Interplay between Cdh1 and major regulatory pathways in human cancer

描述(申请人提供)：有缺陷的细胞周期调节导致基因组不稳定，最终导致癌症的发展。E3泛素连接酶APC/CDH1是细胞周期进程的主要调节因子，与DNA损伤修复和肿瘤抑制有关，尽管其确切作用尚不清楚。在这个计划中，我们将探索CDH1与人类癌症中经常改变的多个主要抑癌和致癌途径的相互作用，以确定CDH1的新功能和调控机制。我们先前报道，CDH1靶向Skp2癌蛋白进行蛋白降解；我们的初步数据表明，PI3K/Akt通路保护Skp2免受CDH1介导的破坏。在目标1中，我们将利用遗传和生化方法来揭示Akt1控制Skp2丰度和细胞定位的潜在分子机制。这些研究将为Skp2在人类癌症中的过度表达提供一种新的机制。这也将促进我们对特定的激酶信号级联如何影响由APC/CDH1复合体控制的蛋白降解的了解，并为开发Akt1特异性抑制剂作为有效的抗癌药物提供理论基础。在目标2中，我们将确定CDH1缺失激活P53和Rb通路的分子机制，以及它们在CDH1缺失诱导的过早衰老中的作用。我们发现，通过控制Claspin的破坏，CDH1调节Chk1的活性，并进一步影响P53通路，暗示CDH1在DNA损伤修复中发挥作用。我们还发现，CDH1可以影响Rb/E2F1的功能。因此，原代人成纤维细胞中CDH1的缺失通过激活P53和Rb通路而导致过早衰老的开始。其他肿瘤抑制基因的失活，包括PTEN和VHL，也会导致过早衰老，这被认为是一种内在的故障安全机制，可以防止癌症的发展。我们在AIM 2中提出的工作将为肿瘤细胞中较少观察到的CDH1丢失提供可能的潜在分子机制，这可能进一步暗示CDH1丢失是肿瘤发展的晚期事件。最后，我们的提案探讨了CDH1本身是如何受到监管的。我们的初步数据表明，在G1/S晚期，MDM2癌蛋白控制着CDH1的稳定性。这一发现扩大了我们对CDH1和P53/MDM2通路之间相互作用的理解，并进一步证明了主要细胞周期调节因子的功能相互交织，以实现协同效应。我们在AIM 3中提出的工作将为CDH1稳定性控制提供一种新的调控机制，从而揭示MDM2致癌功能的另一层机制，并支持MDM2抑制剂用于癌症治疗。综上所述，这些研究将极大地扩展我们目前对CDH1在细胞周期控制中的经典作用之外的重要功能的认识，通过深入了解CDH1如何整合到主要的肿瘤抑制因子(P53和Rb)和癌基因(Akt和MDM2)网络中，不仅控制细胞周期的进展，而且维持基因组的稳定性和参与肿瘤抑制。

DESCRIPTION (provided by applicant): Defective cell cycle regulation leads to genomic instability and ultimately cancer development. The E3 ubiquitin ligase APC/Cdh1 is a major regulator of cell cycle progression and has been implicated in DNA damage repair and tumor suppression, although its exact roles remain unclear. In this proposal, we will explore the interaction of Cdh1 with multiple major tumor suppressor and oncogenic pathways frequently altered in human cancers to define the novel functions as well as regulatory mechanisms for Cdh1. We previously reported that Cdh1 targets the Skp2 oncoprotein for proteolysis; our preliminary data showed that the PI3K/Akt pathway protected Skp2 from Cdh1-mediated destruction. In Aim 1, we will utilize both genetic and biochemical approaches to reveal the underlying molecular mechanisms by which Akt1 controls Skp2 abundance and cellular localization. The proposed studies will provide a novel mechanism for Skp2 overexpression in human cancers. It will also advance our knowledge of how specific kinase signaling cascades influence proteolysis governed by the APC/Cdh1 complex, and provide the rationale for developing Akt1-specific inhibitors as potent anti-cancer drugs. In Aim 2, we will define the molecular mechanisms by which loss of Cdh1 activates the p53 and Rb pathways, and their contribution to Cdh1 loss-induced premature senescence. We found that by controlling Claspin destruction, Cdh1 regulates Chk1 activity and further influences the p53 pathway, implicating a role for Cdh1 in DNA damage repair. We also found that Cdh1 could affect Rb/E2F1 function. As a result, depletion of Cdh1 in primary human fibroblasts led to the onset of premature senescence by activating both the p53 and Rb pathways. Inactivation of other tumor suppressors, including PTEN and VHL, also induced premature senescence, which has been proposed as a built-in fail-safe mechanism against cancer development. Our proposed work in Aim 2 will provide the possible underlying molecular mechanism for the less frequently observed Cdh1 loss in tumor cells, which could further imply Cdh1 loss as a late event in tumor development. Lastly, our proposal explores how Cdh1 itself is regulated. Our preliminary data indicated that the Mdm2 oncoprotein controls the stability of Cdh1 in late G1/S phase. This finding extends our understanding of the interplay between the Cdh1 and p53/Mdm2 pathways, and provides further evidence that the functions of major cell cycle regulators are interwoven to achieve synergized effects. Our proposed work in Aim 3 will provide a novel regulatory mechanism for Cdh1 stability control, thus uncovering another layer of mechanism for the oncogenic function of Mdm2 and supporting the use of Mdm2 inhibitors for cancer treatment. Altogether, these studies will significantly expand our current knowledge of the important functions of Cdh1 outside its classic role in cell cycle control by providing insight into how Cdh1 integrates into the network of major tumor suppressor (p53 and Rb) and oncogene (Akt and Mdm2) pathways to not only govern cell cycle progression, but also maintain genomic stability and participate in tumor suppression.