcate (with ALDH positivity) the tumor initiating cell compartment of the non dysp lastic 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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17 CHAPTER 1 BACKGROUND AND SIGNI FICANCE 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 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 co lon 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 mate rial (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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18 Microarchitecture of Colon The wall of the colon is made up of four distinct layers: the mucosa, the submucosa, the musc ularis 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 laye r 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 m ucosa. 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 a nd 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 ru n 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 p art 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 t ypes 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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19 like goblet cells that produce mucin and enteroendocrine cells that produce certain di gestive 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 expressi on 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, whi ch 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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20 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 (C RC). 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 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 eventual ly 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 case s. 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 co lorectal 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 c ases 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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21 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 m ovement 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 i s 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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22 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 cancero us 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 chemo therapeutic 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 t he 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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23 sample of colon tissue using a microscope. IBD is chronic inflammation of entire or certain parts of Inflammatory Bowel Disease conditions, which constitute IBD. UC is always restricted to the colon and rectum and 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, C 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 100,0 00 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 co lon 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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24 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 ha s 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 inflamma tion driven genomic stress that leads to CRC (19) Although these studies have shed light on unders tanding 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 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 (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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25 4. Oligopotent stem cells: These cells diffe rentiate 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 adul t 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 morphol ogy 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 po tential for personalized medicine.

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26 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, h air follicles, skin, skeletal muscle, adipose tissue and blood (37) Colon Stem cells: Colon stem cells are a type of adult intestinal ste m 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 (3 9 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) D uring 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

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27 represent the crypt stem cell population, which were originally identifie d 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 st rands (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 + 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 requi red. 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 cell s. Based on positional information Dcamkl1 and Musashi 1 were also indicated as stem cell markers (51) In h uman 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 tumorigenes is and differentiate into various lineages of cells that constitute the tumor (57) Two models have been proposed for tumor heterogeneity 1) the Stochasti c 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, o nly 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

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28 subset of cancer cells that had the abil ity 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 brea st, 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 ad herent 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 fo r 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 tha t 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 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)

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29 Precursor colon cancer stem cells Similar to CCSCs, recently, we have identified ALDH high population in the normal appearing, non dysplastic colonic epithelium of UC patients. This suggested for the first time that CA C 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 ca ncer 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 Ce ll 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 t hese 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 mo re 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 differe ntial proliferative potential of SC (70) This assay therefore could be used for CSCs too.

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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 IL R / mice are more permissive to tumor formation when compared to NOD SCID mice (72) Wnt Signali ng 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 seq uences 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 inhib it the Wnt

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31 activity. Based on the functional activity of the Wnts, it is distributed into two groups: 1. that activates canonical Wnt/ 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 catenin c omplexes. This constitutes canonical Wnt / catenin signaling pathway (76) On the othe r hand, decreased LRP expression or its down regulated through secreted factors such as Dickkopfs, channelizes the binding of Wnt to Fz and activates Tcf/ 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) catenin and transcription factor Tcf (T cell factor) are the main players of Wnt/ catenin signaling pathway. In the absence of Wnt, catenin is targeted to proteosomal degradation by catenin destruction complex. This destruction complex is made up o f scaffold proteins Axin and APC and Catenin phosphorylating proteins CKI (Casein Kinase I) (at ser 45 of catenin) and GSK3 (Glycogen synthase kinase 3 ) (at serine 33, 37 and threonine 41 residues of catenin after the priming phosphorylation by CKI). The catenin phosphorylated by destruction complex is recognized by F box containing catenin. This results in low levels of cytoplasmic catenin and repression of TCF i nduced transcription (Figure 1 7). However in the presence of Wnt, the cytoplasmic tail

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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 catenin. Also on binding of Wnt to Fz and LRP, DSH (Dishivelled) associates with Fz and GSK3 binding protein, Frat. Thus DSH is thought to be involved in the process of stabilizing the catenin in the cytoplasm (76) Free 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/ 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, a nd in various colon disorders. Wnt/ catenin signaling in colon development Wnt signaling plays a vital role in the gut epithelial development. Gut originates from endoderm and in ascidan embryos 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.

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33 Wnt/ catenin signaling in adult colon Wnt/ catenin /Tcf4 pathway is critical in maintaining the prol iferative compartment of the adult gut epithelium. In the colon, catenin is present in all epithelial cell membranes along the crypt. However, nuclear accumulation of catenin is specifically found in the epithelial cells located at the bottom of coloni c 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/ catenin Signaling in Colon Diseases Most of the colon diseases are associated with mutation in one or the other Wnt signaling components. FA P 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 colo n diseases, which involve aberrant Wnt signaling, are CRC and colitis associated cancers. Wnt/ 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 carcinom a sequence of the sporadic CRC (Figure 1 8) (89) Other components of the Wnt pathway like catenin and axins are also found to be mutated in sporadic CRCs (88)

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34 W nt/ 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 demo nstrated activation of 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.

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35 Figure 1 1. Colon in digestive system. ( www.kolorektum.cz/index en.php?pg=for the public -colorectal cancer )

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36 Figure 1 2. Microarchitechture o f colon. (Redrawn from www.afritz.org/ casebook /samplepage _anatomy.pdf)

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37 Figure 1 3 Crypt of Colon (Medema JP and Vermuelen L, 2011 Nature)

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38 Figure 1 4. Eitiology of colon cancer (Modified from www.genomedical.com/you_and_your_family/colon_cance r_risk.cfm ) Figure 1 5. Colonoscopic images of normal and ulcerative colon. (Virtual Medical centre and Mohammad F El Baba, Medscape )

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39 Figure 1 6. Models of heterogeneity in solid tumors. a. Cancer cells of different phenotypes have the potential t o 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 limite d proliferative potential. (Reya T et al. 2011 Nature)

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40 Figure 1 7. Wnt signaling pathway. Left: In the absence of the Wnt ligand Right: In the presence of the Wnt ligand.

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41 Figure 1 8. Mutations that occur in adenoma to carcinoma sequence in CRC and d ysplasia to carcinoma sequence in CAC. (Jianlin Xie et al. 2008 World J Gastroenterol)

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42 CHAPTER 2 MATERIALS AND METHOD S The optimized materials and methods described in this chapter have been developed and adapted over a number of years from multiple prev ious 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 colo n 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 A nimal Care Committee. Cell Culture ALDH high 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 gen erous 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 flas h (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

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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 ALDH high 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 cu lture was TTLG transduced. These TTLG eGFP high (Wnt high ) cells were expanded in vitro so that cultured cells mimicked the TTLG transduced parental ALDH high population with heterogeneous levels of eGFP expression. It is this expanded TTLG eGFP high cells tha t are used throughout the study to further isolate Wnt high or Wnt low 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 is olate 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 c ytometry. Murine cells were eliminated by staining for mouse major histocompatibility complex, H2K d (1:40) (Southern Biotech, Birmingham, Alabama). To detect H2K d PE Cy5 secondary antibody was used at 1:200 dilutions. Flow cytometry was performed on a FAC S Aria

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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 Wnt high and Wnt low 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 ea ch 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 ALDH high Wnt high spheroidal c ultures 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 ALDH high Wnt high colitis and colon cancer sphere cells with the 2% lowest and the 2% highest eGFP intensities, and corresponding to ALDH high Wnt high and ALDH high Wnt low cells (Figure 2 2), were deposited by FACS, onto a 96 well plate containing defined medium admixed with Matrige l 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 ALDH high Wnt high and ALDH high Wnt low 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 Wnt high and Wnt low were injected into NOD SCID mice as described above (Figure 2 3). Single cell injections were also carried out as secondary tumors with both Wnt high an d Wnt low cells derived from primary tumors of indicated sphere isolates.

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45 Also single cell secondary tumors of corresponding isolate were established with ALDH high cells derived from primary ALDH high tumor. Tertiary tumors were generated with 10, 100 and 10 00 ESA+/H2Kd cells with the 10% highest and lowest eGFP intensities from Wnt high 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 mac hine with the following parameters: 95 o C, 30 sec for one cycle followed by 40 cycles of 95 o C, 5 sec for denaturation, and 60 o C, 10 sec for annealing and extension. The melt curve was at 65 95 o C for 5 sec/ cycle. The primers used for different Wnt target ge CCG TCC ATG CGG AAG ATC ATG GCC AGC GGG AAG AC 2. c TCA AGA GGC GAA CAC ACA AC GGC CTT TTC ATT GTT TTC CA CTT CCA ACC TCA GCG TCT T C TTT CCC GCA AGA CGT AAC TC TTA TGC TTT GCA CTA CGT CCC TCC A 3' and (antisense) 5' CGC AAC ATG GTC AAC CCT CAG AC GCT CAC CAT GGA TGA TGA TAT CGC GAC CTG GCC GTC AGG CAG CTC G

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46 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 co ntaining 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 inc ubated overnight at 37 o C. 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 h igh 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 a t 80 o C. The ALDH high /Wnt high 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).

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47 In vivo Indomethacin Treatment and Catenin Knockdown Tumors One hundred ALDH high Wnt high sphere cells with the 2% lowest and highest eGFP intensities (corresponding to ALDH high Wnt high and ALDH high Wnt low cells), admixed with Matrigel at 1:1 rati o 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. Fo catenin knockdown tumors 100 each of control (scrambled) shRNA and #2 shRNA treated Wnt high 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 dilut ions 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) catenin, dephosphorylated on Ser37 or Thr41) was used at a 1:100 dilution on cytospun TTLG eGFP high and TTLG eGFP low colitic pCCSC and CCSCs. Cytospun cells were fixed using 1:1 methanol and acetone for 30 min at 20 o C. All the secondary antibodies were used at 1:500 dilutions. Visual ization was carried out under the same microscopy

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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 Tabl e 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 m M 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 pr otein 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 significan ce was defined as p < 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

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49 explanatory variables. We in cluded subject (mouse) as a random effect, and we assumed a compound symmetric covariance structure.

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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

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51 Figure 2 1. Lentivi ral constructs used in the study. Constructs include A. TTLG Wnt reporter, B. TLG negative control and C. The transduction control (TC)

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52 Figure 2 1. Continued

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53 Figure 2 1. Continued

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54 Figure 2 2. A representative image of the FACS histogram. Unfil led histogram is the autoFluorescent peak of the cells. The gates on either side of the filled histogram which is an eGFP peak represents the Wnt low and Wnt high population that were sorted

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55 Figure 2 3. Overview of experimental design. This figure d escribes the in vitro clonogenicity and in vivo tumorigenicity assay conducted at different generations using indicated cell fractions.

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56 CHAPTER 3 EARLY ACTIVATION OF WNT/ CATENIN SIGNALING IN INFLAMATION DYSPLASIA CARCINOMA SEQUENCE I S ASSOCIATED WITH CO LITIS 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 coli tis 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 ge ne frequently found mutated in the adenoma to carcinoma sequence in CRC is Adenomatous Polyposis Coli (APC), an important component of the catenin signaling pathway (89, 97) Also catenin sig naling is involved in the maintenance of adult intestinal homeostasis and crypt structure, and the proliferation of intestinal epithelial progenitor cells (98, 99) However, i n colitis, the role catenin sig naling is poorly understood. Initial mutational studies examining the pathogenesis of the colitis to cancer transition suggested that over activation of the catenin pathway is much less frequent in the pathogenesis of CAC than in sporadic CRC and occ urs later in the pathogenic cascade (90) However, in this study we re catenin on human tis sue 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/ Catenin Signaling in Non Dysplastic Colitic Colon catenin signaling in the colitis to cancer catenin in colon samples from

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57 healthy controls colitis patients with non dysplastic colon, and sporadic CRC patients. catenin staining (100) In an unstimulated cell catenin is found on the catenin and E cadherin to help arbitrate cell adhesion (100, 101) catenin in the cells we sco red the 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 co lon (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 catenin signaling in pCCSCs during the colitis to cancer transition. Catenin Signaling with pCCSC Marker ALDH in Non Dysplastic Colitic Col on 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 compa red to normal colon and colon cancer (69) (Figures 3 3, 3 4). This prompted us to perform the co imm 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 repr esent a major subpopulation

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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 express ion 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 canonic al 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 (luminesce nt 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, fir efly 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

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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 d etermine 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 act ive 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 lumin ometer 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 analysi s 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

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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 passa ges, 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 eG FP 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 (F igure 3 10). TTLG specificity was verified by comparing Wnt target genes (Figure 3 11, 3 12, 3 13) in TTLG eGFP high and TTLG eGFP low populations of pCCSCs and CCSCs. These correspond, respectively, to Wnt high and Wnt low populations of cells. TTLG specifici ty was confirmed by nuclear and/or cytoplasmic catenin staining (using anti catenin signaling (Figure 3 14). Using quantitative PCR, Wnt high 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 t hat decrease in expression of some Wnt target genes in Wnt high 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

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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.

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62 Figure 3 1. Wnt/ catenin signaling in normal, colitis and CRC colon. Nuclear/ cat) (white arrows) staining suggesting Wnt catenin limited to membranes indicates low/no Wnt activity. Scale

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63 Figure 3 2. Active Wnt signal ing pathway as a percentage of crypt epithelial cells. Bars indicate meanSEM (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.

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64 Figure 3 catenin signaling in AL DH+ cells of normal, colitis and CRC colon. Immunohistochemistry shows co localization (white arrows) of ALDH+ cells catenin staining as a percentage of crypt epithelial cells. Scale bar

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65 Figure 3 4. ALDH+ and nuclear/ catenin staining as a percentage of crypt epithelial cells. Bars indicate meanSEM (normal colon n=3; colitis n=5; CRC n=4 4000 6000 epithelial cells were counted per condition ), *p<0.05

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66 Figure 3 catenin staining on SW480 and HEK293 cells

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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.

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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.

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69 Figure 3 7. Continued:

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70 Figure 3 8. The eGFP expression (Wnt activity) in TTLG and TLG transduced colitis sphere cells (pCCSC) showing successf ul transduction. Scale bar: 50 m

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71 Figure 3 9. The eGFP expression (Wnt activity) in TTLG and TLG transduced colon cancer sphere cells (CCSC) showing successful transduction. Scale bar: 50 m

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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.

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73 Figure 3 11. Real time PCR of Wnt target genes in Wnt high and Wnt low fractions of CT 2 pCCSCs. Real time PCR of W nt 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 eGFP high (Wnt high ) and the TTLG eGFP low (Wnt low ) fractions. Error bars denote m ean+SEM.

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74 Figure 3 12. Real time PCR of Wnt target genes in Wnt high and Wnt low 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 th e fold change in expression of 4 genes in TTLG eGFP high (Wnt high ) and the TTLG eGFP low (Wnt low ) fractions. Error bars denote mean+SEM.

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75 Figure 3 13. Real time PCR of Wnt target genes in Wnt high and Wnt low 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 eGFP high (Wnt high ) and the TTLG eGFP low (Wnt low ) fractions. Error bars denote mean+SEM.

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76 Figure 3 14. TTLG eGFP fractions (2% highest and lowest) of the indicated sphere isolates stained for ac catenin (ABC) localized to nucleus and/or

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77 CHAPTER 4 HIGH WNT ACTIVITY CO NFERS SUSTAINED TUMO R INITIATING POTENTI AL ON PRECURSSOR COLON CAN CER STEM CELL As described in Ch apter 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/ catenin signaling (106, 107) pCCSCs (Figures 3 3, 3 catenin signaling (68) catenin signaling is associated wit h 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. Wnt high pCCSCs Exhibit CCSC Properties While Wnt low pCCSCs C orrespond to Transit Amplifying Cell Population To determine the functional significance of Wnt/ catenin signaling in pCCSCs and CCSCs, we subjected ALDH high Wnt high and ALDH high Wnt low 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). Tum ors 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 attribu ted to a starting cell population of ALDH high 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

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78 be absolute, and therefore, some finite lev el of heterogeneity in the Wnt high and Wnt low populations may be present. This heterogeneity was confirmed by eGFP expression studies: FACS analysis revealed heterogeneity of eGFP expression in ALDH high Wnt low and ALDH high Wnt high tumors (Figure 4 8A) wher e some cells in the ALDH high Wnt low tumor had increased eGFP expression corresponding to high Wnt activity. However, total catenin staining in these tumors revealed a significantly greater percentage of Wnt active cells in ALDH high Wnt high tumors than in ALDH high Wnt low tumors (Figure 4 4). Also, the resulting ALDH high Wnt low tumors phenocopied the histological appearance of ALDH high Wnt high 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) 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 ALDH high Wn t high xenografts. Simultaneously, an in vitro LDA was performed to determine clonogenicity (Figure 2 3). Clonal frequency correlated well with tumor forming potential wherein Wnt high cells formed tumors at a greater frequency than Wnt low cells (Figures 4 3 B, 4 3C, 4 6, Tables 1 and 2 Secondary and Tertiary tumors). Wnt low cells largely failed to grow with subsequent passages

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79 (Tables 4 1,4 2 and 4 3 Secondary and Tertiary tumors). Also, tumors derived from Wnt high 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 ALDH high Wnt low tumors of CT 2 (Figure 4 9). This indicates that pCCSCs generate self renewing Wnt high 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 ALDH high Cells To test whether high Wnt activity convenes an additional level of enrichment to already existing pCCSC marker ALDH high we performed single cell injections of ALDH high cells derived from ALDH high primary tumor and Wnt high and Wnt low cells derived from ALDH hig h Wnt high primary tumor. We had 25% (5 of 20 injected) success rate with tumor formation from single Wnt high cell, whereas none of the ALDH high or the Wnt low single cells developed into a palpable mass (Figure 4 10 and Table 4 2). Resulting Wnt high tumor d isplayed histological characteristics of well differentiated adenocarcinoma in contrast to the poorly differentiated adenocarcinoma phenotype of primary ALDH high tumor and the Wnt high tumors at the primary stage (Figure 4 11 and 4 5). Furthermore, besides 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 a ctivity on ALDH high cells.

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80 Figure 4 1. No difference in clonogenic potential of CT 2 ALDH high Wnt high and ALDH high Wnt low cells. A. Wnt high and Wnt low c ell 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).

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81 Figure 4 2. No difference in clonogenic potential of CA 1 ALDH high Wnt high and ALDH high Wnt low cells. Wnt high a nd Wnt low c ell 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).

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82 Figure 4 3. Clonogenic potential of CT 1 Wnt high and Wnt low cells. Wnt high and Wnt low c ell 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. I o : cell subsets enriched from CT 1 primary tumors; C. 2 o : cell subsets enriched from CT 1 secondary tumors.

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83 Figure 4 4. Wn t activity in Wnt high and Wnt low colitic primary tumors. Upper Panel: Immunohistochemistry. Lower Panel: Quantification of nuclear and/or catenin staining in Wnt high and Wnt low primary xenografts (CT 2). Scale bar: 25um. Data are mean+SEM

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84 Figure 4 5 Histology of ALDH hi gh Wnt low and ALDH high Wnt high 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 cel ls.

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85 Figure 4 6. Clonogenic potential of Wnt high and Wnt low cells derived from CT 2 primary and secondary tumors. Wnt high and Wnt low c ell fractions were plated for limiting dilution assays. The y axis indicates clonogenic potentials mathematic ally defined as the minimum number of cells required to form a single sphere plotted with 95% Confidence Intervals (error bars). A. I o : cell subsets enriched from CT 2 primary tumors; B. 2 o : cell subsets derived from CT 2 secondary tumors

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86 Figure 4 7. C lonogenic potential of Wnt high and Wnt low cells derived from CA 1 primary and secondary tumors. Wnt high and Wnt low c ell fractions were plated for limiting dilution assays. The y axis indicates clonogenic potentials mathematically defined as the minimum num ber of cells required to form a single sphere plotted with 95% Confidence Intervals (error bars). A. I o : cell subsets enriched from CA 1 primary tumors; B. 2 o : cell subsets derived from CA 1 secondary tumors

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87 Figure 4 8. At lower dilutions, Wnt high col itic secondary tumors grew faster than Wnt high CT 2 primary tumors. Tumor growth curves of Wnt high primary tumors (CT 2) and Wnt high secondary tumors (CT 2) all generated from injections of 10 cells/recipient mouse

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88 Figure 4 9. Primary colitic Wnt low tumor creates a phenocopy of a Wnt high tumor. A. Overlay of FACS profiles based on the intensity of eGFP expression from H2Kd /ESA+ cells derived from CT 2 primary Wnt high vs Wnt low tumors. B. Left: Schematic of the experimental design. Right top: Clonoge nic potential of Wnt high vs Wnt low cells derived from CT 2 primary Wnt low tumors. Right bottom: Tumorigenic potential of Wnt high vs Wnt low cells derived from CT 2 primary Wnt low tumors induced by injection of 100 cells Bar graphs show mean + SEM, ***p<0.000 1.

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89 Figure 4 10. Tumor from single Wnt high cell derived from primary ALDH high Wnt high xenograft. First two panels show the ALDH high Wnt high single cell with green fluorescence indicative of high Wnt activity. Panel 3 shows the tumor that is generated from ALDH high Wnt high single cell.

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90 Figure 4 11. Histology of single Wnt high cell derived tumor (left) and 500 cells derived ALDH high primary tumor (right). Right the tumor is poorly differentiated with only occasional lumens, which are the holes in t he columns of cells. Scale bar: 50um.

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91 Figure 4 catenin and Muc 2 staining in single cell tumor. Differential expression of catenin (left) and Muc 2 (right) in single Wnt high cell derived tumor. Scale bar: 25um. Single cell tumor study was perfo rmed with CT 2 pCCSCs.

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92 Table 4 1. Tumorigenic and self renewal potential of Wnt high vs Wnt low cells derived from CT 1 pCCSCs. Xenograft Passage Subset Cells Injected 1000 100 10 Primary Tumor (I o ) Wnt high (I) 6/6 2/7 1/7 Wnt low (I) 4/6 7/7 3/7 Se condary Tumor (2 o ) Wnt high (I o ) 3/3 6/6 4/6 Wnt low (I o ) 0/3 0/6 0/6 Tertiary Tumor (3 o ) Wnt high (2 o ) 6/6 5/6 3/6 Wnt low (2 o ) 1/6 0/6 0/6 Enriched cell subsets obtained from pCCSCs were injected into the flanks of NOD SCID mice as indicated. Ratio s 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 Wnt high and Wnt low In column 2: "I" indicates cell subsets derived from CT 1 ALDH+ sphe re cells; "I o indicates cell subsets enriched from CT 1 primary tumors; "2 o indicates cell subsets derived from the CT 1 secondary tumors. Table 4 2. Tumorigenic and self renewal potential of Wnt high vs Wnt low cells derived from CT 2 pCCSCs. Xenograft Passage Subset Cells Injected 1000 100 10 1 Primary Tumor (I o ) Wnt high (I) 3/3 5/7 3/6 ND Wnt low (I) 3/3 5/7 3/6 ND Secondary Tumor (2 o ) Wnt high (I o ) 5/6 6/6 2/6 4/20 Wnt low (I o ) 5/6 2/6 0/6 0/20 ALDH high 1 0 ) ND ND ND 0/20 Tertiary Tumor (3 o ) Wnt high 2 o ) 5/6 4/6 2/6 ND Wnt low (2 o ) 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 Wnt high and Wnt low In column 2: "I" indicates cell subsets derived from CT 2 ALDH+ sphere cells; "I o indicates cell subsets enriched from CT 2 primary tumors; "2 o indicat es cell subsets derived from the CT 2 secondary tumors.

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93 Table 4 3. Tumorigenic and self renewal potential of Wnt high vs Wnt low cells derived from CA 1 pCCSCs. Xenograft Passage Subset Cells Injected 1000 100 10 Primary Tumor (I o ) Wnt high (I) 5/6 2/ 6 2/6 Wnt low (I) 5/6 4/6 1/6 Secondary Tumor (2 o ) Wnt high (I o ) 5/5 7/8 3/9 Wnt low (I o ) 3/5 1/8 0/9 Tertiary Tumor (3 o ) Wnt high (2 o ) 6/6 5/6 1/6 Wnt low (2 o ) 0/6 0/6 0/6 Enriched cell subsets obtained from pCCSCs were injected into the flanks o f 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 Wnt high and Wnt low In column 2: "I" indicates cell s ubsets derived from CA 1 ALDH+ sphere cells; "I o indicates cell subsets enriched from CA 1 primary tumors; "2 o indicates cell subsets derived from the CA 1 secondary tumors.

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94 CHAPTER 5 INHIBITION OF SUSTAI NED WNT ACTIVITY IN WNT HIGH PCCSC REDUCES TUMOR G ROWTH 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 rol e 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 vel. However, oligodeoxyribonucleic acids, ribozymes and small interfering RNA (siRNA) / transcriptional level (110) Inhibition of catenin reduces the canonical Wnt signaling activity (111) Here we set 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 catenin at transcriptional level was not a suitable option and hence we catenin at post transcriptional level using shRNA. The process of silencing the gene using siRNA/ shRNA is termed a s RNA interference (RNAi). Further, to elevate the relevance and significance of this study in cl inical context we used a pharmac ological catenin inhibitor (113, 114) indomethacin (indo), which is an FDA approved non steroidal anti inflammatory drug (NSAID). Catenin Knockdown Decreases the Tumor Growth Rate So far we have shown high Wnt signaling pathway as sociates with increased tumorigeni city and self renewal, thus correlating to CSC property of pCCSCs. To tes t the idea that high level of sustained Wnt activity is necessary for tumor initiation and growth in colitis we used RNA interference catenin

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95 using two shRNAs, #1 and #2 (115) Scamble d 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 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 Wnt high pCCSCs and CCSCs grew at significantly slower rate compared to Sc treated Wnt high 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/ catenin expression (113, 114) Wnt high tumors in vehicle treated mice grew significantly faster (p< 0.005) than the Indomethacin treated animals (Figure 5 4). The decre ased 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 o catenin signaling may serve to delay or mitigate the progression of colitis to cancer.

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96 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 exp ression

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97 Figure 5 catenin in CT 2 TTLG eGFP cells transduced with shRNA Sc, #1 and #2.

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98 Figure 5 catenin decreases the tumor growth rate. Tumor growth curves of shRNA Sc and #2 treated A LDH high Wnt high 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 meanSEM.

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99 Figure 5 4. Inhibition of tumor growth rate by indomethacin. A. Tumor growth c urves of indomethacin and DMSO treated ALDH high Wnt high primary tumors (CT 2) generated by injection of 100 cells/recipient mouse. Bars indicate meanSEM. B. Decreased Wnt active cells were detected in Indomethacin treated tumors when compared to DMSO tre catenin. Scale bar: 25um

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100 CHAPTER 6 DISCUSSION In the presen t study, we have demonstrated catenin signaling in the CCT based on immunohistochemical analysis, as well as in vitro and in vivo funct ional 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 enr ich pCCSCs within the ALDH high 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 postulate s were origi nally posited as applicable to bacterial agents that are responsible for major disease outbreaks (116) Koch s postulate s 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 disea ses (117) In this study, in addition to satisfying the criteria for cancer stem cell s we worked towards fulfilling the Koch s postulate s in establishing the role of high W nt signaling in the colitis to cancer transition. Corresponding to each element of the Koch s postulate s we have shown that 1) high Wnt activity is prevalent in pCCS Cs 2) Wnt high cells could be isolated and cultured in vitro 3) Wnt high cells could be introduced as secondary and tertiary tumors 4) Wnt high cells could be recovered from

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101 these tumors each time. Apart from satisfying these postulates, we have also shown th at inhi catenin in Wnt high cells reduced the tumor growth rate. Th catenin pathway activation to the colitis to cancer transition has been controversial. While initial reports suggested that activation of 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 dat a Furthermore we found no mutations in Wnt target genes including APC and 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 catenin signaling in the development of dysplasia (92) Moreover activation of catenin signaling results through cross talk with other pathways such as TGF /BMP, Hedgehog (Hh), Notch, and mitogen activated protein kinase (MAPK) during development, adult homeostasis, stem cell maintenance and in disease s (122 126) catenin in CCT could be the result of pathway cross talk rather than simply mutations in the pathway components. In this stud y 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 fr om ALDH high cells derived from non dysplastic colitic colon, which is

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1 02 a pproximately 5 13% (69) The frequency of sphere formation from colitis derived ALDH high cells is similar to the incidence of colitis associate d 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 through out the study s ince CCSCs with 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 s elf 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 Wnt high pCCSCs exhibit CSC proper ties (Figure 6 1). In primary tumors, we observed that Wnt low pCCSCs were as clonogenic and tumorigenic as Wnt high 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, t heir report was based on single xenograft passage. We attribute the equal clonogenic and tumorigenic potential of Wnt high and Wnt low cells to stem/transit amplifying cell s from an enriched ALDH high starting population, which was confirmed by serial passagi ng of Wnt high as well as Wnt low tumors. Failure of Wnt low 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

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103 verifying the co 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 Wnt high 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 Wnt high 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 Wnt hi gh colon cancer cells to self renew (68) In this study, we compared the tumorigenic potential of colitis derived (CT 2) ALDH high and ALDH high Wnt high cells at single cell level and demonstrated that a single ALDH high Wnt high cell was sufficient to initiate the tumor formation and satisfy all the three criteria of CSC. Despite the presence of Wnt high cells in ALDH high cell population, the ALDH high single cell failed to generate a tumor, as we have previously shown that the minimum number of colitis derived ALDH high cells that will lead to xenograft tumor formation in mice is 50 (69) Therefore, having an additional level of high Wnt activity enrichment to ALDH high cells confe rs more efficient CCSC activity to ALDH high cells. Thereafter, we tested whether high Wnt activity could be used as a therapeutic catenin using RNAi strategy to reduce the Wnt activity. We observ ed reduction in tumorigenicity of Wnt high catenin knockdown, which was further confirmed by pharmacological ca tenin using NSAID, indomethacin Previously, indomethacin was used as a pharmacotherapy for the treatment of UC. Howe ver, one particular case reports described relapse of IBD in 22 patien ts treated with indomethacin (128 130) But later on it was found that, out of the 22 patients, only 2 patients showed a significant

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104 associati on of drug treatment to relapse thus weak en ing 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/ 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 ALDH high / Wnt high as a marker may provide a more specific method of screening for pCCSCs in patients with colitis. This combination of markers ALDH high + Wnt high 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 exhibit ing high Wnt activation might be best served by a prophylactic colectomy. Additional clinical studies are warranted to rigorously demonstrate such a correlation.

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105 Figure 6 1. A schematic diagram that suggests the CSC hierarchy in CAC. In CSC model, AL DH high Wnt high cell is at the top of the hierarchy, which has the ability to self renew and maintain sustained tumorigenicity.

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116 BIOGRAPHICAL SKETCH Anitha Shenoy was born in March of 197 9 i n 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 b iochemistry from Manipal University. She then served as l ecturer in Department of Biochemistry at Sri Devraj Urs Medical College Kolar, India for 2 years. Following tha t, she worked for a year as a research s cientist in Technology Information, Forecasting & Assessment Council Centres of Releva nce & 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 200 7 She joined the laboratory of Dr. Edward Scott and studied about col on 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 Res earch c onferences.