021 - The transcription factor E2F1 controls the alternative splicing of VEGF-A in human lung carcinoma cell lines - 05/12/08

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 VEGF_xxx, 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 VEGF_xxxb 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 VEGF_xxx isoforms, at both mRNA and protein levels. More importantly, we further provide evidence that E2F1 increases the level of anti-angiogenic VEGF_xxxb mRNAs, as well as leads to the accumulation of the VEGF_165b 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 VEGF_xxxb 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.