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.

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.

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.

Inhibition of liver methionine adenosyltransferase gene expression by 3-methylcolanthrene: protective effect of S-adenosylmethionine.

Methionine adenosyltransferase (MAT) is an essential enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet), the most important biological methyl donor. Liver MAT I/III is the product of the MAT1A gene. Hepatic MAT I/III activity and MAT1A expression are compromised under pathological conditions such as alcoholic liver disease and hepatic cirrhosis, and this gene is silenced upon neoplastic transformation of the liver. In the present work, we evaluated whether MAT1A expression could be targeted by the polycyclic arylhydrocarbon (PAH) 3-methylcholanthrene (3-MC) in rat liver and cultured hepatocytes. MAT1A mRNA levels were reduced by 50% following in vivo administration of 3-MC to adult male rats (100 mg/kg, p.o., 4 days' treatment). This effect was reproduced in a time- and dose-dependent fashion in cultured rat hepatocytes, and was accompanied by the induction of cytochrome P450 1A1 gene expression. This action of 3-MC was mimicked by other PAHs such as benzo[a]pyrene and benzo[e]pyrene, but not by the model arylhydrocarbon receptor (AhR) activator 2,3,7,8-tetrachlorodibenzo-p-dioxin. 3-MC inhibited transcription driven by a MAT1A promoter-reporter construct transfected into rat hepatocytes, but MAT1A mRNA stability was not affected. We recently showed that liver MAT1A expression is induced by AdoMet in cultured hepatocytes. Here, we observed that exogenously added AdoMet prevented the negative effects of 3-MC on MAT1A expression. Taken together, our data demonstrate that liver MAT1A gene expression is targeted by PAHs, independently of AhR activation. The effect of AdoMet may be part of the protective action of this molecule in liver damage.

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.

Identification of argininosuccinate lyase as a hypoxia-responsive gene in rat hepatocytes.

BACKGROUND/AIMS: The differential oxygenation of periportal and perivenous hepatocytes has been demonstrated as a major determinant in the zonated expression of certain metabolic pathways in the liver. We have searched for novel genes whose expression could be modulated by hypoxia in cultured rat hepatocytes. METHODS: Primary cultures of rat hepatocytes were incubated under normoxic (21% oxygen) or hypoxic (3% oxygen) conditions for 6 h. Differences in gene expression under both conditions were analyzed using the technique of differential display by means of PCR. RESULTS: We have identified the enzyme argininosuccinate lyase (ASL) as being downregulated by hypoxia. ASL is a cytosolic protein which participates in urea metabolism. ASL expression was time-dependently reduced in hypoxia. Hypoxia modulated the responses of this gene to the two main hormonal signals which induce ASL mRNA: glucocorticoids and cAMP. ASL mRNA levels decreased in response to ATP-reducing agents. CoCl2 mimicked the effect of hypoxia, suggesting the implication of a hemoprotein in this response. Hypoxia did not affect ASL mRNA stability, indicating that this effect occurs at the transcriptional level. CONCLUSIONS: Our observations suggest that differences in oxygen levels across the hepatic parenchyma could participate in the zonated expression of ASL.

DNA methylation and histone acetylation of rat methionine adenosyltransferase 1A and 2A genes is tissue-specific.

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet). In mammals MAT activity derives from two separate genes which display a tissue-specific pattern of expression. While MAT1A is expressed only in the adult liver, MAT2A is expressed in non-hepatic tissues. The mechanisms behind the selective expression of these two genes are not fully understood. In the present report we have evaluated MAT1A and MAT2A methylation in liver and in other tissues, such as kidney, by methylation-sensitive restriction enzyme digestion of genomic DNA. Our data indicate that MAT1A is hypomethylated in liver and hypermethylated in non-expressing tissues. The opposite situation is found for MAT2A. Additionally, histones associated to MAT1A and MAT2A genes showed enhanced levels of acetylation in expressing tissues (two-fold for MAT1A and 3.5-fold for MAT2A liver and kidney respectively). These observations support a role for chromatin structure and its modification in the tissue-specific expression of both MAT genes.

Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation.

Methionine adenosyltransferase (MAT) is the enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main donor of methyl groups in the cell. In mammals MAT is the product of two genes, MAT1A and MAT2A. MAT1A is expressed only in the mature liver whereas fetal hepatocytes, extrahepatic tissues and liver cancer cells express MAT2A. The mechanisms behind the tissue and differentiation state specific MAT1A expression are not known. In the present work we examined MAT1A promoter methylation status by means of methylation sensitive restriction enzyme analysis. Our data indicate that MAT1A promoter is hypomethylated in liver and hypermethylated in kidney and fetal rat hepatocytes, indicating that this modification is tissue specific and developmentally regulated. Immunoprecipitation of mononucleosomes from liver and kidney tissues with antibodies mainly specific to acetylated histone H4 and subsequent Southern blot analysis with a MAT1A promoter probe demonstrated that MAT1A expression is linked to elevated levels of chromatin acetylation. Early changes in MAT1A methylation are already observed in the precancerous cirrhotic livers from rats, which show reduced MAT1A expression. Human hepatoma cell lines in which MAT1A is not expressed were also hypermethylated at this locus. Finally we demonstrate that MAT1A expression is reactivated in the human hepatoma cell line HepG2 treated with 5-aza-2'-deoxycytidine or the histone deacetylase inhibitor trichostatin, suggesting a role for DNA hypermethylation and histone deacetylation in MAT1A silencing.