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: 7965428
• 负责人: thomas j hornyak
• 金额: $ 47.13万
• 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/7965428
• 关键词: Affect; Agar; Amino Acids; BRAF gene; Behavior; CCR; Candidate Disease Gene; 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; Laboratories; Lysine; Macromolecular Complexes; Malignant - descriptor; Melanocytic nevus; Melanoma Cell; Metastatic Melanoma; Molecular; Morphology; Mus; Nevi and Melanomas; Nevus; Nevus Cell; Nucleosome Core Particle; Oncogenes; Oncogenic; Operative Surgical Procedures; PRC1 Protein; Pathogenesis; Patients; Play; Polycomb; Principal Investigator; Proteins; Proto-Oncogene Proteins B-raf; Protocols documentation; RNA Interference; Role; S-Phase Fraction; Skin; Specific qualifier value; Specimen; Testing; Tumorigenicity; Work; beta-Galactosidase; design; gene repression; histone methyltransferase; in vivo; inhibitor/antagonist; melanocyte; melanoma; member; neoplastic cell; protein expression; protein function; research study; senescence; tumor

This year our efforts have been focused upon characterizing expression of key polycomb proteins in human melanocytes, melanocytic nevi, and melanoma cells. 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. We propose that in melanoma cells, EZH2 represses a gene or set of genes that are themselves determinants of oncogene-induced senescence. We are currently examining the effect of EZH2 depletion on candidate genes previously implicated in oncogene-induced senescence, and also evaluating other genes identified through a microarray screen for their activities. We have also performed experiments to evaluate the activity of BMI-1 in melanoma cells. We have identified 2 human melanoma cell lines sensitive to depletion of BMI-1. In these cells, BMI-1 depletion results in a decrease of the rate of cellular proliferation. We are currently planning experiments to determine whether some of the same characteristics of tumor cells that we have shown are affected by depletion of EZH2 are also affected by depletion of BMI-1. 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，我们的努力集中在表征人类黑色素细胞，黑素细胞和黑色素瘤细胞中关键多膜蛋白的表达。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。我们提出，在黑色素瘤细胞中，EZH2抑制了一组基因或一组基因，它们本身是癌基因诱导的衰老的决定因素。目前，我们正在研究EZH2耗竭对先前与癌基因诱导的衰老有关的候选基因的影响，并评估通过微阵列筛选其活动的其他基因。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2种对BMI-1耗竭敏感的人黑色素瘤细胞系。在这些细胞中，BMI-1消耗导致细胞增殖率降低。我们目前正在计划实验，以确定我们所显示的肿瘤细胞的某些特征是否受EZH2耗竭的影响也受BMI-1耗竭的影响。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。