【作者】 苏庆波；

【导师】 胡三元；

【作者基本信息】 山东大学 ， 外科学， 2009， 博士

【摘要】 研究背景乳腺癌是危及女性的最常见的恶性肿瘤之一,其发病率在欧美发达国家多居女性全部肿瘤的首位,国内统计,近年来我国乳腺癌的发病率持续明显上升,目前已跃居我国女性恶性肿瘤的第二位,且发病年龄呈年轻化。随着对乳腺癌生物学特性的深入研究,对乳腺癌的认识也有了全新的概念。目前研究认为乳腺癌自发病即为全身性疾病,其治疗观念由最初的单纯手术治疗发展到以手术为主辅以放疗化疗内分泌治疗,再到目前的综合治疗理论。综合治疗理论中,手术治疗的主导地位被动摇,而内分泌治疗因治疗时间长且效应持久,已超越化疗成为乳腺癌最重要的治疗手段之一。多项研究证明,内分泌治疗的应用大大提高了早期乳腺癌的治疗效果,而内分泌治疗在晚期乳腺癌的合理应用,也显著改善了晚期患者的生活质量并延长了患者的生存期。1896年Beatson首先将卵巢切除术用于治疗绝经期前妇女的乳腺癌,开创了乳腺癌内分泌治疗的先河。上世纪60年代,Toft分离出小鼠雌激素受体（ER）蛋白,70年代ICI46 474（Tamoxifen）被发现对乳腺癌具有治疗作用。三苯氧胺于1977年被FDA批准用于进展期乳腺癌,于1986年用于原发性乳腺癌的辅助治疗,在1990年用于淋巴结阴性的绝经前和绝经后乳腺癌的辅助治疗。对于ER阳性的乳腺癌患者,三苯氧胺可以使年复发率下降50%,死亡率下降28%。以三苯氧胺为代表的选择性雌激素受体调节剂应用于乳腺癌已有30余年的历史,是所有激素受体阳性乳腺癌内分泌治疗的首选药物,并一直作为乳腺癌内分泌治疗的金标准。三苯氧胺和雌激素（E）竞争性与ER的激素结合区结合后,形成激素-受体复合物,引起ER构像改变,使DNA结合区不能暴露,不能激活基因转录,从而拮抗E的许多活性。三苯氧胺可应用于ER（+）、淋巴结阴性和/或阳性的乳腺癌辅助内分泌治疗,也可用作高危妇女预防乳腺癌。目前发现了多种新的内分泌治疗药物,但三苯氧胺的标准治疗的地位仍无法被完全取代。三苯氧胺与ER的特性仍是当前研究热点之一。一项大规模随机临床试验证实,有数十万应用三苯氧胺的乳腺癌患者至今仍保持无病存活。另有研究报道,ER阳性乳腺癌术后辅助内分泌治疗效果优于或等于化疗。然而,三苯氧胺敏感的ER阳性患者仍可能发生三苯氧胺耐药。研究发现ER（+）的乳腺癌患者经过5年的三苯氧胺辅助治疗,可获得长期生存率,其效果可以延续到停药后5年。但是超过5年的使用将使乳腺癌产生耐药。临床上也只有50%的ER（+）的乳腺癌患者起始对抗激素治疗有效,之后也会逐渐耐药,导致肿瘤进展和死亡。因此,研究内分泌治疗耐药的机制、寻找克服耐药的方法有十分重要的临床意义。乳腺癌扩增性抗原1（amplified in breast cancer 1,AIBI）为近年来发现的细胞核激素受体共激活剂P160 SRC家族成员,是新定义的一个原癌基因。AIBI基因定位于人染色体20q12,因最初发现于人乳腺癌细胞株扩增的染色体区,故被命名为乳腺癌扩增性抗原1（AIB1）。体外转染实验显示P160甾体激素受体共激活剂基因家族能增强细胞核受体（nuclear receptor,NR）的活性,通过补充组蛋白乙酰转移酶和组蛋白甲基转移酶发挥重要作用。研究显示AIBI在许多生物学过程中起重要作用,如细胞的增殖、迁移、分化、性成熟以及一些癌症的发生发展。但是,目前关于AIBI功能的作用机制还知之甚少。在人上皮性肿瘤进展过程中,AIB1是SRC家族中唯一扩增和高表达的成员,它能增强三苯氧胺的雌激素样作用。AIB1还是重要的ER辅助激活因子,在转基因动物模型中高表达时,具有促进肿瘤形成的作用。AIB1丢失后通过抑制受体的降解和灭活转录的启动而影响ER的信号途径。作为ER的辅助激活因子,AIB1只在ER阳性的乳腺癌细胞系中表达,而在ER阴性的乳腺癌细胞系中均不表达。临床研究也证实AIB1普遍存在于ER阳性的肿瘤中。而且,AIB1与ERα成正相关,与ERβ成负相关。激素依赖性乳腺癌中,E与ER结合是促进乳腺癌增殖作用的主要途径,然而除了E-ER信号途径之外,EGFR家族的信号途径也是乳腺癌发病的另一个重要机制。HER2作为EGFR家族的重要成员之一,是广泛应用于临床的预测乳腺癌患者内分泌治疗和肿瘤复发的肿瘤标记物。Kent Osborne等已证实AIB1和HER2共同作用降低三苯氧胺对乳腺癌病人的疗效。他们对316例乳腺癌患者术后进行长期随访,发现没用三苯氧胺治疗的AIB1高表达的乳腺癌患者预后较好,无病生存期较长。相反,接受三苯氧胺治疗的AIB1高表达的乳腺癌患者无病生存期短。研究结果表明AIB1和三苯氧胺之间存在密切的相互作用。另有研究表明在AIB1和HER2高表达的乳腺癌患者中,HER2通过激活MAPK,而使AIB1和ER磷酸化,导致三苯氧胺的抗雌激素作用明显减弱,而雌激素作用增强,从而导致耐药。这不仅使三苯氧胺的抗肿瘤作用消失,而且因三苯氧胺的雌激素作用还加快了肿瘤的生长。最近报道AIB1在HER1-3阳性的乳腺癌中表达可以预测乳腺癌的早期复发和死亡。这对乳腺癌的内分泌治疗及预后具有重要的指导意义。这些研究均表明AIB1在乳腺癌抗雌激素治疗耐药中发挥重要作用。然而,到目前为止人们还没有弄清楚AIB1是如何将三苯氧胺的雌激素拮抗作用转为激活作用的。本课题的目的就是为了阐明在AIB1和HER2高表达的雌激素受体阳性的乳腺癌中三苯氧胺的耐药机制。我们研究了在雌激素受体阳性的肿瘤细胞中AIB1对三苯氧胺诱导的雌激素样作用,从而为其临床应用提供试验基础。第一部分:AIB1基因的RNAj真核表达载体的构建及鉴定目的利用RNA干扰技术,构建针对AIB1的shRNA真核表达载体。方法（1）根据AIB1特异的siRNA序列和阴性对照干扰序列合成相应的4条寡核苷酸;（2）将两两互补的四条寡核苷酸链进行磷酸化,并退火连接形成互补的双链;（3）将质粒pGenesil-1-U6用BamHⅠ和HindⅢ双酶切,使其线性化;（4）用T4 DNA连接酶将互补的siRNA模板链与线性化的质粒载体连接;（5）用大肠杆菌DH5α制备感受态细胞,将连接好的重组质粒转化到感受态细胞内;（6）扩增大肠杆菌,用质粒提取试剂盒提取细菌内的质粒;（7）提取到的质粒DNA用琼脂糖凝胶电泳和紫外分光光度计测其浓度和纯度;（8）重组质粒用SalI酶切鉴定,酶切产物行琼脂糖凝胶电泳;（9）将经酶切鉴定的阳性菌液收集1ml,交由武汉晶赛生物技术公司行DNA测序。结果（1）构建的pGenesil-shAIB1和pGenesil-shControl的质粒表达载体,用SalI酶切后行琼脂糖凝胶电泳,紫外线下可看到一条长约400bp的DNA条带,说明我们构建的质粒表达载体符合设计要求。（2）将酶切鉴定的阳性菌液送武汉晶赛生物公司行DNA测序,测序结果证实构建的质粒载体含有插入的目的片段,与我们设计的序列完全一致。结论成功构建了目的基因pGenesil-shAIB1和阴性对照pGenesil-shControl的质粒表达载体。第二部分:AIB1在ER阳性的乳腺癌细胞中对三苯氧胺耐药的作用机制目的（1）研究AIB1的RNAi表达载体在BT474乳腺癌细胞中对AIB1基因的抑制作用;（2）观察RNAi沉默AIB1基因表达后,乳腺癌BT474细胞生物学行为的变化,并通过测定一些肿瘤标记物的表达水平,探讨AIB1在ER阳性乳腺癌细胞中对三苯氧胺耐药的作用机制。方法（1）常规培养人乳腺癌BT474细胞,利用Lipofectamine 2000将pshAIB1载体和pshControl载体转染入BT474细胞中,用G418筛选出稳定表达AIB1 shRNA的细胞株;（2）用RT-PCR和Western blot法检测BT474/shAIB1和BT474/shControl细胞中AIB1 mRNA和蛋白的表达水平;（3）台盼蓝拒染测定BT474/shAIB1和BT474/shControl细胞的的增殖情况,并绘制细胞生长曲线;（4）测定MCF-7,BT474,BT474/shAIB1和BT474/shControl四种细胞在不同浓度的Tam刺激下的增殖情况,以确定各细胞对Tam的敏感性;并检测干扰前后细胞对E₂的敏感性变化;（5）流式细胞仪检测BT474/shAIB1和BT474/shControl细胞在激素和无激素环境中细胞周期的分布情况;（6） Western blot检测BT474细胞在干扰前后及Tam和E₂刺激下AIB1,ERα,HER2和pS2蛋白的表达变化。结果（1）经Lipofectamine 2000介导成功将pshAIB1和pshControl质粒载体转染入BT474细胞,并用G418筛选获得阳性克隆细胞株。（2）与BT474和BT474/shControl细胞相比,BT474/shAIB1细胞中AIB1mRNA和蛋白的表达水平明显降低,而在BT474和BT474/shControl中表达无明显差异。（3）细胞生长曲线结果显示:BT474与BT474/shControl细胞的生长变化不大,差异无统计学意义（P＞0.05）,而BT474/shAIB1细胞生长明显减慢,与其他两组差异具有统计学意义（P＜0.05）。（4）以三苯氧胺敏感的MCF-7细胞为对照,ER（+）的乳腺癌BT474细胞对Tam是原发耐药的,而AIB1基因沉默后的BT474/shControl细胞则对Tam敏感;转染阴性对照载体的BT474/shControl细胞对Tam仍然耐药。（5）细胞周期结果显示:在无激素和雌激素存在情况下与BT474/shControl细胞相比,BT474/shAIB1细胞进入S期的数量均明显受抑制。（6） Western blot结果:ERα在BT474/shControl和BT474/shAIB1细胞中的表达水平是相同的;E₂治疗后导致了ERα的降解,而Tam能够稳定ERα使其不被降解。当AIB1基因通过RNAi沉默后,E₂则不能再降解ERα;BT474/shControl和BT474/shAIB1这两种细胞中,Tam能增强AIB1的表达。BT474/shControl和BT474/shAIB1细胞中HER2的表达水平相同。同样,给予E₂或Tam治疗后,HER2在这两种细胞中的表达也无明显改变。而AIB1沉默后导致了pS2表达的下降。E₂则增强了pS2在这两种细胞中的表达,Tam本身对pS2蛋白的表达没有影响。结论（1）构建的AIB1 RNAi表达载体经证实对乳腺癌BT474细胞中的AIB1mRNA及蛋白的表达具有干扰效应;（2） AIB1基因与乳腺癌BT474细胞的增殖密切相关,并且是激素非依赖性的;AIB1刺激细胞增殖的作用,部分是通过加快细胞周期的进程而实现的。（3）在AIB1和HER2高表达的ER阳性的乳腺癌BT474细胞中;三苯氧胺表现为雌激素激动剂作用,从而导致了原发耐药;AIB1受抑制后能够消除这种雌激素激动剂作用,从而恢复了三苯氧胺的抗肿瘤作用。更多还原

【Abstract】 BackgroundBreast cancer is one of the most common malignant tumors in women,and its incidence is the highest for all the female tumors in European and American developed countries.According to national statistics,in recent years the incidence of breast cancer in China continued to rise significantly,which located in the second place of female malignant tumors in China.With the in-depth study for the biological characteristics of breast cancer,we also have a new concept for breast cancer.Since the current study suggest that breast cancer is a systemic disease from it begin,and the treatment regimen is transited from initial simple surgical treatment to adjuvant radiotherapy and chemotherapy for surgical treatment,and then to current comprehensive treatment theory.In the comprehensive treatment,the position of dominant surgery was shaken,while endocrine therapy has suppressed chemotherapy and became one of the most important treatment ways due to the long lasting effect of the endocrine therapy.A number of studies have proved that endocrine therapy greatly improved the curative effect of early breast cancers.And it also significantly improved the life quality and extended the survival time of the patients because of the rational application of the endocrine therapy in advanced breast cancers.Oophorectomy was first used for treatment of pre-menopausal breast cancer patients by Beatson in 1896,which created a precedent for endocrine treatment of breast cancer.In 1960s,Toft isolated mouse ER protein.Tamoxifen was found to have therapeutic effects on breast cancer in 1970s.And it has been approved by FDA in 1977 for the treatment of woman with advanced breast cancer.Then it was applied for adjuvant treatment of primary breast cancer in 1986.For ER positive breast cancer patients,the recurrence rate could be decreased to 50%and the motality could be reduced by 28%by use of tamoxifen.Tamoxifen has been used to treat breast cancer for more than 30 years,which is the preferred endocrine therapy for all the ER-positive and post-menopausal breast cancers,and also has been the gold standard for endocrine therapy of breast cancer. Tamoxifen and estrogen compete to bind ER hormone binding domain,then the tamoxifen-ER complex was formed which then induced the changes of ER conformation,and closed the DNA binding domain,so that the gene transcription could not be activated,thus inhibited the estrogen’s activities.Tamoxifen can be used as adjuvant endocrine therapy for ER positive breast cancer patients with node negative and/or positive.A wide range of endocrine therapy drugs has been found currently,whereas the standard treatment status of tamoxifen still can not be completely replaced.Nowadays,the characteristics of tamoxifen and ER are still the research hotspot.A large-scale randomized clinical trial confirmed that hundreds of thousands of breast cancer patients have remained disease free survival（DFS） because of using tamoxifen.Some other studies reported that postoperative adjuvant endocrine therapy is better than or equal to chemotherapy for ER positive breast cancers.However,ER positive breast cancer patients who is primary sensitive to tamoxifen may still become tamoxifen resistance.Lots of studies found that 5 years of tamoxifen treatment for ER positive breast cancer could acquire long term survival rate and its effect can be extended to 5 years after stopping the drug.However, tamoxifen could acquire resistance to breast cancer for more than 5 years of application.Only 50%of ER positive breast cancer was initially sensitive to tamoxifen therapy in the clinic,and all of them would become tamoxifen resistance, and finally resulted in tumor progress and death.Therefore,it has great clinical significance to study the resistance mechanisms of endocrine therapy and to find ways to overcome the resistance.AIB1 gene is p160 steroid receptor transcription coactivator（SRC） family mumber which is a new definite proto-oncogene discovered in recent years.Because it was first found in amplified chromosome area of human breast cancer,so it was first named to be amplified in breast cancer 1（AIB1）.AIB 1 is the only amplified member of SRC family in the progression of human epithelial tumors,which can enhance the estrogen agonist activity of tamoxifen.The transfection experiments in vitro showed that P160 SRCs could enchance the activity of the nuclear recptor.Lots of studies suggested that AIB1 played an important role on many biological processes,such as cell proliferation,migration,differentiation,sexual maturation,as well as the occurrence and development of some cancers.However,at present,we have known little about the mechanism of AIB1 function.In human epithelial tumors,AIB1 is the unique overexpressed members in SRC family,which could enchance the estrogen-agonist activity of tamoxifen.AIB1 is also an important coactivator for ER, which could stimulate tumorigensis in transgenic animal models when AIB1 is overexpressed.The loss of AIB1 will influence ER signals via inhibiting its receptor degradation and deactivating transcription.As the coactivator of the ER,AIB1 was only overexpressed in ER positive breast cancer cell lines,and was not expressed in ER negative breast cancer cell lines.Clinical studies also proved that ABI1 was widely existed in ER positive breast tumors.Moreover,the expression of AIB1 was positively correlated with ERαand negatively correlated with ERβ.The binding of estrogen and ER was the main pathways to promote the proliferation of breast cancer in estrogen dependent breast cancers.However,besides the E-ER signal pathways,the EGFR family signal pathways were also another important mechanism.As one of the important EGFR family numbers,HER2 was a widely used tumor marker in clinical to predict endocrine therapy and recurrence of breast cancer. Kent Osborne has proved that AIB1 and HER2 is worked together to decrease the effect of tamoxifen therapy for breast cancer patients.AIB1 and HER2 protein levels in tumors from 316 breast cancer patients were determined using western blot analysis; they found that high AIB1 expression in patients who had not received adjuvant tarnoxifen therapy was associated with better prognosis and longer DFS.In contrast, for patients who had received tamoxifen therapy,high AIB1 expression was associated with worse DFS,which is indicative of tamoxifen resistance.In multivariable analyses of these primarily ER positive patients,AIB1 expression was an even more important predictor of tamoxifen responsiveness than expression of PR or HER2.Another study suggests that the antagonist activity of tamoxifen is reduced and its agonist activity is enhanced by the phosphorylation of AIB1 and ER via activating MAPK by HER2 in the patients with high level of AIB1 and HER2,which resulted in tamoxifen resistance.In this situation,tamoxifen not only fails to provide the protective antitumor signals but may instead even stimulate the tumor because of its agonist activity.A recent report showed that the expression of AIB1 in HER1-3 positive breast cancers would predict early relapse and death of the breast cancers, which acquires important guiding significance for the endocrine therapy and prognosis of breast cancers.These studies showed that AIB1 played an important role in antiestrogen-resistant breast tumors.However,a unifying mechanism of how AIB1 alters tamoxifen activity from an antagonist to an agonist has not been elucidated.The purpose of the present study was to identify the mechanism for the tamoxifen resistance displayed by ER positive tumors that express high levels of both AIB1 and HER2.We examined the effect of AIB1 on estrogen agonist activity of Tam-bound ER in tumor cells induced by tamoxifen,in order to take the experimental basis for clinical application.PartⅠConstruction and identification of RNAi eukaryotic expression vectors AIB1ObjectiveAIB1 specific shRNA expressing vector is constructed by the technique of gene engineer,so as to study its inhibition role on AIB1 gene in breast cancer BT474 cell line.Methods（1） Four chains of oligonucleotides were synthesized correspondingly according to the AIB1-specific siRNA sequence and the negative control sequence;（2） The complementary 4 oligonucleotides chains were phosphorylated,and then the complementary double strands were formed after the annealing;（3） The linerarization of plasmid vector pGenesil-1-U6 was formed after the digestion by both BamHI and HindⅢenzyme;（4） The complementary template strand of siRNA and the linear plasmid vector was ligated by T4 DNA ligating enzyme;（5） The competence cell was prepared by the bacillus coli DH5α,and the recombinant plasmids were transformed into the competence;（6） The bacillus coli were amplified,and the depurated plasmid was extracted from the bacterium by the plasmid extraction kit;（7） The extracted plasmids DNA were performed for electrophoresis under agarose gel,the concentration and purity of the plasmids were analyzed by ultraviolet photometer;（8） The recombinant plasmids were identified by SalI enzyme digestion,and the digested products were performed for electrophoresis under agarose gel;（9） The identified positive bacterium was sent to Wuhan Crystal Biotech Company for DNA sequencing.Results（1） The constructed pGenesil-shAIB1 and pGenesil-shControl plasmid expression vector was digested by SalI enzyme and performed for agarose gel electrophoresis,a 400bp of DNA band can be seen under the ultraviolet,which showed that the constructed plasmid vector was identical to the design requirement;（2） The enzyme-identified positive bacterium was sent to Wuhan Crystal Biotech Company for DNA sequencing,the results showed that the inserted segments in the recombinant plasmids were in accordance with the designed sequences.ConclusionsWe have successfully constructed the RNAi expression vector of target gene pGenesil- shAIB1 and negative control pGenesil-shControl.PartⅡRole of AIB1 for tamoxifen resistance in ER-positive breast cancer cellsObjective（1） To explore the role of AIB1-specific RNAi expression vector on inhibition of AIBI gene in human breast cancer BT474 cells;（2） To observe the changes of biological characteristics of BT474 cells after the AIB1 is silenced by RNAi,and to explore the mechanisms of AIB1 on tamoxifen resistance in ER positive breast cancer cells by evaluating the expression of AIB1,ERα,HER2 and pS2 proteins in BT474 cells.Methods（1） BT474 cell line is cultured routinely,and the pshAIB1 and pshControl vector was transfected into the BT474 cells by Lipofectamine 2000.The cells with stable expression of AIB1 shRNA was screened by G418.（2） The expression level of AIB1 mRNA and protein was analyzed in BT474, BT474/shAIB1 and BT474/shControl cells by RT-PCR and Western blot analysis.（3） The cells were counted using a Vi-cell XR automated cell viability analyzer according to the trypan blue dye exclusion method,and then drew the cell growth curves.（4） The cell proliferation of MCF-7,BT474,BT474/shAIB1 and BT474/shControl cells were determined in the presence of different concentrations of tamoxifen,so as to identify the tamoxifen responsiveness of these cells.And the changes of estradiol sensitivity on the cells were determined between pre- and post-RNAi.（5） The cell cycle distribution of the BT474/shAIB1 and BT474/shControl cells was assessed in absence or presence of estradiol by flow cytometry.（6） The expression of AIB1,ERa,HER2 and pS2 protein in BT474/shAIB1 and BT474/shControl cells was evaluated in the presence of Tam and E₂ by Western blot analysis.Results（1） The vector was successfully transfected into the BT474 cells by Lipofectamine 2000 and the positive clone cell lines was screened by G418.（2） Compared to BT474 and BT474/shControl cells,the expression of AIB1 mRNA and protein was significantly decreased in BT474/shAIB1 cells,while their expression level has no difference between BT474 and BT474/shControl cells.（3） The cell growth curve showed that there is no significant difference between on the cell growth of BT474 and BT474/shControl cells（p＞0.05）;while the growth of BT474/shAIB1 cells was significantly reduced compared to the other two groups （p＜0.05）.（4） ER-positive breast cancer BT474 cells are primary resistance to tamoxifen contrasted by the tamoxifen-sensitive MCF-7 cells.BT474/shControl cells become sensitive to tamoxifen with the inhibition of AIB1;whereas the BT474/shControl cells which transfected negative control vector is still resistance to tamoxifen.（5） The cell cycle analysis showed that the cell entry into the S phase was strongly inhibited in BT474/shAIB1 cells compared with the BT474/shControl cells both in the absence or presence of E₂.（6） Western bolt analysis showed that the expression level of ERαwas comparable in BT474/shControl and BT474/shAIB1 cells;E₂ treatment resulted in degradation of ERα,whereas tamoxifen stabilized the receptor.When the AIB1 levels were reduced by RNAi,ERαwas stabilized in the presence of E₂.The antiestrogen tamoxifen upregulated the expression of AIB1 in both of the BT474/shControl and BT474/shAIB1 cells.Compared to BT474/shControl cells,the BT474/shAIB1 cells expressed a considerable level of HER2.There are also no significant changes in HER2 expression in E₂ or tamoxifen treated cells.Knockdown of AIB1 resulted in a concomitant loss of expression of pS2 in BT474 cells.E₂ treatment increased pS2 expression in both of the BT474/shControl and BT474/shAIB1 cell lines,and tamoxifen alone has no significant effect on pS2 protein expression.Conclusions（1） The constructed RNAi expression vector to AIB1 was demonstrated to have inhibiting effect on the expression of AIB1 mRNA and protein in breast cancer BT474 cells;（2） AIB1 gene is intimate associated with the cell growth of BT474 cells,and the effect is hormone independent;the role of AIB1 on stimulating the cell growth is partly caused by accelerating the cell cycle progression.（3） Tamoxifen behaves like an estrogen agonist in ER-positive breast cancer cells that express high levels of both AIB1 and HER2,resulting in de novo resistance. Knockdown of AIB1 can eliminate this cross talk and restore the antitumor effects of tamoxifen.更多还原