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      Store-Operated Ca 2+ Entry Is Remodelled and Controls In Vitro Angiogenesis in Endothelial Progenitor Cells Isolated from Tumoral Patients

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          Abstract

          Background

          Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca 2+ entry (SOCE), which is activated by a depletion of the intracellular Ca 2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca 2+-sensor, Stim1, and the plasmalemmal Ca 2+ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca 2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.

          Methodology/Principal Findings

          The present study employed Ca 2+ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La 3+ and Gd 3+. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca 2+ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca 2+ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.

          Conclusions

          SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.

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          Most cited references 83

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          Store-operated calcium channels.

          In electrically nonexcitable cells, Ca(2+) influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and proliferation, and apoptosis. The major Ca(2+) entry pathway in these cells is the store-operated one, in which the emptying of intracellular Ca(2+) stores activates Ca(2+) influx (store-operated Ca(2+) entry, or capacitative Ca(2+) entry). Several biophysically distinct store-operated currents have been reported, but the best characterized is the Ca(2+) release-activated Ca(2+) current, I(CRAC). Although it was initially considered to function only in nonexcitable cells, growing evidence now points towards a central role for I(CRAC)-like currents in excitable cells too. In spite of intense research, the signal that relays the store Ca(2+) content to CRAC channels in the plasma membrane, as well as the molecular identity of the Ca(2+) sensor within the stores, remains elusive. Resolution of these issues would be greatly helped by the identification of the CRAC channel gene. In some systems, evidence suggests that store-operated channels might be related to TRP homologs, although no consensus has yet been reached. Better understood are mechanisms that inactivate store-operated entry and hence control the overall duration of Ca(2+) entry. Recent work has revealed a central role for mitochondria in the regulation of I(CRAC), and this is particularly prominent under physiological conditions. I(CRAC) therefore represents a dynamic interplay between endoplasmic reticulum, mitochondria, and plasma membrane. In this review, we describe the key electrophysiological features of I(CRAC) and other store-operated Ca(2+) currents and how they are regulated, and we consider recent advances that have shed insight into the molecular mechanisms involved in this ubiquitous and vital Ca(2+) entry pathway.
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            Orai1 and STIM1 are critical for breast tumor cell migration and metastasis.

            Tumor metastasis is the primary cause of death of cancer patients. Understanding the molecular mechanisms underlying tumor metastasis will provide potential drug targets. We report here that Orai1 and STIM1, both of which are involved in store-operated calcium entry, are essential for breast tumor cell migration in vitro and tumor metastasis in mice. Reduction of Orai1 or STIM1 by RNA interference in highly metastatic human breast cancer cells or treatment with a pharmacological inhibitor of store-operated calcium channels decreased tumor metastasis in animal models. Our data demonstrate a role for Orai1 and STIM1 in tumor metastasis and suggest store-operated calcium entry channels as potential cancer therapeutic targets.
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              Calcium and cancer: targeting Ca2+ transport.

              Ca2+ is a ubiquitous cellular signal. Altered expression of specific Ca2+ channels and pumps are characterizing features of some cancers. The ability of Ca2+ to regulate both cell death and proliferation, combined with the potential for pharmacological modulation, offers the opportunity for a set of new drug targets in cancer. However, the ubiquity of the Ca2+ signal is often mistakenly presumed to thwart the specific therapeutic targeting of proteins that transport Ca2+. This Review presents evidence to the contrary and addresses the question: which Ca2+ channels and pumps should be targeted?
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                25 September 2012
                : 7
                : 9
                Affiliations
                [1 ]Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia, Italy
                [2 ]Section of Human Physiology, Department of Molecular Medicine, University of Pavia, Pavia, Italy
                [3 ]Clinical Epidemiology Laboratory Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
                [4 ]Department of Pharmaceutical Sciences, University of Eastern Piedmont “Amedeo Avogadro”, Novara, Italy
                [5 ]Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America
                [6 ]Department of Health Sciences, University of Molise, Campobasso, Italy
                [7 ]Medical Oncology IRCCS Policlinico San Matteo, Pavia, Italy
                [8 ]Laboratory of Biotechnology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
                University of Florida, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Performed the experiments: FL UL EB DL SD CB HLO CG M. Massa M. Manzoni VR FM. Analyzed the data: FL UL EB DL SD CB GG CG M. Massa AAG VR PP FT FM CP. Contributed reagents/materials/analysis tools: UL M. Massa M. Manzoni ISA AAG VR PP FT FM CP. Wrote the paper: VR PP FT FM CP.

                ¶ These authors also contributed equally to this work.

                Article
                PONE-D-12-05946
                10.1371/journal.pone.0042541
                3458053
                23049731

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 21
                Funding
                This work was supported by a grant from Ricerca Corrente, #674, IRCCS Policlinico San Matteo Foundation, Pavia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Anatomy and Physiology
                Cardiovascular System
                Cell Physiology
                Molecular Cell Biology
                Signal Transduction
                Signaling in Cellular Processes
                Calcium Signaling
                Cell Division
                Neuroscience
                Neuroimaging
                Calcium Imaging
                Medicine
                Cardiovascular
                Vascular Biology
                Hematology
                Oncology
                Basic Cancer Research
                Urology
                Renal Cancer

                Uncategorized

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