MECHANISMS OF SINGLE IG IL-1-RELATED RECEPTOR MEDIATED SUPPRESSION OF COLON TUMORIGENESIS by JUNJIE ZHAO Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor Xiaoxia Li, Ph.D. Department of Molecular Medicine CASE WESTERN RESERVE UNIVERSITY May 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Junjie Zhao Candidate for the degree of Ph.D.* Committee Chair Brian Cobb, Ph.D. Research Advisor Xiaoxia Li, Ph.D. Committee Member Donal Luse, Ph.D. Committee Member Booki Min, Ph.D. Committee Member Bo Shen, M.D. Date of Defense March 8, 2016 *We also certify that written approval has been obtained for any proprietary contained therein. ii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................. 1 LIST OF FGIURES ................................................................................................................ 2 ABSTRACT ........................................................................................................................... 4 Chapter 1 Introduction To The Role Of Toll-Like Receptor And Interleukin 1 Receptors In Colon Tumorigenesis SIGIRR, A NEGATIVE REGULATOR OF TLR-IL-1R SIGNALING .............................................. 8 TLR-IL-1R SUPERFAMILY AND COLON TUMORIGENESIS .................................................... 10 SIGIRR IN INTESTINAL EPITHELIAL CELLS ......................................................................... 14 SIGIRR IN ADAPTIVE IMMUNE CELLS ................................................................................ 16 SUMMARY ....................................................................................................................... 19 Chapter 2 Human Colon Tumors Express A Dominant-Negative Form Of Sigirr That Promotes Inflammation And Colitis-Associated Colon Cancer In Mice Loss Of N-Linked Glycosylation On Sigirr In Colorectal Cancer .............................. 23 SIGIRRΔE8 Is A Common Variant In Colon Cancer Cells With Reduced Complex Glycan Modification ..................................................................................................... 26 Rna Sequencing Reveals Increased Expression Of SIGIRRΔE8 In Colorectal Cancer .. 29 SIGIRRΔE8 Is A Dominant Negative Mutant Retained In The Er And Traps Full-Length Sigirr ......................................................................................................... 33 Loss Of Modification By Complex Glycan Is Sufficient To Inactivate SIGIRR In Vivo 39 iii DISCUSSION ................................................................................................................... 48 MATERIALS AND METHODS ........................................................................................... 53 Chapter 3 Il-17-Signaling Induces Plet1 Expression In Lgr5-Positive Cells Contributing To Tumorigenesis Il17rc Deficiency Attenuates Colon Tumor Development .......................................... 63 Il-17rc-Deficiency Impairs Regenerative Response Following Dss-Induced Injury .. 63 Il-17 Novel Target Genes Are Highly Induced In Regenerating And Tumor Tissues . 68 Plet1+Lgr5+ Mark A Highly Proliferative Cell Population Expressing Il-17-Target Genes .............................................................................................................................. 69 Plet1 Promotes Erk1/2 Activation, Tissue Repair And Tumorigenesis ........................ 72 DISCUSSION ................................................................................................................... 75 MATERIALS AND METHODS ........................................................................................... 79 Chapter 4 Summary And Future Directions THE ROLE OF SIGIRR IN COLONIC EPITHELIAL CELLS ................................................... 85 THE ROLE OF SIGIRR IN COLONIC EPITHELIAL CELLS ................................................... 87 BIBLIOGRAPHY ............................................................................................................... 88 iv List of Tables Table 1.1 Summary of evidence for receptors inhibited by SIGIRR……………………11 1 List of Figures Figure 2.1 SIGIRR loses modification in colon cancer.…………………………….….24 Figure 2.2 SIGIRR is N-linked glycosylated protein….……………………………….25 Figure 2.3 SIGIRRΔE8 is a common variant in colon cancer cells………………………27 Figure 2.4 Standard Curve of the assay..………………………………………………28 Figure 2.5 RNA sequencing revealed increased expression of SIGIRRΔE8……………30 Figure 2.6 Normalized exon8 expression using……………..…………………………32 Figure 2.7 SIGIRR expression pattern in colon…..……………………………………34 Figure 2.8 SIGIRRΔE8 is a dominant negative mutant …...……………………………36 Figure 2.9 Colon cancer array data.……………………………………………………40 Figure 2.10 SIGIRR co-localization quantification…...…………………………….…42 Figure 2.11 Loss of glycan is sufficient to inactivate SIGIRR.………………………..44 Figure 2.12 AOM/DSS model………………………..………………………………..45 Figure 2.13 Dominant negative impact in vivo………………………………………..47 Figure 2.14 Model of SIGIRR mediated tumor suppression…………………………..50 Figure 3.1 IL-17RC mediates colon tumorigenesis in CAC model..………….………....…64 Figure 3.2 IL-17RC-deficiency impacts wound healing…………………….…………65 Figure 3.3 IL-17RC-dependent gene analysis ……………………………………………67 Figure 3.4 PLET1 is induced from colonic crypts during DSS treatment………………..…70 Figure 3.5 Lgr5 specific ablation of ACT1 impairs CAC tumorigenesis.……………..71 Figure 3.6 PLET1 is an ERK5-target and promotes P-ERK1/2 signaling …….………73 Figure 3.7 The Cas9/gRNA-targeting site in mouse Plet1………….……………….…74 2 Figure 3.8 Model for IL-17 signaling in tissue repair and colon tumorigenesis.......…76 3 Mechanisms of Single Ig Il-1-Related Receptor Mediated Suppression of Colon Tumorigenesis Abstract by JUNJIE ZHAO Inappropriate activation of the Toll-IL-1R (TL-IL-1) signaling by commensal bacteria contributes to the pathogenesis of inflammatory bowel diseases and colitis-associated cancer. SIGIRR is negative regulator of TL-IL-1 signaling. It is highly expressed in colonic epithelial cells, and Th17 cells, serving as a brake for both innate and adaptive immune system. SIGIRR deficient mice are highly susceptible to both chemical-induced and genetically based tumorigenesis, indicating that SIGIRR functions as a suppressor of colon tumorigenesis. In this treatment, we examined the cellular and molecular mechanisms of SIGIRR mediated suppression of tumor suppression. While it has been shown that epithelial-cell derived SIGIRR inhibits colon tumorigensis in mouse models, the role of SIGIRR expression in human colonic cells during the carcinogenesis remains elusive. Most SIGIRR detected in human colon cancer tissues was cytoplasmic, whereas in non-tumor tissues it was found at the cell membrane. RNA sequence analyses revealed increased expression of a SIGIRR mRNA isoform, SIGIRRΔE8, in colorectal cancer tissues compared to paired non-tumor tissues. SIGIRRΔE8 functions as a dominant negative mutant that traps full-length SIGIRR in the cytoplasm and prevents it from trafficking to the membrane. SIGIRR deficiency in T cell leads to hyper Th17 activity. Abrogation of the signaling of IL-17, the signature cytokine of 4 Th17, attenuated the tumorigenic process by limiting the tissue repair activity. A effector downstream gene, Plet1 was identified as an IL-17 target gene that is highly induced in response to tumorigenic treatment and was required for the tissue repair process and subsequent tumorigenesis. The studies presented herein identify SIGIRR as a negative regulator of human colorectal cancer through its impact on both T cells and colonic epithelial cells. 5 Chapter 1: Introduction to The Role of Toll-like Receptor and Interleukin 1 Receptors in Colon Tumorigenesis Portions of this Chapter are published in: Zhao J, Zepp J, Bulek K, Li X. SIGIRR a negative regulator of colon tumorigenesis Drug Discov Today Dis Mech. 2011 Winter;8(3-4):e63-e69. Epub 2012 Mar 15. 6 The intestinal tract is inhabited by trillions of microbes collectively referred to as commensal microflora. The host has developed a mutualism with commensal bacteria where they are beneficial to each other. An important aspect of this relationship is the “microbial tolerance” installed by host immune system. A group of membrane proteins, named Toll-like Receptors (TLRs) can recognize molecules derived intestinal-resident and pathogenic bacteria and initiate inflammatory response, mounting the defense attack. Inappropriate activation of these receptors is implicated in the pathogenesis of inflammatory bowl disease (IBD). Patients with ulcerative colitis (UC), a type of inflammatory bowl disease, stand a higher risk of developing colon cancer, indicating an association of inflammation and tumorigensis . The chronic inflammation process is commonly believed to be the cause of neoplastic transformation of the intestinal epithelium. Therefore, the “microbial tolerance” of the intestinal epithelial layer is not only important for controlling local inflammation but also for preventing tumorigenesis in the colon. Many mechanisms have been proposed to explain how the host immune system selectively ignores resident bacteria but retains its ability to fight pathogenic invasion. A popular model suggests that the temporally and spatially regulated expression of TLRs can restrict their exposure to pathogen associated pattern molecules (PAMPs),