A group of novel VEGF splice variants as alternative therapeutic targets in renal cell carcinoma

Abstract

The efficacy of anti-angiogenic treatment by targeting VEGF/VEGF receptors in metastatic clear cell renal cell carcinoma (ccRCC) varies from patient to patient. Discovering the reasons behind this variability could lead to the identification of relevant therapeutic targets. Thus, we investigated the novel splice variants of VEGF that are less efficiently inhibited by anti-VEGF/VEGFR targeting than the conventional isoforms. By in silico analysis, we identified a novel splice acceptor in the last intron of the VEGF gene resulting in an insertion of 23 bp in VEGF mRNA. Such an insertion can shift the open-reading frame in previously described splice variants of VEGF (VEGF_XXX ), leading to a change in the C-terminal part of the VEGF protein. Next, we analysed the expression of these alternatively spliced VEGF new isoforms (VEGF_XXX/NF ) in normal tissues and in RCC cell lines by qPCR and ELISA, and we investigated the role of VEGF_222/NF (equivalent to VEGF₁₆₅ ) in physiological and pathological angiogenesis. Our in vitro data demonstrated that recombinant VEGF_222/NF stimulated endothelial cell proliferation and vascular permeability by activating VEGFR2. In addition, VEGF_222/NF overexpression enhanced proliferation and metastatic properties of RCC cells, whereas downregulation of VEGF_222/NF resulted in cell death. We also generated an in vivo model of RCC by implanting RCC cells overexpressing VEGF_222/NF in mice, which we treated with polyclonal anti-VEGF_XXX/NF antibodies. VEGF_222/NF overexpression enhanced tumour formation with aggressive properties and a fully functional vasculature, while treatment with anti-VEGF_XXX/NF antibodies slowed tumour growth by inhibiting tumour cell proliferation and angiogenesis. In a patient cohort from the NCT00943839 clinical trial, we investigated the relationship between plasmatic VEGF_XXX/NF levels, resistance to anti-VEGFR therapy and survival. High plasmatic VEGF_XXX/NF levels correlated with shorter survival and lower efficacy of anti-angiogenic drugs. Our data confirmed the existence of new VEGF isoforms that could serve as novel therapeutic targets in patients with RCC that are resistant to anti-VEGFR therapy.

Keywords: VEGF; alternative splicing; angio/lymphangiogenesis; renal cell carcinoma; resistance to antiangiogenics.