VEGF-induced intracellular Ca2+ oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells

Abstract

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 Ca²⁺ 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 Ca²⁺ signalling to control angiogenesis in BC-derived ECFCs (BC-ECFCs). Although VEGFR-2 was normally expressed, VEGF failed to induce proliferation and <i>in vitro</i> tubulogenesis in BC-ECFCs. Likewise, VEGF did not trigger robust Ca²⁺ oscillations in these cells. Similar to normal cells, VEGF-induced intracellular Ca²⁺ oscillations were triggered by inositol-1,4,5-trisphosphate-dependent Ca²⁺ release from the endoplasmic reticulum (ER) and maintained by store-operated Ca²⁺ entry (SOCE). However, InsP₃-dependent Ca²⁺ release was significantly lower in BC-ECFCs due to the down-regulation of ER Ca²⁺ levels, while there was no remarkable difference in the amplitude, pharmacological profile and molecular composition of SOCE. Thus, the attenuation of the pro-angiogenic Ca²⁺ response to VEGF was seemingly due to the reduction in ER Ca²⁺ concentration, which prevents VEGF from triggering robust intracellular Ca²⁺ oscillations. However, the pharmacological inhibition of SOCE prevented BC-ECFC proliferation and <i>in vitro</i> 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.