Tomio Matsumura

NFκB2 Gene as a Novel Candidate that Epigenetically Responds to Interval Walking Training.

Physical fitness has been reported to decrease the risk of lifestyle-related diseases. The present study evaluated genome-wide methylation under the hypothesis that interval walking training (IWT) imparted beneficial effects on health, particularly by epigenetically ameliorating susceptibility to inflammation. We screened DNA from peripheral blood samples via genome-wide microarray for genes whose methylation was affected by IWT, paying special attention to promoter regions, and identified over 40 hyper- or hypo-methylated genes following IWT that were not witnessed in controls. We next selected genes in which the degree of methylation change in the promoter region was correlated with energy consumption following IWT. In this way, we found the NFκB2 gene to have increased methylation in multiple regions of its promoter sequence following participation in an exercise regimen. Next, IWT-induced NFκB2 hyper-methylation was confirmed by a quantitative PyroSequencing assessment of methylation in samples obtained from independent subjects who also underwent IWT. The increase in NFκB2 gene promoter methylation by IWT indicates that this regimen may suppress pro-inflammatory cytokines. Thus, these results provide an additional line of evidence that IWT is advantageous in promoting health from an epigenetic perspective by ameliorating susceptibility to inflammation.

Clinical significance of apoptosis-associated speck-like protein containing a caspase recruitment domain in oral squamous cell carcinoma

OBJECTIVES To assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) expression in oral squamous cell carcinoma (OSCC) and analyze its clinical and pathological significance. STUDY DESIGN ASC expression was studied using immunohistochemistry in 119 OSCCs patients. The relationships between ASC expression and clinical and pathological parameters were statistically analyzed. In addition, the relationships between ASC expression and cell differentiation [IVL (involcrin) expression] and apoptosis (TUNEL [TdT-mediated dUTP nick end labeling] positive cell number) were investigated. RESULTS ASC expression showed significant correlations with parameters including clinical tumor stage, mode of invasion, and histological differentiation, and had a significant impact on survival of OSCC. The distribution of ASC correlated well with that of IVL. ASC expression was significantly correlated with the TUNEL-positive cell number. CONCLUSIONS Lower ASC expression correlates with clinical and pathological malignancy and, consequently, poor prognosis of OSCC. ASC has a close association with cell differentiation and apoptosis.

ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication

ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy.

Novel role of ASC as a regulator of metastatic phenotype.

Disorders of cytoskeletal remodeling and signal transduction are frequently involved in cancer progression. In particular, apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (ASC) has been reported a proapoptotic molecule that is epigenetically silenced in several human cancers. ASC is a well‐characterized adaptor protein involved in the formation of multiprotein oligomers, called inflammasomes, and plays a crucial role in the activation and secretion of interleukin‐1β and interleukin‐18 in innate immune cells. However, the function of ASC in the regulation of tumor progression remains elusive. The present investigation examined the involvement of ASC in cancer progression and the acquisition of metastatic ability. To determine the effect of ASC depletion in in vitro and in vivo model systems, ASC was stably knocked down in B16 murine melanoma cell lines using retroviral transduction of shRNA. ASC suppression increased the motility of B16BL6 cells in scratch assays and augmented invasiveness as assessed by a Matrigel‐coated transwell system. Invadopodia formation and Src phosphorylation level were markedly enhanced in ASC‐knockdown cells as well. Since caspase‐8 has been reported to enhance cellular migration by Tyr380 phosphorylation via Src, we examined Tyr380 phosphorylation of caspase‐8 in ASC‐knockdown cells and found it to be elevated in ASC‐knockdown cells but attenuated by z‐VAD‐fmk or z‐IETD‐fmk. Moreover, ASC ablation increased pulmonary metastasis in mice after intravenous injection of B16BL6 cells. Our cumulative findings indicate that ASC suppresses cancer metastasis and progression via the modulation of cytoskeletal remodeling and the Src‐caspase‐8 signaling pathway.

Survival pathway of cholangiocarcinoma via AKT/mTOR signaling to escape RAF/MEK/ERKpathway inhibition by sorafenib

Cholangiocarcinoma (CCC) is a strongly aggressive malignancy for which surgical resection is the only potential curative therapy. Sorafenib, a multikinase inhibitor of the RAF/MEK/ERK pathway, is a molecular-targeted drug that is approved for hepatocellular carcinoma (HCC) but not for CCC. The differences in signaling pathway characteristics under sorafenib treatment between HCC (HLF, Huh7, PLC/PRF/5) and CCC (RBE, YSCCC, Huh28) cell lines were therefore investigated using cell proliferation, western blotting, and apoptosis analyses. Sorafenib inhibited cell growth significantly less in CCC cells than in HCC cells, with lower suppression of ERK phosphorylation. Significantly decreased AKT Ser473 phosphorylation in HCC cells, and conversely enhanced phosphorylation of AKT Ser473 and mTORC2 in CCC cells, were observed with sorafenib treatment. Disassembly of the mTORC2 complex in RBE cells with siRNA targeting Rictor resulted in the downregulation of AKT Ser473 phosphorylation and enhanced apoptosis presumably via increased FOXO1, which consequently suppressed RBE cell proliferation. Phosphorylation of mTORC1 and autophagy were not influenced by sorafenib in CCC cells. Simultaneous administration of everolimus to suppress activated mTORC1 in RBE cells revealed that combined everolimus and sorafenib treatment under mTORC2 disassembly could enhance growth inhibition through the suppression of both sorafenib- and everolimus-dependent AKT Ser473 phosphorylation in addition to the inhibition of mTORC1 phosphorylation. Prevention of escape by AKT/mTOR signaling from the RAF/MEK/ERK pathway in sorafenib treatment by suppressing mTORC2 activity may lead to promising new approaches in CCC therapy.

Potential Role of ASC, a Proapoptotic Protein, for Determining the Cisplatin Susceptibility of Lung Cancer Cells

Primary lung cancer is the most frequent cause of cancer-related deaths worldwide. Cisplatin has been used as a key drug in the treatment for patients with lung cancer; however, most of the patients failed to respond to cisplatin within several months, and the mechanisms underlying the cisplatin resistance have not been fully elucidated. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a key adaptor protein in the formation of inflammasomes. ASC is also involved in apoptotic signaling. Importantly, ASC expression is decreased in lung cancer and various cancers, but its precise function in tumor progression remains unknown. To explore the hitherto unknown role of ASC in lung cancer, we initially searched for lung cancer cell lines with higher expression levels of ASC using Cancer Cell Line Encyclopedia (CCLE) database, thereby identifying the A549 human non-small cell lung cancer cell line. Accordingly, with retroviral shRNA, the expression of ASC was forced to decrease in A549 cells. Stable ASC-knockdown cells, thus established, showed the increased activities of proliferation, motility, and invasion, compared with control cells. Importantly, ASC-knockdown cells also became resistant to cisplatin, but not to other anti-cancer agents, 5-fluorouracil and paclitaxel. Bcl-2 and phospho-Src levels were increased in ASC-knockdown cells. A Bcl-2 inhibitor, ABT-199, induced an apoptotic response in ASC-knockdown cells, and dasatinib, a Src inhibitor, blocked cell invasiveness. Thus, ASC may be involved in tumor suppression and cell death via Bcl-2 and pSrc. Targeting Bcl-2 and Src in ASC-downregulated populations of lung cancer may improve treatment outcome.

Fascin1 Suppresses RIG-I-like Receptor Signaling and Interferon-β Production by Associating with IκB Kinase ϵ (IKK ϵ) in Colon Cancer

Fascin1 is an actin-bundling protein involved in cancer cell migration and has recently been shown also to have roles in virus-mediated immune cell responses. Because viral infection has been shown to activate immune cells and to induce interferon-β expression in human cancer cells, we evaluated the effects of fascin1 on virus-dependent signaling via the membrane- and actin-associated protein RIG-I (retinoic acid-inducible gene I) in colon cancer cells. We knocked down fascin1 expression with shRNA retrovirally transduced into a DLD-1 colon cancer and L929 fibroblast-like cell lines and used luciferase reporter assays and co-immunoprecipitation to identify fascin1 targets. We found that intracellular poly(I·C) transfection to mimic viral infection enhances the RIG-I/MDA5 (melanoma differentiation-associated gene 5)-mediated dimerization of interferon regulatory factor 3 (IRF-3). The transfection also significantly increased the expression levels of IRF-7, interferon-β, and interferon-inducible cytokine IP-10 in fascin1-deleted cells compared with controls while significantly suppressing cell growth, migration, and invasion. We also found that fascin1 constitutively interacts with IκB kinase ϵ (IKKϵ) in the RIG-I signaling pathway. In summary, we have identified fascin1 as a suppressor of the RIG-I signaling pathway associating with IκB kinase ϵ in DLD-1 colon cancer cells to suppress immune responses to viral infection.

Abstract A23: Anti-PD-1 antibody scFv producing recombinant Bifidobacterium exerts antitumor effect in a larger fraction of the treated mice compared to full-length anti-PD-1 antibody

Anti-PD-1 therapy has improved therapeutic outcomes of patients in multiple cancer types. However, the therapy has demonstrated clinical benefits in only a small fraction of patients. The reason of the limited response in clinical practice is not fully understood. In an aim to improve anti-cancer drug delivery and potency, we have been developing in situ delivery and production system (i-DPS) by modifying a non-pathogenic anaerobic bacterium, Bifidobacterium, which localizes and proliferates only in the hypoxic environment like solid tumors after intravenous administration, produces anticancer proteins, enzymes or other pharmacologically active molecules selectively at the tumor site. Here we present anti-human PD-1 antibody scFv producing i-DPS in cancer immunotherapy, which could be specifically delivered to and amplified only at the hypoxic sites of solid tumors. A series of in vitro assays has been performed to confirm the stable expression and secretion of anti-human PD-1 scFv by recombinant Bifidobacterium, the binding inhibition of PD-1/PD-L1 interaction and elevated IFN gamma production in mixed lymphocyte culture by anti-human PD-1 scFv secreted from recombinant Bifidobacterium. Anti-murine PD-1 scFv producing Bifidobacterium as surrogate systemically administered to the syngeneic mice model demonstrated significant tumor growth inhibition. Of particular interest, the suppression of tumor growth was observed in a larger fraction of the treated mice while the control anti-PD-1 antibody showed the effect on only a few mice. The analysis of tumor infiltrating lymphocytes and myeloid cells will be presented as well. Taken together, i-DPS for anti-PD-1 antibody provides a new promising immune-therapeutic modality to target hypoxic solid tumors and also provides a unique insight for antibody drug delivery in cancer immunotherapy. Citation Format: Koichiro Shioya, Li Wang, Tomio Matsumura, Hitomi Shimizu, Yasuyoshi Kanari, Yuji Seki, Yuko Shimatani, Shun’ichiro Taniguchi, Shiro Kataoka. Anti-PD-1 antibody scFv producing recombinant Bifidobacterium exerts antitumor effect in a larger fraction of the treated mice compared to full-length anti-PD-1 antibody [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A23.

Inhibition of PC3 human prostate cancer cell proliferation, invasion and migration by eicosapentaenoic acid and docosahexaenoic acid

The n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), found in fish oil, exert a number of beneficial effects, and they are used in the treatment of hyperlipidemia. In recent years, EPA and DHA have been found to affect cancer cell proliferation. In the present study, PC3 cells, which are androgen-independent prostate cancer cells that resemble castration-resistant prostate cancer cells, were used to investigate a possible novel treatment for castration-resistant prostate cancer. The PC3 cells were cultured and incubated with various concentrations of EPA or DHA. Cancer proliferation was confirmed by trypan blue microscopy. Invasion and migration assays were used in the upper chamber in PC3 cells, and serum-free medium and various concentrations of EPA or DHA were placed in the lower chamber in serum-containing medium. EPA and DHA decreased PC3 cell proliferation, invasion and migration. The effect of EPA on PC3 cells was dose-dependent and significant differences were observed at concentrations of 100 and 200 µg/ml. The effect of DHA on PC3 cells was similar to that of EPA. In the migration assay, EPA exerted almost no effects at 25 µg/ml, but migration was reduced at 50 µg/ml. Similar to EPA, DHA exerted almost no effects at 25 µg/ml, but further reduction was observed at the 50 µg/ml concentration. In the invasion assay, EPA at 25 µg/ml was not significantly different from the control, but suppressed invasion at 50 µg/ml. DHA decreased invasion compared with the control at 25 µg/ml, whereas invasion was significantly reduced at a DHA concentration of 50 µg/ml. In conclusion, it was demonstrated that EPA and DHA were effective in decreasing the proliferation, invasion and migration of prostate PC3 cancer cells. However, the detailed underlying mechanisms have not yet been fully elucidated.

Abstract A29: Anti-CTLA-4 antibody scFv producing recombinant Bifidobacterium secretes CTLA-4 blocker specifically inside hypoxic tumor and suppresses tumor growth in syngeneic mice model

Success of immune checkpoint antibody drugs has provided broad perspective in cancer immunotherapy. Anti-PD-1 antibody and anti-CTLA-4 antibody showed notable efficacy and the combination complimentarily enhanced antitumor benefit. Nevertheless, either single-agent therapy or combination therapy is facing immune-related adverse events (irAEs), which is major cause of treatment discontinuation. Also, still large fraction of cancer patients doesn9t respond to the antibody immunotherapy. Since non-specific systemic activation of normal immune system by the therapy is one of major reasons to cause the above problems, approaches to target the hypoxic condition of tumors may help increasing the drug9s tumor-targeting specificity, which results in minimizing the irAEs and improving the response rate in patients. In an aim to improve anti-cancer drug delivery efficacy, we have been developing in situ Delivery and Production System ( i -DPS) by modifying a non-pathogenic anaerobic bacterium, Bifidobacterium , which localizes and proliferates only in the hypoxic environment like solid tumors after intravenous administration, produces anticancer proteins, enzymes or other pharmacologically active molecules selectively at the tumor site. Here we present anti-human CTLA-4 antibody scFv producing i -DPS in cancer immunotherapy, which could be specifically delivered to and amplified only at the hypoxic sites of solid tumors. A series of in vitro assays has been performed to confirm the stable expression and secretion of anti-hCTLA-4 scFv by recombinant Bifidobacterium , the binding affinity of purified anti-hCTLA-4 scFv and the IC50 competing with hCTLA-4/hCD80 or hCD86 interaction as well. Anti-murine CTLA-4 scFv producing Bifidobacterium as surrogate systemically administered to the syngeneic mice model demonstrated significant tumor growth inhibition. Moreover, the combination therapy of anti-mCTLA-4 scFv producing Bifidobacterium and anti-murine PD-1 antibody was more effective than each of monotherapy. It is noteworthy that scFv was only detected in tumor tissue but not in blood proved by immunoprecipitation assay. Altogether, i -DPS for anti-CTLA-4 antibody provide a new promising immune-therapeutic modality to target hypoxic solid tumors and also provide a unique insight for antibody drug delivery in cancer immunotherapy. Citation Format: Koichiro Shioya, Shiro Kataoka, Li Wang, Tomio Matsumura, Hitomi Shimizu, Yasuyoshi Kanari, Yuji Seki, Yuko Shimatani, Minoru Fujimori, Shun9ichiro Taniguchi. Anti-CTLA-4 antibody scFv producing recombinant Bifidobacterium secretes CTLA-4 blocker specifically inside hypoxic tumor and suppresses tumor growth in syngeneic mice model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A29.