Mechanism of Merlin-mediated contact inhibition and tumor suppression.

Merlin 介导的接触抑制和肿瘤抑制机制。

• 批准号: 8613469
• 负责人: FILIPPO G GIANCOTTI
• 金额: $ 46.47万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8613469
• 关键词: Actins; Affinity Chromatography; Agar; Apoptotic; Binding; Biochemical; Biochemical Reaction; Biological; Breeding; C-terminal; Cell Cycle; Cell Nucleus; Cell membrane; Cells; Contact Inhibition; Down-Regulation; Gene Expression; Genes; Genetic; Growth; In Vitro; Integrins; Intercellular Junctions; Knock-out; Laboratories; Light; Malignant Conversion; Mass Spectrum Analysis; Mediating; Methods; Modeling; Monitor; Morphogenesis; Mus; Mutant Strains Mice; Neurilemmoma; Neurofibromin 2; Nude Mice; Oncogenes; Oncogenic; Pathogenesis; Pathway interactions; Physiological; Physiology; Proliferating; Proteins; Role; Schwann Cells; Signal Transduction; Site; Tertiary Protein Structure; Testing; Tissues; Tumor Suppression; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor-Derived; Up-Regulation; Xenograft procedure; cell type; conformer; ezrin; in vivo; knock-down; meningioma; moesin; mouse model; mutant; neoplastic cell; novel; overexpression; programs; public health relevance; radixin protein; receptor; repaired; subcutaneous; tumor; tumor growth; tumorigenesis; ubiquitin-protein ligase

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.

描述（由申请人提供）：目前的模型表明，由肿瘤抑制因子NF2编码的FERM结构域蛋白Merlin抑制质膜处或附近的有丝分裂信号传导。我们发现封闭的生长抑制形式的Merlin在细胞核中积累并结合到DCAF1上，DCAF1是E3泛素连接酶CRL4DCAF1的受体成分。遗传和生化证据表明，Merlin是CRL4DCAF1的负调控因子。在梅林缺陷细胞中，DCAF1的缺失阻断了接触抑制的退出和细胞周期的进展。Merlin的表达和DCAF1的沉默诱导了大量重叠的基因表达程序，其中包括生长停滞和促凋亡基因的上调以及有丝分裂和生存基因的下调。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。这些发现有力地表明，Merlin通过转运到细胞核抑制crl4dcaf1依赖性基因的表达来抑制肿瘤发生。我们建议实现四个具体目标。I)为了确定Merlin是否通过抑制CRL4DCAF1介导接触抑制，我们将检验一种Merlin不敏感形式的DCAF1的表达是否会导致接触抑制的丧失。此外，我们将检查肿瘤来源的错义突变体是否失去了抑制CRL4DCAF1的能力，无法介导接触抑制。II)为了检验Merlin是否通过抑制CRL4DCAF1在体内抑制肿瘤发生，我们将在其雪旺细胞中产生缺乏DCAF1的小鼠，并将其繁殖成NF2小鼠模型。我们将检查对照小鼠和DCAF1突变小鼠的肿瘤发生和进展，正如我们之前在整合素信号传导研究中所做的那样。III)为了鉴定CRL4DCAF1的生理底物并检查其在梅林介导的肿瘤抑制中的作用，我们将使用野生型或突变失活的DCAF1进行串联亲和纯化，然后进行质谱分析。或者，我们将对照和dcaf1沉默细胞进行全局稳定性分析，正如Elledge实验室最近所描述的那样。IV)为了检验CRL4DCAF1是否显示出促癌活性，我们将检验过表达DCAF1是否增强了缺乏梅林、重新表达梅林或两种类型的神经鞘瘤细胞的体外增殖能力以及它们在体内形成肿瘤的能力。此外，我们将研究编码DCAF1的基因是否在nf2相关或散发性脑膜瘤和神经鞘瘤中被C末端缺失扩增或组成性激活。综上所述，这些研究应该有助于阐明梅林介导接触抑制和抑制肿瘤发生的机制。