The transcription factor E2F1 controls the alternative splicing of VEGF-A in human lung carcinoma cell lines

Introduction The angiogenesis is an absolute requirement for tumor survival and progression. The Vascular Endothelial Growth Factor (VEGF-A) is one of the most potent angiogenic factor and is upregulated in many tumors. VEGF-A exists in most tissues as multiple pro-angiogenic isoforms, termed VEGFxxx, generated by alternative splicing of a single gene product. A novel family of splice variants formed by the use of a distal splice acceptor site in the terminal exon 8 of the VEGF-A gene has been recently described. These isoforms, termed VEGFxxxb possess anti-angiogenic properties and are downregulated in tumors. To date, the cellular signaling pathways controlling this VEGF-A splicing switch remain unknown. In this study, we have investigated the potential role of the transcription factor E2F1 in the control of VEGF-A alternative splicing. Methods To analyze VEGF mRNA and protein levels, RT-PCR, RT-qPCR, ELISA assays, RNA interference and WB experiments were performed in stable inducible clones derived from H358 pulmonary adenocarcinoma cells, in which E2F1 is overexpressed upon doxycyclin addition. Results In our cells, deprived of p53 function, we provide evidence that E2F1, but not the mutant E2F1(E132) unable to bind DNA, down-regulates the VEGF promoter activity in normoxic conditions, leading to a decrease of all VEGFxxx isoforms, at both mRNA and protein levels. More importantly, we further provide evidence that E2F1 increases the level of anti-angiogenic VEGFxxxb mRNAs, as well as leads to the accumulation of the VEGF165b protein isoform. These data indicate that E2F1 selectively enhances the inclusion of VEGF-A alternative exon 8b. We recently identified the splicing factor SC35, a member of the Ser-Rich Arg (SR) proteins family that play a key role in constitutive and alternative splicing events, as a new direct transcriptional target of E2F1. Finally, we demonstrate that SC35 is absolutely required for upregulation of VEGFxxxb splice variants in response to E2F1, while it is dispensable for E2F1-mediated VEGF-A transcriptional repression. Conclusion These findings demonstrate that E2F1 regulates the angiogenic switch of VEGF-A, in favor of anti-angiogenic splice variants, and identify the splicing factor SC35 as a key component of this process. Such effects could have a physiological relevance during tumoral neo-angiogenesis of lung carcinoma, in which E2F1 is abnormally expressed.