锌转运体SLC39A6遗传变异影响食管癌预后的作用及机制研究

Esophageal cancer is a common and specific cancer in China. Surgery and chemoradiotherapy are the most common treatments for esophageal cancer patients. However,the survival of esophageal cancer patients after treatment is still dismal with great heterogeneity. Because few biomakers can be used to predict the progrosis of esophageal cancer, optimal individualized treatment is difficult to achive. The prognosis of human cancer differs significantly from individual to individual. Clarifying the mechanism of individual difference has profound significance for the understanding of cancer progression and for the prevention and personalized medicine of cancer. Recently, we employed GWAS to examine genetic variants for survival of esophageal squamous-cell carcinoma (ESCC). By using two-stage analysis of prognostic loci associated with overall survival of ESCC patients, we have identified rs1050631 and rs7241481 SNPs in the SLC39A6 gene on chromosome 18q12 significantly associated with overall survival in patients with ESCC. SLC39A6 comprises an entire subfamily of zinc transporters, the literature often uses ZIP6 (SLC39A6) and LIV-1 interchangeably in reference to the specific transporter. Likewise, it has been proposed that zinc transporters such as SLC39A6 may be mediators of key intracellular growth regulating signaling pathways. There was no clear delineation of the molecular mechanism underlying SLC39A6 function in the development and prognosis of ESCC. In this proposal, based on our previous findings, we plan to conduct a series of biochemical assays to investigate the effects of the associated SNPs on SLC39A6 gene function such as its expression. We also want to investigate the effects of SLC39A6 on phenotypes of ESCC, such as proliferation, migration, metastasis and epithelial-to-mesenchymal transition (EMT) in vitro and in vivo in animals. The aim of this study is to clarify the mechanism of genetic variations of SLC39A6 impacted on the gene expression and association with survival of ESCC patients, which is believe to be important in better understanding the progression of ESCC and to have potential utility in personalized clinical care of the cancer.

食管癌是我国特色肿瘤，尽管目前有外科手术和放化疗，但其预后仍令人沮丧。食管癌患者的预后存在巨大个体差异，已知患者的年龄、性别以及一些临床特征如肿瘤分期、淋巴结转移等影响患者生存，但不能解释巨大的异质性。目前临床上鲜有可判断患者预后的分子标志，以致难以实现理想的个体化治疗。我们前期通过大样本食管癌病例进行全基因组关联分析，发现SLC39A6基因rs7242481 SNP是食管癌患者生存差异的重要因素。该SNP位于基因5'调控区，可能影响转录和表达。初步实验还表明SLC39A6在大多数食管癌中高表达。但该基因对食管癌预后的作用机制尚不清楚。本项目在这些前期工作基础上，研究该SNP对基因表达的影响，并着重研究SLC39A6对食管癌细胞恶性表型包括增殖、迁移、转移以及上皮-间质细胞转化等作用，以揭示其对食管癌预后的作用机制，为食管癌个体化治疗提供新思路。

食管癌是最常见的消化道恶性肿瘤之一，研究表明遗传因素与肿瘤患者的预后密切相关。我们前期通过全基因组关联研究和独立样本验证，发现位于染色体18q12上SLC39A6基因的遗传变异与食管癌患者生存相关，位于5'UTR的变异（rs7242481 G>A）破坏了转录抑制因子结合位点，导致SLC39A6基因的异常表达。我们采取细胞体外实验和动物体内实验系统探究了SLC39A6的生物学功能。通过慢病毒感染的方法在食管癌细胞KYSE30 和 KYSE450及HeLa细胞构建了SLC39A6稳定过表达与敲降细胞系。敲降SLC39A6抑制食管鳞癌细胞的增殖、迁移和侵袭能力，而过表达SLC39A6促进食管癌细胞的增殖、迁移和侵袭能力。提示SLC39A6对食管癌细胞体内增殖是必需的，SLC39A6可能作为一个癌基因发挥重要作用。利用本实验室2010年到2014年收集的94对食管癌和癌旁组织、TCGA和SurvExpress数据库进行临床相关性分析，发现SLC39A6在淋巴结转移阳性的食管癌中高表达，并且与食管癌、肾癌、结肠癌、肺癌和乳腺癌等患者生存相关，提示SLC39A6可作为预后的分子标志物。.运用锌离子特异性荧光探针Zinpyr-1首次证实，SLC39A6表达与胞内锌离子浓度呈正相关，提示SLC39A6参与调控胞内锌离子稳态。当向培养基中添加锌离子螯合剂TPEN降低胞内锌离子浓度时，食管癌细胞的迁移和侵袭能力显著降低；而向培养基中添加ZnCl2提高胞内锌离子浓度时，显著促进食管癌细胞的迁移和侵袭能力。蛋白免疫印迹实验发现，当胞内锌离子浓度降低时，磷酸化的Akt（Ser308）及ERK1/2含量降低，同时信号转导通路下游与细胞恶性表型相关的靶蛋白MMP1、MMP3、MYC和SLUG表达量也降低；而胞内锌离子浓度升高时，则活化相应信号转导通路。锌离子通过活化PI3K/Akt和MAPK/ERK信号转导通路促进食管癌细胞的迁移和侵袭。我们还发现在食管鳞癌细胞系及食管鳞癌组织中SLC39A6与MMP1、MMP3、MYC和SLUG表达呈正相关关系。SLC39A6在食管癌组织中表达升高，导致胞内锌离子累积，从而激活PI3K/Akt和MAPK/ERK信号转导通路，促进细胞增殖、侵袭和转移。从生物学功能和作用机制上进一步证明SLC39A6基因在食管癌发展中起重要作用，可作为食管癌治疗靶点。