Phosphorylation Of Ezrin Enhances The Metastatic Potential Of Breast Invasive Ductal Carcinoma Via P38MAPK Pathway

IntroductionEzrin-Radixin-Moesin(ERM) protein controls the cytolergy by composing cellular membrane-cytoskeleton complex,which played a important role in invasive and migratory of cancer.Cytoskeleton linking protein ezrin belonged to ERM family,which had been mapped to chromasome 6q25.2-6q26.Recently,there have been some reports about over-expression of ezrin had positive correlation with the invasive and migratory of cancer.The inactivation state of ezrin formed a head-to-tail ligation by N-tip and C-tip interaction,which shielded the binding site in molecular surface.Fievet found that phospho-Thr567 of ezrin and PIP2 were both needed in activating of ezrin. Then the activated ezrin effected by linking F-actin to CD44,E-cadherin,ICAM 1/2 et al.While to comprehend the function of ezrin wasn't our purpose,but to make clear of its upstream regulating factors.Because those factors may be the key-point to control the metabasis of cancer.Mitogen Activated Protein Kinase(MAPK) was one of important signal transducting systems,and p38 was of the subgroup of MAPK.Such growth factors,inflammatory factors and alarm reactions can active it.Then the actived-p38 changed the genetic transcription,protein synthesis,cell motility and cytoskeleton structure that were correlated with the potency of invasive and migratory.Mengdong Lan found that When in the oncogenic Raf-1-transfected mouse hepatic cell line,epithelial to mesenchymal transition(EMT) indicated as down regulation of E-cadherin and up-regulation of Snail occurred,loss of microvilli and down regulation of ezrin were also observed.In the Raf-1 transfectants treated with the p38 MAP-kinase inhibitor SB203580,the numbers of microvilli and the expression of ezrin,E-cadherin and Snail were recovered.More interestingly,treatment with SB203580 induced elongation of microvilli and increased phosphorylation of ezrin(at Thr-567 and Tyr-353).These results suggested that phosphorylation of ezrin via the p38 MAP-kinase signaling pathway might be involved in the formation of microvilli during development of epithelial cell polarization.While which were different from our hypothesis.We think that p38MAPK signal pathway may take part in phosphorylating ezrin C-tip(Thr567),up-regulate p-ezrin,elongation of microvilli,and then enhance the potency of invasive and migratory in cancer.Which were also the questions we tried to answer in our experiment.Materials and Methods1,Samples(1)80 breast cancer tissues smples(80 invasive ductal carcinoma(IDC)),and 50 fresh breast tissues(30 IDC and 20 breast fibroadenoma)were obtained from the First Affiliated Hospital of China Medical University.(2)3 breast cell lines(MCF-10A are no-cancer cell line,MCF-7 has lower potential of invasive and migratory and MDA-MB-435s has the higher potential).(3)The anti-human p-p38 monoclonal antibody(#9216) and p-ezrin monoclonal antibody(#3149) were purchased from Cell signaling.SB203580,the specifical inhibitor of p38MAPK was provided by Calbiochem company,and Phalloidin conjugated tetramethylrhodamineisothiocyanate(TRITC- Phalloidin) was purchased from Sigma company,and the transwell chamber was provided by Corning company.2,Methods(1)Immunohistochmistry:Detect the expression of p-p38 and p-ezrin by Immunohistochmistry S-P methold.(2)Western blot:Membranes were incubated with primary antibody(p-p38 1:400,p-ezrin1:400),then incubated with corresponding secondary antibody(1:4000). For negative controls,PBS was used instead of primary antibody.(3)Cell immunoflorescence:435s cells blocked were incubated with primary antibody(p-p38 1:100,p-ezrin1;100) overnight at 4℃,then incubated with FITC-conjugated secondary antibody(1:50)and TRITC- conjugated Phalloidin(1:50) away from light,30min,they were incubated with Hoechest for nuclear counterstaining.(4)Scanning electronic microscope:435s cells were inoculated on coverslips, treated with different concentrations of SB203580 24h,fixed with 1%osmic acid,dried by CO₂ critical point,plated with gold,at last examined with scanning electronic microscope(JEOL SEM-T300).(5)Matrigel Invasion Assay:Inoculated 435s cells in up-cave,then added SB203580,incubated at 37℃,24h.Wiped out the cells in up-cave,fixed,stained and counted the cells in down-cave.3,Statistical analysisAll the data are analyzed with SPSS for Windows 13.0 software.We useχ² test to analyze the correlation between clinic pathologic parameters of breast cancer and expression of p-p38 and p-ezrin,we use the permutation test for the Spearman correlation coefficient to analyze the correlation between expression of p-p38 and p-ezrin.The result of Western Blot is analyzed by t-test.The statiscal significance is defined as P＜0.05.Results1,Expression of p-p38 and p-ezrin protein in breast samples and their relationship with clinicopathological characteristic(1)Breast tissues Immunohistochmistry shows:The positive expressions of p-p38, p-ezrin were 61.3%(49/80),42.5%(34/80) in IDC tissues and p-p38 mainly localized in nucleus and manipulus in cytoplasm.While p-ezrin expressed strongly in cell membrane.These changes were correlated with the clinical TNM staging (P=0.012,P=0.002) and lymphatic metastasis(P=0.015,P=0.001) in IDC.P-p38 and p-ezrin protein has a positive correlation in IDC samples(r=0.269,P=0.016).(2)Fresh breast tisues Western Blot shows:The expression of p-p38 and p-ezrin protein in IDC samples were significantly higher than those in breast fibroadenoma tissues(P=0.015,P=0.000),while the expression in TNM stagingâ…¢-â…£was higher than which inâ… -â…¡(P=0.023,P=0.000),and the expression in tissues with lymphatic metastasis was higher then which without lymphatic metastasis(P=0.028,P=0.002).2,Expression of p-p38 and p-ezrin protein in cell lines(1)Cell Western Blot shows:The expression of p-p38 and p-ezrin in cancer-cell line were higher than no-cancer cell line(all of the P＜0.05).(2)435s cells Immunoflorescence shows:p-p38 mainly localized in nucleus and manipulus in cytoplasm.P-ezrin,F-actin were co-localized in cell membrane and cytoplasm.3,The changes of 435s cells dealed with SB203580,the specifical inhibitor of p38MAPK after 24h(1)The 435s cells which were incubated with SB203580 decreased the expression of p-ezrin with a dose-dependent manner(P＜0.05).But the expression of p-p38 had no change(P＞0.05).(2)The SB203580 made pseudopodia fewer and shortener,and inhibitted the potential of invasion and migratory of 435s(P＜0.05).ConclusionThe over-expression of p-p38 and p-ezrin were correlated with the clinical TNM staging and lymphatic metastasis in IDC.P-ezrin enhanced the metastatic potential of breast IDC,which may via the p38MAPK pathway.