Mechanisms of ATF3-induced mammary tumorigenesis

项目概述：ATF 3是一种高度保守的转录因子，在细胞中普遍上调， 哺乳动物对DNA损伤的反应初步数据显示，ATF 3在两种细胞中均显著上调， 许多人类乳腺肿瘤中的mRNA和蛋白质水平。ATF 3在角蛋白5表达细胞中的过表达 ATF 3转基因小鼠的BK5.ATF3细胞，包括乳腺的肌上皮细胞，导致高表达。 过表达β-连环蛋白、K5、K6和K8的乳腺癌的发生率，与 Wnt/β-连环蛋白通路的遗传改变。假设ATF 3的致癌作用 在该模型中的表达涉及Wnt/β-连环蛋白信号通路的上调，并且实验 来检验这个假设。在特定目标1中，TOPGAL报告基因将用于确定 Wnt/β-连环蛋白信号传导在BK5.ATF3转基因小鼠中产生的乳腺肿瘤中是否活跃。这些 肿瘤还将在mRNA水平上表征Wnt/β-连环蛋白和其他重要的 信号通路在特定目标2中，未孕乳腺中出现的癌前化生病变， 还将对BK 5-ATF 3转基因小鼠进行分子学检查，以获得Wnt/β-连环蛋白途径被抑制的证据。 使用同样的技术，冷冻切片的激光捕获显微切割将用于 分离病变用于RNA制备。在具体目标3中，将开发转基因小鼠，其中Wnt/p- 可以有条件地消除表达K5的细胞中的连环蛋白信号传导。这种基因改变将是 与BK5.ATF3转基因联合使用，以及对鳞状化生和 将确定病变和腺鳞癌。在具体目标4中， 将测定野生型和转基因小鼠腺体中的干/祖细胞群。的影响 在ATF 3诱导的乳腺肿瘤中，已知Wnt/β-连环蛋白诱导的乳腺肿瘤所需的Sdd基因缺失， 将确定肿瘤发生。这些研究的结果有望提供线索， 将ATF 3与Wnt/β-连环蛋白信号转导联系起来的机制，并提出了新的机制， 环境致癌物与人类肿瘤发生的关系。这将确定ATF 3的相关性 已知与Wnt信号传导相关的几种肿瘤类型，特别是基底样人乳腺癌 肿瘤，一个预后不良且与ATF 3诱导的小鼠乳腺肿瘤表型相似的亚组。 相关性：乳腺癌困扰着九分之一的美国女性。只有10%的癌症可以解释 遗传的癌症基因;其余的可能是由于暴露于DMA破坏性的环境因素， 遗传易感因素ATF 3在DNA损伤应答中的已知重要性以及ATF 3在DNA损伤应答中的作用， 在目前的模型中，ATF 3的致癌特性可能提供了环境与肿瘤之间的机制联系。 暴露和人类乳腺癌易感性，特别是预后不良的基底细胞样肿瘤。

Project Summary: ATF3 is a highly conserved transcription factor that is ubiquitously upregulated in the mammalian response to DNA damage. Preliminary data shows that ATF3 is significantly upregulated at both the mRNA and protein levels in many human breast tumors. Overexpression of ATF3 in keratin 5-expressing cells of BK5.ATF3 transgenic mice, including myoepithelial cells of the mammary gland, results in a high incidence of mammary carcinomas that overexpress p-catenin, K5, K6 and K8, similar to tumors induced by genetic alterations of the Wnt/p-catenin pathway. It is hypothesized that the oncogenic effects of ATF3 expression in this model involve upregulation of the Wnt/p-catenin signaling pathway, and experiments are proposed to test this hypothesis. In Specific Aim 1, the TOPGAL reporter gene will be used to determine whether Wnt/p-catenin signaling is active in mammary tumors arising in BK5.ATF3 transgenic mice. These tumors will also be characterized at the mRNA level for activation of the Wnt/p-catenin and other important signaling pathways. In Specific Aim 2, premalignant, metaplastic lesions arising in virgin mammary glands of BK5-ATF3 transgenic mice will also be examined molecularly for evidence that the Wnt/p-catenin pathway is upregulated, using the same techniques. Laser capture microdissection of frozen sections will be used to isolate the lesions for RNA preparation. In Specific Aim 3, transgenic mice will be developed in which Wnt/p- catenin signaling in K5-expressing cells can be conditionally eliminated. This genetic alteration will be combined with the BK5.ATF3 transgene and the effect on development of both squamous metaplastic lesions and adenosquamous carcinomas will be determined. In Specific Aim 4, the proportion of putative stem/progenitor cell populations in the glands of wild-type and transgenic mice will be assayed. The effect of loss of the Sdd gene, known to be required for Wnt/p-catenin-induced mammary tumors, on ATF3-induced tumorigenesis will be determined. The results of these studies are expected to provide clues to the mechanism that links ATF3 with Wnt/p-catenin signaling and to suggest new mechanisms for the involvement of environmental carcinogens with human tumorigenesis. This will establish relevance of ATF3 to several tumor types with known relationships to Wnt signaling, and particularly to basal-like human breast tumors, a subset with poor prognosis and phenotypic similarity to ATF3-induced mouse mammary tumors. Relevance: Breast cancer afflicts 1 in 9 American women. Only about 10% of these cancers are explained by inherited cancer genes; the rest are likely due to exposure to DMA-damaging environmental factors and genetic susceptibility factors. The known importance of ATF3 in the response to DNA damage and the oncogenic properties of ATF3 in the current model may provide a mechanistic link between environmental exposures and human breast cancer susceptibility, especially for basal-like tumors with poor prognosis.