The Effects Of ERβ On BCRP-mediated Drug Resistance And Cell Proliferation And Apoptosis In Breast Cancer Of Different Molecular Subtypes

[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.