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/Tgfαtransgenic 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/Tgfαphenotype 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/Tgfαmice 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]
