Background: Overexpression of the bZip transcription factor, ATF3, in basal epithelial cells of transgenic mice under the control of the bovine cytokeratin-5 (CK5) promoter has previously been shown to induce epidermal hyperplasia, hair follicle anomalies and neoplastic lesions of the oral mucosa including squamous cell carcinomas. CK5 is known to be expressed in myoepithelial cells of the mammary gland, suggesting the possibility that transgenic BK5.ATF3 mice may exhibit mammary gland phenotypes.

Female transgenic mice that constitutively overexpress the transcription factor ATF3 in the basal epithelium of the mammary gland develop mammary carcinomas with high frequency, but only if allowed to mate and raise pups early in life. This transgenic mouse model system reproduces some features of human breast cancer in that about 20% of human breast tumor specimens exhibit overexpression of ATF3 in the tumor cells. The ATF3-induced mouse tumors are phenotypically similar to mammary tumors induced by overexpression of activating Wnt/β-catenin pathway genes. We now show that the Wnt/β-catenin pathway is indeed activated in ATF3-induced tumors. β-catenin is transcriptionally up-regulated in the tumors, and high levels of nuclear β-catenin are seen in tumor cells. A reporter gene for Wnt/β-catenin pathway activity, TOPGAL, is up-regulated in the tumors and several downstream targets of Wnt signaling, including Ccnd1, Jun, Axin2 and Dkk4, are also expressed at higher levels in ATF3-induced tumors compared to mammary glands of transgenic females. Several positive-acting ligands for this pathway, including Wnt3, Wnt3a, Wnt7b, and Wnt5a, are significantly overexpressed in tumor tissue, and mRNA for Wnt3 is about 5-fold more abundant in transgenic mammary tissue than in non-transgenic mammary tissue. Two known transcriptional targets of ATF3, Snai1 and Snai2, are also overexpressed in the tumors, and Snail and Slug proteins are found to be located primarily in the nuclei of tumor cells. In vitro knockdown of Atf3 expression results in significant decreases in expression of Wnt7b, Tcf7, Snai2 and Jun, suggesting that these genes may be direct transcriptional targets of ATF3 protein. By chromatin immunoprecipitation analysis, both ATF3 and JUN proteins appear to bind to a particular subclass of AP-1 sites upstream of the transcriptional start sites of each of these genes.

Basal-like breast cancer (BLBC) is an aggressive and deadly subtype of human breast cancer that is highly metastatic, displays stem-cell like features, and has limited treatment options. Therefore, developing and characterizing preclinical mouse models with tumors that resemble BLBC is important for human therapeutic development. ATF3 is a potent oncogene that is aberrantly expressed in most human breast cancers. In the BK5.ATF3 mouse model, overexpression of ATF3 in the basal epithelial cells of the mammary gland produces tumors that are characterized by activation of the Wnt/β-catenin signaling pathway. Here, we used RNA-Seq and microRNA (miRNA) microarrays to better define the molecular features of BK5.ATF3-derived mammary tumors. These analyses showed that these tumors share many characteristics of human BLBC including reduced expression of Rb1, Esr1, and Pgr and increased expression of Erbb2, Egfr, and the genes encoding keratins 5, 6, and 17. An analysis of miRNA expression revealed reduced levels of Mir145 and Mir143, leading to the upregulation of their target genes including both the pluripotency factors Klf4 and Sox2 as well as the cancer stem-cell-related gene Kras. Finally, we show through knock-down experiments that ATF3 may directly modulate MIR145/143 expression. Taken together, our results indicate that the ATF3 mouse mammary tumor model could provide a powerful model to define the molecular mechanisms leading to BLBC, identify the factors that contribute to its aggressiveness, and, ultimately, discover specific genes and gene networks for therapeutic targeting.

The Wnt/β-catenin signaling pathway is up-regulated in mammary tumors that arise with 70% incidence in parous female BK5.ATF3 transgenic mice. Nuclear accumulation of β-catenin is seen in both basal and supra-basal tumor cells in this model, whereas nuclear expression of the transcription factor ATF3 is only seen in the basal cell layer of the tumors. If ATF3 acted as a positive regulator of one or more Wnt ligands in the basal cells, one might expect paracrine upregulation of the Wnt/β-catenin pathway in neighboring supra-basal cells. To test this, we measured mRNA expression levels for 9 Wnt genes in BK5.ATF3 induced mammary tumors and normal mammary glands. The genes for five known positive acting Wnt ligands, Wnt3, Wnt3a, Wnt5a, Wnt7b, and Wnt10b, were significantly upregulated in tumors as compared to transgenic mammary glands. One of these, Wnt3, was significantly down-regulated in non-transgenic glands. In a mouse mammary tumor cell line, EMT6, in which the endogenous Atf3 gene is overexpressed, the only Wnt gene for which expression could be detected by qPCR was Wnt7b. ChIP assays demonstrated binding of both Atf3 and Jun proteins to two putative AP-1 sites upstream of the Wnt7b transcription start site in these cells. ATF3 and JUN are known in other systems to form heterodimers that exhibit positive transactivation through AP-1 sites, and we have identified a physical interaction between ATF3 and JUN in BK5.ATF3-induced tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2022. doi:10.1158/1538-7445.AM2011-2022

We previously showed that in a transgenic model (BK5.ATF3) in which the transcription factor ATF3 is overexpressed in cytokeratin 5 (CK5)-expressing cells, parous female mice develop mammary carcinomas with about 70% incidence between 6 months and one year of age. In these mice, ATF3 is overexpressed in cell nuclei in the myoepithelial cell layer (basal layer) of normal mammary glands, and in the basal cell layers of the tumors, in addition to expression in epidermis and other CK5-expressing epithelia. Non-parous, female transgenic mice exhibited a high incidence of squamous metaplastic lesions in the mammary glands between 14 and 32 weeks of age. These lesions showed dysregulation of cytokeratin expression, but no other molecular defects in non-parous mammary glands have been described. Several genes in the EGFR-MAPK pathway and in the Wnt/β-catenin pathway are misregulated in the mammary tumors that arise in parous transgenic mice. When expression of these genes was compared between normal mammary glands from transgenic and non-transgenic mice, the only genes that showed significant differences were Lef1 and Tcf7, two members of the family of transcription factors that dimerize with β-catenin to mediate Wnt-induced transcription. These genes were overexpressed by 2.5-3-fold in the transgenic mammary glands. A mouse mammary tumor cell line, EMT6, was found to have high levels of expression of both Atf3 and Tcf7, but not Lef1. Expression of Atf3 was knocked down by 97% in EMT6 cells 48 hrs post-treatment with Atf3-specific siRNA. This resulted in about a 70% decrease in expression of Tcf7, and a similar decrease in expression of cJun, a known target of Wnt/β-catenin signaling. This is consistent with the possibility that Tcf7 is a target gene for Atf3, and may be involved in the early stages of tumorigenesis in this model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1241.