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，通常导致 癌基因的激活和衰老，随后逃避衰老和抑制肿瘤的丧失。 I类Mage基因家族的成员通常仅在发育生殖细胞和胎盘中表达 但是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类法法蛋白表达 与临床不良结局和对治疗的抵抗力以及法师蛋白的敲低有关 在体外增加黑色素瘤细胞凋亡，并降低MAGE（+）人类黑色素瘤细胞的生长 免疫功能低下的小鼠，表明法师蛋白在黑色素瘤中起重要功能作用 生物学。 MAGE蛋白与引起的脚手架蛋白和泛素E3岩酶结合并调节KAP1 当局部染色质压实和抑制基因表达时，当通过 Krab-Zinc手指转录因子（KZFS），脊椎动物中最大的转录因子群。法师 蛋白质修饰KZF-KAP1与特定基因的结合和抑制，MAGE表达会影响ID1，AP2 和p16，所有众所周知都参与黑素细胞转化。但是，法师蛋白的特定作用 在黑色素瘤的生物学中，它们的作用机理以及将其用于治疗的方法尚未 未完全表征。我们的总体目标是了解I级法师表达在黑色素瘤中的作用 发展。我们将检验以下假设：法师蛋白通过 调节合作的基因表达，以促进从衰老中逃脱并增加 黑素细胞增殖和组织侵袭。 目标1：确定法师如何影响黑素细胞的生长和组织侵袭。 AIM 1A将测试法师是否通过通过抑制p16通过 ID1，使用BRAFV600E（+）细胞中的内源性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。法师的表观遗传效应将通过法师的芯片确定 亮氨酸9（H3ME3K9），KAP1诱导基因抑制的分子特征。 AIM 1B将使用类似策略来测试AP2法师调节是否促进迁移和入侵 博伊登室和体外伤口分析的黑素细胞。 目标2：建立与黑素细胞转化的法师表达的体内关联。 该目标将使用独特的组织微阵列使用最新的多光谱免疫组织学来测试 假设法师在黑素细胞转化早期表达，而法师蛋白共定位 带有增殖标记（KI67），并与衰老标记的表达成反比。 目标3：检验法术表达促进体内黑素细胞转化的假设。 AIM 3A将确定法师是否会使用使用素质腺细胞迁移或使用UTERO中的黑素细胞迁移或组织入侵 免疫组织学和独特的转基因小鼠，表达黑素细胞特异性，强力霉素诱导法师。 AIM 3B将检验以下假设，即法师通过跨越黑色素的法师促进黑素细胞的转化 （+）将基于癌基因诱导黑素细胞NEVI的基于BRAFV600E或NRASQ61K的小鼠。 AIM 3C将通过跨越法师转基因来确定MAGE-A3表达在早期黑色素瘤中的影响 进入表达BRAFV600E或NRASQ61K的黑色素瘤模型并显示肿瘤抑制剂损失PTEN或 P16。结果包括结果的转化率，数量和组织学特征 黑色素瘤。评估单个基因的法师抑制作用将使用芯片，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目标来使退伍军人受益。