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Ppp2r2a Prostate Cancer Haploinsufficiency Is Associated

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PPP2R2A PROSTATE CANCER HAPLOINSUFFICIENCY IS ASSOCIATED WITH WORSE PROGNOSIS AND A HIGH VULNERABILITY TO B55/PP2A RECONSTITUTION THAT TRIGGERS CENTROSOME DESTABILIZATION AND INHIBITS CELL INVASION A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Ziran Zhao May 2020 Examining Committee Members: Xavier Graña, PhD, Advisory Chair, Fels Institute and Dept. of Biochemistry Scott K. Shore, PhD, Fels Institute and Dept. of Biochemistry Dale S. Haines, PhD, Fels Institute and Dept. of Biochemistry M. Raza Zaidi, PhD, Fels Institute and Dept. of Biochemistry Bojana Gligorijevic, PhD, External Member, College of Engineering ABSTRACT The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through multiple phases in the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase to anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity. ii With that aim and considering the limitations of conventional 2D cell culture in mimicking the tumor environment and predicting drug responses in animal models and humans, we also established 3D organoid cultures of PCa cells and immortalized human prostate epithelial cells (hPrEC) in Matrigel. This allowed us to explore cell to extracellular matrix (ECM) interactions. PC3 cells initially form round organoids in Matrigel, followed by an invasive switch to where cell protrusions invade the surrounding ECM. Strikingly, B55α reconstitution, dramatically suppressed rupture of the basement lamina and ECM invasion, while proliferation appeared not affected. Tracking organoid growth at defined time points or using live imaging, shows that protrusions in PC3 organoids are very dynamic and resemble invadopodia. Interestingly, reconstitution of B55α in PC3 organoids just prior the invasive switch results in reduction of invasive leads and those protrusions that appear to initiate keep forming and collapsing, with most organoids remain round. Our previous phosphoproteomics data in 2D culture suggests that cell-to-ECM signaling is likely altered with B55α reconstitution, identifying potential B55α/PP2A substrates among key mediators of integrin signaling. In sum, reconstitution of B55α suppresses invasion in PC3 organoids, possibly by regulating potential B55α substrates in focal adhesion signaling, such as Paxillin and/or Talin. Alternatively, centrosomal defects due to dephosphorylation of B55α substrates (e.g. HAUS6, NEDD1) may cause microtubule defects that preclude invasion. Further studies are required to identify the mechanism. Moreover, because our studies presented above are based on prostate cancer cell lines with undefined genetic alterations, we have immortalized primary human PrEC by a novel approach to generate a cell model to study cooperation of PPP2R2A loss with step- wise introduction of specific oncogenes and/or tumor suppressor gene alterations in iii transformation, tumorigenicity and invasion. Our newly develop method combines expression of hTERT, which appears insufficient for immortalization of hPrEC with CDKN2A knockout, which we predicted will prevent stress-induced replicative senescence. We have obtained two independent immortalized clones (hPrEC-T-ΔN2A) using this method and confirmed their identity using PCR and western blot analyses. Although cytogenetic analysis showed these two clones are of mixed population with minor alterations in karyotype, 4 out of 11 cells examined in clone 1 appear completely normal. We also find that the clones exit the cell cycle upon contact inhibition and induce p53 expression when treated with flavopiridol, further supporting that hPrEC-T-ΔN2A clones exhibit the features of normal cells. Characterization in 3D culture reveals that the clones are likely of basal epithelial origin. Finally, soft agar and clonogenic assays show hPrEC-T-ΔN2A clones are highly proliferative but not transformed. We are using these cell models to dissect the role of PPP2R2A depletion in step-wise transformation of immortalized PrEC and hope to develop a defined 3D organoid system to study invasion, which could also be suitable for drug screens. Altogether our work has very significantly advanced our understanding of B55α in suppressing transformation in prostate cancer cells and has developed novel tools for further mechanistic characterization of PPP2R2A haploinsufficiency and the development of potential pharmacologic therapeutic agents. iv I dedicate this thesis in memory of my grandmother, Zhongfeng Zhao and my grandfather, Honglin Zhao, who inspired me in scientific research and nurtured me with tremendous love. v ACKNOWLEDGMENTS I would like to thank my mentor Dr Xavier Graña wholeheartedly for teaching and training me during the past seven years, starting from when I was a master student. As an international student, it was tough at the beginning of my study and research. I feel very grateful for him for always being patient with me, showing me how to perform experiments, encouraging me to think more critically and guiding me through all the challenges in research. Thanks for putting the immeasurable trust in me and making me always feel valued and confident. All what I learn from him would influence me for life long. I would like to thank my committee members Dr Scott Shore, Dr Dale Haines and Dr Raza Zaidi for reviewing my project every six months. Their insightful questions and valuable suggestions for the past years have helped me progress through my project with essential checkpoints. I grew to be a more mature researcher thanks to their extreme patience and collective guidance. Moreover, I want to give special thanks to Dr Bojana Gligorijevic for agreeing to be my external committee member and reading my thesis. I also owe many thanks to my great lab members from past to present, Dr Alison Kurimchak, Holly Fowle, Felicity Feiser and Jason Wasserman, as well as many impressive colleagues I have worked with, including but not limited to Megan Connors, Tinsa Varughese, Yu Qing Xu and Morgan Pantuck. They not only provided massive help in work but also are of amazing personality in befriending with me. Thanks for expanding my English vocabulary my dear friends! I'm deeply grateful of our collaborators as without their help many works cannot be done. Thanks Dr Erica Golemis for reading and revising our manuscript many times. vi Thanks Dr Bojana Gligorijevic and Dr Edna Cukierman for generating valuable ideas in organoid culture experiments. I also want to thank U54 grant for giving me the opportunity to complete the work described in Chapter 5 with the support of the prepilot award. I also feel appreciative to everyone in Fels for always being kind and understanding personally, and for helping us with experiments and reagents professionally. Thanks to the Pomerantz lab for the assistance with the radioactive experiments. Thanks Yuanyuan Tian for being my on-call technique support when setting up flow cytometry, and a good friend when not. It's very pleasant for being here as part of Fels. I want to thank my college friends, Dr Qiwen Dong, Dr Lu Sun, Dr Yuan Wang, Dr Lu Zhang and Liang Wen. Words are too limited and pale to express my appreciation for all their company and love. It has become an irreplaceable part of my life like the sunshine. This is the friendship I will proudly take larger than life. Especially, I want to thank my parents, Yongxia Cai and Yingqiang Zhao. Thanks for approving and encouraging me, the only child of the family, to chase her dream so far away from home on the other side of the Earth. It takes unimaginable love, selflessness, and courage, that I will always hold deeply in my heart. Their love and support are the fundamental source of my happiness and will power me through anything in life. It has been a long journey. And there’s more to come. I wish the best for everyone with future endeavors! vii TABLE OF CONTENTS Page ABSTRACT .......................................................................................................................
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