Background The distant metastasis of cancer cells is a risk factor for tumor lethality and poor prognosis in non-small-cell lung carcinoma (NSCLC). Increased SOX9 expression has been associated with clinical stage and poor prognosis in NSCLC, but the molecular mechanisms by which SOX9 promotes metastasis in NSCLC are still unknown. Methods The relationship between SOX9 expression and T, N, M classification was assessed using the χ 2 test and Spearman’s analysis in 142 immunohistochemically diagnosed specimens of NSCLC. We also generated SOX9-overexpression and SOX9-knockdown cells lines and their corresponding control cell lines by transfection with lentiviral constructs. In vivo assay, SOX9-overexpressing and SOX9-knockdown NSCLC cells were injected in zebrafish to examine distance metastasis. Gene set enrichment analysis (GSEA) was applied to analysis the correlation between SOX9 overexpression and Wnt/β-catenin pathway. Luciferase assay was used to check transcriptional activity of TCF/LEF and western blot and immunofluorescence was employed to detect β-catenin translocation in SOX9-overexpression, SOX9-knockdown and their corresponding control cell lines. Results We found that SOX9 overexpression correlates with the T, N and M stage significantly ( p = 0.03, 0.000, and 0.032 respectively) in 142 immunohistochemically diagnosed specimens of NSCLC. SOX9 overexpression was found to decrease the expression of the epithelial cell markers E-cadherin and γ-catenin and increase the expression of the mesenchymal cell markers N-cadherin and vimentin. An in vivo assay showed distant metastasis of the SOX9-overexpressing cells, which was not observed in the SOX9-knockdown cells. These findings indicate that SOX9 promotes distant metastasis by promoting EMT in NSCLC cells. GSEA showed that SOX9 overexpression was significantly correlated with the Wnt/β-catenin pathway which was corroborated by the expression of EMT-associated proteins in this pathway and its downstream target genes. SOX9 overexpression was also found to enhance the transcriptional activity of TCF/LEF, promote the nuclear translocation of β-catenin and increase the phosphorylation of GSK3β at Ser9. Further, inhibition of β-catenin suppressed the metastasis-promoting effects of SOX9 overexpression. Conclusions This study is the first to report that SOX9 is associated with clinical TNM stage and indicates that SOX9 promotes migration, invasion and the EMT process through the Wnt/β-catenin pathway.
BackgroundSex determining region Y (SRY)-related high mobility groupbox 9 (SOX9) is an important transcription factor required for development, which regulates the expression of target genes in the associated pathway. The aim of this study was to describe the expression of SOX9 in human non-small cell lung cancer (NSCLC) and to investigate the association between SOX9 expression and progression of NSCLC.MethodsSOX9 protein and mRNA expression in normal human pneumonocytes, lung cancer cell lines, and eight pairs of matched lung cancer tissues and their adjacent normal lung tissues were detected by Western blotting and real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry was used to determine SOX9 protein expression in 142 cases of histologically characterized NSCLC. Statistical analyses were applied to test for prognostic and diagnostic associations.ResultsSOX9 in lung cancer cell lines was upregulated at both mRNA and protein levels, and SOX9 mRNA and protein were also elevated in NSCLC tissues compared with levels in corresponding adjacent non-cancerous lung tissues. Immunohistochemical analysis demonstrated a high expression of SOX9 in 74/142 (52.1%) paraffin-embedded archival lung cancer biopsies. Statistical analysis indicated that upregulation of SOX9 was significantly correlated with the histological stage of NSCLC (P = 0.017) and that patients with a high SOX9 level exhibited a shorter survival time (P < 0.001). Multivariate analysis illustrated that SOX9 upregulation might be an independent prognostic indicator for the survival of patients with NSCLC.ConclusionsThis work shows that SOX9 may serve as a novel and prognostic marker for NSCLC, and play a role during the development and progression of the disease.
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