Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer

Aromatase inhibitors (AIs) reduce estrogen levels up to 98% as the standard practice to treat postmenopausal women with estrogen receptor-positive (ER+) breast cancer. However, approximately 30% of ER+ breast cancers develop resistance to treatment. Enhanced interferon-alpha (IFNα) signaling is upregulated in breast cancers resistant to AIs, which drives expression of a key regulator of survival, interferon-induced transmembrane protein 1 (IFITM1). However, how upregulated IFNα signaling mediates AI resistance is unknown. In this study, we utilized MCF-7:5C cells, a breast cancer cell model of AI resistance, and demonstrate that these cells exhibit enhanced IFNα signaling and ligand-independent activation of the estrogen receptor (ERα). Experiments demonstrated that STAT1, the mediator of intracellular signaling for IFNα, can interact directly with ERα. Notably, inhibition of IFNα signaling significantly reduced ERα protein expression and ER-regulated genes. In addition, loss of ERα suppressed IFITM1 expression, which was associated with cell death. Notably, chromatin immunoprecipitation experiments validated that both ERα and STAT1 associate with ERE sequences in the IFITM1 promoter. Overall, hyperactivation of IFNα signaling enhances ligand-independent activation of ERα, which promotes ER-regulated, and interferon stimulated gene expression to promote survival in AI-resistant breast cancer cells.

Phosphorylation of human placental aromatase CYP19A1

Aromatase CYP19A1 catalyzes the synthesis of estrogens in endocrine, reproductive and central nervous systems. Higher levels of 17β-estradiol (E2) are associated with malignancies and diseases of the breast, ovary and endometrium, while low E2 levels increase the risk for osteoporosis, cardiovascular diseases and cognitive disorders. E2, the transcriptional activator of the estrogen receptors, is also known to be involved in non-genomic signaling as a neurotransmitter/neuromodulator, with recent evidence for rapid estrogen synthesis (RES) within the synaptic terminal. Although regulation of brain aromatase activity by phosphorylation/dephosphorylation has been suggested, it remains obscure in the endocrine and reproductive systems. RES and overabundance of estrogens could stimulate the genomic and non-genomic signaling pathways, and genotoxic effects of estrogen metabolites. Here, by utilizing biochemical, cellular, mass spectrometric, and structural data we unequivocally demonstrate phosphorylation of human placental aromatase and regulation of its activity. We report that human aromatase has multiple phosphorylation sites, some of which are consistently detectable. Phosphorylation of the residue Y361 at the reductase-coupling interface significantly elevates aromatase activity. Other sites include the active site residue S478 and several at the membrane interface. We present the evidence that two histidine residues are phosphorylated. Furthermore, oxidation of two proline residues near the active site may have implications in regulation. Taken together, the results demonstrate that aromatase activity is regulated by phosphorylation and possibly other post-translational modifications. Protein level regulation of aromatase activity not only represents a paradigm shift in estrogen-mediated biology, it could also explain unresolved clinical questions such as aromatase inhibitor resistance.

MIR2052HG regulates ERα levels and aromatase inhibitor resistance through LMTK3 by recruiting EGR1

Our previous GWAS using the MA.27 aromatase inhibitors (AIs) adjuvant trial identified SNPs in the lncRNA MIR2052HG associated with breast cancer free interval. Here we report that MIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing PKC activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor - dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibility of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.