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雌激素受体蛋白表达和启动子甲基化与女性散发性乳腺癌发生发展的相关性研究
The Study on the Relationship between Protein Expression and Promoter Hypermethylation of Estrogen Receptor and Progression in Women Sporadic Breast Carcinomas
【作者】 赵琳；
【导师】 魏敏杰；
【作者基本信息】 中国医科大学 ， 药理学， 2009， 博士
【摘要】 目的雌激素受体（Estrogen Receptors,ER）属核受体超家族成员,目前为止发现有雌激素受体α（Estrogen Receptorα,ERα）、雌激素受体β（Estrogen Receptorβ,ERβ）及雌激素受体γ（Estrogen Receptorγ,ERγ）3种亚型存在,在人类乳腺组织中仅见ERα和ERβ两种ER亚型表达。研究表明ERα和ERβ的结构、功能存在差异,因此推测两者在乳腺癌的发生发展中发挥不同的作用,且研究表明ERα/β启动子甲基化与ERα/β蛋白失表达相关。本实验研究在中国女性散发性乳腺癌组织中ERα/β的蛋白表达及与乳腺癌患者临床病理参数的相关性,并检测ERα/β基因启动子甲基化水平,探讨两种雌激素受体亚型在中国女性散发性乳腺癌中的表达特征及调控机制,同时就乳腺癌中耐药相关蛋白表达与ERα/β蛋白表达及启动子甲基化的相关性进行分析,以阐明雌激素受体的失表达及启动子甲基化在女性散发性乳腺癌发生发展中的重要意义。材料与方法采用免疫组织化学（immunohistochemistry,IHC）染色法,检测214例散发性乳腺癌和25例乳腺纤维腺瘤组织中ERα、ERβ、BCRP、MRP、LRP、P-gP、PR、HER-2、P53蛋白的表达,通过甲基化特异性PCR（Methylation specific PCR,MSP）法检测138例乳腺浸润性导管癌肿瘤及14例乳腺纤维腺瘤组织中ERα基因启动子区四个CpG岛密集区域甲基化及ERβ启动子区甲基化水平,分析ERα/β蛋白表达与临床病理学参数之间的相关性;分析乳腺癌组织中ERα/β基因启动子区甲基化水平及与ERα/β蛋白表达的相关性;分析ERα/β蛋白表达与启动子甲基化与乳腺癌耐药相关蛋白的相关性;通过逆转录PCR（Reverse transcript PCR,RT-PCR）方法检测5’Aza-dC、E2处理后ER阴性MDA-MB-435s、ER阳性T47D乳腺癌细胞株ERα、ERβ、BCRP、MRP、LRP、P-gP mRNA变化情况,Westernblot方法检测BCRP蛋白表达的变化。结果1、女性散发性乳腺癌中ERα/β蛋白表达与临床病理参数相关性（1）ERα、ERβ在乳腺癌组织中蛋白表达情况:乳腺癌中ERα、ERβ蛋白表达阳性率分别为56.5%（121/214）、62.1%（133/214）,乳腺纤维腺瘤中ERα、ERβ蛋白表达阳性率分别76%（19/25）、84%（21/25）,与乳腺纤维腺瘤组织比较,乳腺癌组织ERα蛋白阳性表达率无差异;乳腺癌组织ERβ蛋白阳性表达率显著低于乳腺纤维腺瘤组织（X2=4.664,P=0.03 1）。ERβ蛋白表达阳性表达率略高于ERα,但未见统计学意义,ERα和ERβ蛋白表达呈正相关（X2=13.243,P＜0.0001）;ERα、ERβ蛋白表达均阳性的乳腺癌组织为41.1%（88/214）,仅表达ERα乳腺癌组织为15.4%（33/214）,仅表达ERβ乳腺癌组织为21%（45/214）;ERα和ERβ蛋白表达均阴性的乳腺癌组织为22.4%（48/214）。（2）ERα、ERβ蛋白表达与乳腺癌临床病理参数相关性:在乳腺癌组织中,ERα蛋白表达水平与患者年龄、病理类型、临床分级及淋巴结转移、HER-2、P53蛋白表达均无关,ERα蛋白表达与PR蛋白表达正相关（84.2%vs.46.1%,X2=8.137,P＜0.0001）。ERβ蛋白表达与患者年龄和绝经状态相关,＞50岁患者中ERβ蛋白表达阳性率明显小于＜50岁患者（72.4%vs.51.2%,X2=8.170,P=0.004）;绝经后的患者ERβ蛋白表达阳性率明显低于未绝经患者（71.2%vs.49.7%,X2=8.137,P=0.004）;在不同的病理类型中,浸润性小叶癌的ERβ蛋白表达阳性率明显高于其它类型肿瘤（X2=9.008,P=0.029）。ERβ蛋白表达与患者年龄临床分级、淋巴结转移情况、HER-2、P53蛋白表达比较均未见相关性。（3）ERα/ERβ表达临床病理参数相关性:ERα/ERβ表达与患者年龄、绝经状态、病理分型、PR蛋白表达相关,＞50岁患者中ERα/ERβ均为阳性表达的发生率明显小于＜50岁患者（71.2%vs.50.9%,X2=4.505,P=0.034）,绝经后的患者ERα/ERβ均为阳性表达的发生率明显低于未绝经患者（71.4%vs.47.8%,X2=8.137,P=0.015）;在不同的病理类型中,浸润性小叶癌的ERα/ERβ均为阳性表达的发生率明显高于其它类型肿瘤（X2=11.528,P=0.009）。PR蛋白表达阳性乳腺癌组织中ERα/ERβ均为阳性表达的发生率明显高于PR蛋白表达阴性乳腺癌组织（90.6%vs.44.6%,X2=18.882,P＜0.0001）。ERα/ERβ蛋白表达与肿瘤大小、临床分级、淋巴结转移情况、P53和HER-2蛋白表达比较均未见相关性。2、女性散发性乳腺癌中ERα/β蛋白表达与基因启动子甲基化相关性研究（1）乳腺癌与纤维腺瘤组织中的ERα/β基因启动子甲基化水平:乳腺癌组织中ERα、ERβ总甲基化发生率分别为60.1%（83/138）、53.6%（74/138）;纤维腺瘤组织中,ERα、ERβ甲基化发生率分别为28.6%（3/14）、14.3%（2/14）;乳腺癌组织ERα、ERβ甲基化发生率均显著高于纤维腺瘤组织（60.1%vs.28.6%,X2=5.117,P=0.023;53.6%vs.14.3%,X2=7.867,P=0.005）。（2）乳腺癌中ERα/β基因启动子甲基化与ERα/β蛋白表达相关性:ERα蛋白表达阳性的乳腺癌组织中ERα基因启动子甲基化发生率为37.7%（26/69）,而在ERα蛋白表达阴性组织总甲基化发生率82.6%（57/69）,说明ERα蛋白表达水平与ERα基因启动子异常甲基化呈负相关。进一步针对四个CpG岛密集区域异常甲基化检测发生率,发现在ERα蛋白表达阳性的乳腺癌组织中ERα基因启动子甲基化发生率分别为14.5%、18.8%、7.2%、17.4%,在ERα蛋白表达阴性的乳腺癌组织中ERα基因启动子甲基化发生率明显升高,分别为55.1%、52.2%、58%、56.5%;在ERα蛋白表达阳性组织各检测区域间甲基化发生率未见显著性差别。以ERα表达水平与甲基化检出率做Spearman相关性分析,二者呈显著负相关（r=-0.469,P＜0.0001）;在ERβ蛋白表达阳性的乳腺癌组织中ERβ基因启动子甲基化发生率为35.4%（29/82）,而在ERβ蛋白表达阴性组织总甲基化发生率80.4%（45/56）。以ERβ蛋白表达水平与ERβ基因启动子甲基化发生率做Spearman相关性分析,二者呈显著负相关（r=-0.493,P＜0.0001）。（3）乳腺癌中ERα/β基因启动子甲基化与临床病理参数的相关性:ERα基因启动子甲基化检出率与患者年龄、绝经状况、临床分级、有否淋巴结转移、病理类型均未见相关性。PR蛋白表达阳性患者ERα甲基化发生率为24.1%,PR蛋白表达阴性患者ERα甲基化发生率为76%,两者相比有显著性差异（X2=8.427,P＜0.0001）;ERβ基因启动子甲基化发生率与发病年龄、临床分期具有相关性:年龄＞50岁的患者ERβ基因启动子甲基化发生率明显高于年龄≤50患者（72.4%vs.52.2%,X2=12.617,P＜0.00001）;随着临床分期的进展,ERβ基因启动子甲基化发生率逐渐加大（X2=8.727,P=0.029）;ERβ基因启动子甲基化发生率与PR蛋白表达、绝经状况、临床分级、有否淋巴结转移、病理类型均未见相关性。（4） 5-Aza-dC对ERα/βmRNA表达的影响:与溶剂处理组比较,不同浓度5-Aza-dC均可明显抑制DNMT1mRNA表达,20uM的5-Aza-dC对DNMT1mRNA抑制作用最为明显;采用1,2.5,5,10,20uM终浓度的5-Aza-dC处理MDA-MB-435s细胞96h后,ERα、ERβmRNA表达水平随5-Aza-dC浓度增大而增加,差别具有显著性（P＜0.05）;3、乳腺癌耐药相关蛋白表达水平与ERα/β蛋白表达及启动子甲基化相关性研究（1）乳腺癌组织中耐药相关蛋白表达水平与ERα/β蛋白表达及启动子甲基化相关性:乳腺癌组织中ERα/β蛋白表达与BCRP、MRP、P-gP、LRP蛋白表达具有正相关性（P＜0.05）,ERα/β基因启动子甲基化与BCRP、MRP、P-gP、LRP无关。（2） E2对乳腺癌细胞中耐药相关蛋白表达的影响:ER阳性T47D细胞,2.5、5、10、20nM E2不同浓度处理T47D乳腺癌细胞株,与溶剂处理组相比BCRP、MRP、LRP、P-gPmRNA表达均呈浓度依赖性增加。在ER阴性MDA-MB-435S细胞中,与溶剂组相比,不同浓度E2处理组,上述耐药蛋白mRNA表达均未发生明显变化。Western blot检测细胞中BCRP蛋白的表达,结果发现在ER阳性T47D细胞,2.5、5、10、20nM E2不同浓度处理T47D乳腺癌细胞株,与溶剂处理组相比BCRP蛋白表达呈浓度依赖性增加。在ER阴性MDA-MB-435S细胞中,与溶剂组相比,不同浓度E2处理组BCRP蛋白表达未发生明显变化。（3） 5-Aza-dC对MDA-MB-435S乳腺癌细胞株BCRP蛋白表达的影响:1、2.5、5、10、20uM 5-Aza-dC不同浓度处理MDA-MB-435S乳腺癌细胞株,与溶剂处理组相比BCRP蛋白表达增加,1、2.5、5、10、20uM 5-Aza-dC分别使BCRP表达增加了2.3、2.6、2.7、3.1、3.5倍,无浓度依赖性。（4）5-Aza-dC与E2联合应用后对MDA-MB-435S乳腺癌细胞株BCRP mRNA表达的影响:2.5、5、10、20uM终浓度的5-Aza-dC分别与3nM的E2联合处理ER阴性的MDA-MB-435S乳腺癌细胞株,发现与单独E2处理组比较,不同浓度的5-Aza-dC与E2联合处理后BCRP的mRNA表达无明显差别。结论1、乳腺癌组织ERα蛋白表达与乳腺纤维腺瘤组织比较无显著差异;乳腺癌组织中ERα蛋白表达与患者年龄、病理类型、临床分期、淋巴结转移、P53、HER-2蛋白无关,与PR蛋白表达正相关。乳腺癌组织中ERβ蛋白表达与乳腺纤维腺瘤组织比较明显降低;乳腺癌组织中ERβ蛋白表达与患者年龄、绝经状态、病理类型相关,与患者临床分级、淋巴结转移情况、PR、P53和HER-2蛋白表达无关。2、乳腺癌组织ERα基因启动子总甲基化发生率、4个检测区域（ER1、ER3、ER4和ER5）甲基化发生率与乳腺纤维腺瘤组织比较均明显升高,ERα蛋白表达阴性乳腺癌组织中总甲基化发生率及4个检测区域启动子甲基化发生率均明显高于ERα蛋白表达阳性乳腺癌组织,各检测区域间甲基化发生率未见显著性差别,ERα蛋白表达水平与ERα基因启动子甲基化发生率呈显著负相关,ERα基因启动子甲基化发生率与患者年龄、绝经状况、临床分级、淋巴结转移、病理类型比较均未见相关性,ERα蛋白表达阴性、PR蛋白表达阴性乳腺癌组织中ERα基因启动子甲基化发生率最高;乳腺癌组织ERβ基因启动子甲基化发生率与乳腺纤维腺瘤组织比较明显升高,ERβ蛋白表达阴性的乳腺癌组织中ERβ基因启动子总甲基化发生率明显高于ERα蛋白表达阳性的乳腺癌组织,ERβ蛋白表达水平与ERβ基因启动子甲基化发生率呈显著负相关,乳腺癌组织ERβ启动子甲基化发生率与患者发病年龄、临床分期具有相关性,与肿瘤大小、绝经状况、有否淋巴结转移、病理类型、PR蛋白表达比较均未见相关性;DNMT抑制剂5-Aza-dC可以浓度依赖性恢复ER阴性细胞中ERα/βmRNA表达,说明ERα/β基因启动子甲基化是引起ERα/β蛋白失表达的重要机制。3、乳腺癌组织中,ERα/β蛋白表达与BCRP、MRP、P-gP和LRP蛋白表达正相关,ERα/β启动子甲基化与BCRP、MRP、P-gP、LRP蛋白表达不具有相关性;ER阳性T47D乳腺癌细胞中,E2显著上调BCRP、MRP、P-gP和LRP mRNA表达,ER阴性MDA-MB-435s乳腺癌细胞E2对BRCP等耐药蛋白mRNA表达无影响,提示E2通过ERα调节BCRP等耐药蛋白的表达。
【Abstract】 ObjectiveEstrogen receptors belong to the member of nuclear superfamily.There was three subgroup,ERα,ERβand ERγ,but there is only ERα,ERβtwo subtypes in human mammry gland.It is known that ERαstatus is a well established predictor of response to endocrine therpy and related closely with prognosis in breast cancer.ERα,ERβhas different structure and function,it is proposed that ERα,ERβhave different role in breast cancer progression and endocrine therpy.One potential mechanism leading to loss of ERα,ERβis through reversible epigenetic modifications including DNA methylation. However,the methylation status of ERα/βhas not been elucidated in sporadic breast cancer in China,so our objective is to investigate the features of estrogen receptor alpha/beta（ERα/β） expression and its relationship with ERα/βgene promoter methylation status of sporadic breast cancers in Chinese women,analyze the relationship between drug-resistance related protein and ERα/βprotein expression,methylation of ERα/βpromoter.To discuss the possible mechanism of ERα/βloss expression.Elucidate the role of the protein expression and promoter methylation of estrogen receptor in the progression of women sporadic breast cancers.Materials and MethodsThe protein expression of ERα,ERβ,BCRP,MRP,LRP,P-gp,PR,HER-2 and P53 in 214 primary breast cancers and 25 fibroadenoma tissues were examined using immunohistochemistry.Following DNA extraction from 138 sporadic breast tumors, methylation-specific polymerase chain reaction（MS-PCR） was performed to analyze the promoter methylation status of ERα/β.Correlation analysis between methylation of promoter of ERα/βgene and its’ expression as well as clinical parameter was performed. The level of ERα/βand BCRP,MRP,LRP,P-gP mRNA in ER negative MDA-MB-435s breast cancer cell line before or after treated with 5-Aza-dC and E2 by RT-PCR and western blot.Results1.ERα,ERβprotein expression and its correlations with clinicopathological parameters in women sporadic breast cancers.（1）The expression of ERα,ERβprotein in breast cancer tissues:the positive immunostaining rate of ERα,ERβin breast cancer were 56.5%（121/214）,62.1% （133/214）,the positive immunostaining rate of ERα,ERβin fibroadenoma tissues were 76%（19/25）,84%（21/25）.The positive immunostaining rate of ERβsignificantly lower than that in fibroadenoma tissues（84%vs.62.1%,X2=4.664,P=0.031）,There was no significant difference of ERαprotein expression in breast carcinoma samples compared with benign breast hyperplasia.The ERβexpression was higher than ERα,but it didn’t show statistically difference.There was positively correlation between ERαand ERβexpression（X2=13.243,P＜0.0001）.41.1%（88/214） breast cancers was positive for ERαpuls ERβexpression,15.4%（33/214） breast cancers was only positive for ERαexpression,15.4%（33/214） breast cancers was only positive for ERαexpression, 21%（45/214） breast cancers was only positive for ERβexpression,22.4%（48/214） breast cancers was negative for ERαand ERβexpression.（2）The correlation of ERα,ERβprotein expression with clinicopathological parameters in sporadic breast cancers:the expression of ERαin breast cancer tissues was not associated with histological types,age,menopause status,clinical stage,lymph node metastases state,P53 and HER-2 expression.There was significantly lower in ERβpositive pro-menopause patients（71.2%vs.49.7%,X2=8.137,P=0.004）.Among different histologic type,86.4%infiltrating lobular carcinoma showed ERP positive higher than other histologic type tumors,In contrast,no correlation was found between the ERβpositive and tumor size,clinical stage lymph node metastases state,P53 and HER-2 expression.（3）The correlation of ERαplus ERβprotein expression with clinicopathological parameters in sporadic breast cancers:Correlation was found between ERβplus ERαexpression and histological types,age,menopause status,PR expression.There was significantly lower in ERβplus ERαpositive in＜50 years patients（71.2%vs.50.9%, X2=4.505,P=0.034）,infiltrating lobular carcinoma showed ERβpositive higher than other histologic type tumors（X2=11.528,P=0.009）,there was higher ERβplus ERαpositive expression in PR positive breast cancers compared with PR negative breast cancers.The expression of ERαplus ERβin breast cancer tissues was not associated with clinical stage,lymph node metastases state,P53 and HER-2 expression.2.Methylation status of the ERα,ERβpromoter in sporadic breast carcinomas and its correlation with ERα,ERβprotein expression.（1）Methylation status of the ERα,ERβpromoter in sporadic breast carcinomas:In 138 breast cancer tissues,we detected ERαmethylation in 83 of 138（60.1%） sporadic breast tumors and 4 of 14（28.6%） benign breast hyperplasia.Specifically within each region the methylation was as follows:methylation of the ER1 region was detected in 34.8%of cases,ER3 region detected in 35.5%,ER4 region detected in 39.1%,and ER5 region detected in 36.9%,there were no significant difference in the overall average percent methylation between the four primers in the sporadic breast tumors.（2）Downregulation of ERα,ERβexpression in breast cancer by methylation:A strong correlation was found between ERαmethylated with ERα-negative cases,82.6% ERα-negative being methylated vs 37.7%ERα-positive cases being methylated （P＜0.0001）,specifically within each region,hypermethylation frequencies were 55.1%for ER1,52.2%for ER3,58%for ER4 and 56.5%for ER5 respectively in ERα-negative cases and were significantly higher than ERα-positive cases（P＜0.0001）.In spearman’s correlation test,analyses of the correlation between intensity of ERαstaining by IHC and frequency of ERαpromoter methylation,statistically significant inverse correlation was found（P＜0.0001,r=-0.469）.We also found the strong correlation of ERβmethylation with ERβexpression,80.4%ERβ-negative being methylated vs 35.4%ERβ-positive cases being methylated（P＜0.0001）,In spearman’s correlation test,statistically significant inverse correlation was found between intensity of ERβstaining by IHC and frequency of ERβ（r=-0.493,P＜0.0001）.（3）Correlation of ERα,ERβpromoter methylation with clinicpathological features of breast tumors:No correlation was found between the ERαmethylation status and age, histologic type,menopausal status,lymph-node number,tumor size,TNM stage.A significant correlation was found between ERαmethylation and the reduced PgR expression,most of the cases with PgR-negative presented hypermethylation of ERαpromoter（57 out of 75 cases）,only 7 out of 29 PgR-positive tumors showed ERαunmethylation（76%vs.24.1%;P＜0.00001）.Aberrant methylation of ERβwas found in 52.2%of tumors from patients aged＜50 years,and 72.4%in those of＞50 years（72.4%vs.52.2%,X2=12.617,P＜0.00001）.ERβmethylation was observed higher in advanced stages（X2=8.727,P=0.029）.No correlation was found between the ERβmethylation status and histologic type,menopausal status, lymph-node number,tumor size and PR expression.（4）Effect of 5-Aza-dC on the ERα,ERβexpression in breast cancer cells:Compared with vehical group,cells treated with different concentration of 5-Aza-dC（1,2.5,5,10, 20uM） decreased DNMT1 mRNA expression in a dose dependent manner.5-Aza-dC resored the ERα,ERβmRNA expression in ER-negative cell line MDA-MB-435S..3.The relationship of estrogen receptor isoforms with BCRP,MRP,P-gP,LRP expression:（1）The relationship of estrogen receptor isoforms with BCRP,MRP,P-gP,LRP expression in breast cancer tissues:ERα/βprotein expression positively correlated with BCRP,MRP,P-gP and LRP statues.ERα/βgeng promoter methylation didn’t show any correlation with BCRP,MRP,P-gP and LRP statues in breast cancer tissues.（2）The effect of E2 on drug resistance related protein in breast cancer cell line:After exposed to E2 for 96h,the expression of BCRP,MRP,P-gP,LRP mRNA didn’t changed in ER-negative MDA-MB-435S cell line,but in ER-positive cell line T47D,E2 could increase the expression of BCRP,MRP,P-gP,LRP mRNA in a dose dependent manner. The results by estern blot analysis showed E2 increased the protein expression of BCRP in ER-negative MDA-MB-435S cell line.（3）The effect of 5-Aza-dC on BCRP protein expression in MDA-MB-435S cell line: After treated with 1、2.5、5、l0、20uM 5-Aza-dC,BCRP protein expression was increased by 2.3、2.6、2.7、3.1、3.5 fold,but it didn’t showed dose dependent.（4）The effect of 5-Aza-dC and E2 on the BCRP mRNA expression:in MDA-MB-435S cell line:After been exposed to 2.5、5、10、20uM 5-Aza-dC respectively in combination with 3nM E2 for 96h,the expressions of BCRP mRNA in MDA-MB-435s didn’t show any change when compared with E2 treated group. Conclusions1.There was no difference between breast cancer and fibro adenoma tissues in ERαexpression.The expression of ERαin breast cancer tissues was not associated with histological types,age,clinical stage,lymph node metastases state,HER-2 and P53 protein expression.ERαpositively correlated with PR statues.The protein level of ERβin breast cancer was lower than that in fibroadenoma tissues and positively correlated with age,menopause state and pathlogical type but no correlation with clinical stage, lymph node metastases state,HER-2 and P53 protein expression.2.In general,there was a higher degree of ERαtotal methylation and four regions detected in breast carcinoma samples compared with benign breast hyperplasia.Most of the ERα-negative cases presented hypermethylation compared with ERα-positive cases. Analyses of the correlation between intensity of ERαstaining and frequency of ERαpromoter methylation,statistically significant inverse correlation was found.No correlation was found between the ERαmethylation status and age,histologic type, menopausal status,lymph-node number,tumor size,TNM stage.The highest methylation was observed in ERα-positive and PgR-positive tumors.There was a higher degree of ERβmethylation in breast carcinoma samples compared with benign breast hyperplasia. In ERβ-negative cases,the frequency of ERβmethylation increased compared with ERβ-positive cases.Analyses of the correlation between intensity of ERβstaining and frequency of ERβpromoter methylation,statistically significant inverse correlation was found.ERβpromoter methylation correlated with age,TNM stage.No correlation was found between the ERβmethylation status and histologic type,menopausal status, lymph-node number,tumor size.The expression of ERα/βmRNA in ER negative breast cancer cell line coud be resorted by 5-Aza-dC in a dose dependent manner.So,aberrant methylation was one of the main mechanisms of inducing the loss of ERα/βexpression.3.In breast cancer tissues,ERα/βprotein expression positively correlated with BCRP,MRP,P-gP and LRP statues,ERα/βgene promoter methylation didn’t show any correlation with BCRP,MRP,P-gP and LRP statues.E2 increased the expression of BCRP,MRP,P-gP and LRP mRNA in ER-positive T47D breast cancer cell line,but it had no effect on the expression of BCRP,MRP,P-gP and LRP mRNA in ER-negative MDA-MB-435S breast cancer cell line.ERαplayed an importante role in the regulation of the expression of BCRP,MRP,P-gP and LRP.