DISCOVERY AND CHARACTERIZATION OF LONG NON-CODING RNAs IN PROSTATE CANCER by John R. Prensner A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Cellular Pathology) in the University of Michigan 2012 Doctoral Committee: Professor Arul M. Chinnaiyan, Chair Professor David Beer Professor Kathleen Cho Professor David Ginsburg Assistant Professor Yali Dou © John R. Prensner All Rights Reserved 2012 ACKNOWLEDGEMENTS This work represents the combined efforts of many talented individuals. First, my mentor, Arul Chinnaiyan, deserves much credit for guiding me through this experience and pushing me to produce the best science that I could. My thesis committee—Kathy Cho, David Beer, David Ginsburg, and Yali Dou—was an integral part of my development and I am truly grateful for their participation in my education. I am also indebted to the many collaborators who have contributed to this work. Foremost, Matthew Iyer has been responsible for much of my progress and has been an invaluable colleague. Saravana M. Dhanasekaran mentored me and taught me much of what I know. Wei Chen has spent countless hours working on these projects. I have also been fortunate to collaborate with Felix Feng, Qi Cao, Alejandro Balbin, Sameek Roychowdhury, Brendan Veeneman, Lalit Patel, Anirban Sahu, Chad Brenner, Irfan Asangani, and Xuhong Cao who have helped to make this thesis possible. I would like to extend my appreciation to my wonderful support network at MCTP and the MSTP office: Karen Giles, Christine Betts, Dianna Banka, Ron Koenig, Ellen Elkin, and Hilkka Ketola, who have been immensely supportive and helpful during my training. I would also like to thank my funding sources: the University of Michigan Cancer Biology Training Grant, the Department of Defense Predoctoral Grant, and the Department of Defense Idea Award supported me financially. Finally, my life has depended on my friends and family. My wife, Sarah Barbrow, has been a source of enduring kindness that has lifted my spirits on innumerable occasions. I also would like to thank my parents for supporting my decision to remain a student in perpetuity. ii TABLE OF CONTENTS Acknowledgements…………………………………………………………………………ii List of Figures………………………………………………………………………………v List of Tables……………………………………………………………………………….ix List of Appendices………………………………………………………………………….x Abstract……………………………………………………………………………………..xi Chapter 1. Introduction: Long non-coding RNAs in biology and disease………………...1 THE CENTRAL DOGMA OF MOLECULAR BIOLOGY………………1 NON-CODING RNAs: A NEW KIND OF GENE………………………..3 LONG NON-CODING RNAs IN CANCER……………………………..7 FUNCTIONS AND MECHANISMS OF LONG NON-CODING RNAs……………………………………………………………………..15 METHODS TO DISCOVER LONG NON-CODING RNAs……............23 PROSTATE CANCER………………………………………………….. 25 CONCLUSION………………………………………………………….. 33 FIGURES……………............................................................................... 35 TABLES………………………………………………………………… 42 REFERENCES………………………………………………………….. 45 2. Transcriptome sequencing across a cohort of prostate cancers identifies long non-coding RNAs associated with disease progression…………………….. 50 SUMMARY……………………………………………………………... 50 INTRODUCTION………………………………………………………. 51 RESULTS..……………………………………………………………… 52 DISCUSSION…………………………………………………………… 57 MATERIALS AND METHODS………………………………………... 58 FIGURES………………………………………………………………... 68 TABLES………………………………………………………………… 82 REFERENCES…………………………………………………………...88 3. Characterization of PCAT-1 in prostate cancer………………………………90 iii SUMMARY……………………………………………………….......... 90 INTRODUCTION…………………………..……………………………91 RESULTS……………………………………………………………….. 92 DISCUSSION…………………………………………………………… 96 MATERIALS AND METHODS………………………………………... 98 FIGURES………………………………………………………………. 106 TABLES………………………………………………………………...118 REFERENCES………………………………………………………… 122 4. A lineage-specific long non-coding RNA controls homologous recombination in cancer……………………………………………………………………. 123 SUMMARY……………………………………………………………. 123 INTRODUCTION……………………………………………………... 124 RESULTS……………………………………………………………… 125 DISCUSSION………………………………………………………….. 130 MATERIALS AND METHODS……………………………………… 133 FIGURES………………………………………………………………. 140 REFERENCES………………………………………………………… 157 5. A novel, lineage-specific long non-coding RNA coordinates prostate cancer aggressiveness .…………………………………………………………… 158 SUMMARY...……………………….……………..…………………... 158 INTRODUCTION.……………………………………………………. 159 RESULTS……………………………………………………………... 160 DISCUSSION…………………………………………………..……... 169 MATERIALS AND METHODS……………………………………... 171 FIGURES……………………………………………………………... 174 REFERENCES……………………………………………….……….. 198 6. Conclusion: Future directions for long non-coding RNA research in cancer……………………………………………………………………… 199 SUMMARY OF THESIS WORK……………………………………. 199 EMERGING DIRECTIONS IN LONG NON-CODING RNA RESEARCH…………………………………………………………... 201 IMPLICATIONS OF LONG NON-CODING RNAs IN CANCER MANAGEMENT……………………………………………………... 207 ARE LNCRNAs REALLY NON-CODING?........................................ 210 CONCLUSION……………………………………………………….. 212 FIGURES……………………………………………………………... 214 REFERENCES………………………………………………………... 217 Appendices……………………………………………………………………………. 219 iv LIST OF FIGURES Figure 1.1 A schematic of the central dogma of molecular biology, wherein DNA is transcribed into RNA, and RNA is translated into proteins 35 1.2 MicroRNA-mediated pathways in cancer 36 1.3 Gene expression regulation by lncRNAs 37 1.4 Mechanisms of lncRNA function 38 1.5 Major molecular events in prostate cancer progression 40 1.6 Schematic of urine sample analysis 41 2.1 Analysis of transcriptome data for the detection of unannotated transcripts. 68 2.2 Transcript assembly of known genes 69 2.3 Prostate cancer transcriptome sequencing reveals dysregulation of novel transcripts 70 2.4 Analysis of EST support for novel transcripts 71 2.5 Analysis of coding potential of unannotated transcripts 72 2.6 Overlap of unannotated transcripts with ChIP-Seq data 73 2.7 Intergenic unannotated genes are located halfway between annotated genes 74 2.8 Unannotated intergenic transcripts differentiate prostate cancer and benign prostate samples 75 2.9 Validation of novel transcripts in prostate cell lines 77 2.10 Prostate-specificity of PCATs in RNA-Seq compendium 78 2.11 PCAT-14 is upregulated by androgen signaling 79 v 2.12 PCAT-1 and PCAT-14 are upregulated in matched tumor tissues 80 2.13 The SChLAP1 locus spans >500 kb 81 3.1 Analysis of PCAT-1 and PCAT-14 transcript structure 106 3.2 PCAT-1 is a marker of aggressive cancer and a PRC2-repressed ncRNA 107 3.3 In vitro translation of PCAT-1 confirms ncRNA status 108 3.4 PCAT-1 and EZH2 expression is not exclusive with other Chr8q genes 109 3.5 Genomic amplification of Chr8q is not required for PCAT-1 upregulation 110 3.6 HOTAIR is not upregulated in prostate cancer 112 3.7 PCAT-1 transcript associates with the PRC2 complex in VCaP cells. 113 3.8 PCAT-1 promotes cell proliferation 114 3.9 PCAT-1 is not a PRC2 target in LNCaP cells 115 3.10 Prostate cancer tissues recapitulate PCAT-1 signaling 116 4.1 PCAT-1 expression leads to defective homologous recombination in prostate cells 140 4.2 Effect of PCAT-1 on RAD51 foci formation post-ABT-888 treatment 141 4.3 PCAT-1 modulates g-H2AX foci formation following genotoxic stress 142 4.4 PCAT-1 expression alters clonogenic survival following PARP inhibition 143 4.5 PCAT-1 expression alters clonogenic survival following radiation 144 4.6 PCAT-1 expression results in prostate cell sensitivity to PARP inhibition 145 4.7 PCAT-1 overexpression in RWPE engenders sensitivity to genotoxic stress 147 4.8 RWPE-PCAT1 cells do not show altered sensitivity to rapamycin 148 4.9 Modulation of PCAT1 expression does not impact cell cycle distribution 149 4.10 Treatment of PCAT-1-expressing tumors with PARP inhibitors leads to in vivo tumor regression 150 vi 4.11 Expression of PCAT-1 regulated genes in RWPE isogenic cell lines 151 4.12 BRCA2 mRNA is repressed by PCAT-1 but not by an epigenetic or STAU1- mediated mechanism 152 4.13 The 5’ terminus of PCAT-1 represses BRCA2 via the 3’UTR of BRCA2 154 4.14 PCAT-1 expression does not correlate with ETS status 155 4.15 A model of PCAT-1 function in homologous recombination 156 5.1 Discovery of SChLAP-1 as a prostate cancer lncRNA 174 5.2 A chromosome 2 region contains prostate cancer-associated transcripts 176 5.3 The structure and sequence of SChLAP1 177 5.4 Expression of SChLAP-1 across cancers 178 5.5 In vitro translation assays for SChLAP-1 179 5.6 SChLAP-1 coordinates prostate cancer cell invasion 180 5.7 Knockdown of SChLAP-1 impairs prostate cancer cell growth 182 5.8 Overexpression of SChLAP-1 does not increase cell proliferation 183 5.9 SChLAP-1 overexpression in breast cells does not increase invasion 184 5.10 Structural predictions of SChLAP-1 isoforms and deletion constructs 185 5.11 Intracardiac injection of SChLAP-1 knockdown cells impairs cancer cell metastasis 186 5.12 Knockdown of SChLAP-1 delays tumor take but not tumor growth kinetics 187 5.13 SChLAP-1 antagonizes SWI/SNF complex function 188 5.14 Knockdown of SWI/SNF components produces overlapping sets of regulated genes 190 5.15 Knockdown of SWI/SNF components increases cell proliferation and invasion in 22Rv1 cells 191 5.16 Summarized GSEA results for SChLAP-1 and SWI/SNF knockdowns 192 vii 5.17 SChLAP-1 does not impact SWI/SNF protein expression 193 5.18 Immunoprecipitation of AR and SNRNP70 controls for RNA-IP experiments 194 5.19 SChLAP-1 expression characterizes aggressive prostate cancers 195 5.20 SChLAP-1 expression