Identification and characterization of pro-oncogenic low frequency VEGFR2 mutations

Background Cancer is a multi-step process during which cells accumulate modifications in the genetic programs. We have focused on the alteration of the Vascular Endothelial Growth Factor receptor 2 (VEGFR2). Although no oncogenetic mutations have been reported in VEGFR2 using the traditional mutation-detection strategies, Next-generation sequencing (NGS) technology identified several low frequency mutations in the receptor sequence. Here we assess the possibility that activating VEGFR2 gene mutations may affect tumor progression. Methods We used a new computational tool for low frequency mutation analysis (LowMACA) developed in collaboration with Dr L.Zammataro (Yale University). Briefly, LowMACA tool is based on the concept that non-synonymous mutations that affect one gene of the RTK family have a high probability to be conserved in other genes of the same family, increasing the statistical power of the analysis. We introduced the identified mutations in VEGFR2 sequence, and analyzed their effects in breast cancer MCF7 cells. Results The multiple analysis of tyrosine kinase domain (TK) of tyrosine receptors (RTK) using LowMACA allowed us to identify six conserved low frequency mutations in cancer samples. We focused our attention on the positions 255, 256, 304 of the TK consensus domain (TKCD). Positions 255 and 256 of TKCD are frequently substituted in FLT3 in acute myeloid leukemia, while no data are available for the position 304. The substitutions of D255 and R256 residues in VEGFR2 induce a faster and a long last activation of VEGFR2. Also the expression of these mutants in MCF7 cells supports a more aggressive phenotype in terms of proliferation, migration and invasion. Instead, the substitution of S304 prevents the VEGF-dependent phosphorylation of VEGFR2 conferring a less aggressive phenotype to MCF7. Conclusion In conclusion low frequency mutations may actively play in cancer. Further analysis are needed to better describe the evolution of cancer and provide clinically interesting prognostic or predictive informations.