【作者】 李魏玮；

【导师】 周庚寅；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 [研究背景]迄今为止,乳腺癌仍旧是全世界女性死亡的首要原因之一。流行病学及实验研究揭示高雌激素水平与乳腺癌发生密切相关。这种关联的一种可能解释是雌激素通过作用于其受体,调控下游抑癌原癌基因表达,促肿瘤发生发展。雌激素受体包含两种亚型,ERa和ERβ。众所周知,ERa对高雌激素所致的乳腺癌有促进作用,而ERβ在乳腺癌中的作用还存在诸多争议。大部分研究称ERβ可负性调节ERα,是预后较好的指标。然而,这些报道都局限在对ERa阳性乳腺癌中所表达的ERβ功能的研究。与此相反,多项调查发现在ERa阴性乳腺癌中,ERβ表达与预后不良的分子亚型呈正相关,比如ERβ可促进癌细胞增生,ERβ在基底细胞样亚型乳腺癌中的表达增多等等。我们知道肿瘤的恶行性转化体现在肿瘤的耐药性增强,增殖速度加快及更容易侵袭和发生远处转移。多药耐药的发生可降低化疗的成功率,增加患者死亡率,逆转肿瘤的多药耐药是十分重要的研究方向。目前,雌激素受体,尤其是ERβ亚型与乳腺癌多药耐药关系的报道还比较少,机制尚不明确。细胞膜耐药蛋白高表达是导致肿瘤多药耐药的重要原因之一。乳腺癌耐药蛋白(BCRP)是耐药蛋白家族中的一员,它的表达可介导米托蒽醌,阿霉素,表柔比星,拓扑替康,甲氨蝶呤,5氟尿嘧啶等多种化疗药物的耐药。由于BCRP广泛表达于多种乳腺癌细胞及乳腺癌组织中,它对于乳腺癌多药耐药的发生有极为重要的意义。我们前期研究发现,BCRP基因的启动子区含有一个雌激素反应元件(ERE),配体-ERα复合物可与之结合,激活经典的基因组通路,调节BCRP的转录表达。因此,我们十分感兴趣是否ERβ对BCRP基因表达也有调控作用。鉴于ERβ在ERα阳性及阴性乳腺癌中的作用报道不一致,我们选取不同分子亚型的乳腺癌细胞系(ERα阳性细胞系MCF-7以及ERα阴性细胞系MDA-MB-453和MDA-MB-468)和169例乳腺癌组织标本(99例ERα阴性标本及70例ERα阳性标本),对其中表达的ERβ在乳腺癌耐药蛋白(BCRP)介导的多药耐药和肿瘤生长方面的作用进行研究。[实验方法]应用雌激素及雌激素拮抗剂(TAM或TOR)处理各组细胞,RT-PCR和western blot法检测各组细胞BCRP基因转录翻译水平的变化。通过米托葸醌外排实验,检测细胞表达的BCRP是否具有泵活性。MTT法检测各组细胞对化疗药的耐药活性。此外,通过EMSA及ChIP实验,证实ERβ调节BCRP基因表达的结合位点。通过分析PCNA蛋白表达及EdU方法,检测ERβ结合不同配体对癌细胞增殖所起的作用；通过观察Caspase-3活化片段生成及AnnexinV/PI双染流式观察ERβ在促细胞凋亡方面的作用。免疫组化染色分析组织样本中ERβ与BCRP,增殖指标(Ki67和肿瘤大小)及侵袭转移的关系。[实验结果]99例ERα/PR阴性乳腺癌标本的免疫组化示ERβ表达与BCRP表达及增殖指标Ki67和肿瘤大小呈正相关。在70例ERα阳性标本中,发现ERβ表达与ERα高表达呈正相关,与BCRP和淋巴结转移呈负相关。体外细胞实验的研究发现,在ERα阴性的乳腺癌细胞系中(MDA-MB-453和MDA-MB-468),生理浓度雌激素作用下,ERp表达促进BCRP的生成,米托葸醌外排显示生成的BCRP具泵活性,导致BCRP介导的耐药增强。MTT进一步检测了细胞在化疗药作用后生存率升高。此外,ERβ表达导致癌细胞中PCNA增殖蛋白合成增多,EdU示ERp高表达组较干扰组及不表达组的增殖细胞比例增多,提示ERp与雌激素作用后刺激癌细胞增生。而在ERa阳性细胞系中(MCF-7),干扰掉ERa后,细胞内源性ERβ与雌激素作用可下调BCRP表达,从而逆转BCRP介导的耐药。与此同时,western blot检测到Caspase-3活化片段出现,AnnexinV/PI双染显示早期凋亡细胞所占比例增多,提示诱导癌细胞凋亡。进一步干扰掉ERβ后,上述作用不明显。在ERα阴性的乳腺癌细胞系中,雌激素拮抗剂与ERβ作用可逆转雌激素导致的耐药性增强及癌细胞增生的情况；而在ERα阳性细胞系中,雌激素拮抗剂与ERβ结合却会诱导耐药及促癌细胞增殖。传统认为,雌激素拮抗剂是不能单独刺激雌激素受体的,我们的结果却有不同的发现。与此同时,应用EMSA和ChIP技术,证实ERp与其配体复合物是通过结合BCRP基因上游启动子区的雌激素反应元件(ERE),启动经典基因组通路调节BCRP的表达的。[结论]乳腺癌病人治疗中出现的一大障碍,就是化疗中发生的多药耐药。多药耐药的产生通常与细胞膜上高表达ABC转运蛋白密切相关,这种蛋白具泵功能,可将细胞内的底物药物泵出,直接减少胞内药物浓度引发耐药。BCRP作为ABC转运蛋白家族中的一员,涉及多种抗癌药物的自发性和继发性耐药。其中某些药物并不是严格意义上BCRP的底物,却也能发生交叉耐药。尽管BCRP在细胞浆中合成,但它只有转移到胞膜上才有泵功能。因此BCRP在细胞中的定位直接影响其功能活性。本实验免疫组化评分,我们仅将胞膜染色的BCRP视为阳性。先前研究发现激素核受体与BCRP基因表达相关。有报道称,雌激素与ERa结合可通过一种转录后机制下调BCRP的表达。Wang等人则发现,在人胎盘BeWo细胞中,雌激素可下调BCRP,但通过ERβ而非ERa。直到BCRP基因启动子区的结构特征研究明确后,我们发现BCRP启动子区含有雌激素／孕激素反应元件。我们实验组对此进行了大量研究,证实了雌激素-ERa复合物通过结合于BCRP启动子区的雌激素反应元件,上调BCRP的表达：此外,孕激素-PR复合物结合孕激素反应元件后,下调BCRP。然而,ERβ与BCRP的关系至今仍不明确。仅有两篇血脑屏障的文献称,雌激素与ERβ结合后,可通过非基因组通路下调BCRP的表达。鉴于之前关于ERβ作用报道的矛盾不统一,我们将在ERa阳性及阴性的不同类型乳腺癌中,探究ERβ的效用。首先,在ERα/PR均阴性的乳腺癌中,免疫组化结果示有近半数的病例BCRP膜阳性,提示这些病人可能存在BCRP介导的多药耐药。且BCRP阳性与ERβ核阳性呈正相关。同时,我们发现ERβ与Ki67及肿瘤大小也呈正相关,提示ERβ与ERa/PR均阴性乳腺癌细胞的增殖密切相关。而在ERa阳性乳腺癌中,ERα高表达伴随着ERβ表达,且二者都与BCRP及淋巴结转移呈负相关。我们知道在细胞试验中,雌激素与ERa阳性乳腺癌细胞作用促进细胞增殖,转移；而临床病理诊断ERa阳性的乳腺癌细胞分化程度高,预后好。这些结果的矛盾性,是否可用ERβ表达后负性调节ERa,逆转了ERa的促癌作用来解释呢?大部分研究都赞同ERp在ERa阳性乳腺癌中起抑制增殖的作用。此外少数研究报道在ERa阴性的乳腺癌组织中,ERp与增殖指标正相关,这些结果与我们的发现一致。综上,免疫组化结果表明,ERa阴性的乳腺癌组织中,ERβ表达提示与化疗耐药和肿瘤增殖相关。ERα阳性乳腺癌中,ERβ与耐药及淋巴结转移呈负相关。为了深入了解ERβ作用机制,我们构建了不同的细胞模型。对ERa/ERβ/PR均阴性的453细胞,我们外源性过表达ERβ及BCRP(此BCRP的表达由含ERE的启动子启动P-BCRP)。雌激素(E2)处理后,453/ERβ/P-BCRP细胞的BCRP基因RNA及蛋白水平增高,对照组则无改变。上述结果证明BCRP的调控依赖ERβ和雌激素反应元件的同时存在。在内源性表达ERβ和BCRP的ERα阴性的468细胞中,我们再次印证了上述结论。此外,E2结合ERβ上调BCRP的作用可被雌激素拮抗剂三苯氧胺(TAM)抵消。另外,我们还发现内源性和外源性ERβ在雌激素作用下都可促进ERα阴性癌细胞增生,这些结果都与免疫组化结论一致。综上发现,抗雌激素治疗不仅适用于ERa/PR阳性的乳腺癌,也适用于ERa/PR阴性但ERβ阳性的乳腺癌。因此,检测ERa/PR阴性乳癌患者ERβ的表达,对指导内分泌治疗有一定意义。对于ERβ阳性乳腺癌的研究,我们选取了ERa/PR/ERβ阳性的MCF-7细胞系。由于细胞中ERβ表达低于ERα,直接给予雌激素或其拮抗剂,均表现ERa的作用。干扰ERα后,ERβ的作用凸显。ERβ与雌激素作用后下调BCRP表达,效果与ERα相反,也与ERα阴性乳腺癌中ERβ作用相反。并且,我们发现原本起治疗作用的TAM和托瑞米芬(TOR),与ERα阳性乳癌细胞中ERβ结合后,促进了BCRP介导的耐药及癌细胞增生。另外,我们应用EMSA及ChIP在细胞内和细胞外证实ERβ对BCRP的调节作用是通过结合雌激素反应元件,经由经典的基因组通路达成的。综上所述,我们通过分类研究,发现ERβ在ERα阳性和阴性乳腺癌中的作用相反。ERβ在ERα阴性乳腺癌中起促肿瘤作用；而在ERα阳性病例中则负性调节ERα,起抑制肿瘤的作用。ERβ作用的双面性在治疗病人,尤其对选择有效的内分泌治疗有一定的价值。ERβ成为常规乳腺癌指标检测的意义不容忽视。更多还原

【Abstract】 [Background and objective]Breast cancer continues to be a leading cause of women death worldwide. It is well known that estrogen exposure is closely associated with breast cancer in both epidemiological and experimental studies. One potential explanation of this association is that estrogen induced activity of estrogen receptors (ERs) stimulates tumor growth and development. ERs include ERa and ERβ. The harmful role of ERa in estrogen-mediated breast cancer has been well-accepted, whereas the effect of ERP on breast cancer remains controversial. Most studies have provided evidence that ERP acts as a negative modulator of ERa and protends a good prognosis with prolonged disease free survival. However, these studies have predominantly focused on ERa+breast cancer and the role of ERβ co-expression with ERa. Several investigators have reported that there is a positive correlation between ERβ expression and poor prognostic phenotypes, such as accelerated proliferation and basal phenotype in ERa-negative breast cancer. Moreover, the highest content of proliferating cells was seen in ERa-/ERpβ+cancer cells.There have been few reports on the relationship between ERs and multidrug resistance (MDR) in breast cancer, particularly for ERβ.It is known that MDR as well as proliferation and invasion/metastasis represent malignant transformation of tumors. MDR is a major obstacle of successful chemotherapy and decreases the survival rate in patients with breast cancer. Breast cancer resistance protein (BCRP), as a member of the ATP-binding cassette (ABC) superfamily, has been verified to result in MDR to various chemotherapeutic agents, such as adriamycin, epirubicin, mitoxantrone (MX), topotecan, methotrexate, doxorubicin and flavopiridol. Its overexpression has been observed in many cell lines and tissue samples of breast cancer. Our previous study found that regulation of BCRP relied on ligand-ERa complex binding to the estrogen response element (ERE) of BCRP promoter via the classical genomic pathway. Therefore, it is of interest to investigate whether ERβ also plays a role in regulation of BCRP expression.Herein we we chose breast cancer cells (MCF-7with high ERa expression and MDA-MB-453, MDA-MB-468without ERa expression) and samples (99ERα/PR-negative and70ERa-positive breast cancer samples) of ERa negative or positive subtypes to identify the role of ERβ in BCRP-mediated drug resistance and tumor growth.[Materials and methods]Several different human breast cancer cell models were treated with TAM, TOR or E2. Thereafter, BCRP levels were evaluated by semi-quantitative RT-PCR and Western blot. The pump function of BCRP was detected by the mitoxantrone (MX) efflux assay. MTT assay was used to assess the effect of ERβ on the chemosensitivity of BCRP expressing cells to anticancer drugs and on the survival rate of different cell groups.Proliferation of cells in different groups was assessed using the MTT assay, EdU assay and Western blot for proliferation cell nuclear antigen (PCNA). Apoptosis was also analyzed with the MTT assay and Western blot for caspase-3and by flow cytometry with AnnexinV and propidium iodide (PI). Moreover, an electrophoretic mobility shift assay (EMSA) and ChIP were performed to verify specific binding between ERβ and the estrogen response element (ERE) in the BCRP promoter region.[Results]In the immunohistochemistry study of99ERa/PR-negative breast cancer samples, nuclear expression of ERβ was positively associated with membranous expression of breast cancer resistance protein (BCRP), Ki67(proliferation marker) and tumor size. In70ERa-positive breast cancer samples, ERP was positively associated with high ERa expression, and was negatively associated with BCRP expression and LN. Moreover, ERβ has opposite roles in ERa-and ERα-breast cancer cells. In the presence of17β-estradiol (E2), ERP enhanced BCRP expression and cell proliferation of ERa/PR-negative cells (including MDA-MB-453and MDA-MB-468cell lines), while downregulated BCRP and induced cell apoptosis of ERa-positive cells (MCF-7cell). Additionally, these effects were reversed by additional use of tamoxifen (TAM) or toremifene (TOR). Interestingly, treatment with TAM or TOR alone resulted in the opposite effects compared with E2treatment of the cell lines mentioned above. In addition, the regulation of BCRP via specific binding between ERP and estrogen response element (ERE) was demonstrated in the EMS A and ChIP assay.[Conclusion]The major obstacle in the treatment of breast cancer is the rapid development of multidrug resistance (MDR) in patients. MDR is usually related to the elevated expression of ABC transporters, which can pump substrate drugs out of cells, reducing intracellular drug concentration. BCRP, as a primary type of ABC transporters, is involved in MDR to several kinds of anticancer drugs, in which some are not the specific substrates of BCRP. Although the BCRP protein is synthesized in the cytoplasm, it must be transported to the cell membrane for its pump function. Therefore, the location of BCRP may impact its function. In this experiment, only the brown staining on cell membrane was judged as BCRP positive.Prior studies have found that hormone nuclear receptors were involved in the regulation of BCRP. It was reported that estrogen downregulated BCRP expression by novel posttranscriptional mechanisms through binding to ERa. Wang et al. also found that E2downregulated BCRP expression through an ER, but possibly ERβ in human placental BeWo cells. After the genomic structure and characterization of the BCRP promoter was demonstrated, a novel estrogen/progesterone response element (ERE/PRE) was also revealed in the promoter region of BCRP. It has also been reported that E2-ERa complex upregulated, while progesterone-PR complex downregulated, BCRP expression through binding to ERE/PRE in breast cancer cells. Nevertheless, the correlation of ERP and BCRP has not previously been reported in breast cancer. Only two studies determined the role of E2in downregulation of BCRP through binding to ERβ via a non-genomic pathway at blood-brain barrier.Because of discrepancy about the effect of ERβ, we chose ERa-negative and positive breast cancer cells and samples to identify the role of ERβ in BCRP-mediated drug resistance and tumor growth. In the ERa-/PR-breast cancer samples, the immunohistochemistry results show that membranous BCRP expression accounts for nearly50%in ERa-/PR-breast invasive ductal carcinoma samples, and positively correlates with the nuclear expression of ERβ. We also observed that ERβ was positively associated with Ki67and tumor size. It has previously been demonstrated that ERa induces proliferation of breast cancer cells in presence of E2. Published data from several studies have demonstrated that ERβ has an anti-proliferative function when re-introduced into ERα-breast cancer cells. However, it has also been reported that in ERα-breast cancers, ERβ correlated with the proliferation marker Ki67, and highest content of proliferative cells was seen in ERa-/ERβ+cancers, which were consistent with our findings. Taken together, these findings implicate ERP as a marker for chemotherapy resistance and cell proliferation of breast cancer. However, in the ERa+breast cancer samples, there was no obvious relationship between ERβ and the markers mentioned above. The reason might be that the expression of ERa affected or surpassed the function of ERβ.To further elaborate the molecular mechanisms by which ERβ regulated BCRP expression and cell proliferation, we constructed different cell models. In ERa-breast cancer cell lines, ERβ and BCRP plasmids were transfected into MDA-MB-453(ERa-/ERβ-/PR-) cells. By treating the transfected cells with a various range of E2, the dose-dependent upregulation of BCRP level was observed in453/ERβ/P-BCRP cells, but not in453/ERβ/C-BCRP,453/P-BCRP and MDA-MB-453cells. These findings indicate that the regulation of BCRP depends on both ERβ and ERE. Furthermore, we verify that endogenous ERP has the same effect on BCRP expression in MDA-MB-468cells (ERa-/ERβ+/PR-/BCRP+). However, no remarkable change of the basal BCRP level was observed after knockdown of ERβ in MDA-MB-468cells, which indicates that while the presence of the receptor alone might not affect BCRP expression, the E2-ERβ complex can. Additionally, the role of E2in upregulating BCRP was suppressed by combined treatment of tamoxifen (TAM). Apart from this, we also found that both exogenous and endogenous ERβ induced proliferation of ERa-/PR-breast cancer cells (453/ERβ and468) in the presence of E2, which provides confirmation to the immunohistochemistry result about the relationship between ERP and Ki67/tumor size. Moreover, additional use of TAM reversed E2-induced cellular proliferation. Collectively, these findings demonstrated that antiestrogen therapy might also be valuable for ERa-/PR-but ERβ+breast cancer. Nowadays, endocrine therapy is not routinely used for ERa-/PR-breast cancer patients. However, our results suggest that detection of ERβ might be meaningful for endocrine therapy of these patients. In ERα+breast cancer cells, we chose MCF-7cell. Because the expression of ERβ is lower than that of ERa in this cell line, the effect of ERa surpassed ERβ when treated with E2or antiestrogens. After knowckdown of ERa, ERβ downregulated the expression of BCRP in the cells treated with estrogen, which effect was opposite to ERa and ERP expressed in ERα-breast cancer cell groups. In addition, we found that TAM or TOR binding with ER(3co-expressed with ERa induced BCRP-mediated drug resistance and cell proliferation.Collectively, our findings manifest that ERβ might act as a tumor promoter of cell proliferation and BCRP-mediated drug resistance in ERa-negative breast cancer and a tumor suppressor in ERa-positive breast cancer. The role of ER(3in breast cancer depends on many aspects, such as the type of ligands and the expression of other hormone receptors (ERa/PR). TAM or TOR routinely used for patients with ERa+/PR+breast cancer might also be effective in ERa-/PR-but ERβ+breast cancer. However, whether TAM or TOR plays a harmful role in breast cancer cells with high ERP meanwhile low ERa expression needs further study. Anyway, the detection of ER(3in clinic is valuable and should not be neglected in breast cancer.更多还原