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Identification of Important Regulators of Colon Cancer Tumorigenesis by Functional Screening

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Identification of Important Regulators of Colon Cancer Tumorigenesis by Functional Screening University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-6-2017 Identification of Important Regulators of Colon Cancer Tumorigenesis By Functional Screening Haowei Yi University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Cancer Biology Commons Recommended Citation Yi, Haowei, "Identification of Important Regulators of Colon Cancer Tumorigenesis By Functional Screening" (2017). Theses & Dissertations. 192. https://digitalcommons.unmc.edu/etd/192 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. IDENTIFICATION OF IMPORTANT REGULATORS OF COLON CANCER TUMORIGENESIS BY FUNCTIONAL SCREENING by Haowei Yi A DISSERTATION Presented to the Faculty of The University of Nebraska Graduate College in Partial Fulfillment of Requirements for the Degree of Doctor of Philosophy Genetic, Cell Biology & Anatomy Graduate Program Under the Supervision of Professor Jenny Wang University of Nebraska Medical Center Omaha, Nebraska April, 2017 Supervisory Committee: Andrew Dudley, Ph.D. Jennifer Black, Ph.D. Shantaram Joshi, Ph.D. Xu Luo, Ph.D. i ACKNOWLEDGMENTS I would like to owe my sincere gratitude to my adviser, Dr. Jenny Wang, for her patience, constant support, encouragement, and scientific guidance throughout my studies. Her continuous support helped me to overcome stressful situations, and to complete this dissertation successfully. I would also like to thank my committee members, Dr. Michael Brattain, Dr. Runqing Lu, Dr. Samming Wang, Dr. Jennifer Black, Dr. Xu Luo, Dr. Shantaram Joshi, Dr. Andrew Dudley for their suggestions, thoughtful ideas, and scientific evaluations throughout my study. Their insightful comments and criticisms helped me to enthusiastically focus my research. I am thankful to my colleague Dr. Liying Geng, for her constant help and constructive advice that helped me to sort out technical difficulties and conceptualize my work. I am also thankful to my pervious lab mates, Dr. Yang Zhang, Dr. Xiaolin Zhou for their support all along. I would thank Dr. Geoffrey Talmon and Dr. Adrian Black for their kind help on the histology part of my study. I would like to thank all faculties and staffs in the Department of GCBA for their help on my study in this department. I especially thank and appreciate my wife, Ting Jia for her love, spiritual support to me. I sincerely thank my parents, for their love, encouragement and support. I also offer my sincere thanks to all my friends, for their help on my work and on my life. Lastly, I would like to thank my country, China. I am thankful to the China Scholarship Council for providing financial support to pursue my PhD program. ii ABSTRACT TGFβ signaling is an important regulator in colon cancer. miRNAs regulate TGFβ signaling at multiple levels. In this study, through a functional screening, we identified miR- 487b-3p and miR-656-3p, which modulate TGFβ effect in colon cancer cells. Further studies revealed that GRM3 and VGLUT3 are their respective targets. GRM3, a Metabotropic glutamate receptor in glutamatergic pathway is significantly upregulated in majority of human colonic adenocarcinomas tested and colon cancer cell lines. Knockdown of GRM3 expression or inhibition of GRM3 activation in colon cancer cells reduces cell survival and anchorage-independent growth in vitro and inhibits tumor growth in vivo. Mechanistically, GRM3 antagonizes TGFβ-mediated activation of protein kinase A and inhibition of AKT. In addition, TGFβ signaling increases GRM3 protein stability and knockdown of GRM3 enhances TGFβ-mediated tumor suppressor function. Since miR-487b- 3p directly targets GRM3, overexpression of miR-487b-3p mimics the effects of GRM3 knockdown in vitro and in vivo. Expression of miR-487b-3p is decreased in colon adenocarcinomas and inversely correlates with GRM3 expression. VGLUT3, a vesicular glutamate transporter, is also markedly upregulated in human colonic adenocarcinomas and colon cancer cell lines. Knockdown of VGLUT3 expression in colon cancer cells reduces cell survival and anchorage-independent growth in vitro and inhibits tumor growth in vivo. Mechanistically, VGLUT3 antagonizes TGFβ-mediated suppression of cell survival and clonogenicity by maintaining AKT activation. MiR-656-3p represses VGLUT3 expression and mimics the effects of VGLUT3 knockdown in vitro and in vivo. iii Moreover, expression of miR-656-3p is decreased in colon cancer specimens and inversely correlates with VGLUT3 expression. This is particularly interesting and important from a therapeutic standpoint because numerous glutamatergic signaling inhibitor, many of which have been found unsuitable for treatment of neuropsychiatric disorders for reasons such as inability to readily penetrate blood brain barriers. Since GRM3 and VGLUT3 are upregulated in colon cancer, but rarely expressed in normal peripheral tissues, targeting GRM3 and VGLUT3 with such agents would not likely cause adverse neurological or peripheral side effects, making them attractive and specific molecular targets for colon cancer treatment. iv TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................................. i ABSTRACT ..................................................................................................................................... ii TABLE OF CONTENTS ................................................................................................................ iv LIST OF TABLE ........................................................................................................................... viii LIST OF FIGURES ......................................................................................................................... ix LIST OF ABBREVIATIONS .......................................................................................................... xi CHAPTER 1：INTRODUCTION ................................................................................................... 1 Colorectal cancer ........................................................................................................................... 1 TGFβ and cancer ........................................................................................................................... 1 MiRNAs ........................................................................................................................................ 6 miRNAs and cancer ...................................................................................................................... 9 MiRNAs and TGFβ ..................................................................................................................... 10 Glutamate and its signaling ......................................................................................................... 11 CHARAPTER 2：MATERIAL AND METHODS ........................................................................ 18 Cell lines and reagents ................................................................................................................ 18 Western blot analysis, RT-PCR and Q-PCR assays ..................................................................... 18 Apoptosis assays ......................................................................................................................... 19 Soft agarose assays ...................................................................................................................... 19 v Plasmids construction and lentiviral infection ............................................................................ 19 Luciferase Assays ........................................................................................................................ 20 In vivo xenograft model .............................................................................................................. 20 IHC staining of GRM3/VGLUT3 in xenograft tumors and human patient samples ................... 21 TUNEL assay and Ki67 staining ................................................................................................. 21 In Situ hybridization .................................................................................................................... 22 Transwell assays .......................................................................................................................... 22 Statistical analysis ....................................................................................................................... 22 CHAPTER 3: MIRNA LIBRARY SCREENING. .......................................................................... 25 CHAPTER 4 ：THE MIR-487B-3P/GRM3/TGFΒ SIGNALING AXIS IS AN IMPORTANT REGULATOR OF COLON CANCER TUMORIGENESIS .......................................................... 28 INTRODUCTION ....................................................................................................................... 28 RESULTS .................................................................................................................................... 30 Expression of GRM3 is markedly increased in colon cancer specimens. ............................... 30 GRM3 is critical for tumor
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