Novel regulation of the CDK4/Cdh1/Pin1 signaling axis for targeted breast cancer therapies

项目摘要/摘要： 后期促进复合体/环体(APC/C)是一种定义明确的多亚基E3泛素 通过泛素蛋白酶体途径调节靶细胞周期调节因子降解的连接酶 (UPP)，促进细胞周期从中期向后期进展，并参与G1期 维修。APC/C E3连接酶复合体在进化上是保守的，依赖于两种接头蛋白， CDC20和CDH1，识别不同的靶蛋白，调节细胞周期进程。然而， 与受到CDH1介导的破坏的CDC20相比，对E3连接酶活性的调节 CDH1目前还不为人所知。先前的研究表明，在蛋白质表面有19个丝氨酸和苏氨酸残基 在体内可以被多个激酶磷酸化的CDH1，表明CDH1的磷酸化调节是 要复杂得多。在目前的方案中，我发现CDK4在体外可以磷酸化CDH1，并且 调节其E3连接酶的活性。此外，我们还发现CDK4对CDH1的磷酸化可以是 被Pin1脯氨酸异构酶识别，促进CDH1-Pin1复合体的形成。与此相适应 认为，使用CDK4/6抑制剂或突变磷酸化位点可以破坏CDH1-Pin1 互动。因此，CDH1可以介导Pin1的多泛素化和降解。因此，耗尽 内源CDH1可阻断Pin1抑制剂处理诱导的细胞Pin1降解。重要的是 在CDH1缺失的MEF中，Pin1抑制剂诱导的细胞增殖抑制也被取消，这表明 在Pin1抑制剂诱导的细胞增殖抑制中，CDH1的功能性存在是必需的。在……里面 此外，CDK4/6抑制剂和Pin1抑制剂的组合显示出显著增强的抑制作用 对乳腺癌细胞的影响。在这项提议的第一个目的中，我将探索CDK4激酶在 调节CDH1的E3连接酶活性(目标1)。因此，这项提案的第二个目标将是 CDH1在介导Pin1抑制剂诱导Pin1蛋白破坏中的潜在作用 (目标2)。综上所述，这些结果暗示了CDK4/CDH1/Pin1信号轴在调节 细胞增殖，为联合应用CDK4/6抑制剂和Pin1抑制剂治疗乳腺疾病提供了依据 癌症。

Project Summary/Abstract: The anaphase-promoting complex/Cyclosome (APC/C) is a well-defined multi-subunit E3 ubiquitin ligase that regulates targeted cell cycle regulators for degradation by the Ubiquitin Proteasome Pathway (UPP), promoting cell cycle progression from metaphase to anaphase and being involved in G1 phase maintenance. The APC/C E3 ligase complex is evolutionarily conserved and relies on two adaptor proteins, Cdc20 and Cdh1, to recognize different target proteins and regulate cell cycle progression. However, compared to Cdc20 that is subjected to Cdh1-mediated destruction, regulation of the E3 ligase activity of Cdh1 is not well known yet. Previous study has shown that there were 19 serine and threonine residues on Cdh1 that can be phosphorylated by multi-kinases in vivo, indicating that the phosphoregulation of Cdh1 is much more complex. In the present proposal, I found that CDK4 can phosphorylates Cdh1 in vitro and modulates its E3 ligase activity. Furthermore, we found that the phosphorylation of Cdh1 by CDK4 can be recognized by the Pin1 proline isomerase, facilitating Cdh1-Pin1 complex formation. In keeping with this notion, employment of the CDK4/6 inhibitor or mutating the phosphorylation sites can disrupt the Cdh1-Pin1 interaction. Consequently, Cdh1 can mediate Pin1 for polyubiquitination and degradation. As such, depletion of endogenous Cdh1 abolished the Pin1 inhibitor treatment induced Pin1 degradation in cells. Importantly, the Pin1 inhibitor-induced cell proliferation suppression was also abolished in Cdh1-null MEFs, suggesting the functional presence of Cdh1 is required for Pin1 inhibitor-induced cell proliferation suppression. In addition, combination of the CDK4/6 inhibitor and Pin1 inhibitor exhibits significantly enhanced suppressing effect in breast cancer cells. In the first Aim of this proposal, I am going to explore the role of CDK4 kinase in regulating the E3 ligase activity of Cdh1 (Aim #1). Therefore, the second Aim in this proposal will be exploring the potential role of Cdh1 in mediating Pin1 inhibitor treatment induced Pin1 protein destruction (Aim 2). Together, these results implicate a functional role of the CDK4/Cdh1/Pin1 signaling axis in regulating cell proliferation, and provide rational for combining the CDK4/6 inhibitor and Pin1 inhibitor to treat breast cancer.