Effect Of ERα On Drug Resistance Of Paclitaxel In Breast Cancer Cells And Its Mechanism

Breast cancer is one of the most common female malignant tumours. Millions of women are suffering from it every year worldwide. Presently, chemotherapy is the main supplementary treatment for breast cancer. However, drug resistance of tumor cells is the main reason causing the failure of chemotherapy. The relationship between drug resistance and the expression level of estrogen receptor (ER) become increasingly apparent and needed further research.Taxol is a fist-line drug used in chemotherapy for breast cancer treatment. Both clinical and experimental studies showed that the level of ERα expression in breast cancer cells and the toleration of pacilitaxel was positively correlated, our study confirmed paclitaxel could induce the expression of ERα, which suggested that ERα played an important role in causing drug-resistance of breast cancer cells. ERα can regulate gene expression through classical and non-classical models. In non-classical model, the protein complex containing ERα and the transcription factor Sp1 or Ap1 indirectly binds to non-ERα recognition sequence, or with atypical ERα recognition sequence (ERE1/2), in order to regulate the transcriptional activity of target genes. High expression of MDR1 gene is the important resistant mechanism of tumor cells. Pgp, the product of MDR1 gene, is an ATP-dependent membrane protein, which could pump an enormous number of substrates, while paclitaxel is one of the substrates of Pgp. MDR1 gene is regulated by the transcription factor Sp1 and Ap1, the bioinformatics analysis also showed that the MDR1 promoter contained ERE1/2 sequence. On this basis, to explore whether ERa direct regulate the expression of MDR1 gene, Western blot, Realtime-PCR and Luciferase reporter assay were conducted to analysis the regulatory function of the ERa on MDR1 gene, we confirmed that ovER expression of ERa could increased both activity and expression of MDR1 and knockdown of ERa decreased both activity and expression of MDR1. Then, we confirmed that ERa could form a protein complex with Spl protein and regulated the expression of MDR1 gene via bridge binding to (GC rich)-(N)x-(EREl/2) sequence with quantitative ChIP and EMSA expERiments. FurthERmore, using MTT, quantitative ChIP and animal expERiments, we validated that sensitivity of breast cancER cells to chemothERapy could be improved by paclitaxel treatment combined with antagonizing the effect of ERa, ICI othER than MPP could eliminate the regulation of non-classical model of ERa.In summary, we first confirmed that ERa forming a protein complex with Spl positively regulated MDR1 gene through non-classical model, enhancing the resistance of breast cancER cells to paclitaxel. Then we suggested that ER antagonist (ICI) can improve the sensitivity of MCF-7 cells to paclitaxel in animal model. These findings provided a theoretical basis for clinical breast cancER treatment.Estrogen receptor a (ERa) plays an important role in chemotherapy failure of breast cancer cell. ERa is one member of steroid hormone receptor family, expressing in about 65% of breast cancer patients. The clinical data showed that ERa-positive (ER+) breast cancer patients are more resistant to chemotherapy drugs than ERa-negative (ER-) breast cancer patients. However, the drug-resistant molecular mechanism of ERa is still not clear.Recent studies have shown that abnormal methylation of genomic DNA are closely related with tumor cells resistance to drugs, steroid hormones and their receptors could be involved in the regulation of DNA methyltransferase expression and activity. We previously established paclitaxel-resistant breast cancer cell lines (MCF-7(ER+)/PTX) and (MDA-MB-231(ER-)/PTX), and confirmed that paclitaxel could induce the expression level of ERa in MCF-7(ER+) cells. The LINE-1 repetitive sequences is up to 18% in the proportion of human global genome, we chose the methylation level of LINE-1 promoter to represent the methylation level of global genomic DNA using methylation-specific PCR. We showed that the methylation level of genomic DNA in MCF-7(ER+)/PTX cells was significantly higher than that in MCF-7 cells, the methylation level of genomic DNA in MDA-MB-231(ER-)/PTX cells was lower than that in MDA-MB-231 cells. Correspondingly, the change of DNMT1 in transcription level and protein level were consistent with the change of the global genomic DNA methylation in the two drug-resistant cell lines, but DNMT3b expression level was increased in both two resistant cells, DNMT3a expression was no significant change. In order to investigate whether ERa could regulate the DNMT1 gene for the drug resistance phenotype in ER+ breast cancer, we used Western blot and Luciferase reporter assay to analyze the regulatory function of ERa on DNMT1 gene. We confirmed that over expression of ERa protein could increase DNMTl promoter activity and knockdown of ERa protein decreased the DNMT1 promoter activity. ERa could combine to the ERE sequence in the promoter region of DNMT1 gene, which is upstream of the-734bp ～-714bp and-114bp～-92bp of the transcription initiation site. For the first time, we proved that ERa could regulate DNMT1 transcriptional activity by binding to its promoter, suggesting that ERa enhanced resistance phenotype of breast cancer cells by changing the level of genomic DNA methylation. These findings provided a new approach to overcome clinical breast cancer resistance.