Abstract 2853: Sunitinib induces anti-angiogenic splice-variant VEGF165b through induction of CLK1 and CLK4 kinases in prostate cell line DU-145

Abstract

Abstract Sunitinib is an oral anti-tumor agent active in renal carcinoma and GIST, which inhibits specific receptor tyrosine kinases that includes targeting vascular endothelial growth factor receptors (VEGFR). To understand mechanisms which may drive this agent's activity, transcriptional profiling (GeneChip® HT Human Genome U133 Array Plate Set) of the NCI-60 human tumor cell lines treated with 200 and 2000 nM sunitinib was completed. Examination of the data indicated the CDC2-like genes, CLK1 and CLK4, were upregulated in multiple cell lines after 2 h drug incubation, with a decreasing level of expression over 6 and 24 h. These kinases are implicated in regulation of alternative splicing of mRNA and may favor selection of the distal splice site of exon 8 in the VEGFA gene. These splice variants of VEGFA are designated VEGFAxxxb and have been shown to have anti-angiogenic properties; regulation of this form of alternative splicing has been identified as a potential novel strategy for anti-angiogenic therapeutics. To confirm whether sunitinib-induced CLK1 and CLK4 expression was indicative of a change in the splice variants of VEGFA, specific primers were used in TaqMan® Q-RT/PCR to distinguish the exon 8 distal splice variant VEGFA165b (anti-angiogenic) from the proximal splice variant VEGFA165a (pro-angiogenic) of the dominant mRNA isoform, VEGFA165. VEGFA165b variant gene expression was induced 2-4 fold over 24h, compared to < 2-fold change in the VEGFA165a expression in 2 cell lines (DU-145 and NCI-H322M) with robust sunitinib-induced CLK1 and 4 expression compared to no change in VEGFA165b expression in a cell line with no increase in CLK1 and 4 after sunitinib treatment. Using DU-145 cells, we also demonstrated a sunitinib-induced, 2-fold increase in VEGFA165b protein expression over 24 h. Inhibition of CLK 1 and 4 kinases with a specific CLK inhibitor, TG003, decreased constitutive gene expression of VEGFA165b in DU-145, indicating the importance of the CLK genes in regulating distal splice variants of the VEGFA165 mRNA isoform. In combination with sunitinib, CLK inhibition by TG003, prevented sunitinib-induced upregulation of the VEGFA165b splice variant. Thus we have uncovered a novel mechanism underlying the activity of the VEGFR inhibitor, sunitinib, where induction of CLK kinases change the balance of VEGFA splice variants towards expression of anti-angiogenic species through which modulation of angiogenesis may occur. Funded by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2853. doi:10.1158/1538-7445.AM2011-2853