Integrative Pathway Analysis of Eqigenomic/transcriptional Alteration in HNSCC

描述(申请人提供)：本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全基因组表观遗传学变化的标准范例一直集中在发现特定启动子高甲基化的肿瘤抑制基因上。我们最近证实，在头颈部鳞状细胞癌(HNSCC)中，全基因组的表观遗传去掩蔽和启动子低甲基化经常激活候选原癌基因和肿瘤特异性癌症睾丸抗原。过去的发现技术主要集中在基于细胞系的药物去甲基化策略上，引入了对发现肿瘤表观遗传学改变的偏见。在这个项目中，我们将全基因组启动子甲基化的直接测量和表达分析结合在一起，以同时确定与激活的癌基因相关的去甲基化启动子，以及在HNSCC中失活肿瘤抑制基因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变化放在特定生物途径的背景下。该项目将促进从有限的单基因观点过渡到基于途径的HNSCC生物学和治疗开发的观点。假设：候选原癌基因和肿瘤抑制基因在转录上被激活和抑制，与基因特异的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来识别，2)可以以协调的方式改变特定的生物通路。具体目的1：利用表观遗传学启动子阵列分析，确定一组潜在的表观原癌基因和抑癌基因。这些候选基因将与同步的全基因组表达阵列分析相结合，并通过启动子甲基化和在原发肿瘤中的表达分析来验证。理论基础：利用药物去甲基化的细胞系模型，我们以前已经发表了全基因组的发现方法来定义HNSCC中的启动子超甲基化、失活的抑癌基因，最近描述了一种识别由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集方面是有效的。在原发HNSCC中同时使用基因组启动子甲基化和表达阵列将允许在全基因组基础上直接测量这些变化，而不需要细胞培养和药物治疗偏向。确定显著的个体启动子甲基化-转录相关性将确定候选原癌基因和肿瘤抑制基因，这些基因由启动子甲基化改变而改变。具体目标2：在目标1中同时使用原发HNSCC的表观基因组和转录分析，我们将1)识别甲基化调节因子的转录靶标，2)识别在单个肿瘤样本中发生协调变化的生物途径。选定的协调、改变的途径将在原发HNSCC中得到验证。理论基础：我们最近定义了HNSCC中候选原癌基因激活的协调表观遗传调控，通过关键的转录元件。将基因特定的表观遗传改变置于全基因组表达改变的背景下，将使我们能够将表观遗传和转录改变置于共同的、共享的生物途径的背景下。这将有助于设计针对HNSCC的更高影响的治疗策略，这些策略是特定于途径的，而不是局限于针对单基因改变的靶点。约翰霍普金斯大学的机构每年在马里兰州直接创造约100亿美元的经济活动，比2002年的70亿美元增加了43%，相当于今天该州经济每24美元中就有1美元。2008年，约翰霍普金斯大学提供了4.5万个工作岗位，自2002年以来，每年创造700个新工作岗位。约翰霍普金斯大学的机构直接和间接地支持了马里兰州10万多个工作岗位，该州每29个工作岗位中就有一个。仅在巴尔的摩市，约翰斯·霍普金斯就直接和间接支持了60,000个工作岗位，占整个城市就业人数的16.7%。该应用程序将创造或保留四个工作岗位。 公共卫生相关性：人类癌症全基因组表观遗传学改变的标准范例一直集中在发现特定启动子超甲基化的肿瘤抑制基因上。在这个项目中，我们将全基因组启动子甲基化的直接测量和表达分析结合在一起，以同时确定与激活的癌基因相关的去甲基化启动子，以及在HNSCC中失活肿瘤抑制基因的异常甲基化启动子。

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.