FoxM1 promotes epithelial-mesenchymal transition, invasion, and migration of tongue squamous cell carcinoma cells through a c-Met/AKT-dependent positive feedback loop

Abstract

Forkhead box protein M1 (FoxM1) has been associated with cancer progression and metastasis. However, the function of FoxM1 in tongue squamous cell carcinoma (TSCC) remains largely unknown. The purpose of this study was to determine the role of FoxM1 in regulation of epithelial-mesenchymal transition (EMT) and migration of TSCC cells. We found that FoxM1 induced EMT and increased invasion/migration capacity in SCC9 and SCC25 cells. FoxM1 stimulation increased c-Met, pAKT, and vimentin levels but decreased E-cadherin level. Chromatin immunoprecipitation assay established that FoxM1 is bound to the promoter of c-Met to activate its transcription. In turn, c-Met promoted the expression of FoxM1 and pAKT. Blocking AKT signaling attenuated the invasion and migration of SCC9 and SCC25 cells stimulated by FoxM1 or c-Met. These results indicate that a positive feedback loop controls the EMT and migration of TSCC cells induced by FoxM1 and c-Met through AKT. Furthermore, the expression levels of FoxM1, pAKT, and c-Met were found to significantly increase in TSCC tissues compared with normal tissues, and these three biomarkers were concomitantly expressed in TSCC tissues. Clinical association analyses indicated that the expression of FoxM1, c-Met, and pAKT was associated with clinicopathological characteristics of patients with TSCC including tumor stage, tumor size, and lymph node metastasis. Taken together, our findings suggest that FoxM1 promotes the EMT, invasion and migration of TSCC cells, and cross-talks with c-Met/AKT signaling to form a positive feedback loop to promote TSCC development.

Fig. 1

The effects of FoxM1 knockdown on the expression of pc-Met, c-Met, pAKT, AKT, E-cadherin, and vimentin and the abilities of migration and invasion of tongue squamous cell carcinoma cells. (a) SCC9 and SCC25 cells were transfected with FoxM1 shRNA or shNC, and the protein levels of FoxM1, pc-Met, c-Met, pAKT and AKT, E-cadherin, and vimentin were analyzed by western blot analysis. (b) The mRNA levels of FoxM1 and c-Met were analyzed by quantitative real-time PCR analysis. (c, d) The effects of FoxM1 knockdown on the abilities of migration and invasion of SCC9 and SCC25 cells were measured by transwell assay (**P<0.01, ***P<0.001).

Fig. 2

The effects of FoxM1 overexpression and LY294002 on the expression of pc-Met, c-Met, pAKT, AKT, E-cadherin, and vimentin and the abilities of migration and invasion of tongue squamous cell carcinoma cells. (a) SCC9-FoxM1 and SCC25-FoxM1 cells were treated with LY294002 for 12 h, and the protein levels of FoxM1, pc-Met, c-Met, pAKT and AKT, E-cadherin, and vimentin were analyzed by western blot analysis. (b) The mRNA levels of FoxM1 and c-Met were analyzed by quantitative real-time PCR analysis. (c, d) The effects of FoxM1 overexpression and LY294002 on the abilities of migration and invasion of SCC9 and SCC25 cells were measured by transwell assay (*P<0.05, **P<0.01, ***P<0.001).

Fig. 3

The effects of c-Met knockdown on the expression of FoxM1, pc-Met, pAKT, AKT, E-cadherin, and vimentin and the abilities of migration and invasion of tongue squamous cell carcinoma cells. (a) SCC9 and SCC25 cells were transfected with c-Met shRNA or shNC, and the protein levels of FoxM1, pc-Met, c-Met, pAKT and AKT, E-cadherin, and vimentin were analyzed by western blot analysis. (b) The mRNA levels of FoxM1 and c-Met were analyzed by quantitative real-time PCR analysis. (c, d) The effects of c-Met knockdown on the abilities of migration and invasion of SCC9 and SCC25 cells were measured by transwell assay (**P<0.01, ***P<0.001).

Fig. 4

The effects of c-Met overexpression and LY294002 on the expression of FoxM1, pc-Met, pAKT, AKT, E-cadherin, and vimentin and the abilities of migration and invasion of tongue squamous cell carcinoma cells. (a) SCC9-c-Met and SCC25-c-Met cells were treated with LY294002 for 12 h, and the protein levels of FoxM1, pc-Met, c-Met, pAKT and AKT, E-cadherin, and vimentin were analyzed by western blot analysis. (b) The mRNA levels of FoxM1 and c-Met were analyzed by quantitative real-time PCR analysis. (c, d) The effects of c-Met overexpression and LY294002 on the abilities of migration and invasion of SCC9 and SCC25 cells were measured by transwell assay (*P<0.05,**P<0.01, ***P<0.001).

Fig. 5

FoxM1 binds to human c-Met promoter and directly enhances its transcription. (a) A putative FoxM1-binding site in the c-Met promoter and construction of reporter plasmids. (b) Chromatin immunoprecipitation analysis of the c-Met promoter using antibodies against FoxM1 in SCC9 and SCC25 cells. (c) The promoter activity of two truncated constructs was measured in SCC9 and SCC25 cells when cotransfected with the control plasmid or FoxM1 shRNA plasmid. (d) The transcriptional activity of FoxM1 on c-Met-luc wild-type (WT) or mutants (MT) was analyzed by luciferase reporter assay in SCC9 and SCC25 cells. (e) The promoter activity of two truncated constructs was measured in SCC9 and SCC25 cells when cotransfected with the control plasmid or pcDNA3.1-FoxM1 plasmid. Promoter activity was examined using a dual luciferase assay kit. The data represent three independent experiments; each bar represents mean±SD. P values were calculated using Student’s t-test (*P<0.05, **P<0.01, ***P<0.001).

Fig. 6

The coordinate expression of FoxM1, c-Met, and pAKT in tongue squamous cell carcinoma tissues. (a) Representative immunohistochemical staining images of FoxM1, c-Met, and pAKT by using consecutive tissue sections from the same patient with tongue squamous cell carcinoma (scale bars, 100 μm). (b) The relationship between the expression of FoxM1, c-Met, and pAKT was analyzed based on immunohistochemical staining. Note that some of the dots on the graphs represent more than one specimen.

Fig. 7

Correlation of the expression of FoxM1, c-Met, and pAKT with clinicopathological characteristics of tongue squamous cell carcinoma. The expression of FoxM1 (a), c-Met (b), and pAKT (c) was positively correlated with disease stage, tumor size, and lymph node metastasis (*P<0.05, **P<0.01, ***P<0.001). (TNM staging T: size and/or wedge of primary tumor; T1: ≤2 cm; T2: >3 to ≤4 cm; T3: >4 cm; T4: locally invasive tumor, N: regional lymph node; N0: no involvement; N1: a single lateral lymph node metastasis, diameter≤3 cm; N2: lymph node metastasis, diameter≤6 cm; N3: lymph node metastasis, diameter≥6 cm, M: metastasis. I: T1N0M0; II: T2N0M0; III: T3N0M0 or T1/T2/T3N1M0; IV: T4N0/N1M0 or TxN3M0 or TxNxM1).