Functional analysis of the CYLD tumor suppressor

CYLD抑癌基因的功能分析

• 批准号: 7194303
• 负责人: Julide T. Celebi
• 金额: $ 12.4万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7194303
• 关键词: 16q12; Ablation; Affect; Alleles; Amino Acids; Animal Model; Animals; Apoptosis; Behavior; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Cell Cycle Regulation; Cell Nucleus; Cell Proliferation; Cells; Childhood; Chromosomes; Clinical; Clinical Research; Code; Correlation Studies; Cutaneous; Cytoplasm; Cytoplasmic Protein; Data; Defect; Deubiquitinating Enzyme; Development; Disease; Distant Metastasis; Dominant-Negative Mutation; Eccrine Spiradenoma; Embryo; Epidermis; Exhibits; Exons; Family; Generations; Genes; Genomics; Genotype; Germ-Line Mutation; Goals; Hair Follicle Neoplasm; Hair follicle structure; Hela Cells; Heterogeneity; Histology; Human; Hydrolase; Immunohistochemistry; Individual; Inherited; Invasive; Knock-out; Lead; Life; Loss of Heterozygosity; Malignant - descriptor; Mediating; Microtubules; Missense Mutation; Molecular; Mus; Mutant Strains Mice; Mutation; Neoplasms; Nonsense Codon; Nuclear; Nuclear Export; Numbers; Organogenesis; Pathway interactions; Patients; Personal Satisfaction; Phenotype; Phosphotransferases; Play; Principal Investigator; Proliferation Marker; Proteins; Proto-Oncogenes; Protocols documentation; Regulation; Reporting; Research Personnel; Role; Scalp structure; Signal Pathway; Signal Transduction; Signaling Molecule; Skin; Skin Appendage Neoplasm; Skin Neoplasms; Sweat Gland Neoplasms; Sweat Glands; Syndrome; Thinking; Tissues; Trichoepithelioma; Tumor Suppression; Tumor Suppressor Genes; Tumor Suppressor Proteins; Ubiquitin; appendage; base; clinical phenotype; embryonic stem cell; gain of function; homologous recombination; human disease; insertion/deletion mutation; insight; interest; leptomycin B; loss of function; mRNA Decay; member; mouse model; novel; p65; positional cloning; programs; size; tumor; tumorigenesis

DESCRIPTION (provided by applicant): CYLD is a novel tumor suppressor gene that was discovered by positional cloning of the linkage interval for Familial Cylindromatosis(FC) on chromosome 16q12. FC is an autosomal dominantly inherited syndrome characterized by disfiguring skin appendage tumors, such as cylindromas, trichoepitheliomas and spiradenomas. Typically these tumors are located on the scalp and face,appear in childhood or early adulthood, and gradually increase in size and number throughout life. Moreover, malignant transformation of these tumors with locally invasive behavior as well as distant metastasis can occur. Germline mutations in CYLD have been demonstrated in families with FC, and loss of heterozygosity at the CYLD locus has been found in these neoplasms, suggesting that CYLD functions as a tumor suppressor. The protein product of CYLD is 956 amino acids and contains sequence motifs found in deubiquitinating enzymes and microtubule binding proteins. It is expressed in a variety of tissues, and of interest, its expression in the skin is observed in the epidermis as well as in the skin appendages. Mutations leading to gain of function of proto-oncogenes or loss of function of tumor suppressor genes result in tumor development. Tumor suppressor genes either inhibit proliferation, promote apoptosis, or enhance differentiation, and maintain genomic integrity via regulation of distinct cellular pathways, one of which is the NF-KB signaling pathway. Recent data suggests that CYLD has enzymatic activity to deubiquitinate target proteins. It has been shown to interact with several members of the NF-KB signaling pathway, such as TRAF-2, TRIP, and IKKy/NEMO, and negatively regulate NF-KB activation. However, the molecular and cellular mechanism(s) of CYLD tumor suppression is largely unknown. NF-KB signaling is essential for ectodermal organogenesis. NF-KB suppression results in severe defects in the development of epidermal appendages including hair follicles and sweat glands. In addition, abnormalities in NF-KB signaling play a role in epidermal neoplasia. However, the mechanisms of tumor development related to NF-KB signaling, and in particular the role of CYLD-dependent tumorigenesis, are not well understood. Our overall goal is to define the functions of CYLD in cutaneous tumorigenesis. We have identified a variety of mutations in the CYLD gene in patients with FC. However, the mutational data on CYLD is currently limited. In Aim 1, we will evaluate genotype and phenotype correlation in FC that will lead to a molecular-based understanding of the skin appendage tumors. As a crucial step in defining the mechanisms of CYLD-mediated tumor suppression, we will establish a mouse model for FC in Aim 2. And lastly, our Preliminary Studies demonstrate that CYLD is present in both the nucleus and the cytoplasm of HeLa cells at steady state and that leptomycin B treatment increases its nuclear localization. This observation suggests that CYLD constitutively shuttles between cytoplasmic and nuclear compartments in a CRMl-dependent manner. However, its role in the nucleus has not been defined. In Aim 3, we first plan to identify a functional nuclear export signal responsible for nucleo-cytoplasmic shuttling of CYLD and evaluate localization of CYLD during NF-KB activation. Second, to provide insights into its nuclear role, we will attempt to identify its interaction partners in the nucleus. We anticipate that significant insights into the pathway of CYLD regulated tumor suppression will arise in the course of these studies, thereby extending our understanding of tumorigenesis.

描述(申请人提供)：CyLD是一种新的肿瘤抑制基因，通过对染色体16q12上的家族性纤维母细胞瘤病(FC)连锁区间的定位克隆而发现。FC是一种常染色体显性遗传综合征，其特征是皮肤附件肿瘤毁容，如圆柱瘤、毛发上皮瘤和螺旋腺瘤。这些肿瘤通常位于头皮和面部，出现在儿童或成年早期，并在一生中逐渐增大大小和数量。此外，这些具有局部侵袭行为和远处转移的肿瘤也可能发生恶变。在FC家系中发现了CyLD的胚系突变，并在这些肿瘤中发现了CyLD基因的杂合性丢失，这表明CyLD具有肿瘤抑制作用。CyLD的蛋白质产物由956个氨基酸组成，含有脱泛素化酶和微管结合蛋白中的序列基序。它在多种组织中表达，有趣的是，它在皮肤中的表达在表皮和皮肤附件中都有观察到。导致原癌基因功能增强或抑癌基因功能丧失的突变会导致肿瘤的发生。肿瘤抑制基因通过调节不同的细胞通路来抑制增殖、促进细胞凋亡或促进分化，并维持基因组的完整性，其中之一就是核因子-kB信号通路。最近的数据表明，CyLD具有脱泛素化靶蛋白的酶活性。已有研究表明，它与TRAF-2、TRIP、Ikky/Nemo等多个核因子-KB信号通路成员相互作用，负向调节核因子-KB的激活。然而，抑制CyLD肿瘤的分子和细胞机制(S)在很大程度上是未知的。核因子-KB信号通路在外胚层器官发生中起着至关重要的作用。抑制核因子-KB会导致包括毛囊和汗腺在内的表皮附属物发育严重缺陷。此外，核因子-KB信号的异常在表皮肿瘤的发生中也起着一定的作用。然而，与核因子-kB信号相关的肿瘤发生机制，尤其是依赖细胞色素脱氢酶的肿瘤发生机制，目前还不是很清楚。我们的总体目标是确定CyLD在皮肤肿瘤发生中的作用。我们已经在FC患者中发现了CyLD基因的各种突变。然而，目前关于CyLD的突变数据有限。在目标1中，我们将评估FC中的基因型和表型相关性，这将导致对皮肤附件肿瘤的分子基础的了解。作为确定CyLD介导的肿瘤抑制机制的关键一步，我们将在Aim 2中建立FC的小鼠模型。最后，我们的初步研究表明，CyLD稳定存在于HeLa细胞的胞核和胞浆中，并且瘦素B治疗增加了其核定位。这一观察结果表明，CyLD以CRM1依赖的方式在细胞质和核间穿梭。然而，它在核中的作用还没有确定。在目标3中，我们首先计划确定一个负责CyLD核质穿梭的功能性核输出信号，并评估CyLD在核因子-KB激活过程中的定位。其次，为了提供对其核作用的洞察，我们将尝试确定其在核中的交互伙伴。我们预计，在这些研究过程中，将出现对CyLD调控的肿瘤抑制途径的重要见解，从而扩大我们对肿瘤发生的理解。