Masahide Mori

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.

Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer.

Background:Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC).Methods:Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT–PCR.Results:Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001).Conclusions:Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.

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.

Role of multidrug resistance protein 2 (MRP2) in chemoresistance and clinical outcome in oesophageal squamous cell carcinoma.

Background:Although multidrug resistance protein 2 (MRP2) confers chemoresistance in some cancer types, its implication on oesophageal squamous cell carcinoma (ESCC) remains unclear.Methods:We evaluated MRP2 expression by immunohistochemistry and RT–PCR using 81 resected specimens from ESCC patients who did or did not receive neo-adjuvant chemotherapy (NACT), including 5-fluorouracil, doxorubicin, and cisplatin (CDDP). Correlation between MRP2 expression and response to chemotherapy was also examined in 42 pre-therapeutic biopsy samples and eight ESCC cell lines.Results:MRP2-positive immunostaining was more frequently observed in ESCCs with NACT than in those without NACT (27.3 vs 5.4%). The MRP2-positive patients showed poorer prognosis than MRP2-negative patients (5-year survival rate, 25.6 vs 55.7%). Concordantly, ESCC with NACT showed 2.1-fold higher mRNA expression of MRP2 than those without NACT (P=0.0350). In pre-therapeutic biopsy samples of patients with NACT, non-responders showed 2.9-fold higher mRNA expression of MRP2 than responders (P=0.0035). Among the panel of ESCC cell lines, TE14 showed the highest MRP2 mRNA expression along with the strongest resistance to CDDP. Inhibition of MRP2 expression by small-interfering RNA reduced chemoresistance to CDDP.Conclusion:Our data suggested that MRP2 is one of molecules, which regulate the sensitivity to chemotherapy including CDDP in advanced ESCC patients.

Sustained interleukin-6 signalling leads to the development of lymphoid organ-like structures in the lung

A variety of pathological changes are seen in lymphoproliferative disorders of the lung but the histogenesis of these abnormalities is not yet fully understood. We previously showed that adenovirus vector‐mediated transient expression of both the human interleukin‐6 (IL‐6) and IL‐6 receptor (IL‐6R) genes, but not the IL‐6 gene alone, in the rat lung induced lymphocytic alveolitis. In the present study, we explored the lung pathology of human IL‐6 and IL‐6R double transgenic mice to elucidate the effects of prolonged IL‐6 signalling on the lung. The transgenic animals developed mononuclear cell accumulation in peribronchovascular regions, but little infiltration into alveolar spaces. Immunohistochemical analysis revealed that the cellular accumulations contained not only mixtures of inflammatory cells but also lymphoid tissue‐like structures. As the expression of CXCL13/BLC, the indispensable chemokine for lymphoid organogenesis, was recognized in the B cell follicles of the pulmonary lesions, we speculate that this chemokine plays an inductive role in the development of the lymphoid tissue‐like structures. These structures were distinguished from bronchus‐associated lymphoid tissues (BALTs) by their location and by the lack of lymphoepithelium, which is a characteristic of BALT. These findings imply that IL‐6 signalling may play a role in the pathogenesis of lymphoproliferative disorders of the lung. Copyright

Rapamycin Causes Upregulation of Autophagy and Impairs Islets Function Both In Vitro and In Vivo

Autophagy is a lysosomal degradation process of redundant or faulty cell components in normal cells. However, certain diseases are associated with dysfunctional autophagy. Rapamycin, a major immunosuppressant used in islet transplantation, is an inhibitor of mammalian target of rapamycin and is known to cause induction of autophagy. The objective of this study was to evaluate the in vitro and in vivo effects of rapamycin on pancreatic β cells. Rapamycin induced upregulation of autophagy in both cultured isolated islets and pancreatic β cells of green fluorescent protein–microtubule‐associated protein 1 light chain 3 transgenic mice. Rapamycin reduced the viability of isolated β cells and down‐regulated their insulin function, both in vitro and in vivo. In addition, rapamycin increased the percentages of apoptotic β cells and dead cells in both isolated and in vivo intact islets. Treatment with 3‐methyladenine, an inhibitor of autophagy, abrogated the effects of rapamycin and restored β‐cell function in both in vitro experiments and animal experiments. We conclude that rapamycin‐induced islet dysfunction is mediated through upregulation of autophagy, with associated downregulation of insulin production and apoptosis of β cells. The results also showed that the use of an autophagy inhibitor abrogated these effects and promoted islet function and survival. The study findings suggest that targeting the autophagy pathway could be beneficial in promoting islet graft survival after transplantation.

Elongation factor 1 gamma mRNA expression in oesophageal carcinoma.

Elongation factor 1 gamma (EF1 gamma) is known to be a subunit of EF1, one of the G proteins that mediate the transport of aminoacyl tRNA to 80S ribosomes during translation. As little is known regarding the expression of EF1 gamma in human oesophageal carcinoma, this study looked at its expression using a northern blot analysis. Thirty six cases of oesophageal carcinoma and 15 oesophageal carcinoma cell lines were studied. The EF1 gamma mRNA overexpression at a level of twofold or more was seen in five (14%) of 36 carcinomatous tissues compared with the normal counterparts. All five overexpressed cases showed severe lymph node metastases compared with the non-overexpressed cases, and the difference was significant (p = 0.028). The stage of the disease of these five cases was far advanced compared with the nonoverexpressed cases (p = 0.012). All 15 oesophageal carcinoma cells expressed EF1 gamma mRNA relatively lower than the gastric or pancreatic carcinoma cell lines, in which EF1 gamma was originally isolated. As the expression of EF1 gamma mRNA could be detected even in the biopsy specimens, its overexpression in tumour tissue may provide preoperative useful information for predicting the aggressiveness of tumours.

Dukes's classification: a valid prognostic indicator for gastric cancer.

Dukess classification for colorectal cancer is simple, and correlates well with survival. This study assessed whether the principles of Dukess classification applied to gastric cancer resulted in the separation of prognostically useful groups. The cumulative survival curves determined by Dukess, Astler-Collers (another classification for colorectal cancer), and the Japanese classification for gastric cancer were evaluated using 476 curatively treated patients with gastric cancer. Of the three staging systems, both Dukess and the Japanese classifications showed a step-wise relationship between the stage of the tumour and the survival of patients. The prognostic value of Dukess classification was reinforced when Dukess C cases were subdivided according to the number of positive nodes (1-6 v > or = 7) or the level of positive nodes (n1 v n2,n3) but not when the subdivision was made according to the depth of wall invasion. A modified Dukess classification in which Dukess cases are subdivided according to the number of metastatic nodes (Dukess Ca = 1-6 nodes, Dukess Cb > 6 nodes) could be both simply and accurately applied to gastric cancer.

Cytokeratins 18 and 8 are poor prognostic markers in patients with squamous cell carcinoma of the oesophagus

Background:Cytokeratins (CKs) are structural marker proteins specific for epithelial cells. However, recent studies indicate their involvement in cancer progression.Methods:We evaluated CK18 and its filament partner, CK8 expression, by immunohistochemistry in 210 resected specimens from patients with oesophageal squamous cell carcinoma (OSCC). We also analysed the relationship between their expression and various clinicopathological parameters including prognosis.Results:Neither CK18 nor CK8 was expressed in non-cancerous squamous epithelium whereas proper oesophageal glands expressed both CKs. Ninety (42.9%) tumours were CK18 positive and 85 (40.5%) CK8 positive, and the concordance rate for immunohistochemical classification for CK18 and CK8 was 82.4%. CK18 expression correlated with poorly differentiated tumours, use of neo-adjuvant chemotherapy, and advanced stage. Prognosis of patients with CK18-positive tumours was poorer than that of patients with negative OSCC (P<0.001). A similar trend was noted for CK8 expression. Multivariate analysis identified pT (P=0.020), pN number (P=0.001), and CK18 expression (P=0.004) as independent prognostic factors. CK18 expression in 83 pretreatment biopsy specimens was detected in 47 cases (56.6%) and also correlated with prognosis (P=0.045).Conclusion:CK18/CK8 expression correlated with progression of OSCC. The significant correlation with prognosis and stable expression in biopsy specimen suggest usefulness of CK18 in selection of treatment strategies for OSCC.

Cell Surface Tetraspanin CD9 Mediates Chemoresistance in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive malignancy with extremely high mortality due to the appearance of widespread metastases early in its clinical course and rapid acquisition of chemoresistance after initial therapy. A theory of cell adhesion-mediated drug resistance is thought to be a principal mechanism in which extracellular matrix proteins provide a survival advantage against cytotoxic drug-induced apoptosis. We found that the tetraspanin family member CD9 was expressed preferentially in SCLC tumors and metastases from three of seven relapsed patients, whereas chemonaïve primary tumors from 16 patients were CD9 negative with only one exception. Additionally, CD9 was highly expressed on SCLC cell lines rendered resistant to cisplatin or etoposide, and was upregulated in parental chemosensitive cells within 48 hours after exposure to either of these compounds. CD9-expressing chemoresistant SCLC cells adhered more tightly to fibronectin via β1 integrin, but they were less motile than the respective chemosensitive parental lines. Notably, treatment of the chemoresistant cells with chemokine CXCL12 downregulated CD9 and transiently restored motility. Moreover, selective targeting of CD9 by treatment with specific monoclonal antibody ALB6 or a small interfering RNA triggered apoptosis in the chemoresistant cells. Taken together, our findings implicate CD9 in the cell adhesion-mediated drug resistance mechanism, highlighting CD9 as an attractive therapeutic target to improve therapeutic outcomes in SCLC.