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.

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.

Cheng X ，Wei L ，Huang X ，Zheng J ，Shao M ，Feng T ，Li J ，Han Y ，Tan W ，Tan W ，Lin D ，Wu C ... -  《-》

BRCA1-Associated Protein Increases Invasiveness of Esophageal Squamous Cell Carcinoma.

We performed a screen for genes whose expression correlates with invasiveness of esophageal squamous cell carcinoma (ESCC) cells. We studied the effects of overexpression and knockdown of these genes in cell lines and expression levels in patient samples. We selected genes for analysis from 11 loci associated with risk of ESCC. We analyzed the effects of knocking down expression of 47 of these genes using RNA interference on-chip analysis in ESCC cells and HeLa cells. Cells with gene overexpression and knockdown were analyzed in migration and invasion assays or injected into nude mice and metastasis of xenograft tumors was quantified. We collected ESCC and non-tumor esophageal tissues from 94 individuals who underwent surgery in China from 2010 and 2014; clinical information was collected and survival time was measured from the date of diagnosis to the date of last follow-up or death. Levels of messenger RNAs (mRNAs) were quantified by RNA sequencing, and levels of proteins were determined from immunoblot analyses. Patient survival was compared with mRNA levels using Kaplan-Meier methods and hazard ratios were calculated by Cox models. We identified 8 genes whose disruption increased migration and 10 genes whose disruption reduced migration. Knockdown of BRCA1-associated protein gene (BRAP) significantly reduced migration of KYSE30, KYSE150, and HeLa cells. In patient tumors, 90% of ESCCs examined had higher levels of BRAP protein than paired non-tumor tissues, and 63.8% had gains in BRAP DNA copy number. Levels of BRAP mRNA in ESCC tissues correlated with patient survival time, and high expression increased risk of death 2.4-fold compared with low expression. ESCCs that had metastasized to lymph node had significantly higher levels of BRAP mRNA than tumors without metastases. Knockdown of BRAP in ESCC and HeLa cell lines significantly reduced migration and invasiveness; these cell lines formed less metastases in mice than control cells. Nuclear translocation of the nuclear factor-κB (NF-κB) P65 subunit and phosphorylation of inhibitor of NF-κB kinase subunit β (IKBKB or IKKβ) increased in cells that overexpressed BRAP and decreased in cells with BRAP knockdown. In immunoprecipitation assays, BRAP interacted directly with IKKβ. Expression of matrix metalloproteinase 9 and vascular epithelial growth factor C, which are regulated by NF-κB, was significantly reduced in cells with knockdown of BRAP and significantly increased in cells that overexpressed BRAP. Expression of BRAP is increased in ESCC samples compared with non-tumor esophageal tissues; increased expression correlates with reduced patient survival time and promotes metastasis of xenograft tumors in mice. BRAP overexpression leads to increased activity of NF-κB and expression of matrix metalloproteinase 9 and vascular epithelial growth factor C.

Zhao Y ，Wei L ，Shao M ，Huang X ，Chang J ，Zheng J ，Chu J ，Cui Q ，Peng L ，Luo Y ，Tan W ，Tan W ，Lin D ，Wu C ... -  《-》

Nuclear Localization of DNAJB6 Is Associated With Survival of Patients With Esophageal Cancer and Reduces AKT Signaling and Proliferation of Cancer Cells.

The DnaJ (Hsp40) homolog, subfamily B, member 6 (DNAJB6) is part of a family of proteins that regulates chaperone activities. One of its isoforms, DNAJB6a, contains a nuclear localization signal and regulates β-catenin signaling during breast cancer development. We investigated the role of DNAJB6 in the pathogenesis of esophageal squamous cell carcinoma (ESCC). We performed immunohistochemical analyses of primary ESCC samples and lymph node metastases from a cohort of 160 patients who underwent esophagectomy with no preoperative chemoradiotherapy at Hong Kong Queen Mary Hospital. Data were collected on patient outcomes over a median time of 12.1 ± 2.9 months. Retrospective survival association analyses were performed. Wild-type and mutant forms of DNAJB6a were overexpressed in cancer cell lines (KYSE510, KYSE 30TSI, KYSE140, and KYSE70TS), which were analyzed in proliferation and immunoblot assays, or injected subcutaneously into nude mice. Levels of DNAJB6 were knocked down in ESCC cell lines (KYSE450 and T.Tn), immortalized normal esophageal epithelial cell lines (NE3 and NE083), and other cells with short hairpin RNAs, or by genome engineering. Bimolecular fluorescence complementation was used to study interactions between proteins in living cells. In primary ESCC samples, patients whose tumors had high nuclear levels of DNAJB6 had longer overall survival times (19.2 ± 1.8 months; 95% confidence interval [CI], 15.6-22.8 mo) than patients whose tumors had low nuclear levels of DNAJB6 (12.6 ± 1.4 mo; 95% CI, 9.8-15.4 mo; P = .004, log-rank test). Based on Cox regression analysis, patients whose tumors had high nuclear levels of DNAJB6 had a lower risk of death than patients with low levels (hazard ratio, 0.562; 95% CI, 0.379-0.834; P = .004). Based on log-rank analysis and Cox regression analysis, the combination of the nuclear level of DNAJB6 and the presence of lymph node metastases at diagnosis could be used to stratify patients into groups with good or bad outcomes (P < .0005 for both analyses). There was a negative association between the nuclear level of DNAJB6 and the presence of lymph node metastases (P = .022; Pearson χ(2) test). Cancer cell lines that overexpressed DNAJB6a formed tumors more slowly in nude mice than control cells or cells that expressed a mutant form of DNAJB6a that did not localize to the nucleus. DNAJB6 knockdown in cancer cell lines promoted their growth as xenograft tumors in mice. A motif of histidine, proline, and aspartic acid in the J domain of DNAJB6a was required for its tumor-suppressive effects and signaling via AKT1. Loss of DNAJB6a resulted in up-regulation of AKT signaling in cancer cell lines and immortalized esophageal epithelial cells. Expression of a constitutively active form of AKT1 restored proliferation to tumor cells that overexpressed DNAJB6a, and DNAJB6a formed a complex with AKT1 in living cells. The expression of DNAJB6a reduced the sensitivity of ESCC to AKT inhibitors; the expression level of DNAJB6a affected AKT signaling in multiple cancer cell lines. Nuclear localization of DNAJB6 is associated with longer survival times of patients with ESCC. DNAJB6a reduces AKT signaling, and DNAJB6 expression in cancer cells reduces their proliferation and growth of xenograft tumors in mice. DNAJB6a might be developed as a biomarker for progression of ESCC.

Yu VZ ，Wong VC ，Dai W ，Ko JM ，Lam AK ，Chan KW ，Samant RS ，Lung HL ，Shuen WH ，Law S ，Chan YP ，Lee NP ，Tong DK ，Law TT ，Lee VH ，Lung ML ... -  《-》

MMP1 promotes tumor growth and metastasis in esophageal squamous cell carcinoma.

Matrix metalloproteinases play an essential role in the progression of esophageal squamous cell carcinoma (ESCC). Here, we show that MMP1 expression was markedly increased in a majority of ESCC compared with nontumorous tissue. High expressions of MMP1 were closely associated with lymph node metastasis, microvessel density and advanced TNM stage. Kaplan-Meier and multivariate analyses indicated MMP1 as an independent factor for overall survival in two independent cohorts of 613 patients with ESCC. In vitro studies demonstrated that MMP1 overexpression resulted in enhanced cell viability, abilities of colony formation and cell migration. The knockdown of MMP1 in ESCC cells resulted in the opposite phenomenon. Consistently, in vivo data showed that ectopic expression of MMP1 promoted tumor growth and metastasis. Further study revealed that MMP1 facilitated ESCC through the activation of the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway by LY294002 significantly attenuated MMP1-mediated cell proliferation and migration. Taken together, our data suggest that MMP1 functions as an oncogene and serves as a prognostic biomarker and a potential therapeutic target in ESCC.

Liu M ，Hu Y ，Zhang MF ，Luo KJ ，Xie XY ，Wen J ，Fu JH ，Yang H ... -  《-》

Identification of PTK6, via RNA sequencing analysis, as a suppressor of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most commonly observed histologic subtype of esophageal cancer. ESCC is believed to develop via accumulation of numerous genetic alterations, including inactivation of tumor suppressor genes and activation of oncogenes. We searched for transcripts that were altered in human ESCC samples compared with nontumor tissues. We performed integrative transcriptome sequencing (RNA-Seq) analysis using ESCC samples from 3 patients and adjacent nontumor tissues to identify transcripts that were altered in ESCC tissue. We performed molecular and functional studies of the transcripts identified and investigated the mechanisms of alteration. We identified protein tyrosine kinase 6 (PTK6) as a transcript that was significantly down-regulated in ESCC tissues and cell lines compared with nontumor tissues or immortalized normal esophageal cell lines. The promoter of the PTK6 gene was inactivated in ESCC tissues at least in part via hypermethylation and histone deacetylation. Knockdown of PTK6 in KYSE30 ESCC cells using small hairpin RNAs increased their ability to form foci, migrate, and invade extracellular matrix in culture and form tumors in nude mice. Overexpression of PTK6 in these cells reduced their proliferation in culture and tumor formation in mice. PTK6 reduced phosphorylation of Akt and glycogen synthase kinase (GSK)3β, leading to activation of β-catenin. PTK6 was identified as a transcript that is down-regulated in human ESCC tissues via epigenetic modification at the PTK6 locus. Its product appears to regulate cell proliferation by reducing phosphorylation of Akt and GSK3β, leading to activation of β-catenin. Reduced levels of PTK6 promote growth of xenograft tumors in mice; it might be developed as a marker of ESCC.

Ma S ，Bao JYJ ，Kwan PS ，Chan YP ，Tong CM ，Fu L ，Zhang N ，Tong AHY ，Qin YR ，Tsao SW ，Chan KW ，Lok S ，Guan XY ... -  《-》