Shigeo Takaishi

IFN-γ Inhibits Gastric Carcinogenesis by Inducing Epithelial Cell Autophagy and T-Cell Apoptosis

IFN-γ mediates responses to bacterial infection and autoimmune disease, but it is also an important tumor suppressor. It is upregulated in the gastric mucosa by chronic Helicobacter infection; however, whether it plays a positive or negative role in inflammation-associated gastric carcinogenesis is unexplored. To study this question, we generated an H(+)/K(+)-ATPase-IFN-γ transgenic mouse that overexpresses murine IFN-γ in the stomach mucosa. In contrast to the expected proinflammatory role during infection, we found that IFN-γ overexpression failed to induce gastritis and instead inhibited gastric carcinogenesis induced by interleukin-1beta (IL-1β) and/or Helicobacter infection. Helper T cell (Th) 1 and Th17 immune responses were inhibited by IFN-γ through Fas induction and apoptosis in CD4 T cells. IFN-γ also induced autophagy in gastric epithelial cells through increased expression of Beclin-1. Finally, in the gastric epithelium, IFN-γ also inhibited IL-1β- and Helicobacter-induced epithelial apoptosis, proliferation, and Dckl1(+) cell expansion. Taken together, our results suggest that IFN-γ coordinately inhibits bacterial infection and carcinogenesis in the gastric mucosa by suppressing putative gastric progenitor cell expansion and reducing epithelial cell apoptosis via induction of an autophagic program.

Clinical outcomes of a novel therapeutic vaccine with Tax peptide-pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study

Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type‐I (HTLV‐I)‐infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV‐I Tax‐specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti‐ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate‐ to high‐risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax‐specific CTL responses were observed with peaks at 16–20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide‐pulsed DC vaccine is a safe and promising immunotherapy for ATL.

Human STEAP3 maintains tumor growth under hypoferric condition.

Iron is essential in cellular proliferation and survival based on its crucial roles in DNA and ATP synthesis. Tumor cells proliferate rapidly even in patients with low serum iron, although their actual mechanisms are not well known. To elucidate molecular mechanisms of efficient tumor progression under the hypoferric condition, we studied the roles of six-transmembrane epithelial antigen of the prostate family member 3 (STEAP3), which was reported to facilitate iron uptake. Using Raji cells with low STEAP3 mRNA expression, human STEAP3-overexpressing cells were established. The impact of STEAP3 expression was analyzed about the amount of iron storage, the survival under hypoferric conditions in vitro and the growth of tumor in vivo. STEAP3 overexpression increased ferritin, an indicator of iron storage, in STEAP3-overexpressing Raji cells. STEAP3 gave Raji cells the resistance to iron deprivation-induced apoptosis. These STEAP3-overexpressing Raji cells preserved efficient growth even in hypoferric mice, while parental Raji cells grew less rapidly. In addition, iron deficiency enhanced STEAP3 mRNA expression in tumor cells. Furthermore, human colorectal cancer tissues exhibited more STEAP3 mRNA expression and iron storage compared with normal colon mucosa. These findings indicate that STEAP3 maintains iron storage in human malignant cells and tumor proliferation under the hypoferric condition.

The phase-I study of a therapeutic vaccine to ATL patients with autologous dendritic cells pulsed with peptides corresponding to Tax-specific CTL epitopes

In the last decade, various kinds of clinical trials of hematopoietic stem cell transplantation (HSCT) have been performed for ATL patients, and showed therapeutic effects with long-term remission in a part of recipients. Our previous finding of activation of CD8+ Tax-specific cytotoxic T-lymphocytes (CTL) in post-HSCT ATL patients indicated the presence of Tax expression in vivo and potential contributions of CTL to GVL effects. These findings let us attempt developing an anti-ATL therapeutic vaccine consisting of autologous dendritic cells (DC) pulsed with Tax peptides corresponding to the major epitopes of Tax-specific CTL previously identified from HLA-A2, A24 or A11-possessing post-HSCT ATL patients. In preliminary experiments, the DC induced with a conventional method showed matured phenotype and produced IL-12 in 2 of 3 ATL patients tested. Under the official approval by the institutional ethical committees, we conducted the phase-I clinical study of anti-ATL immunotherapy for ATL patients at stable conditions after other therapy. The peptide-pulsed DC was subcutaneously injected for three times with 2 weeks intervals. The first patient showed a significant reduction in the proviral loads in 1 week after administration of DC, which gradually increase during the treatment, then decreased again at 8 weeks after the initiation of the therapy. Reduction in the size of surface lymph nodes by CT confirmed the therapeutic effects. So far two patients completed the course of the study without severe side effects except for fever and skin rash, and their clinical outcomes were partial remission and stable disease respectively.

Efficacy and safety of an increased-dose of dexamethasone in patients receiving fosaprepitant chemotherapy in Japan

BACKGROUND Antiemetic triplet therapy including dexamethasone (DEX) is widely used for patients receiving highly emetogenic chemotherapy (HEC). In Japan, the appropriate dose of DEX has not been established for this combination. MATERIALS AND METHODS To assess the efficacy and safety of increased-dose DEX, we retrospectively examined patients receiving HEC with antiemetic triplet therapy. RESULTS Twenty-four patients (fosaprepitant group) were given an increased-dose of DEX (average total dose: 45.8mg), fosaprepitant, and 5-HT3 antagonist. A lower-dose of DEX (33.6mg), oral aprepitant, and 5-HT3 antagonist were administered to the other 48 patients (aprepitant group). The vomiting control rates in the fosaprepitant and aprepitant groups were 100% and 85.4% in the acute phase, and were 75.0% and 64.6% in the delayed phase. The incidences of toxicity were similar comparing the two groups. CONCLUSIONS Triplet therapy using an increased-dose of DEX is suggested to be safe and effective for patients receiving HEC.

E-cadherin regulates proliferation of colorectal cancer stem cells through NANOG

Cancer stem cells (CSCs) possess a self-renewal ability and display tumorigenic potential in immunodeficient mice. Colorectal CSCs are thought to be a uniform population and no functionally distinct subpopulations have been identified. Because E-cadherin is an essential molecule for self-renewal of embryonic stem cells, we examined E-cadherin expression, which may play a role in maintaining the properties of CSCs, in EpCAMhigh/CD44+ colorectal CSCs from human primary colorectal cancers. We obtained 18 surgical specimens of human primary colorectal cancer. CD44, EpCAM, and E-cadherin expression were analyzed by fluorescence-activated cell sorting. Sorted EpCAMhigh/CD44+ colorectal CSCs were injected into immunodeficient mice to estimate the tumorigenic potential. Genetic profiles were analyzed by cDNA microarray. Notably, colorectal CSCs could be divided into two populations based on the E-cadherin expression status, and they exhibited different pathological characteristics. Compared to E-cadherin-negative colorectal CSCs, E-cadherin-positive (EC+) colorectal CSCs demonstrated higher tumor growth potential in vivo. EC+ colorectal CSCs revealed a higher expression of the pluripotency factor NANOG, which contributed to the higher tumor growth potential of EC+ colorectal CSCs through control of cyclin D1 expression. These findings are the first demonstration of functionally distinct subpopulations of colorectal CSCs in human clinical samples.

Abstract 714: Macrophage-to-fibroblast transition promotes cancer progression in peritoneal carcinomatosis of gastrointestinal cancer patient

[Background] Cancer stromal cell plays an important role in cancer progression. Fibroblasts localized in tumor are especially called cancer-associated fibroblasts (CAFs). CAFs and inflammatory cells form tumor microenvironment and promote cancer growth through the direct or indirect interaction between cancer cells and stromal cells. However, the origin of CAF is not fully understood. Malignant ascites contains not only cancer cells but inflammatory cells including macrophage. Accumulation of macrophages and fibrosis has close relationship. In the research field of fibrotic diseases such as renal fibrosis, some reports indicated macrophages were able to change to fibroblasts phenotypically. Peritoneal carcinomatosis also develops peritoneal fibrosis. We demonstrate that malignant ascites are abundant in macrophages and these macrophages changed to CAFs which promote cancer progression in vivo. [Material and method] Ascitic samples from 44 peritoneal carcinomatosis patients due to gastrointestinal cancer were collected at 5 institutions. This study was approved by each institutional review board. Ascites was separated into cell fraction and supernatant by centrifugation. Supernatant was stored at -20°C. Cells were sorted by FACS using anti-CD45, anti-CD14, anti-CD163 and anti-CD90 antibodies. CD45+CD14+ macrophages were cultured in RPMI medium containing fetal bovine serum (FBS) or supernatant of ascites. Human colorectal cancer cell line DLD-1 cells in combination with the cultured cells from ascites were inoculated to immunodeficient mice subcutaneously. All experiments were conducted following the guidelines of the institutional animal committee of Kyushu University. [Result] CD45+CD14+ macrophage was most frequently observed in CD45+ leukocyte fraction from ascites. Most of macrophages expressed M2 marker (CD163). Some of these macrophages changed to CD45-CD90+ fibroblast-like cells which form spindle shape after 2-3 weeks culture. These fibroblast-like cells expressed fibroblast specific genes such as COL3A1, ACTA2 and FAP. These changes were enhanced by ascites supernatant-containing medium compared with FBS-containing medium. DLD-1 cells with the fibroblast-like cells formed larger tumors in immunodeficient mice, compared with DLD-1 cells alone. [Conclusion] In peritoneal carcinomatosis, macrophage is a potential source of CAF. This macrophage-to-CAF transition is enhanced by malignant ascitic environment. As CAF induced from macrophage enhances tumor progression, inhibition of this transition could be possible therapeutic strategy. Citation Format: Mamoru Tanaka, Michitaka Nakano, Hiroshi Ariyama, Kyoko Inadomi, Risa Tanaka, Shigeo Takaishi, Hitoshi Kusaba, Eishi Baba, Koichi Akashi. Macrophage-to-fibroblast transition promotes cancer progression in peritoneal carcinomatosis of gastrointestinal cancer patient. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 714.

Abstract 1707: Epithelial mesenchymal transition generates cancer stem cells in CD44- colorectal cancer cells

Background: EpCAMhigh CD44+colorectal cancer (CRC) cells are thought to be cancer stem cells. Recently CD44- CRC cells are also suggested to acquire a property of cancer stem cells. Epithelial-mesenchymal transition (EMT) is a possible process of acquisition of cancer stem cell (CSC)-like properties. However, it is unclear whether EMT can be induced in primary human CRC cells. Patients and Methods: We obtained surgical specimens from 51 CRC patients, and cultured isolated cancer cells on matrigel-coated dish with medium containing growth factors. For induction of EMT, TGF-beta was added into the culture medium. Otherwise, TWIST1 expression was enforced in the cells by using lentiviral transduction. Immunocytochemical analysis, flow cytometric analysis and single cell PCR analysis using 24-genes set containing embryonic stem cell (ES)-related and EMT-related genes were performed. PCR analysis was carried out by C1 single cell auto prep system. CD44- CRC cells with or without enforced expression of TWIST1 were injected into immunodeficient mice. Results: Fifteen out of 51 samples of cancer cells formed sphere (>50 μm in diameter) after one week culture. The sphere-forming ability was related with clinical stage (Stage 1 and 2;16.7%, Stage 3 and 4;41.3%). Sorted single CD44+ cell had higher sphere-forming ability, compared to CD44- cell. The higher expression of ES- and EMT-related genes was observed in short term culture than in long-term culture, suggesting that differentiation occurred in sphere cells after long-term culture. Single cell PCR analysis revealed that sphere-forming cells were classified into 2 different populations on the basis of primary component analysis. Correlation analysis showed expression of TWIST1 and ES-related genes were correlated. In addition, flow cytometric analysis revealed that sphere forming CD44+ cells gave rise to CD44- cells. These results suggest that CD44+ cells have an ability to reconstruct the heterogeneous population, and EMT is involved in acquisition of CSC-like properties. TGF-beta increased the number of CD44+ cells in CD44- cells, and enhanced sphere-forming ability of CD44- cells. TGF-beta also induced expression of ES-related genes and TWIST1. Enforced expression of TWIST1 induced sphere-forming ability and tumorigenicity in CD44- cells. Conclusions: We established the culture system to observe the differentiation of CSCs and revealed that EMT might be involved in maintenance of CSCs. We firstly demonstrated that primary CD44- CRC cells undergo EMT and become CD44+ cells by TGF-beta treatment or enforced expression of TWIST1. Citation Format: Michitaka Nakano, Mamoru Tanaka, Taichi Isobe, Kohta Miyawaki, Yoshikane Kikushige, Hitoshi Kusaba, Shigeo Takaishi, Takashi Ueki, Eishi Baba, Koichi Akashi. Epithelial mesenchymal transition generates cancer stem cells in CD44- colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1707.

Abstract 4795: CCK2R marks gastric antral stem cell and mediates antral carcinogenesis

Gastrin is a hormone that binds to the CCK2 receptor and promotes proximal gastric cancer, but inhibits distal gastric cancer development. However, the precise roles of the CCK2 receptor, and its alternative ligand, progastrin, in gastric carcinogenesis have not been clarified. In this study, we found that progastrin accelerated antral proliferation and carcinogenesis through CCK2R+ antral stem cell expansion, using mouse gastric cancer models and transgenic mice lines including human progastrin-overexpressing (hGAS) mice, CCK2R knockout mice, Lgr5-CreERT-IRES-GFP knockin mice, and newly generated CCK2R-BAC-CreERT mice. Progastrin promoted gastric antral cancer development induced by MNU and/or H. felis. During carcinogenesis, progastrin increased the expression of Lgr5 and gland fission in response to the chemical carcinogen MNU. Genetic ablation of CCK2R diminished these progastrin-mediated effects. In vitro 3D culture experiments revealed that progastrin, but not amidated gastrin, significantly increased gastric organoid formation and growth in Noggin-free condition, effects that were ablated by a CCK2R inhibitor YF-476 or CCK2R gene deletion. In the antrum, CCK2R was expressed in an Lgr5low cell population that displayed stemness, which could be enhanced by progastrin. In the presence of progastrin, Lgr5lowCCK2R+ cells interconverted to Lgr5hi cells. Finally, we generated a new BAC-transgenic CCK2R-CreERT murine line, and lineage tracing experiments showed that CCK2R+ cells, which reside slightly above the base of the antrum, contained long-lived stem cells in vivo and in vitro. Chemical inhibition of CCK2R attenuated progastrin-dependent cancer development in mice. In conclusion, CCK2R labels Lgr5low antral stem cells that can be activated and expanded by progastrin. These findings may help the understanding of the underlying mechanism in gastric stem cell regulation by a CCK2R signal. Citation Format: Hayakawa Yoku, Guanchun Jin, Hongshan Wang, Xiaowei Chen, Christoph B. Westphalen, Samuel Asfaha, Daniel L. Worthley, Bernhard Renz, Hiroshi Ariyama, Zinaida A. Dubeykovskaya, Yoshihiro Takemoto, Ashlesha Mulay, Yagnesh Tailor, Duan Chen, Sureshkumar Muthupalani, James G. Fox, Shigeo Takaishi, Timothy C. Wang. CCK2R marks gastric antral stem cell and mediates antral carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4795. doi:10.1158/1538-7445.AM2014-4795