Estrogen receptor (ER)-positive breast cancer recurrence is thought to be driven by tumor-initiating cells (TIC). TICs are enriched by endocrine therapy through NOTCH signaling. Side effects have limited clinical trial testing of NOTCH-targeted therapies. Death-associated factor 6 (DAXX) is a newly identified marker whose RNA expression inversely correlates with NOTCH in human ER⁺ breast tumor samples. In this study, knockdown and overexpression approaches were used to investigate the role of DAXX on stem/pluripotent gene expression, TIC survival in vitro, and TIC frequency in vivo, and the mechanism by which DAXX suppresses TICs in ER⁺ breast cancer. 17β-Estradiol (E₂)-mediated ER activation stabilized the DAXX protein, which was required for repressing stem/pluripotent genes (NOTCH4, SOX2, OCT4, NANOG, and ALDH1A1), and TICs in vitro and in vivo. Conversely, endocrine therapy promoted rapid protein depletion due to increased proteasome activity. DAXX was enriched at promoters of stem/pluripotent genes, which was lost with endocrine therapy. Ectopic expression of DAXX decreased stem/pluripotent gene transcripts to levels similar to E₂ treatment. DAXX-mediated repression of stem/pluripotent genes and suppression of TICs was dependent on DNMT1. DAXX or DNMT1 was necessary to inhibit methylation of CpGs within the SOX2 promoter and moderately within the gene body of NOTCH4, NOTCH activation, and TIC survival. E₂-mediated stabilization of DAXX was necessary and sufficient to repress stem/pluripotent genes by recruiting DNMT1 to methylate some promoters and suppress TICs. These findings suggest that a combination of endocrine therapy and DAXX-stabilizing agents may inhibit ER⁺ tumor recurrence. SIGNIFICANCE: Estradiol-mediated stabilization of DAXX is necessary and sufficient to repress genes associated with stemness, suggesting that the combination of endocrine therapy and DAXX-stabilizing agents may inhibit tumor recurrence in ER⁺ breast cancer.

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