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: 9898234
• 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 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898234
• 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; Regulation; Repression; Research; Resistance; Reverse Transcription; Risk; Role; Scaffolding Protein; Services; Site; Skin; Skin Cancer; Somatic Cell; Testing; Time; Tissue Microarray; Tissues; Transgenic Mice; Transgenic Model; Transgenic Organisms; Tumor Cell Line; Tumor Suppression; Tumor Suppressor Proteins; UV Radiation Exposure; Ultraviolet Rays; Vertebrates; Veterans; Xenograft Model; Zinc Fingers; base; beta-Galactosidase; cell growth; effective therapy; enhancer-binding protein AP-2; 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; recruit; senescence; spatiotemporal; transcription factor; tumor; ubiquitin-protein ligase; wound; wound healing

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.

黑色素瘤的发展是由于黑素细胞积累了遗传和表观遗传异常，通常会导致 癌基因的激活和衰老，随之而来的是逃避衰老和肿瘤抑制的丧失。 I类MAGE基因家族的成员通常只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都被激活。I类MAGE蛋白的表达 与不良的临床结果和对治疗的抗药性以及MAGE蛋白的敲除有关 体外诱导黑色素瘤细胞凋亡及抑制MAGE(+)人黑素瘤细胞生长 免疫受损的小鼠，提示MAGE蛋白在黑色素瘤中发挥重要功能 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，导致 当被募集到特定的DNA位点时，染色质的局部紧致和抑制基因的表达 Krab-锌指转录因子(KZF)是脊椎动物中最大的一组转录因子。法师 蛋白修饰KZF-KAP1与特定基因的结合和抑制，MAGE的表达影响ID1、AP2 和p16，都是已知参与黑素细胞转化的基因。然而，MAGE蛋白的特定作用 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还没有被研究过。 不完全有特点的。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。我们将验证MAGE蛋白通过以下方式促进黑素细胞转化的假设 调节促进逃避衰老的基因的表达，并增加 黑素细胞增殖和组织侵袭。 目的1：探讨MAGE对黑素细胞生长和组织侵袭的影响。 Aim 1a将测试MAGE是否通过抑制p16而促进逃避BRAFV600E诱导的衰老 Id1，在BRAFV600E(+)细胞中选择性地敲除内源性Id1、p16和MAGE，或通过异位 BRAFV600E、Id1、p16和MAGE的表达及细胞生长和衰老的检测 记号笔。MAGE的表观遗传效应将通过MAGE和三甲基化的组蛋白3的芯片来确定 亮氨酸9(H3me3K9)，KAP1诱导基因抑制的分子标志。 Aim 1b将使用类似的策略来测试MAGE对AP2的调节是否促进肿瘤的迁移和侵袭 Boyden小室黑素细胞和体外创面分析。 目的2：在体内建立MAGE表达与黑素细胞转化的关系。 这个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 MAGE在黑素细胞转化早期表达和MAGE蛋白共定位的假说 与增殖标记物(Ki67)呈负相关，与衰老标记物的表达呈负相关。 目的3：验证MAGE在体内表达促进黑素细胞转化的假说。 Aim 3a将确定MAGE是否影响子宫内黑素细胞的迁移或组织侵袭 免疫组织学和表达黑素细胞特异性、强力霉素诱导的MAGE的独特转基因小鼠。 Aim 3b将验证MAGE通过与黑素细胞杂交促进黑素细胞转化的假设 (+)小鼠进入基于BRAFV600E或NRASQ61K的致癌基因诱导的黑素细胞痣小鼠模型。 目的3c将通过杂交MAGE转基因基因来确定MAGE-A3在早期黑色素瘤中的表达效果 转化为黑色素瘤模型，表达BRAFV600E或NRASQ61K并显示肿瘤抑制基因PTEN或 第16页。结果包括转化率、转移率、转移率和组织学特征。 黑色素瘤。评估MAGE对单个基因的抑制作用将使用芯片，实时逆转录 定量聚合酶链式反应(RT-qPCR)和蛋白免疫印迹。 这些研究将通过确定治疗和预防黑色素瘤的新靶点使退伍军人受益。