A Practical Risk Classification of Early Recurrence in Hepatocellular Carcinoma Patients with Microvascular invasion after Hepatectomy: A Decision Tree Analysis.

PURPOSE: This study was designed to establish risk classifications for early recurrence in hepatocellular carcinoma (HCC) patients with microvascular invasion (MVI) after hepatectomy. METHODS: The data of 563 HCC patients with MVI after hepatectomy from two hospitals were retrospectively reviewed. Kaplan-Meier curves and Cox proportional hazards regression models were used to analyse early recurrence. The risk classification for early recurrence was established by using classification and regression tree (CART) analysis and validated by using two independent validation cohorts from two hospitals. RESULTS: Multivariate analysis revealed that four indices, namely, infection of chronic viral hepatitis, MVI classification, tumour size, and serum alpha-fetoprotein (AFP), were independent prognostic factors for early recurrence in HCC patients with MVI. By CART analysis, MVI classification and serum AFP became the nodes of a decision tree and 3-stratification classifications that satisfactorily determined the risk of early recurrence were established. The area under the time-dependent receiver operating characteristic curve (AUC) values of the classification for early recurrence at 0.5, 1.0, and 2.0 years were 0.75, 0.73, and 0.71, respectively, which were all significantly higher than three common classic HCC stages (BCLC stage, Chinese stage, and TNM stage). The calibration curves showed good agreement between predictions by classification for early recurrence and actual survival outcomes. These prediction results also were confirmed in the independent internal and external validation cohorts. CONCLUSIONS: The 3 stratification classifications enabled satisfactory risk evaluation of early recurrence in HCC patients with MVI after hepatectomy.

PPARγ Coactivator-1α Suppresses Metastasis of Hepatocellular Carcinoma by Inhibiting Warburg Effect by PPARγ-Dependent WNT/ß-Catenin/Pyruvate Dehydrogenase Kinase Isozyme 1 Axis.

BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator-1α (PGC1α) is a key regulator of mitochondrial biogenesis and respiration. PGC1α is involved in the carcinogenesis, progression, and metabolic state of cancer. However, its role in the progression of hepatocellular carcinoma (HCC) remains unclear. APPROACH AND RESULTS: In this study, we observed that PGC1α was down-regulated in human HCC. A clinical study showed that low levels of PGC1α expression were correlated with poor survival, vascular invasion, and larger tumor size. PGC1α inhibited the migration and invasion of HCC cells with both in vitro experiments and in vivo mouse models. Mechanistically, PGC1α suppressed the Warburg effect through down-regulation of pyruvate dehydrogenase kinase isozyme 1 (PDK1) mediated by the WNT/ß-catenin pathway, and inhibition of the WNT/ß-catenin pathway was induced by activation of PPARγ. CONCLUSIONS: Low levels of PGC1α expression indicate a poor prognosis for HCC patients. PGC1α suppresses HCC metastasis by inhibiting aerobic glycolysis through regulating the WNT/ß-catenin/PDK1 axis, which depends on PPARγ. PGC1α is a potential factor for predicting prognosis and a therapeutic target for HCC patients.

Phosphoglucomutase 1 inhibits hepatocellular carcinoma progression by regulating glucose trafficking.

Glycogen metabolism commonly altered in cancer is just beginning to be understood. Phosphoglucomutase 1 (PGM1), the first enzyme in glycogenesis that catalyzes the reversible conversion between glucose 1-phosphate (G-1-P) and glucose 6-phosphate (G-6-P), participates in both the breakdown and synthesis of glycogen. Here, we show that PGM1 is down-regulated in hepatocellular carcinoma (HCC), which is associated with the malignancy and poor prognosis of HCC. Decreased PGM1 expression obstructed glycogenesis pathway, which leads to the increased flow of glucose into glycolysis, thereby promoting tumor cell proliferation and HCC development. The loss of forkhead box protein J2 (FOXJ2), at least partly due to low genomic copy number in HCC, releases cellular nucleic acid-binding protein (CNBP), a nucleic acid chaperon, to bind to and promote G-quadruplex formation in PGM1 promoter and therefore decreases PGM1 expression. In addition, integrated analyses of PGM1 and FOXJ2 expression provide a better prediction for the malignance and prognosis of HCC. This study establishes a tumor-suppressive role of PGM1 by regulating glucose trafficking and uncovers a novel regulatory mechanism of PGM1 expression.

Postoperative adjuvant transcatheter arterial chemoembolization improves the prognosis of patients with huge hepatocellular carcinoma.

BACKGROUND: Surgical resection of huge hepatocellular carcinoma (HCC, ≥ 10 cm) is potentially curative. More adjuvant treatments are needed to reduce relapses in these patients. We evaluated the influence of postoperative adjuvant transcatheter arterial chemoembolization (PA-TACE) on the prognosis of huge HCC. METHODS: Data from consecutive patients who underwent curative resection for huge HCC in our center were retrospectively collected. Recurrence-free survival (RFS) and overall survival (OS) were compared between patients who did and did not undergo PA-TACE. Propensity score matching (PSM) was used. RESULTS: Among the 255 enrolled patients, 93 underwent PA-TACE. The clinical outcomes were significantly better in the PA-TACE group than those in the non PA-TACE group (5-year RFS rate: 33.5% vs. 18.0%; 5-year OS rate: 47.0% vs. 28.0%, all P < 0.001). After PSM, similar results were obtained (5-year RFS rate: 28.8% vs. 17.6%, P < 0.001; 5-year OS rate: 42.5% vs. 25.0%, P = 0.004). PA-TACE decreased the possibility of early recurrence (< 2 years, crude cohort: P < 0.001, PSM cohort: P < 0.001) but not late recurrence (≥ 2 years, crude cohort: P = 0.692, PSM cohort: P = 0.325). Multivariable Cox regression analysis suggested that PA-TACE was an independent protective factor prolonging early RFS, RFS and OS. CONCLUSIONS: PA-TACE is a safe intervention for huge HCC patients after liver resection and improves outcomes.

An Eastern Hepatobiliary Surgery Hospital Microvascular Invasion Scoring System in Predicting Prognosis of Patients with Hepatocellular Carcinoma and Microvascular Invasion After R0 Liver Resection: A Large-Scale, Multicenter Study.

BACKGROUND: Microvascular invasion (MVI) is associated with poor postoperative survival outcomes in patients with hepatocellular carcinoma (HCC). An Eastern Hepatobiliary Surgery Hospital (EHBH) MVI scoring system was established to predict prognosis in patients with HCC with MVI after R0 liver resection (LR) and to supplement the most commonly used classification systems. MATERIALS AND METHODS: Patients with HCC with MVI who underwent R0 LR as an initial therapy were included. The EHBH-MVI score was developed from a retrospective cohort from 2003 to 2009 to form the training cohort. The variables associated with overall survival (OS) on univariate analysis were subsequently investigated using the log-rank test, and the EHBH-MVI score was developed using the Cox regression model. It was validated using an internal prospective cohort from 2011 to 2013 as well as three independent external validation cohorts. RESULTS: There were 1,033 patients in the training cohort; 322 patients in the prospective internal validation cohort; and 493, 282, and 149 patients in the three external validation cohorts, respectively. The score was developed using the following factors: α-fetoprotein level, tumor encapsulation, tumor diameter, hepatitis B e antigen positivity, hepatitis B virus DNA load, tumor number, and gastric fundal/esophageal varicosity. The score differentiated two groups of patients (≤4, >4 points) with distinct long-term prognoses outcomes (median OS, 55.8 vs. 19.6 months; p < .001). The predictive accuracy of the score was greater than the other four commonly used staging systems for HCC. CONCLUSION: The EHBH-MVI scoring system was more accurate in predicting prognosis in patients with HCC with MVI after R0 LR than the other four commonly used staging systems. The score can be used to supplement these systems. IMPLICATIONS FOR PRACTICE: Microvascular invasion (MVI) is a major determinant of survival outcomes after curative liver resection for patients with hepatocellular carcinoma (HCC). Currently, there is no scoring system aiming to predict prognosis of patients with HCC and MVI after R0 liver resection (LR). Most of the widely used staging systems for HCC do not use MVI as an independent risk factor, and they cannot be used to predict the prognosis of patients with HCC and MVI after surgery. In this study, a new Eastern Hepatobiliary Surgery Hospital (EHBH) MVI scoring system was established to predict prognosis of patients with HCC and MVI after R0 LR. Based on the results of this study, postoperative adjuvant therapy may be recommended for patients with HCC and MVI with an EHBH-MVI score >4. This score can be used to supplement the currently used HCC classifications to predict postoperative survival outcomes in patients with HCC and MVI.

Hippo Cascade Controls Lineage Commitment of Liver Tumors in Mice and Humans.

Primary liver cancer consists mainly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). A subset of human HCCs expresses a ICC-like gene signature and is classified as ICC-like HCC. The Hippo pathway is a critical regulator of normal and malignant liver development. However, the precise function(s) of the Hippo cascade along liver carcinogenesis remain to be fully delineated. The role of the Hippo pathway in a murine mixed HCC/ICC model induced by activated forms of AKT and Ras oncogenes (AKT/Ras) was investigated. The authors demonstrated the inactivation of Hippo in AKT/Ras liver tumors leading to nuclear localization of Yap and TAZ. Coexpression of AKT/Ras with Lats2, which activates Hippo, or the dominant negative form of TEAD2 (dnTEAD2), which blocks Yap/TAZ activity, resulted in delayed hepatocarcinogenesis and elimination of ICC-like lesions in the liver. Mechanistically, Notch2 expression was found to be down-regulated by the Hippo pathway in liver tumors. Overexpression of Lats2 or dnTEAD2 in human HCC cell lines inhibited their growth and led to the decreased expression of ICC-like markers, as well as Notch2 expression. Altogether, this study supports the key role of the Hippo cascade in regulating the differentiation status of liver tumors.

Microvascular invasion predicts a poor prognosis of solitary hepatocellular carcinoma up to 2 cm based on propensity score matching analysis.

AIM: Microvascular invasion (MVI) is not discussed for solitary hepatocellular carcinoma (HCC) up to 2 cm in the 8th Edition of the American Joint Committee on Cancer Staging Manual. The present study aimed to reappraise the influence of MVI on solitary HCC up to 2 cm in diameter. METHODS: Between January 2010 and December 2012, a retrospective cohort of 496 HCC patients from the Eastern Hepatobiliary Surgery Hospital was analyzed. Propensity score matching was carried out to balance the baseline characteristics. Survival analysis was carried out using the Kaplan-Meier method. Risk factors were evaluated using the Cox proportional hazards model. Multivariate logistic regression was used to identify the risk factors associated with MVI. RESULTS: All patients were classified into either an MVI-negative group (n = 332) or an MVI-positive group (n = 164). The MVI-positive group had poorer recurrence-free survival and overall survival before and after propensity score matching. The multivariate analysis showed that MVI; being male; increased total bilirubin levels, alanine transaminase levels and γ-glutamyl transpeptidase levels; decreased albumin levels; and HBV DNA load >103  IU/mL were risk factors for recurrence-free survival. MVI, older age, lower albumin levels, and cirrhosis were risk factors for overall survival. Age <50 years, alpha-fetoprotein >20 ng/mL, and lack of or an incomplete capsule were significantly independent predictors for MVI. CONCLUSIONS: MVI had a negative impact on the prognosis of solitary HCC up to 2 cm after curative hepatectomy. The 8th edition of the American Joint Committee on Cancer staging system could be improved by subdividing solitary HCC up to 2 cm according to MVI.

Overexpression of MTHFD1 in hepatocellular carcinoma predicts poorer survival and recurrence.

Aim: MTHFD1 was the enzyme providing one-carbon derivatives of tetrahydrofolate. We sought to investigate the impact of MTHFD1 on hepatocellular carcinoma (HCC). Methods: Bioinformatic analysis, western blot and immunohistochemistry were conducted to detect MTHFD1 expression in HCC. The relationships between MTHFD1 and prognosis of 172 HCCs were analyzed by Kaplan-Meier method and Cox proportional hazards model. Results: High MTHFD1 expression in HCC represented poor prognosis (overall survival p = 0.025; time to recurrence p = 0.044). Combining MTHFD1 with serum AFP, survival analysis demonstrated the prognosis of the MTHFD1 low expression and AFP ≤20 ng/ml group was better than that of the MTHFD1 high expression or AFP >20 ng/ml group and the MTHFD1 high expression and AFP >20 ng/ml group (overall survival p < 0.0001; time to recurrence p < 0.0001). Conclusion: High MTHFD1 expression in HCC indicated poorer prognosis. Combining MTHFD1 with serum AFP improved the accuracy of prognostic prediction.

Postoperative adjuvant sorafenib improves survival outcomes in hepatocellular carcinoma patients with microvascular invasion after R0 liver resection: a propensity score matching analysis.

BACKGROUND: Microvascular invasion (MVI) is a major determinant of survival outcome for hepatocellular carcinoma (HCC). This study aimed to investigate the efficacy of postoperative adjuvant Sorafenib (PA-Sorafenib) in HCC patients with MVI after R0 liver resection (LR). METHODS: The data of patients who underwent R0 LR for HCC with histologically confirmed MVI at the Eastern Hepatobiliary Surgery Hospital were retrospectively analyzed. The survival outcomes for patients who underwent PA-Sorafenib were compared with those who underwent R0 LR alone. Propensity score matching (PSM) analysis was performed. RESULTS: 728 HCC patients had MVI in the resected specimens after R0 resection, with 581 who underwent LR alone and 147 patients who received in additional adjuvant sorafenib. PSM matched 113 patients in each of these two groups. The overall survival (OS) and recurrence free survival (RFS) were significantly better for patients in the PA-sorafenib group (for OS: before PSM, P = 0.003; after PSM, P = 0.007), (for RFS: before PSM, P = 0.029; after PSM, P = 0.001), respectively. Similar results were obtained in patients with BCLC 0-A, BCLC B and Child-Pugh A stages of disease. CONCLUSIONS: PA-Sorafenib was associated with significantly better survival outcomes than LR alone for HCC patients with MVI.

Antiviral therapy improves survival in patients with HBV infection and intrahepatic cholangiocarcinoma undergoing liver resection.

BACKGROUND & AIMS: The impact of hepatitis B virus (HBV) infection on outcomes after resection of intrahepatic cholangiocarcinoma (ICC) has not been reported. The aim of this study was to examine the impact of antiviral therapy on survival outcomes after liver resection for patients with ICC and underlying HBV infection. METHODS: Data on 928 patients with ICC and HBV infection who underwent liver resection at two medical centers between 2006 and 2011 were analyzed. Data on viral reactivation, tumor recurrence, cancer-specific survival (CSS) and overall survival (OS) were obtained. Survival rates were analyzed using the time-dependent Cox regression model adjusted for potential covariates. RESULTS: Postoperative viral reactivation occurred in 3.3%, 8.3% and 15.7% of patients who received preoperative antiviral therapy, who did not receive preoperative antiviral therapy with a low, or a high HBV-DNA level (< or ≥2,000 IU/ml), respectively (p <0.001). A high viral level and viral reactivation were independent risk factors of recurrence (hazard ratio [HR] 1.22 and 1.34), CSS (HR 1.36 and 1.46) and OS (HR1.23 and 1.36). Five-year recurrence, CSS and OS were better in patients who received antiviral therapy (70.5%, 46.9% and 43.0%) compared with patients who did not receive antiviral therapy and had a high viral level (86.5%, 20.9% and 20.5%, all p <0.001), respectively. The differences in recurrence, CSS and OS were minimal compared with no-antiviral therapy patients with a low viral level (71.7%, 35.5% and 33.5%, p = 0.057, 0.051 and 0.060, respectively). Compared to patients with a high viral level who received no antiviral therapy, patients who initiated antiviral therapy either before or after surgery had better long-term outcomes (HR 0.44 and 0.54 for recurrence; 0.38 and 0.57 for CSS; 0.46 and 0.54 for OS, respectively). CONCLUSIONS: Viral reactivation was associated with worse prognoses after liver resection for HBV-infected patients with ICC. Antiviral therapy decreased viral reactivation and prolonged long-term survival for patients with ICC and a high viral level. LAY SUMMARY: Postoperative hepatitis B virus reactivation was associated with an increased complication rate and a decreased survival rate after liver resection in patients with ICC and hepatitis B virus infection. Antiviral therapy before liver resection reduced the risk of postoperative viral reactivation. Both pre- and postoperative antiviral therapy was effective in prolonging patient survival.

Regulator of Calcineurin 1 Gene Isoform 4, Down-regulated in Hepatocellular Carcinoma, Prevents Proliferation, Migration, and Invasive Activity of Cancer Cells and Metastasis of Orthotopic Tumors by Inhibiting Nuclear Translocation of NFAT1.

BACKGROUND & AIMS: Individuals with Down syndrome have a low risk for many solid tumors, prompting the search for tumor suppressor genes on human chromosome 21 (HSA21). We aimed to identify and explore potential mechanisms of tumor suppressors on HSA21 in hepatocellular carcinoma (HCC). METHODS: We compared expression of HSA21 genes in 14 pairs of primary HCC and adjacent noncancer liver tissues using the Affymetrix HG-U133 Plus 2.0 array (Affymetrix, Santa Clara, CA). HCC tissues and adjacent normal liver tissues were collected from 108 patients at a hospital in China for real-time polymerase chain reaction and immunohistochemical analyses; expression levels of regulator of calcineurin 1 (RCAN1) isoform 4 (RCAN1.4) were associated with clinical features. We overexpressed RCAN1.4 from lentiviral vectors in MHCC97H and HCCLM3 cells and knocked expression down using small interfering RNAs in SMMC7721 and Huh7 cells. Cells were analyzed in proliferation, migration, and invasion assays. HCC cells that overexpressed RCAN1.4 or with RCAN1.4 knockdown were injected into livers or tail veins of nude mice; tumor growth and numbers of lung metastases were quantified. We performed bisulfite pyrosequencing and methylation-specific polymerase chain reaction analyses to analyze CpG island methylation. We measured phosphatase activity of calcineurin in HCC cells. RESULTS: RCAN1.4 mRNA and protein levels were significantly decreased in primary HCC compared with adjacent noncancer liver tissues. Reduced levels of RCAN1.4 mRNA were significantly associated with advanced tumor stages, poor differentiation, larger tumor size, and vascular invasion. Kaplan-Meier survival analysis showed that patients with HCCs with lower levels of RCAN1.4 mRNA had shorter time of overall survival and time to recurrence than patients whose tumors had high levels of RCAN1.4 mRNA. In HCC cell lines, expression of RCAN1.4 significantly reduced proliferation, migration, and invasive activity. HCC cells that overexpressed RCAN1.4 formed smaller xenograft tumors, with fewer metastases and blood vessels, than control HCC cells. In HCC cells, RCAN1.4 inhibited expression of insulin-like growth factor 1 and vascular endothelial growth factor A by reducing calcineurin activity and blocking nuclear translocation of nuclear factor of activated T cells (NFAT1). HCC cells incubated with the calcineurin inhibitor cyclosporin A had decreased nuclear level of NFAT1. HCC cells had hypermethylation of a CpG island in the 5' regulatory region of RCAN1.4, which reduced its expression. CONCLUSIONS: RCAN1.4 is down-regulated in HCC tissues, compared with non-tumor liver tissues. RCAN1.4 prevents cell proliferation, migration, and invasion in vitro; overexpressed RCAN1.4 in HCC cells prevents growth, angiogenesis, and metastases of xenograft tumors by inhibiting calcineurin activity and nuclear translocation of NFAT1.

Chronic inflammation-elicited liver progenitor cell conversion to liver cancer stem cell with clinical significance.

The substantial heterogeneity and hierarchical organization in liver cancer support the theory of liver cancer stem cells (LCSCs). However, the relationship between chronic hepatic inflammation and LCSC generation remains obscure. Here, we observed a close correlation between aggravated inflammation and liver progenitor cell (LPC) propagation in the cirrhotic liver of rats exposed to diethylnitrosamine. LPCs isolated from the rat cirrhotic liver initiated subcutaneous liver cancers in nonobese diabetic/severe combined immunodeficient mice, suggesting the malignant transformation of LPCs toward LCSCs. Interestingly, depletion of Kupffer cells in vivo attenuated the LCSC properties of transformed LPCs and suppressed cytokeratin 19/Oval cell 6-positive tumor occurrence. Conversely, LPCs cocultured with macrophages exhibited enhanced LCSC properties. We further demonstrated that macrophage-secreted tumor necrosis factor-α triggered chromosomal instability in LPCs through the deregulation of ubiquitin D and checkpoint kinase 2 and enhanced the self-renewal of LPCs through the tumor necrosis factor receptor 1/Src/signal transducer and activator of transcription 3 pathway, which synergistically contributed to the conversion of LPCs to LCSCs. Clinical investigation revealed that cytokeratin 19/Oval cell 6-positive liver cancer patients displayed a worse prognosis and exhibited superior response to sorafenib treatment. CONCLUSION: Our results not only clarify the cellular and molecular mechanisms underlying the inflammation-mediated LCSC generation but also provide a molecular classification for the individualized treatment of liver cancer. (Hepatology 2017;66:1934-1951).

Loss of FBP1 facilitates aggressive features of hepatocellular carcinoma cells through the Warburg effect.

Reprogrammed metabolism has been identified as an emerging hallmark in cancer cells. It has been demonstrated that fructose-1, 6-bisphosphatase 1 (FBP1) as a rate-limiting enzyme in gluconeogenesis plays critical roles in tumor initiation and progression in several cancer types. However, function of FBP1 in hepatocellular carcinoma (HCC) is still not clear. In this study, we observed that the expression of FBP1 was obviously downregulated in the cell lines and tissues of HCC. Downregulation of FBP1 in HCC tissues was correlated with a lower overall survival rate and had a relatively higher tendency of tumor recurrence (n = 224). Silencing FBP1 could significantly promote colony formation, proliferation and metastasis of HCC cells, while ectopic overexpression of FBP1 resulted in impaired abilities of colony formation, proliferation and metastasis in vitro and in vivo. Mechanistically, silencing FBP1 facilitated glycolysis in HCC cell lines, which may be responsible for aggressiveness of HCC cells. We further found that targeting the Warburg effect using the specific inhibitor FX11 could suppress the aggressiveness of HCC cells which was mediated by loss of FBP1. These findings indicate that FBP1 appears to be a tumor suppressor in HCC. Strategies to restore the levels and activities of FBP1 might be developed to treat patients with HCC.

Pan-mTOR inhibitor MLN0128 is effective against intrahepatic cholangiocarcinoma in mice.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model. METHODS: We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study. RESULTS: Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation. CONCLUSIONS: This study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 vs. gemcitabine/oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to gemcitabine/oxaliplatin-based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.

Interferon-microRNA signalling drives liver precancerous lesion formation and hepatocarcinogenesis.

OBJECTIVE: Precancerous lesion, a well-established histopathologically premalignant tissue with the highest risk for tumourigenesis, develops preferentially from activation of DNA damage checkpoint and persistent inflammation. However, little is known about the mechanisms by which precancerous lesions are initiated and their physiological significance. DESIGN: Laser capture microdissection was used to acquire matched normal liver, precancerous lesion and tumour tissues. miR-484(-/-), Ifnar1(-/-) and Tgfbr2(△hep) mice were employed to determine the critical role of the interferon (IFN)-microRNA pathway in precancerous lesion formation and tumourigenesis. RNA immunoprecipitation (RIP), pull-down and chromatin immunoprecipitation (ChIP) assays were applied to explore the underlying mechanisms. RESULTS: miR-484 is highly expressed in over 88% liver samples clinically. DEN-induced precancerous lesions and hepatocellular carcinoma were dramatically impaired in miR-484(-/-) mice. Mechanistically, ectopic expression of miR-484 initiates tumourigenesis and cell malignant transformation through synergistic activation of the transforming growth factor-ß/Gli and nuclear factor-κB/type I IFN pathways. Specific acetylation of H3K27 is indispensable for basal IFN-induced continuous transcription of miR-484 and cell transformation. Convincingly, formation of precancerous lesions were significantly attenuated in both Tgfbr2(△hep) and Ifnar1(-/-) mice. CONCLUSIONS: These findings demonstrate a new protumourigenic axis involving type I IFN-microRNA signalling, providing a potential therapeutic strategy to manipulate or reverse liver precancerous lesions and tumourigenesis.

LIFR functions as a metastasis suppressor in hepatocellular carcinoma by negatively regulating phosphoinositide 3-kinase/AKT pathway.

Hepatocellular carcinoma (HCC) is one of the leading causes for cancer related mortality worldwide. Poor prognosis of HCC patients is mainly due to frequent metastasis and recurrence. Deregulation of metastasis suppressors in malignant cells plays critical roles during cancer metastasis. Thus, novel metastasis suppressors are urgently needed to be uncovered to shed new light on molecular mechanisms driving HCC metastasis. In the present study, decreased expression of leukemia inhibitory factor receptor (LIFR) was demonstrated in HCC, and its expression levels were even lower in HCC with metastasis. Downregulated LIFR expression predicted poor prognosis in HCC patients. LIFR was an independent and significant risk factor for their recurrence and survival. Silencing LIFR resulted in forced metastasis of HCC cells, whereas ectopic overexpression of LIFR attenuated migration and invasion of HCC cells in vitro and in vivo. Moreover, LIFR knockdown could activate phosphoinositide 3-kinase/V-akt Murine Thymoma Viral Oncogene Homolog (PI3K/AKT) signaling through enhancing phosphorylation of Janus kinase 1 (JAK1), which successively promoted matrix metalloproteinase 13 (MMP13) expression and HCC metastasis. Combination of LIFR and p-AKT or MMP13 was a more powerful predictor of poor prognosis for HCC patients. Together, these findings conclude that LIFR functions as a novel metastasis suppressor in HCC and may serve as a prognostic biomarker for HCC patients.

PTPN11/Shp2 overexpression enhances liver cancer progression and predicts poor prognosis of patients.

BACKGROUND & AIMS: We have previously reported that Shp2, a tyrosine phosphatase previously known as a pro-leukemogenic molecule, suppresses the initiation of hepatocellular carcinoma (HCC). However, the role of Shp2 in HCC progression remains obscure. METHODS: Shp2 expression was determined in human HCC using real-time PCR, immunoblotting and immunohistochemistry. Clinical significance of Shp2 expression was analyzed in 301 HCC tissues with clinico-pathological characteristics and follow-up information. Short hairpin RNA was utilized to investigate the function of Shp2 in hepatoma cell behavior. Role of Shp2 in HCC progression was monitored through nude mice xenograft assay. Kinase activity assay and co-immunoprecipitation were used for mechanism analysis. RESULTS: Elevated expression of Shp2 was detected in 65.9% (394/598) of human HCCs, and its levels were even higher in metastasized foci. Overexpression of Shp2 correlated well with the malignant clinico-pathological characteristics of HCC and predicted the poor prognosis of patients. Interference of Shp2 expression suppressed the proliferation of hepatoma cells in vitro and inhibited the growth of HCC xenografts in vivo. Down-regulation of Shp2 attenuated the adhesion and migration of hepatoma cells and diminished metastasized HCC formation in mice. Our data demonstrated that Shp2 promotes HCC growth and metastasis by coordinately activating Ras/Raf/Erk pathway and PI3-K/Akt/mTOR cascade. Moreover, down-regulation of Shp2 enhanced the sensitivity of hepatoma cells upon sorafenib treatment, and patients with low Shp2 expression exhibited superior prognosis to sorafenib. CONCLUSIONS: Shp2 promotes the progression of HCC and may serve as a prognostic biomarker for patients.

Hepatocyte nuclear factor 4α-nuclear factor-κB feedback circuit modulates liver cancer progression.

UNLABELLED: Hepatocyte nuclear factor 4α (HNF4α) is a liver enriched transcription factor and is indispensable for liver development. However, the role of HNF4α in hepatocellular carcinoma (HCC) progression remains to be elucidated. We report that reduced HNF4α expression correlated well with the aggressive clinicopathological characteristics of HCC and predicted poor prognosis of patients. HNF4α levels were even lower in metastatic HCCs, and ectopic HNF4α expression suppressed the metastasis of hepatoma cells both in vitro and in vivo. Forced HNF4α expression attenuated the expression and nuclear translocation of RelA (p65) and impaired NF-κB activation through an IKK-independent mechanism. Blockage of RelA robustly attenuated the suppressive effect of HNF4α on hepatoma cell metastasis. MicroRNA (miR)-7 and miR-124 were transcriptionally up-regulated by HNF4α, which repressed RelA expression by way of interaction with RelA-3' untranslated region (UTR). In addition, nuclear factor kappa B (NF-κB) up-regulated the expression of miR-21 in hepatoma cells, resulting in decreased HNF4α levels through down-regulating HNF4α-3'UTR activity. CONCLUSIONS: Collectively, an HNF4α-NF-κB feedback circuit including miR-124, miR-7, and miR-21 was identified in HCC, and the combination of HNF4α and NF-κB exhibited more powerful predictive efficiency of patient prognosis. These findings broaden the knowledge of hepatic inflammation and cancer initiation/progression, and also provide novel prognostic biomarkers and therapeutic targets for HCC.

[Association of chromosome 17q copy number variation with overall survival of patients with hepatocellular carcinoma and screening of potential target genes].

OBJECTIVE To assess the association of copy number variations (CNVs) in chromosome 17q with the overall survival(OS) of patients with hepatocellular carcinoma(HCC), and to screen for target genes contained in the OS-related CNVs. METHODS A total of 174 HCC cases were enrolled. For 66 patients, the follow-up data was available. High-resolution Agilent Hu-244A array comparative genomic hybridization (aCGH) and Affymetrix U133 Plus 2.0 expression arrays were used to detect CNVs and gene expression of genes from the 17q region, respectively. The association of CNVs and OS was assessed with Log-rank test, Kaplan-Meier survival analysis, and Cox proportional hazards models. The gene expression in HCCs with 17q gain, HCCs without, and non-tumor liver tissues were compared with a Mann-Whitney U test. RESULTS Univariate association analysis showed that copy number gain in 17q25.1-25.3 was significantly associated with reduced OS (Log-rank test, P = 0.00002), and HCC cases with 17q25.1-25.3 gain had a 4.76-fold (95%CI: 2.31-9.81) increased hazard ratio (HR) for death from HCC, as compared to those without the gain. Multivariate Cox proportional hazards regression model revealed 17q25.1-25.3 gain to be an independent prognostic marker for poor OS (HR = 3.17, 95%CI: 1.39-7.26, P = 0.006). The expression levels of 18 genes in 17q25.1-25.3 including SLC9A3R1, GRB2, and TK1 were significantly increased in HCCs with gain than in those without (all P < 0.01) and non-tumor liver tissues (all P < 0.01). CONCLUSION The association of 17q25.1-25.3 gain with reduced OS has indicated that it is a prognostic marker for poor patient survival in HCC, for which SLC9A3R1, GRB2, and TK1 are candidate genes.