Details of a Researcher - Sato, Toshiro

Papers - Sato, Toshiro

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Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways

Kikuchi Ippei, Takahashi-Kanemitsu Atsushi, Sakiyama Natsuki, Tang Chao, Tang Pei Jung, Noda Saori, Nakao Kazuki, Kassai Hidetoshi, Sato Toshiro, Aiba Atsu, Hatakeyama Masanori

Nature Communications 7 2016.09

Research paper (scientific journal), Joint Work, ISSN 2041-1723

Evolutionally conserved Wnt, Hedgehog (Hh) and Notch morphogen pathways play essential roles in the development, homeostasis and pathogenesis of multicellular organisms. Nevertheless, mechanisms that intracellularly coordinate these signal inputs remain poorly understood. Here we found that parafibromin, a component of the PAF complex, competitively interacts with β-catenin and Gli1, thereby potentiating transactivation of Wnt- and Hh-target genes in a mutually exclusive manner. Parafibromin also binds to the Notch intracellular domain (NICD), enabling concerted activation of Wnt- and Notch-target genes. The transcriptional platform function of parafibromin is potentiated by tyrosine dephosphorylation, mediated by SHP2 phosphatase, while it is attenuated by tyrosine phosphorylation, mediated by PTK6 kinase. Consequently, acute loss of parafibromin in mice disorganizes the normal epithelial architecture of the intestine, which requires coordinated activation/inactivation of Wnt, Hh and/or Notch signalling. Parafibromin integrates and converts signals conveyed by these morphogen pathways into appropriate transcriptional outputs in a tyrosine phosphorylation/dephosphorylation-regulated manner.

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Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation

Dominguez-Brauer Carmen, Hao Zhenyue, Elia Andrew J., Fortin Jérôme M., Nechanitzky Robert, Brauer Patrick M., Sheng Yi, Mana Miyeko D., Chio Iok In Christine, Haight Jillian, Pollett Aaron, Cairns Robert, Tworzyanski Leanne, Inoue Satoshi, Reardon Colin, Marques Ana, Silvester Jennifer, Cox Maureen A., Wakeham Andrew, Yilmaz Omer H., Sabatini David M., van Es Johan H., Clevers Hans, Sato Toshiro, Mak Tak W.

Cell Stem Cell 19 ( 2 ) 205 - 216 2016.08

Research paper (scientific journal), Joint Work, ISSN 1934-5909

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.

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A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis

Fujii Masayuki, Shimokawa Mariko, Date Shoichi, Takano Ai, Matano Mami, Nanki Kosaku, Ohta Yuki, Toshimitsu Kohta, Nakazato Yoshihiro, Kawasaki Kenta, Uraoka Toshio, Watanabe Toshiaki, Kanai Takanori, Sato Toshiro

Cell Stem Cell 18 ( 6 ) 827 - 838 2016.06

Research paper (scientific journal), Joint Work, ISSN 1934-5909

Summary Colorectal tumor is a heterogeneous disease, with varying clinical presentation and prognosis in patients. To establish a platform encompassing this diversity, we generated 55 colorectal tumor organoid lines from a range of histological subtypes and clinical stages, including rare subtypes. Each line was defined by gene expression signatures and optimized for organoid culture according to niche factor requirements. In vitro and in xenografts, the organoids reproduced the histopathological grade and differentiation capacity of their parental tumors. Notably, we found that niche-independent growth is predominantly associated with the adenoma-carcinoma transition reflecting accumulation of multiple mutations. For matched pairs of primary and metastatic organoids, which had similar genetic profiles and niche factor requirements, the metastasis-derived organoids exhibited higher metastatic capacity. These observations underscore the importance of genotype-phenotype analyses at a single-patient level and the value of our resource to provide insights into colorectal tumorigenesis and patient-centered therapeutic development.

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Inhibition of DNA Methylation Suppresses Intestinal Tumor Organoids by Inducing an Anti-Viral Response

Saito Yoshimasa, Nakaoka Toshiaki, Sakai Kasumi, Muramatsu Toshihide, Toshimitsu Kohta, Kimura Masaki, Kanai Takanori, Sato Toshiro, Saito Hidetsugu

Scientific Reports 6 2016.05

Joint Work

Recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-responsive genes via dsRNAs-containing endogenous retroviruses. Recently, a 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established organoids from intestinal tumors of Apc^Min/+ (Min) mice and subjected them to 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-responsive genes were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response. These findings indicate that DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-responsive genes. Treatment with DNA methylation inhibitors to activate a growth-inhibiting immune response may be an effective therapeutic approach for colon cancers.

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SETD7 Controls Intestinal Regeneration and Tumorigenesis by Regulating Wnt/β-Catenin and Hippo/YAP Signaling

Oudhoff Menno J., Braam Mitchell J S, Freeman Spencer A., Wong Denise, Rattray David G., Wang Jia, Antignano Frann, Snyder Kimberly, Refaeli Ido, Hughes Michael R., McNagny Kelly M., Gold Michael R., Arrowsmith Cheryl H., Sato Toshiro, Rossi Fabio M V, Tatlock John H., Owen Dafydd R., Brown Peter J., Zaph Colby

Developmental Cell 37 ( 1 ) 47 - 57 2016.04

Research paper (scientific journal), Joint Work, ISSN 1534-5807

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/β-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and β-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of β-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/β-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis. The Wnt/β-catenin and Hippo/YAP pathways are intimately linked in structure and function. Oudhoff et al. provide evidence that the lysine methyltransferase SETD7 is a central link between these pathways. SETD7-dependent methylation of YAP is required for optimal β-catenin-dependent gene expression. Accordingly, loss of SETD7 attenuates Wnt-dependent intestinal tumorigenesis and regeneration.

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Active and water-soluble form of lipidated wnt protein is maintained by a serum glycoprotein afamin/α-albumin

Mihara Emiko, Hirai Hidenori, Yamamoto Hideki, Tamura-Kawakami Keiko, Matano Mami, Kikuchi Akira, Sato Toshiro, Takagi Junichi

eLife 5 ( FEBRUARY2016 ) 2016.02

Research paper (scientific journal), Joint Work, ISSN 2050-084X

Wnt plays important role during development and in various diseases. Because Wants are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestinal stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, ataman complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years.

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EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

Nakajo Atsuhiro, Yoshimura Shin ichiro, Togawa Hiroko, Kunii Masataka, Iwano Tomohiko, Izumi Ayaka, Noguchi Yuria, Watanabe Ayako, Goto Ayako, Sato Toshiro, Harada Akihiro

Journal of Cell Biology 212 ( 3 ) 297 - 306 2016

Research paper (scientific journal), Joint Work, ISSN 0021-9525

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1-or Bin1-depleted or dynamininhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

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Mini-Gut Organoids

Date Shoichi, Sato Toshiro

Annual Review of Cell and Developmental Biology 31 269 - 289 2015.11

Research paper (scientific journal), Joint Work, ISSN 1081-0706

In the adult mammalian body, self-renewal of tissue stem cells is regulated by extracellular niche environments in response to the demands of tissue organization. Intestinal stem cells expressing Lgr5 constantly self-renew in their specific niche at the crypt bottom to maintain rapid turnover of the epithelium. Niche-regulated stem cell self-renewal is perturbed in several mouse genetic models and during human tumorigenesis, suggesting roles for EGF, Wnt, BMPTGF-β, and Notch signaling. In vitro niche reconstitution capitalizing on this knowledge has enabled the growth of single intestinal stem cells into mini-gut epithelial organoids comprising Lgr5⁺ stem cells and all types of differentiated lineages. The mini-gut organoid culture platform is applicable to various types of digestive tissue epithelium from multiple species. The mechanism of self-renewal in organoids provides novel insights for organogenesis, regenerative medicine, and tumorigenesis of the digestive system.

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Efficient genetic engineering of human intestinal organoids using electroporation

Fujii Masayuki, Matano Mami, Nanki Kosaku, Sato Toshiro

Nature Protocols 10 ( 10 ) 1474 - 1485 2015.10

Research paper (scientific journal), Joint Work, ISSN 1754-2189

Gene modification in untransformed human intestinal cells is an attractive approach for studying gene function in intestinal diseases. However, because of the lack of practical tools, such studies have largely depended upon surrogates, such as gene-engineered mice or immortalized human cell lines. By taking advantage of the recently developed intestinal organoid culture method, we developed a methodology for modulating genes of interest in untransformed human colonic organoids via electroporation of gene vectors. Here we describe a detailed protocol for the generation of intestinal organoids by culture with essential growth factors in a basement membrane matrix. We also describe how to stably integrate genes via the piggyBac transposon, as well as precise genome editing using the CRISPR-Cas9 system. Beginning with crypt isolation from a human colon sample, genetically modified organoids can be obtained in 3 weeks.

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SnapShot: Growing Organoids from Stem Cells.

Sato Toshiro, Clevers Hans

Cell (Elsevie) 161 ( 7 ) 1700 - 1700.e1 2015.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0092-8674

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Ink4a/arf-dependent loss of parietal cells induced by oxidative stress promotes CD44-dependent gastric tumorigenesis

Seishima Ryo, Wada Takeyuki, Tsuchihashi Kenji, Okazaki Shogo, Yoshikawa Momoko, Oshima Hiroko, Oshima Masanobu, Sato Toshiro, Hasegawa Hirotoshi, Kitagawa Yuko, Goldenring James R., Saya Hideyuki, Nagano Osamu

Cancer Prevention Research 8 ( 6 ) 492 - 501 2015.06

Research paper (scientific journal), Joint Work, ISSN 1940-6207

Loss of parietal cells initiates the development of spasmolytic polypeptide-expressing metaplasia (SPEM), a precancerous lesion in stomach. CD44 variant (CD44v) that enhances the ability to defend against reactive oxygen species (ROS) in epithelial cells is expressed de novo in SPEM of K19-Wnt1/C2mE mice, a transgenic model of gastric tumorigenesis, and is required for the efficient development of SPEM and gastric tumor in these animals. The role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown, however. With the use of the K19-Wnt1/C2mE mouse, we now show that parietal cells in the inflamed stomach are highly sensitive to oxidative stress and manifest activation of p38MAPK signaling by ROS. Oral treatment with the antioxidant ascorbic acid or genetic ablation of the Ink4a/Arf locus, a major downstream target of ROS-p38MAPK signaling, inhibited parietal cell loss and the subsequent gastric tumorigenesis. Our results indicate that signaling activated by oxidative stress in parietal cells plays a key role in CD44-dependent gastric tumorigenesis.

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Combination therapy with infliximab and thiopurine compared to infliximab monotherapy in maintaining remission of postoperative crohn's disease

Sakuraba Atsushi, Okamoto Susumu, Matsuoka Katsuyoshi, Sato Toshiro, Naganuma Makoto, Hisamatsu Tadakazu, Iwao Yasushi, Ogata Haruhiko, Kanai Takanori, Hibi Toshifumi

Digestion 91 ( 3 ) 233 - 238 2015.05

Research paper (scientific journal), Joint Work, ISSN 0012-2823

Background and Aims: Infliximab is an efficacious agent used for the induction and maintenance of remission in Crohn's disease (CD), and recent studies suggested that it may also prevent the recurrence of this disease after surgery. The present study was performed to assess the efficacy and safety of infliximab in the postoperative setting, and to identify whether combination treatment with thiopurines had any additional beneficial effect as compared to mono-therapy. Methods: We performed a retrospective cohort study to compare the efficacy of infliximab mono-therapy and combination treatment with a thiopurine in preventing recurrence after surgery. Results: Forty-one patients who received infliximab as maintenance treatment following surgery from May 2002 to April 2010 were identified. Twenty-four were naive to infliximab, and 17 who underwent surgery during infliximab treatment were continued on it following surgery. The median follow-up period was 27 months (range 12-66 months). All patients continued infliximab as maintenance treatment, but 10 required dose intensification due to clinical recurrence. Kaplan-Meier analysis demonstrated that the use of concomitant thiopurine was correlated with the continuation of infliximab treatment at an 8-week interval (log-rank test p = 0.018). The rate of adverse event was 9.8% with no patient experiencing severe adverse reactions. Conclusion: Infliximab appears to be safe and it prevented clinical recurrence after surgery. Concomitant thiopurine use predicted response toward continuation of therapy at an 8-week interval. Prospective controlled studies to assess the efficacy of combination treatment in the postoperative setting are warranted.

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Suppressing TGFβ signaling in regenerating epithelia in an inflammatory microenvironment is sufficient to cause invasive intestinal cancer

Oshima Hiroko, Nakayama Mizuho, Han Tae Su, Naoi Kuniko, Ju Xiaoli, Maeda Yusuke, Robine Sylvie, Tsuchiya Kiichiro, Sato Toshiro, Sato Hiroshi, Taketo Makoto Mark, Oshima Masanobu

Cancer Research 75 ( 4 ) 766 - 776 2015.02

Research paper (scientific journal), Joint Work, ISSN 0008-5472

Genetic alterations in the TGFβ signaling pathway in combination with oncogenic alterations lead to cancer development in the intestines. However, the mechanisms of TGFβ signaling suppression in malignant progression of intestinal tumors have not yet been fully understood. We have examined Apc^Δ716 TGFβr2ΔIEC compound mutant mice that carry mutations in Apc and TGFβr2 genes in the intestinal epithelial cells. We found inflammatory microenvironment only in the invasive intestinal adenocarcinomas but not in noninvasive benign polyps of the same mice. We thus treated simple TGFβr2ΔIEC mice with dextran sodium sulfate (DSS) that causes ulcerative colitis. Importantly, these TGFβr2ΔIEC mice developed invasive colon cancer associated with chronic inflammation.Wealso found that TGFβ signaling is suppressed in human colitis-associated colon cancer cells. In the mouse invasive tumors, macrophages infiltrated and expressed MT1-MMP, causingMMP2activation. These results suggest that inflammatory microenvironment contributes to submucosal invasion of TGFβ signaling-repressed epithelial cells through activation of MMP2. We further found that regeneration was impaired in TGFβr2ΔIEC mice for intestinal mucosa damaged by DSS treatment or X-ray irradiation, resulting in the expansion of undifferentiated epithelial cell population. Moreover, organoids of intestinal epithelial cells cultured from irradiated TGFβr2ΔIEC mice formed "long crypts" in Matrigel, suggesting acquisition of an invasive phenotype into the extracellular matrix. These results, taken together, indicate that a simple genetic alteration in the TGFβ signaling pathway in the inflamed and regenerating intestinal mucosa can cause invasive intestinal tumors. Such a mechanism may play a role in the colon carcinogenesis associated with inflammatory bowel disease in humans.

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An individual based computational model of intestinal crypt fission and its application to predicting unrestrictive growth of the intestinal epithelium

Pin Carmen, Parker Aimee, Gunning A. Patrick, Ohta Yuki, Johnson Ian T., Carding Simon R., Sato Toshiro

Integrative Biology (United Kingdom) 7 ( 2 ) 213 - 228 2015.02

Research paper (scientific journal), Joint Work, ISSN 1757-9694

Intestinal crypt fission is a homeostatic phenomenon, observable in healthy adult mucosa, but which also plays a pathological role as the main mode of growth of some intestinal polyps. Building on our previous individual based model for the small intestinal crypt and on in vitro cultured intestinal organoids, we here model crypt fission as a budding process based on fluid mechanics at the individual cell level and extrapolated predictions for growth of the intestinal epithelium. Budding was always observed in regions of organoids with abundant Paneth cells. Our data support a model in which buds are biomechanically initiated by single stem cells surrounded by Paneth cells which exhibit greater resistance to viscoelastic deformation, a hypothesis supported by atomic force measurements of single cells. Time intervals between consecutive budding events, as simulated by the model and observed in vitro, were 2.84 and 2.62 days, respectively. Predicted cell dynamics was unaffected within the original crypt which retained its full capability of providing cells to the epithelium throughout fission. Mitotic pressure in simulated primary crypts forced upward migration of buds, which simultaneously grew into new protruding crypts at a rate equal to 1.03 days(-1) in simulations and 0.99 days(-1) in cultured organoids. Simulated crypts reached their final size in 4.6 days, and required 6.2 days to migrate to the top of the primary crypt. The growth of the secondary crypt is independent of its migration along the original crypt. Assuming unrestricted crypt fission and multiple budding events, a maximal growth rate of the intestinal epithelium of 0.10 days(-1) is predicted and thus approximately 22 days are required for a 10-fold increase of polyp size. These predictions are in agreement with the time reported to develop macroscopic adenomas in mice after loss of Apc in intestinal stem cells.

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Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids

Matano Mami, Date Shoichi, Shimokawa Mariko, Takano Ai, Fujii Masayuki, Ohta Yuki, Watanabe Toshiaki, Kanai Takanori, Sato Toshiro

Nature Medicine 21 ( 3 ) 256 - 262 2015

Research paper (scientific journal), Joint Work, ISSN 1078-8956

Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

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Back to 2D Culture for Ground State of Intestinal Stem Cells

Shimokawa Mariko, Sato Toshiro

Cell Stem Cell 17 ( 1 ) 5 - 7 2015

Research paper (scientific journal), Joint Work, ISSN 1934-5909

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Classical Th1 cells obtain colitogenicity by co-existence of RORγt-expressing T cells in experimental colitis

Saigusa Keiichiro, Hisamatsu Tadakazu, Handa Tango, Sujino Tomohisa, Mikami Yohei, Hayashi Atsushi, Mizuno Shinta, Takeshita Kozue, Sato Toshiro, Matsuoka Katsuyoshi, Kanai Takanori

Inflammatory Bowel Diseases 20 ( 10 ) 1820 - 1827 2014.10

Research paper (scientific journal), Joint Work, ISSN 1078-0998

Background: Both Th1 and Th17 cell types are involved in the pathogenesis of chronic intestinal inflammation. We recently demonstrated that retinoid-related orphan receptor gamma t (RORγt)-expressing Th17 cells are progenitor cells for alternative Th1 cells, which have the potential to induce colitis. However, the involvement of classical Th1 (cTh1) cells generated directly from naive T cells without RORγt expression in the pathogenesis of colitis remains poorly understood. Methods: We performed a series of in vivo experiments using a murine chronic colitis model induced by adoptive transfer of splenic CD4+CD45RBhigh T cells obtained from wild-type, RORγtgfp/gfp, or RORγtgfp/+ mice into RAG-2-/- mice. Results: RAG-2-/- mice receiving transfer of in vitro-manipulated RORγtgfp/gfp Th1 cells developed colitis. RAG-2-/- mice co-transferred with splenic CD4+CD45RBhigh T cells obtained from wild-type mice and RORγtgfp/gfp mice developed colitis with a significant increase in RORγtgfp/gfp cTh1 cell numbers when compared with noncolitic mice transferred with splenic CD4+CD45RBhigh T cells obtained from RORγtgfp/gfp mice. Furthermore, RAG-2-/- mice transferred with in vivo-manipulated RORγtgfp/gfp cTh1 cells developed colitis with a significant increase in RORγtgfp/gfp cTh1 cell numbers. Conclusions: These findings indicate that both alternative Th1 cells and cTh1 cells have the potential to be colitogenic in an adaptive transfer model. The development of cTh1 cells was dependent on the co-existence of RORγt-expressing T cells, suggesting a critical role for the interactions of these cell types in the development of chronic intestinal inflammation.

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KLF5 regulates the integrity and oncogenicity of intestinal stem cells

Nakaya Takeo, Ogawa Seishi, Manabe Ichiro, Tanaka Masami, Sanada Masashi, Sato Toshiro, Taketo Makoto M., Nakao Kazuki, Clevers Hans, Fukayama Masashi, Kuroda Masahiko, Nagai Ryozo

Cancer Research 74 ( 10 ) 2882 - 2891 2014.05

Research paper (scientific journal), Joint Work, ISSN 0008-5472

The intestinal epithelium maintains homeostasis by a self-renewal process involving resident stem cells, including Lgr5+ crypt-base columnar cells, but core mechanisms and their contributions to intestinal cancer are not fully defined. In this study, we examined a hypothesized role for KLF5, a zinc-finger transcription factor that is critical to maintain the integrity of embryonic and induced pluripotent stem cells, in intestinal stem-cell integrity and cancer in the mouse. Klf5 was indispensable for the integrity and oncogenic transformation of intestinal stem cells. In mice, inducible deletion of Klf5 in Lgr5+ stem cells suppressed their proliferation and survival in a manner associated with nuclear localization of β-catenin (Catnb), generating abnormal apoptotic cells in intestinal crypts. Moreover, production of lethal adenomas and carcinomas by specific expression of an oncogenic mutant of β-catenin in Lgr5+ stem cells was suppressed completely by Klf5 deletion in the same cells. Given that activation of the Wnt/β-catenin pathway is the most frequently altered pathway in human colorectal cancer, our results argue that KLF5 acts as a fundamental core regulator of intestinal oncogenesis at the stem-cell level, and they suggest KLF5 targeting as a rational strategy to eradicate stem-like cells in colorectal cancer.

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Macrophages and dendritic cells emerge in the liver during intestinal inflammation and predispose the liver to inflammation

Mikami Yohei, Mizuno Shinta, Nakamoto Nobuhiro, Hayashi Atsushi, Sujino Tomohisa, Sato Toshiro, Kamada Nobuhiko, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Ebinuma Hirotoshi, Hibi Toshifumi, Yoshimura Akihiko, Kanai Takanori

PLoS One 9 ( 1 ) 2014.01

Joint Work

The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2^-/- mice adoptively transferred with CD4⁺CD45RB^high T cells; and IL-10 ^-/- mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b^-CD11c^lowPDCA-1⁺ plasmacytoid dendritic cells (DCs) abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4⁺CD45RB^high T cell-transferred RAG-2^-/- mice and IL-10^-/- mice in parallel with the emergence of macrophages (Mφs) and conventional DCs (cDCs). Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4⁺ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS)-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation.

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Continuous low-dose irradiation by I-125 seeds induces apoptosis of gastric cancer cells regardless of histological origin

Takabayashi Kaoru, Kashiwagi Kazuhiro, Kawata Tetsuya, Sato Toshiro, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Takaishi Hiromasa, Hibi Toshifumi, Ogata Haruhiko, Yahagi Naohisa, Kitagawa Yuko, Shigematsu Naoyuki, Kanai Takanori

Cancer Biology and Therapy 15 ( 1 ) 81 - 88 2014.01

Research paper (scientific journal), Joint Work, ISSN 1538-4047

The efficacy of conventional radiation therapy for gastric cancer is controversial. In this study, we evaluated the in vitro and in vivo effects of continuous low-dose-rate irradiation by I-125 seeds on different histological types of gastric cancer cell lines. Three human gastric cancer cell lines (MKN74, MKN45, and NUGC4) were treated with or without continuous low-dose irradiation by I-125 seeds in vitro and in vivo. Cell viability, apoptosis, caspase-3 assay, and cell-cycle distribution were examined in vitro. Body weight and tumor volumes of BALB/c nude mice bearing MKN74, MKN45, and NUGC4 gastric cancer xenografts were measured, and in vivo cell proliferation and apoptosis assays were performed by Ki67 and TUNEL staining, respectively. Continuous low-dose-rate irradiation by I-125 seeds reduced cell viability and induced cell apoptosis through the activation of caspase-3, and led to the accumulation of cells in the G2/M phase in vitro. It also suppressed the growth of gastric cancer xenografts in nude mice, while inhibiting cell proliferation and inducing apoptosis as demonstrated by Ki67 and TUNEL staining. Therefore, our data suggest that continuous low-dose-rate irradiation by I-125 seeds could be a promising new option for gastric cancer treatment, regardless of histological origin.

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