Determinants of Melanocyte Transformation and Melanoma Progression

先前，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。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polycomb蛋白的表达。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。我们正试图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。我们利用取自正常人类志愿者的人类皮肤、来自患有多发性黑素细胞痣的人类志愿者的黑素细胞痣和来自患有转移性疾病的患者的恶性黑素瘤组织来研究黑素细胞中恶性进展期间BMI-1和EZH 2的表达。正常皮肤是根据CCR皮肤科分支综合方案获得的，痣是从我设计的临床方案06-C-0060中招募的患有大量黑色素细胞痣的患者中获得的，转移性黑色素瘤标本是从CCR外科分支获得的。对人皮肤沿着的六个痣样本和六个恶性黑素瘤样本进行免疫荧光分析，以比较这些样本中的表达和表达水平。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。在培养的黑素细胞和人黑色素瘤细胞系中的结果相似，除了EZH 2在培养的人黑素细胞中以低水平表达。BMI-1和EZH 2共定位于人黑素瘤细胞中，并且RNAi介导的EZH 2敲低降低了组蛋白3赖氨酸27三甲基化的总体核水平。这些结果表明polycomb系统在人黑素瘤细胞中是有活性的。此外，癌前细胞中不存在EZH 2表明，在这些细胞中通过EZH 2表达激活多梳系统可能导致BMI-1募集到靶位点，诱导选择性基因抑制并促进细胞永生化或恶性转化。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干扰减少这些细胞中的BMI-1和EZH 2表达。BMI-1 RNA干扰实验的初步结果表明，BMI-1表达的损失单独对免疫功能低下小鼠的黑色素瘤细胞增殖、软琼脂集落形成或肿瘤生长没有实质性影响。我们目前正在进行的实验相结合的BMI-1敲除与DNA甲基转移酶抑制剂或敲除其他polycomb因子的细胞治疗，以确定功能性作用的polycomb因子过表达的恶性黑色素瘤。我们还计划检验这样的假设，即在致癌衰老的人黑素细胞中诱导EZH 2表达，将BMI-1招募到重要的肿瘤抑制基因座并促进恶性转化，该细胞被认为是体内人黑素细胞痣细胞的代表。

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.