The Study On The Relationship Between Protein Expression And Promoter Hypermethylation Of Estrogen Receptor And Progression In Women Sporadic Breast Carcinomas

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.