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.
