Jingbo Pan

Genetic mechanisms of hepatocarcinogenesis.

The development of hepatocellular carcinoma (HCC) is a multistep process associated with changes in host gene expression, some of which correlate with the appearance and progression of tumor. Preneoplastic changes in gene expression result from altered DNA methylation, the actions of hepatitis B and C viruses, and point mutations or loss of heterozygosity (LOH) in selected cellular genes. Tumor progression is characterized by LOH involving tumor suppressor genes on many chromosomes and by gene amplification of selected oncogenes. The changes observed in different HCC nodules are often distinct, suggesting heterogeneity on the molecular level. These observations suggest that there are multiple, perhaps redundant negative growth regulatory pathways that protect cells against transformation. An understanding of the molecular pathogenesis of HCC may provide new markers for tumor staging, for assessment of the relative risk of tumor formation, and open new opportunities for therapeutic intervention.

Hepatitis B virus X protein protects against anti-Fas-mediated apoptosis in human liver cells by inducing NF-κB

The hepatitis B virus-encoded X antigen (HBxAg) may contribute to the development of liver cancer, in part, by stimulating the growth and survival of infected cells in the face of ongoing immune responses. Given that the Fas ligand/receptor system contributes to the pathogenesis of chronic hepatitis B, experiments were designed to test the hypothesis that HBxAg mediates resistance of liver cells to anti-Fas killing. Accordingly, when HBxAg was introduced into HepG2 cells, it rendered these cells partially resistant to killing by anti-Fas. In HepG2 cells replicating virus, protection against anti-Fas killing was also observed, but to a lesser extent. Survival correlated with the activation of nuclear factor kappa B (NF-κB) by HBxAg. Sensitivity to anti-Fas was observed in control cells, and was re-established in HepG2X cells stably transfected with the dominant negative inhibitor of NF-κB, IκBα. HBxAg activation of NF-κB was also associated with decreased levels of endogenous IκBα mRNA. Hence, HBxAg stimulation of NF-κB promotes the survival of liver cells against Fas killing. This may contribute to the persistence of infected hepatocytes during chronic infection.

The translation initiation factor, hu-Sui1 may be a target of hepatitis B X antigen in hepatocarcinogenesis.

The role of hepatitis B virus X antigen in the development of hepatocellular carcinoma was explored by stably transfecting HepG2 cells with an X antigen expression vector, and identifying the differences in gene expression that distinguish X positive from X negative cells by subtractive PCR. One differentially expressed gene, the human homolog of sui1 (hu-sui1), encodes a translation initiation factor whose expression was suppressed by X antigen in HepG2 cells. Hu-Sui1 was also expressed in nontumor liver but not in tumor cells from patients with hepatocellular carcinoma. Introduction of hu-sui1 into HepG2 cells inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice. Hence, the suppression of hu-sui1 by X antigen may result in the abrogation of negative growth regulation and contribute to the development of hepatocellular carcinoma.

Early molecular and genetic determinants of primary liver malignancy

Although the overview above provides a partial molecular picture of the early stages of stepwise hepatocarcinogenesis. it should be emphasized that tumor and nontumor liver contain multiple changes, and that there is variability in their profile among different patients even within single studies. Variability in the number and types of genetic changes has also been observed geographically, and may be dependent upon the etiology of the tumor (viral, chemical or both). Interestingly, HBxAg inactivates tumor suppressors (such as p53 [by direct binding] and Rb [by stimulating its phosphorylation]) early in carcinogenesis that are mutated later during tumor progression. HBxAg also constitutively activates signal transduction pathways, such as those involving c-jun and ras, and activates oncogenes,such as c-nloc, that are otherwise activated by 3-catenin mutations. These findings suggest common molecular targets in hepatocarcinogenesis, despite different mechanisms of activation or inactivation. These observations need to be exploited in future drug discovery and in the development of new therapeutics. Heterogeneity in the mechanisms of tumor development, evidenced by the differences in the up- and down regulated genes reported in micro array analyses, as well as in the genetic loci that undergo mutation or LOH indifferent reports, has now been well documented. This suggests that there are multiple pathways to HCC, and that there is redundancy in the pathways that regulate cell growth and survival. These findings also reflect that,although hepatocarcinogenesis is multistep, the molecular changes that underpin histopathological changes in tumor development are likely to be different or only partially overlapping in individual tumors. Overall, the consequences of these changes suggest that the pathogenesis of HCC is accompanied by a progressive loss of differentiation, loss of normal cell adhesion, loss of the ECM, and constitutive activation of selected signal transduction pathways that promote cell growth and survival. Although mechanisms are important, attention also has to be paid to the target genes whose altered expression actually mediate the neoplastic phenotype. Other key avenues of work need to be explored. For example, it will be important to try to identify germline mutations in HBV-infected patients that are passed on to their children, resulting in the development of HCC in childhood. Clinical materials will also be important for the validation of new markers with diagnostic or prognostic potential. In this context, there is an urgent need to establish simple and low-cost tests based upon molecular changes that are hallmarks of HCC development. Identification of patients with early HCC will also significantly increase survival through its impact upon treatment. The discovery and validation of HCC markers may permit accurate staging of lesions, determine the proximity of such lesions to malignancy, and determine whether lesions with a particular genetic profile are still capable of remodeling through appropriate therapeutic intervention. The efficient reintroduction of the relevant tumor suppressors, or the inhibition of oncogene expression by siRNA, provide just some of the additional opportunities that will ultimately be useful in patient treatment. Together, these approaches will go far in reducing the very high morbidity and mortality associated with HCC.

Putative roles of hepatitis B x antigen in the pathogenesis of chronic liver disease

Under most circumstances, hepatitis B virus (HBV) is noncytopathic. However, hepatocellular regeneration that accompanies each bout of hepatitis appears to be associated with increased integration of HBV DNA fragments expressing the virus encoded hepatitis B x antigen (HBxAg). Intrahepatic HBxAg staining correlates with the intensity and progression of chronic liver disease (CLD), and additional work has shown that HBxAg blocks immune mediated killing by Fas and by tumor necrosis factor alpha (TNFalpha). This is not only associated with the blockage of caspase activities by HBxAg, but also by the constitutive stimulation of hepatoprotective pathways, such as nuclear factor kappa B (NF-kappaB), phosphoinositol 3-kinase (PI3K), and beta-catenin (beta-catenin). HBxAg also appears to promote fibrogenesis, by stimulating the production of fibronectin. HBxAg also stimulates the production and activity of transforming growth factor beta1 (TGFbeta1) by several mechanisms, thereby promoting the profibrogenic and tumorigenic properties of this important cytokine. In addition, HBxAg appears to remodel the extracellular matrix (ECM) by altering the expression of several matrix metalloproteinases (MMPs), which may promote tumor metastasis. Hence, HBxAg appears to promote chronic infection by preventing immune mediated apoptosis of infected hepatocytes, by promoting the establishment and persistence of fibrosis and cirrhosis preceding the development of HCC, and by promoting the remodeling of EMC during tumor progression.

Hepatitis C virus replication in stably transfected HepG2 cells promotes hepatocellular growth and tumorigenesis

HepG2 cells stably transfected with a full‐length, infectious hepatitis C virus (HCV) cDNA demonstrated consistent replication of HCV for more than 3 years. Intracellular minus strand HCV RNA was present. Minus strand synthesis was NS5B dependent, and was sensitive to interferon alpha (IFNα) treatment. NS5B and HCV core protein were detectable. HCV stimulated HepG2 cell growth and survival in culture, in soft agar, and accelerated tumor growth in SCID mice. These mice became HCV RNA positive in blood, where the virus was also sensitive to IFNα. The RNA banded at the density of HCV, and was resistant to RNase prior to extraction. Hence, HCV stably replicates in HepG2 cells, stimulates hepatocellular growth and tumorigenesis, and is susceptible to IFNα both in vitro and in vivo.

Mechanisms of HBx Mediated Liver Cancer: Multiple Pathways and Opportunities

Chronic hepatitis B virus (HBV) infection is associated with a high risk for the development of chronic liver diseases (CLDs) which include hepatitis, cirrhosis and hepatocellular carcinoma (HCC). HCC is among the top five most prevalent tumor types worldwide, has few effective treatment options, and is highly lethal. The pathogenesis of CLD and HCC is immune mediated, and the virus has developed a number of defense mechanisms that essentially prevent infected cells from being effectively eliminated by the immune system. This, in part, involves the sustained, high level expression of the virus encoded protein, hepatitis B x antigen (HBx). Recent work has shown that HBx blocks pathways of innate immunity (Kumar et al., 2011; Wei et al., 2010), thereby blunting the development of adaptive immunity that is central to virus elimination. In addition, HBx inhibits immune mediated apoptosis by multiple pathways, including those mediated by Fas and tumor necrosis factor alpha (TNF). In this context, HBx has been shown to up-regulate TNF expression (Lara-Pezzi et al., 1998), which is thought to kill uninfected hepatocytes more readily than infected cells, thereby promoting expansion of the virus within the liver, since virus infected hepatocytes would preferentially regenerate following a bout of chronic hepatitis. HBx also switches the growth signals mediated by elevated transforming growth factor beta 1 (TGF1) from that of negative growth regulation to that of positive growth regulation. TGF1 is a transcriptional target of HBx (Yoo et al., 1996), suggesting that HBx expression in the liver promotes fibrogenesis and the development of cirrhosis. Within the infected hepatocyte, HBx blocks the action of tumor suppressors, such as p53 and Rb (Feitelson et al., 2008), and up-regulates the expression of selected host genes that strongly promote hepatocarcinogenesis even in the absence of HBx (see below). Recent work has also

12 - Immunohistochemical Detection of Host Gene Products Up-Regulated by Hepatitis B Virus X Antigen and Their Putative Roles in Hepatocellular Carcinoma

Chronic hepatitis B virus (HBV) infection is associated with the development of chronic liver diseases (CLDs), including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). A protein encoded by HBV, known as X antigen, or hepatitis B virus X antigen (HB XAg), contributes importantly to the development of HCC. As a transactivating protein, HB XAg contributes to the development of HCC, at least in part by upregulating the expression of host genes that promote cell growth and survival, while it turns off genes that keep cell growth in check. To identity these genes, subtraction hybridization was conducted on RNAs from HB XAg-positive and compared to HB XAg-negative cells in an experiment described in the chapter. The results revealed a list of cellular gene products that were expressed at elevated levels in HB XAg-positive hepatocytes from HBV carriers who developed HCC. These markers consistently showed elevated levels of expression in peritumor hepatocytes but often lower levels in adjacent HCC cells. In vitro characterization of these proteins showed that they stimulated hepatocellular growth and survival as well as promoted tumorigenesis, suggesting that they were early markers of HCC.