Xin W. Wang

TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the major risk factors include chronic infections with the hepatitis B (HBV) or C (HCV) virus, and exposure to dietary aflatoxin B1 (AFB1) or alcohol consumption. Multiple genetic and epigenetic changes are involved in the molecular pathogenesis of HCC, for example, somatic mutations in the p53 tumor suppressor gene (TP53) and the activation of the WNT signal transduction pathway. AFB1 frequently induces G:C to T:A transversions at the third base in codon 249 of TP53 and cooperates with HBV in causing p53 mutations in HCC. The detection of TP53 mutant DNA in plasma is a biomarker of both AFB1 exposure and HCC risk. Chronic infection with HBV and HCV viruses, and oxyradical disorders including hemochromatosis, also generate reactive oxygen/nitrogen species that can both damage DNA and mutate cancer-related genes such as TP53. Certain mutant p53 proteins may exhibit a ‘gain of oncogenic function’. The p53 biological network is a key responder to this oxidative and nitrosative stress. Depending on the extent of the DNA damage, p53 regulates the transcription of protective antioxidant genes and with extensive DNA damage, transactivates pro-oxidant genes that contribute to apoptosis. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC and the integrated HBx is frequently mutated. Mutant HBx proteins still retain their ability to bind to p53, and attenuate DNA repair and p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology of TP53 mutations during the molecular pathogenesis of HCC.

DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53

The p33ING1 protein is a regulator of cell cycle, senescence, and apoptosis. Three alternatively spliced transcripts of p33ING1 encode p47ING1a, p33ING1b, and p24ING1c. We cloned an additional ING family member, p33ING2/ING1L. Unlike p33ING1b, p33ING2 is induced by the DNA-damaging agents etoposide and neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell growth and survival in a p53-dependent manner through induction of G1-phase cell-cycle arrest and apoptosis. p33ING2 strongly enhances the transcriptional-transactivation activity of p53. Furthermore, p33ING2 expression increases the acetylation of p53 at Lys-382. Taken together, p33ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation.

Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells.

Prostaglandin A2 (PGA2) suppresses tumor growth in vivo, is potently antiproliferative in vitro, and is a model drug for the study of the mammalian stress response. Our previous studies using breast carcinoma MCF-7 cells suggested that p21(Waf1/Cip1) induction enabled cells to survive PGA2 exposure. Indeed, the marked sensitivity of human colorectal carcinoma RKO cells to the cytotoxicity of PGA2 is known to be associated with a lack of a PGA2-mediated increase in p21(Waf1/Cip1) expression, inhibition of cyclin-dependent kinase activity, and growth arrest. To determine if cell death following exposure to PGA2 could be prevented by forcing the expression of p21(Waf1/Cip1) in RKO cells, we utilized an adenoviral vector-based expression system. We demonstrate that ectopic expression of p21(Waf1/Cip1) largely rescued RKO cells from PGA2-induced apoptotic cell death, directly implicating p21(Waf1/Cip1) as a determinant of the cellular outcome (survival versus death) following exposure to PGA2. To discern whether p21(Waf1/Cip1)-mediated protection operates through the implementation of cellular growth arrest, other growth-inhibitory treatments were studied for the ability to attenuate PGA2-induced cell death. Neither serum depletion nor suramin (a growth factor receptor antagonist) protected RKO cells against PGA2 cytotoxicity, and neither induced p21(Waf1/Cip1) expression. Mimosine, however, enhanced p21(Waf1/Cip1) expression, completely inhibited RKO cell proliferation, and exerted marked protection against a subsequent PGA2 challenge. Taken together, our results directly demonstrate a protective role for p21(Waf1/Cip1) during PGA2 cellular stress and provide strong evidence that the implementation of cellular growth arrest contributes to this protective influence.

Molecular pathogenesis of human hepatocellular carcinoma.

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, the viral-chemical etiology as well as molecular mechanisms of HCC pathogenesis remains largely unknown. Recent studies in our laboratory have identified several potential factors that may contribute to the pathogenesis of HCC. Oxidative stress and chronic inflammation have been linked to an increased risk of liver cancer. For example, oxyradical overload diseases such as Wilson disease and hemochromatosis result in the generation of oxygen/nitrogen species that can cause mutations in the p53 tumor suppressor gene. The Hepatitis B virus X gene (HBx), a viral transactivator with oncogenic potentials, has been shown to bind to and inactivate p53-mediated apoptosis. HBx mutants derived from HCC have a diminished ability to act as a transactivator. However, they still retain the ability to bind to and abrogate p53-mediated apoptosis. The comparison of gene expression profiles between HBx-expressing primary human hepatocytes and HBV-infected liver samples by cDNA microarrays indicate a unique alteration of a subset of oncogenes and tumor suppressor genes including p53. Our studies implicate both viral and endogenous chemical processes in the etiology of HCC, and p53 may be a common target for the inactivation during liver carcinogenesis.

Involvement of Crm1 in hepatitis B virus X protein-induced aberrant centriole replication and abnormal mitotic spindles.

ABSTRACT Hepatitis B virus (HBV) includes an X gene (HBx gene) that plays a critical role in liver carcinogenesis. Because centrosome abnormalities are associated with genomic instability in most human cancer cells, we examined the effect of HBx on centrosomes. We found that HBx induced supernumerary centrosomes and multipolar spindles. This effect was independent of mutations in the p21 gene. Furthermore, the ability of HBV to induce supernumerary centrosomes was dependent on the presence of physiological HBx expression. We recently showed that HBx induces cytoplasmic sequestration of Crm1, a nuclear export receptor that binds to Ran GTPase, thereby inducing nuclear localization of NF-κB. Consistently, supernumerary centrosomes were observed in cells treated with a Crm1-specific inhibitor but not with an HBx mutant that lacked the ability to sequester Crm1 in the cytoplasm. Moreover, a fraction of Crm1 was found to be localized at the centrosomes. Immunocytochemical and ultrastructural examination of these supernumerary centrosomes revealed that inactivation of Crm1 was associated with abnormal centrioles. The presence of more than two centrosomes led to an increased frequency of defective mitoses and chromosome transmission errors. Based on this evidence, we suggest that Crm1 is actively involved in maintaining centrosome integrity and that HBx disrupts this process by inactivating Crm1 and thus contributes to HBV-mediated carcinogenesis.

Defective Interplay of Activators and Repressors with TFIIH in Xeroderma Pigmentosum

Inherited mutations of the TFIIH helicase subunits xeroderma pigmentosum (XP) B or XPD yield overlapping DNA repair and transcription syndromes. The high risk of cancer in these patients is not fully explained by the repair defect. The transcription defect is subtle and has proven more difficult to evaluate. Here, XPB and XPD mutations are shown to block transcription activation by the FUSE Binding Protein (FBP), a regulator of c-myc expression, and repression by the FBP Interacting Repressor (FIR). Through TFIIH, FBP facilitates transcription until promoter escape, whereas after initiation, FIR uses TFIIH to delay promoter escape. Mutations in TFIIH that impair regulation by FBP and FIR affect proper regulation of c-myc expression and have implications in the development of malignancy.

p53 tumor-suppressor gene: Clues to molecular carcinogenesis

This article is a US Government work and, as such, is in the public domain in the United States of America.

The clinical potential of microRNAs

MicroRNAs are small noncoding RNAs that function to control gene expression. These small RNAs have been shown to contribute to the control of cell growth, differentiation and apoptosis, important features related to cancer development and progression. In fact, recent studies have shown the utility of microRNAs as cancer-related biomarkers. This is due to the finding that microRNAs display altered expression profiles in cancers versus normal tissue. In addition, microRNAs have been associated with cancer progression. In this review, the mechanisms to alter microRNA expression and their relation to cancer will be addressed. Moreover, the potential application of microRNAs in clinical settings will also be highlighted. Finally, the challenges regarding the translation of research involving microRNAs to the clinical realm will be discussed.

Hepatitis B virus X mutants derived from human hepatocellular carcinoma retain the ability to abrogate p53-induced apoptosis

Chronic hepatitis B virus (HBV) infection and the integration of its X gene (HBx) are closely associated with the development of hepatocellular carcinoma (HCC). The integrated HBx frequently is truncated or contains point mutations. Previous studies indicated that these HBx mutants have a diminished co-transactivational activity. We have compared the effects of wild-type (wt) HBx and its naturally occurring mutants derived from human HCCs on transcriptional co-transactivation, apoptosis and interactive effects with p53. We demonstrated that overexpression of mutant, but not wt HBx, is defective in transcriptional co-transactivation of the NF-κB-driven luciferase reporter. By using a microinjection technique, the HBx mutants were shown to have an attenuated pro-apoptotic activity. This deficiency may be attributed to multiple mutations in the co-transactivation domain of HBx, that leads to decreased stability of the translated product. However, wt or mutant HBx bind to p53 in vitro and retain their ability to block p53-mediated apoptosis in vivo, which has been implicated as its major tumor suppressor function. The abrogation of p53-mediated apoptosis by integrated HBx mutants may provide a selective clonal advantage for preneoplastic or neoplastic hepatocytes and contribute to hepatocellular carcinogenesis.

Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a malignant cancer with an observable heterogeneity and microRNAs are functionally associated with the tumorigenesis of HCC. We recently identified that EpCAM (CD326)-positive cells isolated from alpha-fetoprotein (AFP)-positive HCC samples are hepatic cancer stem cells (HepCSCs). EpCAM+AFP+ HepCSCs have an activated Wnt/β-catenin signaling with a parallel increased expression of all four microRNA-181 family members. We hypothesized that Wnt/β-catenin signaling transcriptionally activates microRNA-181s in HCC.ResultsUsing both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines. MicroRNA-181 expression could be directly induced upon an activation of Wnt/β-catenin signaling, which includes Wnt10B overexpression, inhibition of GSK3β signaling by LiCl, or forced expression of β-catenin/Tcf4. Moreover, microRNA-181 expression was inhibited upon an inactivation of Wnt/β-catenin signaling by an induction of adenomatosis polyposis coli (APC) expression or silencing β-catenin via RNA interference. In addition, seven putative β-catenin/Tcf4 binding sites were identified in the promoter region of the microRNA-181a-2 and microRNA-181b-2 transcripts. Consistently, we found that Tcf4 interacted with these regions in vivo using chromatin immunoprecipitation assay.ConclusionsTaken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.