Dissertation submitted to the Combined Faculty of Natural Sciences and Mathematics of the Ruperto Carola University Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by: Arlou Kristina J. Angeles, M.Sc. born in Manila, Philippines Oral examination: 19 July 2019 The ERG-driven long non-coding RNA LINC00920 promotes cell proliferation and migration in prostate cancer cells by modulating FOXO activity through a direct interaction with 14-3-3ε Referees: PD Dr. Odilia Popanda Prof. Dr. Holger Sültmann Declaration I hereby declare that the submitted dissertation entitled “The ERG-driven long non-coding RNA LINC00920 promotes cell proliferation and migration in prostate cancer cells by modulating FOXO activity through a direct interaction with 14-3-3ε” is the product of my own work and that all assistance received in preparing this thesis and manuscript have been acknowledged. I have not applied to be examined at any other institution, nor have I submitted this dissertation to any other faculty. Furthermore, I took reasonable care to ensure that the work is original, and, to the best of my knowledge, does not breach copyright law, and has not been taken from other sources except where such work has been cited and acknowledged within the text. ________________________ ________________________ Place, Date Arlou Kristina Angeles Summary This thesis describes LINC00920, a tumor-associated lncRNA identified in the transcriptome dataset of the International Cancer Genome Consortium-Early Onset Prostate Cancer (ICGC-EOPC) cohort. SiRNA-mediated knockdown of LINC00920 negatively affected proliferation, colony formation, and migration of PC-3 prostate cancer cells. Gene set enrichment analysis of microarray expression data revealed perturbation of pathways related to cell cycle, cell division, apoptosis, and cell movement. Focused pathway analysis of the top LINC00920-deregulated genes showed an inverse relationship between the lncRNA expression and FOXO signaling. Furthermore, as measured by qPCR, knockdown of LINC00920 activated canonical FOXO targets GADD45A, BCL2L11, and PMAIP1 while overexpression of the lncRNA reversed this effect. In both The Cancer Genome Atlas-Prostate Adenocarcinoma (TCGA-PRAD) and ICGC-EOPC cohorts, LINC00920 positively correlated with ERG overexpression. The regulatory influence of ERG on the lncRNA was then established using cell line models of ERG overexpression, chromatin immunoprecipitation (ChIP) of ERG at the LINC00920 promoter, and promoter luciferase assays using wild-type and mutant promoter fragments. To address the question of how LINC00920 elicits its associated cellular phenotypes with consideration to its presence across cytosolic, nucleoplasmic, and chromatin compartments, chromatin isolation by RNA purification (ChIRP) followed by high throughput DNA sequencing (ChIRP-seq) and mass spectrometry (ChIRP-MS) were conducted. At the chromatin level, LINC00920 was found primarily associating with heterochromatin regions. LINC00920 occupancy was also be detected in a subset of promoter regions and putative enhancer loci. Interestingly, the lncRNA trace across the mappable genome bore a resemblance to that of the enhancer-associated histone mark H3K4me1, suggesting a role for LINC00920 at enhancer elements. At the protein level, most of the identified LINC00920 interacting partners are well established RNA binding proteins typically associated with the process of transcription. Among the LINC00920-precipitated proteins robustly identified in three biological replicates were two 14-3-3 isoforms—14-3-3ε and 14-3-3ζ. Binding of LINC00920 to 14-3- 3ε but not to 14-3-3ζ was validated by RNA immunoprecipitation (RIP) and affinity purification of recombinant 14-3-3ε on streptavidin beads using biotinylated LINC00920. FOXO activity is mitigated by AKT phosphorylation. FOXO phosphorylation triggers 14-3-3/FOXO complex formation, leading to nuclear exportation. Current results indicate the repressive influence of LINC00920 on FOXO signaling as well as the positive interaction between the transcript and 14-3-3ε. Considering these observations, a rational hypothesis emerged wherein LINC00920/14-3-3ε binding further stabilizes the 14-3-3ε/FOXO complex, resulting in a more efficient sequestration and consequent deactivation of FOXO. i Altogether, this thesis contributes a novel mechanism for a tumor-associated lncRNA in the context of ERG-overexpressing prostate cancer cells. Beginning with the transcriptome analysis of the ICGC- EOPC cohort, and later the TCGA-PRAD dataset, LINC00920 was identified to be an ERG-driven transcript. Ultimately, molecular characterization of LINC00920 by ChIRP-MS has revealed its apparent role in modulating FOXO in conjunction with 14-3-3ε, resulting in reduced expression of a subset of tumor suppressive FOXO targets. Since ERG fusions are clonal events while PTEN deletions are subclonal, driving LINC00920 transcription could be a strategy, in part, for ERG-positive cells to alleviate the influence of an intact PTEN, paving the way for tumorigenesis. ii Zusammenfassung Diese Dissertation beschreibt LINC00920, eine tumorassoziierte lncRNA, die im Transkriptom- Datensatz der International Cancer Genome Consortium-Early Onset Prostate Cancer (ICGC-EOPC) Kohorte identifiziert wurde. SiRNA-vermittelter Knockdown von LINC00920 reduzierte die Proliferation, Koloniebildung und Migration von PC-3-Zellen. Eine Gen-Set-Anreicherungsanalyse von Microarray-Expressionsdaten zeigte, dass Gene, die im Zellzyklus, sowie bei Zellteilung, Apoptose und Zellbewegung eine Rolle spielen, dereguliert waren. Die am stärksten von LINC00920 deregulierten Gene waren invers mit Aktivität des FOXO Signalweges azzoziiert. Knockdown von LINC00920 führte zu einer erhöhten Transkription der kanonischen FOXO Zielgene GADD45A, BCL2L11 und PMAIP1, während die Überexpression von LINC00920 diesen Effekt umkehrte. Sowohl im „The Cancer Genome Atlas-Prostate Adenocarcinoma“ (TCGA-PRAD) Datensatz als auch in der ICGC-EOPC Kohorte korrelierte LINC00920 mit dem TMPRSS2/ERG-Fusionssstatus der Tumoren, d.h. ERG-Überexpression. Die Regulation von LINC00920 durch ERG wurde mittels Chromatin-Immunpräzipitation (ChIP) und Luciferase-Tests mit Wildtyp- und Mutanten- Promotor- Sequenzen nachgewiesen. Um die Bindungspartner von LINC00920 im Zyto- bzw. Nukleoplasma sowie im Chromatin zu identifizieren wurden Chromatinisolierung durch RNA-Präzipitation (ChIRP), gefolgt von Hochdurchsatz DNA-Sequenzierung (ChIRP-Seq) bzw. Massenspektrometrie (ChIRP-MS) durchgeführt. Hierbei wurde LINC00920 primär mit Heterochromatin assoziiert gefunden und war v.a. in Promotor- und Enhancerregionen angereichert. Die Genom-weite Verteilung von LINC00920 zeigte eine Ähnlichkeit mit der Histonmarkierung H3K4me1, was auf eine regulatorische Rolle von LINC00920 in Enhancer-Elementen hinweist. Auf Proteinebene waren die meisten der identifizierten LINC00920 Interaktionspartner gut etablierte RNA-Bindungsproteine, welche typischerweise mit dem Transkriptionsprozess verbunden sind. Zu den LINC00920-präzipitierten Proteinen gehörten zwei 14- 3-3 Isoformen: 14-3-3ε und 14-3-3ζ. Die Bindung von LINC00920 an 14-3-3ε, aber nicht an 14-3-3ζ wurde durch RNA-Immunpräzipitation (RIP) und Affinitätsreinigung von rekombinantem 14-3-3ε auf Streptavidin-Beads mittels biotinylierter LINC00920 nachgewiesen. Die Aktivität von FOXO wird durch AKT Phosphorylierung reduziert, welche wiederum die Bildung von 14-3-3/FOXO-Komplexen auslöst, was zu einem Kernexport führt. Die Ergebnisse deuten auf den repressiven Einfluss von LINC00920 auf den FOXO-Signalweg durch Bindung an das 14-3-3ε Protein hin. Basierend auf diesen Beobachtungen wurde gezeigt, dass die LINC00920/14-3-3ε- Bindung den 14-3-3ε/FOXO-Komplex stabilisiert, was zu einem erhöhten Abbau von FOXO und erhöhter Aktivität des AKT-Signalweges führt. Da ERG-Fusionen klonale Ereignisse darstellen, während PTEN-Deletionen subklonal sind, könnte die erhöhte LINC00920-Transkription eine iii Strategie ERG-positiver Zellen sein, um den Einfluss der Tumorsuppression durch PTEN zu reduzieren und den Weg für die Tumorentstehung oder -progression zu ebnen. iv List of Abbreviations Abbreviation Definition ADT androgen deprivation therapy AMPK AMP-activated protein kinase APA alternative polyadenylation AR androgen receptor ARE androgen response element ARLNC1 Androgen Receptor Regulated Long Non-coding RNA 1 ASO antisense oligonucleotide cDNA complementary DNA CHART capture hybrid analysis of RNA targets CHGA chromogranin A CHGB chromogranin B ChIP chromatin immunoprecipitation ChIRP chromatin isolation by RNA purification circRNA circular RNA CLIP crosslinked immunoprecipitation co-IP co-immunoprecipitation Cp crossing point-PCR-cycle CPAT Coding Potential Assessment Tool CPC Coding Potential Calculator CRPC castration resistant prostate cancer CTBP1-AS C-Terminal Binding Protein 1 Antisense DHT 5α-dihydrotestosterone DNA deoxyribonucleic acid Dox doxycycline European Association of Urology-European Society for Radiotherapy and EAU-ESTRO-SIOG Oncology-International Society of Geriatric Oncology EBRT external beam radiation therapy EDTA ethylenediaminetetraacetic acid EMT epithelial-to-mesenchymal transition eRNA enhancer-associated RNA ETS E26 transformation specific or E-twenty six FBS fetal bovine serum FH forkhead FHD forkhead domain FOXO forkhead box class O FRE forkhead response element FPKM fragments per kilobase per million mapped reads GM-CSF granulocyte-macrophage colony-stimulating factor GnRH gonadotropin-releasing