Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27KIP1 Regulation.

Background and Objectives: Aberrant upregulation of fatty acid synthase (FASN), catalyzing de novo synthesis of fatty acids, occurs in various tumor types, including human hepatocellular carcinoma (HCC). Although FASN oncogenic activity seems to reside in its pro-lipogenic function, cumulating evidence suggests that FASN's tumor-supporting role might also be metabolic-independent. Materials and Methods: In the present study, we show that FASN inactivation by specific small interfering RNA (siRNA) promoted the downregulation of the S-phase kinase associated-protein kinase 2 (SKP2) and the consequent induction of p27KIP1 in HCC cell lines. Results: Expression levels of FASN and SKP2 directly correlated in human HCC specimens and predicted a dismal outcome. In addition, forced overexpression of SKP2 rendered HCC cells resistant to the treatment with the FASN inhibitor C75. Furthermore, FASN deletion was paralleled by SKP2 downregulation and p27KIP1 induction in the AKT-driven HCC preclinical mouse model. Moreover, forced overexpression of an SKP2 dominant negative form or a p27KIP1 non-phosphorylatable (p27KIP1-T187A) construct completely abolished AKT-dependent hepatocarcinogenesis in vitro and in vivo. Conclusions: In conclusion, the present data indicate that SKP2 is a critical downstream effector of FASN and AKT-dependent hepatocarcinogenesis in liver cancer, envisaging the possibility of effectively targeting FASN-positive liver tumors with SKP2 inhibitors or p27KIP1 activators.

TEA Domain Transcription Factor 4 Is the Major Mediator of Yes-Associated Protein Oncogenic Activity in Mouse and Human Hepatoblastoma.

Hepatoblastoma (HB) is the most common type of pediatric liver cancer. Activation of yes-associated protein (YAP) has been implicated in HB molecular pathogenesis. The transcriptional co-activator Yap regulates downstream gene expression through interaction with the TEA domain (TEAD) proteins. Nonetheless, YAP also displays functions that are independent of its transcriptional activity. The underlying molecular mechanisms by which Yap promotes HB development remain elusive. In the current study, we demonstrated that blocking TEAD function via the dominant-negative form of TEAD2 abolishes Yap-driven HB formation in mice and restrains human HB growth in vitro. When TEAD2 DNA-binding domain was fused with virus protein 16 transcriptional activation domain, it synergized with activated ß-catenin to promote HB formation in vivo. Among TEAD genes, silencing of TEAD4 consistently inhibited tumor growth and Yap target gene expression in HB cell lines. Furthermore, TEAD4 mRNA expression was significantly higher in human HB lesions when compared with corresponding nontumorous liver tissues. Human HB specimens also exhibited strong nuclear immunoreactivity for TEAD4. Altogether, data demonstrate that TEAD-mediated transcriptional activity is both sufficient and necessary for Yap-driven HB development. TEAD4 is the major TEAD isoform and Yap partner in human HB. Targeting TEAD4 may represent an effective treatment option for human HB.

The mTORC2-Akt1 Cascade Is Crucial for c-Myc to Promote Hepatocarcinogenesis in Mice and Humans.

Hepatocellular carcinoma (HCC) is a deadly form of liver cancer with limited treatment options. The c-Myc transcription factor is a pivotal player in hepatocarcinogenesis, but the mechanisms underlying c-Myc oncogenic activity in the liver remain poorly delineated. Mammalian target of rapamycin complex 2 (mTORC2) has been implicated in cancer by regulating multiple AGC kinases, especially AKT proteins. In the liver, AKT1 and AKT2 are widely expressed. While AKT2 is the major isoform downstream of activated phosphoinositide 3-kinase and loss of phosphatase and tensin homolog-induced HCC, the precise function of AKT1 in hepatocarcinogenesis is largely unknown. In the present study, we demonstrate that mTORC2 is activated in c-Myc-driven mouse HCC, leading to phosphorylation/activation of Akt1 but not Akt2. Ablation of Rictor inhibited c-Myc-induced HCC formation in vivo. Mechanistically, we discovered that loss of Akt1, but not Akt2, completely prevented c-Myc HCC formation in mice. Silencing of Rictor or Akt1 in c-Myc HCC cell lines inhibited phosphorylated forkhead box o1 expression and strongly suppressed cell growth in vitro. In human HCC samples, c-MYC activation is strongly correlated with phosphorylated AKT1 expression. Higher expression of RICTOR and AKT1, but not AKT2, is associated with poor survival of patients with HCC. In c-Myc mice, while rapamycin, an mTORC1 inhibitor, had limited efficacy at preventing c-Myc-driven HCC progression, the dual mTORC1 and mTORC2 inhibitor MLN0128 effectively promoted tumor regression by inducing apoptosis and necrosis. Conclusion: Our study indicates the functional contribution of mTORC2/Akt1 along c-Myc-induced hepatocarcinogenesis, with AKT1 and AKT2 having distinct roles in HCC development and progression; targeting both mTORC1 and mTORC2 may be required for effective treatment of human HCC displaying c-Myc amplification or overexpression.

Functional role of SGK3 in PI3K/Pten driven liver tumor development.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer related deaths worldwide. The PI3K cascade is one of the major signaling pathways underlying HCC development and progression. Activating mutations of PI3K catalytic subunit alpha (PIK3CA) and/or loss of Pten often occur in human HCCs. Serum and glucocorticoid kinase 3 (SGK3) belongs to the SGK family of AGK kinases and functions in parallel to AKT downstream of PI3K. Previous studies have shown that SGK3 may be the major kinase responsible for the oncogenic potential of PIK3CA helical domain mutants, such as PIK3CA(E545K), but not kinase domain mutants, such as PIK3CA(H1047R). METHODS: We investigated the functional contribution of SGK3 in mediating activated PIK3CA mutant or loss of Pten induced HCC development using Sgk3 knockout mice. RESULTS: We found that ablation of Sgk3 does not affect PIK3CA(H1047R) or PIK3CA(E545K) induced lipogenesis in the liver. Using PIK3CA(H1047R)/c-Met, PIK3CA(E545K)/c-Met, and sgPten/c-Met murine HCC models, we also demonstrated that deletion of Sgk3 moderately delays PIK3CA(E545K)/c-Met driven HCC, while not affecting PIK3CA(H1047R)/c-Met or sgPten/c-Met HCC formation in mice. Similarly, in human HCC cell lines, silencing of SGK3 reduced PIK3CA(E545K) -but not PIK3CA(H1047R)- induced accelerated tumor cell proliferation. CONCLUSION: Altogether, our data suggest that SGK3 plays a role in transducing helical domain mutant PIK3CA signaling during liver tumor development.

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.

A functional mammalian target of rapamycin complex 1 signaling is indispensable for c-Myc-driven hepatocarcinogenesis.

Amplification and/or activation of the c-Myc proto-oncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc-dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of mTORC1, strongly inhibits c-Myc liver tumor formation. Also, the p70 ribosomal S6 kinase/ribosomal protein S6 and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed up-regulation of multiple amino acid transporters, including solute carrier family 1 member A5 (SLC1A5) and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1 as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human hepatocellular carcinoma specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6. CONCLUSION: Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis; thus, targeting the mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of hepatocellular carcinoma with activated c-Myc signaling. (Hepatology 2017;66:167-181).

Pan-mTOR inhibitor MLN0128 is effective against intrahepatic cholangiocarcinoma in mice.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model. METHODS: We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study. RESULTS: Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation. CONCLUSIONS: This study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 vs. gemcitabine/oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to gemcitabine/oxaliplatin-based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.

Inactivation of fatty acid synthase impairs hepatocarcinogenesis driven by AKT in mice and humans.

BACKGROUND & AIMS: Cumulating evidence underlines the crucial role of aberrant lipogenesis in human hepatocellular carcinoma (HCC). Here, we investigated the oncogenic potential of fatty acid synthase (FASN), the master regulator of de novo lipogenesis, in the mouse liver. METHODS: FASN was overexpressed in the mouse liver, either alone or in combination with activated N-Ras, c-Met, or SCD1, via hydrodynamic injection. Activated AKT was overexpressed via hydrodynamic injection in livers of conditional FASN or Rictor knockout mice. FASN was suppressed in human hepatoma cell lines via specific small interfering RNA. RESULTS: Overexpression of FASN, either alone or in combination with other genes associated with hepatocarcinogenesis, did not induce histological liver alterations. In contrast, genetic ablation of FASN resulted in the complete inhibition of hepatocarcinogenesis in AKT-overexpressing mice. In human HCC cell lines, FASN inactivation led to a decline in cell proliferation and a rise in apoptosis, which were paralleled by a decrease in the levels of phosphorylated/activated AKT, an event controlled by the mammalian target of rapamycin complex 2 (mTORC2). Downregulation of AKT phosphorylation/activation following FASN inactivation was associated with a strong inhibition of rapamycin-insensitive companion of mTOR (Rictor), the major component of mTORC2, at post-transcriptional level. Finally, genetic ablation of Rictor impaired AKT-driven hepatocarcinogenesis in mice. CONCLUSIONS: FASN is not oncogenic per se in the mouse liver, but is necessary for AKT-driven hepatocarcinogenesis. Pharmacological blockade of FASN might be highly useful in the treatment of human HCC characterized by activation of the AKT pathway.

Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: novel insights from interspecies comparison.

Hepatocellular carcinoma is a frequent and fatal disease. Recent researches on rodent models and human hepatocarcinogenesis contributed to unravel the molecular mechanisms of hepatocellular carcinoma dedifferentiation and progression, and allowed the discovery of several alterations underlying the deregulation of cell cycle and signalling pathways. This review provides an interpretive analysis of the results of these studies. Mounting evidence emphasises the role of up-regulation of RAS/ERK, P13K/AKT, IKK/NF-kB, WNT, TGF-ß, NOTCH, Hedgehog, and Hippo signalling pathways as well as of aberrant proteasomal activity in hepatocarcinogenesis. Signalling deregulation often occurs in preneoplastic stages of rodent and human hepatocarcinogenesis and progressively increases in carcinomas, being most pronounced in more aggressive tumours. Numerous changes in signalling cascades are involved in the deregulation of carbohydrate, lipid, and methionine metabolism, which play a role in the maintenance of the transformed phenotype. Recent studies on the role of microRNAs in signalling deregulation, and on the interplay between signalling pathways led to crucial achievements in the knowledge of the network of signalling cascades, essential for the development of adjuvant therapies of liver cancer. Furthermore, the analysis of the mechanisms involved in signalling deregulation allowed the identification of numerous putative prognostic markers and novel therapeutic targets of specific hepatocellular carcinoma subtypes associated with different biologic and clinical features. This is of prime importance for the selection of patient subgroups that are most likely to obtain clinical benefit and, hence, for successful development of targeted therapies for liver cancer.

Pleiotropic effects of methionine adenosyltransferases deregulation as determinants of liver cancer progression and prognosis.

Downregulation of liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and upregulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Being inhibited by its reaction product, MATII isoform upregulation cannot compensate for MATI/III decrease. Therefore, MAT1A:MAT2A switch contributes to decrease in SAM level in rodent and human hepatocarcinogenesis. SAM administration to carcinogen-treated rats prevents hepatocarcinogenesis, whereas MAT1A-KO mice, characterized by chronic SAM deficiency, exhibit macrovesicular steatosis, mononuclear cell infiltration in periportal areas, and HCC development. This review focuses upon the pleiotropic changes, induced by MAT1A/MAT2A switch, associated with HCC development. Epigenetic control of MATs expression occurs at transcriptional and post-transcriptional levels. In HCC cells, MAT1A/MAT2A switch is associated with global DNA hypomethylation, decrease in DNA repair, genomic instability, and signaling deregulation including c-MYC overexpression, rise in polyamine synthesis, upregulation of RAS/ERK, IKK/NF-kB, PI3K/AKT, and LKB1/AMPK axis. Furthermore, decrease in MAT1A expression and SAM levels results in increased HCC cell proliferation, cell survival, and microvascularization. All of these changes are reversed by SAM treatment in vivo or forced MAT1A overexpression or MAT2A inhibition in cultured HCC cells. In human HCC, MAT1A:MAT2A and MATI/III:MATII ratios correlate negatively with cell proliferation and genomic instability, and positively with apoptosis and global DNA methylation. This suggests that SAM decrease and MATs deregulation represent potential therapeutic targets for HCC. Finally, MATI/III:MATII ratio strongly predicts patients' survival length suggesting that MAT1A:MAT2A expression ratio is a putative prognostic marker for human HCC.

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

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

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

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

Connective tissue growth factor autocriny in human hepatocellular carcinoma: oncogenic role and regulation by epidermal growth factor receptor/yes-associated protein-mediated activation.

UNLABELLED: The identification of molecular mechanisms involved in the maintenance of the transformed phenotype of hepatocellular carcinoma (HCC) cells is essential for the elucidation of therapeutic strategies. Here, we show that human HCC cells display an autocrine loop mediated by connective tissue growth factor (CTGF) that promotes DNA synthesis and cell survival. Expression of CTGF was stimulated by epidermal growth factor receptor (EGFR) ligands and was dependent on the expression of the transcriptional coactivator, Yes-associated protein (YAP). We identified elements in the CTGF gene proximal promoter that bound YAP-enclosing complexes and were responsible for basal and EGFR-stimulated CTGF expression. We also demonstrate that YAP expression can be up-regulated through EGFR activation not only in HCC cells, but also in primary human hepatocytes. CTGF contributed to HCC cell dedifferentiation, expression of inflammation-related genes involved in carcinogenesis, resistance toward doxorubicin, and in vivo HCC cell growth. Importantly, CTGF down-regulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 expression and was involved in the reduced sensitivity of these cells toward TRAIL-mediated apoptosis. CONCLUSION: We have identified autocrine CTGF as a novel determinant of HCC cells' neoplastic behavior. Expression of CTGF can be stimulated through the EGFR-signaling system in HCC cells in a novel cross-talk with the oncoprotein YAP. Moreover, to our knowledge, this is the first study that identifies a signaling mechanism triggering YAP gene expression in healthy and transformed liver parenchymal cells.

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.

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.

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

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

Blood Cell Count Indexes of Systemic Inflammation in Carotid Artery Disease: Current Evidence and Future Perspectives.

Carotid artery disease is commonly encountered in clinical practice and accounts for approximately 30% of ischemic strokes in the general population. Numerous biomarkers have been investigated as predictors of the onset and progression of carotid disease, the occurrence of cerebrovascular complications, and overall prognosis. Among them, blood cell count (BCC) indexes of systemic inflammation might be particularly useful, from a pathophysiological and clinical point of view, given the inflammatory nature of the atherosclerotic process. The aim of this review is to discuss the available evidence regarding the role of common BCC indexes, such as the neutrophil to lymphocyte ratio (NLR), monocyte to lymphocyte ratio (MLR), platelet to lymphocyte ratio (PLR), mean platelet volume (MPV), platelet distribution width (PDW), and red cell distribution width (RDW), in the diagnosis and risk stratification of carotid artery disease, and their potential clinical applications.