| Resumo: | Introduction: In colon cancer distinct genetic subtypes have been described, one of which involves overexpression of RAC1B, a variant generated by alternative splicing. Aberrant splicing is known to occur in cancer and can be caused by mutation in a gene or splicing factor but also represents a dynamic response to oncogene-induced cellular signaling and in this case it may be pharmacologically targeted. Here we explore how signaling pathways are involved in the deregulation of alternative RAC1B splicing in colorectal tumor cells. Materials and Methods: HT29 colorectal cells represent serrated colorectal tumors with BRAF gene mutation V600E in one allele and RAC1B overexpression. Cells were transfected with shRNA vectors directed against target candidate protein kinase transcripts and their effects on RAC1B levels analyzed 24h later by Western Blot and qRT-PCR. Treatment with kinase inhibitors or anti-inflammatory drugs was performed 24h prior to cell lysis. Results and Discussion: Two kinases, SRPK1 and GSK3β, were found required to sustain RAC1B levels and both were shown to act upon the phosphorylation of splicing factor SRSF1, which binds to and promotes the inclusion of the alternative exon in RAC1B. SRPK1 knockdown or pharmacological inhibition reduced SRSF1 phosphorylation decreasing its nuclear translocation and concomitantly RAC1B splicing. The same regulatory pathway was also found to be controlled by GSK3β. Interestingly, GSK3β phosphorylation was identified to serve as target for the anti-inflammatory drug ibuprofen, decreasing SRPK1 nuclear translocation and inhibiting RAC1B overexpression. Together, our results demonstrate that oncogenic signal transduction pathways deregulate alternative splicing and this may be drug revertable.
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