| BackgroundCholangiocarcinoma (CCA) has one of the highest mortalities because of stronginvasiveness and early metastasization. Surgical resection provides the opportunity forlong-term survival. However, lymphnodal or distant metastasis or micrometastasis ispresent early in CCA patients, limiting the surgery option. Besides, despite comprehensivepreoperative staging to select patients for surgery, many patients present with recurrentdisease within two years after tumor resection, and their survival varies in a large extent.Therefore, investigating the molecular mechanisms of CCA invasion and metastasisremains a priority. Besides, identifying biomarkers, which have prognostic value for CCAsafter surgery, may help the patients to choose suitable follow-up interval and subsequenttherapy.GATA binding protein6(GATA6) is a member of an evolutionarily conserved familyof zinc-finger transcription factors which play important roles in development. Asmalignancy progression shares some common regulatory genes with development, GATAproteins are aberrantly expressed in tumors and play important roles during theirprogression. Recent studies have reported that overexpression of GATA6is involved in cellinvasion and tumor growth in colon cancer, pancreatic cancer and esophagealadenocarcinoma. Furthermore, GATA6expression correlated with poor survival inpancreatic cancer and esophageal adenocarcinoma. However, some other studies reportedthat loss of GATA6was involved in malignant transformation in ovarian cancer andastrocytoma. Therefore, GATA6functions both as a tumor promoter and suppressor. Up topresent, the expression pattern of GATA6and its clinical significance in CCA has not beenexplored. Besides, its role in CCA invasion and metastasis is unknown.GATA6regulates several tissue-specific genes in digestive epithelia by affecting the transcriptional activity through binding to the promoter region.67kDa laminin receptor(67LR) is a cell surface non-integrin receptor expressed in the epithelial cells mediating cellattachment to laminin. An increase of67LR expression has been found in a variety ofcancers and promotes cancer cell invasion and metastasis though several mechanisms.Therefore,67LR is considered a molecular marker of metastatic behavior. Our previousstudies have demonstrated67LR was overexpressed in CCA and promoted CCA cellinvasion and metastasis.67LR can be regulated both at the transcriptional andposttranscriptional levels. At the transcriptional level, it can be regulated by severaltranscription factors. We analyzed the67LR gene promoter region using GPMiner, andGATA6binding sites were predicted in this region, indicating that GATA6has thepossibility to bind to67LR gene promoter. Based on the observations, we hypothesized67LR might be a new GATA6target gene and GATA6might impact CCA invasion andmetastasis through regulating67LR expression.HypothesisGATA6might promote invasion and metastasis through regulating67LR expression incholangiocarcinoma.Materials and Methods1. A total of87CCA patients undergoing surgical resection between2005and2010, inSouthwest Hospital were included.87cancerous samples,24pericancerous samples,32lymph node metastatic samples and8liver metastatic samples from87CCAs werecollected. Expression of GATA6on paraffin clinical samples were investigated by IHC.Correlations between GATA6expression and clinical features were analyzed.2. Overall survival was calculated from the date of surgery until the date of last contact.Recurrence-free survival was defined at the time of surgery to tumor recurrence.Correlations between GATA6and overall survival and recurrence were analyzed by usingKaplan-Meier and Cox models.3. Two human CCA cell lines, QBC939and RBE, were cultured in present study. Cellswere transfected with lentiviral vectors encoding short hairpin RNA targeting humanGATA6for GATA6knockdown (shGATA6) or a scrambled shRNA as control (shControl). Cells were transfected with GATA6plasmids for GATA6overexpression (ExGATA6) orempty plasmid (ExControl). Impact of intervening GATA6expression on cell migration,invasion and metastasis was evaluated by cell migration (wound healing) assay,cellinvasion (transwell) assay and xenotransplantation of CCA cells in nude mice, respectively.4. Expression of67LR on paraffin clinical samples were investigated by IHC.Correlations between GATA6and67LR expression were analyzed. Impact of interveningGATA6expression on67LR expression was evaluated by real-time PCR, western blot, andflow cytometry. Chromatin Immunoprecipitation (ChIP) was performed to study whetherGATA6bound to the promoter region of67LR gene in CCA cells.Results1. By IHC,39of87CCA cancerous tissues (45%) were defined as GATA6positivestaining. Among positive samples,32(37%) were strong positive and7(8%) were weakpositive. However, all24matched paracancerous samples showed negative GATA6staining on the bile ducts. Expression of GATA6was significantly associated with lymphnode metastasis, but not with age, gender, tumor location, histological grade, Tclassification and distant metastasis.23of32lymph-node metastatic tissues (72%) showedGATA6positive staining, which was significantly higher than it in primary canceroustissues.5of8liver metastatic samples (63%) showed GATA6positive staining. Together,the data suggested that aberrant expression of GATA6was significantly associated with anenhanced metastatic behavior in CCAs.2. Kaplan-Meier analysis showed expression of GATA6significantly correlated with asignificant reduction of overall survival (log-rank p=0.002) and an increased risk ofrecurrence (log-rank p=0.002) in87CCAs. Multifactorial Cox analysis showed thatexpression of GATA6is an independent predictor for poor overall survival andrecurrence-free survival (HR=2.015,95%CI=1.188-3.418, p=0.01; HR=2.132,95%CI=1.271-3.576, p=0.004). Together, our data revealed GATA6expression was a riskfactor indicating poor prognosis in CCA patients following surgical resection.3. Both mRNA and nuclear protein levels of GATA6were significantly higher inQBC939cells than in RBE cells, therefore, knockdown of GATA6was performed in QBC939cells and overexpression of GATA6was performed in RBE cells, respectively. Bytranswell assay, the number of invading cells was remarkably reduced by knockdown ofGATA6in QBC939cells and was remarkably increased by overexpression of GATA6inRBE cells. Consistently, cell migration was remarkably reduced by GATA6knockdownand enhanced by GATA6overexpression by wound-healing assay. Then impact of GATA6on CCA cell metastasis was performed following xenotransplantation into nude mice byintrasplenic injection. The number of distant masses from spleen was significantly reducedby GATA6knockdown in QBC939cells one month after injection. Together, the datasuggested a promotive role of GATA6in CCA cell invasion and metastasis.4.35of87cancerous samples (40%) showed67LR positive staining. Expression ofGATA6significantly correlated with67LR expression in87CCAs. The mRNA and proteinlevels of67LR were significantly reduced by GATA6knockdown in QBC939cells andincreased by GATA6overexpression in RBE cells. Consistently, flow cytometry showedmembrane expression of67LR was reduced by GATA6knockdown and increased byGATA6overexpression in CCA cells. ChIP analysis showed that GATA6bound to67LRgene promoter region in QBC939and RBE cells. Together, our data suggested that GATA6regulated67LR expression by binding to its promoter region in CCA cells.ConclusionOur results suggested that aberrant expression of GATA6correlates with poorprognosis and promotes cell invasion and metastasis in CCA. In addition, it mightcontribute to CCA invasion and metastasis by upregulating67LR expression throughbinding to its promoter region. |
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