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

基本信息

批准号：
7592767
负责人：
thomas j hornyak
金额：
$ 66.48万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7592767
关键词：
17-(Allylamino)-17-demethoxygeldanamycin Agar Amino Acids BRAF gene CCR Cell Line Cell Proliferation Cells Clinical Protocols Complex DNA Methyltransferase Inhibitor Dermatology Development Disease Drosophila genus EZH2 gene Enrollment Epigenetic Process Evaluation Gene Expression Genes Genetic Goals Histones Human Human Volunteers Immunocompromised Host Immunofluorescence Immunologic Intervention Lentivirus Vector Localized Lysine Macromolecular Complexes Maintenance Malignant - descriptor Mediating Melanocytic nevus Melanoma Cell Metastatic Melanoma Molecular Mus Nevi and Melanomas Nevus Nevus Cell Nuclear Nucleosome Core Particle Operative Surgical Procedures PRC1 Protein Pathogenicity Patients Play Polycomb Premalignant Process Protein Overexpression Proteins Protocols documentation RNA Interference Recruitment Activity Research Role Skin Solutions Specimen System Testing Therapeutic Tissues Tumor Suppressor Proteins cancer cell design gene repression histone methyltransferase in vivo melanocyte melanoma member research study senescence tumor growth

项目摘要

Previously, 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. 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 attempting to determine 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. We utilized human skin taken from normal human volunteers, melanocytic nevi from human volunteers with multiple melanocytic nevi, and malignant melanoma tissue from patients with metastatic disease to study the expression of BMI-1 and EZH2 during malignant progression in melanocytes. 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, that I devised, and metastatic melanoma specimens were obtained from the CCR Surgery Branch. Immunofluorescence analysis of human skin along with six nevus specimens and six malignant melanoma specimens was performed to compare expression and levels of expression among these specimens. 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. Results in cultured melanocytes and human melanoma cell lines were similar except that EZH2 was expressed at low levels in cultured human melanocytes. BMI-1 and EZH2 are co-localized in human melanoma cells, and RNAi-mediated knockdown of EZH2 decreases global nuclear levels of histone 3 lysine 27 trimethylation. These results suggest that the polycomb system is active in human melanoma cells. Moreover, the absence of EZH2 from pre-malignant cells suggests that activation of the polycomb system by EZH2 expression in these cells might result in BMI-1 recruitment to target loci, inducing selective gene repression and facilitating cellular immortalization or malignant transformation. We have developed retroviral and lentiviral vectors that permit us to reduce BMI-1 and EZH2 expression in these cells using RNA interference. Preliminary results of experiments with BMI-1 RNA interference suggest that the loss of BMI-1 expression alone does not have substantive effects upon melanoma cell proliferation, soft agar colony formation, or tumor growth in immunocompromised mice. We are currently conducting experiments combining BMI-1 knockdown with either treatment of cells with DNA methyltransferase inhibitors or knockdown of other polycomb factors to determine the functional role of polycomb factor overexpression in malignant melanoma. We also plan to test the hypothesis that induction of EZH2 expression in oncogenically-senescent human melanocytes, considered representative of human melanocytic nevus cells in vivo, recruits BMI-1 to important tumor suppressor loci and facilitates malignant transformation.

以前，我们发现17-AAG可以通过诱导BRAF，BRAF和CRAF的降解或通过HSP90：BRAF复合物抑制BRAF活性来抑制5/5人黑色素瘤细胞系中黑色素瘤细胞的增殖。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。我们正在尝试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作用。 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。我们利用了从正常的人类志愿者那里采取的人类皮肤，来自多个黑色素细胞NEVI的人类志愿者的黑素细胞NEVI以及转移性疾病患者的恶性黑色素瘤组织来研究黑色素细胞中恶性进展过程中BMI-1和EZH2的表达。根据CCR皮肤病学分支综合方案获得了正常的皮肤，NEVI是从我设计的临床方案06-C-0060的大量黑素细胞NEVI的患者中获得的，并从CCR手术分支中获得了转移性黑色素瘤标本。对人皮肤的免疫荧光分析以及六个nevus标本和六个恶性黑色素瘤标本进行了比较这些标本中的表达和表达水平。 BMI-1在黑素细胞，黑素细胞梗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。培养的黑素细胞和人黑色素瘤细胞系的结果相似，除了EZH2在培养的人类黑色素细胞中以低水平表达。 BMI-1和EZH2在人黑色素瘤细胞中共定位，RNAi介导的EZH2敲低降低了组蛋白3赖氨酸27三甲基化的全球核水平。这些结果表明，多肉液系统在人黑色素瘤细胞中具有活性。此外，在临时细胞中不存在EZH2表明，在这些细胞中EZH2表达对Polycomb系统的激活可能会导致BMI-1募集以靶向基因座，从而诱导选择性基因抑制并促进细胞的不朽或恶性转化。我们已经开发了逆转录病毒和慢病毒载体，使我们能够使用RNA干扰降低这些细胞中的BMI-1和EZH2表达。 BMI-1 RNA干扰实验的初步结果表明，仅BMI-1表达的丧失对黑色素瘤细胞增殖，软琼脂菌落的形成或免疫受损小鼠的肿瘤生长没有实质性影响。我们目前正在进行实验，将BMI-1敲低与DNA甲基转移酶抑制剂的细胞处理或其他PolyComb因子敲低，以确定PolyComb因子过表达在恶性黑色素瘤中的功能作用。我们还计划检验以下假设：肿瘤性人类黑素细胞中EZH2表达的诱导，被认为是体内人类黑色素细胞瘤的代表，招募BMI-1来招募BMI-1来抑制重要的肿瘤基因座并促进恶性转化。