Molecular diagnosis of chronic liver disease and hepatocellular carcinoma: the potential of gene expression profiling.

Gene expression profile analysis through DNA microarrays and other high-throughput technologies permit simultaneous investigation of all genes within a biologic sample, providing a snapshot of the transcriptional state of healthy or diseased tissue. Although most of the current applications are still geared toward research (study of disease mechanisms/signaling pathways involved, identification of novel oncogenes/tumor suppressor genes), clinical diagnostic applications of this approach are beginning to enter more routine molecular usage. There are clear examples where a group of genes, or "signature," can provide clinically useful information (e.g., cancer prognosis and response to treatment). Importantly, the identification of genes that are up-regulated during tumorigenesis is heralding a new era of targeted molecular therapies in oncology. In addition, the high capacity of the technologies available allow for the identification of new biomarkers for early diagnosis. In the future, gene expression profiling ("disease fingerprinting") is likely to complement liver biopsy in the molecular differential diagnosis of chronic liver diseases and hepatocellular carcinoma (HCC). There has already been some success in the identification of subtypes of HCC based on derived sets of signature gene clusters. Data recently reported have been able to provide the first molecular classification of HCC, although more comprehensive studies are required to confirm these results and translate them into the clinical practice. In fact, variations in gene expression in normal and diseased livers, as well technical factors, are obstacles to the routine use of microarray-based methods in the liver clinic. Continued progress is anticipated in the practical application of gene array methods to refine diagnosis and therapy of HCC.

Activation of the canonical Wnt/beta-catenin pathway confers growth advantages in c-Myc/E2F1 transgenic mouse model of liver cancer.

BACKGROUND/AIMS: Previously, we showed that activation of the beta-catenin/Wnt pathway is a dominant event during c-Myc/E2F1 hepatocarcinogenesis. Majority of c-Myc/E2F1 HCCs displayed nuclear accumulation of beta-catenin in the absence of beta-catenin mutations, suggesting that alterations in other members of the Wnt pathway might be responsible for nuclear localization of beta-catenin. Here, we investigated the mechanisms responsible for nuclear translocation of wild-type beta-catenin and addressed the potential contribution of the Wnt pathway in c-Myc/E2F1 hepatocarcinogenesis. METHODS: Status of the members of the Wnt pathway was determined through microsatellite and Western blot analysis. RESULTS: Majority of c-Myc/E2F1 HCCs exhibited multiple abnormalities in the Wnt pathway regardless of the presence of beta-catenin mutations. The observed abnormalities included overexpression of Wnt-1, Frizzled 1 and 2 receptors, Dishevelled-1, downregulation of Secreted frizzled-related protein-1, GSK-3beta inactivation, microsatellite instability at the Axin locus as well as induction of beta-catenin target genes, such as glutamine synthetase, glutamate transporter-1, and Wisp-1. HCCs with beta-catenin activation displayed significantly higher proliferation rate and larger tumor size when compared with beta-catenin negative tumors. CONCLUSIONS: The data demonstrate that multiple abnormalities in the members of the Wnt pathway lead to nuclear accumulation of beta-catenin and suggest that activation of Wnt pathway provides proliferative advantages in c-Myc/E2F1-driven hepatocarcinogenesis.

Intact signaling by transforming growth factor beta is not required for termination of liver regeneration in mice.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF-beta signaling in liver regeneration in vivo, the TGF-beta type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing "floxed" Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver-specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF-beta1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7-fold higher in R2LivKO mice compared with controls. Accelerated S-phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF-beta regulates G(1) to S phase transition of hepatocytes, but intact signaling by TGF-beta is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF-beta pathway is abolished, and may be a principal factor in the termination of liver regeneration.

Fumonisin B1-induced hepatocellular and cholangiocellular tumors in male Fischer 344 rats: potentiating effects of 2-acetylaminofluorene on oval cell proliferation and neoplastic development in a discontinued feeding study.

Fumonisin B1 (FB1) is a naturally occurring mycotoxin produced by Fusarium verticillioides. Dietary exposure to FB1 has been linked to human cancer in certain parts of the world, and treatment with FB1 causes oval cell proliferation and liver tumors in rats. To study the potential role of oval (liver progenitor) cells in the cellular pathogenesis of FB1-induced liver tumors, we gave male F344 rats prolonged treatment with FB1 for 25 weeks, followed by return to control diet until 50 weeks ('stop study'). The time course of FB1-induced liver lesions was followed by examination of serial liver biopsies at set time intervals and post-mortem liver tissue at the end of the study. The effects of different FB1 treatment regimens (5 versus 25 weeks), as well as the modulating effect of 2-acetylaminofluorene (2-AAF), on the kinetics of oval cell proliferation and development of liver tumors were compared. Prolonged treatment with FB1 in normal diet caused persistent oval cell proliferation and generation of both hepatic adenomas and cholangiofibromas (CFs). These liver lesions occurred in the setting of chronic toxic hepatitis and liver fibrosis/cirrhosis, similar to that seen in human hepatocarcinogenesis. Some adenomas and CFs were dysplastic, and one post-mortem liver contained a hepatocellular carcinoma. OV-6+ oval cells were noted in close relation to proliferative neoplastic liver lesions, and some of these lesions expressed OV-6, suggesting that all these cell types were derived from a common progenitor cell. 2-AAF enhanced the size of FB1-induced glutathione S-transferase pi+ hepatocellular lesions and the incidence of CFs in post-mortem liver specimens, but this was not statistically significant. In conclusion, this study supports the involvement of dietary FB1 in liver carcinogenesis in male F344 rats. Oval cells may be the source of both the hepatocellular and cholangiocellular tumors induced by prolonged treatment with FB1. 2-AAF appears to have an enhancing effect on FB1-induced liver tumors, presumably due to its potent inhibitory effects on hepatocyte regeneration.

E2F1 blocks and c-Myc accelerates hepatic ploidy in transgenic mouse models.

Previously, we have shown that over-expression of either E2F1 or c-Myc promotes hepatocarcinogenesis and that E2F1 mice acquire HCC more rapidly than c-Myc transgenic mice. We also found that co-expression of E2F1/c-Myc further accelerates liver cancer development. Here we describe that the deregulated expression of these two transcription factors also affects hepatic ploidy during post-natal liver growth and before the onset of tumors. Oncogenic activity of E2F1 and/or c-Myc was associated with a persistent increase in hepatocyte proliferation. However, E2F1-mediated cell proliferation favored the predominance of diploid cells characteristic of pre-neoplastic type of liver growth whereas c-Myc functioned to accelerate age-related hepatocyte polyploidization. Similarly, proliferative advantage conferred by co-expression of E2F1 and c-Myc increased the frequency of diploid cells at a young age. Thus, the opposing effects of E2F1 and c-Myc on hepatocyte ploidy suggest that these two transcription factors have different mechanisms by which they control liver proliferation/maturation and ultimately, carcinogenesis.