Takehiro Noda

Activation of Wnt/ β -catenin signalling pathway induces chemoresistance to interferon- α /5-fluorouracil combination therapy for hepatocellular carcinoma

Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-α/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-α/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/β-catenin signalling pathway contributed to resistance to IFN-α/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/β-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/β-catenin signalling by glycogen synthesis kinase-3 inhibitor (6-bromoindirubin-3′-oxime (BIO)) induced chemoresistance to IFN-α/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-α/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/β-catenin signalling pathway induces chemoresistance to IFN-α/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.

Molecular prediction of early recurrence after resection of hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) remains poor. Vascular invasion, tumour multiplicity and large tumour size are the conventional poor prognostic indicators related to early recurrence. However, it is difficult to predict prognosis of each HCC in the absence of these indicators. The purpose of this study is to predict early recurrence of HCC after radical resection based on whole human gene expression profiling. Microarray analyses were performed in 139 HCC primary tumours. A total of 88 cases lacking the conventional poor prognostic indicators were analysed to establish a molecular prediction system characteristic for early recurrence in 42 training cases with two polarised prognoses, and to test its predictive performance in 46 independent cases (group C). Subsequently, this system was applied to another 51 independent cases with some poor prognostic indicators (group D). The molecular prediction system accurately differentiated HCC cases into poor and good prognoses in both the independent group C (disease-free survival [DFS]: p=0.029, overall survival [OS]: p=0.0043) and independent group D (DFS: p=0.0011, OS, p=0.035). Multivariate Cox regression analysis indicated that the clinical value of molecular prediction system was an independent prognostic factor (p<0.0001, hazard ratio=3.29). Gene expression pattern related to early intrahepatic recurrence inherited in the primary HCC tumour can be useful for the prediction of prognosis.

PLOD2 induced under hypoxia is a novel prognostic factor for hepatocellular carcinoma after curative resection

Under hypoxia, tumour cells undergo genetic and adaptive changes that allow their survival. Previously, we reported that high expression of hypoxia‐inducible factor (HIF)‐1 was a significant predictive factor for recurrence in hepatocellular carcinoma (HCC). Hypoxia also stimulates expression of procollagen‐lysine, 2‐oxoglutarate 5‐dioxygenase (PLOD) genes via the HIF‐1 pathway.

MicroRNA-155 controls exosome synthesis and promotes gemcitabine resistance in pancreatic ductal adenocarcinoma

The cancer drug gemcitabine (GEM) is a key drug for treating pancreatic ductal adenocarcinoma (PDAC), but PDAC cells develop chemoresistance after long-term administration. Since the tolerance was immediately spread to every PDAC tissue in a patient, it is assumed that some certain efficient mechanisms underlay in the development of chemoresistance. Changes in the levels of particular microRNAs or alterations in intercellular communication play a dominant role in chemoresistance development, and recent data also suggest that exosomes play an important role in this process. In this study, we revealed that the loop conferred chemoresistance in PDAC cells. The loop was as follows; 1, The long-term exposure of GEM increased miR-155 expression in PDAC cells. 2, The increase of miR-155 induced two different functions; exosome secretion and chemoresistance ability via facilitating the anti-apoptotic activity. 3, Exosome deliver the miR-155 into the other PDAC cells and induce the following function. The target therapy to miR-155 or the exosome secretion effectively attenuated the chemoresistance, and these results were validated with both clinical samples and in vivo experiments. This mechanism represents a novel therapeutic target in GEM treatment to PDAC.

Interferon alpha receptors are important for antiproliferative effect of interferon‐α against human hepatocellular carcinoma cells

Aim:  Interferon (IFN)‐α is a promising drug for the prevention and treatment of hepatocellular carcinoma (HCC). We reported that responders to IFN‐α/5‐fluorouracil combination therapy expressed higher IFN alpha receptor (IFNAR)2 in tumor. Herein we studied involvement of IFNARs in response to IFN‐α in HCC cells.

Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells.

BackgroundThe combination therapy of interferon (IFN)-alpha and 5-fluorouracil (5-FU) improved the prognosis of the patients with hepatocellular carcinoma (HCC). To determine the molecular mechanisms of the anti-tumor and anti-angiogenic effects, we examined the direct anti-proliferative effects on human umbilical vein endothelial cells (HUVEC) and indirect effects by regulating secretion of angiogenic factors from HCC cells.MethodsThe direct effects on HUVEC were examined by TUNEL, Annexin-V assays and cell cycles analysis. For analysis of the indirect effects, the apoptosis induced by the conditioned medium from HCC cell treated by IFN-alpha/5-FU and expression of angiogenic factors was examined.ResultsIFN-alpha and 5-FU alone had anti-proliferative properties on HUVEC and their combination significantly inhibited the growth (compared with control, 5-FU or IFN alone). TUNEL and Annexin-V assays showed no apoptosis. Cell cycle analysis revealed that IFN-alpha and 5-FU delayed cell cycle progression in HUVEC with S-phase accumulation. The conditioned medium from HuH-7 cells after treatment with IFN/5-FU significantly inhibited HUVEC growth and induced apoptosis, and contained high levels of angiopoietin (Ang)-1 and low levels of vascular endothelial growth factor (VEGF) and Ang-2. Knockdown of Ang-1 in HuH-7 cells abrogated the anti-proliferative effects on HUVEC while knockdown of Ang-2 partially rescue the cells.ConclusionThese results suggested that IFN-alpha and 5-FU had direct growth inhibitory effects on endothelial cells, as well as anti-angiogenic effects through regulation of angiogenic factors released from HCC cells. Modulation of VEGF and Angs secretion by IFN-alpha and 5-FU may contribute to their anti-angiogenic and anti-tumor effects on HCC.

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells

Background:A striking efficiency of interferon (IFN)-based anticancer therapy for advanced hepatocellular carcinoma (HCC) has been reported. Because its clinical efficiency greatly depends on each patients local response, prediction of local response is crucial.Methods:Continuous exposure of IFN-α to parental PLC/PRF/5 cells (PLC-P) and a limiting dilution method resulted in the establishment of IFN-resistant cell clones (PLC-Rs). Microarray analyses of PLC-P and PLC-Rs identified insulin-like growth factor-binding protein 7 (IGFBP7) as one of the most significantly downregulated genes in PLC-Rs. Changes in anticancer effects of IFN-α were examined in HCC cells after genetic manipulation of IGFBP7 expression. The correlation between immunohistochemically determined IGFBP7 expression and the response to IFN-α/5-fluorouracil (5-FU) therapy was investigated in surgically resected HCC specimens.Results:PLC-R cells showed a remarkable downregulation of IGFBP7 and resistance to IFN-α, compared with PLC-P. Parental PLC/PRF/5 cells transfected with short hairpin RNA against IGFBP7 showed a significant resistance to IFN-α relative to control cells (IC50 fold increase=14.38 times). Insulin-like growth factor-binding protein 7 transfection into PLC-R restored sensitivity to IFN-α. In resected specimens, IGFBP7 expression significantly correlated with the response to IFN-α/5-FU therapy.Conclusion:IGFBP7 could be a useful predictor of the response to IFN-based therapy in advanced HCC.

A Histone Deacetylase Inhibitor Suppresses Epithelial-Mesenchymal Transition and Attenuates Chemoresistance in Biliary Tract Cancer

Epithelial-mesenchymal transition (EMT) is involved in the characteristics of malignancy, such as invasion, metastasis, and chemoresistance. In biliary tract cancer (BTC), EMT is induced by transforming growth factor-beta 1 (TGF-β1). The EMT is reversible; therefore, it is conceivable that it could be related to some epigenetic changes. We focused on histone deacetylase (HDAC) inhibitors as regulators of TGF-β1 signaling, and investigated their effect on EMT and chemoresistance. We employed four BTC cell lines (MzChA-1, gemcitabine-resistant MzChA-1, TFK-1, and gemcitabine-resistant TFK-1) and used vorinostat as the HDAC inhibitor. The relative mRNA expression of an epithelial marker (CDH1) and mesenchymal markers (CDH2, vimentin, SNAI1) were measured by qRT-PCR to evaluate factors associated with EMT. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the chemoresistance of each cell line. In addition, NOD/SCID mice were used to evaluate the effect of vorinostat in vivo. In the parent MzChA-1 and TFK-1 cell lines, TGF-β1 induced EMT and chemoresistance; while vorinostat inhibited the EMT and chemoresistance induced by TGF-β1. In gemcitabine-resistant cell lines that highly expressed TGF-β1, vorinostat inhibited EMT and attenuated chemoresistance. We showed that vorinostat inhibits nuclear translocation of SMAD4 which is a signaling factor of TGF-β1, and this is one of the mechanisms by which vorinostat regulates EMT. We also showed that vorinostat attenuates the binding affinity of SMAD4 to the CDH1-related transcription factors SNAI1, SNAI2, ZEB1, ZEB2, and TWIST. Furthermore, combination therapy with vorinostat and gemcitabine improved survival time in the mice xenografted with gemcitabine resistant MzChA-1 cells. In conclusion, vorinostat regulated TGF-β1-induced EMT and chemoresistance through inhibition of SMAD4 nuclear translocation.

Prognosis of hepatocellular carcinoma with biliary tumor thrombi after liver surgery

BACKGROUND The incidence of biliary tumor thrombi (BTT) in hepatocellular carcinoma (HCC) is very low and operative outcomes are poor. There is little information on the postoperative prognostic factors for patients with HCC with BTT. We analyzed retrospectively the clinicopathologic features of 22 patients with HCC with BTT to identify the prognostic factors associated with operative outcome. METHODS Of the 551 patients who underwent hepatic resection for HCC between 1988 and 2007, 22 (4.0%) had HCC with BTT. Their clinicopathologic features were compared with those with HCC but no BTT. The survival rates were also calculated for various clinicopathologic factors. RESULTS Of the 22 patients, 13 (59%) also had portal or hepatic vein tumor thrombosis. Multiple HCC tumors were identified in 91% of patients and 20 patients were classified as stage IVA. The 1- and 3-year overall survival rates were 89% and 73% for patients with HCC free of BTT, respectively, and 62% and 30%, respectively, for those of HCC with BTT (P < .0001). Portal or hepatic vein tumor thrombosis was the only significant determinant of poor prognosis in 22 patients with HCC with BTT. The 1- and 3-year cumulative survival rates were 89% and 52%, respectively, for patients with only BTT but worse in those of HCC with both BTT and portal or hepatic vein tumor thrombosis (43% and 17%, respectively). CONCLUSION The clinicopathologic features of HCC patients with BTT indicated advanced-stage disease and poor operative outcomes. Portal or hepatic vein tumor thrombosis was the only significant determinant of poor prognosis of HCC patients with BTT.

Tyrosine kinase inhibitor PTK/ZK enhances the antitumor effects of interferon-α/5-fluorouracil therapy for hepatocellular carcinoma cells.

PurposeThere is no standardized treatment for hepatocellular carcinoma (HCC) with portal vein tumor thrombus. We previously reported the efficacy of interferon-alpha and 5-fluorouracil combination (IFN/5-FU) therapy for these patients and the potential mechanism via the regulation of vascular endothelial growth factor (VEGF). In this study, we showed the VEGF-related effects of IFN/5-FU therapy using VEGF-receptor (VEGFR) selective inhibitor, PTK787/ZK222584 (PTK/ZK), in HCC cells.MethodsUsing two VEGF secreting and VEGFR expressing human HCC cell lines, PLC/PRF/5 and HuH7, we performed growth inhibitory assays in vitro and in vivo, apoptosis assay, cell cycle analysis, and Western blot analysis for the mechanism, with or without PTK/ZK in IFN/5-FU therapy.ResultsThe combination of PTK/ZK and IFN/5-FU significantly inhibited cell growth in vitro and tended to reduce tumor growth in vivo in a HuH7 xenograft model in nude mice—in both cases without affecting VEGF secretion. PTK/ZK enhanced the IFN/5-FU induced apoptosis, based on increased proteins levels of Bax and reduced Bcl-xL and Bcl-2. Cell cycle analysis showed different results between the HCC cell lines following the combination therapy, possibly due to differences in p21 protein.ConclusionsVEGF signaling inhibition would support an antitumor effect of IFN/5-FU therapy against HCC cell lines via induction of apoptosis and cell cycle delay.