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

甘丙氨酸受体1 （GALR1）位于头颈部鳞状细胞癌（HNSCC） 18q缺失的共同区域，经常被甲基化灭活。为了研究GALR1及其信号通路的作用，我们在不表达内源性GALR1的人口腔癌细胞系（UM-SCC-I-GALR1）中稳定表达ha标记的GALR1。在UM-SCC-1-GALR1细胞中，甘丙氨酸诱导细胞外调节蛋白激酶1/2 （ERK1/2）的激活并抑制增殖，而在模拟转染细胞中则没有。丙氨酸刺激还导致细胞周期蛋白依赖性激酶抑制剂（CKI） p27Kip1和p57Kip2的表达增加，细胞周期蛋白Dl的表达降低。用erk1 /2特异性抑制剂U0126预处理UM-SCC-I-GALR1细胞可阻止这些丙氨酸诱导的效应。甘丙氨酸处理后，UM-SCC-1-GALR1和um - scc -1模拟细胞的PI3K通路激活无显著差异。使用Giα蛋白特异性抑制剂-百日毒和PI3K特异性抑制剂- LY294002，我们发现甘丙氨酸和GALR1通过Giα途径诱导ERK1/2激活，而不是通过与Gβγ亚基相关的PI3K途径。Galanin和GALR1在体内也抑制菌落的形成和异种移植肿瘤的生长。综上所述，我们的研究结果确定了一种新的机制，即GALR1，一种Giα蛋白偶联受体，通过ERK1/2激活抑制细胞增殖。

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.