Study On The Relationship Between BCRP Expression And Methylation Of Promoter Of ERα Gene In Sporadic Breast Cancers

ObjectiveTo discuss the relationship of the expression of breast cancer resistance protein (BCRP) and Estrogen receptor alpha(ERα) with the methylation feature of promoter of ERαgene in breast cancer.Investigate the changes of methylation state of promoter of ERαand the level of ERαand BCRP mRNA in ERαnegative MDA-MB-435s breast cancer cell line before or after treated with methyltransferases inhibitor 5'-Aza-deoxycytidine(5'-Aza-dC).To investigate the correlation between the expression of BCRP and estrogen signaling and observe the possible effect of demethylation drugs on antihormone therapy to breast cancer,to elucidate the possible internal relation between endocrine therapy resistance and multidrug resistance as well as different pathological profiles in breast cancer.MethodsThe protein expression of BCRP,ER,PR,HER-2 and P53 in 146 sporadic breast cancers and 22 fibroadenoma tissues were examined using immunohistochemistry.The correlations of BCRP with the clinical data as well as the pathological parameters were analyzed by correlate test.the methylation states of four CpG compact sites in promoter region of ERαgene in 60 infitrating ductal carcinomas(IDCs) and 14 fibro adenomas(FAs) were detected by methylation specific PCR(MSP),correlation analysis between methylation of promoter of ERαgene and its' expression as well as clinical parameter was performed.The methylation state of promoter of ERαand the level of ERαand BCRP mRNA in ERαnegative MDA-MB-435s breast cancer cell line before or after be treated with 5'-Aza-dC by MSP and RT-PCR.ResultsThe positive immunostaining rate of BCRP in breast cancer was significantly higher than that in fibroadenoma tissues(60.3%vs.31.8%,X²=6.301,P=0.012).The expression of BCRP in breast cancer tissues was not associated with histological types, age,clinical stage or lymph node metastases state,but was significantly lower in ERαpositive pre menopause patients(38.5%,X²=9.301,P=0.007).The expression of BCRP in HER-2 positive pateins were significantly higher than that in HER-2 negative pateins (81.0%vs.46.6%,X²=17.321,P=0.001),but no correlation with P53 or PR.The expression of ERαprotein in IDCs didn't show statistical difference compared with that in AFs;the methylation rate in IDCs were significantly higher than that in AFs(83.3%vs.28.6%,X²=17.260,P=0.001);the methylation rates of checking site ER2,ER3,ER4 in ERαnegative IDCs(63.3%,58.1%,83.9%) were totally higher than that in ERαpositive IDCs(24.1%,17.2%,13.8%);The methylation frequency of the promoter region of ERαgene was dramatically negative correlated with expression of ERα(r=-0.713,P=0.001) but modest positive correlated with expression of BCRP (r=0.254,P=0.050) in IDCs;the methylation rate of ER4 was significantly higher than that of other three check sites in ERαnegative IDCs(X²=8.321,P=0.004);the methylation frequency of the promoter region of ERαgene in progesterone receptor (PR)positive IDCs was significantly lower than that in PR negative IDCs(X²=9.598, P=0.002),but not correlated with age,clinical stage,lymph node metastases or HER-2 state.Three of four methylation detection site were demethylated in MDA-MB-435s cell line which after been treated with 10 ng/ml 5'-Aza-dC and all the four sites were demethylated in that treated with 50 or 100ng/ml 5'-Aza-dC for 96h.The expression of ERαmRNA was increased for 16.3 fold in MDA-MB-435s after been treated only with 100 ng/ml 5'-Aza-dC for 96h(0.814±0.031 vs.0.050±0.006);After been exposed to 10, 50 or 100ng/ml 5'-Aza-dC respectively in combination with 3nmol/l E₂ for 96h,the expressions of ERαmRNA in MDA-MB-435s were correspondingly increased for 11.3 fold(0.563±0.007 vs.0.050±0.006),13.2 fold(0.661±0.008 vs.0.050±0.006) and 16.0 fold(0.799±0.051 vs.0.050±0.006);the expressions of ERαmRNA increased followed the concentration of 5'-Aza-dC.Positive stain band of BCRP mRNA were present in every group,the expression of ERαmRNA didn't changed in MDA-MB-435s after been treated singly with 100 ng/ml 5'-Aza-dC or 3nmol/ml E₂ for 96h(F=3.256, P=0.425);After been exposed to 10,50 or 100ng/ml 5'-Aza-dC respectively in combination with 3nmol/l E₂ for 96h,the expressions of BCRP mRNA in MDA-MB-435s were correspondingly decreased to 81.5%(0.551±0.0177 vs.0.676±0.028),51.7%(0.350±0.024 vs.0.676±0.028),42.8%(0.289±0.077 vs.0.676±0.028) when compared with the control group,the expressions of BCRP mRNA decreased contrast with the concentration of 5'-Aza-dC.Conclusion1.the protein level of BCRP in sporadic breast cancer was higher than that in fibroadenoma tissues and negatively correlated with ERαstatues in pre-menopause pateints but positively correlated with HER-2 statues all the menopause state;The expression of BCRP in breast cancer tissues was not associated with histological types, age,clinical stage or lymph node metastases state.2.The aberrant methylation of the promoter region of ERαgene was detected in IDC and this aberrant methylation contribute to the loss of ERαexpression;the effect of the methylation on protein expression depends on the frequency of methylation or specific methylated CpG Island;PR expression was correlated with methylation of the promoter region of ERαgene.3.5'Aza-dC can not only efficaciously induced demethylation of the promoter of ERαgene but also restore the ERαexpression in a concentration dependent manner in ERαnegative breast cancer cell line.When used in combine with 5'Aza-dC,E₂ dramatically induced BCRP Expression down regulation in ERαnegative breast cancer cell line.These datas sugest that methylation of promoter of ERαgene might contrubite to MDR development in ERαnegative breast cancer due to Estrogen signaling deficiency caused BCRP regulation malfunction...