Epigenetic Control of Melanoma by MAGE Transcription Factors

MAGE 转录因子对黑色素瘤的表观遗传控制

• 批准号: 9241735
• 负责人: B Jack Longley
• 金额: --
• 依托单位: WM S. MIDDLETON MEMORIAL VETERANS HOSP
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9241735
• 关键词: Affect; Apoptosis; Binding; Binding Proteins; Biological Assay; Biology; Birth; Cell Aging; Cells; Characteristics; Chromatin; Clinical; Co-Immunoprecipitations; Cutaneous; DNA; Data; Development; Doxycycline; ETS1 gene; Early Diagnosis; Ectopic Expression; Epigenetic Process; Exposure to; Gamma-H2AX; Gene Expression; Gene Family; General Population; Genes; Genetic; Germ Cells; Goals; Growth; Health; Heterochromatin; Histologic; Histology; Histones; Human; Immunoblotting; Immunocompromised Host; In Vitro; Incidence; Individual; Leucine; Location; Malignant Neoplasms; Measurement; Mediating; Melanocytic Neoplasm; Melanocytic nevus; Melanoma Cell; Military Personnel; Modeling; Molecular Profiling; Morphology; Mus; Nevus; Oncogene Activation; Oncogenes; Outcome; PTEN gene; Pattern; Placenta; Play; Prevention; Proliferation Marker; Proteins; RNA Interference; Recording of previous events; Recruitment Activity; Regulation; Repression; Research; Resistance; Reverse Transcription; Risk; Role; Scaffolding Protein; Services; Site; Skin; Skin Cancer; Somatic Cell; Testing; Time; Tissue Microarray; Tissues; Transcription Factor AP-2 Alpha; Transgenic Mice; Transgenic Model; Transgenic Organisms; Tumor Cell Line; Tumor Suppression; Tumor Suppressor Proteins; Ultraviolet Rays; Vertebrates; Veterans; Wound Healing; Xenograft Model; Zinc Fingers; base; beta-Galactosidase; cell growth; effective therapy; experimental study; gene repression; in utero; in vivo; interest; knock-down; loss of function; melanocyte; melanoma; member; migration; mouse model; mutant; novel; novel strategies; prevent; protein expression; senescence; spatiotemporal; transcription factor; tumor; ubiquitin-protein ligase; wound

Melanoma develops as melanocytes accumulate genetic and epigenetic abnormalities, typically causing oncogene activation and senescence, followed by escape from senescence and loss of tumor suppression. Members of the Class I MAGE gene family are normally expressed only in developing germ cells and placenta but their expression is activated in many malignancies including melanoma. Class I MAGE protein expression is associated with poor clinical outcomes and resistance to treatment, and knockdown of MAGE proteins increases melanoma cell apoptosis in vitro and decreases growth of MAGE (+) human melanoma cells in immunocompromised mice, suggesting that MAGE proteins play important functional roles in melanoma biology. MAGE proteins bind to and regulate KAP1, a scaffolding protein and ubiquitin E3-ligase that causes localized chromatin compaction and represses gene expression when recruited to specific DNA sites by KRAB-zinc finger transcription factors (KZFs), the largest group of transcription factors in vertebrates. MAGE proteins modify KZF-KAP1 binding to and repression of specific genes, and MAGE expression affects Id1, AP2 and p16, all known to be involved in melanocytic transformation. However, the specific roles of MAGE proteins in the biology of melanoma, their mechanisms of action, and ways to exploit them for treatment have not been not fully characterized. Our overall goal is to understand the role of Class I MAGE expression in melanoma development. We will test the hypothesis that MAGE proteins facilitate melanocyte transformation by regulating expression of genes that cooperate to promote escape from senescence, and increase melanocyte proliferation and tissue invasion. Aim 1: To determine how MAGE affects melanocyte growth and tissue invasion. Aim 1a will test whether MAGE promotes escape from BRAFV600E induced senescence by suppressing p16 via Id1, using selective knockdown of endogenous Id1, p16 and MAGE in BRAFV600E (+) cells, or by ectopic expression of BRAFV600E , Id1, p16, and MAGE, followed by measurement of cell growth and senescence markers. Epigenetic effects of MAGE will be determined by ChIP for MAGE and histone 3 trimethylated on leucine 9 (H3me3K9), a molecular signature of KAP1 induced gene repression. Aim 1b will use a similar strategy to test whether MAGE regulation of AP2 promotes migration and invasion of melanocytes in Boyden chamber and in vitro wound assays. Aim 2: To establish in vivo associations of MAGE expression with melanocyte transformation. This Aim will use state of the art multispectral immunohistology with a unique tissue microarray to test the hypothesis that MAGE is expressed early in melanocyte transformation and that MAGE proteins co-localize with markers of proliferation (Ki67) and are inversely correlated with expression of senescence markers. Aim 3: To test the hypothesis that MAGE expression promotes melanocyte transformation in vivo. Aim 3a will determine whether MAGE affects melanocyte migration or tissue invasion in utero using immunohistology and unique transgenic mice that express melanocyte specific, doxycycline inducible MAGE. Aim 3b will test the hypothesis that MAGE promotes melanocyte transformation by crossing melanocyte MAGE (+) mice into BRAFV600E or NRASQ61K based murine models of oncogene induced melanocytic nevi. Aim 3c will determine the effects of MAGE-A3 expression in early melanoma by crossing MAGE transgenics into melanoma models expressing BRAFV600E or NRASQ61K and showing loss of tumor suppressors PTEN or p16. Outcomes include the rate of transformation, number, and histologic characteristics of resulting melanomas. Assessment of MAGE repression of individual genes will use ChIP, reverse transcription real time quantitative PCR (RT-qPCR) and protein immunoblotting. These studies will benefit veterans by identifying novel targets for treatment and prevention of melanoma.

黑色素瘤随着黑色素细胞积累遗传和表观遗传异常而发展，通常会导致 癌基因激活和衰老，然后逃避衰老并失去肿瘤抑制作用。 I 类 MAGE 基因家族的成员通常仅在发育中的生殖细胞和胎盘中表达 但它们的表达在包括黑色素瘤在内的许多恶性肿瘤中被激活。 I 类 MAGE 蛋白表达 与不良临床结果、治疗耐药性以及 MAGE 蛋白敲低相关 增加体外黑色素瘤细胞凋亡并降低 MAGE (+) 人类黑色素瘤细胞的生长 免疫功能低下的小鼠，表明 MAGE 蛋白在黑色素瘤中发挥重要的功能作用 生物学。 MAGE 蛋白结合并调节 KAP1，KAP1 是一种支架蛋白和泛素 E3 连接酶，可导致 当被招募到特定 DNA 位点时，局部染色质压缩并抑制基因表达 KRAB-锌指转录因子 (KZF)，脊椎动物中最大的转录因子组。法师 蛋白质修饰 KZF-KAP1 与特定基因的结合和抑制，MAGE 表达影响 Id1、AP2 和 p16，均已知参与黑素细胞转化。然而，MAGE 蛋白的具体作用 在黑色素瘤的生物学中，它们的作用机制以及利用它们进行治疗的方法尚未被阐明。 没有完全表征。我们的总体目标是了解 I 类 MAGE 表达在黑色素瘤中的作用 发展。我们将通过以下方式检验 MAGE 蛋白促进黑素细胞转化的假设： 调节协同促进逃避衰老的基因表达，并增加 黑素细胞增殖和组织侵袭。 目标 1：确定 MAGE 如何影响黑素细胞生长和组织侵袭。 目标 1a 将测试 MAGE 是否通过抑制 p16 来促进逃避 BRAFV600E 诱导的衰老 Id1，使用 BRAFV600E (+) 细胞中内源 Id1、p16 和 MAGE 的选择性敲低，或通过异位 BRAFV600E、Id1、p16 和 MAGE 的表达，然后测量细胞生长和衰老 标记。 MAGE 的表观遗传效应将通过 ChIP 确定 MAGE 和组蛋白 3 三甲基化 亮氨酸 9 (H3me3K9)，KAP1 诱导基因抑制的分子特征。 目标1b将使用类似的策略来测试AP2的MAGE调节是否促进AP2的迁移和侵袭 博伊登室和体外伤口测定中的黑素细胞。 目标 2：建立 MAGE 表达与黑素细胞转化的体内关联。 该目标将使用最先进的多光谱免疫组织学和独特的组织微阵列来测试 假设 MAGE 在黑素细胞转化早期表达并且 MAGE 蛋白共定位 与增殖标记物 (Ki67) 相关，并与衰老标记物的表达呈负相关。 目标 3：检验 MAGE 表达促进体内黑素细胞转化的假设。 目标 3a 将确定 MAGE 是否影响子宫内黑素细胞迁移或组织侵袭 免疫组织学和表达黑素细胞特异性、多西环素诱导型 MAGE 的独特转基因小鼠。 目标 3b 将检验 MAGE 通过与黑素细胞 MAGE 交叉促进黑素细胞转化的假设 (+) 将小鼠放入基于癌基因诱导的黑素细胞痣的 BRAFV600E 或 NRASQ61K 小鼠模型中。 目标 3c 将通过杂交 MAGE 转基因来确定 MAGE-A3 表达对早期黑色素瘤的影响 进入表达 BRAFV600E 或 NRASQ61K 并显示肿瘤抑制因子 PTEN 丢失的黑色素瘤模型或 第 16 页。结果包括转化率、数量和所得组织学特征 黑色素瘤。评估单个基因的 MAGE 抑制将使用 ChIP、实时逆转录 定量 PCR (RT-qPCR) 和蛋白质免疫印迹。 这些研究将通过确定治疗和预防黑色素瘤的新靶标使退伍军人受益。