The Oncogenic Significance of Cyclin Overexpression and Smad 3 Tumor Suppression

描述(申请人提供)：美国每年约有44,000名女性死于乳腺癌，乳腺癌是继肺癌之后女性最常见的癌症死亡原因。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成分Smads有关。我们工作的长期目标是调查Smad3信号转导的变化如何影响乳腺癌的进展，以帮助建立疾病的分子分期，并促进发现导致疾病消退的新的治疗策略。这一假设认为，在高表达细胞周期蛋白的乳腺癌中，CDK4/2的激活导致Smad3的磷酸化和抑制，从而释放细胞周期停滞，促进细胞增殖和转移。我们的假说基于以下观察：1)Smad3信号通过转录细胞周期蛋白依赖性激酶(CDK)抑制物转录和抑制细胞周期有丝分裂原c-myc而导致G1期细胞周期停滞；2)Smad3信号被CDK4/2磷酸化后被抑制，CDK4/2是由细胞周期蛋白D和E调节的激酶；3)某些侵袭性、基底层类型的乳腺癌过度表达细胞周期蛋白，与高转移性疾病相关的预后不良。基于这些发现，本研究的三个具体目标将直接研究乳腺癌中细胞周期蛋白过度表达和Smad3抑制之间的相互作用，具体如下：目的1：研究细胞周期蛋白介导的Smad3抑制机制及其对G1期抑制的影响。这一目标将测试CDK磷酸化在Smad3介导的细胞周期控制中的作用，并利用转录因子报告阵列来评估周期蛋白在乳腺癌细胞中过度表达的下游后果。目的：研究CDK抑制对高表达细胞周期蛋白的乳腺癌细胞体外及原发和转移性异种移植瘤增殖的影响。这一目标将在体外培养和体内小鼠模型中检验CDK4/2抑制Smad3磷酸化降低乳腺癌细胞增殖的假设。目的：研究Smad3、Cyclin D、Cyclin E、CDK4和CDK2在1、2、3级乳腺癌、基底细胞样癌和正常乳腺组织中的表达。这一目标将测试乳腺癌亚型是否存在Smad3、Cyclins和CDKs的模式表达，这些表达与更多已建立的乳腺癌预后标志相关。 公共卫生相关性：对Smad3等新的肿瘤抑制因子的研究可能会通过促进疾病的有组织的分子分期来扩大乳腺癌的传统分期和分级。这些努力最终将使个体化预后标记物的进一步发展成为可能，以实施针对患者的治疗策略。靶向CDK/Cyclin活性以恢复Smad3肿瘤抑制可能为Cyclin过表达和基底细胞样乳腺癌患者带来希望，这些患者目前的疾病亚型预后最差。

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.