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，仅次于肺癌，乳腺癌是女性癌症死亡的最常见原因。乳腺癌发病和疾病进展的几个方面与TGF 2超家族成员及其相关的下游信号组分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，并促进发现新的治疗策略，从而导致疾病消退。这一建议的假设是，在cyclin过表达的乳腺癌中，CDK 4/2的激活导致磷酸化并抑制Smad 3，从而释放细胞周期停滞并促进细胞增殖和转移。我们的假设基于以下观察：1）Smad 3信号传导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的抑制而有助于G1细胞周期停滞，2）Smad 3作用在磷酸化后被CDK 4/2抑制，CDK 4/2是由细胞周期蛋白D和E调节的激酶，3）某些侵袭性，基底型乳腺癌过表达细胞周期蛋白，并且具有与高转移性疾病相关的不良结果。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表达和Smad 3抑制之间的相互作用，具体如下：目标1：研究细胞周期蛋白介导的Smad 3抑制机制及其对G1期阻滞的影响。这个目标将测试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阵列来评估乳腺癌细胞中细胞周期蛋白过表达的下游后果。目标二：研究CDK抑制对细胞周期蛋白过表达乳腺癌细胞体外增殖的影响，以及使用小鼠模型在原发性和转移性异种移植物中的影响。这一目的将检验CDK 4/2抑制Smad 3磷酸化降低体外培养和体内小鼠模型中乳腺癌细胞增殖的假设。目标3：研究Smad 3、cyclin D、cyclin E、CDK 4和CDK 2在1、2、3级人乳腺癌组织、基底细胞样乳腺癌和正常乳腺组织中的表达模式。这一目标将测试是否存在具有Smad 3、细胞周期蛋白和CDK模式表达的乳腺癌亚型，这些亚型与更确定的乳腺癌预后标志物相关。 公共卫生相关性：对新的肿瘤抑制因子如Smad 3的研究可能通过促进疾病的组织化分子分期来扩展乳腺癌的常规分期和分级。这些努力最终将允许进一步开发个性化的预后标志物，以实现患者特异性治疗策略。靶向CDK/细胞周期蛋白活性以恢复Smad 3肿瘤抑制的治疗可能为细胞周期蛋白过表达和基底样乳腺癌患者带来希望，这些患者目前患有预后最差的疾病亚型。

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.