University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2014-03-28 Identification and Characteristics of Factors Regulating Hepatocellular Carcinoma Progression and Metastasis: A Dissertation Leanne G. Ahronian University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cancer Biology Commons, Digestive System Diseases Commons, Molecular Genetics Commons, and the Neoplasms Commons Repository Citation Ahronian LG. (2014). Identification and Characteristics of Factors Regulating Hepatocellular Carcinoma Progression and Metastasis: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/ M2002W. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/705 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. IDENTIFICATION AND CHARACTERISTICS OF FACTORS REGULATING HEPATOCELLULAR CARCINOMA PROGRESSION AND METASTASIS A Dissertation Presented By LEANNE G. AHRONIAN Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY MARCH 28, 2014 CANCER BIOLOGY IDENTIFICATION AND CHARACTERISTICS OF FACTORS REGULATING HEPATOCELLULAR CARCINOMA PROGRESSION AND METASTASIS A Dissertation Presented By LEANNE G. AHRONIAN The signatures of the Dissertation Defense Committee signify completion and approval as to the style and content of the Dissertation _______________________________________________________________ Brian C. Lewis, Ph.D., Thesis Advisor _______________________________________________________________ JeanMarie Houghton, M.D., Ph.D., Member of Committee _______________________________________________________________ Alec Kimmelman, M.D., Ph.D., Member of Committee _______________________________________________________________ Karl Simin, Ph.D., Member of Committee _______________________________________________________________ Scot Wolfe, Ph.D., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee _______________________________________________________________ Leslie Shaw, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school. _______________________________________________________________ Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Cancer Biology March 28, 2014 iv Acknowledgements I would like to thank Brian Lewis for his mentorship during my graduate studies. His encouragement and insights during this journey were indispensable, and I value all of the time he invested in my training. I also appreciate the members of the Lewis Lab, past and present, for their technical support and insights. Particularly, I would like to thank Jiufeng Cai and Victor Adelanwa for their technical assistance. Experimental contributions from YaGWen Chen were critical to the KLF6 project, and I also thank her for taking the time to share her expertise with me after I joined the lab. Lihua (Julie) Zhu provided me with excellent suggestions for experimental design, and I thank her for the bioinformatics analysis and support that she provided. Leslie Shaw, Karl Simin, and Scot Wolfe provided excellent advice and comments throughout my graduate studies, and I truly appreciate the time they dedicated to considering my work. I would also like to thank Justine Landis, Brian Quattrochi, and Kirsten Tracy for providing comments on portions of my thesis. Of course, I must thank my friends and family for the unwavering support they continue to provide. v Abstract Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancerGrelated death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of KrüppelGlike factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of KrüppelGlike factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. SingleGcopy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6Gmediated tumor and metastasis suppression, we performed gene expression profiling and ChIPGsequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 vi and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6Gmediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gainGofG function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gainGofGfunction properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformationGassociated phenotypes in these cells. However, p53R172HGexpressing mice did not have enhanced tumor formation or metastasis compared to p53Gnull mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gainGofGfunction mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gainGofGfunction effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53Gnull and p53R172HGexpressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. vii Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients. viii Table of Contents Title Page ii Signature Page iii Acknowledgments iv Abstract v Table of Contents viii List of Tables x List of Figures xi List of Abbreviations xvi CHAPTER I: Introduction 1 CHAPTER II 51 KLF6 is a repressor of HCC cell migration, tumor formation, and metastasis Introduction 53 Results 57 Materials and Methods 96 Discussion 105 CHAPTER III 111 KLF6 suppresses RHO family GTPase activity through transcriptional repression of VAV3 and CDC42EP3 Introduction 113 Results 118 Materials and Methods 140 ix Discussion 148 CHAPTER IV 152 The p53R172H mutant does not enhance hepatocellular carcinoma development and progression Introduction 154 Results 159 Materials and Methods 177 Discussion 183 CHAPTER V: Discussion 187 Appendix A 217 Appendix B 227 Bibliography 237 x List of Tables Table 2.1 64 Gene Ontology terms present in the subpopulation gene expression profiling dataset. Table 2.2 103 Antibody conditions used for Western blotting and immunohistochemistry Table 2.3 104 Primers used for amplification of target genes by qRTGPCR. Table 3.1 129 Transcriptional targets of KLF6 as detected by ChIPGsequencing and gene expression profiling of KLF6 knockdown cells Table 3.2 147 Antibodies and conditions used for Western blotting Table 4.1 166 The incidence of HCC or gross metastases is not different at any period of time in HCC development. Table 4.2 182 Antibodies and conditions used for Western blotting and immunohistochemistry Table 4.3 182 Primer sequences used for detection of transcripts by qRTGPCR xi List of Figures Figure 1.1 5 Development of hepatocellular carcinoma Figure 1.2 26 The Klf6 locus is alternatively spliced to generate four known variants Figure 1.3 41 p53 incorporates cellular stress signals and transcribes a myriad of target genes to regulate multiple cell processes Figure 2.1 59 Subpopulations of an HCC cell line display increased proliferation, transformation and migration Figure 2.2 61