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

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.