ALDOB/KAT2A interactions epigenetically modulate TGF-ß expression and T cell functions in hepatocellular carcinogenesis.

BACKGROUND AND AIMS: Cross talk between tumor cells and immune cells enables tumor cells to escape immune surveillance and dictate responses to immunotherapy. Previous studies have identified that downregulation of the glycolytic enzyme fructose-1,6-bisphosphate aldolase B (ALDOB) in tumor cells orchestrated metabolic programming to favor HCC. However, it remains elusive whether and how ALDOB expression in tumor cells affects the tumor microenvironment in HCC. APPROACH AND RESULTS: We found that ALDOB downregulation was negatively correlated with CD8 + T cell infiltration in human HCC tumor tissues but in a state of exhaustion. Similar observations were made in mice with liver-specific ALDOB knockout or in subcutaneous tumor models with ALDOB knockdown. Moreover, ALDOB deficiency in tumor cells upregulates TGF-ß expression, thereby increasing the number of Treg cells and impairing the activity of CD8 + T cells. Consistently, a combination of low ALDOB and high TGF-ß expression exhibited the worst overall survival for patients with HCC. More importantly, the simultaneous blocking of TGF-ß and programmed cell death (PD) 1 with antibodies additively inhibited tumorigenesis induced by ALDOB deficiency in mice. Further mechanistic experiments demonstrated that ALDOB enters the nucleus and interacts with lysine acetyltransferase 2A, leading to inhibition of H3K9 acetylation and thereby suppressing TGFB1 transcription. Consistently, inhibition of lysine acetyltransferase 2A activity by small molecule inhibitors suppressed TGF-ß and HCC. CONCLUSIONS: Our study has revealed a novel mechanism by which a metabolic enzyme in tumor cells epigenetically modulates TGF-ß signaling, thereby enabling cancer cells to evade immune surveillance and affect their response to immunotherapy.

The POR rs10954732 polymorphism decreases susceptibility to hepatocellular carcinoma and hepsin as a prognostic biomarker correlated with immune infiltration based on proteomics.

The effect of the cytochrome P450 oxidoreductase (POR) rs10954732 (G > A) polymorphism on hepatocellular carcinoma (HCC) susceptibility is unknown. Here we found that A allele carriers showed a 69% decrease in susceptibility to HCC with overall survival (OS) prolonged to 199%, accompanied by lower activity for cytochrome P450 2E1. A total of 222 differentially expressed proteins were mainly enriched in neutrophil and T cell activation and involved in the immune and inflammatory responses, constituting the altered immune tumor microenvironment related with A allele by proteomics analysis. Hepsin (HPN) showed significant down-regulation in HCC and up-regulation in A allele carriers. A lower HPN level was associated with increased susceptibility to HCC and a worse prognosis. Moreover, HPN is a potential independent prognostic biomarker for HCC and is strongly associated with clinicopathological features, tumor-infiltrating status of immune cells both in our discovery cohort and database surveys. Our findings provide a new potential mechanism by which HPN may play an important role in the susceptibility of rs10954732 A allele carriers to HCC and their prognosis through tumor immune infiltration, thus offering potential insights for future studies on tumor immunotherapy.

Fructose-1,6-Bisphosphate Aldolase B Depletion Promotes Hepatocellular Carcinogenesis Through Activating Insulin Receptor Signaling and Lipogenesis.

BACKGROUND AND AIMS: Insulin receptor (IR) transduces cell surface signal through phosphoinositide 3-kinase (PI3K)-AKT pathways or translocates to the nucleus and binds to the promoters to regulate genes associated with insulin actions, including de novo lipogenesis (DNL). Chronic activation of IR signaling drives malignant transformation, but the underlying mechanisms remain poorly defined. Down-regulation of fructose-1,6-bisphosphate aldolase (ALDO) B in hepatocellular carcinoma (HCC) is correlated with poor prognosis. We aim to study whether and how ALDOB is involved in IR signaling in HCC. APPROACH AND RESULTS: Global or liver-specific ALDOB knockout (L-ALDOB-/- ) mice were used in N-diethylnitrosamine (DEN)-induced HCC models, whereas restoration of ALDOB expression was achieved in L-ALDOB-/- mice by adeno-associated virus (AAV). 13 C6 -glucose was employed in metabolic flux analysis to track the de novo fatty acid synthesis from glucose, and nontargeted lipidomics and targeted fatty acid analysis using mass spectrometry were performed. We found that ALDOB physically interacts with IR and attenuates IR signaling through down-regulating PI3K-AKT pathways and suppressing IR nuclear translocation. ALDOB depletion or disruption of IR/ALDOB interaction in ALDOB mutants promotes DNL and tumorigenesis, which is significantly attenuated with ALDOB restoration in L-ALDOB-/- mice. Notably, attenuated IR/ALDOB interaction in ALDOB-R46A mutant exhibits more significant tumorigenesis than releasing ALDOB/AKT interaction in ALDOB-R43A, whereas knockdown IR sufficiently diminishes tumor-promoting effects in both mutants. Furthermore, inhibiting phosphorylated AKT or fatty acid synthase significantly attenuates HCC in L-ALDOB-/- mice. Consistently, ALDOB down-regulation is correlated with up-regulation of IR signaling and DNL in human HCC tumor tissues. CONCLUSIONS: Our study reports a mechanism by which loss of ALDOB activates IR signaling primarily through releasing IR/ALDOB interaction to promote DNL and HCC, highlighting a potential therapeutic strategy in HCC.

Use of proteomics to identify mechanisms of hepatocellular carcinoma with the CYP2D6*10 polymorphism and identification of ANGPTL6 as a new diagnostic and prognostic biomarker.

BACKGROUND: Although an association between the cytochrome P4502D6 (CYP2D6) *10 (100C>T) polymorphism and hepatocellular carcinoma (HCC) is known, the mechanism remains unclear. Here we aimed to explore mechanisms of CYP2D6*10 (100C>T) polymorphism conferring to HCC, and screen markers for HCC. METHODS: Label-free global proteome profiling with 34 normal livers and peritumor tissue from 61 HCC patients was performed, and angiopoietin-like protein-6 (ANGPTL6) was evaluated in 2 liver samples validation cohorts and 2 blood specimens validation cohorts. RESULTS: We found a significantly decreased frequency of TT in HCC patients which reduced HCC susceptibility by 69.2% and was accompanied by lowered enzymatic activity for CYP2D6. Proteomic analysis revealed 1342 differentially expressed proteins (DEPs) that were associated with HCC and 88 DEPs were identified as 100 TT-related proteins, likely underlying the susceptibility to HCC. Twenty-two upregulated DEPs and 66 downregulated DEPs were mainly related to lipid metabolism and the extracellular matrix, respectively. High ANGPTL6 was associated with a higher risk to HCC and worse prognosis. ANGPTL6 was both an independent risk factor and an independent prognostic factor for HCC and exhibited strong potential for predicting HCC occurrence, with comparable AUC values and higher sensitivity compared with alpha-fetoprotein. CONCLUSIONS: The TT genotype-associated decreased risk of HCC appears to be related to lowered CYP2D6 activity and altered protein expression in the tumor microenvironment, and ANGPTL6 is a promising new diagnostic and prognostic biomarker for HCC. Our findings reveal new mechanistic insights for polymorphisms related to HCC risk and provide avenues for screening for HCC.

The CD68+ macrophages to CD8+ T-cell ratio is associated with clinical outcomes in hepatitis B virus (HBV)-related hepatocellular carcinoma.

BACKGROUND: Tumor microenvironment plays an essential role during the progression of hepatocellular carcinoma (HCC). Tumor infiltrating immune cells (TILs) was an important component of tumor microenvironment. However, whether TIL features are correlated with the prognosis of HCC patients remains unclear. METHODS: Cancer tissue and paired paracancerous tissues from 220 stage II∼III HBV-related HCC patients were collected. TILs were analyzed using a tyramide signal amplification system combined with immunohistochemistry. Kaplan-Meier survival analysis was conducted to investigate the associations between the prognosis and the infiltrating pattern of TILs. RESULTS: The patients were classified into three distinct subgroups (Clusters (C)1-3) with different overall survival (OS) and disease-free survival (DFS) according to the distribution pattern of TILs. The CD68/CD8 ratio in the cancer SA was correlated with the prognosis. Patients with a higher CD68/CD8 ratio exhibited poorer OS and DFS than those with a lower ratio. The CD68/CD8 ratio in the cancer SA was an independent factor for OS prediction but not DFS. CONCLUSION: CD68+ macrophages and CD8+ T-cells are essential immunological determinants for HBV-related HCC prognosis, and the CD68/CD8 ratio in cancer SA is a novel, prognostic factor for OS prediction in HBV-related HCC patients.

A20 suppresses hepatocellular carcinoma proliferation and metastasis through inhibition of Twist1 expression.

BACKGROUND: Aberrant expression of A20 has been reported in several human malignancies including hepatocellular carcinoma (HCC). However, its clinical relevance and potential role in HCC remain unknown. METHODS: Quantitative PCR, Western blots and immunohistochemistry analyses were used to quantify A20 expression in HCC samples and cell lines. The correlation of A20 expression with clinicopathologic features was analyzed in a cohort containing 143 patients with primary HCC. Kaplan-Meier curves were used to evaluate the association between A20 expression and patient survival. Functional studies were performed to determine the effects of A20 on proliferation and metastasis of HCC cells in vitro and in vivo. RESULTS: Expression of A20 was increased in HCC tissues and cell lines. Increased expression of A20 was negatively correlated with the tumor size, TNM stage, tumor thrombus formation, capsular invasion and serum AFP levels. Patients with higher A20 expression had a prolonged disease-free survival and overall survival than those with lower A20 expression. Forced expression of A20 significantly inhibited the proliferative and invasive properties of HCC cells both in vitro and in vivo, whereas knockdown of A20 expression showed the opposite effects. Further studies revealed that expression of A20 was inversely correlated with Twist1 levels and NF-κB activity in HCC tissues and cell lines. A20-induced suppression of proliferation and migration of HCC cells were mainly mediated through inhibition of Twist1 expression that was regulated at least partly by A20-induced attenuation of NF-κB activity. CONCLUSIONS: Our results demonstrate that A20 plays a negative role in the development and progression of HCC probably through inhibiting Twist1 expression. A20 may serve as a novel prognostic biomarker and potential therapeutic target for HCC patients.

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA-bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 µg/ml, respectively, with a dynamic range between 0.19 and 2.04 µg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 µg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.

A novel risk scoring system predicts overall survival of hepatocellular carcinoma using cox proportional hazards machine learning method.

BACKGROUND: Robust and practical prognosis prediction models for hepatocellular carcinoma (HCC) patients play crucial roles in personalized precision medicine. MATERIAL AND METHODS: We recruited two independent HCC cohorts (discovery cohort and validation cohort), totally consisting of 222 HCC patients undergone surgical resection. We quantified the expressions of immune-related proteins (CD8, CD68, CD163, PD-1 and PD-L1) in paired HCC tissues and non-tumor liver tissues from these HCC patients using immunohistochemistry (mIHC) assays. We constructed the HCC prognosis prediction model using five different machine learning methods based on the patients in the discovery cohort, such as Cox proportional hazards (CoxPH). RESULTS: We identified 19 features that were associated with overall survival of HCC patients in the discovery cohort (p < 0.1), such as immune-related features CD68+ and CD8+ cell infiltration. We constructed five HCC prognosis prediction models using five different machine learning methods. Among the five different machine learning models, the CoxPH model achieved the best performance (area under the curve [AUC], 0.839; C-index, 0.779). According to the risk score from CoxPH model, we divided HCC patients into high-risk group/low-risk group. In both discovery cohort and validation cohort, the patients in low-risk group showed longer overall survival compared with those in high-risk group (p = 1.8 × 10-7 and 3.4 × 10-5, respectively). Moreover, our novel scoring system efficiently predicted the 6, 12, and 18 months survival rate of HCC patients with AUC >0.75 in both discovery cohort and validation cohort. In addition, we found that the scoring system could also distinguish the patients with high/low risks of relapse in both discovery cohort and validation cohort (p = 0.00015 and 0.00012). CONCLUSION: The novel CoxPH-based risk scoring model on clinical, laboratory-testing and immune-related features showed high prediction efficiencies for overall survival and recurrence of HCCs undergone surgical resection. Our results may be helpful to optimize clinical follow-up or therapeutic interventions.

Mechanism of HBx carcinogenesis interaction with non-coding RNA in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an extremely malignant tumor that affects individuals throughout the world. One of the main causes of HCC is hepatitis B virus (HBV). Therefore, it is crucial to understand the mechanisms underlying HBV carcinogenesis. Increasing evidence suggests that the HBV X protein (HBx), which is encoded by HBV, plays a significant role in cell apoptosis, DNA damage repair, and cell cycle regulation. This ultimately leads to the development of HCC. Additionally, recent studies have shown that non-coding RNA (ncRNA) also contributes to the carcinogenesis and pathogenesis of different of tumors. ncRNA plays a significant role in the formation of HCC by regulating the inflammatory signaling pathway, activating immune cells, and modifying epigenetics. However, it remains unclear whether ncRNA is involved in the regulation of the carcinogenic mechanisms of HBx. This article reviews the carcinogenic mechanism of HBx and its interaction with ncRNA, providing a novel strategy for the clinical diagnosis and treatment of HCC.

Identification of Cytochrome P450 2E1 as a Novel Target in Glioma and Development of Its Inhibitor as an Anti-Tumor Agent.

Glioblastoma (GBM) is a devastating inflammation-related cancer for which novel therapeutic targets are urgently required. Previous studies of the authors indicate Cytochrome P450 2E1 (CYP2E1) as a novel inflammatory target and develop a specific inhibitor Q11. Here it is demonstrated that CYP2E1 overexpression is closely related to higher malignancy in GBM patients. CYP2E1 activity is positively correlated with tumor weight in GBM rats. Significantly higher CYP2E1 expression accompanied by increased inflammation is detected in a mouse GBM model. Q11, 1-(4-methyl-5-thialzolyl) ethenone, a newly developed specific inhibitor of CYP2E1 here remarkably attenuates tumor growth and prolongs survival in vivo. Q11 does not directly affect tumor cells but blocks the tumor-promoting effect of microglia/macrophage (M/Mφ) in the tumor microenvironment through PPARγ-mediated activation of the STAT-1 and NF-κB pathways and inhibition of the STAT-3 and STAT-6 pathways. The effectiveness and safety of targeting CYP2E1 in GBM are further supported by studies with Cyp2e1 knockout rodents. In conclusion, a pro-GBM mechanism in which CYP2E1-PPARγ-STAT-1/NF-κB/STAT-3/STAT-6 axis fueled tumorigenesis by reprogramming M/Mφ and Q11 as a promising anti-inflammatory agent for GBM treatment is uncovered.

The HBV Specially-Related Long Noncoding RNA HBV-SRL Involved in the Pathogenesis of Hepatocellular Carcinoma.

Hepatitis B virus (HBV) is one of the major risk factors for HCC (hepatocellular carcinoma) occurrence with a diverse role in the pathogenesis of HCC. More works need to be performed to elucidate a more thorough understanding of the molecular mechanisms involving in HBV-induced HCC, although some mechanisms such as genome integration have been reported. In the present study, aberrantly expressed lncRNAs were identified between HCC tumor tissues with or without HBV infection. Among these molecules, HBV specially-related long noncoding RNA (HBV-SRL) was further found to correlate with poor prognosis and a shorter overall survival time in HCC patients with HBV infection. Additionally, HBV-SRL was found function as oncogene by upregulating the NF-κB2 expression. These data suggest that HBV infection altered gene expression pattern in liver cells which contributed to HBV-related HCC development, and HBV-SRL may serve as a new molecular marker or potential therapeutic target of HBV-related HCC.

Investigation of Plasma cell-free cancer genome chromosomal instability as a tool for targeted minimally invasive biomarkers for primary liver cancer diagnoses.

PURPOSE: To characterize plasma cell-free cancer genome chromosomal instabilities (CIN) in patients with liver cancer and to evaluate the potential of CIN as minimally invasive biomarkers for primary liver cancer (PLC) diagnoses. EXPERIMENTAL DESIGN: We collected 196 plasma samples from 172 individuals in two cohorts, a discovery cohort of surgery ineligible PLC patients and a validation cohort of hepatectomy patients with pathological disease confirmations. All samples were subjected to HiSeq X10 sequencing followed by a customized bioinformatics workflow Ultrasensitive Chromosome Aneuploidy Detection (UCAD). RESULTS: In the discovery cohort, 29 significant copy number changes were identified in plasma from surgery-ineligible PLC. Twenty-two (95.7%) surgery-ineligible liver cancers were identified as harboring copy number changes in at least 1 of 29 segments. Meanwhile 40/41 (97.6%) noncancers harbored no changes. In the validation cohort, 54 (69.4%) surgery-eligible liver cancers were identified with positive screening, all of which were subsequently confirmed as cancer by pathological examination. Moreover, 26/27 = 96.3% noncancers were identified with negative screening. UCAD-positive screening was significantly associated with microvascular invasion (OR > 10, 95% CI:[2.53,]), tumor stages B and C (OR = 8.59, 95% CI [1.07, 400]), and tumor size ≥ 3 cm (OR = 5.68, 95% CI [1.43, 28.1]). Furthermore, we collected 29 followed-up plasma samples from 19 postsurgery patients. Nine (31.0%) postsurgery samples from 6 (31.5%) patients were identified with positive screening. Among them, 3 patients (50.0%) with positive screening were then confirmed as having disease recurrences. CONCLUSIONS: In addition to AFP, plasma cell-free DNA sequencing is a useful tool for primary liver cancer diagnoses.

Development and evaluation of immunoassay for zeranol in bovine urine.

A high affinity polyclonal antibody-based enzyme linked immunosorbent assay (ELISA) was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1:5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 microg/ml, and the detection limit was 0.02 microg/ml for the assay. The overall recoveries and the coefficients of variation (CVs) were in the range of 82% to approximately 127% and 3.5% to approximately 8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol (ranging from 0.2 to 10 microg/ml) were detected by the ELISA and liquid chromatography (LC) method, and good correlations were obtained between the two methods (R(2)=0.9643). We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an alternative for the conventional LC method for zeranol in bovine urine.

Bif-1 promotes tumor cell migration and metastasis via Cdc42 expression and activity.

Tumor metastasis is the process by which tumor cells disseminate from tumors and enter nearby and distant microenvironments for new colonization. Bif-1 (BAX-interacting factor 1), which has a BAR domain and an SH3 domain, has been reported to be involved in cell growth, apoptosis and autophagy. However, the influence of Bif-1 on metastasis has been less studied. To understand the role of Bif-1 in metastasis, we studied the expression levels of Bif-1 in human HCC specimens using immunohistochemistry, a tissue microarray and quantitative PCR. The function of Bif-1 was assessed in migration and translocation assays and the pulmonary metastatic animal model. The relationship between Bif-1 and the Rho family was determined using immunoblot analyses and chromatin immunoprecipitation. The results showed that the expression of Bif-1 was higher in hepatocellular carcinoma (HCC) than matched adjacent non-tumor liver tissues. Increased Bif-1 expression was associated with tumor size and the intercellular spread and metastasis of HCC. Analysis of the relationship between Bif-1 expression and patients' clinical characteristics revealed that patients with higher levels of Bif-1 had shorter disease-free and overall survival rates. Knockdown of Bif-1 with RNAi suppressed the migration of HCC cells and pulmonary metastasis and decreased the expression of Cdc42, a member of the Rho family. Bif-1 localized to the cytosol and nucleus and interacted with the promoter transcription region of Cdc42, which may regulate Cdc42 expression. Our results demonstrate a novel role of Bif-1 in HCC, in which Bif-1 promotes cell metastasis by regulating Cdc42 expression and activity.

Roles of dopamine receptors and their antagonist thioridazine in hepatoma metastasis.

Tumor metastasis is the most common cause of death and poor prognosis for cancer patients. Therapeutics that prevent tumor metastasis are the key to prolonging the lifespan of cancer patients. Cancer stem cells are believed to be critical in the metastatic process. Recently, drug screening for cancer stem cells reports that antipsychotic drugs displayed potential anticancer activity. Thioridazine, one of the antipsychotic drugs for dopamine receptors (DRs), is shown to induce the differentiation of cancer stem cells in leukemic disease and breast cancer, but it is not known if this drug would affect liver cancer. In this study, expression of DR5 was higher in tumors than in nontumor adjacent tissues, while DR1 was lower in human hepatocellular carcinoma (HCC) than those in the adjacent tissues. Other DRs were very low or undetectable. Treatment of HCC cells with thioridazine displays a dose-dependent response in HCC cell lines SNU449, LM3, and Huh7. Thioridazine treatment reduced cell viability and sphere formation of HCC cell lines through induction of G0/G1 cell cycle arrest and suppression of stemness genes CD133, OCT4, and EpCam. It also inhibited cell migration via suppression of epithelial-mesenchymal transition (EMT)-related genes such as twist2 and E-cadherin. Thioridazine-pretreated LM3 cells decreased the capacity of tumorigenesis in nude mice. Taken together, our data suggest that thioridazine may have the potential role in treatment of HCC.

A splicing variant of Merlin promotes metastasis in hepatocellular carcinoma.

Merlin, which is encoded by the tumour suppressor gene Nf2, plays a crucial role in tumorigenesis and metastasis. However, little is known about the functional importance of Merlin splicing forms. In this study, we show that Merlin is present at low levels in human hepatocellular carcinoma (HCC), particularly in metastatic tumours, where it is associated with a poor prognosis. Surprisingly, a splicing variant of Merlin that lacks exons 2, 3 and 4 ((Δ2-4)Merlin) is amplified in HCC and portal vein tumour thrombus (PVTT) specimens and in the CSQT2 cell line derived from PVTT. Our studies show that (Δ2-4)Merlin interferes with the capacity of wild-type Merlin to bind ß-catenin and ERM, and it is expressed in the cytoplasm rather than at the cell surface. Furthermore, (Δ2-4)Merlin overexpression increases the expression levels of ß-catenin and stemness-related genes, induces the epithelium-mesenchymal-transition phenotype promoting cell migration in vitro and the formation of lung metastasis in vivo. Our results indicate that the (Δ2-4)Merlin variant disrupts the normal function of Merlin and promotes tumour metastasis.

Low glucose promotes CD133mAb-elicited cell death via inhibition of autophagy in hepatocarcinoma cells.

CD133 on cancer stem cells is a potential therapeutic target. In this study, CD133 antibody (CD133mAb) treatment resulted in cell death in hepatoma LM3, HepG2, Hep3B and Huh-7 cells, especially under low glucose condition. The treatment also inhibited formation of spheroids, colonies, and xenograft tumors. Ectopic CD133 enabled hepatocyte L02 to be suppressed by CD133mAb and increased spheroid formation. CD133mAb caused cell death in primary HCC cells and sensitized them to Doxorubicin and Cisplatin. The antibody effect was attributed to suppressing autophagy and promoting necrotic cell death. Therefore, targeting CD133 under low glucose condition is a potential therapeutic approach for hepatocarcinomas.