The Study Of Regulation Mechanism And Significance Between P120ctn And Kaiso In Lung Cancer

The catenin p120(hereafter pl20ctn) is an Armadillo protein first identified as a prominent tyrosine kinase substrate implicated in cell transformation by Src,and in ligand-induced receptor signaling through various tyrosine kinase receptors.pl20ctn binds to the juxtamembrane domain of classical cadherins,where it modulates cell-cell adhesion by regulating cadherin turnover and stability at the cell surface.The recent identification of the transcription factor Kaiso as a binding partner for pl20ctn,suggest that pl20ctn may contribute to tumorigenesis and adhesion defects by regulating Kaiso.Kaiso is a nuclear protein that plays a role in transcription repression.It contains an amino-terminal BTB/POZ domain(Broad Complex,Tramtrak,Brie a' brac/Pox virus and Zinc finger) and a carboxyl-terminal region with three zinc finger motifs of the C2H2 type.Whether Kaiso inhibits or promotes oncogenesis cannot be stated conclusively.It was considered as a negative regulator of Wnt signaling,since several target genes of the canonical Wnt signaling pathway(such as MTA2 and matrilysiri) and the non-canonical Wnt signaling pathway(such as Wnt 11)are inhibited by Kaiso and may function as a tumor suppressor.However,a recent report showed that Kaiso-null mice were healthy and did not develop tumors.Interestingly,when Kaiso-null mice were crossed with the well-characterized Apcmin/+ mice that develop intestinal polyps, polyp formation was delayed in the progeny.Furthermore,a newly performed study suggested that Kaiso was a methylation-dependent "opportunistic" oncogene that silences tumor suppressor genes in colon cancer cell lines.Despite these advances,the involvement of Kaiso in tumor,especially in lung cancer,is largely unknown.There has been only one report examining the Kaiso expression in lung cancer,and the authors reported no Kaiso expression in 2 cases of lung squamous cell carcinomas. These situations promoted us to clarify the expression profile of Kaiso proteins as well as its relationship with p120ctn in lung cancer with a large sample size.1.Patients and specimensThe primary tumors specimens were from patients with lung squamous cell carcinomas 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.Immunohistochemical study and result assessmentStaining scores were determined by the percentage of positive cells per slide for membranous and cytoplasmic staining separately.As proposed previously,normal expression was defined when over 90%of the tumor cells showed cell membrane staining of p120ctn.When less than 90%of the tumor cells were stained for p120ctn at the cell membrane,the sample was labeled with "reduced membranous expression". When more than 10%of the tumor cells stained for cytoplasmic p120ctn expression, the sample was labeled with "ectopic cytoplasmic expression".A designation of either "reduced membranous expression" or "ectopic cytoplasmic expression" was defined as abnormal expression of p120ctn.For evaluation of the staining of Kaiso in tissue sections,we use the criteria as follows:0:less than 25%;1:26%-50%;2:51%-75%; and 3:more than 75%.The staining intensity was categorized by relative intensity as follows:l(weak);2(intermediate) and 3(strong).The proportion and intensity scores were then multiplied to obtain a total score.Score less than 1 was considered as negative,while scores of 2 or more were considered as positive.Cases were scored nuclear positive when≥5%of the cells reacted with the anti-Kaiso antibody.3.Immunofluorescent stainingTissues and cells grown on glass coverslips were fixed with ice-cold 100% methanol for 15 minutes at -20℃,followed by permeabilization with 0.2%Triton X-100.Kaiso was detected using two mouse monoclonal antibodies,and a polyclonal antibodyand pl20ctn was detected using mouse monoclonal antibodies.Primary antibodies were applied overnight at 4℃followed by incubation with secondary antibody conjugated to rhodamine/fluorescein isothiocyanate(FITC)-labeled.The nuclei were counterstained with propidium iodide/ 4,6 diamidino-2-phenylindole.The cells were examined with an Olympus IX51 fluorescent microscope(Olympus,Tokyo, Japan),and images were recorded with a CoolPIX 5400 camera(Nikon,Japan).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 and Kaiso.After incubation with peroxidase-coupled anti-mouse IgG at 37℃for 2 hours,the proteins were visualized using DAB/ECL.5.RT-PCRRT-PCR was performed with the RNA PCR Kit(AMV) Version 3.0,according to the manufacturer's instructions.6.Cell cultureBE1,SPC,A549 cells were cultured in RPMI 1640 medium,containing 10%fetal calf serum,1OOIU/ml penicillin,and 100μg/ml streptomycin.Cells were grown on sterilized culture dishes glass and were passaged every 2 days with 0.25%trypsin.7.Plasmid construction and transfectionThe shRNA-Kaiso plasmids transfections were carried out using the Arrest-In^TM Transfection Reagent,according to the manufacturer's instructions. The cells were stably transfected with the pl20ctn-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 andG418.8.Matrigel invasion assay Following 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 200x magnifications per filter.9.3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay Cell proliferation was evaluated each day for four days after the MTT treatment. The absorbance,which is directly proportional to the number of living cells in the culture,was measured at 570 nm using a microplate reader(Model 550,Bio-Rad, Hercules,CA,USA).A blank with dimethyl sulfoxide(DMSO) alone was taken and subtracted from all values.10.Cellular FractionationCellular fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents(Thermo Fisher Scientific Inc.Meridian Rd.,Rockford,USA) according to the manufacturer's instructions.Antibodies against a-tubulin and Lamin B 1 were used in control experiments. 11.ImmunoprecipitationCells were washed twice with 5 ml of PBS followed by incubation on ice with lysis buffer containing 0.5%NP-40,50 mM Tris,150 Mm NaCl,1 mM phenylmethylsulfonyl fluoride,5 mg/ml leupeptin,2mg/ml aprotinin,1 mM sodium orthovanadate,and 1 mM EDTA for 5 minutes.Cells were harvested from the plates, and transferred to a 1.5 ml tube.The lysate was centrifuged at 16,000 g for 5 minutes at 4℃and the supernatant transferred to a new tube.Lysates were quantified by Bradford assay and equal amounts of total protein were used for immunoprecipitation with the anti-pl20ctn or anti-Kaiso pAb.The immunocomplexes were then subjected to SDS-PAGE.12.Cell Synchronization(1)GO synchronization(seum starvation method) Cells in the exponential growth phase were incubated at 37℃in 5%CO²/95%air until they reached 30% confluence.The cells were harvested by centrifugation at 1000xg for 5min,washed with preheated PBS twice then seeded in the serum-free RPMI-1640 medium for further culturing for 36h.The cells that were cultured in serum-containing medium for 36 h at 37℃in 5%CO²/95%air,harvested for testing after 24h,48h,and 72h.(2)S phase Synchronization(Double Thymidine Block) Cells in the exponential growth phase had thymidine added at a final concentration of 2 mmol/L and were cultured at 37℃in 5%CO²/95%air for 18 h.The cultures were centrifuged at 1000xg for 5min and the supernatant was discarded.The cells were washed 3 times with pre-heated PBS to remove the thymidine.Following resuspension,the cells were seeded in a culture flask and incubated for 16 h.Thymidine was added again at a final concentration of 2 mmol/L,and the cells were cultured for 12 h.After the cells were digested with 0.25%trypsin to remove them from the flask and fresh medium was added,followed by centrifugation at 1000xg,the cells were washed with pre-heated PBS twice and then seeded in another flask for an additional 2 h of culture. 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 (l×l0⁶) were centrifuged at 1500 rpm 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.14.Statistical analysis All statistical calculations were performed by SPSS 13.0 for Windows software,p values less than 0.05 were considered statistically significant. Results1.Kaiso was expressed in the cytoplasm of lung cancer cells and is associated with the malignancy of NSCLCKaiso was primarily expressed in the cytoplasm of lung cancer tissues.Overall positive cytoplasmic expression rate was 63.61%(187/294).The positive cytoplasmic expression of Kaiso was higher in III+IV TNM stages of NSCLC,compared to I+II stages(ρ=0.019).The western blot analysis revealed that Kaiso expression was significant higher in tumorous tissues(t=10.610,n=20,ρ=0.000).A correlation between cytoplasmic Kaiso expression and lymph node metastasis was found(ρ=0.003).In paired cases,cytoplasmic expression of Kaiso was 72.7%in primary sites and 90.9%in lymph node metastases(ρ=0.001).The lung cancer-related 5-year survival rate was significantly lower in patients who were cytoplasmic Kaiso-positive (22.22%),compared to those with cytoplasmic Kaiso-negative tumors(64.00%)(ρ=0.005).Cytoplasmic Kaiso status may be an independent prognostic factor for the p-value(ρ=0.054).2.Cytoplasmic Kaiso and p120ctn expression in lung cancer was correlated;the Kaiso-pl20ctn complex can be detected in lung cancer tissuesCytoplasmic Kaiso and pl20ctn expression in lung cancer was related.In the 196 cases of primary lung cancer tissues,cytoplasmic expression of Kaiso correlated with that of cytoplasmic pl20ctn(Kappa=0.269,ρ=0.000).The same was true for the primary cancer and lymph node metastasis samples of the 55 paired(Kappa=0.267,ρ=0.037)(Kappa=0.393,ρ=0.00l).To verify that Kaiso was a bona fide pl20ctn binding partner in lung cancer tissues,we performed coprecipitation studies to determine whether pl20ctn and Kaiso interacted in vivo.Immunohistochemical study already showed that Kaiso and pl20ctn were synergistically expressed in 13 of 20 cases of lung cancer tissues,both in primary and lymph node metastases samples. Furthermore,co-immunoprecipitation results confirmed that Kaiso coprecipitated efficiently with pl20ctn-specific mAb from all these 13 paired lung cancer tissues.3.Down-regulating nuclear Kaiso increases matrilysin transcription and enhances the proliferative and invasive abilities of lung cancer cellsSince Kaiso primarily localized to the nucleus in vitro,it was conceivable to explore the biological role of nuclear Kaiso in lung cancers using in vitro cultured cells. The MTT assay results demonstrated that after down-regulating nuclear Kaiso by transfecting shRNA-Kaiso,the levels of proliferation were significantly higher in the shRNA-Kaiso group cells,compared to the control cells[ρ>0.05(day 1);ρ<0.05(day 2-3),n=3].For the Kaiso antibody addition groups,the growth rates were markedly different from the control cells at all three days.Meanwhile,the shRNA-Kaiso cells and the Kaiso antibody-treated cells showed increased invasion onto the lower surfaces of the Transwell filters,compared to control cells(ρ<0.0l).To further confirm whether the enhancement of proliferative and invasive abilities contributes to Kaiso down-regulation in the nucleus,matrilysin mRNA levels were detected by RT-PCR.The results demonstrated that matrilysin mRNA increased significantly in both shRNA-Kaiso and Kaiso antibody-treated cells,compared with controls(ρ<0.01).4.Expression and localization of Kaiso protein is affected by its binding partner,pl20ctn.Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1We knocked down pl20ctn using siRNA in BE1 and SPC cells.Ablation of pl20ctn reduced the levels of Kaiso proteins,but not true for the mRNA of Kaiso. Furthermore,pl20ctn ablation altered the subcellular localization of Kaiso.The nuclear localizaiton of Kaiso was significantly reduced in siRNA-BEl cells,while that was significantly increased in siRNA-SPC cells.RhoA,but increased the activity of Cdc42 and Racl,and promoted proliferation and the invasive ability of lung cancer cells both in vitro and in vivo.We overexpressed pl20ctn lA and 3A in A549 cells.Overexpression of pl20ctn 1A and 3A significantly increased the levels of Kaiso proteins,but not true for the mRNA of Kaiso.After pl20ctn 1A or 3A stable overexpression,more Kaiso can be observed in both cytoplasm and nucleus.But difference existed between pl20ctn-lA and pl20ctn-3A cells.In pl20ctn-lA cells,although more Kaiso can be coprecipitated from both cytoplasmic and nuclear extract,significant difference only found in cytoplasmic extract.As for pl20ctn 3A overexpression A549 cells,significant difference existed in both cytoplasmic and nuclear extract.Besides,Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1.5.Subcellular loacalization of Kaiso may affected by cell cycle progress. We noticed that Kaiso subcelluar localization altered in different in vitro culture time points.More Kaiso was found in the cytoplasm of the BE1 and siRNA-BEl cells at stage Go,while more Kaiso was found in the nucleus at stageS. 1.Different subcellular localizations of Kaiso may play differential biological roles in NSCLC.2.Expression and localization of Kaiso protein is affected by its binding partner, pl20ctn.Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1.3.Subcellular loacalization of Kaiso may affected by cell cycle progress.