Mechanism of Merlin-mediated contact inhibition and tumor suppression.

描述(申请人提供)：目前的模型表明，由肿瘤抑制基因NF2编码的FERM结构域蛋白Merlin抑制质膜或质膜附近的有丝分裂信号。我们发现，封闭的、生长抑制形式的Merlin聚集在细胞核中，并与E3泛素连接酶CRL4DCAF1的受体成分DCAF1结合。遗传和生化证据表明，Merlin是CRL4DCAF1的负调控因子。在Merlin缺失的细胞中，DCAF1的缺失阻止了接触抑制的退出和细胞周期的进展。Merlin的表达和DCAF1的沉默在很大程度上诱导了基因表达的重叠，其中包括生长停滞和原凋亡基因的上调，有丝分裂和存活基因的下调。肿瘤来源的Merlin突变体不能积聚到细胞核中，不能与DCAF1结合，也不能抑制CRL4DCAF1。最后，DCAF1的缺失抑制了Merlin缺陷的肿瘤细胞在软琼脂中生长和在裸鼠体内形成肿瘤的能力。这些发现强烈表明，Merlin通过移位到细胞核来抑制CRL4DCAF1依赖的基因表达，从而抑制肿瘤的发生。我们建议追求四个具体目标。I)为了确定Merlin是否通过抑制CRL4DCAF1来介导接触抑制，我们将检测Merlin不敏感的DCAF1的表达是否会导致接触抑制的丧失。此外，我们将检查已经失去抑制CRL4DCAF1能力的肿瘤来源的Merlin错义突变体是否无法介导接触抑制。Ii)为了检验Merlin是否通过抑制CRL4DCAF1在体内抑制肿瘤形成，我们将在它们的Schwann细胞中产生缺乏DCAF1的小鼠，并将它们培育成NF2的小鼠模型。我们将检测对照和DCAF1突变小鼠的肿瘤发生和发展，就像我们之前在整合素信号研究中所做的那样。Iii)为了鉴定CRL4DCAF1的生理底物并研究它们在Merlin介导的肿瘤抑制中的作用，我们将进行串联亲和纯化，然后使用野生型或突变失活的DCAF1进行质谱分析。或者，我们将使控制和DCAF1沉默的细胞接受全球稳定性分析，正如Elledge实验室最近所描述的那样。IV)为了检测CRL4DCAF1是否具有促肿瘤活性，我们将检测DCAF1的过表达是否增强Merlin缺陷、Merlin重新表达或这两种类型的神经鞘瘤细胞在体外增殖的能力以及它们在体内形成肿瘤的能力。此外，我们将检查编码DCAF1的基因在NF2相关或散发性脑膜瘤和神经鞘瘤中是否因C末端缺失而被扩增或结构性激活。综上所述，这些研究应该有助于阐明Merlin介导接触抑制和抑制肿瘤形成的机制。 公共卫生相关性：接触抑制生长对于适当的组织形态形成和修复至关重要，它有助于抑制肿瘤的形成。Ezrin Radisin Moesin(ERM)蛋白Merlin由肿瘤抑制基因NF2编码，定位于细胞膜，介导接触性生长抑制。我们发现Merlin也在细胞核中积累，在那里它与新的E3泛素连接酶CRL4DCAF1结合并抑制。我们的初步研究表明，Merlin需要移位到细胞核并抑制CRL4DCAF1，以抑制肿瘤的发生。我们打算测试这一假设，即Merlin通过抑制CRL4DCAF1来调节接触性生长抑制，并将这种生化反应置于细胞生长抑制的途径中。我们还希望测试CRL4DCAF1的抑制是否会阻止体内肿瘤的生长，以及DCAF1是否是一个新的癌基因。这些研究将有助于阐明Merlin介导接触抑制和肿瘤抑制的机制。希望从机制上理解Merlin的功能将有助于揭示接触抑制和NF2突变肿瘤的发病机制。

DESCRIPTION (provided by applicant): Current models suggest that the FERM domain protein Merlin, encoded by the tumor suppressor NF2, inhibits mitogenic signaling at or near the plasma membrane. We have discovered that the closed, growth inhibitory form of Merlin accumulates in the nucleus and binds to DCAF1, the receptor component of the E3 ubiquitin ligase CRL4DCAF1. Genetic and biochemical evidence indicates that Merlin functions as a negative regulator of CRL4DCAF1. Depletion of DCAF1 blocks exit from contact inhibition and progression through the cell cycle in Merlin-deficient cells. Expression of Merlin and silencing of DCAF1 induce a largely overlapping program of gene expression, which includes the upregulation of growth arrest and proapototic genes and the downregulation of mitogenic and survival genes. Tumor-derived mutants of Merlin fail to accumulate into the nucleus, to bind to DCAF1, or to inhibit CRL4DCAF1. Finally, depletion of DCAF1 suppresses the ability of Merlin-deficient tumor cells to grow in soft agar and to form tumors in nude mice. These findings strongly suggest that Merlin suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4DCAF1-dependent gene expression. We propose to pursue four Specific Aims. I) To determine if Merlin mediates contact inhibition through inhibition of CRL4DCAF1, we will examine if expression of a Merlin-insensitive form of DCAF1 causes loss of contact inhibition. In addition, we will examine if the tumor-derived missense mutants of Merlin, which have lost the ability to suppress CRL4DCAF1, are unable to mediate contact inhibition. II) To examine if Merlin suppresses tumorigenesis in vivo through inhibition of CRL4DCAF1, we will generate mice lacking DCAF1 in their Schwann cells and breed them to mouse models of NF2. We will examine tumor onset and progression in control and DCAF1 mutant mice, as we have done previously in our studies on integrin signaling. III) To identify the physiological substrates of CRL4DCAF1 and to examine their involvement in Merlin-mediated tumor suppression, we will conduct Tandem Affinity Purification followed by mass spectrometry using wild type or mutationally inactivated DCAF1. Alternatively, we will subject control and DCAF1-silenced cells to Global Stability Profiling, as recently described by the Elledge laboratory. IV) To examine if CRL4DCAF1 displays pro- oncogenic activity, we will examine if overexpression of DCAF1 enhances the ability of Merlin-deficient, Merlin- re-expressing, or both types of schwannoma cells to proliferate in vitro as well as their ability to form tumors in vivo. In addition, we will examine if the gene encoding DCAF1 is amplified or constitutively activated by C- terminal deletion in NF2-related or sporadic meningiomas and in Schwannomas. Taken together, these studies should help to elucidate the mechanism through which Merlin mediates contact inhibition and suppresses tumorigenesis. PUBLIC HEALTH RELEVANCE: Contact inhibition of growth is critical for proper tissue morphogenesis and repair and it contributes to suppress tumorigenesis. The Ezrin Radixin Moesin (ERM) protein Merlin, encoded by the tumor suppressor gene NF2, localizes at the plasma membrane and mediates contact inhibition of growth. We have found that Merlin accumulates also in the nucleus, where it binds to and inhibits the novel E3 ubiquitin ligase CRL4DCAF1. Our Preliminary Studies indicate that Merlin needs to translocate to the nucleus and to inhibit CRL4DCAF1 in order to suppress tumorigenesis. We intend to test the hypothesis that Merlin mediates contact inhibition of growth by inhibiting CRL4DCAF1 and to place this biochemical reaction within a cellular pathway of growth inhibition. We also wish to test if inhibition of CRL4DCAF1 blocks tumor growth in vivo and if DCAF1 is a novel oncogene. These studies will contribute to elucidate the mechanisms through which Merlin mediates contact inhibition and tumor suppression. It is expected that a mechanistic understanding of Merlin's function will shed light on contact inhibition and on the pathogenesis of NF2 mutant tumors.