VEGF-induced intracellular Ca(2+) oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells

Endothelial colony forming cells (ECFCs) represent a population of truly endothelial precursors that promote the angiogenic switch in solid tumors, such as breast cancer (BC). The intracellular Ca(2+) toolkit, which drives the pro-angiogenic response to VEGF, is remodelled in tumor-associated ECFCs such that they are seemingly insensitive to this growth factor. This feature could underlie the relative failure of anti-VEGF therapies in cancer patients. Herein, we investigated whether and how VEGF uses Ca(2+) signalling to control angiogenesis in BC-derived ECFCs (BC-ECFCs). Although VEGFR-2 was normally expressed, VEGF failed to induce proliferation and in vitro tubulogenesis in BC-ECFCs. Likewise, VEGF did not trigger robust Ca(2+) oscillations in these cells. Similar to normal cells, VEGF-induced intracellular Ca(2+) oscillations were triggered by inositol-1,4,5-trisphosphate-dependent Ca(2+) release from the endoplasmic reticulum (ER) and maintained by store-operated Ca(2+) entry (SOCE). However, InsP(3)-dependent Ca(2+) release was significantly lower in BC-ECFCs due to the down-regulation of ER Ca(2+) levels, while there was no remarkable difference in the amplitude, pharmacological profile and molecular composition of SOCE. Thus, the attenuation of the pro-angiogenic Ca(2+) response to VEGF was seemingly due to the reduction in ER Ca(2+) concentration, which prevents VEGF from triggering robust intracellular Ca(2+) oscillations. However, the pharmacological inhibition of SOCE prevented BC-ECFC proliferation and in vitro tubulogenesis. These findings demonstrate for the first time that BC-ECFCs are insensitive to VEGF, which might explain at cellular and molecular levels the failure of anti-VEGF therapies in BC patients, and hint at SOCE as a novel molecular target for this disease.