Genetic effectors of TGF-beta induced growth and arrest.

乳腺癌是美国癌症死亡的主要原因，有约20万新发病例， 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子 将能够识别新的治疗靶点。这项建议的目的是 识别和验证TGF-β信号通路的关键成分， 开发用于治疗乳腺癌的特定疗法。目的1：分析cdc 25 A在肿瘤细胞中的作用 TGF-β介导的乳腺上皮细胞生长刺激。我们的初步研究表明，TGF- β介导的生长刺激与cdc 25 A酪氨酸表达增加相关 磷酸酶基因的细胞缺乏p21。在这个目标中，我们将进一步分析cdc 25 A介导 使用RNA干扰、启动子/突变/ChIP分析和p21检测TGF-β诱导细胞增殖 cDNA突变研究，结合使用组织微阵列分析人类乳腺癌。目的 2：鉴定TGF-β诱导的细胞增殖的介质。在目标2中，我们假设 其它整合基因，其上调是TGF-β诱导的生长刺激所需的。我们将使用 微阵列表达分析以使用我们先前表征的p21-/-细胞系鉴定这些基因。在 为了证实这些基因是TGF-β刺激细胞增殖的真正介质，RNA 干扰基因“敲低”实验和通过同源重组的靶向基因消除将 被执行。目的3：鉴定介导TGF-β生长反应的遗传效应子。在这 目的，我们将使用功能性遗传学方法鉴定TGF-β介导的生长抑制的其他效应物。 屏幕我们将联合收割机外显子捕获与遗传不稳定性相结合，分离出失活导致 对TGF-β介导的生长抑制的抗性。分离的基因将由以下人员进行验证和确认： 恢复假定的基因，并与免疫组织化学研究使用组织芯片。

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.