Splicing regulator SLU7 preserves survival of hepatocellular carcinoma cells and other solid tumors via oncogenic miR-17-92 cluster expression.

Resisting death is a central hallmark of cancer cells. Tumors rely on a number of genetic mechanisms to avoid apoptosis, and alterations in mRNA alternative splicing are increasingly recognized to have a role in tumorigenesis. In this study, we identify the splicing regulator SLU7 as an essential factor for the preservation of hepatocellular carcinoma (HCC) cells viability. Compared with hepatocytes, SLU7 expression is reduced in HCC cells; however, further SLU7 depletion triggered autophagy-related cellular apoptosis in association with the overproduction of reactive oxygen species. Remarkably, these responses were not observed in primary human hepatocytes or in the well-differentiated HepaRG cell line. Mechanistically, we demonstrate that SLU7 binds the C13orf25 primary transcript in which the polycistronic oncomir miR-17-92 cluster is encompassed, and is necessary for its processing and expression. SLU7 knockdown altered the splicing of the C13orf25 primary transcript, and markedly reduced the expression of its miR-17, miR-20 and miR-92a constituents. This led to the upregulation of CDKN1A (P21) and BCL2L11 (BIM) expression, two bona fide targets of the miR-17-92 cluster and recognized mediators of its pro-survival and tumorigenic activity. Interestingly, altered splicing of miR-17-92 and downregulation of miR-17 and miR-20 were not observed upon SLU7 knockdown in non-transformed hepatocytes, but was found in other (HeLa, H358) but not in all (Caco2) non-hepatic tumor cells. The functional relevance of miR-17-92 dysregulation upon SLU7 knockdown was established when oxidative stress, autophagy and apoptosis were reversed by co-transfection of HCC cells with a miR-17 mimic. Together, these findings indicate that SLU7 is co-opted by HCC cells and other tumor cell types to maintain survival, and identify this splicing regulator as a new determinant for the expression of the oncogenic miR-17-92 cluster. This novel mechanism may be exploited for the development of antitumoral strategies in cancers displaying such SLU7-miR-17-92 crosstalk.

Inflammation and liver cancer: new molecular links .

A connection between inflammation and cancer has been long suspected. Epidemiological studies have established that many tumors occur in association with chronic infectious diseases, and it is also known that persistent inflammation in the absence of infections increases the risk and accelerates the development of cancer. One clear example of inflammation-related cancer is hepatocellular carcinoma (HCC). HCC is a type tumor that slowly unfolds on a background of chronic inflammation mainly triggered by exposure to infectious agents (hepatotropic viruses) or to toxic compounds (ethanol). The molecular links that connect inflammation and cancer are not completely known, but evidences gathered over the past few years are beginning to define the precise mechanisms. In this article we review the most compelling evidences on the role of transcription factors such as NF-kappaB and STAT3, cytokines like IL-6 and IL-1alpha, ligands of the EGF receptor and other inflammatory mediators in cancer development, with special emphasis in HCC. The molecular dissection of the pathways connecting the inflammatory reaction and neoplasia will pave the way for better therapies to treat cancers.

Epidermal growth factor receptor ligands in murine models for erythropoietic protoporphyria: potential novel players in the progression of liver injury.

Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.

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Interleukin-15 liver gene transfer increases the number and function of IKDCs and NK cells.

The surface phenotype CD3-NK1.1+DX5+CD11c(int)B220+GR1- has been recently ascribed to a novel subset of mouse leukocytes termed interferon (IFN)-producing killer dendritic cells (IKDCs) that shares functions with natural killer (NK) cells and DCs. Interleukin-15 (IL-15) is critical for NK cells but its relationship with IKDC remained unexplored. An expression cassette encoding human IL-15 (hIL-15) has been transferred by hydrodynamic injection into the liver of mice, resulting in transient expression of the cytokine that is detectable during the first 48 h. hIL-15 hydrodynamic gene transfer resulted in an expansion of NK cells and IKDCs. Relative expansions of IKDCs were more dramatic in the IL-15 gene-transferred hepatic tissue than in the spleen. Adoptively transferred DX5+ cells comprising both NK cells and IKDCs proliferated in response to hydrodynamic injection of hIL-15, indicating that quantitative increases are at least in part the result of proliferation from already differentiated cells. Expansion is accompanied by enhanced cytolytic activity and increased expression of TRAIL and CD137 (4-1BB), without augmenting interferon-gamma production. The effects of a single hydrodynamic injection surpassed those of two intraperitoneal doses of the recombinant protein. The novel functional link between circulating IL-15 and IKDCs opens new possibilities to study the biology and applications of this minority cell subset.

Amphiregulin: a new growth factor in hepatocarcinogenesis.

Amphiregulin (AR) is a member of the epidermal growth factor family and a ligand of the epidermal growth factor receptor (EGFR). As other ligands of the EGFR, AR is synthesized as a precursor that is shed from the plasma membrane by metalloproteases. Hyperactive autocrine loops involving AR production have been described in a variety of tumors, and this growth factor is thought to play a non-redundant role in cancer development. AR expression is not detected in the normal liver, however it is readily induced during acute liver injury and behaves as a potent pro-regenerative and survival factor. Increased AR expression is also detected in human chronic liver injury (liver cirrhosis), which is considered a pre-neoplastic condition. Recent evidences suggest that AR can play a unique role in liver tumorigenesis and in the maintenance of the neoplastic phenotype of hepatocarcinoma cells. In this review, we summarize some aspects of AR patho-biology and the rationale behind its definition as a novel target in hepatocarcinoma therapy.

New therapies for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is often diagnosed at an advanced stage when most potentially curative therapies such as resection, transplantation or percutaneous and transarterial interventions are of limited efficacy. The fact that HCC is resistant to conventional chemotherapy, and is rarely amenable to radiotherapy, leaves this disease with no effective therapeutic options and a very poor prognosis. Therefore, the development of more effective therapeutic tools and strategies is much needed. HCCs are phenotypically and genetically heterogeneous tumors that commonly emerge on a background of chronic liver disease. However, in spite of this heterogeneity recent insights into the biology of HCC suggest that certain signaling pathways and molecular alterations are likely to play essential roles in HCC development by promoting cell growth and survival. The identification of such mechanisms may open new avenues for the prevention and treatment of HCC through the development of targeted therapies. In this review we will describe the new potential therapeutic targets and clinical developments that have emerged from progress in the knowledge of HCC biology, In addition, recent advances in gene therapy and combined cell and gene therapy, together with new radiotherapy techniques and immunotherapy in patients with HCC will be discussed.

Influence of impaired liver methionine metabolism on the development of vascular disease and inflammation.

Methionine (Met) metabolism involves the sequential formation of S-adenosylmethionine (SAM, the main biological methyl donor), S-adenosylhomocysteine (SAH) and homocysteine (Hcy). Hcy can be remethylated to Met or catabolized through the trans-sulfuration pathway. In mammals, as much as 48% of Met metabolism and up to 85% of all transmethylation reactions occur in the liver. These figures underscore the central role played by this organ in Met metabolism. Maintaining the homeostasis of this metabolic cycle has proved to be essential for the preservation of liver function up to the point of preventing its neoplastic transformation. However, an adequate hepatic metabolism of Met is not only important for the liver parenchymal cell. Evidence has accumulated over the past few years supporting the involvement of Met-derived metabolites in the triggering or attenuation of pathological processes with systemic implications. This is best illustrated by the fact that a deteriorated liver function has emerged as a major factor in the development of hyperhomocysteinemia. Elevated plasma levels of Hcy have been related to several disorders including cardiovascular and cerebrovascular diseases. On the other end, liver damage also leads to deficient SAM synthesis. Among the consequences of impaired SAM synthesis in liver tissue are the enhanced production of pro-inflammatory cytokines and mediators. In this review, we will address the mechanisms and consequences of abnormal Met metabolism in liver injury, the systemic implications of such impairment and finally the potential therapeutic interventions.

Hyperhomocysteinemia in liver cirrhosis: mechanisms and role in vascular and hepatic fibrosis.

Numerous clinical and epidemiological studies have identified elevated homocysteine levels in plasma as a risk factor for atherosclerotic vascular disease and thromboembolism. Hyperhomocysteinemia may develop as a consequence of defects in homocysteine-metabolizing genes; nutritional conditions leading to vitamin B(6), B(12), or folate deficiencies; or chronic alcohol consumption. Homocysteine is an intermediate in methionine metabolism, which takes place mainly in the liver. Impaired liver function leads to altered methionine and homocysteine metabolism; however, the molecular basis for such alterations is not completely understood. In addition, the mechanisms behind homocysteine-induced cellular toxicity are not fully defined. In the present work, we have examined the expression of the main enzymes involved in methionine and homocysteine metabolism, along with the plasma levels of methionine and homocysteine, in the liver of 26 cirrhotic patients and 10 control subjects. To gain more insight into the cellular effects of elevated homocysteine levels, we have searched for changes in gene expression induced by this amino acid in cultured human vascular smooth muscle cells. We have observed a marked reduction in the expression of the main genes involved in homocysteine metabolism in liver cirrhosis. In addition, we have identified the tissue inhibitor of metalloproteinases-1 and alpha1(I)procollagen to be upregulated in vascular smooth muscle cells and liver stellate cells exposed to pathological concentrations of homocysteine. Taken together, our observations suggest (1) impaired liver function could be a novel determinant in the development of hyperhomocysteinemia and (2) a role for elevated homocysteine levels in the development of liver fibrosis.

S-adenosylmethionine regulates MAT1A and MAT2A gene expression in cultured rat hepatocytes: a new role for S-adenosylmethionine in the maintenance of the differentiated status of the liver.

Methionine metabolism starts with the formation of S-adenosylmethionine (AdoMet), the most important biological methyl donor. This reaction is catalyzed by methionine adenosyltransferase (MAT). MAT is the product of two different genes: MAT1A, which is expressed only in the adult liver, and MAT2A, which is widely distributed, expressed in the fetal liver, and replaces MAT1A in hepatocarcinoma. In the liver, preservation of high expression of MAT1A and low expression of MAT2A is critical for the maintenance of a functional and differentiated organ. Here we describe that in cultured rat hepatocytes MAT1A expression progressively decreased, as described for other liver-specific genes, and MAT2A expression was induced. We find that this switch in gene expression was prevented by adding AdoMet to the culture medium. We also show that in cultured hepatocytes with decreased MAT1A expression AdoMet addition markedly increased MAT1A transcription in a dose-dependent fashion. This effect of AdoMet was mimicked by methionine, and blocked by 3-deazaadenosine and L-ethionine, but not D-ethionine, indicating that the effect was specific and mediated probably by a methylation reaction. These findings identify AdoMet as a key molecule that differentially regulates MAT1A and MAT2A expression and helps to maintain the differentiated status of the hepatocyte.

Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma.

BACKGROUND/AIMS: It has been known for at least 50 years that alterations in methionine metabolism occur in human liver cirrhosis. However, the molecular basis of this alteration is not completely understood. In order to gain more insight into the mechanisms behind this condition, mRNA levels of methionine adenosyltransferase (MAT1A), glycine methyltransferase (GNMT), methionine synthase (MS), betaine homocysteine methyltransferase (BHMT) and cystathionine beta-synthase (CBS) were examined in 26 cirrhotic livers, five hepatocellular carcinoma (HCC) tissues and ten control livers. METHODS: The expression of the above-mentioned genes was determined by quantitative RT-PCR analysis. Methylation of MAT1A promoter was assessed by methylation-sensitive restriction enzyme digestion of genomic DNA. RESULTS: When compared to normal livers MAT1A, GNMT, BHMT, CBS and MS mRNA contents were significantly reduced in liver cirrhosis. Interestingly, MAT1A promoter was hypermethylated in the cirrhotic liver. HCC tissues also showed decreased mRNA levels of these enzymes. CONCLUSIONS: These findings establish that the abundance of the mRNA of the main genes involved in methionine metabolism is markedly reduced in human cirrhosis and HCC. Hypermethylation of MAT1A promoter could participate in its reduced expression in cirrhosis. These observations help to explain the hypermethioninemia, hyperhomocysteinemia and reduced hepatic glutathione content observed in cirrhosis.

Pathological and virological findings in patients with persistent hypertransaminasaemia of unknown aetiology.

BACKGROUND: The histopathological spectrum and role of hepatitis viruses in cases of hypertransaminasaemia of unknown aetiology have not been correctly analysed in a sufficiently large number of patients. METHODS: We studied 1075 consecutive patients referred for liver biopsy because of elevation of alanine aminotransferase (ALT) levels for more than six months. From this population we selected those cases in whom the aetiology could not be defined from clinical, biochemical, and serological data obtained before biopsy. In these patients liver biopsies were reviewed, and hepatitis B virus (HBV)-DNA and hepatitis C virus (HCV)-RNA were assayed in serum by polymerase chain reaction (PCR). Serum hepatitis G virus (HGV)-RNA was determined by PCR in 74 patients. RESULTS: Of 1075 patients studied, the cause of the increased serum ALT levels remained elusive after appropriate testing in 109 patients (10.1%). Liver biopsies from these patients showed non-specific changes in 32.7% of cases, non-alcoholic steatohepatitis (NASH) in 15.8%, and chronic hepatitis or cirrhosis in 51.5%. HBV-DNA and/or HCV-RNA was detected more frequently in cryptogenic liver disease than in healthy blood donors (26.7% v 3.4%; p<0.001). HGV-RNA was found in only one patient. The proportion of cases with detectable HBV-DNA or HCV-RNA was 14.3% in patients with non-specific changes or NASH, 30.7% in patients with chronic hepatitis, and 61.5% in patients with cirrhosis. Cirrhosis was found more frequently in patients with positive HBV-DNA and/or HCV-RNA in serum than in those who tested negatively (p=0.005). CONCLUSIONS: In our series, patients in whom biochemical and serological data did not determine the aetiology of the disease represented 10% of all cases referred for liver biopsy for persistent elevation of serum transaminases. Approximately 50% of patients had chronic hepatitis or cirrhosis and the remainder had NASH or non-specific changes. Occult viral infections were found in a high proportion of cases in the first group and in a low percentage of patients in the second.

Immunogenicity of variable regions of hepatitis C virus proteins: selection and modification of peptide epitopes to assess hepatitis C virus genotypes by ELISA.

The immunogenicity of variable regions of hepatitis C virus (HCV) proteins was studied by ELISA by using 543 synthetic peptides from 120 variable regions and 90 sera from HCV-infected patients. Some regions from certain genotypes were less immunogenic, or even non-immunogenic, compared with their equivalents in other genotypes. However, the mean recognition of all peptides from genotypes 1a, 1b and 3 by sera infected with genotypes 1a, 1b and 3, respectively, showed no significant differences, suggesting a similar overall immunogenicity of variable regions from these genotypes. Proteins NS4a, NS4b and NS5a were found to be the most immunogenic. Recognition of individual peptides by the sera of infected patients showed that the humoral response against HCV is patient-dependent. The work shows that 15-mer peptides may encompass several B-cell epitopes. These epitopes may lie in slightly different positions in different genotypes. Thirty-one percent of the 543 peptides were recognized by some of the 35 healthy donors. This may be a reflection of the large number of antigens to which they had been exposed, but it may also reflect a strategy of HCV to respond to immune pressure. After selection and modification, a set of 40 peptides was used to assess genotypes 1a, 1b, 1, 2 and 3 in the sera of HCV-infected patients, with sensitivities of 34.1, 48.5, 68.8, 58.3 and 48.9% and specificities of 100, 99.1, 97.1, 99.5 and 99%, respectively. The overall sensitivity and specificity for the assessment of genotypes 1, 2 and 3 were 64 and 98%, respectively.

Oncogenic activation of a human cyclin A2 targeted to the endoplasmic reticulum upon hepatitis B virus genome insertion.

Cyclins are major cell cycle regulators which role in malignant transformation remains controversial. In this report we describe a new mechanism of cyclin oncogenic activation. We demonstrate that an altered form of cyclin A2 (S2A) which N-terminal part is replaced by the hepatitis B virus envelope protein transforms normal rat kidney cells and cooperates with ras to transform rat embryo fibroblasts. In contrast, neither the viral moiety, nor a full length or N-terminally deleted cyclin A2 show these oncogenic properties. S2A oncogenicity arises from its binding to cyclin dependent kinases, since mutation in the MRAIL sequence abolishes transformation and correlates with an abnormal cellular localization in the endoplasmic reticulum membrane. Together, these results implicate modification in the cellular distribution of a cell cycle regulator as a mechanism of virally-induced transformation.

Simple strategy to induce antibodies of distinct specificity: application to the mapping of gp120 and inhibition of HIV-1 infectivity.

In this study 96 15-mer peptides encompassing the entire sequence of HIV-1 gp120 were synthesized and used to immunize BALB/c mice (i) alone or (ii) in conjunction with the T helper cell determinant FISEAIIHVLHSR (FIS) from sperm whale myoglobin, which is well recognized by major histocompatibility complex (MHC) class II molecules of BALB/c. Of these peptides 39 were immunogenic per se and 57 were not. Out of the 57 non-immunogenic peptides 53 could be rendered immunogenic with the second immunization protocol. With the exception of 4 cases, the anti-peptide antibody titers induced in (ii) were equal (14 cases) or higher (78 cases) than those induced in (i). From the 96 anti-peptide antibodies tested, 12 were able to recognize recombinant gp120 with good antibody titers, a result in agreement with previously identified B cell epitopes from gp120 by anti-peptide antibodies induced with longer peptides conjugated to a carrier protein. Moreover, 4 of the 12 anti-peptide antisera that recognized gp120 were able to neutralize HIV-1 infectivity in vitro, showing that the strategy of co-immunization with FIS may afford functional antibodies.

Overcoming class II-linked non-responsiveness to hepatitis B vaccine.

This work shows that class II-linked humoral lack of response to an antigen can be overcome by joint immunization with the antigen and a T-helper cell determinant (TDh) well recognized by class II molecules of a non-responder individual. Thus, SJL/J mice (H-2s), which are non-responders to the S region of hepatitis B virus surface antigen (HBsAg), were rendered responders by joint immunization with a recombinant surface antigen, only composed of the S region, and a short synthetic TDh peptide well recognized by the H-2s restriction. By contrast, when this peptide is not recognized as TDh, as in B10M mice (H-2f restricted and also non-responders to the S region), no humoral response could be induced against the S region. These results have important implications for therapy and vaccination against hepatitis B virus as well as in enhancing the immunogenicity of other antigens.

Detection of anti-hepatitis C virus antibodies by ELISA using synthetic peptides.

A novel ELISA assay for the detection of anti-hepatitis C virus antibodies in the sera of infected individuals is described. This assay is based on a mixture of three 15-amino acid synthetic peptides encompassing regions of core and NS4 proteins of hepatitis C virus. Comparison with other available ELISA assays based on recombinant polypeptides shows that, short synthetic peptides have the advantage over some larger recombinant peptides by giving higher specificity without loss of sensitivity.