Filamin A Regulates The Activation Of Epidermal Growth Factor Receptors And Enhances Breast Cancer Cells Proliferation And Migration

Objective: Breast cancer is one of the most important malignant tumorswhich threating to contemporary women's health. A survey carried out by theMinistry of Health in2007had showed that the clinical incidence of breastcancer was the highest in all female malignant tumors. According to statistics,the global incidence and mortality of breast cancer were increasing year byyear, and its pathogenesis has not been completely elucidated. In view of thissituation, exploring new ideas and more effective therapeutic targets for breastcancer treatment are still research focuses.The epidermal growth factor receptor (EGFR) family, a receptor tyrosinekinase family, comprises four members of erbB-1/EGFR, erbB-2/HER2,erbB-3/HER3and erbB-4/HER4, and closely related to breast cancer. Tyrosinephosphorylation induced by these receptors binding to its ligands may regulatecell proliferation, differentiation and movement, which plays more importantrole in normal mammary gland development, maturation and degradation.However, Four receptors usually overexpress in breast cancinoma. Manystudies had confirmed that HER-2positive was a major reason of poorprognostic in breast cancer patients and EGFR overexpression was also anadverse sign in prognosis of patients with breast cancer, but it has not beenfound the significant influence of ErbB-3and ErbB-4overexpression in theprognosis of breast cancer. A lot of molecular targeting drugs against EGFR,HER-2had been applied in the clinical treatment of breast cancer, such ascetuximab, trastuzumab, lapatinib and so on, but the efficiency was not high.So it will be imperative to exploit more efficient drugs which against EGFR orHER-2.Acting as actin-binding protein, filamin A not only cross-links actin filaments to form a stable cytoskeleton, but also binds a variety of cellmembrane proteins, therefore, it can involve in cell proliferation, adhesion,migration, invasion and other behaviors. Moreover, FLNa also interferes withthe expresstion of many intracellular receptors, thus affects the multiple signaltransduction pathways and involves in tumorigenesis and development. So far,it has been discovered that expression of FLNa increased in lung cancer,metastatic breast carcinoma, poorly differentiated astrocytomas, prostatecancer, melanoma and other tumors. Some studies have shown the expresstionof FLNa was closely related to malignant degree of breast cancinoma, but theexact mechanism is still not clear. Weather FLNa affects the activation ofEGFR and HER-2in breast cancer cells has not been reported.In the current study, three kinds of human breast cancer cell lines werecultured in vitro.(1)The expression of FLNa,EGFR and HER-2proteins indifferent breast cancer cell lines were detected by Western-blot.(2)Theproliferation and migration of different breast cancer cell lines were observedby MTT, flow cytometry and wound healing assay,respctively.(3)Theproliferation, the levels of EGFR, HER-2phosphorylation and the changes ofdownstream signal transduction molecular were examined. The aim of thisstudy is to analyze the effects of FLNa on proliferation, migration, EGFR andHER-2phosphorylation and related signal transduction pathway in breastcancer cells. It will provide a new target for the treatement of breast cancer.Methods:1The levels of FLNa, EGFR and HER-2proteins in different breast cancercell lines were detected by Western blotHuman breast cancer MDA-MB-436cells, MDA-MB-231cells andSKBR-3cells were cultured for48h in vitro, then proteins were extracted fromcells, and the levels of FLNa, EGFR and HER-2proteins were assessed byWestern-blot.2The proliferative capacity of different breast cancer cells were examined byMTT colorimetric assayThree breast cancer cell lines were digested and transferred into96well plates, respectively. The cells proliferative capacity was examined by MTTcolorimetric assay after EGF (20nM) treatment for24h.3The cell cycle distribution of different breast cancer cells was examined byflow cytometryAfter culturing for48h, three kinds of breast cancer cells were collectedand fixed by70%ethanol overnight at4℃. The distribution state of breastcancer cell cycles were detected by utilizing flow cytometry next day.4The migration of different breast cancer cells were examined by wound-healing assayAs described above,Three kinds of breast cancer cells were cultured toconfluence or near confluence (>90%). Images of the same field were taken at0h,6h and12h after scratching under inverted microscope. The scratch widthwas measured by using of appropriate software.5The proliferative capacity of breast cancer cells were examined by MTTcolorimetric assay after plasmid transfectionThrough above experiments, we selected MDA-MB-436cells (highFLNa expression) and SKBR-3cells (low FLNa expression) to perform thetransient transfections with FLNa-siRNA or pREP4/FLNa plasmids to knockdown or increase the expression of FLNa, respctively. The cells proliferationwas examined by MTT colorimetric assay after stimulating by differentconcentrations of EGF for24h.6The localization of FLNa, EGFR, HER-2in different breast cancer cells wasobserved by confocal laser scanning microscopyHuman breast cancer MDA-MB-436cells and SKBR-3cells werecultrued. We stained cells with anti-FLNa, anti-EGFR or anti-FLNa andanti-HER-2, as well as fluorescent secondary antibodies. The localizations ofEGFR and FLNa, FLNa and of HER-2were observed by confocal laserscanning microscopy.7The levels of different protein in breast cancer cells were examined byWestern-blot after plasmid transfectionMDA-MB-436cells and SKBR-3cells were cultured and transient transfections were performed with FLNa-siRNA or pREP4/FLNa plasmids toknock down or increase the expresstion of FLNa. Proteins were extracted fromthese cells treated with EGF (20nM) for0min,5min,10min and30min. Thelevels of FLNa, EGFR phosphorylation (p-EGFR), HER-2phosphorylation(p-HER-2), EGFR, HER-2, ERK phosphorylation (p-ERK), and ERK wereassessed by western-blot at each time point.Results:1Comparison of protein expression and biological behavior in different breastcancer cells1.1Comparison of protein expression in different breast cancer cellsWe utilized ImageJ software to scan the protein bands. There was nostatistically significant difference in the expression of FLNa (0.20±0.01,0.19±0.00), EGFR (0.41±0.02,0.40±0.01) and HER-2(0.02±0.00,0.02±0.00)between MDA-MB-436cells and MDA-MB-231cells. But in SKBR-3cells,the expressions of FLNa (0.08±0.00) was significantly lower than that inMDA-MB-436cells and MDA-MB-231cells (P＜0.01). On the contrary,Compired with MDA-MB-436and MDA-MB-231cells, the expressions ofEGFR (0.48±0.03) and HER-2(0.12±0.03) were higher in SKBR-3cells, thedifference was statistically significant (P＜0.01).1.2Comparison of proliferative capacity in different breast cancer cellsThere was no statistically significant difference in the increase ratebetween the MDA-MB-436cells (30.35±9.18%) and MDA-MB-231cells(31.71±2.87%). However, both were higher than SKBR-3cells (8.13±2.61%)in cell growth speed, there was statistical difference (P＜0.01).1.3Comparison of the cell cycle distribution in different breast cancer cellsThere was no statistically significant difference in the proliferation indexbetween the MDA-MB-436cells (57.03±2.63%) and MDA-MB-231cells(52.97±3.58%). But the proliferation index in SKBR-3cells (30.30±0.56%)was lower than that in MDA-MB-436group and MDA-MB-231group, thedifference was statistically significant (P＜0.01).1.4Comparison of migration capacity in different breast cancer cells There were no statistically significant difference in the migration rates at6h,12h after scratching between the MDA-MB-436cells group (15.22±4.18%,31.82±3.36%) and MDA-MB-231group (14.84±2.96%,29.12±5.65%).However, both were higher than SKBR-3group (1.70±0.94%,2.78±1.20%) incell mobility speed, there was statistical difference (P＜0.01).2Comparison of proliferative capacity in different breast cancer cells afterplasmid transfectionWe carried out statistical analysis in OD values detected by MTT. Theproliferation levels of FLNa-siRNA group (1.00±0.10,1.18±0.25,1.28±0.16)were lower than the nonsilencing control siRNA group (1.38±0.19,1.60±0.20,1.73±0.20) after EGF (4nM,20nM,100nM) stimulation in MDA-MB-436cells. There was statistical difference between two groups (P＜0.05). Theproliferation levels of pREP4/FLNa group (1.19±0.20,1.36±0.20,1.59±0.13)were higher than pREP4group (0.87±0.16,1.00±0.19,1.26±0.14) in SKBR-3cells. There was statistical difference between two groups (P＜0.05). Theproliferation levels of four groups rose with the increase of EGFconcentration.3The analysis of mechanisms in FLNa3.1The localization of FLNa, EGFR, HER-2in different breast cancer cellsThe results of immunocytochemical staining were observed byImmunocytochemical staining: in human breast carcinoma cell lineMDA-MB-436, FLNa mainly existed in cytoplasm and cytoplasmicmembrane, EGFR mainly existed in cytoplasmic membrane, they co-localizein cytoplasmic membrane of MDA-MB-436cells. On the contrary, FLNa andEGFR, FLNa and HER-2were existed and co-localized in the cytoplasmicmembrane of SKBR-3cells.3.2Effects of FLNa silence on EGFR and ERK phosphorylation in humanbreast carcinoma MDA-MB-436cellsWe examined expresstion of different protein by western blot afterFLNa-siRNA plasmid transfection in MDA-MB-436cells, each group wasstimulated by EGF (20nM) for0min,5min,10min and30min. The expressions of FLNa in FLNa-siRNA group (0.43±0.07,0.52±0.03,0.49±0.05,0.26±0.03) were lower than nonsilencing control siRNA group (0.93±0.11,1.08±0.20,1.18±0.20,0.89±0.13) at each time point. There was a statisticaldifference between two groups (P＜0.05or P＜0.01). The levels of bothp-EGFR (0.81±0.04,0.80±0.0,0.14±0.01) and p-ERK (0.71±0.17,0.77±0.03,0.81±0.02) in FLNa-siRNA group were lower than nonsilencing controlsiRNA group (1.01±0.02,1.22±0.12,0.68±0.04),(1.61±0.07,1.67±0.22,1.17±0.10). There was statistical difference between two groups (P＜0.05or P＜0.01).3.3Effects of FLNa overexpression on EGFR, HER2and ERKphosphorylation in human breast carcinoma SKBR-3cellsThe expresstion of different proteins were examined by western blot inSKBR-3cells transfected with pREP4/FLNa plasmid, each group wasstimulated by EGF (20nM) for0min,5min,10min and30min. The levels ofFLNa in pREP4/FLNa transfetion group (1.05±0.10,1.20±0.05,1.04±0.09,1.34±0.03) were higher than pREP4control group (0.62±0.01,0.82±0.08,0.58±0.02,0.44±0.01) at each time point. There was statistically significantdifference between two groups (P＜0.05or P＜0.01). The levels of p-EGFR(0.95±0.10,0.74±0.02,0.38±0.09), p-HER-2(1.17±0.12,1.29±0.15,2.20±0.23) and p-ERK (1.49±0.05,1.41±0.07,1.41±0.02) in pREP4/FLNatransfection group were higher than that in pREP4control group (0.73±0.06,0.41±0.01,0.20±0.02),(0.79±0.09,0.83±0.04,1.65±0.14),(1.32±0.06,1.06±0.08,0.80±0.05). The differences were statistically significant (P＜0.05or P＜0.01).Conclusions:1The level of FLNa expression was closely related to proliferation andmigration capacity in breast cancer cells. FLNa silencing could decrease theproliferation capacity of breast cells, inversely, the cells proliferationcapacity increased when FLNa overexpressed. These results suggest thatFLNa has promotive role in proliferation of breast cancer cells.2The quantities of EGFR, HER-2expression are not the decisive factors in evaluating the prognosis of breast cancer. The activation degree of receptorsis the key to the development of breast carcinoma. The activity ofRas/Raf/MEK/ERK signal transduction pathway could be mediated byFLNa affecting the phosphorylation levels of EGFR, HER-2in breastcancer cells, thereby FLNa has significant effects on the proliferation ofbreast cancer cells.