P01.18AUTOCRINE VEGFC-VEGFR2 SIGNALING IS OF IMPORTANCE FOR THE GROWTH OF GLIOBLASTOMA MULTIFORME

BACKGROUND: Recent research shows that the angiogenic mediator Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is expressed in Glioblastoma Multiforme (GBM) cells and that inhibition of VEGFR2 results in reduced growth of GBM cells. Although autocrine VEGFA-VEGFR2 signaling has been identified in these GBM cells, VEGFA inhibition has very limited effect on growth in the cells, indicating that activation of VEGFR2 in GBM cells not solely is dependent on VEGFA. METHODS: Material consisted of 5 GBM cell cultures established under stem cell conditions and tumors from 18 GBM patients. Angiogenesis-related genes were deregulated using SU1498, recombinant VEGFA-165 and VEGFC protein and VEGFC specific siRNA and a corresponding control. Cell viability was measured using MTT assay, mRNA expression by Q-PCR, protein expression by western blotting and tumor growth by bioluminescence imaging. RESULTS: Examination of a panel of GBM cell cultures identified one culture positive for VEGFR2, while the receptor was undetectable in the remaining cultures. As shown by others, we found that inhibition of receptor phosphorylation by SU1498 resulted in significantly reduced proliferation of the VEGFR2-positive cells. Although VEGFR2 phoshorylation could be stimulated by recombinant VEGFA, inhibition of the VEGFA expressed by the cells using Bevacizumab only had minimal effect on proliferation. Examination of the expression of a range of genes revealed that the VEGFR2 positive cells also were positive for the VEGF variant VEGFC. Addition of recombinant VEGFC protein to the VEGFR2 positive cells resulted in VEGFR2 phosphorylation, while inhibition of VEGFC using specific siRNA constructs resulted in significantly reduced in vitro growth of VEGFR2 positive cells. Moreover, when transplanted as orthotopic brain tumors significantly reduced tumor growth was seen for VEGFC siRNA transfected cells as compared to control cells. In parallel, the median survival was increased from 34 days in mice injected with control cells to 55 days in mice injected with VEGFC siRNA transfected cells. Further, to establish the relevance of VEGFC in GBM patient tumors in general, we measured the VEGFC mRNA level in 18 GBM tumors. Although the level was varying, all examined tumors were positive for VEGFC expression. CONCLUSION: GBM cells can in addition to VEGFA also be dependent on VEGFC for VEGFR2 activation, cell viability and tumor growth.