S-Adenosylmethionine: From the Discovery of Its Inhibition of Tumorigenesis to Its Use as a Therapeutic Agent.

Alterations of methionine cycle in steatohepatitis, cirrhosis, and hepatocellular carcinoma induce MAT1A decrease and MAT2A increase expressions with the consequent decrease of S-adenosyl-L-methionine (SAM). This causes non-alcoholic fatty liver disease (NAFLD). SAM administration antagonizes pathological conditions, including galactosamine, acetaminophen, and ethanol intoxications, characterized by decreased intracellular SAM. Positive therapeutic effects of SAM/vitamin E or SAM/ursodeoxycholic acid in animal models with NAFLD and intrahepatic cholestasis were not confirmed in humans. In in vitro experiments, SAM and betaine potentiate PegIFN-alpha-2a/2b plus ribavirin antiviral effects. SAM plus betaine improves early viral kinetics and increases interferon-stimulated gene expression in patients with viral hepatitis non-responders to pegIFNα/ribavirin. SAM prevents hepatic cirrhosis, induced by CCl4, inhibits experimental tumors growth and is proapoptotic for hepatocellular carcinoma and MCF-7 breast cancer cells. SAM plus Decitabine arrest cancer growth and potentiate doxorubicin effects on breast, head, and neck cancers. Furthermore, SAM enhances the antitumor effect of gemcitabine against pancreatic cancer cells, inhibits growth of human prostate cancer PC-3, colorectal cancer, and osteosarcoma LM-7 and MG-63 cell lines; increases genomic stability of SW480 cells. SAM reduces colorectal cancer progression and inhibits the proliferation of preneoplastic rat liver cells in vivo. The discrepancy between positive results of SAM treatment of experimental tumors and modest effects against human disease may depend on more advanced human disease stage at moment of diagnosis.

Nuclear localization dictates hepatocarcinogenesis suppression by glycine N-methyltransferase.

BACKGROUND: GNMT (glycine N-methyltransferase) is a tumor suppressor gene, but the mechanisms mediating its suppressive activity are not entirely known. METHODS: We investigated the oncosuppressive mechanisms of GNMT in human hepatocellular carcinoma (HCC). GNMT mRNA and protein levels were evaluated by quantitative RT-PCR and immunoblotting. GNMT effect in HCC cell lines was modulated through GNMT cDNA induced overexpression or anti-GNMT siRNA transfection. RESULTS: GNMT was expressed at low level in human HCCs with a better prognosis (HCCB) while it was almost absent in fast-growing tumors (HCCP). In HCCB, the nuclear localization of the GNMT protein was much more pronounced than in HCCP. In Huh7 and HepG2 cell lines, GNMT forced expression inhibited the proliferation and promoted apoptosis. At the molecular level, GNMT overexpression inhibited the expression of CYP1A (Cytochrome p450, aromatic compound-inducible), PREX2 (Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2), PARP1 [Poly (ADP-ribose) polymerase 1], and NFKB (nuclear factor-kB) genes. By chromatin immunoprecipitation, we found GNMT binding to the promoters of CYP1A1, PREX2, PARP1, and NFKB genes resulting in their strong inhibition. These genes are implicated in hepatocarcinogenesis, and are involved in the GNMT oncosuppressive action. CONCLUSION: Overall, the present data indicate that GNMT exerts a multifaceted suppressive action by interacting with various cancer-related genes and inhibiting their expression.

Genetic Predisposition to Hepatocellular Carcinoma.

Liver preneoplastic and neoplastic lesions of the genetically susceptible F344 and resistant BN rats cluster, respectively, with human HCC with better (HCCB) and poorer prognosis (HCCP); therefore, they represent a valid model to study the molecular alterations determining the genetic predisposition to HCC and the response to therapy. The ubiquitin-mediated proteolysis of ERK-inhibitor DUSP1, which characterizes HCC progression, favors the unrestrained ERK activity. DUSP1 represents a valuable prognostic marker, and ERK, CKS1, or SKP2 are potential therapeutic targets for human HCC. In DN (dysplastic nodule) and HCC of F344 rats and human HCCP, DUSP1 downregulation and ERK1/2 overexpression sustain SKP2-CKS1 activity through FOXM1, the expression of which is associated with a susceptible phenotype. SAM-methyl-transferase reactions and SAM/SAH ratio are regulated by GNMT. In addition, GNMT binds to CYP1A, PARP1, and NFKB and PREX2 gene promoters. MYBL2 upregulation deregulates cell cycle and induces the progression of premalignant and malignant liver. During HCC progression, the MYBL2 transcription factor positively correlates with cells proliferation and microvessel density, while it is negatively correlated to apoptosis. Hierarchical supervised analysis, regarding 6132 genes common to human and rat liver, showed a gene expression pattern common to normal liver of both strains and BN nodules, and a second pattern is observed in F344 nodules and HCC of both strains. Comparative genetics studies showed that DNs of BN rats cluster with human HCCB, while F344 DNs and HCCs cluster with HCCP.

The Warburg Effect 97 Years after Its Discovery.

The deregulation of the oxidative metabolism in cancer, as shown by the increased aerobic glycolysis and impaired oxidative phosphorylation (Warburg effect), is coordinated by genetic changes leading to the activation of oncogenes and the loss of oncosuppressor genes. The understanding of the metabolic deregulation of cancer cells is necessary to prevent and cure cancer. In this review, we illustrate and comment the principal metabolic and molecular variations of cancer cells, involved in their anomalous behavior, that include modifications of oxidative metabolism, the activation of oncogenes that promote glycolysis and a decrease of oxygen consumption in cancer cells, the genetic susceptibility to cancer, the molecular correlations involved in the metabolic deregulation in cancer, the defective cancer mitochondria, the relationships between the Warburg effect and tumor therapy, and recent studies that reevaluate the Warburg effect. Taken together, these observations indicate that the Warburg effect is an epiphenomenon of the transformation process essential for the development of malignancy.

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.

Alterations of Methionine Metabolism as Potential Targets for the Prevention and Therapy of Hepatocellular Carcinoma.

Several researchers have analyzed the alterations of the methionine cycle associated with liver disease to clarify the pathogenesis of human hepatocellular carcinoma (HCC) and improve the preventive and the therapeutic approaches to this tumor. Different alterations of the methionine cycle leading to a decrease of S-adenosylmethionine (SAM) occur in hepatitis, liver steatosis, liver cirrhosis, and HCC. The reproduction of these changes in MAT1A-KO mice, prone to develop hepatitis and HCC, demonstrates the pathogenetic role of MAT1A gene under-regulation associated with up-regulation of the MAT2A gene (MAT1A:MAT2A switch), encoding the SAM synthesizing enzymes, methyladenosyltransferase I/III (MATI/III) and methyladenosyltransferase II (MATII), respectively. This leads to a rise of MATII, inhibited by the reaction product, with a consequent decrease of SAM synthesis. Attempts to increase the SAM pool by injecting exogenous SAM have beneficial effects in experimental alcoholic and non-alcoholic steatohepatitis and hepatocarcinogenesis. Mechanisms involved in hepatocarcinogenesis inhibition by SAM include: (1) antioxidative effects due to inhibition of nitric oxide (NO•) production, a rise in reduced glutathione (GSH) synthesis, stabilization of the DNA repair protein Apurinic/Apyrimidinic Endonuclease 1 (APEX1); (2) inhibition of c-myc, H-ras, and K-ras expression, prevention of NF-kB activation, and induction of overexpression of the oncosuppressor PP2A gene; (3) an increase in expression of the ERK inhibitor DUSP1; (4) inhibition of PI3K/AKT expression and down-regulation of C/EBPα and UCA1 gene transcripts; (5) blocking LKB1/AMPK activation; (6) DNA and protein methylation. Different clinical trials have documented curative effects of SAM in alcoholic liver disease. Furthermore, SAM enhances the IFN-α antiviral activity and protects against hepatic ischemia-reperfusion injury during hepatectomy in HCC patients with chronic hepatitis B virus (HBV) infection. However, although SAM prevents experimental tumors, it is not curative against already established experimental and human HCCs. The recent observation that the inhibition of MAT2A and MAT2B expression by miRNAs leads to a rise of endogenous SAM and strong inhibition of cancer cell growth could open new perspectives to the treatment of HCC.

MicroRNA-203 impacts on the growth, aggressiveness and prognosis of hepatocellular carcinoma by targeting MAT2A and MAT2B genes.

Hepatocellular carcinoma (HCC) is characterized by the down-regulation of the liver-specific methyladenosyltransferase 1A (MAT1A) gene, encoding the S-adenosylmethionine synthesizing isozymes MATI/III, and the up-regulation of the widely expressed methyladenosyltransferase 2A (MAT2A), encoding MATII isozyme, and methyladenosyltransferase 2B (MAT2B), encoding a ß-subunit without catalytic action that regulates MATII enzymatic activity. Different observations showed hepatocarcinogenesis inhibition by miR-203. We found that miR-203 expression in HCCs is inversely correlated with HCC proliferation and aggressiveness markers, and with MAT2A and MAT2B levels. MiR-203 transfection in HepG2 and Huh7 liver cancer cells targeted the 3'-UTR of MAT2A and MAT2B, inhibiting MAT2A and MAT2B mRNA levels and MATα2 and MATß2 protein expression. These molecular events were paralleled by an increase in SAM content and were associated with growth restraint and apoptosis, inhibition of cell migration and invasiveness, and suppression of the expression of CD133 and LIN28B stemness markers. In contrast, MAT2B transfection in the same cell lines led to a rise of both MATß2 and MATα2 expression, associated with increases in cell growth, migration, invasion and overexpression of stemness markers and p-AKT. Altogether, our results indicate that the miR-203 oncosuppressor activity may at least partially depend on its inhibition of MAT2A and MAT2B and show, for the first time, an oncogenic activity of MAT2B linked to AKT activation.

Alterations of methionine metabolism in hepatocarcinogenesis: the emergent role of glycine N-methyltransferase in liver injury.

The methionine and folate cycles play a fundamental role in cell physiology and their alteration is involved in liver injury and hepatocarcinogenesis. Glycine N-methyltransferase is implicated in methyl group supply, DNA methylation, and nucleotide biosynthesis. It regulates the cellular S-adenosylmethionine/S-adenosylhomocysteine ratio and S-adenosylmethionine-dependent methyl transfer reactions. Glycine N-methyltransferase is absent in fast-growing hepatocellular carcinomas and present at a low level in slower growing HCC ones. The mechanism of tumor suppression by glycine N-methyltransferase is not completely known. Glycine N-methyltransferase inhibits hepatocellular carcinoma growth through interaction with Dep domain-containing mechanistic target of rapamycin (mTor)-interacting protein, a binding protein overexpressed in hepatocellular carcinoma. The interaction of the phosphatase and tensin homolog inhibitor, phosphatidylinositol 3,4,5-trisphosphate-dependent rac exchanger, with glycine N-methyltransferase enhances proteasomal degradation of this exchanger by the E3 ubiquitin ligase HectH. Glycine N-methyltransferase also regulates genes related to detoxification and antioxidation pathways. It supports pyrimidine and purine syntheses and minimizes uracil incorporation into DNA as consequence of folate depletion. However, recent evidence indicates that glycine N-methyltransferase targeted into nucleus still exerts strong anti-proliferative effects independent of its catalytic activity, while its restriction to cytoplasm prevents these effects. Our current knowledge suggest that glycine N-methyltransferase plays a fundamental, even if not yet completely known, role in cellular physiology and highlights the need to further investigate this role in normal and cancer cells.

Deregulation of methionine metabolism as determinant of progression and prognosis of hepatocellular carcinoma.

The under-regulation of liver-specific MAT1A gene codifying for S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and the up-regulation of widely expressed MAT2A, MATII isozyme occurs in hepatocellular carcinoma (HCC). MATα1:MATα2 switch strongly contributes to the fall in SAM liver content both in rodent and human liver carcinogenesis. SAM administration to carcinogen-treated animals inhibits hepatocarcinogenesis. The opposite occurs in Mat1a-KO mice, in which chronic SAM deficiency is followed by HCC development. This review focuses upon the changes, induced by the MATα1:MATα2 switch, involved in HCC development. In association with MATα1:MATα2 switch there occurs, in HCC, global DNA hypomethylation, decline of DNA repair, genomic instability, and deregulation of different signaling pathways such as overexpression of c-MYC (avian myelocytomatosis viral oncogene homolog), increase of polyamine (PA) synthesis and RAS/ERK (Harvey murine sarcoma virus oncogene homolog/extracellular signal-regulated kinase), IKK/NF-kB (I-k kinase beta/nuclear factor kB), PI3K/AKT, and LKB1/AMPK axes. Furthermore, a decrease in MATα1 expression and SAM level induces HCC cell proliferation and survival. SAM treatment in vivo and enforced MATα1 overexpression or MATα2 inhibition, in cultured HCC cells, prevent these changes. A negative correlation of MATα1:MATα2 and MATI/III:MATII ratios with cell proliferation and genomic instability and a positive correlation with apoptosis and global DNA methylation are present in human HCC. Altogether, these data suggest that the decrease of SAM level and the deregulation of MATs are potential therapeutic targets for HCC.

DNA-PKcs: A promising therapeutic target in human hepatocellular carcinoma?

Hepatocellular carcinoma (HCC) is a frequent and deadly disease worldwide. The absence of effective therapies when the tumor is surgically unresectable leads to an extremely poor outcome of HCC patients. Thus, it is mandatory to elucidate the molecular pathogenesis of HCC in order to develop novel therapeutic strategies against this pernicious tumor. Mounting evidence indicates that suppression of the DNA damage response machinery might be deleterious for the survival and growth of the tumor cells. In particular, DNA dependent protein kinase catalytic subunit (DNA-PKcs), a major player in the non-homologous end-joining (NHEJ) repair process, seems to represent a valuable target for innovative anti-neoplastic therapies in cancer. DNA-PKcs levels are strongly upregulated and associated with a poor clinical outcome in various tumor types, including HCC. Importantly, DNA-PKcs not only protects tumor cells from harmful DNA insults coming either from the microenvironment or chemotherapeutic drug treatments, but also possesses additional properties, independent from its DNA repair activity, that provide growth advantages to cancer cells. These properties (metabolic and gene reprogramming, invasiveness and metastasis, resistance to apoptosis, etc.) have started to be elucidated. In the present review, we summarize the physiologic and oncogenic roles of DNA-PKcs, with a special emphasis on liver cancer. In particular, this work focuses on the molecular mechanism whereby DNA-PKcs exerts its pro-tumorigenic activity in cancer cells. In addition, the upstream regulator of DNA-PKcs activation as well as its downstream effectors thus far identified are illustrated. Furthermore, the potential therapeutic strategies aimed at inhibiting DNA-PKcs activity in HCC are discussed.

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.

Simultaneous detection of multiple lower genital tract pathogens by an impedimetric immunochip.

Lower genital tract infections caused by both sexually and not-sexually transmitted pathogens in women are a key public health priority worldwide, especially in developing countries. Since standard analyses are time-consuming, appropriate therapeutic intervention is often neglected or delayed. Lab-on-chips and biosensors open new perspectives and offer innovative tools to simplify the diagnosis by medical staff, especially in countries with inadequate resources. Here we report a biosensing platform based on Electrochemical Impedance Spectroscopy (EIS) that allows multiplexed detection of Candida albicans, Streptococcus agalactiae and Chlamydia trachomatis with a single biochip, enabling a quick screening thanks to the presence of different immobilized antibodies, each specific for one of the different target pathogens.

Portable gliadin-immunochip for contamination control on the food production chain.

Celiac disease (CD) is one of the most common digestive disorders caused by an abnormal immune reaction to gluten. So far there are no available therapies, the only solution is a strict gluten-free diet, which however could be very challenging as gluten can be hidden in many food products. Furthermore an additional problem is related to cross-contamination of nominal gluten-free foods with gluten-based ones during manufacturing. Here we propose a lab on chip platform as a powerful tool to help food manufacturers to evaluate the real amount of gluten in their products by an accurate in-situ control of the production chain and maybe to specify the real gluten content in packages labeling. Our portable gliadin-immunochips, based on an electrochemical impedance spectroscopy transduction method, were first calibrated and then validated for both liquid and solid food matrixes by analyzing different beers and flours. The high specificity of our assay was also demonstrated by performing control experiments on rice and potatoes flours containing prolamin-like proteins. We achieved limit of quantification of 0.5 ppm for gliadin that is 20 times lower than the worldwide limit established for gluten-free food while the method of analysis is faster and cheaper than currently employed ELISA-based methods. Moreover our results on food samples were validated through a mass spectrometry standard analysis.

Multifocal hepatocellular carcinoma: intrahepatic metastasis or multicentric carcinogenesis?

Multifocal Hepatocellular carcinoma (HCC) may be multiple HCCs of multicentric origin (MO) or intrahepatic metastases (IM) arising from a primary HCC. Numerous attempts to differentiate the two types of multifocal HCC have been made including the valuation of the clinicopathologic characteristics of MO and IM patients and the recurrence time, loss-of-heterozygosity analysis of specific DNA microsatellite loci to distinguish multiclonal MO from IM of monoclonal origin, and the research of diagnostic and progression markers through genomic and proteomic analyses. These approaches, however, have been unsatisfactory hitherto. Recently, a multi-omic analysis of HBV-related multifocal HCCs, including intergraded genomics and transcriptomics, was performed and the results, validated by a cohort of 174 HCC patients, were correlated with HCC clinicopathological data. The two multifocal HCC types were effectively discerned by multi-omics profiling that could predict HCC clonality and aggressiveness. Further, the dual-specificity protein kinase TTK was recognized as a prognostic marker for HCC. Multi-omics strategy potentially opens new perspectives for the diagnosis, prognosis and personalized treatment of multi-focal HCC. Further work aimed at extending this strategy to HCC with other etiology, simplifying the analysis, and reducing its costs is necessary for its routine clinical application.

New insights on the role of epigenetic alterations in hepatocellular carcinoma.

Emerging evidence assigns to epigenetic mechanisms heritable differences in gene function that come into being during cell development or via the effect of environmental factors. Epigenetic deregulation is strongly involved in the development of hepatocellular carcinoma (HCC). It includes changes in methionine metabolism, promoter hypermethylation, or increased proteasomal degradation of oncosuppressors, as well as posttranscriptional deregulation by microRNA or messenger RNA (mRNA) binding proteins. Alterations in the methylation of the promoter of methyl adenosyltransferase MAT1A and MAT2A genes in HCC result in decreased S-adenosylmethionine levels, global DNA hypomethylation, and deregulation of signal transduction pathways linked to methionine metabolism and methyl adenosyltransferases activity. Changes in S-adenosylmethionine levels may also depend on MAT1A mRNA destabilization associated with MAT2A mRNA stabilization by specific proteins. Decrease in MAT1A expression has also been attributed to miRNA upregulation in HCC. A complex deregulation of miRNAs is also strongly involved in hepatocarcinogenesis, with up-regulation of different miRNAs targeting oncosuppressor genes and down-regulation of miRNAs targeting genes involved in cell-cycle and signal transduction control. Oncosuppressor gene down-regulation in HCC is also induced by promoter hypermethylation or posttranslational deregulation, leading to proteasomal degradation. The role of epigenetic changes in hepatocarcinogenesis has recently suggested new promising therapeutic approaches for HCC on the basis of the administration of methylating agents, inhibition of methyl adenosyltransferases, and restoration of the expression of tumor-suppressor miRNAs.