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Purpose: The Hippo pathway is a tumor suppressor in the liver. However, the clinical significance of Hippo pathway inactivation in HCC is not clearly defined. We analyzed genomic data from human and mouse tissues to determine clinical relevance of Hippo pathway inactivation in HCC.Experimental Design: We analyzed gene expression data from Mst1/2−/− and Sav1−/− mice and identified a 610-gene expression signature reflecting Hippo pathway inactivation in the liver [silence of Hippo (SOH) signature]. By integrating gene expression data from mouse models with those from human HCC tissues, we developed a prediction model that could identify HCC patients with an inactivated Hippo pathway and used it to test its significance in HCC patients, via univariate and multivariate Cox analyses.Results: HCC patients (National Cancer Institute cohort, n = 113) with the SOH signature had a significantly poorer prognosis than those without the SOH signature [P < 0.001 for overall survival (OS)]. The significant association of the signature with poor prognosis was further validated in the Korean (n = 100, P = 0.006 for OS) and Fudan University cohorts (n = 242, P = 0.001 for OS). On multivariate analysis, the signature was an independent predictor of recurrence-free survival (HR, 1.6; 95% confidence interval, 1.12–2.28: P = 0.008). We also demonstrated significant concordance between the SOH HCC subtype and the hepatic stem cell HCC subtype that had been identified in a previous study (P < 0.001).Conclusions: Inactivation of the Hippo pathway in HCC is significantly associated with poor prognosis. Clin Cancer Res; 22(5); 1256–64. ©2015 AACR.

Purpose: Intrahepatic cholangiocarcinoma (ICC), a highly malignant hepatobiliary cancer, has a poor prognosis and is refractory to conventional therapies. The aim of this study is to discover a novel molecular target for the treatment of ICC.Experimental Design: To discover novel cancer-associated membrane antigens expressed in ICC cells, we generated monoclonal antibodies (mAb) by immunizing mice with intact ICC cell lines and screened for those that bind to the plasma membrane of ICC cells but not to normal cells. The mAb A10-A3 was selected and its target antigen was identified as the L1 cell adhesion molecule. Expression of L1 in ICC was evaluated by immunohistochemical analysis of tumor samples from 42 ICC patients. The functional significance of L1 expression in the tumor progression of ICC was investigated by L1 suppression, L1 overexpression, and antibody treatment.Results: L1 was not expressed in normal hepatocytes and intrahepatic bile duct epithelium but highly expressed in 40.5% of ICC patients, remarkably at the invasive front of the tumors. Suppression of L1 with short hairpin RNA significantly decreased proliferation, migration, and invasion of ICC cells in vitro. Consistently, L1 overexpression in ICC cells enhanced proliferation, migration, invasion, and apoptosis resistance. In addition, L1 short hairpin RNA or anti-L1 mAb significantly reduced the tumor growth in nude mice bearing ICC xenograft.Conclusions: We identified that L1 is expressed in ICC. L1 plays an important role in the tumor progression of ICC by enhancing cell proliferation, migration, invasion, and survival. L1 may represent a novel therapeutic target for ICC. Clin Cancer Res; 16(14); 3571–80. ©2010 AACR.

Figure S1: Significant concordance between silence of Hippo signature and YAP1 expression in human hepatocellular carcinoma (HCC); Figure S2: Venn diagram of gene lists from two different gene expression signatures; Table S1: Genes in the SOH signature; Table S2: Functional categories of genes in the SOH signature; Table S3: Canonical Pathways enriched in the SOH signature; Table S4: Significant association between the SOH subtype and YAP1 protein expression in HCC; Table S5: Univariate and multivariate Cox regression analyses of recurrence-free survival with continuous SOH probability; Table S6: Univariate and multivariate Cox regression analyses of overall survival; Table S7: Univariate and multivariate Cox regression analyses of overall survival with continuous SOH probability; Table S8: Drop in concordance index in multivariable analysis; Table S9: Concordance of silence of Hippo signature with other prognostic HCC gene expression signatures in the NCI cohort; Table S10: Characteristics of HCC patients in the MSH cohort, stratified by SOH signature.

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Despite the epidemiological association between intrahepatic cholangiocarcinoma (iCCA) and HBV infection, little is known about the relevant oncogenic effects. We sought to identify the landscape and mechanism of HBV integration, along with the genomic architecture of HBV-infected iCCA (HBV-iCCA) tumors.We profiled a cohort of 108 HBV-iCCAs using whole-genome sequencing, deep sequencing, and RNA sequencing, together with preconstructed data sets of HBV-infected HCC (HBV-HCC; n = 167) and combined hepatocellular cholangiocarcinoma (HBV-cHCC/CCA; n = 59), and conventional (n = 154) and fluke-related iCCAs (n = 16). Platforms based on primary iCCA cell lines to evaluate the functional effects of chimeric transcripts were also used. We found that HBV had inserted at multiple sites in the iCCA genomes in 45 (41.7%) of the tumors. Recurrent viral integration breakpoints were found at nine different sites. The most common insertional hotspot (7 tumors) was in the TERT (telomerase reverse transcriptase) promoter, where insertions and mutations (11 tumors) were mutually exclusive, and were accompanied by promoter hyperactivity. Recurrent HBV integration events (5 tumors) were also detected in FAT2 (FAT atypical cadherin 2), and were associated with enrichment of epithelial-mesenchymal transition-related genes. A distinctive intergenic insertion (chr9p21.3), between DMRTA1 (DMRT like family A1) and LINC01239 (long intergenic non-protein coding RNA 1239), had oncogenic effects through activation of the mammalian target of rapamycin (mTOR)/4EBP/S6K pathway. Regarding the mutational profiles of primary liver cancers, the overall landscape of HBV-iCCA was closer to that of nonviral conventional iCCA, than to HBV-HCC and HBV-cHCC/CCA.Our findings provide insight into the behavior of iCCAs driven by various pathogenic mechanisms involving HBV integration events and associated genomic aberrations. This knowledge should be of use in managing HBV carriers.

Hepatitis B virus (HBV) X protein (HBx) is associated with hepatocarcinogenesis. E2-EPF ubiquitin carrier protein (UCP) catalyzes ubiquitination of itself and von Hippel–Lindau protein (pVHL) for degradation and associates with tumor growth and metastasis. However, it remains unknown whether HBx modulates the enzyme activity of UCP and thereby influences hepatocarcinogenesis. Here, we show that UCP is highly expressed in liver tissues of HBx-transgenic mice, but not non-transgenic mice. UCP was more frequently expressed in HBV-positive liver cancers than in HBV-negative liver cancers. HBx binds to UCP specifically and serotype independently, and forms a ternary complex with UCP and pVHL. HBx inhibits self-ubiquitination of UCP, but enhances UCP-mediated pVHL ubiquitination, resulting in stabilization of hypoxia-inducible factor-1α and -2α. HBx and UCP stabilize each other by mutually inhibiting their ubiquitination. HBx promotes cellular proliferation and metastasis via UCP. Our findings suggest that UCP plays a key role in HBV-related hepatocarcinogenesis. Electronic supplementary material The online version of this article (10.1007/s00018-019-03066-9) contains supplementary material, which is available to authorized users.

Altered expression of suppressor of cytokine signaling (SOCS) is found in various tumors. However, regulation of SOCS2 by upstream molecules has yet to be clearly elucidated, particularly in tumor cells. SCOCS2 expression was examined in tumor cells transfected with an inducible p53 expression system. The impact of SOCS2 on cell proliferation was measured with in vitro assays. Inhibition of tumorigenicity by SOCS2 knockdown was assessed via a mouse model. Expression profiles were compared and genes differentially expressed were identified using four types of p53-null cells (Saos, HLK3, PC3, and H1299) and the same cells stably expressing p53. Twelve kinds of target genes were simultaneously upregulated or downregulated by p53 in three or more sets of p53-null cells. SOCS2 expression was reciprocally inhibited by inducible p53 expression in p53-null cells, even colon cancer cells. SOCS2 promoter activity was inhibited by wild type but not mutant p53. SOCS2 knockdown inhibited tumor growth in vitro and in an animal xenograph model. SOCS2 overexpression was detected in a murine model of azoxymethane/dextran sulfate sodium-induced colitis-associated colon cancer compared to mock-treated controls. SOCS2 expression was heterogeneously upregulated in some human colon cancers. Thus, SOCS2 was upregulated by p53 dysfunction and seemed to be associated with the tumorigenic potential of colon cancer., Colon cancer: A delicate balance to regulate growth Insights into a signaling protein’s role in cell growth could inform new therapeutic strategies for treating colon cancer. SOCS-2 acts as an ‘off switch’ for cell signaling pathways. It has been identified as possibly protective against many cancers, although some cancers are associated with elevated SOCS-2 levels. Researchers led by Daeghon Kim at Chonbuk National University Hospital in South Korea have now shown that the effects of SOCS-2 are apparently dependent on how much of it is present. Moderate levels of SOCS-2 can suppress growth in colon cancer cells, but Kim’s team showed that excessive SOCS-2 has the opposite effect, promoting proliferation. The researchers also identified a gene commonly mutated in cancer cells that can drive overproduction of SOCS-2. Drugs that inhibit SOCS-2 or block its production may therefore offer useful treatments for colorectal cancer.

Background/Aims Long-term data on antiviral therapy in Korean patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) are limited. This study evaluated the efficacy and safety of entecavir (ETV) and lamivudine (LAM) over 240 weeks. Methods Treatment-naive patients with HBeAg-negative CHB were randomized to receive ETV 0.5 mg/day or LAM 100 mg/day during the 96 week double-blind phase, followed by open-label treatment through week 240. The primary endpoint was the proportion of patients with virologic response (VR; hepatitis B virus [HBV] DNA16 years old) were included (ETV, n=56; LAM, n=64). Baseline characteristics were comparable between the two groups. A significantly higher proportion of ETV-treated patients achieved VR compared to LAM at week 24 (92.9% vs. 67.2%, P=0.0006), week 96 (94.6% vs. 48.4%, P

CTHRC1 expression is involved in invasion and metastasis in various tumors. However, the molecules involved in its signaling pathways in hepatocellular carcinoma (HCC) remain elusive. The migration and invasion abilities of HCC cells stably expressing CTHRC1 were assessed in vitro and in vivo with a mouse model. Moreover, signaling pathways involved in invasion and metastasis were analyzed. CTHRC1 was abundantly expressed in HCC cell lines and HCC tissues. CTHRC1 was also detectable in the serum of HCC patients, compared with non-tumor controls. CTHRC1 mRNA was positively correlated with large tumor size (p

AIM To assess the effect of long-term oral nucleos(t)ide analogues (NUCs) therapy on liver volume change in patients with suppress hepatitis B virus (HBV)-related liver cirrhosis. METHODS We reviewed the data of naïve patients with HBV-related liver cirrhosis, who had taken oral NUCs therapy, between 2003 and 2007 at Chonbuk University Hospital. We analyzed two consecutive sets of abdominal computerized tomography scans-one at the time of treatment initiation and another at the second-year follow-up. Liver volume was calculated by 3-dimensional liver extraction volumetry program. RESULTS A total of 55 patients (34 males) were included. There was 114.3 mL ± 167.8 mL (12.9% ± 17.9%) of increase in liver volume during the two years of NUCs therapy (993.8 mL ± 242.8 mL at baseline vs 1108.1 mL ± 263.3 mL at two-year follow-up, P < 0.001). The ratio of the measured baseline liver volume to the estimated standard liver volume was improved from 70.8% to 78.0%. An increase in liver volume was shown not only in patients with compensated cirrhosis (P = 0.046) but also in those with decompensated cirrhosis (P < 0.001). Significant factors for volume increases were Child-Turcotte-Pugh grade and model for end-stage liver disease score improvement without virological breakthrough. In multiple linear regression analysis, delta albumin and delta alanine aminotransferase levels showed a significant association with the increase in liver volume (P = 0.002 and 0.005, respectively). CONCLUSION Long-term oral NUCs therapy in patients with HBV-related liver cirrhosis lead to significant increase in liver volume assessed with 3-dimensional liver extraction volumetry program.

Genome-wide association study in diffusely infiltrating type cholangiocarcinoma (CC) can be limited due to the difficulty of obtaining tumor tissue. We aimed to evaluate the genomic alterations of diffusely infiltrating type CC using next-generation sequencing (NGS) of bile and to compare the variations with those of mass-forming type CC. A total of 24 bile samples obtained during endoscopic retrograde cholangiopancreatography (ERCP) and 17 surgically obtained tumor tissue samples were evaluated. Buffy coat and normal tissue samples were used as controls for a somatic mutation analysis. After extraction of genomic DNA, NGS analysis was performed for 48 cancer related genes. There were 27 men and 14 women with a mean age of 65.0±11.8years. The amount of extracted genomic DNA from 3cm(3) of bile was 66.0±84.7μg and revealed a high depth of sequencing coverage. All of the patients had genomic variations, with an average number of 19.4±2.8 and 22.3±3.3 alterations per patient from the bile and tumor tissue, respectively. After filtering process, damaging SNPs (8 sites for each type of CC) were predicted by analyzing tools, and their target genes showed relevant differences between the diffusely infiltrating and mass-forming type CC. Finally, in somatic mutation analysis, tumor-normal paired 14 tissue and 6 bile samples were analyzed, genomic alterations of EGFR, FGFR1, ABL1, PIK3CA, and CDKN2A gene were seen in the diffusely infiltrating type CC, and TP53, KRAS, APC, GNA11, ERBB4, ATM, SMAD4, BRAF, and IDH1 were altered in the mass-forming type CC group. STK11, GNAQ, RB1, KDR, and SMO genes were revealed in both groups. The NGS analysis was feasible with bile sample and diffusely infiltrating type CC revealed genetic differences compared with mass-forming type CC. Genome-wide association study could be performed using bile sample in the patients with CC undergoing ERCP and a different genetic approach for accurate diagnosis, pathogenesis study, and targeted therapy will be needed in diffusely infiltrating type CC.

BACKGROUND AND AIM Epithelial-mesenchymal transition (EMT) of biliary epithelial cells (BECs) plays an important role in biliary fibrosis. This study investigated the effects of simvastatin on the lipopolysaccharide (LPS)-induced EMT and related signal pathways in BECs. METHODS Biliary epithelial cells were exposed to LPS (2 µg/mL) or transforming growth factor β1 (TGF-β1) (5 ng/mL) for 5 days. The EMT was assessed by a gain of mesenchymal cell markers (vimentin, N-cadherin, slug, and Twist-1) and a loss of epithelial cell markers (E-cadherin). The effects of simvastatin on the EMT induced by LPS or TGF-β1 were determined by the changes in the levels of EMT markers and TLR4 and in the c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB) signaling pathways. RESULTS Compared with the BECs treated with LPS alone, co-treatment with simvastatin and LPS induced an increase in the expression of E-cadherin and decreases in the expression levels of mesenchymal cell markers. The LPS-induced TLR4 expression level was slightly decreased by co-treatment with simvastatin. LPS-induced BEC growth was markedly inhibited by co-treatment with simvastatin. Furthermore, pretreatment with simvastatin inhibited the LPS-induced EMT in BECs by downregulating NF-κB and JNK phosphorylation. The suppressive effects of simvastatin pretreatment on the induction of the EMT by TGF-β1 were also demonstrated in H69 cells. CONCLUSIONS Our results demonstrate that LPS or TGF-β1 promote the EMT in BECs that that pretreatment with simvastatin inhibited the induced EMT by downregulating toll-like receptor 4 and NF-κB phosphorylation. This finding suggests that simvastatin can be considered a new agent for preventing biliary fibrosis associated with the EMT of BECs.

Previously, our data indicated that both cAMP and MAP kinase signaling play important roles in microalgal physiology as well as in lipid or carotenoid biosynthesis. In order to understand downstream genes of these signaling pathways, we employed proteomics approach. Both signal pathways were first altered with specific signaling inhibitors or modulators. Treatment of specific inhibitors changed microalgal size and increased lipid contents. With the microalgal cells after treatments of specific signaling inhibitor or modulators, we performed the proteomics analysis to identify downstream genes responsible for these phenotypes. Interestingly, multiple photosynthesis genes were identified, particularly proteins associated with PSII. Our data suggested that MAP kinase and cAMP signaling affect the photosynthesis, thereby leading to microalgal lipid or carotenoid biosynthesis.

Different ligands can lead to the activation of epidermal growth factor receptor, and the subsequent signal transduction leads to an increase in cellular motility, proliferation, invasion, and blocking of apoptosis, and all of this contributes to the development and progression of cancer. Our studies clarified; 1) The EGFR expression level is reduced during tumor progression or during ligand-induced EGFR activation. 2) ANXA8 and its regulatory pathway may well be alternative, strategic targets for inhibiting tumor metastasis. 3) The molecular pathway of EphA2 signaling and provide insight into the mechanisms that control cancer progression and metastatic spread in cholangiocarcinoma (CC). Therefore, therapeutic strategies that target EphA2 and its downstream effectors may be useful to control CC. Korean J Pancreatobiliary 2015;20(4):175-189

Purpose In response to the challenges and difficulties imposed by rare cancers, multi-stakeholder initiatives dedicated to improving rare cancer care was launched, and several recommendations were made by professional societies. However, these primarily reflect the view of the advocates and supporters, and may not represent the views of the “average” clinician or researcher. In this study, we sought to investigate perceived difficulties with regard to rare cancer care and potential solutions endorsed by oncologists. Materials and Methods A representative sample of 420 oncologists recruited in 13 cancer centers participated in a nationwide survey. Results Oncologists faced various difficulties in treatment of patients with rare cancers, including the lack of clinical practice guidelines (65.7%) and personal experience (65.2%), lack of approved treatment options (39.8%), and reimbursement issues (44.5%). They were generally supportive of recent recommendations by multi-stakeholder initiatives as well as professional societies for development of clear clinical practice guidelines (66.0%), flexible reimbursement guidelines (52.9%), and a national rare cancer registry (47.4%). However, there was only moderate endorsement for referrals to high-volume centers (35.5%) and encouragement of off-label treatments (21.0%). Conclusion Insights into the general attitudes of oncologists gained through our nationwide survey of representative samples would be helpful in development of clinical practices and public health policies in rare cancer treatment and research.

Background Objective evaluation tools for assessing the effectiveness of stenting in palliative treatment of malignant biliary obstruction are not satisfactory. Effects of biliary stenting on liver volume change have never been studied. Objective We aimed to use volumetry to analyze liver volume changes after endoscopic stenting in bile duct cancer according to the location and number of stents. Design Retrospective review. Setting University hospital. Patients Patients with a diagnosis of hilar or distal bile duct cancer and who underwent biliary metal stenting. Interventions ERCP with self-expandable metal stent placement. Main Outcome Measurements Liver volume change after biliary stenting and its comparison according to the location (hilar vs distal common bile duct) and number (hilar bilateral vs hilar unilateral). Results There were 60 patients; 31 were treated for hilar bile duct cancer (13 for bilateral stent and 18 for unilateral stent) and 29 for distal bile duct cancer. Overall mean follow-up duration was 11.7 ± 4.9 weeks. Liver volume increased 17.4 ± 24.1%. The rate of liver growth was rapid during the early period from 4 to 8 weeks. Stenting in hilar bile duct cancer tended to increase liver volume more than distal biliary stents (22.5% vs 11.9%, P = .091). In hilar bile duct cancer, unilateral and bilateral stents showed similar liver volume increases (20.1% and 25.8%, respectively; P = .512). Limitations Single center, retrospective. Conclusions Biliary stenting markedly increased liver volume in both hilar and distal bile duct cancer. Our data suggest that liver volume assessment could be a useful tool for evaluating stent efficacy.

Parthenolide (PT) is responsible for the bioactivities of feverfew (Tanacetum parthenium). Apart from its potent anti-inflammatory effects, this compound has been reported to induce apoptosis in various cancer cells. However, little is known about its role in the process of tumor angiogenesis. In the present study, we investigated the effects and potential mechanisms of action of PT on angiogenesis in human colorectal cancer (CRC). The anti-angiogenic effects of PT were evaluated in cultured human umbilical vein endothelial cells (HUVECs) and in the human CRC cell lines, HT-29, SW620 and HCT116. PT markedly inhibited vascular cell migration and capillary-like structure formation even at a dose which had not effects on cell viability. PT also suppressed the expression of angiogenic biomarker proteins [vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1 and VEGFR2] in both the HUVECs and CRC cells. Additionally, PT effectively inhibited tumor neovascularization in a HT-29 xenograft model. These results indicate that PT suppresses angiogenesis by reducing the expression of VEGF and its receptors and may be a viable drug candidate in anti-angiogenesis therapies for human CRC.

A facile method of preparing water-dispersible sulfonated graphene (SPG) using sulfonated poly(ether–ether–ketone) organic polymer as a modifier was realized. A glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) on the surface of AuNPs used to modify SPG and chitosan (CH) deposited on an indium tin-oxide (ITO) glass electrode by a solution casting method. Morphological and structural characterizations confirm that the AuNPs can be efficiently applied to the SPG–CH matrix. The amperometric response of the GOx/SPG–AuNPs–CH/ITO bioelectrode shows a broad linear range of 0.5 to 22.2 mM, with a limit of detection of 0.13 mM and a high sensitivity of 6.51 μA/(mM cm2). The excellent performance of the constructed biosensor is attributed to the large surface-to-volume ratio and electron transfer ability of SPG, the high catalytic activity of the AuNPs, and the good biocompatibility of CH. In addition, the sensor has important advantages, such as its simple preparation, fast response time (10 s), good stability (70 days), and high reproducibility. Favorable results upon examining the electrochemical response for the determination of glucose in human blood serum were obtained, without the assistance of a negligible effect of interfering bio-analytes. The results of studies show that the ternary SPG–AuNPs–CH nanocomposite may offer a new approach for developing novel types of highly sensitive and stable electrochemical biosensors.

Lipocalin-2 (Lcn2) is preferentially expressed in hepatocellular carcinoma (HCC). However, the functional role of Lcn2 in HCC progression is still poorly understood, particularly with respect to its involvement in invasion and metastasis. The purpose of this study was to investigate whether Lcn2 is associated with the epithelial-mesenchymal transition (EMT) in HCC and to elucidate the underlying signaling pathway(s). Lcn2 was preferentially expressed in well-differentiated HCC versus liver cirrhosis tissues, and its expression was positively correlated with the stage of HCC. The characteristics of EMT were reversed by adenoviral transduction of Lcn2 into SH-J1 cells, including the down-regulation of N-cadherin, vimentin, alpha-smooth muscle actin, and fibronectin, and the concomitant up-regulation of CK8, CK18, and desmoplakin I/II. Knockdown of Lcn2 by short hairpin RNA (shRNA) in HKK-2 cells expressing high levels of Lcn2 was associated with EMT. Epidermal growth factor (EGF) or transforming growth factor beta1 (TGF-β1) treatment resulted in down-regulation of Lcn2, accompanied by an increase in Twist1 expression and EMT in HCC cells. Stable Lcn2 expression in SH-J1 cells reduced Twist1 expression, inhibited cell proliferation and invasion in vitro, and suppressed tumor growth and metastasis in a mouse model. Furthermore, EGF or TGF-β1 treatment barely changed EMT marker expression in SH-J1 cells ectopically expressing Lcn2. Ectopic expression of Twist1 induced EMT marker expression even in cells expressing Lcn2, indicating that Lcn2 functions downstream of growth factors and upstream of Twist1. Conclusion: Together, our findings indicate that Lcn2 can negatively modulate the EMT in HCC cells through an EGF (or TGF-β1)/Lcn2/Twist1 pathway. Thus, Lcn2 may be a candidate metastasis suppressor and a potential therapeutic target in HCC. (Hepatology 2013;58:1349–1361)

Wilson's disease (WD) is an autosomal recessive inherited disorder of copper metabolism that results in the accumulation of copper in the body and primarily in the liver, brain, and cornea. Copper is a toxic metal and might be associated with cancer induction. Most malignancies associated with WD are hepatocellular carcinoma and cholangiocarcinoma. Other intra-abdominal malignancies have been only rarely reported. To our knowledge, this is the first report to suggest that patients with WD may be vulnerable to a malignant change in the colonic mucosa during long-term copper chelating therapy. We report a case of colonic adenocarcinoma in a patient with WD and review the related literature.

Parthenolide (PT), a NF-κB inhibitor, has recently been demonstrated as a promising anticancer agent that promotes apoptosis of cancer cells. 5-fluorouracil (5-FU) has been a drug of choice for treatment of colorectal cancer (CRC). Unfortunately, many of the therapies that use 5-FU alone or in combination with other agents are likely to become ineffective due to drug resistance. In the present study, we investigated the antitumor effect of PT combined with 5-FU on a human CRC cell line, SW620. The results demonstrated that combination of PT and 5-FU induced apoptosis which was determined using MTT, cell cycle analysis, annexin-V assay, and Hoechst 33258 staining. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, cytochrome C release, and activation of caspase 3 and 9. Moreover, intra-peritoneal injection of PT and 5-FU showed significant inhibition of tumor growth in the xenograft model. These results demonstrate that PT exhibits anticancer activity in human colorectal cancer in vitro and in vivo. These findings provide an efficacious strategy to overcome 5-FU resistance in certain CRC.

Eph receptor 2 (EphA2) overexpression is frequently accompanied by the loss of its cognate ligand during tumor progression. However, the molecular mechanism of this ligand-independent promotion of tumor by EphA2 remains unclear in highly malignant and fatal cholangiocarcinoma (CC). We examined the biological role of EphA2 in tumor growth and metastasis in CC tissues and cells according to the degree of differentiation and we explored the downstream signaling pathways of EphA2. Growth factor-mediated EphA2 overexpression itself leads to the activation of the mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase (ERK) pathways through ligand-independent activation of EphA2 (phosphorylation of S897). An in vitro soft agar assay and in vivo orthotopic or subcutaneous tumor model showed that EphA2 enhanced colony formation and accelerated tumor growth, and which seemed to be mainly associated with Akt (T308)/mTORC1 activation. Aberrant expression and activation of EphA2 was also associated with poorer differentiation and higher metastatic ability. Enhanced metastatic ability was also observed in an orthotopic tumor model or lung metastasis model, correlating with Pyk2(Y402)/c-Src/ERK activation in addition to activation of the canonical Raf/MEK/ERK pathway. The mTORC1 and Raf/Pyk2 pathways also appeared to affect each other. These results suggest that growth factor-mediated EphA2 might be involved in tumor growth and metastasis through activation of the mTORC1 and Raf/Pyk2 pathways. Therapeutic strategies that target EphA2 and its downstream effectors may be useful to control CC. (HEPATOLOGY 2013;57:2248-2260).

Although increased evidence has suggested that epithelial-mesenchymal transition has been implicated in cancer invasion and is associated with poor prognosis, its significance in cholangiocarcinoma remains unclear. We evaluated the levels of expression of epithelial-mesenchymal transition-related genes and proteins in 2 established human cholangiocarcinoma cell lines with different morphological characteristics and performed transwell cell invasion assays. Furthermore, we investigated the association between altered expression of 6 epithelial-mesenchymal transition-related proteins and clinical outcomes in human cholangiocarcinoma patients (n = 119) by immunohistochemistry using a tissue microarray approach. Comparative analysis of protein and messenger RNA expression revealed that the cell line with less differentiation (JCK) showed increased expression of mesenchymal markers and zinc-finger proteins and decreased expression of epithelial markers. The invasion activity of JCK cells was significantly higher than that of cells from OZ cell lines. Tissue microarray analysis revealed that the combined expression pattern of 6 epithelial-mesenchymal transition-related proteins predicted shortened disease-free survival (13.0 versus 22.0 months, P = .033) and overall survival (23.0 versus 63.0 months, P = .003) and was confirmed as an independent unfavorable prognostic factor for survival in multivariate survival analysis (disease-free survival, P = .028 for the 3 epithelial-mesenchymal transition-related markers; overall survival, P = .010 for the 6 epithelial-mesenchymal transition-related markers). In conclusion, our results suggest that altered expression of a number of epithelial-mesenchymal transition-related genes in tumor cells with poor differentiation may explain their increased invasive ability. Our results also suggest that altered expression of a suite of epithelial-mesenchymal transition-related proteins could be used as a tool to predict poor outcomes in human cholangiocarcinoma patients.

// Hua Li 1 , Jun Zhang 1 , Mi-Jin Lee 1 , Goung-Ran Yu 1 , Xueji Han 2 , Dae-Ghon Kim 1 1 Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Research Institute of Clinical Medicine, Biomedical Research Institute Chonbuk National University Medical School and Hospital, Jeonju, Jeonbuk, Republic of Korea 2 Department of Infectious Disease, Yanbian University Hospital, Yanji, Jilin Province, China Correspondence to: Dae-Ghon Kim, email: daeghon@chonbuk.ac.kr Keywords: OIP5, miR-15b-5p, hepatocellular carcinoma, tumor growth, metastasis Received: June 29, 2016 Accepted: January 16, 2017 Published: February 08, 2017 ABSTRACT Opa interacting protein 5 (OIP5) is upregulated in some types of human cancers, but the biological implications of its upregulation have not yet been clarified in human hepatocellular carcinoma (HCC). In this study, the signaling pathway downstream of OIP5 was analyzed by proteome kinase profiling. A putative microRNA targeting OIP5 was identified using a miRNA PCR array. Tumorigenicity and metastatic ability were examined in an orthotopic animal model. OIP5 expression was strongly detected in early and advanced tumors via gene expression profiling and immunohistochemical staining analyses. Cells with knockdown of OIP5 via target shRNA exhibited reduced hepatic mass formation and metastatic tumor nodules in an orthotopic mouse model. OIP5-induced AKT activation was mediated by both mTORC2 and p38/PTEN activation. AKT activation was linked to mTORC1 and GSK-3β/β-catenin signaling, which are primarily associated with tumor cell growth and metastasis, respectively. miR-15b-5p, which targets OIP5, efficiently inhibited OIP5-mediated mTORC1 and GSK-3β/β-catenin signaling. These findings suggest that OIP5 may be involved in HCC growth and metastasis and that miR-15b-5p inhibits OIP5-mediated oncogenic signaling in HCC.

When patients with advanced gastric cancer experience active bleeding, gastroenterologists normally choose between two treatment modalities, endoscopic hemostasis and transarterial embolization (TAE). In patients with advanced gastric cancer with bleeding, the predictive factors for endoscopic hemostatic failure are still unknown. Thus, the purpose of this study was to evaluate predictive factors for endoscopic hemostasis failure and to differentiate which hemostasis procedure is more effective for advanced gastric cancer with bleeding.We reviewed the medical records of patients who were diagnosed with advanced gastric cancer and acute non-variceal gastric bleeding from January 2006 to August 2011. Forty-five patients were enrolled in this study and they were divided into a group of 14 patients who had experienced successful endoscopic hemostasis and a group of 31 patients who had had unsuccessful hemostasis with the first endoscopy and then underwent TAE.Lesion size and bleeding condition of Forrest class 1a or 1b were statistically significant predictive factors for endoscopic hemostatic failure (P = 0.023 and P = 0.017, respectively). On multivariate logistic regression analysis, size (lesion2 cm) was a significant predictive factor for endoscopic hemostatic failure [adjusted odds ratio (aOR) 8.056; 95% confidence interval (CI) 1.329-48.846].We determined that small bleeding lesions (2 cm) and exposed vessels in the bleeding site with gastric cancer indicated that endoscopic hemostasis would be an effective hemostatic modality to choose. Particularly, in the opposite condition, the presence of large bleeding lesions (2 cm) and non-exposed vessel bleeding with a tumor, endoscopic hemostasis failure is predicted and TAE could be recommended.

Background/Aims: Clevudine (CLV) has potent antiviral activity against chronic hepatitis B (CHB) virus infection. The long-term efficacy and safety of CLV therapy in naive patients with CHB were investigated. Methods: In this retrospective study, 152 naive Korean patients with CHB who received 30 mg of CLV once daily for at least 12 months were investigated. Results: The cumulative rates at months 12, 24, and 36, respectively, were 65.8%, 74.7%, and 74.7% for undetectable serum hepatitis B virus (HBV) DNA (

Cholangiocarcinoma (CC) is a malignant epithelium neoplasm that originates from the bile epithelium and for which there are few therapeutic strategies. The mTOR pathway involved in many cellular processes was reported to be up-regulated in various cancers. We investigated the activation of the AKT/mTOR pathway in CC cell lines with different degrees of dedifferentiation and found that rapamycin could suppress the motility and the peritoneal dissemination of sarcomatoid SCK cells. Inhibition of the mTOR pathway with rapamycin decreased significantly the number of tumor nodules and prolonged the survival rates of nude mice inoculated with sarcomatoid CC cells. Prolonged treatments with rapamycin were found to disrupt the mTORC2 assembly and to reduce the phosphorylation of STAT3 at Ser 727. Rapamycin decreased both mRNA and protein levels of MMP2 and Twist1, which are regulated by STAT3 and associated with cancer metastasis. The overexpression of STAT3 S727A lacking the phosphorylation site resulted in significantly less sensitivity to rapamycin than the overexpression of STAT3 WT. Taken together, our results suggest that rapamycin could suppress the motility of sarcomatoid CC by down-regulating p-STAT3 (S727) through the impairment of mTORC2 assembly.

Parthenolide (PT), a principal active component in medicinal plants, has been used conventionally to treat migraine and inflammation. This component has recently been reported to induce apoptosis in cancer cells, through mitochondrial dysfunction. In the present study, we investigated PT-mediated cell death signaling pathway by focusing on the involvement of Bcl-2 family members in human colorectal cancer cells. We also investigated the inhibitory effect of PT on tumor growth in xenografts. Using the human colorectal cancer cell lines HT-29, SW620 and LS174T, we demonstrated that treatment of these cancer cells with PT induces apoptosis using MTT, Annexin V assay and Hoechst 33258 staining. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, cytochrome c release and caspase activation. Moreover, intraperitoneal injection of PT showed significant inhibition of tumor growth, angiogenesis in the xenograft model. These results demonstrate that PT exhibits anti-cancer activity in human colorectal cancer in vitro and in vivo. These findings may also provide a novel approach for the treatment of colorectal cancer.

Background The prevalence of hepatitis C virus (HCV) infections in elderly patients has been increasing in a number of countries. A few reports concerning pegylated interferon-α (PEG-IFN-α)-based combination treatment in elderly chronic hepatitis C (CHC) patients have been published, with slightly different treatment outcomes. Objectives We investigated the treatment response and safety of PEG-IFN-α plus ribavirin combination therapy in elderly patients with CHC. Patients and Methods Among a total of 181 treatment-naïve CHC patients (60 patients with genotype 1, 121 patients with genotype 2 or 3), 38 were aged ≥ 65 years (defined as the elderly group) and 143 were aged < 65 years (defined as the non-elderly group). Results The overall sustained virologic response (SVR) was lower in the elderly group than in the non-elderly group, but it was not significantly different (65.8 % vs. 76.2 %, P = 0.15). In a subgroup analysis, among patients with genotype 1, the elderly group had a significantly lower SVR rate than the non-elderly group (30.8 % vs. 66.0 %, P = 0.03). However, the SVR rate in patients with HCV genotype 2 or 3 was comparable between the two groups (84.0 % vs. 81.3 %, P = 0.85). HCV genotype was significantly associated with SVR in the elderly patients (genotype 1 vs. 2 or 3, odds ratio: 0.18, 95% confidence interval: 0.000-0.869, P = 0.03). The incidence of premature discontinuation of treatment (21.1 % vs. 9.1 %, P = 0.05) and dose modification (52.6 % vs. 31.5 %; P = 0.02) due mainly to adverse events or laboratory abnormalities, were higher in the elderly group than in the non-elderly group. Conclusions PEG-IFN-α plus ribavirin combination therapy might be considered for elderly CHC patients, especially for genotype 2 or 3, with vigilant monitoring of adverse events.

Parthenolide (PT), a sesquiterpene lactone derived from the plant feverfew, has pro-apoptotic activity in a number of cancer cell types. We assessed whether PT induces the apoptosis of hepatic stellate cells (HCSs) and examined its effects on hepatic fibrosis in an in vivo model. The effects of PT on rat HSCs were investigated in relation to cell growth inhibition, apoptosis, NF-κB binding activity, intracellular reactive oxygen species (ROS) generation, and glutathione (GSH) levels. In addition, the anti-fibrotic effects of PT were investigated in a thioacetamide-treated rat model. PT induced growth inhibition and apoptosis in HSCs, as evidenced by cell growth inhibition and apoptosis assays. PT increased the expression of Bax proteins during apoptosis, but decreased the expression of Bcl-2 and Bcl-X(L) proteins. PT also induced a reduction in mitochondrial membrane potential, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. PT inhibited TNF-α-stimulated NF-κB binding activity in HSCs. The pro-apoptotic activity of PT in HSCs was associated with increased intracellular oxidative stress as evidenced by increased intracellular ROS levels and depleted intracellular GSH levels. Furthermore, PT ameliorated hepatic fibrosis significantly in a thioacetamide- treated rat model. In conclusion, PT exhibited pro-apoptotic effects in rat HSCs and ameliorated hepatic fibrosis in a thioacetamide-induced rat model.

Clinical application of the prognostic gene expression signature has been delayed due to the large number of genes and complexity of prediction algorithms. In the current study we aimed to develop an easy-to-use risk score with a limited number of genes that can robustly predict prognosis of patients with hepatocellular carcinoma (HCC). The risk score was developed using Cox coefficient values of 65 genes in the training set (n = 139) and its robustness was validated in test sets (n = 292). The risk score was a highly significant predictor of overall survival (OS) in the first test cohort (P = 5.6 × 10−5, n = 100) and the second test cohort (P = 5.0 × 10−5, n = 192). In multivariate analysis, the risk score was a significant risk factor among clinical variables examined together (hazard ratio [HR], 1.36; 95% confidence interval [CI], 1.13-1.64; P = 0.001 for OS). Conclusion: The risk score classifier we have developed can identify two clinically distinct HCC subtypes at early and late stages of the disease in a simple and highly reproducible manner across multiple datasets. (HEPATOLOGY 2011)

Intrahepatic cholangiocarcinoma (ICC) is refractory to conventional chemotherapy. We previously generated chemoresistant ICC (SCK(R)) cells and showed that AKT and ERK signaling conferred cisplatin resistance. Here, we report that epidermal growth factor receptor (EGFR) signaling and L1 cell adhesion molecule (L1CAM) conferred cisplatin resistance in SCK(R) cells in an additive fashion. Activation of EGFR connected to AKT and ERK signaling pathways may induce anti-apoptosis and promote cell proliferation, while L1CAM promoted cell proliferation by mainly activating ERK signaling. Inhibition of EGFR activation or L1ACM greatly sensitized the cells to cisplatin. EGFR and L1CAM may be important targets for ICC therapy.

NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). There is abundant mRNA expression of NM23-H1, NM23-H2, or a read through transcript (NM23-LV) in the primary sites of hepatocellular carcinoma (HCC). Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study was to examine whether NM23-H2 is associated with hepatocarcinogenesis. The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Its subcellular localization was confined to mainly the cytoplasm and to a lesser extent in the nucleus. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes showed a transformed morphology, enhanced focus formation, and allowed anchorage-independent growth. Finally, NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice and showed c-Myc over-expression. In addition, NF-κB and cyclin D1 expression were also increased by NM23-H2. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Collectively, these results indicate that NM23-H2 may be pro-oncogenic in hepatocarcinogenesis.

The cell-cycle regulator Cks1 has recently been implicated in Skp2-mediated ubiquitination of the tumor suppressor protein p27. In this article, we report that Cks1 exerts a Skp2-independent regulation of NF-κB that promotes interleukin-8 (IL-8) expression, which is critical to hepatocellular carcinoma (HCC) growth. Cks1 was upregulated frequently in human HCC tissues and cell lines. Cks1 knockdown in HCC cells elevated p27 levels and decreased tumorigenicity in a manner that was also associated with a strong downregulation of IL-8 expression. IL-8 downregulation was not phenocopied by either RNAi-mediated knockdown of Skp2 or ectopic overexpression of p27. However, attenuation of IL-8 expression itself was sufficient to blunt HCC growth. Mechanistic investigations revealed that IL-8 was controlled at a transcriptional level by Cks1 targeting of the NF-κB regulator IκBα, which led to NF-κB activation and IL-8 expression, through a p27-independent regulation of IκB kinase complex components. Collectively, our findings support the hypothesis that Cks1 supports hepatocarcinogenesis by NF-κB–mediated regulation of IL-8 expression, broadening the function of Cks1 in cancer beyond its role as a Skp2 cofactor in p27 ubiquitination. Cancer Res; 71(21); 6827–35. ©2011 AACR.

The human E3 ubiquitin ligase murine double minute 2 (MDM2) targets the tumor suppressor p53 for ubiquitination and degradation but also promotes its own ubiquitination and subsequent degradation. As the balance between MDM2 and p53 levels plays a crucial role in regulating cell proliferation and apoptosis, we sought to identify factors selectively inhibiting MDM2 self-ubiquitination. Here we have shown that the LIM domain protein Enigma directly interacts with MDM2 to form a ternary complex with p53 in vitro and in human hepatoma and colon carcinoma cell lines and mouse embryonic fibroblasts. We found that Enigma elicited p53 degradation by inhibiting MDM2 self-ubiquitination and increasing its ubiquitin ligase activity toward p53 in cells. Moreover, mitogenic stimuli such as serum, FGF, and HGF increased Enigma transcription via induction of serum response factor (SRF), leading to MDM2 stabilization and subsequent p53 degradation. We observed similar results in the livers of mice treated with HGF. In humans, we found SRF and Enigma coexpressed with MDM2 but not p53 in several liver and stomach tumors. Finally, we showed that Enigma promoted cell survival and chemoresistance by suppressing p53-mediated apoptosis in both cell lines and a mouse xenograft model. Our findings suggest a role for Enigma in tumorigenesis and uncover a mechanism whereby mitogens attenuate p53 antiproliferative activity through an SRF/Enigma/MDM2 pathway.

The authors assessed the efficacy and antiviral resistance of 48-week clevudine therapy versus lamivudine in treatment of naive patients with HBeAg positive chronic hepatitis B. In this retrospective study, a total of 116 HBeAg positive patients, who received 30 mg of clevudine once daily (n=53) or 100 mg of lamivudine once daily (n=63) for 48 weeks, were included. At week 48, clevudine therapy produced a significantly greater mean reductions in serum HBV DNA levels from baseline than lamivudine therapy (-5.2 vs. -4.2 log(10)IU/mL; P=0.005). Furthermore, a significantly higher proportion of patients on clevudine achieved negative serum HBV DNA by PCR (

Objectives: The authors compared the efficacies and tolerabilities of pegylated interferon-α2a (PEG-IFN-α2a) + ribavirin and pegylated interferon-α2b (PEG-IFN-α2b) + ribavirin for the initial treatment of chronic hepatitis C. Methods: A total of 126 treatment-naive patients (29.4% genotype 1, 70.6% genotype non-1) were treated with PEG-IFN-α2a 180 µg/week (group A, n = 79) or PEG-IFN-α2b 1.5 µg/kg/week (group B, n = 47) with ribavirin (800 mg/day for genotype non-1 or 1,000–1,200 mg/day for genotype 1) for 24 (genotype non-1) or 48 weeks (genotype 1). Results: End-of-treatment virologic response, sustained virologic response, and biochemical response were not significantly different in groups A and B (84.8 vs. 89.4%, 70.9 vs. 72.3%, and 70.9 vs. 74.5%, respectively; p > 0.05). In patients with the HCV genotype 1 or non-1, treatment responses were not significantly different. Multivariate analysis showed that HCV genotype only was an independent factor that affected sustained virologic response (p = 0.048). The proportions of treatment discontinuations in groups A and B were similar (10.1 vs. 10.6%; p = 1.000). Conclusions: PEG-IFN-α2a or PEG-IFN-α2b + ribavirin combination therapies showed similar efficacies and tolerabilities as initial treatments for chronic hepatitis C.