Genetic effectors of TGF-beta induced growth and arrest.

描述(申请人提供)：乳腺癌是美国癌症死亡的主要原因，每年约有20万新病例和约4万人死亡。更好地了解肿瘤生长和抑制的遗传效应将有助于识别新的治疗靶点。这项建议的目的是确定和验证转化生长因子-β信号通路的关键组成部分，这些关键成分将导致乳腺癌治疗的特定疗法的开发。目的1：分析cdc25A在转化生长因子-β介导的乳腺上皮细胞生长刺激中的作用。我们的初步研究表明，转化生长因子-β介导的生长刺激与缺乏p21的细胞中cdc25A酪氨酸磷酸酶基因的表达增加有关。为此，我们将通过RNA干扰、启动子/突变/芯片分析和p21cDNA突变研究，结合组织微阵列对人类乳腺癌的分析，进一步分析cdc25A介导转化生长因子-β诱导的细胞增殖的能力。目的2：确定转化生长因子-β诱导细胞增殖的介质。在目标2中，我们假设还有其他整合基因的上调是转化生长因子-β诱导的生长刺激所必需的。我们将使用微阵列表达分析来识别这些基因，使用我们之前描述的p21-/-细胞系。为了验证这些基因是转化生长因子-β刺激的细胞增殖的真正介体，将进行RNA干扰基因“敲除”实验和通过同源重组进行靶向基因消融。目的3：确定介导对转化生长因子-β的生长反应的遗传效应。在这一目标中，我们将使用功能基因筛选来确定转化生长因子-β介导的生长抑制的其他效应物。我们将结合外显子捕获和遗传不稳定性来分离其失活导致抵抗转化生长因子-β介导的生长抑制的基因。分离的基因将通过修复假定的基因进行验证和确认，并与使用组织微阵列的免疫组织化学研究相关联。

DESCRIPTION (provided by applicant): Breast Cancer is a leading cause of cancer death in the United States with ~200,000 new cases and ~40,000 deaths each year. A better understanding of the genetic effectors of tumor growth and suppression would enable the identification of new targets for therapy. The aims of this proposal are directed at identifying and validating key components of the TGF-beta signaling pathway that would lead to the development of specific therapies for the treatment of breast cancer. AIM 1: Analyzing the role of cdc25A in TGF-beta mediated growth stimulation of breast epithelial cells. Our initial studies demonstrated that TGF- beta mediated growth stimulation correlated with an increased expression of the cdc25A tyrosine phosphatase gene in cells lacking p21. In this aim we will further analyze the ability of cdc25A to mediate TGF-beta induced cellular proliferation using RNA interference, promoter/mutational/ChIP assays, and p21 cDNA mutational studies, coupled with an analysis of human breast cancers using tissue microarrays. AIM 2: Identifying mediators of TGF-beta induced cellular proliferation. In Aim 2, we hypothesize that there are other integral genes whose upregulation is needed for TGF-beta induced growth stimulation. We will use microarray expression analysis to identify these genes using our previously characterized p21-/- cell lines. In order to validate that these genes are true mediators of TGF-beta stimulated cellular proliferation, RNA interference gene "knock down" experiments and targeted gene ablation via homologous recombination will be performed. AIM 3: Identifying genetic effectors that mediate the growth response to TGF-beta. In this aim, we will identify additional effectors of TGF-beta mediated growth inhibition using a functional genetic screen. We will combine exon trapping with genetic instability to isolate genes whose inactivation confers resistance to TGF-beta mediated growth suppression. Isolated genes will be verified and validated by restoration of the putative gene and correlated with immunohistochemical studies using tissue microarrays.