Zhaorui Lian

Enhanced cell survival of Hep3B cells by the hepatitis B x antigen effector, URG11, is associated with upregulation of β‐catenin

Intrahepatic expression of hepatitis B x antigen (HBxAg) is associated with the development of hepatocellular carcinoma (HCC), perhaps through trans‐activation of selected cellular genes. When this was examined by PowerBlot analysis, upregulated levels of β‐catenin and several known β‐catenin effectors were observed in HBxAg‐positive compared with HBxAg‐negative HepG2 cells. When HBxAg was introduced into Hep3B cells, upregulated expression of wild‐type β‐catenin was observed. This was also observed in Hep3B cells overexpressing the HBxAg upregulated gene, URG11. Upregulated expression of URG11 and β‐catenin correlated with HBxAg trans‐activation function. Transient transfection assays with fragments of the β‐catenin promoter showed that it was activated by both HBxAg and URG11 and inhibited by URG11‐specific small inhibitory RNA. The latter also inhibited the growth of Hep3BX cells in a serum‐free medium, which correlated with depressed levels of β‐catenin. Activation of β‐catenin effector genes was observed in cells stably expressing HBxAg or overexpressing URG11 compared with control cells transfected with the pTOPFLASH reporter plasmid. Extensive costaining between HBxAg, URG11, and β‐catenin was observed in infected liver and HCC nodules, suggesting a close relationship in vivo. In conclusion, wild‐type β‐catenin is activated by HBxAg, in part, through the upregulated expression of the HBxAg effector URG11. URG11 stimulates the β‐catenin promoter and hepatocellular growth and survival. These observations also suggest that URG11 may be a regulatory element in the β‐catenin signaling pathway and may be a target for chemoprevention of HCC. (HEPATOLOGY 2006;43:415–424.)

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.

Upregulated Expression of a Unique Gene by Hepatitis B x Antigen Promotes Hepatocellular Growth and Tumorigenesis

Hepatitis B x antigen (HB x Ag) is a trans-activating protein that may be involved in hepatocarcinogenesis, although few natural effectors of HB x Ag that participate in this process have been identified. To identify additional effectors, whole cell RNA isolated from HB x Ag-positive and HB x Ag-negative HepG2 cells were compared by polymerase chain reaction select cDNA subtraction, and one clone, upregulated gene, clone 11 (URG11), was chosen for further characterization. Elevated levels of URG11 mRNA and protein were observed in HB x Ag-positive compared to HB x Ag-negative HepG2 cells. Costaining was observed in infected liver (P < 0.01). URG11 stimulated cell growth in culture (P < 0.01), anchorage-independent growth in soft agar (P < 0.001), and accelerated tumor formation (P < 0.01), and yielded larger tumors (P < 0.02) in SCID mice injected subcutaneously with HepG2 cells. These data suggest that URG11 is a natural effector of HB x Ag that may promote the development of hepatocellular carcinoma.

Hepatitis B x antigen up-regulates vascular endothelial growth factor receptor 3 in hepatocarcinogenesis

Hepatitis B x antigen (HBxAg) is a trans‐activating protein that contributes to liver cancer, in part, by altering the expression of cellular genes. However, few natural effectors of HBxAg have been identified. Hence, HBxAg positive and negative HepG2 cells were prepared and analyzed by PCR select cDNA subtraction. The results identified elevated vascular endothelial growth factor receptor‐3 short form splice variant (VEGFR‐3S) expression in HBxAg positive compared to negative cells. Normally, VEGFR‐3 activates Akt signaling in lymphatic endothelial cells, resulting in lymphangiogenesis. In contrast, the results here show that the expression of VEGFR‐3S is up‐regulated in >75% of HBxAg positive hepatocellular carcinoma (HCC) nodules. VEGFR‐3S up‐regulation correlates with the expression of HBxAg, is associated with decreased survival in tumor bearing patients, and when over‐expressed in HepG2 cells, strongly stimulated cell growth in culture, in soft agar, and accelerated tumor formation in a ligand independent manner. VEGFR‐3S siRNA partially blocked the ability of HBxAg to promote hepatocellular growth. In conclusion, HBxAg may short circuit VEGFR‐3S signaling in liver cancer. Blocking VEGFR‐3S signaling may be effective in preventing tumor development and/or prolonging survival in tumor bearing patients. (HEPATOLOGY 2007;45:1390–1399.)

URG11 promotes gastric cancer growth and invasion by activation of β‐catenin signalling pathway

Upregulated gene 11 (URG11), a new gene upregulated by Heptatitis B Virus X protein (HBx), was previously shown to activate β‐catenin and promote hepatocellular growth and tumourigenesis. Although the oncogenic role of URG11 in the development of hepatocellular carcinoma has been well documented, its relevance to other human malignancies and the underlying molecular mechanisms remain largely unknown. Here we reported a novel function of URG11 to promote gastric cancer growth and metastasis. URG11 was found to be highly expressed in gastric cancer tissues compared with adjacent nontumourous ones by immunohistochemical staining and western blot. Knockdown of URG11 expression by small interfering RNA (siRNA) effectively attenuated the proliferation, anchorage‐independent growth, invasiveness and metastatic potential of gastric cancer cells. URG11 inhibition led to decreased expression of β‐catenin and its nuclear accumulation in gastric cancer cells and extensive costaining between URG11 and β‐catenin was observed in gastric cancer tissues. Transient transfection assays with the β‐catenin promoter showed that it was inhibited by URG11‐specific small inhibitory RNA. Moreover, suppression of endogenous URG11 expression results in decreased activation of β‐catenin/TCF and its downstream effector genes, cyclinD1 and membrane type 1 matrix metallopeptidase (MT1‐MMP), which are known to be involved in cell proliferation and invasion, respectively. Taken together, our data suggest that URG11 contributes to gastric cancer growth and metastasis at least partially through activation of β‐catenin signalling pathway. These findings also propose a promising target for gene therapy in gastric cancer.

Human S15a expression is upregulated by hepatitis B virus X protein

The hepatitis B virus (HBV)–encoded X antigen (HBxAg) may contribute to the development of hepatocellular carcinoma (HCC) through the upregulated expression of selected cellular genes. To identify these genes, RNAs isolated from HBxAg‐positive and ‐negative HepG2 cells were compared by PCR select cDNA subtraction. One gene overexpressed in HBxAg‐positive cells by Northern and Western blotting is the ribosomal protein S15a. The S15a mRNA is 535 base pairs, encoding a protein 130 amino acids long with a molecular weight of 14.3 kDa. S15a expression was upregulated in HBV‐infected livers, where it costained with HBxAg. Overexpression of S15a stimulated cell growth, colony formation in soft agar, and tumor formation in SCID mice. Hence, HBxAg upregulated the expression of S15a, the latter of which participates in the development of HCC, perhaps by altering the integrity of translation.