VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy

Abstract

Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF₁₆₅ and antiangiogenic VEGF₁₆₅b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF_xxxb, but there was a variable upregulation of VEGF_xxx and downregulation of VEGF_xxxb in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF_xxxb, whereas colonic carcinoma cells expressed predominantly VEGF_xxx. However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF_xxxb to predominantly VEGF_xxx. VEGF₁₆₅b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF₁₆₅b bound to bevacizumab with similar affinity as VEGF₁₆₅. However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF₁₆₅, it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF₁₆₅b. Both bevacizumab and anti-VEGF₁₆₅b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF₁₆₅b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.