Abstract 5259: Role of Orai1 and STIM1 in store-operated Ca2+ entry and cell migration in melanoma

Abstract

Abstract Background: Melanoma has poor prognosis due to its strong metastatic ability that is mainly controlled by cell migration. Store operated Ca2+ entry (SOCE), which is defined as Ca2+ entry from extracellular space triggered with depletion of Ca2+ store in the endoplasmic reticulum (ER), largely regulates Ca2+ homeostasis in non-excitable cells. However, little is known about the role of SOCE in melanoma. Here we report that expressions of SOCE modulators, i.e., ORAI calcium release-activated calcium modulator (Orai), which is the plasma membrane Ca2+ channel, and stromal interaction molecule (STIM), an activator of Orai in the ER, in cultured melanoma cells and human melanoma tissues. We also examined the effect of these modulators on SOCE and cell migration in melanoma cells Method: Western blot analysis in SK-Mel-2 cells, and immunohistochemistry in human tissue microarray, were performed to examine expressions of Orai1 or STIM1. Intracellular Ca2+ was measured with fluo-4 AM, Ca2+-sensing fluorescent dye, in human metastatic melanoma cell lines (SK-MEL-2, SK-MEL-24 and C8161 cells), a mouse melanoma cell line (B16 cells), a human melanocyte cell line, (HEMA-LP), and a mouse melanocyte cell line (Melan-A). Cell migration was examined with the Boyden chambers. In order to ablate Orai1 or STIM1, shRNA for each protein was induced in SK-Mel-2 cells with lentiviral infection. Results: Expressions of Orai1 and STIM1 were observed in melanoma cells by western blot, and in human melanoma tissues by immunohistochemistry. SOCE, as demonstrated by enhancement of Ca2+ enhancement after Ca2+ depletion with thapsigargin, was clearly observed in melanoma cell lines, but not in melanocytes, suggesting that SOCE is enhanced in melanoma cells compared to melanocytes. When Orai1 was ablated in SK-MEL-2 cells, the peak Ca2+ signal within SOCE was reduced by 75 % compared to control (2.78±0.44 (control shRNA) vs. 1.44±0.18 (Orai1 shRNA) F/F0 ratio normalized to baseline, p<0.05, n=5). Similarly, ablation of STIM1 reduces the peak Ca2+ signal within SOCE by 37 % compared to control (2.78±0.44 (control shRNA) vs. 2.12±0.63 (STIM1 shRNA) F/F0 ratio normalized to baseline, F/F0, normalized to baseline, p<0.05, n=10). These data suggest that Orai1 and STIM1 are involved in SOCE in melanoma cells. We next examined the role of Orai1 and STIM1 in melanoma cell migration. Ablation of Orai1 reduced cell migration by 46 % (48±4.6 (control shRNA) vs. 26±3.2 (Orai1 shRNA) cells/field, p<0.05, n=4). Similarly, ablation of STIM1 decreased cell migration by 40 % (48±4.6 (control shRNA) vs. 29±4.8 (STIM1 shRNA) cells/field, p<0.05, n=4). These data suggest that Orai1 and/or STIM1 are involved in basal cell migration in melanoma. Conclusion: SOCE, which is regulated by Orai1 and STIM1 in melanoma cells, plays a major role in Ca2+ homeostasis, and cell migration in melanoma, and thus potentially metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5259. doi:10.1158/1538-7445.AM2011-5259