ERa/PR crosstalk is altered in the context of the ERa Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation

Background: The constitutively active ESR1 Y537S mutation is associated with endocrine therapy resistance and progression of metastatic breast cancer through its effects on estrogen receptor (ERa) gene regulatory functions. However, the complex relationship between ERa and the progesterone receptor (PR), known as ERa/PR crosstalk, has yet to be characterized in the context of the ERa Y537S mutation. This study aimed to elucidate the effects of the ERa Y537S mutation on ERa/PR crosstalk and resultant transcriptional activity and to identify potential therapeutic sensitivities that may offer novel treatment options to patients with endocrine therapy-resistant breast cancer. Methods: Proximity-based interactions of ERa and PR were assessed via proximity ligation assay (PLA). Gene expression in MCF7 and T47D cells was assessed by RNA-seq analysis with comparison to publicly available patient tumor transcriptome data. Chromatin immunoprecipitation (ChIP)-qPCR and immunoblotting were used to assess ERa/PR-associated gene expression and protein expression, respectively. Data were analyzed by ordinary two-way ANOVA (a = 0.05) with Tukey's multiple comparisons tests. Results: Physical interaction of ERa and PR was increased in the context of the ERa Y537S mutation, including in the nucleus where this interaction may translate to altered gene expression. As such, more than 30 genes were differentially expressed in both patient tumor and cell line data (MCF7 and/or T47D cells) in the context of the ERa Y537S mutation compared to ERa WT. Of these, IRS1 stood out as a gene of interest, and ERa and PR occupancy at chromatin binding sites along IRS1 were uniquely altered in the context of ERa Y537S. Furthermore, siRNA knockdown of IRS1 or treatment with the IRS1 inhibitor NT-157 indicated a significant anti-proliferative effect of IRS1 depletion or inhibition in ERa Y537S cell lines. Conclusions: Previous research has characterized gene regulatory changes associated with the ERa Y537S mutation from the viewpoint of ERa. Here, we identify consequential changes to both ERa and PR transcription factor activity, including at chromatin binding sites for the signaling adaptor protein IRS1. We identified a significant dependence of ERa Y537S-expressing cells on IRS1 for proliferation, implicating IRS1 as a potential therapeutic target for restoring treatment sensitivity to patients with breast cancers harboring ERa Y537S mutations. Overall design: MCF7 and T47D cell variants (ERa WT, ERa Y537S-heterozygous, or ERa Y537S-homozygous) were hormone-deprived in charcoal-stripped media for 48 hours. Cells were then treated with vehicle or 10 nM R5020 for PR stimulation and collected via trypsinization after 2 hours of treatment. RNA was extracted using the Qiagen RNeasy Plus kit (#74104) according to the manufacturer's protocol. RNA concentrations were quantified by Nanodrop nucleic acid measurement. RNA library preparation for sequencing was completed using the KAPA mRNA HyperPrep Kit (#KR1352) according to the manufacturer's protocol. Sequencing was completed on the Illumina NovaSeq 6000 by the University of Chicago Functional Genomics core (RRID: SCR_019196). DESeq2 was used to determine differentially expressed genes between each cell variant and between each treatment.