Supplementary Materialsoncotarget-08-103415-s001. primary luminal breast cancers. Addback of mutant recapitulated mutation-specific gene expression changes and enhanced soft agar colony formation, suggesting a gain of function for the mutant protein. BMN673 inhibitor gene, encoding a transcription factor crucial for breast development, occur in 15% of estrogen receptor-positive (ER+), or luminal-type, breast cancers [1, 2]. Mutations in are heterogeneous, but almost all of the mutations affect splice sites or are insertions/deletions (indels) that result in translational frameshifts. Many of these BMN673 inhibitor mutations result in premature termination of translation and truncated proteins lacking all or part of the second zinc finger, which mediates DNA binding [3]. Another cluster of mutations occurs near the C-terminus of Gata3, and it is not clear whether these mutations affect Gata3 function in the same manner as truncating mutations. Some of the breast cancer-associated truncating mutations cluster in the same region as mutations in the HDR syndrome (hypoparathyroidism, sensorineural deafness, and renal insufficiency), an autosomal dominant disorder ascribed to Gata3 haploinsufficiency [4]. However, mutant transcripts and proteins are highly expressed, and the mutational bias toward the distal part of the protein suggests that these mutations do not cause a simple loss of function. Gata3 expression is highest in hormone receptor-positive breast cancers. Several studies have shown that Gata3 BMN673 inhibitor expression correlates with a better prognosis, which is not surprising given the tight correlation between Gata3 expression and ER expression ( 90% co-expression) [5C9]. Since mutations in most cases examined do BMN673 inhibitor not lead to loss of transcript or protein, they are not identified by prognostic studies using gene expression microarrays or immunohistochemistry. The METABRIC study reported that mutant tumors have a favorable prognosis compared to wild type ER+ breast cancers [1]. However, the prevalence of GATA3 mutations in a population of treatment refractory metastatic breast cancers was identical to that reported in primary tumors (12%), suggesting that GATA3 mutant tumors are not especially favorable [10]. Studies using human breast cancer cell lines show that Gata3 co-regulates certain genes with the estrogen receptor alpha (ER) and that Rabbit Polyclonal to PKR there may be reciprocal regulation between Gata3 and ER [11, 12]. A BMN673 inhibitor gene expression signature enriched for genes induced by both estrogen and Gata3 defined a good prognosis subgroup of breast cancer patients, however Gata3-regulated genes were defined as those induced by overexpression of Gata3 in HEK-293 kidney cells, rather than in breast epithelial cells [13]. Several Gata3 target genes have been proposed, including [14C16], however the target genes affected by Gata3 mutations in human breast cancers have not been elucidated. Prior work by others has demonstrated a range of phenotypes with ectopic Gata3 overexpression or knockdown. Studies examining the effect of expressing wild type Gata3 in ER-negative cell lines such as MDA-MB-231 have shown that Gata3 favors expression of epithelial over mesenchymal markers and negatively regulates breast cancer metastasis [17C21]. However, such studies, while suggestive, do not address the function of Gata3 in the luminal breast cell types where it is highly expressed and frequently mutated. Ectopic overexpression or gene knockdown do not always recapitulate the phenotypes generated by physiologic expression of cancer-associated mutations using gene editing [22C24]. Here, we have utilized gene editing in human ER+ breast cancer cell lines to identify phenotypes and transcriptional targets dependent on mutant mutations In order to study the functional consequences of mutations in a human breast cancer system, we utilized the MCF-7 cell line, widely used as a representative model for ER+, luminal-type breast cancer. MCF-7 cells have a naturally occurring mutation, a G insertion in exon 5, leading to a frameshift and premature truncation of the translated polypeptide (D336Gfs*17, Figure ?Figure1)1) [4]. This mutation occurs in the second zinc finger of the Gata3 protein, and such mutations have been shown to disrupt binding to GATA motifs in DNA [3, 25]. This mutation is a recurrent hotspot in primary human breast cancers, having been reported 16 times in the TCGA and METABRIC datasets. Therefore, MCF-7 is a relevant model to understand the functional consequences of truncating mutations that occur almost exclusively in ER+ breast cancers. We confirmed.