ANGI-11VEGF-C CONTRIBUTES TO AUTOCRINE VEGFR2 SIGNALING AND AFFECTS CELL VIABILITY AND TUMOR GROWTH IN GLIOBLASTOMA

The overexpression of VEGF-A in glioblastoma (GBM) and recently reported autocrine VEGF-A-VEGFR2 signaling in GBM cells, make this deadly disease an ideal candidate for anti-angiogenic therapies. However, despite initial enthusiasm, the use of the humanized anti-VEGFA antibody Bevacizumab offered only limited benefit. Moreover, contradicting reports are existent regarding the inhibition VEGF-A and VEGFR2 in GBM cells, implying that VEGFR2 in GBM may also be activated by other factor(s) than VEGF-A. With the aim to further investigate the regulation of VEGFR2 autocrine signaling, a panel of GBM cell cultures was examined for VEGFR2 expression and a number of both positive and negative cultures were identified, which all were shown to express VEGF-A. In line with others, we found that VEGFR2 phosphorylation in the GBM cells could be stimulated by recombinant VEGF-A protein, and observed that inhibition of receptor activation by SU1498 resulted in reduced proliferation of the VEGFR2-positive cells, while neither addition of VEGF-A protein nor Bevacizumab impacted the cell viability. Our further experiments revealed that the VEGFR2-positive cell cultures also expressed another VEGF-variant VEGF-C, but not VEGFR3, which is associated with VEGF-C signaling. Addition of recombinant VEGF-C protein to the VEGFR2-positive cells stimulated VEGFR2 phosphorylation, while the inhibition of VEGF-C (using siRNA-mediated knockdown) resulted in reduced in vitro growth of VEGFR2-positive cells. Furthermore, when injected into the brains of immunocompromised mice, VEGF-C-siRNA transfected cells exhibited reduced tumor growth and subsequently resulted in increased survival of mice as compared to control cells. Taking into consideration that both VEGFR2 and VEGF-C expression also could be found when examining GBM patient samples, our study indicates that targeting of VEGF-C could be of high clinical relevance. In conclusion, the results show that VEGF-C is of importance for GBM cell viability and tumor growth presumable due to its ability to stimulate autocrine activation of VEGFR2.