Activation Of PI3K/mTOR Pathway Regulates Epithelial Mesenchymal Transition In Squamous Cell Carcinoma Tongue

ObjectivesSquamous cell carcinoma (SCC) constitutes about90%of the different types of head and neck cancers and tongue squamous cell carcinoma (TSCC) is the highest incidence in head and neck squamous cell carcinoma. EMT occurs during development and cancer progression to metastasis and results in enhanced cell motility and invasion.1.To investigate the expression of Akt, mTOR and E-cadherin,(3-catenin, Vimentin in TSCC tissues.2.We examined the protein change of Akt, mTOR, the epithelial cell marker E-cadherin,(3-catenin and mesenchymal cell marker vimentin in Tb3.1cell that undergo EMT.3.We observe that how to regulate EMT process through PI3K/mTOR signal pathways in TSCC.Methods1. Studied on the expression of Akt, mTOR and E-cadherin,β-catenin, Vimentin in TSCC tissues.41cases of TSCC with positive lymph node tissues were collected to be studied and41cases of TSCC with negative lymph node tissues were uses as control.①The expression of Akt, mTOR and E-cadherin,3-catenin, Vimentin were determined by immunohistochemistry;②The relationship between them and lymphatic metastasis were studied.2. Tb3.1cells were plated at2×109cell/well in6well plates in DMEM without FBS overnight. After overnight incubation, cell were treated with EGF (0,5,10,20,40ng/ml) dissolved in DMSO for Oh,24h,48h and cell morphology were observed under invert phase-contrast microscope. The experiment is divided into five groups:①control②20ng/ml EGF group③20ng/ml EGF+200nM/L Rapamycin group④20ng/ml EGF+20μM/L LY294002group⑤20ng/ml EGF+200nM/L Rapamycin+20μM/L LY294002group. Morphological conversion occurred when cells were cultured in PI3K and mTOR inhibitor; wound healing assay was conducted as described the cell motility; we also detected the expression levels of E-cadherin,β-catenin, Vimentin, P-Akt/Akt and P-mTOR/mTOR with specific antibodies. Result1. The active expression rate of Akt in TSCC with positive lymph node tissues was80.5%(33/41), and mTOR was73.2%(30/41), and E-cadherin was17.1%(7/41), and p-catenin was26.8%(11/41), and Vimentin was82.9%(34/41); The active expression rate of Akt in TSCC with negative lymph node tissues was24.4%(10/41), and mTOR was31.7%(13/41), and E-cadherin was63.4%(26/41), and β-catenin was65.9%(27/41), and Vimentin was19.5%(8/41); the active expression rate of five protein in TSCC were significantly different (P<0.05).2. Exposure to EGF at concentrations ranging from5to10ng/ml did not induce obvious morphological changes in Tb3.1cells. However, morphological conversion occurred when cells were cultured in20and40ng/ml of EGF, and begun to change at20ng/ml. Tb3.1cells had an elongatedshape, assuming a fibroblast-like appearance. In contrast, LY294002and (or) mTOR treated Tb3.1cells seemed to restore their epithelial morphology of a polygonal shape, but their did not affect by EGF. Wound healing assay demonstrated that EGF was able to enhance cell movement and inhibitor reduced tumor cell movement in Tb3.1cells.We detected the protein expression by Western blot in response to EGF(20ng/ml) treatment, E-cadherin and β-catenin decreased with the treatment, whereas Vimentin, P-Akt and P-mTOR increased. However, the protein expression instead changes were detected in cells that treated by LY294002and (or) mTOR. The inhibition of PI3K and (or) mTOR activity induced the localization of E-cadherin and β-catenin, and decreased that of Vimentin, as seen in the immunofluorescence analysis. When the two inhibitor combined each other the results were more obvious.Conclusions1. This study identified that PI3K/mTOR signaling pathway is actived and EMT occurs in TSCC with positive lymph node tissues.2. After treatment with EGF, the expression of E-cadherin was decreased, while increased expression of Vimentin was observed, corresponding increased in migration of ability. 3. In this experiment, LY294002and Rapamycin can inhibit the occurrence of EMT in TSCC.4. EGF induced EMT is mediated by PI3K/mTOR signaling in TSCC.5. PI3K/mTOR signaling plays a pivotal role in TSCC EMT process, suggesting that this pathway could be a potential target for cancer therapeutics.