VEGF-induced intracellular Ca2+ oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells
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Francesco Lodola1,11,*, Umberto Laforenza2,*, Fabio Cattaneo3,*, Federico Alessandro Ruffinatti4, Valentina Poletto5, Margherita Massa6, Richard Tancredi7, Estella Zuccolo1, Dlzar Alì Khdar1, Alberto Riccardi7,8, Marco Biggiogera9, Vittorio Rosti5,**, Germano Guerra10,** and Francesco Moccia1**
1Laboratory of General Physiology, Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia 27100, Italy
2Department of Molecular Medicine, University of Pavia, Pavia 27100, Italy
3Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples 80131, Italy
4Department of Life Sciences and Systems Biology, Turin 10123, Italy
5Laboratory of Biochemistry, Biotechnology and Advanced Diagnosis, Foundation IRCCS Policlinico San Matteo, Pavia 27100, Italy
6Laboratory of Immunology Transplantation, Foundation IRCCS Policlinico San Matteo, Pavia 27100, Italy
7Medical Oncology Unit, Foundation IRCCS Salvatore Maugeri, Pavia 27100, Italy
8Department of Internal Medicine, University of Pavia, Pavia 27100, Italy
9Laboratory of Cell Biology and Neurobiology, Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia 27100, Italy
10Department of Medicine and Health Sciences “Vincenzo Tiberio”, University of Molise, Campobasso 86100, Italy
11Current 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
Francesco Moccia, email: email@example.com
Germano Guerra, email: firstname.lastname@example.org
Keywords: VEGF, breast cancer, endothelial colony forming cells, intracellular Ca2+ oscillations, angiogenesis
Received: June 01, 2017 Accepted: July 12, 2017 Published: August 14, 2017
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 in vitro 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 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.
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