New splice variants of VEGF as relevant targets for the treatment of Renal Cell Carcinoma

Abstract Background The efficacy of anti-VEGF/VEGF receptors in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) varies from patient to patient. Discovering the reasons for this variability could lead to the identification of relevant therapeutic targets. We have investigated the possibility of splicing events leading to new forms of VEGF that are less efficiently inhibited by anti-VEGF/VEGFR targeting the conventional forms. Methods In silico analysis identified the presence of an unknown splice acceptor in the last intron of the VEGF gene resulting in an insertion of 23 bases in VEGF mRNA. Such an insertion can occur in previously described splice variants of VEGF (VEGFXXX) and shift the open reading frame, leading to a change in the c-terminal part of VEGF. We investigated the role of the resulting new major form of VEGF, VEGF222NF, in physiological and pathological angiogenesis. We analyzed the expression of these new alternatively spliced forms in normal tissue and in a series of RCC cells by qPCR and ELISA. We generated experimental RCC in mice by implanting ccRCC cells overexpressing VEGF222NF. The experimental RCC were also treated with polyclonal anti-VEGF/NF antibodies. The relationship between plasmatic VEGF/NF levels and resistance to anti-VEGFR and survival was also investigated in a cohort of patients from the NCT00943839 clinical trial. Results VEGF222/NF stimulated endothelial cell proliferation and vascular permeability through activation of VEGFR2. Overexpression of VEGF222/NF stimulated proliferation and metastatic properties of RCC cells, whereas its downregulation resulted in cell death. RCC cells overexpressing VEGF222/NF generated aggressive experimental tumors that developed functional blood and lymphatic vessels. Anti-VEGFXXX/NF antibodies slowed the growth of experimental RCC by inhibiting tumor cell proliferation and the development of blood and lymphatic vessels. High plasmatic VEGFXXX/NF levels correlated with shorter survival and lower efficacy of anti-angiogenic drugs. Conclusions The existence of new VEGF isoforms has shed new light on the VEGF field.