2009—2019年研究所代表性英文论文摘要(IF≥5)

十、2009—2019年研究所代表性英文论文摘要(IF≥5)

1.Capn4 overexpression underlies tumor invasion and metastasis after liver transplantation for hepatocellular carcinoma　(IF 14.971)

Bai DS,Dai Z,Zhou J,Liu YK,Qiu SJ,Tan CJ,Shi YH,Huang C,Wang Z,He YF,Fan J

Liver transplantation(LT)is one of the best therapeutic options for nonresectable hepatocellular carcinoma(HCC).Unfortunately,some HCC patients succumb to the disease after LT,which reduces long-and medium-term survival.To identify the proteins associated with HCC invasion and metastasis,HCC patients undergoing LT with complete follow-up data were included in this study and were categorized into recurrence and nonrecurrence groups.We extracted the total protein from the acquired homogeneous tumor cells and applied a cleavable isotope-coded affinity tag technology to quantitate relative changes in protein levels between the two groups.We identified a total of 149 proteins with two-dimensional liquid chromatography coupled with tandem mass spectrometry,including 52 differentially expressed proteins by at least two-fold.Among them,calpain small subunit 1(Capn4),a protein with relevant interactions with many migration-invasion-related proteins,has attracted more attention.First,Capn4 overexpression in the recurrence group was confirmed via real-time polymerase chain reaction and western blotting in another cohort of 40 HCC patients undergoing LT.Second,Capn4 was associated with enhanced invasiveness in vitro.The small interfering RNA-mediated knockdown expression of Capn4 in HCC cell lines significantly inhibited its mobile and invasive ability.Tissue microarray in a further 192 cases revealed that Capn4 significantly correlated with invasive phenotype of HCC,and univariate and multivariate analyses indicated that Capn4 is an independent prognostic factor for recurrence and survival of HCC patients.CONCLUSION:Our study revealed that Capn4 overexpression underlies invasion and metastasis after LT for HCC and might be a candidate biomarker for future diagnosis and a target for therapy.

(Hepatology,2009,49:460-470)

2.Human leukocyte antigen-G protein expression is an unfavorable prognostic predictor of hepatocellular carcinoma following curative resection　(IF 8.911)

Cai MY,Xu YF,Qiu SJ,Ju MJ,Gao Q,Li YW,Zhang BH,Zhou J,Fan J

PURPOSE:Human leukocyte antigen-G(HLA-G)is a tumor-associated immunosuppressive molecule involved in tumor escape mechanisms.The aim of this study is to elucidate its prognostic significance in hepatocellular carcinoma(HCC).EXPERIMENTAL DESIGN:Immunohistochemical staining of HLA-G expression as well as tumor-infiltrating Fox P3+regulatory(Tregs)and CD8+cytotoxic T cells was carried out on tissue microarrays containing 173 HCC tissue specimens.Membrane-bound HLA-G1 protein expression in five human HCC cell lines was detected by Western blot.RESULTS:HLA-G expression was associated with HCC prognosis,especially in early-stage diseases,with high expression independently associated with shortened overall survival(P=0.041)and increased tumor recurrence(P=0.023).HLA-G level was positively related to Tregs/CD8+ratio and their combination served as a better prognosticator,patients having concurrent high levels of both variables at more than three times of risk of death and tumor relapse than those with concurrent low levels(both P＜0.001).In addition,HLA-G1 expression increased in a concordant manner with the increase of metastatic potential in human HCC cell lines.CONCLUSIONS:Overexpression of HLA-G protein in HCC was an independent indicator for poor outcome especially in early-stage disease.The combination of HLA-G expression and Tregs/CD8+ratio added the prognostic power to both variables,offering a possible strategy of tumor-stroma interaction-oriented cancer immunotherapy.

(Clin Cancer Res,2009,15:4686-4693)

3.Liver-intestine cadherin predicts microvascular invasion and poor prognosis of hepatitis B viruspositive hepatocellular carcinoma　(IF 6.102)

Ding ZB,Shi YH,Zhou J,Shi GM,Ke AW,Qiu SJ,Wang XY,Dai Z,Xu Y,Fan J

BACKGROUND:Liver-intestine cadherin(LI-cadherin;CDH-17)is a new member of the cadherin superfamily with distinct structural and functional features.The study was designed to investigate the role of LI-cadherin in tumor invasion and prognosis of human hepatitis B virus(HBV)-positive hepatocellular carcinoma(HCC).METHODS:LI-cadherin expression in HBV-positive hepatocellular carcinoma cell lines with low-and high-invasive potentials was evaluated by Westernblot,immunofluorescence,and real-time polymerase chain reaction(PCR)analyses.The role of LIcadherin in tumor invasion was also evaluated in vitro by a small-interfering ribonucleic acid(siRNA)-mediated approach.The prognostic significance of LI-cadherin was validated in a cohort of HBV-positive HCC patients by immunohistochemistry and Western-blot.RESULTS:Significant high levels of LI-cadherin m RNA and protein were found in the high-invasive HCCLM3 as compared with those in low-invasive PLC/PRF/5 and Hep3B cell line.Cell migration,adhesion to extracellular matrix,and matrigel invasion were significantly reduced after LI-cadherin knockdown in HCCLM3 cells.Immunohistochemical analysis of 255 HBV-positive HCC cases showed that overexpression of LI-cadherin was well correlated with microvascular invasion,which was confirmed by Western-blot in 32 tumor tissues,and its overexpression was strongly associated with shorter overall survival as well as higher incidence of tumor recurrence.CONCLUSIONS:LI-cadherin is predictive of microvascular invasion and poor prognosis of HBV-positive HCC,and would be a potential useful intervention target for HCC.

(Cancer,2009,115:4753-4765)

4.Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma　(IF 8.911)

Gao Q,Wang XY,Qiu SJ,Yamato I,Sho M,Nakajima Y,Zhou J,Li BZ,Shi YH,Xiao YS,Xu Y,Fan J

PURPOSE:The aberrant expression of programmed cell death 1 ligands 1 and 2(PD-Ls)on tumor cells dampens antitumor immunity,resulting in tumor immune evasion.In this study,we investigated the expression of PD-Ls in human hepatocellular carcinoma(HCC)to define their prognostic significance after curative surgery.EXPERIMENTAL DESIGN:Immunohistochemistry was used to investigate PD-Ls expression as well as granzyme B+cytotoxic and Fox P3+regulatory T cell infiltration on tissue microarrays containing 240 randomly selected HCC patients who underwent surgery.The results were further verified in an independent cohort of 125 HCC patients.PD-Ls expression on HCC cell lines was detected by Western blot assay.RESULTS:Patients with higher expression of PD-L1 had a significantly poorer prognosis than patients with lower expression.Although patients with higher expression of PD-L2 also had a poorer survival,the difference in recurrence was not statistically significant.Multivariate analysis identified tumor expression of PDL1 as an independent predictor for postoperative recurrence.No correlation was found between PDLs expression and granzyme B+lymphocyte infiltration,whereas a significant positive correlation was detected between PD-Ls expression and Fox P3+lymphocyte infiltration.In addition,tumorinfiltrating cytotoxic and regulatory T cells were also independent prognosticators for both survival and recurrence.The prognostic value of PD-L1 expression was validated in the independent data set.CONCLUSION:Our data suggest for the first time that PD-L1 status may be a new predictor of recurrence for HCC patients and provide the rationale for developing a novel therapy of targeting the PD-L1/PD-1 pathway against this fatal malignancy.

(Clin Cancer Res,2009,15:971-979)

5.MicroRNA expression,survival,and response to interferon in liver cancer　(IF 70.670)

Ji J,Shi J,Budhu A,Yu Z,Forgues M,Roessler S,Ambs S,Chen Y,Meltzer PS,Croce CM,Qin LX,Man K,Lo CM,Lee J,Ng IO,Fan J,Tang ZY,Sun HC,Wang XW

BACKGROUND:Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men.We examined micro RNA expression patterns,survival,and response to interferon alfa in both men and women with the disease.METHODS:We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003.MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women.In addition,to validate our findings,we used quantitative reversetranscriptase-polymerase-chain-reaction assays to measure micro RNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent,prospective,randomized,controlled trials of adjuvant interferon therapy.RESULTS:In patients with hepatocellular carcinoma,the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men.Tumors had reduced levels of miR-26 expression,as compared with paired noncancerous tissues,which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma.Moreover,tumors with reduced miR-26 expression had a distinct transcriptomic pattern,and analyses of gene networks revealed that activation of signaling pathways between nuclear factor kappaB and interleukin-6 might play a role in tumor development.Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA.CONCLUSIONS:The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma.The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa.

(N Engl J Med,2009,361:1437-1447)

6.Preoperative serum gamma-glutamyl transferase to alanine aminotransferase ratio is a convenient prognostic marker for Child-Pugh A hepatocellular carcinoma after operation　(IF 5.130)

Ju MJ,Qiu SJ,Fan J,Zhou J,Gao Q,Cai MY,Li YW,Tang ZY

BACKGROUND:The ratio of serum gamma-glutamyl transferase(GGT)to alanine aminotransferase(ALT)is a parameter for assessing responses to antiviral therapies.The relationship between the prognosis of hepatitis B-associated hepatocellular carcinoma(HCC)and preoperative GGT/ALT is studied in hepatectomized Child-Pugh A patients.METHODS:Two hundred and nineteen patients with hepatitis B virus-related HCC were included.Serum ALT and GGT levels were examined preoperatively and the GGT/ALT ratios were calculated.Their prognostic capabilities were evaluated by Cox-regression model.RESULTS:As dichotomized variables,GGT and ALT were distributed unequally(P＜0.001).Most patients had high levels of GGT(n=110)but low levels of ALT(n=185).ALT displayed no relation to recurrence or survival,while GGT was independently associated with survival(P=0.002).The GGT/ALT ratio could predict survival precisely either in a continuous or dichotomized fashion(P＜0.001 and 0.001,respectively),and also related to recurrence when dichotomized(P=0.002).Additionally,high GGT/ALT ratio was associated with high early recurrence rates,more recurrence-related deaths and various aggressive tumor characteristics such as larger tumor size,vascular invasion,poor encapsulation and advanced BCLC stage.In further stratified analyses,this ratio could discriminate the outcomes of patients with high-or low-alpha-fetoprotein level.CONCLUSIONS:The elevated GGT/ALT ratio was a promising predictor for poor prognosis of Child-Pugh A HCC patients after operation mainly through its tight relevance to the primary tumor burden.

(J Gastroenterol,2009,44:635-642)

7.Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma(IF 14.971)

Ke AW,Shi GM,Zhou J,Wu FZ,Ding ZB,Hu MY,Xu Y,Song ZJ,Wang ZJ,Wu JC,Bai DS,Li JC,Liu KD,Fan J

It has been reported that tetraspanin CD151 acts as a promoter of metastasis in several tumors and plays an important role in c-Met/hepatocyte growth factor signaling.However,the role of CD151 alone and coexpression of CD151/c-Met in hepatocellular carcinoma(HCC)remains unclear.We found that expression of CD151 was positively related to metastatic potential of HCC cell lines,and modified cells with CD151(high)showed higher secretion of matrix metalloproteinase 9 and aggressiveness in vitro and higher metastatic ability in vivo.Furthermore,HCC patients with vascular invasion,large tumors,multiple tumors,high tumor-node-metastasis stage,and undifferentiated tumor were prone to have higher CD151 expression.The postoperative 3-,5-,and 7-year overall survival(OS)of patients in HCCs with CD151(high)were significantly lower than those in the CD151(low)group,and correspondingly cumulative recurrence rates in HCCs with CD151(high)were significantly higher than those in the CD151(low)group.Both CD151 and c-Met were remarkably overexpressed in HCCs,compared with adjacent nontumorous and normal liver tissues.Pearson correlation analysis showed a slight correlation between CD151 and c-Met in HCCs.Importantly,the 5-and 7-year OS rates in CD151(high)/c-Met(high)patients were 50.5%and 37.8%,respectively,significantly lower than those of CD151(low)/c-Met(low)patients(63.9%and 54.6%,respectively).Five-and 7-year cumulative recurrence rates in CD151(high)/c-Met(high)patients were 53.3%and 71.9%,respectively,markedly higher than those of CD151(low)/c-Met(low)patients(39.0%and 52.5%,respectively).Multivariate analysis revealed that CD151 and combination of CD151/c-Met were independent prognostic indicators for OS and cumulative recurrence.CONCLUSION:CD151 is positively associated with invasiveness of HCC,and CD151 or combination of CD151/c-Met is a novel marker in predicting the prognosis of HCC and a potential therapeutic target.

(Hepatology,2009,49:491-503)

8.Prognostic significance of Beclin 1-dependent apoptotic activity in hepatocellular carcinoma(IF 11.059)

Shi YH,Ding ZB,Zhou J,Qiu SJ,Fan J

Autophagy is believed to be important in tumorigenesis and tumor progression.However,the role of autophagy in hepatocellular carcinoma(HCC),and especially the prognostic value of autophagic proteins,has not been investigated.Our studies described here show decreased basal expression of autophagic genes and their corresponding autophagic activity under conditions of starvation in HCC cell lines,and the autophagy defect correlated well with the highly malignant phenotype of HCC.In addition,in a tissue microarray study of HCC patients who underwent resection,the expression of the autophagy-related protein Beclin 1 was extremely low in tumors,where Beclin 1 could predict the prognosis of HCC patients only in a Bcl-X(L)-positive expression background.Based on our results,we propose that autophagy defects that synergize with altered apoptotic activity might facilitate tumor progression and poor prognosis of HCC,due to the fact that autophagy may interact with apoptosis in the regulation of HCC.

(Autophagy,2009,5:380-382)

9.Human hepatocellular carcinoma tumor-derived endothelial cells manifest increased angiogenesis capability and drug resistance compared with normal endothelial cells　(IF 8.911)

Xiong YQ,Sun HC,Zhang W,Zhu XD,Zhuang PY,Zhang JB,Wang L,Wu WZ,Qin LX,Tang ZY PURPOSE:Increasing evidence indicates that tumor-derived endothelial cells(TEC)possess a distinct and unique phenotype compared with endothelial cells(NEC)from adjacent normal tissue and may be able to acquire resistance to drugs.The aim of this study was to investigate the angiogenesis activity and response to drug treatment of TECs and NECs derived from human hepatocellular carcinoma(HCC).EXPERIMENTAL DESIGN:TECs or NECs were isolated from HCC or adjacent normal liver tissue using anti-CD105 antibody coupled to magnetic beads.The phenotypic and functional properties of endothelial cells were characterized by testing the expression of CD105,CD31,CD144,vascular endothelial growth factor receptor-1,vascular endothelial growth factor receptor-2,and von Willebrand factor,and the ability of DiI-Ac-LDL-uptake and tube formations.CD105(+)TECs were compared with CD105(+)NECs and human umbilical vein endothelial cells(HUVEC)by examining their ability to proliferate,motility,ability to adhere to tumor cells,response to tumor conditioned medium,and reactions to the chemotherapy drugs Adriamycin and 5-fluorouracil and the antiangiogenic drug sorafenib.RESULTS:Compared with CD105(+)NECs and HUVECs,CD105(+)TECs showed increased apoptosis resistance and motility and proangiogenic properties.Meanwhile,CD105(+)TECs had a greater ability to adhere to tumor cells and survive in the tumor environment.Moreover,CD105(+)TECs acquired more resistance to Adriamycin,5-fluorouracil,and sorafenib than CD105(+)NECs and HUVECs.CONCLUSIONS:TECs possessed enhanced angiogenic activity and resistance to chemotherapeutic drugs and an angiogenesis inhibitor,and may provide a better tool for studying tumor angiogenesis and antiangiogenesis drugs in HCC.

(Clin Cancer Res,2009,15:4838-4846)

10.CD24 is a novel predictor for poor prognosis of hepatocellular carcinoma after surgery(IF 8.911)

Yang XR,Xu Y,Yu B,Zhou J,Li JC,Qiu SJ,Shi YH,Wang XY,Dai Z,Shi GM,Wu B,Wu LM,Yang GH,Zhang BH,Qin WX,Fan J

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,beta-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 relapsefree survival.CD24 expression was correlated with poor prognosis independent of alpha-fetoprotein,tumor-node-metastasis stage,and Edmondson stage.High CD24 expression was significantly associated with cytoplasmic and nuclear accumulation of beta-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/beta-catenin pathway.CD24 may be a novel predictor for poor prognosis of HCC patients after surgery.

(Clin Cancer Res,2009,15:5518-5527)

11.Elevated expression of DKK1 is associated with cytoplasmic/nuclear beta-catenin accumulation and poor prognosis in hepatocellular carcinomas　(IF 18.946)

Yu B,Yang X,Xu Y,Yao G,Shu H,Lin B,Hood L,Wang H,Yang S,Gu J,Fan J,Qin W

BACKGROUND/AIMS:To assess the value of Dickkopf-1(DKK1)for predicting clinical outcome of human hepatocellular carcinoma(HCC)in patients with HCC.METHODS:Expression of DKK1 and beta-catenin was investigated in HCC cell lines using qRT-PCR,Western blotting and immunofluorescence.Tissue microarrays representing 314 HCC patients were used to determine the expression patterns of DKK1 and beta-catenin by immunohistochemistry,and prognostic significance was assessed by using Kaplan-Meier survival estimates and log-rank tests.RESULTS:The expression level of DKK1 was associated with the staining pattern of beta-catenin in HCC cell lines,and DKK1 overexpression correlated with beta-catenin cytoplasmic/nuclear accumulation in clinical HCC samples(P=0.011,correlation coefficient=0.144).High DKK1 expression predicted unfavorable prognosis in HCC patients,especially in early stage patients and those with normal AFP levels.In multivariate analyses,DKK1 was an independent predictor for overall survival(OS)(P=0.002)and disease-free survival(DFS)(P=0.002)of HCC patients.Furthermore,the HCC patients with high DKK1 expression and cytoplasmic/nuclear beta-catenin accumulation had very poor prognosis.CONCLUSIONS:Elevated expression of DKK1 is a critical event in patients with HCC that indicates poor clinical outcome.DKK1,alone or combined with beta-catenin,is a novel prognostic predictor for HCC patients.

(J Hepatol,2009,50:948-957)

12.Transcription factor c-Myb promotes the invasion of hepatocellular carcinoma cells via increasing osteopontin expression　(IF 5.646)

Chen RX,Xia YH,Xue TC,Ye SL

BACKGROUND:Specific gene expression is tightly regulated by various transcription factors.Osteopontin(OPN)is a phosphoprotein that mediates hepatocellular carcinoma(HCC)progression and metastasis.However,the mechanism of OPN up-regulation in HCC metastasis remains to be clarified.METHODS:Oligonucleotide array-based transcription factor assays were applied to compare different activities of transcription factors in two human HCC cell lines with different OPN expression levels.The effects of one selected transcription factor on OPN expression were further evaluated.RESULTS:Eleven transcription factors were over-expressed in metastatic HCC cell line HCCLM6 cells whereas twelve transcription factors were down-regulated.Electrophoretic mobility shift assays(EMSA)and reporter gene assays showed that one of up-regulated transcription factors c-Myb could bind the OPN promoter and increase its transcription activity.In addition,small interfering RNA targeting c-Myb could inhibit OPN expression and significantly decrease migration and invasion of HCCLM6 cells in vitro.CONCLUSION:Our data first demonstrate that c-Myb has a functionally important role in the regulation of OPN expression in HCC cells,suggesting that c-Myb might be a new target to control HCC metastasis.

(J Exp Clin Cancer Res,2010,29:172)

13.Overexpression of CD151 as an adverse marker for intrahepatic cholangiocarcinoma patients(IF 6.102)

Huang XY,Ke AW,Shi GM,Ding ZB,Devbhandari RP,Gu FM,Li QL,Dai Z,Zhou J,Fan J

BACKGROUND:Previous studies have indicated that CD151,a hydrophobic protein,forms a functional complex with the proto-oncogene that encodes an N-methyl-N'-nitro-N-nitroso-guanidine(MET)protein(c-Met),and CD151 overexpression reportedly is involved in metastasis/invasion of several tumors.The objective of the current study was to investigate the expression and role of CD151 and/or c-Met in intrahepatic cholangiocarcinoma(ICC).METHODS:Sixty ICC tissues with matched nontumorous tissues and 20 normal liver tissues were used to analyze CD151 expression at the level of messenger RNA(m RNA)and protein.Then,the expression of CD151 in an ICC cell line was interrupted using a specific lentiviral-mediated small hairpin RNA(sh RNA)-CD151,and the role of CD151 in the proliferation,metastasis,and invasion of ICC cells was assessed.The expression of CD151/c-Met was examined further by immunohistochemistry in a tissue microarray(TMA)that included 140 samples of ICC,and the prognostic role of CD151 and/or c-Met in ICC was evaluated in Kaplan-Meier and Cox regression analyses.RESULTS:The expression of CD151 in ICC tissues was much higher than that in nontumorous samples and normal liver;and,after the down-regulation of CD151,HCCC-9810 cells had decreased capability for metastasis/invasion in vitro.CD151 overexpression was correlated significantly with larger tumors,poor differentiation,multiple nodular,microvascular/bile duct invasion,and lymphatic metastasis(P＜0.05).The postoperative 2-year and 5-year overall survival(OS)rates for patients with low CD151 expression(＜50%tumor staining)and/or low c-Met expression(＜20%tumor staining)were higher than the rates for patients with high CD151 expression(≥50%tumor staining)and/or high c-Met expression(≥20%tumor staining).Multivariate analysis revealed that CD151 overexpression and c-Met overexpression were independent prognostic markers for ICC.CONCLUSIONS:Overexpression of CD151 was implicated in metastasis/invasion of ICC,and both CD151 overexpression and c-Met overexpression may be potential molecular therapeutic targets for ICC.

(Cancer,2010,116:5440-5451)

14.High expression of macrophage colony-stimulating factor-1 receptor in peritumoral liver tissue is associated with poor outcome in hepatocellular carcinoma after curative resection　(IF 5.252)

Jia JB,Wang WQ,Sun HC,Zhu XD,Liu L,Zhuang PY,Zhang JB,Zhang W,Xu HX,Kong LQ,Lu L,Wu WZ,Wang L,Tang ZY

BACKGROUND:Macrophage colony-stimulating factor 1 receptor(CSF-1R)expression in hepatocellular carcinoma(HCC)and its prognostic values are unclear.This study evaluated the prognostic values of the intratumoral and peritumoral expression of CSF-1R in HCC patients after curative resection.METHODS:Tissue microarrays containing material from cohort 1(105 patients)and cohort 2(32 patients)were constructed.Immunohistochemistry was performed and prognostic values of these and other clinicopathological data were evaluated.The CSF-1R m RNA level was assessed by quantitative real-time polymerase chain reaction in cohort 3(52 patients).RESULTS:Both the CSF-1R density and its m RNA level were significantly higher in peritumoral liver tissue than in the corresponding tumor tissue.CSF-1R was distributed in a gradient in the longdistance peritumoral tissue microarray,with its density decreasing as the distance from the tumor margin increased.High peritumoral CSF-1R was significantly associated with more intrahepatic metastases and poorer survival.Peritumoral CSF-1R was an independent prognostic factor for both overall survival and time to recurrence and affected the incidence of early recurrence.However,intratumoral CSF-1R did not correlate with any clinicopathological feature.Peritumoral CSF-1R was also associated with both overall survival and time to recurrence in a subgroup with small HCCs(≤5 cm).CONCLUSIONS:Peritumoral CSF-1R is associated with intrahepatic metastasis,tumor recurrence,and patient survival after hepatectomy,highlighting the critical role of the peritumoral liver milieu in HCC progression.CSF-1R may become a potential therapeutic target for postoperative adjuvant treatment.

(Oncologist,2010,15:732-743)

15.Up-regulation of Kruppel-like factor 8 promotes tumor invasion and indicates poor prognosis for hepatocellular carcinoma　(IF 19.233)

Li JC,Yang XR,Sun HX,Xu Y,Zhou J,Qiu SJ,Ke AW,Cui YH,Wang ZJ,Wang WM,Liu KD,Fan J

BACKGROUND&AIMS:The transcription factor Kruppel-like factor 8(KLF8)has a role in tumor development,growth,and metastasis,but its role in hepatocellular carcinoma(HCC)is not clear.METHODS:KLF8 expression in human HCC cell lines and tumor tissues was measured by quantitative real-time polymerase chain reaction,immunoblot,and immunochemical analyses.The effects of KLF8 depletion or overexpression in HCC cells were observed in cultured cells and in mice.Changes in gene expression patterns in HCC cells in which levels of KLF8 were reduced using small interfering RNA were investigated by microarray analysis.The clinical significance of KLF8 expression levels were validated using tissue microarray analysis of surgical samples from 314 HCC patients.RESULTS:KLF8 was overexpressed in highly metastatic HCC cell lines and in samples from patients with recurrent HCC.In cultured cells,KLF8 up-regulation promoted cell proliferation and invasion;inhibited apoptosis;down-regulated N-cadherin,vimentin,and fibronectin;and upregulated E-cadherin.In mice,overexpression of KLF8 increased HCC progression and metastasis.Microarray analysis showed that reduction of KLF8 in HCC cells down-regulated expression of multiple genes involved in tumor progression and metastasis.KLF8 expression was a significant predictor of overall survival(P=0.040)and time to HCC recurrence(P=0.006)and was associated with early tumor recurrence(P=0.001).CONCLUSIONS:KLF8 promotes HCC cell proliferation and invasion,inhibits apoptosis,and induces the epithelial-to-mesenchymal transition.KLF8 up-regulation might be used to indicate poor prognosis or early recurrence of cancer in patients who have had surgery for HCC.

(Gastroenterology,2010,139:2146-2157 e2112)

16.Activation of beta-catenin by hypoxia in hepatocellular carcinoma contributes to enhanced metastatic potential and poor prognosis　(IF 8.911)

Liu L,Zhu XD,Wang WQ,Shen Y,Qin Y,Ren ZG,Sun HC,Tang ZY

PURPOSE:Aberrant activation of beta-catenin contributes to the malignant phenotype in hepatocellular carcinoma(HCC).Hypoxia is also known to promote HCC invasion and metastasis.However,the association between beta-catenin and the proinvasive role of hypoxia remains unclear.We investigated the role of beta-catenin in the proinvasive consequences of hypoxia in HCC.EXPERIMENTAL DESIGN:We established in vitro and in vivo hypoxic models to investigate the expression of beta-catenin in hypoxic HCC cells and its role in hypoxia-induced aggressiveness.The clinical significance of beta-catenin and/or hypoxia-induced factor-1alpha(HIF-1alpha)was evaluated using HCC tissue microarrays.RESULTS:Hypoxia induced beta-catenin overexpression and/or intracellular accumulation in four HCC cell lines through downregulating the endogenous degradation machinery,and promoted in vitro invasion and in vivo metastasis of MHCC97 and Hep3B cells.Besides morphologic changes,hypoxic MHCC97 and Hep3B cells exhibited molecular alterations consistent with epithelial-mesenchymal transition,characterized by the loss of epithelial cell markers(E-cadherin and plakoglobin)and upregulation of mesenchymal markers(vimentin and N-cadherin),as well as the increase of matrix metalloproteinase 2.However,silencing beta-catenin in these hypoxic cells reversed epithelial-mesenchymal transition and repressed metastatic potential.Positive expression of beta-catenin in HCC tissue microarray was associated with the expression of HIF-1alpha(P=0.034),and coexpression of beta-catenin and HIF-1alpha in HCC was correlated with shorter overall survival and time to recurrence.CONCLUSION:beta-Catenin in HCC is activated by hypoxia and contributes to hypoxia-induced metastatic potential.

(Clin Cancer Res,2010,16:2740-2750)

17.CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma　(IF 14.971)

Shi GM,Ke AW,Zhou J,Wang XY,Xu Y,Ding ZB,Devbhandari RP,Huang XY,Qiu SJ,Shi YH,Dai Z,Yang XR,Yang GH,Fan J

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 3beta(GSK-3beta)/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 CD151(high)/MMP9(high)/MVD(high)were significantly lower than those of the CD151(low)/MMP9(low)/MVD(low)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 CD151(high)/MMP9(high)/MVD(high)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-3beta/Snail pathway.CD151-dependent neoangiogenesis appeared to promote the progression of HCC,and this suggests that CD151 may be useful as a highpriority therapeutic target for antiangiogenesis in HCC.

(Hepatology,2010,52:183-196)

18.PEBP1 downregulation is associated to poor prognosis in HCC related to hepatitis B infection(IF 18.946)

Xu YF,Yi Y,Qiu SJ,Gao Q,Li YW,Dai CX,Cai MY,Ju MJ,Zhou J,Zhang BH,Fan J

BACKGROUND&AIMS:Phosphatidylethanolamine-binding protein 1(PEBP1,also RKIP)plays a pivotal role in cancer by regulating multiple cellular signaling processes and suppressing metastasis in animal models.We examined whether PEBP1 expression in hepatocellular carcinoma(HCC)correlated with the risk of recurrence and survival after resection.METHODS:A randomly selected cohort of 240 Chinese HCC patients,predominantly hepatitis B related,formed the basis of the study.PEBP1 expression levels were evaluated by immunohistochemistry and real-time reversetranscriptase PCR.Survival analysis was performed by univariate and multivariate analyses.The results were further validated in an independent series of 403 patients.The relevance of PEBP1 to phospho-ERK was determined by Western blot analysis on clinical samples and hepatoma cell lines.RESULTS:PEBP1,prevalently down-regulated in HCC,was significantly associated with tumor invasive characteristics(such as vascular invasion,lack of encapsulation,poor differentiation and large size).Both PEBP1 protein and m RNA levels were independent predictors for tumor recurrence(hazard ratio(HR)=1.877,P=0.001;HR=2.633,P=0.001;respectively),and patient survival(HR=1.796,P=0.004;HR=1.730,P=0.044;respectively).The prognostic value of PEBP1 was then confirmed in the validation cohort.In addition,Western blot suggested that loss of PEBP1 led to hyperactivity of MAPK signaling.CONCLUSIONS:Down-regulation of PEBP1 in HCC indicated aggressive tumor behaviors and predicted a worse clinical outcome,which may be a useful biomarker to identify the patients at high risk of post-operative recurrence.

(J Hepatol,2010,53:872-879)

19.Thrombin is a therapeutic target for metastatic osteopontin-positive hepatocellular carcinoma(IF 14.971)

Xue YH,Zhang XF,Dong QZ,Sun J,Dai C,Zhou HJ,Ren N,Jia HL,Ye QH,Qin LX

We previously identified osteopontin(OPN)as a promoter and thus a potential therapeutic target for hepatocellular carcinoma(HCC)metastasis.The serine protease thrombin interacts with OPN and can modify its biological activity.To explore the role of thrombin alone or in conjunction with OPN in HCC,we studied the correlation of thrombin levels to HCC prognosis in patients with various OPN levels,and evaluated the effects of OPN fragments generated by thrombin cleavage on proliferation and adhesion of HCC cells.We found that the thrombin level was strongly associated with the metastatic potential of HCC cell lines,and that thrombin was remarkably overexpressed in HCC tissue compared with adjacent nontumor tissue.In addition,HCC tissue from patients with recurrent disease displayed much higher thrombin levels,particularly in those with elevated OPN levels.Only HCCs with elevated OPN levels had a significant correlation between high thrombin levels and overall survival(OS;P＜0.01),or time to recurrence(TTR;P＜0.000 1)of HCC.Multivariate analysis revealed that thrombin was an independent prognostic indicator.In vitro assays demonstrated that thrombin promotes the proliferation and adhesion of OPN(+)HCC cells.Furthermore,thrombin activated the focal adhesion kinase(FAK)pathway of OPN(+)HCC cells,which was blocked by the inhibition of integrin beta1.CONCLUSION:Thrombin plays an important role in OPN-mediated aggressive phenotype of HCC through activation of integrin beta1-FAK signaling,and is an independent poor prognostic factor for HCC.Thus,thrombin may be a potential therapeutic target to inhibit HCC metastasis in OPN(+)patients.

(Hepatology,2010,52:2012-2022)

20.High expression levels of putative hepatic stem/progenitor cell biomarkers related to tumour angiogenesis and poor prognosis of hepatocellular carcinoma　(IF 17.943)

Yang XR,Xu Y,Yu B,Zhou J,Qiu SJ,Shi GM,Zhang BH,Wu WZ,Shi YH,Wu B,Yang GH,Ji Y,Fan J

BACKGROUND/AIMS:To investigate the prognostic values of putative hepatic stem/progenitor cell(HSC/HPC)biomarkers in patients with hepatocellular carcinoma(HCC).METHODS:Fourteen biomarkers related to HSCs/HPCs or tumour angiogenesis were assessed by qRT-PCR and then validated by tissue microarrays(TMAs)in three independent cohorts of patients with HCC undergoing curative resection(n=67,314 and 73).RESULTS:Most of the biomarkers were found to be overexpressed in patients with recurrent HCC by quantitative reverse transcription-PCR(qRTPCR).The HSC/HPC biomarkers cytokeratin 19,ATP-binding cassette subfamily G member 2(ABCG2),CD133,Nestin and CD44,and the markers of angiogenesis microvessel density(MVD,determined by CD34 immunostaining),vascular endothelial growth factor(VEGF)and plateletderived endothelial cell growth factor(PD-ECGF)were confirmed as significant predictors for overall survival(OS)and/or relapse-free survival(RFS)in TMA analysis.As compared with the low HSC/HPC profile group,patients with a high HSC/HPC profile who had higher VEGF levels(P=0.012)and MVD(P=0.030)in tumours had significantly lower OS and RFS(P＜0.000 1).Based on Cox regression,a simplified model including CD133,CD44,Nestin and MVD was constructed and confirmed as an independent predictor for OS(P＜0.000 1)and RFS(P＜0.000 1),regardless of alpha-fetoprotein level,tumour stage and recurrence time(P＜0.000 1 for all).CONCLUSION:High expression levels of HSC/HPC biomarkers are related to tumour angiogenesis and poor prognosis of HCC.The simplified model based on the HSC/HPC and tumour angiogenesis profile can be used to classify patients with HCC with a high risk of tumour recurrence after surgery.

(Gut,2010,59:953-962)

21.Current approaches to the treatment of early hepatocellular carcinoma　(IF 5.252)

Ye SL,Takayama T,Geschwind J,Marrero JA,Bronowicki JP

For patients with early-stage hepatocellular carcinoma(HCC),potentially curative treatment options exist,including liver transplantation,surgical resection,and ablation therapy.These treatments are associated with survival benefits,and outcomes are optimized by identification of appropriate patients.However,further studies are needed to definitively confirm optimal treatment approaches for all patients.Treatment patterns vary in different parts of the world as a result of geographic differences in the incidence and presentation of the disease.In particular,because of successful screening programs,a high proportion of tumors that are identified in Japan are amenable to curative treatments,which are appropriate in a smaller proportion of patients in the west,although screening is now widely carried out in industrialized countries.Differences in the applicability of transplantation are also evident between the west and Asia.Although existing treatments for early-stage HCC are supported by considerable evidence,there remain significant data gaps.For example,further data,ideally from randomized controlled trials,are needed regarding:the use of neoadjuvant and adjuvant therapy to decrease the rate of recurrence after resection or ablation,further investigation of the role of chemoprevention following resection,and prospective analysis of outcomes of living donor compared with deceased donor liver transplantation.

[Oncologist,2010,15(Suppl 4):34-41]

22.Interferon alpha inhibits hepatocellular carcinoma growth through inducing apoptosis and interfering with adhesion of tumor endothelial cells　(IF 6.508)

Zhang T,Sun HC,Zhou HY,Luo JT,Zhang BL,Wang P,Wang L,Qin LX,Ren N,Ye SL,Li Q,Tang ZY

The aim of this study was to observe the effect of interferon alpha(IFNalpha)on tumor endothelial cells(TECs)in highly metastatic hepatocellular carcinoma(HCC)model,and to investigate the underlying mechanism.Nude mice with HCC xenograft were treated with IFNalpha.Gene expression profiles of TECs were analyzed by utilizing cDNA microarray.The differentiation of tumor blood vessels was evaluated by CD31/alphaSMA dual immunohistochemistry.Apoptosis of TECs was determined by CD31/TUNEL double staining.The functions of TECs in adhesion and uptake of acetylated low-density lipoprotein were observed in vitro.Results showed that IFNalpha effectively inhibited HCC tumor growth,with decreased microvessel density,increased apoptosis in TECs and normalized tumor blood vessels.cDNA microarray analysis revealed differential gene expression patterns in TECs under the treatment of IFNalpha.The cell-cell contact distribution of VE-Cadherin and uptake of acetylated low-density lipoprotein were significantly inhibited by IFNalpha in cultivated TECs.These results suggest that IFNalpha may induce apoptosis and interfere with hemophilic adhesion of TECs.The changes of gene expression in TECs contribute essentially to its effect of anti-angiogenesis and the subsequent inhibition of tumor progression.

(Cancer Lett,2010,290:204-210)

23.Depletion of tumor-associated macrophages enhances the effect of sorafenib in metastatic liver cancer models by antimetastatic and antiangiogenic effects　(IF 8.911)

Zhang W,Zhu XD,Sun HC,Xiong YQ,Zhuang PY,Xu HX,Kong LQ,Wang L,Wu WZ,Tang ZY

PURPOSE:To investigate the role of macrophages in tumor progression under sorafenib treatment and to explore whether combination of drugs that deplete macrophages improved the antitumor effect of sorafenib.EXPERIMENTAL DESIGN:Tumor growth,lung metastasis,and tumor angiogenesis were observed in HCCLM3-R and SMMC7721,two human hepatocellular carcinoma xenograft nude mouse models,when treated with sorafenib(30 mg/kg daily,n=6 per group)or a vehicle as control.Macrophage infiltration was measured in the peripheral blood and in sorafenibtreated tumor by immunohistochemistry and flow cytometry with F4/80 antibody and CD11b antibody.The effect of macrophage depletion on tumor angiogenesis and metastasis after sorafenib treatment,using two drug target macrophages,zoledronic acid(ZA)and clodrolip,was measured in the two models of hepatocellular carcinoma.RESULTS:Although sorafenib significantly inhibited tumor growth and lung metastasis,it induced a significant increase in peripheral recruitment and intratumoral infiltration of F4/80-and CD11b-positive cells,which was accompanied with elevation of colony-stimulating factor-1,stromal-derived factor 1alpha,and vascular endothelial growth factor in the tumor and elevation of plasma colony-stimulating factor-1 and mouse vascular endothelial growth factor in peripheral blood,suggesting the role of macrophages in tumor progression under sorafenib treatment.Depletion of macrophages by clodrolip or ZA in combination with sorafenib significantly inhibited tumor progression,tumor angiogenesis,and lung metastasis compared with mice treated with sorafenib alone.ZA was more effective than clodrolip.CONCLUSIONS:Macrophages may have an important role in tumor progression under sorafenib treatment.ZA is promising when combined with sorafenib to enhance its antitumor effect.

(Clin Cancer Res,2010,16:3420-3430)

24.Prognostic value of interleukin 2 and interleukin 15 in peritumoral hepatic tissues for patients with hepatitis B-related hepatocellular carcinoma after curative resection　(IF 17.943)

Zhou H,Huang H,Shi J,Zhao Y,Dong Q,Jia H,Liu Y,Ye Q,Sun H,Zhu X,Fu L,Guo K,Gao D,Sun J,Yan Z,Ren N,Tang Z,Qin L

BACKGROUND AND AIMS:Th1/Th2-like cytokine m RNA levels in non-cancerous hepatic tissues from patients with hepatocellular carcinoma(HCC)are associated with metastases and recurrence.This study evaluated the prognostic values of intratumoral and peritumoral Th1/Th2 cytokine protein levels in patients with HCC after curative resection.METHODS:Two independent cohorts(A and B)of 453 patients with HCC were enrolled.Twelve Th1/Th2 cytokines in tumour and peritumoral hepatic tissues from cohort A(n=192)were quantified with enzyme-linked immunosorbent assays.This cohort was split into training and test sets which were used to identify and verify the prognostic cytokines.The prognostic values of identified cytokines were further validated in cohort B(n=261)using tissue microarray and immunohistochemical staining.RESULTS:In the training set,higher interleukin(IL)-2 and IL-15 levels in peritumoral liver tissues,but not in tumour tissues,were significantly associated with a decreased incidence of recurrence of intrahepatic tumour and a prolonged overall survival.This association was verified in the testing set and further validated in patients in cohort B.Importantly,this correlation remained significant in patients with early HCC.Univariate and multivariate analyses indicated that the prognostic performance of peritumoral IL-2(HR for recurrence=0.4,95%CI 0.3 to 0.6,P＜0.000 1;HR for death=0.6,95%CI 0.4 to 0.8,P=0.005)and IL-15(HR for recurrence=0.7,95%CI 0.5 to 0.95,P=0.025)was independent of other clinicopathological factors.CONCLUSION:Peritumoral IL-2 and IL-15 levels are useful for stratifying patients,even those with early-stage HCC,into subgroups with different prognoses after curative resection.

(Gut,2010,59:1699-1708)

25.Autophagy activation in hepatocellular carcinoma contributes to the tolerance of oxaliplatin via reactive oxygen species modulation　(IF 8.911)

Ding ZB,Hui B,Shi YH,Zhou J,Peng YF,Gu CY,Yang H,Shi GM,Ke AW,Wang XY,Song K,Dai Z,Shen YH,Fan J

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,2011,17:6229-6238)

26.IL-17 induces AKT-dependent IL-6/JAK2/STAT3 activation and tumor progression in hepatocellular carcinoma　(IF 10.679)

Gu FM,Li QL,Gao Q,Jiang JH,Zhu K,Huang XY,Pan JF,Yan J,Hu JH,Wang Z,Dai Z,Fan J,Zhou J

BACKGROUND:The 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).METHODS:Effects 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.RESULTS:Although 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.CONCLUSIONS:IL-17 mediated tumor-promoting role involves a direct effect on HCC cells through IL-6/JAK2/STAT3 induction by activating the AKT pathway.

(Mol Cancer,2011,10:150)

27.CD151 amplifies signaling by integrin alpha6beta1 to PI3K and induces the epithelial-mesenchymal transition in HCC cells　(IF 19.233)

Ke AW,Shi GM,Zhou J,Huang XY,Shi YH,Ding ZB,Wang XY,Devbhandari RP,Fan J

BACKGROUND&AIMS:Overexpression of CD151 is associated with poor prognosis for hepatocellular carcinoma(HCC),yet its role in pathogenesis is not known.METHODS:We analyzed the expression of the integrin subunit alpha6 by quantitative,real-time polymerase chain reaction and immunoblot analyses of 120 HCC tissue samples;its clinical significance was investigated using tissue microarray(TMAs)analysis of samples from 335 patients with HCC.Immunoprecipitation was used to assess the relationship between alpha6 and CD151.The molecular effects of high expression levels of alpha6 and CD151 in HCC cells were determined using RNA interference and pharmacologic approaches.RESULTS:Overexpression of alpha6 correlated with poor prognosis of patients with HCC;alpha6 formed a complex with endogenous CD151 in HCC cells.In cells that expressed high levels of alpha6 and CD151,laminin-5 promoted cell spreading by inducing the epithelial-mesenchymal transition(EMT);this effect was not observed in cells that expressed high levels of only alpha6 or CD151.Cells that expressed high levels of alpha6 and CD151 underwent the EMT in response to laminin-5,through hyperactivation of phosphatidylinositol-3-kinase(PI3K),primarily induced via the PI3K-protein kinase B(Akt)-Snail-phosphatase and tensin homolog feedback pathway.The EMT was reversed by PI3K inhibitors and antibodies against CD151 or alpha6 in vitro,and was delayed by specific interference with CD151 and alpha6 in vivo.CONCLUSIONS:High expression levels of CD151 and alpha6 promote invasiveness of HCC cells.Either of these proteins,or PI3K signaling,might be targets for therapeutics for subgroups of patients with HCC.

(Gastroenterology,2011,140:1629-1641,e1615)

28.Intratumoral neutrophils:a poor prognostic factor for hepatocellular carcinoma following resection(IF 18.946)

Li YW,Qiu SJ,Fan J,Zhou J,Gao Q,Xiao YS,Xu YF

BACKGROUND&AIMS:Neutrophil infiltration has been linked to clinical outcome of various cancer types.However,its role in hepatocellular carcinoma(HCC)is unclear.In this study,we investigated prognostic values for intratumoral and peritumoral neutrophils in HCC patients undergoing curative resection.METHODS:The expression of CD66b,CD8,TGF-beta,and CD34 was assessed by immunohistochemistry in tissue microarrays containing paired intratumoral and peritumoral tissues from 197 patients receiving curative resection for HCC.Prognostic values for these and other clinicopathologic factors were evaluated.RESULTS:Intratumoral CD66b(+)neutrophils significantly correlated with CD8(+)T cells(r=0.240,P=0.004),TGF-beta expression(P=0.012),BCLC stage(P=0.016),and early recurrence(P=0.041).Increased intratumoral neutrophils were significantly associated with decreased RFS/OS(P=0.001 and P＜0.001,respectively)in univariate analysis and were identified as an independent prognostic factor(HR=1.845,95%CI=1.169-2.911,P=0.008 for RFS;HR=2.578,95%CI=1.618-4.106,P＜0.001 for OS)in multivariate analysis.Intratumoral neutrophil-to-CD8(+)T cell ratio(iNTR)better predicted the outcome in terms of minimum P values.Intratumoral neutrophils were also demonstrated to be statistically predictive for RFS/OS in the normal AFP subgroup,small HCC subgroup,and validation cohort.However,peritumoral neutrophils were not associated with the outcome of HCC.CONCLUSIONS:The presence of intratumoral neutrophils was a poor prognostic factor for HCC after resection.Intratumoral neutrophil-to-CD8(+)T cell ratio was a better predictor of outcome.

(J Hepatol,2011,54:497-505)

29.Association of specific genotypes in metastatic suppressor HTPAP with tumor metastasis and clinical prognosis in hepatocellular carcinoma　(IF 8.378)

Ren N,Wu JC,Dong QZ,Sun HJ,Jia HL,Li GC,Sun BS,Dai C,Shi J,Wei JW,Sheng YY,Zhou HJ,Ye QH,Qin LX

The phosphatidic acid phosphatase HTPAP has been defined as a metastatic suppressor of hepatocellular carcinoma(HCC),but little is known about its function or potential applications as a prognostic marker.In this study,we analyzed patterns of HTPAP genetic variation and gene expression in 864 patients who underwent HCC resection,assessing these patterns for correlations to tumor metastasis potential.Focusing on two tagSNPs that were selected(+357G/C and+1 838A/G),we found that only the+357G/C genotype was significantly associated with HTPAP m RNA and protein expression levels and the probability of metastasis.In an independent cohort of 665 HCC patients,we determined that the+357G/C genotype was associated with shorter time to recurrence and overall survival.Together,these results indicated that the HTPAP tagSNP+357 GG+GC genotypes may influence HCC metastatic potential and clinical prognosis by downregulating HTPAP expression.Extending these results,a global expression profiling analysis identified 41 genes including the pro-inflammatory genes IL-8 and TLR2 that were significantly overexpressed in the+357 GG+GC group,as possible coregulated markers with HTPAP.Together,our findings identify an HTPAP genotype and associated gene expression pattern that favors metastasis progression and that could be used to predict tumor metastasis and prognosis in HCC patients.

(Cancer Res,2011,71:3278-3286)

30.Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis　(IF 11.059)

Shi YH,Ding ZB,Zhou J,Hui B,Shi GM,Ke AW,Wang XY,Dai Z,Peng YF,Gu CY,Qiu SJ,Fan J

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.

(Autophagy,2011,7:1159-1172)

31.Identification of transaldolase as a novel serum biomarker for hepatocellular carcinoma metastasis using xenografted mouse model and clinic samples　(IF 6.508)

Wang C,Guo K,Gao D,Kang X,Jiang K,Li Y,Sun L,Zhang S,Sun C,Liu X,Wu W,Yang P,Liu Y

Hepatocellular carcinoma(HCC)is one of serious disorders with the highest morbidities and mortalities worldwide.Metastasis is the major concern that causes death in HCC.The goal of this study was to screen and identify potential serum proteins indicating HCC metastasis.Serum samples collected from control and HCCLM3-R metastatic HCC tumor model at specific stages of metastasis(1 wk,3 wks and 6 wks)were subjected to i TRAQ labeling followed by 2DLC-ESI-MS/MS analysis.A total of 554 proteins were identified and 80 proteins were differential expressed at least between one adjacent time points.Among them,expression level of transaldolase(TALDO)was validated in mouse and human serum.The level of TALDO protein was found to be higher in metastatic mice serum compared to that of non-metastatic mice.Human specific TALDO was then identified in mouse serum through human specific peptides.Immunohistochemical and western blot analysis showed that the expression of TALDO in human HCC tissues and HCC cell lines was associated with its metastatic behavior.Subsequent screening of TALDO expression in 72 clinical serum samples(comprising 36 non-metastatic HCC and 36 metastatic HCC samples)revealed higher TALDO level in the serum of metastatic HCC patients.A receiver operating characteristic(ROC)curve estimated a maximal sensitivity of 77.8%and 86.1%specificity for TALDO in detection of HCC metastasis.The present results demonstrated that the nude mouse xenograft model is an efficient system for performing metastasis-related biomarker discovery.TALDO may be useful biomarkers for the detection of HCC metastasis.

(Cancer Lett,2011,313:154-166)

32.Metadherin promotes hepatocellular carcinoma metastasis through induction of epithelialmesenchymal transition　(IF 8.911)

Zhu K,Dai Z,Pan Q,Wang Z,Yang GH,Yu L,Ding ZB,Shi GM,Ke AW,Yang XR,Tao ZH,Zhao YM,Qin Y,Zeng HY,Tang ZY,Fan J,Zhou J

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(sh RNA)-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 MTDH(high)group than for the MTDH(low)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 sh RNA-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 beta-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,2011,17:7294-7302)

33.CXCR6 upregulation contributes to a proinflammatory tumor microenvironment that drives metastasis and poor patient outcomes in hepatocellular carcinoma　(IF 8.378)

Gao Q,Zhao YJ,Wang XY,Qiu SJ,Shi YH,Sun J,Yi Y,Shi JY,Shi GM,Ding ZB,Xiao YS,Zhao ZH,Zhou J,He XH,Fan J

CXC chemokines and their cognate receptors have been implicated widely in cancer pathogenesis.In this study,we report a critical causal relationship between CXCR6 expression and tumorigenesis in the setting of human hepatocellular carcinoma(HCC).Among the CXC chemokine receptors,only CXCR6 was detected in all the hepatoma cell lines studied.Moreover,in HCC tissue,CXCR6 expression was significantly higher than in noncancerous liver tissues.Reduction of CXCR6 or its ligand CXCL16 in cancer cells reduced cell invasion in vitro and tumor growth,angiogenesis,and metastases in vivo.Importantly,loss of CXCR6 led to reduced Gr-1+neutrophil infiltration and decreased neoangiogenesis in hepatoma xenografts via inhibition of proinflammatory cytokine production.Clinically,high expression of CXCR6 was an independent predictor of increased recurrence and poor survival in HCCs.Human HCC samples expressing high levels of CXCR6 also contained an increased number of CD66b+neutrophils and microvessels,and the combination of CXCR6 and neutrophils was a superior predictor of recurrence and survival than either marker used alone.Together,our findings suggest that elevated expression of CXCR6 promotes HCC invasiveness and a protumor inflammatory environment and is associated with poor patient outcome.These results support the concept that inhibition of the CXCR6-CXCL16 pathway may improve prognosis after HCC treatment.

(Cancer Res,2012,72:3546-3556)

34.Decreased selenium-binding protein 1 enhances glutathione peroxidase 1 activity and downregulates HIF-1alpha to promote hepatocellular carcinoma invasiveness　(IF 8.911)

Huang C,Ding G,Gu C,Zhou J,Kuang M,Ji Y,He Y,Kondo T,Fan J

PURPOSE:We aimed to characterize the role of selenium-binding protein 1(SBP1)in hepatocellular carcinoma(HCC)invasiveness and underlying clinical significance.EXPERIMENTAL DESIGN:SBP1 expression was measured in stepwise metastatic HCC cell lines by Western blotting.The role of SBP1 in HCC was investigated using siRNA.Immunofluorescence analyses were used to detect the interaction between SBP1 and glutathione peroxidase 1(GPX1).Nineteen fresh tumor tissues and 323 paraffin-embedded samples were used to validate in vitro findings and to detect the prognostic significance of SBP1,respectively.RESULTS:Inhibition of SBP1 effectively increased cell motility,promoted cell proliferation,and inhibited apoptosis only under oxidative stress;it also greatly enhanced GPX1 activity without altering GPX1 expression and downregulated hypoxia-inducible factor-1alpha(HIF-1alpha)expression.SBP1 and GPX1 formed nuclear bodies and colocalized under oxidative stress.In freshly isolated clinical HCC tissues,decreased SBP1 was linked with increased GPX1 activity and correlated with vascular invasion.Tumor tissue microarrays indicated that SBP1 was an independent risk factor for overall survival and disease recurrence;patients with lower SBP1 expression experienced shorter overall survival periods and higher rates of disease recurrence(P＜0.001).Further analyses indicated that the predictive power of SBP1 was more significant for patients beyond the Milan criteria than patients within the Milan criteria.CONCLUSIONS:Decreased expression of SBP1 could promote tumor invasiveness by increasing GPX1 activity and diminishing HIF-1alpha expression in HCC;SBP1 could be a novel biomarker for predicting prognosis and guiding personalized therapeutic strategies,especially in patients with advanced HCC.

(Clin Cancer Res,2012,18:3042-3053)(J Exp Clin Cancer Res,2012,31:93)

35.TGF beta1 and related-Smads contribute to pulmonary metastasis of hepatocellular carcinoma in mice model　(IF 5.646)

Li GC,Ye QH,Dong QZ,Ren N,Jia HL,Qin LX

BACKGROUND:Recent studies indicate that Transforming Growth Factor beta(TGF beta)correlated with pulmonary metastasis of cancers.However,the correlation between TGF beta and pulmonary metastasis of hepatocellular carcinoma(HCC)is till unknown.METHODS:We detected the in vitro and in vivo expression levels of TGF beta1/Smads by Real-time PCR and Western blot in MHCC97-H and MHCC97-L cell lines,which are HCC cell lines and have higher and lower pulmonary metastatic potential respectively.RESULTS:TGF beta1 m RNA level in MHCC97-L tumors were higher than that in MHCC97-H tumors,(2.81+/-1.61 vs.1.24+/-0.96,P=0.002),TGF beta1 protein level in MHCC97-L tumors were also higher than that in MHCC97-H tumors(1.37+/-0.95 vs.0.32+/-0.22,P＜0.001).In addition,the TGF beta1 m RNA level positively correlated with pulmonary metastasis,and the relations between TGF beta1 and Smads were also found(R2=0.12 and 0.40,respectively).CONCLUSIONS:Our results suggest that TGF beta/Smads promote pulmonary metastasis of HCC.

36.Tanshinone IIA inhibits metastasis after palliative resection of hepatocellular carcinoma and prolongs survival in part via vascular normalization　(IF 8.731)

Wang WQ,Liu L,Sun HC,Fu YL,Xu HX,Chai ZT,Zhang QB,Kong LQ,Zhu XD,Lu L,Ren ZG,Tang ZY

BACKGROUND:Promotion of endothelial normalization restores tumor oxygenation and obstructs tumor cells invasion,intravasation,and metastasis.We therefore investigated whether a vasoactive drug,tanshinone IIA,could inhibit metastasis by inducing vascular normalization after palliative resection(PR)of hepatocellular carcinoma(HCC).METHODS:A liver orthotopic double-tumor xenograft model in nude mouse was established by implantation of HCCLM3(high metastatic potential)and Hep G2 tumor cells.After removal of one tumor by PR,the effects of tanshinone IIA administration on metastasis,tumor vascularization,and survival were evaluated.Tube formation was examined in mouse tumor-derived endothelial cells(TECs)treated with tanshinone IIA.RESULTS:PR significantly accelerated residual hepatoma metastases.Tanshinone IIA did not inhibit growth of single-xenotransplanted tumors,but it did reduce the occurrence of metastases.Moreover,it inhibited PR-enhanced metastases and,more importantly,prolonged host survival.Tanshinone IIA alleviated residual tumor hypoxia and suppressed epithelial-mesenchymal transition(EMT)in vivo;however,it did not downregulate hypoxia-inducible factor 1alpha(HIF-1alpha)or reverse EMT of tumor cells under hypoxic conditions in vitro.Tanshinone IIA directly strengthened tube formation of TECs,associated with vascular endothelial cell growth factor receptor 1/platelet derived growth factor receptor(VEGFR1/PDGFR)upregulation.Although the microvessel density(MVD)of residual tumor tissue increased after PR,the microvessel integrity(MVI)was still low.While tanshinone IIA did not inhibit MVD,it did dramatically increase MVI,leading to vascular normalization.CONCLUSIONS:Our results demonstrate that tanshinone IIA can inhibit the enhanced HCC metastasis associated with PR.Inhibition results from promoting VEGFR1/PDGFRrelated vascular normalization.This application demonstrates the potential clinical benefit of preventing postsurgical recurrence.

(J Hematol Oncol,2012,5:69)

37.Polymeric nanoparticle-encapsulated hedgehog pathway inhibitor HPI-1(NanoHHI)inhibits systemic metastases in an orthotopic model of human hepatocellular carcinoma　(IF 8.911)

Xu Y,Chenna V,Hu C,Sun HX,Khan M,Bai H,Yang XR,Zhu QF,Sun YF,Maitra A,Fan J,Anders RA

PURPOSE:To illustrate the prognostic significance of hedgehog(Hh)signaling in patients with hepatocellular carcinoma(HCC)and to evaluate the efficacy of a novel nanoparticle-encapsulated inhibitor of the Hh transcription factor,Gli1(Nano HHI)using in vitro and in vivo models of human HCCs.EXPERIMENTAL DESIGN:Patched1(Ptch1)expression was detected in tumor tissue microarrays of 396 patients with HCC who underwent curative surgical resection during February 2000 to December 2002.Prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests.The effects of Nano HHI alone and in combination with sorafenib were investigated on HCC cell lines.Primary HCC tumor growth and metastasis were examined in vivo using subcutaneous and orthotopic HCC xenografts in nude mice.RESULTS:Elevated expression of Ptch1 in HCC tissues was significantly related to disease recurrence,as well as a shorter time to recurrence in patients with HCC.In vitro,Nano HHI significantly inhibited the proliferation and invasion of HCC cell lines.Nano H HI potently suppressed in vivo tumor growth of HCC xenografts in both subcutaneous and orthotopic milieus,and in contrast to sorafenib,resulted in significant attenuation of systemic metastases in the orthotopic setting.Furthermore,Nano H HI significantly decreased the population of CD133-expressing HCC cells,which have been implicated in tumor initiation and metastases.CONCLUSION:Downstream Hh signaling has prognostic significance in patients with HCC as it predicts early recurrence.Gli inhibition through Nano H HI has profound tumor growth inhibition and antimetastatic effects in HCC models,which may provide a new strategy in the treatment of patients with HCC and prevention post-operative recurrence.

(Clin Cancer Res,2012,18:1291-1302)

38.HIWI is associated with prognosis in patients with hepatocellular carcinoma after curative resection(IF 6.102)

Zhao YM,Zhou JM,Wang LR,He HW,Wang XL,Tao ZH,Sun HC,Wu WZ,Fan J,Tang ZY,Wang L

BACKGROUND:PIWI protein family was found to play an important role in stem cell selfrenewal.Overexpression of HIWI,the human homolog of PIWI family proteins,was found in several solid tumors,although the role of HIWI in hepatocellular carcinoma(HCC)and its prognostic value remain unclear.METHODS:HIWI expression was measured in stepwise metastatic HCC cell lines(HCCLM3,MHCC97 H,MHCC97L,SMMC7721,and Hep G2),the normal liver cell line(L02),and HCC tissue samples(n=20).Proliferation and invasion were investigated in HCC cell lines undergoing HIWI target small interfering RNA transfection.Also explored was HIWI expression in HCC tissue microarrays(n=168)for survival analysis.RESULTS:Levels of HIWI protein and m RNA were up-regulated in highly metastatic HCC cell lines(HCCLM3,MHCC97 H,and MHCC97L),whereas their proliferation and invasion significantly decreased after depletion of HIWI.Intratumoral HIWI expression was higher than that of peritumoral tissue(P＜0.001)and positively associated with proliferating cell nuclear antigen expression(P＜0.001).Positive expression of intratumoral HIWI was associated with larger tumor size(P=0.047)and intrahepatic metastasis(P=0.027)and was an independent risk factor for overall survival(P=0.007)and recurrence-free survival(P=0.036),particularly in patients with low serum alpha-fetoprotein and low Edmondson-Steiner grade.CONCLUSIONS:HIWI may play a key role in HCC proliferation and metastasis and can be a potential prognostic factor for HCC after curative resection,particularly with well-differentiated HCC.

(Cancer,2012,118:2708-2717)

39.Overexpression of CXCL5 mediates neutrophil infiltration and indicates poor prognosis for hepatocellular carcinoma　(IF 14.971)

Zhou SL,Dai Z,Zhou ZJ,Wang XY,Yang GH,Wang Z,Huang XW,Fan J,Zhou J

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(m RNA)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)

40.Osteopontin promoter polymorphisms at locus 443 significantly affect the metastasis and prognosis of human hepatocellular carcinoma　(IF 14.971)

Dong QZ,Zhang XF,Zhao Y,Jia HL,Zhou HJ,Dai C,Sun HJ,Qin Y,Zhang WD,Ren N,Ye QH,Qin LX

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:-1 748A/-616G/-443T/-155*[*indicates base deletion];Ht3:-1 748A/-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.

(Hepatology,2013,57:1024-1034)

41.alphaB-crystallin complexes with 14-3-3zeta to induce epithelial-mesenchymal transition and resistance to sorafenib in hepatocellular carcinoma　(IF 14.971)

Huang XY,Ke AW,Shi GM,Zhang X,Zhang C,Shi YH,Wang XY,Ding ZB,Xiao YS,Yan J,Qiu SJ,Fan J,Zhou J

The overall survival of patients with hepatocellular carcinoma(HCC)remains poor,and the molecular pathogenesis remains incompletely defined in HCC.Here we report that increased expression of alphaB-Crystallin in human HCC predicts poor survival and disease recurrence after surgery.Multivariate analysis identifies alphaB-Crystallin expression as an independent predictor for postoperative recurrence and overall survival.We show that elevated expression of alphaB-Crystallin promotes HCC progression in vivo and in vitro.We demonstrate that alphaB-Crystallin overexpression fosters HCC progression by inducing epithelial-mesenchymal transition(EMT)in HCC cells through activation of the extracellular-regulated protein kinase(ERK)cascade,which can counteract the effect of sorafenib.alphaB-Crystallin complexes with and elevates 14-3-3zeta protein,leading to up-regulation of ERK1/2 activity.Moreover,overexpression of alphaB-Crystallin in HCC cells induces EMT progression through an ERK1/2/Fra-1/slug signaling pathway.Clinically,our data reveal that overexpression of both alphaB-Crystallin and 14-3-3zeta correlates with the HCC poorest survival outcomes,and sorafenib response is impaired in patients with alphaB-Crystallin overexpression.CONCLUSION:These data suggest that the alphaB-Crystallin-14-3-3zeta complex acts synergistically to promote HCC progression by constitutively activating ERK signaling.This study reveals alphaB-Crystallin as a potential therapeutic target for HCC and a biomarker for predicting sorafenib treatment response.

(Hepatology,2013,57:2235-2247)

42.High expression of IL-17 and IL-17RE associate with poor prognosis of hepatocellular carcinoma　(IF 5.646)

Liao R,Sun J,Wu H,Yi Y,Wang JX,He HW,Cai XY,Zhou J,Cheng YF,Fan J,Qiu SJ

Hepatocellular carcinoma(HCC)is a typical malignancy in a background of chronic inflammation.Th17 cells(a major source of IL-17)constitute crucial components of infiltrating inflammatory/immune cells in HCC and can amplify inflammatory response via binding to interleukin-17 receptor(IL-17R).Thus,we investigated the expression and clinical significance of IL-17 and IL-17 receptor family cytokines in HCC.METHODS:The expression and prognostic value of IL-17 and IL-17R(A-E)were examined in 300 HCC patients after resection.Six Th17 associated cytokines in serum(n=111)were quantified using enzyme-linked immunosorbent assays.Phenotypic features of IL-17+CD4+T cells were determined by flow cytometry analysis.RESULTS:High expression of intratumoral IL-17 and IL1-7RE were significantly associated with poorer survival(P=0.016 and＜0.001,respectively)and increased recurrence(both P＜0.001)of HCC patients.Moreover,intratumoral IL-17,individually or synergistically with IL-17RE,could predict HCC early recurrence and late recurrence.Also,peritumoral IL-17RE showed the prognostic ability in HCC(P＜0.001 for OS/TTR).Furthermore,expression levels of Th17 associated cytokines including IL-6,-22,-17R and TNF-alpha were increased in serum of HCC patients compared to haemangioma patients.Importantly,activated human hepatic stellate cells induced in vitro expansion of IL-17+CD4+T cells.CONCLUSIONS:High expression of IL-17 and IL-17RE were promising predictors for poor outcome of HCC patients.The protumor power of IL-17 producing CD4+T cells was probably involved in the crosstalk with different types of inflammatory/immune cells in HCC.

(J Exp Clin Cancer Res,2013,32:3)

43.Clinical significance and gene expression study of human hepatic stellate cells in HBV relatedhepatocellular carcinoma　(IF 5.646)

Liao R,Wu H,Yi Y,Wang JX,Cai XY,He HW,Cheng YF,Zhou J,Fan J,Sun J,Qiu SJ

BACKGROUND:Peritumoral activated hepatic stellate cells(HSCs)are versatile myofibroblastlike cells closely related with hepatocellular carcinoma(HCC)progression.So far,comprehensive comparison of gene expression of human HSCs during hepatocarcinogenesis is scanty.Therefore,we identified the phenotypic and genomic characteristics of peritumoral HSCs to explore the valuable information on the prognosis and therapeutic targets of HBV related HCC.METHODS:A tissue microarray containing 224 HBV related HCC patients was used to evaluate the expression of phenotype markers of HSCs including alpha-SMA,glial fibrillary acidic protein(GFAP),desmin,vinculin and vimentin.HSCs and cancer associated myofibroblasts(CAMFs)were isolated from normal,peritumoral human livers and cancer tissues,respectively.Flow cytometry and gene microarray analysis were performed to evaluate the phenotypic changes and gene expression in HCC,respectively.RESULTS:Peritumoral alpha-SMA positive HSCs showed the prognostic value in time to recurrence(TTR)and overall survival(OS)of HCC patients,especially in early recurrence and AFP-normal HCC patients.Expression of GFAP positive HSCs cell lines LX-2 was significantly decreased after stimulation with tumor conditioned medium.Compared with quiescent HSCs,peritumoral HSCs and intratumoral CAMFs expressed considerable up-and down-regulated genes associated with biological process,cellular component,molecular function and signaling pathways involved in fibrogenesis,inflammation and progress of cancer.CONCLUSIONS:Peritumoral activated HSCs displayed prognostic value in HBV related-HCC,and their genomic characteristics could present rational biomarkers for HCC risk and promising therapeutic targets.

(J Exp Clin Cancer Res,2013,32:22)

44.PROX1 promotes hepatocellular carcinoma metastasis by way of up-regulating hypoxia-inducible factor 1alpha expression and protein stability　(IF 14.971)

Liu Y,Zhang JB,Qin Y,Wang W,Wei L,Teng Y,Guo L,Zhang B,Lin Z,Liu J,Ren ZG,Ye QH,Xie Y

Hepatocellular carcinoma(HCC)is one of the most common cancers and the third leading cause of death from cancer worldwide.HCC has a very poor prognosis because of tumor invasiveness,frequent intrahepatic spread,and extrahepatic metastasis.The molecular mechanism of HCC invasiveness and metastasis is poorly understood.The homeobox protein PROX1 is required for hepatocyte migration during mouse embryonic liver development.In this study,we show that high PROX1 protein expression in primary HCC tissues is associated with significantly worse survival and early tumor recurrence in postoperative HCC patients.Knockdown of PROX1 expression in HCC cells inhibited cell migration and invasiveness in vitro and HCC metastasis in nude mice while overexpression of PROX1 in HCC cells promoted these processes.PROX1's prometastasis activity is most likely attributed to its up-regulation of hypoxia-inducible factor 1alpha(HIF-1alpha)transcription and stabilization of HIF-1alpha protein by recruiting histone deacetylase 1(HDAC1)to prevent the acetylation of HIF-1alpha,which subsequently induces an epithelial-mesenchymal transition response in HCC cells.We further demonstrated the prognostic value of using the combination of PROX1 and HDAC1 levels to predict postoperative survival and early recurrence of HCC.CONCLUSION:PROX1 is a critical factor that promotes HCC metastasis.

(Hepatology,2013,58:692-705)

45.Autophagy inhibition suppresses pulmonary metastasis of HCC in mice via impairing anoikis resistance and colonization of HCC cells　(IF 11.059)

Peng YF,Shi YH,Ding ZB,Ke AW,Gu CY,Hui B,Zhou J,Qiu SJ,Dai Z,Fan J Metastasis is one of the main causes of poor prognosis for hepatocellular carcinoma(HCC),which has been linked to cell-death resistance.Autophagy is an important survival mechanism under conditions of cell stress.We hypothesized that autophagy may play a role in HCC metastasis due to its prosurvival effect.Highly metastatic HCC cell lines with stable autophagy inhibition were established via lentivirus-mediated silencing of BECN1 and ATG5 genes.Mouse models of pulmonary metastasis were then developed using the cells with or without autophagy inhibition.The analysis of lung metastasis by histopathological examination and small animal imaging showed that autophagy inhibition significantly decreased the incidence of pulmonary metastases in vivo.Further invasion,migration,detachment,lung colonization,and epithelial-mesenchymal transition(EMT)assays indicated that autophagy inhibition did not affect cell invasiveness,migration or EMT but attenuated the anoikis-resistance and lung colonization of HCC cells.Investigation of the molecular mechanisms underlying showed that the autophagy-inhibition-mediated anoikis-resistance attenuation was associated with the regulation of apoptotic signaling.As autophagy inhibition was shown to be able to suppress HCC metastasis,an autophagy-based HCC tissue-specific target therapy system(AFP-Cre/Lox P-sh RNA)was constructed.In vitro and in vivo analyses showed that the system was able to efficiently inhibit autophagy of HCC cells and tissue in a tissue-specific manner.Further in vivo metastasis assay showed that intratumoral administration of the system could significantly suppress lung metastasis.Together,our findings suggest that autophagy may be involved in HCC metastasis through facilitating anoikis resistance and lung colonization of HCC cells.Autophagy-based HCC tissue-specific target therapy may be a new strategy for the management of HCC metastasis.

(Autophagy,2013,9:2056-2068)

46.Margin-infiltrating CD20(+)B cells display an atypical memory phenotype and correlate with favorable prognosis in hepatocellular carcinoma　(IF 8.911)

Shi JY,Gao Q,Wang ZC,Zhou J,Wang XY,Min ZH,Shi YH,Shi GM,Ding ZB,Ke AW,Dai Z,Qiu SJ,Song K,Fan J

PURPOSE:The role of infiltrating B cells in hepatocellular carcinoma has been overlooked for many years.This study is aimed to delineate the distribution,prognostic value,and functional status of B cells in human hepatocellular carcinoma.EXPERIMENTAL DESIGN:Immunohistochemistry was used to investigate the distribution and clinical significance of infiltrating CD20(+)B cells in a series of 120 patients with hepatocellular carcinoma.The results were further tested in an independent series of 200 patients with hepatocellular carcinoma.The functional status of CD20(+)B cells was determined by flow cytometry,immunofluorescence,and in vitro coculture assay.RESULTS:Infiltrating CD20(+)B cells were predominantly concentrated in the tumor invasive margin,compared with the peri-and intratumor areas.High density of margin-infiltrating B lymphocytes(MIL-B)positively correlated with small tumor size,absence of vascular invasion,and increased density of CD8(+)T cells(P＜0.05).Survival analyses revealed that increased number of MIL-Bs and their penetration through the tumor capsule were significantly associated with improved overall and recurrence-free survival,and were identified as independent prognosticators for patients with hepatocellular carcinoma(P＜0.05).Importantly,the results were further validated in another independent hepatocellular carcinoma cohort.Moreover,we found that MIL-Bs featured an atypical memory phenotype[IgD(-)IgG(+)CD27(-)CD38(-)],expressed surface markers characteristic of antigen-presenting cells,possessed tumor-killing potential by producing IFNgamma,interleukin 12p40(IL-12p40),granzyme B,and TRAIL,and acted in cooperation with CD8(+)T cells.CONCLUSIONS:The profile of CD20(+)B cells in situ is a new predictor of prognosis for patients with hepatocellular carcinoma and provides a novel target for an optimal immunotherapy against this fatal malignancy.

(Clin Cancer Res,2013,19:5994-6005)

47.High expression of Dickkopf-related protein 1 is related to lymphatic metastasis and indicates poor prognosis in intrahepatic cholangiocarcinoma patients after surgery　(IF 6.102)

Shi RY,Yang XR,Shen QJ,Yang LX,Xu Y,Qiu SJ,Sun YF,Zhang X,Wang Z,Zhu K,Qin WX,Tang ZY,Fan J,Zhou J

BACKGROUND:Dickkopf-related protein 1(DKK1)has been reported involved in metastasis and invasion in several tumors.This study sought to investigate the prognostic value of DKK1 in intrahepatic cholangiocarcinoma(ICC)and its role in promoting ICC metastasis.METHODS:Tissue microarrays of 138 ICC patient samples were employed to detect DKK1,vascular endothelial growth factor C(VEGF-C),and matrix metalloproteinase 9(MMP9)expression using immunohistochemistry.The prognostic significances were assessed by Kaplan-Meier survival estimates.DKK1 expression was measured in an ICC cell line(HCCC-9810)and ICC tissues by immunofluorescence assay,quantitative real-time polymerase chain reaction,and western blot.Serum levels of DKK1 from 37 ICC patients were tested by enzyme-linked immunosorbent assay.The role of DKK1 in proliferation,migration,invasion,and gene expression regulation was assessed by DKK1 depletion using small interfering RNA.RESULTS:Multivariate analyses revealed that DKK1 was an unfavorable predictor for overall survival and time to recurrence.The prognostic significance was retained in ICC patients with low recurrence risk(P＜0.05).DKK1 expression was elevated in an ICC cell line,tumor samples,and patient sera.High levels of DKK1 in ICC tissues correlated with elevated MMP9,VEGF-C,and metastasis of hepatic hilar lymph nodes.DKK1 depletion caused a decrease in cell migration and invasiveness,and down-regulation of MMP9 and VEGF-C expression.CONCLUSIONS:DKK1 is a novel prognostic biomarker for ICC,and it enhances tumor cell invasion and promotes lymph node metastasis of ICC through the induction of MMP9 and VEGF-C.DKK1 may be a potential therapeutic target for ICC.

(Cancer,2013,119:993-1003)

48.Hypoxia inducible factor 2 alpha inhibits hepatocellular carcinoma growth through the transcription factor dimerization partner 3/E2F transcription factor 1-dependent apoptotic pathway(IF 14.971)

Sun HX,Xu Y,Yang XR,Wang WM,Bai H,Shi RY,Nayar SK,Devbhandari RP,He YZ,Zhu QF,Sun YF,Hu B,Khan M,Anders RA,Fan J

Hypoxia inducible factors(HIFs)are activated in many tumors and show either promoter or suppressor activity,depending on tumor cell biology and background.However,the role of HIF member HIF-2alpha remains unclear in hepatocellular carcinoma(HCC).Here,HIF-2alpha expression was measured in HCC and paired peritumoral tissues by quantitative real-time polymerase chain reaction,western blotting,and immunofluorescence assays,and the clinical significance was explored in 246 HCC patients.In cell culture,HIF-2alpha levels were up-regulated or downregulated by use of expression or short hairpin RNA recombinant plasmid,respectively.Cells were analyzed by immunoblotting,chromatin immunoprecipitation coupled with microarray,coimmunoprecipitation,and immunohistochemical staining.In vivo tumor growth was analyzed in nude mice.We found that the average expression of HIF-2alpha was relatively low in HCC tissues,and the decreased level was associated with lower overall survival(P=0.006).High HIF-2alpha expression in HCC cells induced higher levels of apoptosis and expression of proapoptotic proteins and inhibited cell and tumor growth.Furthermore,HIF-2alpha inhibited expression of the novel target gene,transcription factor dimerization partner 3(TFDP3).TFDP3 protein was found to bind with E2F transcription factor 1(E2F1)and inhibit its transcriptional activity through both p53-dependent and-independent pathways.Reintroduction of TFDP3 expression reversed HIF-2alphainduced apoptosis.CONCLUSIONS:Data gathered from cell lines,tumorigenicity studies,and primary HCC samples demonstrate a negative role of HIF-2alpha in tumors,which is mediated by the TFDP3/E2F1 pathway.Our study provides evidence supporting a possible tumor-suppressor role for HIF-2alpha and has uncovered a mechanism that links HIF-2alpha to a fundamental biological regulator,E2F1.

(Hepatology,2013,57:1088-1097)

49.Circulating stem cell-like epithelial cell adhesion molecule-positive tumor cells indicate poor prognosis of hepatocellular carcinoma after curative resection　(IF 14.971)

Sun YF,Xu Y,Yang XR,Guo W,Zhang X,Qiu SJ,Shi RY,Hu B,Zhou J,Fan J

Epithelial cell adhesion molecule-positive(Ep CAM+)hepatocellular carcinoma(HCC)cells may constitute a tumor-initiating subpopulation in tumorigenic cell lines and HCC specimens.In the present study,Ep CAM+circulating tumor cells(CTCs)were identified prospectively in HCC patients undergoing curative resection,and the prognostic significance and their stem cell-like characteristics were investigated further.Blood samples from 123 HCC patients were tested prior to resection and 1 month thereafter.CTCs were present in 66.67%of patients,and the cell count measured in 7.5 m L of blood(CTC(7.5))ranged between 1 and 34.Fifty-one patients had CTC(7.5)of≥2 preoperatively,and these patients developed tumor recurrence earlier than those with CTC(7.5)of＜2 CTCs(P＜0.001).A preoperative CTC(7.5)of≥2 was an independent prognostic factor for tumor recurrence(P＜0.001).Its prognostic significance also applied to patients with alpha-fetoprotein(AFP)levels of≤400 ng/m L or subgroups with low recurrence risk(all P＜0.05).A significant decrease of CTC-positive rates(66.67%to 28.15%,P＜0.05)and CTC(7.5)values(2.60+/-0.43 to 1.00+/-0.36,P＜0.05)was observed 1 month after resection.Patients with consistent CTC(7.5)＜2 had lower recurrence rates than those with values consistently≥2(15.5%vs.87.50%,P＜0.001).Ep CAM+CTCs displayed cancer stem cell biomarkers(CD133 and ABCG2),epithelial-mesenchymal transition,Wnt pathway activation,high tumorigenic potential,and low apoptotic propensity.CONCLUSION:Stem cell-like phenotypes are observed in Ep CAM+CTCs,and a preoperative CTC(7.5)of≥2 is a novel predictor for tumor recurrence in HCC patients after surgery,especially in patient subgroups with AFP levels of≤400 ng/m L or low tumor recurrence risk.Ep CAM+CTCs may serve as a real-time parameter for monitoring treatment response and a therapeutic target in HCC recurrence.

(Hepatology,2013,57:1458-1468)

50.miR-612 suppresses the invasive-metastatic cascade in hepatocellular carcinoma　(IF 10.892)

Tao ZH,Wan JL,Zeng LY,Xie L,Sun HC,Qin LX,Wang L,Zhou J,Ren ZG,Li YX,Fan J,Wu WZ

Micro RNAs(miRNAs)play a critical role in tumor metastasis.In this study,we identified a set of 32 miRNAs involved in hepatocellular carcinoma(HCC)metastasis.Among them,miR-612 was shown for the first time to have inhibitory effects on HCC proliferation,migration,invasion,and metastasis.AKT2 was verified to be one of the direct targets of miR-612,through which the epithelial-mesenchymal transition(EMT)and metastasis were inhibited.The level of miR-612 in HCC patients was inversely associated with tumor size,stage,EMT,and metastasis.Of particular importance,miR-612 is involved in both the initial and final steps of the metastatic cascade,by suppressing local invasion and distant colonization.The pleiotropic roles of miR-612 in the HCC metastatic cascade suggest that it could be an effective target for both early and advanced HCC.

(J Exp Med,2013,210:789-803)

51.Mutations in isocitrate dehydrogenase 1 and 2 occur frequently in intrahepatic cholangiocarcinomas and share hypermethylation targets with glioblastomas　(IF 6.634)

Wang P,Dong Q,Zhang C,Kuan PF,Liu Y,Jeck WR,Andersen JB,Jiang W,Savich GL,Tan TX,Auman JT,Hoskins JM,Misher AD,Moser CD,Yourstone SM,Kim JW,Cibulskis K,Getz G,Hunt HV,Thorgeirsson SS,Roberts LR,Ye D,Guan KL,Xiong Y,Qin LX,Chiang DY Mutations in the genes encoding isocitrate dehydrogenase,IDH1 and IDH2,have been reported in gliomas,myeloid leukemias,chondrosarcomas and thyroid cancer.We discovered IDH1 and IDH2 mutations in 34 of 326(10%)intrahepatic cholangiocarcinomas.Tumor with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels,as well as increased dimethylation of histone H3 lysine 79(H3K79).Mutations in IDH1 or IDH2 were associated with longer overall survival(P=0.028)and were independently associated with a longer time to tumor recurrence after intrahepatic cholangiocarcinoma resection in multivariate analysis(P=0.021).IDH1 and IDH2 mutations were significantly associated with increased levels of p53 in intrahepatic cholangiocarcinomas,but no mutations in the p53 gene were found,suggesting that mutations in IDH1 and IDH2 may cause a stress that leads to p53 activation.We identified 2309 genes that were significantly hypermethylated in 19 cholangiocarcinomas with mutations in IDH1 or IDH2,compared with cholangiocarcinomas without these mutations.Hypermethylated Cp G sites were significantly enriched in Cp G shores and upstream of transcription start sites,suggesting a global regulation of transcriptional potential.Half of the hypermethylated genes overlapped with DNA hypermethylation in IDH1-mutant gliobastomas,suggesting the existence of a common set of genes whose expression may be affected by mutations in IDH1 or IDH2 in different types of tumors.

(Oncogene,2013,32:3091-3100)

52.Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-kappaB pathways induced by paracrine cytokines　(IF 5.646)

Wang YH,Dong YY,Wang WM,Xie XY,Wang ZM,Chen RX,Chen J,Gao DM,Cui JF,Ren ZG

BACKGROUND:It is well documented that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks.However,little is known about the effects of endothelial cells on the behavior of tumor cells.The study here was to determine the roles of endothelial cells in HCC cell growth,migration and invasion.METHODS:A mixture of highly metastatic MHCC97H cells and HUVEC cells,as well as MHCC97H cells alone were subcutaneously injected into nude mice to observe the effects of HUVECs on HCC growth.The biological characteristics of MHCC97H cells respectively treated with conditioned medium(CM)derived from HUVECs and endothelial cell basal medium(EBM)in vitro,such as proliferation,migration and invasion,invasion/metastasis associated gene expression,were comparatively analyzed.Differential cytokines between CM and EBM were screened and identified using human cytokine array.Effects of the interested differential cytokine CCL2,IL-8 and CXCL16 and its related signaling pathways were further investigated in HCC cells.RESULTS:Subcutaneous tumorigenicity of MHCC97H cells in nude mice was promoted by HUVECs and its invasion/metastasis associated genes were significantly upregulated.The in vitro,proliferation,migration and invasion of HCC cells treated with CM were all significantly enhanced as compared to those with EBM stimulation.Simultaneously,PI3K/Akt and ERK1/2 pathway in HCC cells were activated by CM.Total of 25 differential cytokines were identified between CM and EBM such as angiopoietin-2,CCL2(MCP-1),uPA,endostatin,CXCL16,IL-8,pentraxin 3 etc.The selected differential cytokines CCL2,IL-8 and CXCL16 all modulated the expressions of HCCinvasion/metastasis genes,especially MMP2 and MMP9.In exposure to CCL2 or CXCL16 alone,upregulation in AKT phosphorylation but no change in ERK phosphorylation were found in MHCC97H cells,moreover the contents of nuclear transcription factor NF-kappaB were increased as compared to the control.However,no effects on the activation of Akt and ERK pathway in MHCC97H were found in exposure to IL-8.CONCLUSION:This study expands the contribution of endothelial cells to the progression of HCC.It unveils a new paradigm in which endothelial cells function as initiators of molecular crosstalks that enhance survival,migration and invasion of HCC cells.

(J Exp Clin Cancer Res,2013,32:51)

53.MicroRNA-26a suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting interleukin-6-Stat3 pathway　(IF 14.971)

Yang X,Liang L,Zhang XF,Jia HL,Qin Y,Zhu XC,Gao XM,Qiao P,Zheng Y,Sheng YY,Wei JW,Zhou HJ,Ren N,Ye QH,Dong QZ,Qin LX

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 upregulation.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.

(Hepatology,2013,58:158-170)

54.The functional impairment of HCC-infiltrating gammadelta T cells,partially mediated by regulatory T cells in a TGFbeta-and IL-10-dependent manner　(IF 18.946)

Yi Y,He HW,Wang JX,Cai XY,Li YW,Zhou J,Cheng YF,Jin JJ,Fan J,Qiu SJ

BACKGROUND&AIMS:The immunosuppressive network within the tumor microenvironment is one of the major obstacles to the success of cancer immunotherapy.gammadelta T cells are attractive effectors for cancer immunotherapy.Nevertheless,the promising anti-tumor effect in vitro is partially if not totally mitigated in vivo.Thus,understanding the immune status of tumorinfiltrating gammadelta T cells is essential for orchestrating effective immunotherapy strategies.In this study,we have investigated the immunophenotype and function of gammadelta T cells in hepatocellular carcinoma(HCC)patients.METHODS:The phenotype of gammadelta T cells in peripheral blood,and peritumoral and tumoral tissues of HCC patients(n=61)was characterized by flow cytometry.Functional analysis of the HCC-infiltrating gammadelta T cells was conducted directly after gammadelta T cell isolation.RESULTS:The infiltration of gammadelta T cells in tumoral tissues was significantly reduced compared to paired peritumoral tissues.Impairment in degranulation of the granule pathway and downregulation of IFN-gamma secretion were also demonstrated in HCC-infiltrating gammadelta T cells,which was in agreement with the results of gene microarray analysis,and further strengthened by the compromised specific cytotoxicity and IFN-gamma secretion in vitro.Moreover,isolated HCC-infiltrating CD4(+)CD25(+)regulatory T cells(Treg cells)directly suppressed the cytotoxic function and IFN-gamma secretion of gammadelta T cells in a TGFbeta-and IL-10-dependent manner.CONCLUSIONS:The effector function of gammadelta T cells was substantially impaired in HCC,which is partially mediated by Treg cells.We propose a new mechanism by which immune privilege develops within the tumor milieu.

(J Hepatol,2013,58:977-983)

55.Prognostic value of peritumoral heat-shock factor-1 in patients receiving resection of hepatocellular carcinoma　(IF 5.416)

Zhang JB,Guo K,Sun HC,Zhu XD,Zhang B,Lin ZH,Zhang BH,Liu YK,Ren ZG,Fan J

BACKGROUND:The cross-talk of hepatocellular carcinoma(HCC)cells and abnormal metabolic signals in peritumoral microenvironment modifies our knowledge of hepatocarcinogenesis.As an indispensable modulator of various stresses,the clinical significance of heat-shock transcription factor-1(HSF1)in HCC microenvironment has never been defined.METHODS:Hepatocellular carcinoma and matched peritumoral liver tissues(n=332)were semiquantitatively analysed for HSF1 expression,followed by correlation with clinicopathological parameters(patient outcomes).Moreover,the effects of HSF1 deficiency in L02 on monocarboxylate transporter-4(MCT4)and HCC cells'colonisation and proliferation were investigated.RESULTS:High expression of HSF1 in peritumoral tissue but not in HCC tissue was associated with poorer overall survival(OS)and time to recurrence(TTR),especially early recurrence(ER),which was further reconfirmed in validation cohort.Multivariate analysis showed that prognostic performance of peritumoral HSF1 was independent of other clinicopathological factors(hazard ratio for OS=2.60,P=0.002,for TTR=2.52,P＜0.001).Notably,downregulation of HSF1 in L02 decreased MCT4 expression significantly.The supernatant from L02-sh RNA-HSF1 in hypoxia,NOT normoxia condition,inhibited HCC cell colonisation and proliferation.Moreover,the combination of peritumoral HSF1 and MCT4 was the best predictor for ER and OS.CONCLUSION:High peritumoral HSF1 expression can serve as a sensitive‘readout’for high-risk HCC ER,and could be a potential metabolic intervention target following curative resection.

(Br J Cancer,2013,109:1648-1656)

56.Evaluation of midkine as a diagnostic serum biomarker in hepatocellular carcinoma(IF 8.911)

Zhu WW,Guo JJ,Guo L,Jia HL,Zhu M,Zhang JB,Loffredo CA,Forgues M,Huang H,Xing XJ,Ren N,Dong QZ,Zhou HJ,Ren ZG,Zhao NQ,Wang XW,Tang ZY,Qin LX,Ye QH

PURPOSE:To evaluate the value of serum midkine(MDK)as a diagnostic biomarker in hepatocellular carcinoma,particularly for those with negative alpha-fetoprotein(AFP)and at an early stage.EXPERIMENTAL DESIGN:MDK expression in tumors was assessed by immunohistochemistry from 105 patients with hepatocellular carcinomas or liver cirrhosis.Serum MDK levels were detected by ELISA in 933 participants including hepatocellular carcinomas and hospital controls from different medical centers.Sensitivities and specificities of serum MDK in diagnosing hepatocellular carcinoma according to AFP level and Barcelona Clinic Liver Cancer(BCLC)stage were analyzed.RESULTS:MDK levels were significantly elevated in hepatocellular carcinoma tissues as well as serum samples.The sensitivity of serum MDK for hepatocellular carcinoma diagnosis was much higher than that of AFP(86.9%vs.51.9%)with similar specificities(83.9%vs.86.3%).Notably,serum MDK had an outstanding performance in distinguishing AFP-negative hepatocellular carcinomas from different controls:In those AFPnegative hepatocellular carcinomas,the sensitivity could reach as high as 89.2%.Moreover,receiver operating characteristic(ROC)curve analysis also showed that serum MDK had a better performance compared with AFP in distinguishing early-stage hepatocellular carcinomas as well as small hepatocellular carcinomas.Even in very early-stage hepatocellular carcinomas,MDK showed an obviously higher sensitivity compared with AFP(80%vs.40%).Furthermore,serum MDK level was significantly decreased in patients with hepatocellular carcinomas after curative resection and re-elevated when tumor relapse occurred.CONCLUSIONS:Serum MDK is significantly elevated in most hepatocellular carcinomas,including those with negative AFP and at an early stage,which may serve as a novel diagnostic marker in early diagnosis and postoperative monitoring of hepatocellular carcinomas.

(Clin Cancer Res,2013,19:3944-3954)

57.Capn4 contributes to tumour growth and metastasis of hepatocellular carcinoma by activation of the FAK-Src signalling pathways　(IF 5.942)

Dai Z,Zhou SL,Zhou ZJ,Bai DS,Xu XY,Fu XT,Chen Q,Zhao YM,Zhu K,Yu L,Yang GH,Wang Z,Wu WZ,Zhou J,Fan J

Calpain small subunit 1(Capn4)has been identified as a major gene that promotes metastasis of hepatocellular carcinoma(HCC).However,the mechanism by which Capn4 promotes progression of HCC is not understood.In this study,we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue.Over-expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness,tumourigenicity and lung metastasis in vivo.Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4.Interestingly,Capn4 was found to physically associate with FA K and promoted hyperactivity of the FAK-Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK,Src and p130Cas.Knock-down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FA K-Src signalling pathway.Furthermore,Capn4-mediated invasion and metastasis of HCC cells required up-regulation of matrix metalloproteinase-2(M MP2)through activation of this signalling pathway.Our clinical data revealed that Capn4 expression correlated well with the levels of phospho-FA K,and over-expression of both Capn4 and phospho-FA K correlates with the poorest survival outcomes in HCC.In conclusion,our data showed that Capn4 can contribute to HCC grow th and metastasis via activation of the FA K-Src signalling pathway and M MP2.

(J Pathol,2014,234:316-328)

58.Amplification and over-expression of MAP3K3 gene in human breast cancer promotes formation and survival of breast cancer cells　(IF 5.942)

Fan Y,Ge N,Wang X,Sun W,Mao R,Bu W,Creighton CJ,Zheng P,Vasudevan S,An L,Yang J,Zhao YJ,Zhang H,Li XN,Rao PH,Leung E,Lu YJ,Gray JW,Schiff R,Hilsenbeck SG,Osborne CK,Yang J,Zhang H

Gene amplifications in the 17q chromosomal region are observed frequently in breast cancers.An integrative bioinformatics analysis of this region nominated the MAP3K3 gene as a potential therapeutic target in breast cancer.This gene encodes mitogen-activated protein kinase kinase kinase 3(MAP3K3/MEKK3),which has not yet been reported to be associated with cancer-causing genetic aberrations.We found that MAP3K3 was amplified in approximately 8%～20%of breast cancers.Knockdown of MAP3K3 expression significantly inhibited cell proliferation and colony formation in MAP3K3-amplified breast cancer cell lines MCF-7 and MDA-MB-361 but not in MAP3K3 nonamplified breast cancer cells.Knockdown of MAP3K3 expression in MAP3K3-amplified breast cancer cells sensitized breast cancer cells to apoptotic induction by TNFalpha and TRAIL,as well as doxorubicin,VP-16 and fluorouracil,three commonly used chemotherapeutic drugs for treating breast cancer.In addition,ectopic expression of MAP3K3,in collaboration with Ras,induced colony formation in both primary mouse embryonic fibroblasts and immortalized human breast epithelial cells(MCF-10A).Combined,these results suggest that MAP3K3 contributes to breast carcinogenesis and may endow resistance of breast cancer cells to cytotoxic chemotherapy.Therefore,MAP3K3 may be a valuable therapeutic target in patients with MAP3K3-amplified breast cancers,and blocking MAP3K3 kinase activity with a small molecule inhibitor may sensitize MAP3K3-amplified breast cancer cells to chemotherapy.

(J Pathol,2014,232:75-86)

59.Activating mutations in PTPN3 promote cholangiocarcinoma cell proliferation and migration and are associated with tumor recurrence in patients　(IF 19.233)

Gao Q,Zhao YJ,Wang XY,Guo WJ,Gao S,Wei L,Shi JY,Shi GM,Wang ZC,Zhang YN,Shi YH,Ding J,Ding ZB,Ke AW,Dai Z,Wu FZ,Wang H,Qiu ZP,Chen ZA,Zhang ZF,Qiu SJ,Zhou J,He XH,Fan J

BACKGROUND&AIMS:The pathogenesis of intrahepatic cholangiocarcinoma(ICC),the second most common hepatic cancer,is poorly understood,and the incidence of ICC is increasing worldwide.We searched for mutations in human ICC tumor samples and investigated how they affect ICC cell function.METHODS:We performed whole exome sequencing of 7 pairs of ICC tumors and their surrounding nontumor tissues to detect somatic alterations.We then screened 124 pairs of ICC and nontumor samples for these mutations,including 7 exomes.We compared mutations in PTPN3 with tumor recurrence in 124 patients and PTPN3 expression levels with recurrence in 322 patients(the combination of both in 86 patients).The functional effects of PTPN3 variations were determined by RNA interference and transgenic expression in cholangiocarcinoma cell lines(RBE,HCCC-9810,and Huh28).RESULTS:Based on exome sequencing,pathways that regulate protein phosphorylation were among the most frequently altered in ICC samples and genes encoding protein tyrosine phosphatases(PTPs)were among the most frequently mutated.We identified mutations in 9 genes encoding PTPs in 4 of 7 ICC exomes.In the prevalence screen of 124 paired samples,51.6%of ICCs contained somatic mutations in at least 1 of 9 PTP genes;41.1%had mutations in PTPN3.Transgenic expression of PTPN3 in cell lines increased cell proliferation,colony formation,and migration.PTPN3(L232R)and PTPN3(L384 H),which were frequently detected in ICC samples,were found to be gain-of-function mutations;their expression in cell lines further increased cell proliferation,colony formation,and migration.ICC-associated variants of PTPN3 altered phosphatase activity.Patients whose tumors contained activating mutations or higher levels of PTPN3 protein than nontumor tissues had higher rates of disease recurrence than patients whose tumors did not have these characteristics.CONCLUSIONS:Using whole exome sequencing of ICC samples from patients,we found that more than 40%contain somatic mutations in PTPN3.Activating mutations in and high expression levels of PTPN3 were associated with tumor recurrence.

(Gastroenterology,2014,146:1397-1407)

60.Clinical significance of EpCAM mRNA-positive circulating tumor cells in hepatocellular carcinoma by an optimized negative enrichment and qRT-PCR-based platform　(IF 8.911)

Guo W,Yang XR,Sun YF,Shen MN,Ma XL,Wu J,Zhang CY,Zhou Y,Xu Y,Hu B,Zhang X,Zhou J,Fan J

PURPOSE:This study aimed to construct a novel platform for the detection of circulating tumor cells(CTC)in patients with hepatocellular carcinoma(HCC)and to investigate the clinical significance of epithelial cell adhesion molecule m RNA-positive[Ep CAM(m RNA+)]CTCs using this platform.EXPERIMENTAL DESIGN:An optimized platform for CTC detection was constructed by evaluating different negative enrichment,m RNA isolation,and cDNA synthesis procedures and compared with the CellSearch system.A total of 299 patients with HCC were recruited into this prospective study;of these,157 who received curative resection,76 who received transcatheter arterial chemoembolization(TACE),and 66 who received radiotherapy were tested using our platform.The diagnostic value of Ep CAM(m RNA+)CTCs was investigated in 122 patients with HCC who underwent resection and 120 control subjects.RESULTS:The optimized negative enrichment and quantitative real-time PCR(qRT-PCR)-based CTC detection platform had high sensitivity,specificity,and reproducibility and a low sample volume requirement.This platform showed a potential diagnostic value in patients with HCC and exhibited 76.7%consistency with the CellSearch system(r=0.54,P＜0.050).Pretreatment CTC level showed prognostic significance in patients with HCC treated with resection,TACE,and radiotherapy(all P＜0.050).Most of the patients showed a decrease in CTC levels after treatment that reflected tumor response.In contrast,patients with an increased CTC level showed disease progression after treatment.CONCLUSIONS:We established an optimized platform based on negative enrichment and qRTPCR for highly sensitive,specific,and reproducible CTC detection.This platform might be clinically useful in auxiliary diagnosis,treatment response assessment,and early decision-making to tailor the most effective antitumor strategies.

(Clin Cancer Res,2014,20:4794-4805)

61.Systemic immune-inflammation index predicts prognosis of patients after curative resection for hepatocellular carcinoma　(IF 8.911)

Hu B,Yang XR,Xu Y,Sun YF,Sun C,Guo W,Zhang X,Wang WM,Qiu SJ,Zhou J,Fan J

PURPOSE:We developed a novel systemic immune-inflammation index(SII)based on lymphocyte,neutrophil,and platelet counts and explored its prognostic value in hepatocellular carcinoma(HCC).EXPERIMENTAL DESIGN:The SII was developed based on a retrospective study of 133 patients with HCC undergoing resection between 2005 and 2006,and validated in a prospective study of 123 patients enrolled from 2010 to 2011.The circulating tumor cell(CTC)level in the validation cohort was measured using the CellSearch system.Prediction accuracy was evaluated with area under the receiver operating characteristic curve(AUC).RESULTS:An optimal cutoff point for the SII of 330×10(9)stratified the patients with HCC into high(≥330)and low SII(＜330)groups in the training cohort.Univariate and multivariate analyses revealed the SII was an independent predictor for overall survival and relapse-free survival,and prognostic for patients with negative alpha-fetoprotein and Barcelona Clinic Liver Cancer stage 0+A.The AUCs of the SII for survival and recurrence were higher than other conventional clinical indices.An SII≥330 was significantly associated with vascular invasion,large tumors,and early recurrence.CTC levels were significantly higher in the SII≥330 group(1.71+/-0.34 vs.4.37+/-1.04,P=0.029).In patients with detectable CTCs,those with SII≥330 had higher recurrence rates and shorter survival time than patients with SII＜330.CONCLUSION:The SII was a powerful prognostic indicator of poor outcome in patients with HCC and is a promising tool for HCC treatment strategy decisions.The dismal outcome in patients with high SII scores might be related to higher CTC levels.

(Clin Cancer Res,2014,20:6212-6222)

62.Clinical significance of the ubiquitin ligase UBE3C in hepatocellular carcinoma revealed by exome sequencing　(IF 14.971)

Jiang JH,Liu YF,Ke AW,Gu FM,Yu Y,Dai Z,Gao Q,Shi GM,Liao BY,Xie YH,Fan J,Huang XW,Zhou J

Virus-induced hepatocarcinogenesis involves a series of histological developmental processes with the stepwise acquisition of several genetic changes that are necessary for the malignant transformation of hepatocytes.Although genetic alterations are known to be involved in the pathogenesis of hepatocellular carcinoma(HCC),little is known about the contributions of specific genes to this process.To gain insight into the genetic alterations involved in the neoplastic evolution from chronic hepatitis B virus infection to dysplastic nodules(DN)to HCC,we captured and sequenced the exomes of four DNA samples:one DN sample,two HCC samples,and one control peripheral blood sample from a single HCC patient.Mutations in the UBE3C gene(encoding ubiquitin ligase E3C)were observed in both tumor tissues.Then we resequenced the UBE3C gene in a cohort of 105 HCC patients and identified mutations in 17 out of a total of 106(16.0%)HCC patients.The subsequent experiments showed that UBE3C promoted HCC progression by regulating HCC cells epithelialmesenchymal transition.Clinically,a tissue microarray study of a cohort containing 323 HCC patients revealed that the overexpression of UBE3C in primary HCC tissues correlated with decreased survival(hazard ratio[HR]=1.657,95%confidence interval[CI]=1.220-2.251,P=0.001)and early tumor recurrence(HR=1.653,95%CI=1.227-2.228,P=0.001)in postoperative HCC patients.CONCLUSION:Our findings indicate that UBE3C is a candidate oncogene involved in tumor development and progression and therefore a potential therapeutic target in applicable HCC patients.

(Hepatology,2014,59:2216-2227)

63.High expression of 5-hydroxymethylcytosine and isocitrate dehydrogenase 2 is associated with favorable prognosis after curative resection of hepatocellular carcinoma　(IF 5.646)

Liu WR,Tian MX,Jin L,Yang LX,Ding ZB,Shen YH,Peng YF,Zhou J,Qiu SJ,Dai Z,Fan J,Shi YH

BACKGROUND:The 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.METHODS:The 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.RESULTS:We discovered that low 5-hmC and IDH2 expression was associated with malignant behaviors.Low 5-hmC or IDH2 expression alone and combined 5-hm C and IDH2 expression were associated with lower overall survival(OS)rates and higher cumulative recurrence rates.Multivariate analysis indicated that 5-hm C 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.CONCLUSIONS:5-hm C 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.

(J Exp Clin Cancer Res,2014,33:32)

64.MicroRNA-26a suppresses angiogenesis in human hepatocellular carcinoma by targeting hepatocyte growth factor-c Met pathway　(IF 14.971)

Yang X,Zhang XF,Lu X,Jia HL,Liang L,Dong QZ,Ye QH,Qin LX

Micro RNA(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(c Met)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-c Met signaling,and it is a new hopeful therapeutic target and prognostic marker for HCC.

(Hepatology,2014,59:1874-1885)

65.Arsenic trioxide induces differentiation of CD133+hepatocellular carcinoma cells and prolongs posthepatectomy survival by targeting GLI1 expression in a mouse model　(IF 8.731)

Zhang KZ,Zhang QB,Zhang QB,Sun HC,Ao JY,Chai ZT,Zhu XD,Lu L,Zhang YY,Bu Y,Kong LQ,Tang ZY

BACKGROUND:Cancer stem cells(CSCs)play a key role in the posthepatectomy recurrence of hepatocellular carcinoma(HCC).CD133+HCC cells exhibit liver CSC-like properties,and CSC differentiation-inducing therapy may lead these cells to lose their self-renewal ability and may induce terminal differentiation,which may in turn allow their malignant potential to be controlled.Because arsenic trioxide(As(2)O(3))increases remission rates and prolongs survival among patients with acute promyelocytic leukemia by inducing differentiation and apoptosis of leukemic cells,we hypothesized that As(2)O(3)might also inhibit HCC recurrence and prolong survival time after hepatectomy by inducing differentiation of HCC CSCs.METHODS:We evaluated the As(2)O(3)induced differentiation of human HCC CSCs and its mechanism in vitro,and we investigated the effects of treatment with As(2)O(3)on recurrence rates and median survival in a mouse xenograft model.RESULTS:We found that As(2)O(3)induced HCC CSC differentiation by down-regulating the expression of CD133 and some stemness genes,thus inhibiting the cells'self-renewal ability and tumorigenic capacity without inhibiting their proliferation in vitro.In vivo experiments indicated that As(2)O(3)decreased recurrence rates after radical resection and prolonged survival in a mouse model.As(2)O(3),which shows no apparent toxicity,may induce HCC CSC differentiation by down-regulating the expression of GLI1.CONCLUSIONS:We found that As(2)O(3)induced HCC CSC differentiation,inhibited recurrence,and prolonged survival after hepatectomy by targeting GLI1expression.Our results suggest that the clinical safety and utility of As(2)O(3)should be further evaluated.

(J Hematol Oncol,2014,7:28)

66.HNRNPAB induces epithelial-mesenchymal transition and promotes metastasis of hepatocellular carcinoma by transcriptionally activating SNAIL　(IF 8.378)

Zhou ZJ,Dai Z,Zhou SL,Hu ZQ,Chen Q,Zhao YM,Shi YH,Gao Q,Wu WZ,Qiu SJ,Zhou J,Fan J

Expression of heterogeneous nuclear ribonucleoprotein AB(HNRNPAB)has been reported to be dysregulated in tumors,but its specific contributions to tumor formation and progression are not fully understood.Here,we demonstrate that HNRNPAB is overexpressed in highly metastatic cells and tumor tissues from patients with hepatocellular carcinoma(HCC)with recurrence.We found that HNRNPAB overexpression promoted epithelial-mesenchymal transition(EMT)in a manner associated with HCC metastasis in vitro and in vivo.RNA interference-mediated silencing of the EMT factor SNAIL attenuated HNRNPAB-enhanced cell invasion in vitro and lung metastasis in vivo.Mechanistically,HNRNPAB acted to transactivate SNAIL1 transcription,which in turn inhibited transcription of the pivotal SNAIL target gene E-cadherin.Overexpression of HNRNPAB in HCC samples correlated with higher SNAIL levels,shorter overall survival,and higher tumor recurrence.HNRNPAB overexpression,alone or in combination with SNAIL,was found to be a significant independent risk factor for recurrence and survival after curative resection.In conclusion,our findings define HNRNPAB as an activator of EMT and metastasis in HCC that predicts poor clinical outcomes.

(Cancer Res,2014,74:2750-2762)

67.Ubiquitin-specific protease 7 accelerates p14(ARF)degradation by deubiquitinating thyroid hormone receptor-interacting protein 12 and promotes hepatocellular carcinoma progression(IF 14.971)

Cai JB,Shi GM,Dong ZR,Ke AW,Ma H H,Gao Q,Shen ZZ,Huang XY,Chen H,Yu DD,Liu LX,Zhang PF,Zhang C,Hu MY,Yang LX,Shi YH,Wang XY,Ding ZB,Qiu SJ,Sun HC,Zhou J,Shi YG,Fan J

The prognosis for hepatocellular carcinoma(HCC)remains dismal in terms of overall survival(OS),and its molecular pathogenesis has not been completely defined.Here,we report that expression of deubiquitylase ubiquitin-specific protease 7(USP7)is higher in human HCC tissues than in matched peritumoral tissues.Ectopic USP7 expression promotes growth of HCC cells in vivo and in vitro.Mechanistically,USP7 overexpression fosters HCC cell growth by forming a complex with and stabilizing thyroid hormone receptor-interacting protein 12(TRIP12),which induces constitutive p14(ARF)ubiquitination.Clinically,USP7 overexpression is significantly correlated with a malignant phenotype,including larger tumor size,multiple tumor,poor differentiation,elevated alpha-fetoprotein,and microvascular invasion.Moreover,overexpression of USP7 and/or TRIP12 correlates with shorter OS and higher cumulative recurrence rates of HCC.CONCLUSION:USP7 stabilizes TRIP12 by deubiquitination,thus constitutively inactivating p14(ARF)and promoting HCC progression.This represents a novel marker for predicting prognosis and a potential therapeutic target for HCC.

(Hepatology,2015,61:1603-1614)

68.microRNA-26a suppresses recruitment of macrophages by down-regulating macrophage colonystimulating factor expression through the PI3K/Akt pathway in hepatocellular carcinoma(IF 8.731)

Chai ZT,Zhu XD,Ao JY,Wang WQ,Gao DM,Kong J,Zhang N,Zhang YY,Ye BG,Ma DN,Cai H,Sun HC

BACKGROUND:micro RNAs(miRNAs)have been reported to modulate macrophage colonystimulating factor(M-CSF)and macrophages.The aim of this study was to find whether miR-26a can suppress M-CSF expression and the recruitment of macrophages.METHODS:Hepatocellular carcinoma(HCC)cell lines with decreased or increased expression of miR-26a were established in a previous study.M-CSF expression by tumor cells was measured by enzyme-linked immunosorbent assay,and cell migration assays were used to explore the effect of HCC cell lines on macrophage recruitment in vitro.Real-time PCR measured a panel of m RNAs expressed by macrophages.Xenograft models were used to observe tumor growth.Immunohistochemistry was conducted to study the relation between miR-26a expression and M-CSF expression and macrophage recruitment in patients with HCC.RESULTS:Ectopic expression of miR-26a reduced expression of M-CSF.The conditioned medium(CM)from Hep G2 cells that overexpressed miR-26a reduced the migration ability of THP-1 cells stimulated by phorbol myristate acetate(PMA)increased expression of interleukin(IL)-12b or IL-23 m RNA and decreased expression of chemokine(C-C motif)ligand(CCL)22,CCL17,and IL-10 m RNA,in comparison to the medium from the parental Hep G2 cells.These effects could be interrupted by the PI3K/Akt pathway inhibitor LY294002.Ectopic expression of miR-26a in HCC cells suppressed tumor growth,M-CSF expression,and infiltration of macrophages in tumors.Similar results were also found when using HCCLM3 cells.Furthermore,the expression of miR-26a was inversely correlated with M-CSF expression and macrophage infiltration in tumor tissues from patients with HCC.CONCLUSIONS:miR-26a expression reduced M-CSF expression and recruitment of macrophages in HCC.

(J Hematol Oncol,2015,8:56)

69.MiR-199a-5p is negatively associated with malignancies and regulates glycolysis and lactate production by targeting hexokinase 2 in liver cancer　(IF 14.971)

Guo W,Qiu Z,Wang Z,Wang Q,Tan N,Chen T,Chen Z,Huang S,Gu J,Li J,Yao M,Zhao Y,He X

Cancer cells possess a unique metabolic phenotype that allows them to preferentially utilize glucose through aerobic glycolysis.This phenomenon is referred to as the“Warburg effect.”Accumulating evidence suggests that microRNAs(miRNAs),a class of small noncoding regulatory RNAs,interact with oncogenes/tumor suppressors and induce such metabolic reprograming in cancer cells.To systematically study the metabolic roles of miRNAs in cancer cells,we developed a gain-offunction miRNA screen in HeLa cells.Subsequent investigation of the characterized miRNAs indicated that miR-199a-5p acts as a suppressor for glucose metabolism.Furthermore,miR-199a-5p is often down-regulated in human liver cancer,and its low expression level was correlated with a low survival rate,large tumor size,poor tumor differentiation status,high tumor-node-metastasis stage and the presence of tumor thrombus of patients.MicroRNA-199a-5p directly targets the 3'-untranslated region of hexokinase 2(HK2),an enzyme that catalyzes the irreversible first step of glycolysis,thereby suppressing glucose consumption,lactate production,cellular glucose-6-phosphate and adenosine triphosphate levels,cell proliferation,and tumorigenesis of liver cancer cells.Moreover,HK2 is frequently up-regulated in liver cancer tissues and associated with poor patient outcomes.The up-regulation of hypoxia-inducible factor-1alpha under hypoxic conditions suppresses the expression of miR-199a-5p and promotes glycolysis,whereas reintroduction of miR-199a-5p interferes with the expression of HK2,abrogating hypoxia-enhanced glycolysis.CONCLUSION:miR-199a-5p/HK2 reprograms the metabolic process in liver cancer cells and provides potential prognostic predictors for liver cancer patients.

(Hepatology,2015,62:1132-1144)

70.Hypoxia upregulates Rab11-family interacting protein 4 through HIF-1alpha to promote the metastasis of hepatocellular carcinoma　(IF 6.634)(https://www.daowen.com)

Hu F,Deng X,Yang X,Jin H,Gu D,Lv X,Wang C,Zhang Y,Huo X,Shen Q,Luo Q,Zhao F,Ge T,Zhao F,Chu W,Shu H,Yao M,Fan J,Qin W Hypoxic microenvironment is a powerful driving force for the invasion and metastasis of hepatocellular carcinoma(HCC).Hypoxia-inducible factor 1alpha(HIF-1alpha),as a crucial regulator of transcriptional responses to hypoxia,induces the expression of multiple target genes involved in different steps of HCC metastatic process.It is critical to find target genes associated with metastasis under hypoxia for shedding new light on molecular mechanism of HCC metastasis.In this study,we uncovered that hypoxia could induce the upregulation of Rab11-family interacting protein 4(Rab11-FIP4)and activation of Rab11-FIP4 promoter by HIF-1alpha.The overexpression of Rab11-FIP4 significantly enhanced the mobility and invasiveness of HCC cells in vitro,also contributed to distant lung metastasis in vivo,whereas silencing of Rab11-FIP4 decreased the ability of migration and invasion in HCC cells in vitro and suppressed lung metastasis in vivo.Rab11-FIP4 facilitated HCC metastasis through the phosphorylation of PRAS40,which was regulated by m TOR.Furthermore,the expression level of Rab11-FIP4 was significantly increased in HCC tissues and high expression of Rab11-FIP4 was closely correlated with vascular invasion and poor prognosis in HCC patients.A markedly positive correlation between the expression of Rab11-FIP4 and HIF-1alpha was observed in HCC tissues and combination of Rab11-FIP4 and HIF-1alpha was a more valuable predictor of poor prognosis for HCC patients.In conclusion,Rab11-FIP4 is a target gene of HIF-1alpha and has a pro-metastatic role in HCC,suggesting that Rab11-FIP4 may be a promising candidate target for HCC treatment.

(Oncogene,2015,34:6007-6017)

71.PROX1 promotes hepatocellular carcinoma proliferation and sorafenib resistance by enhancing betacatenin expression and nuclear translocation　(IF 6.634)

Liu Y,Ye X,Zhang JB,Ouyang H,Shen Z,Wu Y,Wang W,Wu J,Tao S,Yang X,Qiao K,Zhang J,Liu J,Fu Q,Xie Y

Aberrant activation of the Wnt/beta-catenin pathway is frequent in hepatocellular carcinoma(HCC)and contributes to HCC initiation and progression.This abnormal activation may result from somatic mutations in the genes of the Wnt/beta-catenin pathway and/or dysregulation of the Wnt/beta-catenin pathway.The mechanism for the latter remains poorly understood.Prospero-related homeobox 1(PROX1)is a downstream target of the Wnt/beta-catenin pathway in human colorectal cancer and elevated PROX1 expression promotes malignant progression.However,the Wnt/betacatenin pathway does not regulate PROX1 expression in the liver and HCC cells.Here we report that PROX1 promotes HCC cell proliferation in vitro and tumor growth in HCC xenograft mice.PROX1 and beta-catenin levels are positively correlated in tumor tissues as well as in cultured HCC cells.PROX1 can upregulate beta-catenin transcription by stimulating the beta-catenin promoter and enhance the nuclear translocation of beta-catenin in HCC cells,which leads to the activation of the Wnt/beta-catenin pathway.Moreover,we show that increase in PROX1 expression renders HCC cells more resistant to sorafenib treatment,which is the standard therapy for advanced HCC.Overall,we have pinpointed PROX1 as a critical factor activating the Wnt/beta-catenin pathway in HCC,which promotes HCC proliferation and sorafenib resistance.

(Oncogene,2015,34:5524-5535)

72.Randomized controlled trial of the prophylactic effect of urea-based cream on sorafenib-associated hand-foot skin reactions in patients with advanced hepatocellular carcinoma　(IF 28.245)

Ren Z,Zhu K,Kang H,Lu M,Qu Z,Lu L,Song T,Zhou W,Wang H,Yang W,Wang X,Yang Y,Shi L,Bai Y,Guo X,Ye SL

PURPOSE:To assess whether urea-based cream(UBC)has prophylactic benefits on sorafenibinduced hand-foot skin reaction(HFSR)in patients with advanced hepatocellular carcinoma(HCC).PATIENTS AND METHODS:In this randomized,open-label trial,871 patients with advanced HCC throughout China were treated with 10%UBC three times per day plus best supportive care(BSC;n=439)or BSC alone excluding all creams(n=432),starting on day 1 of sorafenib treatment,for up to 12 weeks.HFSR was assessed every 2 weeks and at 14 weeks for patients completing the study.Once HFSR occurred,patients were allowed any cream,including a UBC.RESULTS:The 12-week incidence of any grade HFSR was significantly lower in the UBC group versus the BSC-alone group(56.0%vs.73.6%,respectively;odds ratio[OR],0.457;95%CI,0.344 to 0.608;P＜0.001),as was the incidence of grade≥2 HFSR(20.7%vs.29.2%,respectively;OR,0.635;95%CI,0.466 to 0.866;P=0.004).Median time to first occurrence of HFSR was significantly longer in the UBC group than the BSC-alone group(84 vs.34 days,respectively;hazard ratio,0.658;95%CI,0.541 to 0.799;P＜0.001).Elevated AST was associated with increased risk of HFSR but did not alter the treatment effect of UBC.UBC plus BSC,compared with BSC alone,did not affect the sorafenib dose reduction or interruption rate(9.1%vs.11.8%,respectively;P=0.193 7),response rate(11.1%vs.10.1%,respectively;P=0.667 4),or disease control rate(98.8%vs.98.2%,respectively;P=0.535 0)at week 12.CONCLUSION:UBC prophylaxis in patients with advanced HCC starting sorafenib reduced HFSR rates,extended the time to first occurrence of HFSR,and improved patient quality of life compared with BSC.Blinded,randomized,placebo-controlled trials to determine the role of UBC on the incidence and severity of HFSR are warranted.

(J Clin Oncol,2015,33:894-900)

73.CC chemokine receptor-like 1 functions as a tumour suppressor by impairing CCR7-related chemotaxis in hepatocellular carcinoma　(IF 5.942)

Shi JY,Yang LX,Wang ZC,Wang LY,Zhou J,Wang XY,Shi GM,Ding ZB,Ke AW,Dai Z,Qiu SJ,Tang QQ,Gao Q,Fan J

Atypical chemokine receptors(ACRs)have been discovered to participate in the regulation of tumour behaviour.Here we report a tumour-suppressive role of a novel ACR member,CC chemokine receptor like 1(CCRL1),in human hepatocellular carcinoma(HCC).Both m RNA and protein expressions of CCRL1 correlated with the malignant phenotype of HCC cells and were significantly down-regulated in tumour tissue compared with paired normal liver tissue.In both the initial and validation cohorts(n=240 and n=384,respectively),CCRL1 deficiency was associated with advanced tumour stage and was an independent index for worse survival and increased recurrence.Furthermore,knock-down or forced expression of CCRL1 revealed that CCRL1 suppressed the proliferation and invasion of HCC cells in vitro and reduced tumour growth and lung metastasis in vivo,with depressed levels of CCL19 and CCL21.By sequestrating CCL19 and CCL21,CCRL1 reduced their binding to CCR7 and consequently mitigated the detrimental impact of CCR7,including Akt-GSK3beta pathway activation and nuclear accumulation of beta-catenin in tumour cells.Clinically,the prognostic value of the CCR7 expression in HCC depended on the expression level of CCRL1,suggesting that CCRL1 may serve as an upstream switch for the CCR7 signalling cascade.Together,our findings suggest that CCRL1 impairs chemotactic events associated with CCR7 in the progression and metastasis of HCC.Our results also show a potential interplay between typical and atypical chemokine receptors in human cancer.

(J Pathol,2015,235:546-558)

74.Hepatic stellate cells activated by acidic tumor microenvironment promote the metastasis of hepatocellular carcinoma via osteopontin　(IF 6.508)

Song J,Ge Z,Yang X,Luo Q,Wang C,You H,Ge T,Deng Y,Lin H,Cui Y,Chu W,Yao M,Zhang Z,Gu J,Fan J,Qin W

Extracellular p H of solid tumor is generally acidic due to excessive glycolysis and poor perfusion.But whether acidic tumor microenvironment influenced the stromal cells infiltrating in tumor remains unknown.As the predominant progenitor of stromal cells in liver,the number of activated hepatic stellate cells(HSCs)was found positively correlated to the acidification level in the tumor tissues of HCC patients in our study.Whereas,in vitro acidic culture condition and in vivo coimplanting xenograft model were adopted to study the response of HSCs and its influence on HCC progression.HSCs were activated under acidic culture condition depending on the phosphorylation of cellular signal-regulated kinase(ERK).Acidity-activated HSCs promoted HCC metastasis in vitro and in vivo.Osteopontin(OPN)excretion from HSCs was increased under acidic condition and proved to promote the migration of HCC cells.Furthermore,the expression level of OPN was significantly associated with myofibroblasts and the combination of alpha-SMA with OPN was a powerful predictor for poor prognosis of HCC patients.Activation of HSCs in acidic tumor microenvironment represents a novel mechanism for HCC metastasis and provides a potential therapeutic strategy for HCC.

(Cancer Lett,2015,356:713-720)

75.The nanomechanical signature of liver cancer tissues and its molecular origin　(IF 6.970)

Tian M,Li Y,Liu W,Jin L,Jiang X,Wang X,Ding Z,Peng Y,Zhou J,Fan J,Cao Y,Wang W,Shi Y

Patients with cirrhosis are at higher risk of developing hepatocellular carcinoma(HCC),the second most frequent cause of cancer-related deaths.Although HCC diagnosis based on conventional morphological characteristics serves as the“gold standard”in the clinic,there is a high demand for more convenient and effective diagnostic methods that employ new biophysical perspectives.Here,we show that the nanomechanical signature of liver tissue is directly correlated with the development of HCC.Using indentation-type atomic force microscopy(ITAFM),we demonstrate that the lowest elasticity peak(LEP)in the Young's modulus distribution of surgically removed liver cancer tissues can serve as a mechanical fingerprint to evaluate the malignancy of liver cancer.Cirrhotic tissues shared the same LEP as normal tissues.However,a noticeable downward shift in the LEP was detected when the cirrhotic tissues progressed to a malignant state,making the tumor tissues more prone to microvascular invasion.Cell-level mechanistic studies revealed that the expression level of a Rho-family effector(m Dia1)was consistent with the mechanical trend exhibited by the tissue.Our findings indicate that the mechanical profiles of liver cancer tissues directly varied with tumor progression,providing an additional platform for the future diagnosis of HCC.

(Nanoscale,2015,7:12998-13010)

76.Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor-induced epithelial-mesenchymal transition(IF 14.971)

Wang ZC,Gao Q,Shi JY,Guo WJ,Yang LX,Liu XY,Liu LZ,Ma LJ,Duan M,Zhao YJ,Wu YN,Gao DM,Wang XY,Shi GM,Ding ZB,Ke AW,Tang QQ,Cao Y,Zhou J,Fan J

Hepatocellular carcinoma(HCC)is the third-most lethal cancer worldwide.Understanding the molecular pathogenesis of HCC recurrence and metastasis is the key to improve patients'prognosis.In this study,we report that protein tyrosine phosphatase receptor S(PTPRS)is significantly down-regulated in nearly 80%of HCCs,and its expression negatively correlates with aggressive pathological features,such as larger tumor size and advanced stage.In addition,PTPRS deficiency is independently associated with shorter survival and increased recurrence in patients,although 16.7%of HCCs show intratumor heterogeneous expression of PTPRS.Restoration of wild-type,but not mutant,PTPRS expression significantly inhibits HCC cell migration and invasion in vitro as well as lung metastasis in vivo,whereas knockdown of its expression significantly promotes invasion and metastasis.Notably,PTPRS-regulated HCC invasiveness is accompanied by typical changes of epithelial-mesenchymal transition(EMT).Moreover,PTPRS forms a complex with epithermal growth factor receptor(EGFR)and regulates its tyrosine residues'phosphorylation.Ectopic expression of EGFR reverses the metastasis-inhibiting effects of PTPRS,whereas silencing of EGFR or inhibiting phosphorylation of key molecules in EGFR downstream pathways reinhibits EMT and metastasis caused by PTPRS down-regulation.Meanwhile,promoter hypermethylation of PTPRSis frequently detected in HCC samples and cell lines.Treatment with a demethylation agent,5-aza-2'-deoxycytidine,recovers PTPRS expression in a dose-dependent manner.CONCLUSIONS:Epigenetic inactivation of PTPRS may increase phosphorylation and activity of EGFR signaling to promote EMT and metastasis in HCC.

(Hepatology,2015,62:1201-1214)

77.Mitogen-activated protein kinase kinase kinase 4 deficiency in intrahepatic cholangiocarcinoma leads to invasive growth and epithelial-mesenchymal transition　(IF 14.971)

Yang LX,Gao Q,Shi JY,Wang ZC,Zhang Y,Gao PT,Wang XY,Shi YH,Ke AW,Shi GM,Cai JB,Liu WR,Duan M,Zhao YJ,Ji Y,Gao DM,Zhu K,Zhou J,Qiu SJ,Cao Y,Tang QQ,Fan J

The molecular pathogenesis of intrahepatic cholangiocarcinoma(iCCA)is poorly understood,and its incidence continues to increase worldwide.Deficiency of mitogen-activated protein kinase kinase kinase 4(MAP3K4)has been reported to induce the epithelial-mesenchymal transition(EMT)process of placental and embryonic development,yet its role in human cancer remains unknown.MAP3K4 has somatic mutation in iCCA so we sequenced all exons of MAP3K4 in 124 iCCA patients.We identified nine somatic mutations in 10(8.06%)patients,especially in those with lymph node metastasis and intrahepatic metastasis.We also showed that messenger RNA and protein levels of MAP3K4 were significantly reduced in iCCA versus paired nontumor tissues.Furthermore,knockdown of MAP3K4 in cholangiocarcinoma cells markedly enhanced cell proliferation and invasiveness in vitro and tumor progression in vivo,accompanied by a typical EMT process.In contrast,overexpression of MAP3K4 in cholangiocarcinoma cells obviously reversed EMT and inhibited cell invasion.Mechanistically,MAP3K4 functioned as a negative regulator of EMT in iCCA by antagonizing the activity of the p38/nuclear factor kappaB/snail pathway.We found that the tumor-inhibitory effect of MAP3K4 was abolished by inactivating mutations.Clinically,a tissue microarray study containing 322 iCCA samples from patients revealed that low MAP3K4 expression in iCCA positively correlated with aggressive tumor characteristics,such as vascular invasion and intrahepatic or lymph node metastases,and was independently associated with poor survival and increased recurrence after curative surgery.CONCLUSIONS:MAP3K4,significantly down-regulated,frequently mutated,and potently regulating the EMT process in iCCA,was a putative tumor suppressor of iCCA.

(Hepatology,2015,62:1804-1816)

78.Coexpression of gene Oct4 and Nanog initiates stem cell characteristics in hepatocellular carcinoma and promotes epithelial-mesenchymal transition through activation of Stat3/Snail signaling(IF 8.731)

Yin X,Zhang BH,Zheng SS,Gao DM,Qiu SJ,Wu WZ,Ren ZG

BACKGROUND:Oct4 and Nanog are key regulatory genes that maintain the pluripotency and selfrenewal properties of embryonic stem cells.We previously reported that the two stemness markers were tightly associated with cancer progression and poor outcomes of hepatocellular carcinoma.In this study,we demonstrate that coexpression of Oct4/Nanog modulates activation of signal transducer and activator of transcription 3(Stat3),an oncogenic transcription factor that is activated in many human malignancies including hepatocellular carcinoma(HCC),as well as the expression of Snail,a key regulator implicated in epithelial-mesenchymal transition and tumor metastasis.METHODS:Oct4 and Nanog were ectopic expressed in MHCC97-L cell lines via lentiviral gene transfection.The stemness characteristics including self-renewal,proliferation,chemoresistance,and tumorigenicity were assessed.The effect of coexpression of Oct4 and Nanog on epithelialmesenchymal transition change,and the underlying molecular signaling was investigated.RESULTS:Ectopic coexpression of Oct4 and Nanog empowered MHCC97-L cells with cancer stem cell(CSC)properties,including self-renewal,extensive proliferation,drug resistance,and high tumorigenic capacity.Significantly,Oct4 and Nanog encouraged epithelial-mesenchymal transition change contributing to tumor migration,invasion/metastasis in vitro and in vivo.Following molecular mechanism investigation indicated Oct4/Nanog-regulated epithelial-mesenchymal transition change through Stat3-dependent Snail activation.Moreover,silencing Stat3 abrogates Oct4/Nanog-mediated epithelial-mesenchymal transition(EMT)change and invasion/metastasis in HCC.CONCLUSIONS:We delineate Oct4 and Nanog initiate stem cell characteristics in hepatocellular carcinoma and promote epithelial-mesenchymal transition through activation of Stat3/Snail signaling.Our findings propose Stat3/Snail pathway as a novel therapeutic target for the treatment of progression and metastasis of HCC with CSC-like signatures and epithelialmesenchymal transition phenotype.

(J Hematol Oncol,2015,8:23)

79.Promyelocytic leukemia protein induces arsenic trioxide resistance through regulation of aldehyde dehydrogenase 3 family member A1 in hepatocellular carcinoma　(IF 6.508)

Zhang X,Yang XR,Sun C,Hu B,Sun YF,Huang XW,Wang Z,He YF,Zeng HY,Qiu SJ,Cao Y,Fan J,Zhou J

Clinical response of hepatocellular carcinoma(HCC)to arsenic trioxide(ATO)has been poor.Promyelocytic leukemia protein(PML)is central to ATO treatment efficacy of acute promyelocytic leukemia.We examine impacts of PML expression on the effectiveness of ATO treatment in HCC.We show that increased PML expression predicts longer survival and lower cancer recurrence rates after HCC resection.However,high PML expression dampens the anti-tumor effects of ATO in HCC cells.Gene microarray analysis shows that reduced PML expression significantly downregulates expression of aldehyde dehydrogenase 3 family member A1(ALDH3A1).ALDH3A1 depression facilitates accumulation of ATO-induced reactive oxygen species.Chromatin immunoprecipitation analysis and promoter activity assays confirm that PML regulates ALDH3A1 expression through binding to the promoter region of ALDH3A1.Clinically,ATO treatment decreases the disease progression rate in advanced HCC patients with negative PML expression.In conclusion,PML confers a favorable prognosis in HCC patients,but it induces ATO resistance through ALDH3A1 up-regulation in HCC cells.ATO is effective for HCC patients with negative PML expression.Combined with an ALDH3A1 inhibitor,ATO may be efficacious in patients with positive PML expression.

(Cancer Lett,2015,366:112-122)

80.CXCR2/CXCL5 axis contributes to epithelial-mesenchymal transition of HCC cells through activating PI3K/Akt/GSK-3beta/Snail signaling　(IF 6.508)

Zhou SL,Zhou ZJ,Hu ZQ,Li X,Huang XW,Wang Z,Fan J,Dai Z,Zhou J

Upregulation of CXCR2 in tumor cells has been documented in several types of cancer.As one of its ligands,CXCL5 is associated with neutrophil infiltration and poor prognosis in hepatocellular carcinoma(HCC).However,little is known about the role of the CXCR2/CXCL5 axis in the invasion and metastasis of HCC cells.In this study,we examined CXCR2 expression in human HCC cell lines and in three independent cohorts of HCC patients.The molecular effects of high expression levels of CXCR2 and CXCL5 in HCC cells were determined using qRT-PCR,western blot analysis,immunofluorescence,matrigel invasion assay,and xenograft mouse models.We found that high levels of CXCR2 correlated with progression and poor prognosis in human HCC.CXCR2/CXCL5 together promoted cell spreading by inducing the epithelial-mesenchymal transition(EMT)through activation of the PI3K/Akt/GSK-3beta/Snail signaling pathway.In clinical HCC samples,high expression of both CXCR2 and CXCL5 showed a significant correlation with the activation of PI3K/Akt/GSK-3beta/Snail signaling and EMT phenotype.In conclusion,our data showed that the CXCR2/CXCL5 axis contributes to EMT of HCC cells through activating PI3K/Akt/GSK-3beta/Snail signaling,and it may serve as a potential therapeutic target.

(Cancer Lett,2015,358:124-135)

81.Activation of the JNK-c-Jun pathway in response to irradiation facilitates Fas ligand secretion in hepatoma cells and increases hepatocyte injury　(IF 5.646)

Dong Y,Shen X,He M,Wu Z,Zheng Q,Wang Y,Chen Y,Wu S,Cui J,Zeng Z

BACKGROUND:It is well established that some irradiated liver non-parenchymal cells secrete proinflammatory cytokines to facilitate the development of radiation-induced liver disease.However,little is known on whether the irradiated hepatoma cells-mediated non-irradiated hepatocyte injury occurs in HCC patients.Here,we elucidated the roles of the irradiated hepatoma cells in driving non-irradiated hepatocyte injury and its underlying mechanism.METHODS:SMMC7721 cells were cultured and divided into irradiated(4Gy X-ray,R)and non-irradiated(NR)groups.At 24th hour after irradiation,conditioned medium(CM)from these cultures was mixed with normal culture medium in specific proportions,and termed as 7721-R-CM and 7721-NR-CM.Following incubation with these CM compound,the biological characteristics of L02 cells related to liver cell injury including viability,apoptosis and liver dysfunction indices were comparatively analyzed.Simultaneously,the levels of proliferation-and apoptosis-related cytokines in irradiated and nonirradiated SMMC7721 cells were also measured.Fas L as a cytokine with significantly differential expression,was selected to clarify its effects on L02 apoptosis.Subsequently,Fas L expression following irradiation was examined in SMMC7721 and other HCC cells with varying malignant potentials,as well as in HCC tissues,the related mechanism of higher expression of Fas L in irradiated HCC cells was further investigated.RESULTS:Apoptosis and liver dysfunction indices were all significantly enhanced in L02 cells treated with 7721-R-CM,whereas viability was suppressed,compared to those with 7721-NR-CM stimulation.Fas L was identified as a leading differential cytokine in the irradiated SMMC7721 cells.Higher proportion of apoptosis was also found in L02 cells following Fas L incubation.A recombinant Fas-Fc protein,which blocks Fas-Fas L interaction,ameliorated 7721-R-CM-induced apoptosis in L02 cells.Fas L was highly expressed in a dose-dependent manner,and peaked at the 24th hour post-irradiation in different HCC cells and their culture supernatant.Meanwhile,phosphorylation levels of JNK,ERK,Akt,and p38 were all upregulated significantly in irradiated HCC cells.But,only JNK inhibition was validated to block radiation-induced Fas L expression in HCC cells.c-Jun,the target transcription factor of JNK,was also activated.CONCLUSION:In HCC cells,the JNK-c-Jun pathway plays an important role in mediating irradiation-induced Fas L expression,which may be critical in determining non-irradiated hepatocyte injury.

(J Exp Clin Cancer Res,2016,35:114)

82.Overexpression of interleukin-35 associates with hepatocellular carcinoma aggressiveness and recurrence after curative resection　(IF 5.416)

Fu YP,Yi Y,Cai XY,Sun J,Ni XC,He HW,Wang JX,Lu ZF,Huang JL,Cao Y,Zhou J,Fan J,Qiu SJ

BACKGROUND:Aberrant expression of interleukin-35(IL-35)has been implicated in dampening antitumour immunity.The aim of this study was to explore the prognostic significance of IL-35 expression in patients with hepatocellular carcinoma(HCC)following curative resection.Furthermore,we aimed to formulate an effective prognostic nomogram for HCC after hepatectomy.METHODS:Immunohistochemistry was applied to explore IL-35 expression as well as CD39(+)Foxp3(+)and Foxp3(+)regulatory T cell(Treg)infiltration in tissue microarrays in primary cohort comprising 210 randomly selected HCC patients who underwent curative resection.The results were further verified in an independent validation cohort of 138 HCC patients.RESULTS:Patients with higher expression of IL-35 are more likely to suffer postoperative recurrence.Interleukin-35 was also identified as an independent prognostic factor for recurrence free survival in multivariate analysis.No correlation was detected between IL-35 expression and Foxp3(+)Treg infiltration,whereas significant positive correlation was found between IL-35 expression and CD39(+)Foxp3(+)Treg infiltration.In addition,CD39(+)Foxp3(+)Treg infiltration was also an independent predictor for postoperative recurrence.The nomogram comprising tumour size,tumour vascular invasion,IL-35 and CD39(+)Foxp3(+)Tregs had better predictive accuracy when compared with BCLC stage for RFS.These results were further validated in the validation cohort.CONCLUSIONS:Our data suggest for the first time that IL-35 expression correlates with HCC aggressiveness and emerged as a novel independent prognostic factor for recurrence,thus conferring the rationale to develop a novel therapy of targeting IL-35.Furthermore,IL-35 should be incorporated into nomogram to generate a more accurate predictive model.

(Br J Cancer,2016,114:767-776)

83.miR-612 suppresses stem cell-like property of hepatocellular carcinoma cells by modulating Sp1/Nanog signaling　(IF 5.959)

Liu Y,Liu DL,Dong LL,Wen D,Shi DM,Zhou J,Fan J,Wu WZ

In our previous study we found that miR-612 negatively regulated stem cell-like property and tumor metastasis of hepatocellular carcinoma cells(HCC).In this study,we try to elucidate underlying mechanism of the regulation,and find that miR-612 inversely modulate the m RNA and protein level of epithelial cell adhesion molecule as well as CD133,negatively regulate the numbers and sizes of tumor spheres,directly inhibit the protein level of Sp1,and subsequently reduce transcription activity of Nanog.Of importance,the higher levels of Sp1 and Nanog in biopsies are the more unfavorable prognoses of HCC patients are found after tumor resection.Taken together,miR-612 has a suppressive role on HCC stemness via Sp1/Nanog signaling pathway.

(Cell Death Dis,2016,7:e2377)

84.MicroRNA-26a suppresses epithelial-mesenchymal transition in human hepatocellular carcinoma by repressing enhancer of zeste homolog 2　(IF 8.731)

Ma DN,Chai ZT,Zhu XD,Zhang N,Zhan DH,Ye BG,Wang CH,Qin CD,Zhao YM,Zhu WP,Cao MQ,Gao DM,Sun HC,Tang ZY

BACKGROUND:Our previous study reported that microRNA-26a(miR-26a)inhibited tumor progression by inhibiting tumor angiogenesis and intratumoral macrophage infiltration in hepatocellular carcinoma(HCC).The direct roles of miR-26a on tumor cell invasion remain poorly understood.In this study,we aim to explore the mechanism of miR-26a in modulating epithelialmesenchymal transition(EMT)in HCC.METHODS:In vitro cell morphology and cell migration were compared between the hepatoma cell lines HCCLM3 and Hep G2,which were established in the previous study.Overexpression and down-regulation of miR-26a were induced in these cell lines,and Western blot and immunofluorescence assays were used to detect the expression of EMT markers.Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis.Immunohistochemical assays were conducted to study the relationships between miR-26a expression and enhancer of zeste homolog 2(EZH2)and E-cadherin expression in human HCC samples.RESULTS:Down-regulation of miR-26a in HCCLM3 and Hep G2 cells resulted in an EMT-like cell morphology and high motility in vitro and increased in tumor growth and pulmonary metastasis in vivo.Through down-regulation of EZH2 expression and up-regulation of E-cadherin expression,miR-26a inhibited the EMT process in vitro and in vivo.Luciferase reporter assay showed that miR-26a directly interacted with EZH2 messenger RNA(m RNA).Furthermore,the expression of miR-26a was positively correlated with E-cadherin expression and inversely correlated with EZH2 expression in human HCC tissue.CONCLUSIONS:miR-26a inhibited the EMT process in HCC by down-regulating EZH2 expression.

(J Hematol Oncol,2016,9:1)

85.Moderate swimming suppressed the growth and metastasis of the transplanted liver cancer in mice model:with reference to nervous system　(IF 6.634)

Zhang QB,Zhang BH,Zhang KZ,Meng XT,Jia QA,Zhang QB,Bu Y,Zhu XD,Ma DN,Ye BG,Zhang N,Ren ZG,Sun HC,Tang ZY

Physical activity has been shown to suppress tumor initiation and progression.The neurotransmitter dopamine(DA)is closely related to movement and exhibits antitumor properties.However,whether the suppressive effects of physical activity on tumors was mediated by the nervous system via increased DA level remains unknowns.Here we show that regular moderate swimming(8 min/day,9 weeks)raised DA levels in the prefrontal cortex,serum and tumor tissue,suppressed growth,reduced lung metastasis of transplanted liver cancer,and prolonged survival in a C57BL/6 mouse model,while overload swimming(16 and 32 min/day,9 weeks)had the opposite effect.In nude mice that were orthotopically implanted with human liver cancer cell lines,DA treatment significantly suppressed growth and lung metastasis by acting on the D2 receptor(DR2).Furthermore,DR2 blockade attenuated the suppressive effect of moderate swimming on liver cancer.Both moderate swimming and DA treatment suppressed the transforming growth factor-beta(TGF-beta1)-induced epithelial-mesenchymal transition of transplanted liver cancer cells.At the molecular level,DR2 signaling inhibited extracellular signal-regulated kinase phosphorylation and expression of TGF-beta1 in vitro.Together,these findings demonstrated a novel mechanism by which the moderate exercise suppressed liver cancer through boosting DR2 activity,while overload exercise had the opposite effect,highlighting the possible importance of the dopaminergic system in tumor growth and metastasis of liver cancer.

(Oncogene,2016,35:4122-4131)

86.miR-28-5p-IL-34-macrophage feedback loop modulates hepatocellular carcinoma metastasis(IF 14.971)

Zhou SL,Hu ZQ,Zhou ZJ,Dai Z,Wang Z,Cao Y,Fan J,Huang XW,Zhou J

Micro RNAs(miRNAs)play a critical role in regulation of tumor metastasis.However,the role of these molecules in hepatocellular carcinoma(HCC)has not been fully elucidated.In this study,we employed miRNA-sequencing and identified 22 miRNAs involved in HCC metastasis.One of these,miR-28-5p,was down-regulated in HCCs.This down-regulation correlated with tumor metastasis,recurrence,and poor survival.Biofunctional investigations revealed that miR-28-5p deficiency promoted tumor growth and metastasis in nude mice without altering the in vitro biological characteristics of HCC cells.Through gene expression profiles and bioinformatics analysis,we identified interleukin-34(IL-34)as a direct target of miR-28-5p,and the effects of miR-28-5p deficiency on HCC growth and metastasis was dependent on IL-34-mediated tumor-associated macrophage(TAM)infiltration.Moreover,we found that TAMs induced by miR-28-5p-IL-34 signaling inhibit miR-28-5p expression on HCC cells by transforming growth factor beta 1,resulting in an miR-28-5p-IL-34-macrophage-positive feedback loop.In clinical HCC samples,miR-28-5p levels were inversely correlated with IL-34 expression and the number of TAMs.Patients with low miR-28-5p expression,high IL-34 levels,and high numbers of TAMs had a poor prognosis with shorter overall survival and time to recurrence.CONCLUSION:A miR-28-5p-IL-34-macrophage feedback loop modulates HCC metastasis and serves as a novel prognostic factor as well as a therapeutic target for HCC.

(Hepatology,2016,63:1560-1575)

87.Tumor-Associated Neutrophils Recruit Macrophages and T-Regulatory Cells to Promote Progression of Hepatocellular Carcinoma and Resistance to Sorafenib　(IF 19.233)

Zhou SL,Zhou ZJ,Hu ZQ,Huang XW,Wang Z,Chen EB,Fan J,Cao Y,Dai Z,Zhou J

BACKGROUND&AIMS:Neutrophils can either promote or inhibit tumor progression,depending on the tumor microenvironment,via release of cytokines.Neither the factors produced by tumorassociated neutrophils(TANs)nor their effects on tumor progression have been characterized.We investigated the roles of TANs in progression of hepatocellular carcinoma(HCC)using cell lines and immune cells isolated from patients.METHODS:We performed studies with Hep G2,PLC/PRF/5,MHCC97 H,and HCCLM3 human and Hepa1-6 and H22 mouse HCC cell lines;expression of chemokines and cytokines were knocked down with small hairpin RNAs.Cells were analyzed in chemotaxis assays and as growth as tumors in mice.HCC tissues and peripheral blood were collected from 20 patients undergoing curative resection or 20 healthy individuals(controls)in 2012 at Zhongshan Hospital in China.TANs and peripheral blood neutrophils(PBNs)were isolated and exposed to conditioned media from HCC cell lines;reverse-transcription polymerase chain reaction was used to quantify the expression of cytokines and chemokines.We collected neutrophils from another 60 patients undergoing curative resection for HCC in 2012 to measure the production of C-C motif chemokine ligand 2(CCL2)and CCL17.Patients were followed up until March 15,2014.For immunohistochemical analyses,we collected HCC tissues and paired,adjacent,nontumor cirrhotic liver tissues from 832 HCC patients undergoing curative resection from 2006 through 2008.All patients were followed up until March 15,2013.To study the effects of sorafenib,we collected clinical and pathology data from 46 patients who underwent curative resection in 2010.RESULTS:CCL2 and CCL17 were the cytokines most highly expressed by TANs and HCC cell-activated PBNs.Levels of CCL2 and CCL17 messenger RNAs and proteins were significantly higher in TANs than in PBNs,and increased in patients with HCC recurrence.CCL2 and CCL17 messenger RNA and proteins also increased when PBNs were exposed to conditioned media from HCC cell lines.Immunohistochemical analysis of a tissue microarray showed that CCL2+and CCL17+cells,which also expressed the neutrophil marker CD66b,were distributed throughout the HCC stroma,but not in tumor cells or the adjacent nontumor liver cells.The number of CCL2(+)or CCL17(+)TANs correlated with tumor size,microvascular invasion,tumor encapsulation,tumor differentiation,and stage.Patients whose tumors had lower levels of CCL2(+)or CCL17(+)cells had longer survival times than those with higher numbers of these cells.TAN-conditioned media,as well as recombinant CCL2 and CCL17,increased the migratory activity of the macrophages and Tregulatory(Treg)cells from patients or mice with HCC to a greater extent that PBN-conditioned media.Neutralizing antibodies against CCL2 and CCL17,or their receptors C-C chemokine receptor 2 and C-C chemokine receptor 4,reduced the migratory activities of macrophage and Treg cells.HCC cell lines injected into mice formed larger tumors when they were co-injected with TANs and formed more pulmonary metastases;these tumors were infiltrated by Ly6G(+)cells,F4/80+macrophages,and Foxp3(+)Treg cells.In a phosphokinase array of human PBNs,levels of phosphorylated AKT and P38 increased after exposure to conditioned media from all 4 HCC cell types.Pharmacologic inhibitors of AKT and P38 inhibited secretion of CCL2 and CCL17 by these PBNs.In tumor-bearing mice,sorafenib increased the numbers of TANs and levels of CCL2 and CCL17 in tumors.HCC tissues from patients who received sorafenib before surgery contained more TANs than tissues from patients who did not receive sorafenib.In knockdown cells,HCC cell-derived CXCL5 was the strongest effector of neutrophil migration under hypoxic conditions.In mice,the combination of sorafenib and TAN depletion inhibited tumor growth and neovascularization to a greater extent than sorafenib alone.CONCLUSIONS:TANs recruit macrophages and Treg cells to HCCs to promote their growth,progression,and resistance to sorafenib.

(Gastroenterology,2016,150:1646-1658,e1617)

88.Colony-stimulating factor-1-induced AIF1 expression in tumor-associated macrophages enhances the progression of hepatocellular carcinoma　(IF 5.333)

Cai H,Zhu XD,Ao JY,Ye BG,Zhang YY,Chai ZT,Wang CH,Shi WK,Cao MQ,Li XL,Sun HC

M2-polarized(alternatively activated)macrophages play an important role in the progression of hepatocellular carcinoma(HCC).Allograft inflammatory factor 1(AIF1)is overexpressed in M2-polarized macrophages.This study explored the role of AIF1 in tumor-associated macrophages in HCC.Macrophages were stimulated with colony-stimulating factor 1(CSF1)to characterize the regulatory pathway of AIF1 in macrophages.The chromatin immunoprecipitation and luciferase reporter gene assay were conducted to examine transcription factors associated with AIF1 expression.AIF1 was down or upregulated,and the effects on tumor progression were evaluated by using in vitro and in vivo co-culture systems.A cytokine array was performed to screen the downstream functional components of AIF1.Tumor tissue from 206 patients with HCC were used to explore the clinical significance of AIF1.AIF1 induced a M2-like phenotype of macrophages.By facilitating the binding of c-Jun to the promoter of AIF1,CSF1 secreted from hepatoma cells increased AIF1 expression through the CSF1R-MEK1/2-Erk1/2-c-Jun axis.AIF1 expressed in macrophages promoted the migration of hepatoma cells in co-culture system of RAW264.7 and Hepa1-6 and tumor growth in an animal model.The cytokine array showed that CXCL16 was increased in RAW264.7 cells with overexpressed AIF1,leading to enhanced tumor cell migration.In human HCC tissue,AIF1-positive macrophages in the adjacent microenvironment was associated with microvascular invasion and advanced TNM stages and with patients'overall and disease-free survival(P=0.002 for both).AIF1 expression in macrophages plays a pivotal role in the interaction between macrophages and hepatoma cells.

(Oncoimmunology,2017,6:e1333213)

89.MicroRNA-29a induces loss of 5-hydroxymethylcytosine and promotes metastasis of hepatocellular carcinoma through a TET-SOCS1-MMP9 signaling axis　(IF 5.959)

Chen Q,Yin D,Zhang Y,Yu L,Li XD,Zhou ZJ,Zhou SL,Gao DM,Hu J,Jin C,Wang Z,Shi YH,Cao Y,Fan J,Dai Z,Zhou J

Ten eleven translocation(TET)enzymes convert 5-methylcytosine(5-mC)to 5-hydroxymethylcytosine(5-hmC)and have crucial roles in biological and pathological processes by mediating DNA demethylation,however,the functional role of this epigenetic mark and the related enzymes in hepatocellular carcinoma(HCC)progression remains unknown.Here,we demonstrated that TETfamily enzymes downregulation was one likely mechanism underlying 5-hm C loss in HCC.We found that miR-29a overexpression increased DNA methylation of suppressor of cytokine signaling 1(SOCS1)promoter was associated with HCC metastasis in vitro and in vivo.Furthermore,miR-29a silenced anti-metastatic SOCS1 through direct TET-family targeting,resulting in SOCS1 promoter demethylation inhibition.Chromatin immunoprecipitation analyses confirmed that TET1 regulated SOCS1 expression through binding to the promoter region of SOCS1.Finally,miR-29a overexpression correlated with poor clinical outcomes and TET-SOCS1-matrix metalloproteinase(MMP)9 axis silencing in HCC patients.In conclusion,our findings demonstrate that 5-hmC loss is an epigenetic hallmark of HCC,and miR-29a is an important epigenetic modifier,promoting HCC metastasis through TET-SOCS1-MMP9 axis silencing.The results offer a new strategy for epigenetic cancer therapy.

(Cell Death Dis,2017,8:e2906)

90.Prognostic Nomograms Stratify Survival of Patients with Hepatocellular Carcinoma Without Portal Vein Tumor Thrombosis After Curative Resection　(IF 5.252)

Fu YP,Yi Y,Huang JL,Jing CY,Sun J,Ni XC,Lu ZF,Cao Y,Zhou J,Fan J,Qiu SJ

BACKGROUND:The prognosis of patients with hepatocellular carcinoma(HCC)without portal vein tumor thrombosis(PVTT)after curative resection is at variance.We identified the risk factors of poor postoperative prognosis and consequently developed prognostic nomograms generating individual risk of death and recurrence for this subgroup of patients with HCC.METHODS:The risk factors were identified and nomograms were developed based on a retrospective study of 734 patients in the primary cohort who underwent curative resection for HCC from 2010 to 2012.The predictive accuracy and discriminative ability of the nomograms were determined by concordance index(C-index)and calibration curve and compared with traditional staging systems of HCC.The results were validated in an independent cohort of 349 patients operated at the same institution in 2007.RESULTS:All of the independent factors for survival in multivariate analysis in the primary cohort were selected into the nomograms.The calibration curve for probability of survival showed good agreement between prediction by nomograms and actual observation.The C-indices of the nomograms for predicting overall survival and recurrence-free survival were 0.755(95%confidence interval[CI],0.752-0.758)and 0.665(95%CI,0.662-0.668),respectively,which were statistically higher than the C-indices of other HCC prognostic models.The results were further confirmed in the validation cohort.CONCLUSION:The proposed nomograms resulted in more accurate prognostic prediction for patients with HCC without PVTT after curative resection.The Oncologist 2017;22:561-569 IMPLICATIONS FOR PRACTICE:Hepatocellular carcinoma(HCC)poses a great therapeutic challenge due to the poor prognosis in patients underwent surgical resection.The portal vein tumor thrombosis(PVTT)as a robust risk factor for survival has been routinely integrated to staging systems.Nonetheless,the prognosis stratification for patients without PVTT was neglected to some extent.Herein,independent risk factors of OS and RFS in HCC patients without PVTT were reconfirmed.A predictive nomogram was constructed on these risk factors and was demonstrated to be a more accurate predictive model in HCC patients without PVTT,compared with the traditional staging systems.

(Oncologist,2017,22:561-569)

91.Cell Culture System for Analysis of Genetic Heterogeneity Within Hepatocellular Carcinomas and Response to Pharmacologic Agents　(IF 19.233)

Gao Q,Wang ZC,Duan M,Lin YH,Zhou XY,Worthley DL,Wang XY,Niu G,Xia Y,Deng M,Liu LZ,Shi JY,Yang LX,Zhang S,Ding ZB,Zhou J,Liang CM,Cao Y,Xiong L,Xi R,Shi YY,Fan J

BACKGROUND&AIMS:No targeted therapies have been found to be effective against hepatocellular carcinoma(HCC),possibly due to the large degree of intratumor heterogeneity.We performed genetic analyses of different regions of HCCs to evaluate levels of intratumor heterogeneity and associate alterations with responses to different pharmacologic agents.METHODS:We obtained samples of HCCs(associated with hepatitis B virus infection)from 10 patients undergoing curative resection,before adjuvant therapy,at hospitals in China.We collected 4-9 spatially distinct samples from each tumor(55 regions total),performed histologic analyses,isolated cancer cells,and carried them low-passage culture.We performed whole-exome sequencing,copy-number analysis,and high-throughput screening of the cultured primary cancer cells.We tested responses of an additional 105 liver cancer cell lines to a fibroblast growth factor receptor(FGFR)4 inhibitor.RESULTS:We identified a total of 3 670 non-silent mutations(3 192 missense,94 splice-site variants,and 222 insertions or deletions)in the tumor samples.We observed considerable intratumor heterogeneity and branched evolution in all 10 tumors;the mean percentage of heterogeneous mutations in each tumor was 39.7%(range,12.9%～68.5%).We found significant mutation shifts toward C＞T and C＞G substitutions in branches of phylogenetic trees among samples from each tumor(P＜0.000 1).Of note,14 of the 26 oncogenic alterations(53.8%)varied among subclones that mapped to different branches.Genetic alterations that can be targeted by existing pharmacologic agents(such as those in FGF19,DDR2,PDGFRA,and TOP1)were identified in intratumor subregions from 4 HCCs and were associated with sensitivity to these agents.However,cells from the remaining subregions,which did not have these alterations,were not sensitive to these drugs.High-throughput screening identified pharmacologic agents to which these cells were sensitive,however.Overexpression of FGF19 correlated with sensitivity of cells to an inhibitor of FGFR 4;this observation was validated in 105 liver cancer cell lines(P=0.002 4).CONCLUSIONS:By analyzing genetic alterations in different tumor regions of 10 HCCs,we observed extensive intratumor heterogeneity.Our patient-derived cell line-based model,integrating genetic and pharmacologic data from multiregional cancer samples,provides a platform to elucidate how intratumor heterogeneity affects sensitivity to different therapeutic agents.

(Gastroenterology,2017,152:232-242 e234)

92.Pro-inflammation NF-kappaB signaling triggers a positive feedback via enhancing cholesterol accumulation in liver cancer cells　(IF 5.646)

He M,Zhang W,Dong Y,Wang L,Fang T,Tang W,Lv B,Chen G,Yang B,Huang P,Xia J

BACKGROUND:Hepatocellular carcinoma(HCC)develops in a complex microenvironment characterized by chronic inflammation.In recent years,cholesterol metabolic abnormalities have been implicated the importance in cancer cell physiology.This study was designed to investigate the relationship between inflammation and cholesterol accumulation in HCC cells.METHODS:Human HCC cells Hep G2 and Huh7 were cultured and stimulated with lipopolysaccharide(LPS)for 24 h.The changes of HCC cells related to cholesterol metabolism including intracellular cholesterol concentrations,cholesterol uptake,and the expression of cholesterol-related genes 3-hydroxy-3-methylglutaryl-Co A reductase(HMGCR),LDL receptor(LDLR),sterol regulatory elementbinding transcription factor 2(SREBF2),and proprotein convertase subtilisin/kexin 9(PCSK9)were comparatively analyzed.Simultaneously,the effects of nuclear factor-kappa B(NF-kappaB)signaling pathway on cholesterol metabolism were clarified by knocking-down of nuclear factor kappa-B kinase subunit alpha(IKKalpha)and TGF-beta-activated kinase 1 and MAP3K7-binding protein 3(TAB3)via RNAi and microRNA(miR)-195.Subsequently,the roles of cholesterol accumulation in LPS induced pro-inflammatory effects were further investigated.RESULTS:Proinflammatory factor LPS significantly increased intracellular cholesterol accumulation by upregulating the expression of HMGCR,LDLR,and SREBF2,while downregulating the expression of PCSK9.These effects were revealed to depend on NF-kappaB signaling pathway by knockingdown and overexpression of IKKalpha and TAB3.Additionally,miR-195,a regulator directly targeting IKKalpha and TAB3,blocked the effects of cholesterol accumulation,further supporting the critical role of pro-inflammation NF-kappaB signaling in regulating cholesterol accumulation.Intriguingly,the accumulation of cholesterol conversely exerted an augmented pro-inflammation effects by further activating NF-kappaB signaling pathway.CONCLUSIONS:These results indicated that pro-inflammation effects of NF-kappaB signaling could be augmented by a positive feedback via enhancing the cholesterol accumulation in liver cancer cells.

(J Exp Clin Cancer Res,2017,36:15)

93.Circumventing intratumoral heterogeneity to identify potential therapeutic targets in hepatocellular carcinoma　(IF 18.946)

Huang A,Zhao X,Yang XR,Li FQ,Zhou XL,Wu K,Zhang X,Sun QM,Cao Y,Zhu HM,Wang XD,Yang HM,Wang J,Tang ZY,Hou Y,Fan J,Zhou J

BACKGROUND&AIMS:Identifying target genetic mutations in hepatocellular carcinoma(HCC)for therapy is made challenging by intratumoral heterogeneity.Circulating cell-free DNAs(cf DNA)may contain a more complete mutational spectrum compared to a single tumor sample.This study aimed to identify the most efficient strategy to identify all the mutations within heterogeneous HCCs.METHODS:Whole exome sequencing(WES)and targeted deep sequencing(TDS)were carried out in 32 multi-regional tumor samples from five patients.Matched preoperative cf DNAs were sequenced accordingly.Intratumoral heterogeneity was measured using the average percentage of non-ubiquitous mutations(present in parts of tumor regions).Profiling efficiencies of single tumor specimen and cf DNA were compared.The strategy with the highest performance was used to screen for actionable mutations.RESULTS:Variable levels of heterogeneity with branched and parallel evolution patterns were observed.The heterogeneity decreased at higher sequencing depth of TDS compared to measurements by WES(28.1%vs.34.9%,P＜0.01)but remained unchanged when additional samples were analyzed.TDS of single tumor specimen identified an average of 70%of the total mutations from multi-regional tissues.Although genome profiling efficiency of cf DNA increased with sequencing depth,an average of 47.2%total mutations were identified using TDS,suggesting that tissue samples outperformed it.TDS of single tumor specimen in 66 patients and cf DNAs in four unresectable HCCs showed that 38.6%(26/66 and 1/4)of patients carried mutations that were potential therapeutic targets.CONCLUSIONS:TDS of single tumor specimen could identify actionable mutations targets for therapy in HCC.cf DNA may serve as secondary alternative in profiling HCC genome.LAY SUMMARY:Targeted deep sequencing of single tumor specimen is a more efficient method to identify mutations in hepatocellular carcinoma made from mixed subtypes compared to circulating cell-free DNA in blood.cf DNA may serve as secondary alternative in profiling HCC genome.Identifying mutations may help clinicians choose targeted therapy for better individual treatments.

(J Hepatol,2017,67:293-301)

94.Programmed cell death-1(PD-1)checkpoint blockade in combination with a mammalian target of rapamycin inhibitor restrains hepatocellular carcinoma growth induced by hepatoma cell-intrinsic PD-1　(IF 14.971)

Li H,Li X,Liu S,Guo L,Zhang B,Zhang J,Ye QH

Inhibitors of programmed cell death 1(PD-1)administered as single agents have resulted in durable tumor regression in advanced cancer patients.However,only a minority of cancer patients respond to anti-PD-1 immunotherapy.Here,we show that PD-1 expression in hepatocellular carcinoma promotes tumor growth independently of adaptive immunity.Knockdown of PD-1 suppresses tumor growth,whereas PD-1 overexpression enhances tumorigenesis in immunodeficient xenografted mice.Mechanistically,PD-1 binds the downstream mammalian target of rapamycin effectors eukaryotic initiation factor 4E and ribosomal protein S6,thus promoting their phosphorylation.Moreover,combining mammalian target of rapamycin inhibition with anti-PD-1 antibody treatment results in more durable and synergistic tumor regression than either single agent alone,each of which presents only modest efficacy.CONCLUSION:Targeting mammalian target of rapamycin pathways in combination with PD-1 may result in increased antitumor efficacy in cancer patients.

(Hepatology 2017;66:1920-1933).

95.Chemerin has a protective role in hepatocellular carcinoma by inhibiting the expression of IL-6 and GM-CSF and MDSC accumulation　(IF 6.634)

Lin Y,Yang X,Liu W,Li B,Yin W,Shi Y,He R

Hepatocellular carcinoma(HCC)is linked to inflammation and immunosuppression.Chemerin is highly expressed in the liver and implicated in the regulation of inflammation.However,the role of chemerin in HCC remains unclear.In this study,we aimed to investigate whether chemerin is able to influence HCC progression by regulating tumor-associated inflammation.Here we demonstrated that chemerin significantly decreased in blood and tumor tissues of HCC patients,and tumor chemerin levels were inversely associated with the prognosis.In an orthotopic mouse model of HCC,Rarres2(-/-)mice exhibited aggressive tumor growth and lung metastasis,whereas chemerin overexpression greatly inhibited tumor growth.The tumor-inhibitory effect of chemerin was accompanied by a shift in tumor-infiltrating immune cells from myeloid-derived suppressive cells(MDSCs)to interferon-gamma(+)T cells and decreased tumor angiogenesis.Furthermore,we demonstrated that the tumor-inhibitory effect of chemerin was partly dependent on T cells,as chemerin overexpression could inhibit tumor growth,albeit to a lesser extent,in Rag1(-/-)mice when compared with wild-type controls.Mechanistically,chemerin inhibited nuclear factor-kappaB activation and the expression of granulocyte-macrophage colony-stimulating factor(GM-CSF)and interleukin-2(IL-6)by tumor cells and tumor-associated endothelial cell,respectively,via its receptors,and consequently,MDSC induction was impaired,leading to restoration of antitumor Tcell response and decreased tumor angiogenesis.Clinically,systemic and tumor levels of chemerin were found to inversely correlate with circulating concentrations of GM-CSF or IL-6 and tumorinfiltrating myeloid cells,respectively,in HCC patients.Moreover,neutralization of GM-CSF and IL-6 abrogated HCC progression and MDSC accumulation in Rarres2(-/-)mice.In conclusion,our study reveals the tumor-inhibitory effect of chemerin by suppressing inflammatory tumor microenvironment with therapeutic implications for inflammation-associated cancer-like HCC.

(Oncogene,2017,36:3599-3608)

96.Telomere length variation in tumor cells and cancer-associated fibroblasts:potential biomarker for hepatocellular carcinoma　(IF 5.942)

Ma LJ,Wang XY,Duan M,Liu LZ,Shi JY,Dong LQ,Yang LX,Wang ZC,Ding ZB,Ke AW,Cao Y,Zhang XM,Zhou J,Fan J,Gao Q

The role of telomere dysfunction and aberrant telomerase activities in hepatocellular carcinoma(HCC)has been overlooked for many years.This study aimed to delineate the variation and prognostic value of telomere length in HCC.Telomere-specific fluorescence in situ hybridization(FISH)and qPCR were used to evaluate telomere length in HCC cell lines,tumor tissues,and isolated non-tumor cells within the tumor.Significant telomere attrition was found in tumor cells and cancer-associated fibroblasts(CAFs)compared to their normal counterparts,but not in intratumor leukocytes or bile duct epithelial cells.Clinical relevance and prognostic value of telomere length were investigated on tissue microarrays of 257 surgically treated HCC patients.Reduced intensity of telomere signals in tumor cells or CAFs correlated with larger tumor size and the presence of vascular invasion(P＜0.05).Shortened telomeres in tumor cells or CAFs associated with reduced survival and increased recurrence,and were identified as independent prognosticators for HCC patients(P＜0.05).These findings were validated in an independent HCC cohort of 371 HCC patients from The Cancer Genome Atlas(TCGA)database,confirming telomere attrition and its prognostic value in HCC.We also showed that telomerase reverse transcriptase promoter(TERTp)mutation correlated with telomere shortening in HCC.Telomere variation in tumor cells and non-tumor cells within the tumor microenvironment of HCC was a valuable prognostic biomarker for this fatal malignancy.(c)2017 The Authors.The Journal of Pathology published by John Wiley&Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

(J Pathol,2017,243:407-417)

97.Upregulation of B7-H4 promotes tumor progression of intrahepatic cholangiocarcinoma(IF 5.959)

Xie N,Cai JB,Zhang L,Zhang PF,Shen YH,Yang X,Lu JC,Gao DM,Kang Q,Liu LX,Zhang C,Huang XY,Zou H,Zhang XY,Song ZJ,Sun HX,Fu BM,Ke AW,Shi GM

Recent reports show that B7-H4 is highly expressed in a variety of tumor cells,functions as a negative regulator of T cells and then promotes tumor progression.However,its expression and role in intrahepatic cholangiocarcinoma(ICC)remain unclear.In present study,B7-H4 expression in ICC and peritumoral tissues was determined at the level of m RNA and protein,and its bioactivity in ICC cells was studied after modification of B7-H4 expression.Then,the mechanism related to tumor progression induced by B7-H4 expression in ICC cells was explored.Finally,clinical significance of B7-H4 expression in ICC patients was further analyzed.The results showed that B7-H4 expression in ICC was much higher than that in peritumoral tissues at the level of both m RNA and protein.The high level of B7-H4 in ICC cells induced epithelial-to-mesenchymal transitions and promoted invasion and metastasis of tumor cells through activation of ERK1/2 signaling.The elevated B7-H4 expression was associated with the downregulated Bax,upregulated Bcl-2 expression,and activation of caspase-3.Clinically,high B7-H4 expression in tumor samples was significantly related to malignant phenotype,such as lymph node metastasis,high tumor stage,and poor differentiation.ICC patients with high expression of B7-H4 had shorter overall survival(OS)and disease-free survival.Moreover,the B7-H4 expression was an independent prognostic factor for predicting OS and tumor recurrence of ICC patients after operation.In conclusion,high expression of B7-H4 promotes tumor progression of ICC and may be a novel therapeutic target for ICC patients.

(Cell Death Dis,2017,8:3205)

98.JCAD Promotes Progression of Nonalcoholic Steatohepatitis to Liver Cancer by Inhibiting LATS2 Kinase Activity　(IF 8.378)

Ye J,Li TS,Xu G,Zhao YM,Zhang NP,Fan J,Wu J

Nonalcoholic steatohepatitis-associated hepatocellular carcinoma(NASH-HCC)is a malignancy whose incidents are rapidly increasing.However,the mechanisms that drive development of HCC in a steatotic microenvironment remain unknown.Here we report that the obesity-associated protein JCAD is expressed at significantly higher levels in human NASH-HCC specimens compared with pericarcinoma specimens.High JCAD expression was verified in multiple hepatoma cell lines.Forced overexpression of JCAD in hepatoma cells promoted tumor growth and proliferation,whereas JCAD silencing yielded opposite effects.JCAD interacted with the kinase domain of the tumor suppressor kinase LATS2,a core component of the Hippo signaling pathway.JCAD overexpression inhibited the ability of LATS2 to phosphorylate YAP in this pathway,in turn upregulating CCND1 and GLI2 to promote hepatoma cell proliferation.JCAD was induced by fatty acid overload in hepatic cells and was highly expressed in a mouse model of NASH-precarcinoma lesions,where the ratio of phospho-YAP to YAP was decreased.In human NASH-HCC specimens,JCAD expression and YAP phosphorylation patterns paralleled with the mouse model.Our findings illuminate a new role for JCAD and its critical interplay in the Hippo signaling cascade during the transition of NASH to HCC,with potential implications for therapeutic development in this setting.

(Cancer Res,2017,77:5287-5300)

99.ID1 promotes hepatocellular carcinoma proliferation and confers chemoresistance to oxaliplatin by activating pentose phosphate pathway　(IF 5.646)

Yin X,Tang B,Li JH,Wang Y,Zhang L,Xie XY,Zhang BH,Qiu SJ,Wu WZ,Ren ZG

BACKGROUND:Drug resistance is one of the major concerns in the treatment of hepatocellular carcinoma(HCC).The aim of the present study was to determine whether aberrant high expression of the inhibitor of differentiation 1(ID1)confers oxaliplatin-resistance to HCC by activating the pentose phosphate pathway(PPP).METHODS:Aberrant high expression of ID1 was detected in two oxaliplatin-resistant cell lines MHCC97 H-OXA(97 H-OXA)and Hep3B-OXA(3B-OXA).The lentiviral sh RNA or control sh RNA was introduced into the two oxaliplatin-resistant cell lines.The effects of ID1 on cell proliferation,apoptosis and chemoresistance were evaluated in vitro and vivo.The molecular signaling mechanism underlying the induction of HCC proliferation and oxaliplatin resistance by ID1 was explored.The prognostic value of ID1/G6PD signaling in HCC patients was assessed using the Cancer Genome Atlas(TCGA)database.RESULTS:ID1 was upregulated in oxaliplaitin-resistant HCC cells and promoted HCC cell proliferation and oxaliplatin resistance.Silencing ID1 expression in oxaliplaitin-resistant HCC cell lines inhibited cell proliferation and sensitized oxaliplaitin-resistant cells to death.ID1 knockdown significantly decreased the expression of glucose-6-phosphate dehydrogenase(G6PD),a key enzyme of the PPP.Silencing ID1 expression blocked the activation of G6PD,decreased the production of PPP NADPH,and augmented reactive oxygen and species(ROS),thus inducing cell apoptosis.Study of the molecular mechanism showed that ID1 induced G6PD promoter transcription and activated PPP through Wnt/beta-catenin/c-MYC signaling.In addition,ID1/G6PD signaling predicted unfavorable prognosis of HCC patients on the basis of TCGA.CONCLUSIONS:Our study provided the first evidence that ID1 conferred oxaliplatin resistance in HCC by activating the PPP.This newly defined pathway may have important implications in the research and development of new more effective anti-cancer drugs.

(J Exp Clin Cancer Res,2017,36:166)

100.Polymeric immunoglobulin receptor promotes tumor growth in hepatocellular carcinoma(IF 14.971)

Yue X,Ai J,Xu Y,Chen Y,Huang M,Yang X,Hu B,Zhang H,He C,Yang X,Tang W,Peng X,Dong L,Wang H,Fan J,Ding J,Geng M

Deregulation of the immune system is believed to contribute to cancer malignancy,which has led to recent therapeutic breakthroughs facilitating antitumor immunity.In a malignant setting,immunoglobulin receptors,which are fundamental components of the human immune system,fulfill paradoxical roles in cancer pathogenesis.This study describes a previously unrecognized prooncogenic function of polymeric immunoglobulin receptor(pIgR)in the promotion of cell transformation and proliferation.Mechanistically,pIg R overexpression is associated with YES proto-oncogene 1,Src family tyrosine kinase(Yes)activation,which is required for p Ig R-induced oncogenic growth.Specifically,p Ig R activates the Yes-DNAX-activating protein of 12 k Da-spleen tyrosine kinase-Rac1/CDC42-MEK(extracellular signal-regulated kinase kinase)/ERK(extracellular signal-regulated kinase)cascade in an immunoreceptor tyrosine-based activating motif(ITAM)-dependent manner to promote cell transformation and tumor growth,although p Ig R itself does not contain an ITAM sequence.Additionally,the combination of pIgR and phosphorylated Yes(p-Yes)levels serves as a prognostic biomarker for hepatitis B surface antigen-positive and earlystage hepatocellular carcinoma(HCC)patients.Moreover,pharmacological targeting of MEK/ERK or Yes represents a therapeutic option for the subgroup of patients with pIgR/p-Yes-positive HCC based on our results with both cancer cell-line-based xenografts and primary patient-derived xenografts.CONCLUSION:Our findings reveal the molecular mechanism by which pIg R promotes cancer malignancy,suggest the clinical potential of targeting this pathway in HCC,and provide new insight into the oncogenic role of immunoglobulin receptors.

(Hepatology,2017,65:1948-1962)

101.Protein glycosylation in viral hepatitis-related HCC:Characterization of heterogeneity,biological roles,and clinical implications　(IF 6.508)

Zhang S,Cao X,Gao Q,Liu Y

Protein glycosylation is one of the most frequent and well-known posttranslational modifications,playing important roles in physiopathological processes.Glycosylation is catalyzed by enzymatic additions of heterogeneous glycans with specific linkage of monosaccharides and,in a preformed fashion,to specific amino acids within glycoproteins.Altered glycan macroheterogeneity and microheterogeneity have been reported in glycoproteins produced mainly by the liver,facilitating tumorigenesis,progression,and metastasis.Characterizing the heterogeneity and biological functions of glycans in liver diseases would lead to a better understanding of the molecular pathogenesis of liver damage and cancer,providing novel diagnostic,prognostic,and therapeutic clues.Technical advances in glycoproteomics and glycomics have allowed a more comprehensive and deeper understanding of diseaserelated glycosylation events.Here,we briefly review the structural diversity of glycans in viral hepatitis-related hepatocellular carcinoma,discuss their roles in regulating the initiation and development of liver cancer,and introduce potential clinical applications.

(Cancer Lett,2017,406:64-70)

102.miR-182-5p promotes hepatocellular carcinoma progression by repressing FOXO3a(IF 8.731)

Cao MQ,You AB,Zhu XD,Zhang W,Zhang YY,Zhang SZ,Zhang KW,Cai H,Shi WK,Li XL,Li KS,Gao DM,Ma DN,Ye BG,Wang CH,Qin CD,Sun HC,Zhang T,Tang ZY

BACKGROUND:High frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma(HCC).microRNA(miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis.Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown.METHODS:Real-time PCR(RT-PCR)and in situ hybridization(ISH)were conducted to assess the expression of miR-182-5p in HCC cells and tissues.Cell Counting Kit-8(CCK-8),transwell assays were performed to detected cells proliferation and migration ability.Flow cytometry assays were used to detect cell apoptosis rate,and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis.The target of miR-182-5p was validated with a dualluciferase reporter assay and western blotting.Immunohistochemistry,immumoblotting,and immunoprecipitation were performed to test relative protein expression.RESULTS:We showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery.miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo.miR-182-5p directly targets 3'-UTR of FOXO3a and repressed FOXO3a expression,activating AKT/FOXO3a pathway to promote HCC proliferation.Notably,miR-182-5p activated Wnt/beta-catenin signaling by inhibiting the degradation of beta-catenin and enhancing the interaction between beta-catenin and TCF4 which was mediated by repressed FOXO3a.CONCLUSIONS:Consistently,miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery,and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

(J Hematol Oncol,2018,11:12)

103.BAP1 acts as a tumor suppressor in intrahepatic cholangiocarcinoma by modulating the ERK1/2 and JNK/c-Jun pathways　(IF 5.959)

Chen XX,Yin Y,Cheng JW,Huang A,Hu B,Zhang X,Sun YF,Wang J,Wang YP,Ji Y,Qiu SJ,Fan J,Zhou J,Yang XR

Current therapeutic options for intrahepatic cholangiocarcinoma(ICC)are very limited,which is largely attributed to poor understanding of molecular pathogenesis of ICC.Breast cancer type 1 susceptibility protein-associated protein-1(BAP1)has been reported to be a broad-spectrum tumor suppressor in many tumor types,yet its role in ICC remains unknown.The aim of this study was to investigate the clinical implications and biological function of BAP1 in ICC.Our results showed that the messenger RNA and protein levels of BAP1 were significantly downregulated in ICC versus paired non-tumor tissues.Overexpression of wild-type but not mutant BAP1 significantly suppressed ICC cell proliferation,cell cycle progression,and invasion in vitro,as well as tumor progression in vivo.Conversely,knockdown of BAP1 yielded opposing effects.Mechanistically,BAP1 functioned as a tumor suppressor in ICC by inhibiting the extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase/c-Jun pathways,and this function was abolished by inactivating mutations.Clinically,low BAP1 expression was positively correlated with aggressive tumor characteristics,such as larger tumor size,presence of lymphatic metastasis,and advanced tumor node metastasis stage.Survival analysis revealed that low BAP1 expression was significantly and independently associated with poor overall survival and relapse-free survival after curative surgery.In conclusion,BAP1 is a putative tumor suppressor of ICC,and may serve as a valuable prognostic biomarker as well as potential therapeutic target for ICC.

(Cell Death Dis,2018,9:1036)

104.Phosphorothioate-Modified AP613-1 Specifically Targets GPC3 when Used for Hepatocellular Carcinoma Cell Imaging　(IF 5.919)

Dong L,Zhou H,Zhao M,Gao X,Liu Y,Liu D,Guo W,Hu H,Xie Q,Fan J,Lin J,Wu W

Glypican-3(GPC3),the cellular membrane proteoglycan,has been established as a tumor biomarker for early diagnosis of hepatocellular carcinoma(HCC).GPC3 is highly expressed in more than 70%HCC tissues detected by antibody-based histopathological systems.Recently,aptamers,a short single-strand DNA or RNA generated from systematic evolution of ligands by exponential enrichment(SELEX),were reported as potential alternatives in tumor-targeted imaging and diagnosis.In this study,a total of 19 GPC3-bound aptamers were successfully screened by capillary electrophoresis(CE)-SELEX technology.After truncated,AP613-1 was confirmed to specifically target GPC3 with a dissociation constant(KD)of 59.85 n M.When modified with a phosphorothioate linkage,APS613-1 targeted GPC3 with a KD of 15.48 n M and could be used as a specific probe in living Huh7 and PLC/PRF/5 imaging,GPC3-positive cell lines,but not in L02 or A549,two GPC3-negative cell lines.More importantly,Alexa Fluor 750-conjugated APS613-1 could be used as a fluorescent probe to subcutaneous HCC imaging in xenograft nude mice.Our results indicated that modified AP613-1,especially APS613-1,was a potential agent in GPC3-positive tumor imaging for HCC early diagnosis.

(Mol Ther Nucleic Acids,2018,13:376-386)

105.Diverse modes of clonal evolution in HBV-related hepatocellular carcinoma revealed by single-cell genome sequencing　(IF 17.848)

Duan M,Hao J,Cui S,Worthley DL,Zhang S,Wang Z,Shi J,Liu L,Wang X,Ke A,Cao Y,Xi R,Zhang X,Zhou J,Fan J,Li C,Gao Q

Hepatocellular carcinoma(HCC)is a cancer of substantial morphologic,genetic and phenotypic diversity.Yet we do not understand the relationship between intratumor heterogeneity and the associated morphologic/histological characteristics of the tumor.Using single-cell whole-genome sequencing to profile 96 tumor cells(30-36 each)and 15 normal liver cells(5 each),collected from three male patients with HBV-associated HCC,we confirmed that copy number variations occur early in hepatocarcinogenesis but thereafter remain relatively stable throughout tumor progression.Importantly,we showed that specific HCCs can be of monoclonal or polyclonal origins.Tumors with confluent multinodular morphology are the typical polyclonal tumors and display the highest intratumor heterogeneity.In addition to mutational and copy number profiles,we dissected the clonal origins of HCC using HBV-derived foreign genomic markers.In monoclonal HCC,all the tumor single cells exhibit the same HBV integrations,indicating that HBV integration is an early driver event and remains extremely stable during tumor progression.In addition,our results indicated that both models of metastasis,late dissemination and early seeding,have a role in HCC progression.Notably,early intrahepatic spreading of the initiating clone leads to the formation of synchronous multifocal tumors.Meanwhile,we identified a potential driver gene ZNF717 in HCC,which exhibits a high frequency of mutation at both single-cell and population levels,as a tumor suppressor acting through regulating the IL-6/STAT3 pathway.These findings highlight multiple distinct tumor evolutionary mechanisms in HCC,which suggests the need for specific treatment strategies.

(Cell Res,2018,28:359-373)

106.NOD-like receptor X1 functions as a tumor suppressor by inhibiting epithelial-mesenchymal transition and inducing aging in hepatocellular carcinoma cells　(IF 8.731)

Hu B,Ding GY,Fu PY,Zhu XD,Ji Y,Shi GM,Shen YH,Cai JB,Yang Z,Zhou J,Fan J,Sun HC,Kuang M,Huang C

BACKGROUND:This study was performed to investigate the role of nucleotide-binding oligomerization domain(NOD)-like receptor X1(NLRX1)in regulating hepatocellular carcinoma(HCC)progression.METHODS:Expression 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 beta-galactosidase assays.Kaplan-Meier analyses and Cox regression models were used for prognostic evaluation.RESULTS:NLRX1 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.CONCLUSIONS:NLRX1 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.

(J Hematol Oncol,2018,11:28)

107.Prognostic Nomogram Based on Histological Characteristics of Fibrotic Tumor Stroma in Patients Who Underwent Curative Resection for Intrahepatic Cholangiocarcinoma　(IF 5.252)

Jing CY,Fu YP,Huang JL,Zhang MX,Yi Y,Gan W,Xu X,Shen HJ,Lin JJ,Zheng SS,Zhang J,Zhou J,Fan J,Ren ZG,Qiu SJ,Zhang BH

BACKGROUND:Fibrotic tumor stroma(FTS)has been implicated in cancer promotion in several neoplasms.The histological features of FTS are convenient and easily accessible in clinical routine in intrahepatic cholangiocarcinoma(ICC)specimens.The goal of this study was to explore prognostic impacts of the quantity and maturity of FTS on surgical ICC patients.Moreover,we aimed to propose an efficient prognostic nomogram for postoperative ICC patients.MATERIALS AND METHODS:The clinical profiles of 154 consecutive postoperative ICC patients were retrospectively analyzed.Tumor-stroma ratio and morphological maturity of FTS were evaluated on hematoxylin and eosin-stained tumor sections.CD3,CD8,and alpha-smooth muscle actin(alpha-SMA)staining were performed on corresponding tissue microarrays.The nomogram was established on variables selected by multivariate analyses and was validated in 10-fold cross-validation.RESULTS:Rich tumor stroma and strong alpha-SMA expression were associated with poor overall survival(OS).However,in multivariate analyses,these two biomarkers failed to stratify both OS and recurrencefree survival(RFS).Immature FTS was correlated with tumor multiplicity,advanced clinical stage,and sparser CD3 and CD8 positive tumor-infiltrating lymphocytes(TILs)and was identified as an independent prognostic indicator for both OS and RFS.The nomogram comprising FTS maturity,tumor number,microvascular invasion,and lymph node metastasis possessed higher predictive power relative to conventional staging systems.CONCLUSION:Immature FTS was an independent risk factor for survival and was associated with sparser CD3 and CD8 positive TILs in ICC.The prognostic nomogram integrating the maturity of FTS offers a more accurate risk stratification for postoperative ICC patients.IMPLICATIONS FOR PRACTICE:Accumulating evidence has suggested that fibrotic components in tumor microenvironment(TME)play a complicated and vital role in TME reprogramming and cancer progression.However,in clinical practice,the evaluation of fibrotic tumor stroma(FTS)is still neglected to some extent.This study's findings indicated that,in intrahepatic cholangiocarcinoma(ICC),the histological maturity of FTS is a robust prognostic indicator for patients who underwent curative resection.Moreover,prognostic nomogram constructed on the maturity of FTS possessed higher predictive power relative to the conventional tumor-node-metastasis staging systems.Taken together,the evaluation of FTS should be emphasized in clinical routine for more accurate prognostic prediction in postoperative ICC patients.

(Oncologist,2018,23:1482-1493)

108.The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma　(IF 11.878)

Li Z,Zhang J,Liu X,Li S,Wang Q,Di C,Hu Z,Yu T,Ding J,Li J,Yao M,Fan J,Huang S,Gao Q,Zhao Y,He X

Recurrent chromosomal aberrations have led to the discovery of oncogenes or tumour suppressors involved in carcinogenesis.Here we characterized an oncogenic long intergenic non-coding RNA in the frequent DNA-gain regions in hepatocellular carcinoma(HCC),LINC01138(long intergenic non-coding RNA located on 1q21.2).The LINC01138 locus is frequently amplified in HCC;the LINC01138 transcript is stabilized by insulin like growth factor-2 m RNA-binding proteins 1/3(IGF2BP1/IGF2BP3)and is associated with the malignant features and poor outcomes of HCC patients.LINC01138 acts as an oncogenic driver that promotes cell proliferation,tumorigenicity,tumour invasion and metastasis by physically interacting with arginine methyltransferase 5(PRMT5)and enhancing its protein stability by blocking ubiquitin/proteasome-dependent degradation in HCC.The discovery of LINC01138,a promising prognostic indicator,provides insight into the molecular pathogenesis of HCC,and the LINC01138/PRMT5 axis is an ideal therapeutic target for HCC treatment.

(Nat Commun,2018,9:1572)

109.Restoration of FBP1 suppressed Snail-induced epithelial to mesenchymal transition in hepatocellular carcinoma　(IF 5.959)

Liu GM,Li Q,Zhang PF,Shen SL,Xie WX,Chen B,Wu J,Hu WJ,Huang XY,Peng BG

Fructose-1,6-bisphosphatase(FBP1),one of the rate-limiting gluconeogenic enzymes,plays critical roles in several cancers and is treated as a tumour suppressor.However,its role in hepatocellular carcinoma(HCC)is unclear.Here,we demonstrated that FBP1 was significantly inhibited during Snail-induced epithelial to mesenchymal transition(EMT)and tissues in HCC.Restoration of FBP1 expression in HCC cancer cells suppressed EMT phenotype,tumour migration and tumour growth induced by Snail overexpression in SMMC-7721 cells.Gene set enrichment analyses revealed significantly enriched terms,including WNT,Notch,ESC,CSR and PDGF,in the group with high Snail and low FBP1 compared with those with low Snail and high FBP1.Low FBP1 expression was significantly correlated with higher AFP level,satellite nodules,portal vein tumour thrombus,and advanced tumour stage.Survival analyses showed that FBP1 was an independent prognostic factor for overall survival and recurrence-free survival.In conclusion,our study revealed a vital role for FBP1 in Snail-induced EMT and prognostic prediction in HCC.

(Cell Death Dis,2018,9:1132)

110.Metabolic reprogramming by PCK1 promotes TCA cataplerosis,oxidative stress and apoptosis in liver cancer cells and suppresses hepatocellular carcinoma　(IF 6.634)

Liu MX,Jin L,Sun SJ,Liu P,Feng X,Cheng ZL,Liu WR,Guan KL,Shi YH,Yuan HX,Xiong Y

Phosphoenolpyruvate carboxykinase(PEPCK or PCK)catalyzes the first rate-limiting step in hepatic gluconeogenesis pathway to maintain blood glucose levels.Mammalian cells express two PCK genes,encoding for a cytoplasmic(PCPEK-C or PCK1)and a mitochondrial(PEPCK-M or PCK2)isoforms,respectively.Increased expressions of both PCK genes are found in cancer of several organs,including colon,lung,and skin,and linked to increased anabolic metabolism and cell proliferation.Here,we report that the expressions of both PCK1 and PCK2 genes are downregulated in primary hepatocellular carcinoma(HCC)and low PCK expression was associated with poor prognosis in patients with HCC.Forced expression of either PCK1 or PCK2 in liver cancer cell lines results in severe apoptosis under the condition of glucose deprivation and suppressed liver tumorigenesis in mice.Mechanistically,we show that the pro-apoptotic effect of PCK1 requires its catalytic activity.We demonstrate that forced PCK1 expression in glucose-starved liver cancer cells induced TCA cataplerosis,leading to energy crisis and oxidative stress.Replenishing TCA intermediate alpha-ketoglutarate or inhibition of reactive oxygen species production blocked the cell death caused by PCK expression.Taken together,our data reveal that PCK1 is detrimental to malignant hepatocytes and suggest activating PCK1 expression as a potential treatment strategy for patients with HCC.

(Oncogene,2018,37:1637-1653)

111.microRNA-501-3p suppresses metastasis and progression of hepatocellular carcinoma through targeting LIN7A　(IF 5.959)

Luo CB,Yin D,Zhan H,Borjigin U,Li C,Zhou Z,Hu Z,Wang P,Sun Q,Fan J,Zhou J,Wang X,Zhou S,Huang XW

Increasing numbers of evidences have demonstrated that micro RNAs(miRNAs)are implicated in metastasis and progression of hepatocellular carcinoma(HCC).However,their detailed expression levels and actual functions in HCCs have not been fully clarified yet.Results from our recent study revealed that some miRNAs were particularly related to metastasis of HCCs.As one of these newly found miRNAs,miR-501-3p showed to highly involve into metastatic process of HCCs.Here we reported that the expression of miR-501-3p was decreased in both metastatic HCC cell lines and tissue samples from HCC patients with recurrence and metastasis.Downregulation of miR-501-3p correlated with tumor progression and poor prognosis in the HCC patients.Results of functional analyses revealed that overexpression of miR-501-3p in HCCLM3 cancer cells inhibited their proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT),while miR-501-3p loss in PLC/PRF/5 cancer cells facilitated all these cellular activities.In addition,Lin-7 homolog A(LIN7A)was directly targeted by miR-501-3p to mediate the suppression effects on metastasis in HCC cells.miR-501-3p suppresses metastasis and progression of HCCs through targeting LIN7A.This finding suggests that miR-501-3p could be used as a potential prognostic predictor as well as a potential therapeutic tool for HCC therapies.

(Cell Death Dis,2018,9:535)

112.Clinical significance of PD-1/PD-Ls gene amplification and overexpression in patients with hepatocellular carcinoma　(IF 8.063)

Ma LJ,Feng FL,Dong LQ,Zhang Z,Duan M,Liu LZ,Shi JY,Yang LX,Wang ZC,Zhang S,Ding ZB,Ke AW,Cao Y,Zhang XM,Zhou J,Fan J,Wang XY,Gao Q

Background:The remarkable clinical activity of PD-1 antibody in advanced hepatocellular carcinoma(HCC)highlights the importance of PD-1/PD-L1-mediated immune escape as therapeutic target in HCC.However,the frequency and prognostic significance of PD-Ls genetic alterations in HCC remain unknown.Methods:Fluorescence in situ hybridization were used to determine PD-Ls genetic alterations,and qPCR data coupled with immunofluorescence were used to measure the m RNA and protein levels of PD-Ls.Clinical relevance and prognostic value of 9p24.1 genetic alterations were investigated on tissue microarray containing three independent cohorts of 578 HCC patients.The results were further validated in an independent cohort of 442 HCC patients from The Cancer Genome Atlas(TCGA)database.Results:In total,7.1%～15.0%for amplification and 15.8%～31.3%for polysomy of 9p24.1 were revealed in three cohorts of HCC patients,similar to the objective response rate of PD-1 antibody in HCC.Patients with 9p24.1 genetic alterations significantly and independently correlated with unfavorable outcomes than those without.FISH and qPCR data coupled with immunofluorescence revealed that genetic alterations of 9p24.1 robustly contributed to PD-L1 and PD-L2 upregulation.In addition,increased expression of PD-L1 instead of PD-L2 also predicted poor survival by multivariate analyses.Meanwhile,high infiltration of PD-1(+)immune cells also indicated dismal survival in HCC.Conclusions:Amplification or higher expression of PD-L1 significantly and independently correlated with unfavorable survival in HCC patients,authenticating the PD-1/PD-L1 axis as rational immunotherapeutic targets for HCC.

(Theranostics,2018,8:5690-5702)

113.miR-296-5p suppresses EMT of hepatocellular carcinoma via attenuating NRG1/ERBB2/ERBB3 signaling　(IF 5.646)

Shi DM,Li LX,Bian XY,Shi XJ,Lu LL,Zhou HX,Pan TJ,Zhou J,Fan J,Wu WZ

BACKGROUND:Accumulation of evidence indicates that miRNAs have crucial roles in the regulation of EMT-associated properties,such as proliferation,migration and invasion.However,the underlying molecular mechanisms are not entirely illustrated.Here,we investigated the role of miR-296-5p in hepatocellular carcinoma(HCC)progression.METHODS:In vitro cell morphology,proliferation,migration and invasion were compared between HCC cell lines with up-or downregulation of miR-296-5p.Immunofluorescence and Western blot immunofluorescence assays were used to detect the expression of EMT markers.Bioinformatics programs,luciferase reporter assay and rescue experiments were used to validate the downstream targets of miR-296-5p.Xenograft nude mouse models were established to observe tumor growth and metastasis.Immunohistochemical assays were conducted to study the relationships between miR-296-5p expression and Neuregulin-1(NRG1)/EMT markers in human HCC samples and mice.RESULTS:miR-296-5p was prominently downregulated in HCC tissues relative to adjacent normal liver tissues and associated with favorable prognosis.Overexpression of miR-296-5p inhibited EMT along with migration and invasion of HCC cells via suppressing NRG1/ERBB2/ERBB3/RAS/MAPK/Fra-2 signaling in vitro.More importantly,miR-296-5p disrupted intrahepatic and pulmonary metastasis in vivo.NRG1,as a direct target of miR-296-5p,mediates downstream biological responses.In HCC tissues from patients and mice,the levels of miR-296-5p and NRG1 also showed an inverse relationship.CONCLUSIONS:miR-296-5p inhibited EMT-related metastasis of HCC through NRG1/ERBB2/ERBB3/RAS/MAPK/Fra-2 signaling.

(J Exp Clin Cancer Res,2018,37:294)

114.PFKFB3 blockade inhibits hepatocellular carcinoma growth by impairing DNA repair through AKT　(IF 5.959)

Shi WK,Zhu XD,Wang CH,Zhang YY,Cai H,Li XL,Cao MQ,Zhang SZ,Li KS,Sun HC

Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3),a key molecule of glucose metabolism in cytoplasm,has been found in various tumors.Emerging evidence has suggested that PFKFB3 is also located in the nucleus and apparent in regulatory functions other than glycolysis.In this study,we found that PFKFB3 expression is associated with hepatocellular carcinoma(HCC)growth and located mainly in the nucleus of tumor cells.PFKFB3 overexpression was associated with large tumor size(P=0.04)and poor survival of patients with HCC(P=0.027).Knockdown of PFKFB3 inhibited HCC growth,not only by reducing glucose consumption but also by damaging the DNA repair function,leading to G2/M phase arrest and apoptosis.In animal studies,overexpression of PFKFB3 is associated with increased tumor growth.Mechanistically,PFKFB3 silencing decreased AKT phosphorylation and reduced the expression of ERCC1,which is an important DNA repair protein.Moreover,PFK15,a selective PFKFB3 inhibitor,significantly inhibited tumor growth in a xenograft model of human HCC.PFKFB3 is a potential novel target in the treatment of HCC.

(Cell Death Dis,2018,9:428)

115.Circulating Tumor Cells from Different Vascular Sites Exhibit Spatial Heterogeneity in Epithelial and Mesenchymal Composition and Distinct Clinical Significance in Hepatocellular Carcinoma(IF 8.911)

Sun YF,Guo W,Xu Y,Shi YH,Gong ZJ,Ji Y,Du M,Zhang X,Hu B,Huang A,Chen GG,Lai PBS,Cao Y,Qiu SJ,Zhou J,Yang XR,Fan J

Purpose:The spatial heterogeneity of phenotypic and molecular characteristics of CTCs within the circulatory system remains unclear.Herein,we mapped the distribution and characterized biological features of CTCs along the transportation route in hepatocellular carcinoma(HCC).Experimental Design:In 73 localized HCC patients,blood was drawn from peripheral vein(PV),peripheral artery(PA),hepatic veins(HV),infrahepatic inferior vena cava(IHIVC),and portal vein(Po V)before tumor resection.Epithelial and mesenchymal transition(EMT)phenotype in CTCs were analyzed by a 4-channel immunofluorescence CellSearch assay and microfluidic quantitative RTPCR.The clinical significance of CTCs from different vascular sites was evaluated.Results:The CTC number and size gradient between tumor efferent vessels and postpulmonary peripheral vessels was marked.Tracking the fate of CTC clusters revealed that CTCs displayed an aggregatedsingular-aggregated manner of spreading.Single-cell characterization demonstrated that EMT status of CTCs was heterogeneous across different vascular compartments.CTCs were predominantly epithelial at release,but switched to EMT-activated phenotype during hematogeneous transit via Smad2 and beta-catenin related signaling pathways.EMT activation in primary tumor correlated with total CTC number at HV,rather than epithelial or EMT-activated subsets of CTCs.Follow-up analysis suggested that CTC and circulating tumor microemboli burden in hepatic veins and peripheral circulation prognosticated postoperative lung metastasis and intrahepatic recurrence,respectively.Conclusions:The current data suggested that a profound spatial heterogeneity in cellular distribution and biological features existed among CTCs during circulation.Multivascular measurement of CTCs could help to reveal novel mechanisms of metastasis and facilitate prediction of postoperative relapse or metastasis pattern in HCC.

(Clin Cancer Res,2018,24:547-559)

116.Phospho-ERK is a biomarker of response to a synthetic lethal drug combination of sorafenib and MEK inhibition in liver cancer　(IF 18.946)

Wang C,Jin H,Gao D,Lieftink C,Evers B,Jin G,Xue Z,Wang L,Beijersbergen RL,Qin W,Bernards R

BACKGROUND&AIMS:Treatment of liver cancer remains challenging because of a paucity of drugs that target critical dependencies.Sorafenib is a multikinase inhibitor that is approved as the standard therapy for patients with advanced hepatocellular carcinoma,but it only provides limited survival benefit.In this study we aimed to identify potential combination therapies to improve the clinical response to sorafenib.METHODS:To investigate the cause of the limited therapeutic effect of sorafenib,we performed a CRISPR-Cas9 based synthetic lethality screen to search for kinases whose knockout synergizes with sorafenib.Synergistic effects of sorafenib and selumetinib on cell apoptosis and phospho-ERK(p-ERK)were analyzed by caspase-3/7 apoptosis assay and western blot,respectively.p-ERK was measured by immunochemical analysis using a tissue microarray containing 78 liver cancer specimens.The in vivo effects of the combination were also measured in two xenograft models.RESULT:We found that suppression of ERK2(MAPK1)sensitizes several liver cancer cell lines to sorafenib.Drugs inhibiting the MEK(MEK1/2[MAP2K1/2])or ERK(ERK1/2[MAPK1/3])kinases reverse unresponsiveness to sorafenib in vitro and in vivo in a subset of liver cancer cell lines characterized by high levels of active p-ERK,through synergistic inhibition of ERK kinase activity.CONCLUSION:Our data provide a combination strategy for treating liver cancer and suggest that tumors with high basal p-ERK levels,which are seen in approximately 30%of liver cancers,are most likely to benefit from such combinatorial treatment.LAY SUMMARY:Sorafenib is approved as the standard therapy for patients with advanced hepatocellular carcinoma,but only provides limited survival benefit.Herein,we found that inhibition of the kinase ERK2 increases the response to sorafenib in liver cancer.Our data indicate that a combination of sorafenib and a MEK inhibitor is most likely to be effective in tumors with high basal phospho-ERK levels.

(J Hepatol,2018,69:1057-1065)

117.Adjuvant Transarterial Chemoembolization for HBV-Related Hepatocellular Carcinoma After Resection:A Randomized Controlled Study　(IF 8.911)

Wang Z,Ren Z,Chen Y,Hu J,Yang G,Yu L,Yang X,Huang A,Zhang X,Zhou S,Sun H,Wang Y,Ge N,Xu X,Tang Z,Lau W,Fan J,Wang J,Zhou J

Purpose:The survival of patients with hepatocellular carcinoma(HCC)recurrence after curative resection is usually poor.We sought to evaluate the safety and efficacy of adjuvant transarterial chemoembolization(TACE)in HBV-related HCC patients with an intermediate(a single tumor larger than 5 cm without microvascular invasion)or high risk(a single tumor with microvascular invasion,or two or three tumors)of recurrence.Experimental Design:In this randomized phase 3 trial,280 eligible patients were assigned to adjuvant TACE(n=140)or no adjuvant treatment(control;n=140)groups.The primary endpoint was recurrence-free survival(RFS);secondary endpoints included overall survival(OS)and safety.Multivariable Cox-proportional hazards model was used to determine the independent impact of TACE on patients'outcomes.Results:Patients who received adjuvant TACE had a significantly longer RFS than those in the control group[56.0%vs.42.1%,P=0.01;HR,0.68;95%confidence interval(CI),0.49-0.93].Patients in the adjuvant TACE group had 7.8%higher 3-year OS rate than the control group(85.2%vs.77.4%;P=0.04;HR,0.59;95%CI,0.36-0.97).The impact of adjuvant TACE on RFS and OS remained significant after controlling for other known prognostic factors(HR,0.67;P=0.01 for RFS;and HR,0.59;P=0.04 for OS).There was no grade 3 or 4 toxicity after adjuvant TACE.Conclusions:For patients with HBV-related HCC who had an intermediate or high risk of recurrence after curative hepatectomy,our study showed adjuvant TACE significantly reduced tumor recurrence,improved RFS and OS,and the procedure was well tolerated.

(Clin Cancer Res,2018,24:2074-2081)

118.Glucose-regulated phosphorylation of TET2 by AMPK reveals a pathway linking diabetes to cancer(IF 43.070)

Wu D,Hu D,Chen H,Shi G,Fetahu IS,Wu F,Rabidou K,Fang R,Tan L,Xu S,Liu H,Argueta C,Zhang L,Mao F,Yan G,Chen J,Dong Z,Lv R,Xu Y,Wang M,Ye Y,Zhang S,Duquette D,Geng S,Yin C,Lian CG,Murphy GF,Adler GK,Garg R,Lynch L,Yang P,Li Y,Lan F,Fan J,Shi Y,Shi YG

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications(1,2).Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer(3-5).High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer,but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype.Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome.We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase(AMPK),which phosphorylates TET2 at serine 99,thereby stabilizing the tumour suppressor.Increased glucose levels impede AMPKmediated phosphorylation at serine 99,which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine(5hmC)and the tumour suppressive function of TET2 in vitro and in vivo.Treatment with the anti-diabetic drug metformin protects AMPKmediated phosphorylation of serine 99,thereby increasing TET2 stability and 5hm C levels.These findings define a novel‘phospho-switch’that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC,which connects diabetes to cancer.Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression.Thus,this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state,offering a potential strategy for cancer prevention and treatment.

(Nature,2018,559:637-641)

119.Matrix stiffness-upregulated LOXL2 promotes fibronectin production,MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation　(IF 5.646)

Wu S,Zheng Q,Xing X,Dong Y,Wang Y,You Y,Chen R,Hu C,Chen J,Gao D,Zhao Y,Wang Z,Xue T,Ren Z,Cui J

BACKGROUND:Higher matrix stiffness affects biological behavior of tumor cells,regulates tumor-associated gene/miRNA expression and stemness characteristic,and contributes to tumor invasion and metastasis.However,the linkage between higher matrix stiffness and pre-metastatic niche in hepatocellular carcinoma(HCC)is still largely unknown.METHODS:We comparatively analyzed the expressions of LOX family members in HCC cells grown on different stiffness substrates,and speculated that the secreted LOXL2 may mediate the linkage between higher matrix stiffness and pre-metastatic niche.Subsequently,we investigated the underlying molecular mechanism by which matrix stiffness induced LOXL2 expression in HCC cells,and explored the effects of LOXL2 on pre-metastatic niche formation,such as BMCs recruitment,fibronectin production,MMPs and CXCL12 expression,cell adhesion,etc.RESULTS:Higher matrix stiffness significantly upregulated LOXL2 expression in HCC cells,and activated JNK/c-JUN signaling pathway.Knockdown of integrin beta1 and alpha5 suppressed LOXL2 expression and reversed the activation of above signaling pathway.Additionally,JNK inhibitor attenuated the expressions of p-JNK,p-c-JUN,c-JUN and LOXL2,and sh RNA-c-JUN also decreased LOXL2 expression.CM-LVLOXL2-OE and rh LOXL2 upregulated MMP9 expression and fibronectin production obviously in lung fibroblasts.Moreover,activation of Akt pathway contributed to LOXL2-induced fibronectin upregulation.LOXL2 in CM as chemoattractant increased motility and invasion of BMCs,implicating a significant role of LOXL2 in BMCs recruitment.Except that,CM-LV-LOXL2-OE as chemoattractant also increased the number of migrated HCC cells,and improved chemokine CXCL12 expression in lung fibroblasts.The number of HCC cells adhered to surface of lung fibroblasts treated with CM-LV-LOXL2-OE was remarkably higher than that of the control cells.These results indicated that the secreted LOXL2 facilitated the motility of HCC cells and strengthened CTCs settlement on the remodeled matrix“soil”.CONCLUSION:Integrin beta1/alpha5/JNK/c-JUN signaling pathway participates in higher matrix stiffness-induced LOXL2 upregulation in HCC cells.The secreted LOXL2 promotes fibronectin production,MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation.

(J Exp Clin Cancer Res,2018,37:99)

120.Dynamic network biomarker indicates pulmonary metastasis at the tipping point of hepatocellular carcinoma　(IF 11.878)

Yang B,Li M,Tang W,Liu W,Zhang S,Chen L,Xia J

Developing predictive biomarkers that can detect the tipping point before metastasis of hepatocellular carcinoma(HCC),is critical to prevent further irreversible deterioration.To discover such early-warning signals or biomarkers of pulmonary metastasis in HCC,we analyse time-series gene expression data in spontaneous pulmonary metastasis mice HCCLM3-RFP model with our dynamic network biomarker(DNB)method,and identify CALML3 as a core DNB member.All experimental results of gain-of-function and loss-of-function studies show that CALML3 could indicate metastasis initiation and act as a suppressor of metastasis.We also reveal the biological role of CALML3 in metastasis initiation at a network level,including proximal regulation and cascading influences in dysfunctional pathways.Our further experiments and clinical samples show that DNB with CALML3 reduced pulmonary metastasis in liver cancer.Actually,loss of CALML3 predicts shorter overall and relapse-free survival in postoperative HCC patients,thus providing a prognostic biomarker and therapy target in HCC.

(Nat Commun,2018,9:678)

121.CD31 regulates metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma via the ITGB1-FAK-Akt signaling pathway　(IF 6.508)

Zhang YY,Kong LQ,Zhu XD,Cai H,Wang CH,Shi WK,Cao MQ,Li XL,Li KS,Zhang SZ,Chai ZT,Ao JY,Ye BG,Sun HC

Platelet endothelial cell adhesion molecule-1(PECAM-1 or CD31)is a well-known marker of endothelial cells and a key factor for adhesion and accumulation of platelets.CD31 plays roles in cell proliferation,apoptosis,migration,and cellular immunity.CD31 is also expressed on tumor cells,such as breast cancer cells and non-Hodgkin's lymphomas,and contributes to tumor cell invasion.Here,our experiments show that CD31 promotes metastasis by inducing the epithelial-mesenchymal transition in hepatocellular carcinoma by up-regulating integrin beta1 via the FAK/Akt signaling pathway.

(Cancer Lett,2018,429:29-40)

122.Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China(2017 Edition)(IF 5.944)

Zhou J,Sun HC,Wang Z,Cong WM,Wang JH,Zeng MS,Yang JM,Bie P,Liu LX,Wen TF,Han GH,Wang MQ,Liu RB,Lu LG,Ren ZG,Chen MS,Zeng ZC,Liang P,Liang CH,Chen M,Yan FH,Wang WP,Ji Y,Cheng WW,Dai CL,Jia WD,Li YM,Li YX,Liang J,Liu TS,Lv GY,Mao YL,Ren WX,Shi HC,Wang WT,Wang XY,Xing BC,Xu JM,Yang JY,Yang YF,Ye SL,Yin ZY,Zhang BH,Zhang SJ,Zhou WP,Zhu JY,Liu R,Shi YH,Xiao YS,Dai Z,Teng GJ,Cai JQ,Wang WL,Dong JH,Li Q,Shen F,Qin SK,Fan J

Background:Hepatocellular carcinoma(HCC)(about 85%～90%of primary liver cancer)is particularly prevalent in China because of the high prevalence of chronic hepatitis B infection.HCC is the fourth most common malignancy and the third leading cause of tumor-related deaths in China.It poses a significant threat to the life and health of Chinese people.Summary:This guideline presents official recommendations of the National Health and Family Planning Commission of the People's Republic of China on the surveillance,diagnosis,staging,and treatment of HCC occurring in China.The guideline was written by more than 50 experts in the field of HCC in China(including liver surgeons,medical oncologists,hepatologists,interventional radiologists,and diagnostic radiologists)on the basis of recent evidence and expert opinions,balance of benefits and harms,cost-benefit strategies,and other clinical considerations.Key Messages:The guideline presents the Chinese staging system,and recommendations regarding patients with HCC in China to ensure optimum patient outcomes.

(Liver Cancer,2018,7:235-260)

123.Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma　(IF 17.943)

Cai J,Chen L,Zhang Z,Zhang X,Lu X,Liu W,Shi G,Ge Y,Gao P,Yang Y,Ke A,Xiao L,Dong R,Zhu Y,Yang X,Wang J,Zhu T,Yang D,Huang X,Sui C,Qiu S,Shen F,Sun H,Zhou W,Zhou J,Nie J,Zeng C,Stroup EK,Zhang X,Chiu BC,Lau WY,He C,Wang H,Zhang W,Fan J OBJECTIVE:The lack of highly sensitive and specific diagnostic biomarkers is a major contributor to the poor outcomes of patients with hepatocellular carcinoma(HCC).We sought to develop a noninvasive diagnostic approach using circulating cell-free DNA(cf DNA)for the early detection of HCC.DESIGN:Applying the 5hm C-Seal technique,we obtained genome-wide 5-hydroxymethylcytosines(5hmC)in cf DNA samples from 2 554 Chinese subjects:1 204 patients with HCC,392 patients with chronic hepatitis B virus infection(CHB)or liver cirrhosis(LC)and 958 healthy individuals and patients with benign liver lesions.A diagnostic model for early HCC was developed through case-control analyses using the elastic net regularisation for feature selection.RESULTS:The 5hmC-Seal data from patients with HCC showed a genome-wide distribution enriched with liver-derived enhancer marks.We developed a 32-gene diagnostic model that accurately distinguished early HCC(stage 0/A)based on the Barcelona Clinic Liver Cancer staging system from non-HCC(validation set:area under curve(AUC)=88.4%;(95%CI 85.8%to 91.1%)),showing superior performance over alpha-fetoprotein(AFP).Besides detecting patients with early stage or small tumours(eg,≤2.0 cm)from non-HCC,the 5hmC model showed high capacity for distinguishing early HCC from high risk subjects with CHB or LC history(validation set:AUC=84.6%;(95%CI 80.6%to 88.7%)),also significantly outperforming AFP.Furthermore,the 5hm C diagnostic model appeared to be independent from potential confounders(eg,smoking/alcohol intake history).CONCLUSION:We have developed and validated a noninvasive approach with clinical application potential for the early detection of HCC that are still surgically resectable in high risk individuals.

(Gut,2019,[Epub ahead of print])

124.The miR-561-5p/CX3CL1 Signaling Axis Regulates Pulmonary Metastasis in Hepatocellular Carcinoma Involving CX3CR1(+)Natural Killer Cells Infiltration　(IF 8.063)

Chen EB,Zhou ZJ,Xiao K,Zhu GQ,Yang Y,Wang B,Zhou SL,Chen Q,Yin D,Wang Z,Shi YH,Gao DM,Chen J,Zhao Y,Wu WZ,Fan J,Zhou J,Dai Z

Natural killer(NK)cell can inhibit tumor initiation and regulates metastatic dissemination,acting as key mediators of the innate immune response.Intrinsic factors modulating NK cells infiltration and its anticancer activity remain poorly characterized.We investigated the roles of dysregulation of micro(mi)RNAs and NK cells in progression of hepatocellular carcinoma(HCC).Methods:Small RNA sequencing were used to detect the miRNA profiles of tumor tissues from HCC patients with(n=14)or without(n=13)pulmonary metastasis and HCC cell lines with different pulmonary metastatic potentials.Chemokine expression profiling and bioinformatics were used to detect the downstream target of candidate target.In gain-and loss-of-function assays were used to investigate the role of miRNA in HCC progression.Different subsets of NK cells were isolated and used for chemotaxis and functional assays in vivo and in vitro.In situ hybridization and immunohistochemical analyses were performed to detect the expression of miRNA in tumor tissues from 242 HCC patients undergoing curative resection from 2010.Results:Three miRNAs(miR-137,miR-149-5p,and miR-561-5p)were identified to be associated with pulmonary metastasis in patients with HCC.miR-561-5p was most highly overexpressed in metastatic HCC tissues and high-metastatic-potential HCC cell lines.In gain-and loss-of-function assays in a murine model,miR-561-5p promoted tumor growth and spread to the lungs.Yet,miR-561-5p did not appear to affect cellular proliferation and migration in vitro.Bioinformatics and chemokine expression profiling identified chemokine(C-X3-C motif)ligand 1(CX3CL1)as a potential target of miR-561-5p.Furthermore,miR-561-5p promoted tumorigenesis and metastasis via CX3CL1-dependent regulation of CX3CR1(+)NK cell infiltration and function.CX3CR1(+)NK cells demonstrated stronger in vivo anti-metastatic activity relative to CX3CR1(-)NK cells.CX3CL1 stimulated chemotactic migration and cytotoxicity in CX3CR1(+)NK cells via STAT3 signaling.Blockade of CX3CL1,CX3CR1,or of pSTAT3 signaling pathways attenuated the antitumor responses.Clinical samples exhibited a negative correlation between miR-561-5p expression and levels of CX3CL1 and CX3CR1(+)NK cells.High miR-561-5p abundance,low CX3CL1 levels,and low numbers of CX3CR1(+)NK cells were associated with adverse prognosis.Conclusion:We delineated a miR-561-5p/CX3CL1/NK cell axis that drives HCC metastasis and demonstrated that CX3CR1(+)NK cells serve as potent antitumor therapeutic effectors.

(Theranostics,2019,9:4779-4794)

125.Activated and Exhausted MAIT Cells Foster Disease Progression and Indicate Poor Outcome in Hepatocellular Carcinoma　(IF 8.911)

Duan M,Goswami S,Shi JY,Wu LJ,Wang XY,Ma JQ,Zhang Z,Shi Y,Ma LJ,Zhang S,Xi RB,Cao Y,Zhou J,Fan J,Zhang XM,Gao Q

PURPOSE:Innate immunity is an indispensable arm of tumor immune surveillance,and the liver is an organ with a predominance of innate immunity,where mucosal-associated invariant T(MAIT)cells are enriched.However,little is known about the phenotype,functions,and immunomodulatory role of MAIT cells in hepatocellular carcinoma(HCC).Experimental Design:The distribution,phenotype,and function of MAIT cells in patients with HCC were evaluated by both flow cytometry(FCM)and in vitro bioassays.Transcriptomic analysis of MAIT cells was also performed.Prognostic significance of tumor-infiltrating MAIT cells was validated in four independent cohorts of patients with HCC.RESULTS:Despite their fewer densities in HCC tumor than normal liver,MAIT cells were significantly enriched in the HCC microenvironment compared with other mucosa-associated organs.Tumor-derived MAIT cells displayed a typical CCR7(-)CD45RA(-)CD45RO(+)CD95(+)effector memory phenotype with lower costimulatory and effector capabilities.Tumor-educated MAIT cells significantly upregulated inhibitory molecules like PD-1,CTLA-4,TIM-3,secreted significantly less IFNgamma and IL17,and produced minimal granzyme B and perforin while shifting to produce tumor-promoting cytokines like IL8.Transcriptome sequencing confirmed that tumor-derived MAIT cells were reprogrammed toward a tumor-promoting direction by downregulating genes enriched in pathways of cytokine secretion and cytolysis effector function like NFKB1 and STAT5B and by upregulating genes like IL8,CXCL12,and HAVCR2(TIM-3).High infiltration of MAIT cells in HCC significantly correlated with an unfavorable clinical outcome,revealed by FCM,q RT-PCR,and multiplex IHC analyses,respectively.CONCLUSIONS:HCC-infiltrating MAIT cells were functionally impaired and even reprogrammed to shift away from antitumor immunity and toward a tumor-promoting direction.See related commentary by Carbone,p.3199.

(Clin Cancer Res,2019,25:3304-3316)

126.Clinical Characteristics and Prognostic Factors of Patients with Intrahepatic Cholangiocarcinoma with Fever:A Propensity Score Matching Analysis　(IF 5.252)

Gong ZJ,Cheng JW,Gao PT,Huang A,Sun YF,Zhou KQ,Hu B,Qiu SJ,Zhou J,Fan J,Yang XR

BACKGROUND:Patients with intrahepatic cholangiocarcinoma(ICC)rarely present fever as the initial symptom.We aimed to identify clinical characteristics and prognostic factors for these feverish patients.SUBJECTS,MATERIALS,AND METHODS:This study retrospectively reviewed 31 patients with ICC with fever(≥38.0 degrees C)treated at our hospital between January 2002 and December 2014.A propensity score was used to match patients with and without fever at a ratio of 1∶2.RESULTS:Patients with ICC with fever had higher serum gamma-glutamyl transferase and carcinoembryonic antigen levels,larger tumors,poorer tumor differentiation,and worse prognosis(all P＜0.05)than those without fever.This was supported by propensity score matching(PSM)analysis.Univariate and multivariate analyses indicated that microvascular invasion,hilar lymph node metastasis,and temperature≥38.6 degrees C were related to prognosis.Patients with ICC with fever had higher levels of leucocytes,neutrophils,neutrophil-to-lymphocyte ratio(NLR),and platelet-to-lymphocyte ratio(PLR)in peripheral blood before and after PSM analysis.Body temperature positively correlated with leucocytes(r=0.599,P＜0.001),neutrophils(r=0.644,P＜0.001),NLR(r=0.681,P＜0.001),and PLR(r=0.457,P=0.010).CONCLUSION:Patients with ICC with fever≥38.0 degrees C and≥38.6 degrees C had poor and extremely poor prognosis,respectively.Radical surgical treatment may improve the prognosis of patients with ICC with fever＜38.6 degrees C.However,systemic therapy(e.g.,anti-inflammatory and immune therapy)may be preferable to surgery for these patients with fever≥38.6 degrees C.IMPLICATIONS FOR PRACTICE:Patients with intrahepatic cholangiocarcinoma(ICC)with fever(≥38.0 degrees C)as the initial symptom are extremely rare.Because their symptoms are similar to those of liver abscess,diagnosis is challenging,and most of these patients are already at an advanced stage at the time of diagnosis.Patients with ICC with fever had different clinical characteristics and worse prognosis than those without fever.The prognosis of those with temperature＜38.6 degrees C would be improved by timely surgical intervention.Those with fever≥38.6 degrees C had an extremely dismal outcome,although they all received radical surgical treatment.New therapeutic strategies are needed to improve survival for patients with ICC with temperature≥38.6 degrees C.

(Oncologist,2019,24:997-1007)

127.HHLA2 in intrahepatic cholangiocarcinoma:an immune checkpoint with prognostic significance and wider expression compared with PD-L1　(IF 8.676)

Jing CY,Fu YP,Yi Y,Zhang MX,Zheng SS,Huang JL,Gan W,Xu X,Lin JJ,Zhang J,Qiu SJ,Zhang BH

BACKGROUND:Intrahepatic cholangiocarcinoma(ICC)is a highly mortal malignancy with limited therapeutic options.Immunotherapies targeting PD-1/PD-L1 pathway represent a promising treatment for ICC.However,PD-L1 expression and microsatellite instability are not common in ICC.This study aimed to investigate whether H HLA2,a newly identified B7 family immune checkpoint for T cells,could be a therapeutic target next to PD-L1 in ICC.METHODS:Expression levels of PD-L1 and H HLA2 as well as infiltrations of CD3(+),CD8(+),CD4(+)Foxp3(+),CD68(+),CD163(+)and CD20(+)cells were evaluated by immunohistochemistry in 153 resected ICC samples.Comprehensive comparisons were made between PD-L1 and HHLA2 in terms of the expression rates,clinicopathological features and infiltrations of different immune cells.The expression level and prognostic significance of H HLA2 were further validated in an independent cohort.RESULTS:Expression of HHLA2 is more frequent than PD-L1 in ICC(49.0%vs.28.1%).Co-expression of both immune checkpoints was infrequent(13.1%)and 50%PD-L1 negative cases were with elevated H HLA2.HHLA2 overexpression was associated with sparser CD3(+)tumor infiltrating lymphocytes(TILs),CD8(+)TILs and a higher CD4(+)Foxp3(+)/CD8(+)TIL ratio,whereas PD-L1 expression was associated with prominent T cells and CD163+tumor associated macrophages infiltrations.PD-L1 failed to stratify overall survival(OS)but H HLA2 was identified as an independent prognostic indicator for OS in two independent cohorts.CONCLUSIONS:Compared with PD-L1,H HLA2 is more prevalent and possesses more explicit prognostic significance,which confer the rationale for HHLA2 as a potential immunotherapeutic target next to PD-L1 for ICC patients.

(J Immunother Cancer,2019,7:77)

128.CCL15 Recruits Suppressive Monocytes to Facilitate Immune Escape and Disease Progression in Hepatocellular Carcinoma　(IF 14.971)

Liu LZ,Zhang Z,Zheng BH,Shi Y,Duan M,Ma LJ,Wang ZC,Dong LQ,Dong PP,Shi JY,Zhang S,Ding ZB,Ke AW,Cao Y,Zhang XM,Xi R,Zhou J,Fan J,Wang XY,Gao Q

Chemokines play a key role in orchestrating the recruitment and positioning of myeloid cells within the tumor microenvironment.However,the tropism regulation and functions of these cells in hepatocellular carcinoma(HCC)are not completely understood.Herein,by scrutinizing the expression of all chemokines in HCC cell lines and tissues,we found that CCL15 was the most abundantly expressed chemokine in human HCC.Further analyses showed that CCL15 expression was regulated by genetic,epigenetic,and microenvironmental factors,and negatively correlated with patient clinical outcome.In addition to promoting tumor invasion in an autocrine manner,CCL15 specifically recruited CCR1(+)cells toward HCC invasive margin,approximately 80%of which were CD14(+)monocytes.Clinically,a high density of marginal CCR1(+)CD14(+)monocytes positively correlated with CCL15 expression and was an independent index for dismal survival.Functionally,these tumor-educated monocytes directly accelerated tumor invasion and metastasis through bursting various pro-tumor factors and activating signal transducer and activator of transcription 1/3,extracellular signal-regulated kinase 1/2,and v-akt murine thymoma viral oncogene homolog signaling in HCC cells.Meanwhile,tumor-derived CCR1(+)CD14(+)monocytes expressed significantly higher levels of programmed cell death-ligand 1,B7-H3,and Tcell immunoglobulin domain and mucin domain-3 that may lead to immune suppression.Transcriptome sequencing confirmed that tumor-infiltrating CCR1(+)CD14(+)monocytes were reprogrammed to upregulate immune checkpoints,immune tolerogenic metabolic enzymes(indoleamine and arginase),inflammatory/pro-angiogenic cytokines,matrix remodeling proteases,and inflammatory chemokines.Orthotopic animal models confirmed that CCL15-CCR1 axis forested an inflammatory microenvironment enriched with CCR1(+)monocytes and led to increased metastatic potential of HCC cells.Conclusion:A complex tumor-promoting inflammatory microenvironment was shaped by CCL15-CCR1 axis in human HCC.Blockade of CCL15-CCR1 axis in HCC could be an effective anticancer therapy.

(Hepatology,2019,69:143-159)

129.Tumor Size Affects Efficacy of Adjuvant Transarterial Chemoembolization in Patients with Hepatocellular Carcinoma and Microvascular Invasion　(IF 5.252)

Liu S,Li H,Guo L,Zhang B,Zhou B,Zhang W,Zhou J,Fan J,Ye QH

BACKGROUND:Patients with hepatocellular carcinoma(HCC)and microvascular invasion(m VI)have shown dismal postoperative prognosis;however,whether adjuvant transarterial chemoembolization(TACE)can improve their outcomes remains unclear.MATERIALS AND METHODS:We retrospectively identified 549 eligible patients to form the crude cohort and adopted propensity score matching method to assemble another cohort of 444 patients with similar baseline characteristics.We assessed the effects of adjuvant TACE by stratified analyses and multivariate Cox analyses in two cohorts.RESULTS:There was significant interaction between tumor size and adjuvant TACE with respect to overall survival(OS;P=0.006 for interaction).In the matched cohort,patients who received adjuvant TACE showed higher rates of 5-year OS(72.4%vs.50.9%,P=0.005)and 5-year recurrence-free survival(50.5%vs.36.4%,P=0.003)in the tumor≤5 cm subgroup,but not in the tumor＞5 cm subgroup(32.3%vs.24.9%,P=0.350 and 18.8%vs.19.7%,P=0.180).The independent protective role of adjuvant TACE on OS was observed in patients with tumor≤5 cm(adjusted odds ratio[OR]=0.59,95%confidence interval[CI]0.36-0.97)but not in patients with tumor＞5 cm(adjusted OR=1.17,95%CI 0.84-1.62).The effects of adjuvant TACE did not change materially while the analysis was performed in the crude cohort.CONCLUSION:For patients with HCC and m VI,adjuvant TACE was associated with improved outcomes,but not for those with tumor＞5 cm,according to the current protocol.IMPLICATIONS FOR PRACTICE:The outcomes of patients with hepatocellular carcinoma and microvascular invasion who received adjuvant transarterial chemoembolization were inconsistent in this study.According to the current protocol,adjuvant transarterial chemoembolization was associated with improved prognosis in patients with microvascular invasion,except for those with tumor＞5 cm.Multivariate Cox models confirmed adjuvant transarterial chemoembolization was an independent protective factor in the tumor≤5 cm subgroup but not in the tumor＞5 cm subgroup.

(Oncologist,2019,24:513-520)

130.Distinct PD-L1/PD1 Profiles and Clinical Implications in Intrahepatic Cholangiocarcinoma Patients with Different Risk Factors　(IF 8.063)

Lu JC,Zeng HY,Sun QM,Meng QN,Huang XY,Zhang PF,Yang X,Peng R,Gao C,Wei CY,Shen YH,Cai JB,Dong RZ,Shi YH,Sun HC,Shi YG,Zhou J,Fan J,Ke AW,Yang LX,Shi GM

Rationale:PD1/PD-L1 immune checkpoint inhibitors have shown promising results for several malignancies.However,PD1/PD-L1 signaling and its therapeutic significance remains largely unknown in intrahepatic cholangiocarcinoma(ICC)cases with complex etiology.Methods:We investigated the expression and clinical significance of CD3 and PD1/PD-L1 in 320 ICC patients with different risk factors.In addition,we retrospectively analyzed 7 advanced ICC patients who were treated with PD1 inhibitor.Results:The cohort comprised 233 patients with HBV infection,18 patients with hepatolithiasis,and 76 patients with undetermined risk factors.PD-L1 was mainly expressed in tumor cells,while CD3 and PD1 were expressed in infiltrating lymphocytes of tumor tissues.PD1/PD-L1 signals were activated in tumor tissues,and expression was positively correlated with HBV infection and lymph node invasion.More PD1(+)T cells and higher PD-L1 expression were observed in tumor tissues of ICC patients with HBV infection compared to patients with hepatolithiasis or undetermined risk factors.More PD1(+)T cells and/or high PD-L1 expression negatively impacted the prognosis of patients with HBV infection but not those with hepatolithiasis.Multivariate analysis showed PD1/PD-L1 expression was an independent indicator of ICC patient prognosis.Advanced ICC patients with HBV infection and less PD1(+)T cells tended to have good response to anti-PD1 therapy.Conclusion:Hyperactivated PD1/PD-L1 signals in tumor tissues are a negative prognostic marker for ICCs after resection.HBV infection-and hepatolithiasis-related ICCs have distinct PD1/PD-L1 profiles.Further,PD1(+)T cells could be used as a biomarker to predict prognosis and assay the efficiency of anti-PD1 immunotherapy in ICC patients with HBV infection.

(Theranostics,2019,9:4678-4687)

131.N(6-)Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation(IF 12.154)

Ma H,Wang X,Cai J,Dai Q,Natchiar SK,Lv R,Chen K,Lu Z,Chen H,Shi YG,Lan F,Fan J,Klaholz BP,Pan T,Shi Y,He C

N(6)-Methyladenosine(m(6)A)RNA modification is present in messenger RNAs(m RNA),ribosomal RNAs(r RNA),and spliceosomal RNAs(sn RNA)in humans.Although m RNA m(6)A modifications have been extensively studied and shown to play critical roles in many cellular processes,the identity of m(6)A methyltransferases for r RNAs and the function of r RNA m(6)A modifications are unknown.Here we report a new m(6)A methyltransferase,ZCCHC4,which primarily methylates human 28S r RNA and also interacts with a subset of m RNAs.ZCCHC4 knockout eliminates m(6)A4220 modification in 28S r RNA,reduces global translation,and inhibits cell proliferation.We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors,and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model.Our results highlight the functional significance of an r RNA m(6)A modification in translation and in tumor biology.

(Nat Chem Biol,2019,15:88-94)

132.CD73 promotes hepatocellular carcinoma progression and metastasis via activating PI3K/AKT signaling by inducing Rap1-mediated membrane localization of P110beta and predicts poor prognosis　(IF 8.731)

Ma XL,Shen MN,Hu B,Wang BL,Yang WJ,Lv LH,Wang H,Zhou Y,Jin AL,Sun YF,Zhang CY,Qiu SJ,Pan BS,Zhou J,Fan J,Yang XR,Guo W

BACKGROUND:Hepatocellular carcinoma(HCC)is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence.Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer.However,the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive.METHODS:CD73 expression in HCC cell was determined by RT-PCR,Western blot,and immunohistochemistry staining.Clinical significance of CD73 was evaluated by Cox regression analysis.Cell counting kit-8 and colony formation assays were used for proliferation evaluation.Transwell assays were used for motility evaluations.Co-immunoprecipitation,cytosolic and plasma membrane fractionation separation,and ELISA were applied for evaluating membrane localization of P110beta and its catalytic activity.NOD/SCID/gammac(null)(NOG)mice model was used to investigate the in vivo functions of CD73.RESULTS:In the present study,we demonstrate that CD73 was crucial for epithelialmesenchymal transition(EMT),progression and metastasis in HCC.CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics.Clinically,CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival.CD73 knockdown dramatically inhibits HCC cells proliferation,migration,invasion,and EMT in vitro and hinders tumor growth and metastasis in vivo.Opposite results could be observed when CD73 is overexpressed.Mechanistically,adenosine produced by CD73 binds to adenosine A2A receptor(A2AR)and activates Rap1,which recruits P110beta to the plasma membrane and triggers PIP3 production,thereby promoting AKT phosphorylation in HCC cells.Notably,a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone.CONCLUSIONS:CD73 promotes progression and metastasis through activating PI3K/AKT signaling,indicating a novel prognostic biomarker for HCC.Our data demonstrate the importance of CD73 in HCCin addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.

(J Hematol Oncol,2019,12:37)

133.Overexpression of RNF38 facilitates TGF-beta signaling by Ubiquitinating and degrading AHNAK in hepatocellular carcinoma　(IF 5.646)

Peng R,Zhang PF,Yang X,Wei CY,Huang XY,Cai JB,Lu JC,Gao C,Sun HX,Gao Q,Bai DS,Shi GM,Ke AW,Fan J

BACKGROUND:RING finger protein 38(RNF38),a member of the RNF protein family,has just emerged as a vital driver of cancer progression.However,the oncogenic mechanisms of RNF38 remain unexplored.METHODS:Using frozen tumor tissue and tissue microarray from hepatocellular carcinoma(HCC)patients,we tried to probe the expression of RNF38 in HCC and its clinical value.Then the biological functions of RNF38 were analyzed in vivo and vitro.Stable isotope labeling with amino acids(SILAC)in cell culture and co-immunoprecipitation proteomic analyses were combined to reveal the potential mechanism of RNF38 in HCC progression.RESULTS:We report that RNF38 expression was markedly higher in HCC tissues than in peritumor tissues.Correspondingly,RNF38 overexpression promoted the HCC cell migration and invasion and inhibited apoptosis both in vitro and in vivo.And elevated RNF38 expression induced HCC cell epithelial-mesenchymal transition by facilitating transforming growth factor-beta(TGFbeta)signaling via ubiquitinating and degrading neuroblast differentiation-associated protein(AHNAK),a well-established inhibitor of TGF-beta signaling.Furthermore,AHNAK interference restored the HCC cell invasion and metastasis deprived by RNF38 downregulation.Clinically,elevated RNF38 and transforming growth factor beta receptor 1(TGFBR1)expression was related to short overall survival(OS)and high cumulative recurrence rates in HCC patients.CONCLUSIONS:High levels of RNF38 promote HCC by facilitating TGF-beta signaling and are a novel marker for predicting the prognosis of HCC patients and a potential therapeutic target in HCC.

(J Exp Clin Cancer Res,2019,38:113)

134.Circular RNA circTRIM33-12 acts as the sponge of MicroRNA-191 to suppress hepatocellular carcinoma progression　(IF 10.679)

Zhang PF,Wei CY,Huang XY,Peng R,Yang X,Lu JC,Zhang C,Gao C,Cai JB,Gao PT,Gao DM,Shi GM,Ke AW,Fan J

BACKGROUND:Recently,the dysregulation of circular RNA(circ RNA)have been shown to have important regulatory roles in cancer development and progression,including hepatocellular carcinoma(HCC).However,the roles of most circRNAs in HCC are still unknown.METHODS:The expression of circular tripartite motif containing 33-12(circ TRIM33-12)in HCC tissues and cell lines was detected by q RT-PCR.The role of circ TRIM33-12 in HCC progression was assessed by western blotting,CCK-8,flow cytometry,transwell and a subcutaneous tumor mouse assays both in vitro and in vivo.In vivo circ RNA precipitation,RNA immunoprecipitation,luciferase reporter assays were performed to evaluate the interaction between circ TRIM33-12 and miR-191.RESULTS:Here,we found that circ TRIM33-12,is downregulated in HCC tissues and cell lines.The downregulation of circ TRIM33-12 in HCC was significantly correlated with malignant characteristics and served as an independent risk factor for the overall survival(OS)and recurrencefree survival(RFS)of patients with HCC after surgery.The reduced expression of circ TRIM33-12 in HCC cells increases tumor proliferation,migration,invasion and immune evasion.Mechanistically,we demonstrated that circ TRIM33-12 upregulated TET1 expression by sponging miR-191,resulting in significantly reduced 5-hydroxymethylcytosine(5hmC)levels in HCC cells.CONCLUSIONS:These results reveal the important role of circ TRIM33-12 in the proliferation,migration,invasion and immune evasion abilities of HCC cells and provide a new perspective on circRNAs in HCC progression.

(Mol Cancer,2019,18:105)

135.Landscape of infiltrating B cells and their clinical significance in human hepatocellular carcinoma(IF 5.333)

Zhang Z,Ma L,Goswami S,Ma J,Zheng B,Duan M,Liu L,Zhang L,Shi J,Dong L,Sun Y,Tian L,Gao Q,Zhang X

As a major cellular component in tumor microenvironment,the distribution,frequency,and prognostic significance of infiltrating B cell subsets in hepatocellular carcinoma(HCC)remain controversial.Using tyramide signal amplification(TSA)based fluorescent multiplexed immunohistochemistry in situ,we evaluated the distribution and frequency of B cell subsets in two independent HCC cohorts(n=619).The results were further confirmed by flow cytometry.Correlations of B cell subsets with clinicopathologic features and patient prognosis were analyzed.Five B cell subsets were defined by multiplexed immunohistochemistry and each subset was clearly separated by t-SNE dimension reduction analysis.Notably,the densities of all B cell subsets were significantly decreased in the tumor.The frequency of plasma cells within B cells was most abundant in the tumor.In training cohort(n=258),high densities of tumor-infiltrating CD20(+)B cells,naive B cells,Ig M(+)memory B cells,CD27(-)isotype-switched memory B cells,and plasma cells were associated with superior survival.Multivariate analysis further identified CD20(+)B cells,naive B cells,and CD27(-)isotype-switched memory B cells as independent prognosticators for survival.Unsupervised cluster analysis confirmed increased B cell subsets harbored superior survival.In addition,high density of B cells was correlated with smaller tumor size and well differentiation.The results were validated in the independent cohort of 361 HCC patients.Intratumor infiltration of B cells is significantly impaired during HCC progression.High densities of tumor-infiltrating B cells imply a better clinical outcome.Therapies designed to target B cells may be a novel strategy in HCC.

(Oncoimmunology,2019,8:e1571388)

136.A Positive Feedback Loop Between Cancer Stem-Like Cells and Tumor-Associated Neutrophils Controls Hepatocellular Carcinoma Progression　(IF 14.971)

Zhou SL,Yin D,Hu ZQ,Luo CB,Zhou ZJ,Xin HY,Yang XR,Shi YH,Wang Z,Huang XW,Cao Y,Fan J,Zhou J

Tumor-associated neutrophils(TANs)play a crucial role in tumor development and progression in the cancer microenvironment.Despite increased understanding of TAN contributions to hepatocellular carcinoma(HCC)progression and prognosis,the direct interaction between TANs and HCC cells is not fully understood.In this study,we tested the effect of TANs on HCC cells in vitro and in vivo and investigated the mechanism of interaction between them.Our results showed that TANs secreted bone morphogenetic protein 2 and transforming growth factor beta 2 and triggered micro RNA 301b-3p(miR-301-3p)expression in HCC cells,subsequently suppressed gene expression of limbic system-associated membrane protein(LSAMP)and CYLD lysine 63 deubiquitinase(CYLD),and increased stem cell characteristics in HCC cells.These TAN-induced HCC stem-like cells were hyperactive in nuclear factor kappa B signaling,secreted higher levels of chemokine(C-X-C motif)ligand 5(CXCL5),and recruited more TAN infiltration,suggesting a positive feedback loop.In clinical HCC samples,increased TANs correlated with elevated miR-301b-3p,decreased LSAMP and CYLD expression,and increased nuclear p65 accumulation and CXCL5 expression,all of which predicted patient outcome.Conclusion:Our work identified a positive feedback loop governing cancer stem-like cells and TANs in HCC that controls tumor progression and patient outcome.

(Hepatology,2019,[Epub ahead of print])

137.Genomic Sequencing Identifies WNK2 as a Driver in hepatocellular carcinoma and a Risk Factor for Early Recurrence　(IF 18.946)

Zhou SL,Zhou ZJ,Hu ZQ,Song CL,Luo YJ,Luo CB,Xin HY,Yang XR,Shi YH,Wang Z,Huang XW,Cao Y,Fan J,Zhou J

BACKGROUND&AIMS:Early recurrence of hepatocellular carcinoma(HCC)after curative resection is common.However,the association between genetic mechanisms and HCC early recurrence,especially in Chinese patients,remains largely unknown.METHODS:We performed whole-genome sequencing(49 cases),whole-exome sequencing(18 cases),and deep targeted sequencing(115 cases)on 182 primary HCC samples.Focusing on WNK2,we used Sanger sequencing and qPCR to evaluate all the coding exons and copy numbers of that gene in an additional 554 HCC samples.We also explored the functional effect and mechanism of WNK2 on tumor growth and metastasis.RESULTS:We identified five genes(WNK2,RUNX1T1,CTNNB1,TSC1,and TP53)harboring somatic mutations that correlated with early tumor recurrence after curative resection in 182 primary HCC samples.Focusing on WNK2,the overall somatic mutation and copy number loss occurred in 5.3%(39/736)and 27.2%(200/736),respectively,of the total 736 HCC samples.Both types of variation were associated with lower WNK2 protein levels,higher rates of early tumor recurrence,and shorter overall survival.Biofunctional investigations revealed a tumor-suppressor role of WNK2:its inactivation led to ERK1/2 signaling activation in HCC cells,tumor-associated macrophage infiltration,and tumor growth and metastasis.CONCLUSIONS:Our results delineate genomic events that characterize Chinese HCCs and identify WNK2 as a driver of early HCC recurrence after curative resection.LAY SUMMARY:We applied next-generation sequencing and conducted an in-depth genomic analysis of hepatocellular carcinomas(HCCs)from a Chinese patient cohort.The results delineate the genomic events that characterize Chinese HCCs and identify WNK2 as a driver associated with early tumor recurrence after curative resection.

(J Hepatol,2019,[Epub ahead of print])

138.Integrated Proteogenomic Characterization of HBV-Related Hepatocellular Carcinoma.(IF 36.2)

Gao Q,Zhu H,Dong L,Shi W,Chen R,Song Z,Huang C,Li J,Dong X,Zhou Y,Liu Q,Ma L,Wang X,Zhou J,Liu Y,Boja E,Robles AI,Ma W,Wang P,Li Y,Ding L,Wen B,Zhang B,Rodriguez H,Gao D,Zhou H,Fan J.

We performed the first proteogenomic characterization of hepatitis B virus(HBV)-related hepatocellular carcinoma(HCC)using paired tumor and adjacent liver tissues from 159 patients.Integrated proteogenomic analyses revealed consistency and discordance among multi-omics,activation status of key signaling pathways,and liver-specific metabolic reprogramming in HBVrelated HCC.Proteomic profiling identified three subgroups associated with clinical and molecular attributes including patient survival,tumor thrombus,genetic profile,and the liver-specific proteome.These proteomic subgroups have distinct features in metabolic reprogramming,microenvironment dysregulation,cell proliferation,and potential therapeutics.Two prognostic biomarkers,PYCR2 and ADH1A,related to proteomic subgrouping and involved in HCC metabolic reprogramming,were identified.CTNNB1 and TP53 mutation-associated signaling and metabolic profiles were revealed,among which mutated CTNNB1-associated ALDOA phosphorylation was validated to promote glycolysis and cell proliferation.Our study provides a valuable resource that significantly expands the knowledge of HBV-related HCC and may eventually benefit clinical practice.

[Cell,2019,179(2):561-577.e22.]