Abstract 5244: Store-operated Ca2+ entry (SOCE) regulates melanoma progression.
Authors: Umemura Masanari , Balijinnyam Erdene , Feske Stefan , De Lorenzo Mariana S. , Xie Lai-Hua , Ishikawa Yoshihiro , Iwatsubo Kousaku 
Published: August 2014

Abstract
Background: Melanoma is the worst poor-prognosis skin cancer in the U.S. Although Ca2+ is well recognized as a major second messenger, the role of Ca2+ remains poorly understood in the cancer research field including melanoma. Store-operated calcium entry (SOCE) is the mechanism of Ca2+ entry from the extracellular space in response to Ca2+ depletion in the endoplasmic reticulum (ER). SOCE is regulated by interaction between STIM1 (stromal interaction molecule 1) in the ER and Orai (ORAI calcium release-activated calcium modulator) in the plasma membrane. Our previous studies have demonstrated the presence of SOCE is observed in melanoma cells, and potentially regulates cell migration and cell cycle. In the present study, we extensively examined the effects of SOCE on migration and proliferation in multiple melanoma and melanocyte cell lines as well as on metastasis in mice.
Method: Changes in intracellular Ca2+ level were measured by Fluo4-AM, a Ca2+-sensing fluorescent dye. In order to ablate STIM-1 or Orai-1, shRNA for each protein was induced with lentiviral infection in SK-Mel-2, SK-Mel-24 and C8161 cells. Proliferation was measured by the MTT assay. Migration was examined with the Boyden chambers and the scratch assay. Immunocytochemistry was performed to count the number of lamellipodia with F-actin staining. The experimental metastasis assay was performed as follows. Three weeks after the intravenous injection of SK-Mel-2 cells with knockdown of Orai1 or STIM1 in Balb/c nu/nu mice, the lungs were removed and fixed with picric acid. The number of metastatic colonies in the lung surface was counted.
Results: SOCE, as demonstrated by enhancement of Ca2+ entry from extracellular space after Ca2+ depletion in the ER, was observed in 5 primary and 3 metastatic human melanoma cell lines, a mouse melanoma cell line, human and mouse melanocyte cell lines. In addition, metastatic, but not primary, melanoma cell lines showed significantly greater SOCE compared to melanocytes, suggesting that SOCE may positively correlate with malignancy of melanoma. Knockdown of STIM1 inhibited proliferation in metastatic melanoma cells (SK-Mel-2, SK-Mel-24 and C8161) (p&lt;0.01), suggesting that SOCE activates proliferation of melanoma. Inhibition of SOCE with knockdown of Orai1 or STIM1 suppressed migration in cell lines tested (SK-Mel-2 and SK-Mel-24) (p&lt;0.01) and the SOCE inhibitors suppressed it in cell lines (SK-Mel-2 and C8161) (p&lt;0.01 for both cell lines), indicating that SOCE regulates melanoma migration. Indeed, the number of lamellipodia, which reflects the activity of migration, was decreased by deletion of STIM1 or Orai1 (p&lt;0.01). The experimental metastasis assay demonstrated that the number of lung colonies was reduced by knockdown of STIM1 or Orai1 (p&lt;0.01).
Conclusion: Our results demonstrated that inhibition of SOCE suppressed proliferation, migration, and metastasis in melanoma, thus SOCE could be a new target for melanoma therapy.
Citation Format: Masanari Umemura, Erdene Balijinnyam, Stefan Feske, Mariana S. De Lorenzo, Lai-Hua Xie, Yoshihiro Ishikawa, Kousaku Iwatsubo. Store-operated Ca2+ entry (SOCE) regulates melanoma progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2013-5244