ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation. ERα/PR crosstalk is altered in the context of the ERα 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 (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 endocrine therapy-resistant breast cancer. Methods: Proximity-based interactions of ERα 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 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. Results: Physical interaction of ERα and PR was increased in the context of the ERα 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 ERα Y537S mutation compared to ERα WT. Of these, IRS1 stood out as a gene of interest, and ERα and PR occupancy at chromatin binding sites along IRS1 were uniquely altered in the context of ERα 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 ERα Y537S cell lines. Conclusions: Previous research has characterized gene regulatory changes associated with the ERα Y537S mutation from the viewpoint of ERα. Here, we identify consequential changes to both ERα and PR transcription factor activity, including at chromatin binding sites for the signaling adaptor protein IRS1. We identified a significant dependence of ERα Y537S-expressing cells on IRS1 for proliferation, implicating IRS1 as a potential therapeutic target for restoring treatment sensitivity to patients with breast cancers harboring ERα Y537S mutations. Overall design: MCF7 and T47D cell variants (ERα WT, ERα Y537S-heterozygous, or ERα 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.

背景：组成型激活的ESR1 Y537S突变通过影响雌激素受体（ERα）的基因调控功能，与内分泌治疗耐药及转移性乳腺癌进展密切相关。然而，ERα与孕激素受体（PR）之间的复杂相互作用（即ERα/PR串扰）在ERα Y537S突变背景下尚未得到系统阐明。本研究旨在明确ERα Y537S突变对ERα/PR串扰及其介导的转录活性的影响，并鉴定潜在的治疗敏感性靶点，为内分泌治疗耐药性乳腺癌患者提供全新的治疗选择。
方法：通过邻近连接实验（PLA）检测ERα与PR的邻近相互作用；采用RNA测序（RNA-seq）分析MCF7和T47D细胞的基因表达，并与公开的患者肿瘤转录组数据进行比对；分别采用染色质免疫沉淀-定量PCR（ChIP-qPCR）与免疫印迹实验，检测ERα/PR相关的基因表达与蛋白表达。数据采用普通双因素方差分析（α=0.05）结合Tukey多重比较检验进行统计学分析。
结果：在ERα Y537S突变背景下，ERα与PR的物理相互作用显著增强，该相互作用发生于细胞核内，可进而导致基因表达谱改变。据此，与ERα野生型（WT）相比，ERα Y537S突变背景下的患者肿瘤及细胞系（MCF7和/或T47D细胞）数据中均有超过30个基因出现差异表达。其中，IRS1为重点关注基因，且ERα与PR在IRS1染色质结合位点的占据情况在ERα Y537S背景下发生了特异性改变。此外，通过小干扰RNA（siRNA）敲低IRS1或采用IRS1抑制剂NT-157处理，均证实IRS1敲低或抑制对ERα Y537S细胞系具有显著的抗增殖作用。
结论：既往研究多从ERα单一角度阐明ERα Y537S突变相关的基因调控改变。本研究则揭示了ERα与PR两类转录因子活性的实质性变化，包括信号衔接蛋白IRS1的染色质结合位点处的调控变化。我们证实表达ERα Y537S的细胞增殖对IRS1存在显著依赖性，提示IRS1可作为潜在治疗靶点，用于恢复携带ERα Y537S突变乳腺癌患者的治疗敏感性。
整体实验设计：将MCF7和T47D细胞的不同变体（ERα野生型、ERα Y537S杂合型或ERα Y537S纯合型）在活性炭吸附培养基中进行激素剥夺处理48小时。随后分别采用溶剂对照或10 nM R5020处理以刺激PR，处理2小时后通过胰酶消化收集细胞。按照试剂盒说明书，采用Qiagen RNeasy Plus试剂盒（货号#74104）提取RNA，通过Nanodrop核酸定量仪检测RNA浓度。按照试剂盒说明书，采用KAPA mRNA HyperPrep试剂盒（货号#KR1352）完成测序用RNA文库构建。测序工作由芝加哥大学功能基因组学中心（RRID: SCR_019196）在Illumina NovaSeq 6000平台上完成。采用DESeq2软件分析不同细胞变体之间以及不同处理组之间的差异表达基因。