Role Of Store-Operated Ca2+Entry In Proliferation And Cell Cycle In Melanoma

Background: Melanoma has a poor prognosis due to its rapid progression and a high metastatic ability. Ca2+ homeostasis plays a pivotal role in proliferation in cancer cells, but little is known about the role of Ca2+ in melanoma. Store-operated calcium entry (SOCE) is defined as Ca2+ entry from extracellular space triggered when Ca2+ store in the endoplasmic reticulum (ER) is depleted. SOCE is regulated by coordination between Orai (ORAI calcium release-activated calcium modulator) in the plasma membrane, and STIM1 (stromal interaction molecule 1) in the ER. We examined the role of Ca2+ in melanoma cells especially focusing on SOCE. Method: Intracellular Ca2+ level was measured by using Fluo4-AM, a Ca2+-sensing fluorescent dye. Cell proliferation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were stained with propidium iodide and cell cycle was assessed using flow cytometer. Protein phosphorylation microarray was performed by using a commercially available kit (Cancer Signaling Phospho-Antibody Array). Results: SOCE, as demonstrated by enhancement of Ca2+ entry from extracellular space after Ca2+ depletion in the ER, was observed in SK-MEL-2, C8161, SK-MEL-24, UACC257 (human) and B16 (mouse) melanoma cell lines, suggesting that the existence of SOCE is universal among melanoma cells. SOCE was attenuated by Pyr3, a pyrazole compound which is known to inhibit SOCE, as well as by EGTA that chelates extracellular Ca2+. Pyr3 inhibits cellular proliferation in SK-MEL-2 cells (IC50: 5.1uM) and C8161 cells (IC50: 2.1uM). Cell cycle was analyzed in C8161 cells, and the cell number in the G2 phase was increased by both Pyr3 and EGTA, indicating that SOCE plays a role in the transition of G2/S phase. Protein microarray data showed that Pyr3 reduces phosphorylation of signal transducers and activators of transcription 5 (STAT5) by 93%, suggesting that the JAK/STAT pathway may be involved in SOCE-induced alteration of cell cycle. Conclusion: Our results showed that inhibition of SOCE suppresses proliferation in melanoma, thus it could be a new strategy for melanoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1864. doi:1538-7445.AM2012-1864