Role of transcriptional and posttranscriptional regulation of methionine adenosyltransferases in liver cancer progression.

UNLABELLED: Down-regulation of the liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and up-regulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Here we found Mat1A:Mat2A switch and low SAM levels, associated with CpG hypermethylation and histone H4 deacetylation of Mat1A promoter, and prevalent CpG hypomethylation and histone H4 acetylation in Mat2A promoter of fast-growing HCC of F344 rats, genetically susceptible to hepatocarcinogenesis. In HCC of genetically resistant BN rats, very low changes in the Mat1A:Mat2A ratio, CpG methylation, and histone H4 acetylation occurred. The highest MAT1A promoter hypermethylation and MAT2A promoter hypomethylation occurred in human HCC with poorer prognosis. Furthermore, levels of AUF1 protein, which destabilizes MAT1A messenger RNA (mRNA), Mat1A-AUF1 ribonucleoprotein, HuR protein, which stabilizes MAT2A mRNA, and Mat2A-HuR ribonucleoprotein sharply increased in F344 and human HCC, and underwent low/no increase in BN HCC. In human HCC, Mat1A:MAT2A expression and MATI/III:MATII activity ratios correlated negatively with cell proliferation and genomic instability, and positively with apoptosis and DNA methylation. Noticeably, the MATI/III:MATII ratio strongly predicted patient survival length. Forced MAT1A overexpression in HepG2 and HuH7 cells led to a rise in the SAM level, decreased cell proliferation, increased apoptosis, down-regulation of Cyclin D1, E2F1, IKK, NF-κB, and antiapoptotic BCL2 and XIAP genes, and up-regulation of BAX and BAK proapoptotic genes. In conclusion, we found for the first time a post-transcriptional regulation of MAT1A and MAT2A by AUF1 and HuR in HCC. Low MATI/III:MATII ratio is a prognostic marker that contributes to determine a phenotype susceptible to HCC and patients' survival. CONCLUSION: Interference with cell cycle progression and I-kappa B kinase (IKK)/nuclear factor kappa B (NF-κB) signaling contributes to the antiproliferative and proapoptotic effect of high SAM levels in HCC.

Activation of v-Myb avian myeloblastosis viral oncogene homolog-like2 (MYBL2)-LIN9 complex contributes to human hepatocarcinogenesis and identifies a subset of hepatocellular carcinoma with mutant p53.

UNLABELLED: Up-regulation of the v-Myb avian myeloblastosis viral oncogene homolog-like2 B-Myb (MYBL2) gene occurs in human hepatocellular carcinoma (HCC) and is associated with faster progression of rodent hepatocarcinogenesis. We evaluated, in distinct human HCC prognostic subtypes (as defined by patient survival length), activation of MYBL2 and MYBL2-related genes, and relationships of p53 status with MYBL2 activity. Highest total and phosphorylated protein levels of MYBL2, E2F1-DP1, inactivated retinoblastoma protein (pRB), and cyclin B1 occurred in HCC with poorer outcome (HCCP), compared to HCC with better outcome (HCCB). In HCCP, highest LIN9-MYBL2 complex (LINC) and lowest inactive LIN9-p130 complex levels occurred. MYBL2 positively correlated with HCC genomic instability, proliferation, and microvessel density, and negatively with apoptosis. Higher MYBL2/LINC activation in HCC with mutated p53 was in contrast with LINC inactivation in HCC harboring wildtype p53. Small interfering RNA (siRNA)-mediated MYBL2/LINC silencing reduced proliferation, induced apoptosis, and DNA damage at similar levels in HCC cell lines, irrespective of p53 status. However, association of MYBL2/LINC silencing with doxorubicin-induced DNA damage caused stronger growth restraint in p53(-/-) Huh7 and Hep3B cells than in p53(+/+) Huh6 and HepG2 cells. Doxorubicin triggered LIN9 dissociation from MYBL2 in p53(+/+) cell lines and increased MYBL2-LIN9 complexes in p53(-/-) cells. Doxorubicin-induced MYBL2 dissociation from LIN9 led to p21(WAF1) up-regulation in p53(+/+) but not in p53(-/-) cell lines. Suppression of p53 or p21(WAF1) genes abolished DNA damage response, enhanced apoptosis, and inhibited growth in doxorubicin-treated cells harboring p53(+/+) . CONCLUSION: We show that MYBL2 activation is crucial for human HCC progression. In particular, our data indicate that MYBL2-LIN9 complex integrity contributes to survival of DNA damaged p53(-/-) cells. Thus, MYBL2 inhibition could represent a valuable adjuvant for treatments against human HCC with mutated p53.

Mybl2 expression is under genetic control and contributes to determine a hepatocellular carcinoma susceptible phenotype.

BACKGROUND & AIMS: MYBL2 is implicated in human malignancies and over expressed in hepatocellular carcinoma (HCC). We investigated Mybl2 role in the acquisition of susceptibility to HCC and tumor progression. METHODS: MYBL2 mRNA and protein levels were evaluated by quantitative RT-PCR and immunoblotting, respectively. MYBL2 expression in HCC cell lines was controlled through MYBL2 cDNA or anti-MYBL2 siRNA transfection. Gene expression profile of cells transfected with MYBL2 was analyzed by microarray. RESULTS: Low induction of Mybl2 and its target Clusterin mRNAs, in low-grade dysplastic nodules (DN), progressively increased in fast growing high-grade DN and HCC of F344 rats, susceptible to hepatocarcinogenesis, whereas no/lower increases occurred in slow growing lesions of resistant BN rats. Highest Mybl2 protein activation, prevalently nuclear, occurred in F344 than BN lesions. Highest Mybl2, Clusterin, Cdc2, and Cyclin B1 expression occurred in fast progressing DN and HCC of E2f1 transgenics, compared to c-Myc transgenics, and anti-Mybl2 siRNA had highest anti-proliferative and apoptogenic effects in cell lines from HCC of E2f1 transgenics. MYBL2 transfected HepG2 and Huh7 cells exhibited increased cell proliferation and G1-S and G2-M cell cycle phases. The opposite occurred when MYBL2 was silenced by specific siRNA. MYBL2 transfection in Huh7 cells led to upregulation of genes involved in signal transduction, cell proliferation, cell motility, and downregulation of oncosuppressor and apoptogenic genes. CONCLUSIONS: mybl2 expression and activation are under genetic control. Mybl2 upregulation induces fast growth and progression of premalignant and malignant liver, through cell cycle deregulation and activation of genes and pathways related to tumor progression.

The degradation of cell cycle regulators by SKP2/CKS1 ubiquitin ligase is genetically controlled in rodent liver cancer and contributes to determine the susceptibility to the disease.

Previous work showed a genetic control of cell cycle deregulation during hepatocarcinogenesis. We now evaluated in preneoplastic lesions, dysplastic nodules and hepatocellular carcinoma (HCC), chemically induced in genetically susceptible F344 and resistant Brown Norway (BN) rats, the role of cell cycle regulating proteins in the determination of a phenotype susceptible to HCC development. p21(WAF1), p27(KIP1), p57(KIP2) and p130 mRNA levels increased in fast growing lesions of F344 rats. Lower/no increases occurred in slowly growing lesions of BN rats. A similar behavior of RassF1A mRNA was previously found in the 2 rat strains. However, p21(WAF1), p27(KIP1), p57(KIP), p130 and RassF1A proteins exhibited no change/low increase in the lesions of F344 rats and consistent rise in dysplastic nodules and HCC of BN rats. Increase in Cks1-Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rat lesions, indicating that posttranslational modifications of cell cycle regulators are under genetic control and contribute to determine a phenotype susceptible to HCC. Moreover, proliferation index of 60 human HCCs was inversely correlated with protein levels but not with mRNA levels of P21(WAF1), P27(KIP1), P57(KIP2) and P130, indicating a control of human HCC proliferation by posttranslational modifications of cell cycle regulators.

SKP2 and CKS1 promote degradation of cell cycle regulators and are associated with hepatocellular carcinoma prognosis.

BACKGROUND & AIMS: The cell cycle regulators P21(WAF1), P27(KIP1), P57(KIP2), P130, RASSF1A, and FOXO1 are down-regulated during hepatocellular carcinoma (HCC) pathogenesis. We investigated the role of the ubiquitin ligase subunits CKS1 and SKP2, which regulate proteasome degradation of cell cycle regulators, in HCC progression. METHODS: Human HCC tissues from patients with better (HCCB, >3 years survival) and poorer prognosis (HCCP, <3 years survival) and HCC cell lines were analyzed. RESULTS: The promoters of P21(WAF1), P27(KIP1), and P57(KIP2) were more frequently hypermethylated in HCCP than HCCB. Messenger RNA levels of these genes were up-regulated in samples in which these genes were not methylated; protein levels increased only in HCCB because of CKS1- and SKP2-dependent ubiquitination of these proteins in HCCP. The level of SKP2 expression correlated with rate of HCC cell proliferation and level of microvascularization of samples and was inversely correlated with apoptosis and survival. In HCCB, SKP2 activity was balanced by degradation by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-CDH1 and up-regulation of SKP2 suppressor histidine triad nucleotide binding protein 1 (HINT1). In HCCP, however, SKP2 was not degraded because of down-regulation of the phosphatase CDC14B, CDK2-dependent serine phosphorylation (which inhibits interaction between CDH1 and SKP2), and HINT1 inactivation. In HCC cells, small interfering RNA knockdown of SKP2 reduced proliferation and ubiquitination of the cell cycle regulators, whereas SKP2 increased proliferation and reduced expression of cell cycle regulators. CONCLUSIONS: Ubiquitination and proteasome degradation of P21WAF1, P27KIP1, P57KIP2, P130, RASSF1A, and FOXO1 and mechanisms that prevent degradation of SKP2 by APC/C-CDH1 contribute to HCC progression. CKS1-SKP2 ligase might be developed as a therapeutic target or diagnostic marker.

Experimental Models to Define the Genetic Predisposition to Liver Cancer.

Hepatocellular carcinoma (HCC) is a frequent human cancer and the most frequent liver tumor. The study of genetic mechanisms of the inherited predisposition to HCC, implicating gene-gene and gene-environment interaction, led to the discovery of multiple gene loci regulating the growth and multiplicity of liver preneoplastic and neoplastic lesions, thus uncovering the action of multiple genes and epistatic interactions in the regulation of the individual susceptibility to HCC. The comparative evaluation of the molecular pathways involved in HCC development in mouse and rat strains differently predisposed to HCC indicates that the genes responsible for HCC susceptibility control the amplification and/or overexpression of c-Myc, the expression of cell cycle regulatory genes, and the activity of Ras/Erk, AKT/mTOR, and of the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways, the methionine cycle, and DNA repair pathways in mice and rats. Comparative functional genetic studies, in rats and mice differently susceptible to HCC, showed that preneoplastic and neoplastic lesions of resistant mouse and rat strains cluster with human HCC with better prognosis, while the lesions of susceptible mouse and rats cluster with HCC with poorer prognosis, confirming the validity of the studies on the influence of the genetic predisposition to hepatocarinogenesis on HCC prognosis in mouse and rat models. Recently, the hydrodynamic gene transfection in mice provided new opportunities for the recognition of genes implicated in the molecular mechanisms involved in HCC pathogenesis and prognosis. This method appears to be highly promising to further study the genetic background of the predisposition to this cancer.

Aberrant iNOS signaling is under genetic control in rodent liver cancer and potentially prognostic for the human disease.

Mounting evidence underlines the role of inducible nitric oxide synthase (iNOS) in hepatocellular carcinoma (HCC) development, but its functional interactions with pathways involved in HCC progression remain uninvestigated. Here, we analyzed in preneoplastic and neoplastic livers from Fisher 344 and Brown Norway rats, possessing different genetic predisposition to HCC, in transforming growth factor-alpha (TGF-alpha) and c-Myc-TGF-alpha transgenic mice, characterized by different susceptibility to HCC, and in human HCC: (i) iNOS function and interactions with nuclear factor-kB (NF-kB) and Ha-RAS/extracellular signal-regulated kinase (ERK) during hepatocarcinogenesis; (ii) influence of genetic predisposition to liver cancer on these pathways and role of these cascades in determining a susceptible or resistant phenotype and (iii) iNOS prognostic value in human HCC. We found progressive iNos induction in rat and mouse liver lesions, always at higher levels in the most aggressive models represented by HCC of rats genetically susceptible to hepatocarcinogenesis and c-Myc-TGF-alpha transgenic mice. iNOS, inhibitor of kB kinase/NF-kB and RAS/ERK upregulation was significantly higher in HCC with poorer prognosis (as defined by patients' survival length) and positively correlated with tumor proliferation, genomic instability and microvascularization and negatively with apoptosis. Suppression of iNOS signaling by aminoguanidine led to decreased HCC growth and NF-kB and RAS/ERK expression and increased apoptosis both in vivo and in vitro. Conversely, block of NF-kB signaling by sulfasalazine or short interfering RNA (siRNA) or ERK signaling by UO126 caused iNOS downregulation in HCC cell lines. These findings indicate that iNOS cross talk with NF-kB and Ha-RAS/ERK cascades influences HCC growth and prognosis, suggesting that key component of iNOS signaling could represent important therapeutic targets for human HCC.

Ras-driven proliferation and apoptosis signaling during rat liver carcinogenesis is under genetic control.

Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1-S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf-1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/Mst1-driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1-dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC.

Inhibition of HSF1 suppresses the growth of hepatocarcinoma cell lines in vitro and AKT-driven hepatocarcinogenesis in mice.

Upregulation of the heat shock transcription factor 1 (HSF1) has been described as a frequent event in many cancer types, but its oncogenic role in hepatocellular carcinoma (HCC) remains poorly delineated. In the present study, we assessed the function(s) of HSF1 in hepatocarcinogenesis via in vitro and in vivo approaches. In particular, we determined the importance of HSF1 on v-Akt murine thymoma viral oncogene homolog (AKT)-induced liver cancer development in mice. We found that knockdown of HSF1 activity via specific siRNA triggered growth restraint by suppressing cell proliferation and inducing massive cell apoptosis in human HCC cell lines. At the molecular level, HSF1 inhibition was accompanied by downregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) cascade and related metabolic pathways. Most importantly, overexpression of a dominant negative form of HSF1 (HSF1dn) in the mouse liver via hydrodynamic gene delivery led to the inhibition of mouse hepatocarcinogenesis driven by overexpression of AKT. In human liver cancer specimens, we detected that HSF1 is progressively induced from human non-tumorous surrounding livers to HCC, reaching the highest expression in the tumors characterized by the poorest outcome (as defined by the length of patients' survival). In conclusion, HSF1 is an independent prognostic factor in liver cancer and might represent an innovative therapeutic target in HCC subsets characterized by activation of the AKT/mTOR pathway.

Deregulated c-Myc requires a functional HSF1 for experimental and human hepatocarcinogenesis.

Deregulated activity of the c-Myc protooncogene is a frequent molecular event underlying mouse and human hepatocarcinogenesis. Nonetheless, the mechanisms sustaining c-Myc oncogenic activity in liver cancer remain scarcely delineated. Recently, we showed that the mammalian target of rapamycin complex 1 (mTORC1) cascade is induced and necessary for c-Myc dependent liver tumor development and progression. Since the heat shock factor 1 (HSF1) transcription factor is a major positive regulator of mTORC1 in the cell, we investigated the functional interaction between HSF1 and c-Myc using in vitro and in vivo approaches. We found that ablation of HSF1 restrains the growth of c-Myc-derived mouse hepatocellular carcinoma (HCC) cell lines, where it induces downregulation of c-Myc levels. Conversely, silencing of c-Myc gene in human and mouse HCC cells led to downregulation of HSF1 expression. Most importantly, overexpression of a dominant negative form of HSF1 (HSF1dn) in the mouse liver via hydrodynamic gene delivery resulted in the complete inhibition of mouse hepatocarcinogenesis driven by overexpression of c-Myc. Altogether, the present results indicate that a functional HSF1 is necessary for c-Myc-driven hepatocarcinogenesis. Consequently, targeting HSF1 might represent a novel and effective therapeutic strategy for the treatment of HCC subsets with activated c-Myc signaling.

Chromosome mapping of multiple loci affecting the genetic predisposition to rat liver carcinogenesis.

Previous studies on (BNxF344)F1 (BFF1) rat model of genetic predisposition to hepatocarcinogenesis led to the identification, in BFF1xF344 backcross progeny, of two hepatocarcinogenesis susceptibility (Hcs) and three resistance (Hcr) loci affecting the progression of neoplastic liver nodules. To evaluate the presence of other hepatocarcinogenesis-related loci in the BFF1 genome, nodule induction by resistant hepatocyte model in 116 male BFF2 rats 32 weeks after initiation with diethylnitrosamine was subjected to quantitative trait loci analysis. The rats were typed with 179 genetic markers, and linkage analysis identified three loci on chromosomes 1, 16, and 6, in significant linkage with nodule mean volume (V), volume fraction, and number, respectively, and two loci on chromosomes 4 and 8 in suggestive linkage with V. These loci were differently positioned with respect to Hcs and Hcr loci mapped previously in backcross rats. On the basis of phenotypic and allele distribution patterns of BFF2 rats, loci on chromosomes 1 and 16 were identified as Hcs3 and Hcs4, and loci on chromosomes 4, 8, and 6 as Hcr4, Hcr5, and Hcr6. Additive interactions occurred between Hcs3 and Hcs4, and Hcr4 and a locus on chromosome 3 with less than suggestive linkage with V. All of the loci were in chromosomal regions syntenic to mouse and/or human chromosomal segments showing allelic gain or loss in hepatocellular carcinomas. These data indicate that inheritance of predisposition to rat liver tumor is characterized by the interplay of several genetic factors and suggest some possible mechanisms of polygenic control of human liver cancer.

Post-translational deregulation of YAP1 is genetically controlled in rat liver cancer and determines the fate and stem-like behavior of the human disease.

Previous studies showed that YAP1 is over-expressed in hepatocellular carcinoma (HCC). Here we observed higher expression of Yap1/Ctgf axis in dysplastic nodules and HCC chemically-induced in F344 rats, genetically susceptible to hepatocarcinogenesis, than in lesions induced in resistant BN rats. In BN rats, highest increase in Yap1-tyr357, p73 phosphorylation and Caspase 3 cleavage occurred. In human HCCs with poorer prognosis (< 3 years survival after partial liver resection, HCCP), levels of YAP1, CTGF, 14-3-3, and TEAD proteins, and YAP1-14-3-3 and YAP1-TEAD complexes were higher than in HCCs with better outcome (> 3 years survival; HCCB). In the latter, higher levels of phosphorylated YAP1-ser127, YAP1-tyr357 and p73, YAP1 ubiquitination, and Caspase 3 cleavage occurred. Expression of stemness markers NANOG, OCT-3/4, and CD133 were highest in HCCP and correlated with YAP1 and YAP1-TEAD levels. In HepG2, Huh7, and Hep3B cells, forced YAP1 over-expression led to stem cell markers expression and increased cell viability, whereas inhibition of YAP1 expression by specific siRNA, or transfection of mutant YAP1 which does not bind to TEAD, induced opposite alterations. These changes were associated, in Huh7 cells transfected with YAP1 or YAP1 siRNA, with stimulation or inhibition of cell migration and invasivity, respectively. Furthermore, transcriptome analysis showed that YAP1 transfection in Huh7 cells induces over-expression of genes involved in tumor stemness. In conclusion, Yap1 post-translational modifications favoring its ubiquitination and apoptosis characterize HCC with better prognosis, whereas conditions favoring the formation of YAP1-TEAD complexes are associated with aggressiveness and acquisition of stemness features by HCC cells.

An expression signature of phenotypic resistance to hepatocellular carcinoma identified by cross-species gene expression analysis.

BACKGROUND AND AIMS: Hepatocarcinogenesis is under polygenic control. We analyzed gene expression patterns of dysplastic liver nodules (DNs) and hepatocellular carcinomas (HCCs) chemically-induced in F344 and BN rats, respectively susceptible and resistant to hepatocarcinogenesis. METHODS: Expression profiles were performed by microarray and validated by quantitative RT-PCR and Western blot. RESULTS: Cluster analysis revealed two distinctive gene expression patterns, the first of which included normal liver of both strains and BN nodules, and the second one F344 nodules and HCC of both strains. We identified a signature predicting DN and HCC progression, characterized by highest expression of oncosuppressors Csmd1, Dmbt1, Dusp1, and Gnmt, in DNs, and Bhmt, Dmbt1, Dusp1, Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13 in HCCs of resistant rats. Integrated gene expression data revealed highest expression of proliferation-related CTGF, c-MYC, and PCNA, and lowest expression of BHMT, DMBT1, DUSP1, GADD45g, and GNMT, in more aggressive rat and human HCC. BHMT, DUSP1, and GADD45g expression predicted patients' survival. CONCLUSIONS: Our results disclose, for the first time, a major role of oncosuppressor genes as effectors of genetic resistance to hepatocarcinogenesis. Comparative functional genomic analysis allowed discovering an evolutionarily conserved gene expression signature discriminating HCC with different propensity to progression in rat and human.

Dual-specificity phosphatase 1 ubiquitination in extracellular signal-regulated kinase-mediated control of growth in human hepatocellular carcinoma.

Sustained activation of extracellular signal-regulated kinase (ERK) has been detected previously in numerous tumors in the absence of RAS-activating mutations. However, the molecular mechanisms responsible for ERK-unrestrained activity independent of RAS mutations remain unknown. Here, we evaluated the effects of the functional interactions of ERK proteins with dual-specificity phosphatase 1 (DUSP1), a specific inhibitor of ERK, and S-phase kinase-associated protein 2 (SKP2)/CDC28 protein kinase 1b (CKS1) ubiquitin ligase complex in human hepatocellular carcinoma (HCC). Levels of DUSP1, as assessed by real-time reverse transcription-PCR and Western blot analysis, were significantly higher in tumors with better prognosis (as defined by the length of patients' survival) when compared with both normal and nontumorous surrounding livers, whereas DUSP1 protein expression sharply declined in all HCC with poorer prognosis. In the latter HCC subtype, DUSP1 inactivation was due to either ERK/SKP2/CKS1-dependent ubiquitination or promoter hypermethylation associated with loss of heterozygosity at the DUSP1 locus. Noticeably, expression levels of DUSP1 inversely correlated with those of activated ERK, as well as with proliferation index and microvessel density, and directly with apoptosis and survival rate. Subsequent functional studies revealed that DUSP1 reactivation led to suppression of ERK, CKS1, and SKP2 activity, inhibition of proliferation and induction of apoptosis in human hepatoma cell lines. Taken together, the present data indicate that ERK achieves unrestrained activity during HCC progression by triggering ubiquitin-mediated proteolysis of its specific inhibitor DUSP1. Thus, DUSP1 may represent a valuable prognostic marker and ERK, CKS1, or SKP2 potential therapeutic targets for human HCC.

Role of HSP90, CDC37, and CRM1 as modulators of P16(INK4A) activity in rat liver carcinogenesis and human liver cancer.

Current evidence indicates that neoplastic nodules induced in liver of Brown Norway (BN) rats genetically resistant to hepatocarcinogenesis are not prone to evolve into hepatocellular carcinoma. We show that BN rats subjected to diethylnitrosamine/2-acetylaminofluorene/partial hepatectomy treatment with a "resistant hepatocyte" protocol displayed higher number of glutathione-S-transferase 7-7(+) hepatocytes when compared with susceptible Fisher 344 (F344) rats, both during and at the end of 2-acetylaminofluorene treatment. However, DNA synthesis declined in BN but not F344 rats after completion of reparative growth. Upregulation of p16(INK4A), Hsp90, and Cdc37 genes; an increase in Cdc37-Cdk4 complexes; and a decrease in p16(INK4A)-Cdk4 complexes occurred in preneoplastic liver, nodules, and hepatocellular carcinoma of F344 rats. These parameters did not change significantly in BN rats. E2f4 was equally expressed in the lesions of both strains, but Crm1 expression and levels of E2f4-Crm1 complex were higher in F344 rats. Marked upregulation of P16(INK4A) was associated with moderate overexpression of HSP90, CDC37, E2F4, and CRM1 in human hepatocellular carcinomas with a better prognosis. In contrast, strong induction of HSP90, CDC37, and E2F4 was paralleled by P16(INK4A) downregulation and high levels of HSP90-CDK4 and CDC37-CDK4 complexes in hepatocellular carcinomas with poorer prognosis. CDC37 downregulation by small interfering RNA inhibited in vitro growth of HepG2 cells. In conclusion, our findings underline the role of Hsp90/Cdc37 and E2f4/Crm1 systems in the acquisition of a susceptible or resistant carcinogenic phenotype. The results also suggest that protection by CDC37 and CRM1 against growth restraint by P16(INK4A) influences the prognosis of human hepatocellular carcinoma.

Chemopreventive N-(4-hydroxyphenyl)retinamide (fenretinide) targets deregulated NF-{kappa}B and Mat1A genes in the early stages of rat liver carcinogenesis.

Cell-cycle deregulation is an early event of hepatocarcinogenesis. We evaluated the role of changes in activity of nuclear factor kappaB (NF-kappaB) and some related pathways in this alteration, and the interference of N-(4-hydroxyphenyl)retinamide (HPR), a retinoid chemopreventive for various cancer types, with these molecular mechanisms and the evolution of preneoplastic liver to cancer. Male F344 rats, initiated according to the 'resistant hepatocyte' model of liver carcinogenesis, received weekly 840 nmol of liposomal HPR (SL-HPR)/100 g body wt or empty liposomes, between 5 and 25 weeks after initiation. Inhibition of DNA synthesis and induction of apoptosis occurred in pre-cancerous lesions, 7-147 days after starting SL-HPR, and a decrease in carcinoma incidence and multiplicity was observed 25 weeks after arresting treatment. An increase in NF-kappaB expression and binding activity, and under-expression of the inhibitor kappaB-alpha (IkappaB-alpha) were found in preneoplastic liver and neoplastic nodules, 5 and 25 weeks after initiation, respectively. These lesions also showed low expression of Mat1A and low activity of methionine adenosyltransferase I/III, whose reaction product, S-adenosyl-l-methionine, enhances IkappaB-alpha expression. SL-HPR prevented these changes and induced a decrease in expression of iNos, c-myc, cyclin D1 and Vegf-A genes, that were over-expressed in preneoplastic liver and nodules, and a decrease in Bcl-2/Bax, Bcl-2/Bad and Bcl-xL/Bax mRNA ratios with respect to the lesions of control rats. Liposomes alone did not influence the parameters tested. These results indicate that signal transduction pathways controlled by NF-kappaB, nitric oxide and S-adenosyl-l-methionine are deregulated in pre-cancerous lesions. Recovery from these alterations by SL-HPR is associated with chemoprevention of hepatocarcinogenesis. Overall, these studies elucidate some molecular changes, in early stages of hepatocarcinogenesis, and underline their pathogenetic role. Moreover, they demonstrate a partially new mechanism of HPR chemopreventive effect and indicate the potential clinical relevance of this compound for prevention of hepatocellular carcinoma.

Cell cycle deregulation in liver lesions of rats with and without genetic predisposition to hepatocarcinogenesis.

Preneoplastic and neoplastic hepatocytes undergo c-Myc up-regulation and overgrowth in rats genetically susceptible to hepatocarcinogenesis, but not in resistant rats. Because c-Myc regulates the pRb-E2F pathway, we evaluated cell cycle gene expression in neoplastic nodules and hepatocellular carcinomas (HCCs), induced by initiation/selection (IS) protocols 40 and 70 weeks after diethylnitrosamine treatment, in susceptible Fisher 344 (F344) rats, and resistant Wistar and Brown Norway (BN) rats. No interstrain differences in gene expression occurred in normal liver. Overexpression of c-myc, Cyclins D1, E, and A, and E2F1 genes, at messenger RNA (mRNA) and protein levels, rise in Cyclin D1-CDK4, Cyclin E-CDK2, and E2F1-DP1 complexes, and pRb hyperphosphorylation occurred in nodules and HCCs of F344 rats. Expression of Cdk4, Cdk2, p16(INK4A), and p27(KIP1) did not change. In nodules and/or HCCs of Wistar and BN rats, low or no increases in c-myc, Cyclins D1, E, and A, and E2F1 expression, and Cyclin-CDKs complex formation were associated with p16(INK4A) overexpression and pRb hypophosphorylation. In conclusion, these results suggest deregulation of G1 and S phases in liver lesions of susceptible rats and block of G1-S transition in lesions of resistant strains, which explains their low progression capacity.

Phenotypic reversion of rat neoplastic liver nodules is under genetic control.

Low DNA synthesis and high redifferentiation (remodeling) characterize neoplastic nodules induced by chemical carcinogens in hybrid BFF1 rats, generated by crossing the susceptible F344 and resistant BN strains. We performed whole-genome scanning of BFF2 rats to identify loci controlling remodeling of nodules induced, 32 weeks after initiation with diethylnitrosamine, by the RH protocol. Remodeling nodules were identified as areas lacking uniformity of GST-P immunostaining and with irregular margins. Two loci in suggestive linkage with the percentage of remodeling nodules were identified on chromosomes 7 and 1 (LOD scores 3.85 and 2.9 at D7Rat25 and D1Mgh14). Significant dosage-negative effect of the B allele on remodeling and additive interaction between these loci were found. Significant epistatic interactions, showing a recessive, remodeling-enhancing effect of B alleles, occurred between D1Mit3 and D11Rat11 (corrected p = 0.0013) and between D6Rat14 and D8Rat46 (corrected p = 0.028). These data show that remodeling of neoplastic nodules during rat hepatocarcinogenesis is under genetic control. Loci affecting remodeling map to chromosomal regions syntenic to chromosomal segments of human HCC showing structural abnormalities.

Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells.

A number of genetic interactions are involved in the control of cell cycle, but their role and nature have not been completely clarified. The knowledge of the behavior of these interactions in hepatocellular carcinoma, could optimize preventive and therapeutic strategies based on cell cycle restraint. We studied downstream events following c-MYC and CYCLIN D1 gene inhibition, by lipoplex-delivered MYC and CYCLIN D1 antisense oligodeoxy nucleotides (aODNM, aODND1), in in vitro cultured human HepG2 and rat Morris 5123 hepatoma cells. 0.5-20 micro M aODN(M) and aODND1 inhibited in vitro growth of both cell types. Scramble oligomer (SCR) and sense ODNs had no or relatively poor effect. Ten micromolar aODNM and aODND1, but not SCR, also induced a significant increase in the apoptotic index of HepG2 and 5123 cells, and inhibited colony formation in soft agar by HepG2 cells. Treatment of the cells with aODNM plus aODND1 had no additive effect on growth and apoptosis. aODNM and aODND1 induced >50% decrease in c-MYC and CYCLIN D1 gene expression, respectively, at both mRNA and protein level. The inhibition of gene expression by aODNs was highly specific, and SCR was without effect. The reduction in c-MYC and CYCLIN D1 expression by aODNs, was associated with a >50% decrease in E2F1 mRNA and protein production, without changes in CYCLIN A and CYCLIN E expression. These results suggest the involvement of both c-MYC and CYCLIN D1 on E2F1 gene function, and indicate that aODNM and aODND1 may inhibit hepatoma cell growth through down-regulation of the E2F1 gene. The inhibition of E2F1 gene expression by E2F1 aODN, was associated with strong growth restraint of HepG2 cells. Thus, interactions of c-MYC and CYCLIN D1 with E2F1 gene are essential for cell cycle activity in hepatoma cells, and their inhibition may have a therapeutic effect.