Effect And Mechanism Of WWTR1Promoting Cell Proliferation And Inhibiting Apoptosis In Non-small Cell Lung Cancer

Objective:Lung cancer is one of the leading causes of all cancer-related deaths worldwide. Majority of the diagnosed lung cancer cases are non-small-cell lung cancers (NSCLCs). Tumor proliferation and decreased apoptosis are main cause of malignant progression. The identification of biomolecules playing a direct role in tumor proliferation and cell survival is an important step toward rational design of drugs for cancer treatment.The Hippo tumor suppressor pathway plays a major role in development and organ size control, and its dysregulation contributes to tumorigenesis. WWTRl (transcriptional co-activator with PDZ-binding motif; also known as TAZ) is a transcription co-activator acting downstream of the Hippo pathway. WWTRl is proposed as a candidate cancer-associated gene and increased WWTRl protein levels have been shown in various human cancers. Previous studies have shown that higher WWTRl mRNA or protein levels were associated with the aggressiveness of colorectal, breast and lung cancers. Although some reports showed that WWTRl functions an oncogene in non-small cell lung cancer (NSCLC), the mechanism of WWTRl regulating lung cancer cell proliferation and apoptosis remains unclear. In order to address the issue, we examined WWTRl expression in non-small-cell lung cancer cell lines. We investigated the role of WWTRl on the cell cycle progression and apoptosis of lung cancer cells and explored the potential underlying mechanism.Materials and Methods:(1) A total of105cases of NSCLC and10cases of normal control paraffin-embeded tissue were retrieved from the Pathology Archive of Chinese PLA General Hospital from2007to2009. The histological diagnosis and grade of differentiation were evaluated by two independent pathologists according to theWorld Health Orgnanization (WHO) guidelines of classification (2004). Clinicopathological information of the patients was obtained from patient records. All of the enrolled patients underwent curative surgical resection without having prior chemotherapy or radiation therapy. The study has been approved by the hospital ethical committee. Inaddition we analyzed the relationship between WWTR1expression and theclinicopathologic factors.(2)We used western blot and Real-time PCR to detect the expression of WWTR1inthe normal bronchial epithelium cell HBE and a series of lung cancer cell lines (LK2H1299H460H446A549) both on protein and RNA level.(3)We transfected WWTR1siRNA in high expressed WWTR1cell line A549andoverexpressed WWTR1in HBE cells with low expression of WWTR1.(4) Determined by MTT cell proliferation technology,we tested the proliferation ofA549and HBE cells.(5) Annexin V/PI flow cytometry analysis was used to examine the effect ofWWTR1on apoptosis of A549and HBE cells.6) Using flow cytometry to detect the role of WWTR1in cell cycle progression.(7) We used western blot and Real-time PCR to determine the possible downstreamtarget of WWTR1in NSCLC.(8) The application of SPSS software was utilized for statistical results.Results:(1) We investigated WWTR1expression in NSCLC tissues and normal lung tissuesby immunohistochemistry. Normal bronchial epithelia and pneumocytes showednegative or weak WWTR1staining. Of105NSCLC patients,44(41.90%) werescored as positive for WWTR1, which was localized in nuclear and/or cytoplasmiccompartments of tumor cells.(2) The relationship between WWTR1expression and various clinicopathologicfeatures was investigated. As shown in Table1, no statistical difference was foundbetween the positive WWTR1expression and such clinical parameters as age(p=0.7060), gender (p=0.1510), histology (Adenocarcinoma versus Squamous cellcarcinoma, p=0.4608), and differentiation(well versus moderate-poor, p=0.9166).WWTR1overexpression significantly correlated with TNM stage (Ⅰv ersus Ⅱ+Ⅲ,p=0.0174); WWTR1overexpression significantly correlated with tumor status(T1+T2 versus T3, p=0.0370); WWTR1overexpression significantly correlated with nodalstatus (N0versus N1+N2, p=0.0723).(3) WWTR1expression was analyzed by western blotand Real-time PCR in a panelof lung cancer cell lines. We found that levels of WWTR1were higher in lung cancercell lines than in the normal bronchial cell line HBE. Extremely high expression ofWWTR1was observed in A549cells. In order to explore the biological function ofWWTR1in lung cancer, a pool consisting of three WWTR1-targeting siRNAs wasemployed in A549cell line.(4) We tested the effect of WWTR1on cell growth in A549cells and HBE cells. Theproliferation ability was determined by the MTT. We found that treatment ofWWTR1-specific siRNA resulted in a significant decrease in proliferation comparedto control siRNA.(5) To examine whether the reduced cell proliferation in WWTR1knockdown cellswas due to increased cell apoptosis, the rate of apoptosis was evaluated using AnnexinV/PI double staining-coupled flow cytometry analysis. We characterized the effect ofWWTR1on lung cancer cell apoptosis after paclitaxel treatment (10nmol/L,24hours).Clearly, a significant increase of early and late apoptosis was observed in cells withWWTR1knockdown compared with scramble controls, while WWTR1overexpression inhibited apoptosis in HBE cell lines.(6) To ascertain whether the decreased cell proliferation induced by knockdown ofWWTR1is due to cell cycle arrest, we determined the cell cycle distribution inWWTR1depleted cells (Figure2B). Flow cytometric analysis revealed that theG0/G1-phase DNA content was increased in WWTR1knocked-down cells (A549siRNA vs Neg control:64.6%vs57%) and decreased in cells transfected withWWTR1plasmid (HBE WWTR1vs empty vector:68.1%vs72.1%). While theS-phase DNA content was upregulated in knocked-down cells (A549siRNA vs Negcontrol:24.9%vs34.5%) and downregulated in WWTR1transfected HBE cells (HBEWWTR1vs empty vector:28.1%vs23.1%).(7)To understand the underlying mechanism of the proliferation and apoptosisinhibition effects of WWTR1, we analyzed several potential genes (cyclinA, CTGF and c-Myc, Bcl-2, Bcl-xl, XIAP, Bax, cyclinD1, cyclinE). Both Real-time PCR andwestern blot analysis showed that cyclin A and CTGF were significantly repressed inWWTR1knocked-down cells and overexpressed in cells transfected with WWTR1plasmid, indicating the regulation of cyclinA and CTGF may, at least partly,contribute to WWTR1-mediated growth and apoptosis modulation.Conclusion:(1) WWTR1was overexpressed in NSCLCs and WWTR1overexpression correlatedclosely with TNM stage, tumor status as well as nodal status.(2) Overexpression of WWTR1promoted proliferation while depletion of WWTR1inhibited cell proliferation of NSCLC cells.(3) Overexpression of WWTR1inhibited apoptosis, while depletion of WWTR1promoted apoptosis of NSCLC cells.(4) Overexpression of WWTR1facilitated cell cycle progression, while depletion ofWWTR1induced cell cycle arrest.(5) WWTR1-mediated growth and apoptosis modulation in NSCLC, at least partly,dependent on the regulation of cyclinA and CTGF.