Caveolin-1Regulates The Activation Of Epidermal Growth Factor Receptors And Suppresses Breast Cancer Cells Proliferation And Migration

Objective：Breast cancer is the most common cancer and its morbidity isthe highest in women. According to the latest statistics, the global incidenceand mortality of breast cancer are increasing year by year. An estimated400000women worldwide were died of breast cancer every year. Therefore,finding effective treatment for breast cancer is imperative.The epidermal growth factor receptor (EGFR) family of receptor tyrosinekinases encompasses four members: ErbB-1/EGFR, ErbB-2/HER-2, ErbB-3and ErbB-4that control important aspects of cell proliferation, differentiation,motility, and survival, and their deregulation is implicated in oncogenesis.HER-2plays important role in the pathogenesis of breast cancer and hasalready been recognized as an important prognostic factor in breast cancer.HER-2gene amplification and/or protein over-expression has been associatedwith increased breast cancer cell proliferation, tumor invasiveness. Afterdimerization, tyrosine phosphorylation in HER-2receptor will initate a seriesof intracellular signals activation, which will involved in the regulation oftumor cell growth and differentiation. Drugs targeting to HER-2have alreadybeen applied in the clinical treatment of breast cancer, but the efficiency wasvery low, so it is very imperative to explore new therapeutic target for thetreatment of breast cancer.Some investigators have found that EGFR is also an indicator of poorprognosis in patients with breast cancer. Over-expression of EGFR wasidentified in35%～50%breast cancer patients. The frequency of EGFRpositivity in triple negative breast cancers is60%. After binding with EGF,EGFR dimerization and tyrosine phosphorylation lead to activate theMEK/ERK signal pathway which involved in the breast cancer cell proliferation, migration and invasion.Caveolin-1is a principal component of caveolae, invaginations of theplasma membrane that are enriched in cholesterol and sphingolipids. Studieshave shown that caveolin-1acts as a tumor suppressor protein in human breastcancer, but the mechanisms has not been completely elucidated. Caveolin-1associates and interacts with a variety of signaling molecules via thecaveolin-scaffolding domain. Previous evidence suggested that Caveolin-1binds directly to EGFR and inhibits its phosphorylation. Whether Caveolin-1binds directly to HER-2remains unclear.The aim of this study is to investigate the role of Caveolin-1on the cellproliferation through regulating EGFR and HER-2phosphorylation in breastcancer cells.Methods:1Cells cultureHuman breast carcinoma cells MDA-MB-453, MDA-MB-436, SKBR-3were cultured at37℃,5%CO2in1640media containing10%fetal calf serum.2The invasion capacity of different breast cancer cellsHuman breast cancer cells MDA-MB-453, MDA-MB-436, SKBR-3werecultured into35mm dish, then culture cells24hours to confluence (80%-90%).After that, cells have been cultured in1640media for24hours without fetalcalf serum. The activities of MMP2and MMP9in different breast cancer cellswere examined by Gelatin zymography.3The migration of different breast cancer cellsHuman breast cancer cells MDA-MB-453, MDA-MB-436, SKBR-3werecultured into35mm dish, Cells have been cultured to confluence (80%-90%),then in1640media without fetal calf serum for4hours. After scratch a line onthe dish using a sterile200ul pipet, the cells have been observed under10×inverted microscope at6and12hours later separately4The expression of Caveolin-1, EGFR, HER-2proteins in different breastcancer cellsHuman breast cancer cells MDA-MB-453, MDA-MB-436, SKBR-3were cultured into35mm dish, Cells have been cultured to confluence (80%), thenculture them in1640media without fetal calf serum for4hours. Aftercollected and extracted protein, the expression levels of Caveolin-1, EGFR,HER-2were assessed by western-blot.5The localization of Caveolin-1and EGFR in breast cancer cellsHuman breast cancer MDA-MB-436cells were cultured into35mm dishthat equipped with a sterile coverslip. Immunocytochemical stain has beenused to double stain cells, then the localization of Caveolin-1and EGFR wasobserved by Confocal Microscopy.6Effects of Caveolin-1silencing or overexpression on EGFR, HER-2andERK phosphorylation in breast cancer cell linesBreast cancer cells were cultured in35mm dishs. Experimental groupswere as following: MDA-MB-453and SKBR-3cells were divided intoCaveolin-1pcDNA and pcDNA3.1transfection groups; MDA-MB-436cellswere divided into Caveolin-1siRNA and Shut transfection groups. Cells ineach group were stimulated by EGF for omin,5min,10min,30min, after thatcells were collected and proteins were extracted. The expression of Caveolin-1,HER-2, p-HER-2, EGFR, p-EGFR, ERK, p-ERK were assessed by WesternBlot.7Effects of Caveolin-1silencing or overexpression on cell proliferation inbreast cancer cell linesBreast cancer cells were cultured in96well plates. Experimental groupswere as following: MDA-MB-453and SKBR-3cells were divided intoCaveolin-1pcDNA and pcDNA3.1transfection groups; MDA-MB-436cellswere divided into Caveolin-1siRNA and Shut1transfection groups. Cell ineach group were stimulated by EGF(100nM) for24hours, the cellsproliferation were examined by MTT colorimetric assay.Result:1The invasion capacity of different breast cancer cellsThe activity of MMP9in breast cancer MDA-MB-436(1.44±0.15)cellswas higher than that in MDA-MB-453(0.31±0.04) and SKBR-3(0.37±0.08) cells, the different was statistically significant(P＜0.01). The activity ofMMP2in breast cancer MDA-MB-436(1.65±0.37)cells was higher than thatin MDA-MB-453(0.40±0.11) and SKBR-3(0.38±0.09) cells, the differencewas statistically significant(P＜0.01).2The migration rate of different breast cancer cellsAfter scratching, there was no statistically significant difference in themigration rates at6h,12h between MDA-MB-453(1.82±0.79,3.37±1.36) andSKBR-3(2.41±0.92,4.52±0.89) breast cancer cells, but they were both lowerthan that in MDA-MB-436(18.71±4.06,36.80±3.42) cells, the difference werestatistically significant(P＜0.01, P＜0.01), respectively.3The expression of Caveolin-1, EGFR, HER-2proteins in different breastcancer cellsThe expression of Caveolin-1in MDA-MB-436(2.40±0.39) cells washigher than that in MDA-MB-453(0.03±0.01)cells and SKBR-3(0.03±0.00)cells, the difference was statistically significant(P＜0.01). The expression ofHER-2in MDA-MB-453(1.05±0.13) cells and SKBR-3(2.12±0.32)cells washigher than that in MDA-MB-436(0.03±0.02) cells, the difference werestatistically significant(P＜0.01, P＜0.01), respectively. The expression ofEGFR in MDA-MB-436(0.75±0.07) cells and SKBR-3(0.97±0.10) cells washigher than that in MDA-MB-453(0.01±0.00)cells, the difference wasstatistically significant(P＜0.05).4The localization of Caveolin-1and EGFR in breast cancer cellsConfocal microscopy observation showed that Caveolin-1and EGFR areco-localized in the membrane of MDA-MB-436cells.5Effects of Caveolin-1silencing or overexpression on EGFR, HER-2andERK phosphorylation in breast cancer cell lines5.1Effects of Caveolin-1overexpression on EGFR and HER-2phosphorylation in breast cancer cellsThe expression of Caveolin-1in MDA-MB-453cells transfected withCaveolin-1pcDNA plasmids were higher than that in pcDNA3.1transfectiongroups. After stimulating by EGF (20nM) for5min,10min,30min, the expression of p-HER-2(0.48±0.05vs0.76±0.03,0.57±0.09vs0.87±0.06,0.40±0.02vs0.65±0.12); p-ERK(0.65±0.04vs1.11±0.02,0.84±0.03vs1.32±0.05,0.07±0.02vs0.16±0.02)were lower compared with pcDNA3.1transfection groups(P＜0.05, respectively).The expression of Caveolin-1in SKBR-3cells transfected withCaveolin-1pcDNA plasmids were higher than that in pcDNA3.1transfectiongroups. After stimulating by EGF (20nM) for5min,10min,30min, theexpression of p-HER-2(0.32±0.02,0.43±0.03,0.19±0.03), p-EGFR(0.24±0.02,0.37±0.03,0.13±0.02), p-ERK (0.06±0.02,0.40±0.16,0.36±0.04) were lower than pcDNA3.1transfection group p-HER-2(0.45±0.02,0.55±0.03,0.28±0.02), p-EGFR(0.55±0.03,0.68±0.03,0.25±0.02), p-ERK(0.40±0.02,0.93±0.09,0.45±0.03), the differences werestatistically significant(P＜0.05, respectively).5.2Effect of Caveolin-1silencing on EGFR phosphorylation in breastcancer cellsThe expression of Caveolin-1in MDA-MB-436cells transfected withCaveolin-1siRNA plasmids were lower than in Shut1transfection groups.After stimulating by EGF(20nM) for5min,10min,30min, the expressionsof p-EGFR(1.15±0.02,1.27±0.02,0.72±0.03), p-ERK(0.71±0.02,0.85±0.02,0.61±0.03)were higher than in Shut1transfection groups p-EGFR(0.53±0.02,0.66±0.03,0.20±0.02), p-ERK(0.41±0.02,0.50±0.03,0.31±0.01), thedifferences were statistically significant(P＜0.01, respectively).6Effects of Caveolin-1silencing or overexpression on cell proliferation inbreast cancer cell linesCaveolin-1pcDNA plasmid transfection breast cancer MDA-MB-453andSKBR-3cells, Caveolin-1siRNA plasmid transfection breast cancerMDA-MB-436cells,each group was stimulated by100nM EGF. Whencompared with pcDNA3.1transfection groups, the proliferation levels ofCaveolin-1pcDNA transfection groups (0.87±0.16vs1.31±0.08) inMDA-MB-453, Caveolin-1pcDNA transfection groups (0.99±0.18vs1.29±0.05)in SKBR-3cells were lower(P＜0.05, respectively). The proliferation levels of Caveolin-1siRNA transfection group(s0.84±0.03)werehigher than Shut1transfection groups (0.68±0.03)(P＜0.01).Conclusion:1. In breast cancer cells, the expression of EGFR and HER-2proteins do notrepresent the activation of EGFR and HER-2.2. Caveolin-1influences the proliferation through regulating the EGFR,HER-2phosphorylation and the activity of downstream signal transductionpathway (Ras/Raf/MEK/ERK) in breast cancer cells.