Genomic Classification of Human Liver Cancer

人类肝癌的基因组分类

• 批准号: 7966159
• 负责人: Snorri Thorgeirsson
• 金额: $ 81.4万
• 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/7966159
• 关键词: Adult; Affect; Apoptosis; Apoptotic; Belgium; Biological; Bioluminescence; Blood specimen; CD34 gene; Cell Cycle Arrest; Cell Line; Cell Transplantation; Cells; Characteristics; China; Cholangiocarcinoma; Chromosomes; Classification; Clinic; Clinical; Clinical Oncology; Clinical/Radiologic; Collaborations; Collection; Communities; Complex; DNA copy number; Data; Databases; Derivation procedure; Detection; Development; Diagnosis; Disease; Down-Regulation; Dysplasia; Employee Strikes; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor Receptor; Epithelial-Stromal Communication; Evaluation; Event; Exhibits; Extramural Activities; Future; G1 Phase; Gene Expression; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Genomics; Glycoproteins; Goals; Growth; HSF1; Hematopoietic stem cells; Hepatic; Hepatitis B Virus; Hepatitis C virus; Hepatocarcinogenesis; Hepatocyte; Heterogeneity; Human; Image; Immunodeficient Mouse; In Vitro; Informed Consent; Injection of therapeutic agent; Institutes; KRT19 gene; Knowledge; Laboratories; Lasers; Lesion; Link; Lipids; Liver; Luciferases; Malignant Conversion; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Messenger RNA; Microarray Analysis; Modality; Molecular; Molecular Genetics; Molecular Profiling; Monitor; Neoplasms; Neoplastic liver; Nodule; Normal tissue morphology; Nucleic Acids; Oncogenes; Oncogenic; Outcome; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Play; Primary carcinoma of the liver cells; Protein p53; Proteins; Proteomics; Proto-Oncogene Protein c-kit; Relapse; Reporting; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Scientist; Screening procedure; Signal Pathway; Signal Transduction; Small Interfering RNA; Spleen; Staging; Stem cells; System; TACSTD1 gene; TP53 gene; Tail; Technology; Testing; The Sun; Time; Tissue Sample; Tissues; Transcription Alteration; Translating; Transplantation; Ubiquitin; United States National Institutes of Health; Variant; Veins; Western Blotting; Xenograft Model; biobank; cell growth; cholangiocyte; dosage; embryonic stem cell; human FRAP1 protein; in vivo; insight; interest; molecular phenotype; mouse model; neoplastic; novel; outcome forecast; particle; prognostic; regenerative; therapeutic target; tool; trait; transcriptomics; tumor; tumor progression

We have performed an integrative analysis of DNA copy numbers and gene expression profiles in hepatocellular carcinoma (HCC) in an attempt to uncover most provable genomic regions of concomitant copy number and transcription alterations. Unbiased screening of these regions identifies 50 potential driver genes with significant prognostic relevance.Our integrative analyses of genomic data also identified novel potential driver genes for HCC development including NCSTN and SCRIB. Particularly, NCSTN, encoding Type I transmembrane glycoprotein, has never been known to be associated with cancers. SCRIB has recently been reported to be associated with human cancers, but its oncogenic role has not been fully studied yet. Moreover, our analysis provided a novel notion i.e. a connection of EGFR, AMPK, and mTOR with the 50 genes. Recently, HSF1, one of the 50 genes, was reported to have oncogenic potential, and which function is mediated by mTOR and EGFR signaling. Similarly, we also demonstrated that the oncogenic activity of NCSTN and SCRIB is possibly mediated via the mTOR pathway. From these results, we postulate the existence of a multifaceted association of the 50 driver genes with three signaling pathways in cancer progression, although further elucidation will be necessary. We conclude our data demonstrate that a systems-level integrative analysis of genome scale data independently collected from a small group of patients provide a striking advantage for identifying genes likely to drive neoplastic development in the liver, and provides new biological and clinical insights for the development of novel treatment modalities. To decode the molecular events during early stages of liver carcinogenesis, we performed gene expression profiling on cirrhotic (regenerative) and dysplastic nodules as well as early HCC. Although considerable heterogeneity was observed at the regenerative and dysplastic stages, a clear difference was detected between dysplastic nodules and eHCC which included 460 differentially expressed genes. Functional analysis of the significant gene set identified the MYC oncogene as a plausible driver gene for malignant conversion of the dysplastic nodules. In addition, gene set enrichment analysis (GSEA) revealed a remarkable enrichment of MYC up-regulated gene sets in eHCC versus dysplasia. Presence of the MYC signature significantly correlated with increased expression of CSN5 as well as with the higher overall transcription rate of genes located in the 8q chromosome region. Furthermore, a classifier constructed from MYC target genes could robustly discriminate eHCC from HGDN and LGDN. In conclusion, our study identified unique expression patterns associated with the transition of high-grade dysplastic nodules to early HCC and demonstrated that activation of the MYC transcription signature is critical for the malignant conversion of pre-neoplastic liver lesions. The fifth subunit of COP9 signalosome CSN5 complex targets p53 tumor suppressor for its degradation through the ubiquitin system in coordination with Mdm2. Our recent microarray analysis identified the elevated expression of CSN5 in early HCC as compared to dysplastic stage, implying that CSN5 is one of the early markers of malignant conversion. We have now tested if targeting of CSN5 can affect the course of HCC progression in vitro and in vivo. To inactivate CSN5 gene expression, Huh7 and HepG2 cells were treated with three different siRNAs. The down-regulation of CSN5 in both mRNA and protein level was confirmed by quantitative real-time RT-PCR and Western blotting. Cell growth was analyzed by MTT and FACS analysis, and apoptosis was estimated by ELISA for detection of ssDNA. For in vivo evaluation of CSN5 as a therapeutic target, a bioluminescent Huh7 cell line permanently expressing luciferase was transplanted into the spleen of immunodeficient mice to establish Huh7-luc+ HCC orthotopic xenograft model. Chemically modified CSN5siRNA was systematically delivered to liver through tail vein injection of stable nucleic acid lipid particle (SNALP). Tumor relapses were persistently monitored by bioluminescence imaging, up to 28 days after cell transplantation. Huh7 and HepG2 cells transfected with CSN5-siRNA showed 68% and 77% growth inhibition, respectively, associated with cell-cycle arrest in G1 phase. CSN5-deficient cells also exhibited a 1.8 fold increase in apoptosis as compared with negative control siRNA-treated cells. We also have demonstrated that, when the HCC cells were undergoing apoptotic progression through downregulation of CSN5 protein following the siRNA treatment, the levels of p53, its responder p21 and p27 were restored. CSN5siRNA, a chemically modified variant of CSN5-2siRNA, was selected for in vivo application in terms of growth inhibition of Compared to SNALP-control siRNA treatment, four times injection of SNALP-CSN5siRNA at a dosage of 2 mg/kg effectively suppressed neoplastic growth in the mouse model of metastatic human liver cancer. Taken together, the results suggest that CSN5 is an important regulator of HCC cell growth and survival, and may be an attractive target for treatment of HCC disease. Further, SNALP technology for in vivo delivery of siRNA may be effective in treating human HCC. HCC and ICC are two major primary liver cancers in adults. HCC have enormously heterogeneous phenotypes with dismal outcome, while ICC are more difficult to diagnose and to treat than HCC. In addition, a rare form of combined hepatocellular cholangiocarcinoma (CHC) has been reported to have intermediate characteristics between HCC and ICC. Although HCCs and ICCs are believed to be derived from hepatocytes and cholangiocytes, respectively, the expression of bipotential traits in CHC may suggest the origin from hepatic progenitor cells (HPC). HPC can be identified by coexpression of hepatocytic and cholangiocytic lineage markers (HepPar1 and CK19) as well as hematopoietic stem cell markers (c-kit and CD34). Thus, phenotypic classification of ICC, CHC and HCC may not be evident for tumors co-expressing HPC-like traits. The goal of this project is to characterize the molecular phenotypes of ICC, CHC and HCC by comparing gene expression profiles and clinical features. Current results show that ICC has a more aggressive clinical outcome than HCC, and CHC displays an intermediate expression pattern and clinical outcome closer to ICC than HCC. In addition, we identified a novel HCC subtype, ICC-like HCC (CLHCC), which expressed ICC-like traits (ICC signature) including KRT19, TACSTD1 and PROM1. The expression of ICC signature in HCC was strongly associated with HCC prognosis. Moreover, the ICC signature-expressing HCCs concomitantly expressed the embryonic stem cell-like expression traits (ES signatures), suggesting derivation from the bipotent hepatic progenitor cells. The prognostic value of the ICC signature was independent from ES-like, hepatoblast-like, or proliferation-related gene expression traits. Furthermore, the combined CC and ES signature was a strong determinant of the clinical outcome of HCC, indicating their cellular origins from different developmental stages. The data suggest that the attainment of ICC-like-expression traits plays a critical role in the progression and clinical outcomes of liver cancer. Our future course includes (i) expanding the ICC database in LEC and comprehensive transcriptomic classification of ICC tumors; (ii) detailed analysis of the stromal-epithelial interactions (using laser micro-dissec [summary truncated at 7800 characters]

我们对肝细胞癌 (HCC) 中的 DNA 拷贝数和基因表达谱进行了综合分析，试图揭示伴随拷贝数和转录改变的最可证明的基因组区域。对这些区域的公正筛选确定了 50 个具有显着预后相关性的潜在驱动基因。我们对基因组数据的综合分析还确定了 HCC 发展的新型潜在驱动基因，包括 NCSTN 和 SCRIB。特别是，编码 I 型跨膜糖蛋白的 NCSTN 从未被发现与癌症有关。最近有报道称 SCRIB 与人类癌症有关，但其致癌作用尚未得到充分研究。此外，我们的分析提供了一个新的概念，即 EGFR、AMPK 和 mTOR 与 50 个基因的联系。最近，HSF1（50 个基因之一）被报道具有致癌潜力，其功能由 mTOR 和 EGFR 信号传导介导。同样，我们还证明了 NCSTN 和 SCRIB 的致癌活性可能是通过 mTOR 途径介导的。根据这些结果，我们假设 50 个驱动基因与癌症进展中的三个信号通路之间存在多方面的关联，尽管还需要进一步阐明。我们得出的结论是，我们的数据表明，对从一小群患者独立收集的基因组规模数据进行系统级综合分析，为识别可能驱动肝脏肿瘤发展的基因提供了显着的优势，并为该发展提供了新的生物学和临床见解。的新治疗方式。为了解码肝癌早期阶段的分子事件，我们对肝硬化（再生）和发育不良结节以及早期 HCC 进行了基因表达谱分析。尽管在再生和发育不良阶段观察到相当大的异质性，但在发育不良结节和 eHCC 之间检测到明显的差异，其中包括 460 个差异表达基因。对重要基因集的功能分析确定了 MYC 癌基因是发育不良结节恶性转化的合理驱动基因。此外，基因集富集分析 (GSEA) 显示，与不典型增生相比，eHCC 中 MYC 上调基因集显着富集。 MYC 特征的存在与 CSN5 表达的增加以及位于 8q 染色体区域的基因较高的总体转录率显着相关。此外，由 MYC 靶基因构建的分类器可以稳健地区分 eHCC 与 HGDN 和 LGDN。总之，我们的研究确定了与高级不典型增生结节向早期 HCC 转变相关的独特表达模式，并证明 MYC 转录特征的激活对于肿瘤前肝脏病变的恶性转化至关重要。 COP9 信号体 CSN5 复合物的第五个亚基以 p53 肿瘤抑制因子为目标，通过泛素系统与 Mdm2 协同降解。我们最近的微阵列分析发现，与发育不良阶段相比，早期 HCC 中 CSN5 的表达升高，这意味着 CSN5 是恶性转化的早期标志物之一。我们现在已经测试了 CSN5 的靶向是否可以影响体外和体内 HCC 进展的过程。为了灭活 CSN5 基因表达，用三种不同的 siRNA 处理 Huh7 和 HepG2 细胞。通过定量实时 RT-PCR 和蛋白质印迹证实了 CSN5 在 mRNA 和蛋白质水平上的下调。通过MTT和FACS分析来分析细胞生长，并通过用于检测ssDNA的ELISA来估计细胞凋亡。为了体内评估 CSN5 作为治疗靶点，将永久表达荧光素酶的生物发光 Huh7 细胞系移植到免疫缺陷小鼠的脾脏中，建立 Huh7-luc+ HCC 原位异种移植模型。通过尾静脉注射稳定核酸脂质颗粒（SNALP），将化学修饰的 CSN5siRNA 系统地递送至肝脏。通过生物发光成像持续监测肿瘤复发情况，直至细胞移植后 28 天。转染 CSN5-siRNA 的 Huh7 和 HepG2 细胞分别显示 68% 和 77% 的生长抑制，与细胞周期停滞在 G1 期相关。 与阴性对照 siRNA 处理的细胞相比，CSN5 缺陷细胞的细胞凋亡也增加了 1.8 倍。我们还证明，当 HCC 细胞在 siRNA 治疗后通过下调 CSN5 蛋白而经历凋亡进展时，p53、其反应物 p21 和 p27 的水平得到恢复。 CSN5siRNA是CSN5-2siRNA的化学修饰变体，在生长抑制方面被选择用于体内应用。与SNALP对照siRNA治疗相比，四次注射2mg/kg剂量的SNALP-CSN5siRNA可有效抑制肿瘤生长在转移性人类肝癌的小鼠模型中。综上所述，结果表明 CSN5 是 HCC 细胞生长和存活的重要调节因子，并且可能是治疗 HCC 疾病的一个有吸引力的靶标。此外，用于体内递送siRNA的SNALP技术可能有效治疗人类HCC。 HCC 和 ICC 是成人中两种主要的原发性肝癌。 HCC 具有极其异质的表型，结果令人沮丧，而 ICC 比 HCC 更难以诊断和治疗。此外，据报道，一种罕见的混合型肝细胞性胆管癌 (CHC) 具有介于 HCC 和 ICC 之间的中间特征。尽管 HCC 和 ICC 被认为分别源自肝细胞和胆管细胞，但 CHC 中双电位特征的表达可能表明起源于肝祖细胞 (HPC)。 HPC 可以通过肝细胞和胆管细胞谱系标记物（HepPar1 和 CK19）以及造血干细胞标记物（c-kit 和 CD34）的共表达来识别。因此，ICC、CHC 和 HCC 的表型分类对于共表达 HPC 样特征的肿瘤可能并不明显。该项目的目标是通过比较基因表达谱和临床特征来表征 ICC、CHC 和 HCC 的分子表型。目前的结果表明，ICC 比 HCC 具有更具侵袭性的临床结果，而 CHC 表现出中间表达模式，临床结果更接近 ICC，而不是 HCC。此外，我们还发现了一种新的 HCC 亚型，即 ICC 样 HCC (CLHCC)，其表达 ICC 样特征（ICC 特征），包括 KRT19、TACSTD1 和 PROM1。 HCC 中 ICC 特征的表达与 HCC 预后密切相关。此外，表达 ICC 特征的 HCC 同时表达胚胎干细胞样表达特征（ES 特征），表明源自双能肝祖细胞。 ICC 特征的预后价值独立于 ES 样、肝母细胞样或增殖相关基因表达特征。此外，CC 和 ES 的组合特征是 HCC 临床结果的重要决定因素，表明它们的细胞起源于不同的发育阶段。数据表明，ICC 样表达特征的获得在肝癌的进展和临床结果中发挥着关键作用。我们未来的课程包括（i）扩展LEC中的ICC数据库和ICC肿瘤的综合转录组学分类； (ii) 基质-上皮相互作用的详细分析（使用激光显微切割[摘要截断为 7800 个字符]