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: 8349051
• 负责人: thomas j hornyak
• 金额: $ 45.83万
• 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/8349051
• 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 are planning to complete experiments that will specify the mechanism for these effects.

今年，我们已经巩固了我们在人类黑素细胞，黑素细胞痣和黑色素瘤细胞中表达和功能关键polycomb蛋白的发现，并提交了这项工作的初步产品供出版。Polycomb蛋白是一种表观遗传基因阻遏物，与果蝇发育过程中抑制Hox基因表达的蛋白质相关，通过与特定组蛋白氨基酸残基相互作用或修饰而发挥功能。我们正在确定其中两种蛋白质BMI-1和EZH 2在恶性黑色素瘤中的功能作用。BMI-1和EZH 2分别是大分子复合物Polycomb抑制复合物（PRC）-1和-2的成员。PRC 2中EZH 2的组蛋白甲基转移酶活性在核心核小体的组蛋白3上产生赖氨酸27的稳定三甲基化衍生物，其被含有BMI-1的PRC 1识别，导致表观遗传基因抑制。 以前，我们对体内正常黑素细胞、黑素细胞痣和黑色素瘤中polycomb蛋白表达的分析表明，BMI-1在黑素细胞、黑素细胞痣细胞和转移性黑色素瘤中表达。相反，EZH 2仅在黑色素瘤细胞中表达，而不在黑色素细胞或痣中表达。正常皮肤是根据CCR皮肤科分支综合方案获得的，痣是从入组临床方案06-C-0060的患有大量黑素细胞痣的患者中获得的（托马斯·J·霍尼亚克，医学博士，哲学博士、主要研究者），转移性黑色素瘤标本从NCI/CCR外科分支获得。EZH 2在正常和痣相关黑色素细胞和恶性黑色素瘤细胞之间表达的显著差异使我们假设EZH 2表达是黑色素瘤恶性进展的决定因素。今年投入了大量的努力来验证这一假设。 使用从MIT的Robert温伯格博士实验室获得的基因工程转化的人黑素细胞系，我们研究了使用RNA干扰从这些细胞中消除EZH 2的效果。我们发现EZH 2耗尽的细胞表现出细胞增殖速率降低和细胞衰老标记衰老相关β-半乳糖苷酶（SA β-gal）表达增加。它们还表现出变宽、变平的形态。EZH 2耗尽的细胞在软琼脂中生长时也形成较少的细胞集落，并且在引入免疫功能低下的小鼠后形成肿瘤更慢。这些结果表明，EZH 2在转化的人黑素细胞中的表达是这些细胞的致瘤性的重要因素。 为了概括这些发现，我们扩展了这些实验以确定EZH 2消耗在已建立的人黑素瘤细胞系中的作用。我们现在已经确定，在人黑素瘤细胞系的一个子集中，减少EZH 2表达会降低其增殖速率并增加SA β-gal的表达。目前，我们的努力集中在验证EZH 2的这些作用的机制上。EZH 2应答基因似乎介导这些细胞的大量衰老表型。这项工作于2011年初发表在《分子癌症研究》杂志上。 我们还进行了实验以评估BMI-1在黑素瘤细胞中的活性。我们已经鉴定了对BMI-1耗竭敏感的人黑素瘤细胞系。我们目前正在研究这些作用的机制，部分是为了确定EZH 2和BMI-1是否通过重叠或冗余机制在黑色素瘤细胞中发挥作用。 在此之前，我们启动了一个项目，旨在探讨热休克蛋白90（HSP 90）抑制剂17-烯丙氨基-17-去甲氧基格尔德霉素（17-AAG）对黑色素瘤细胞中致癌激酶BRAF和CRAF的影响。我们发现17-AAG可以通过诱导BRAF、BRAF和CRAF的降解或通过HSP 90：BRAF复合物抑制BRAF活性来抑制5/5人黑素瘤细胞系中的黑素瘤细胞增殖。我们正在计划完成实验，以详细说明这些效应的机制。