title: Solute Carrier Family 39 Member 6 Gene Promotes Aggressiveness of Esophageal Carcinoma Cells by Increasing Intracellular Levels of Zinc, Activating Phosphatidylinositol 3-Kinase Signaling, and Up-regulating Genes That Regulate Metastasis.
abstract: 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.
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Lixuan
authors.initials: L
authors.lastname: Wei
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Xudong
authors.initials: X
authors.lastname: Huang
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Shao
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Feng
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Yaling
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Wenle
authors.initials: W
authors.lastname: Tan
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: tanwen68@hotmail.com.
authors.forename: Wen
authors.initials: W
authors.lastname: Tan
authors.affiliation: 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; Collaborative Innovation Center for Cancer Medicine, Guangzhou, China; Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China. Electronic address: lindx@cicams.ac.cn.
authors.forename: Dongxin
authors.initials: D
authors.lastname: Lin
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: chenwu@cicams.ac.cn.
authors.forename: Chen
authors.initials: C
authors.lastname: Wu
mesh_terms: D000368:Aged; D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000201:enzymology* / Q000235:genetics / Q000401:mortality / Q000556:secondary; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement; D049109:Cell Proliferation; D030541:Databases, Genetic; D004938:Esophageal Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000401:mortality / Q000473:pathology; D000077277:Esophageal Squamous Cell Carcinoma; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D008207:Lymphatic Metastasis; D008297:Male; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction; D016019:Survival Analysis; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden; D015854:Up-Regulation; D015032:Zinc / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D027682:Cation Transport Proteins; D009363:Neoplasm Proteins; C418627:SLC39A6 protein, human; D058539:Phosphatidylinositol 3-Kinase; D015032:Zinc
keywords: AKT; ERK; Esophageal Cancer; PI3K