FoxM1 Promotes Epithelial-mesenchymal Transition, Cell Invasion And Migration Of Tongue Squamous Cell Carcinoma Via A C-Met-dependent Positive Feedback Loop

Oral cancer is one of the most common cancer worldwide, and tongue squamous cell carcinoma (TSCC) is the most common pathological type of oral cancer. The incidence of cervical lymph node metastasis is quite high during the early period of TSCC. Invasion and migration are major causes of poor prognosis in TSCC. Recently, more and more researches have found that epithelial-mesenchymal transition (EMT) plays an important role in tumour migration, invasion and metastasis. As a typical proliferation-associated transcription factor, FoxMl mainly exerts its function in tumorigenesis through transcriptional regulation of its target genes to initiate various cellular responses. FoxMl is frequently overexpressed in many human cancers, and its expression is associated with poor cancer outcomes. Several studies have shown that FoxMl plays an important role in the EMT, invasion and migrationof cancer cells. However, the molecular mechanism of EMT, invasion and migration induced by FoxM1 in TSCC are not fully understood.In this study, our results that FoxMl lies both downstream and upstream of the c-Met signaling pathway strongly suggest that FoxMl overexpression accounts for a novel mechanism of consecutive c-Met/AKT activation, which may be a critical mechanism for the EMT, invasion and migration of TSCC cells.We firstly investigated the expression of FoxMl, c-Met and pAKT in a total of 58 pairs of TSCC specimens and adjacent non-cancerous specimens by immunohistochemical staining. The expressions of FoxMl, c-Met and pAKT were confirmed to be higher in TSCC specimens than in adjacent non-cancerous specimens. Spearman rank correlation analysis showed significant positive correlations between FoxMl and c-Met protein levels, FoxMl and pAKT protein levels, c-Met and pAKT protein levels. Moreover, the expression level of FoxM1, c-Met and pAKT in TSCC patients were correlated with T staging, TNM staging and lymph node metastasis.Secondly, to determine whether FoxM1 is a key mediator of the c-Met signaling pathway, negative control shRNA (shNC) and FoxM1 shRNA (shFOXM1) were transfected into SCC9 and SCC25 cells. The western blot assays showed that the protein levels of FoxM1, pc-Met, c-Met, pAKT and Vimentin were significantly decreased, but E-cadherin increased by FoxM1 knockdown in SCC9 and SCC25 cells. In line with our above results, quantitative real-time PCR analyses showed that FoxM1 and c-Met mRNA levels were significantly decreased by FoxM1 knockdown in SCC9 and SCC25 cells. The transwell migration and invasion assays showed that knockdown of FoxM1 significantly inhibited the migration and invasion of TSCC cells. Futhermore, the western blot assays showed that the protein levels of FoxM1, pc-Met, c-Met, pAKT and Vimentin were significantly increased, but E-cadherin decreased by FoxM1 overexpression in SCC9 and SCC25 cells, and this effect was reversed byLY294002 treatment. In line with our above results, quantitative real-time PCR analyses showed that FoxM1 and c-Met mRNA levels were significantly increased by FoxM1 overexpression in SCC9 and SCC25 cells. The transwell migration and invasion assays showed that FoxM1 overexpression significantly promoted the migration and invasion of TSCC cells, and this effect was reversed byLY294002 treatment. To explore whether FoxMl directly regulates c-Met, we first performed ChIP assays in SCC9 and SCC25 cells. The results suggested that c-Met chromatins were specifically immunoprecipitated with antibody against FoxM1, compared with the IgG control. The luciferase reporter gene assay showed that FoxM1 regulated c-Met expression at the transcriptional level.Thirdly, to further reveal the crosstalk between FoxMl and the c-Met pathway, we examined the effect of the c-Met signaling pathway on FoxM1. The western blot assays showed that the protein levels of FoxM1, pc-Met, c-Met, pAKT and Vimentin were significantly decreased, but E-cadherin increased by c-Met knockdown in SCC9 and SCC25 cells. In line with our above results, quantitative real-time PCR analyses showed that FoxMl and c-Met mRNA levels were significantly decreased by c-Met knockdown in SCC9 and SCC25 cells. The transwell migration and invasion assays showed that knockdown of c-Met significantly inhibited the migration and invasion of TSCC cells. Futhermore, the western blot assays showed that the protein levels of FoxM1, pc-Met, c-Met, pAKT and Vimentin were significantly increased, but E-cadherin decreased by c-Met overexpression in SCC9 and SCC25 cells, and this effect was reversed byLY294002 treatment. In line with our above results, quantitative real-time PCR analyses showed that FoxM1 and c-Met mRNA levels were significantly increased by c-Met overexpression in SCC9 and SCC25 cells. The transwell migration and invasion assays showed that c-Met overexpression significantly promoted the migration and invasion of TSCC cells, and this effect was reversed byLY294002 treatment.Taken together, FoxMl bound directly to the c-Met promoter regions and regulated the promoter activities and the expression of c-Met at the transcriptional level. We further demonstrate that the expression levels of FoxMl, pAKT and c-Met are significantly increased in TSCC tissues relative to normal tissues, and these three biomarkerswere concomitantly overexpressed in TSCC tissues. Importantly, our results indicate that FoxMlpromotes EMT, invasion and metastasis of TSCC cells, and FoxM1 crosstalks with c-Met/AKT signalingto form a positive feedback loop to promote TSCCdevelopment.