Down-regulation Of FAP Suppresses Cell Migration And Invasion Through PTEN/PI3K/AKT And Ras-ERK Signaling In Oral Squamous Cell Carcinoma

BACKGROUND&OBJECTIVEHead and neck squamous cell carcinoma is the most common type of cancer,More than90%of head and neck cancer,Meanwhile head and neck squamous cancer accounts for7-8%of body.Oral squamous cell carcinoma of head and neck cancer incidence is the first one,Its high degree of malignancy, fast growth, invasion strong, prone to cervical lymph node metastasis,Local recurrence and lymph node metastasis is the main reason for his death,Despite the advances in therapeuticapproaches, percentages of morbidity and mortality of OSCChave still not significantly improved during the last30yearswith the5-year overall survival rate for patients with OSCCvarying between40and50%.Advanced oral squamous cell carcinoma mostly invasive growth,Infiltration of surrounding tissue has already occurred and metastatic tumor surgical resection difficult to complete,And the effect can hardly be satisfied with conventionalRadiotherapy and chemotherapy,So for the presence of extensive infiltration of surrounding and distant metastasis of oral squamous cell carcinoma elucidate the pathogenesis,Is very important to find new therapeutic targets.Tumor invasion and metastasis is the tumor cells from their primary site,Spread to the surrounding, normal tissue infiltration,And transported through the blood circulation, lymphatic drainage, planting, etc. to the secondary target tissue or organ growth continued to proliferate,The process of formation of secondary tumors and primary tumors of the same nature.Tumor invasion and metastasis are multiple genes involved in complex multi-step process to complete, Weakened adhesion and tumor cell motility of tumor cells is the basis for enhanced inter-tumor invasion and metastasis.A number of studies have demonstrated that epithelial-mesenchymal transition in tumor invasion and metastasis plays an important role.EMT concept in1982first proposed by Greenbwg and Hay,Refers to certain specific physiological or pathological conditions, the epithelial cells lose their polarity with the polarity, having activity to convert the process can move freely between the extracellular matrix in stromal cells;Under normal circumstances, EMT seen in early embryonic development gastrulation, neural crest formation, heart valve formation and wound healing process,The embryonic development, wound healing and tumor during the process of EMT are different,Therefore, some scholars have suggested that EMT is divided into three types:That development type (type Ⅰ), fibrosis, wound healing, and Type (type Ⅱ) and cancerous (type Ⅲ).EMT are various types of epithelial cell polarity loss and gain of mesenchymal characteristics important feature,Specifically include:①cell adhesion molecule (E-cadherin) decreased expression, resulting in the disintegration of intercellular connections, cell dispersion;②keratin-based cytoskeleton into vimentin-based cytoskeleton;Cytoskeletal rearrangement, forming cells-matrix adhesion, causing changes in cell phenotype, cell motility enhancing;So metastasis of tumor cells of epithelial origin may cause by EMT. EMT phenomenonhave been found in breast cancer、lung cancer、ovarian cancer、colon cancer、prostate cancer、esophageal cancer、 head and neck Squamous cell carcinoma are present in many human tumors.The EMT is the result of interaction with the cell microenvironment,Extracellular stimuli to promote EMT occur under normal circumstances,Cell membrane or intracellular receptors,ntracellular signal transduction and transcription factors are likely in the tumor microenvironment,Due to improper expression and regulation caused pathological EMT.In recent years, epithelial-mesenchymal transformation in the process of invasion and metastasis of malignant tumors may play a key role attracted widespread attention.Fibroblast activation protein (FAP) as a member of the serine protease family,In expressing stromal activation, participate in various physiological and pathophysiological processes,Such as embryonic development, wound healing, chronic inflammation and malignant tumors. Once was considered to be a F19cell surface antigen,Is an inducible cell surface glycoprotein,It was originally developed in1986by the monoclonal antibody F19in cultured fibroblasts found.1994, F19cell surface antigen was named fibroblast activation protein (FAP).In1990, a molecular weight of170kDa collagenase been identified in the human melanoma cell lines LOX.In1994, the170kDa collagenase is named Seprase.Molecular cloning study for Seprase and FAP found that they are the same a cell surface serine protease,Gene is located at2q23.FAP is a membrane-bound glycoprotein, Belongs to the class of serine proteases, The FAPa (molecular weight95kDa) and FAPβ (molecular weight of105kDa) two subunits of a molecular weight of170kDa dimer. FAP with two proteolytic activity. First, collagenase activity, followed by dipeptidyl peptidase activity. Since the FAP is a serine protease, Capable of degrading many dipeptide and Collagen tapy Ⅰ, It’s such a proteolytic activity seen in primary human epithelial tumors, So FAP may play an important role on the Tumor stroma formation, remodeling and maintenance. FAP expression in the tumor interstitial fibroblast-like cells can promote tumor cell proliferation、invasion and apoptosis, And closely associated with poor prognosis in cancer patients. For tumor stroma FAP new cancer treatment strategies, Attracting more and more researchers attention. FAP is generally considered mainly expressed in epithelial tumor stroma fibroblasts activated, are not found in normal fibroblasts and Cancer cells or other normal tissue, Its existence relate to tumor invasion、tumor angiogenesis and Subsequent growth and metastasis. However, recently FAP has also been reported the expression of gastric、colorectal、 breast、cervix and prostate cancer cells, Current research about FAP in oral squamous cell carcinoma reported only a few Chinese study. Mainly in oral squamous cell carcinoma tissues and its clinical significance, Confirmed expression Inter-cancer substance Fibroblasts, However, we found that oral cancer cell lines KB also expressed FAP, And the first to explore its role and its molecular mechanisms in oral cancer cells, For further study FAP, Applied to provide a basis for the diagnosis, treatment and prognosis.Are FAP with oral squamous cell carcinoma EMT about? RhoA is an important factor involved in the regulation of tumor EMT reported in the literature. Results after colorectal cancer cell FAP interference,its morphological changes, By the spindle morphology similar to fibroblasts become typical round or oval, change of the form may be the mesenchymal-epithelial transition (MET) caused. Migration and invasion experimental and animal transferred model results after FAP interference show that ability of invasion and migration had a significant change in oral squamous cell carcinoma cell, Mainly related to the P13K-AKT and Ras-MEK-ERK two signaling pathways. Thus, we speculate that FAP may play an important role in the EMT in oral squamous cell carcinoma. This study focuses on the relationship between the FAP gene and its oral squamous cell signaling pathways involved in EMT, Aims to reveal the way of FAP participate in oral squamous cell carcinoma metastasis. Addition new knowledge of the unknown function gene FAP, provide new clues for the study of the mechanism of tumor metastasis.Method:1. FAP expression and its clinical significance in oral squamous cell carcinomaImmunohistochemical S-P method. For detect FAP protein expression situation,84cases of oral squamous cell carcinoma and the corresponding12cases of normal oral mucosa detection by the of Stomatology of Nanfang Hospital between February2010to July2012,calculate the relationship between oral squamous cell carcinoma FAP expression and clinicopathological parameters,and statistical analysis.Cell immunofluorescence detection FAP expression and localization in oral squamous cell carcinoma cell lines KB and Tca-8113.2. FAP interferenceing effect on oral squamous cell’s EMTFAP interference vector has been constructedand use of the lentiviral vectors packaged it transfectedoral squamous cell carcinoma cells KB,get FAP interference monoclonal selected by GFP-labeled sorting.and expand its training,then detection its stability interference effect by fluorescence quantitative RT-PCR and Western blotting;While transiently transfected siRNA use of cationic liposomes, then detection its transient interference effect by fluorescence quantitative RT-PCR and Western blotting. The two most efficient of interference fragments stably transfected transiently transfected cell clone were screened respectively,transwell、 Boyden、scratches experiments、cell adhesion experiments、liver tumor metastasis model in nude mice to detect the changes of invasion and metastasis in oral squamous cell carcinoma cell,but detected the changes in cell morphology by TRITC-phalloidin immunofluorescence.3. The signal path way may be FAP participate in oral squamous cell’s EMT.Use Western Blotting detecting oral squamous cell carcinoma EMT-related signaling proteins Such as PI3K, AKT, ERK, MEK and the corresponding phosphorylated protein P-P13K, P-AKT, P-ERK, P-MEK,metastasis-related tumor suppressor PTEN,nuclear transcription factor Snail、Slug,epithelial marker E-cadherin and mesenchymal marker vimentin、N-cadherin and expression of matrix metalloproteinases after FAP interference.Result:1. FAP expression and its clinical significance in oral squamous cell carcinomaProtein expression levels of FAP weremeasured in samples of84archived paraffin-embeddedOSCC tissues and12non-cancerous oral cavity epitheliumtissues using immunohistochemical staining.Furthermore, FAP expression in OSCC samples wasrelatively higher than in benign samples (P=0.0001). Further, we analyzed the relationship betweenclinicopathologic characteristics and FAP expression levelsin individuals with OSCC,Although we did not find asignificant association of FAP expression levels with patient’sage、sex、smoking status、alcohol、recurrence、 tumor location、differentiation、and distant metastasis (M classification), we observed that the elevated expression level of FAP wasmarkedly correlated with tumor size (T classification)(T1-T2versus T3-T4)(P=0.027), lymph-node metastasis (Nclassification)(N0-N1versus N2-N3)(P=0.015) andclinical stage (Ⅰ-Ⅱ versus Ⅲ-Ⅳ)(P=0.014) in OSCC patients.To investigate the prognostic value of FAPexpression for OSCC, the follow-up data of84OSCCpatients for up to5years were used to assess the value of FAP for predicting patient survival in OSCC patients.Kaplan-Meier survival analysis revealed a correlationbetween FAP expression level and overall survival times(P=0.005).Immunofluorescence showed FAP expression in oral squamous cell carcinoma cell lines mainly located in the cell membrane and cytoplasm. 2. The impact of FAP interference on oral squamous cell carcinoma’s EMTFAP interference efficiency of75%or more,oral squamous cell morphology changed,by the spindle、similar shape of fibroblasts to a typical round or oval;transwell chamber experiments, scratches experiments, cell adhesion experiments, nude mice with liver metastasis model are all showed cell invasion and migration exercise capacity was significantly reduced after the FAP silence (P <0.05).These results indicate that endogenous FAP can induce knot of oral squamous cell carcinoma cells EMT.3. The signal path way may be FAP participate in oral squamous cellcarcinomaEMT.EMT-related signaling molecules PI3K, AKT, ERK, MEK, GSK-3P phosphorylation of proteins P-PI3K, P-AKT, P-ERK, P-MEK, up to P-GSK-3p protein expression were reduced after FAP interference, and metastasis suppressor gene PTEN expression increases,nuclear transcription factor Snail and Slug were down-regulation,epithelial marker E-cadherin increases,however,mesenchymal marker vimentin、N-cadherin down-regulation,matrix metalloproteinases MMP-2and MMP-9were down-regulation. These results suggest that FAP may promote the occurrence of EMT on oral squamous cellcarcinoma by PTEN/PI3K/AKT and Ras/MEK/ERK two signaling pathways.Conclusion:1. FAP associated with oral squamous cellcarcinomaEMT;2. FAP promote the occurrence of EMT on oral squamous cellcarcinoma by PTEN/PI3K/AKT and Ras/MEK/ERK two signaling pathways.The innovation of this study:1. First discovered oral squamous cell carcinoma cells also express FAP;2. The first time to clarify the role of FAP participate in oral squamous cell EMT mechanism;3. To study the mechanism of oral squamous cell carcinoma metastasis provides a new target, for further study FAP and used in the diagnosis、treatment and prognosis provide a basis.