Preneoplastic Liver Research Articles

Abstract 176: A characteristic set of microRNAs are deregulated in pre-neoplastic liver exposed to chemical carcinogens

Abstract This study aimed to identify microRNAs (miRNAs) that were deregulated in pre-neoplastic liver exposed to chemical hepatocarcinogens. Expressions of miRNAs are dysregulated in liver diseases, including hepatocellular carcinomas (HCC). However, the hepatic miRNA profiles at the early stages of liver carcinogenesis, which may be important in cancer initiation and progression, have not so far been thoroughly examined. Male rats were dietary exposed to the maximum tolerated dose of eight compounds, six carcinogens and two non-carcinogens, for a period of 90 days. The selected carcinogens were a diverse group including a genotoxic carcinogen, one carcinogen with a cytotoxic mode of action, a PPARa agonist, a CAR activator, and two chemicals with unknown carcinogenic mechanisms. The non carcinogens were one CAR activator and a chemical with no liver effects. MiRNA microarrays were then used to profile the animals’ liver It was found that at 90 days all tested carcinogens resulted in altered expression of liver miRNAs. It is predicted that some miRNAs altered by a given carcinogen will be chemical-specific and irrelevant to the process of carcinogenesis. Conversely, miRNAs that were deregulated by several carcinogens are more likely to be involved in liver carcinogenesis. A set of ten miRNAs that were deregulated by two or more of the tested carcinogens were identified. These included the miR-200a/200b/429 cluster (induced by five carcinogens), miR-34a (induced by four carcinogens), and the miR-182/96 cluster (induced by four carcinogens). The biggest induction of the miR-200a/200b/429 and miR-182/96 clusters was observed in the livers of animals treated with the CAR activator phenobarbital (PB). To uncover the temporal and dose pattern of the deregulation of these clusters their hepatic expression was assessed in animals treated with one carcinogenic and two non-carcinogenic doses of PB for 1,3,7, and 14 days. A progressive pattern of deregulation was found, with both clusters showing the greatest induction at 90 days, a smaller induction at 14 days, and little or no deregulation at earlier timepoints. Moreover, it was found that induction of the miR-200a/200b/429 and miR-182/96 clusters was specific to the carcinogenic dose of PB. Therefore, the effect of PB on these clusters is both time and dose dependent. Induction of miR-200b was confirmed by qPCR and immunoblotting of its predicted target proteins zeb1 and zeb2. In conclusion, the diverse chemical carcinogens tested here result in the deregulation of a characteristic set of miRNAs in pre-neoplastic liver. These characteristic set did not include a number of miRNAs that have previously been reported to be deregulated in later stages of HCC development (e.g. miR-122, miR-21, miR-221 and let-7). It is possible that the miRNAs identified here are involved in the earliest stages of chemical hepatocarcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 176. doi:10.1158/1538-7445.AM2011-176

Indole-3-carbinol enhances oxidative stress responses resulting in the induction of preneoplastic liver cell lesions in partially hepatectomized rats initiated with diethylnitrosamine

The liver tumor-promoting effects of indole-3-carbinol (I3C), a cytochrome P450 (CYP) 1A inducer found in cruciferous vegetables, were investigated using a medium-term hepatocarcinogenesis model in rats. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing 0 (DEN-alone), 0.25, 0.50 or 1.0% of I3C for 8 weeks from 2 weeks after DEN-initiation. The number and area of liver cell foci positive for glutathione S-transferase placental form (GST-P) significantly increased in the livers of rats given 0.5% I3C or more, compared to those in the DEN-alone group. The number of GST-P positive foci also increased in the 0.25% I3C group. The number of liver cells positive for proliferating cell nuclear antigen (PCNA) significantly increased in all I3C groups compared to that in the DEN-alone group. Real-time RT-PCR analysis showed that I3C increased transcript levels of not only Cyp1a1 but also aryl hydrocarbon receptor (AhR) and/or nuclear factor (erythroid-derived 2)-like 2 (Nrf2) gene batteries, such as Cyp1a2, Cyp1b1, Ugt1a6, Nrf2, Nqo1, Gsta5, Gstm2, Ggt1and Gpx2. Reactive oxygen species (ROS) in the microsomal fraction significantly increased in all I3C-treated groups compared to the DEN-alone group, and thiobarbituric acid-reactive substances (TBARS) levels and 8-hydroxy-2′-deoxyguanosine (8-OHdG) content significantly increased in all of the I3C-treated groups and 1.0% I3C group, respectively. These results suggest that I3C is an AhR activator and enhances microsomal ROS production resulting in the upregulation of Nrf2 gene batteries, but the oxidative stress generated overcomes the antioxidant effect of Nrf2-related genes. Such ‘a redox imbalance’ subsequently induces liver tumor-promoting effects by enhancing cellular proliferation in rats.

Activation of the SKP2 protooncogene is a common effector pathway in hepatocarcinogenesis and contributes to malignant transformation of the liver

We have recently shown that upregulation of SKP2 protooncogene is associated with the development and progression of human hepatocellular carcinoma (HCC). In the present study, we further investigated the functional role of SKP2 by using in vitro and in vivo approaches. Forced overexpression of a number of known liver oncogenes, including AKT, E2F1, N-Ras, ERK2, STAT5b, and mutated beta-catenin in human HCC cell lines led to increased proliferation, which was associated with the rise in the levels and activation of SKP2 (as assessed by SKP2-mediated degradation of p27, p57, and p130 target genes). Although the protooncogene FOXM1 has been shown to transcriptionally activate SKP2, induction of SKP2 was mediated by FOXM1 only in N-Ras- and ERK2-overexpressing cells. On the other hand, the growth promoting effects driven by AKT, ERK2, E2F1, N-Ras, STAT5b, and mutated beta-catenin overexpression in vitro were significantly decreased when their overexpression was paralleled by siRNA mediated silencing of SKP2. Forced induction of SKP2 gene via hydrodynamic gene delivery did not induce significant morphologic alterations in the mouse liver, similar to that occurring in mice transfected with the N-Ras gene. However, when SKP2 and N-Ras genes were co-injected via hydrodynamic transfection, preneoplastic liver lesions developed, which finally evolved to HCC 18 weeks after injection. Furthermore, SKP2 accelerated hepatocarcinogenesis induced by the AKT protooncogene when both genes were co-injected in the mouse liver. In conclusion, the in vitro and in vivo data obtained in this study provide solid evidence that SKP2 functions as an oncogene in liver cancer, acting downstream of many signaling pathways involved in human HCC development. Therapeutic approaches aimed at inhibiting SKP2 might be highly beneficial for the treatment of human HCC.

Glycine soya diet synergistically enhances the suppressive effect of tamoxifen and inhibits tamoxifen-promoted hepatocarcinogenesis in 7,12-dimethylbenz[α]anthracene-induced rat mammary tumor model

There is increasing interest in phytoestrogens as potential alternatives to synthetic selective estrogen receptor modulators (SERMs) in the prevention and therapy of breast cancer. The present study is aimed at determining whether dietary glycine soya (Glycine max seeds; GS), which is rich in phytoestrogens, can enhance the anti breast cancer efficacy of the SERM tamoxifen (TAM) and the effect of TAM and GS, either alone or in combination, on DMBA-initiated hepatocarcinogenesis in rat. For determination of enhancing effect, rats bearing palpable 7, 12-dimethylbenz[α] anthracene (DMBA)-induced mammary tumors were treated with TAM (10mgkg−1/day) while being fed AIN-93G diet with or without added GS (3×104mgkg−1), and the tumor growth was monitored up to 5weeks of treatment. For determining the effect on hepatocarcinogenesis, DMBA-initiated rats were exposed to TAM and dietary GS as above for 6weeks during promotion stage in a medium-term bioassay, and the development of placental form of glutathione-S-transferase (GST-P)-expressing preneoplastic liver lesions was quantified. Exposure to both TAM and dietary GS enhanced the anti tumor efficacy of TAM via a combination of tumor cell apoptosis (determined by TUNEL) and inhibition of tumor cell proliferation (determined by PCNA immunostaining) and suppressed the growth of GST-P-positive liver lesions. The findings show that dietary GS enhances the therapeutic efficacy of TAM against mammary tumors and minimizes TAM’s hepatocarcinogenesis promotion potential.

Plk4 is required for cytokinesis and maintenance of chromosomal stability

Aneuploidy is a characteristic feature of established cancers and can promote tumor development. Aneuploidy may arise directly, through unequal distribution of chromosomes into daughter cells, or indirectly, through a tetraploid intermediate. The polo family kinase Plk4/Sak is required for late mitotic progression and is haploinsufficient for tumor suppression in mice. Here we show that loss of heterozygosity (LOH) occurs at the Plk4 locus in 50% of human hepatocellular carcinomas (HCC) and is present even in preneoplastic cirrhotic liver nodules. LOH at Plk4 is associated with reduced Plk4 expression in HCC tumors but not with mutations in the remaining allele. Plk4(+/-) murine embryonic fibroblasts (MEFs) at early passage show a high incidence of multinucleation, supernumerary centrosomes, and a near-tetraploid karyotype. Underlying these phenotypes is a high rate of primary cytokinesis failure, associated with aberrant actomyosin ring formation, reduced RhoA activation, and failure to localize the RhoA guanine nucleotide exchange factor Ect2 to the spindle midbody. We further show that Plk4 normally localizes to the midbody and binds to and phosphorylates Ect2 in vitro. With serial passaging Plk4(+/-) MEFs rapidly immortalize, acquiring an increasing burden of nonclonal and clonal gross chromosomal irregularities, and form tumors in vivo. Our results indicate that haploid levels of Plk4 disrupt RhoGTPase function during cytokinesis, resulting in aneuploidy and tumorigenesis, thus implicating early LOH at Plk4 as one of the drivers of human hepatocellular carcinogenesis. These findings represent an advance in our understanding of genetic predisposition to HCC, which continues to increase in incidence globally and particularly in North America.

DNA oxidative damage of neoplastic rat liver lesions

Hepatic lesions, experimentally-induced in Fisher 344 (F344) and Brown Norway (BN) rats, respectively, susceptible and resistant to liver carcinogenesis, progress differently to hepatocellular carcinoma (HCC). The mechanisms responsible for the acquisition of the resistant phenotype are not completely clear. Herein, we show that in F344 rats subjected to carcinogenic treatment, angiogenesis and DNA oxidation markers increase in preneoplastic and neoplastic liver lesions. On the contrary, in the HCCs of treated BN rats, angiogenesis and a minor DNA oxidation are accompanied by an attempt of tissue remodelling. This study suggests that DNA oxidation might be an important factor in the initiation and promotion of the events of hepatocarcinogenesis. On the other hand, the enhancement of GSH levels and the down-regulation of superoxide dismutase (SOD) expression in both rat strains suggest that antioxidant response is not involved in the acquisition of resistant phenotype.

Tumor promotion by copper-overloading and its enhancement by excess iron accumulation involving oxidative stress responses in the early stage of a rat two-stage hepatocarcinogenesis model

To investigate liver tumor promotion mechanisms of copper (Cu)- and iron (Fe)-overloading, immunolocalization of metal-related biomolecules and lipid peroxidation end products was examined in preneoplastic liver cell foci that expressed glutathione S-transferase placental form (GST-P) in early-stage tumor promotion over 6 weeks in a rat two-stage hepatocarcinogenesis model. Gene expression and concentrations of thiobarbituric acid-reactive substance (TBARS) in the liver were also analyzed. Cu-overloading alone exerted a weak promoting activity, which was enhanced by Fe-overloading. By Cu-overloading, GST-P(+) foci that co-expressed transferrin receptors or downregulated ceruloplasmin increased, suggesting preneoplastic lesion-specific enhancement of oxidative cellular stress. Cu-overloading also increased transcripts of antioxidant enzymes (Gstm3 and Gst Yc2 subunit), cell proliferation, and numbers of single liver cells expressing GST-P or heme oxygenase-1 (HO-1) in the liver, suggesting that oxidative stress induces single-cell toxicity, with the ensuing regeneration contributing to tumor promotion. Fe-overloading increased liver TBARS and HO-1-expressing Kupffer cells, the latter suggesting protection against inflammatory stimuli causing fluctuating proinflammatory cytokine mRNA levels. By co-overloading of Cu and Fe, Cu-overload-related single liver cell toxicity and regeneration increased, as did cytokine imbalances involving increased cyclooxygenase-2-producing Kupffer cells and accumulation of malondialdehyde within GST-P(+) foci. These results suggest an involvement of oxidative stress responses in Cu-induced tumor promotion and Fe-induced enhancement by increasing cytokine imbalances and GST-P(+) foci-specific lipid peroxidation.

The role of epigenetic events in genotoxic hepatocarcinogenesis induced by 2-acetylaminofluorene

It is well established that genotoxic reactivity of chemical carcinogens or their metabolites is a critical event in the initiation of tumorigenesis. However, the underlying mechanisms of events following initiation are less well understood, and with respect to genotoxic liver carcinogenesis, it is largely unknown how the initiated cells progress to form preneoplastic hepatic foci. In the present study, we investigated the underlying events associated with tumor-promoting activity of 2-acetylaminofluorene (2-AAF), a powerful complete genotoxic rat liver carcinogen. Male Sprague–Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 24 weeks, and the status of cytosine DNA methylation, histone methylation, and microRNA expression was determined in the livers of control and 2-AAF-fed rats. The results demonstrate that stages of multistage carcinogenesis following the initiation are driven primarily by carcinogen-induced epigenetic alterations. This was evidenced by altered global histone lysine methylation patterns, increased histone H3 lysine 9 and histone H3 lysine 27 trimethylation in the promoter regions of Rassf1a, p16 INK4a, Socs1, Cdh1, and Cx26 tumor suppressor genes, early Rassf1a and p16 INK4a promoter CpG island hypermethylation, and altered microRNA expression in preneoplastic livers of rats exposed to 2-AAF. These changes were accompanied by dysregulation of the balance between cell proliferation and apoptosis, a fundamental pro-tumorigenic event in hepatocarcinogenesis. These results signify the fundamental role of epigenetic alterations in genotoxic liver carcinogenesis.

Attenuation of the Wnt/β-catenin/TCF pathway by in vivo interferon-α2b (IFN-α2b) treatment in preneoplastic rat livers

Wnt/β-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, promoting cell growth and proliferation. Forkhead box containing protein class O (FOXO) transcription factors compete with TCF for β-catenin binding, particularly under cellular oxidative stress conditions. Contrary to β-catenin/TCF, β-catenin/FOXO promotes the transcription of genes involved in cell cycle arrest and apoptosis. We have previously demonstrated that in vivo interferon-α2b (IFN-α2b) administration induces apoptosis in preneoplastic livers, a mechanism mediated by reactive oxygen species (ROS) and transforming growth factor-β1 (TGF-β1). This study was aimed to assess the status of the Wnt/β-catenin/TCF pathway in a very early stage of rat hepatocarcinogenesis and to further evaluate the effects of in vivo IFN-α2b treatment on it. We demonstrated that the Wnt/β-catenin/TCF pathway is activated in preneoplastic rat livers. More important, in vivo IFN-α2b treatment inhibits Wnt/β-catenin/TCF pathway and promotes programed cell death possibly providing a link with FOXO pathway.

Identifying genes progressively silenced in preneoplastic and neoplastic liver tissues

High-throughput genomic technologies are increasingly being used to identify therapeutic targets and risk factors for specific diseases. Using 116 independent liver samples, we identified 793 probe sets that demonstrated a significant association in the frequency of absent calls as tissues progressed from normal to pre-neoplastic to neoplastic, followed by a bioinformatic approach which identified that 78.9% of the significant probe sets contained at least one CpG island in the gene promoter region compared with 58.9% of the remaining genes examined. Our results indicate that further high-throughput methylation studies to more fully characterize molecular events involved in hepatocarcinogenesis are warranted.

Hepatocellular carcinoma occurring in a Crohn’s disease patient

We report a case of hepatocellular carcinoma (HCC) occurring in a patient with Crohn's disease (CD) without chronic hepatitis or liver cirrhosis, and review the clinicopathological features of HCC in CD patients. A 37-year-old Japanese man with an 8-year history of CD and a medication history of azathioprine underwent resection of a liver tumor. The histopathology of the liver tumor was pseudoglandular type HCC. In the non-neoplastic liver, focal hepatocyte glycogenosis (FHG) was observed, however, there was no evidence of liver cirrhosis or primary sclerosing cholangitis. Only nine cases of HCC in CD patients have been reported previously in the English-language literature. Eight of 10 cases (including the present case) had received azathioprine treatment, and four of these cases also showed FHG, which is considered a preneoplastic liver lesion, within the non-neoplastic liver. Although the precise mechanism of the development of HCC in CD patients is controversial, these results suggest that azathioprine therapy and FHG in the non-neoplastic liver contribute to the development of HCC. These findings also indicate that it is important to survey CD patients treated with prolonged azathioprine therapy for potential liver tumors.

Assessment of the “fish tumors or other deformities” beneficial use impairment in brown bullhead ( Ameiurus nebulosus): II. Liver neoplasia

Liver pathology of fishes, including neoplastic and preneoplastic lesions, is widely used as an indicator of exposure to anthropogenic contaminants. By definition, the “fish tumor or other deformities” beneficial use impairment (BUI) at Great Lakes Areas of Concern (AOC) includes neoplastic and preneoplastic liver lesions in brown bullhead ( Ameiurus nebulosus) or suckers. Unfortunately, adequate guidelines for defining neoplastic and preneoplastic liver lesions or determining rates at unimpacted control sites were not provided and different criteria have been used. In some cases, only neoplastic changes were used to calculate tumor prevalence, in some both neoplastic and preneoplastic changes and in some it is difficult to determine which changes were included. Using standardized criteria, the prevalence of liver neoplasia was compared at eight AOC during 1998–2000. The Cuyahoga River had the highest prevalence (25.0%), while the Maumee River had the lowest (3.9%). The Buffalo (4.8%), Detroit (5.9%), Ashtabula (6.8%), Niagara (7.5%) and Black (8.9%) rivers were intermediate, as was Presque Isle Bay (7.1%). From 2002 to 2007 the prevalence of liver neoplasia at Presque Isle Bay ranged from a low of 2.1% (2002) to a high of 12.0% (2007). Non-AOC sites, as potential reference sites, also were monitored during this time. By combining years and sites, the prevalence of liver neoplasia in bullhead (aged 2 to 12 years) at inland lakes was 0.7%, at bays/harbors was 1.6% and at tributary sites was 4.1%. This is the same trend (inland lakes < bays/harbors < tributaries < Presque Isle Bay) noted for orocutaneous neoplasms.

A historical perspective on the “fish tumors or other deformities” beneficial use impairment at Great Lakes Areas of Concern

The Great Lakes Water Quality Agreement defines Areas of Concern as geographic areas that fail to meet the general or specific objectives of the agreement where such failure has caused or is likely to cause impairment of beneficial use of the area's ability to support aquatic life. One of the beneficial use impairments, fish tumors or other deformities, is defined by the International Joint Commission to occur when the incidence rate of fish tumors and other deformities exceeds rates at unimpacted or control sites, or when survey data confirm the presence of neoplastic or preneoplastic liver tumors in bullhead or suckers. Brown bullhead, a benthic species with a limited home range, have frequently been used as indicator species in U.S. Areas of Concern. While there is strong field evidence for an association between PAH exposure and hepatic neoplasia in brown bullhead, laboratory investigations would strengthen the association. There is less evidence linking specific classes of chemicals in the environment to orocutaneous neoplasia in brown bullhead. Studies on orocutaneous neoplasia of brown bullhead should focus on assessing the presence or absence of viruses and on epidermal exposure to specific chemicals and chemical mixtures. Lastly, the effects of covariates such as length, age, and gender on the prevalence of liver and skin neoplasms should be investigated. This paper reviews the state of science on the fish tumors or other deformities beneficial use impairment. Subsequent papers address specific issues related to this impairment and provide recommendations for standardized criteria.

The Ras inhibitor farnesylthiosalicyclic acid (FTS) prevents nodule formation and development of preneoplastic foci of altered hepatocytes in rats

Background Aberrant activation of oncogenes, such as Ras, likely contributes to the development of hepatocarcinoma (HCC). Aims/methods We evaluated in vivo the effect of intraperitoneal injections of the Ras inhibitor S-trans, trans-farnesylthiosalicyclic acid (FTS) on Ras activation and the development of preneoplastic liver lesions in rats receiving weekly diethylnitrosamine (DEN) injections for 16 weeks. Western blotting, quantitative PCR, immunohistochemistry, Tunel and caspase activity assays were used. Results FTS prevents liver nodule formation and reduces foci expressing the tumour marker GSTp. FTS abrogates DEN-induced Ras membrane activity, increases Tunel positive cells in transformed, GSTp-expressing hepatocytes, up-regulates caspase 3 and 8 activity, induces Fas, Fas ligand and JNK phosphorylation that occurs independently of TNFα and Trail. Cytochrome C release, Bax, Bcl2, Bcl-xl, Ki67 and nuclear cyclin D expression is not affected by FTS. Conclusions FTS inhibits Ras activation and prevents preneoplastic liver nodule development by inducing apoptosis in transformed hepatocytes through activation of the Fas/Fas ligand system. FTS might be new molecule for HCC treatment.

Utility of glypican‐3 and survivin in differentiating hepatocellular carcinoma from benign and preneoplastic hepatic lesions and metastatic carcinomas in liver fine‐needle aspiration biopsies

Glypican-3 (GPC-3), a membrane-anchored heparin sulfate proteoglycan, has been shown to be expressed in approximately 80% of hepatocellular carcinoma (HCC) but not in benign hepatic lesions. Survivin, a novel inhibitor of apoptosis, and a prognostic marker, has also been expressed in HCC. We evaluated these two immunomarkers (GPC-3 and survivin) in differentiating HCC from benign and preneoplastic hepatic lesions and metastatic carcinomas, comparing them to HepPar-1 (hepatocyte paraffin-1) in liver fine-needle aspiration biopsies (FNAB).Immunohistochemistry for GPC-3, survivin and HepPar-1 was performed on 92 FNAB including HCC, hepatic cirrhosis, focal nodular hyperplasia (FNH), hepatic adenoma, dysplastic hepatic nodules and metastatic carcinomas. Immunostaining was scored as positive, if > or =10% of tumor cells stained.GPC-3 is immunoexpressed in 56.8% of HCC, but not in benign and preneoplastic hepatic lesions, or metastatic carcinomas; whereas survivin is expressed in HCC (86.4%), benign hepatic lesions (85.7%), dysplastic hepatic nodules (100%) and metastatic carcinomas (94.3%). HepPar-1 is immunoexpressed in HCC (72.7%), benign hepatic lesions (100%), dysplastic nodules (100%) and metastatic carcinomas (2.9%). The sensitivity and specificity of GPC-3, survivin and HepPar-1 for detection of HCC are 56.8 and 100%, 86.4 and 6.3%, 72.7 and 70.8%, respectively.GPC-3 is a reliable and more specific immunohistochemical marker than survivin for the diagnosis of HCC in FNAB. HepPar-1, although a more sensitive marker than GPC-3, has a lower specificity for detection of HCC. Our data supports the potentially significant diagnostic utility of GPC-3 in FNABs in differentiating primary malignant from benign and preneoplastic liver lesions, and metastatic carcinomas.

Recurrent and nonrandom DNA copy number and chromosome alterations in Myc transgenic mouse model for hepatocellular carcinogenesis: implications for human disease

Mouse models for hepatocellular carcinoma (HCC) provide an experimental ground for dissecting the genetic and biological complexities of human liver cancer and contribute to our ability to gain insights into the relevance of candidate cancer genes. We examined, using spectral karyotyping (SKY) and array-based CGH (aCGH), seven cell lines derived from HCC spontaneously developed in transgenic Myc mice (Myc), and four cell lines established from tumors induced in nude mice by inoculation with the original Myc cells (nuMyc). All the cell lines exhibited gain of material from chromosomes 5, 6, 8, 10, 11, 15, and 19 and DNA copy-number loss from chromosomes 2, 4, 7, 9, 12, 14, and X. In addition, several recurrent chromosome reorganizations were found, including del(3), t(3;8), del(4), t(4;11), t(6;5), del(7), del(8), del(9), t(10;14), del(11), and del(16). Chromosome breakpoints underlying rearrangements clustered in the regions previously identified as important for the early stages of Myc-induced hepatocarcinogenesis. The results strongly suggest the importance of recurrent breakage and loss of chromosomes 4, 9, and 14 and gain of chromosomes 15 and 19 in mouse liver neoplasia. Genomic changes observed in Myc HCC cell lines are also recurrent in HCC developed in other transgenic mouse models, in mouse spontaneous HCC and derivative cell lines, and in preneoplastic liver lesions induced with chemical carcinogens. Overall, the present results document selective, nonrandom genomic changes involving chromosomal regions homologous to those implicated in human HCC.

Maté attenuates DNA damage and carcinogenesis induced by diethylnitrosamine and thermal injury in rat esophagus

Drinking hot maté has been associated with risk for esophageal cancer in South America. Thus, the aims of this study were to evaluate the modifying effects of maté intake on DNA damage and esophageal carcinogenesis induced by diethylnitrosamine (DEN) and thermal injury (TI) in male Wistar rats. At the initiation phase of carcinogenesis, rats were treated with DEN (8 × 80 mg/kg) and submitted to TI (water at 65 °C, 1 ml/rat, instilled into the esophagus). Concomitantly, the animals received maté (2.0% w/v) for 8 weeks. Samples of peripheral blood were collected 4 h after the last DEN application for DNA damage analysis. At weeks 8 and 20, samples from esophagus and liver were also collected for histological and immunohistochemical analysis. Maté significantly decreased DNA damage in leukocytes, cell proliferation rates in both esophagus and liver and the number of preneoplastic liver lesions from DEN/TI-treated animals at week 8. A significant lower incidence of esophageal papillomas and liver adenomas and tumor multiplicity was observed in the animals previously treated with maté at week 20. Thus, maté presented protective effects against DNA damage and esophageal and liver carcinogenesis induced by DEN.

Central Role of c-Myc during Malignant Conversion in Human Hepatocarcinogenesis

Hepatocarcinogenesis is a multistage process in which precursor lesions progress into early hepatocellular carcinomas (eHCC) by sequential accumulation of multiple genetic and epigenetic alterations. To decode the molecular events during early stages of liver carcinogenesis, we performed gene expression profiling on cirrhotic (regenerative) and dysplastic nodules (DN), as well as eHCC. Although considerable heterogeneity was observed at the regenerative and dysplastic stages, overall, 460 differentially expressed genes were detected between DN and eHCC. Functional analysis of the significant gene set identified the MYC oncogene as a plausible driver gene for malignant conversion of the DNs. In addition, gene set enrichment analysis revealed global activation of the MYC up-regulated gene set in eHCC versus dysplasia. Presence of the MYC signature significantly correlated with increased expression of CSN5, as well as with higher overall transcription rate of genes located in the 8q chromosome region. Furthermore, a classifier constructed from MYC target genes could robustly discriminate eHCC from high-grade and low-grade DNs. In conclusion, our study identified unique expression patterns associated with the transition of high-grade DNs into eHCC and showed that activation of the MYC transcription signature is strongly associated with the malignant conversion of preneoplastic liver lesions.

Cross-talk between IFN-α and TGF-β1 signaling pathways in preneoplastic rat liver

Interferon-γ/transforming growth factor-β (IFN-γ/TGF-β) pathways have opposite effects on diverse cellular functions. However, little is known about interactions between IFN-α/TGF-β. In previous studies, we showed that IFN-α2b increases TGF-β1 production and secretion in hepatocytes from preneoplastic rat livers. Here, the interaction between IFN-α/TGF-β1 pathways was explored. We observed a positive cross-talk between IFN-α and TGF-β1 signaling, with activation of both pathways. p300 protein levels in hepatocytes from preneoplastic livers were enough to interact with both activated Stat1 and Smad2/3. Besides, Smad7 was not directly related with TGF-β1 and IFN-α signals. Interestingly, we reported the novel finding that the autocrine TGF-β1 up-regulates TGF-βRII at protein and mRNA levels. In conclusion, the intracellular signals triggered by IFN-α2b and by autocrine TGF-β1 are integrated at the nuclear level, where activated Stat1 and Smad2/3 are capable of interact with p300, present in no restrictive cellular amounts.

Interferon-α2b (IFN-α2b)-induced apoptosis is mediated by p38 MAPK in hepatocytes from rat preneoplastic liver via activation of NADPH oxidase

It is still unclear how Interferon-alfa (IFN-α) acts on preventing the appearance of hepatocarcinogenesis. We have demonstrated that IFN-α2b induces hepatocytic transforming growth factor-beta1 (TGF-β1) production and secretion by inducing reactive oxygen species (ROS) formation through the activation of NADPH oxidase. This TGF-β1, alters antioxidant defences and induces programmed cell death. Since it was demonstrated that IFN-α induces apoptosis through the activation of p38 mitogen-activated protein kinase (p38 MAPK), this study was aimed to assess the role of this kinase in the IFN-α2b-induced apoptosis in rat liver preneoplasia; and to further evaluate the participation of NADPH oxidase. p38 MAPK pathway was activated during the IFN-α2b-induced apoptosis in rat liver preneoplasia. This activation was accompanied with phosphorylation of different transcription factors, depending on the time of IFN-α2b stimulus. Our data suggest that NADPH oxidase is activated by IFN-α2b through p38 MAPK. p38 MAPK-induced activation of NADPH oxidase is accomplished by a two-step pathway: first, ROS-independent and second ROS- and TGF-β1-dependent.