Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, China.
Gastroenterology. 2017 Jun;152(8):1985-1997.e12. doi: 10.1053/j.gastro.2017.02.006. Epub 2017 Feb 14.
BACKGROUND & AIMS: A common variant in the solute carrier family 39 member 6 gene (SLC39A6) has been associated with survival times of patients with esophageal squamous cell carcinoma (ESCC). We investigated the function of SLC39A6 and ways in which this variant affects tumor progression by studying ESCC samples and cell lines.
SLC39A6 was expressed or knocked down by expression of short hairpin RNAs in ESCC cells (KYSE30 and KYSE450) and HeLa cells using lentiviral vectors; we analyzed effects on proliferation, colony formation, migration, and invasion in vitro. Cells were grown as xenograft tumors in nude mice and tumor volume and metastases were quantified; tumors were collected and analyzed histologically. Cells were also analyzed for levels of intracellular zinc and messenger RNA (mRNA) expression patterns. We obtained ESCC and adjacent normal esophageal tissues from 94 patients who underwent esophagectomy in China from 2010 through 2014. Survival times of patients were measured from the date of diagnosis to the date of last follow-up or death. We sequenced mRNAs and compared levels between tumor and non-tumor tissues using the Wilcox rank-sum test. Total proteins in cell lines or tissue samples were measured by immunoblotting. We searched publicly available databases for variants of SLC39A6 in human tumor and non-tumor tissues.
Knockdown of SLC39A6 reduced proliferation of ESCC cells in culture and metastasis of xenograft tumors in mice. Cells that overexpressed SLC39A6 had significant increases in intracellular levels of zinc and were more invasive in assays, activating phosphatidylinositol 3-kinase signaling to AKT serine/threonine kinase 1 and mitogen-activated protein kinase 1. Cells that overexpressed SLC39A6 had increased expression of mRNAs and proteins associated with metastasis, such as matrix metalloproteinase (MMP) 1, MMP3, MYC, and snail family transcriptional repressor 2 (SNAI2 or SLUG). Levels of MMP1, MMP3, MYC, and SLUG mRNAs correlated with levels of SLC39A6 mRNA in ESCC samples from patients. ESCC tissues had increased levels of SLC39A6 mRNA compared with non-tumor tissues; the increase correlated with tumor metastasis to lymph node and reduced patient survival time.
In an analysis of ESCC samples and cell lines, we associated increased expression of SLC39A6 with tumor invasiveness, intracellular level of zinc, and patient survival time. ESCC cell lines that overexpress SLC39A6 up-regulate expression MMP1, MMP3, MYC, and SLUG and form metastatic xenograft tumors in mice. Up-regulation of SLC39A6 might be used to determine prognoses of patients with ESCC or as a therapeutic target.
溶质载体家族 39 成员 6 基因（SLC39A6）的常见变体与食管鳞状细胞癌（ESCC）患者的存活时间有关。我们通过研究 ESCC 样本和细胞系，研究了 SLC39A6 的功能以及该变体如何影响肿瘤进展。
通过慢病毒载体在 ESCC 细胞（KYSE30 和 KYSE450）和 HeLa 细胞中表达或敲低 SLC39A6 的短发夹 RNA，分析对体外增殖、集落形成、迁移和侵袭的影响。将细胞作为异种移植肿瘤在裸鼠中生长，并定量肿瘤体积和转移；收集并分析肿瘤的组织学。还分析了细胞内锌的水平和信使 RNA（mRNA）表达模式。我们从 2010 年至 2014 年在中国接受食管切除术的 94 名患者中获得 ESCC 和相邻正常食管组织。从诊断日期到最后随访或死亡日期测量患者的存活时间。我们对来自肿瘤和非肿瘤组织的 mRNA 进行测序，并使用 Wilcox 秩和检验比较水平。通过免疫印迹法测量细胞系或组织样本中的总蛋白。我们在公共数据库中搜索 SLC39A6 在人类肿瘤和非肿瘤组织中的变体。
SLC39A6 敲低可降低 ESCC 细胞在培养中的增殖和异种移植肿瘤在小鼠中的转移。过表达 SLC39A6 的细胞细胞内锌水平显著增加，在侵袭性测定中更具侵袭性，激活磷脂酰肌醇 3-激酶信号转导至 AKT 丝氨酸/苏氨酸激酶 1 和丝裂原活化蛋白激酶 1。过表达 SLC39A6 的细胞表达与转移相关的 mRNA 和蛋白质增加，例如基质金属蛋白酶（MMP）1、MMP3、MYC 和蜗牛家族转录阻遏物 2（SNAI2 或 SLUG）。来自患者的 ESCC 样本中 MMP1、MMP3、MYC 和 SLUG mRNA 的水平与 SLC39A6 mRNA 的水平相关。ESCC 组织中的 SLC39A6 mRNA 水平高于非肿瘤组织；这种增加与淋巴结转移和患者生存时间缩短有关。
在对 ESCC 样本和细胞系的分析中，我们将 SLC39A6 的表达增加与肿瘤侵袭性、细胞内锌水平和患者生存时间联系起来。过表达 SLC39A6 的 ESCC 细胞系上调 MMP1、MMP3、MYC 和 SLUG 的表达，并在小鼠中形成转移性异种移植肿瘤。SLC39A6 的上调可用于确定 ESCC 患者的预后或作为治疗靶点。
