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.

黑色素瘤的发展是由于黑色素细胞积累遗传和表观遗传异常，通常引起 癌基因激活和衰老，随后逃避衰老和肿瘤抑制的丧失。 I类法师基因家族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素瘤在内的许多恶性肿瘤中被激活。I类法师蛋白表达 与不良临床结果和治疗耐药性以及法师蛋白的敲除相关 增加体外黑素瘤细胞凋亡并降低法师（+）人黑素瘤细胞在 提示法师蛋白在黑色素瘤中发挥重要的功能作用 生物学法师蛋白结合并调节KAP 1，KAP 1是一种支架蛋白和泛素E3连接酶， 局部染色质压缩和抑制基因表达时，招募到特定的DNA位点， KRAB-锌指转录因子（KZF），是脊椎动物中最大的一类转录因子。法师 蛋白质修饰KZF-KAP 1与特定基因的结合和抑制，法师表达影响Id 1、AP 2 和p16，所有这些都已知参与黑素细胞转化。然而，法师蛋白的特定作用 在黑色素瘤的生物学中，它们的作用机制以及利用它们进行治疗的方法还没有被 没有完全特征化。我们的总体目标是了解I类法师表达在黑色素瘤中的作用 发展我们将通过以下方法检验法师蛋白促进黑素细胞转化的假设： 调节基因的表达，这些基因协同促进逃避衰老， 黑素细胞增殖和组织侵袭。 目的1：确定法师如何影响黑素细胞的生长和组织侵袭。 目的1a将测试法师是否通过抑制p16来促进逃避BRAFV 600 E诱导的衰老， Id 1，在BRAFV 600 E（+）细胞中使用内源性Id 1、p16和法师的选择性敲低，或通过异位 BRAFV 600 E、Id 1、p16和法师的表达，随后测量细胞生长和衰老 标记。通过ChIP测定法师和组蛋白3三甲基化对法师的表观遗传效应。 亮氨酸9（H3 me 3 K9），KAP 1诱导的基因阻遏的分子标记。 目标1b将使用类似的策略来测试AP 2的法师调节是否促进了细胞的迁移和侵袭。 在Boyden室和体外伤口测定中的黑素细胞。 目的2：建立体内法师表达与黑素细胞转化的关系。 本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检测 假设法师在黑素细胞转化早期表达且法师蛋白共定位 与增殖标志物（Ki 67）相关，并且与衰老标志物的表达负相关。 目的3：验证法师基因表达促进黑素细胞转化的假说。 目的3a将确定法师是否影响黑素细胞迁移或组织入侵在子宫内使用 免疫组织学和表达黑素细胞特异性、多西环素诱导法师的独特转基因小鼠。 目的3b将检验法师通过与黑素细胞法师交叉促进黑素细胞转化的假设 （+）小鼠转化为基于BRAFV 600 E或NRASQ 61 K的致癌基因诱导的黑素细胞痣的鼠模型。 目的3c通过交叉法师转基因，研究MAGE-A3在早期黑色素瘤中的表达 表达BRAFV 600 E或NRASQ 61 K并显示肿瘤抑制因子PTEN或 第16页。结果包括转化率，数量，和组织学特征， 黑素瘤。单个基因的法师抑制的评估将使用ChIP，逆转录真实的时间 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点使退伍军人受益。