Majority Of Human Hepatocellular Carcinoma Research Articles

Prohibitin 1 in liver injury and cancer.

Prohibitin 1 is an evolutionary conserved and ubiquitously expressed protein that exerts different biological functions depending on its subcellular localization. The role of prohibitin 1 in liver cancer is controversial as it can be pro- or anti-tumorigenic. However, most of the studies to date have described prohibitin 1 primarily as a tumor suppressor in the liver. Its deficiency sensitizes the liver to cholestatic liver injury, non-alcoholic fatty liver disease, inflammatory insults, and cancer. Liver-specific Phb1-knockout mice spontaneously develop hepatocellular carcinoma, Phb1 heterozygotes are more susceptible to develop cholangiocarcinoma, and the majority of human hepatocellular carcinomas and cholangiocarcinomas have reduced prohibitin 1 expression. Consistent with a tumor suppressive role in the liver, prohibitin 1 negatively regulates proliferation in hepatocytes and human hepatocellular carcinoma and cholangiocarcinoma cell lines, and multiple oncogenic signaling pathways are activated when prohibitin 1 is deficient. Although best known as a mitochondrial chaperone, prohibitin 1 can protect the liver by mitochondrial-independent mechanisms. This review summarizes what’s known about prohibitin 1’s role in liver pathology, with the focus on hepatoprotection and carcinogenesis. Impact statement This review summarizes the last decades of research on PHB1 in liver pathobiology. PHB1 is a key player for liver health as it is hepatoprotective and tumor suppressive. We highlight the importance of PHB1’s subcellular localization, post-translational modifications, and interacting proteins as major determinants of PHB1 cytoprotective function and anti-tumor activity in the liver.

MiR-199a-3p inhibits cell proliferation and induces apoptosis by targeting YAP1, suppressing Jagged1-Notch signaling in human hepatocellular carcinoma.

BackgroundmiR-199a-3p was significantly downregulated in the majority of human hepatocellular carcinoma (HCC) tissues and HCC cell lines. Yes associated protein 1 (YAP1) was overexpressed in human HCC, which promoted HCC development and progression by upregulating Jagged1 and activating the Notch pathway. We searched potential targets of miR-199a-3p with DIANA, TargetScan and PicTar tools, and found that YAP1 is one of the potential targets. Based on these findings, we speculated that miR-199a-3p might suppress HCC growth by targeting YAP1, downregulating Jagged1 and suppressing the Notch pathway.ResultsWe determined the expression of miR-199a-3p and YAP1 by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found downregulation of miR-199a-3p and upregulation of YAP1 in HCC cell lines. Cell proliferation and apoptosis assays showed that miR-199a-3p suppresses HCC cell proliferation and promotes apoptosis, and knockdown of YAP1 has similar role. Furthermore, we verified that miR-199a-3p can directly target YAP1. We further investigated and confirmed that miR-199a-3p and YAP1 regulate HCC cell proliferation and apoptosis through Jagged1-Notch signaling.ConclusionmiR-199a-3p targets YAP1, downregulates Jagged1 and suppresses the Notch signaling to inhibit HCC cell proliferation and promote apoptosis. These findings provide new insights into the mechanism by which miR-199a-3p suppresses HCC cell proliferation and induces apoptosis.

Glutaminase 2 negatively regulates the PI3K/AKT signaling and shows tumor suppression activity in human hepatocellular carcinoma.

The tumor suppressor p53 and its signaling pathway play a critical role in tumor prevention. As a direct p53 target gene, the role of glutaminase 2 (GLS2) in tumorigenesis is unclear. In this study, we found that GLS2 expression is significantly decreased in majority of human hepatocellular carcinoma (HCC). Restoration of GLS2 expression in HCC cells inhibits the anchorage-independent growth of cells and reduces the growth of HCC xenograft tumors. Interestingly, we found that GLS2 negatively regulates the PI3K/AKT signaling, which is frequently activated in HCC. Blocking the PI3K/AKT signaling in HCC cells largely abolishes the inhibitory effect of GLS2 on the anchorage-independent cell growth and xenograft tumor growth. The GLS2 promoter is hypermethylated in majority of HCC samples. CpG methylation of GLS2 promoter inhibits GLS2 transcription, whereas reducing the methylation of GLS2 promoter induces GLS2 expression. Taken together, our results demonstrate that GLS2 plays an important role in tumor suppression in HCC, and the negative regulation of PI3K/AKT signaling contributes greatly to this function of GLS2. Furthermore, hypermethylation of GLS2 promoter is an important mechanism contributing to the decreased GLS2 expression in HCC.

MicroRNA‐494 within an oncogenic microRNA megacluster regulates G 1 /S transition in liver tumorigenesis through suppression of mutated in colorectal cancer

Hepatocellular carcinoma (HCC) is associated with poor survival for patients and few effective treatment options, raising the need for novel therapeutic strategies. MicroRNAs (miRNAs) play important roles in tumor development and show deregulated patterns of expression in HCC. Because of the liver’s unique affinity for small nucleic acids, miRNA-based therapy has been proposed in the treatment of liver disease. Thus, there is an urgent need to identify and characterize aberrantly expressed miRNAs in HCC. In our study, we profiled miRNA expression changes in de novo liver tumors driven by MYC and/or RAS, two canonical oncogenes activated in a majority of human HCCs. We identified an up-regulated miRNA megacluster comprised of 53 miRNAs on mouse chromosome 12qF1 (human homolog 14q32). This miRNA megacluster is up-regulated in all three transgenic liver models and in a subset of human HCCs. An unbiased functional analysis of all miRNAs within this cluster was performed. We found that miR-494 is overexpressed in human HCC and aids in transformation by regulating the G1/S cell cycle transition through targeting of the Mutated in Colorectal Cancer tumor suppressor. miR-494 inhibition in human HCC cell lines decreases cellular transformation, and anti-miR-494 treatment of primary MYC-driven liver tumor formation significantly diminishes tumor size. Conclusion: Our findings identify a new therapeutic target (miR-494) for the treatment of HCC. (Hepatology 2014;58:202–215)

The ubiquitin ligase SIAH-1 mediates overexpression of nucleic-acid binding protein FBP-3 and promotes HCC cell proliferation

Background/Aims: Far upstream element (FUSE) binding protein (FBP)-1 and FBP-2 are potent regulators of hepatocellular carcinoma (HCC) cell proliferation and cell motility, respectively. In this study we aimed to analyze the expression, functional relevance, and regulation of FBP-3, another FBP family member in human hepatocarcinogenesis. Methods: Expression of FBP-3 was analyzed in normal livers, dysplastic nodules and HCCs. Functional consequences after RNAi-mediated reduction of FBP-3 on HCC cell proliferation, viability, and migration were analyzed. In order to identify potential upstream regulators of FBP-3, explorative statistical analyses using HCC-protein arrays were performed. Results: A strong nuclear overexpression of FBP-3 was observed in 74% of all analyzed human HCCs which significantly correlated with tumor dedifferentiation, tumor cell proliferation (p<0.001), and poor cumulative survival (p<0.05). FBP-3 predominantly supported proliferation but not cell migration in different HCC cell lines. Based on explorative analyses, nuclear expression of the E3-ubiquitin ligase seven in absentia homologue (SIAH)-1 significantly correlated with FBP-3 expression in dedifferentiated HCCs (p<0.01). Indeed, the knock-down of nuclear SIAH-1 as well as treatment with proteasome inhibitors drastically reduced FBP-3 transcript and protein levels. Because inhibition of SIAH-1 also reduced HCC cell viability, we tested whether exogenous FBP-3 might rescue from SIAH-1-mediated effects. Indeed, vector-based overexpression of FBP-3 partially rescued HCC cells from diminished cell viability after SIAH-1 knock-down. Conclusion: FBP-3 is strongly induced in the majority of human HCCs and represents a potent inducer of HCC cell proliferation. Its accumulation is regulated by proteasome-dependent effector mechanisms comprising nuclear activity of the ubiquitin ligase SIAH-1.

The imprinting status of genetic imprinted gene PEG10 in human hepatocellular carcinomas

To study the imprinting status of genetic imprinted gene PEG10 (perternally expressed gene 10) in human hepatocellular carcinoma (HCC) tissues and liver cancer cell lines. Genomic DNA was extracted from 20 HCC tissues and its adjacent tissues, 15 normal liver tissues, 5 liver cancer cell lines (PLC/PRF/5, smmc-7721, HepG2, Hep3B, SK-HEP-1) and 2 normal human liver cell lines (changliver, HL7702). The DNA fragments containing single nucleotide polymorphism (SNP) site of PEG10 were amplified by polymerase chain reaction (PCR) and the genotype of samples was detected by DNA sequencing. Total RNA was extracted from heterozygous samples, the imprinting status and expression level of PEG10 were evaluated by quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) and DNA sequencing. It was found that 16/40 of HCC and its adjacent tissues were heterozygous, 3/15 of normal liver tissue were heterozygous. A site of heterozygous mutation was found in HepG2 cells by DNA sequencing. The other liver tissues and cell lines were all homozygous. PEG10 was biallelically expressed and showed loss of imprinting (LOI) in 82.4% (14/17) liver cancer samples (16 HCC tissues and HepG2), however it was a monoallelic expression and showed genomic imprinting in17.6% (3/17) liver cancer samples. In HCC, the expression levels of PEG10 were increased apparently, but it was negative expressed in cancer adjacent tissues and normal liver tissues. Expression levels of PEG10 were not significantly different between imprinted HCC tissues and HCC tissues with LOI (t = 1.311, P value is more than 0.05). PEG10 imprinting is lost in a majority of HCC and no correlation exists between the imprinting status of PEG10 and its expressions in HCC tissues.

Abnormal nitrosothiol metabolism in hepatocellular carcinoma

University Clinic for Visceral Medicineand Surgery, Inselspital, University of Bern, SwitzerlandCOMMENTARY ON:S-nitrosylation from GSNOR deficiency impairs DNA repair andpromotes hepatocarcinogenesis. Wei W, Li B, Hanes MA, KakarS, Chen X, Liu L. Sci Transl Med 2010;2(19):19ra13. Copyright(2010). Abstract reprinted with permission from AAAS.http://www.ncbi.nlm.nih.gov/pubmed/20371487Abstract: Human hepatocellular carcinoma (HCC) is associatedwith elevated expressions of inducible nitric oxide synthase (iNOS)but the role of nitric oxide in the pathogenesis of HCC remainsunknown. We found that the abundance and activity of S-nitrosoglu-tathione reductase (GSNOR), a protein critical for control of proteinS-nitrosylation, was significantly decreased in approximately 50%of patients with HCC. GSNOR-deficient mice were very susceptibleto spontaneous and carcinogen-induced HCC. During inflammatoryresponses, the livers of GSNOR-deficient mice exhibited substantialS-nitrosylation and proteasomal degradation of the key DNA repairprotein, O(6)-alkylguanine-DNA alkyltransferase. As a result, repairof carcinogenic O(6)-alkylguanines in GSNOR-deficient mice was sig-nificantly impaired. Predisposition to HCC, S-nitrosylation and deple-tion of alkylguanine-DNA alkyltransferase, and accumulation ofO(6)-alkylguanines were all abolished in mice deficient in bothGSNOR and iNOS. Thus, our data suggest that GSNOR deficiency,through dysregulated S-nitrosylation, may promote HCC, possiblyby inactivating a DNA repair system. 2010 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.Hepatocellular carcinoma is comprised of a heterogeneous groupof tumors [1]. Complex transcriptomic analyses have led to themolecular classification of hepatocellular tumors into severalsubtypes [2–4]. The multiplicity of tumor subtypes renders thedevelopment of treatment strategies especially challengingbecause the molecules that can be targeted by drugs will differaccording to the subtype. In fact, there are few target moleculesactive in the pathogenesis of HCC that are shared among tumorsubtypes. mTOR is one targetable molecule that is shared bymany HCC subtypes; it is inhibited by sirolimus and everolimusand is active in almost half of all HCCs studied [5]. Another inter-esting target molecule is Ras, which phosphorylates Raf, the tar-get of sorafenib, and is active in the majority of HCCs [6].Asapossible addition to this short list, Limin Liu and co-workers haveuncovered a new carcinogenic pathway with therapeuticpotential.S-nitroso-glutathione (GSNO) is a metabolite, which is formedby the nitrosylation of GSH by nitric oxide (NO) in the presence ofoxygen and metabolized by GSNO reductase (GSNOR). GSNO actsas a NO donor, a reservoir of nitrosyl residues capable of formingproteins adducts. Nitrosylation of Cys residues in proteins servesa post-translational regulatory function in many processes,including the inactivation of the DNA repair enzyme, O

Abstract 5009: Analyses of human specimens and knockout mouse model reveal a tumor suppressor function of EphA2 in liver

Abstract EphA2 kinase is highly expressed in normal mammalian liver, but its functions are poorly defined. Here we show that EphA2 expression was down-regulated in majority of human hepatocellular carcinoma (HCC) from three different populations. In mice, EphA2 deletion led to enhanced susceptibility to hepatocarcinogenesis induced by diethylnitrosamine (DEN) in tumor multiplicity and burden compared with wild type mice. Acute DEN treatment induced EphA2 expression upregulation, concomitant with mitotic arrest in wild type liver. EphA2-null liver displayed significantly increased apoptosis compared with wild type. Further, acute DEN treatment caused substantially elevated Akt and ERK1/2 kinase activities in EphA2-null but not in wild type liver. Ephrin-A1 stimulation inhibited AKT and ERK1/2 and suppressed cell proliferation in vitro. Re-expression of EphA2 fully restored its function. Liver development displayed stable EphA2 expression and kinase activation increase by age coinciding with upregulated ephrin-A1. Human HCC and the mouse model show that EphA2 suppresses hepatocarcinogenesis by facilitating mitotic arrest in response to carcinogenic insults; inhibition of Akt and ERK is one possible mechanism involved in tumor progression. Together, these finding provide a mechanistic link between EphA2 and liver tumor development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5009.

Oncogenic Potential of Retinoic Acid Receptor-γ in Hepatocellular Carcinoma

Retinoic acid receptors (RAR; alpha, beta, and gamma), members of the nuclear receptor superfamily, mediate the pleiotropic effects of the vitamin A metabolite retinoic acid (RA) and derivatives (retinoids) in normal and cancer cells. Abnormal expression and function of RARs are often involved in the growth and development of cancer. However, the underlying molecular mechanisms remain largely elusive. Here, we report that levels of RARgamma were significantly elevated in tumor tissues from a majority of human hepatocellular carcinoma (HCC) and in HCC cell lines. Overexpression of RARgamma promoted colony formation by HCC cells in vitro and the growth of HCC xenografts in animals. In HepG2 cells, transfection of RARgamma enhanced, whereas downregulation of RARgamma expression by siRNA approach impaired, the effect of RA on inducing the expression of alpha-fetoprotein, a protein marker of hepatocarcinogenesis. In studying the possible mechanism by which overexpression of RARgamma contributed to liver cancer cell growth and transformation, we observed that RARgamma resided mainly in the cytoplasm of HCC cells, interacting with the p85alpha regulatory subunit of phosphatidylinositol 3-kinase (PI3K). The interaction between RARgamma and p85alpha resulted in activation of Akt and NF-kappaB, critical regulators of the growth and survival of cancer cells. Together, our results show that overexpression of RARgamma plays a role in the growth of HCC cells through nongenomic activation of the PI3K/Akt and NF-kappaB signaling pathways.

Factors of transforming growth factor beta signalling are co-regulated in human hepatocellular carcinoma.

Transforming growth factor beta (TGFbeta) is a central mitoinhibitory factor for epithelial cells, and alterations of TGFbeta signalling have been demonstrated in many different human cancers. We have analysed human hepatocellular carcinomas (HCCs) for potential pro-tumourigenic alterations in regard to expression of Smad4 and mutations and expression changes of the pro-oncogenic transcriptional co-repressors Ski and SnoN, as well as mRNA levels of matrix metalloproteinase-2 (MMP2), which is transcriptionally regulated by TGFbeta. Smad4 mRNA was detected in all HCCs; while, using immunohistology, loss of Smad4 expression was found in 10% of HCCs. Neither mutations in the transformation-relevant sequences nor significant pro-tumourigenic expression changes of the Ski and SnoN genes were detected. In HCC cell lines, expression of both genes was regulated, potentially involving phosphorylation. Ski showed a distinct nuclear speckled pattern, indicating recruitment to active transcription complexes. MMP2 mRNA levels were increased in 19% of HCCs, whereas MMP2 mRNA was not detectable in HCC cell lines, suggesting that MMP2 was derived only from tumour stroma cells. Transcript levels of Smad4, Ski, SnoN and MMP2 correlated well. These data argue against a significant role of Ski and SnoN in human hepatocarcinogenesis and suggest that, in the majority of HCCs, the analysed factors are co-regulated by an upstream mechanism, potentially by TGFbeta itself.

Decreased expression of the CCAAT/enhancer binding protein α gene involved in hepatocyte proliferation in human hepatocellular carcinomas

CCAAT/enhancer binding protein alpha (C/EBPalpha), expressed at a high level in liver, plays an important role in proliferation and differentiation of hepatocytes. Previous studies showed that an expression level of the C/EBPalpha gene in hepatocytes was downregulated in response to proliferation signals and that forced expression of the C/EBPalpha gene in a number of cells caused cell cycle arrest. We compared the expression level of the C/EBPalpha gene in surgical specimens between hepatocellular carcinoma and non-tumorous regions of the same patients. In 9 out of 13 cases, the expression level in the tumors was decreased compared with that in corresponding non-tumorous regions. Transfection of the C/EBPalpha gene into C/EBPalpha-negative human hepatocellular carcinoma HLF cells, however, did not influence the rate of cell proliferation or cell cycle. Our present data suggest that the expression of the C/EBPalpha gene was downregulated in the majority of human hepatocellular carcinoma but the expression may not be directly associated with impaired proliferative activity of hepatocellular carcinoma cells.

Disruption of the p16/Cyclin D1/Retinoblastoma Protein Pathway in the Majority of Human Hepatocellular Carcinomas

p16, cyclin D1 and retinoblastoma protein (pRB) regulate G₁ to S transition and are commonly targeted in various cancers. However, few studies have simultaneously examined all components of the p16/cyclin D1/pRB pathway (RB pathway) in hepatocellular carcinoma (HCC). To clarify the role of the disruption of the RB pathway in HCC, we analyzed p16, pRB and cyclin D1 in 47 HCCs. Inactivation of p16 was detected in 30 of 47 HCCs (64%) by Western blot analysis and significantly correlated with hypermethylation of the promoter of this gene. pRB expression was found to be absent in 13 of 47 HCCs (28%) by immunohistochemistry. We found that 38 of 47 HCCs (81%) contained at least one inactivation in either pRB or p16. Furthermore, there was a significant inverse correlation between p16 and pRB inactivation (p = 0.041). Overexpression of cyclin D1 was detected in 5 of 47 HCCs (11%) by immunohistochemistry. The cases with cyclin D1 overexpression exhibited an advanced clinicopathological appearance and also contained inactivation of pRB and/or p16. These findings suggest that inactivation of pRB and/or p16 is a major event in human hepatocarcinogenesis, while cyclin D1 overexpression may confer additional growth advantages to the tumor in addition to pRB and/or p16 inactivation in HCC.

A t(3;8) chromosomal translocation associated with hepatitis B virus intergration involves the carboxypeptidase N locus.

Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocation present in a hepatocellular carcinoma developed in noncirrhotic liver tissue. One side of the translocation, in 8p23, is shown to be in the vicinity of the carboxypeptidase N gene, a locus that is heavily transcribed in liver tissue and frequently deleted in hepatocellular carcinomas and other epithelial tumors. The other side of the translocation, in 3q27-29, is widely implicated in several types of translocations occurring in different malignancies, such as large-cell lymphomas. The present data strongly support a model in which HBV-induced chromosomal rearrangements play a key role during multistep liver oncogenesis.

HLA expression in human hepatocellular carcinoma.

This study examines the expression of MHC class I and II antigens and their related invariant chains in 70 cases of human hepatocellular carcinoma (HCC), using monoclonal (Mabs) and polyclonal antibodies. In comparison with normal hepatocytes, the majority (94.3%) of HCCs show enhancement or acquisition of HLA-A, B, C in either a cytoplasmic or membranous distribution, with staining being uniformly distributed throughout the specimen. HLA-A, B, C was accompanied by beta 2-microglobulin expression in all but two cases. Although 44.9% of specimens showed HLA-DR expression, positively staining tumour cells were often sparse and heterogeneously distributed. By contrast, the invariant (I) chain, present in 47.1% of cases, was frequently intensively stained and extensive in distribution. HLA-DR staining was usually cytoplasmic although two cases showed faint membranous enhancement. In addition to HLA-DR and I-chain, two cases also showed HLA-DQ staining. Display of MHC antigens was not related to tumour differentiation or size of the lesion (resected vs. advanced tumours). It is possible that the acquisition of class I antigens by the majority of HCCs may influence tumour behaviour.