Abstract
The selective estrogen receptor (ER) modulator tamoxifen inhibits ER signaling in breast cancer cells, and it is used for the treatment of ER-positive breast cancer. However, this type of cancer often acquires resistance to tamoxifen, and a better understanding of the molecular mechanisms underlying tamoxifen resistance is required. In this study, we established tamoxifen-resistant (TAM-R) breast cancer cells by long-term tamoxifen treatment of ER-positive breast cancer MCF7 cells. In TAM-R cells, expression of not only ERα, a major form of ER in breast cancer, but also its transcriptional partner forkhead box protein A1 (FOXA1) was found to be reduced. In contrast, activation of the transcription factor nuclear factor-κB (NF-κB) and expression of its target IL6 were increased in these cells. Stable expression of FOXA1, but not ERα, reduced the expression of IL6 in the FOXA1- and ERα-negative breast cancer MDA-MB-231 cells and TAM-R cells, without affecting the activation of the NF-κB signaling pathways. Conversely, FOXA1 knockdown induced IL6 expression in MCF7 cells. Chromatin immunoprecipitation assays revealed that FOXA1 bound to the promoter region of IL6 and repressed recruitment of the NF-κB complex to this region. TAM-R cells were found to have high mammosphere-forming activity, characteristics of cancer stem cells, and this activity was suppressed by NF-κB and IL6 signaling inhibitors. Taken together, these results suggest that FOXA1 suppresses expression of IL6 through inhibition of NF-κB recruitment to the IL6 promoter in an ERα-independent manner and that reduction in FOXA1 expression induces IL6 expression and contributes to cancer stem cell-like properties in TAM-R cells.
Highlights
The selective estrogen receptor (ER) modulator tamoxifen inhibits estrogen receptors (ERs) signaling in breast cancer cells, and it is used for the treatment of ER-positive breast cancer
These results suggest that acquired tamoxifen resistance significantly reduces the expression levels of ER␣, forkhead box protein A1 (FOXA1), and ER␣ targets
We found an increase in tumor necrosis factor-␣ (TNF-␣)-induced phosphorylation of IKK, IB␣, and RelA in TAM-R cells (Fig. 2C), which is characteristic of activation of the canonical nuclear factor-B (NF-B) pathway
Summary
We established the TAM-R breast cancer cell line by treating ER␣-positive breast cancer MCF7 cells with tamoxifen (4-OHT, 5 M) for more than 1 year (Fig. 1A). ER␣ knockdown had no apparent effect on IL6 protein expression, and FOXA1 knockdown was sufficient for IL6 induction Taken together, these results suggest that expression of FOXA1 represses mRNA expression of IL6 independently of ER␣ expression without affecting activation of the canonical NF-B pathway in breast cancer cells. ChIP assays revealed that FOXA1 knockdown promotes the binding of RelA to the IL6 promoter region in MCF7 cells and that stable expression of FOXA1 suppressed this binding in MDA-MB-231 cells and TAM-R cells (Fig. 6, C–E) These results suggest that FOXA1 functions as a transcriptional repressor of both IL6 and NIK by binding to their promoter regions and that the binding of FOXA1 to the IL6 promoter region represses the recruitment of the canonical NF-B complex to this region. These results suggest that about half of breast tumors with acquired resistance to tamoxifen may have increased IL6 expression caused by reduction in expression of FOXA1 and ER␣ as seen in TAM-R cells established in this study
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