Yoshito Tomimaru

Circulating microRNA-21 as a novel biomarker for hepatocellular carcinoma.

BACKGROUND & AIMS Several groups have reported the significance of circulating microRNA as a biochemical marker of cancer. To our knowledge, however, there are no reports on the significance of circulating microRNA in hepatocellular carcinoma. The aim of this study was to evaluate the significance of plasma microRNA-21 level as a biochemical marker for hepatocellular carcinoma. METHODS Plasma microRNA-21 level was measured by qRT-PCR in 10 patients before and after curative resection of hepatocellular carcinoma. Plasma microRNA-21 was also compared in other groups of: 126 patients with hepatocellular carcinoma, 30 patients with chronic hepatitis, and 50 healthy volunteers. The power of microRNA-21 in differentiating hepatocellular carcinoma from chronic hepatitis or from healthy volunteers was compared to that of α-fetoprotein. RESULTS In the 10-patient group, plasma microRNA-21 levels significantly diminished after surgery compared with the pre-operative values (p=0.0125). Plasma microRNA-21 level in the 126 patients with hepatocellular carcinoma was significantly higher than in patients with chronic hepatitis and healthy volunteers (p<0.0001, p<0.0001, respectively). ROC analysis of plasma microRNA-21 yielded an AUC of 0.773 with 61.1% sensitivity and 83.3% specificity when differentiating hepatocellular carcinoma from chronic hepatitis, and an AUC of 0.953 with 87.3% sensitivity and 92.0% specificity when differentiating hepatocellular carcinoma from healthy volunteers. Both sets of values were superior to α-fetoprotein and improved for the combination of microRNA-21 and α-fetoprotein. CONCLUSIONS Plasma microRNA-21 level is a promising biochemical marker for hepatocellular carcinoma.

MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells

Background:We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.Methods:Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT–PCR, and response to the therapy was also investigated in clinical HCC specimens.Results:Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate.Conclusions:The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.

Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice

The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mdm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

TIE2-expressing monocytes as a diagnostic marker for hepatocellular carcinoma correlates with angiogenesis.

Angiogenesis is a critical step in the development and progression of hepatocellular carcinoma (HCC). Myeloid lineage cells, such as macrophages and monocytes, have been reported to regulate angiogenesis in mouse tumor models. TIE2, a receptor of angiopoietins, conveys pro‐angiogenic signals and identifies a monocyte/macrophage subset with pro‐angiogenic activity. Here, we analyzed the occurrence and kinetics of TIE2‐expressing monocytes/macrophages (TEMs) in HCC patients. This study enrolled 168 HCV‐infected patients including 89 with HCC. We examined the frequency of TEMs, as defined as CD14+CD16+TIE2+ cells, in the peripheral blood and liver. The localization of TEMs in the liver was determined by immunofluorescence staining. Micro‐vessel density in the liver was measured by counting CD34+ vascular structures. We found that the frequency of circulating TEMs was significantly higher in HCC than non‐HCC patients, while being higher in the liver than in the blood. In patients who underwent local radio‐ablation or resection of HCC, the frequency of TEMs dynamically changed in the blood in parallel with HCC recurrence. Most TEMs were identified in the perivascular areas of tumor tissue. A significant positive correlation was observed between micro‐vessel density in HCC and frequency of TEMs in the blood or tumors, suggesting that TEMs are involved in HCC angiogenesis. Receiver operating characteristic analyses revealed the superiority of TEM frequency to AFP, PIVKA‐II and ANG‐2 serum levels as diagnostic marker for HCC. Conclusion: TEMs increase in patients with HCC and their frequency changes with the therapeutic response or recurrence. We thus suggest that TEM frequency can be used as a diagnostic marker for HCC, potentially reflecting angiogenesis in the liver. (HEPATOLOGY 2013)

Epithelial-mesenchymal transition with expression of SNAI1-induced chemoresistance in colorectal cancer.

BACKGROUND Previous reports have demonstrated that SNAI1 plays a role in epithelial-mesenchymal transition (EMT) through the suppression of CDH1. Its role in the pathology and regulation of EMT expression to chemoresistance in colorectal cancer (CRC) has not yet been fully elucidated. METHODS Immunohistochemistry was performed to evaluate the expression of Snai1 protein in 30 primary CRC samples. The biological significance of Snai1 expression was studied by induction of the wild-type (WT) and mutant SNAI1 gene in CRC SW480 cells. RESULTS Examination of 20 surgical specimens of CRC indicated that Snai1 protein expression was localized outer regions of invasive tumors. Introduction of phosphorylation-defective active EMT forms, SNAI1-6SA and SNAI1-8SA, caused downregulation of CDH1 and upregulation of VIM compared with SNAI1-WT and the negative control (NC). Chemoresistance to 5-fluorouracil (IC50) was higher in SNAI1-6SA and SNAI1-8SA transfectants compared with SNAI1-WT and NC. All the above results were significantly different. CONCLUSION The present study demonstrated that Snai1 plays a role in CRC invasion through phosphorylation, suggesting a plausible mechanism for overcoming chemoresistance that will lead to the development of effective treatments for CRC.

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer

Background:Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance.Methods:We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining.Results:The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients.Conclusions:miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

miR-146a suppresses the sensitivity to interferon-α in hepatocellular carcinoma cells

BACKGROUND Interferon-based (IFN-based) therapy is effective in the treatment of advanced hepatocellular carcinoma (HCC). However, the issue of resistance to this therapy remains to be solved. The aim of this study was to identify microRNAs (miRNAs) that govern the sensitivity to IFN-α in HCC cells. METHODS miRNA microarray analysis using IFN-α-resistant clones of PLC/PRF/5 (PLC-Rs) and their parental cells (PLC-P) was conducted. Changes in the anti-cancer effects of IFN-α were studied after gain-of-function and loss-of-function of the candidate miRNA. RESULTS miR-146a expression was significantly higher in PLC-Rs than in PLC-P. miR-146a decreased the sensitivity to IFN-α through the suppression of apoptosis. Further experiments showed that miR-146a-related resistance to IFN-α was mediated through SMAD4. CONCLUSIONS The results indicated that miR-146a regulated the sensitivity of HCC cells to the cytotoxic effects of IFN-α through SMAD4, suggesting that this miRNA could be suitable for prediction of the clinical response and potential therapeutic target in HCC patients on IFN-based therapy.

miR-320c regulates gemcitabine-resistance in pancreatic cancer via SMARCC1

Background:Gemcitabine-based chemotherapy is the standard treatment for pancreatic cancer. However, the issue of resistance remains unresolved. The aim of this study was to identify microRNAs (miRNAs) that govern the resistance to gemcitabine in pancreatic cancer.Methods:miRNA microarray analysis using gemcitabine-resistant clones of MiaPaCa2 (MiaPaCa2-RGs), PSN1 (PSN1-RGs), and their parental cells (MiaPaCa2-P, PSN1-P) was conducted. Changes in the anti-cancer effects of gemcitabine were studied after gain/loss-of-function analysis of the candidate miRNA. Further assessment of the putative target gene was performed in vitro and in 66 pancreatic cancer clinical samples.Results:miR-320c expression was significantly higher in MiaPaCa2-RGs and PSN1-RGs than in their parental cells. miR-320c induced resistance to gemcitabine in MiaPaCa2. Further experiments showed that miR-320c-related resistance to gemcitabine was mediated through SMARCC1, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. In addition, clinical examination revealed that only SMARCC1-positive patients benefited from gemcitabine therapy with regard to survival after recurrence (P=0.0463).Conclusion:The results indicate that miR-320c regulates the resistance of pancreatic cancer cells to gemcitabine through SMARCC1, suggesting that miR-320c/SMARCC1 could be suitable for prediction of the clinical response and potential therapeutic target in pancreatic cancer patients on gemcitabine-based therapy.

MicroRNA-29a induces resistance to gemcitabine through the Wnt/β-catenin signaling pathway in pancreatic cancer cells

Although we studied previously the mechanisms of resistance of pancreatic cancer cells to gemcitabine (GEM), prediction of the response to GEM remains unsatisfactory. The aim of this study was to investigate the relationship between miR-29a expression and the response to GEM in pancreatic cancer cells. Changes in the growth-inhibitory effect of pancreatic cancer cells (MIAPaCa-2, PSN-1, BxPC-3 and Panc-1) to GEM were examined after overexpression or suppression of miR-29a. We also examined the effect of miR-29a on the Wnt/β-catenin signaling pathway and investigated whether the altered growth-inhibitory effect by miR-29a suppression was weakened after the addition of Wnt3a, a Wnt/β-catenin signaling activator. MIAPaCa-2 and PSN-1 cells transfected with anti-miR-29a showed significantly lower resistance to GEM. In the anti-miR-29a-transfected cells, GEM induced significantly larger numbers of apoptotic cells and S phase accumulation compared to control cells, demonstrated by Annexin V assay and flow cytometric analysis of the cell cycle, respectively. The transfected cells showed overexpression of putative target molecules including Dkk1, Kremen2 and sFRP2 and lower activation of the Wnt/β-catenin signaling pathway. The addition of Wnt3a weakened the augmented growth-inhibitory effect of anti-miR-29a transfection. Our findings suggest that miR-29a expression correlates significantly with the growth-inhibitory effect of GEM and that activation of the Wnt/β-catenin signaling pathway mediated the miR-29a-induced resistance to GEM in pancreatic cancer cell lines.

Plasma miR-21 is a novel diagnostic biomarker for biliary tract cancer

Biliary tract cancer (BTC) has a generally poor prognosis. Furthermore, it is difficult to distinguish BTC from benign biliary disease (BBD) with commonly used modalities. Therefore, a novel biomarker to facilitate cancer detection is highly desirable. Recent studies have reported the use of circulating microRNAs (miRNAs) as biomarkers for cancers. The purpose of this study was to evaluate whether circulating miRNA‐21 (miR‐21) could be used as a biomarker for BTC. Plasma samples were obtained from 94 BTC patients, 50 healthy volunteers (HVs), and 23 BBD patients. miR‐21 levels in the samples were measured by qRT‐PCR. Plasma miR‐21 levels in patients with BTC were significantly higher than in HVs or in patients with BBD (P < 0.0001 for both). Receiver–operator curve (ROC) curve analysis in differentiating BTC patients from HVs indicated that area under the curve (AUC), optimal sensitivity and specificity was 0.93, 85.1% and 100%, respectively, and those in differentiating BTC patients from BBD patients was 0.83, 72.3%, 91.3%, respectively. Validation of these results indicated that the negative predictive value, positive predictive value, sensitivity, specificity, and accuracy in differentiating BTC patients from HVs was 76.6%, 98.6%, 84.0%, 98.0%, and 88.9%, respectively, and those in differentiating BTC patients from BBD patients was 42.2%, 93.0%, 71.2%, 82.6%, and 72.6%, respectively. These sets of values were improved by combining miR‐21 and CA19‐9 measurements. Plasma miR‐21 is a novel diagnostic biomarker for BTC, and may be useful in distinguishing between BTC and BBD patients.