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Background Recently, the dysregulation of circular RNA (circRNA) 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 (circTRIM33–12) in HCC tissues and cell lines was detected by qRT-PCR. The role of circTRIM33–12 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 circRNA precipitation, RNA immunoprecipitation, luciferase reporter assays were performed to evaluate the interaction between circTRIM33–12 and miR-191. Results Here, we found that circTRIM33–12, is downregulated in HCC tissues and cell lines. The downregulation of circTRIM33–12 in HCC was significantly correlated with malignant characteristics and served as an independent risk factor for the overall survival (OS) and recurrence-free survival (RFS) of patients with HCC after surgery. The reduced expression of circTRIM33–12 in HCC cells increases tumor proliferation, migration, invasion and immune evasion. Mechanistically, we demonstrated that circTRIM33–12 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 circTRIM33–12 in the proliferation, migration, invasion and immune evasion abilities of HCC cells and provide a new perspective on circRNAs in HCC progression. Electronic supplementary material The online version of this article (10.1186/s12943-019-1031-1) contains supplementary material, which is available to authorized users.

Lymphoid‐specific helicase (LSH) is overexpressed in tumor tissues and its overexpression is associated with poor prognosis in several cancers. However, the role and molecular mechanism of LSH in hepatocellular carcinoma (HCC) remains largely unknown. Herein, we report that LSH was overexpressed in tumor tissues of HCC, and overexpression of LSH was associated with poor prognosis from a public HCC database, and validated by clinical samples from our department. Ectopic LSH expression promoted the growth of HCC cells in vivo and in vitro. Mechanistically, LSH overexpression promoted tumor growth by activating transcription of centromere protein F (CENPF). Clinically, overexpression of LSH and/or CENPF correlated with shorter overall survival and higher cumulative recurrence rates of HCC. In conclusion, LSH promotes tumor growth of HCC through transcriptional regulation of CENPF expression. Therefore, LSH may be a novel predictor for prognosis and a potential therapeutic target for HCC.

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-β (TGF-β) signaling via ubiquitinating and degrading neuroblast differentiation-associated protein (AHNAK), a well-established inhibitor of TGF-β 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-β signaling and are a novel marker for predicting the prognosis of HCC patients and a potential therapeutic target in HCC. Electronic supplementary material The online version of this article (10.1186/s13046-019-1113-3) contains supplementary material, which is available to authorized users.

Lysine acetylation (Kac) as an important posttranslational modification of histones is essential for the regulation of gene expression in hepatocellular carcinoma (HCC). However, the atlas of whole acetylated proteins in HCC tissues and the difference in protein acetylation between normal human tissues and HCC tissues are unknown. In this report, we characterized the proteome and acetyl proteome (acetylome) profile of normal, paracancerous, and HCC liver tissues in human clinical samples by quantitative proteomics techniques. We identified 6781 acetylation sites of 2582 proteins and quantified 2492 acetylation sites of 1190 proteins in normal, paracancerous, and HCC liver tissues. Among them, 15 proteins were multiacetylated with more than 10 lysine residues. The histone acetyltransferases p300 and CBP were found to be hyperacetylated in hepatitis B virus pathway. Moreover, we found that 250 Kac sites of 214 proteins were upregulated and 662 Kac sites of 451 proteins were downregulated in HCC compared with normal liver tissues. Additionally, the acetylation levels of lysine 120 in histone H2B (H2BK120ac), lysine 18 in histone H3.3 (H3.3K18ac), and lysine 77 in histone H4 (H4K77ac) were increased in HCC. Interestingly, the higher levels of H2BK120ac, H3.3K18ac, and H4K77ac were significantly associated with worse prognosis, such as poorer survival and higher recurrence in an independent clinical cohort of HCC patients. Overall, this study lays a foundation for understanding the functions of acetylation in HCC and provides potential prognostic factors for the diagnosis and therapy of HCC., Our analysis showed a landscape of lysine acetylation in HCC. And we clearly identified significantly altered acetylated sites in normal, paracancerous and HCC liver tissues and validated the clinical significance of three histone acetylation sites using an independent cohort.

Objective Natural killer (NK) cells play a critical role in the innate antitumor immune response. Recently, NK cell dysfunction has been verified in various malignant tumors, including hepatocellular carcinoma (HCC). However, the molecular biological mechanisms of NK cell dysfunction in human HCC are still obscure. Methods The expression of circular ubiquitin-like with PHD and ring finger domain 1 RNA (circUHRF1) in HCC tissues, exosomes, and cell lines was detected by qRT-PCR. Exosomes were isolated from the culture medium of HCC cells and plasma of HCC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit and then characterized by transmission electronic microscopy, NanoSight and western blotting. The role of circUHRF1 in NK cell dysfunction was assessed by ELISA. In vivo circRNA precipitation, RNA immunoprecipitation, and luciferase reporter assays were performed to explore the molecular mechanisms of circUHRF1 in NK cells. In a retrospective study, the clinical characteristics and prognostic significance of circUHRF1 were determined in HCC tissues. Results Here, we report that the expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues. Increased levels of circUHRF1 indicate poor clinical prognosis and NK cell dysfunction in patients with HCC. In HCC patient plasma, circUHRF1 is predominantly secreted by HCC cells in an exosomal manner, and circUHRF1 inhibits NK cell-derived IFN-γ and TNF-α secretion. A high level of plasma exosomal circUHRF1 is associated with a decreased NK cell proportion and decreased NK cell tumor infiltration. Moreover, circUHRF1 inhibits NK cell function by upregulating the expression of TIM-3 via degradation of miR-449c-5p. Finally, we show that circUHRF1 may drive resistance to anti-PD1 immunotherapy in HCC patients. Conclusions Exosomal circUHRF1 is predominantly secreted by HCC cells and contributes to immunosuppression by inducing NK cell dysfunction in HCC. CircUHRF1 may drive resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for patients with HCC.

Background Amplification of chromosome 7q21-7q31 is associated with tumor recurrence and multidrug resistance, and several genes in this region are powerful drivers of hepatocellular carcinoma (HCC). We aimed to investigate the key circular RNAs (circRNAs) in this region that regulate the initiation and development of HCC. Methods We used qRT-PCR to assess the expression of 43 putative circRNAs in this chromosomal region in human HCC and matched nontumor tissues. In addition, we used cultured HCC cells to modify circRNA expression and assessed the effects in several cell-based assays as well as gene expression analyses via RNA-seq. Modified cells were implanted into immunocompetent mice to assess the effects on tumor development. We performed additional experiments to determine the mechanism of action of these effects. Results circMET (hsa_circ_0082002) was overexpressed in HCC tumors, and circMET expression was associated with survival and recurrence in HCC patients. By modifying the expression of circMET in HCC cells in vitro, we found that circMET overexpression promoted HCC development by inducing an epithelial to mesenchymal transition and enhancing the immunosuppressive tumor microenvironment. Mechanistically, circMET induced this microenvironment through the miR-30-5p/Snail/ dipeptidyl peptidase 4(DPP4)/CXCL10 axis. In addition, the combination of the DPP4 inhibitor sitagliptin and anti-PD1 antibody improved antitumor immunity in immunocompetent mice. Clinically, HCC tissues from diabetic patients receiving sitagliptin showed higher CD8+ T cell infiltration than those from HCC patients with diabetes without sitagliptin treatment. Conclusions circMET is an onco-circRNA that induces HCC development and immune tolerance via the Snail/DPP4/CXCL10 axis. Furthermore, sitagliptin may enhance the efficacy of anti-PD1 therapy in a subgroup of patients with HCC.

The incidence of combined hepatocellular carcinoma-intrahepatic cholangiocarcinoma (cHCC-ICC) is relatively low, and the knowledge about the prognosis of cHCC-ICC remains obscure. In the study, we aimed to screen existing primary liver cancer staging systems and shed light on the prognosis and risk factors for cHCC-ICC.We retrospectively reviewed 206 cHCC-ICC patients who received curative surgical resection from April 1999 to March 2017. The correlation of survival measures with the histological types or with tumor staging systems was determined and predictive values of tumor staging systems with cHCC-ICC prognosis were compared.The histological type was not associated with overall survival (OS) (P = 0.338) or disease-free survival (DFS) (P = 0.843) of patients after curative surgical resection. BCLC, TNM for HCC, and TNM for ICC stages correlated with both OS and DFS in cHCC-ICC (all P 0.05). The predictive values of TNM for HCC and TNM for ICC stages were similar in terms of predicting postoperative OS (P = 0.798) and DFS (P = 0.191) in cHCC-ICC. TNM for HCC was superior to BCLC for predicting postoperative OS (P = 0.022) in cHCC-ICC.The TNM for HCC staging system should be prioritized for clinical applications in predicting cHCC-ICC prognosis.

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 β-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. Electronic supplementary material The online version of this article (10.1186/s13045-018-0573-9) contains supplementary material, which is available to authorized users.

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 mRNA 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 mRNA 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.

The ubiquitin-dependent proteasomal degradation of proteins controls signaling and cellular survival. In this study, we found that ubiquitin associated protein 2 (UBAP2) was significantly downregulated in hepatocellular carcinoma (HCC) tissues compared with adjacent normal tissues. Furthermore, higher expression of UBAP2 in cancer tissues was correlated with good prognosis in HCC patients. Knockdown of UBAP2 significantly enhanced the invasion and proliferation of HCC cells in vitro and promoted tumor growth in vivo, while enforced expression of UBAP2 impaired the aggressive ability and tumor growth of HCC cells. Mechanistically, UBAP2 formed a complex with Annexin A2 and promoted the degradation of Annexin A2 protein by ubiquitination, and then inhibited HCC progression. Collectively, UBAP2 appears as a novel marker for predicting prognosis and a therapeutic target for HCC.

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 p14ARF 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 p14ARF and promoting HCC progression. This represents a novel marker for predicting prognosis and a potential therapeutic target for HCC. (Hepatology 2015;61:1603-1614)

The loss of 5-hydroxymethylcytosine (5-hmC) has been identified as an epigenetic hallmark in several malignancies. However, its role in intrahepatic cholangiocarcinoma (ICC) is still unknown. Our study aims to investigate the level of 5-hmC in diagnosis and prognosis prediction of ICC. The 5-hmC levels were detected using dot blot, tissue microarray technique and immunohistochemical method, and the correlation between 5-hmC level and ICC clinicopathological parameters was analysed. Compared with matched liver tissues, most of ICC tissues presented with the loss of 5-hmC. Furthermore, the subgroups of cirrhotic and poor differentiation tissues showed the lowest level of 5-hmC. We found that 5-hmC level in non-elevated ICC patients was significantly related to lymph node metastasis and TNM stage and not related to vessel invasion, sex, age, HBV, cirrhosis or degree of differentiation. ICC patients with high TNM stage (stages III and IV) and lymph node metastases had significantly lower 5-hmC level than those with low TNM stage (stages I and II) and no lymph node metastases. Further analysis showed that low 5-hmC level is significantly correlated with worse overall survival (OS) and disease-free survival (DFS). Importantly, multivariate analysis indicated that 5-hmC level, tumour diameter, lymphatic metastasis and tumour differentiation could be used as independent prognostic factors for ICC. The loss of 5-hmC is implicated in the progression of ICC. Our results can contribute to the diagnostic ability and postoperative surveillance of ICC patients.

Background Microvascular invasion (MVI) is recognized as a prognostic factor associated with poor outcome in hepatocellular carcinoma (HCC) patients after curative resection. It remains unclear, however, whether MVI can provide prognostic information for patients at a specific tumor stage. Methods Consecutive HCC patients who underwent curative resection in years of 2007 and 2008 (discovery cohort) were enrolled in this retrospective study. Patients were stratified by the Barcelona Clinic Liver Cancer (BCLC) staging system. The prognostic significance of MVI for overall survival (OS) and recurrence-free survival (RFS) was studied in each subgroup. The clinical significance of MVI was validated in another cohort of patients underwent curative surgery in the year of 2006 (validation cohort). Results Of the 1540 patients in the discovery cohort, 389 (25.3%) patients had detectable MVI. Occurrence rates of MVI in the BCLC stage 0, A, and B subgroups were 12.4, 26.2, and 34.4%, respectively. In univariate analysis, MVI was associated with poor OS and RFS (P

The 14-3-3 proteins are highly conserved molecules that are involved in many vital biologic processes and are associated with the progression of cancer. The role of 14-3-3ζ, a dimeric isoform of 14-3-3, in intrahepatic cholangiocarcinoma (ICC) was investigated in this study. The expression of 14-3-3ζ in tumour samples from patients with ICC was examined by Western blot and immunohistochemistry, and the correlation between its expression and various clinicopathological features was determined. Then, the capacity for invasion, migration and proliferation as well as the expression of epithelial-mesenchymal transition (EMT)-related markers in ICC cells were assessed after 14-3-3ζ depletion. Finally, the prognostic significance of 14-3-3ζ in patients with ICC was further evaluated by Kaplan-Meier and Cox regression analyses. The expression of 14-3-3ζ was significantly higher in ICC tissues compared to peritumoural tissues. High expression of 14-3-3ζ positively correlated with lymphatic metastasis and tumour-node-metastasis (TNM) stage. The inhibition of 14-3-3ζ expression was able to impair the invasion, migration and proliferation of ICC cells in vitro. The expression of 14-3-3ζ was significantly correlated with the expression of the EMT-related markers snail and E-cadherin in ICC samples. Moreover, the down-regulation of 14-3-3ζ also decreased the phosphorylation of extracellular signal-regulated kinase (ERK) in ICC cells. Clinically, patients with ICC with high 14-3-3ζ expression demonstrated a poor prognosis in terms of a short overall survival and a high recurrence rate of the disease. A multivariate analysis revealed that 14-3-3ζ overexpression was an independent prognostic indicator for patients with ICC. 14-3-3ζ may enhance the invasive and proliferative capacity of tumour cells and thus prompt the progression of ICC via the activation of ERK signalling and the induction of EMT. The overexpression of 14-3-3ζ may be used as a prognostic biomarker and therapeutic target in patients with ICC.

Studies have highlighted important features of the nucleocytoplasmic transport of mRNAs and proteins. Nuclear RNA export factor 3 (NXF3) is a member of the nuclear RNA export factor family that plays a role in mediating the export of cellular mRNA from the nucleus to the cytoplasm for translation. However, little is known about the clinical significance of NXF3 in human tumors. To evaluate the prognostic significance of NXF3 in hepatocellular carcinoma (HCC), the expression levels of NXF3 in a cohort of 112 patients with primary HCC who had undergone hepatectomy for histologically confirmed HCC were assessed by immunohistochemistry. It was identified that the expression levels of NXF3 were higher in the primary HCC tissues compared with those in paired peritumoral liver tissues. The overexpression of NXF3 in the HCC tissues was correlated with decreased survival time [hazard ratio (HR) = 1.954, 95% confidence interval (CI) = 1.034–3.695, P=0.039] and earlier tumor recurrence (HR = 2.101, 95% CI = 1.186–3.722, P=0.011) in postoperative patients with HCC. Notably, overexpression of NXF3 was correlated with a poor survival time and increased recurrence following HCC resection in male patients (P=0.020 and P=0.007, respectively) but not in female patients (P=0.916 and P=0.821, respectively). In conclusion, the findings provide evidence that implicates NXF3 as a prospective predictor of HCC prognosis as well as a potential therapeutic target for cancer treatment.

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 IFN-γ, 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; 19(21); 5994–6005. ©2013 AACR.

Aims The prognosis of patients with intrahepatic cholangiocarcinoma (ICC) remains poor in terms of overall survival (OS) and recurrence rate. Mortalin, a stress chaperone, has been reported to be involved in carcinogenesis and metastasis. However, its role in ICC has not been defined. Methods Mortalin expression in tumour samples from patients with ICC was examined by Western blot and immunohistochemistry, and correlation between its expression and clinicopathological features was assessed. In addition, invasion, migration proliferation and apoptosis, and the expression of epithelial-mesenchymal transition (EMT)-related markers in ICC cells were assessed after mortalin depletion. Finally, the prognostic significance of mortalin in patients with ICC was further evaluated by Kaplan–Meier and Cox regression analysis. Results We provide evidence that expression of mortalin in human ICC tissues is higher than that in matched peritumoural tissues. The interference of mortalin expression inhibited the proliferation and invasion of ICC cells in vitro. Mechanistically, inhibition of mortalin expression in ICC cells upregulated E-cadherin expression and decreased vimentin and snail expression. Clinically, a high level of mortalin in ICC samples was associated with loss of E-cadherin, and increased expression of vimentin and snail. Patients with ICC and high mortalin expression had a shorter OS and a higher recurrence rate. Multivariate analysis revealed that mortalin overexpression was an independent prognostic indicator for patients with ICC. Conclusions Mortalin may promote cell proliferation and invasion via induction of EMT of ICC cells. A high level of mortalin may be used as a prognostic biomarker and therapeutic target for patients with ICC.

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; 72(14); 3546–56. ©2012 AACR.

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 (mRNA) and protein. Then, the expression of CD151 in an ICC cell line was interrupted using a specific lentiviral-mediated small hairpin RNA (shRNA)-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

To investigate the prognostic values of putative hepatic stem/progenitor cell (HSC/HPC) biomarkers in patients with hepatocellular carcinoma (HCC).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).Most of the biomarkers were found to be overexpressed in patients with recurrent HCC by quantitative reverse transcription-PCR (qRT-PCR). 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 platelet-derived 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 (p0.0001). Based on Cox regression, a simplified model including CD133, CD44, Nestin and MVD was constructed and confirmed as an independent predictor for OS (p0.0001) and RFS (p0.0001), regardless of alpha-fetoprotein level, tumour stage and recurrence time (p0.0001 for all).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.

Purpose: Cytokeratin 10 (CK10) was found to be expressed differently in human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials in our previous research. The aim of this study was to assess the value of CK10 alone or in combination with cytokeratin 19 (CK19) in predicting tumor recurrence after curative resection in HCC patients. Experimental Design: CK10 expression in stepwise metastatic HCC cell lines and tumor tissues from 50 HCC patients was investigated using immunofluorescence assay, quantitative real-time reverse transcription-PCR, and Western blot analyses. Tumor tissue microarrays of 300 HCC patients who underwent curative resection between 1997 and 2000 were used to detect the expressions of CK10 and CK19. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Results: CK10 was overexpressed in the high metastatic HCC cell line and in tumor tissues of recurrent patients. Both univariate and multivariate analyses revealed that CK10 was a significant predictor for overall survival (OS) and disease-free survival, and that CK19 was a significant predictor for OS. CK10 expression was correlated with poor prognosis regardless of α-fetoprotein, tumor-node-metastasis stage, and vascular invasion. The 7-year OS and disease-free survival rates in CK10+ and/or CK19+ patients were 30.0% and 37.6%, respectively, which were significantly lower than that of CK10−/CK19− patients (56.1% and 60.0%, respectively; P < 0.001). Conclusion: CK10 is associated with HCC invasiveness. CK10 alone, or in combination with CK19, can be a novel predictor for poor prognosis of HCC patients after curative resection.

Background The aim of this study is to investigate the expression profile of multiple epithelial mesenchymal transition (EMT)-related molecules in intrahepatic cholangiocarcinoma (ICC) and the related prognostic significance. Methods Immunohistochemistry was performed to determine the expression of E-cadherin, Vimentin, Snail, slug and β-catenin in a tissue microarray consisting of tumor tissues of 140 ICC patients undergoing curative resection. The correlation between the expression of these molecules and the clinicopathological characteristics of ICC patients was analyzed, and their prognostic implication was evaluated. Results Reduced E-cadherin and increased Vimentin expression, the characteristic changes of EMT, identified in 55.0% and 55.7% of primary ICCs, respectively, were correlated with lymphatic metastasis and poorer overall survival (OS) and disease-free survival (DFS) of ICCs. The overexpression of snail and nonmembranous β-catenin, which are the major regulators of the EMT, were identified in 49.2% and 45.7% of primary ICCs, while little slug expression was detected in ICCs. Cytoplasmic/nuclear β-catenin did not significantly predict worse DFS and was not related with E-cadherin loss. The overexpression of snail predicted worse OS and DFS. Snail overexpression correlated with the down-regulation of E-cadherin and the up-regulation of Vimentin. Inhibition of snail in an ICC cell line decreased the expression of E-cadherin, enhanced the expression of Vimentin and impaired the invasion and migration ability of ICC cells. Conclusions These data support the hypothesis that EMT plays vital roles in ICC progression and suggest that snail but not slug and β-catenin plays a crucial role in the EMT induction of ICC.

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 epithelial-mesenchymal 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)

BACKGROUND: Calpain small subunit 1 (Capn4) has been shown to correlate with the metastasis/invasion of hepatocellular carcinoma. This study aimed to investigate the role of Capn4 in intrahepatic cholangiocarcinoma (ICC). METHODS: Capn4 expression was measured in 33 ICC tissues by quantitative real-time polymerase chain reaction and western blot. The role of Capn4 in the migration, invasion and proliferation of ICC cells and matrix metalloproteinase 2 (MMP2) expression were assessed after Capn4 depletion by specific small interfering RNA. Capn4 expression was further examined by immunohistochemistry in a tissue microarray consisting of 140 ICC patients and 13 normal liver tissues, and the prognostic role of Capn4 in ICC was evaluated by Kaplan-Meier and Cox regression analyses. RESULTS: Capn4 expression was significantly higher in the ICC tissues compared to the peritumor tissues. Capn4 down-regulation impaired the migration/invasion ability of HCCC-9810 and QBC939 cells in vitro and decreased MMP2 expression. Capn4 overexpression significantly correlated with the presence of lymphatic metastasis of ICC (p = 0.026) and the tumor-node-metastasis (TNM) stage (p = 0.009). The postoperative 2- and 5-year overall survivals in patients with Capn4(low) were higher than those in the Capn4(high) group. The cumulative recurrence rate in patients with Capn4(low) was much lower than in the Capn4(high) group. Multivariate analysis showed that Capn4 overexpression was an independent prognostic marker in ICC. CONCLUSIONS: Capn4 overexpression was implicated in ICC metastasis/invasion, and Capn4 overexpression may be used as a molecular therapeutic target for ICC.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC). METHODS: LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens. RESULTS: Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set. CONCLUSIONS: LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.

Inflammatory reactions at a tumor site have both detrimental and beneficial effects on tumor progression. This study was designed to assess the clinical significance of tumor-infiltrating inflammatory cells in patients with intrahepatic cholangiocarcinoma (ICC).A total of 123 consecutive ICC patients who underwent curative resection were enrolled. Tissue microarray and immunohistochemistry were used to analyze the distribution and clinical relevance of IL-17(+), FOXP3(+), CD8(+), CD66b(+) cells, and microvessel density (CD34) in different microanatomical areas.IL-17(+) cells, FOXP3(+) lymphocytes, CD66b(+) neutrophils, and microvessels were enriched predominantly in intratumor (IT) area, whereas CD8(+) lymphocytes were most abundant in tumor invasive front. On univariate analyses, increasing IL-17 (IT) (+) and neutrophils(IT) were significantly associated with worse patient survival. Multivariate analyses revealed that IL-17 (IT) (+) (hazard ratio [HR] = 1.59; 95% confidence interval [CI], 1.05-2.41; P = 0.028), neutrophils(IT) (HR = 1.76; 95% CI, 1.16-2.65; P = 0.007), and their combination (HR 2.8; 95% CI 1.72-4.57; P0.001) were independent prognostic factors, which were superior to conventional clinicopathologic features, such as intrahepatic metastasis and TNM stage. IL-17 (IT) (+) significantly correlated with the presence of lymph node metastasis, intrahepatic metastasis, and advanced stages, whereas neutrophils(IT) correlated with the presence of vascular invasion. In addition, significant positive correlations were detected among densities of IL-17(+) cells, neutrophils, and microvessel density.Our data suggested that intratumor IL-17(+) cells, neutrophils are novel, powerful predictors of prognosis in patients with ICC.

Tetraspanin CD151 has been implicated in metastasis through forming complexes with different molecular partners. In this study, we mapped tetraspanin web proteins centered on CD151, in order to explore the role of CD151 complexes in the progression of hepatocellular carcinoma (HCC). Immunoprecipitation was used to isolate tetraspanin complexes from HCCLM3 cells using a CD151 antibody, and associated proteins were identified by mass spectrometry. The interaction of CD151 and its molecular partners, and their roles in invasiveness and metastasis of HCC cells were assayed through disruption of the CD151 network. Finally, the clinical implication of CD151 complexes in HCC patients was also examined. In this study, we identified 58 proteins, characterized the tetraspanin CD151 web, and chose integrin β1 as a main partner to further investigate. When the CD151/integrin β1 complex in HCC cells was disrupted, migration, invasiveness, secretion of matrix metalloproteinase 9, and metastasis were markedly influenced. However, both CD151 and integrin β1 expression were untouched. HCC patients with high expression of CD151/integrin β1 complex had the poorest prognosis of the whole cohort of patients. Together, our data show that CD151 acts as an important player in the progression of HCC in an integrin β1-dependent manner.

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

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 Western-blot, immunofluorescence, and real-time polymerase chain reaction (PCR) analyses. The role of LI-cadherin 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 mRNA 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. © 2009 American Cancer Society.

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 CD151high 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 CD151high were significantly lower than those in the CD151low group, and correspondingly cumulative recurrence rates in HCCs with CD151high were significantly higher than those in the CD151low 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 CD151high/c-Methigh patients were 50.5% and 37.8%, respectively, significantly lower than those of CD151low/c-Metlow patients (63.9% and 54.6%, respectively). Five- and 7-year cumulative recurrence rates in CD151high/c-Methigh patients were 53.3% and 71.9%, respectively, markedly higher than those of CD151low/c-Metlow 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 2008.)

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The role of autophagy and the prognostic value of autophagic genes are largely unknown in HCC. Here, we showed decreased expression of autophagic genes and their corresponding autophagic activity and increased expression of the antiapoptotic gene Bcl-xL in HCC cell lines compared with a normal hepatic cell line. We also found decreased expression of the autophagic gene Beclin 1 in 44 HCC tissue samples compared with adjacent nontumor tissues. In addition, we found that the most aggressive malignant HCC cell lines and HCC tissues with recurrent disease displayed much lower autophagic levels, especially when Bcl-xL was overexpressed. Interestingly, in a tissue microarray study consisting of 300 HCC patients who underwent curative resection, the expression of Beclin 1 was only significantly correlated with disease-free survival (DFS; P < 0.0001) and overall survival (OS; P < 0.0001) in the Bcl-xL+ group. Multivariate and univariate analyses also revealed that Beclin 1 expression was an independent predictor for DFS and OS in Bcl-xL+ patients. In addition, we found a significant correlation between Beclin 1 expression and tumor differentiation in Bcl-xL+ but not in Bcl-xL− HCC patients. In conclusion, our data showed expression of autophagic genes and their corresponding autophagic activities were suppressed in HCC. The autophagy defects synergized with altered apoptotic activity might facilitate tumor malignant differentiation, which results in a more aggressive cancer cell phenotype and poor prognosis of HCC. [Cancer Res 2008;68(22):9167–75]

Background. Osteopontin (OPN) plays important roles in tumor progression and metastasis through binding to CD44 and integrin. The goal of this study was to elucidate the prognostic significance of OPN and CD44 in hepatocellular carcinoma patients. Methods. Tumor tissue microarray was used to detect the expression levels of OPN and CD44 in 302 hepatocellular carcinoma patients undergoing curative resection between 1997 and 2000 at our institute. Clinicopathologic data for these patients were investigated. The prognostic effects of OPN and CD44 were evaluated using the Kaplan–Meier method and compared using the log-rank test. The Spearman rank test and Fisher's exact test were applied to demonstrate correlations. Results. Both OPN and CD44 were independent predictors for overall survival and disease-free survival. When OPN and CD44 were taken into consideration together, the predictive range was extended and the sensitivity was improved, especially for those patients with normal serum α-fetoprotein levels. The 8-year overall survival and disease-free survival rates in OPN+ and/or CD44+ patients were 28.2% and 25.6%, respectively, which were significantly lower than those of OPN−CD44− patients (52.1% and 51.6%, respectively). Conclusions. OPN combined with CD44 is a promising independent predictor of tumor recurrence and survival in hepatocellular carcinoma patients.

To identify the side population (SP) cells from four hepatocellular carcinoma (HCC) cell lines with stepwise metastatic potentials.SP cells were sorted from HCCLM3, MHCC97-H, MHCC97-L and Hep3B by flow cytometry, and then analyzed by differentiation study, clonogenic assay, chemoresistance study and tumorigenicity assay in vivo. The expression of ABCG(2) in SP cells was detected by immunocytochemistry, western blotting and real-time quantitative PCR, respectively.There was significant difference in SP proportion among HCCLM3, MHCC97-H, MHCC97-L and Hep3B (28.7 +/- 1.6%, 14.5 +/- 0.6%, 4.2 +/- 0.4%, 0.9 +/- 0.1%, respectively, P0.01). All the SP cells showed similar characteristics of self-renewal, high clonogenicity, remarkable chemo-resistance and high expression of ABCG(2). As low as 2,000 SP cells could initiate tumors in non-obese diabetic/severe combined immunodeficiency mice successfully.SP cells purified from HCC cell lines harbors cancer stem cell-like properties, and may be related to the metastatic potentials and therapeutic-resistance of HCC.

Background Recurrence prediction of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) present a great challenge because of a lack of biomarkers. Genetic variations play an important role in tumor development and metastasis. Methods Oligonucleotide microarrays were used to evaluate the genetic characteristics of tumor DNA in 30 HBV-related HCC patients who were underwent LT. Recurrence-related single-nucleotide polymorphism were selected, and their prognostic value was assessed and validated in two independent cohorts of HCC patients (N = 102 and N = 77), using pretransplant plasma circulating DNA. Prognostic significance was assessed by Kaplan-Meier survival estimates and log-rank tests. Multivariate analyses were performed to evaluate prognosis-related factors. Results rs894151 and rs12438080 were significantly associated with recurrence (P = .003 and P = .004, respectively). Multivariate analyses demonstrated that the co-index of the 2 SNPs was an independent prognostic factor for recurrence (P = .040). Similar results were obtained in the third cohort (N = 77). Furthermore, for HCC patients (all the 3 cohorts) exceeding Milan criteria, the co-index was a prognostic factor for recurrence and survival (P