Ecd as a regulator of cell cycle and breast oncogenesis

描述(申请人提供)：精确调控的细胞增殖对于胚胎发育以及成人器官和组织的动态平衡是必不可少的，而不受控制的细胞增殖是癌症的标志。因此，阐明细胞周期机制是如何被控制的是癌细胞生物学的一个重要研究领域。大量证据表明，视网膜母细胞瘤(RB)蛋白家族与E2F转录因子家族共同参与了细胞周期调控的基本范式。在G0/G1期，低磷酸化的Rb蛋白与E2F相互作用，阻止E2F靶基因的转录。在细胞周期过程中产生的细胞周期蛋白-CDK复合体过度磷酸化Rb，导致Rb从E2F中释放；这使得E2F靶基因转录和细胞周期进展。 我们发现哺乳动物中的无蜕皮激素(ECD)蛋白是Rb-E2F依赖的细胞周期进程中一种新的和重要的调节因子。ECD的丢失延缓了Rb和E2F的分离，阻止了G1/S交界处的细胞，并阻止了细胞周期的进展。这些发现导致了一个新的模型，它代表了依赖RB-E2F的细胞周期控制范式的根本转变。值得注意的是，ECD在乳腺癌细胞系以及乳腺导管原位癌和浸润性导管癌中过表达。值得注意的是，ECD过表达产生了两种相反的表型：与缺乏p16的永生人乳腺上皮细胞(HMECs)相比，成纤维细胞中P53依赖的衰老和细胞周期的快速传递；ECD和激活的RAS共表达诱导了三维培养中hMECs的显著过度增殖和异常分支。这些特征使人想起RAS等癌基因引起的衰老。这些发现导致我们假设ECD是RB-E2F依赖的细胞周期进程控制的新的和必要的组件，并且ECD水平和/或功能的改变有助于致癌转化。在这里，我们将使用我们团队建立的独特和创新的细胞和动物模型来解决这些假说。我们将研究ECD在细胞周期进程中的作用及其调控的结构基础。我们将描述ECD诱导的细胞衰老的特征。我们将利用可诱导的转基因小鼠，在体外和体内分析ECD过表达在促进乳腺肿瘤发生中的后果。最后，我们将确定ECD是否是由人类乳腺癌相关癌基因ErbB2驱动的乳腺癌发生、发展和维持所必需的，在ECD-FLOX小鼠中使用乳腺特异的ECD缺失。如果我们的研究取得成功，将阐明一种新的细胞周期调控因子在乳腺癌中的作用，对人类癌症的发生具有广泛的意义，并可能有助于将ECD确立为癌症的潜在治疗靶点。

DESCRIPTION (provided by applicant): Precisely regulated cell proliferation is essential for embryonic development as well as homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer. Thus, elucidating how the cell cycle machinery is controlled is an important area of research in cancer cell biology. A large body of evidence has established a basic paradigm of the control of cell cycle progression involving the Retinoblastoma (Rb) protein family in conjunction with the E2F family of transcription factors. During G0/G1, interaction of hypo-phosphorylated Rb proteins with E2Fs prevents the transcription of E2F target genes. Cyclin-CDK complexes generated during cell cycle progression hyper-phosphorylate Rb, leading to release of Rb from E2Fs; this allows E2F target gene transcription and cell cycle progression. We have identified the mammalian ortholog of Drosophila ecdysoneless (Ecd) protein as a novel and essential regulator of Rb-E2F-dependent cell cycle progression. Loss of Ecd retards the separation of Rb from E2F, arrests cells at G1/S boundary and prevents cell cycle progression. These findings have led to a new model that represents a fundamental shift in the Rb-E2F-dependent cell cycle control paradigm. Notably, Ecd is overexpressed in breast cancer cell lines as well as in ductal carcinoma in situ and infiltrating ductal carcinomas of the breast. Notably, Ecd overexpression produced two opposite phenotypes: p53-dependent senescence in fibroblasts, compared to rapid transit through cell cycle in immortal human mammary epithelial cells (hMECs) that lack p16; and co-overexpression of Ecd with activated Ras induced a dramatic hyper-proliferation and aberrant branching of hMECs in three-dimensional culture. These features are reminiscent of senescence induced by oncogenes, such as Ras. These findings lead us to hypothesize that Ecd is a novel and essential component of Rb-E2F-dependent control of cell cycle progression, and alterations in the levels and/or function of Ecd contribute to oncogenic transformation. Here, we will address these hypotheses using unique and innovative cellular and animal models established by our team. We will examine the structural basis of the role of Ecd in cell cycle progression and its regulation. We will characterize Ecd-induced cellular senescence. We will analyze the consequences of Ecd overexpression in promoting mammary oncogenesis in vitro and in vivo using inducible transgenic mice. Finally, we will determine if Ecd is essential for mammary tumor initiation, progression and maintenance driven by a human breast cancer-relevant oncogene ErbB2 using mammary-specific deletion of Ecd in Ecd-floxed mice. A successful outcome of our studies will elucidate the role of a novel cell cycle control regulator in breast cancer with broad implications for oncogenesis in human cancer, and could help establish Ecd as a potential therapeutic target in cancer.