Molecular Classification of Human and Mouse Hepatocellular Carcinoma by Gene Exp

肝癌的发生通常被描述为一个多阶段的过程，其中前体病变可以通过积累多种遗传和表观遗传改变进展为早期肝细胞癌（HCC）。为了破译早期肝癌发生过程中的分子事件，我们对49个结节性肝脏病变（24个肝硬化结节，3个低级别，12个高级别发育不良结节和10个早期HCC）进行了微阵列分析。尽管在再生和发育不良阶段观察到相当大的异质性，但我们发现发育不良结节和早期HCC之间存在明显的表达差异，包括460个差异表达基因。重要基因组的功能分析也暗示MYC癌基因可能是许多转录组改变的原因。因此，为了探索MYC激活在肿瘤前病变恶性转化中的潜在作用，我们转向了比较基因组方法。通过基因集富集分析（GSEA）评估来自转基因小鼠和人细胞系的MYC和RAS靶基因集的表达。值得注意的是，MYC上调的基因组在早期HCC和非典型增生中表现出富集。MYC标记的存在与CSN5的表达增加以及8q染色体区域基因的总体转录率升高显著相关。此外，由MYC靶基因构建的基因组分类器可以区分早期HCC与HGDN和LGDN，准确率为75-85%。总之，我们的研究成功地确定了与早期肝癌发生进展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病变的恶性转化是必不可少的。由DNA低甲基化和/或启动子基因CpG高甲基化组成的异常甲基化与包括HCC在内的多种实体肿瘤的发展有关。我们分析了DNA甲基化的全球水平以及105个推定的肿瘤抑制基因的甲基化状态，发现全基因组低甲基化和CpG高甲基化的程度与HCC患者的生物学特征和临床结局相关。在所有HCC中，由于Ras通路抑制剂的表观遗传沉默，我们发现Ras和下游Ras效应物（ERK、AKT和RAL）被激活。此外，SPRY1和-2、DAB2、SOCS4和-5基因以及血管生成抑制剂（BNIP3、BNIP3L、IGFBP3和EGLN2）的选择性失活与不良预后相关。重要的是，在HCC中发现的几种表观遗传沉默的推定肿瘤抑制基因在非肿瘤肝脏中也失活。我们的研究结果为人类HCC的甲基化模式赋予了治疗和化学预防的意义，并开启了使用分子靶点（包括本研究中发现的分子靶点）有效抑制HCC发展和进展的可能性。尽管癌症基因表达谱数据快速增长，但由于许多基因在癌症中发生改变，确定少数候选基因作为诊断标记或治疗靶点仍然是一个挑战。基因改变的全基因组筛选已经确定了许多具有DNA拷贝数变化的复发性基因组位点，这些位点可能包含肿瘤发生的潜在驱动基因。然而，当我们有数百个候选基因组位点时，对这些基因座中的基因进行功能验证是非常耗时且不切实际的。我们探索了基因表达和拷贝数的基因组相关性，发现了肝细胞癌（HCC）中50个潜在的驱动基因。使用三种独立的方法，我们进行了详尽的搜索基因，其表达与拷贝数改变最相关，并与患者的预后最相关。此外，对与50种驱动基因显著相关的基因表达模式进行无偏筛选，预测EGFR是HCC的最佳治疗靶点之一。组蛋白的翻译后修饰导致染色质重塑在基因表达调控中起着关键作用。此外，已经确定的是，组蛋白H4的整体低乙酰化是人类肿瘤的共同标志，H4乙酰化的变化可能发生在肿瘤发生的早期。我们最近在原发性人肝细胞癌（HCC）和HCC衍生细胞系中发现了组蛋白去乙酰化酶2 （HDAC2）基因的表达升高。我们现在研究了HDAC2基因敲低对人类HCC细胞系细胞生长和凋亡的影响。为了沉默HDAC2基因的表达，用5 20 nM的三种不同的sirna （HDAC2-1、HDAC2-2和HDAC2-3）靶向HDAC2处理Huh7和HepG2细胞。MTT和FACS分析细胞生长情况，ELISA检测ssDNA和caspase活化，检测细胞凋亡情况。采用实时荧光定量RT-PCR检测HDAC2靶基因的表达。在三种测试的siRNA分子中，与对照治疗（未治疗和阴性对照siRNA治疗）相比，HDAC2-1siRNA在抑制HCC细胞生长方面最有效。15 nM HDAC2-1siRNA转染4 d后，Huh7和HepG2细胞的生长抑制率分别为68%和71%，通常伴有G2/M细胞数量的减少。抑制肝癌细胞生长是由于凋亡细胞死亡率增加，比对照细胞高约1.9倍。凋亡水平升高与caspase-3激活约65%、靶mRNA水平降低约75%直接相关。综上所述，这些结果表明HDAC2是HCC细胞生长和存活的重要调节因子，因此可能代表人类HCC治疗的潜在靶点。肝细胞癌（HCC）患者预后的可变性表明，HCC可能包括几种不同的生物学表型。这些表型可能是由于肿瘤发生过程中不同致癌途径的激活和/或来自不同的起源细胞。我们已经讨论了HCC的转录特征是否可以为肿瘤的细胞起源提供见解。我们整合了来自人类和小鼠肝癌模型的大鼠胎儿肝母细胞和成年肝细胞的基因表达数据。与胎儿肝母细胞有相同基因表达模式的HCC患者预后较差。将该亚型与其他类型HCC区分开来的基因表达程序包括肝卵圆细胞标记物，表明该亚型HCC可能起源于肝祖细胞。基因网络分析表明，AP-1转录因子的激活在这种新发现的HCC亚型中可能在肿瘤发展中起关键作用。在这项工作中，我们已经证明，通过应用两个独立的基因表达特征，我们能够将HCC患者分为三个亚组，这些亚组的临床结果具有统计学上的显著差异。这些发现支持了多个分子途径决定HCC的发展和不同临床结果的观点。我们的发现还表明，HCC的分子特征，如预后基因表达特征，在诊断时就存在。因此，基因表达谱的应用有望改善HCC的分子分类和预后预测。此外，将HCC患者分子分层为同质亚组可能为开发新的治疗方式提供机会

Hepatocarcinogenesis has been often described as a multi-stage process where precursor lesions could progress into early hepatocellular carcinomas (HCC) by accumulating multiple genetic and epigenetic alterations. In order to decipher the molecular events during early liver carcinogenesis we performed microarray profiling on forty-nine nodular liver lesions (24 cirrhotic nodules, 3 low-, 12 high-grade dysplastic nodules and 10 early HCC). In spite of the considerable heterogeneity observed in the regenerative and dysplastic stages, we detected clear expression differences between dysplastic nodules and early HCC including 460 differentially expressed genes. Functional analysis of the significant gene set also implicated that the MYC oncogene might be responsible for many of transcriptomic alterations. Thus, to explore the potential role of MYC activation in the malignant conversion of the pre-neoplastic lesions, we turned to a comparative genomic approach. Expression of MYC and RAS target gene sets derived from transgenic mice as well as from human cell lines was assessed with gene set enrichment analysis (GSEA). Remarkably, the MYC up-regulated gene sets showed enrichment in early HCC 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 genomic classifier constructed from MYC target genes could discriminate early HCC from HGDN and LGDN with the accuracy 75-85%. In conclusion, our study successfully identified unique expression patterns associated the progression of early hepatocarcinogenesis and demonstrated that activation of the MYC transcription signature is indispensable for the malignant conversion of pre-neoplastic lesions. Aberrant methylation, consisting of DNA hypomethylation and/or promoter gene CpG hypermethylation, is implicated in the development of a variety of solid tumors, including HCC. We analyzed the global levels of DNA methylation as well as the methylation status of 105 putative tumor suppressor genes and found that the extent of genome-wide hypomethylation and CpG hypermethylation correlates with biological features and clinical outcome of HCC patients.We identified activation of Ras and downstream Ras effectors (ERK, AKT, and RAL) due to epigenetic silencing of inhibitors of the Ras pathway in all HCC. Further,selective inactivation of SPRY1 and -2, DAB2, and SOCS4 and -5 genes and inhibitors of angiogenesis (BNIP3,BNIP3L, IGFBP3, and EGLN2) was associated with poor prognosis. Importantly,several epigenetically silenced putative tumor suppressor genes found in HCC were also inactivated in the nontumorous liver. Our results assign both therapeutic and chemopreventive significance to methylation patterns in human HCC and open the possibility of using molecular targets, including those identified in this study, to effectively inhibit HCC development and progression. Despite rapid growth in gene expression profile data of cancer, identification of few candidate genes for diagnostic markers or therapeutic targets remains a challenge due to the fact that many genes are altered in cancer. Genome-wide screening of genetic alteration has identified many recurrent genomic loci with DNA copy number changes that might harbor potential driver genes for tumorigenesis. However, functional validation of genes reside in these loci is very time-consuming and is impractical when we have hundreds of candidate genomic loci. We have explored genomic correlates of gene expression and copy number and discovered 50 potential driver genes in hepatocellular carcinoma (HCC). Using three independent approaches, we carried out exhaustive search for genes whose expression is best correlated with copy-number alteration and best associated with prognosis of the patients. In addition, unbiased screening of gene expression patterns that are significantly correlated with the 50 driver genes predicted EGFR as one of best therapeutic targets for HCC. Post-translational modification of histones resulting in chromatin remodelling plays a key role in the regulation of gene expression. Also, it is well established that global hypoacetylation of histone H4 is a common hallmark of human tumors and changes in H4 acetylation may occur early in the tumorigenic process. We have recently identified an elevated expression of histone deacetylase 2 (HDAC2) gene in primary human hepatocellular carcinoma (HCC) and HCC derived cell lines. We have now examined the effects of HDAC2 gene knockdown on cell growth and apoptosis in human HCC cell lines. For silencing of HDAC2 gene expression, Huh7 and HepG2 cells were treated with 5 20 nM of three different siRNAs (HDAC2-1, HDAC2-2, and HDAC2-3) directed against HDAC2. Cell growth was then analyzed by MTT and FACS analysis, and apoptosis was estimated by ELISA for detection of ssDNA and caspase activation. The expression of HDAC2 target gene was determined by quantitative real-time RT-PCR. Among the three tested siRNA molecules, the HDAC2-1siRNA was the most effective in inhibiting HCC cell growth when compared to control treatments (untreated and treated with a negative control siRNA). Huh7 and HepG2 cells transfected with 15 nM of HDAC2-1siRNA for 4 days showed about 68% and 71% growth inhibition, respectively, commomly associated accompanying with decreased G2/M cell populations. Inhibition of liver cancer cell growth was due to increased rate of apoptotic cell death which was about 1.9-fold higher than in control cells. The increased level of apoptosis directly correlated with about 65% activation of caspase-3 and about 75% reduction of target mRNA level. Taken together, these results show that HDAC2 is an important regulator of HCC cell growth and survival, and therefore may represent a potential target for human HCC treatment. The variability in the prognosis of individuals with hepatocellular carcinoma (HCC) suggests that HCC may comprise several distinct biological phenotypes. These phenotypes may result from activation of different oncogenic pathways during tumorigenesis and/or from a different cell of origin. We have address whether the transcriptional characteristics of HCC can provide insight into the cellular origin of the tumor. We integrated gene expression data from rat fetal hepatoblasts and adult hepatocytes with HCC from human and mouse models. Individuals with HCC who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis. The gene expression program that distinguished this subtype from other types of HCC included markers of hepatic oval cells, suggesting that HCC of this subtype may arise from hepatic progenitor cells. Analyses of gene networks showed that activation of AP-1 transcription factors in this newly identified HCC subtype might have key roles in tumor development. In this work we have shown that by applying two independent gene expression signatures, we were able to divide individuals with HCC into three subgroups characterized by statistically significant differences in clinical outcome. These findings support the notion that multiple molecular pathways dictate the development and different clinical outcomes of HCC. Our finding also indicates that the molecular features of HCC such as prognostic gene expression signatures are present at the time of diagnosis. Therefore, the use of gene expression profiling promises to improve molecular classification and prediction of outcomes in HCC. Furthermore, molecular stratification of individuals with HCC into homogeneous subgroups may provide opportunities for the development of new treatment modalities