ASB3 promotes hepatocellular carcinoma progression by mediating DR5 ubiquitination in TRAIL resistance.

Ankyrin-repeat proteins with a suppressor of cytokine signaling box (ASB) proteins belong to the E3 ubiquitin ligase family. 18 ASB members have been identified whose biological functions are mostly unexplored. Here, we discovered that ASB3 was essential for hepatocellular carcinoma (HCC) development and high ASB3 expression predicted poor clinical outcomes. ASB3 silencing induced HCC cell growth arrest and apoptosis in vitro and in vivo. Liver-specific deletion of Asb3 gene suppressed diethylnitrosamine (DEN)-induced liver cancer development. Mechanistically, ASB3 interacted with death receptor 5 (DR5), which promoted ubiquitination and degradation of DR5. We further showed that ASB3 knockdown stabilized DR5 and increased the sensitivity of liver cancer cells to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a DR5-dependent manner in cellular and in animal models. In summary, we demonstrated that ASB3 promoted ubiquitination and degradation of DR5 in HCC, suggesting the potential of targeting ASB3 to HCC treatment and overcome TRAIL resistance.

Disruption of SLFN11 Deficiency-Induced CCL2 Signaling and Macrophage M2 Polarization Potentiates Anti-PD-1 Therapy Efficacy in Hepatocellular Carcinoma.

BACKGROUND & AIMS: The therapeutic effect of immune checkpoint inhibitors (ICIs) is poor in hepatocellular carcinoma (HCC) and varies greatly among individuals. Schlafen (SLFN) family members have important functions in immunity and oncology, but their roles in cancer immunobiology remain unclear. We aimed to investigate the role of the SLFN family in immune responses against HCC. METHODS: Transcriptome analysis was performed in human HCC tissues with or without response to ICIs. A humanized orthotopic HCC mouse model and a co-culture system were constructed, and cytometry by time-of-flight technology was used to explore the function and mechanism of SLFN11 in the immune context of HCC. RESULTS: SLFN11 was significantly up-regulated in tumors that responded to ICIs. Tumor-specific SLFN11 deficiency increased the infiltration of immunosuppressive macrophages and aggravated HCC progression. HCC cells with SLFN11 knockdown promoted macrophage migration and M2-like polarization in a C-C motif chemokine ligand 2-dependent manner, which in turn elevated their own PD-L1 expression by activating the nuclear factor-κB pathway. Mechanistically, SLFN11 suppressed the Notch pathway and C-C motif chemokine ligand 2 transcription by binding competitively with tripartite motif containing 21 to the RNA recognition motif 2 domain of RBM10, thereby inhibiting tripartite motif containing 21-mediated RBM10 degradation to stabilize RBM10 and promote NUMB exon 9 skipping. Pharmacologic antagonism of C-C motif chemokine receptor 2 potentiated the antitumor effect of anti-PD-1 in humanized mice bearing SLFN11 knockdown tumors. ICIs were more effective in patients with HCC with high serum SLFN11 levels. CONCLUSIONS: SLFN11 serves as a critical regulator of microenvironmental immune properties and an effective predictive biomarker of ICIs response in HCC. Blockade of C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 signaling sensitized SLFN11low HCC patients to ICI treatment.

An Angiogenic Gene Signature for Prediction of the Prognosis and Therapeutic Responses of Hepatocellular Carcinoma.

Among cancer-related deaths worldwide, hepatocellular carcinoma (HCC) ranks second. The hypervascular feature of most HCC underlines the importance of angiogenesis in therapy. This study aimed to identify the key genes which could characterize the angiogenic molecular features of HCC and further explore therapeutic targets to improve patients' prognosis. Public RNAseq and clinical data are from TCGA, ICGC, and GEO. Angiogenesis-associated genes were downloaded from the GeneCards database. Then, we used multi-regression analysis to generate a risk score model. This model was trained on the TCGA cohort (n = 343) and validated on the GEO cohort (n = 242). The predicting therapy in the model was further evaluated by the DEPMAP database. We developed a fourteen-angiogenesis-related gene signature that was distinctly associated with overall survival (OS). Through the nomograms, our signature was proven to possess a better predictive role in HCC prognosis. The patients in higher-risk groups displayed a higher tumor mutation burden (TMB). Interestingly, our model could group subsets of patients with different sensitivities to immune checkpoint inhibitors (ICIs) and Sorafenib. We also predicted that Crizotinib, an anti-angiogenic drug, might be more sensitive to these patients with high-risk scores by the DEPMAP. The inhibitory effect of Crizotinib in human vascular cells was obvious in vitro and in vivo. This work established a novel HCC classification based on the gene expression values of angiogenesis genes. Moreover, we predicted that Crizotinib might be more effective in the high-risk patients in our model.

MFN1-dependent alteration of mitochondrial dynamics drives hepatocellular carcinoma metastasis by glucose metabolic reprogramming.

BACKGROUND: Mitochondrial dynamics plays an important role in tumour progression. However, how these dynamics integrate tumour metabolism in hepatocellular carcinoma (HCC) metastasis is still unclear. METHODS: The mitochondrial fusion protein mitofusin-1 (MFN1) expression and its prognostic value are detected in HCC. The effects and underlying mechanisms of MFN1 on HCC metastasis and metabolic reprogramming are analysed both in vitro and in vivo. RESULTS: Mitochondrial dynamics, represented by constant fission and fusion, are found to be associated with HCC metastasis. High metastatic HCC displays excessive mitochondrial fission. Among genes involved in mitochondrial dynamics, MFN1 is identified as a leading downregulated candidate that is closely associated with HCC metastasis and poor prognosis. While promoting mitochondrial fusion, MFN1 inhibits cell proliferation, invasion and migration capacity both in vitro and in vivo. Mechanistically, disruption of mitochondrial dynamics by depletion of MFN1 triggers the epithelial-to-mesenchymal transition (EMT) of HCC. Moreover, MFN1 modulates HCC metastasis by metabolic shift from aerobic glycolysis to oxidative phosphorylation. Treatment with glycolytic inhibitor 2-Deoxy-D-glucose (2-DG) significantly suppresses the effects induced by depletion of MFN1. CONCLUSIONS: Our results reveal a critical involvement of mitochondrial dynamics in HCC metastasis via modulating glucose metabolic reprogramming. MFN1 may serve as a novel potential therapeutic target for HCC.

MET Inhibitors Promote Liver Tumor Evasion of the Immune Response by Stabilizing PDL1.

BACKGROUND & AIMS: Inhibitors of MET have not produced satisfactory outcomes in trials of patients with liver cancer. We investigated the mechanisms of liver tumor resistance to MET inhibitors in mice. METHODS: We tested the effects of MET inhibitors tivantinib and capmatinib in the mouse hepatocellular carcinoma (HCC) cell line HCA-1 and in immune-competent and immunodeficient mice with subcutaneous tumors grown from this cell line. Tumors were collected from mice and tumor cells were analyzed by time-of-flight mass cytometry. We used short hairpin RNAs to weaken expression of MET in Hep3B, SK-HEP-1, HA59T, and HA22T liver cancer cell lines and analyzed cells by immunoblot, immunofluorescence, and immunoprecipitation assays. Mass spectrometry was used to assess interactions between MET and glycogen synthase kinase 3ß (GSK3B), and GSK3B phosphorylation, in liver cancer cell lines. C57/BL6 mice with orthotopic tumors grown from Hep1-6 cells were given combinations of capmatinib or tivantinib and antibodies against programmed cell death 1 (PDCD1; also called PD1); tumors were collected and analyzed by immunofluorescence. We analyzed 268 HCCsamples in a tissue microarray by immunohistochemistry. RESULTS: Exposure of liver cancer cell lines to MET inhibitors increased their expression of PD ligand 1 (PDL1) and inactivated cocultured T cells. MET phosphorylated and activated GSK3B at tyrosine 56, which decreased the expression of PDL1 by liver cancer cells. In orthotopic tumors grown in immune-competent mice, MET inhibitors decreased the antitumor activity of T cells. However, addition of anti-PD1 decreased orthotopic tumor growth and prolonged survival of mice compared with anti-PD1 or MET inhibitors alone. Tissue microarray analysis of HCC samples showed an inverse correlation between levels of MET and PDL1 and a positive correlation between levels of MET and phosphorylated GSK3B. CONCLUSIONS: In studies of liver cancer cell lines and mice with orthotopic tumors, MET mediated phosphorylation and activated GSK3B, leading to decreased expression of PDL1. Combined with a MET inhibitor, anti-PD1 and anti-PDL1 produced additive effect to slow growth of HCCs in mice.

Disruption of tumour-associated macrophage trafficking by the osteopontin-induced colony-stimulating factor-1 signalling sensitises hepatocellular carcinoma to anti-PD-L1 blockade.

OBJECTIVE: In the tumour microenvironment, critical drivers of immune escape include the oncogenic activity of the tumour cell-intrinsic osteopontin (OPN), the expression of programmed death ligand 1 (PD-L1) and the expansion of tumour-associated macrophages (TAMs). We investigated the feasibility of targeting these pathways as a therapeutic option in hepatocellular carcinoma (HCC) mouse models. DESIGN: We analysed the number of tumour-infiltrating immune cells and the inflammatory immune profiles in chemically induced liver tumour isolated from wild-type and OPNknockout (KO) mice. In vitro cell cocultures were further conducted to investigate the crosstalk between TAMs and HCC cells mediated by OPN, colony stimulating factor-1 (CSF1) and CSF1 receptor (CSF1R). The in vivo efficacy of anti-PD-L1 and CSF1/CSF1R inhibition was evaluated in OPN overexpressing subcutaneous or orthotopic mouse model of HCC. RESULTS: The numbers of TAMs, as well as the expression levels of M2 macrophage markers and PD-L1 were significantly decreased, but the levels of cytokines produced by T-helper 1 (Th1) cells were upregulated in tumour tissues from OPN KO mice compared with that from the controls. In addition, we observed a positive association between the OPN and PD-L1 expression, and OPN expression and TAM infiltration in tumour tissues from patients with HCC. We further demonstrated that OPN facilitates chemotactic migration, and alternative activation of macrophages, and promotes the PD-L1 expression in HCC via activation of the CSF1-CSF1R pathway in macrophages. Combining anti-PD-L1 and CSF1R inhibition elicited potent antitumour activity and prolonged survival of OPNhigh tumour-bearing mice. Histological, flow cytometric and ELISA revealed increased CD8+ T cell infiltration, reduced TAMs and enhanced Th1/Th2 cytokine balance in multiple mouse models of HCC. CONCLUSIONS: OPN/CSF1/CSF1R axis plays a critical role in the immunosuppressive nature of the HCC microenvironment. Blocking CSF1/CSF1R prevents TAM trafficking and thereby enhances the efficacy of immune checkpoint inhibitors for the treatment of HCC.

Targeting cancer stem cells and their niche: perspectives for future therapeutic targets and strategies.

A small subpopulation of cells within the bulk of tumors share features with somatic stem cells, in that, they are capable of self-renewal, they differentiate, and are highly resistant to conventional therapy. These cells have been referred to as cancer stem cells (CSCs). Recent reports support the central importance of a cancer stem cell-like niche that appears to help foster the generation and maintenance of CSCs. In response to signals provided by this microenvironment, CSCs express the tumorigenic characteristics that can drive tumor metastasis by the induction of epithelial-mesenchymal-transition (EMT) that in turn fosters the migration and recolonization of the cells as secondary tumors within metastatic niches. We summarize here recent advances in cancer stem cell research including the characterization of their genetic and epigenetic features, metabolic specialities, and crosstalk with aging-associated processes. Potential strategies for targeting CSCs, and their niche, by regulating CSCs plasticity, or therapeutic sensitivity is discussed. Finally, it is hoped that new strategies and related therapeutic approaches as outlined here may help prevent the formation of the metastatic niche, as well as counter tumor progression and metastatic growth.

Mutated EPHA2 is a target for combating lymphatic metastasis in intrahepatic cholangiocarcinoma.

Exploring the genetic aberrations favoring metastasis is important for understanding and developing novel strategies to combat cancer metastasis. It remains lack of effective treatment for the dismal prognosis of intrahepatic cholangiocarcinoma (ICC). Here, we aimed to study genetic alternations during lymph node metastasis of ICC and investigate potential mechanisms and clinical strategy focused on mutations. We performed whole-exome sequencing and transcriptome sequencing on samples from 30 ICC patients, including lymph node metastases from five of the patients. We identified the alterations of genetic pattern related to lymph node metastases of ICC. EPHA2, a member of the tyrosine kinase family, was found to be frequently mutated in ICC. Correlation analysis indicated that EPHA2 mutations were closely associated with lymph node metastasis of ICC. In vitro and in vivo experiments revealed that EPHA2 mutations could lead to ligand independent phosphorylation of Ser897, and promote lymphatic metastasis of ICC, in which NOTCH1 signaling pathway played an important role. In both in vitro assays and patient-derived xenografts, an inhibitor of Ser897 phosphorylation effectively suppressed the metastasis of ICC with mutated EPHA2. Our findings demonstrated that EPHA2 mutants may be an attractive therapeutic target for lymphatic metastasis of ICC.

Current perspectives of cancer-associated fibroblast in therapeutic resistance: potential mechanism and future strategy.

The goal of cancer eradication has been overshadowed despite the continuous improvement in research and generation of novel cancer therapeutic drugs. One of the undeniable existing problems is drug resistance due to which the paradigm of killing all cancer cells is ineffective. Tumor microenvironment plays a crucial role in inducing drug resistance besides cancer development and progression. Recently, many efforts have been devoted to understand the role of tumor microenvironment in cancer drug resistance as it provides the shelter, nutrition, and paracrine niche for cancer cells. Cancer-associated fibroblasts (CAFs), one major component of tumor microenvironment, reside in symbiotic relationship with cancer cells, supporting them to survive from cancer drugs. The present review summarizes the recent understandings in the role of CAFs in drug resistance in various tumors. Acknowledging the fact that drug resistance depends not only upon cancer cells but also upon the microenvironment niche could guide us to formulate novel cancer drugs and provide the optimal cancer treatment.

Genome-Wide Association Study Identifies a Genetic Prediction Model for Postoperative Survival in Patients with Hepatocellular Carcinoma.

BACKGROUND As an important aspect of tumor heterogeneity, genetic variation may influence susceptibility and prognosis in different types of cancer. By exploring the prognostic value of genetic variation, this study aimed to establish a model for predicting postoperative survival and assessing the impact of variation on clinical outcomes in patients with hepatocellular carcinoma (HCC). MATERIAL AND METHODS A genome-wide association study of 367 patients with HCC was conducted to identify single nucleotide polymorphisms (SNPs) associated with prognosis. Identified predictors were further evaluated in 758 patients. Two prognostic models were established using Cox proportional hazards regression and Nomogram strategy, and validated in another 316 patients. The effect of the SNP rs2431 was analyzed in detail. RESULTS A prognostic model including 5 SNPs (rs10893585, rs2431, rs34675408, rs6078460, and rs6766361) was established and exhibited high predictive accuracy for HCC prognosis. The panel combined with tumor node metastasis (TNM) stage resulted in a significantly higher c-index (0.723) than the individual c-index values. Stratified by the Nomogram prediction model, the median overall survival for the low-risk and high-risk groups were 100.1 versus 30.8 months (P<0.001) in the training set and 82.2 versus 22.5 months (P<0.001) in the validation set. A closer examination of rs2431 revealed that it may regulate the expression of FNDC3B by disrupting a microRNA-binding site. CONCLUSIONS This study established prediction models based on genetic factors alone or in combination with TNM stage for postoperative survival in patients with HCC, and identified FNDC3B as a potential therapeutic target for combating HCC metastasis.

C-C chemokine receptor type 1 mediates osteopontin-promoted metastasis in hepatocellular carcinoma.

In the hepatocellular carcinoma (HCC) microenvironment, chemokine receptors play a critical role in tumorigenesis and metastasis. Our previous studies have found that osteopontin (OPN) is a promoter for HCC metastasis. However, the role of chemokine receptors in OPN-induced HCC metastasis remains unclear. In this study, we demonstrate that OPN is dramatically elevated in HCC tissues with metastasis and that high expression of OPN correlates with poorer overall survival and higher recurrence rate. OPN upregulates chemokine receptor expression, migration, invasion and pulmonary metastasis in HCC. We find that C-C chemokine receptor type 1 (CCR1) and C-X-C chemokine receptor type 6 (CXCR6) are the most upregulated chemokine receptors induced by OPN. CCR1 knockdown results in reduction of migration, invasion and pulmonary metastasis induced by OPN in vitro and in vivo, whereas CXCR6 knockdown does not reverse OPN-promoted migration and invasion. Moreover, OPN upregulates the expression of CCR1 through activating phosphoinositide 3-kinase (PI3K)/AKT and hypoxia-inducible factor 1α (HIF-1α) in HCC cells. Furthermore, blockade of OPN-CCR1 axis with CCR1 antagonist significantly restrains the promoting effects of OPN on HCC progression and metastasis. In human HCC tissues, OPN expression shows significantly positive correlation with CCR1 expression, and the patients with high levels of both OPN and CCR1 have the most dismal prognosis. Collectively, our results indicate that the OPN-CCR1 axis in HCC is important for accelerating tumor metastasis and that CCR1 is a potential therapeutic target for controlling metastasis in HCC patients with high OPN.

RA190, a Proteasome Subunit ADRM1 Inhibitor, Suppresses Intrahepatic Cholangiocarcinoma by Inducing NF-KB-Mediated Cell Apoptosis.

BACKGROUND/AIMS: Effective drug treatment for intrahepatic cholangiocarcinoma (ICC) is currently lacking. Therefore, there is an urgent need for new targets and new drugs that can prolong patient survival. Recently targeting the ubiquitin proteasome pathway has become an attractive anti-cancer strategy. In this study, we aimed to evaluate the therapeutic effect of and identify the potential mechanisms involved in targeting the proteasome subunit ADRM1 for ICC. METHODS: The expression of ADRM1 and its prognostic value in ICC was analyzed using GEO and TCGA datasets, tumor tissues, and tumor tissue arrays. The effects of RA190 on the proliferation and survival of both established ICC cell lines and primary ICC cells were examined in vitro. Annexin V/propidium iodide staining, western blotting and immunohistochemical staining were performed. The in vivo anti-tumor effect of RA190 on ICC was validated in subcutaneous xenograft and patient-derived xenograft (PDX) models. RESULTS: ADRM1 levels were significantly higher in ICC tissues than in normal bile duct tissues. ICC patients with high ADRM1 levels had worse overall survival (hazard ratio [HR] = 2.383, 95% confidence interval [CI] =1.357 to 4.188) and recurrence-free survival (HR = 1.710, 95% CI =1.045 to 2.796). ADRM1 knockdown significantly inhibited ICC growth in vitro and in vivo. The specific inhibitor RA190 targeting ADRM1 suppressed proliferation and reduced cell vitality of ICC cell lines and primary ICC cells significantly in vitro. Furthermore, RA190 significantly inhibited the proteasome by inactivating ADRM1, and the consequent accumulation of ADRM1 substrates decreased the activating levels of NF-κB to aggravate cell apoptosis. The therapeutic benefits of RA190 treatment were further demonstrated in both subcutaneous implantation and PDX models. CONCLUSIONS: Our findings indicate that up-regulated ADRM1 was involved in ICC progression and suggest the potential clinical application of ADRM1 inhibitors (e.g., RA190 and KDT-11) for ICC treatment.

Whole-exome mutational and transcriptional landscapes of combined hepatocellular cholangiocarcinoma and intrahepatic cholangiocarcinoma reveal molecular diversity.

BACKGROUND: Primary liver cancer (PLC) is the third largest contributor to cancer mortality in the world. PLC is a heterogeneous disease that encompasses several biologically distinct subtypes including hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC). CHC is a distinct, albeit rare, subtype of PLC and is comprised of cells with histopathological features of both HCC and ICC. Several studies have focused on the mutation and expression landscapes of HCC and ICC. However, studies of CHC were rare. OBJECTIVE: The aim of the current study was to identify genetic and gene expression alterations in the carcinogenesis and development of CHC and ICC in the Chinese population. Unraveling both similar and differing patterns among these subtypes may help to identify personalized medicine approaches that could improve patient survival. METHODS: Whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-seq were performed on 10 ICC and 10 CHC samples, matched with adjacent non-tumor liver tissue specimens. Comparative analysis was performed using HCC datasets from The Cancer Genome Atlas (TCGA). RESULTS: Mutational and transcriptional landscapes of CHC and ICC were clearly delineated. TP53 and CTNNB1 were identified as exhibiting mutations in CHC. ARID1A, PBRM1, and IDH1 were frequently mutated in ICC. RYR3, FBN2, and KCNN3 are associated with cell migration and metastasis and might be driver genes in CHC. KCNN3 was identified as also exhibiting mutations in ICC. The ECM-receptor interaction pathway associated fibrogenic hepatic progenitor cell differentiation and liver fibrosis may play an important role in carcinogenesis of PLC. Chromatin remodeling and chromosome organization are key processes in carcinogenesis and development in PLC. P53 related pathways showed alterations in CHC and HCC. Inflammation may be a key factor involved in ICC carcinogenesis. CONCLUSION: CHC and ICC are different subtypes of PLC. This study discusses predominantly the molecular genetic details of PLC subtypes and highlights the need for an accurate diagnosis and treatment of specific PLC subtypes to optimize patient management.

Prospero-related homeobox 1 drives angiogenesis of hepatocellular carcinoma through selectively activating interleukin-8 expression.

Angiogenesis has been proven to play an important role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanism underlying HCC angiogenesis is not well understood. In this study, Prospero-related homeobox 1 (PROX1) was identified as a novel proangiogenic factor in HCC cell lines and tissues. A strong positive correlation was found between the levels of PROX1 and microvessel density in HCC tissues. Knockdown of PROX1 expression in HCC cells significantly inhibited the in vitro capillary tube formation by human vascular endothelial cells and in vivo angiogenesis of HCC, while overexpression of PROX1 in HCC cells induced the opposite effects. PROX1 and nuclear factor κB p65 expression levels were positively correlated in both HCC tissues and cell lines. PROX1 enhances the nuclear accumulation of p65 and stabilizes p65 by recruiting ubiquitin-specific protease 7 to prevent p65 ubiquitination. Consequently, PROX1 activated nuclear factor κB signaling and selectively promoted expression of the proangiogenic interleukin-8 (IL-8) by epigenetically stimulating the IL-8 promoter. Finally, progression of high PROX1 expression HCC in tumor xenograft mice could be effectively contained by an anti-IL-8 monoclonal antibody. CONCLUSIONS: We have identified PROX1 as a crucial promoter of HCC angiogenesis; our study provides an insight into PROX1's function in HCC progression and the potential therapeutic application of anti-IL-8 antibody in high PROX1 expression HCC patients. (Hepatology 2017;66:1894-1909).

Hepatic IFIT3 predicts interferon-α therapeutic response in patients of hepatocellular carcinoma.

Adjuvant interferon-α (IFN-α) therapy is used to control certain types of cancer in clinics. For hepatocellular carcinoma (HCC), IFN-α therapy is effective in only a subgroup of patients; therefore, identifying biomarkers to predict the response to IFN-α therapy is of high significance and clinical utility. As the induced IFN-stimulated gene expression following IFN-α treatment plays pivotal roles in IFN-α effects, we screened IFN-stimulated gene expression in HCC tissues and found that several IFN-stimulated genes were significantly decreased in HCC. Interestingly, expression of IFN-induced protein with tetratricopeptide repeats (IFIT) family members, including IFIT1, IFIT2, IFIT3, and IFIT5, was decreased in HCC tissues. We further analyzed the expression of IFIT family members in HCC and their roles in patients' responses to IFN-α therapy in two independent randomized controlled IFN-α therapy clinical trials of HCC patients. We found that higher expression of IFIT3, but not other IFITs, in HCC tissues predicts better response to IFN-α therapy, suggesting that IFIT3 may be a useful predictor of the response to IFN-α therapy in HCC patients. Mechanistically, IFIT3 enhanced the antitumor effects of IFN-α by promoting IFN-α effector responses both in vitro and in vivo. IFIT3 could bind signal transducer and activator of transcription 1 (STAT1) and STAT2 to enhance STAT1-STAT2 heterodimerization and nuclear translocation upon IFN-α treatment, thus promoting IFN-α effector signaling. CONCLUSION: Higher IFIT3 expression in HCC tissues predicts better response to IFN-α therapy in HCC patients; IFIT3 promotes IFN-α effector responses and therapeutic effects by strengthening IFN-α effector signaling in HCC. (Hepatology 2017;66:152-166).

Elevated G6PD expression contributes to migration and invasion of hepatocellular carcinoma cells by inducing epithelial-mesenchymal transition.

Altered metabolism is one of the hallmarks of cancer cells. Pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, is elevated in many cancers and contributes to tumor growth by producing ribose-5-phosphate and NADPH through PPP. However, the role of G6PD in hepatocellular carcinoma (HCC) metastasis and the clinical significance of G6PD in HCC progression and prognosis have not been well determined. In this study, by investigating tissue samples from HCC patients and HCC cell lines, we found that elevated G6PD expression is significantly associated with HCC metastasis and poor prognosis of HCCs, and that knockdown of G6PD inhibits in vitro proliferation, migration and invasion of HCC cell lines. Further studies reveal that G6PD contributes to HCC migration and invasion of hepatocellular carcinoma cells by inducing epithelial-mesenchymal transition through activation of signal transducer and activator of transcription 3 (STAT3) pathway. Our findings suggest that targeting G6PD could open up possibilities for metastasis intervention and improve the patients' outcomes for HCC.

MicroRNA-26a suppresses angiogenesis in human hepatocellular carcinoma by targeting hepatocyte growth factor-cMet pathway.

UNLABELLED: MicroRNA (miR)-26a can suppress tumor growth and metastasis of hepatocellular carcinoma (HCC). Since angiogenesis is important for tumor growth and metastasis, we investigated the possible roles of miR-26a in tumor angiogenesis. Down-regulation of miR-26a was found to correlate with an increased angiogenic potential of HCC. Through gain- and loss-of-function studies, miR-26a was demonstrated to significantly inhibit vascular endothelial growth factor A (VEGFA) expression in HCC cells and then suppress the promoting effects of HCC cells on in vitro proliferation, migration, and capillary tube formation of endothelial cells, as well as in vivo tumor angiogenesis of HCC. Hepatocyte growth factor (HGF) was identified as a target of miR-26a. HGF simulation antagonized the effects induced by miR-26a up-regulation. In contrast, silencing HGF induced similar effects to miR-26a. We further found that miR-26a exerted its antiangiogenesis function, at least in part, by inhibiting HGF-hepatocyte growth factor receptor (cMet) and its downstream signaling pathway, in turn, suppressing VEGFA production in HCC cells and impairing VEGFR2-signaling in endothelial cells. HCC patients who had high miR-26a, low HGF, low VEGFA, or low microvessel density (MVD) in tumor tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR-26a, or in combination with HGF, was demonstrated to be an independent prognostic indicator for OS and TTR of HCC patients. CONCLUSION: miR-26a could suppress tumor angiogenesis of HCC through HGF-cMet signaling, and it is a new hopeful therapeutic target and prognostic marker for HCC.

Osteopontin promoter polymorphisms at locus -443 significantly affect the metastasis and prognosis of human hepatocellular carcinoma.

UNLABELLED: Osteopontin (OPN) plays a crucial role in hepatocellular carcinoma (HCC) metastasis. However, little is known about the impact of OPN polymorphisms on cancer progression. In this study, we first identified the single nucleotide polymorphisms (SNPs) in the OPN promoter region by direct sequencing in 30 HCCs, and then evaluated the prognostic values of the selected ones in two large cohorts of 826 HCC patients. The identified SNPs were functionally analyzed using in vitro and in vivo assays and their correlations with OPN levels were also evaluated. Only SNP at locus -443 and their related haplotypes (Ht2: -1748A/-616G/-443T/-155* [*indicates base deletion]; Ht3: -1748A/-616G/-443C/-155*) were significantly associated with overall survival (OS) and time to recurrence (TTR). The patients with the -443TT/TC genotype or Ht2 had a shorter OS and TTR compared with those with -443CC genotype or Ht3. This was further confirmed in the validation cohort. Moreover, this correlation remained significant in patients with small HCCs (≤5 cm). Multivariate analyses indicated that the prognostic performance of the -443 genotypes (OS, P=0.031; TTR, P=0.005) and their related haplotypes (OS, P=0.002; TTR, P=0.001) was independent of other clinicopathological factors. The Ht2 and -443TT genotype could significantly increase the promoter transcriptional activity and expression level of OPN compared with the Ht3 or -443CC genotype, and lead to an obvious increase in both in vitro invasion and in vivo tumor growth and lung metastasis of HCC cells (P<0.05). CONCLUSION: The genetic variation at locus -443 of the OPN promoter plays important roles in the regulation of OPN expression and cancer progression of HCCs, which is a novel determinant and target for HCC metastasis and prognosis.

MicroRNA-26a suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting interleukin-6-Stat3 pathway.

UNLABELLED: Down-regulation of microRNA-26a (miR-26a) is associated with poor prognosis of hepatocellular carcinoma (HCC), but its functional mechanism in HCC remains unclear. In this study, we investigated the roles of miR-26a in tumor growth and metastasis of HCC and found that miR-26a was frequently down-regulated in HCC tissues. Down-regulation of miR-26a correlated with HCC recurrence and metastasis. Through gain- and loss-of-function studies, miR-26a was demonstrated to significantly inhibit in vitro cell proliferation, migration, and invasion. In addition, miR-26a induced G1 arrest and promoted apoptosis of HCC cells. Importantly, miR-26a suppressed in vivo tumor growth and metastasis in nude mice models bearing human HCC. Interleukin-6 (IL-6) was identified as a target of miR-26a. Knockdown of IL-6 induced effects on HCC cells similar to those induced by miR-26a. In contrast, IL-6 treatment abrogated the effects induced by miR-26a up-regulation. Moreover, miR-26a dramatically suppressed expression of signal transducer and activator of transcription 3 (Stat3) target genes, including Bcl-2, Mcl-1, cyclin D1, and MMP2. IL-6 treatment antagonized this effect, while knockdown of IL-6 by IL-6 short hairpin RNA (shIL-6) induced inhibitory effects on the expression of p-Stat3 and its main target genes, similar to miR-26a. The messenger RNA and protein levels of IL-6 inversely correlated with miR-26a in HCCs. Patients with high miR-26a or low IL-6 in HCC tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR-26a, IL-6, and their combination were demonstrated to be independent prognostic indicators for OS and TTR of HCC patients. CONCLUSION: miR-26a could suppress tumor growth and metastasis of HCC through IL-6-Stat3 signaling and is a novel prognostic marker and therapeutic target for HCC.