Comparative Functional Genomics

比较功能基因组学

• 批准号: 7966157
• 负责人: Snorri Thorgeirsson
• 金额: $ 40.7万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7966157
• 关键词: Address; Adopted; Adult; Age; Alleles; Animals; Apoptosis; Apoptotic; Binding Sites; Biogenesis; Blood Vessels; Breeding; Categories; Cell Cycle; Cell Line; Characteristics; Classification; Cluster Analysis; Code; Collaborations; Conserved Sequence; Cytoskeletal Modeling; Cytoskeleton; DLEC1 gene; DNA Repair; DNA Repair Gene; DNA biosynthesis; Data; Defect; Development; Diagnostic Neoplasm Staging; Elements; Embryo; Exhibits; Exons; FOS Family Genes; Fatty Acids; Future; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Targeting; Genes; Genomic Instability; Genomics; Goals; Growth; Hepatocarcinogenesis; Hepatocyte; Human; Immunologic Surveillance; In Vitro; Investigation; Knock-out; Knockout Mice; Lesion; Ligands; Liver; MAPK8 gene; Malignant Neoplasms; Malignant neoplasm of liver; Mean Survival Times; Metastatic Neoplasm to the Liver; Microarray Analysis; Modeling; Molecular; Molecular Classification of Tumors; Molecular Genetics; Molecular Profiling; Mouse Strains; Mus; Mutation; Natural Immunity; Natural Killer Cells; Neoplasm Metastasis; Nucleic Acid Regulatory Sequences; Oncogenic; Orthologous Gene; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Predictive Value; Pregnancy; Prevention; Production; Promoter Regions; Property; Proteins; Reactive Oxygen Species; Recurrence; Regulation; Regulatory Element; Reporting; Repression; Research; Role; Sampling; Side; Signal Pathway; Site; Staging; Subgroup; Testing; Time; Tissues; Transcription Factor AP-1; Transforming Growth Factor beta; Transgenic Mice; Transgenic Organisms; Translations; Tumor stage; Tumor-Derived; Up-Regulation; Work; angiogenesis; base; biological adaptation to stress; c-myc Genes; cell motility; clinically relevant; clinically significant; comparative; density; detoxication; functional genomics; human cancer mouse model; human data; in vivo; indexing; insight; meetings; mouse model; mutant; neoplastic; novel; outcome forecast; oxidation; statistics; trait; transmission process; tumor; tumor progression

Identification of specific gene expression signatures characteristic of oncogenic pathways is an important step toward molecular classification of human malignancies. Aberrant activation of the Met signaling pathway is frequently associated with tumor progression and metastasis. We have defined the Met-regulated gene expression signature using global gene expression profiling of WT and Met-deficient primary mouse hepatocytes. Novel transcriptional targets of Met pathway were identified that included genes involved in the regulation of oxidative stress responses as well as cell motility, cytoskeletal organization, and angiogenesis. To assess the importance of Met-regulated gene expression signature, a comparative functional genomics approach was applied to 242 human HCCs and 7 metastatic liver lesions. Cluster analysis revealed that a subset of human HCC and all liver metastases shared a Met-induced expression signature. Furthermore, the presence of Met signature showed a significant correlation with increased vascular invasion rate, microvessel density, and decreased mean survival of HCC patients. We conclude that the genetically defined gene expression signatures, in combination with comparative functional genomics, constitute an attractive paradigm for defining both the function of oncogenic pathways and the clinically relevant subgroups of human cancers. Transforming growth factor-beta (TGF-beta) is known to exhibit tumor stage dependent suppressive (growth inhibition) and oncogenic (invasiveness) properties. Based on these considerations, we hypothesized that the TGF-beta gene expression signature established under well-controlled experimental conditions in vitro may contain gene sets characteristic of both tumor suppressive and oncogenic properties and thus be relevant for the molecular classification of tumors (69). Using the comparative functional genomics approach, we demonstrated that a time related TGF-beta gene expression signature established in mouse primary hepatocytes successfully discriminated distinct subgroups of HCC. The TGF-beta positive cluster included two novel homogeneous groups of HCC associated with early and late TGF-beta signatures. Kaplan-Meier plots and logrank statistics indicated that patients with the late TGF-beta signature showed significantly (P < 0.005) shortened mean survival time (16.25.3 months) compared to patients with the early TGF-beta signature (60.716.1 months). Significantly, tumors expressing the late TGF-beta-responsive genes displayed invasive phenotype and increased tumor recurrence. Also, late TGF-beta signature accurately predicted liver metastasis and discriminated HCC cell lines by degree of invasiveness. In addition, the TGF-beta gene expression signature possessed a predictive value for tumors other than HCC. These data demonstrate the clinical significance of the genes embedded in the TGF-beta expression signature for the molecular classification of HCC, further supporting the validity of the comparative functional genomics approach. Activation of AP-1 transcription factors is a characteristic feature of a HCC subtype expressing hepatoblast traits. The goal of this project was to evaluate the relevance of AP-1 (Jun/Fos) gene expression signature for human HCC. Accomplishments and future plans. We performed gene expression profiling of liver samples (normal and tumors) derived from WT (Junfl/fl) and Jun-KO mice. In total, 364 genes were found to be differentially expressed (P<0.01) in tumors derived from Jun-KO vs. WT animals, with 60% being down-regulated. The up-regulated genes were strongly associated with apoptosis. This subset of genes was referred to as "Jun signature in HCC". Applying a comparative functional genomics approach, we found orthologous genes of the Jun signature in human, and then integrated the gene profiles from mouse tumor samples and human HCC (n=139). The Jun-signature successfully discriminated human HCC into clusters displaying either WT or Jun-KO signatures. Notably, integration of the Jun signature with the human data revealed a clear and significant association of Jun-KO and Jun-WT signatures, respectively, with either Hepatocyte (HC) or Hepatoblast (HB) signatures which have been described previously. The Jun signature was significantly associated with patient survival. The gene expression signature in tumors derived from Jun-KO livers was close to the group of human HCC with a better survival. These tumors exhibited a greater apoptotic index. In the future, we will validate the Jun signature by analysis of enrichment of specific binding sites in the promoter regions of this subset of genes. A similar approach is currently used in JNK conditional knockout mice. We have previously reported that the gene expression profiles of HCC derived from c-Myc/Tgfα transgenic mice closely reproduce those observed in a subset of human HCC with poor prognosis. Our goal is to achieve a comprehensive characterization of the multiple-step tumor progression by profiling gene expression from the early to late stages of HCC development. Significantly, gene expression data were highly consistent with the c-Myc/Tgfα phenotype described in our previous work. Starting from 3 wk of age, microarray analysis revealed upregulation of genes involved in DNA replication (Mcm6, Mcm7, Myc), DNA repair (Ddb2, Rad51l1) and translation (Eef1b2, Eef2, Rpl13). By 3 m of age, the list of differentially expressed DNA repair genes was increased to include Ddb2, Mlh1, Msh3, Rad51ap1, along with upregulation of genes involved in fatty acid beta-oxidation (Acaa1b, Acadm), and detoxication (Gsta1, Ugt1a9) as a reflection of increasing genomic instability and production of reactive oxygen species. Moreover, a global gene expression analysis at the early stage of hepatocarcinogenesis revealed a previously unrecognized dysregulation of genes involved in innate immunity. In particular, we found an induction of several ligands for natural killer cells (Raet1a-e) and simultaneous repression of MHC-I. We confirmed these results using FACS analysis of hepatocytes isolated from 3-m-old dysplastic livers. However, the most dramatic changes of gene expression were found in tumors. The functional categories of differentially expressed genes involved translation, cell cycle, DNA repair, detoxication and cytoskeleton biogenesis. We conclude from these data that tumor development in c-Myc/Tgfα mice results from a progressive accumulation of genetic alterations which culminate in HCC. Furthermore, our results strongly suggest that a disruption of the innate immune surveillance may contribute to a rapid selection of tumor clones at the early stages of hepatocarcinogenesis. This aspect is under investigation. To study the in vivo effects of DLC1 deficiency, we had previously generated a Dlc1 knockout mouse strain. Homozygous mutant embryos died by 10 days of gestation with defects in several tissues, demonstrating DLC1s essential role for normal development. To address the role of DLC1 in adult tissues, we set out to generate a conditional Dlc1 knockout mouse. In collaboration with the Gene Targeting Facility at NCI-Frederick, mice with germline transmission of the conditional knockout allele (Dlc1neo) were obtained, and the frt-flanked neo cassette was removed by breeding with Flpe transgenic mice, to generate the floxed Dlc1 allele (Dlc1fl) with single loxP sites on both sides of exon 4. Both the Dlc1neo/neo and Dlc1fl/fl mice are viable. The condit [summary truncated at 7800 characters]

鉴定致癌途径的特定基因表达特征是迈向人类恶性肿瘤分子分类的重要一步。 MET信号通路的异常激活通常与肿瘤进展和转移有关。我们已经使用WT和MET缺陷原代小鼠肝细胞的全局基因表达分析定义了受元调节的基因表达特征。确定了MET途径的新型转录靶标，其中包括涉及调节氧化应激反应以及细胞运动，细胞骨架组织和血管生成的基因。为了评估元调节基因表达特征的重要性，将比较功能基因组学方法应用于242个人HCC和7个转移性肝病变。聚类分析表明，人类HCC和所有肝转移的子集具有MET诱导的表达特征。此外，MET签名的存在显示与HCC患者的血管浸润率，微血管密度和平均存活率降低显着相关。我们得出的结论是，遗传定义的基因表达特征与比较功能基因组学结合使用，构成了一个有吸引力的范式，用于定义致癌途径的功能和人类癌症的临床相关亚组。已知转化生长因子β（TGF-β）表现出肿瘤阶段依赖性抑制（生长抑制）和致癌性（侵入性）特性。基于这些考虑，我们假设在体外控制良好的实验条件下建立的TGF-β基因表达签名可能包含肿瘤抑制和致癌特性的特征，因此与肿瘤的分子分类有关（69）。使用比较功能基因组学方法，我们证明了在小鼠原代肝细胞中建立的时间相关的TGF-β基因表达特征成功地歧视了HCC的不同亚组。 TGF-beta阳性簇包括与早期和晚期TGF-beta签名相关的两个新型HCC组。 Kaplan-Meier情节和Logrank统计数据表明，与患有早期TGF-BetA签名早期（60.716.1个月）的患者相比，TGF-β晚期签名患者的平均生存时间（16.25.3个月）显着缩短（P <0.005）。值得注意的是，表达晚期TGF-β响应基因的肿瘤表现出侵入性表型和增加的肿瘤复发。同样，晚期TGF-beta签名准确地预测了肝转移，并按照侵入性区分了HCC细胞系。另外，TGF-β基因表达特征对HCC以外的肿瘤具有预测值。这些数据证明了嵌入HCC分子分类的TGF-β表达特征中嵌入的基因的临床意义，进一步支持了比较功能基因组学方法的有效性。 AP-1转录因子的激活是表达肝母细胞性状的HCC亚型的特征。该项目的目的是评估人类HCC的AP-1（JUN/FOS）基因表达签名的相关性。成就和未来计划。我们对源自WT（JUNFL/FL）和JUN-KO小鼠的肝样品（正常和肿瘤）进行了基因表达分析。总共发现364个基因在Jun-Ko与WT动物的肿瘤中差异表达（P <0.01），其中60％的基因被下调。上调的基因与凋亡密切相关。该基因的子集被称为“ HCC中的Jun Signature”。采用比较功能基因组学方法，我们在人类中发现了JUN Signature的直系同源基因，然后整合了小鼠肿瘤样品和人HCC的基因谱（n = 139）。 Jun-Signature成功地将人类HCC分为群集，显示WT或Jun-Ko签名。值得注意的是，JUN签名与人类数据的整合表明，Jun-Ko和Jun-WT签名的明确而显着的关联与肝细胞（HC）或肝类分类（HB）（HB）签名的相关性。 JUN签名与患者存活显着相关。来自Jun-Ko肝脏的肿瘤中的基因表达特征与人类HCC的群体接近，生存期更好。这些肿瘤表现出更大的凋亡指数。 将来，我们将通过分析该基因子集的启动子区域中特定结合位点的富集来验证JUN签名。 JNK条件敲除小鼠目前使用了类似的方法。我们以前已经报道过，源自C-MYC/TGF＆＃945;的HCC的基因表达谱。转基因小鼠在预后不良的人类HCC子集中亲密地再现了那些。我们的目标是通过从HCC发育的早期到晚期进行基因表达来分析基因表达，从而实现多步肿瘤进展的全面表征。值得注意的是，基因表达数据与C-MYC/TGF＆＃945;我们以前的工作中描述的表型。从3周开始，微阵列分析表明，与DNA复制（MCM6，MCM7，MYC），DNA修复（DDB2，RAD51L1）和翻译（EEF1B2，EEF2，EEF2，EEF2，RPL13）有关的基因上调。到3 m年龄，增加了差异表达的DNA修复基因列表，包括DDB2，MLH1，MSH3，RAD51AP1，以及与脂肪酸β-氧化涉及的基因的上调（ACAAA1B，ACADM）和解毒（GSTA1，UGT1A9），以及增长的基因和生成基因并添加了基因的基因并添加了基因构成。此外，在肝癌发生早期的全球基因表达分析表明，先前未识别的与先天免疫的基因失调。特别是，我们发现了几种天然杀伤细胞（RAET1A-E）和同时抑制MHC-I的配体的诱导。我们使用从3米的发育不良肝分离的FACS分析进行了FACS分析来证实这些结果。然而，在肿瘤中发现了基因表达的最大变化。差异表达基因的功能类别涉及翻译，细胞周期，DNA修复，解毒和细胞骨架生物发生。从这些数据中，我们得出结论，C-MYC/TGF中的肿瘤发展。小鼠是由于遗传改变的逐渐积累而导致的，这些改变在HCC中达到了最终。此外，我们的结果强烈表明，在肝癌发生的早期阶段，先天免疫监测的破坏可能导致肿瘤克隆的快速选择。这方面正在研究中。为了研究DLC1缺乏症的体内效应，我们以前已经产生了DLC1敲除小鼠菌株。纯合突变胚胎死于妊娠10天，在几个组织中缺陷，这表明DLC1s在正常发育中的重要作用。为了解决DLC1在成人组织中的作用，我们着手生成条件DLC1敲除小鼠。通过与NCI-Frederick的基因靶向设施合作，获得了条件敲除等位基因（DLC1NEO）的生殖线传播的小鼠，并通过用FLPE转基因小鼠育种Flpe Transenic小鼠将FRT浮装的Neo Cassette与floxed DLC1等位基因（DLC1FL）一起繁殖，从DLC1NEO/NEO和DLC1FL/FL小鼠可行。孔[摘要以7800个字符截断]