Hooper Robert, Zhang Xuexin, Webster Marie, Go Christina, Kedra Joseph, Marchbank Katie, Gill Donald L, Weeraratna Ashani T, Trebak Mohamed, Soboloff Jonathan
Mol Cell Biol. 2015 Aug;35(16):2790-8. doi: 10.1128/MCB.01500-14.
The incidence of malignant melanoma, a cancer of the melanocyte cell lineage, has nearly doubled in the past 20 years. Wnt5A, akey driver of melanoma invasiveness, induces Ca2 signals. To understand how store-operated calcium entry (SOCE) contributes to Wnt5A-induced malignancy in melanoma models, we examined the expression and function of STIM1 and Orai1 in patient-derived malignant melanoma cells, previously characterized as either highly invasive (metastatic) or noninvasive. Using both fluorescence microscopy and electrophysiological approaches, we show that SOCE is greatly diminished in invasive melanoma compared to its level in noninvasive cell types. However, no loss of expression of any members of the STIM and Orai families was observed in invasive melanoma cells. Moreover, overexpressed wild-type STIM1 and Orai1 failed to restore SOCE in invasive melanoma cells, and we observed no defects in their localization before or after store depletion in any of the invasive celllines. Importantly, however, we determined that SOCE was restored by inhibition of protein kinase C, a known downstream target of Wnt5A. Furthermore, coexpression of STIM1 with an Orai1 mutant insensitive to protein kinase C-mediated phosphorylation fully restored SOCE in invasive melanoma. These findings reveal a level of control for STIM/Orai function in invasive melanoma not previously reported.
恶性黑色素瘤是一种源自黑素细胞谱系的癌症，其发病率在过去20年中几乎翻了一番。Wnt5A是黑色素瘤侵袭性的关键驱动因子，可诱导Ca2信号。为了了解在黑色素瘤模型中，储存式钙内流（SOCE）如何促进Wnt5A诱导的恶性肿瘤，我们检测了源自患者的恶性黑色素瘤细胞中STIM1和Orai1的表达及功能，这些细胞先前被鉴定为高侵袭性（转移性）或非侵袭性。通过荧光显微镜和电生理方法，我们发现与非侵袭性细胞类型相比，侵袭性黑色素瘤中的SOCE大大降低。然而，在侵袭性黑色素瘤细胞中未观察到STIM和Orai家族任何成员的表达缺失。此外，过表达的野生型STIM1和Orai1未能恢复侵袭性黑色素瘤细胞中的SOCE，并且在任何侵袭性细胞系中，我们在储存耗尽前后均未观察到它们的定位缺陷。然而，重要的是，我们确定通过抑制蛋白激酶C（Wnt5A的已知下游靶点）可恢复SOCE。此外，将STIM1与对蛋白激酶C介导的磷酸化不敏感的Orai1突变体共表达，可完全恢复侵袭性黑色素瘤中的SOCE。这些发现揭示了侵袭性黑色素瘤中STIM/Orai功能的一种前所未有的调控水平。
