Mechanism of Merlin-mediated contact inhibition and tumor suppression.

描述（由申请人提供）：目前的模型表明，由肿瘤抑制因子NF 2编码的FERM结构域蛋白Merlin抑制质膜处或附近的促有丝分裂信号传导。我们已经发现，封闭的，生长抑制形式的梅林积累在细胞核中，并结合DCAF 1，E3泛素连接酶CRL 4DCAF 1的受体成分。遗传和生物化学证据表明，Merlin作为CRL 4DCAF 1的负调节剂发挥作用。在Merlin缺陷细胞中，DCAF 1的耗尽阻断了接触抑制的退出和细胞周期的进展。Merlin的表达和DCAF 1的沉默诱导了一个很大程度上重叠的基因表达程序，包括生长停滞和促凋亡基因的上调以及促有丝分裂和存活基因的下调。Merlin的肿瘤衍生突变体不能积聚到细胞核中，不能结合DCAF 1或抑制CRL 4DCAF 1。最后，DCAF 1的耗竭抑制Merlin缺陷型肿瘤细胞在软琼脂中生长和在裸鼠中形成肿瘤的能力。这些发现有力地表明，Merlin通过转移到细胞核抑制CRL 4DCAF 1依赖性基因表达来抑制肿瘤发生。我们提出了四个具体目标。I）为了确定Merlin是否通过抑制CRL 4DCAF 1介导接触抑制，我们将检查Merlin不敏感形式的DCAF 1的表达是否导致接触抑制的丧失。此外，我们将检查是否梅林的肿瘤来源的错义突变体，已经失去了抑制CRL 4DCAF 1的能力，不能介导接触抑制。II）为了检查Merlin是否通过抑制CRL 4DCAF 1来抑制体内肿瘤发生，我们将产生在其雪旺细胞中缺乏DCAF 1的小鼠，并将它们培育成NF 2的小鼠模型。我们将检查对照组和DCAF 1突变小鼠的肿瘤发病和进展，正如我们以前在整合素信号转导研究中所做的那样。III）为了鉴定CRL 4DCAF 1的生理底物并检查它们在梅林介导的肿瘤抑制中的参与，我们将使用野生型或突变失活的DCAF 1进行串联亲和纯化，然后进行质谱分析。或者，我们将对照和DCAF 1沉默细胞进行整体稳定性分析，如Elledge实验室最近所述。IV）为了检查CRL 4DCAF 1是否显示促癌活性，我们将检查DCAF 1的过表达是否增强Merlin缺陷型、Merlin再表达型或这两种类型的神经鞘瘤细胞体外增殖的能力以及它们体内形成肿瘤的能力。此外，我们将检查编码DCAF 1的基因在NF 2相关或散发性脑膜瘤和神经鞘瘤中是否被扩增或由C-末端缺失组成性激活。总之，这些研究应有助于阐明梅林介导接触抑制和抑制肿瘤发生的机制。 公共卫生相关性：生长的接触抑制对于适当的组织形态发生和修复是至关重要的，并且它有助于抑制肿瘤发生。由肿瘤抑制基因NF 2编码的Ezrin Radixin Moesin（ERM）蛋白Merlin定位于质膜并介导生长的接触抑制。我们已经发现Merlin也在细胞核中积累，在那里它结合并抑制新的E3泛素连接酶CRL 4DCAF 1。我们的初步研究表明，Merlin需要易位到细胞核并抑制CRL 4DCAF 1以抑制肿瘤发生。我们打算检验Merlin通过抑制CRL 4DCAF 1介导接触性生长抑制的假设，并将该生化反应置于生长抑制的细胞途径内。我们还希望测试抑制CRL 4DCAF 1是否在体内阻断肿瘤生长，以及DCAF 1是否是一种新的致癌基因。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。预期对Merlin功能的机制理解将阐明接触抑制和NF 2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.