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. 2016 Apr 12;7(15):20966-80.
doi: 10.18632/oncotarget.8038.

ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

Xinxin Si  1   2 , Yue Liu  1   2 , Jinghuan Lv  3 , Haijian Ding  1   2 , Xin A Zhang  4 , Lipei Shao  1   2 , Nan Yang  1 , He Cheng  1   2 , Luan Sun  1   2 , Dongliang Zhu  2 , Yin Yang  1   2 , Andi Li  1   2 , Xiao Han  1 , Yujie Sun  1   5   6   2
Affiliations

ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

Xinxin Si et al. Oncotarget. .

Abstract

Drug-induced aberrant DNA methylation is the first identified epigenetic marker involved in chemotherapy resistance. Understanding how the aberrant DNA methylation is acquired would impact cancer treatment in theory and practice. In this study we systematically investigated whether and how ERα propelled aberrant global DNA hypermethylation in the context of breast cancer drug resistance. Our data demonstrated that anticancer drug paclitaxel (PTX) augmented ERα binding to the DNMT1 and DNMT3b promoters to activate DNMT1 and DNMT3b genes, enhancing the PTX resistance of breast cancer cells. In support of these observations, estrogen enhanced multi-drug resistance of breast cancer cells by up-regulation of DNMT1 and DNMT3b genes. Nevertheless, the aberrant global DNA hypermethylation was dominantly induced by ERα-activated-DNMT1, since DNMT1 over-expression significantly increased global DNA methylation and DNMT1 knockdown reversed the ERα-induced global DNA methylation. Altering DNMT3b expression had no detectable effect on global DNA methylation. Consistently, the expression level of DNMT1 was positively correlated with ERα in 78 breast cancer tissue samples shown by our immunohistochemistry (IHC) analysis and negatively correlated with relapse-free survival (RFS) and distance metastasis-free survival (DMFS) of ERα-positive breast cancer patients. This study provides a new perspective for understanding the mechanism underlying drug-resistance-facilitating aberrant DNA methylation in breast cancer and other estrogen dependent tumors.

Keywords: DNMT1; DNMT3b; ERα; breast cancer chemoresistance; global DNA hypermethylation.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. The expression of ERα was positively correlated with that of the DNMT1 and DNMT3b in breast cancer cell lines
A. Western blot analysis of the ERα expression in PTX-resistant breast cancer cell lines and their paired parental cell lines (left); the histogram depicting the relative ERα protein levels (right). B. Western blot analysis of the DNMTs protein levels in the two pairs of PTX-resistant breast cancer cells (left); the histogram depicting the relative expression levels of DNMTs proteins (right). C. Real-time PCR was performed to check the DNMTs transcriptional products.
Figure 2
Figure 2. ERα activated DNMT1 and DNMT3b genes in ERα-positive breast cancer cells
A. Western blot was performed to check the expression levels of ERα and DNMTs in MCF-7 cells transfected with ERα expression vectors. B. Luciferase reporter assay showed that over-expression of ERα enhanced the promoter activities of DNMT1 and DNMT3b, but not DNMT3a, in MCF-7 cells. C. Real-time PCR showed that over-expression of ERα up regulated the intracellular mRNA levels of DNMT1 and DNMT3b, but not DNMT3a, in MCF-7 cells. D. Western blot was performed to check the expression levels of ERα and DNMTs in MCF-7/PTX cells transfected with ERα-shRNA plasmids. E. Luciferase reporter assays showed that knockdown of ERα reduced DNMT1 and DNMT3b promoter activities in MCF-7/PTX cells. F. Real-time PCR showed that knockdown of ERα reduced the DNMT1 and DNMT3b intracellular mRNA levels in MCF-7/PTX cells.
Figure 3
Figure 3. ERα occupancy on the DNMT1 and DNMT3b promoters was significantly increased in PTX-resistant breast cancer cells
A. Diagram of the ERα binding sites in the human DNMT1 and DNMT3b gene promoters indicated by bioinformatics analysis. B. ChIP assay revealed that the DNMT1-S2 and DNMT1-S3 and the DNMT3b-S1 and DNMT3b-S3 were immunoprecipitated with ERα antibody, confirming ERα binds to theses sequences in breast cancer cells. C. qChIP assay indicated that the bindings of ERα to the DNMT1 and DNMT3b promoters were significantly increased in MCF-7/PTX cells when compared with the parental MCF-7 cells. D. The qChIP assay was repeated in ZR-75-1/PTX cells and similar results were obtained, indicating that anticancer drug exposure enhanced binding of ERα to the DNMT1 and DNMT3b promoters.
Figure 4
Figure 4. DNMT1 or DNMT3b expression enhanced drug resistance of breast cancer cells and was negatively correlated with the prognosis of breast cancer patients
A. Western blot analysis of DNMT1 expression in MCF-7 cells transiently transfected with DNMT1 expression plasmids (upper panel). MTT assay indicated that over-expression of DNMT1 increased viability of breast cancer cells under the stress of PTX treatment (lower panel). B. Similar experiments were performed to test the effect of DNMT3b on the response of breast cancer cells to PTX. DNMT3b over-expression increased the cell survival in the presence of PTX. C, D. Western blot analysis of DNMT1 expression in MCF-7/PTX (upper left) or ZR-75-1/PTX (upper right) cells transiently transfected with DNMT1-shRNA plasmids (upper). MTT assay was performed to determine cell viabilities of MCF-7/PTX (lower left) or ZR-75-1/PTX cells (lower right) treated with PTX at different concentrations. E, F. Kaplan-Meier analysis revealed negative correlation between DNMT1 and RFS and DMFS of ERα-positive breast cancer patients. G, H. Kaplan-Meier analysis displayed the similar results regarding the correlation between DNMT3b and the RFS and DMFS.
Figure 5
Figure 5. DNMT1 and DNMT3b were downstream target genes of ERα in ERα-mediated chemoresistance
A, B. Western blot was performed to determine the ERα knockdown efficiency in MCF-7/PTX (upper left) or ZR-75-1/PTX cells (upper right) transfected with ERα-shRNA. MTT assay showed that knockdown of ERα partly restored the sensitivity of PTX drug resistant breast cancer cells (lower panel), indicating ERα contributed to breast cancer drug resistance. C, D. Western blot was performed to check the transfection efficiencies in MCF-7 cells transfected with ERα expression plasmids together with either DNMT1-shRNA plasmids (C, upper panel) or DNMT3b-shRNA plasmids (D, upper panel). MTT assays were performed to examine the cell viability. Knockdown of DNMT1 (C, lower panel) or DNMT3b (D, lower panel) reduced the effect of ERα over-expression on the drug resistance of the MCF-7 cells. E. MTT assays showed that double knockdown of DNMT1 and DNMT3b restricted the effect of ERα over-expression on drug resistance more than the single DNMT knockdown.
Figure 6
Figure 6. Estrogen increased DNMT1 and DNMT3b expression and enhanced the multi-drug resistance of ERα-positive breast cancer cells
A. Luciferase reporter assay was performed to detect DNMTs promoter activities following treatment with graded concentrations of estrogen. B. Real-time PCR was performed to detect transcriptional levels of DNMTs in MCF-7 cells treated with estrogen. C. Western blot was performed to detect the expression levels of DNMT1 and DNMT3b in MCF-7 cells treated with estrogen. D. qChIP assay confirmed that estrogen treatment increased the bindings of ERα to DNMT1 and DNMT3b promoters in MCF-7 cells. E–J. MTT assay showed that estrogen increased the resistance of MCF-7 and ZR-75-1 cells to multi anticancer drugs, including PTX (E, H), EPI (F, I) and VCR (G, J).
Figure 7
Figure 7. ERα elevated the global DNA methylation level through DNMT1
Quantitative methylation-sensitive PCR (qMSP) was performed to detect the genomic DNA methylation levels. A. qMSP showed that global methylation level was increased in MCF-7 cells transfected with ERα expression plasmids and was decreased in MCF-7/PTX cells transfected with ERα-shRNA plasmids when compared with their controls. B. qMSP showed that global methylation level was increased in MCF-7 cells transfected with DNMT1 expression plasmids compared with the control. C. qMSP showed that knockdown of DNMT1 in MCF-7 cells significantly restrained the ERα-induced global hypermethylation. D, E. qMSP showed that over-expression or knockdown of DNMT3b had no detectable effect on global DNA methylation level.
Figure 8
Figure 8. ERα expression was positively correlated with DNMT1 expression in breast cancer patients
Representative immunohistochemical staining pictures of ERα, DNMT1 and DNMT3b in breast cancer tissues. The upper panel represented the strong positive staining and the lower panel represented the weak positive staining. The level of ERα in breast cancer tissues showed a statistically positive correlation with DNMT1, while no significant correlation with DNMT3b was observed.
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