Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells.

BACKGROUND: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. METHODS: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. RESULTS: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. CONCLUSIONS: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy.

CDC2-like (CLK) protein kinase inhibition as a novel targeted therapeutic strategy in prostate cancer.

Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.

Autoregulation of the human splice factor kinase CLK1 through exon skipping and intron retention.

Alternative splicing is a key process required for the regulation of gene expression in normal development and physiology. It is regulated by splice factors whose activities are in turn regulated by splice factor kinases and phosphatases. The CDC-like protein kinases are a widespread family of splice factor kinases involved in normal physiology and in several diseases including cancer. In humans they include the CLK1, CLK2, CLK3 and CLK4 genes. The expression of CLK1 is regulated through alternative splicing producing both full-length catalytically active and truncated catalytically inactive isoforms, CLKT1 (arising from exon 4 skipping) and CLKT2 (arising from intron 4 retention). We examined CLK1 alternative splicing in a range of cancer cell lines, and report widespread and highly variable rates of exon 4 skipping and intron 4 retention. We also examined the effect of severe environmental stress including heat shock, osmotic shock, and exposure to the alkaloid drug harmine on CLK1 alternative splicing in DU145 prostate cancer cells. All treatments rapidly reduced exon 4 skipping and intron 4 retention, shifting the balance towards full-length CLK1 expression. We also found that the inhibition of CLK1 with the benzothiazole TG003 reduced exon 4 skipping and intron 4 retention suggesting an autoregulatory mechanism. CLK1 inhibition with TG003 also resulted in modified alternative splicing of five cancer-associated genes.