Expression And Significance Of Integrin β1, FAK And PY397FAK In Non-small Cell Lung Carcinoma

BACHGROUND&OBJECTIBE: Focal adhesion kinase (FAK) is a non-receptor protein kinase which plays important roles in integrin-stimulated signaling network between cells and extracellular cell matrix and participates in intracellular signaling events that are necessary for cell growth, apoptosis, adhesion and migration as well. Recently a growing number of reports have revealed that abnormal expression of integrin and FAK can attribute to tumorigenesis and progression. Our study is to explore the relationship between integrin β1, FAK and its phosphorylated form, i.e. pY397FAK by detecting their expression in human normal bronchial epithelium, primary Non-small Cell Lung(NSCLC) tissues and analyze their correlation to clinicopathological characteristics. In addition, altered expression levels of FAK and pY397FAK and the behavioral changes in biology of lung adenocarcinoma cell line A549 were observed after treatment of different concentration of RGDS (Arg-Gly-Asp-Ser) tetrapeptide. The aim is to define the possible effect of integrin and FAK on the development of NSCLC.METHODS: Using immunohistochemical method, the expression of integrinβ1, FAK, pY397FAK and Ki-67 in 60 NSCLC tissues and 19 normal bronchial epithelia was measured. The relationship between integrinβ1, FAK, pY397FAK expression and clinicopathological characteristics was analyzed; MTT assay was applied to measure the altered adhesion capability of A549 cell to Fibronectin after treatment of different concentration of RGDS tetrapeptide; a Transwell cell culture chamber was used to assay effects of RGDS tetrapeptide on A549 cell chemotactic motility and invasion on reformed basement membrane; the altered expression levels of FAK and pY397FAK were detected in A549 cell with laser scanning confocal microscopy (LSCM) and immunocytochemical method.RESULTS: 1. Immunohistochemistry revealed that elevated levels of FAK and pY397FAK expression were detected in primary cancerous tissues (83.3%, 55.0%)compared with normal lung epithelium(57.9%, 21.1%) (PO.05). There was no significant difference for integrinpl expression between NSCLC(81.7%) and normal lung epithelium(68.4%) (P>0.05). Most of NSCLC showed moderate or strong integrinpl protein expression(29/60), while most of normal bronchial epitheliums showed weak expression(13/19), the expression intensity in NSCLC was higher than that in normal samples with statistic significance (P<0.05).2. Expression of integrinpl was correlated with differentiation and TNM stage in NSCLC (PO.05). Overexpression of FAK was associated with differentiation, TNM stage and LN metastasis (PO.05). Elevated levels of pY397FAK expression were correlated with TNM stage and LN metastasis (PO.05). No relationship was found between integrinpl, FAK, pY397FAK and other clinical characteristics, such as age, gender, tumor histology (P>0.05).3. No linear correlation fashion was found between FAK, pY397FAK and integrinpl (P>0.05).Though the expression of integrinpl, FAK, pY397FAK was higher in group of Ki-67 positive than that in negative ones, no statistic significance was found between them (P>0.05).4. There were dramatic decreases in the capability of adhesion to FN, chemotactic mobility and invasion in cultured A549 cell line after RGDS tetrapeptide treatment. RGDS tetrapeptide at the concentrations of 25pg.mL"1, SOpig.mL'1 and lOOug.mL"1 inhibited A549 cells attachment to fibronection with the inhibited rates of 59.12%, 70.52%, 86.30%, respectively; and this inhibition was concentration-dependent manners. The inhibited rates of chemotactic mobility and invasion were respectively 43.6%, 75.6% and 60.1%, 71.3% at the RGDS tetrapeptide concentration of SOug.mL"1 and lOOug.mL"1. All the rates above mentioned were higher that those in control group (PO.01). After exposure of A549 cell to RGDS tetrapeptide in concentration between 50 and lOOug.mL"1, FAK protein remained unchanged with optical density value at 0.1475±0.01708, 0.1550±0.02082 comparing that in control (0.1600±0.01414) (P>0.05) while pY397FAK protein down-regulated significantly with optical density value at 0.1275±0.01258,0.1050±0.01291 compared with that in controlO (0.1603±0.00732) (PO.01) by the means ofimmunocytochemistry. LSCM also showed the similar altered expression of pY397FAK protein in different RGDS tetrapeptide treatment (PO.01).CONCLUSIONS: (1) Abnormal expression of Integrinpi, FAK existed in NSCLC; (2) FAK signaling stimulated by Integrins clustering may play distinct roles in carcinogenesis and tumor progessions; (3) RGDS tetrapeptide can disturb FAK tyrosine phosphorylation. FAK, pY397FAK may be useful indexes for biologic behavior and important targets in NSCLS.