41P - Signal Transduction Pathways Regulating Alternative Splicing of Tumor-Related RAC1b

Date 11 September 2017
Event ESMO 2017 Congress
Session Poster display session
Topics Basic Science
Presenter Vânia Gonçalves
Citation Annals of Oncology (2017) 28 (suppl_5): v1-v21. 10.1093/annonc/mdx361
Authors V. Gonçalves, P. Matos, A.A. Henriques, J. Pereira, P. Jordan
  • Human Genetics Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 - Lisboa/PT

Abstract

Background

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.

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

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, which inhibits RAC1b overexpression.

Conclusions

Together, our results demonstrate that oncogenic signal transduction pathways deregulate alternative splicing and this may be drug revertable.

Clinical trial identification

Legal entity responsible for the study

Peter Jordan

Funding

Fundação para a Ciência e Tecnologia

Disclosure

All authors have declared no conflicts of interest.