【作者】 徐嘉雯；

【导师】 许祖德；

【作者基本信息】 复旦大学 ， 病理学， 2011， 博士

【摘要】 表皮生长因子受体(epidermal growth factor receptor, EGFR)是表皮生长因子(EGF)参与细胞增殖和信号传导的受体,属于HER家族的成员之一。EGFR的高表达与肿瘤细胞增殖、血管生成、肿瘤侵袭、转移及细胞凋亡的抑制有关。本课题组前期研究发现：①EGFR在阿霉素耐药乳腺癌细胞株MCF7/Adr (adriamycin resistant MCF7)中高表达,但在其相应敏感株MCF7中表达量很低；②P-糖蛋白(P-glycoprotein, P-gp)相关化疗药物在体外可以增强耐药乳腺癌细胞株MCF7/Adr中细胞外基质金属蛋白酶诱导物(extracellular matrix metalloproteinase inducer, EMMPRIN)和基质金属蛋白酶(matrix metal loproteinases, MMPs)的基因转录和蛋白表达,增强耐药乳腺癌细胞的侵袭能力,而该过程可以被EGFR抑制剂阻断。提示EGFR在调控耐药乳腺癌细胞的侵袭迁移能力方面发挥着重要的作用,但具体作用及其机制国内外尚未见系统性的研究报道。化疗耐药是乳腺癌治疗面临的最大难题之一,其中最重要的机制即为ATP结合盒膜转运蛋白超家族的表达,包括P-gp、乳腺癌耐药蛋白(Breast Cancer Resistance Protein, BCRP,又名ABCG2)等,其产物具有ATP依赖性的药物泵作用,能将多种结构和作用机制不同的化疗药物转运出细胞,使细胞内药物浓度降低,导致耐药。本课题组前期发现在耐药乳腺癌细胞株MCF7/Adr培养基中加入不同浓度的P-gp底物化疗药物后,EGFR的表达水平明显高于对照组,说明乳腺癌化疗药物耐药与EGFR之间也许存在着某些关联；我们同时也发现处于不同细胞周期的MCF7细胞在P-gp底物化疗药物诱导下侵袭迁移能力不同,提示细胞周期的改变不仅与肿瘤的侵袭能力有关,而且可能参与了化疗药物的耐受调控。有文献报道在加入EGFR酪氨酸激酶抑制剂AG1478后平滑肌瘤细胞周期分布发生明显改变,但EGFR在此过程中作用的具体机制未见报道。我们早期发现阿霉素耐药乳腺癌细胞株MCF7/Adr的增殖能力明显高于敏感株MCF7,通常这是由于细胞周期G1/S转换加快导致的,所以我们将致力于阐明EGFR在介导乳腺癌耐药细胞周期改变中发挥的作用,并且探索其是否与化疗药物的耐受性有关。泛素羧基末端水解酶1 (ubiquitin carboxy terminal hydrolase 1, UCH-L1)为泛素羧基末端水解酶家族(ubiquitin carboxy terminal hydrolases, UCHs)中的一个成员,它除了传统认为的去泛素化作用外,还具有泛素连接酶和稳定细胞内泛素单体的功能。我们课题组的前期实验中发现：①耐药乳腺癌细胞株MCF7/Adr细胞比敏感株MCF7细胞表达UCH-L1水平较高；②在阿霉素诱导MCF7细胞耐药过程中,UCH-L1的表达上调与细胞耐药性的增强和侵袭、迁移能力的提高正相关；③UCH-L1可以通过调节P-gp、EMMPRIN和MMPs的表达及P-gp和EMMPRIN的泛素化降解,参与调控人乳腺癌细胞的耐药和侵袭迁移。有文献报道,EGFR的单泛素化对于诱导其内化和降解是必需的,阻断泛素化的过程可以阻抑EGFR的内化,而去泛素化酶是否参与了耐药乳腺癌中EGFR的降解及其相关机制未见系统报道。基于UCH-L1与EGFR在耐药乳腺癌细胞MCF7/Adr和敏感株MCF7中表达水平的对应性,我们推测耐药乳腺癌细胞中EGFR的降解可能受到了泛素蛋白酶体途径调节。根据上述陈述,我们有以下几个推测：①EGFR参与人耐药乳腺癌细胞的侵袭迁移能力的调控；②EGFR促进了细胞周期的转换,最终导致乳腺癌细胞耐药性的改变；③耐药乳腺癌细胞中EGFR的降解受到了泛素蛋白酶体途径的调节。为了证实我们的推测,本课题设想通过EGFR siRNA干扰CF7/Adr细胞和pcDNA3.0-EGFR质粒转染MCF7细胞,阐明EGFR对人耐药乳腺癌细胞增殖、侵袭迁移、化疗耐受性和耐药相关蛋白的影响及其机制；将MCF7细胞同步化于不同的细胞周期观察化疗药物敏感性和耐药相关蛋白的变化,验证EGFR是否可以通过调控细胞周期来介导人乳腺癌细胞耐药性的改变；并进一步观察P-gp底物化疗药物作用下UCH-L1和EGFR的表达,通过pIRES2-UCH-L1-EGFP (Enhanced Green Fluorecent protein, EGFP,增强型绿色荧光蛋白)质粒转染MCF7细胞和在MCF7/Adr细胞加入泛素蛋白酶体抑制剂MG-132阻断泛素蛋白酶降解途径来观察EGFR的改变,初步探讨UCH-L1对EGFR的调控作用、泛素降解系统对EGF-EGFR的调节及其可能的机制。以上研究有助于进一步明确肿瘤耐药和浸润转移的相关关系及其调控机制,为临床乳腺癌治疗中EGFR靶向治疗使用提供理论支持,故本研究是一项具有显著的临床和社会效益的重大课题。第一部分EGFR促进人耐药乳腺癌细胞的侵袭迁移和增殖目的明确EGFR对人耐药乳腺癌细胞侵袭、迁移和增殖的影响。方法采用EGFR siRNA双链寡核糖核苷酸干扰耐药乳腺癌细胞MCF7/Adr和pcDNA3.0-EGFR真核表达质粒瞬时转染敏感乳腺癌MCF7细胞,应用细胞免疫荧光法检测转染效率；应用transwell侵袭实验研究EGFR对人乳腺癌细胞体外侵袭、迁移运动能力的影响；应用western blot的方法检测EGFR、EMMPRIN、MMPs的表达；应用细胞生长曲线法、流式细胞仪检测细胞的生长和凋亡。结果EGFR siRNA干扰可以下调耐药乳腺癌细胞MCF7/Adr细胞内EGFR、EMMPRIN、MMP2和MMP9的蛋白表达,同时细胞体外侵袭、迁移能力均有所减弱,生长速度显著减慢；而进行转染pcDNA3.0-EGFR质粒可以上调MCF7细胞内EGFR、EMMPRIN、MMP2和MMP9的蛋白表达,同时细胞体外侵袭、迁移能力也均有所增强,生长速度显著加快。小结EGFR可以促进人耐药乳腺癌细胞的增殖,并通过上调EMMPRIN、MMP2和MMP9的表达,增强人耐药乳腺癌细胞的侵袭转移能力。第二部分EGFR通过调控细胞周期介导人乳腺癌细胞化疗耐药性的改变目的明确EGFR与人乳腺癌细胞化疗耐药之间的关系及其机制。方法采用EGFR siRNA双链寡核糖核苷酸干扰耐药乳腺癌细胞MCF7/Adr和pcDNA3.0-EGFR真核表达质粒瞬时转染敏感乳腺癌MCF7细胞；应用流式细胞仪检测细胞周期；应用MTT检测药敏活性；应用血清饥饿法和胸腺嘧啶双阻断法分别同步化细胞周期于G1和S期;应用western blot的方法检测P-gp、ABCG2、MRP1、cyclinD1、CDK4、p21、p27和PCNA的表达。结果进行EGFR siRNA干扰后的MCF7/Adr细胞内S期比例明显下降,G1期比例明显增高,P-gp、ABCG2、cyclinD1、CDK4的蛋白表达明显下调,p21和p27的表达水平明显上调,细胞对阿霉素和紫杉醇的耐受性明显降低,MRP1和PCNA表达水平未见明显改变；转染pcDNA3.0-EGFR质粒后MCF7细胞内S期比例明显增高,G1期比例明显下降,P-gp、ABCG2、cyclin D1、CDK4的蛋白表达明显上调,p21和p27的表达水平明显下调,细胞对阿霉素和紫杉醇的耐受性明显增高,但MRP1和PCNA表达同样未见明显改变。将MCF7细胞分别同步化于G1和S期后,S期的细胞内P-gp、ABCG2的表达水平以及对阿霉素和紫杉醇的耐药性也明显高于G1期和未同步化对照组。小结EGFR通过上调cyclin D1、CDK4,下调p21和p27促进了细胞周期G1/S的转换,使S期细胞比例升高,从而促进了P-gp、ABCG2的表达,增强人乳腺癌细胞耐药性。第三部分UCH-L1对人耐药乳腺癌细胞中EGFR表达的调控目的初步探讨P-gp底物化疗药物对耐药乳腺癌细胞UCH-L1和EGFR表达的影响,并明确EGFR的降解是否受到泛素蛋白酶体途径的调节。方法在耐药乳腺癌细胞株MCF7/Adr及其敏感乳腺癌细胞株MCF7培养过程中分别加入不同剂量的P-gp底物化疗药物(阿霉素和紫杉醇)以及非P-gp底物化疗药物(博莱霉素),检测癌细胞UCH-L1、EGFR、EMMPRIN、MMP2和MMP9表达水平的改变；采用pIRES2-UCH-L1-EGFP (EGFP:增强型绿色荧光蛋白)真核表达质粒瞬时转染MCF7细胞和在MCF7/Adr细胞中加入泛素蛋白酶体抑制剂MG-132,观察EGFR表达量的变化。结果分别加入不同浓度的阿霉素和紫杉醇之后,耐药乳腺癌细胞株MCF7/Adr中的UCH-L1、EGFR、EMMPRIN、MMP2和MMP9蛋白水平显著上调,这种作用不具有剂量依赖性,而敏感乳腺癌细胞株MCF7没有相应的变化。加入博莱霉素之后,无论MCF7/Adr还是MCF7细胞中的上述各项检测指标均没有显著改变；转染pIRES2-UCH-L1-EGFP后MCF7细胞内的EGFR表达量明显增高；并且与未加药组和溶剂组相比,在耐药乳腺癌细胞株MCF7/Adr中加入泛素蛋白酶体抑制剂MG-132后EGFR的表达量也有显著增加。小结P-gp底物化疗药物可以通过上调耐药乳腺癌细胞UCH-L1、EGFR、EMMPRIN、MMP2和MMP9的表达,增强肿瘤细胞侵袭转移能力；EGFR的降解可能受到了泛素蛋白酶体途径的调节。更多还原

【Abstract】 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:①EGFR expression appears higher level in MCF7/Adr breast cancer cells (adriamycin-resistant MCF7, multidrug resistant breast cancer cells) than in its sensitve MCF7 cells.②Treatment 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:①UCH-L1 expression appears higher level in MCF7/Adr breast cancer cells than in its sensitve MCF7 cells.②during 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;③UCH-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:①EGFR promote the abilities of migration, invasion and proliferation in multidrug resistant breast cancer cells;②EGFR affect the chemotherapy drugs-sensitivity of multidrug resistant breast cancer cells via regulating cell cycles;③PP 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.PartⅢUCH-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.更多还原