TGF-ß1 induces N-cadherin expression by upregulating Sox9 expression and promoting its nuclear translocation in human oral squamous cell carcinoma cells.

Squamous cell carcinoma (SCC) is the most frequent cancer that develops in the oral cavity. Epithelial-mesenchymal transition (EMT) is known to play an important role in the process of metastasis of SCC cells. In our previous study, we demonstrated that TGF-ß1 induced EMT in the human oral SCC (hOSCC) cell line HSC-4. We also found that Slug plays an important role in suppressing E-cadherin expression and promotion of the migratory activity of HSC-4 cells. However, we also demonstrated that Slug does not participate in upregulation of N-cadherin expression, suggesting that EMT-related transcription factors other than Slug also play an important role in the process. In the present study, we aimed to elucidate how the transcription factor Sox9 affects the TGF-ß1-induced upregulation of N-cadherin expression in HSC-4 cells. We found that TGF-ß1 upregulated Sox9 expression in HSC-4 cells. In addition, Sox9 siRNA significantly abrogated the TGF-ß1-induced upregulation of N-cadherin expression and inhibited the TGF-ß1-promoted migratory activity in HSC-4 cells. We also demonstrated that TGF-ß1 upregulated the phosphorylation status of Sox9 and then promoted nuclear translocation of Sox9 from the cytoplasm, possibly resulting in an increase in N-cadherin expression. The cyclic AMP-dependent protein kinase A inhibitor H-89, which is known to suppress phosphorylation of Sox9, significantly abrogated the TGF-ß1-induced upregulation of N-cadherin expression. These results suggested that TGF-ß1 induced N-cadherin expression by upregulating Sox9 expression and promoting its nuclear translocation, which results in EMT progression in hOSCC cells.

Transforming growth factor-ß1 induces invasion ability of HSC-4 human oral squamous cell carcinoma cells through the Slug/Wnt-5b/MMP-10 signalling axis.

Molecular mechanism underlying the invasion of oral cancer cells remains to be clarified. We previously demonstrated that transforming growth factor-ß1 (TGF-ß1) induces the expression of mesenchymal markers in human oral squamous cell carcinoma HSC-4 cells. Intriguingly, the expression of the epithelial-mesenchymal transition-related transcription factor Slug was also significantly upregulated upon TGF-ß1 stimulation. However, the mechanism by which Slug transduces the TGF-ß1-induced signal to enhance the invasiveness of HSC-4 cells is poorly understood. Proteomic analysis revealed that the expression of matrix metalloproteinase (MMP)-10 was upregulated in TGF-ß1-stimulated cells. Additionally, a Boyden chamber assay revealed that the TGF-ß1-induced increase in invasiveness of HSC-4 cells was significantly inhibited by MMP-10 small interfering RNA (siRNA). Intriguingly, Slug siRNA suppressed TGF-ß1-induced expression of MMP-10. These results suggest that TGF-ß1 induces invasion in HSC-4 cells through the upregulation of MMP-10 expression in a Slug-dependent manner. On the other hand, Slug siRNA suppressed TGF-ß1-induced Wnt-5b expression. Wnt-5b significantly induced MMP-10 expression, whereas Wnt-5b siRNA suppressed the TGF-ß1-induced increase in invasiveness, suggesting that TGF-ß1-induced expression of MMP-10 and the resulting upregulation of invasiveness are mediated by Wnt-5b. Overall, these results suggest that TGF-ß1 stimulates HSC-4 cell invasion through the Slug/Wnt-5b/MMP-10 signalling axis.

Transforming growth factor-ß1 induces epithelial-mesenchymal transition and integrin α3ß1-mediated cell migration of HSC-4 human squamous cell carcinoma cells through Slug.

We investigated whether transforming growth factor (TGF)-ß1 promoted epithelial-mesenchymal transition (EMT) and migration of human oral squamous cell carcinoma (hOSCC) cells. Among 6 hOSCC cell lines investigated, Smad2 phosphorylation and TGF-ß target genes expression were most clearly upregulated following TGF-ß1 stimulation in HSC-4 cells, indicating that HSC-4 cells were the most responsive to TGF-ß1. In addition, the expression levels of the mesenchymal markers N-cadherin and vimentin were most clearly induced in HSC-4 cells among the hOSCC cell lines by TGF-ß1 stimulation. Interestingly, E-cadherin and ß-catenin at the cell surface were internalized in HSC-4 cells stimulated with TGF-ß1. In addition, the expression levels of the EMT-related transcription factor Slug was significantly upregulated on TGF-ß1 stimulation. Moreover, the downregulation of Slug by RNA interference clearly inhibited the TGF-ß1-induced expression of mesenchymal marker and the migration of HSC-4 cells. Proteomics analysis also revealed that the expression levels of integrin α3ß1-targeted proteins were upregulated in TGF-ß1-stimulated HSC-4 cells. Neutral antibodies against integrin α3 and ß1, as well as a focal adhesion kinase (FAK) inhibitor, clearly suppressed TGF-ß1-induced cell migration. These results suggest that the EMT and integrin α3ß1/FAK pathway-mediated migration of TGF-ß1-stimulated HSC-4 hOSCC cells is positively controlled by Slug.