Abstract
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.
Highlights
The bZip transcription factor, ATF3, has for some time been known to have the potential to both activate and repress transcription in a context-dependent manner [1,2,3,4,5]
In a transgenic mouse model in which human ATF3 is expressed from the bovine cytokeratin 5 promoter (BK5.ATF3), we reported spontaneous development of oral tumors, including about a 70% incidence of squamous cell carcinomas at 16 months of age [20]
Hai and colleagues have demonstrated overexpression of the ATF3 protein in a majority of human breast cancers [19], and we have found by immunohistochemistry (IHC) that in about 20% of human breast cancers ATF3 is found to be localized to the nuclei of the tumor cells; in the remaining ATF3-positive breast cancers, expression is primarily seen in stromal elements [21]
Summary
The bZip transcription factor, ATF3, has for some time been known to have the potential to both activate and repress transcription in a context-dependent manner [1,2,3,4,5]. IHC analysis indicated robust expression of Snail protein (the product of Snai1) in ATF3-induced mammary tumors (Figure 4A) Nuclei of both basal and supra-basal epithelial tumor cells (arrows and arrowheads, respectively) exhibited Snail expression. QPCR analysis of Snai expression failed to show a significant difference between transgenic mammary glands and mammary tumors (data not shown) This failure to detect increased levels of Snai mRNA may be due to the restricted pattern of expression of Slug in basal tumor cells only, as visualized by IHC. The AP-1 sites upstream of Wnt7b and Snai contained one to three mismatches to the consensus motif (Table 2), and the positive PCR signals obtained in the corresponding ChIP samples (Figure 6, f–h) were somewhat weaker These results are consistent with the suggestion that Snai, Snai, Tcf, Jun and Wnt7b may be direct transcriptional targets of ATF3 and JUN, possibly binding as a heterodimer to upstream enhancer elements
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