Differential requirement for de novo lipogenesis in cholangiocarcinoma and hepatocellular carcinoma of mice and humans.

UNLABELLED: Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most prevalent types of primary liver cancer. These malignancies have limited treatment options, resulting in poor patient outcomes. Metabolism reprogramming, including increased de novo lipogenesis, is one of the hallmarks of cancer. Fatty acid synthase (FASN) catalyzes the de novo synthesis of long-chain fatty acids from acetyl-coenzyme A and malonyl-coenzyme A. Increased FASN expression has been reported in multiple tumor types, and inhibition of FASN expression has been shown to have tumor-suppressing activity. Intriguingly, we found that while FASN is up-regulated in human HCC samples, its expression is frequently low in human ICC specimens. Similar results were observed in mouse ICC models induced by different oncogenes. Ablating FASN in the mouse liver did not affect activated AKT and Notch (AKT/Notch intracellular domain 1) induced ICC formation in vivo. Furthermore, while both HCC and ICC lesions develop in mice following hydrodynamic injection of AKT and neuroblastoma Ras viral oncogene homolog oncogenes (AKT/Ras), deletion of FASN in AKT/Ras mice triggered the development almost exclusively of ICCs. In the absence of FASN, ICC cells might receive lipids for membrane synthesis through exogenous fatty acid uptake. In accordance with the latter hypothesis, ICC cells displayed high expression of fatty acid uptake-related proteins and robust long-chain fatty acid uptake. CONCLUSION: Our data demonstrate that FASN dependence is not a universal feature of liver tumors: while HCC development is highly dependent of FASN and its mediated lipogenesis, ICC tumorigenesis can be insensitive to FASN deprivation; our study supports novel therapeutic approaches to treat this pernicious tumor type with the inhibition of exogenous fatty acid uptake. (Hepatology 2016;63:1900-1913).

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.

Activated mutant forms of PIK3CA cooperate with RasV12 or c-Met to induce liver tumour formation in mice via AKT2/mTORC1 cascade.

BACKGROUND & AIMS: Activating mutations of PIK3CA occur in various tumour types, including human hepatocellular carcinoma. The mechanisms whereby PIK3CA contributes to hepatocarcinogenesis remain poorly understood. METHODS: PIK3CA mutants H1047R or E545K were hydrodynamically transfected, either alone or in combination with NRasV12 or c-Met genes, in the mouse liver. RESULTS: Overexpression of H1047R or E545K alone was able to induce AKT/mTOR signalling in the mouse liver, leading to hepatic steatosis. However, none of the mice developed liver tumours over long term. In contrast, H1047R or E545K cooperated with NRasV12 or c-Met to rapidly induce liver tumour formation in mice. At the molecular level, all the tumour nodules displayed activation of AKT/mTOR and Ras/MAPK cascades. Ablation of AKT2 significantly inhibited hepatic steatosis induced by H1047R or E545K and carcinogenesis induced by H1047R/c-Met or E545K/c-Met. Furthermore, tumourigenesis induced by H1047R/c-Met was abolished in conditional Raptor knockout mice. CONCLUSIONS: Both H1047R and E545K are able to activate the AKT/mTOR pathway. An intact AKT2/mTOR complex 1 cascade is required for tumourigenesis induced by H1047R/c-Met or E545K/c-Met in the liver.

Enzyme Biosensors for Biomedical Applications: Strategies for Safeguarding Analytical Performances in Biological Fluids.

Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented.

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.

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.

Oncogene-dependent addiction to carbohydrate-responsive element binding protein in hepatocellular carcinoma.

Metabolic reprogramming is a hallmark of many cancer types, including hepatocellular carcinoma (HCC). Identifying the critical players in this process might be crucial for the generation of novel and effective anti-neoplastic therapies. In the present investigation, we determined the importance of carbohydrate responsive element binding protein (ChREBP), a central player in the regulation of lipid and glucose metabolism in the liver, on the development of HCC in in vitro and in vivo models. We found that genetic deletion of ChREBP (that will be referred to as ChREBPKO mice) strongly delays or impairs hepatocarcinogenesis driven by AKT or AKT/c-Met overexpression in mice, respectively. In contrast, HCC development was found to be completely unaffected by ChREBP depletion in mice co-expressing AKT and N-Ras protooncogenes. In mouse and human HCC cell lines, suppression of ChREBP via specific small interfering RNAs (siRNAs) resulted in decreased proliferation and induction of apoptosis. Of note, these cellular events were strongly augmented by concomitant inhibition of the mitogen-activated protein kinase (MAPK) pathway. The present data indicate that ChREBP activity might be required or dispensable for HCC growth, depending on the oncogenes involved. In particular, the activation of Ras/MAPK signaling might represent a possible mechanism of resistance to ChREBP depletion in this tumor type. Additional studies are needed to unravel the molecular mechanisms rendering HCC cells insensitive to ChREBP suppression.

Oncogene dependent requirement of fatty acid synthase in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the most frequent primary tumor of the liver, is an aggressive cancer type with limited treatment options. Cumulating evidence underlines a crucial role of aberrant lipid biosynthesis (a process known as de novo lipogenesis) along carcinogenesis. Previous studies showed that suppression of fatty acid synthase (FASN), the major enzyme responsible for de novo lipogenesis, is highly detrimental for the in vitro growth of HCC cell lines. To assess whether de novo lipogenesis is required for liver carcinogenesis, we have generated various mouse models of liver cancer by stably overexpressing candidate oncogenes in the mouse liver via hydrodynamic gene delivery. We found that overexpression of FASN in the mouse liver is unable to malignantly transform hepatocytes. However, genetic deletion of FASN totally suppresses hepatocarcinogenesis driven by AKT and AKT/c-Met protooncogenes in mice. On the other hand, liver tumor development is completely unaffected by FASN depletion in mice co-expressing ß-catenin and c-Met. Our data indicate that tumors might be either addicted to or independent from de novo lipogenesis for their growth depending on the oncogenes involved. Additional investigation is required to unravel the molecular mechanisms whereby some oncogenes render cancer cells resistant to inhibition of de novo lipogenesis.

Inhibition of HSF1 suppresses the growth of hepatocarcinoma cell lines in vitro and AKT-driven hepatocarcinogenesis in mice.

Upregulation of the heat shock transcription factor 1 (HSF1) has been described as a frequent event in many cancer types, but its oncogenic role in hepatocellular carcinoma (HCC) remains poorly delineated. In the present study, we assessed the function(s) of HSF1 in hepatocarcinogenesis via in vitro and in vivo approaches. In particular, we determined the importance of HSF1 on v-Akt murine thymoma viral oncogene homolog (AKT)-induced liver cancer development in mice. We found that knockdown of HSF1 activity via specific siRNA triggered growth restraint by suppressing cell proliferation and inducing massive cell apoptosis in human HCC cell lines. At the molecular level, HSF1 inhibition was accompanied by downregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) cascade and related metabolic pathways. Most importantly, overexpression of a dominant negative form of HSF1 (HSF1dn) in the mouse liver via hydrodynamic gene delivery led to the inhibition of mouse hepatocarcinogenesis driven by overexpression of AKT. In human liver cancer specimens, we detected that HSF1 is progressively induced from human non-tumorous surrounding livers to HCC, reaching the highest expression in the tumors characterized by the poorest outcome (as defined by the length of patients' survival). In conclusion, HSF1 is an independent prognostic factor in liver cancer and might represent an innovative therapeutic target in HCC subsets characterized by activation of the AKT/mTOR pathway.

Deregulated c-Myc requires a functional HSF1 for experimental and human hepatocarcinogenesis.

Deregulated activity of the c-Myc protooncogene is a frequent molecular event underlying mouse and human hepatocarcinogenesis. Nonetheless, the mechanisms sustaining c-Myc oncogenic activity in liver cancer remain scarcely delineated. Recently, we showed that the mammalian target of rapamycin complex 1 (mTORC1) cascade is induced and necessary for c-Myc dependent liver tumor development and progression. Since the heat shock factor 1 (HSF1) transcription factor is a major positive regulator of mTORC1 in the cell, we investigated the functional interaction between HSF1 and c-Myc using in vitro and in vivo approaches. We found that ablation of HSF1 restrains the growth of c-Myc-derived mouse hepatocellular carcinoma (HCC) cell lines, where it induces downregulation of c-Myc levels. Conversely, silencing of c-Myc gene in human and mouse HCC cells led to downregulation of HSF1 expression. Most importantly, overexpression of a dominant negative form of HSF1 (HSF1dn) in the mouse liver via hydrodynamic gene delivery resulted in the complete inhibition of mouse hepatocarcinogenesis driven by overexpression of c-Myc. Altogether, the present results indicate that a functional HSF1 is necessary for c-Myc-driven hepatocarcinogenesis. Consequently, targeting HSF1 might represent a novel and effective therapeutic strategy for the treatment of HCC subsets with activated c-Myc signaling.

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.

Co-activation of AKT and c-Met triggers rapid hepatocellular carcinoma development via the mTORC1/FASN pathway in mice.

Activation of the AKT/mTOR cascade and overexpression of c-Met have been implicated in the development of human hepatocellular carcinoma (HCC). To elucidate the functional crosstalk between the two pathways, we generated a model characterized by the combined expression of activated AKT and c-Met in the mouse liver. Co-expression of AKT and c-Met triggered rapid liver tumor development and mice required to be euthanized within 8 weeks after hydrodynamic injection. At the molecular level, liver tumors induced by AKT/c-Met display activation of AKT/mTOR and Ras/MAPK cascades as well as increased lipogenesis and glycolysis. Since a remarkable lipogenic phenotype characterizes liver lesions from AKT/c-Met mice, we determined the requirement of lipogenesis in AKT/c-Met driven hepatocarcinogenesis using conditional Fatty Acid Synthase (FASN) knockout mice. Of note, hepatocarcinogenesis induced by AKT/c-Met was fully inhibited by FASN ablation. In human HCC samples, coordinated expression of FASN, activated AKT, and c-Met proteins was detected in a subgroup of biologically aggressive tumors. Altogether, our study demonstrates that co-activation of AKT and c-Met induces HCC development that depends on the mTORC1/FASN pathway. Suppression of mTORC1 and/or FASN might be highly detrimental for the growth of human HCC subsets characterized by concomitant induction of the AKT and c-Met cascades.