Genetic effectors of TGF-beta induced growth and arrest.

乳腺癌是美国癌症死亡的主要原因，新增病例约20万例， 每年约有4万人死亡。更好地了解肿瘤生长和抑制的遗传效应 将有助于确定新的治疗靶点。这项建议的目的是 确定和验证转化生长因子-β信号通路的关键成分，这些关键成分将导致 开发治疗乳腺癌的特殊疗法。目标1：分析cdc25A的作用 在转化生长因子-β介导的乳腺上皮细胞生长刺激中。我们的初步研究表明，转化生长因子-2 β介导的生长刺激与cdc25A酪氨酸表达增加相关 缺失p21的细胞中的磷酸酶基因。为此，我们将进一步分析cdc25A的中介能力。 用RNA干扰、启动子/突变/芯片检测和p21研究转化生长因子-β诱导的细胞增殖 Cdna突变研究，同时使用组织微阵列对人类乳腺癌进行分析。目标 2：确定转化生长因子-β诱导细胞增殖的介质。在目标2中，我们假设有 其他整合基因，其上调需要转化生长因子-β诱导的生长刺激。我们将使用 使用我们先前描述的p21-/-细胞系进行微阵列表达分析来识别这些基因。在……里面 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中介，RNA 干扰基因“击倒”实验和同源重组靶向基因消融将 被执行。目的3：确定介导转化生长因子-β的生长反应的遗传效应。在这 目的：我们将利用一种功能基因来鉴定转化生长因子-β介导的生长抑制的其他效应物。 屏幕上。我们将结合外显子捕获和遗传不稳定性来分离失活的基因。 对转化生长因子-β介导的生长抑制的抵抗。分离的基因将被验证和确认为 修复可能的基因，并与使用组织芯片的免疫组织化学研究相关联。

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: Analyzingthe 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 effectorsthat 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.