Estrogen receptor gene mutations in metastatic breast cancer: determining the function of the novel GATA4 isoform

雌激素受体-a（ER）基因突变在20-30%的转移性ER+乳腺癌（BC）中检测到，内分泌治疗进展后。这与1%的原发性疾病患者相比，表明它们是由于内分泌治疗对ER抑制的选择性压力而产生的。突变的ER蛋白在雌激素不存在的情况下是有活性的，并且显示对抗雌激素的敏感性降低。许多研究已经确定，与ER-WT BC相比，ER突变型BC具有增加的转移潜力。在我们实验室中进行的先前工作（Harrod等人，在制备中）使用CRISPR-Cas9基因组编辑来产生MCF 7细胞，其中最常见的ER突变L536 R、Y 537 C、Y 537 N、Y 537 S和D538 G被引入内源性ESRl基因座中。每个突变的多个独立克隆的RNA-seq分析在所有突变株系的每个克隆中仅鉴定出15个表达升高的基因。这15个基因中的一个是GATA 4，它是加塔转录因子家族的成员，该家族包括GATA 3，GATA 3是将ER募集到雌激素应答基因所需的先锋因子（Eeckhoute et al.，2007; Theodorou等人，2013年）的报告。值得注意的是，RNA-seq读取鉴定了先前未报告的GATA 4变体。在表达ER突变体的其他细胞系以及具有ER突变的PDX肿瘤中证实了变体GATA 4的表达增加。在转移性BC患者中升高的GATA 4表达也是明显的。总之，这些发现表明ER突变与BC中GATA 4表达的获得之间存在密切联系。这些结果，连同GATA 4作为ER是成骨细胞的先锋因子的所述作用，表明GATA 4可能参与将ER转录组重塑为更具转移性的状态。为了确定GATA 4的功能，我们已经在ER突变细胞系中产生了可诱导的shRNA和过表达模型。

Estrogen receptor-a (ER) gene mutations are detected in 20-30% of metastatic ER+ breast cancer (BC) following progression on endocrine therapies. This is compared to 1% of patients with primary disease, indicating that they arise due to the selective pressure of ER inhibition by endocrine therapies. The mutant ER proteins are active in the absence of estrogen and show reduced sensitivity to anti-estrogens. Many studies have established that ER mutant BC has increased metastatic potential, compared with ER-WT BC. Previous work conducted in our laboratory (Harrod et al., in preparation) used CRISPR-Cas9 genome editing to generate MCF7 cells in which the most common ER mutations, L536R, Y537C, Y537N, Y537S and D538G, were introduced into the endogenous ESR1 gene locus. RNA-seq analysis of multiple independent clones for each mutation identified just 15 genes with elevated expression in every clone for all mutant lines. One of these 15 genes is GATA4, a member of the GATA transcription factor family that includes GATA3, which is a pioneer factor, required for the recruitment of ER to estrogen-responsive genes (Eeckhoute et al., 2007; Theodorou et al., 2013). Of note, the RNA-seq reads identified a previously unreported GATA4 variant. Increased expression of variant GATA4 was confirmed in other cell lines expressing ER mutants, as well as in PDX tumours with ER mutations. Elevated GATA4 expression is also evident in metastatic BC patients. Together, these findings demonstrate a strong link between ER mutations and acquisition of GATA4 expression in BC. These results, together with a described role for GATA4 as pioneer factor for ER is osteoblasts, suggest that GATA4 may be involved in remodelling the ER transcriptome to a more metastatic state. Towards defining GATA4 function, we have generated inducible shRNA and over-expression models in ER mutant cell lines.