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

随着内分泌治疗的进展，在20-30%的转移性ER+乳腺癌(BC)中检测到雌激素受体-a(ER)基因突变。与1%的原发病患者相比，这表明它们是由于内分泌治疗对内质网抑制的选择性压力而产生的。突变的ER蛋白在没有雌激素的情况下是活跃的，并且对抗雌激素的敏感性降低。许多研究已经证实，与ER-WT BC相比，ER突变BC具有更高的转移潜能。本实验室以前的工作(Harrod等人，正在准备中)使用CRISPR-Cas9基因组编辑方法生成MCF7细胞，其中最常见的ER突变L536R、Y537C、Y537N、Y537S和D538G被引入内源ESR1基因座。对每个突变的多个独立克隆的RNA-seq分析发现，在所有突变株系中，每个克隆中只有15个基因表达上调。这15个基因中的一个是GATA4，它是GATA转录因子家族的成员，包括GATA3，这是雌激素反应基因招募ER所必需的先驱因子(Eeckhoute等人，2007年；Theodorou等人，2013年)。值得注意的是，RNA-seq读数确定了一个以前未报道的GATA4变体。变异型GATA4在其他表达ER突变的细胞系中的表达增加，以及在带有ER突变的PDX肿瘤中得到证实。GATA4表达升高在转移性BC患者中也很明显。综上所述，这些发现表明ER突变和BC中GATA4表达的获得之间存在强烈的联系。这些结果，再加上GATA4作为成骨细胞ER的先驱因子的描述作用，表明GATA4可能参与了ER转录组的重塑，使其处于更转移的状态。为了定义GATA4的功能，我们在ER突变细胞系中建立了可诱导的shRNA和过度表达模型。

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.