Interplay between Cdh1 and major regulatory pathways in human cancer

 描述（由申请人提供）：细胞周期调控缺陷导致基因组不稳定并最终导致癌症发展。后期促进复合物（APC）通过形成两个不同的亚复合物APCCdh 1和APCCdc 20来调节细胞周期进程，以促进各种细胞周期调节剂的及时泛素化和降解。APC激活剂Cdh 1与肿瘤发生有关，但其在肿瘤抑制中的确切作用仍不清楚。因此，本提案的主要目标将是继续我们先前的努力，在体外和体内研究Cdh 1如何通过与多种主要肿瘤抑制因子和致癌途径的相互作用来阻断癌症的发展和进展。我们先前发表了无APC的Cdh 1可以增强含HECT结构域的E3连接酶Smurf 1的催化活性以调节成骨细胞分化。此外，我们获得的初步结果表明，除了Smurf 1，Cdh 1也可以调节E3连接酶活性的WWP 2，另一个NEDD 4家族成员，在APC-独立的方式。更重要的是，我们发现Cdh 1的缺失增强了WWP 2介导的泛素底物PTEN的降解。在目标#1中，我们提出使用多种遗传和生物化学方法来鉴定Cdh 1控制WWP 2 E3连接酶活性以管理PTEN的及时降解的新的APC独立的分子机制，并评估Akt信号传导的升高是否在体内Cdh 1介导的癌症发展的丧失中具有关键作用。在目标#2中，我们将表征Cdh 1在通过抑制BRaf活性来抑制肿瘤发生中的多机制作用。我们的初步数据显示，Cdh 1作为一种新的负调节BRaf/ERK致癌信号通过多种机制在上下文依赖性的方式。在原代细胞中，APCCdh 1标记BRaf用于泛素化介导的破坏，而在癌细胞中，Cdh 1主要通过以APC非依赖性方式破坏BRaf二聚体来抑制BRaf激酶活性。在这个特定的目标，我们将进一步表征的分子机制，APCCdh 1目标BRaf的泛素化介导的降解。我们还将研究无APC的Cdh 1如何抑制BRaf二聚化，导致ERK活性降低，从而影响肿瘤发生。此外，我们还将确定一种可能的双重调节机制，在BRafV 600 E黑色素瘤细胞中，ERK和Cdk 4/CyclinD 1的活性升高可能通过直接磷酸化Cdh 1来抑制APCCdh 1。更重要的是，使用多种体外细胞培养和体内工程动物模型，我们将进一步确定APC依赖性和非依赖性调节途径在介导Cdh 1的肿瘤抑制能力中的意义。总而言之，所提出的研究不仅将为Cdh 1在肿瘤发生中的作用提供新的机制见解，而且还将为靶向治疗的开发提供理论基础和分子基础，以抑制Cdh 1的下游靶点，例如在乳腺癌环境中调节PTEN/Akt信号传导（Aim #1）的WWP 2，以及在黑色素瘤发展中起驱动作用的BRaf/ERK信号传导（Aim #2）。

 DESCRIPTION (provided by applicant): Defective cell cycle regulation leads to genomic instability and ultimately cancer development. The Anaphase Promoting Complex (APC) regulates cell cycle progression by forming two distinct sub-complexes, APCCdh1 and APCCdc20, to promote the timely ubiquitination and degradation of various cell cycle regulators. The APC activator Cdh1 has been implicated in tumorigenesis, while its exact roles in tumor suppression remain largely unclear. Therefore, the primary objective of this proposal will be to continue our previous efforts in examining both in vitro and in vivo how Cdh1 functions to block cancer development and progression by its interplay with multiple major tumor suppressor and oncogenic pathways. We previously published that APC- free Cdh1 could augment the catalytic activity of HECT domain-containing E3 ligase Smurf1 to regulate osteoblast differentiation. Furthermore, we obtained preliminary results showing that in addition to Smurf1, Cdh1 could also modulate the E3 ligase activity of WWP2, another NEDD4 family member, in an APC- independent manner. More importantly, we showed that depletion of Cdh1 enhanced WWP2-mediated degradation of its ubiquitin substrate, PTEN. In Aim #1, we propose to use multiple genetic and biochemical approaches to identify the novel APC-independent molecular mechanism(s) by which Cdh1 controls WWP2 E3 ligase activity to govern timely degradation of PTEN, and to evaluate whether elevation of Akt signaling has a critical role in loss of Cdh1-mediated cancer development in vivo. In Aim #2, we will characterize a multi- mechanistic role for Cdh1 in inhibiting tumorigenesis by suppressing BRaf activity. Our preliminary data reveal Cdh1 as a novel negative regulator of the BRaf/ERK oncogenic signaling through multiple mechanisms in a context-dependent manner. In primary cells, APCCdh1 earmarks BRaf for ubiquitination-mediated destruction, while in cancer cells, Cdh1 inhibits BRaf kinase activity largely by disrupting BRaf dimers in an APC- independent manner. In this Specific Aim, we will further characterize the molecular mechanism by which APCCdh1 targets BRaf for ubiquitination-mediated degradation. We will also examine how APC-free Cdh1 may inhibit BRaf dimerization leading to decreased ERK activity to influence tumorigenesis. Furthermore, we will also identify a possible dual regulatory mechanism in BRafV600E melanoma cells where both elevated activity of ERK and Cdk4/CyclinD1 may inhibit APCCdh1 by directly phosphorylating Cdh1. More importantly, using multiple in vitro cell culture and in vivo engineered animal models, we will further determine the significance of both APC-dependent and -independent regulatory pathways in mediating the tumor suppressive capabilities of Cdh1. Altogether, the proposed studies will not only provide novel mechanistic insights into the role of Cdh1 in tumorigenesis, but will also offer the rationale and molecular basis for the development of targeted therapeutics to inhibit downstream targets of Cdh1 such as WWP2 in modulating PTEN/Akt signaling (Aim #1) in breast cancer setting, and BRaf/ERK signaling (Aim #2) that plays driving roles in melanoma development.