Store-Operated Ca2+ Entry Is Remodelled and Controls In Vitro Angiogenesis in Endothelial Progenitor Cells Isolated from Tumoral Patients

Abstract

BackgroundEndothelial 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 Ca2+ entry (SOCE), which is activated by a depletion of the intracellular Ca2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca2+-sensor, Stim1, and the plasmalemmal Ca2+ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.Methodology/Principal FindingsThe present study employed Ca2+ 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, La3+ and Gd3+. 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 Ca2+ 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 Ca2+ 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.ConclusionsSOCE 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.

Remodelling of Ca2+transport in cancer: how it contributes to cancer hallmarks?

“…Thus, abnormalities in the expression and/or function of STIM1 and ORAI1, which were shown to mediate VEGFevoked calcium entry promoting EC migration, tube formation and angiogenesis [91], and might be involved in the enhancing the pro-angiogenic response of ECs in tumours (figure 4). Indeed, upregulated mRNA and protein levels of ORAI1, STIM1 and TRPC1 (which is also known to contribute to SOCE) accompanied by the increase in CAI-sensitive SOCE were detected in endothelial progenitor cells from patients with renal cellular carcinoma [96].…”

STIM and Orai proteins as novel targets for cancer therapy. A Review in the Theme: Cell and Molecular Processes in Cancer Metastasis

“…Using knockdown and overexpression tools, the authors showed that STIM1 regulates VEGF secretion as well as in vivo tumor angiogenesis in mice (17). Sanchez-Hernandez et al (87) assessed SOCE in EPCs and later on characterized the SOCE players in EPCs derived from renal carcinoma (RCC-EPCs) blood samples (63). The authors further established that STIM1 and Orai1 regulate RCC-EPCs SOCE, proliferation, and tubulogenesis (63).…”

“…Sanchez-Hernandez et al (87) assessed SOCE in EPCs and later on characterized the SOCE players in EPCs derived from renal carcinoma (RCC-EPCs) blood samples (63). The authors further established that STIM1 and Orai1 regulate RCC-EPCs SOCE, proliferation, and tubulogenesis (63). Interestingly, STIM1 and Orai1 expression was higher in RCCEPCs compared with normal EPCs, which in turn resulted in elevated SOCE and angiogenesis (63).…”

STIM and ORAI proteins: crucial roles in hallmarks of cancer

“…Similarly to HUVEC, suppression of ORAI1 in endothelial progenitor cells (EPCs) prevents VEGF-mediated SOCE and tubule formation (22,56). Moreover, EPCs isolated from RCC patients (RCC-EPCs) display an increased SOCE, which correlates with ORAI1, STIM1, and TRPC1 overexpression when compared with EPCs from healthy patients: genetic suppression of STIM1, ORAI1, and TRPC1 affects SOCE in RCC-EPCs (59). It has to be noted that the role of ORAI1 in ECs has been questioned by Antigny et al (4), although they described STIM1 as a key player (together with TRPC1 and TRPC4) in tube formation in both HUVEC and EA.hy926 cells.…”