Genetic effectors of TGF-beta induced growth and arrest

描述（由申请人提供）：乳腺癌是癌症死亡的主要原因， 在美国，每年约有190，000例新病例和约40，000例死亡。更好地了解肿瘤生长和抑制的遗传效应物将能够识别新的治疗靶点。该提案的目的是识别和验证TGF-β信号通路的关键成分，从而开发出治疗乳腺癌的特定疗法。目的1：分析cdc 25 A在TGF-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，TGF β介导的生长刺激与缺乏p21的细胞中cdc 25 A酪氨酸磷酸酶基因表达增加相关。 在这个目标中，我们将进一步分析cdc 25 A介导TGF-β诱导的细胞凋亡的能力。 使用RNA干扰和启动子/突变/ChIP测定结合使用组织微阵列分析人乳腺癌的增殖。目的2：鉴定TGF-β诱导细胞增殖的介质。在目标2中，我们假设有其他的整合基因，其上调是TGF-β诱导的生长刺激所必需的。我们将使用微阵列表达分析，以确定这些基因使用我们以前的特点p21-/-细胞系。为了验证这些基因是TGF-β刺激细胞增殖的真正介质，将进行RNA干扰基因“敲低”实验和通过同源重组进行靶向基因消除。目的3：鉴定介导对TGF-β的生长反应的遗传效应物。在这一目标中，我们将使用功能性遗传筛选来鉴定TGF-β介导的生长抑制的其他效应物。我们将联合收割机外显子捕获与遗传不稳定性相结合，分离出失活导致耐药的基因。 TGF-β介导的生长抑制。分离的基因将通过恢复推定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。

DESCRIPTION (provided by applicant): Breast Cancer is a leading cause of cancer death in the United States with approximately 190,000 new cases and approximately 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 TGFbeta 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 and promoter/mutational/ChIP assays 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.