Galanin and galanin receptor type 1 suppress proliferation in squamous carcinoma cells : activation of the extracellular signal regulated kinase pathway and induction of cyclin-dependent kinase inhibitors

甘丙肽受体1(GALR1)定位于头颈部鳞状细胞癌(HNSCC)常见的18q缺失区域，常因甲基化而失活。为了研究GALR1的作用及其信号通路，我们在一个不表达内源性GALR1的口腔癌细胞系(UM-SCC-I-GALR1)中稳定表达了HA标记的GALR1。在UM-SCC-1-GALR1细胞中，甘丙素诱导细胞外调节蛋白激酶-1/2(ERK1/2)激活并抑制细胞增殖。甘丙素刺激还导致细胞周期蛋白依赖性激酶抑制物(CKI)、p27Kip1和p57Kip2表达增加，细胞周期蛋白D1表达降低。用ERK1/2特异性抑制剂U0126预处理UM-SCC-I-GALR1细胞可阻止甘丙素诱导的这些效应。甘丙素处理后，UM-SCC-1-GALR1和UM-SCC-1-mock细胞的磷脂酰肌醇3-激酶(PI3K)通路激活无明显差异。使用Giα蛋白特异性抑制剂百日咳毒素和PI3K特异性抑制剂LY294002，我们发现甘丙素和Galr1通过Giα途径而不是与Gβγ亚基相连的PI3K途径诱导ERK1/2激活。甘丙素和GALR1在体内也能抑制集落形成和移植瘤生长。综上所述，我们的结果确定了一种新的机制，即Giα蛋白偶联受体Galr1通过激活ERK1/2来抑制细胞增殖。

Galanin receptor 1 (GALR1) maps to a common region of 18q loss on head and neck squamous cell carcinomas (HNSCC) and is frequently inactivated by methylation. To investigate effects of GALR1 and its signaling pathways, we stably expressed HA-tagged GALR1 in a human oral carcinoma cell line (UM-SCC-I-GALR1) that expresses no endogenous GALR1. In UM-SCC-1-GALR1 cells but not in mock transfected cells, galanin induced activation of the extracellular regulated protein kinase-1/2 (ERK1/2) and suppressed proliferation. Galanin stimulation also resulted in increased expression of the cyclin-dependent kinase inhibitors (CKI), p27Kip1 and p57Kip2 and decreased expression of cyclin Dl. Pretreatment of UM-SCC-I-GALR1 cells with the ERK1/2-specific inhibitor U0126 prevented these galanin-induced effects. There was no significant difference in phosphatidylinositol 3-kinase (PI3K) pathway activation between UM-SCC-1-GALR1 and UM-SCC-1-mock cells after galanin treatment-. Using the Giα protein specific inhibitor - pertussis toxin and the PI3K-specific inhibitor - LY294002, we show that galanin and GALR1 induce ERK1/2 activation via the Giα pathway, not the PI3K pathway-linked to the Gβγ subunit. Galanin and GALR1 also inhibit colony formation and xenografted tumor growth in vivo. Taken together, our results identify a novel mechanism whereby GALR1, a Giα protein-coupled receptor, inhibits cell proliferation by ERK1/2 activation.