Tumor Progression In Hepatocellular Carcinoma Research Articles

A pipeline to create predictive functional networks: application to the tumor progression of hepatocellular carcinoma

BackgroundIntegrating genome-wide gene expression patient profiles with regulatory knowledge is a challenging task because of the inherent heterogeneity, noise and incompleteness of biological data. From the computational side, several solvers for logic programs are able to perform extremely well in decision problems for combinatorial search domains. The challenge then is how to process the biological knowledge in order to feed these solvers to gain insights in a biological study. It requires formalizing the biological knowledge to give a precise interpretation of this information; currently, very few pathway databases offer this possibility.ResultsThe presented work proposes an automatic pipeline to extract automatically regulatory knowledge from pathway databases and generate novel computational predictions related to the state of expression or activity of biological molecules. We applied it in the context of hepatocellular carcinoma (HCC) progression, and evaluate the precision and the stability of these computational predictions. Our working base is a graph of 3383 nodes and 13,771 edges extracted from the KEGG database, in which we integrate 209 differentially expressed genes between low and high aggressive HCC across 294 patients. Our computational model predicts the shifts of expression of 146 initially non-observed biological components. Our predictions were validated at 88% using a larger experimental dataset and cross-validation techniques. In particular, we focus on the protein complexes predictions and show for the first time that NFKB1/BCL-3 complexes are activated in aggressive HCC. In spite of the large dimension of the reconstructed models, our analyses over the computational predictions discover a well constrained region where KEGG regulatory knowledge constrains gene expression of several biomolecules. These regions can offer interesting windows to perturb experimentally such complex systems.ConclusionThis new pipeline allows biologists to develop their own predictive models based on a list of genes. It facilitates the identification of new regulatory biomolecules using knowledge graphs and predictive computational methods. Our workflow is implemented in an automatic python pipeline which is publicly available at https://github.com/LokmaneChebouba/key-pipeand contains as testing data all the data used in this paper.

MFAP2 Promotes the Proliferation of Cancer Cells and Is Associated With a Poor Prognosis in Hepatocellular Carcinoma

Backgrounds:Microfibril-associated protein 2 (MFAP2) is an extracellular matrix protein that regulates the function of microfibrils by interacting with fibrillin. MFAP2 has been reported to play an important role in metabolic diseases and has been shown to be significantly overexpressed in head and neck squamous cell carcinoma and Hepatocellular carcinoma (HCC). However, the molecular function and prognostic value of MFAP2 have never been reported in HCC or other tumors.Methods:In the present study, expression characteristics of MFAP2 in HCC, its influence on the development of HCC, as well as its function and potential mechanism in HCC were verified by Quantitative reverse transcription-polymerase chain reaction, bioinformatics data mining and in vitro cell experiments.Results:MFAP2 was prominently high-expressed in HCC and associated with cancer stages. HCC patients with higher MFAP2 expression displayed lower overall survival (OS) and disease-specific survival(DSS), while there was no significant difference in recurrence-free survival (RFS). In vitro experiments showed that downregulation of MFAP2 inhibited proliferation, migration level of HCC cells. Transcription factors, DNA methyltransferases, immune factors may interact with MFAP2 mRNA to promote tumor progression in HCC.Conclusion:These findings suggest that MFAP2 may play a key role in the development of HCC. Therefore, MFAP2 may be a valuable prognostic marker and an effective anticancer target in HCC.

CircRNA hsa_circ_104566 Sponged miR-338-3p to Promote Hepatocellular Carcinoma Progression

Circular RNAs (circRNAs) could sponge micro-RNAs (miRNAs) to regulate tumor progression of hepatocellular carcinoma (HCC). Hsa_circ_104566 contributes to papillary thyroid carcinoma progression. However, the tumorigenic mechanism of hsa_circ_104566 on HCC remains enigmatic. The role of hsa_circ_104566 on HCC was therefore evaluated in this study. First, the high expression of hsa_circ_104566 was found in HCC tissues, which was significantly associated with poor prognosis in HCC patients. Second, Hsa_circ_104566 promoted HCC progression by decreasing apoptosis and E-cadherin, while increasing cell viability, proliferation, migration, invasion, and N-cadherin. On the other hand, HCC progression was suppressed by knockdown of hsa_circ_104566. Hsa_circ_104566 could target miR-338-3p, and its expression was negatively correlated with miR-338-3p in HCC patients. Moreover, miR-338-3p suppressed protein expression of Forkhead box protein 1 (FOXP1) and had a negative correlation with FOXP1 in HCC patients. Functional assay further indicated that the promotion of HCC progression by hsa_circ_104566 was reversed by miR-338-3p, and miR-338-3p inhibitor could counteract the effect of hsa_circ_104566 knockdown on the suppression of HCC progression. In vivo assay indicated that hsa_circ_104566 knockdown suppressed HCC tumor growth and metastasis. In conclusion, hsa_circ_104566 sponged miR-338-3p to promote HCC progression, providing a potential therapeutic target for cancer intervention.

FKBPL Is a Potential Prognostic Biomarker and Correlated with Immune Infiltrates and T Cells Exhaustion in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a malignant tumor with high mortality and poor prognosis. And FKBPL plays a crucial role on the development of cancers. This study aims to explore the prognostic potential of FKBPL and comprehensively analyze the correlations between FKBPL, immune infiltration, and T cells function in HCC. GEO database, Oncomine databases, TIMER2.0 and GEPIA2 were used to analyze FKBPL expression, relationship between FKBPL expression and survival of HCC patients as well as immune infiltration, related gene marker sets and T cells exhaustion in HCC. We used STRING database to analysis PPI (protein-protein interaction) for FKBPL and visualization by Cytoscape. Metascape was used for enrichment analysis. FKBPL upregulates in most of the cancer types including HCC. Survival analysis demonstrated that FKBPL shows significant impact on survival of HCC patients with overall survival and disease-free survival. And survival rate of HCC patients with high FKBPL expression was significantly correlated with race, gender and tumor purity of HCC patients. Moreover, FKBPL expression positively correlates with high immune infiltration levels in most of the immune cells and functional T cells including exhausted T cells, Th1, Th2 and Tfh. Specifically, multiple key genes of exhausted T cells comprising PD-1, CTLA4, LAG3, TIM-3, and GZMB have remarkable interaction with FKBPL. At last, we found10 genes including PRRT1, ANKRD49, RBCK1, CSNK1D, HSP90AA1, HSP90AB1, GTSE1, CDKN1A, AVPR2, and EGFL8 were interacted with FKBPL. Enrichment analysis showed that apoptotic signaling pathway, regulation of myeloid cell differentiation, cell death in response to oxidative stress, PIP3 activates AKT signaling may involve in HCC tumor progression about FKBPL. In conclusion, FKBPL is a potential prognostic biomarker and correlated with immune infiltrates and T cells exhaustion in HCC.

Long non-coding RNA LINC00473 acts as a microRNA-29a-3p sponge to promote hepatocellular carcinoma development by activating Robo1-dependent PI3K/AKT/mTOR signaling pathway.

Background:Long non-coding RNAs have suppressive or oncogenic effects in various types of cancers by serving as competing endogenous RNAs for specific microRNAs. In the present study, we aim to delineate the underlying mechanism by which the LINC00473/miR-29a-3p/Robo1 axis affects cell proliferation, migration, invasion, and metastasis in hepatocellular carcinoma (HCC).Methods:The level of Robo1 was examined in HCC tissues and cells, along with its regulatory effects on proliferation, migration, and invasion of HCC cells. Afterwards, the possible involvement of the PI3K/AKT/mTOR signaling pathway was determined. Next, miR-29a-3p expression was overexpressed or inhibited to investigate its regulatory role on HCC cell activities. The interaction among miR-29a-3p, Robo1, and LINC00473 was further characterized. Finally, a xenograft tumor in nude mice was conducted to measure tumorigenesis and metastasis in vivo.Results:miR-29a-3p was downregulated while Robo1 was upregulated in HCC tissues and cells. miR-29a-3p targeted Robo1 and negatively regulated its expression. In response to miR-29a-3p overexpression, Robo1 silencing or LINC00473 silencing, HCC cell proliferation, migration, invasion, tumor progression, and metastasis were impeded, which was involved with the inactivation of the PI3K/AKT/mTOR signaling pathway. Notably, LINC00473 could competitively bind to miR-29a-3p to upregulate Robo1 expression.Conclusion:LINC00473 might be involved in HCC progression by acting as a miR-29a-3p sponge to upregulate the expression of Robo1 that activates the PI3K/AKT/mTOR signaling pathway, which leads to enhanced cell proliferation, migration, invasion, tumor progression, and metastasis in HCC.

Apoptosis induction and ERK/NF-κB inactivation are associated with magnolol-inhibited tumor progression in hepatocellular carcinoma in vivo.

Although hepatitis B and/or hepatitis C virus were recognized as major risk factor for the development of hepatocellular carcinoma (HCC), certain occupational, environmental, and lifestyle factors also play key roles in HCC tumorigenesis. Moreover, in molecular signaling route, extracellular signal-regulated kinase (ERK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was found to be overexpressed and linked to poor prognosis in HCC. Thus, to identify possible nature compound that can suppress ERK/NF-κB may be benefit to HCC patient. Magnolol, a natural compound derived from herbal plant Magnolia officinalis, has been recognized as a liver protection and antitumor reagent. However, whether magnolol-inhibited HCC progression correlates with disruption of ERK/NF-κB signaling is remained unclear. In this studies, we performed SK-Hep1/luc2 HCC bearing animal model to investigate the anticancer efficacy and mechanism of magnolol on tumor progression. Tumor size and tumor growth rate were dramatically suppressed after treatment of magnolol. In addition, expression of phospho-ERK (p-ERK), NF-κB p65 (Ser536), and tumor progression-associated proteins, such as matrix metallopeptidase 9 (MMP-9), vascular endothelial growth factor (VEGF), X-linked inhibitor of apoptosis protein (XIAP), and CyclinD1 were all significantly decreased by magnolol. Most important, major extrinsic and intrinsic apoptosis signaling factors, including active caspase-8 and caspase-9 were both enhanced by magnolol. This study indicated that apoptosis induction through extrinsic/intrinsic pathways and blockage of ERK/NF-κB activation were associated with magnolol-inhibited tumor progression in HCC in vivo.

Neutrophil/Lymphocyte Ratio Predicts Increased Risk of Immediate Progressive Disease following Chemoembolization of Hepatocellular Carcinoma.

To demonstrate that patients with hepatocellular carcinoma (HCC) and elevated baseline neutrophil/lymphocyte ratio (NLR) have a significantly greater risk of progressive disease following initial transarterial chemoembolization. A total of 190 HCC patients (149 male/41 female) treated with transarterial chemoembolization between July 2013 and July 2017 were reviewed. Mean patient age was 62. Child-Pugh grades were 132 A, 61 B, and 4 C. Tracked criteria included etiology of cirrhosis, tumor number, Barcelona Clinic Liver Cancer score, diameter of the largest 2 tumors, and presence ofportal vein thrombosis. Complete blood count with differential before the procedure was used for NLR calculation. Follow-up imaging was performed 2 months after treatment. The modified response evaluation criteria in solid tumors were used to assess response. The association between baseline NLR and tumor response (ordinal modified response evaluation criteria in solid tumors categories) on 2-month follow-up imaging was evaluated using the proportional odds logistic regression model. A total of 194 patients (76.6%) patients had a preprocedural NLR <3.5, and 59 (23%) patients had a preprocedural NLR ≥3.5. There was a statistically significant association between baseline NLR and immediate progression on 2-month follow-up imaging (mean NLR 4.10, 2.76, 2.72, and 2.48 for progressive and stable disease and partial and complete response, respectively; odds ratio 2.1, P= .04). NLR (P= .021) and tumor multiplicity (P= .011) predicted progressive disease at 2-month imaging. Elevated baseline NLR is associated with higher rates of HCC tumor progression at 2-month follow-up imaging after transarterial chemoembolization.

Validation of Hepatocellular Carcinoma Experimental Models for TGF-β Promoting Tumor Progression.

Transforming growth factor beta (TGF-β) is a pleiotropic cytokine with dual role in hepatocellular carcinoma (HCC). It acts as tumor-suppressor and tumor-promoter in the early and late stage respectively. TGF-β influences the tumor-stroma cross-talk affecting the tumoral microenvironment. Therefore, inhibiting the TGF- β mediated pathway alone and/or in combination with chemotherapeutics represents an important therapeutic option. Experimental models to dissect the role of TGF-β in HCC tumor progression as well as the effectiveness of specific inhibitors are tricky. HCC cell lines respond to TGF-β according to their epithelial phenotype. However, the mesenchymal and more aggressive HCC cell lines in vitro, do not develop tumors when transplanted in vivo, thus hampering the understanding of molecular pathways that dictate outcome. In addition, in this model the native immune system is abolished, therefore the contribution of inflammation in hepatocarcinogenesis is unreliable. Different strategies have been set up to engineer HCC animal models, including genetically modified mice, chemically induced HCC, or hydrodynamic techniques. Patient-derived xenograft is currently probably the most fascinating model, keeping in mind that models cannot mirror all the reality. In this context, we discuss the different available HCC mouse models including our experimental model treated with inhibitor of TGF-β receptor Type I kinase (Galunisertib) and a potential role of exosomes in TGF-β moderated tumor progression of HCC. Unfortunately, no positive results were obtained in our treated orthotopic model because it does not reproduce the critical tumor-stroma interactions of the HCC.

1985PD - Aspartate-β-hydroxylase drives hepatocelluar carcinoma progression to metastasis fueling glutamine via HIF1α-mediated mitophagy

BackgroundAspartame (asparagynal)-β-hydroxylase (ASPH) leads to hepatocellular carcinoma (HCC) progression, but the potential molecular mechanism of ASPH on HCC progression to metastasis is still obscure. Our aim was to explore the correlation between ASPH and HIF1α-mediated mitophagy fueling via glutamine during tumor progression to metastasis in HCC. MethodsBioinformatics analysis constructed a composite gene-gene functional interaction network for ASPH regulatory genes to predict potential cellular mechanisms and then verified in vivo and vitro. ASPH+/-expressing stable cell line of HepG2 and Huh7 cell lines were structured by lentivirus encoding the wild-type ASPH open reading frame and ASPH-CRISPR/Cas9 system. 140 cases of HCC patients and a sbcutaneous xenograft HCC tumor model were used to evaluate HCC tumor growth and progression in vivo. The effect of ASPH on mitophagy, glutamine metabolism, angiogenesis and metastasis in HCC were tested via western blot, RT-PCR, immunohistochemical staining, electron microscopy, hematoxylin-eosin (HE) and immunocytofluorescent staining. The HIF1α inhibitor 2-Methoxyestradiol (2-MeOE2) and the serum free cultivation were used to conduct the related rescue experiments in vitro. ResultsIn this study, ASPH improved the expression of LC3-II/LC3-I,Atg7, Atg5 and Beclin1 and down-regulated the expression of TOMM20 and P62. Meanwhile, ASPH also significantly increased the expression of GLS1, which reflects the glutamine metabolism level of tumor cells. In addition, ASPH also significantly inhibited the expressions of HIF1α, BNIP3 and NIX proteins in HCC. Furthermore, a significant association between ASPH and CD34 expression was observed in HCC patients (P=0.024). ConclusionsOur finding suggests that ASPH fuels glutamine via HIF1α-mediated mitophagy to drive cancer angiogenesis and metastasis in HCC. ASPH may act as a promising therapeutic target in HCC patients. Legal entity responsible for the studyThe authors. FundingNational Natural Science Foundation of China (No. 81470877) Beijing Natural Science Foundation (No.7192085). DisclosureAll authors have declared no conflicts of interest.

Down-regulation of MARCO associates with tumor progression in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is a malignant tumor with high mortality due to lack of early diagnostic methods and effective treatments, and the molecular mechanisms are intricate and remain unclear. In the present study, the role of macrophage receptor with collagenous structure (MARCO) in tumor advancement of HCC was investigated. We examined expression level of MARCO in HCC samples, corresponding adjacent nontumor tissues and six hepatoma cell lines by polymerase chain reaction and immunohistochemistry (IHC). Clinical information of HCC patients was also analyzed. The role of MARCO involved in HCC progression via multiple functional experiments in vitro and in vivo was investigated. Bioinformatics analysis was conducted to further explore biological functions of MARCO. We found MARCO was suggestively down-regulated in HCC and associated with favorable prognosis, and MARCO upregulation oppressed tumor cell migration and invasion. Besides, overexpression of MARCO not only promoted apoptosis of hepatoma cells but also suppressed proliferation in vivo and in vitro. Furthermore, gene set enrichment analysis (GSEA) analysis suggested that MARCO may be related to the P53 signaling pathway, and this prediction was confirmed in this study as well. In sum, our study indicated that MARCO was involved in HCC progression and it can be defined as a novel probable biomarker as well as treatment target for HCC.

Eukaryotic elongation factor 2 kinase promotes angiogenesis in hepatocellular carcinoma via PI3K/Akt and STAT3.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with increasing mortality in China. Angiogenesis is crucial for tumor formation, development and metastasis in HCC. Previous studies indicated that high expression levels of elongation factor 2 kinase (eEF2K), a protein kinase that negatively regulates the elongation stage of translation, were associated with poor prognosis of HCC. Here, we show that pharmacological inhibition or knockdown of eEF2K in highly metastatic liver cancer cells inhibits their colony forming and migratory capacities, as well as reducing their invasiveness. Importantly, knocking down eEF2K by lentiviral directed shRNA prevented tumor growth and angiogenesis of HCC in mice. Silencing of eEF2K in endothelial cells (HUVECs) led to a reduction in vascularization, evidenced by a decrease in capillary-like structures in the matrigel. Notably, knocking down eEF2K reduced the expression of angiogenesis-related growth factors in liver cancer cells and the expression of growth factor receptors on HUVECs, and thus restricted signaling crosstalk that promotes angiogenesis between HCC cells and endothelial cells. We also showed that silencing of eEF2K effectively reduced protein levels of SP1/KLF5 transcription factors and hence decreased the levels of bound SP1/KLF5 to the VEGF promoter, resulted in a decrease in VEGF mRNA expression. Knocking down eEF2K also led to a striking decrease in the phosphorylation of PI3K/Akt and STAT3, indicating inactivation of these tumorigenic pathways. Taken together, our data suggest that eEF2K contributes to angiogenesis and tumor progression in HCC via SP1/KLF5-mediated VEGF expression, as well as the subsequent stimulation of PI3K/Akt and STAT3 signaling.

A reliable LC-MS/MS method for the quantification of natural amino acids in mouse plasma: Method validation and application to a study on amino acid dynamics during hepatocellular carcinoma progression

A simple and fast LC-MS/MS method was developed and validated for simultaneous quantification of 20 proteinogenic l-amino acids (AAs) in a small volume (5 μL) of mouse plasma. Chromatographic separation was achieved on an Intrada Amino Acid column within 13 min via gradient elution with an aqueous solution containing 100 mM ammonium formate and an organic mobile phase containing acetonitrile, water and formic acid (v:v:v = 95:5:0.3), at the flow rate of 0.6 mL/min. Individual AAs and corresponding stable-isotope-labeled AAs internal standards were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this LC-MS/MS method as a specific, accurate, and reliable assay. This LC-MS/MS method was thus utilized to compare the dynamics of individual plasma AAs between healthy and orthotopic hepatocellular carcinoma (HCC) xenograft mice housed under identical conditions. Our results revealed that, 5 weeks after HCC tumor progression, plasma l-arginine concentrations were significantly decreased in HCC mice while l-alanine and l-threonine levels were sharply increased. These findings support the utilities of this LC-MS/MS method and the promise of specific AAs as possible biomarkers for HCC.

Long Noncoding RNA MALAT1 Regulates Cancer Glucose Metabolism by Enhancing mTOR-Mediated Translation of TCF7L2.

Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer metabolism at the level of both glycolysis and gluconeogenesis are mostly unknown. We previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here, we investigated the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 and activation of the mTORC1-4EBP1 axis. Pharmacological or genetic inhibition of mTOR and Raptor or expression of a hypophosphorylated mutant version of eIF4E-binding protein (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy polysomes, probably through the TCF7L2 5'-untranslated region (UTR), as determined by polysome fractionation and 5'UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell lines affected their metabolism and abolished their tumorigenic potential, suggesting that the effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken together, our findings suggest that MALAT1 contributes to HCC development and tumor progression by reprogramming tumor glucose metabolism. SIGNIFICANCE: These findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated translation of the transcription factor TCF7L2.

OLFM4 Enhances STAT3 Activation and Promotes Tumor Progression by Inhibiting GRIM19 Expression in Human Hepatocellular Carcinoma.

Olfactomedin 4 (OLFM4) induces signal transducer and activator of transcription 3 (STAT3) activation by inhibiting gene associated with retinoid‐interferon‐induced mortality 19 (GRIM19), a strong STAT3 suppressor gene; however, the mechanisms of OLFM4 for regulating GRIM19‐STAT3 cascade in hepatocellular carcinoma (HCC) remain unclear. The functions and regulations of OLFM4, GRIM19, and STAT3 activation in HCC progression were evaluated using surgical specimens collected from 111 HCC patients or 2 HCC cell lines in vitro. Moreover, the cancer stem cell–like property of OLFM4 mediated by leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5), known as an intestinal stem cell marker, was investigated. OLFM4 was increased in HCC compared with adjacent liver tissue. The multivariate analysis revealed that high OLFM4 expression was an independent factor for poor prognosis. OLFM4 expression was negatively correlated with GRIM19 expression and positively correlated with STAT3 activation in HCC, thereby increasing cell cycle progression. OLFM4 knockdown in HCC cells increased GRIM19 expression and inhibited STAT3 activation; however, after double knockdown of GRIM19 and OLFM4, STAT3 activation decreased by OLFM4 knockdown was increased again. OLFM4 knockdown increased cell apoptosis, inhibited cell proliferation, and suppressed cancer stem cell–like property in HCC cells. The incidence of hematogenous recurrence was higher in HCC patients with high OLFM4 expression, suggesting that anoikis resistance of HCC was enhanced by OLFM4. In clinical cases, LGR5 expression and CD133 expression was correlated with OLFM4 expression in HCC, leading to poor patient prognosis. In vitro, LGR5 enhanced cancer stem cell–like property by up‐regulating OLFM4 through the Wnt signaling pathway. Conclusion: OLFM4 is induced by the LGR5‐Wnt signaling pathway and is strongly associated with aggressive tumor progression and poor prognosis in HCC by regulating STAT3‐induced tumor cell proliferation and cancer stem cell–like property. Therefore, OLFM4 is a novel prognostic predictor and a potential therapeutic target for patients with HCC.

Δ42PD-1 inhibitor acts as a PD-1-independent immunotherapy against human hepatocellular carcinoma

Abstract Pathogen infection-induced chronic inflammation is a key contributor to both carcinogenesis and progression of hepatocellular carcinoma (HCC). Persistent toll-like receptor 4 (TLR4) signaling activation can lead to inflammatory responses and chronic inflammation in the tumor microenvironment. Previously, Δ42PD1, a PD1 isoform, has been shown to directly interact with its cognate receptor TLR4 and contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Here, we aim to determine whether such interaction may also occur in HCC to promote TLR4 signaling activation and in turn induce inflammatory responses, contributing to HCC progression. We studied the relationship between TLR4, Δ42PD1, pro-inflammatory cytokines and tumor development in humanized mouse models and HCC patients. In consistence with others’ findings, TLR4 signal was shown to be associated with chronic inflammation and HCC tumor progression. Furthermore, we demonstrated that Δ42PD1 activated TLR4-mediated inflammatory responses in HCC cells via cJUN MAPK transcriptional activation. Notably, Δ42PD1-TLR4 pathway activation likely formed a feedback loop to induce Δ42PD1-expression CD3+ T cells, a new phenotype of T cell exhaustion, further contributing to tumor development. Importantly, administration of a Δ42PD1-specific monoclonal antibody CH101 interrupted Δ42PD1-TLR4 pathway and displayed therapeutic efficacy in both subcutaneous and orthotopic HCC models. Together, our results demonstrated the importance of Δ42PD1-TLR4-mediated inflammatory responses in promoting HCC and provided conclusive evidence that targeting Δ42PD1-TLR4 pathway reduced inflammatory responses to arrest HCC development.

Nuclear GAPDH is vital for hypoxia-induced hepatic stellate cell apoptosis and is indicative of aggressive hepatocellular carcinoma behavior.

Background/aim: Hepatic stellate cells (HSCs) are critical determinants of liver tumor behavior such as vascular invasion, cell proliferation and migration. The apoptosis of HSCs can inhibit tumor growth and contribute to repressing hepatocellular carcinoma (HCC) progression. Our study aims to investigate the impact of nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on HSCs under hypoxic conditions and the association of nuclear GAPDH with HCC patient outcomes and tumor progression.Patients and methods: Following stable cell passage, 0.3% O2 was used to induce hypoxia. Cell proliferation and apoptosis were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays and flow cytometry, respectively. Proteins expression were detected by extracting nuclear and cytoplasmic proteins and performing Western blots. GAPDH nuclear translocation was blocked by the agent deprenyl. Immunohistochemical staining for GAPDH was investigated in 137 HCC tissue samples from our center. An analysis of the clinicopathological features, Kaplan-Meier analysis and Cox proportional hazards regression analysis were applied.Results: MTT assays and flow cytometry analyses showed that the nuclear accumulation of GAPDH led to the apoptotic death of HSCs, while blockade of this process with deprenyl significantly decreased apoptosis. Western blots revealed that deprenyl inhibited the nuclear translocation of GAPDH. An analysis of the immunohistochemical staining of HSCs in HCC tissue samples (137) revealed that nuclear GAPDH expression was significantly positively correlated with HIF-1α expression. Overall survival (OS) and time-to-recurrence (TTR) estimated by Kaplan-Meier analyses showed that patients with high HIF-1α or low nuclear GAPDH levels in HSCs had significantly poorer prognosis compared with patients with low HIF-1α or high nuclear GAPDH expression in HSCs. Moreover, patients with combined high HIF-1α/low nuclear GAPDH expression in HSCs had the worst prognosis. The Cox regression analysis revealed that the combination of nuclear GAPDH/HIF-1α expression in HSCs was an independent prognostic factor for OS and TTR in HCC patients.Conclusions: These findings provide a novel mechanism underlying the involvement of intranuclear GAPDH in hypoxia-induced HSCs apoptosis and a correlation between nuclear GAPDH levels and the clinical prognosis, which may prompt the development of a novel therapeutic strategy for HCC.

The role of ERCC1 and AFP gene polymorphism in hepatocellular carcinoma.

The aim of this study was to evaluate the effects of polymorphisms in excision repair cross-complementation group 1 (ERCC1) and alpha-fetoprotein (AFP) genes and their haplotypes on the susceptibility to hepatocellular carcinoma (HCC), and to decipher the association between single-nucleotide polymorphisms (SNPs) and clinicopathologic characteristics of HCC.Peripheral blood DNA was extracted from 206 subjects. SNaPshot technique was used for genotyping 5 SNP sites of the ERCC1 rs735482, rs1046282, rs3212948, and AFP rs737241, rs4024 genotypes. Chi-squared test and logistic regression model were used to analyze the relationship of different genotypes or haplotype and the susceptibility and clinicopathologic characteristics of HCC.The frequency of GG.GA and AA genotypes at the AFP rs737241 site in the case and control groups showed statistically significant differences (P < .05). The risk of HCC in subjects carrying mutated allele A (GA+AA) was increased by 0.543-times (P < .05) compared to that in the subjects with the GG genotype. Significant differences were observed in the linkage disequilibrium between 2 of the five SNPs (P < .05); the frequency of ERCC1 C-C and AFP A-A haplotypes was significantly lower in the case group than in the control group (P < .05). The results of clinicopathologic analysis showed that A allele at the rs737241 locus could increase the expression level of AFP (P = .007), the rs1046282 mutation C allele could increase the AFP expression level (P = .011), rs4024 locus mutation A allele could reduce the risk of vascular invasion (P = .013), rs3212948 locus mutation T allele could reduce the differentiation of liver cancer (P = .022), rs1046282 locus C allele could reduce the DNA load of hepatitis B virus (P = .035), and rs735482 A allele could increase the tumor size in HCC (P = .037).The SNPs in rs737241 for AFP gene may correlate with the occurrence of HCC. The SNPs in ERCC1 and AFP genes may affect the prognosis of HCC, offering reliable information for early prediction of tumor progression and diagnosis of HCC.

The long non-coding RNA AK001796 contributes to poor prognosis and tumor progression in hepatocellular carcinoma.

Increasing studies have indicated the important functions of long non-coding RNAs (lncRNAs) in tumorigenesis and progression including hepatocellular carcinoma (HCC). The study aims to explore the role of long non-coding RNA AK001796 in HCC progression. Quantitative Real-time polymerase chain reaction (QRT-PCR) analysis was performed to examine the lncRNA AK001796 expression in 73 cases of human HCC tissue samples and matched adjacent normal tissues. Besides, the relationship between lncRNA AK001796 expression and clinicopathologic characteristics was analyzed. Overall survival (OS) curves of patients were constructed by the Kaplan-Meier methods. Multivariate Cox regression analysis was used to evaluate independent risk factors affecting HCC prognosis. Cell proliferation and invasion abilities are analyzed by cell counting kit-8 (CCK-8) and transwell invasion assays. We showed that the lncRNA AK001796 expression was significantly up-regulated in HCC tissues and cell lines, compared to their controls, respectively. Higher lncRNA AK001796 expression closely correlated with tumor size (p<0.05), TNM stage (p<0.05) and the poor overall survival (OS) rate of HCC patients (p<0.05). Besides, multivariate Cox regression analysis found that lncRNA AK001796 expression was identified as an independent risk factor for HCC prognosis. In vitro, we showed that lncRNA AK001796 knockdown markedly suppressed cell proliferation and cell invasion abilities. Our results suggested that lncRNA AK001796 acts as a predictor of HCC prognosis and may provide an important clinical value for HCC treatment.

Overexpression of miR-664 is associated with poor overall survival and accelerates cell proliferation, migration and invasion in hepatocellular carcinoma.

IntroductionHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. This study aimed to investigate the expression patterns of microRNA-664 (miR-664) in HCC tissues and cells, and assess its clinical significance and functional role in HCC.Patients and methodsOne hundred and thirty-four paired HCC and non-cancerous tissues were collected from patients who underwent surgery in Qianfoshan Hospital affiliated to Shandong University (Shandong, China) between 2009 and 2012. Expression of miR-664 was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Prognostic value of miR-664 in HCC was evaluated using Kaplan–Meier survival analysis and Cox regression analysis. Cell proliferation was analyzed using the CCK-8 assay, and cell migration and invasion of HCC cells was evaluated by the Transwell assay.ResultsExpression of miR-664 was significantly upregulated in HCC tissues and cells when compared with the normal controls (all P<0.05). MiR-664 expression was associated with lymph node metastasis, TNM stage and differentiation (all P<0.05) in the HCC patients. High miR-664 expression predicted poor overall survival (log-rank P=0.004) and acted as an independent prognostic factor (HR =1.945, 95% CI=1.078–3.508, P=0.027). According to cell experiments, the upregulation of miR-664 could promote, whereas the downregulation of miR-664 could inhibit proliferation, migration and invasion of HCC cells (all P<0.05). SIVA1 was predicted as a direct target gene of miR-664 in HCC.ConclusionAll data indicated that overexpression of miR-664 is associated with poor prognosis of HCC patients, and may enhance tumor progression of HCC by targeting SIVA1. MiR-664 may be a candidate therapeutic target for HCC treatment.

Autophagic flux modulation by Wnt/β-catenin pathway inhibition in hepatocellular carcinoma.

Autophagy targets cellular components for lysosomal-dependent degradation in which the products of degradation may be recycled for protein synthesis and utilized for energy production. Autophagy also plays a critical role in cell homeostasis and the regulation of many physiological and pathological processes and prompts this investigation of new agents to effect abnormal autophagy in hepatocellular carcinoma (HCC). 2,5-Dichloro-N-(2-methyl-4-nitrophenyl) benzenesulfonamide (FH535) is a synthetic inhibitor of the Wnt/β-catenin pathway that exhibits anti-proliferative and anti-angiogenic effects on different types of cancer cells. The combination of FH535 with sorafenib promotes a synergistic inhibition of HCC and liver cancer stem cell proliferation, mediated in part by the simultaneous disruption of mitochondrial respiration and glycolysis. We demonstrated that FH535 decreased HCC tumor progression in a mouse xenograft model. For the first time, we showed the inhibitory effect of an FH535 derivative, FH535-N, alone and in combination with sorafenib on HCC cell proliferation. Our study revealed the contributing effect of Wnt/β-catenin pathway inhibition by FH535 and its derivative (FH535-N) through disruption of the autophagic flux in HCC cells.