Kota Iwahori

Identification of CD72 as a lymphocyte receptor for the class IV semaphorin CD100: a novel mechanism for regulating B cell signaling.

We have identified the lymphocyte semaphorin CD100/Sema4D as a CD40-inducible molecule by subtractive cDNA cloning. CD100 stimulation significantly enhanced the effects of CD40 on B cell responses. Administration of soluble CD100 markedly accelerated in vivo antigen-specific antibody responses. CD100 receptors with different binding affinities were detected on renal tubular cells (K(d) = approximately 1 x 10(-9)M) and lymphocytes (K(d) = approximately 3 x 10(-7)M). Expression cloning revealed that the CD100 receptor on lymphocytes is CD72, a negative regulator of B cell responsiveness. CD72 thus represents a novel class of semaphorin receptors. CD100 stimulation induced tyrosine dephosphorylation of CD72 and dissociation of SHP-1 from CD72. Our findings indicate that CD100 plays a critical role in immune responses by the novel mechanism of turning off negative signaling by CD72.

iTRAQ-based proteomic identification of leucine-rich α-2 glycoprotein as a novel inflammatory biomarker in autoimmune diseases

Objective To identify a novel serum biomarker of disease activity in inflammatory autoimmune disorders. Methods Sera obtained from rheumatoid arthritis (RA) patients before and after anti-TNF therapy were analysed by iTRAQ (isobaric tags for relative and absolute quantitation) quantitative proteomic analysis and further validated by ELISA. Results Of 326 proteins identified by proteomic analysis, increased serum levels of leucine-rich α-2 glycoprotein (LRG) was identified in RA patients before therapy. Serum LRG concentrations were significantly elevated in RA patients compared with healthy controls and decreased after anti-TNF therapy. Furthermore, serum LRG concentrations correlated with disease activity in RA and Crohns disease (CD). Interestingly, in a subpopulation of patients with active CD and normal C-reactive protein levels, serum LRG concentrations were elevated. Conclusions LRG represents a novel serum biomarker for monitoring disease activity during therapy in autoimmune patients, particularly useful in patients with active disease but normal CRP levels.

Megakaryocyte potentiating factor as a tumor marker of malignant pleural mesothelioma: Evaluation in comparison with mesothelin

PURPOSE An early and reliable blood test is one deficiency in diagnosis of malignant pleural mesothelioma (MPM). Megakaryocyte potentiating factor (MPF) and mesothelin variants (MSLN), members of the mesothelin gene family, have been studied as candidate serum markers for MPM. We developed a novel enzyme-linked immunosorbent assay (ELISA) system to compare the diagnostic efficacy of MPF and MSLN in MPM and control groups. EXPERIMENTAL DESIGN MPF and MSLN were assayed with ELISA in 27 consecutive MPM patients and 129 controls including patients with lung cancer and asymptomatic asbestos-exposed subjects. RESULTS Statistically significant elevation of serum MPF and MSLN levels was noted in MPM patients in comparison with every control group. The area under the receiver operating characteristic curve (AUC) was calculated for differentiation of MPM and lung cancer, healthy asbestos-exposed subjects, and healthy adults. While the AUC for serum MPF was 0.879, cut-off=19.1ng/ml (sensitivity=74.1%, specificity=90.4%), the AUC for serum MSLN was 0.713, cut-off=93.5ng/ml (sensitivity=59.3%, specificity=86.2%). Comparison between AUC for MPF and MSLN values shows that MPF is significantly superior to MSLN (p=0.025). Finally, there was a significant correlation between MPF and MSLN values for MPM (Pearsons correlation coefficient=0.77; p<0.001). CONCLUSIONS These findings suggest that diagnostic value of MPF for MPM was better than that of MSLN although both markers showed almost equal specificity for MPM.

Overexpression of SOCS3 exhibits preclinical antitumor activity against malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis for which an effective therapy remains to be established. Our study investigated the therapeutic potential of the suppressor of cytokine signaling 3 (SOCS3), an endogenous inhibitor of intracellular signaling pathways, for treatment of MPM. We infected MPM cells (H226, EHMES‐1, MESO‐1 and MESO‐4) with an adenovirus‐expressing SOCS3 (AdSOCS3) to examine the effect of SOCS3 overexpression on MPM cells. SOCS3 overexpression reduced MPM proliferation and induced apoptosis and partial G0/G1 arrest. SOCS3 also inhibited the proliferation of MPM cells via multiple signaling pathways including Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), extracellular signal‐regulated kinase (ERK), focal adhesion kinase (FAK) and p53 pathways. Notably, AdSOCS3 treatment inhibited tumor growth in an MPM pleural xenograft model. These findings demonstrate that overexpression of SOCS3 has a potent antitumor effect against MPM both in vitro and in vivo and indicate the potential for clinical use of SOCS3 for MPM treatment.

Plasma membrane proteomics identifies bone marrow stromal antigen 2 as a potential therapeutic target in endometrial cancer

This report utilizes a novel proteomic method for discovering potential therapeutic targets in endometrial cancer. We used a biotinylation‐based approach for cell‐surface protein enrichment combined with isobaric tags for relative and absolute quantitation (iTRAQ) technology using nano liquid chromatography–tandem mass spectrometry analysis to identify specifically overexpressed proteins in endometrial cancer cells compared with normal endometrial cells. We identified a total of 272 proteins, including 11 plasma membrane proteins, whose expression increased more than twofold in at least four of seven endometrial cancer cell lines compared with a normal endometrial cell line. Overexpression of bone marrow stromal antigen 2 (BST2) was detected and the observation was supported by immunohistochemical analysis using clinical samples. The expression of BST2 was more characteristic of 118 endometrial cancer tissues compared with 59 normal endometrial tissues (p < 0.0001). The therapeutic effect of an anti‐BST2 antibody was studied both in vitro and in vivo. An anti‐BST2 monoclonal antibody showed in vitro cytotoxicity in BST2‐positive endometrial cancer cells via antibody‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity. In an in vivo xenograft model, anti‐BST2 antibody treatment significantly inhibited tumor growth of BST2‐positive endometrial cancer cells in an NK cell‐dependent manner. The anti‐BST2 antibody had a potent antitumor effect against endometrial cancer both in vitro and in vivo, indicating a strong potential for clinical use of anti‐BST2 antibody for endometrial cancer treatment. The combination of biotinylation‐based enrichment of cell‐surface proteins and iTRAQ analysis should be a useful screening method for future discovery of potential therapeutic targets.

ICOS+ Foxp3+ TILs in gastric cancer are prognostic markers and effector regulatory T cells associated with Helicobacter pylori

Regulatory T cells (Tregs) have an immunosuppressive role in the tumor microenvironment. Since effector Tregs (eTregs), which have highly suppressive functions, are located in a subpopulation of Foxp3+ CD4+ Tregs, the TCR‐inducible costimulatory receptor (ICOS) was applied as a marker of eTregs that infiltrated gastric cancer tissue and the induction pathway of ICOS+ Foxp3+ cells was analyzed by flow cytometry and immunohistochemistry. In tumor‐infiltrating lymphocytes (TILs), ICOS+ Foxp3+ CD4+ T cells were abundantly observed in the late stages of gastric cancer. ICOS+ CD4+ TILs exhibited the ability to produce IL‐10, but not IFN‐γ, TNF, or IL‐17 and also to suppress the proliferation of CFSE‐labeled responder CD8+ T cells. With the agonistic ICOS‐L protein (rICOS‐L Ig), ICOS+ Foxp3+ cells were efficiently induced from naive CD4+ T cells under a stimulation with TGF‐β and CD3/CD28 mAbs. Furthermore, when A*0201 PBMCs were cultured with the CMV or Melan‐A antigenic peptide and rICOS‐L Ig, the induction of CMV or Melan‐A tetramer‐binding CD8+ T cells, respectively, was inhibited. The expression of ICOS in Foxp3+ cells was closely related to plasmacytoid dendritic cells (pDCs) and their expression of ICOS‐L and TLR9 as well as Helicobacter pylori infection. Collectively, our results demonstrate the potential of ICOS as a promising target for direct Treg‐targeting therapeutic agents for gastric cancer, and that of eradicating therapy for H. pylori as an indirect immune therapy for gastric cancer.

SOCS‐1 gene delivery cooperates with cisplatin plus pemetrexed to exhibit preclinical antitumor activity against malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis for which an effective therapy remains to be established. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS‐1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of MPM. We infected MPM cells (MESO‐4, H28 and H226) with adenovirus‐expressing SOCS‐1 vector to examine the effect of SOCS‐1 overexpression on MPM cells. We evaluated the antitumor effect of SOCS‐1 gene delivery combined with cisplatin plus pemetrexed by cell proliferation, apoptosis and invasion assay. We also investigated the regulation of NF‐κB and STAT3 signaling related to apoptotic pathways. Furthermore, we evaluated the inhibition of tumor growth by SOCS‐1 gene delivery combined with cisplatin plus pemetrexed in vivo. SOCS‐1 gene delivery cooperated with cisplatin plus pemetrexed to inhibit cell proliferation, invasiveness and induction of apoptosis in MPM cells. SOCS‐1 regulated NF‐κB and STAT3 signaling to induce apoptosis in MESO‐4 and H226 cells. Furthermore, SOCS‐1 gene delivery cooperated with cisplatin plus pemetrexed to regulate NF‐κB signaling and significantly inhibit tumor growth of MPM in vivo. These results suggest that SOCS‐1 gene delivery has a potent antitumor effect against MPM and a potential for clinical use in combination with cisplatin plus pemetrexed.

Molecular mechanism underlying the antiproliferative effect of suppressor of cytokine signaling-1 in non-small-cell lung cancer cells

Lung cancer (LC) is the major cause of death by cancer and the number of LC patients is increasing worldwide. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS‐1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of LC. To examine the antitumor effect of SOCS‐1 overexpression on non‐small‐cell lung cancer (NSCLC) cells, NSCLC cells (A549, LU65, and PC9) were infected with adenovirus‐expressing SOCS‐1 vector. The cell proliferation assay showed that A549 and LU65, but not PC9, were sensitive to SOCS‐1 gene‐mediated suppression of cell growth. Although JAK inhibitor I could also inhibit proliferation of A549 and LU65 cells, SOCS‐1 gene delivery appeared to be more potent as SOCS‐1 could suppress focal adhesion kinase and epidermal growth factor receptor, as well as the JAK/STAT3 signaling pathway. Enhanced phosphorylation of the p53 protein was detected by means of phospho‐kinase array in SOCS‐1 overexpressed A549 cells compared with control cells, whereas no phosphorylation of p53 was observed when JAK inhibitor I was used. Furthermore, treatment with adenoviral vector AdSOCS‐1 in vivo significantly suppressed NSCLC proliferation in a xenograft model. These results suggest that the overexpression of SOCS‐1 gene is effective for antitumor therapy by suppressing the JAK/STAT, focal adhesion kinase, and epidermal growth factor receptor signaling pathways and enhancing p53‐mediated antitumor activity in NSCLC.

NY-ESO-1 Protein Cancer Vaccine With Poly-ICLC and OK-432: Rapid and Strong Induction of NY-ESO-1-specific Immune Responses by Poly-ICLC.

We conducted a clinical trial of a cancer vaccine using NY-ESO-1 protein with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) and/or OK-432 against solid tumors. A total of 15 patients were sequentially enrolled in 4 cohorts. Patients in cohort 1 received NY-ESO-1 protein; cohort 2a received NY-ESO-1 protein+OK-432; cohort 2b received NY-ESO-1 protein+poly-ICLC; cohort 3 received NY-ESO-1 protein+OK-432+poly-ICLC with Montanide ISA-51. The endpoints of this trial were safety, NY-ESO-1 immune responses, and clinical response. Vaccine-related adverse events observed were fever and injection-site reaction (grade 1). Two patients showed stable disease after vaccination. NY-ESO-1 antibodies were observed in 4 patients at the baseline (sero-positive) and augmented in all patients after vaccination. Eleven patients showed a conversion of negative antibody responses at baseline to positive after vaccination (seroconversion). The seroconversions were observed in all 11 sero-negative patients by the fourth immunization; in particular, it was observed by the second immunization in patients with poly-ICLC, and these induced antibody responses were stronger than those in patients immunized without poly-ICLC. The number of NY-ESO-1–specific interferon (IFN)&ggr;-producing T cells was increased in patients immunized with poly-ICLC and/or OK-432, and furthermore, the increase of IFN&ggr;-producing CD8 T cells in patients immunized with poly-ICLC was significantly higher than that in patients without poly-ICLC. Nonspecific activations of T-cell or antigen presenting cells were not observed. Our present study showed that poly-ICLC is a promising adjuvant for cancer vaccines.

Clarifying the biological significance of the CHK2 K373E somatic mutation discovered in The Cancer Genome Atlas database

We identified CHK2 K373E as a recurrent mutation in The Cancer Genome Atlas (TCGA) database. In this study, we demonstrate that the K373E mutation disrupts CHK2 autophosphorylation as well as kinase activity, thus leading to impairment of CHK2 functions in suppressing cell proliferation and promoting cell survival after ionizing radiation. We propose that K373E impairs p53‐independent induction of p21WAF1/CIP1 by CHK2. Our data implicate the K373E mutation of CHK2 in tumorigenesis.