Aidi injection altered the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and diethylnitrosamine-induced hepatocellular carcinoma in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Aidi injection (ADI), a traditional chinese medicine preparation, is widely used in combination with chemotherapy for the treatment of various malignant tumors, such as hepatocellular carcinoma (HCC). Studies have shown that changes in cytochrome P450 (CYP450) activity in disease states would affect the metabolism of drugs in vivo, especially liver diseases. However, the changes of Aidi injection on the activities of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and HCC states are still unknown. AIM OF THE STUDY: The cocktail probe drugs method was used to investigate the effects of ADI on the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and HCC rats. MATERIALS AND METHODS: The HCC rats was induced by diethylnitrosamine (DEN). Then, both normal and HCC rats were randomly divided into 2 groups (n = 6). They were given saline or ADI (10 mL/kg/d, i.p) for 2 weeks, respectively. On the fifteenth day, cocktail probe mixing solution, including metoprolol (10 mg/kg), caffeine (1.0 mg/kg), omeprazole (2.0 mg/kg), midazolam (2.0 mg/kg), chlorzoxazone (4.0 mg/kg) and tolbutamide (0.5 mg/kg), was injected into tail vein of all rats in each group. The blood sample was obtained at specified time. After the protein is precipitated, six probe drugs are analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: Compared with control group, the activity of CYP3A2 and CYP2E1 was significantly lower in the ADI group. Compared with the model group, the activities of CYP1A2, CYP3A2, CYP2E1, and CYP2C11 enzymes in the ADI model group were significantly reduced. Additionally, the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 enzymes in model group was significantly lower than control group. CONCLUSIONS: ADI can inhibit a lot of CYP450 enzyme, so it may reduce the dosage of chemotherapeutic drugs to reach the required plasma concentration of chemotherapeutic drugs, which is of great significance for the combination of anti-tumor chemotherapeutic drugs and is worthy of further in-depth study and clinical attention.

Butyrate-containing structured lipids act on HDAC4, HDAC6, DNA damage and telomerase activity during promotion of experimental hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) presents with a high treatment resistance and poor prognosis. Early diagnosis and preventive approaches such as chemoprevention are essential for the HCC control. Therefore, we evaluated the chemopreventive effects of butyrate-containing structured lipids (STLs) administered during the promotion stage of hepatocarcinogenesis in rats submitted to the 'resistant hepatocyte' (RH) model. Administration of butyrate-containing STLs inhibited the incidence and mean number of visible hepatic nodules per rat and reduced the number and area of glutathione S-transferase placental form-positive (GST-P+) preneoplastic focal lesions in the livers. This was accompanied by the induction of apoptosis and an increased level of hepatic butyric acid. Treatment with butyrate-containing STLs resulted in increased histone H3 lysine 9 (H3K9) acetylation, reduction of total histone deacetylase (HDAC) activity, and lower levels of HDAC4 and HDAC6 proteins. The chemopreventive effect of butyrate-containing STLs was also associated with the increased nuclear compartmentalization of p53 protein and reduced expression of the Bcl-2 protein. In addition, rats treated with butyrate-containing STLs showed decreased DNA damage and telomerase activity in the livers. These results demonstrate that the suppressive activity of butyrate-containing STLs is associated with inhibition of elevated during hepatocarcinogenesis chromatin-modifying proteins HDAC4 and HDAC6, subcellular redistribution of the p53 protein, and decreased DNA damage and telomerase activity.

Glucose deprivation-induced aberrant FUT1-mediated fucosylation drives cancer stemness in hepatocellular carcinoma.

Rapidly growing tumors often experience hypoxia and nutrient (e.g., glucose) deficiency because of poor vascularization. Tumor cells respond to the cytotoxic effects of such stresses by inducing molecular adaptations that promote clonal selection of a more malignant tumor-initiating cell phenotype, especially in the innermost tumor regions. Here, we report a regulatory mechanism involving fucosylation by which glucose restriction promotes cancer stemness to drive drug resistance and tumor recurrence. Using hepatocellular carcinoma (HCC) as a model, we showed that restricted glucose availability enhanced the PERK/eIF2α/ATF4 signaling axis to drive fucosyltransferase 1 (FUT1) transcription via direct binding of ATF4 to the FUT1 promoter. FUT1 overexpression is a poor prognostic indicator for HCC. FUT1 inhibition could mitigate tumor initiation, self-renewal, and drug resistance. Mechanistically, we demonstrated that CD147, ICAM-1, EGFR, and EPHA2 are glycoprotein targets of FUT1, in which such fucosylation would consequently converge on deregulated AKT/mTOR/4EBP1 signaling to drive cancer stemness. Treatment with an α-(1,2)-fucosylation inhibitor sensitized HCC tumors to sorafenib, a first-line molecularly targeted drug used for advanced HCC patients, and reduced the tumor-initiating subset. FUT1 overexpression and/or CD147, ICAM-1, EGFR, and EPHA2 fucosylation may be good prognostic markers and therapeutic targets for cancer patients.

Citral inhibits N-nitrosodiethylamine-induced hepatocellular carcinoma via modulation of antioxidants and xenobiotic-metabolizing enzymes.

Hepatocellular carcinoma (HCC) ranks the sixth position among various cancers worldwide. Recent research shows that natural and dietary compounds possess many therapeutic effects. Citral is a monoterpene aldehyde that contains geranial and neral. The present study was considered to study the role of citral against N-nitrosodiethylamine (NDEA)-induced HCC via modulation of antioxidants and xenobiotic-metabolizing enzymes in vivo. NDEA-alone-administered group II animals profoundly showed increased tumor incidence, reactive oxygen species, liver marker enzyme levels, serum bilirubin levels, tumor markers of carcinoembryonic antigen, α-fetoprotein, proliferative markers of argyrophilic nucleolar organizing regions, proliferating cell nuclear antigen (PCNA) expressions, phase I xenobiotic-metabolic enzymes and simultaneously decreased antioxidants, and phase II enzymes levels. Citral (100 mg/kg b.w.) treatment significantly reverted the levels in group III cancer-bearing animals when compared to group II cancer-bearing animals. In group IV animals, citral-alone administration did not produce any adverse effect during the experimental condition. Based on the results, citral significantly inhibits the hepatocellular carcinogenesis through restoring the antioxidants and phase II xenobiotic-enzyme levels; thereby, it strongly proves as an antiproliferative agent against rat HCC.

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy.

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

Regulation of PKB/Akt-pathway in the chemopreventive effect of lactoferrin against diethylnitrosamine-induced hepatocarcinogenesis in rats.

BACKGROUND: Abnormal activation of protein kinase B (PKB) is associated with many cancers. This makes inhibition of PKB signaling pathway a promising strategy for cancer therapy. Lactoferrin (Lf) has been reported for its inhibition of tumor growth and metastasis, however, the mechanism is not completely understood. Its anti-hepatocarcinogenic activity has not taken the deserved recognition despite the additional advantages of Lf as an antiviral against hepatitis C virus, the main cause of hepatocellular carcinoma (HCC), and as a targeting ligand for delivering chemotherapeutics to hepatoma cells. METHODS: This study evaluated the anti-hepatocarcinogenic effect of Lf, and the role of PKB in this effect using diethylnitrosamine (DENA)-induced HCC rat model, and a primary cell culture prepared from the induced hepatic lesions (DENA-HCC cell culture). RESULTS: Up-regulation of activated PKB in the hepatocytes of rats with DENA-induced HCC was observed, as measured biochemically in the liver homogenate, and localized immunohistochemically. This was accompanied by increment of hepatocytes proliferation, and expression of vascular endothelial growth factor and endothelial nitric oxide synthase. Involvement of PKB in DENA-induced HCC was confirmed by the observed decrease in cell proliferation in DENA-HCC cell culture that was treated with PKB inhibitor. In Lf-treated rats, a dose-dependent chemopreventive effect was observed, with decreased expression and activation of PKB, amelioration of the other DENA-induced alterations, and stimulation of apoptosis. In vitro, Lf blocked PKB activator-induced cell proliferation. CONCLUSION: These findings support the chemopreventive activity of Lf against HCC, and suggest regulation of PKB-pathway as a potential mechanism underlying this effect.

Inhibition of hepatocellular carcinoma by metabolic normalization.

In two different mouse liver cancer models, we recently showed that a switch from oxidative phosphorylation (Oxphos) to glycolysis (the Warburg effect) is invariably accompanied by a marked decline in fatty acid oxidation (FAO) and a reciprocal increase in the activity of pyruvate dehydrogenase (PDH), which links glycolysis to the TCA cycle. We now show that short-term implementation of either medium-chain (MC) or long-chain (LC) high fat diets (HFDs) nearly doubled the survival of mice with c-Myc oncoprotein-driven hepatocellular carcinoma (HCC). Mechanistically, HFDs forced tumors to become more reliant on fatty acids as an energy source, thus normalizing both FAO and PDH activities. More generally, both MC- and LC-HFDs partially or completely normalized the expression of 682 tumor-dysregulated transcripts, a substantial fraction of which are involved in cell cycle control, proliferation and metabolism. That these same transcripts were responsive to HFDs in livers strongly suggested that the changes were the cause of tumor inhibition rather than its consequence. In seven different human cancer cohorts, patients with tumors containing high ratios of FAO-related:glycolysis-related transcripts had prolonged survival relative to those with low ratios. Furthermore, in 13 human cancer types, the expression patterns of transcripts encoding enzymes participating in FAO and/or cholesterol biosynthesis also correlated with significantly prolonged survival. Collectively, our results support the idea that the survival benefits of HFDs are due to a reversal of the Warburg effect and other tumor-associated metabolic and cell cycle abnormalities. They also suggest that short-term dietary manipulation, either alone or in combination with more traditional chemotherapeutic regimens, might be employed as a relatively non-toxic and cost-effective means of enhancing survival in certain cancer types.

Association of p38MAPK-p53-Fas aggregation in S-allyl cysteine mediated regulation of hepatocarcinoma.

Bioactive components of dietary phytochemicals have been reported to possess antitumor activities. Evidences suggested key role of stress responsive p38MAPK in the induction of nutraceuticals mediated apoptosis in hepatocellular carcinoma (HCC). Current study demonstrated detailed molecular bagatelle associated with p38 MAPK mediated effective suppression of cell growth both in HepG2 and chemically induced liver carcinoma after S-allyl cysteine (SAC) treatment. SAC promoted p38MAPK activity responsible for p53 phosphorylation, its stabilization followed by nuclear translocation leading to induction in expression and oligomerization of Fas protein. Distinctive p38MAPK-p53 axis dependent Fas-FasL-FADD mediated caspase activities along with perturbed cell cycling became normalized with continuation of SAC treatment for another month to diethylnitrosamine induced liver carcinoma. Co-treatment with SB203580, the p38MAPK inhibitor, prevented pro-apoptotic effect of SAC by altering p53 phosphorylation and death inducing signaling complex conformation in HepG2 and induced HCC. Collectively study suggested significant contribution of p38MAPK-p53-DISC-Caspase pathway in the regulation of anti-neoplastic activity of SAC against HCC.

HDAC3 Deficiency Promotes Liver Cancer through a Defect in H3K9ac/H3K9me3 Transition.

DNA damage triggers diverse cancers, particularly hepatocellular carcinoma (HCC), but the intrinsic link between DNA damage and tumorigenesis remains unclear. Because of its role as an epigenetic and transcriptional regulator, histone deacetylase 3 (HDAC3) is essential for DNA damage control and is often aberrantly expressed in human HCC. In this study, we used individual class I HDAC member-deficient mice to demonstrate that K9 in histone H3 (H3K9), which is the critical site for the assembly of DNA damage response complexes, is exclusively targeted by HDAC3. Ablation of HDAC3 disrupted the deacetylation and consequent trimethylation of H3K9 (H3K9me3), the first step in double-strand break repair, and led to the accumulation of damaged DNA. Simultaneously, hyperacetylated H3K9 (H3K9ac) served as a transcriptional activator and enhanced multiple signaling pathways to promote tumorigenesis. Together, these results show that HDAC3 targets the H3K9ac/H3K9me3 transition to serve as a critical regulator that controls both DNA damage repair and the transcription of many tumor-related genes. Moreover, these findings provide novel insights into the link between DNA damage and transcriptional reprogramming in tumorigenesis. SIGNIFICANCE: These findings show that HDAC3 exclusively regulates H3K9ac in response to DNA damage, and loss of HDAC3 activity shifts the balance from DNA damage control to protumorigenic transcriptional activity.

Transketolase Deficiency Protects the Liver from DNA Damage by Increasing Levels of Ribose 5-Phosphate and Nucleotides.

De novo nucleotide biosynthesis is essential for maintaining cellular nucleotide pools, the suppression of which leads to genome instability. The metabolic enzyme transketolase (TKT) in the nonoxidative branch of the pentose phosphate pathway (PPP) regulates ribose 5-phosphate (R5P) levels and de novo nucleotide biosynthesis. TKT is required for maintaining cell proliferation in human liver cancer cell lines, yet the role of TKT in liver injury and cancer initiation remains to be elucidated. In this study, we generated a liver-specific TKT knockout mouse strain by crossing TKTflox/flox mice with albumin-Cre mice. Loss of TKT in hepatocytes protected the liver from diethylnitrosamine (DEN)-induced DNA damage without altering DEN metabolism. DEN treatment of TKT-null liver increased levels of R5P and promoted de novo nucleotide synthesis. More importantly, supplementation of dNTPs in primary hepatocytes alleviated DEN-induced DNA damage, cell death, inflammatory response, and cell proliferation. Furthermore, DEN and high-fat diet (HFD)-induced liver carcinogenesis was reduced in TKTflox/floxAlb-Cre mice compared with control littermates. Mechanistically, loss of TKT in the liver increased apoptosis, reduced cell proliferation, decreased TNFα, IL6, and STAT3 levels, and alleviated DEN/HFD-induced hepatic steatosis and fibrosis. Together, our data identify a key role for TKT in promoting genome instability during liver injury and tumor initiation. SIGNIFICANCE: These findings identify transketolase as a novel metabolic target to maintain genome stability and reduce liver carcinogenesis.

Carbonic anhydrase-IX inhibition enhances the efficacy of hexokinase II inhibitor for hepatocellular carcinoma in a murine model.

Hypoxic conditions, which large or infiltrative hypovascular tumors may encounter, also produce acidic environments. Carbonic anhydrase-IX (CA-IX), an enzyme involved in lowering pH, is overexpressed in hepatocellular carcinoma (HCC). In the present study, whether inhibition of CA-IX enhances the efficacy of a hexokinase II inhibitor in an in vivo murine model was examined and its prognostic implication in HCC patients was investigated. CA-IX expression was evaluated using quantitative real-time PCR and western blot analysis using human HCC cell lines. 3-bromopyruvate (3-BP), a hexokinase II inhibitor, and acetazolamide, a carbonic anhydrase inhibitor, were used to target hexokinase II and CA-IX in vitro and in vivo, respectively. A human HCC cell line (Huh-7) was tested as a subcutaneous tumor model in BALB/c nu/nu mice. The prognostic role of CA-IX was evaluated in the TCGA database. Quantitative real-time PCR and western blot analysis revealed that CA-IX expression was activated in the presence of 3-BP. Further analysis showed that introducing an additional stress by treating the orally active CA-IX inhibitor (acetazolamide) can synergistically increase the efficacy of 3-BP in vivo, which was confirmed using a mouse model. We also found that HCC patients with high CA-IX expression show poor overall survival in TCGA database. These results indicate CA-IX is a promising therapeutic target for enhancing the efficacy of 3-BP and can be a prognostic factor for HCC.

Targeting mTOR and Src restricts hepatocellular carcinoma growth in a novel murine liver cancer model.

Liver cancer is a poor prognosis cancer with limited treatment options. To develop a new therapeutic approach, we derived HCC cells from a known model of murine hepatocellular carcinoma (HCC). We treated adiponectin (APN) knock-out mice with the carcinogen diethylnitrosamine, and the resulting tumors were 7-fold larger than wild-type controls. Tumors were disassociated from both genotypes and their growth characteristics evaluated. A52 cells from APN KO mice had the most robust growth in vitro and in vivo, and presented with pathology similar to the parental tumor. All primary tumors and cell lines exhibited activity of the mammalian target of Rapamycin (mTOR) and Src pathways. Subsequent combinatorial treatment, with the mTOR inhibitor Rapamycin and the Src inhibitor Dasatinib reduced A52 HCC growth 29-fold in vivo. Through protein and histological analyzes we observed activation of these pathways in human HCC, suggesting that targeting both mTOR and Src may be a novel approach for the treatment of HCC.

Identification and Therapeutic Intervention of Coactivated Anaplastic Lymphoma Kinase, Fibroblast Growth Factor Receptor 2, and Ephrin Type-A Receptor 5 Kinases in Hepatocellular Carcinoma.

Though kinase inhibitors have been heavily investigated in the clinic to combat advanced hepatocellular carcinoma (HCC), clinical outcomes have been disappointing overall, which may be due to the absence of kinase-addicted subsets in HCC patients. Recently, strategies that simultaneously inhibit multiple kinases are increasingly appreciated in HCC treatment, yet they are challenged by the dynamic nature of the kinase networks. This study aims to identify clustered kinases that may cooperate to drive the malignant growth of HCC. We show that anaplastic lymphoma kinase, fibroblast growth factor receptor 2, and ephrin type-A receptor 5 are the essential kinases that assemble into a functional cluster to sustain the viability of HCC cells through downstream protein kinase B-dependent, extracellular signal-regulated kinase-dependent, and p38-dependent signaling pathways. Their coactivation is associated with poor prognosis for overall survival in about 13% of HCC patients. Moreover, their activities are tightly regulated by heat shock protein 90 (Hsp90). Thereby Combined kinase inhibition or targeting of heat shock protein 90 led to significant therapeutic responses both in vitro and in vivo. Conclusion: Our findings established a paradigm that highlights the cooperation of anaplastic lymphoma kinase, fibroblast growth factor receptor 2, and ephrin type-A receptor 5 kinases in governing the growth advantage of HCC cells, which might offer a conceptual "combined therapeutic target" for diagnosis and subsequent intervention in a subgroup of HCC patients.

Dual Targeting of Histone Methyltransferase G9a and DNA-Methyltransferase 1 for the Treatment of Experimental Hepatocellular Carcinoma.

Epigenetic modifications such as DNA and histone methylation functionally cooperate in fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. We aimed to determine the therapeutic efficacy and potential mechanism of action of our dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of G9a and DNMT1, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs (n = 268), peritumoral tissues (n = 154), and HCC cell lines (n = 32). We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cell growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1, and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6-bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272. Conclusion: Combined targeting of G9a/DNMT1 with compounds such as CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC.

Identification of UAP1L1 as a critical factor for protein O-GlcNAcylation and cell proliferation in human hepatoma cells.

Aged hepatocyte-specific-Mcl-1 knockout (MKO-hep) mice are prone to develop liver tumors mimicking human hepatocellular carcinoma (HCC). Here we reported that a protein named UDP-N-acetylglucosamine pyrophosphorylase-1-like-1 (Uap1l1) is upregulated in the liver of young MKO-hep mice without any macroscopically detectable tumor nodules and is prominently expressed in the hepatic tumors developed in the aged MKO-hep mice. Intriguingly, human UAP1L1 is also significantly upregulated in a distinct subset of HCC tissues and patients with upregulated expression of UAP1L1 appeared to have poor prognosis. Overexpression of UAP1L1 significantly promoted, whereas UAP1L1 knockdown markedly reduced the proliferation of human hepatoma cells both in vitro and in vivo. UAP1L1 shows ~59% sequence identity to UDP-N-acetylglucosamine pyrophosphorylase-1 (UAP1), which is directly involved in the synthesis of the sugar donor (UDP-GlcNac) for N-acetylglucosamine modification (O-GlcNAcylation) of proteins. However, unlike UAP1, UAP1L1 harbors very limited UDP-GlcNAc synthesis activity. Moreover, although both UAP1 and UAP1L1 are required for O-GlcNAc transferase (OGT)-mediated protein O-GlcNAcylation, they appear to function distinctly from each other. UAP1L1 directly interacts with OGT, but does not seem to be an OGT substrate. In addition, UAP1L1 alone is not sufficient to activate OGT activity in vitro, suggesting that UAP1L1 may function together with other proteins to modulate OGT activity in vivo. Lastly, UAP1L1 knockdown attenuated c-MYC O-GlcNAcylation and protein stability, and overexpression of c-MYC significantly rescued the proliferation defect of UAP1L1 knockdown HepG2 cells, suggesting that c-MYC is one downstream target of UAP1L1 that contributes to UAP1L1-mediated cell proliferation, at least in HepG2 cells.

Ribose-5-phosphate isomerase A overexpression promotes liver cancer development in transgenic zebrafish via activation of ERK and ß-catenin pathways.

Dysregulation of the enzymes involved in the pentose phosphate pathway (PPP) is known to promote tumorigenesis. Our recent study demonstrated that ribose-5-phosphate isomerase (RPIA), a key regulator of the PPP, regulates hepatoma cell proliferation and colony formation. Our studies in zebrafish reveal that RPIA-mediated hepatocarcinogenesis requires extracellular signal-regulated kinase (ERK) and ß-catenin signaling. To further investigate RPIA-mediated hepatocarcinogenesis, two independent lines of transgenic zebrafish expressing human RPIA in the liver were generated. These studies reveal that RPIA overexpression triggers lipogenic factor/enzyme expression, steatosis, fibrosis and proliferation of the liver. In addition, the severity of fibrosis and the extent of proliferation are positively correlated with RPIA expression levels. Furthermore, RPIA-mediated induction of hepatocellular carcinoma (HCC) requires the ERK and ß-catenin signaling pathway but is not dependent upon transaldolase levels. Our study presents a mechanism for RPIA-mediated hepatocarcinogenesis and suggests that RPIA represents a valuable therapeutic target for the treatment of HCC.

DNA Damage, Liver Injury, and Tumorigenesis: Consequences of DDX3X Loss.

The pleiotropic roles of DEAD-box helicase 3, X-linked (DDX3X), including its functions in transcriptional and translational regulation, chromosome segregation, DNA damage, and cell growth control, have highlighted the association between DDX3X and tumorigenesis. However, mRNA transcripts and protein levels of DDX3X in patient specimens have shown the controversial correlations of DDX3X with hepatocellular carcinoma (HCC) prevalence. In this study, generation of hepatocyte-specific Ddx3x-knockout mice revealed that loss of Ddx3x facilitates liver tumorigenesis. Loss of Ddx3x led to profound ductular reactions, cell apoptosis, and compensatory proliferation in female mutants at 6 weeks of age. The sustained phosphorylation of histone H2AX (γH2AX) and significant accumulation of DNA single-strand breaks and double-strand breaks in liver indicated that the replicative stress occurred in female mutants. Further chromatin immunoprecipitation analyses demonstrated that DDX3X bound to promoter regions and regulated the expression of DNA repair factors, DDB2 and XPA, to maintain genome stability. Loss of Ddx3x led to decreased levels of DNA repair factors, which contributed to an accumulation of unrepaired DNA damage, replication stress, and eventually, spontaneous liver tumors and DEN-induced HCCs in Alb-Cre/+;Ddx3xflox/flox mice. IMPLICATIONS: These data identify an important role of DDX3X in the regulation of DNA damage repair to protect against replication stress in liver and HCC development and progression.

Diet-induced hepatic steatosis activates Ras to promote hepatocarcinogenesis via CPT1α.

Aberrant activation of the RAS cascade ubiquitously occurs in human hepatocellular carcinomas (HCC), regardless of rare mutations of RAS. However, the association between the Ras cascade and hepatic steatosis during hepatocarcinogenesis remains under-investigated. Here, the variation in the constitutive activity of Ras signaling and HCC incidence was found in a nonalcoholic fatty liver disease (NAFLD)-HCC mouse model, and Ras activity was induced by hepatic steatosis. Even in hepatocyte-specific expression of KrasG12D (Alb-Cre/KrasG12D, Krashep) mice, mutagenic activation of Ras signaling was still significantly enhanced by NAFLD, with downregulation of negative regulators. Interestingly, hepatic steatosis could be alleviated by persistent activation of Ras, whereas Ras accelerated DNA damage and HCC progression through Carnitine palmitoyltransferase 1A (CPT1α). A close correlation between active Ras and CPT1α was also shown in clinical steatosis peri-tumor tissues of HCC samples and experimental models. CPT1α inhibitor etomoxir (ETO) largely ameliorated active Ras-drived HCC. These findings can provide a novel link between steatosis and Ras activity in liver cancer.

Sirtuin 4 Depletion Promotes Hepatocellular Carcinoma Tumorigenesis Through Regulating Adenosine-Monophosphate-Activated Protein Kinase Alpha/Mammalian Target of Rapamycin Axis in Mice.

Sirtuin 4 (SIRT4) has been reported to play a vital role in the maintenance of glutamine catabolism and adenosine triphosphate (ATP) homeostasis, but its character in hepatocellular carcinomas (HCCs) remains obscure. In this study, we observed low expression of SIRT4 in both HCC cell lines and HCCs from patients. Decreased disease-free survival time is associated with low tumor levels of SIRT4 in patients. Deficiency of SIRT4 facilitated liver tumor development and lung metastasis in xenografts and knockout (KO) mice by promoting colony formation and migration of hepatoma cells and enhancing sphere formation of HCCs. Mechanistically, SIRT4 deletion augmented mammalian target of rapamycin (mTOR) signaling by inactivating adenosine-monophosphate (AMP)-activated protein kinase alpha (AMPKα) through regulation of glutamine catabolism and subsequent AM)/liver kinase B1 (LKB1) axis. Blockage of mTOR by rapamycin or inhibition of glutaminolysis abolished the discrepancy in tumorigenic capacity between SIRT4-depleted hepatoma cells and control cells. Suppression of LKB1 or promotion of AMP by metformin also abrogated the hyperproliferative phenotype caused by SIRT4 loss, which further confirmed that the LKB1/AMPKα/mTOR axis is required in SIRT4-deficiency-promoted HCC tumorigenesis. Conclusion: SIRT4 could exert its tumor suppressive function in HCC by inhibiting glutamine metabolism and thereby increasing the adenosine diphosphate (ADP)/AMP levels to phosphorylate AMPKα by LKB1, which blocks the mTOR signaling pathway.

HELLS Regulates Chromatin Remodeling and Epigenetic Silencing of Multiple Tumor Suppressor Genes in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide. Increasing evidence shows that epigenetic alterations play an important role in human carcinogenesis. Deregulation of DNA methylation and histone modifications have recently been characterized in HCC, but the significance of chromatin remodeling in liver carcinogenesis remains to be explored. In this study, by systematically analyzing the expression of chromatin remodeling genes in human HCCs, we found that helicase, lymphoid-specific (HELLS), an SWI2/SNF2 chromatin remodeling enzyme, was remarkably overexpressed in HCC. Overexpression of HELLS correlated with more aggressive clinicopathological features and poorer patient prognosis compared to patients with lower HELLS expression. We further showed that up-regulation of HELLS in HCC was conferred by hyperactivation of transcription factor specificity protein 1 (SP1). To investigate the functions of HELLS in HCC, we generated both gain-of-function and loss-of-function models by the CRISPR activation system, lentiviral short hairpin RNA, and the CRISPR/Cas9 genome editing system. We demonstrated that overexpression of HELLS augmented HCC cell proliferation and migration. In contrast, depletion of HELLS reduced HCC growth and metastasis both in vitro and in vivo. Moreover, inactivation of HELLS led to metabolic reprogramming and reversed the Warburg effect in HCC cells. Mechanistically, by integrating analysis of RNA sequencing and micrococcal nuclease sequencing, we revealed that overexpression of HELLS increased nucleosome occupancy, which obstructed the accessibility of enhancers and hindered formation of the nucleosome-free region (NFR) at the transcription start site. Though this mechanism, up-regulation of HELLS mediated epigenetic silencing of multiple tumor suppressor genes including E-cadherin, FBP1, IGFBP3, XAF1 and CREB3L3 in HCC. Conclusion: Our data reveal that HELLS is a key epigenetic driver of HCC; by altering the nucleosome occupancy at the NFR and enhancer, HELLS epigenetically suppresses multiple tumor suppressor genes to promote HCC progression.