Mechanisms of ATF3-induced mammary tumorigenesis

描述（由申请人提供）：项目概述：ATF 3是一种高度保守的转录因子，在哺乳动物对DNA损伤的反应中普遍上调。初步数据显示，在许多人乳腺肿瘤中，ATF 3在mRNA和蛋白水平上均显著上调。在BK 5.ATF 3转基因小鼠的角蛋白5表达细胞中，包括乳腺的肌上皮细胞中，ATF 3的过表达导致乳腺癌的高发生率，这些乳腺癌过表达B-连环蛋白、K5、K6和K8，类似于Wnt/β-核转录因子的遗传改变诱导的肿瘤。连环蛋白途径假设在该模型中ATF 3表达的致癌作用涉及Wnt/B-连环蛋白信号通路的上调，并且提出实验来检验该假设。在特定目标1中，TOPGAL报告基因将用于确定Wnt/B-连环蛋白信号传导在BK 5.ATF 3转基因小鼠中产生的乳腺肿瘤中是否具有活性。这些肿瘤还将在mRNA水平上表征Wnt/B-连环蛋白和其他重要信号传导途径的活化。在特定目标2中，还将使用相同的技术从分子水平检查BK 5-ATF 3转基因小鼠的处女乳腺中出现的癌前、化生性病变，以获得Wnt/B-连环蛋白途径上调的证据。冷冻切片的激光捕获显微切割将用于分离病变以制备RNA。在特定目标3中，将开发转基因小鼠，其中K5表达细胞中的Wnt/B-连环蛋白信号传导可以被有条件地消除。这种遗传改变将与BK5.ATF3转基因组合，并将确定对鳞状化生性病变和腺鳞癌发展的影响。在特定目标4中，将测定野生型和转基因小鼠腺体中推定干/祖细胞群的比例。将确定已知Wnt/B-连环蛋白诱导的乳腺肿瘤所需的Sdd基因缺失对ATF 3诱导的肿瘤发生的影响。这些研究的结果有望为ATF 3与Wnt/B-catenin信号转导的机制提供线索，并为环境致癌物参与人类肿瘤发生提供新的机制。这将建立ATF 3与已知与Wnt信号传导相关的几种肿瘤类型的相关性，特别是与基底样人乳腺肿瘤的相关性，基底样人乳腺肿瘤是与ATF 3诱导的小鼠乳腺肿瘤具有不良预后和表型相似性的子集。相关性：乳腺癌困扰着九分之一的美国女性。这些癌症中只有约10%是由遗传性癌症基因解释的;其余的可能是由于暴露于DNA损伤的环境因素和遗传易感性因素。已知的重要性，ATF 3在响应DNA损伤和致癌特性的ATF 3在目前的模型中可能提供了一个机制之间的联系，环境暴露和人类乳腺癌的易感性，特别是基底样肿瘤预后不良。

DESCRIPTION (provided by applicant): 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 B-catenin, K5, K6 and K8, similar to tumors induced by genetic alterations of the Wnt/?-catenin pathway. It is hypothesized that the oncogenic effects of ATF3 expression in this model involve upregulation of the Wnt/B-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/B-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/B-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/B-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/B-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/B-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/B-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 DNA-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.