Splicing Regulation of MYC Dependent Exons in Prostate Cancer

The Myc proto-oncogene contributes to the pathogenesis of more than 50 percent of human cancers including prostate cancers. Malignant transformation by Myc is known to transcriptionally upregulate the core pre-mRNA splicing machinery and cause mis-regulation of alternative splicing. However, our understanding of how changes of splice isoforms affect the cancer phenotype is limited and how these changes are directed by Myc remains largely unknown. Here, we performed a signaling pathway-guided differential splicing analysis to identify Myc dependent splicing events, including an HRAS cassette exon that is repressed by Myc transformation across multiple tumor types. To molecularly dissect the regulation of this HRAS cassette exon, we used antisense oligonucleotide tiling to identify intronic splicing enhancers and silencers in the HRAS flanking introns. RNA binding motif prediction indicated the presence of multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream intronic G-rich elements in this H/F mediated splicing activation. Bioinformatic analyses of ENCODE RNAseq datasets also confirmed hnRNP H regulation of HRAS splicing. Consistent with the observed effects of hnRNP H on HRAS splicing, analyses of RNAseq datasets across multiple cancers showed a negative correlation of hnRNP H gene expression with Myc hallmark enrichment. Loss of hnRNP H/F altered the cell cycle progression and induced apoptosis in prostate cancer cells. In the second part of this dissertation, we again applied antisense oligonucleotide tiling to dissect the cis-regulatory elements of another MYC-dependent splicing event, SRSF3 cassette exon, whose inclusion is repressed by Myc transformation in prostate cancer. We identified multiple exonic splicing enhancers as well as downstream intronic splicing silencers. Motif-based RNA binding protein prediction suggested the presence of Sam68 binding motifs in the intronic splicing silencers. siRNA knockdown of Sam68 confirmed it functions as a SRSF3 splicing repressor. Lastly, using RNA-seq data generated from an in vitro model of prostate cancer with inducible Myc expression, we performed RNA binding protein-focused differential gene expression analysis and found majority of differentially expressed RNA binding proteins are downregulated with the depletion of MYC oncoprotein. CRISPRi screening of top Myc-responsive RNA binding proteins suggested that HRAS and SRSF3 are differentially spliced in the loss of RBM28, SNRPD1, EIF3B, and PINX1. Collectively, our results reveal mechanisms for the Myc-dependent regulation of splicing, and point to new possible therapeutic targets in advanced prostate cancers