Zhi Dai

Plasma MicroRNA Panel to Diagnose Hepatitis B Virus–Related Hepatocellular Carcinoma

PURPOSE More than 60% of patients with hepatocellular carcinoma (HCC) do not receive curative therapy as a result of late clinical presentation and diagnosis. We aimed to identify plasma microRNAs for diagnosing hepatitis B virus (HBV) -related HCC. PATIENTS AND METHODS Plasma microRNA expression was investigated with three independent cohorts including 934 participants (healthy, chronic hepatitis B, cirrhosis, and HBV-related HCC), recruited between August 2008 and June 2010. First, we used microarray to screen 723 microRNAs in 137 plasma samples for diagnosing HCC. Quantitative reverse-transcriptase polymerase chain reaction assay was then applied to evaluate the expression of selected microRNAs. A logistic regression model was constructed using a training cohort (n = 407) and then validated using an independent cohort (n = 390). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. RESULTS We identified a microRNA panel (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801) that provided a high diagnostic accuracy of HCC (AUC = 0.864 and 0.888 for training and validation data set, respectively). The satisfactory diagnostic performance of the microRNA panel persisted regardless of disease status (AUCs for Barcelona Clinic Liver Cancer stages 0, A, B, and C were 0.888, 0.888, 0.901, and 0.881, respectively). The microRNA panel can also differentiate HCC from healthy (AUC = 0.941), chronic hepatitis B (AUC = 0.842), and cirrhosis (AUC = 0.884), respectively. CONCLUSION We found a plasma microRNA panel that has considerable clinical value in diagnosing early-stage HCC. Thus, patients who would have otherwise missed the curative treatment window can benefit from optimal therapy.

Association of Autophagy Defect with a Malignant Phenotype and Poor Prognosis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The role of autophagy and the prognostic value of autophagic genes are largely unknown in HCC. Here, we showed decreased expression of autophagic genes and their corresponding autophagic activity and increased expression of the antiapoptotic gene Bcl-xL in HCC cell lines compared with a normal hepatic cell line. We also found decreased expression of the autophagic gene Beclin 1 in 44 HCC tissue samples compared with adjacent nontumor tissues. In addition, we found that the most aggressive malignant HCC cell lines and HCC tissues with recurrent disease displayed much lower autophagic levels, especially when Bcl-xL was overexpressed. Interestingly, in a tissue microarray study consisting of 300 HCC patients who underwent curative resection, the expression of Beclin 1 was only significantly correlated with disease-free survival (DFS; P < 0.0001) and overall survival (OS; P < 0.0001) in the Bcl-xL(+) group. Multivariate and univariate analyses also revealed that Beclin 1 expression was an independent predictor for DFS and OS in Bcl-xL(+) patients. In addition, we found a significant correlation between Beclin 1 expression and tumor differentiation in Bcl-xL(+) but not in Bcl-xL(-) HCC patients. In conclusion, our data showed expression of autophagic genes and their corresponding autophagic activities were suppressed in HCC. The autophagy defects synergized with altered apoptotic activity might facilitate tumor malignant differentiation, which results in a more aggressive cancer cell phenotype and poor prognosis of HCC.

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis.

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.

Overexpression of CXCL5 mediates neutrophil infiltration and indicates poor prognosis for hepatocellular carcinoma

CXCL5 (epithelial neutrophil‐activating peptide‐78) is a member of a proangiogenic subgroup of the CXC‐type chemokine family of small, secreted proteins. Recently, evidence that CXCL5 is involved in carcinogenesis and cancer progression has emerged. To investigate the role of CXCL5 in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC), we examined CXCL5 messenger RNA (mRNA) and protein levels in HCC cell lines with various metastatic potentials and in three independent cohorts of 919 HCC patients. We found that CXCL5 expression was increased in the highly metastatic HCC cell lines and in tumor tissues from patients with recurrent HCC compared to controls. CXCL5 activated the PI3K‐Akt and ERK1/2 signaling pathways in HCC cells and promoted proliferation, migration, and invasion. Furthermore, we found that CXCL5 had a direct chemoattractant effect on neutrophils in vitro. In animal studies, the up‐regulation of CXCL5 in HCC cells promoted tumor growth, lung metastasis, and intratumoral neutrophil infiltration. Conversely, down‐regulation of CXCL5 in HCC cells reduced tumor growth, metastasis, and intratumoral neutrophil infiltration. Immunohistochemical analysis in HCC samples showed that overexpression of CXCL5 was well correlated with intratumoral neutrophil infiltration, shorter overall survival, and tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for overall survival and cumulative recurrence. Conclusion: CXCL5 promotes HCC cell proliferation, invasion, and intratumoral neutrophil infiltration. CXCL5 overexpression, alone or combined with intratumoral neutrophil presence, is a novel prognostic predictor, and CXCL5 is a potential therapeutic target for HCC. (HEPATOLOGY 2012;56:2242–2254)

Autophagy Activation in Hepatocellular Carcinoma Contributes to the Tolerance of Oxaliplatin via Reactive Oxygen Species Modulation

Purpose: Understanding the roles of mammalian autophagy in cancer highlights recent advances in the pharmacologic manipulation of autophagic pathways as a therapeutic strategy for cancer. However, autophagy status and corresponding functions in hepatocellular carcinoma (HCC) after therapeutic stress remain to be clarified. This study was to determine whether the autophagic machinery could be activated after chemotherapy and the contribution of autophagy to tolerance of oxaliplatin in HCC. Experimental Design: Autophagy activation and cell death induced by oxaliplatin were examined in two HCC cell lines as well as in vivo using an HCC model in nude mice. HCC tissue samples with or without locoregional chemotherapy before surgery were also examined by immunohistochemical and electron microscopic analysis. Results: Autophagy was functionally activated in HCC cell lines and xenografts after oxaliplatin treatment. Suppression of autophagy using either pharmacologic inhibitors or RNA interference of essential autophagy gene enhanced cell death induced by oxaliplatin in HCC cells. Generation of reactive oxygen species has an important role in the induction of cell death by oxaliplatin in combination with autophagy inhibitors. Critically, the combination of oxaliplatin with autophagy inhibitor chloroquine resulted in a more pronounced tumor suppression in HCC xenografts. Furthermore, autophagy-specific protein LC3 and autophagic autophagosome formation were induced to a significantly higher level in HCC specimens that had been subjected to locoregional chemotherapy. Conclusions: Autophagy activation under therapy stress contributes to HCC tumor cell survival. Targeting the autophagy pathway is a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes in HCC patients. Clin Cancer Res; 17(19); 6229–38. ©2011 AACR.

IL-17 induces AKT-dependent IL-6/JAK2/STAT3 activation and tumor progression in hepatocellular carcinoma

BackgroundThe Th17 subset and IL-17 have been found in increased frequencies within certain tumors. However, their relevance in cancer biology remains controversial. This study aimed to clarify the biological action of IL-17 on hepatocellular carcinoma (HCC).MethodsEffects and underlying molecular mechanisms of IL-17 on human HCC were explored in vitro using exogenous IL-17 stimulation and in nude mice by implanting IL-17 overexpressed HCC cells. The clinical significance of IL-17 was investigated in tissue microarrays containing HCC tissues from 323 patients following hepatectomy using immunohistochemistry.ResultsAlthough exogenous IL-17 showed no direct effect on the growth rate of HCC cells in vitro, PCR and ELISA showed that IL-17 selectively augmented the secretion of diverse proinvasive factors and transwell showed a direct promotion of invasion of HCC cells by IL-17. Furthermore, transfection of IL-17 into HCC cells significantly promoted neoangiogenesis, neutrophil recruitment and tumor growth in vivo. Using siRNA mediated knockdown of AKT and STAT3, we suggested that the effects of IL-17 were operated through activation of the AKT signaling in HCC, which resulted in IL-6 production. Then, IL-6 in turn activated JAK2/STAT3 signaling and subsequently up-regulated its downstream targets IL-8, MMP2, and VEGF. Supporting these findings, in human HCC tissues, immunostaining indicated that IL-17 expression was significantly and positively associated with STAT3 phosphorylation, neutrophil infiltration and increased tumor vascularity. The clinical significance of IL-17 was authenticated by revealing that the combination of intratumoral IL-17+ cells and phospho-STAT3 served as a better prognosticator for postoperative tumor recurrence than either marker alone.ConclusionsIL-17 mediated tumor-promoting role involves a direct effect on HCC cells through IL-6/JAK2/STAT3 induction by activating the AKT pathway.

CD24 Is a Novel Predictor for Poor Prognosis of Hepatocellular Carcinoma after Surgery

Purpose: To investigate the role of CD24 in tumor invasion and prognostic significance in hepatocellular carcinoma (HCC). Experimental Design: CD24 expression was measured in stepwise metastatic HCC cell lines, tumor, peritumoral tissues, and normal liver tissues by quantitative real-time PCR and Western blot. The role of CD24 in HCC was investigated by CD24 depletion using small interfering RNA. Tumor tissue microarrays of 314 HCC patients who underwent resection between 1997 and 2000 were used to detect expression of CD24, β-catenin, and proliferating cell nuclear antigen. Prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Results: CD24 was overexpressed in the highly metastatic HCC cell line and in tumor tissues of patients with recurrent HCC. Depletion of CD24 caused a notable decrease in cell proliferation, migration, and invasiveness in vitro. Univariate and multivariate analyses revealed that CD24 was a significant predictor for overall survival and relapse-free survival. CD24 expression was correlated with poor prognosis independent of α-fetoprotein, tumor-node-metastasis stage, and Edmondson stage. High CD24 expression was significantly associated with cytoplasmic and nuclear accumulation of β-catenin (P = 0.023), high tumor proliferative status (P = 0.018), and diffused intrahepatic recurrence and distant metastasis (P = 0.026). Adjuvant transcatheter arterial chemoembolization after surgery reduced the rate of early recurrence (≤1 year) in CD24+ HCC patients (P = 0.024) but had no significant effect in CD24− patients (P = 0.284). Conclusions: Overexpression of CD24 in HCC was associated with high invasiveness and metastatic potential, high tumor proliferation status, and activation of the Wnt/β-catenin pathway. CD24 may be a novel predictor for poor prognosis of HCC patients after surgery. (Clin Cancer Res 2009;15(17):5518–27)

Metadherin promotes hepatocellular carcinoma metastasis through induction of Epithelial-Mesenchymal transition

Purpose: To investigate the expression of metadherin (MTDH) for its prognostic value in hepatocellular carcinoma (HCC) and its role in promoting HCC metastasis. Experimental Design: This study employed a tissue microarray containing samples from 323 HCC patients to examine the expression of MTDH and its correlation with other clinicopathologic characteristics. The role of MTDH in the regulation of HCC metastasis was investigated both in vitro and in vivo using short hairpin RNA (shRNA)–mediated downregulation of MTDH in HCC cell lines with various metastatic potentials. Results: The expression of MTDH was markedly higher in HCC tumors than in normal liver tissue. Particularly high MTDH expression was observed in tumors with microvascular invasion, pathologic satellites, poor differentiation, or tumor-node-metastasis stages II to III. Furthermore, the clinical outcome was consistently poorer for the MTDHhigh group than for the MTDHlow group in the 1-, 3-, and 5-year overall survival (OS) rates and in the 1-, 3-, 5-year cumulative recurrence rates. In a nude mice model, the shRNA-mediated downregulation of MTDH resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary and abdominal metastasis. Furthermore, we found that the expression level of MTDH correlated with four epithelial–mesenchymal transition (EMT) markers. Knockdown of MTDH expression in HCC cell lines resulted in downregulation of N-cadherin and snail, upregulation of E-cadherin, and translocation of β-catenin. Conclusions: MTDH may promote HCC metastasis through the induction of EMT process and may be a candidate biomarker for prognosis as well as a target for therapy. Clin Cancer Res; 17(23); 7294–302. ©2011 AACR.

CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

Tetraspanin CD151 is involved in several pathological activities associated with tumor progression, including neoangiogenesis. However, the role and molecular mechanism of CD151 in the neoangiogenesis of hepatocellular carcinoma (HCC) remain enigmatic. We found that the level of expression of matrix metalloproteinase 9 (MMP9) was positively associated with CD151 expression in HCC cells. We developed a zone‐by‐zone blockade and demonstrated that overexpression of CD151 in HCC cells facilitated MMP9 expression through a phosphatidylinositol‐3‐kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase 3β (GSK‐3β)/Snail signaling pathway. In contrast, down‐regulation of CD151 expression impaired the ability of HCC cells to form microvessels in vitro and reduced their in vivo metastatic potential. In a clinical setting, a significant correlation of the expression of CD151 with MMP9 expression and with microvessel density (MVD) was revealed by Pearson correlation analysis of HCC patients. The postoperative 3‐, 5‐, and 7‐year overall survival rates of HCC patients with CD151high/MMP9high/MVDhigh were significantly lower than those of the CD151low/MMP9low/MVDlow group or groups in which only one or two of CD151, MMP9, and MVD were highly expressed. Cumulative recurrence rates were also highest in HCC patients with CD151high/MMP9high/MVDhigh in comparison with the other groups. Multivariate Cox proportional hazards analysis showed that the concomitant overexpression of CD151, MMP9, and MVD was an independent marker for predicting poor prognosis of HCC. Conclusion: Overexpression of CD151 up‐regulated the expression of MMP9 through the PI3K/Akt/GSK‐3β/Snail pathway. CD151‐dependent neoangiogenesis appeared to promote the progression of HCC, and this suggests that CD151 may be useful as a high‐priority therapeutic target for antiangiogenesis in HCC. HEPATOLOGY 2010

Fibroblast growth factor 21 is regulated by the IRE1α-XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress-induced hepatic steatosis.

Background: Although both are involved in metabolic homeostasis, the interconnection between ER stress and FGF21 remains incompletely understood. Results: Directly up-regulated by the IRE1α-XBP1 pathway, FGF21 could alleviate ER stress-induced liver steatosis. Conclusion: FGF21 acts as a metabolic effector of the UPR program, exerting feedback effects upon lipid metabolism. Significance: These findings reveal a regulatory mechanism linking FGF21 actions to metabolic ER stress. Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR) and represents a critical mechanism that underlies metabolic dysfunctions. Fibroblast growth factor 21 (FGF21), a hormone that is predominantly secreted by the liver, exerts a broad range of effects upon the metabolism of carbohydrates and lipids. Although increased circulating levels of FGF21 have been documented in animal models and human subjects with obesity and nonalcoholic fatty liver disease, the functional interconnections between metabolic ER stress and FGF21 are incompletely understood. Here, we report that increased ER stress along with the simultaneous elevation of FGF21 expression were associated with the occurrence of nonalcoholic fatty liver disease both in diet-induced obese mice and human patients. Intraperitoneal administration of the ER stressor tunicamycin in mice resulted in hepatic steatosis, accompanied by activation of the three canonical UPR branches and increased the expression of FGF21. Furthermore, the IRE1α-XBP1 pathway of the UPR could directly activate the transcriptional expression of Fgf21. Administration of recombinant FGF21 in mice alleviated tunicamycin-induced liver steatosis, in parallel with reduced eIF2α-ATF4-CHOP signaling. Taken together, these results suggest that FGF21 is an integral physiological component of the cellular UPR program, which exerts beneficial feedback effects upon lipid metabolism through counteracting ER stress.