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

// Francesco Lodola 1, 11, * , Umberto Laforenza 2, * , Fabio Cattaneo 3, * , Federico Alessandro Ruffinatti 4 , Valentina Poletto 5 , Margherita Massa 6 , Richard Tancredi 7 , Estella Zuccolo 1 , Dlzar Ali Khdar 1 , Alberto Riccardi 7, 8 , Marco Biggiogera 9 , Vittorio Rosti 5, ** , Germano Guerra 10, ** and Francesco Moccia 1 ** 1 Laboratory of General Physiology, Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia 27100, Italy 2 Department of Molecular Medicine, University of Pavia, Pavia 27100, Italy 3 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples 80131, Italy 4 Department of Life Sciences and Systems Biology, Turin 10123, Italy 5 Laboratory of Biochemistry, Biotechnology and Advanced Diagnosis, Foundation IRCCS Policlinico San Matteo, Pavia 27100, Italy 6 Laboratory of Immunology Transplantation, Foundation IRCCS Policlinico San Matteo, Pavia 27100, Italy 7 Medical Oncology Unit, Foundation IRCCS Salvatore Maugeri, Pavia 27100, Italy 8 Department of Internal Medicine, University of Pavia, Pavia 27100, Italy 9 Laboratory of Cell Biology and Neurobiology, Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia 27100, Italy 10 Department of Medicine and Health Sciences “Vincenzo Tiberio”, University of Molise, Campobasso 86100, Italy 11 Current address: Italian Institute of Technology, Center for Nano Science and Technology, Milano 20133, Italy * These authors should be considered as co-first authors ** These authors share the Senior Authorship of the manuscript and should be regarded as co-last authors Correspondence to: Francesco Moccia, email: francesco.moccia@unipv.it Germano Guerra, email: germano.guerra@unipv.it Keywords: VEGF, breast cancer, endothelial colony forming cells, intracellular Ca 2+ oscillations, angiogenesis Received: June 01, 2017 Accepted: July 12, 2017 Published: August 14, 2017 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 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.