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.
BACKGROUND: The ERG oncogene is activated in 50% of prostate cancers. We have designed morpholino-based oligonucleotides that target the ERG oncogene by inducing skipping of exon 4. METHODS: We designed antisense splice-switching morpholino oligonucleotides (SSOs) that target exon 4. We tested their efficacy in ERG-positive VCaP prostate cancer and MG63 osteosarcoma cell lines. We measured their effect on ERG expression, cell proliferation, migration and apoptosis in cell lines, xenografts and a radical prostatectomy sample. RESULTS: SSOs induced exon 4 skipping, reducing ERG levels up to 96 hours following transfection. SSO-induced ERG reduction decreased cell proliferation, cell migration and increased apoptosis. We observed a reduction in cyclin D1, c-Myc, β-catenin and a marker of activated Wnt signalling, p-LRP6. SSOs reduced the growth of MG63 xenografts in mice. We also demonstrated that the SSOs cause a reduction in ERG expression in a patient-derived radical prostatectomy sample ex vivo. CONCLUSIONS: We have successfully tested morpholino-based SSOs that cause a marked reduction in ERG expression, resulting in decreased cell proliferation, a reduced migratory phenotype and increased apoptosis. Our initial tests on mouse xenografts and a human prostate cancer radical prostatectomy specimen indicate that SSOs can be effective for ERG oncogene targeting in vivo.
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