Comparative functional genomics approach to identify best-fit models to study hu

人类肝细胞癌（HCC）的临床异质性以及缺乏良好的诊断标志物和治疗策略使该疾病成为一个重大挑战。HCC患者具有高度可变的临床过程，表明HCC包括几个生物学上不同的亚组。我们假设预后的变异性可能反映了肿瘤的分子异质性。在验证这一假设的过程中，我们询问了TGF-β信号通路特异性的基因表达特征是否可以改善HCC患者的诊断和/或预后预测。由于TGF-β表现出肿瘤阶段依赖性抑制（即生长抑制）和致癌（即侵袭性）特性，TGF-β基因表达标记可能含有这些特性的基因组特征，因此与肿瘤的分子分类相关。应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TGF-β基因表达特征成功地区分了HCC的不同亚组。TGF-β阳性簇突出了两个独立的早期（1-2小时）和晚期（4-24小时）TGF-β特征。为了评价TGF-β标记在HCC分子分类中的临床意义，我们比较了早期或晚期TGF-β标记HCC之间几个临床和病理变量的分布。Kaplan-Meier曲线和对数秩统计表明，与早期（60.7和16.1个月）TGF-β特征的患者相比，具有晚期TGF-β特征的患者的平均生存时间（16.2和5.3个月）显着缩短（P < 0.005）。此外，表达晚期TGF-β反应基因的肿瘤表现出侵袭性表型和增加的肿瘤复发。此外，我们证明了TGF-β基因表达特征对HCC以外的肿瘤具有预测价值，因此为基于TGF-β的新型治疗方法开辟了新的途径。肝细胞癌被认为是一种多血管肿瘤，缺氧诱导因子（HIF）及其靶基因的表达已被报道与预后不良表型相关。使用比较基因组学的方法，我们的特点是缺氧基因表达谱在新鲜分离的小鼠肝细胞，目的是使用此配置文件，以探讨肝癌的缺氧反应的重要性。在缺氧条件下培养小鼠肝细胞超过24小时揭示了超过1800个显著（p<0.001）调节基因。在最初的12小时内，对缺氧条件的最重要的反应是参与血管生成、血管形成和血液凝固的基因的上调，而向无氧代谢的转变似乎是24小时后最重要的反应。选择在低氧和常氧条件之间显示至少2倍表达差异的基因来定义低氧基因表达特征。使用来自低氧标记的504个正向同源基因对139例人HCC进行分层聚类分析。结果，鉴定了两个基因子集。涉及细胞周期和凋亡调节（例如Gadd 45 a、Cdk 4、Map 4k 4、Dusp 1、Csk 2）、血液凝固（例如Plg、F2、F9、F13 b、Hc、Agt、Serpin 1、Serpinf 1）和免疫应答（例如C1 r、C8 a、C8 b、C8 g、C9、Rarres 2）以及其他功能的104个基因的一个子集能够预测HCC，预后良好。另一方面，62个基因的子集，其中一些涉及细胞周期进程（如Cdk 6，Ches 1，Ccng 1，Ngfb），凋亡调控（如Bcl 6 b，Pik 3r 1）和血管生成（如Vegf，Id 3），能够预测HCC预后不良。有趣的是，在这62个基因中，我们鉴定了一些已经被发现预测其他人类癌症中的不良预后的基因（Chin等人，PLoS Medicine. 2006年3月; 3（3））。进一步的研究旨在描述低氧条件下调节这两组基因的机制

The clinical heterogeneity of human hepatocellular carcinoma (HCC) and the lack of good diagnostic markers and treatment strategies have rendered the disease a major challenge. Patients with HCC have a highly variable clinical course indicating that HCC comprises several biologically distinctive subgroups. We hypothesized that the prognostic variability likely reflects a molecular heterogeneity of tumors. In the process of testing this hypothesis we have asked if a gene expression signatures specific for TGF-beta signaling pathway could refine the diagnosis and/or prognostic predictions of HCC patients. Since TGF-beta exhibits tumor stage dependent suppressive (i.e. growth inhibition) and oncogenic (i.e. invasiveness) properties, a TGF-beta&#61472;gene expression signature may contain gene sets characteristic for these properties and thus be relevant for the molecular classification of the tumors. Applying a comparative functional genomic approach we demonstrated that temporal TGF-beta&#61472;gene expression signatures established in mouse primary hepatocytes successfully discriminated distinct subgroups of HCC. The TGF-beta positive cluster highlighted two independent, early (1-2 hours) and late (4-24 hours), TGF-beta signatures. To evaluate the clinical significance of TGF-beta signature in the molecular classification of HCC, we then compared the distribution of several clinical and pathological variables between HCC harboring early or late TGF-beta signatures. Kaplan-Meier plots and log-rank statistics indicated that the patients with a late TGF-beta signature showed a significantly (P < 0.005) shortened mean survival time (16.2 5.3 months) compared to the patients with an early (60.7 16.1 months) TGF-beta signature. Also, tumors expressing late TGF-beta&#61472;responsive genes displayed an invasive phenotype and an increased tumor recurrence. Furthermore, we demonstrated that the TGF-beta gene expression signature possess a predictive value for tumors other than HCC and therefore open new avenues for novel TGF-beta based therapeutic approaches. HCC is considered a hypervascular tumor, and expression of the hypoxia inducible factor (HIF) and its target genes has been reported to be associated with a poor prognosis phenotype. Using a comparative genomics approach, we have characterized the hypoxic gene expression profile in freshly isolated mouse hepatocytes, with the aim of using this profile to explore the importance of the hypoxic response in HCC. Culture of mouse hepatocytes under hypoxic conditions over 24 hours revealed more than 1800 significant (p<0.001) regulated genes. In the first 12 hours the most important response to the hypoxic conditions is the upregulation of genes involved in angiogenesis, blood vessel formation and blood coagulation, while the transition to an anaerobic metabolism seems to be the most important response after 24 hours. Genes that showed at least 2 fold expression difference between hypoxic and normoxic conditions were selected to define the hypoxia gene expression signature. 504 orthologous genes derived from the hypoxic signature were used to perform hierarchical cluster analysis of 139 human HCC. As a result, two subsets of genes were identified. One subset of 104 genes implicated in cell cycle and apoptosis regulation (e.g. Gadd45a, Cdk4, Map4k4, Dusp1, Csk2), blood coagulation (e.g. Plg, F2, F9, F13b, Hc, Agt, Serpinc1, Serpinf1) and immune response (e.g. C1r, C8a, C8b, C8g, C9, Rarres2) among other functions, was able to predict HCC with a good prognosis. On the other hand, a subset of 62 genes, some of them involved in cell cycle progression (e.g. Cdk6, Ches1, Ccng1, Ngfb), apoptosis regulation (e.g. Bcl6b, Pik3r1) and angiogenesis (e.g. Vegf, Id3), was able to predict HCC with a poor prognosis. Interestingly, among these 62 genes, we identified some that have already been found to predict poor prognosis in other human cancers (Chin et al. PLoS Medicine. 2006 Mar; 3(3)). Further studies are aimed at characterizing the mechanisms by wich hypoxic conditions regulate these 2 sets of genes