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TNFα Enhances Tamoxifen Sensitivity through Dissociation of ERα-p53-NCOR1 Complexes in ERα-Positive Breast Cancer.

ABSTRACT: Tamoxifen is widely used as a medication for estrogen receptor α (ERα)-positive breast cancer, despite the ~50% incidence of tamoxifen resistance. To overcome such resistance, combining tamoxifen with other agents is considered an effective approach. Here, through in vitro studies with ER-positive MCF7 cells and ER-negative MDA-MB-231 cells, validated by the use of xenograft mice, we investigated the potential of tumor necrosis factor α (TNFα) to enhance tamoxifen sensitivity and identified NCOR1 as a key downstream regulator. TNFα specifically degraded nuclear receptor corepressor 1 (NCOR1) in MCF7 cells. Moreover, knockdown of NCOR1, similar to TNFα treatment, suppressed cancer cell growth and promoted apoptosis only in MCF7 cells and MCF7 xenograft mice through the stabilization of p53, a tumor suppressor protein. Interestingly, NCOR1 knockdown with TNFα treatment increased the occupancy of p53 at the p21 promoter, while decreasing that of ERα. Notably, NCOR1 formed a complex with p53 and ERα, which was disrupted by TNFα. Finally, combinatorial treatment with tamoxifen, TNFα and short-hairpin (sh)-NCOR1 resulted in enhanced suppression of tumor growth in MCF7 xenograft mice compared to single tamoxifen treatment. In conclusion, TNFα promoted tamoxifen sensitivity through the dissociation of the ERα-p53-NCOR1 complex, pointing at NCOR1 as a putative therapeutic target for overcoming tamoxifen resistance in ERα-positive breast cancer.

SUBMITTER: Kim H

PROVIDER: S-EPMC8199199 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Breast cancer is the most common malignancy, and approximately 70% of breast cancers are estrogen receptor-α (ERα) positive. The anti-estrogen tamoxifen is a highly effective and commonly used treatment for patients with ER+ breast cancer. However, 30% of breast cancer patients fail adjuvant tamoxifen therapy and most of metastatic breast cancer patients develop tamoxifen resistance. Although increasing evidence suggests that microRNA (miRNA) dysregulation influences tamoxifen sensitivity, the mechanism of the cross-talk between miRNA and ERα signaling remains unclear. miR-575 has been reported to be involved in carcinogenesis and progression, however, the role of miR-575 in breast cancer remains limited. The aim of this study was to understand the mechanism of miR-575 in breast cancer tamoxifen resistance. Method: RT-qPCR was employed to assess miR-575 expression in breast cancer tissues and cell lines. The association of miR-575 expression with overall survival in patients with breast cancer was evaluated with KM plotter. Additionally, the effects of miR-575 on breast cancer proliferation and tamoxifen sensitivity were investigated both in vitro and in vivo. Bioinformatic analyses and luciferase reporter assays were performed to validate CDKN1B and BRCA1 as direct targets of miR-31-5p. The ERα binding sites in the miR-575 promoter region was validated with ChIP and luciferase assays. ERα interactions with CDKN1B, cyclin D1 or BRCA1 were determined by IP analysis, and protein expression levels and localization were analyzed by western blotting and immunofluorescence, respectively. Results: miR-575 levels were higher in ER+ breast cancer than in ER- breast cancer and patients with high miR-575 expression had a significantly poorer outcome than those with low miR-575 expression. ERα bound the miR-575 promoter to activate its transcription, and tamoxifen treatment downregulated miR-575 expression in ER+ breast cancer. Overexpression of miR-575 decreased tamoxifen sensitivity by targeting CDKN1B and BRCA1. CDKN1B and BRCA1 were both able to antagonize ERα activity by inhibiting ERα nuclear translocation and interaction with cyclin D1. Furthermore, miR-575 expression was found to be upregulated in ER+ breast cancer cell with acquired tamoxifen resistance, whereas depletion of miR-575 partially re-sensitized these cells to tamoxifen by regulation of CDKN1B. Conclusions: Our data reveal the ERα-miR-575-CDKN1B feedback loop in ER+ breast cancer, suggesting that miR-575 can be used as a prognostic biomarker in patients with ER+ breast cancer, as well as a predictor or a promising target for tamoxifen sensitivity.

Project description:BackgroundThe mechanisms of endocrine resistance are complex, and deregulation of several oncogenic signalling pathways has been proposed. We aimed to investigate the role of the EGFR and Src-mediated STAT3 signalling pathway in tamoxifen-resistant breast cancer cells.MethodsThe ER-positive luminal breast cancer cell lines, MCF-7 and T47D, were used. We have established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen. Cell viability was determined using an MTT assay, and protein expression levels were determined using western blot. Cell cycle and annexin V staining were analysed using flow cytometry.ResultsTamR cells showed decreased expression of estrogen receptor and increased expression of EGFR. TamR cells showed an acceleration of the G1 to S phase transition. The protein expression levels of phosphorylated Src, EGFR (Y845), and STAT3 was increased in TamR cells, while phosphorylated Akt was decreased. The expression of p-STAT3 was enhanced according to exposure time of tamoxifen in T47D cells, suggesting that activation of STAT3 can cause tamoxifen resistance in ER-positive breast cancer cells. Both dasatinib (Src inhibitor) and stattic (STAT3 inhibitor) inhibited cell proliferation and induced apoptosis in TamR cells. However, stattic showed a much stronger effect than dasatinib. Knockdown of STAT3 expression by siRNA had no effect on sensitivity to tamoxifen in MCF-7 cells, while that enhanced sensitivity to tamoxifen in TamR cells. There was not a significant synergistic effect of dasatinib and stattic on cell survival. TamR cells have low nuclear p21(Cip1) expression compared to MCF-7 cells and inhibition of STAT3 increased the expression of nuclear p21(Cip1) in TamR cells.ConclusionsThe EGFR and Src-mediated STAT3 signalling pathway is activated in TamR cells, and inhibition of STAT3 may be a potential target in tamoxifen-resistant breast cancer. An increase in nuclear p21(Cip1) may be a key step in STAT3 inhibitor-induced cell death in TamR cells.

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TNFα Enhances Tamoxifen Sensitivity through Dissociation of ERα-p53-NCOR1 Complexes in ERα-Positive Breast Cancer.

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