Determinants of Melanocyte Transformation and Melanoma Progression

今年，我们的工作重点是表征人类黑素细胞、黑素细胞痣和黑色素瘤细胞中关键多梳蛋白的表达。多梳蛋白是一种表观遗传基因抑制因子，与果蝇发育过程中抑制Hox基因表达的蛋白有关，通过与特定组蛋白氨基酸残基相互作用和修饰起作用。我们正在确定其中两种蛋白质BMI-1和EZH2在恶性黑色素瘤中所起的功能作用。BMI-1和EZH2分别是高分子复合物Polycomb Repressor Complex (PRC)-1和-2的成员。PRC2中EZH2的组蛋白甲基转移酶活性在核心核小体的组蛋白3上产生稳定的赖氨酸27的三甲基化衍生物，该衍生物被含有bmi -1的PRC1识别，导致表观遗传基因抑制。先前，我们对正常黑色素细胞、黑色素细胞痣和黑色素瘤体内多梳蛋白表达的分析表明，BMI-1在黑色素细胞、黑色素细胞痣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，而在黑色素细胞或痣中不表达。正常皮肤是根据CCR皮肤科综合方案获得的，痣是从临床方案中登记的大量黑色素细胞痣患者中获得的，06-C-0060 （Thomas J. Hornyak， M.D, Ph.D.，首席研究员），转移性黑色素瘤标本来自NCI/CCR外科分支。EZH2在正常黑色素细胞和痣相关黑色素细胞以及恶性黑色素瘤细胞中表达的显著差异使我们假设EZH2表达是黑色素瘤恶性进展的决定因素。为了验证这一假设，今年投入了大量的努力。利用麻省理工学院Robert Weinberg博士实验室获得的基因工程改造的人类黑素细胞细胞系，我们研究了使用RNA干扰从这些细胞中消耗EZH2的影响。我们发现ezh2缺失的细胞表现出细胞增殖率降低和细胞衰老标志物衰老相关β -半乳糖苷酶（SA β -gal）的表达增加。它们的形态也变宽变平。ezh2缺失的细胞在软琼脂中培养时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的速度更慢。这些结果提示EZH2在转化的人黑素细胞中的表达是这些细胞致瘤性的重要因素。为了推广这些发现，我们扩展了这些实验，以确定EZH2缺失对已建立的人类黑色素瘤细胞系的影响。我们现在已经确定，在人类黑色素瘤系的一个子集中，减少EZH2的表达会降低其增殖率，并增加SA β -gal的表达。目前，我们的工作重点是验证EZH2这些作用的机制。我们提出，在黑色素瘤细胞中，EZH2抑制一个基因或一组基因，这些基因本身就是致癌基因诱导衰老的决定因素。我们目前正在研究EZH2缺失对先前涉及癌基因诱导衰老的候选基因的影响，并评估通过微阵列筛选鉴定的其他基因的活性。我们还进行了实验来评估BMI-1在黑色素瘤细胞中的活性。我们已经鉴定出2种对BMI-1缺失敏感的人类黑色素瘤细胞系。在这些细胞中，BMI-1的缺失导致细胞增殖速率的降低。我们目前正在计划进行实验，以确定我们所显示的受EZH2缺失影响的肿瘤细胞的一些相同特征是否也受到BMI-1缺失的影响。此前，我们启动了一个项目，旨在探索17-烯丙烯丙烯-17-demethoxygeldanamycin (17-AAG)，一种热休克蛋白90 （HSP90）抑制剂，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降解，或通过HSP90:BRAF复合物抑制BRAF活性，在5/5的人黑色素瘤细胞系中抑制黑色素瘤细胞增殖。我们正计划完成实验，以明确这些效应的机制。

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.