Ying-Hao Shen

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.

Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling

Galectin-1 (Gal-1) is involved in several pathological activities associated with tumor progression and chemoresistance, however, the role and molecular mechanism of Gal-1 activity in hepatocellular carcinoma (HCC) epithelial–mesenchymal transition (EMT) and sorafenib resistance remain enigmatic. In the present study, forced Gal-1 expression promoted HCC progression and sorafenib resistance. Gal-1 elevated αvβ3-integrin expression, leading to AKT activation. Moreover, Gal-1 overexpression induced HCC cell EMT via PI3K/AKT cascade activation. Clinically, our data revealed that Gal-1 overexpression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that Gal-1 may be a potential therapeutic target for HCC and a biomarker for predicting response to sorafenib treatment.

Promoting colonization in metastatic HCC cells by modulation of autophagy.

Background Autophagy is an important adaptive survival mechanism, which has been postulated to be involved in cancer metastasis. The purpose of this study was to investigate autophagy in metastasis of hepatocellular carcinoma (HCC). Methods Immunohistochemical analysis of autophagic activity in metastatic and paired primary HCC tissues using LC3 as autophagosome marker was performed in samples from 216 HCC patients diagnosed with metastasis (including 158 intravascular, 42 intrabiliary, 8 lymph node, 4 bone and 4 lung metastases). Then a mouse model of pulmonary metastasis was established using a highly metastatic HCC cell line (HCCLM3). Autophagy in pulmonary metastases and paired primary tumors were analyzed by LC3 immunohistochemistry, transmission electron microscopy (TEM) and western blot analysis. Further, mouse model of pulmonary metastasis and in vitro cell migration, invasion and detachment models were established using a stable GFP-LC3-expressing HCCLM3 cell line (HCCLM3-GFP-LC3). Autophagic alterations during metastatic colonization, migration, invasion and detachment were determined by GFP-LC3 analysis and western blot analysis. Results LC3 immunohistochemistry of metastases and primary tumors from HCC patients revealed significantly higher LC3 expression in metastases than primary HCC, which suggested a higher level of autophagy in HCC metastases. Further immunohistochemical, TEM, western blot and in vivo GFP-LC3 analyses of lung metastases and primary tumors in mouse model of pulmonary metastasis confirmed that metastatic colonies displayed higher level of autophagy than primary tumors and the early metastatic colonies displayed highest level. The dynamic monitoring of autophagy in cell migration, invasion and detachment showed that autophagy did not significantly alter in those processes. Conclusions Autophagy is activated in metastatic colonization but not in invasion, migration and detachment of HCC cells. Autophagy may play a role in HCC metastasis via promoting metastatic colonization of HCC cells.

High expression of 5-hydroxymethylcytosine and isocitrate dehydrogenase 2 is associated with favorable prognosis after curative resection of hepatocellular carcinoma

BackgroundThe expression of 5-hydroxymethylcytosine (5-hmC) and isocitrate dehydrogenase 2 (IDH2) is frequently downregulated in numerous cancers. 5-hmC and IDH2 expression in hepatocellular carcinoma (HCC) has yet to be determined.MethodsThe immunohistochemical expression of 5-hmC and IDH2 were analyzed in tissue microarrays containing samples from 646 patients who had undergone hepatectomy for histologically proven HCC. The prognostic value of 5-hmC and IDH2 were evaluated by Cox regression and Kaplan-Meier analyses.ResultsWe discovered that low 5-hmC and IDH2 expression was associated with malignant behaviors. Low 5-hmC or IDH2 expression alone and combined 5-hmC and IDH2 expression were associated with lower overall survival (OS) rates and higher cumulative recurrence rates. Multivariate analysis indicated that 5-hmC or IDH2 and 5-hmC/IDH2 were independent prognostic indicators for OS and time to recurrence (TTR), which was confirmed in an independent validation cohort.Conclusions5-hmC and IDH2 correlate with less aggressive tumor behavior in HCC. When 5-hmC and IDH2 are considered together, they serve as a prognostic marker in patients with surgically resected HCCs.

Prediction of Post-Operative Liver Dysfunction by Serum Markers of Liver Fibrosis in Hepatocellular Carcinoma

Aim To investigate the role of biomarkers in predicting postoperative liver dysfunction in patients with hepatocellular carcinoma (HCC). Methods A total of 200 patients operated from July 2009 to June 2010 at Zhongshan Hospital, Fudan University for pathologically confirmed HCC were retrospectively analyzed for clinical data, HBD DNA level and serum biochemical markers for liver fibrosis. The patients were followed up to observersation end point. Correlation of the monitored parameters with postoperative liver dysfunction and patient survival was statistically analyzed. Results Preoperative hepatitis B virus (HBV) DNA level, serum prealbumin (PA) hyaluronic acid (HA), and laminin (LN) levels correlated with postoperative liver dysfunction. A predictive model was generated using these 4 parameters and validated in 89 HCC patients with sensitivity and specificity of 0.625 and 0.912, respectively. However, no correlation was identified between postoperative liver function and overall survival. Conclusion Liver fibrosis markers could be preoperatively used in predicting postoperative liver dysfunction in HCC patients.

Clinical analysis of gastroenteropancreatic neuroendocrine tumor with liver metastasis, compared with primary hepatic neuroendocrine tumor

OBJECTIVE The objective was to study the clinicopathologic features, grading, treatment protocols, and prognostic of gastroenteropancreatic neuroendocrine tumor (NET) with liver metastasis and primary hepatic NET. MATERIALS AND METHODS The clinical data of 34 patients with hepatic NET were retrospectively reviewed. According to the primary tumor location and 2010 World Health Organization classification, the cases were categorized to analyze the clinicopathologic features, treatment condition, and prognostic factors. RESULTS There was a marked male predominance either in gastroenteropancreatic NET liver metastasis group or primary group. Primary hepatic NET is mostly single nodule located in the right lobe of liver, and the metastatic hepatic NET is mostly from pancreas with multiple nodules and metastasizes to both lobes of the liver, with a high degree of malignancy and poor prognosis. There are 17 cases (50%) of NET and 17 cases (50%) of neuroendocrine carcinoma (NEC) in all the 34 patients of this study. The mitotic figure and Ki-67 proliferation index are both higher in NEC group than in NET group, which indicated highly malignancy of the NEC. The 5-year disease-free survival (DFS) rates for primary group and metastatic group were 30% and 40%, respectively (P > 0.05), while the 5-year survival rates were 35% and 66%, respectively (P > 0.05). Different tumor grade was found closely associated with 5-year DFS (P < 0.05) and overall survival (OS) (P < 0.05) in both groups. Furthermore, we found 5-year DFS of patients with primary site of the tumor located in the gastrointestinal tract was much lower than that located in pancreas (P < 0.05), while the 5-year OS showed no significant differences between two groups (P > 0.05). CONCLUSIONS Surgery is an effective method for the treatment of hepatic NET; tumor grading is an important determinant factor of prognosis.

Generation and characterization of a tetraspanin CD151/integrin α6β1-binding domain competitively binding monoclonal antibody for inhibition of tumor progression in HCC

Our previous studies revealed that tetraspanin CD151 plays multiple roles in the progression of hepatocellular carcinoma (HCC) by forming a functional complex with integrin α6β1. Herein, we generated a monoclonal antibody (mAb) that dissociates the CD151/integrin α6β1 complex, and we evaluated its bioactivity in HCCs. A murine mAb, tetraspanin CD151 (IgG1, called CD151 mAb 9B), was successfully generated against the CD151-integrin α6β1 binding site of CD151 extracellular domains. Co-immunoprecipitation using CD151 mAb 9B followed by Western blotting detected a 28 kDa protein. Both immunofluorescent and immunohistochemical staining showed a good reactivity of CD151 mAb 9B in the plasma membrane and cytoplasm of HCC cells, as well as in liver cells. In vitro assays demonstrated that CD151 mAb 9B could inhibit neoangiogenesis and both the mobility and the invasiveness of HCC cells. An in vivo assay showed that CD151 mAb 9B inhibited tumor growth potential and HCC cells metastasis. We successfully produced a CD151 mAb 9B targeting the CD151/integrin α6β1-binding domain, which not only can displayed good reactivity to the CD151 antigen but also prevented tumor progression in HCC.

Dexamethasone for postoperative hyperbilirubinemia in patients after liver resection: An open-label, randomized controlled trial

Background: Although prophylactic glucocorticoids have been used before liver resection to minimize liver dysfunction, it is unknown whether treatment with glucocorticoids will accelerates recovery from hyperbilirubinemia after liver resection. Methods: In this open‐label, randomized, controlled trial, patients with hyperbilirubinemia (>2.5 × and ≤5 × the upper limit of normal) within 7 days after hepatic resection were assigned randomly to the dexamethasone or control groups. For the dexamethasone group, 10 mg, 10 mg, and 5 mg dexamethasone were administered intravenously on days 0, 1, and 2, respectively, after randomization. For the control group, patients received standard treatment only. The primary outcome was time to recovery from hyperbilirubinemia defined as the period from the day of randomization to the day when serum bilirubin decreased to ≤1.5 times that of the upper limit of normal. Secondary outcomes were the prevalence of postoperative complications, postoperative hospital stay, and hospital expense. Results: Between March 2016 and December 2017, 76 participants were enrolled (38 in each group). Median time to recovery from hyperbilirubinemia was less in the dexamethasone group than in the control group (2 vs 4 days, P < .001). Serum bilirubin levels were less in the dexamethasone group on days 1–3 after randomization (P < .05). The prevalence of infection, posthepatectomy liver failure, postoperative hospital stay, and hospital expense were not different between the groups. Conclusion: Dexamethasone accelerated recovery from hyperbilirubinemia and decreased serum bilirubin levels without causing more side effects in patients after hepatectomy.

NOD-like receptor X1 functions as a tumor suppressor by inhibiting epithelial-mesenchymal transition and inducing aging in hepatocellular carcinoma cells

BackgroundThis study was performed to investigate the role of nucleotide-binding oligomerization domain (NOD)-like receptor X1 (NLRX1) in regulating hepatocellular carcinoma (HCC) progression.MethodsExpression levels of NLRX1 in clinical specimens and cell lines were determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB). Transwell assays were conducted to evaluate the effect of NLRX1 on cell invasion, and flow cytometry was used to assess apoptosis. Expression patterns of key molecules in the phosphoinositide 3-kinase (PI3K)-AKT pathways were determined via WB. The effect of NLRX1 on cell senescence was evaluated with β-galactosidase assays. Kaplan-Meier analyses and Cox regression models were used for prognostic evaluation.ResultsNLRX1 was downregulated in tumor tissue compared with adjacent normal liver tissue. Low tumor NLRX1 expression was identified as an independent indicator for HCC prognosis (recurrence: hazard ratio [HR] 1.87, 95% confidence interval [CI] 1.26–2.76, overall survival [OS] 2.26, 95% CI 1.44–3.56). NLRX1 over-expression (OE) significantly inhibited invasiveness ability and induced apoptosis in HCC cells. In vivo experiments showed that NLRX1 knock-down (KD) significantly promoted HCC growth. Mechanistically, NLRX1 exhibited a suppressor function by decreasing phosphorylation of AKT and thus downregulating Snail1 expression, which inhibited epithelial-mesenchymal-transition (EMT) in HCC cells. Moreover, NLRX1 OE could induce cell senescence via an AKT-P21-dependent manner.ConclusionsNLRX1 acted as a tumor suppressor in HCC by inducing apoptosis, promoting senescence, and decreasing invasiveness by repressing PI3K-AKT signaling pathway. Future investigations will focus on restoring expression of NLRX1 to provide new insights into HCC treatment.