Hepatocyte Differentiation and Hepatocellular Carcinoma
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HEPATOCYTE DIFFERENTIATION AND HEPATOCELLULAR CARCINOMA: RATIONALE FOR P53 INDEPENDENT THERAPY by FRANCIS O ENANE Submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy Dissertation Advisor Yogen Saunthararajah, MD Department of Molecular Medicine Cleveland Clinic Lerner College of Medicine CASE WESTERN RESERVE UNIVERSITY May 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Francis O Enane Candindate for Doctor of Philosophy Degree*. Committee Chair: Peter Scacheri, PhD Committee member: Angela Ting PhD Committee member: Xiaoxia Li, PhD Committee member: Alok Khorana, MD Committee member: Yogen Saunthararajah, MD Date of defense: December 19th 2016 *We also certify that written approval has been obtained for any proprietary material contained therein Dedication I dedicate this work to approximately 17.5 million global cancer patient population as of the year 2016. I strongly believe that the scientific and medical communities will continue to work coherently to identify mechanisms to provide better cure rates of cancer, to reduce the economic burden to families affected, and to define psychological and emotional challenges experienced by patients and their families. The work performed in this dissertation is a small contribution to that objective and paves the way to understand new therapeutic mechanisms in hepatocellular carcinoma. In the modern technical and highly skilled society - and with sufficient financial and political support - there will be a time when patients will live long extended lives despite their cancer diagnosis. Table of contents List of Tables ……………………………………………………………………….1 List of Figures ………………………………………………………………………2 Acknowledgements………………………………………………………………...6 Abstract .…………………………………………………………………………….8 Introduction .……………………………………………………………………..10 Hepatocellular carcinoma.……………………………………………………….10 Genomic and epigenetic alterations of hepatocellular carcinoma …………..17 The essential role of epigenetic mechanisms in normal versus cancer cell physiology …………………………………………………………………………20 Current therapies, treatment failures, and opportunities for novel therapeutic strategy ……………………………………………………………………………24 Hepatocyte Differentiation.. .…………………………………………………….26 Chapter 1 …………………………………………………………………………29 Several Genetic Alterations In Hepatocellular Carcinomar Disrupt GATA4- Mediated Transactivation To Suppress Hepatocyte And Preserve Precursor Fate…………………………………………………………………………………29 Abstract ……………………………………………………………………………30 Introduction and Rationale ………………………………………………………30 Results …………………………………………………………………………….38 Discussion ………………………………………………………………………...56 Summary and Significance ……………………………………………………...61 Methods Chapter 1 ……………………………………………………………....64 Chapter 2 …………………………………………………………………………78 Transcriptional Corepressors are Logical Molecular Targets for p53- Independent Differentiation Therapy in Hepatocellular carcinoma …………78 Abstract .…………………………………………………………………………..79 Introduction and Rationale ……………………………………………………...80 Results .……………………………………………………………………………86 Discussion.………………………………………………………………………...98 Summary and Significance .……………………………………………………104 Methods Chapter 2 .…………………………………………………………….106 Future Studies.…………………………………………………………………112 Bibliography.……………………………………………………………………116 List of Tables Table i: Common risk factors associated with liver cancer and geographic distribution………………………………………………………………………………14 Table 1: Liver differentiation genes suppressed in HCC……………………………………………………………………………………..55 1 List of Figures Figure i: Global geographic variations of liver cancer incidence and mortality…………………………………………………………………………………11 Figure ii: Current treatment failures converge to p53 pathway alterations that confer resistance and toxicit…………………………………………………………………………………….23 Figure iii: Key factors involved in the progression of hepatocyte differentiation…………………………………………………………………………...25 Figure 1: Model 1: Genetic alterations of GATA4 mediated transactivation impair hepatocyte differentiation………………………………………………………………………….37 Figure 2: GATA4 is a candidate tumor suppressor gene on chromosome 8p………………………………………………………………………………………..38 Figure 3: GATA4 deletion and MYC gain are hallmarks of HCC………………………………………...............................................................39 Figure 4: Reduced GATA4 expression in HCC from multiple independent studies………………………………………………………………………………......40 Figure 5: Atypical HCC containing a rare germ-line GATA4 mutation…………………………………………………………………………………41 2 Figure 6: Mutant GATA4 does not interact with mediator complex…………………………………….............................................................43 Figure 7: Conditional deletion of one Gata4 allele produced a fatty, proliferative liver phenotype…………………………………………………………………………44 Figure 8: Gene expression analysis demonstrated persistent expression of hepatocyte precursor and impaired expression of hepatocyte genes in Gata4 hapoinsufficient mice…………………………………………………………………46 Figure 9: Hepatocyte precursor genes……………………………………………………………………......................47 Figure 10: Hundreds of liver differentiation genes are also suppressed in HCC compared to normal liver……………………………………………………………………………..............50 Figure 11: Introduction of GATA4 into GATA4-haploinssuficient HCC cells induced terminal hepatocyte differentiation…………………………………………………………………………..51 Figure 12: Frequent inactivation of GATA4 and cofactors in HCC……………………………………………………………………………………..52 3 Figure 13: Liver differentiation genes are suppressed in all histological grades of HCC……………...................................................................................................54 Figure 14: Key transcription factor drivers of hepatocyte terminal differentiation are suppressed in HCC with GATA4 alterations and/or HCC with SWI/SNF alterations………………………………………………………………………………56 Figure 15: Chapter 1 Graphical summary: Several genetic alterations in HCC target the GATA4 transactivation pathway……………………………………………………………………………….62 Figure 16: Curable versus incurable disseminated solid tumors…………………………………………………………………………………..81 Figure 17: Model 2: Copressors aberrantly recruited to FOXA1/2 are logical targets for pharmacologic inhibition………………………………………………………………………………85 Figure 18: FOXA1 and FOXA2 bind to HNF4A enhancer in HCC……………………………………………………………………………………..86 Figure 19: Copy number loss of GATA4 by 8p deletion in PLC……………………………………………………………………………………87 Figure 20: Wild-type GATA4 promotes stronger FOXA2-coactivator interaction in HCC……………………………………………………………………………………..89 4 Figure 21: Wild-type GATA4 impair FOXA1 interaction with DNMT1 in HCC……………………………………………………………………………………..90 Figure 22: Reintroduction of coactivators in HCC promote cell cycle exit by differentiation…………………………………………………………………………91 Figure 23: Inhibition of DNMT1 induce cell cycle exit by differentiation in P53 mutant HCC…………………………………………………………………………….92 Figure 24: Inhibition of DNMT1 shifts balance of corepressor and coactivators towards coactivators in HCC……………………………………………………………………………………..94 Figure 25: Inhibition of DNMT1 does not impair transcription factor interaction in HCC……………………………………………………………………………………..96 Figure 26: Inhibition of DNMT1 induces HCC tumor regression in vivo………………………………………………………………………………………97 Figure 27: Chapter 2 Model Summary……………………………………………………………………..............104 5 Acknowledgements I would like to thank my mother, Jacinta Tionyi and father, Peter Enane for bringing me into this world, and for guiding me into the person I am. Thank you also to my grandmother Eunice Tionyi, for raising me through the roughest times of development. Without you, I will not be here today. Thank you to my mentor Dr. Yogen Saunthararajah, the current and previous members of the lab and to the department of translational hematology and oncology research at the Taussig cancer center of Cleveland Clinic Ohio. Dr. Saunthararajah provided me with the highest level of training together with the necessary funding and resources to succeed in such a complicated area of investigation. His kind, honest and strong scientific rigor has been the greatest influence to my professional development. Special thank you also to Dr. Xiaorong Gu, Dr. Ebrahem Quteba, Dr. Kwok Peng, Dr. Reda Mahfouz and Dr. Shunji Egusa for their everyday input, advice and guidance. Thank you to the collaborators, Dr. Jaraslow Maciejewski, Dr, Tomas Radivoyecitch, Dr. Han Chong, Dr. Timothy Shuein Wai Ho and Dr. Mark Brown. Your leadership has greatly influenced my scientific and academic maturity. Thank you also to my thesis committee members Dr. Peter Scacheri, Dr. Angela Ting, Dr. Xiaoxia Li, Dr. Alok Khorana, and Dr. Pierre Triozzi for their professional input on this dissertation. They have consistently encouraged me and contributed to the strength of the science performed here. Thank you to the 6 molecular medicine PhD program of Cleveland clinic learner college of Medicine of Case Western Reserve University for providing me with the educational platform that contributed to the success of this work. I acknowledge all the friends that I have made in this program and special thank you to Dr. Marcia Jarret, Dr. Robert Fairchild and Dr. Jonathan Smith for their professional development programs. I would like to acknowledge also Dr. Carol De la Mote and her former student Dr. David Hill for introducing me to the molecular medicine program in the summer of 2009 at the Gordon Research Conference on proteoglycans. Your guidance will forever remain with me. Finally, thank you to my beloved wife Dr. Leslie