Multiplexed functional genomic analysis of somatic 5' untranslated region mutations across the spectrum of human prostate cancer (Ribosome Profiling)

The functional consequences of cancer patient-derived genetic variants within the 5’ untranslated regions (UTRs) on a genome-wide scale and their effects on mRNA transcript and translation levels are poorly understood. To systematically interrogate the mutational landscape of 5’ UTRs across cancer patients with localized to metastatic disease, we analyzed the genomes of 226 prostate cancer patients and observed thousands of mutations, many of which impact known cis-regulatory elements. We developed a high-throughput multi-layer massively parallel sequencing-based method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to simultaneously quantify the effects of 545 5’ UTR somatic mutations across recurrently mutated or cancer-related genes from our patient cohort. Our method enabled unprecedented insights into how 5’ UTR mutations can control multiple levels of gene expression simultaneously. In particular, we identified 190 mutations that significantly altered 5’ UTR function, either by creating new DNA binding elements, disrupting known translation regulatory motifs, or simultaneously impacting both transcript levels and mRNA translation. Furthermore, we also determined that 5’ UTR mutations to the MAP kinase signaling pathway are significantly associated with early metastasis. This study is the first to comprehensively interrogate the functional 5’ UTR mutational landscape of a human cancer revealing the importance of untranslated regions in regulating multiple levels of oncogenic gene expression and provides a high-throughput functional genomics solution applicable to many genetically driven diseases. To investigate the functional impact of 5’ UTR mutations in advanced prostate cancer, we will conduct whole exome sequencing utilizing new 5’ UTR exon capture technology on a cohort of end stage castration resistant prostate cancer patient derived xenografts (PDXs). Derived from rapid autopsy specimens, these PDX models have never been cultured, faithfully recapitulate human disease, and represent a nearly inexhaustible supply of tissues for biological replicates. A number of 5’ UTR somatic mutations, insertions, and deletions were identified. To directly correlate these findings with the translation of specific transcripts genome-wide, we will perform ribosome profiling on biological replicates of the same PDXs. Normal human prostate tissue will also be profiled as a comparison to expression in prostate cancer. This will be the very first functional analysis of the 5’ UTR regulome in a human cancer.