The Expression And Significance Of P120-catenin And Its Isoforms In Human Lung Cancer

Introductionp120-catenin(p120ctn)belongs to the Armadillo family of proteins.It influences cell-cell adhesion by directly interacting with E-cadherin and also can regulate cell motility through the actin cytoskeleton via Rho family GTPases.However,the role of p120ctn is currently controversial since evidence suggests that p120ctn can both positively and negatively regulate adhesive activity of cancer cells.One reason for this contradiction may lie in the large number of p120ctn isoforms expressed in different cells and tissues.Owing to alternative splicing and multiple translation initiation codons,several p120ctn isoforms can be expressed,which are tissue and cell specific. These p120ctn isoforms contribute differently to cancer cell adhesion and migration. The present study examined the expression pattern of p120ctn isoforms in lung cancer and their correlation to clinical parameters,the mechanism of p120ctn regulating cancer cell adhesion was also explored.Materials and Methods1.Patients and specimensThe primary tumors specimens were from patients with lung SCC and adenocarcinoma who underwent complete resection in the First Affiliated Hospital of China Medical University None of the patients had received radiotherapy or chemotherapy before surgical resection,and all were treated with routine chemotherapy after the operation.Among these samples,some fresh specimens and corresponding normal tissue samples were stored at -70℃immediately after resection until the extraction of protein and RNA.2.Cell cultureBE1,LH7,SPC,LYE,A549,H460 cells were cultured in RPMI 1640 medium, containing 10%fetal calf serum,100IU/ml penicillin,and 100μg/ml streptomycin. Cells were grown on sterilized culture dishes glass and were passaged every 2 days with 0.25%trypsin.3.Immunohistochemical assessmentImmunostaining was performed by the avidin-biotin-peroxidase complex method. They were then incubated with p120ctn,E-cadherin,RhoA,Cdc42,Rac1 antibody overnight.Five views were examined per slide,and 100 cells were observed per view, at 400x magnification.Labeling scores were determined by the percentage of positive cells per slide.We define no positive cells as(-),positive cells≤25%as(+),26～75% as(++),≥76%as(+++).4.Western Blot50μg proteins were separated by SDS-PAGE.After transferring to polyvinylidene fluoride membrane,the membrane was incubated overnight at 4℃with either the mouse monoclonal antibody against p120ctn,E-cadherin,β-catenin,RhoA,Cdc42 and Rac1.After incubation with peroxidase-coupled anti-mouse IgG at 37℃for 2 hours, the proteins were visualized using DAB/ECL5.RT-PCRRT-PCR was performed with the RNA PCR Kit(AMV)Version 3.0,according to the manufacturer's instructions.6.Plasmid construction and transfectionThe cells were stably transfected with the p120ctn-siRNA plasmids or p120ctn plasmids using Lipofectamine 2000,following the manufacturer's instructions.The empty plasmid was used as a negative control.Selection was accomplished with G418. Transfected cells were cultured in RPMI 1640 medium containing 10%fetal calf serum and G418.7.RhoA,Cdc42,and Rac1 activity assayRhoA activity was measured using colorimetric-based RhoA G-LISA^TMactivation assay according to the manufacturer's instructions.The resulting samples were analyzed by spectrophotometer.Cdc42 and Rac1 activity was measured using a pull-down assay.Cdc42-GTP/Rac1-GTP bound proteins were separated by 12% SDS-PAGE and transferred to PVDF membrane.The membrane was incubated in the anti-Cdc42/Rac1 antibody solutionat 4℃overnight,and detected with the BioImaging System.8.Matrigel invasion assayFollowing the manufacturer's instructions,in the upper chambers,5x10⁵ BE1 cells were grown in serum-free medium on 8μm porous polycarbonate membranes, which were coated with Matrigel basement membrane matrix.The lower chambers were filled with RPMI 1640 medium containing 10%fetal calf serum.After incubation for 6,16,or 24 hours at 37℃in a humid atmosphere of 5%CO2 and 95%air,the cells that had migrated through the pores were fixed with methanol for 30 minutes and stained with hematoxylin.Then the number of cells counted visually using Nikon E200 microscope in five different fields under 200×magnifications per filter.9.Flow cytometry(FCM)After 48 hours of culture,cells from each experimental group were collected and digested with trypsin and fixed with 75%ice-cold ethanol at 4℃overnight.Cells (1×10⁶)were centrifuged at 1500 rpm for 5 minutes,and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis.The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.10.Xenograft to nude miceFour-week-old male BALB/c nude mice were obtained from the animal facility approved by the China Medical University.Each mouse was inoculated subcutaneously in the back with 2.4x10⁶ tumor cells.Mice were sacrificed after 6 weeks.11.Statistical analysisAll statistical calculations were performed by SPSS for Windows software,p values less than 0.05 were considered statistically significant.Results(1)p120ctn and E-cad was expressed mainly in the cell membrane of the normal bronchial epithelium.Of 138 lung cancers,p120ctn or E-cad abnormal expression, including reduced or absent membranous expression and cytoplasmic expression. RhoA,Cdc42,and Rac1 expression is significantly higher in lung cancers in comparison with corresponding normal lung tissues.There was a correlation between abnormal p120ctn expression and overexpression of RhoA,Rac1 and Cdc42,the combination of which associate with poor differentiation,high TNM stage,and lymph node metastasis.(2)We knocked down p120ctn using siRNA in BE1,SPC,LTE cells.Ablation of p120ctn reduced the levels of E-cadherin andβ-catenin proteins,as well as the mRNA ofβ-catenin.Furthermore,p120ctn ablation inactivated RhoA,but increased the activity of Cdc42 and Rac1,and promoted proliferation and the invasive ability of lung cancer cells both in vitro and in vivo.(3)We observed reduced expression or even the absence of p120ctn isoform 1 and 3 in tumor cell compared to normal tissues.The loss of p120ctn isofoms,especially isoform 1 may be responsible for cancer cell development.(4)Different p120ctn isoforms serve in distinct biological functions in lung cancer.Expression of p120ctn isoform 1A upregulated E-cadherin andβ-catenin and downregulated Rac1 activity thus inhibited metastatic ability of lung cancer cell. p120ctn isoform 3A expression resulted in the inactivation of Cdc42 and activation of RhoA,and did not influence metastasis significantly.Flow cytometry showed that p120ctn isoform 3A inhibit cell cycle.In vivo test showed that in nude mice,the mean tumor weight in the p120ctn-3A group was significantly lower than that in p120ctn-1A groups.Conclusions(1)We demonstrate a positive association between increased RhoA,Rac1,Cdc42 expression and abnormal p120ctn expression.In addition,the combination of abnormal p120ctn and Rho family over-expression is associated with high TNM stage and poor differentiation.(2)In lung cancer,p120ctn loss reduced the levels of E-cadherin andβ-catenin, inactivated RhoA,but increased the activity of Cdc42 and Rac1,and promoted proliferation and the invasive ability cancer cells.(3)In normal bronchial epithelial cells,p120ctn isoform 1A and 3A were expressed,which is reduced in lung cancer cells.These p120ctn isoforms contribute differently to cancer cell adhesion and migration.(4)Expression of p120ctn isoform 1A upregulated E-cadherin andβ-catenin and downregulated Rac1 activity thus inhibited metastatic ability of lung cancer cell. p120ctn isoform 3A expression resulted in the inactivation of Cdc42 and activation of RhoA,and did not influence metastasis significantly,p120ctn also induceβ-catenin in lung cancer cell lines,the underlying mechanism is under investigation.