SOX5 Predicts Poor Prognosis In Lung Adenocarcinoma And Promotes Tumor Metastasis Through Epithelial-mesenchymal Transition

Lung cancer has been the most prevalent tumor worldwide,with the first leading status of new cases and deaths among all cancers during 2017,according to the statistics from The American Cancer Society(ACS).In China,lung cancer increased rapidly both in new cases and death cases,which has become the leading cause of cancer-related death.The most common pathologic type among lung cancer is lung adenocarcinoma(LAC),accounting for about 44.21%.Despite the recent progress in comprehensive treatment,5-year survival rate of lung adenocarcinoma is still less than 15%.Tumor metastasis is the main cause of poor prognosis of lung adenocarcinoma.Therefore,it is important for us to find out an effective therapeutic target for LAC treatment.Epithelial to mesenchymal transition(EMT)is a distinguishing feature of tumor progress and metastasis in most cancers.EMT is a developmental process in which epithelial cells lose their epithelial features and develop mesenchymal phenotypes.In LAC,abnormal activation of EMT leads to tumor invasion,metastatic dissemination and acquisition of therapeutic resistance,which result in poor prognosis.EMT is characterized by less expression of epithelial markers(e.g.E-cadherin),up-regulation of mesenchymal markers(e.g.Vimentin)and smooth muscle actin(SMA),acquisition of fibroblast-like morphology with cytoskeleton reorganization,and increases in motility,invasiveness,and metastatic capabilities.Researching the metastatic mechanism of LAC is crucial for earlier detection and intervention of cancer by improving both life quality and prognosis of patients with LAC.Sex determining region Y-box protein 5(SOX5)expression is correlated with various cancers including prostate tumor,breast cancer,glioma,hepatocellular carcinoma and nasopharyngeal carcinoma.Tumors consist not only of malignant cancer cells,but also stromal cells that support the tumor microenvironment.These include fibroblasts and immune cells,as well as endothelial cells and smooth muscle cells that.form blood vessels and provide nourishment for the tumor cells.However,the role of SOX5 in lung adenocarcinoma metastasis and its molecular mechanism have not been reported.Our work found that the expression of SOX5 was significantly increased in lung adenocarcinoma patients,which suggests that the up-regulation of SOX5 may be closely related to the occurrence and metastasis of lung adenocarcinoma.SOX5 may serve as a biomarker to predict the metastatic capacity and prognosis of lung adenocarcinoma,and may be a potential target for the treatment of lung adenocarcinoma.We first analyzed SOX5 expression in four human lung adenocarcinoma(LAC)samples.Quantitative RT-PCR analysis showed that transcriptional expression of SOX5 is higher in tumors than the paired non-tumor controls.Western blot also showed that SOX5 is over-expressed in tumor tissues,whereas it was weakly expressed in respective adjacent mucosas.In vitro studies of LAC cell lines(H1299,95D,A549,H1975)and bronchial epithelium cell line(16HBE)revealed that SOX5 mRNA and protein levels are positively correlated with cells invasive capacity.To investigate the clinicopathological and prognostic significance of SOX5 expression in LAC patients,90 pairs of LAC tissues were examined by immunohistochemical staining.Data were analyzed by SPSS software.It showed that SOX5 expression in lung adenocarcinoma was closely associated with clinical stages(r=0.254,p<0.05),and that SOX5 expression in paracancerous tissues was correlated with tumor size(r=0.211,p<0.05).Kaplan Meier curve showed a negative correlation between high SOX5 expression level and overall survival(OS)in both LAC tissues and the paired adjacent tissues.Univariate and multivariate analyses showed that high SOX5 expression in adjacent non-tumor tissues was an independent prognostic factor for poor survival of LAC patients(p<0.05).All above showed that the expression of SOX5 affects the invasion and migration of LAC cells,leading to a poor prognosis of patients with LAC.Given that SOX5 was up-regulated in LAC patients’ tumoral tissues,we explored the function of SOX5 in LAC cell lines.Using lentivirus shRNA,we silenced SOX5 expression in NCI-H1299 and 95D cells.Western blot,Colony formation assay,CCK8 assay,wound healing assay and transwell assays showed that silencing SOX5 expression can inhibit LAC cell proliferation,migration and invasion ability.To further determine whether SOX5 affected metastasive capacity of LAC cells,we stably transfected lentivirus with an over-expression SOX5 gene into A549 and H1975 cells.Western blot for cell proliferation markers,colony formation assay,CCK8 assay,wound healing assay and cell invasion assay reconfirmed that SOX5 over-expression promoted LAC cells metastasis.We further analyzed SOX5 function in vivo using a zebrafish xenograft model.cell proliferation and metastasis capacity were examined in SOX5 silencing H1299 cells(shSOX5)or control cells(NC).The integrated fluorescence intensity(S)and Cumulative distance of cell migration(L)were calculated to measure tumor growth and metastasis,respectively.It showed the inhibitory rates of tumor growth in H1299 shSOX5-1 and H1299 shSOX5-2 were 36%(p<0.01)and 37%respectively(p<0.001)and the inhibitory rate of metastasis was 85%and 83%in the SOX5 silencing groups,respectively.All above suggests that SOX5 can promote the proliferation,migration and invasion of lung adenocarcinoma cells both in vitro and in vivo.To investigate the effect of SOX5 on the EMT process,the protein expression of mesenchymal phenotype cell biomarkers(ZEB1,Vimentin,N-cadherin,and Twistl)and epithelial phenotype cell biomarker(E-cadherin)were measured by western blot in SOX5-silenced cells,over-expression cells,and the corresponding control cells.SOX5 depletion attenuated the expression level of mesenchymal biomarkers including ZEB1,Vimentin,and N-cadherin,while epithelial marker E-cadherin was up-regulated.Over-expression of SOX5 was followed by increasing expression of Vimentin and twistl and decreasing expression of E-cadherin.Serial sections of tissues of three advanced LAC patients were stained with SOX5,E-cadherin and Vimentin.SOX5 expression in LAC is positively correlated with Vimentin expression and negatively correlated with E-cadherin expression.Therefore,SOX5 affects proliferation and metastasis of lung adenocarcinoma cells by regulating EMT,resulting in the poor prognosis of lung adenocarcinoma patients.SOX5 may become a new therapeutic target for lung adenocarcinoma.