Integrative Pathway Analysis of Epigenomic/transcriptional Alteration in HNSCC

描述（由申请人提供）：该申请涉及广泛的挑战领域（08）基因组学主题：08- de -103：口腔健康和疾病的表观基因组学和表观遗传学。人类癌症中全基因组表观遗传改变的标准范式主要集中在发现特异性启动子高甲基化的肿瘤抑制基因上。我们最近证明，全基因组表观遗传揭开和启动子低甲基化经常激活头颈部鳞状细胞癌（HNSCC）的候选原癌基因和肿瘤特异性癌睾丸抗原。过去的发现技术主要集中在基于细胞系的药理学去甲基化策略上，这对肿瘤表观遗传改变的发现带来了偏见。在这个项目中，我们整合了全基因组启动子甲基化的直接测量和表达分析，同时定义了与活化癌基因相关的去甲基化启动子，以及在HNSCC中使肿瘤抑制基因失活的异常甲基化启动子。对基因表达的协调表观遗传调节模式的分析将把这些改变置于特定生物学途径的背景下。该项目将促进从有限的单基因观点过渡到基于途径的HNSCC生物学和治疗发展的观点。假设：候选原癌基因和肿瘤抑制基因的转录激活和抑制与基因特异性启动子甲基化改变相关，1)可以使用全基因组整合发现技术进行鉴定，2)可以以协调的方式改变特定的生物途径。具体目标1：利用表观遗传启动子阵列分析在HNSCC中定义一组潜在的表观遗传原癌基因和肿瘤抑制基因。这些候选基因将与并发基因组宽表达阵列分析相结合，并通过启动子甲基化和原发肿瘤的表达分析进行验证。原理：利用药理学上的去甲基化细胞系模型，我们之前已经发表了全基因组发现方法来定义HNSCC中启动子高甲基化、灭活的肿瘤抑制基因，并且最近描述了一种鉴定DNA低甲基化激活的候选原癌基因的综合方法。这些技术很麻烦，而且受到细胞培养和去甲基化药物治疗偏见的阻碍，但在确定有限的改变基因组方面是有效的。在原发性HNSCC中使用同步基因组启动子甲基化和表达阵列将允许在全基因组范围内直接测量这些改变，而无需细胞培养和药物治疗偏差。鉴定显著的个体启动子甲基化-转录相关性将鉴定候选原癌基因和肿瘤抑制基因，这些基因会因启动子甲基化变化而改变。特异性目标2：在目标1中，通过对原发性HNSCC的并发表观基因组学和转录分析，我们将1)确定甲基化调节因子的转录靶点，2)确定在个体肿瘤样本中发生协调变化的生物学途径。一个选择的协调的、改变的通路将在原发性HNSCC中得到验证。理由：我们最近通过关键转录元件定义了HNSCC中候选原癌基因激活的协调表观遗传调控。将基因特异性表观遗传改变置于基因组广泛表达改变的背景下，将使我们能够将表观遗传和转录改变置于共同的、共享的生物学途径的背景下。这将有助于为HNSCC设计更高影响的治疗策略，这些策略是通路特异性的，而不是局限于靶向单基因改变。每年约翰霍普金斯大学直接为马里兰州创造了100亿美元的经济活动，比2002年的70亿美元增长了43%相当于今天该州经济中每24美元中的1美元。2008年，约翰霍普金斯大学提供了4.5万个就业岗位，自2002年以来，每年创造700个新就业岗位。约翰霍普金斯大学直接或间接地为马里兰州提供了超过10万个就业岗位，占该州每29个就业岗位的1个。仅在巴尔的摩市，约翰霍普金斯大学就直接或间接地提供了6万个就业岗位，占全市就业岗位的16.7%。此应用程序将创建或保留四个作业。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (08) Genomics Topic: 08-DE-103: Epigenomics and Epigenetics of Oral Health and Disease The standard paradigm for characterization of genome wide epigenetic alteration in human cancers has been focused on discovery of specific promoter hypermethylated tumor suppressor genes. We have recently demonstrated that genome-wide epigenetic unmasking and promoter hypomethylation frequently activate candidate proto-oncogenes and tumor specific cancer testes antigens in head and neck squamous cell carcinoma (HNSCC). Past discovery techniques have focused on cell line based pharmacologic demethylating strategies, introducing bias to discovery of epigenetic alterations in tumors. In this project, we integrate direct measurement of genome wide promoter methylation and expression analysis to concurrently define demethylated promoters associated with activated oncogenes, and aberrantly methylated promoters that inactivate tumor suppressor genes in HNSCC. Analysis of patterns of coordinated epigenetic modulation of gene expression will place these alterations within the context of specific biologic pathways. This project will facilitate transition from a limited single gene view, to a pathway based perspective of HNSCC biology and therapeutic development. Hypothesis: Candidate proto-oncogenes and tumor suppressor genes are transcriptionally activated and repressed in association with gene-specific promoter methylation alterations and 1) can be identified using genome wide integrative discovery techniques, and 2) can alter specific biologic pathways in a coordinated fashion. Specific Aim 1: Define a set of potential epigenetically proto-oncogenes and tumor suppressor genes using epigenetic promoter array analysis in HNSCC. These candidates will be integrated with concurrent genome wide expression array analysis and validated by promoter methylation and expression analysis in primary tumors. Rationale: Using pharmacologically demethylated cell line models, we have previously published genome wide discovery approaches to define promoter hypermethylated, inactivated tumor suppressor genes in HNSCC, and recently described an integrative method identifying candidate protooncogenes activated by DNA hypomethylation. These techniques are cumbersome and hampered by cell culture and demethylating drug treatment bias, but have been effective in defining limited sets of altered genes. Use of concurrent genomic promoter methylation and expression arrays in primary HNSCC will allow for direct measurement of these alterations on a genome wide basis without cell culture and drug treatment bias. Identification of significant individual promoter methylation-transcription correlations will identify candidate proto-oncogenes and tumor suppressor genes that are altered by promoter methylation changes. Specific Aim 2: Using concurrent epigenomic and transcriptional analysis of primary HNSCC in Aim 1, we will 1) identify transcriptional targets of methylated regulatory factors, and 2) identify biologic pathways undergoing coordinated changes across individual tumor samples. A selected coordinated, altered pathway will be validated in primary HNSCC. Rationale: We have recently defined the coordinated epigenetic regulation of candidate proto-oncogene activation in HNSCC, via key transcriptional elements. Placement of gene specific epigenetic alteration within the context of genome wide expression alterations will allow us to place epigenetic and transcriptional alterations within the context of common, shared biologic pathways. This will facilitate design of higher impact therapeutic strategies for HNSCC that are pathway specific rather than limited to targeting of single gene alterations. Every year Johns Hopkins Institutions directly generate about $10 billion in economic activity in the State of Maryland, a 43% increase from the $7 billion generated in 2002 and the equivalent of one of every twenty-four dollars in the state's economy today. In 2008, Johns Hopkins Institutions provided 45,000 jobs and created 700 new jobs each year since 2002. Directly and indirectly, Johns Hopkins Institutions support more than 100,000 jobs in Maryland, one of every 29 in the state. In Baltimore City alone Johns Hopkins directly and indirectly supports 60,000 jobs, or 16.7% of all City employment. This application will create or retain four jobs. PUBLIC HEALTH RELEVANCE: The standard paradigm for characterization of genome wide epigenetic alteration in human cancers has been focused on discovery of specific promoter hypermethylated tumor suppressor genes. In this project, we integrate direct measurement of genome wide promoter methylation and expression analysis to concurrently define demethylated promoters associated with activated oncogenes, and aberrantly methylated promoters that inactivate tumor suppressor genes in HNSCC.