Determinants of Melanocyte Transformation and Melanoma Progression

黑色素细胞转化和黑色素瘤进展的决定因素

• 批准号: 8552734
• 负责人: thomas j hornyak
• 金额: $ 13.09万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552734
• 关键词: Agar; Amino Acids; BRAF gene; CCR; Cell Aging; Cell Line; Cell Proliferation; Cells; Characteristics; Clinical Protocols; Complex; Dermatology; Development; Doctor of Medicine; Doctor of Philosophy; Drosophila genus; EZH2 gene; Engineering; Enrollment; Epigenetic Process; Exhibits; Fostering; Gene Expression; Genes; Genetic; Goals; Heat-Shock Proteins 90; Histones; Human; Immunocompromised Host; Journals; Laboratories; Lysine; Macromolecular Complexes; Malignant - descriptor; Mediating; Melanocytic nevus; Melanoma Cell; Metastatic Melanoma; Molecular; Morphology; Mus; Nevi and Melanomas; Nevus; Nevus Cell; Nucleosome Core Particle; Oncogenic; Operative Surgical Procedures; PRC1 Protein; Pathogenesis; Patients; Phenotype; Play; Polycomb; Principal Investigator; Proteins; Proto-Oncogene Proteins B-raf; Protocols documentation; Publications; Publishing; RNA Interference; Role; Skin; Specific qualifier value; Specimen; Testing; Tumorigenicity; Work; anticancer research; beta-Galactosidase; design; gene repression; histone methyltransferase; in vivo; inhibitor/antagonist; melanocyte; melanoma; member; protein expression; protein function; research study; senescence; tumor

This year we have solidified our findings on the expression and function key polycomb proteins in human melanocytes, melanocytic nevi, and melanoma cells, and submitted the initial products of this work for publication. Polycomb proteins are epigenetic gene repressors, related to proteins repressing Hox gene expression during Drosophila development, that function through interacting with and modifying with specific histone amino acid residues. We are determining the functional role that two of these proteins, BMI-1 and EZH2, play in malignant melanoma. BMI-1 and EZH2 are members of the macromolecular complexes Polycomb Repressor Complex (PRC)-1 and -2, respectively. The histone methyltransferase activity of EZH2 in PRC2 creates the stable trimethylated derivative of lysine 27 on histone 3 of the core nucleosome that is recognized by BMI-1-containing PRC1, leading to epigenetic gene repression. Previously, our analysis of polycomb protein expression in normal melanocytes, melanocytic nevi, and melanomas in vivo showed that BMI-1 was expressed in melanocytes, melanocytic nevus cells, and metastatic melanoma. In contrast, EZH2 was expressed only in melanoma cells, not in melanocytes or nevi. Normal skin was obtained under a CCR Dermatology Branch omnibus protocol, nevi were obtained from patients with numerous melanocytic nevi enrolled in a clinical protocol, 06-C-0060 (Thomas J. Hornyak, M.D., Ph.D., Principal Investigator), and metastatic melanoma specimens were obtained from the NCI/CCR Surgery Branch. The marked difference in EZH2 expression between both normal and nevus-associated melanocytes and malignant melanoma cells led us to hypothesize that EZH2 expression is a determinant of malignant progession in melanoma. A substantial amount of effort was placed this year into testing this hypothesis. Using a genetically engineered transformed human melanocyte cell line, obtained from the laboratory of Dr. Robert Weinberg at MIT, we investigated the effects of depleting EZH2 from these cells using RNA interference. We found that EZH2-depleted cells demonstrated a reduced rate of cell proliferation and increased expression of the cellular senescence marker senescence-associated beta-galactosidase (SA beta-gal). They also exhibited a broadened, flattened morphology. EZH2-depleted cells also form fewer colonies of cells when grown in soft agar and form tumors more slowly following introduction into immunocompromised mice. These results suggest that EZH2 expression in transformed human melanocytes is an important factor in the tumorigenicity of these cells. To generalize these findings, we have extended these experiments to determine the effects of EZH2 depletion in established human melanoma cell lines. We have now determined that reducing EZH2 expression in a subset of human melanoma lines decreases their proliferation rate and increases expression of SA beta-gal. Currently our efforts our focused upon validating a mechanism for these effects of EZH2. An EZH2-responsive gene appears to mediate a substantial amount of the senescent phenotype of these cells. This work was published in early 2011 in the journal Molecular Cancer Research. We have also performed experiments to evaluate the activity of BMI-1 in melanoma cells. We have identified human melanoma cell lines sensitive to depletion of BMI-1. We are currently investigating the mechanism of these effects, in part to determine whether EZH2 and BMI-1 function in melanoma cells via overlapping or redundant mechanisms. Previously, we initiated a project designed to explore the effect of 17-allylaino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein 90 (HSP90), on the oncogenic kinases BRAF and CRAF in melanoma cells. We found that 17-AAG can inhibit melanoma cell proliferation in 5/5 human melanoma cell lines by inducing the degradation of BRAF, BRAF and CRAF, or inhibiting BRAF activity through an HSP90:BRAF complex. We areplanning to complete experiments that will specify the mechanism for these effects.

今年，我们巩固了关于人黑色素细胞，黑色素细胞NEVI和黑色素瘤细胞中表达和功能关键多肉质蛋白的发现，并提交了这项工作的初始产物进行发表。聚癌蛋白是表观遗传基因抑制剂，与果蝇发育过程中抑制HOX基因表达的蛋白有关，该蛋白通过与特定组蛋白氨基酸残基的相互作用并修饰。我们正在确定这些蛋白质中的两种BMI-1和EZH2在恶性黑色素瘤中发挥作用的功能。 BMI-1和EZH2分别是大分子配合物多孔抑制剂复合物（PRC）-1和-2的成员。 EZH2在PRC2中的组蛋白甲基转移酶活性产生了岩心核小体的组蛋白3上赖氨酸27的稳定三甲基化衍生物，该丝蛋白3被含BMI-1的含BMI-1的PRC1识别，从而导致表观遗传抑制。 以前，我们对正常黑素细胞，黑素核NEVI和黑色素瘤在体内的多肉蛋白表达的分析表明，BMI-1在黑素细胞，黑素细胞柳细胞和转移性黑色素瘤中表达。相比之下，EZH2仅在黑色素瘤细胞中表达，而不是在黑色素细胞或NEVI中。根据CCR皮肤病学分支综合方案获得了正常的皮肤，NEVI是从临床方案（06-C-0060）中纳入黑色素细胞NEVI的患者中获得的（Thomas J. Hornyak，M.D.，M.D.，Ph.D.，Primplastal Instuctionator），首席研究员），并从NCI/CCR Surgery获得了Mentastatic Melanoma标本。正常和奈弗相关的黑色素细胞和恶性黑色素瘤细胞之间EZH2表达的显着差异使我们假设EZH2表达是黑色素瘤中恶性进展的决定因素。今年在检验这一假设上付出了大量的努力。 使用从麻省理工学院的罗伯特·温伯格（Robert Weinberg）博士实验室获得的基因工程改造的人类黑色素细胞系，我们使用RNA干扰研究了这些细胞从这些细胞中耗尽EZH2的影响。我们发现，耗尽EZH2的细胞表现出细胞增殖的速度降低，并增加了与细胞衰老标志物衰老相关的β-半乳糖苷酶（SAβ-GAL）的表达增加。他们还表现出扩展的扁平形态。在软琼脂中生长并在引入免疫功能低下的小鼠后，肿瘤生长时，EZH2缺乏的细胞也会形成较少的细胞菌落。这些结果表明，转化的人类黑素细胞中的EZH2表达是这些细胞肿瘤性的重要因素。 为了概括这些发现，我们扩展了这些实验，以确定EZH2耗竭在已建立的人类黑色素瘤细胞系中的影响。现在，我们已经确定在人类黑色素瘤系的一部分中降低EZH2表达会降低其增殖率并增加SAβ-GAL的表达。目前，我们的努力集中在验证EZH2这些影响的机制上。 EZH2响应基因似乎介导了大量这些细胞的衰老表型。这项工作于2011年初发表在《分子癌研究》杂志上。 我们还进行了实验，以评估BMI-1在黑色素瘤细胞中的活性。我们已经确定了对BMI-1耗竭敏感的人黑色素瘤细胞系。 我们目前正在研究这些作用的机制，部分目的是确定黑色素瘤细胞中通过重叠或冗余机制在黑色素瘤细胞中的功能。 以前，我们启动了一个旨在探索17-甲甲基17-甲氧基甘露霉素（17-AAG）的影响，这是一种热休克蛋白90（HSP90），对黑色素瘤细胞中致癌激酶BRAF和CRAF的抑制剂。我们发现17-AAG可以通过诱导BRAF，BRAF和CRAF的降解，或通过HSP90：BRAF复合体抑制BRAF活性，从而抑制5/5人类黑色素瘤细胞系中黑色素瘤细胞的增殖。我们计划完成将指定这些效果机制的实验。