Involvement Of EGFR And UCH-L1 In Regulation Of Drug Resistance, Proliferation, Invasion And Migration In Human Breast Cancer Cells And Their Related Mechanisms

Epidermal growth factor receptor (EGFR) is a member of HER family which can be activated by binding of epidermal growth factor (EGF). EGFR overexpression can lead to increase tumor cell proliferation, angiogenesis, invasion, metastasis and inhibition of apoptosis. Our previous researches suggested:â‘ EGFR expression appears higher level in MCF7/Adr breast cancer cells (adriamycin-resistant MCF7, multidrug resistant breast cancer cells) than in its sensitve MCF7 cells.â‘¡Treatment of (multidrug resistant) MCF7/Adr cells with P-gp substrates could up-regulate the production of EMMPRIN and MMPs, and enhance migratory/invasive abilities in vitro. Moreover, it is too observed that above pathway could be blocked by EGFR inhibitor. These data suggest that EGFR may act as a key role in modulating in vitro metastases in multidrug resistant breast cancer cells. So far, the effect of EGFR expression on multidrug resistant breast cancer cells is not reported.Chemotherapy is one of the major modalities in breast cancer treatment, however, frequent occurrence of drug resistance leads to therapy failure. The principal mechanism of multidrug resistance is the high-expression of the superfamily of ABC (ATP-binding-cassette)-transporters including P-gp, ABCG2 (Breast Cancer Resistance Protein, BCRP), their production can efflux different chemotherapy drugs out of tumor cells, which leads the low concentration of drugs and treatment failure. Our previous study showed that treatment of multidrug resistant MCF7/Adr breast cancer cells with P-gp substrates could up-regulate the production of EGFR, indicating that there maybe some relationship between multidrug resistance and EGFR high-expression. Meanwhile, we also found the invasive ability of MCF7 in Gl/S phase was extremely up-regulated after adriamycin inducement, indicating that the cell cycles change was not only affect tumor migration and invasion, but not play an import role in drugs resistance. The inhibition of EGFR by AG1478 can effectively change the distribution of cell cycles in leiomyoma. Moreover, our previous results showed the proliferative ability of MCF7/Adr was extremely higher than that of MCF7 cells, which may be influenced by triggering rapid G1/S transition. Therefore, in this study, we will focus on find the role of EGFR in regulating the cell cycles in MCF7/Adr cells, and try to clarify the relationship of this role to the multidrug resistance.Ubiquitin carboxy terminal hydrolase 1 (UCH-L1) is a member of UCHs family. In addition to the function of deubiquitination, it can also act as an ubquitin ligase and stabilize mono-ubiquitin. Our recent research observed:â‘ UCH-L1 expression appears higher level in MCF7/Adr breast cancer cells than in its sensitve MCF7 cells.â‘¡during the development of drug resistance to adriamycin in MCF7 cells, the promoted expression of UCH-L1 show positive relation with the elevation of drug resistance as well as enhanced cells invasive and migratory abilities;â‘¢UCH-L1 possess a significant effect on modulating drug resistance, invasion and migration in human breast cancer cells. This effect is carried out by regulating the expression of P-gp, EMMPRIN and MMPs through the ubiquitination and degradation of P-gp and EMMPRIN. EGFR mono-ubiquitination is necessary for its internalization and degradation, and blocking the Ubiquitin-Proteasome Pathway (UPP) can effectively inhibit the internalization of EGFR, while the involvement of deubiquitination in EGFR degradation in multidrug resistant breast cancer cells was not reported. As the high expression of UCH-L1 and EGFR appear in the same cell lines in our recent research, we suppose that the degradation of EGFR maybe is affected by UPP.Since that, we suppose:â‘ EGFR promote the abilities of migration, invasion and proliferation in multidrug resistant breast cancer cells;â‘¡EGFR affect the chemotherapy drugs-sensitivity of multidrug resistant breast cancer cells via regulating cell cycles;â‘¢PP is involved in the degradation of EGFR in multidrug resistant breast cancer cells. In order to confirm above hypothesis, firstly, we transfected EGFR siRNA and pcDNA3.0-EGFR plasmid into MCF7/Adr and MCF7 cells, respectively, to clarify the role of EGFR in regulation of drug resistance, proliferation, invasion and migration and its related mechanisms. Second, to explore the function of EGFR in mediating the multidrug resistance via regulating cell cycles, we synchronized MCF7 cells at different phases and observed the change of drug sensitivity and expression of production of ABC transporters. In addition, we also tested the UCH-L1 and EGFR protein expression under treatment of P-gp substrates, and observing the change of EGFR in MCF7/Adr cells after blocking UPP as well as that in MCF cells which was transfected in pIRES2-UCH-L1-EGFP (Enhanced Green Fluorecent protein), try to find the linkage between the ubiquitin-proteasome degradation and EGF-EGFR pathway. This study not only possess obviously theoretical significance for understanding the biological characteristics of tumors, but also get significantly clinical and social effects.Part I Promotion of Migration, Invasion and Proliferation by EGFR in Multidrug Resistant Breast Cancer CellsPurpose To investigate the effect of EGFR on migration, invasion and proliferation of multidrug resistant breast cancer cells.Methods EGFR siRNA duplex oligoribonucleotides and pcDNA3.0-EGFR eukaryon expression plasmid were transfected into MCF7/Adr and MCF7 cells, respectively. Immunofluorescence was used to detect transfection efficiency. Transwell assay was utilized to evaluate the effects of EGFR on migration and invasion. Western blot were used to analyze protein expression of EGFR, EMMPRTN and MMPs. MTT, flow cytometry analysis were used to analyze cells growth and apoptosis.Results Reducing the expression of EGFR by RNA interference in MCF7/Adr cells decreased protein expressions of EGFR, EMMPRIN, MMP2 and MMP9, and also reduced cells in vitro invasive, migrative and proliferative abilities. Transfected with pcDNA3.0-EGFR plasmid increased protein expressions of EGFR, EMMPRIN, MMP2 and MMP9 in MCF7 cells, and also enhanced cells in vitro invasive, migrative and proliferative abilities.Conclusions EGFR promotes the proliferation of multidrug resistant breast cancer cell and up-regulates migration and invasion via EMMPRIN and MMPs in multidrug resistant breast cancer cells.Part II EGFR mediated Multidrug Resistance in breast cancer cells via Regulating Cell Cycles. Purpose To investigate the relationship between EGFR expression and multidrug resistance in human breast cancer cells and the underlying mechanisms.Methods EGFR siRNA duplex oligoribonucleotides and pcDNA3.0-EGFR eukaryon expression plasmid were transfected into MCF7/Adr and MCF7 cells, respectively. Flow cytometry analysis was used to analyze cell cycles. MTT assay was used to detect drug sensitivity. Serum-free starving and double thymidine block methods were used to cell synchronization. Western blot were used to analyze protein expression of P-gp, ABCG2, MRP1, cyclin D1, CDK4, p21, p27 and PCNA.Results Reducing the expression of EGFR by RNA interference in MCF7/Adr cells decreased accumulation of cells in S-phase, whereas the percentage of cells in G1-phase increased. After interference, the expression of P-gp, ABCG2, cyclin D1,CDK4 and drug resistance activities were down-regulated and p21, p27 were up-regulated as well, but the expression of MRP1 and PCNA were uninfluenced. Transfected with pcDNA3.0-EGFR plasmid promoted accumulation of cells in S-phase,whereas the percentage of cells in G1-phase decreased. After transfection, the expression of P-gp, ABCG2, cyclin D1,CDK4 and drug resistance activities were up-regulated and p21, p27 were down-regulated as well, but the expression of MRP 1 and PCNA were also uninfluenced. The expression level of P-gp, ABCG2, EMMPRIN, MMPs and the drug resistance activities of cells which were synchronizated in S-phase were much higher than that in G1 phase and unsyncronization group.Conclusions EGFR promoted G1/S transition via up-regulating cyclin D1, CDK4 and down-regulating p21, p27 to increase the expression of P-gp, ABCG2, which enhance the multidrug resistance of breast cancer cells in vitro.Partâ…¢UCH-L1 affect the Expression of EGFR in Human Breast Cancer Cells.Purpose To clarify whether P-gp substrates promote UCH-L1 and EGFR expression in multidrug resistant breast cancer cells and elucidate the relationship between EGFR and ubiquitin-proteasome pathway.Methods The expression of UCH-L1, EGFR, EMMPRIN, MMP2 and MMP9 were evaluated in MDR human breast cancer cell line, MCF7/Adr, and its sensitive parental line, MCF7 after treated with various concentrations of P-glycoprotein substrates (adriamycin, paclitaxel) and P-glycoprotein nonsubstrate (bleomycin) in cell culture media. EGFR expression was observed in MCF7 and MCF7/Adr cells which were transfected with pIRES2-UCH-L1-EGFP (EGFP:enhanced green Fluorescent Protein) plasmid or add ubiquitin-proteasome inhibitor MG132, respectively.Results Increased production of UCH-L 1, EGFR, EMMPRIN, MMP2 and MMP9 were observed only in multidrug resistant cancer cells exposed to P-gp substrates. We also found EGFR expression level was increased not only in pIRES2-UCH-L1-EGFP plasmid transfected MCF7 cells, but also in ubiquitin-proteasome inhibited MCF7/Adr cells.Conclusions Treatment of MCF7/Adr cells with P-gp substrates could adversely affect therapeutic outcomes through modulating the production of UCH-L1, EGFR, EMMPRIN, MMP2 and MMP9. The ubiquitin-proteasome pathyway may involved in the process of EGFR degradation.