Ecd as a regulator of cell cycle and breast oncogenesis

描述（由申请人提供）：精确调节的细胞增殖对于胚胎发育以及成体器官和组织的体内平衡至关重要，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控制的是癌细胞生物学研究的一个重要领域。大量的证据已经建立了控制细胞周期进程的基本范例，涉及视网膜母细胞瘤（Rb）蛋白家族与转录因子的E2 F家族的结合。在G 0/G1期，低磷酸化Rb蛋白与E2 F的相互作用阻止E2 F靶基因的转录。在细胞周期进程中产生的细胞周期蛋白-CDK复合物过度磷酸化Rb，导致Rb从E2 F释放;这允许E2 F靶基因转录和细胞周期进程。 我们已经确定了哺乳动物直系同源的果蝇ecdysoneless（Ecd）蛋白作为一种新的和必不可少的调节Rb-E2 F依赖的细胞周期进程。Ecd的缺失阻碍了Rb与E2 F的分离，使细胞停滞在G1/S边界，并阻止细胞周期的进展。这些发现导致了一种新的模型，代表了Rb-E2 F依赖性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的永生人类乳腺上皮细胞（hMEC）中的细胞周期快速转运相比，成纤维细胞中的p53依赖性衰老;以及Ecd与活化Ras的共过表达诱导了三维培养中hMEC的显著过度增殖和异常分支。这些特征使人联想到由癌基因（如Ras）诱导的衰老。这些研究结果使我们假设，Ecd是一种新的和必要的组成部分，Rb-E2 F依赖性控制细胞周期的进展，和改变的水平和/或功能的Ecd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设。我们将研究Ecd在细胞周期进程及其调控中作用的结构基础。我们将表征ECD诱导的细胞衰老。我们将分析Ecd过表达在体外和体内使用诱导型转基因小鼠促进乳腺癌发生的后果。最后，我们将使用Ecd-floxed小鼠中Ecd的乳腺特异性缺失来确定Ecd是否对由人类乳腺癌相关癌基因ErbB 2驱动的乳腺肿瘤起始、进展和维持至关重要。我们的研究的成功结果将阐明一种新型细胞周期控制调节剂在乳腺癌中的作用，对人类癌症的肿瘤发生具有广泛的影响，并有助于将Ecd作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.