Abstract
High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.
Highlights
In eukaryotic cells, the majority of genes harbor intronic sequences that are removed during RNA splicing and transcript maturation (Venter et al, 2001)
In vivo tumors were harvested for DNA isolation when they attained a size of 400 mm3 or more. shRNA hairpin enrichment levels in individual samples were quantified via high-throughput sequencing
We confirmed that the expression of the heterogeneous nuclear ribonucleoprotein M (HNRNPM)-dependent circular RNA (circRNA), which we identified in prostate cancer (PCa), is significantly negatively correlated with HNRNPM expression (R = À0.42, p
Summary
The majority of genes harbor intronic sequences that are removed during RNA splicing and transcript maturation (Venter et al, 2001). Alterations in the expression of specific isoforms of certain genes and of splicing factors themselves can promote cell proliferation (e.g., androgen receptor [AR]; Liu et al, 2014), metastasis (e.g., CD44; Todaro et al, 2014; Xu et al, 2014), or avoid apoptosis (Dewaele et al, 2016; Schwerk and Schulze-Osthoff, 2005) in cancer Some of these changes in isoform ratios may be direct results of mutations in the linear motifs that comprise the splice sites (Puente et al, 2015; Bartram et al, 2017; Jung et al, 2015) or in the splicing machinery itself (Yoshida et al, 2011; Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium et al, 2011; Wang et al, 2011; Graubert et al, 2011)
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