Caffeine induces the splicing factor SC35 and regulates alternative splicing of a subset of cancer-associated genes: The KLF6 story

2575 Alternative pre-mRNA splicing adds to the complexity of gene expression by producing multiple splice variants from one single gene. Importantly, splice variants involved in differentiation, apoptosis, invasion, and metastasis often exist in a delicate equilibrium that is perturbed during tumorigenesis; indeed, efforts to use these splice variants as either biomarkers or drug targets are currently underway. Interestingly, many of the factors that control splicing are also overexpressed in tumors, strongly suggesting that aberrant splicing plays a major role in tumorigenesis and/or progression. To date, however, there is relatively little known about the regulation of alternative splicing either in normal or malignant cells, largely due to a lack of tools to recapitulate alternative splicing in a global system. In the present study, we report that caffeine, a highly consumed stimulant in the American diet and a widely used therapeutic agent, can induce expression of a subset of cancer-related splice variants. Using the tumor suppressor gene KLF6 as a prototype for this cancer gene subclass, we show that caffeine induces the expression of SpKLF6, a novel splice variant of KLF6 in a number of different cell lines. The induction of SpKLF6 (which involves aberrant inclusion of a cryptic exon designated exon 1a) is concentration-, time-dependent, and reversible. Importantly, caffeine induction is due to a direct affect on splice site selection, since other possibilities, such as SpKLF6 RNA stabilization by inhibition of nonsense-mediated decay, have been ruled out . Using a combination of KLF6 minigene deletion/mutation analyses and RNAi assays we have now identified two cis-acting RNA regulatory elements and the cognate splicing factors involved in SpKLF6 exon 1a alternative splicing. We show that the splicing factor SC35 is required for the caffeine-mediated induction of SpKLF6; importantly, SC35 itself is markedly induced by caffeine, implicating this factor in general caffeine-mediated splicing regulation. We hypothesize that caffeine mimics an endogenous and perhaps normal pathway that is activated in certain tumor cells. This novel effect of caffeine provides a valuable tool for dissecting alternative splicing regulation of a cancer-related gene subset, providing insight into the etiology of these tumor-specific variants. Current studies will determine whether caffeine can also regulate alternative splicing in vivo, an effect that could impact the use of cancer-specific splice variants as biomarkers and druggable targets.