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. 2019 Jan 4;11(1):43.
doi: 10.3390/cancers11010043.

Poly-ADP-Ribosylation of Estrogen Receptor-Alpha by PARP1 Mediates Antiestrogen Resistance in Human Breast Cancer Cells

Nicholas Pulliam  1   2 , Jessica Tang  3 , Weini Wang  4 , Fang Fang  5 , Riddhi Sood  6 , Heather M O'Hagan  7 , Kathy D Miller  8 , Robert Clarke  9 , Kenneth P Nephew  10   11   12   13
Affiliations

Poly-ADP-Ribosylation of Estrogen Receptor-Alpha by PARP1 Mediates Antiestrogen Resistance in Human Breast Cancer Cells

Nicholas Pulliam et al. Cancers (Basel). .

Abstract

Therapeutic targeting of estrogen receptor-α (ERα) by the anti-estrogen tamoxifen is standard of care for premenopausal breast cancer patients and remains a key component of treatment strategies for postmenopausal patients. While tamoxifen significantly increases overall survival, tamoxifen resistance remains a major limitation despite continued expression of ERα in resistant tumors. Previous reports have described increased oxidative stress in tamoxifen resistant versus sensitive breast cancer and a role for PARP1 in mediating oxidative damage repair. We hypothesized that PARP1 activity mediated tamoxifen resistance in ERα-positive breast cancer and that combining the antiestrogen tamoxifen with a PARP1 inhibitor (PARPi) would sensitize tamoxifen resistant cells to tamoxifen therapy. In tamoxifen-resistant vs. -sensitive breast cancer cells, oxidative stress and PARP1 overexpression were increased. Furthermore, differential PARylation of ERα was observed in tamoxifen-resistant versus -sensitive cells, and ERα PARylation was increased by tamoxifen treatment. Loss of ERα PARylation following treatment with a PARP inhibitor (talazoparib) augmented tamoxifen sensitivity and decreased localization of both ERα and PARP1 to ERα-target genes. Co-administration of talazoparib plus tamoxifen increased DNA damage accumulation and decreased cell survival in a dose-dependent manner. The ability of PARPi to overcome tamoxifen resistance was dependent on ERα, as lack of ERα-mediated estrogen signaling expression and showed no response to tamoxifen-PARPi treatment. These results correlate ERα PARylation with tamoxifen resistance and indicate a novel mechanism-based approach to overcome tamoxifen resistance in ER+ breast cancer.

Keywords: PARP inhibitor; antiestrogen resistance; breast cancer; estrogen receptor; tamoxifen.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Therapeutic inhibition of PARP1 promotes sensitivity to tamoxifen treatment, in ER+ breast cancer, scale bar: 20 µm. (A) Immunofluorescence staining of 8-hydroxyguanosine (8-oxoG) in MCF7 and MCF7-T cell lines. (B) Basal ROS levels in MCF7 compared MCF7-T cells. Quantification is representative of at least three individual experiments. (C) MCF7 and MCF7-T cells were treated for 24 h with 100 nM tamoxifen (Tamox) and western blot analysis performed against the indicated antibodies. (D) MCF7 and MCF7-T cells were treated with Tamox (24 h, 100 nM) and subjected to PAR ELISA (E) MCF7 and MCF7-T cells were treated with 100 nM Tamox or 1 nM Talaz for 72 h, alone and in combination, and colony formation assay was performed. (F) MCF7 (Top) and MCF7-T (Bottom) cells were treated with Tamox and Talaz for 72 h, alone and in combination, and subjected to clonogenic survival assay to determine drug efficacy; x-axis is indicative of Fraction affected (FA), y-axis is indicative of the combination index (CI). Combinations beneath the black dashed line are synergistic. Results are representative of three independent experiments. (G) MCF7 and MCF7-T cells were treated with 100 nM Tamox or 10 nM veliparib (Velip) for 72 h, alone and in combination, and colony formation assay was performed. PAR, Poly (ADP-ribose). ** p < 0.001, *** p < 0.0001 compared to control, # p < 0.01, ## p < 0.001, ### p < 0.0001 relative to bracketed treatment.
Figure 2
Figure 2
Tamoxifen-talazoparib co-administration induces DNA damage but not RAD51 foci formation. (A) MCF7 and (B) MCF7-T cells were treated with either 100 nM tamoxifen (Tamox) or 1 nM talazoparib (Talaz) for 72 h, alone and in combination. 24 h post treatment RAD51 foci formation assay was performed. Scale bar: 20 µm. (C) Quantification is representative of three individual experiments. (D) MCF7 and (E) MCF7-T cells were treated with 100 nM Tamox, with and without 1 nM Talaz for 48 h. 24 h post treatment cells immunofluorescence staining for γH2 AX was performed. Scale bar: 20 µm. (F) Quantification is representative of three independent experiments. * p < 0.001, *** p < 0.0001 compared to control, # p < 0.01, ## p < 0.001 relative to bracketed treatment.
Figure 3
Figure 3
Tamoxifen-induced ROS accumulation promotes ERα PARylation. (A) MCF7 and MCF7-T cells were treated with the increasing concentrations of tamoxifen (Tamox) for 4 h, and subjected to ROS assay. (B) MCF7 and MCF7-T cells were treated with Tamox (1 μM) in the presence and absence of 1 mM N-acetyl-l-cysteine (NAC). 24 h post treatment immunofluorescence staining of 8-hydroxyguanosine (8-oxoG) was performed. Results are representative of three independent experiments, and quantified below. Scale bar: 20 µm. (C) MCF7 and MCF7-T cells were treated with 100 nM Tamox with or without 1 mM NAC (ROS scavenger). 24 h post treatment cell lysates were subjected to western blot analysis. (D) MCF7-T cells were treated with 2 mM H2O2 for 4 h. Treated cells were subjected to immunoprecipitation (ERα) and western blot analysis against the indicated antibodies. (E) MCF7 and MCF7-T subjected to immunoprecipitation (ERα) and western blot analysis against the indicated antibodies. (F) MCF7-T cells were treated for 4 h with 100 nM Tamox with or without 1 nM talazoparib (Talaz; pre-treat 24 h). Post treatment cells subjected to immunoprecipitation and western blot analysis against the indicated antibodies. * p < 0.01, ** p < 0.001, *** p < 0.0001 compared to control, # p < 0.01, ## p < 0.001 relative to bracketed treatment.
Figure 4
Figure 4
Talazoparib decreases PARP1 and ERα localization to ERα-target genes. Chromatin immunoprecipitation (ChIP) analysis of PARP1 localization to ERα-target genes (RARA, MYC, GREB1, ERBB2, FOS, XBP1) was performed in (A) MCF7 or (B) MCF7-T cells following tamoxifen treatment (Tamox; 100 nM) with or without talazoparib (Talaz; 1 nM). Chromatin immunoprecipitation (ChIP) analysis of ERα localization to ERα-target genes (RARA, MYC, GREB1, ERBB2, FOS, XBP1) was performed in (C) MCF7 or (D) MCF7-T cells following Tamox treatment (100 nM) with or without 1 nM Talaz. * p < 0.01, ** p < 0.001, *** p < 0.0001 compared to control.
Figure 5
Figure 5
miR-222 mediates ER response to tamoxifen, and tamoxifen-mediated PARP1 activation. (A) Basal miR-222 expression was measured in MCF7 and MCF7-T cells by qRT-PCR analysis. (B) MCF7 and MCF7T cells were treated for 4 h with 100 nM tamoxifen (Tamox) and miR-222 expression was measured by qRT-PCR analysis. (C) MC7 cells were treated with the indicated concentration of Tamox for 4 h, with or without miR-222 overexpression. Treated cells were subjected to colony formation assays. Quantification is representative of three independent experiments. (D) MCF7-T cells were treated 72 h with 100 nM Tamox with or without miR-222 inhibition (KD). Treated cells were subjected to colony formation assays. Quantification is representative of three independent experiments. (E) MCF7-T cells were treated for 4 h with 100 nM Tamox with or without miR-222 inhibition. Post treatment cells were subjected to PAR ELISA. Quantification is representative of three independent experiments. (F) miR-222 was overexpressed in MCF7 cells and cell lysates subjected to qRT-PCR and western blot analysis. * p < 0.01, ** p < 0.001, *** p < 0.0001 compared to control, # p < 0.01, ## p < 0.001 relative to bracketed treatment.
Figure 6
Figure 6
Tamoxifen-induced miR-222 expression mediates sensitivity to tamoxifen-talazoparib co-administration. (A) MCF7 and (B) MCF7-Tcells were treated with 1 nM talazoparib (Talaz) for 48 h in the presence or absence of miR-222 overexpression (O/E). Post treatment cells were subjected to RAD51 foci formation assay. Quantification is representative of three independent experiments. Scale bar: 20 µm. (C) MCF7 cells were treated with increasing concentrations of Talaz for 48 h, with or without miR-222 overexpression. Post treatment cells were subjected to colony formation assays. Quantification is representative of three independent experiments. (D) MCF7T cells were treated with 100 nM tamoxifen (Tamox) and increasing concentration of Talaz for 48 h, with or without miR-222 inhibition. Post treatment cells were subjected to colony formation assays. Quantification is representative of three independent experiments. ** p < 0.001, *** p < 0.0001 compared to control, # p < 0.01, ## p < 0.001 relative to bracketed treatment.
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