The Oncogenic Significance of Cyclin Overexpression and Smad 3 Tumor Suppression

描述（由申请人提供）：在美国，每年大约有44,000名妇女死于乳腺癌，而乳腺癌是仅次于肺癌的最常见的女性癌症死亡原因。乳腺癌发病和疾病进展的几个方面与TGF2超家族成员及其相关的下游信号元件Smads有关。我们工作的长期目标是研究Smad 3信号转导的改变如何影响乳腺癌的进展，以帮助建立疾病的分子分期，并促进发现将导致疾病消退的新治疗策略。本研究假设在细胞周期蛋白过表达的乳腺癌中，CDK4/2的激活导致Smad 3的磷酸化和抑制，从而释放细胞周期阻滞，促进细胞增殖和转移。我们的假设基于以下观察结果：1)Smad 3信号通过细胞周期蛋白依赖性激酶（CDK）抑制剂的转录和细胞周期有丝分裂原c-myc的抑制，有助于G1细胞周期阻滞；2)Smad 3的作用在细胞周期蛋白D和E调节的激酶CDK4/2磷酸化时被抑制；3)某些侵袭性基底型乳腺癌过度表达细胞周期蛋白，并且与高转移性疾病相关的预后较差。基于这些发现，本提案的三个具体目的将直接研究乳腺癌中细胞周期蛋白过表达与Smad 3抑制之间的相互作用：目的1：研究细胞周期蛋白介导的Smad 3抑制机制以及这种抑制对G1阻滞的影响。本研究将测试CDK磷酸化对Smad 3介导的细胞周期控制的影响，并利用转录因子报告基因阵列来评估乳腺癌细胞周期蛋白过表达的下游后果。目的2：通过小鼠模型研究CDK抑制对细胞周期蛋白过表达的乳腺癌细胞增殖的影响，以及对原发和转移性异种移植的影响。这一目的将在体外培养和体内小鼠模型中验证CDK4/2抑制Smad 3磷酸化降低乳腺癌细胞增殖的假设。目的3：研究Smad 3、cyclin D、cyclin E、CDK4和CDK2在1级、2级和3级人乳腺癌组织、基底样乳腺癌和正常乳腺组织中的表达模式。这一目标将测试乳腺癌亚型是否存在Smad 3、细胞周期蛋白和CDKs的模式表达，它们与更成熟的乳腺癌预后标志物相关。

DESCRIPTION (provided by applicant): Approximately 44,000 women die of breast cancer in the United States each year, and after lung cancer, breast cancer is the most common cause of cancer death in women. Several aspects of breast cancer onset and disease progression have been linked to members of the TGF2 superfamily and their associated downstream signaling components, the Smads. The long-term goal of our work is to investigate how alterations in Smad 3 signal transduction affect breast cancer progression to help establish a molecular staging of disease and to facilitate the discovery of novel treatment strategies that will result in disease regression. The hypothesis of this proposal is that in cyclin overexpressing breast cancers, activation of CDK4/2 leads to phosphorylation and inhibition of Smad 3, thus releasing cell cycle arrest and promoting cell proliferation and metastasis. Our hypothesis is based on the following observations: 1) Smad 3 signaling contributes to G1 cell cycle arrest through transcription of cyclin dependent kinase (CDK) inhibitors and repression of the cell cycle mitogen c-myc, 2) Smad 3 action is inhibited upon phosphorylation by CDK4/2, kinases that are regulated by cyclins D and E, 3) Certain aggressive, basal-type breast cancers overexpress cyclins and have poor outcomes associated with highly metastatic disease. Predicated on these findings, the three specific aims of this proposal will directly examine the interaction between cyclin overexpression and Smad 3 inhibition in breast cancer as follows: Aim 1: Investigate cyclin-mediated mechanisms of Smad 3 inhibition and the impact of this inhibition on G1 arrest. This aim will test the effects of CDK phosphorylation on Smad 3-mediated cell cycle control and utilize a transcription factor reporter array to assess the downstream consequences of cyclin overexpression in breast cancer cells. Aim 2: Investigate the effect of CDK inhibition on the proliferation of cyclin overexpressing breast cancer cells in vitro and in primary and metastatic xenografts using murine models. This aim will test the hypothesis that CDK4/2 inhibition of Smad 3 phosphorylation decreases breast cancer cell proliferation in in vitro cultures and in vivo murine models. Aim 3: Investigate the expression patterns of Smad 3, cyclin D, cyclin E, CDK4 and CDK2 in grades 1, 2, and 3 human breast cancer tissues, basal-like breast cancers, and normal mammary tissue. This aim will test whether subtypes of breast cancer exist with patterned expression of Smad 3, cyclins and CDKs, which correlate with more established breast cancer prognostic markers. PUBLIC HEALTH RELEVANCE: The study of new tumor suppressors such as Smad 3 may expand the conventional staging and grading of breast cancer by facilitating an organized molecular staging of disease. These efforts will ultimately allow for the further development of individualized prognostic markers to actualize patient-specific treatment strategies. Therapeutic targeting of CDK/cyclin activity to restore Smad 3 tumor suppression may hold promise for patients with cyclin overexpressing and basal-like breast cancers, who currently have the disease sub-type with the worst prognosis.