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Abstract
Background: Half of estrogen receptor (ERα)-positive breast cancer patients treated with endocrine therapies manifest intrinsic or acquired therapy resistance. One-third of these patients present with metastatic tumors containing ERα Y537S mutations. This constitutively activating ERα Y537S mutation is associated with endocrine therapy (ET) resistance and progression of metastatic breast cancer through its effects on ERα gene regulatory functions. However, the complex relationship between ERα and the progesterone receptor (PR), known as ERα/PR crosstalk, has yet to be characterized in the context of the ERα Y537S mutation. This study aimed to elucidate the effects of the ERα Y537S mutation on ERα/PR crosstalk and resultant transcriptional activity, and to identify potential therapeutic sensitivities that may offer novel treatment options to patients with ET-resistant breast cancer.Methods: Proximity-based interactions of ERα and PR were assessed via NanoBRET assays, proximity ligation assays (PLAs), co-immunoprecipitation (CoIP), and sequential chromatin immunoprecipitation (ChIP-reChIP). Gene expression in MCF7 and T47D cells was assessed by RNA-seq analysis with comparison to publicly available patient tumor transcriptome data. siRNA knockdown of differentially regulated genes was used to confirm phenotypic relevance. Chromatin immunoprecipitation (ChIP)-qPCR and immunoblotting were used to assess ERα/PR-associated gene expression and protein expression, respectively. Data were analyzed by ordinary two-way ANOVA (α = 0.05) with Tukey’s multiple comparisons tests or nonlinear regression, where appropriate.
Results: Using a NanoBRET hormone receptor panel, I identified a particularly elevated interaction between ERα and PR, which was further increased in the context of the ERα Y537S mutation. Utilizing PLA, CoIP, and ChIP-reChIP assays, I further confirmed increased proximity-based ERα/PR crosstalk in the context of the constitutively activating ERα Y537S mutation. Of note, ERα Y537S and PR co-occupancy at chromatin binding sites was increased (relative to ERα WT) at several genes implicated in breast cancer progression. Over 30 genes were differentially expressed in both patient tumor and cell line data (MCF7 and/or T47D cells) in the context of the ERα Y537S mutation. siRNA knockdown revealed an ERα Y537S-specific antiproliferative effect of depletion of several candidate genes. Of these, knockdown of the signaling adaptor protein IRS1 had a significant anti-proliferative effect on hormone-deprived MCF7 and T47D cells harboring either heterozygous or homozygous ERα Y537S mutations. Furthermore, ERα and PR occupancy at chromatin binding sites along IRS1 were uniquely altered in the context of ERα Y537S in a cell line-dependent manner. Analysis of the IRS1 inhibitor NT-157 indicates an antiproliferative effect of the compound in ERα Y537S cell lines.
Conclusions: I identified a role of the ERα Y537S mutation beyond that of constitutive activity of the receptor; it also increases ERα/PR crosstalk through both physical interaction and gene regulatory functions. Previous research has characterized gene regulatory changes associated with the ERα Y537S mutation from the frame of ERα. Here, I identify consequential changes to both ERα and PR transcription factor activity, including at chromatin binding sites for the signaling adaptor protein IRS1. I identify a significant dependence of ERα Y537S-expressing cells on IRS1 for proliferation, indicating a potential therapeutic target for restoring treatment sensitivity to patients with breast cancers harboring ERα Y537S mutations.