Estrogen receptor ß target gene expression reveals novel repressive functions in aggressive breast cancer [ChIP-Seq]

Inflammatory breast cancer (IBC) is a highly metastatic breast carcinoma with high frequency of estrogen receptor a (ERa) negativity and limited treatment options. We previously explored the second ER subtype, (ERß) and reported correlation of its tumor expression with reduced metastasis in patients and in preclinical models of the disease. We initially associated the anti-metastatic function of the receptor with the inhibition of actin-based cell migration and the associated Rho GTPase signaling in IBC cells. We have now followed an integrated genomics approach to fully delineate the signaling underlying the anti-metastatic effects of ERß. Through mapping binding sites in IBC cells that express endogenous and transfected ERß in the presence of an agonist and interrogating this information with altered expression of protein coding mRNAs and miRNAs we have defined the interaction of a biologically active receptor with the cancer genome that controls metastatic signals. We reveal key regulatory chromatin binding sites and motifs and identify direct target genes and miRNAs and the associated biological functions. Our new findings implicate the regulation of metabolic pathways and signaling in development and tumor microenvironment in anti-metastatic activity of ERß. Through a rigorous analysis of target genes in clinical datasets we also associate downstream factors with patient outcomes reporting new molecules with targeting potential and strengthening the relevance of ERß signaling for the metastatic process in breast cancer. Our findings thus offer an opportunity to validate the tumor repressive role of ERß through a better understanding of its mechanism of action. Overall design: Analysis of breast cancer cells with different ERß levels and after with treatment with the ERß agonist. KPL4 cells without ERß (ERß knockout cells) vs. KPL4 cells with ERß (?Rß knockout cells with transfected ERß). Wild-type SUM149 cells with endogenous ERß (Control cells) vs. SUM149 cells with overexpressed ERß (Control cells with transfected ERß). KPL4 ?Rß knockout cells with transfected ERß after treatment with vehicle (DMSO) or the ERß agonist LY500307 for 2 hours

炎性乳腺癌（Inflammatory breast cancer, IBC）是一类高转移性乳腺癌，其雌激素受体α（estrogen receptor α, ERα）阴性率高，且治疗选择有限。我们前期针对第二种雌激素受体亚型——雌激素受体β（ERβ）展开研究，报道了该受体在肿瘤组织中的表达与患者及该疾病临床前模型中转移率降低存在相关性。我们最初将该受体的抗转移功能与IBC细胞中基于肌动蛋白的细胞迁移抑制及相关Rho GTP酶信号通路关联起来。如今，我们采用整合基因组学方法，全面阐明了ERβ抗转移效应背后的信号机制。通过在激动剂存在的情况下，于表达内源性及转染型ERβ的IBC细胞中绘制结合位点图谱，并结合蛋白编码mRNA与微小RNA（miRNA）的差异表达信息进行分析，我们明确了具有生物学活性的ERβ与调控转移信号的癌症基因组之间的相互作用。我们揭示了关键的调控性染色质结合位点与基序，鉴定出直接靶基因、miRNA及其相关生物学功能。我们的新发现表明，ERβ的抗转移活性涉及代谢通路调控、发育过程中的信号转导以及肿瘤微环境相关过程。通过对临床数据集的靶基因进行严谨分析，我们还将下游因子与患者预后关联起来，报道了一批具备靶向潜力的新型分子，进一步证实了ERβ信号通路在乳腺癌转移过程中的相关性。综上，我们的研究结果为通过深入解析ERβ的作用机制来验证其肿瘤抑制功能提供了契机。

整体实验设计：对不同ERβ表达水平的乳腺癌细胞，以及经ERβ激动剂处理后的细胞进行分析。具体包括：无ERβ的KPL4细胞（ERβ敲除细胞）与转染了ERβ的KPL4细胞（ERβ敲除并转染ERβ的细胞）；表达内源性ERβ的野生型SUM149细胞（对照细胞）与过表达ERβ的SUM149细胞（转染ERβ的对照细胞）；转染了ERβ的KPL4敲除细胞经溶剂（二甲基亚砜，DMSO）或ERβ激动剂LY500307处理2小时后的组别。