Cyclin D1 and mammary carcinoma

描述（由申请人提供）：我的长期研究目标集中在阐明细胞周期机制感知细胞外信号并调节细胞周期进程的机制。这一信息将为阐明肿瘤生长调控通路是如何被破坏提供必要的框架。我们目前的研究重点是生长信号通路如何调节有丝分裂反应的d型细胞周期蛋白，更具体地说，这些通路如何调节活跃的核细胞周期蛋白D1依赖性激酶的积累。我们的研究表明，细胞在s期从细胞核中去除活性D1/CDK复合物的失败会导致细胞转化，从而强调了阐明调节细胞周期蛋白D1核积累机制的重要性。在细胞周期的G1期，细胞周期蛋白D1在细胞核中聚集，以响应有丝分裂刺激。在s期，细胞周期蛋白D1通过gsk -3 β磷酸化单个苏氨酸残基Thr-286到达细胞质。我们已经确定了一种天然存在的周期蛋白D1异构体D1b，它缺乏周期蛋白核输出所需的关键残基。我们的初步数据表明，这种异构体在癌细胞中特异性表达，可能代表典型细胞周期蛋白D1异构体的致癌变异。此外，在子宫内膜癌中已经报道了细胞周期蛋白D1的c端突变会破坏细胞周期蛋白D1的核输出。我们假设细胞周期蛋白D1b和核细胞周期蛋白D1突变亚型是典型细胞周期蛋白D1 （D1a）的致癌变异，其过表达直接导致肿瘤恶性。为了验证这一假设，我们提出：1)确定cyclin D1b在乳腺癌中的过表达频率；2)确定构成核的cyclin D1突变体驱动乳腺癌的能力；3)描述癌症特异性细胞周期蛋白D1突变体。为了实现这些目标，我们将利用宾夕法尼亚大学的资源来确定核周期蛋白D1变体D1b在原发性人类乳腺癌中的频率，并评估其预后价值。此外，我们将使用新建立的小鼠模型来评估核周期蛋白D1亚型在体内的致癌性。最后，我们建议对新发现的细胞周期蛋白D1突变体进行活性的、依赖crm1的核输出的能力进行表征。虽然野生型细胞周期蛋白D1的表达在体外或动物模型中没有明显的致癌作用，但我的实验室已经证明，组成型核细胞周期蛋白D1亚型具有很强的致癌作用。

DESCRIPTION (provided by applicant): The long-term objective of my research centers on elucidation of the mechanisms whereby extra-cellular signals are sensed by the cell cycle machinery and regulate cell cycle progression. This information will provide the framework necessary to elucidate how growth regulatory pathways are subverted in neoplasia. Our current studies focus on how growth-signaling pathways regulate the mitogenically responsive D-type cyclins and more specifically how these pathways regulate accumulation of an active, nuclear cyclin D1- dependent kinase. The importance of elucidating the mechanisms that regulate nuclear accumulation of cyclin D1 is emphasized by our demonstration that the failure of the cell to remove active D1/CDK complexes from the nucleus during S-phase results in cell transformation. Cyclin D1 accumulates in the nucleus during G1 phase of the cell cycle in response to mitogenic stimulation. During S-phase, cyclin D1 is targeted to the cytoplasm via phosphorylation of the at a single threonyl residue, Thr-286, by GSK-3beta. We have identified a naturally occurring cyclin D1 isoform, D1b, which lacks critical residues necessary for cyclin nuclear export. Our preliminary data indicate that this isoform is specifically expressed in cancer cells and is likely to represent an oncogenic variant of the canonical cyclin D1 isoform. In addition, mutations in the C-terminus of cyclin D1 that will disrupt cyclin D1 nuclear export have been reported in endometrial cancer. We hypothesize that cyclin D1b and mutant nuclear cyclin D1 isoforms are oncogenic variants of canonical cyclin D1 (D1a) whose overexpression contributes directly to neoplastic malignancy. To test this hypothesis, we propose to: 1) Determine the frequency of cyclin D1b overexpression in breast carcinoma; 2) Determine the capacity of cyclin D1 mutants that are constitutively nuclear to drive mammary carcinoma; and 3) Characterize cancer specific cyclin D1 mutants. To accomplish these goals, we will utilize resources here at the University of Pennsylvania to determine the frequency of the nuclear cyclin D1 variant, D1b, in primary human breast cancer and assess its prognostic value. In addition, we will use newly established mouse models to assess the oncogenicity of nuclear cyclin D1 isoforms in vivo. Finally, we propose to characterize newly identified cyclin D1 mutants for their ability to undergo active, CRM1-dependent nuclear export. While expression of wild-type cyclin D1 is not overtly oncogenic in vitro or in animal models, my laboratory has demonstrated that constitutively nuclear cyclin D1 isoforms are strongly oncogenic. Cancer Relevance. Findings from this work would support a model wherein constitutively nuclear cyclin D1 functions as an initiating oncogene and that mechanisms, which regulate its nuclear retention will be targeted during carcinogenesis. Consistent with this, in our preliminary data we provide evidence for a novel, constitutively nuclear cyclin D1 isoform, which is expressed exclusively in cancer. The work proposed herein will establish the relationship between cyclin D1 nuclear retention and the function of cyclin D1 as an oncogenic protein in mammary carcinoma.