Faculty Profiles - ENOMOTO, Atsushi

Neuro-mesenchymal interaction mediated by a β2 adrenergic-nerve growth factor feedforward loop promotes colorectal cancer progression. Reviewed International journal

Hiroki Kobayashi, Tadashi Iida, Yosuke Ochiai, Ermanno Malagola, Xiaofei Zhi, Ruth A White, Jin Qian, Feijing Wu, Quin T Waterbury, Ruhong Tu, Biyun Zheng, Jonathan S LaBella, Leah B Zamechek, Atsushi Ogura, Susan L Woods, Daniel L Worthley, Atsushi Enomoto, Timothy C Wang

Cancer discovery Vol. 15 ( 1 ) page： 202 - 226 2025.1

Language：English Publishing type：Research paper (scientific journal)

Cancer-associated fibroblasts (CAFs) and nerves, components of the tumor microenvironment, have each been shown to directly promote gastrointestinal cancers. However, it remains unknown whether these cells interact with each other to regulate cancer progression. We found that in colorectal cancer (CRC) norepinephrine induces ADRB2-dependent nerve growth factor (NGF) secretion from CAFs, which in turn increases intra-tumor sympathetic innervation and norepinephrine accumulation. Adrenergic stimulation accelerates CRC growth through ADRA2A/Gi-mediated activation of Yes-Associated Protein (YAP). NGF from CAFs directly enhances CRC cell growth via the PI3K/AKT pathway. Treatment with a tropomyosin receptor kinase (Trk) inhibitor decreased YAP and AKT activation and CRC progression in mice. In human CRC, high NGF expression is associated with the mesenchymal-like tumor subtype and poor patient survival. These findings suggest a central role for reciprocal CAF-nerve crosstalk in promoting CRC progression. Blocking this feedforward loop with a Trk inhibitor may represent a potential therapeutic approach for CRC.

DOI： 10.1158/2159-8290.CD-24-0287

ALPK2 prevents cardiac diastolic dysfunction in heart failure with preserved ejection fraction. International journal

Tatsuya Yoshida, Satoya Yoshida, Kohei Inukai, Katsuhiro Kato, Yoshimitsu Yura, Tomoki Hattori, Kentaro Taki, Atsushi Enomoto, Koji Ohashi, Takahiro Okumura, Noriyuki Ouchi, Haruya Kawase, Nina Wettschureck, Stefan Offermanns, Toyoaki Murohara, Mikito Takefuji

FASEB journal : official publication of the Federation of American Societies for Experimental Biology Vol. 38 ( 22 ) page： e70192 2024.11

Language：English Publishing type：Research paper (scientific journal)

Protein phosphorylation, controlled by protein kinases, is central to regulating various pathophysiological processes, including cardiac systolic function. The dysregulation of protein kinase activity plays a significant role in the pathogenesis of cardiac systolic dysfunction. While cardiac contraction mechanisms are well documented, the mechanisms underlying cardiac diastole remain elusive. This gap persists owing to the historical focus on systolic dysfunction in heart failure research. Recently, heart failure with preserved ejection fraction (HFpEF), an age-related disease characterized by cardiac diastolic dysfunction, has emerged as a major public health concern. However, its underlying mechanism remains unclear. In this study, we investigated cardiac protein kinases by analyzing the gene expression of 518 protein kinases in human tissues. We identified alpha-kinase 2 (ALPK2) as a novel cardiac-specific atypical kinase and generated tamoxifen-inducible, cardiomyocyte-specific Alpk2-knockout mice and Alpk2-overexpressing mice. Alpk2 deficiency did not affect cardiac systolic dysfunction in the myocardial infarction model or the pressure-overload-induced heart failure model. Notably, cardiomyocyte-specific Alpk2 deficiency exacerbated cardiac diastolic dysfunction induced by aging and in the HFpEF model. Conversely, Alpk2 overexpression increased the phosphorylation of tropomyosin 1, a major regulator that binds myosin to actin, and mitigated cardiac stiffness in HFpEF. This study provides novel evidence that ALPK2 represents a potential therapeutic target for cardiac diastolic dysfunction in HFpEF and age-related cardiac impairments.

DOI： 10.1096/fj.202402103R

Pulmonary metastases of a renal angiomyolipoma: A case report, with whole-exome sequencing analysis. Reviewed International journal

Yuki Sonoda, Masataka Hirasaki, Yoko Usami, Tomoaki Torigoe, Tomonori Kawasaki, Nobuyuki Suzuki, Yukihiro Shiraki, Atsushi Enomoto, Hiroki Imada, Yasutaka Baba

Radiology case reports Vol. 19 ( 11 ) page： 4963 - 4969 2024.11

Language：English Publishing type：Research paper (scientific journal)

We present a case of pulmonary metastasis originating from renal angiomyolipoma (AML), as evidenced by whole-exome sequencing (WES) analysis. Although AML predominantly arises in the kidneys, it can emerge in various body parts, making it important to distinguish between multicentric development and metastasis. However, previous studies have not distinguished between these conditions. Our case features an 82-year-old woman with a history of renal AML who presented with multiple, randomly distributed, bilateral pulmonary nodules of varying size and pure fat densities. The patient's condition followed a benign course over 10 years. Through WES, we discovered shared mutations in pulmonary lesions that were absent in the patient's blood, including a pathological mutation in TSC2, suggesting a metastatic origin from renal AML. Knowledge of the pulmonary manifestations of AML and their distinctive imaging findings can help radiologists and clinicians diagnose and manage patients with similar presentations.

DOI： 10.1016/j.radcr.2024.07.113

Meflin/ISLR is a marker of adipose stem and progenitor cells in mice and humans that suppresses white adipose tissue remodeling and fibrosis. Reviewed International journal

Toshikazu Ishihara, Katsuhiro Kato, Kotaro Matsumoto, Miyako Tanaka, Akitoshi Hara, Yukihiro Shiraki, Hidenori Morisaki, Yuya Urano, Ryota Ando, Kisuke Ito, Shinji Mii, Nobutoshi Esaki, Kazuhiro Furuhashi, Mikito Takefuji, Takayoshi Suganami, Toyoaki Murohara, Atsushi Enomoto

Genes to cells : devoted to molecular & cellular mechanisms Vol. 29 ( 10 ) page： 902 - 920 2024.10

Language：English Publishing type：Research paper (scientific journal)

Identifying specific markers of adipose stem and progenitor cells (ASPCs) in vivo is crucial for understanding the biology of white adipose tissues (WAT). PDGFRα-positive perivascular stromal cells represent the best candidates for ASPCs. This cell lineage differentiates into myofibroblasts that contribute to the impairment of WAT function. However, ASPC marker protein(s) that are functionally crucial for maintaining WAT homeostasis are unknown. We previously identified Meflin as a marker of mesenchymal stem cells (MSCs) in bone marrow and tissue-resident perivascular fibroblasts in various tissues. We also demonstrated that Meflin maintains the undifferentiated status of MSCs/fibroblasts. Here, we show that Meflin is expressed in WAT ASPCs. A lineage-tracing experiment showed that Meflin+ ASPCs proliferate in the WAT of obese mice induced by a high-fat diet (HFD), while some of them differentiate into myofibroblasts or mature adipocytes. Meflin knockout mice fed an HFD exhibited a significant fibrotic response as well as increases in adipocyte cell size and the number of crown-like structures in WAT, accompanied by impaired glucose tolerance. These data suggested that Meflin expressed by ASPCs may have a role in reducing disease progression associated with WAT dysfunction.

DOI： 10.1111/gtc.13154

Protein kinase N promotes cardiac fibrosis in heart failure by fibroblast-to-myofibroblast conversion. Reviewed International journal

Satoya Yoshida, Tatsuya Yoshida, Kohei Inukai, Katsuhiro Kato, Yoshimitsu Yura, Tomoki Hattori, Atsushi Enomoto, Koji Ohashi, Takahiro Okumura, Noriyuki Ouchi, Haruya Kawase, Nina Wettschureck, Stefan Offermanns, Toyoaki Murohara, Mikito Takefuji

Nature communications Vol. 15 ( 1 ) page： 7638 - 7638 2024.9

Language：English Publishing type：Research paper (scientific journal)

Chronic fibrotic tissue disrupts various organ functions. Despite significant advances in therapies, mortality and morbidity due to heart failure remain high, resulting in poor quality of life. Beyond the cardiomyocyte-centric view of heart failure, it is now accepted that alterations in the interstitial extracellular matrix (ECM) also play a major role in the development of heart failure. Here, we show that protein kinase N (PKN) is expressed in cardiac fibroblasts. Furthermore, PKN mediates the conversion of fibroblasts into myofibroblasts, which plays a central role in secreting large amounts of ECM proteins via p38 phosphorylation signaling. Fibroblast-specific deletion of PKN led to a reduction of myocardial fibrotic changes and cardiac dysfunction in mice models of ischemia-reperfusion or heart failure with preserved ejection fraction. Our results indicate that PKN is a therapeutic target for cardiac fibrosis in heart failure.

DOI： 10.1038/s41467-024-52068-0

Tumor-associated fibrosis: a unique mechanism promoting ovarian cancer metastasis and peritoneal dissemination. Reviewed International journal

Hiroki Fujimoto, Masato Yoshihara, Raymond Rodgers, Shohei Iyoshi, Kazumasa Mogi, Emiri Miyamoto, Sae Hayakawa, Maia Hayashi, Satoshi Nomura, Kazuhisa Kitami, Kaname Uno, Mai Sugiyama, Yoshihiro Koya, Yoshihiko Yamakita, Akihiro Nawa, Atsushi Enomoto, Carmela Ricciardelli, Hiroaki Kajiyama

Cancer metastasis reviews Vol. 43 ( 3 ) page： 1037 - 1053 2024.9

Language：English Publishing type：Research paper (scientific journal)

Epithelial ovarian cancer (EOC) is often diagnosed in advanced stage with peritoneal dissemination. Recent studies indicate that aberrant accumulation of collagen fibers in tumor stroma has a variety of effects on tumor progression. We refer to remodeled fibrous stroma with altered expression of collagen molecules, increased stiffness, and highly oriented collagen fibers as tumor-associated fibrosis (TAF). TAF contributes to EOC cell invasion and metastasis in the intraperitoneal cavity. However, an understanding of molecular events involved is only just beginning to emerge. Further development in this field will lead to new strategies to treat EOC. In this review, we focus on the recent findings on how the TAF contributes to EOC malignancy. Furthermore, we will review the recent initiatives and future therapeutic strategies for targeting TAF in EOC.

DOI： 10.1007/s10555-024-10169-8

骨肉腫微小環境における腫瘍関連マクロファージ由来IL-8の働き

進行卵巣癌でMeflin陽性癌関連線維芽細胞が腫瘍進展、生命予後に与える影響(Meflin-positive cancer-associated fibroblasts exhibit anti-tumor effects in advanced ovarian cancer)

宮本絵美里, 吉原雅人, 小屋美博, 藤本裕基, 伊吉祥平, 茂木一将, 宇野枢, 北見和久, 榎本篤, 梶山広明, 梶山広明

日本癌学会総会記事 Vol. 83回 page： E - 3056 2024.9

辰野力人, 市川二郎, 菰原義弘, 榎本篤, 川崎朋範, 重橋孝洋, 波呂浩孝

日本整形外科学会雑誌 Vol. 98 ( 8 ) page： S1968 - S1968 2024.9

Surface Markers and Chemokines/Cytokines of Tumor-Associated Macrophages in Osteosarcoma and Other Carcinoma Microenviornments-Contradictions and Comparisons. Reviewed International journal

Rikito Tatsuno, Yoshihiro Komohara, Cheng Pan, Tomonori Kawasaki, Atsushi Enomoto, Takahiro Jubashi, Hiroyuki Kono, Masanori Wako, Tomoyuki Ashizawa, Hirotaka Haro, Jiro Ichikawa

Cancers Vol. 16 ( 16 ) 2024.8

Language：English Publishing type：Research paper (scientific journal)

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Prognosis is improving with advances in multidisciplinary treatment strategies, but the development of new anticancer agents has not, and improvement in prognosis for patients with pulmonary metastases has stalled. In recent years, the tumor microenvironment (TME) has gained attention as a therapeutic target for cancer. The immune component of OS TME consists mainly of tumor-associated macrophages (TAMs). They exhibit remarkable plasticity, and their phenotype is influenced by the TME. In general, surface markers such as CD68 and CD80 show anti-tumor effects, while CD163 and CD204 show tumor-promoting effects. Surface markers have potential value as diagnostic and prognostic biomarkers. The cytokines and chemokines produced by TAMs promote tumor growth and metastasis. However, the role of TAMs in OS remains unclear to date. In this review, we describe the role of TAMs in OS by focusing on TAM surface markers and the TAM-produced cytokines and chemokines in the TME, and by comparing their behaviors in other carcinomas. We found contrary results from different studies. These findings highlight the urgency for further research in this field to improve the stalled OS prognosis percentages.

DOI： 10.3390/cancers16162801

Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade. Reviewed International journal

Takayuki Owaki, Tadashi Iida, Yuki Miyai, Katsuhiro Kato, Tetsunari Hase, Makoto Ishii, Ryota Ando, Kunihiko Hinohara, Tomohiro Akashi, Yasuyuki Mizutani, Takuya Ishikawa, Shinji Mii, Yukihiro Shiraki, Nobutoshi Esaki, Masami Yamamoto, Tetsuya Tsukamoto, Sachiyo Nomura, Takashi Murakami, Masahide Takahashi, Yuri Yuguchi, Motohiro Maeda, Tomoyasu Sano, Naoto Sassa, Yoshihisa Matsukawa, Hiroki Kawashima, Shusuke Akamatsu, Atsushi Enomoto

British journal of cancer Vol. 131 ( 2 ) page： 372 - 386 2024.7

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: The proliferation of cancer-associated fibroblasts (CAFs) hampers drug delivery and anti-tumor immunity, inducing tumor resistance to immune checkpoint blockade (ICB) therapy. However, it has remained a challenge to develop therapeutics that specifically target or modulate CAFs. METHODS: We investigated the involvement of Meflin+ cancer-restraining CAFs (rCAFs) in ICB efficacy in patients with clear cell renal cell carcinoma (ccRCC) and urothelial carcinoma (UC). We examined the effects of Am80 (a synthetic retinoid) administration on CAF phenotype, the tumor immune microenvironment, and ICB efficacy in cancer mouse models. RESULTS: High infiltration of Meflin+ CAFs correlated with ICB efficacy in patients with ccRCC and UC. Meflin+ CAF induction by Am80 administration improved ICB efficacy in the mouse models of cancer. Am80 exerted this effect when administered prior to, but not concomitant with, ICB therapy in wild-type but not Meflin-deficient mice. Am80-mediated induction of Meflin+ CAFs was associated with increases in antibody delivery and M1-like tumor-associated macrophage (TAM) infiltration. Finally, we showed the role of Chemerin produced from CAFs after Am80 administration in the induction of M1-like TAMs. CONCLUSION: Our data suggested that Am80 administration prior to ICB therapy increases the number of Meflin+ rCAFs and ICB efficacy by inducing changes in TAM phenotype.

DOI： 10.1038/s41416-024-02734-3

非浸潤性乳管癌に対する乳房温存術後の放射線照射は省略できるか? Reviewed

Correction: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway.

Tatsuno R, Ichikawa J, Komohara Y, Pan C, Kawasaki T, Enomoto A, Aoki K, Hayakawa K, Iwata S, Jubashi T, Haro H

Cell death & disease Vol. 15 ( 7 ) page： 471 2024.7

宮井雄基, 白木之浩, 安藤良太, 榎本篤

実験医学 Vol. 42 ( 11 ) page： 1684 - 1690 2024.7

Language：Japanese Publisher：(株)羊土社

がん関連線維芽細胞(CAF)はがん促進性の機能をもつと長く認識されてきた.一方で,CAFを除去あるいはその増殖を抑制するとがんの増悪が観察されたことから,CAFにはがん抑制性の機能があることも指摘されている.昨今の解析技術の発展により,CAFの多様性やその臨床的意義に関する理解は得られつつあるが,依然,がん抑制性CAFの本態は不明確である.本稿では,がん抑制性CAFに対する理解の現状と難しさについて,われわれの仮説も交えつつ概説したい.(著者抄録)

市原周, 伊藤典子, 森谷鈴子, 岩越朱里, 森田孝子, 大岩幹直, 佐久間貴彦, 中井登紀子, 窪田智行, 水谷三浩, 稲田健一, 川崎朋範, 榎本篤

現代医学 Vol. 71 ( 1 ) page： 104 - 109 2024.6

非浸潤性乳管癌に対する乳房温存術後の放射線照射は省略できるか?

市原周, 伊藤典子, 森谷鈴子, 岩越朱里, 森田孝子, 大岩幹直, 佐久間貴彦, 中井登紀子, 窪田智行, 水谷三浩, 稲田健一, 川崎朋範, 榎本篤

現代医学 Vol. 71 ( 1 ) page： 104 - 109 2024.6

Successful Treatment of Multiple Systemic Artery-to-Pulmonary Artery Fistulas.

Ryo T, Shigemitsu K, Mori S, Enomoto A, Chen-Yoshikawa TF

Annals of thoracic surgery short reports Vol. 2 ( 2 ) page： 172 - 174 2024.6

侵襲性微小乳頭所見と多発リンパ節転移を示す乳房神経内分泌腫瘍(Neuroendocrine tumor of the breast showing invasive micropapillary features and multiple lymph node metastases)

川崎朋範, 田島知明, 村松千左子, 藤本章博, 児玉ひとみ, 市川二郎, 永井宏和, 星田義彦, 谷山清巳, 榎本篤, 川村隆三, 竹見祥大, 鈴木美穂, 村松孝洋, 地主龍平, 宇佐見陽子, 中村泰大, 解良恭一

日本内分泌学会雑誌 Vol. 99 ( 5 ) page： 1674 - 1674 2024.4

Cancer-associated Fibroblast-Derived Itaconate Stimulates Pro-tumorigenic Immune Microenvironment in Cervical Cancer

Nakahara, R; Aki, S; Kato, M; Tsuchida, R; Shimamura, T; Enomoto, A; Osawa, T

CANCER SCIENCE Vol. 115 page： 555 - 555 2024.3

Functional analyses of immune checkpoint molecule, VISTA, in malignant pleural mesothelioma

Okada, H; Yanagisawa, K; Enomoto, A

CANCER SCIENCE Vol. 115 page： 1830 - 1830 2024.3

Exploring the Role of Transgelin in Cancer-associated Fibroblasts in the Development of Pancreatic Adenocarcinoma

Wang, XX; Shinjo, K; Kumegawa, K; Maruyama, R; Mii, S; Murofushi, Y; Suzuki, M; Enomoto, A; Kondo, Y

CANCER SCIENCE Vol. 115 page： 1698 - 1698 2024.3

Cancer-associated fibroblasts as upstream regulators of the tumor immune microenvironment and therapeutic application

Owaki, T; Miyai, Y; Iida, T; Akamatsu, S; Enomoto, A

CANCER SCIENCE Vol. 115 page： 1498 - 1498 2024.3

Meflin, a New Population Maker of Portal Fibroblasts, Suppress Cholangiocarcinoma

Ito, K; Shiraki, Y; Mii, S; Esaki, N; Sunagawa, M; Baba, T; Ebata, T; Enomoto, A

CANCER SCIENCE Vol. 115 page： 2090 - 2090 2024.3

Single-cell colocalization analysis using a deep generative model Reviewed

Yasuhiro Kojima, Shinji Mii, Shuto Hayashi, Haruka Hirose, Masato Ishikawa, Masashi Akiyama, Atsushi Enomoto, Teppei Shimamura

Cell Systems Vol. 15 ( 2 ) page： 180 - 192.e7 2024.2

Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway Reviewed

Rikito Tatsuno, Jiro Ichikawa, Yoshihiro Komohara, Cheng Pan, Tomonori Kawasaki, Atsushi Enomoto, Kaoru Aoki, Keiko Hayakawa, Shintaro Iwata, Takahiro Jubashi, Hirotaka Haro

Cell Death & Disease Vol. 15 ( 2 ) page： 108 2024.2

TGF‐β signaling promotes desmoid tumor formation via CSRP2 upregulation Reviewed

Yu Li, Teruaki Fujishita, Emi Mishiro‐Sato, Yasushi Kojima, Yanqing Niu, Makoto Mark Taketo, Yuya Urano, Tomohisa Sakai, Atsushi Enomoto, Yoshihiro Nishida, Masahiro Aoki

Cancer Science Vol. 115 ( 2 ) page： 401 - 411 2024.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

Abstract

Desmoid tumors (DTs), also called desmoid‐type fibromatoses, are locally aggressive tumors of mesenchymal origin. In the present study, we developed a novel mouse model of DTs by inducing a local mutation in the Ctnnb1 gene, encoding β‐catenin in PDGFRA‐positive stromal cells, by subcutaneous injection of 4‐hydroxy‐tamoxifen. Tumors in this model resembled histologically clinical samples from DT patients and showed strong phosphorylation of nuclear SMAD2. Knockout of SMAD4 in the model significantly suppressed tumor growth. Proteomic analysis revealed that SMAD4 knockout reduced the level of Cysteine‐and‐Glycine‐Rich Protein 2 (CSRP2) in DTs, and treatment of DT‐derived cells with a TGF‐β receptor inhibitor reduced CSRP2 RNA levels. Knockdown of CSRP2 in DT cells significantly suppressed their proliferation. These results indicate that the TGF‐β/CSRP2 axis is a potential therapeutic target for DTs downstream of TGF‐β signaling.

DOI： 10.1111/cas.16037

頭蓋内における間葉系幹細胞マーカーMeflinの発現解析(Expression of Meflin, a Mesenchymal Stem Cell(MSC) Marker, in the Intracranial Region)

白木之浩, 安藤良太, 三井伸二, 榎本篤

日本病理学会会誌 Vol. 113 ( 1 ) page： 439 - 439 2024.2

多発転移を伴う神経内分泌と微小乳頭癌の特徴を示す浸潤性乳癌(Invasive breast cancer showing neuroendocrine and micropapillary features with multiple metastases)

川崎朋範, 田島知明, 村松千左子, 川村隆三, 竹見祥大, 鈴木美穂, 市原周, 榎本篤, 永井宏和, 谷山清巳

日本病理学会会誌 Vol. 113 ( 1 ) page： 302 - 303 2024.2

膵星細胞(PSC)の形態と機能

安藤良太, 渡會蒼大, 白木之浩, 三井伸二, 榎本篤

日本病理学会会誌 Vol. 113 ( 1 ) page： 400 - 400 2024.2

難治性がん腫の克服を目指したがん微小環境を標的とする抗体薬物複合体の研究開発

江崎寛季, 安藤良太, 宮井雄基, 松山誠, 白木之浩, 三井伸二, 高橋雅英, 榎本篤

日本病理学会会誌 Vol. 113 ( 1 ) page： 400 - 400 2024.2

Meflin is a marker of pancreatic stellate cells involved in fibrosis and epithelial regeneration in the pancreas. Reviewed International journal

Ryota Ando, Yukihiro Shiraki, Yuki Miyai, Hiroki Shimizu, Kazuhiro Furuhashi, Shun Minatoguchi, Katsuhiro Kato, Akira Kato, Tadashi Iida, Yasuyuki Mizutani, Kisuke Ito, Naoya Asai, Shinji Mii, Nobutoshi Esaki, Masahide Takahashi, Atsushi Enomoto

The Journal of pathology Vol. 262 ( 1 ) page： 61 - 75 2024.1

Language：English Publishing type：Research paper (scientific journal)

Pancreatic stellate cells (PSCs) are stromal cells in the pancreas that play an important role in pancreatic pathology. In chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC), PSCs are known to get activated to form myofibroblasts or cancer-associated fibroblasts (CAFs) that promote stromal fibroinflammatory reactions. However, previous studies on PSCs were mainly based on the findings obtained using ex vivo expanded PSCs, with few studies that addressed the significance of in situ tissue-resident PSCs using animal models. Their contributions to fibrotic reactions in CP and PDAC are also lesser-known. These limitations in our understanding of PSC biology have been attributed to the lack of specific molecular markers of PSCs. Herein, we established Meflin (Islr), a glycosylphosphatidylinositol-anchored membrane protein, as a PSC-specific marker in both mouse and human by using human pancreatic tissue samples and Meflin reporter mice. Meflin-positive (Meflin+ ) cells contain lipid droplets and express the conventional PSC marker Desmin in normal mouse pancreas, with some cells also positive for Gli1, the marker of pancreatic tissue-resident fibroblasts. Three-dimensional analysis of the cleared pancreas of Meflin reporter mice showed that Meflin+ PSCs have long and thin cytoplasmic protrusions, and are localised on the abluminal side of vessels in the normal pancreas. Lineage tracing experiments revealed that Meflin+ PSCs constitute one of the origins of fibroblasts and CAFs in CP and PDAC, respectively. In these diseases, Meflin+ PSC-derived fibroblasts showed a distinctive morphology and distribution from Meflin+ PSCs in the normal pancreas. Furthermore, we showed that the genetic depletion of Meflin+ PSCs accelerated fibrosis and attenuated epithelial regeneration and stromal R-spondin 3 expression, thereby implying that Meflin+ PSCs and their lineage cells may support tissue recovery and Wnt/R-spondin signalling after pancreatic injury and PDAC development. Together, these data indicate that Meflin may be a marker specific to tissue-resident PSCs and useful for studying their biology in both health and disease. © 2023 The Pathological Society of Great Britain and Ireland.

DOI： 10.1002/path.6211

Impact of elobixibat on liver tumors, microbiome, and bile acid levels in a mouse model of nonalcoholic steatohepatitis

Sugiyama, Y; Yamamoto, K; Honda, T; Kato, A; Muto, H; Yokoyama, S; Ito, T; Imai, N; Ishizu, Y; Nakamura, M; Asano, T; Enomoto, A; Zaitsu, K; Ishigami, M; Fujishiro, M; Kawashima, H

HEPATOLOGY INTERNATIONAL Vol. 17 ( 6 ) page： 1378 - 1392 2023.12

Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway

Nakai, K; Lin, HC; Yamano, S; Tanaka, S; Kitamoto, S; Saitoh, H; Sakuma, K; Kurauchi, J; Akter, E; Konno, M; Ishibashi, K; Kamata, R; Ohashi, A; Koseki, J; Takahashi, H; Yokoyama, H; Shiraki, Y; Enomoto, A; Abe, S; Hayakawa, Y; Ushiku, T; Mutoh, M; Fujita, Y; Kon, S

NATURE COMMUNICATIONS Vol. 14 ( 1 ) page： 7048 2023.11

Neuroendocrine neoplasms in the breast oncology field: dilemmas of nature and morphology

Kawasaki, T; Tashima, T; Enomoto, A; Ichikawa, J; Nagai, H; Muramatsu, C; Nakamura, Y; Kaira, K

FRONTIERS IN ENDOCRINOLOGY Vol. 14 page： 1216424 2023.11

Targeting hyaluronan-mediated motility receptor (HMMR) enhances response to androgen receptor signalling inhibitors in prostate cancer Reviewed International journal

Josephine A. Hinneh, Joanna L. Gillis, Chui Yan Mah, Swati Irani, Raj K. Shrestha, Natalie K. Ryan, Enomoto Atsushi, Zeyad D. Nassar, David J. Lynn, Luke A. Selth, Masashi Kato, Margaret M. Centenera, Lisa M. Butler

British Journal of Cancer Vol. 129 ( 8 ) page： 1350 - 1361 2023.10

Language：English Publishing type：Research paper (scientific journal)

Background: Resistance to androgen receptor signalling inhibitors (ARSIs) represents a major clinical challenge in prostate cancer. We previously demonstrated that the ARSI enzalutamide inhibits only a subset of all AR-regulated genes, and hypothesise that the unaffected gene networks represent potential targets for therapeutic intervention. This study identified the hyaluronan-mediated motility receptor (HMMR) as a survival factor in prostate cancer and investigated its potential as a co-target for overcoming resistance to ARSIs. Methods: RNA-seq, RT-qPCR and Western Blot were used to evaluate the regulation of HMMR by AR and ARSIs. HMMR inhibition was achieved via siRNA knockdown or pharmacological inhibition using 4-methylumbelliferone (4-MU) in prostate cancer cell lines, a mouse xenograft model and patient-derived explants (PDEs). Results: HMMR was an AR-regulated factor that was unaffected by ARSIs. Genetic (siRNA) or pharmacological (4-MU) inhibition of HMMR significantly suppressed growth and induced apoptosis in hormone-sensitive and enzalutamide-resistant models of prostate cancer. Mechanistically, 4-MU inhibited AR nuclear translocation, AR protein expression and subsequent downstream AR signalling. 4-MU enhanced the growth-suppressive effects of 3 different ARSIs in vitro and, in combination with enzalutamide, restricted proliferation of prostate cancer cells in vivo and in PDEs. Conclusion: Co-targeting HMMR and AR represents an effective strategy for improving response to ARSIs. [Figure not available: see fulltext.]

DOI： 10.1038/s41416-023-02406-8

骨肉腫微小環境における腫瘍関連マクロファージ由来ケモカインの働き

腫瘍免疫微小環境の上流制御因子としてのがん関連線維芽細胞、そして臨床応用へ(Cancer-associated fibroblasts as upstream regulators of the tumor immune microenvironment and therapeutic application)

大脇貴之, 宮井雄基, 飯田忠, 赤松秀輔, 榎本篤

日本癌学会総会記事 Vol. 82回 page： 1499 - 1499 2023.9

辰野力人, 市川二郎, 菰原義弘, 榎本篤, 川崎朋範, 波呂浩孝

日本整形外科学会雑誌 Vol. 97 ( 8 ) page： S1891 - S1891 2023.8

骨肉腫微小環境における腫瘍関連マクロファージ由来ケモカインの働き

辰野力人, 市川二郎, 菰原義弘, 榎本篤, 川崎朋範, 波呂浩孝

日本整形外科学会雑誌 Vol. 97 ( 8 ) page： S1891 - S1891 2023.8

免疫チェックポイント阻害剤を用いた膵癌治療:基礎から臨床まで癌関連線維芽細胞をターゲットにした免疫チェックポイント阻害剤感受性改善薬の開発

飯田忠, 榎本篤, 川嶋啓揮

膵臓 Vol. 38 ( 3 ) page： A211 - A211 2023.7

Significance of expression of CD109 in osteosarcoma and its involvement in tumor progression<i> via</i> BMP signaling

Mori, N; Esaki, N; Shimoyama, Y; Shiraki, Y; Asai, N; Sakai, T; Nishida, Y; Takahashi, M; Enomoto, A; Mii, S

PATHOLOGY RESEARCH AND PRACTICE Vol. 245 page： 154443 2023.5

Neuroendocrine neoplasms of the breast: diagnostic confusion and future perspectives. International journal

Tomonori Kawasaki, Tomoaki Tashima, Atsushi Enomoto, Tetsuo Kondo, Hirokazu Nagai, Yasuhiro Nakamura, Jiro Ichikawa, Kiyomi Taniyama, Kyoichi Kaira

Virchows Archiv : an international journal of pathology Vol. 482 ( 5 ) page： 929 - 930 2023.5

Substrate stiffness induces nuclear localization of myosin regulatory light chain to suppress apoptosis. Reviewed International journal

Katsuya Onishi, Seiichiro Ishihara, Masayuki Takahashi, Akihiro Sakai, Atsushi Enomoto, Kentaro Suzuki, Hisashi Haga

FEBS letters Vol. 597 ( 5 ) page： 643 - 656 2023.3

Language：English Publishing type：Research paper (scientific journal)

Stiffness of the extracellular matrix regulates various biological responses, but the response mechanisms are poorly understood. Here, we found that the nuclear diphosphorylated myosin regulatory light chain (2P-MRLC) is a critical mechanomediator that suppresses apoptosis in response to substrate stiffness. Stiff substrates promoted the nuclear localization of 2P-MRLC. Zipper-interacting protein kinase [ZIPK; also known as death-associated protein kinase 3 (DAPK3)], a kinase for MRLC, was localized in the nucleus in response to stiff substrates and promoted the nuclear localization of 2P-MRLC. Moreover, actin fiber formation induced by substrate stiffness promoted the nuclear localization of 2P-MRLC via ZIPK. 2P-MRLC in response to substrate stiffness suppressed the expression of MAF bZIP transcription factor B (MafB) and repressed apoptosis. These findings reveal a newly identified role of MRLC in mechanotransduction.

DOI： 10.1002/1873-3468.14592

The usefulness of meflin expression in cancer-associated fibroblasts as a predictive marker for immune checkpoint inhibitor in clear cell renal cell carcinoma.

Owaki, T; Miyai, Y; Naito, Y; Sano, T; Enomoto, A

JOURNAL OF CLINICAL ONCOLOGY Vol. 41 2023.2

Stiff substrates localize myosin regulatory light chain in nucleus to suppress apoptosis

Katsuya, O; Seiichiro, I; Masayuki, T; Enomoto, A; Haga, H

MOLECULAR BIOLOGY OF THE CELL Vol. 34 ( 2 ) page： 731 - 732 2023.2

TGF-β signaling promotes desmoid tumor formation in a mouse model

Li, Y; Fujishita, T; Mishiro, E; Taketo, MM; Enomoto, A; Nishida, Y; Aoki, M

CANCER SCIENCE Vol. 114 page： 1833 - 1833 2023.2

An Effective Therapy that Antibody-Drug Conjugates Targeting Meflin (+) Cells for Osteosarcoma

Sakoda, T; Esaki, N; Ando, R; Miyai, Y; Iida, T; Matsuyama, M; Shiraki, Y; Mii, S; Nishida, Y; Takahashi, M; Enomoto, A

CANCER SCIENCE Vol. 114 page： 760 - 760 2023.2

Cancer-associated Fibroblast-derived Itaconate in Tumor Microenvironment Promotes Tumor Growth in Cervical Cancer

Nakahara, R; Aki, S; Kato, M; Tsuchida, R; Shimamura, T; Enomoto, A; Osawa, T

CANCER SCIENCE Vol. 114 page： 1779 - 1779 2023.2

Cancer-associated Fibroblast-derived Itaconate in Tumor Microenvironment Promotes Tumor Growth in Cervical Cancer

Nakahara, R; Aki, S; Kato, M; Tsuchida, R; Shimamura, T; Enomoto, A; Osawa, T

CANCER SCIENCE Vol. 114 page： 1923 - 1923 2023.2

Identification of tumor suppressor fibroblasts and therapeutic applications

Iida, T; Mizutani, Y; Kawashima, H; Enomoto, A

CANCER SCIENCE Vol. 114 page： 1650 - 1650 2023.2

Meflin is a good predictive biomarker for ICI in urothelial carcinoma

Owaki, T; Miyai, Y; Iida, T; Esaki, N; Shiraki, Y; Mii, S; Enomoto, A

CANCER SCIENCE Vol. 114 page： 1399 - 1399 2023.2

New horizons for cancer research driven by histopathological approaches

Enomoto, A; Kanai, Y

CANCER SCIENCE Vol. 114 page： 919 - 919 2023.2

Peritoneal restoration by repurposing vitamin D inhibits ovarian cancer dissemination via blockade of TGF-β1/THBS1

Kitami, K; Yoshihara, M; Yamakita, Y; Sugiyama, M; Koya, Y; Miyamoto, E; Fujimoto, H; Iyoshi, S; Uno, K; Mogi, K; Kato, K; Nawa, A; Enomoto, A; Kajiyama, H

CANCER SCIENCE Vol. 114 page： 1047 - 1047 2023.2

Therapeutic Strategies to Overcome Fibrotic Barriers to Nanomedicine in the Pancreatic Tumor Microenvironment. Reviewed International journal

Hiroyoshi Y Tanaka, Takuya Nakazawa, Atsushi Enomoto, Atsushi Masamune, Mitsunobu R Kano

Cancers Vol. 15 ( 3 ) 2023.2

Language：English Publishing type：Research paper (scientific journal)

Pancreatic cancer is notorious for its dismal prognosis. The enhanced permeability and retention (EPR) effect theory posits that nanomedicines (therapeutics in the size range of approximately 10-200 nm) selectively accumulate in tumors. Nanomedicine has thus been suggested to be the "magic bullet"-both effective and safe-to treat pancreatic cancer. However, the densely fibrotic tumor microenvironment of pancreatic cancer impedes nanomedicine delivery. The EPR effect is thus insufficient to achieve a significant therapeutic effect. Intratumoral fibrosis is chiefly driven by aberrantly activated fibroblasts and the extracellular matrix (ECM) components secreted. Fibroblast and ECM abnormalities offer various potential targets for therapeutic intervention. In this review, we detail the diverse strategies being tested to overcome the fibrotic barriers to nanomedicine in pancreatic cancer. Strategies that target the fibrotic tissue/process are discussed first, which are followed by strategies to optimize nanomedicine design. We provide an overview of how a deeper understanding, increasingly at single-cell resolution, of fibroblast biology is revealing the complex role of the fibrotic stroma in pancreatic cancer pathogenesis and consider the therapeutic implications. Finally, we discuss critical gaps in our understanding and how we might better formulate strategies to successfully overcome the fibrotic barriers in pancreatic cancer.

DOI： 10.3390/cancers15030724

【新しい技術で解明されてきた膵臓の病態】癌関連線維芽細胞の形質改変による抗癌剤効果増強剤の開発

飯田忠, 水谷泰之, 山雄健太郎, 石川卓哉, 大野栄三郎, 藤城光弘, 榎本篤, 川嶋啓揮

膵臓 Vol. 38 ( 1 ) page： 37 - 43 2023.2

Language：Japanese Publisher：(一社)日本膵臓学会

膵癌の特徴として間質の線維化とそれに伴う組織硬化がある.組織硬化は間質内圧上昇と血管虚脱を誘導し,その結果抗癌剤の間質浸透と癌細胞への到達を阻害することが知られている.この間質の線維化に中心的な役割を果たすのが癌関連線維芽細胞(CAF)である.このため,これまで様々な方法でCAFをターゲットにする治療が試みられてきたが未だ成功していない.本研究では癌抑制性CAFのマーカーであり機能分子であるMeflinをCAFに発現誘導することで,膵癌における化学療法への感受性を改善させることを明らかにした.さらにスクリーニングにより合成レチノイドであるAM80がCAFにおけるMeflinの発現を効果的に誘導し,ドラッグデリバリーの改善を伴って化学療法に対する感受性を改善させることを明らかにした.筆者らは本結果を受けて切除不能膵癌に対するAM80と従来の抗癌剤の併用効果を検証する医師主導治験を行っている.(著者抄録)

Other Link： https://search.jamas.or.jp/default/link?pub_year=2023&ichushi_jid=J02025&link_issn=&doc_id=20230308380005&doc_link_id=10.2958%2Fsuizo.38.37&url=https%3A%2F%2Fdoi.org%2F10.2958%2Fsuizo.38.37&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Novel Therapeutic Potentials of Taxifolin for Obesity-Induced Hepatic Steatosis, Fibrogenesis, and Tumorigenesis Reviewed

Takayuki Inoue, Bin Fu, Miwako Nishio, Miyako Tanaka, Hisashi Kato, Masashi Tanaka, Michiko Itoh, Hajime Yamakage, Kozue Ochi, Ayaka Ito, Yukihiro Shiraki, Satoshi Saito, Masafumi Ihara, Hideo Nishimura, Atsuhiko Kawamoto, Shian Inoue, Kumiko Saeki, Atsushi Enomoto, Takayoshi Suganami, Noriko Satoh-Asahara

Nutrients Vol. 15 ( 2 ) page： 350 - 350 2023.1

Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

The molecular pathogenesis of nonalcoholic steatohepatitis (NASH) includes a complex interaction of metabolic stress and inflammatory stimuli. Considering the therapeutic goals of NASH, it is important to determine whether the treatment can prevent the progression from NASH to hepatocellular carcinoma. Taxifolin, also known as dihydroquercetin, is a natural bioactive flavonoid with antioxidant and anti-inflammatory properties commonly found in various foods and health supplement products. In this study, we demonstrated that Taxifolin treatment markedly prevented the development of hepatic steatosis, chronic inflammation, and liver fibrosis in a murine model of NASH. Its mechanisms include a direct action on hepatocytes to inhibit lipid accumulation. Taxifolin also increased brown adipose tissue activity and suppressed body weight gain through at least two distinct pathways: direct action on brown adipocytes and indirect action via fibroblast growth factor 21 production in the liver. Notably, the Taxifolin treatment after NASH development could effectively prevent the development of liver tumors. Collectively, this study provides evidence that Taxifolin shows pleiotropic effects for the treatment of the NASH continuum. Our data also provide insight into the novel mechanisms of action of Taxifolin, which has been widely used as a health supplement with high safety.

DOI： 10.3390/nu15020350

Wnt activation-induced disturbance of cell competition causes diffuse invasion of transformed cells through upregulation of NF-κB-mediated MMP21

Kazuki Nakai, Hancheng Lin, Shotaro Yamano, Shinya Tanaka, Sho Kitamoto, Kenta Sakuma, Junpei Kurauchi, Eilma Akter, Masamitsu Konno, Jun Koseki, Hirotaka Takahashi, Hideshi Yokoyama, Yukihiro Shiraki, Atsushi Enomoto, Sohei Abe, Yoku Hayakawa, Tetsuo Ushiku, Michihiro Mutoh, Yasuyuki Fujita, Shunsuke Kon

2022.12

Publisher：Research Square Platform LLC

Abstract

Normal epithelial cells exert their competitive advantage over RasV12-transformed cells and eliminate them into the apical lumen via cell competition. However, the internal or external factors that compromise cell competition and provoke carcinogenesis remains unknown. In this study, we examined the effect of sequential accumulation of gene mutations, mimicking multi-sequential carcinogenesis on RasV12-induced cell competition in intestinal epithelial tissues. Consequently, we found that directionality of RasV12-cell extrusion in Wnt-activated epithelia is reversed, and transformed cells are delaminated into the basal lamina via non-cell autonomous MMP21 upregulation. Subsequently, diffusively infiltrating, transformed cells develop into highly invasive carcinomas. Elevated production of MMP21 is elicited partly through NF-κB signaling, blockage of which restores apical elimination of RasV12 cells. We further found that the NF-κB-MMP21 axis is significantly bolstered in early colorectal carcinoma in humans. Collectively, this study shows that cells with high mutational burdens exploit cell competition for their benefit by behaving as unfit cells, endowing them with an invasion advantage.

DOI： 10.21203/rs.3.rs-2314559/v1

Other Link： https://www.researchsquare.com/article/rs-2314559/v1.html

Girdin Promotes Tumorigenesis and Chemoresistance in Lung Adenocarcinoma by Interacting with PKM2

Cao, FY; Yang, DS; Tang, FY; Lu, C; He, X; Chen, SM; Yang, ZH; Gong, SY; Sun, LQ; Enomoto, A; Takahashi, M; Weng, L

CANCERS Vol. 14 ( 22 ) 2022.11

Mammaglobin 1 mediates progression of trastuzumab-resistant breast cancer cells through regulation of cyclins and NF-κB. Reviewed International journal

Ratih Kusumastuti, Yuji Kumagai, Seiichiro Ishihara, Atsushi Enomoto, Takashi Murakami, Motoaki Yasuda, Hisashi Haga

FEBS open bio Vol. 12 ( 10 ) page： 1797 - 1813 2022.10

Language：English Publishing type：Research paper (scientific journal)

Overexpression of human epidermal growth factor receptor 2 (HER2) in various cancers is correlated with poor patient survival. Trastuzumab, a recombinant humanized monoclonal antibody against HER2, has been considered to be a first-line therapy for HER2-positive breast cancer patients, but its usefulness is limited by the development of resistance. In this study, we established resistant cells by long-term treatment with trastuzumab. These cells showed higher proliferation, invasion, and migration abilities than the wild-type cells. Mammaglobin 1 (MGB1), cyclin D1, E1, A2, and phosphorylated NF-κB (p-p65) were upregulated in resistant cells. These proteins regulate cell proliferation, migration, and invasion of resistant cells. Depletion of MGB1 decreased cyclin and p-p65 expression. Cyclin D1 and A2, but not E1 expression, were affected by p-p65 downregulation. In summary, our results indicate that MGB1 expression is increased in breast cancer cells that have gained resistance to trastuzumab, and suggest that MGB1 promotes aggressiveness through cyclin and NF-κB regulation.

DOI： 10.1002/2211-5463.13468

BMP3b is a Novel Anti-Fibrotic Molecule Regulated by Meflin in Lung Fibroblasts. Reviewed International journal

Atsushi Suzuki, Koji Sakamoto, Yoshio Nakahara, Atsushi Enomoto, Jun Hino, Akira Ando, Masahide Inoue, Yukihiro Shiraki, Norihito Omote, Masahiro Kusaka, Jun Fukihara, Naozumi Hashimoto

American journal of respiratory cell and molecular biology Vol. 67 ( 4 ) page： 446 - 458 2022.10

Language：English Publishing type：Research paper (scientific journal) Publisher：American Thoracic Society

Fibroblasts play a central role in the lung fibrotic process. Our recent study identified a novel subpopulation of lung fibroblasts expressing meflin, anti-fibrotic properties of which were confirmed by murine lung fibrosis model. Meflin expressing fibroblasts were resistant to fibrogenesis induced by transforming growth factor-β (TGF-β), but its underlying mechanisms remain unknown. In this study, evaluation of a silica-nanoparticles-induced lung fibrosis model confirmed the antifibrotic effect of meflin via the regulation of TGF-β signaling. We conducted comparative gene expression profiling in lung fibroblasts, which identified growth differentiation factor 10 (Gdf10) encoding bone morphogenic protein 3b (BMP3b) as the most down-regulated gene in meflin-deficient cells under the profibrotic condition with TGF-β. We hypothesized that BMP3b can be an effector molecule playing an anti-fibrotic role downstream of meflin. As suggested by single-cell transcriptomic data, restricted expressions of Gdf10 (Bmp3b) in stromal cells including fibroblasts were confirmed. We examined possible anti-fibrotic properties of BMP3b in lung fibroblasts and demonstrated that Bmp3b-null fibroblasts were more susceptible to TGF-β-induced fibrogenic changes. Furthermore, Bmp3b-null mice exhibited exaggerated lung fibrosis induced by silica-nanoparticles in vivo. We also demonstrated that treatment with recombinant BMP3B was effective against TGF-β-induced fibrogenesis in fibroblasts, especially in the suppression of excessive extracellular matrix production. These lines of evidence suggested that BMP3b is a novel humoral effector molecule regulated by meflin which exerts anti-fibrotic properties in lung fibroblasts. Supplementation of BMP3B could be a novel therapeutic strategy for fibrotic lung diseases.

DOI： 10.1165/rcmb.2021-0484OC

Insulinoma-associated protein 1 (INSM1) expression in breast carcinomas with neuroendocrine morphologies: application and future perspectives

Kawasaki, T; Tashima, T; Ryozawa, S; Nakamura, Y; Fujimoto, A; Usami, Y; Inozume, T; Saitoh, M; Kondo, T; Enomoto, A; Nagai, H; Taniyama, K; Muramatsu, C; Kaira, K

HISTOPATHOLOGY Vol. 81 page： 31 - 31 2022.10

【免疫チェックポイント阻害薬の"耐性"に挑むがん免疫サイクルから見出す戦略】がん間質の線維芽細胞・腫瘍血管と免疫チェックポイント阻害薬の耐性機構

榎本篤, 宮井雄基, 飯田忠, 大脇貴之

実験医学 Vol. 40 ( 16 ) page： 2580 - 2586 2022.10

Language：Japanese Publisher：(株)羊土社

ヒトのがんの間質の免疫細胞の浸潤、線維化、および腫瘍血管の増生の程度はがんの種類あるいは症例間で実に多様である。同一症例の標本でも視野を少し動かしただけで、同じがんとは思えないような組織像の違いを見せることもある。免疫チェックポイント阻害薬(ICI)の勃興によりがん治療の概念が変わり、それに伴いヒト免疫学も飛躍的に発展したと聞く。しかしながら、一部の特殊なゲノム異常を有するがんを除き、前述の腫瘍内(intratumor)および腫瘍間(intertumor)の多様性がICIの奏功予測や一律な奏効率の押し上げを困難なものにしていると推測される。本稿では、主に線維芽細胞と腫瘍血管に焦点をあて、これらが抗腫瘍免疫応答およびICIの効果に及ぼす影響、およびその知見に基づく治療戦略について概説したい。(著者抄録)

骨肉腫におけるMeflin陽性細胞を標的とした抗体薬物複合体の研究開発(An Effective Therapy that Antibody-Drug Conjugates Targeting Meflin(+) Cells for Osteosarcoma)

迫田朋佳, 江崎寛季, 安藤良太, 宮井雄基, 飯田忠, 松山誠, 白木之浩, 三井伸二, 西田佳弘, 高橋雅英, 榎本篤

日本癌学会総会記事 Vol. 81回 page： E - 2021 2022.9

腫瘍促進的腹膜環境の正常化を標的とした難治性卵巣癌に対する新規治療戦略の開発(Peritoneal restoration by repurposing vitamin D inhibits ovarian cancer dissemination via blockade of TGF-β1/THBS1)

北見和久, 吉原雅人, 山北由彦, 杉山麻衣, 小屋美博, 宮本絵美里, 藤本裕基, 伊吉祥平, 宇野枢, 茂木一将, 加藤一喜, 那波明宏, 榎本篤, 梶山広明

日本癌学会総会記事 Vol. 81回 page： MS1 - 3 2022.9

癌抑制性線維芽細胞の同定と治療応用(Identification of tumor suppressor fibroblasts and therapeutic applications)

飯田忠, 水谷泰之, 川嶋啓揮, 榎本篤

日本癌学会総会記事 Vol. 81回 page： J - 3026 2022.9

Meflinは尿路上皮癌における免疫チェックポイント阻害剤治療の効果予測バイオマーカーである(Meflin is a good predictive biomarker for ICI in urothelial carcinoma)

大脇貴之, 宮井雄基, 飯田忠, 江崎寛季, 白木之浩, 三井伸二, 榎本篤

日本癌学会総会記事 Vol. 81回 page： P - 2185 2022.9

Possible disease-protective roles of fibroblasts in cancer and fibrosis and their therapeutic application Reviewed

Shiraki, Yukihiro, Mii, Shinji, Esaki, Nobutoshi, Enomoto, Atsushi

Vol. 84 ( 3 ) page： 484 - 496 2022.8

【膵臓の発生・生理・解剖から膵炎・膵癌の病態解明に至る最新の知見】がん抑制性線維芽細胞の本態の理解とその治療応用について

水谷泰之, 飯田忠, 石川卓哉, 大野栄三郎, 川嶋啓揮, 藤城光弘, 榎本篤

胆と膵 Vol. 43 ( 8 ) page： 787 - 794 2022.8

Language：Japanese Publisher：医学図書出版(株)

膵癌治療のボトルネックは、その豊富な間質に起因する薬剤送達(drug delivery)の障害であり、これを解決する治療法の開発が強く求められている。膵癌の豊富な間質の原因は、がん関連線維芽細胞(CAF)の増生であり、がんを促進する形質を有することから治療標的として注目されて久しい。しかしながら、線維芽細胞の活性化および増殖を阻害する薬剤を臨床応用する試みはうまくいっていない。それらの結果より近年では、線維芽細胞はがん促進性と抑制性の二つの極性(性質の連続的な差異)を有していること、さらに同細胞の極性はがん発育の文脈に沿って容易に変換されることの2点が原因であると考えられるようになった。われわれは最近、meflin陽性線維芽細胞が、がん抑制性線維芽細胞であり、その機能は従来の活性化線維芽細胞(筋線維芽細胞やCAF)とは異なっていること、および、線維芽細胞の極性を制御することで抗がん剤の送達性の向上が図れることをマウスモデルで示してきた。これらの知見をベースに、現在、われわれは線維芽細胞の極性の変化を誘導する薬剤の効果を検証する医師主導治験を行っている。(著者抄録)

Efficacy of auranofin as an inhibitor of desmoid progression. Reviewed International journal

Kan Ito, Yoshihiro Nishida, Shunsuke Hamada, Koki Shimizu, Tomohisa Sakai, Bisei Ohkawara, Benjamin A Alman, Atsushi Enomoto, Kunihiro Ikuta, Hiroshi Koike, Jiarui Zhang, Kinji Ohno, Shiro Imagama

Scientific reports Vol. 12 ( 1 ) page： 11918 - 11918 2022.7

Language：English Publishing type：Research paper (scientific journal)

Anticancer drugs and molecular targeted therapies are used for refractory desmoid-type fibromatosis (DF), but occasionally cause severe side effects. The purpose of this study was to identify an effective drug with fewer side effects against DF by drug repositioning, and evaluate its efficacy. FDA-approved drugs that inhibit the proliferation of DF cells harboring S45F mutations of CTNNB1 were screened. An identified drug was subjected to the investigation of apoptotic effects on DF cells with analysis of Caspase 3/7 activity. Expression of β-catenin was evaluated with western blot analysis, and immunofluorescence staining. Effects of the identified drug on in vivo DF were analyzed using Apc1638N mice. Auranofin was identified as a drug that effectively inhibits the proliferation of DF cells. Auranofin did not affect Caspase 3/7 activity compared to control. The expression level of β-catenin protein was not changed regardless of auranofin concentration. Auranofin effectively inhibited the development of tumorous tissues by both oral and intraperitoneal administration, particularly in male mice. Auranofin, an anti-rheumatic drug, was identified to have repositioning effects on DF. Since auranofin has been used for many years as an FDA-approved drug, it could be a promising drug with fewer side effects for DF.

DOI： 10.1038/s41598-022-15756-9

Recurrent fulminant non-rheumatic streptococcal myocarditis proven by endomyocardial biopsy and autopsy. Reviewed

Hiroaki Hiraiwa, Ryota Morimoto, Ryota Ando, Ryota Ito, Takashi Araki, Takashi Mizutani, Shingo Kazama, Yuki Kimura, Hideo Oishi, Tasuku Kuwayama, Shogo Yamaguchi, Toru Kondo, Takahiro Okumura, Atsushi Enomoto, Toyoaki Murohara

Journal of cardiology cases Vol. 26 ( 1 ) page： 62 - 65 2022.7

Language：English

A 42-year-old man with a history of acute myocarditis after streptococcal pharyngitis developed recurrent fulminant myocarditis. Endomyocardial biopsy revealed myocyte degeneration, interstitial edema, and neutrophil infiltration. The patient's cardiac function deteriorated rapidly, and he died despite mechanical circulatory support. Autopsy revealed neutrophil infiltration, interstitial edema, and micro-abscesses containing masses of streptococci and neutrophilic phagocytosis within the myocardium. The patient did not meet the diagnostic criteria for acute rheumatic fever; thus, he was diagnosed with non-rheumatic streptococcal myocarditis. Non-rheumatic streptococcal myocarditis rarely recurs, but it can be fulminant upon recurrence. Learning objective: We report a rare case of recurrent fulminant non-rheumatic streptococcal myocarditis. Endomyocardial biopsy and autopsy revealed neutrophil infiltration and micro-abscesses containing bacterial masses of streptococci and neutrophilic phagocytosis in the myocardium. The patient did not meet the diagnostic criteria for acute rheumatic fever; thus, he was diagnosed with non-rheumatic streptococcal myocarditis. Non-rheumatic streptococcal myocarditis rarely recurs, but it can be fulminant upon recurrence.

DOI： 10.1016/j.jccase.2022.02.004

Good and Bad Stroma in Pancreatic Cancer: Relevance of Functional States of Cancer-Associated Fibroblasts. Reviewed International journal

Ryota Ando, Akihiro Sakai, Tadashi Iida, Kunio Kataoka, Yasuyuki Mizutani, Atsushi Enomoto

Cancers Vol. 14 ( 14 ) 2022.7

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

A well-known feature of human pancreatic ductal adenocarcinoma (PDAC) is the extensive proliferation of cancer-associated fibroblasts (CAFs) and highly fibrotic stroma. Recent evidence, based mainly on single-cell analyses, has identified various subsets of CAFs in PDAC mouse models. However, we do not know how these CAF subsets are involved in the progression and drug resistance of human PDAC. Additionally, it remains unclear whether these diverse CAFs have distinct origins and are indicators of genuinely distinct CAF lineages or reflect different states of the same CAFs depending on the tumor microenvironment. Interestingly, recent preclinical studies have started to characterize the nature of cancer-restraining CAFs and have identified their markers Meflin and collagen type I alpha 1. These studies have led to the development of strategies to induce changes in CAF phenotypes using chemical reagents or recombinant viruses, and some of them have been tested in clinical studies. These strategies have the unique potential to convert the so-called bad stroma to good stroma and may also have therapeutic implications for non-cancer diseases such as fibrotic diseases. Together with recently developed sophisticated strategies that specifically target distinct CAF subsets via adoptive cell transfer therapy, vaccination, and antibody-drug conjugates, any future findings arising from these clinical efforts may expand our understanding of the significance of CAF diversity in human PDAC.

DOI： 10.3390/cancers14143315

Correction: Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics. Reviewed International journal

Tadashi Iida, Yasuyuki Mizutani, Nobutoshi Esaki, Suzanne M Ponik, Brian M Burkel, Liang Weng, Keiko Kuwata, Atsushi Masamune, Seiichiro Ishihara, Hisashi Haga, Kunio Kataoka, Shinji Mii, Yukihiro Shiraki, Takuya Ishikawa, Eizaburo Ohno, Hiroki Kawashima, Yoshiki Hirooka, Mitsuhiro Fujishiro, Masahide Takahashi, Atsushi Enomoto

Oncogene Vol. 41 ( 23 ) page： 3302 - 3302 2022.6

Spindle Cell Lipoma with Ossification Mimicking Atypical Lipomatous Tumor/Well-Differentiated Liposarcoma: A Case Report. Reviewed International journal

Jiro Ichikawa, Tomonori Kawasaki, Hiroki Imada, Atsushi Enomoto, Naofumi Taniguchi, Rikito Tatsuno, Satoshi Kanno, Hirotaka Haro

International journal of surgical pathology Vol. 30 ( 4 ) page： 413 - 418 2022.6

Language：English Publishing type：Research paper (scientific journal)

Although spindle cell lipoma (SCL) is a subtype of lipoma, the characteristics of SCL are observed in both lipomatous and non-lipomatous tumors. In this article, we present a case of SCL with ossification mimicking atypical lipomatous tumors/well-differentiated liposarcomas (ALTs/WDLs). Considering the findings of magnetic resonance imaging and needle biopsy, which exhibited ALTs/WDLs, marginal resection was performed. Histopathological findings demonstrated mature adipocytes and spindle cells without atypia and no malignant osteoid tissue in the ossified region. In addition, immunohistochemistry (IHC) showed positive staining for CD34, heterogeneous retinoblastoma protein deficiency, and negative staining for mouse double minute 2 homolog (MDM2) and cyclin-dependent kinase. Fluorescence in-situ hybridization showed negative amplification of MDM2. The final diagnosis of the tumor was established using IHC as an extremely rare SCL with ossification.

DOI： 10.1177/10668969211055798

Letter to Editor Regarding "Nuclear Insulinoma-Associated Protein 1 Expression as a Marker of Neuroendocrine Differentiation in Neoplasms of the Breast" by Seijnhaeve et al. Reviewed International journal

Tomonori Kawasaki, Tomoaki Tashima, Yasuhiro Nakamura, Tetsuo Kondo, Atsushi Enomoto, Kyoichi Kaira

International journal of surgical pathology Vol. 30 ( 4 ) page： 470 - 471 2022.6

The interferon-β/STAT1 axis drives the collective invasion of skin squamous cell carcinoma with sealed intercellular spaces. Reviewed International journal

Yuji Kumagai, Junko Nio-Kobayashi, Seiichiro Ishihara, Atsushi Enomoto, Masashi Akiyama, Ryosuke Ichihara, Hisashi Haga

Oncogenesis Vol. 11 ( 1 ) page： 27 - 27 2022.5

Language：English Publishing type：Research paper (scientific journal)

The process by which cancer cells invade as a cell cluster, known as collective invasion, is associated with metastasis and worse prognosis of cancer patients; therefore, inhibition of collective invasion is considered to improve cancer treatment. However, the cellular characteristics responsible for collective invasion remain largely unknown. Here, we successfully established subclones with various invasive potentials derived from human skin squamous carcinoma cells. The cell cluster of the highly invasive subclone had a hermetically sealed and narrow intercellular space. Interferon-β was localized to the sealed intercellular spaces, leading to collective invasion via the activation of signal transducer and activator of transcription 1 (STAT1). On the other hand, interferon-β was not localized to non-sealed and wide intercellular spaces of the cell cluster of low-invasive subclone with deficient STAT1 activity. In the mixed cell cluster of high- and low-invasive subclones, the high-invasive sub-clonal cells were located at the invasive front of the invasive protrusion, leading to collective invasion by the low-invasive sub-clonal cells. Tissue microarray analysis of human skin squamous cell carcinoma (SCC) also showed enrichment of STAT1 in the invasive front of SCCs. These findings indicate that the intercellular structure controls the potential for collective invasion via STAT1 regulation in SCC.

DOI： 10.1038/s41389-022-00403-9

Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics. Reviewed International journal

Oncogene Vol. 41 ( 19 ) page： 2764 - 2777 2022.5

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

Previous therapeutic attempts to deplete cancer-associated fibroblasts (CAFs) or inhibit their proliferation in pancreatic ductal adenocarcinoma (PDAC) were not successful in mice or patients. Thus, CAFs may be tumor suppressive or heterogeneous, with distinct cancer-restraining and -promoting CAFs (rCAFs and pCAFs, respectively). Here, we showed that induced expression of the glycosylphosphatidylinositol-anchored protein Meflin, a rCAF-specific marker, in CAFs by genetic and pharmacological approaches improved the chemosensitivity of mouse PDAC. A chemical library screen identified Am80, a synthetic, nonnatural retinoid, as a reagent that effectively induced Meflin expression in CAFs. Am80 administration improved the sensitivity of PDAC to chemotherapeutics, accompanied by increases in tumor vessel area and intratumoral drug delivery. Mechanistically, Meflin was involved in the suppression of tissue stiffening by interacting with lysyl oxidase to inhibit its collagen crosslinking activity. These data suggested that modulation of CAF heterogeneity may represent a strategy for PDAC treatment.

DOI： 10.1038/s41388-022-02288-9

The Critical Role of Meflin-Positive Fibroblasts Against Acute Lung Injury

Hashimoto, N; Sakamoto, K; Nakahara, Y; Omote, N; Ando, A; Inoue, M; Suzuki, A; Fukihara, J; Kusaka, M; Wakahara, K; Enomoto, A

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Vol. 205 2022.5

Meflin-BMP3b Axis Is a Novel Anti-Fibrotic Pathway Against TGF-β-Induced Lung Fibrogenesis

Suzuki, A; Sakamoto, K; Nakahara, Y; Enomoto, A; Hino, J; Ando, A; Inoue, M; Omote, N; Kusaka, M; Fukihara, J; Hashimoto, N

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Vol. 205 2022.5

Peritoneal restoration by repurposing vitamin D inhibits ovarian cancer dissemination via blockade of the TGF-β1/thrombospondin-1 axis. Reviewed International journal

Kazuhisa Kitami, Masato Yoshihara, Satoshi Tamauchi, Mai Sugiyama, Yoshihiro Koya, Yoshihiko Yamakita, Hiroki Fujimoto, Shohei Iyoshi, Kaname Uno, Kazumasa Mogi, Yoshiki Ikeda, Akira Yokoi, Nobuhisa Yoshikawa, Kimihiro Nishino, Kaoru Niimi, Akihiro Nawa, Atsushi Enomoto, Hiroaki Kajiyama

Matrix biology : journal of the International Society for Matrix Biology Vol. 109 page： 70 - 90 2022.5

Language：English Publishing type：Research paper (scientific journal)

Ovarian cancer (OvCa), a lethal gynecological malignancy, disseminates to the peritoneum. Mesothelial cells (MCs) act as barriers in the abdominal cavity, preventing the adhesion of cancer cells. However, in patients with OvCa, they are transformed into cancer-associated mesothelial cells (CAMs) via mesenchymal transition and form a favorable microenvironment for tumors to promote metastasis. However, attempts for restoring CAMs to their original state have been limited. Here, we investigated whether inhibition of mesenchymal transition and restoration of MCs by vitamin D suppressed the OvCa dissemination in vitro and in vivo. The effect of vitamin D on the mutual association of MCs and OvCa cells was evaluated using in vitro coculture models and in vivo using a xenograft model. Vitamin D restored the CAMs, and thrombospondin-1 (component of the extracellular matrix that is clinically associated with poor prognosis and is highly expressed in peritoneally metastasized OvCa) was found to promote OvCa cell adhesion and proliferation. Mechanistically, TGF-β1 secreted from OvCa cells enhanced thrombospondin-1 expression in CAMs via Smad-dependent TGF-β signaling. Vitamin D inhibited mesenchymal transition in MCs and suppressed thrombospondin-1 expression via vitamin D receptor/Smad3 competition, contributing to the marked reduction in peritoneal dissemination in vivo. Importantly, vitamin D restored CAMs from a stabilized mesenchymal state to the epithelial state and normalized thrombospondin-1 expression in preclinical models that mimic cancerous peritonitis in vivo. MCs are key players in OvCa dissemination and peritoneal restoration and normalization of thrombospondin-1 expression by vitamin D may be a novel strategy for preventing OvCa dissemination.

DOI： 10.1016/j.matbio.2022.03.003

CD109 expression in tumor cells and stroma correlates with progression and prognosis in pancreatic cancer. Reviewed International journal

Kai Adachi, Yasutaka Sakurai, Masaaki Ichinoe, Masayoshi Tadehara, Akihiro Tamaki, Yurika Kesen, Takuya Kato, Shinji Mii, Atsushi Enomoto, Masahide Takahashi, Wasaburo Koizumi, Yoshiki Murakumo

Virchows Archiv : an international journal of pathology Vol. 480 ( 4 ) page： 819 - 829 2022.4

Language：English Publishing type：Research paper (scientific journal)

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, whose expression is upregulated in some types of malignant tumors. High levels of CD109 in tumor cells have been reported to correlate with poor prognosis; however, significance of CD109 stromal expression in human malignancy has not been elucidated. In this study, we investigated the tumorigenic properties of CD109 in pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analysis of 92 PDAC surgical specimens revealed that positive CD109 expression in tumor cells was significantly associated with poor prognosis (disease-free survival, p = 0.003; overall survival, p = 0.002), and was an independent prognostic factor (disease-free survival, p = 0.0173; overall survival, p = 0.0104) in PDAC. Furthermore, CD109 expression was detected in the stroma surrounding tumor cells, similar to that of α-smooth muscle actin, a histological marker of cancer-associated fibroblasts. The stromal CD109 expression significantly correlated with tumor progression in PDAC (TNM stage, p = 0.033; N factor, p = 0.024; lymphatic invasion, p = 0.028). In addition, combined assessment of CD109 in tumor cells and stroma could identify the better prognosis group of patients from the entire patient population. In MIA PaCa-2 PDAC cell line, we demonstrated the involvement of CD109 in tumor cell motility, but not in PANC-1. Taken together, CD109 not only in the tumor cells but also in the stroma is involved in the progression and prognosis of PDAC, and may serve as a useful prognostic marker in PDAC.

DOI： 10.1007/s00428-021-03230-2

Metastatic Voyage of Ovarian Cancer Cells in Ascites with the Assistance of Various Cellular Components. Reviewed International journal

Kaname Uno, Shohei Iyoshi, Masato Yoshihara, Kazuhisa Kitami, Kazumasa Mogi, Hiroki Fujimoto, Mai Sugiyama, Yoshihiro Koya, Yoshihiko Yamakita, Akihiro Nawa, Tomohiro Kanayama, Hiroyuki Tomita, Atsushi Enomoto, Hiroaki Kajiyama

International journal of molecular sciences Vol. 23 ( 8 ) 2022.4

Language：English Publishing type：Research paper (scientific journal)

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and has a unique metastatic route using ascites, known as the transcoelomic root. However, studies on ascites and contained cellular components have not yet been sufficiently clarified. In this review, we focus on the significance of accumulating ascites, contained EOC cells in the form of spheroids, and interaction with non-malignant host cells. To become resistant against anoikis, EOC cells form spheroids in ascites, where epithelial-to-mesenchymal transition stimulated by transforming growth factor-β can be a key pathway. As spheroids form, EOC cells are also gaining the ability to attach and invade the peritoneum to induce intraperitoneal metastasis, as well as resistance to conventional chemotherapy. Recently, accumulating evidence suggests that EOC spheroids in ascites are composed of not only cancer cells, but also non-malignant cells existing with higher abundance than EOC cells in ascites, including macrophages, mesothelial cells, and lymphocytes. Moreover, hetero-cellular spheroids are demonstrated to form more aggregated spheroids and have higher adhesion ability for the mesothelial layer. To improve the poor prognosis, we need to elucidate the mechanisms of spheroid formation and interactions with non-malignant cells in ascites that are a unique tumor microenvironment for EOC.

DOI： 10.3390/ijms23084383

A novel renal perivascular mesenchymal cell subset gives rise to fibroblasts distinct from classic myofibroblasts. Reviewed International journal

Shun Minatoguchi, Shoji Saito, Kazuhiro Furuhashi, Yuriko Sawa, Masaki Okazaki, Yuko Shimamura, Ahmad Baseer Kaihan, Yusaku Hashimoto, Yoshinari Yasuda, Akitoshi Hara, Yasuyuki Mizutani, Ryota Ando, Noritoshi Kato, Takuji Ishimoto, Naotake Tsuboi, Nobutoshi Esaki, Makoto Matsuyama, Yukihiro Shiraki, Hiroki Kobayashi, Naoya Asai, Atsushi Enomoto, Shoichi Maruyama

Scientific reports Vol. 12 ( 1 ) page： 5389 - 5389 2022.3

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

Perivascular mesenchymal cells (PMCs), which include pericytes, give rise to myofibroblasts that contribute to chronic kidney disease progression. Several PMC markers have been identified; however, PMC heterogeneity and functions are not fully understood. Here, we describe a novel subset of renal PMCs that express Meflin, a glycosylphosphatidylinositol-anchored protein that was recently identified as a marker of fibroblasts essential for cardiac tissue repair. Tracing the lineage of Meflin+ PMCs, which are found in perivascular and periglomerular areas and exhibit renin-producing potential, showed that they detach from the vasculature and proliferate under disease conditions. Although the contribution of Meflin+ PMCs to conventional α-SMA+ myofibroblasts is low, they give rise to fibroblasts with heterogeneous α-SMA expression patterns. Genetic ablation of Meflin+ PMCs in a renal fibrosis mouse model revealed their essential role in collagen production. Consistent with this, human biopsy samples showed that progressive renal diseases exhibit high Meflin expression. Furthermore, Meflin overexpression in kidney fibroblasts promoted bone morphogenetic protein 7 signals and suppressed myofibroblastic differentiation, implicating the roles of Meflin in suppressing tissue fibrosis. These findings demonstrate that Meflin marks a PMC subset that is functionally distinct from classic pericytes and myofibroblasts, highlighting the importance of elucidating PMC heterogeneity.

DOI： 10.1038/s41598-022-09331-5

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis. Reviewed International journal

Hiroki Kobayashi, Krystyna A Gieniec, Tamsin R M Lannagan, Tongtong Wang, Naoya Asai, Yasuyuki Mizutani, Tadashi Iida, Ryota Ando, Elaine M Thomas, Akihiro Sakai, Nobumi Suzuki, Mari Ichinose, Josephine A Wright, Laura Vrbanac, Jia Q Ng, Jarrad Goyne, Georgette Radford, Matthew J Lawrence, Tarik Sammour, Yoku Hayakawa, Sonja Klebe, Alice E Shin, Samuel Asfaha, Mark L Bettington, Florian Rieder, Nicholas Arpaia, Tal Danino, Lisa M Butler, Alastair D Burt, Simon J Leedham, Anil K Rustgi, Siddhartha Mukherjee, Masahide Takahashi, Timothy C Wang, Atsushi Enomoto, Susan L Woods, Daniel L Worthley

Gastroenterology Vol. 162 ( 3 ) page： 890 - 906 2022.3

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

DOI： 10.1053/j.gastro.2021.11.037

Meflin-positive cancer-associated fibroblasts enhance tumor response to immune checkpoint blockade. Reviewed International journal

Yuki Miyai, Daisuke Sugiyama, Tetsunari Hase, Naoya Asai, Tetsuro Taki, Kazuki Nishida, Takayuki Fukui, Toyofumi Fengshi Chen-Yoshikawa, Hiroki Kobayashi, Shinji Mii, Yukihiro Shiraki, Yoshinori Hasegawa, Hiroyoshi Nishikawa, Yuichi Ando, Masahide Takahashi, Atsushi Enomoto

Life science alliance Vol. 5 ( 6 ) 2022.3

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

Cancer-associated fibroblasts (CAFs) are an integral component of the tumor microenvironment (TME). Most CAFs shape the TME toward an immunosuppressive milieu and attenuate the efficacy of immune checkpoint blockade (ICB) therapy. However, the detailed mechanism of how heterogeneous CAFs regulate tumor response to ICB therapy has not been defined. Here, we show that a recently defined CAF subset characterized by the expression of Meflin, a glycosylphosphatidylinositol-anchored protein marker of mesenchymal stromal/stem cells, is associated with survival and favorable therapeutic response to ICB monotherapy in patients with non-small cell lung cancer (NSCLC). The prevalence of Meflin-positive CAFs was positively correlated with CD4-positive T-cell infiltration and vascularization within non-small cell lung cancer tumors. Meflin deficiency and CAF-specific Meflin overexpression resulted in defective and enhanced ICB therapy responses in syngeneic tumors in mice, respectively. These findings suggest the presence of a CAF subset that promotes ICB therapy efficacy, which adds to our understanding of CAF functions and heterogeneity.

DOI： 10.26508/lsa.202101230

Matrix remodeling-associated protein 8 is a marker of a subset of cancer-associated fibroblasts in pancreatic cancer. Reviewed International journal

Ryosuke Ichihara, Yukihiro Shiraki, Yasuyuki Mizutani, Tadashi Iida, Yuki Miyai, Nobutoshi Esaki, Akira Kato, Shinji Mii, Ryota Ando, Masamichi Hayashi, Hideki Takami, Tsutomu Fujii, Masahide Takahashi, Atsushi Enomoto

Pathology international Vol. 72 ( 3 ) page： 161 - 175 2022.3

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

Cancer-associated fibroblasts (CAFs), a compartment of the tumor microenvironment, were previously thought to be a uniform cell population that promotes cancer progression. However, recent studies have shown that CAFs are heterogeneous and that there are at least two types of CAFs, that is, cancer-promoting and -restraining CAFs. We previously identified Meflin as a candidate marker of cancer-restraining CAFs (rCAFs) in pancreatic ductal adenocarcinoma (PDAC). The precise nature of rCAFs, however, has remained elusive owing to a lack of understanding of their comprehensive gene signatures. Here, we screened genes whose expression correlated with Meflin in single-cell transcriptomic analyses of human cancers. Among the identified genes, we identified matrix remodeling-associated protein 8 (MXRA8), which encodes a type I transmembrane protein with unknown molecular function. Analysis of MXRA8 expression in human PDAC samples showed that MXRA8 was differentially co-expressed with other CAF markers. Moreover, in patients with PDAC or syngeneic tumors developed in MXRA8-knockout mice, MXRA8 expression did not affect the roles of CAFs in cancer progression, and the biological importance of MXRA8+ CAFs is still unclear. Overall, we identified MXRA8 as a new CAF marker; further studies are needed to determine the relevance of this marker.

DOI： 10.1111/pin.13198

神経内分泌形態を伴う乳癌におけるインスリノーマ関連タンパク質1(INSM1)の発現応用と将来展望(Insulinoma-associated protein 1(INSM1) expression in breast carcinomas with neuroendocrine morphologies: application and future prospective)

骨肉腫におけるGPIアンカー型膜タンパク質CD109の発現とその機能解析

三井伸二, 森田悠聖, 大河内凱翔, 下山芳江, 高橋雅英, 榎本篤

日本病理学会会誌 Vol. 111 ( 1 ) page： 293 - 293 2022.3

神経内分泌細胞の形態を持つ乳がんにおけるインスリノーマ関連タンパク質1の発現(Insulinoma-associated protein 1 expression in breast carcinomas with neuroendocrine morphologies)

川崎朋範, 解良恭一, 田島知明, 中村泰大, 近藤哲夫, 榎本篤, 村田有也, 星田義彦, 寺本典弘, 谷山清己, NEN project team

日本病理学会会誌 Vol. 111 ( 1 ) page： 254 - 254 2022.3

川崎朋範, 田島知明, 良沢昭銘, 藤本章博, 堀田洋介, 宇佐見陽子, 馬場康貴, 藤田曜, 椙田浩文, 櫻本信一, 濱口哲弥, 石田博徳, 中平光彦, 佐藤弘, 平能康充, 岡本光順, 各務博, 今井久雄, 中村泰大, 解良恭一, 星田義彦, 寺本典弘, 村田有也, 伊東正博, 川島篤弘, 岩下寿秀, 立山義朗, 谷山大樹, 倉岡和矢, 谷山清己, 永井宏和, 村松千左子, 齋藤正夫, 榎本篤, 近藤哲夫, NEN project team

日本内分泌学会雑誌 Vol. 97 ( 5 ) page： 1611 - 1611 2022.3

Insulinoma-associated protein 1(INSM1) expression in breast carcinomas with neuroendocrine morphologies: application and future prospective(和訳中) Reviewed

日本内分泌学会雑誌 Vol. 97 ( 5 ) page： 1611 - 1611 2022.3

Insulinoma-associated protein 1 expression in breast carcinomas with neuroendocrine morphologies(和訳中) Reviewed

川崎朋範, 解良恭一, 田島知明, 中村泰大, 近藤哲夫, 榎本篤, 村田有也, 星田義彦, 寺本典弘, 谷山清己, NENprojectteam

日本病理学会会誌 Vol. 111 ( 1 ) page： 254 - 254 2022.3

Safety and efficacy of MIKE-1 in patients with advanced pancreatic cancer: a study protocol for an open-label phase I/II investigator-initiated clinical trial based on a drug repositioning approach that reprograms the tumour stroma. Reviewed International journal

Yasuyuki Mizutani, Tadashi Iida, Eizaburo Ohno, Takuya Ishikawa, Fumie Kinoshita, Yachiyo Kuwatsuka, Miwa Imai, Shinobu Shimizu, Toshihisa Tsuruta, Atsushi Enomoto, Hiroki Kawashima, Mitsuhiro Fujishiro

BMC cancer Vol. 22 ( 1 ) page： 205 - 205 2022.2

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Cancer-associated fibroblasts (CAFs) are an important component of the tumour microenvironment. Recent studies revealed CAFs are heterogeneous and CAF subset(s) that suppress cancer progression (cancer-restraining CAFs [rCAFs]) must exist in addition to well-characterised cancer-promoting CAFs (pCAFs). However, the identity and specific markers of rCAFs are not yet reported. We recently identified Meflin as a specific marker of rCAFs in pancreatic and colon cancers. Our studies revealed that rCAFs may represent proliferating resident fibroblasts. Interestingly, a lineage tracing experiment showed Meflin-positive rCAFs differentiate into α-smooth muscle actin-positive and Meflin-negative CAFs, which are generally hypothesised as pCAFs, during cancer progression. Using a pharmacological approach, we identified AM80, a synthetic unnatural retinoid, as a reagent that effectively converts Meflin-negative pCAFs to Meflin-positive rCAFs. We aimed to investigate the efficacy of a combination of AM80 and gemcitabine (GEM) and nab-paclitaxel (nab-PTX) in patients with advanced pancreatic cancer. METHODS: The phase I part is a 3 + 3 design, open-label, and dose-finding study. The dose-limiting toxicity (DLT) of these combination therapies would be evaluated for 4 weeks. After the DLT evaluation period, if no disease progression is noted based on the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 or if the patient has no intolerable toxicity, administration of AM80 with GEM and nab-PTX would be continued for up to 24 weeks. The phase II part is an open-label, single-arm study. The maximum tolerated dose (MTD) of AM80 with GEM and nab-PTX, determined in phase I, would be administered until intolerable toxicity or disease progression occurs, up to a maximum of 24 weeks, to confirm efficacy and safety. The primary endpoints are frequency of DLT and MTD of AM80 with GEM and nab-PTX in the phase I part and response rate based on the RECIST in the phase II part. Given the historical control data, we hope that the response rate will be over 23% in phase II. DISCUSSION: Strategies to convert pCAFs into rCAFs have been developed in recent years. We hypothesised that AM80 would be a promising enhancer of chemosensitivity and drug distribution through CAF conversion in the stroma. TRIAL REGISTRATION: Clinicaltrial.gov: NCT05064618 , registered on 1 October 2021. jRCT: jRCT2041210056 , registered on 27 August 2021.

DOI： 10.1186/s12885-022-09272-2

Metabolically distinct glioma stem cells differentially require Girdin for stem cell behavior and tumor development

Watanabe, T; Yanagi, H; Enomoto, A; Sampetrean, O; Saya, H; Shimono, Y

CANCER SCIENCE Vol. 113 page： 988 - 988 2022.2

Fibroblasts positive for meflin have anti-fibrotic properties in pulmonary fibrosis. Reviewed International journal

Yoshio Nakahara, Naozumi Hashimoto, Koji Sakamoto, Atsushi Enomoto, Taylor S Adams, Toyoharu Yokoi, Norihito Omote, Sergio Poli, Akira Ando, Keiko Wakahara, Atsushi Suzuki, Masahide Inoue, Akitoshi Hara, Yasuyuki Mizutani, Kazuyoshi Imaizumi, Tsutomu Kawabe, Ivan O Rosas, Masahide Takahashi, Naftali Kaminski, Yoshinori Hasegawa

The European respiratory journal Vol. 58 ( 6 ) 2021.12

Language：English Publishing type：Research paper (scientific journal)

The prognosis of elderly individuals with idiopathic pulmonary fibrosis (IPF) remains poor. Fibroblastic foci, in which aggregates of proliferating fibroblasts and myofibroblasts are involved, are the pathological hallmark lesions in IPF to represent focal areas of active fibrogenesis. Fibroblast heterogeneity in fibrotic lesions hampers the discovery of the pathogenesis of pulmonary fibrosis. Therefore, to determine of the pathogenesis of IPF, identification of functional fibroblasts is warranted. This study was aimed to determine the role of fibroblasts positive for meflin, identified as a potential marker for mesenchymal stromal cells, during the development of pulmonary fibrosis. We characterised meflin-positive cells in a single cell atlas established by single-cell RNA sequencing (scRNA-seq)-based profiling of 243 472 cells from 32 IPF lungs and 29 normal lung samples. scRNA-seq combined with in situ RNA hybridisation identified proliferating fibroblasts positive for meflin in fibroblastic foci, not dense fibrosis, of fibrotic lungs in IPF patients. We determined the role of fibroblasts positive for meflin using bleomycin (BLM)-induced pulmonary fibrosis. A BLM-induced lung fibrosis model for meflin-deficient mice showed that fibroblasts positive for meflin had anti-fibrotic property to prevent pulmonary fibrosis. Although transforming growth factor-β-induced fibrogenesis and cell senescence with senescence-associated secretory phenotype were exacerbated in fibroblasts via the repression or lack of meflin, these were inhibited in meflin-deficient fibroblasts with meflin reconstitution. These findings provide evidence to show the biological importance of meflin expression on fibroblasts and myofibroblasts in the active fibrotic region of pulmonary fibrosis.

DOI： 10.1183/13993003.03397-2020

Anti-Malignant Effect of Tensile Loading to Adherens Junctions in Cutaneous Squamous Cell Carcinoma Cells. Reviewed International journal

Oleg Dobrokhotov, Masaki Sunagawa, Takeru Torii, Shinji Mii, Keiko Kawauchi, Atsushi Enomoto, Masahiro Sokabe, Hiroaki Hirata

Frontiers in cell and developmental biology Vol. 9 page： 728383 - 728383 2021.11

Language：English Publishing type：Research paper (scientific journal)

Actomyosin contractility regulates various cellular processes including proliferation and differentiation while dysregulation of actomyosin activity contributes to cancer development and progression. Previously, we have reported that actomyosin-generated tension at adherens junctions is required for cell density-dependent inhibition of proliferation of normal skin keratinocytes. However, it remains unclear how actomyosin contractility affects the hyperproliferation ability of cutaneous squamous cell carcinoma (cSCC) cells. In this study, we find that actomyosin activity is impaired in cSCC cells both in vitro and in vivo. External application of tensile loads to adherens junctions by sustained mechanical stretch attenuates the proliferation of cSCC cells, which depends on intact adherens junctions. Forced activation of actomyosin of cSCC cells also inhibits their proliferation in a cell-cell contact-dependent manner. Furthermore, the cell cycle arrest induced by tensile loading to adherens junctions is accompanied by epidermal differentiation in cSCC cells. Our results show that the degree of malignant properties of cSCC cells can be reduced by applying tensile loads to adherens junctions, which implies that the mechanical status of adherens junctions may serve as a novel therapeutic target for cSCC.

DOI： 10.3389/fcell.2021.728383

Newly established patient-derived organoid model of intracranial meningioma. Reviewed International journal

Shintaro Yamazaki, Fumiharu Ohka, Masaki Hirano, Yukihiro Shiraki, Kazuya Motomura, Kuniaki Tanahashi, Takashi Tsujiuchi, Ayako Motomura, Kosuke Aoki, Keiko Shinjo, Yoshiteru Murofushi, Yotaro Kitano, Sachi Maeda, Akira Kato, Hiroyuki Shimizu, Junya Yamaguchi, Alimu Adilijiang, Toshihiko Wakabayashi, Ryuta Saito, Atsushi Enomoto, Yutaka Kondo, Atsushi Natsume

Neuro-oncology Vol. 23 ( 11 ) page： 1936 - 1948 2021.11

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Recent comprehensive studies have revealed several molecular alterations that are frequently found in meningiomas. However, effective treatment reagents targeting specific molecular alterations have not yet been identified because of the limited number of representative research models of meningiomas. METHODS: We performed organoid cultures using meningioma cells and meningioma tumor tissues. Using immunohistochemistry and molecular analyses consisting of whole-exome sequencing, RNA-seq, and DNA methylation analyses, we compared the histological findings and molecular profiling of organoid models with those of parental tumors. Further, using these organoid models together with a public database of meningiomas, we explored molecular alterations, which are a potent treatment target for meningioma. RESULTS: We established 18 organoid models comprising of two malignant meningioma cells (HKBMM and IOMM-Lee), 10 benign meningiomas, four malignant meningiomas, and two solitary fibrous tumors (SFTs). The organoids exhibited consistent histological features and molecular profiles with those of the parental tumors. Using a public database, we identified that upregulated forkhead box M1 (FOXM1) was correlated with increased tumor proliferation. Overexpression of FOXM1 in benign meningioma organoids increased organoid proliferation; depletion of FOXM1 in malignant organoids decreased proliferation. Additionally, thiostrepton, a FOXM1 inhibitor combined with radiation therapy, significantly inhibited the proliferation of malignant meningioma organoid models. CONCLUSIONS: An organoid model for meningioma enabled us to elucidate the tumor biology of meningioma along with potent treatment targets for meningioma.

DOI： 10.1093/neuonc/noab155

Ring artifact removal for differential phase-contrast X-ray computed tomography using a conditional generative adversarial network. Reviewed International journal

Zhuoran Huang, Naoki Sunaguchi, Daisuke Shimao, Atsushi Enomoto, Shu Ichihara, Tetsuya Yuasa, Masami Ando

International journal of computer assisted radiology and surgery Vol. 16 ( 11 ) page： 1889 - 1900 2021.11

Language：English Publishing type：Research paper (scientific journal)

PURPOSE: The integration process used as a pre-processing step in the reconstruction of differential phase-contrast X-ray CT (d-PCCT) causes the measurement noise to propagate throughout the projection image, which is leading to increased ring artifacts (RA) in the reconstructed image. It is difficult to eliminate the RA using conventional RA removal methods that were developed for the absorption-based CT field. We propose an effective method that can remove RA of d-PCCT images. METHODS: The proposed method uses Laplacian images reconstructed from second-derivative projections of d-PCCT. This method is based on a conditional generative adversarial network (cGAN), whose loss function is designed by adding the L1- and L2-norm to the original cGAN. The training data were taken from a numerical phantom generated by a d-PCCT imaging simulator. To validate the applicability of the trained network, we tested its RA removal effect on test data from numerical phantoms generated randomly and actual experimental data. RESULTS: The results of numerical validation using numerical phantoms showed that the proposed method improved the RA removal effect compared to conventional methods. In addition, image comparison by visual evaluation showed that only the proposed method was able to remove RA while preserving original structures in the actual biological d-PCCT images. CONCLUSION: We proposed a cGAN-based method for RA removal that exploits the physical properties of d-PCCT. The proposed method was able to completely remove RA from d-PCCT images on both simulated data and biological data. We believe that this method is useful for the observation of various types of biological soft tissue.

DOI： 10.1007/s11548-021-02500-3

Roles of the Mesenchymal Stromal/Stem Cell Marker Meflin/Islr in Cancer Fibrosis. Reviewed International journal

Masahide Takahashi, Hiroki Kobayashi, Yasuyuki Mizutani, Akitoshi Hara, Tadashi Iida, Yuki Miyai, Naoya Asai, Atsushi Enomoto

Frontiers in cell and developmental biology Vol. 9 page： 749924 - 749924 2021.10

Authorship：Last author Language：English Publishing type：Research paper (scientific journal)

Fibroblasts synthesise the extracellular matrix (ECM) such as collagen and elastin, the excessive accumulation of which can lead to fibrosis and organ dysfunction under pathological conditions. Cancer-associated fibroblasts (CAFs) are major constituents of the tumour microenvironment (TME) that accompany the desmoplastic reaction responsible for anti-cancer treatment resistance. Thus, it is important to dissect the roles of CAFs in the TME to develop new therapeutic strategies for refractory cancers. Recent progress in the studies of CAF biology suggests that the functions of CAFs are complicated and that they are composed of functionally distinct populations, including cancer-promoting CAFs (pCAFs) and cancer-restraining CAFs (rCAFs). We recently identified a new cell surface marker for rCAFs in pancreatic and colon cancers, designated as Meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue)/Islr (immunoglobulin super family containing leucine-rich repeat). Based on the distribution of Meflin/Islr-positive cells, we also considered it a specific candidate marker for mesenchymal stroma/stem cells. Meflin/Islr-positive CAFs have been shown to suppress cancer progression by being involved in regulating collagen structures and BMP signalling in the TME. This review describes the function of Meflin/Islr in cancer fibrosis as well as in cardiac and lung fibrosis and its potential in the development of new cancer therapeutics.

DOI： 10.3389/fcell.2021.749924

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma. Reviewed International journal

Hiroki Kobayashi, Krystyna A Gieniec, Jia Q Ng, Jarrad Goyne, Tamsin R M Lannagan, Elaine M Thomas, Georgette Radford, Tongtong Wang, Nobumi Suzuki, Mari Ichinose, Josephine A Wright, Laura Vrbanac, Alastair D Burt, Masahide Takahashi, Atsushi Enomoto, Daniel L Worthley, Susan L Woods

Journal of visualized experiments : JoVE ( 175 ) 2021.9

Language：English Publishing type：Research paper (scientific journal)

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

DOI： 10.3791/62630

Girdinは代謝特性の異なるグリオーマ幹細胞の幹細胞性、腫瘍形成を特徴的な様式で制御する

渡辺崇, 柳久乃, 榎本篤, サンペトラ・オルテア, 佐谷秀行, 下野洋平

日本癌学会総会記事 Vol. 80回 page： [P11 - 2] 2021.9

神経内分泌細胞の形態を有する乳がんにおけるインスリノーマ関連タンパク質1(INSM1)の発現応用法と将来の展望(Insulinoma-associated protein 1(INSM1) expression in breast carcinomas with neuroendocrine morphologies: application and future prospective)

川崎朋範, 田島知明, 中村泰大, 堀田洋介, 藤本章博, 宮口和也, 宇佐見陽子, 今井久雄, 解良恭一, 村田有也, 谷山清己, 村松千左子, 齋藤正夫, 榎本篤, 近藤哲夫, 良沢昭銘, NEN project team

日本組織細胞化学会総会・学術集会講演プログラム・予稿集 Vol. 62回 page： 72 - 72 2021.9

The sodium-glucose cotransporter-2 inhibitor Tofogliflozin prevents the progression of nonalcoholic steatohepatitis-associated liver tumors in a novel murine model. Reviewed International journal

Naoki Yoshioka, Miyako Tanaka, Kozue Ochi, Akiko Watanabe, Kenji Ono, Makoto Sawada, Tomoo Ogi, Michiko Itoh, Ayaka Ito, Yukihiro Shiraki, Atsushi Enomoto, Masatoshi Ishigami, Mitsuhiro Fujishiro, Yoshihiro Ogawa, Takayoshi Suganami

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Vol. 140 page： 111738 - 111738 2021.8

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Diabetes and obesity contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). However, how diabetes and obesity accelerate liver tumorigenesis remains to be fully understood. Moreover, to verify the therapeutic potential of anti-diabetic drugs, there exists a strong need for appropriate animal models that recapitulate human pathophysiology of NASH and HCC. METHODS: We established a novel murine model of NASH-associated liver tumors using genetically obese melanocortin 4 receptor-deficient mice fed on Western diet in combination with a chemical procarcinogen, and verified the validity of our model in evaluating drug efficacy. FINDINGS: Our model developed multiple liver tumors together with obesity, diabetes, and NASH within a relatively short period (approximately 3 months). In this model, sodium glucose cotransporter 2 inhibitor Tofogliflozin prevented the development of NASH-like liver phenotypes and the progression of liver tumors. Tofogliflozin attenuated p21 expression of hepatocytes in non-tumorous lesions in the liver. INTERPRETATION: Tofogliflozin treatment attenuates cellular senescence of hepatocytes under obese and diabetic conditions. This study provides a unique animal model of NASH-associated liver tumors, which is applicable for assessing drug efficacy to prevent or treat NASH-associated HCC.

DOI： 10.1016/j.biopha.2021.111738

The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia. Reviewed International journal

Kei Kozawa, Miho Sekai, Kenji Ohba, Shoko Ito, Hiroaki Sako, Takeshi Maruyama, Mai Kakeno, Takanobu Shirai, Keisuke Kuromiya, Tomoko Kamasaki, Koki Kohashi, Shinya Tanaka, Susumu Ishikawa, Nanami Sato, Shota Asano, Hironori Suzuki, Nobuyuki Tanimura, Yohei Mukai, Noriko Gotoh, Mishie Tanino, Shinya Tanaka, Ken Natsuga, Tomoyoshi Soga, Tomonori Nakamura, Yukihiro Yabuta, Mitinori Saitou, Takahiro Ito, Kenkyo Matsuura, Makoto Tsunoda, Toyone Kikumori, Tadashi Iida, Yasuyuki Mizutani, Yuki Miyai, Kozo Kaibuchi, Atsushi Enomoto, Yasuyuki Fujita

Current biology : CB Vol. 31 ( 14 ) page： 3086 - + 2021.7

Language：English Publishing type：Research paper (scientific journal)

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

DOI： 10.1016/j.cub.2021.04.078

Meflin defines mesenchymal stem cells and/or their early progenitors with multilineage differentiation capacity. Reviewed International journal

Akitoshi Hara, Katsuhiro Kato, Toshikazu Ishihara, Hiroki Kobayashi, Naoya Asai, Shinji Mii, Yukihiro Shiraki, Yuki Miyai, Ryota Ando, Yasuyuki Mizutani, Tadashi Iida, Mikito Takefuji, Toyoaki Murohara, Masahide Takahashi, Atsushi Enomoto

Genes to cells : devoted to molecular & cellular mechanisms Vol. 26 ( 7 ) page： 495 - 512 2021.7

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

Mesenchymal stem cells (MSCs) are the likely precursors of multiple lines of mesenchymal cells. The existence of bona fide MSCs with self-renewal capacity and differentiation potential into all mesenchymal lineages, however, has been unclear because of the lack of MSC-specific marker(s) that are not expressed by the terminally differentiated progeny. Meflin, a glycosylphosphatidylinositol-anchored protein, is an MSC marker candidate that is specifically expressed in rare stromal cells in all tissues. Our previous report showed that Meflin expression becomes down-regulated in bone marrow-derived MSCs cultured on plastic, making it difficult to examine the self-renewal and differentiation of Meflin-positive cells at the single-cell level. Here, we traced the lineage of Meflin-positive cells in postnatal and adult mice, showing that those cells differentiated into white and brown adipocytes, osteocytes, chondrocytes and skeletal myocytes. Interestingly, cells derived from Meflin-positive cells formed clusters of differentiated cells, implying the in situ proliferation of Meflin-positive cells or their lineage-committed progenitors. These results, taken together with previous findings that Meflin expression in cultured MSCs was lost upon their multilineage differentiation, suggest that Meflin is a useful potential marker to localize MSCs and/or their immature progenitors in multiple tissues.

DOI： 10.1111/gtc.12855

The BMP antagonist gremlin 1 contributes to the development of cortical excitatory neurons, motor balance and fear responses. Reviewed International journal

Mari Ichinose, Nobumi Suzuki, Tongtong Wang, Hiroki Kobayashi, Laura Vrbanac, Jia Q Ng, Josephine A Wright, Tamsin R M Lannagan, Krystyna A Gieniec, Martin Lewis, Ryota Ando, Atsushi Enomoto, Simon Koblar, Paul Thomas, Daniel L Worthley, Susan L Woods

Development (Cambridge, England) Vol. 148 ( 14 ) 2021.7

Language：English Publishing type：Research paper (scientific journal)

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.

DOI： 10.1242/dev.195883

Size and surface modification of silica nanoparticles affect the severity of lung toxicity by modulating endosomal ROS generation in macrophages. Reviewed International journal

Masahide Inoue, Koji Sakamoto, Atsushi Suzuki, Shinya Nakai, Akira Ando, Yukihiko Shiraki, Yoshio Nakahara, Mika Omura, Atsushi Enomoto, Ikuhiko Nakase, Makoto Sawada, Naozumi Hashimoto

Particle and fibre toxicology Vol. 18 ( 1 ) page： 21 - 21 2021.6

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: As the application of silica nanomaterials continues to expand, increasing chances of its exposure to the human body and potential harm are anticipated. Although the toxicity of silica nanomaterials is assumed to be affected by their physio-chemical properties, including size and surface functionalization, its molecular mechanisms remain unclear. We hypothesized that analysis of intracellular localization of the particles and subsequent intracellular signaling could reveal a novel determinant of inflammatory response against silica particles with different physico-chemical properties. RESULTS: We employed a murine intratracheal instillation model of amorphous silica nanoparticles (NPs) exposure to compare their in vivo toxicities in the respiratory system. Pristine silica-NPs of 50 nm diameters (50 nm-plain) induced airway-centered lung injury with marked neutrophilic infiltration. By contrast, instillation of pristine silica particles of a larger diameter (3 μm; 3 μm-plain) significantly reduced the severity of lung injury and neutrophilic infiltration, possibly through attenuated induction of neutrophil chemotactic chemokines including MIP2. Ex vivo analysis of alveolar macrophages as well as in vitro assessment using RAW264.7 cells revealed a remarkably lower cellular uptake of 3 μm-plain particles compared with 50 nm-plain, which is assumed to be the underlying mechanism of attenuated immune response. The severity of lung injury and neutrophilic infiltration was also significantly reduced after intratracheal instillation of silica NPs with an amine surface modification (50 nm-NH2) when compared with 50 nm-plain. Despite unchanged efficacy in cellular uptake, treatment with 50 nm-NH2 induced a significantly attenuated immune response in RAW264.7 cells. Assessment of intracellular redox signaling revealed increased reactive oxygen species (ROS) in endosomal compartments of RAW264.7 cells treated with 50 nm-plain when compared with vehicle-treated control. In contrast, augmentation of endosomal ROS signals in cells treated with 50 nm-NH2 was significantly lower. Moreover, selective inhibition of NADPH oxidase 2 (NOX2) was sufficient to inhibit endosomal ROS bursts and induction of chemokine expressions in cells treated with silica NPs, suggesting the central role of endosomal ROS generated by NOX2 in the regulation of the inflammatory response in macrophages that endocytosed silica NPs. CONCLUSIONS: Our murine model suggested that the pulmonary toxicity of silica NPs depended on their physico-chemical properties through distinct mechanisms. Cellular uptake of larger particles by macrophages decreased, while surface amine modification modulated endosomal ROS signaling via NOX2, both of which are assumed to be involved in mitigating immune response in macrophages and resulting lung injury.

DOI： 10.1186/s12989-021-00415-0

A new primate model of hypophyseal dysfunction. International journal

Teppei Kawabata, Hidetaka Suga, Kazuhito Takeuchi, Yuichi Nagata, Mayu Sakakibara, Kaori Ushida, Chikafumi Ozone, Atsushi Enomoto, Ikuo Kawamoto, Iori Itagaki, Hideaki Tsuchiya, Hiroshi Arima, Toshihiko Wakabayashi

Scientific reports Vol. 11 ( 1 ) page： 10729 - 10729 2021.5

Language：English Publishing type：Research paper (scientific journal)

For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey's general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.

DOI： 10.1038/s41598-021-90209-3

CD4+ T cells are essential for the development of destructive thyroiditis induced by anti-PD-1 antibody in thyroglobulin-immunized mice. Reviewed International journal

Yoshinori Yasuda, Shintaro Iwama, Daisuke Sugiyama, Takayuki Okuji, Tomoko Kobayashi, Masaaki Ito, Norio Okada, Atsushi Enomoto, Sachiko Ito, Yue Yan, Mariko Sugiyama, Takeshi Onoue, Taku Tsunekawa, Yoshihiro Ito, Hiroshi Takagi, Daisuke Hagiwara, Motomitsu Goto, Hidetaka Suga, Ryoichi Banno, Masahide Takahashi, Hiroyoshi Nishikawa, Hiroshi Arima

Science translational medicine Vol. 13 ( 593 ) 2021.5

Language：English Publishing type：Research paper (scientific journal)

Immune-related adverse events induced by anti-programmed cell death-1 antibodies (PD-1-Ab), including destructive thyroiditis (thyroid-irAE), are thought to be caused by activated T cells. However, the T cell subsets that are directly responsible for damaging self-organs remain unclear. To clarify which T cell subsets are involved in the development of thyroid-irAE, a mouse model of thyroid-irAE was analyzed. PD-1-Ab administration 2.5 months after immunization with thyroglobulin caused destructive thyroiditis. Thyroiditis was completely prevented by previous depletion of CD4+ T cells and partially prevented by depleting CD8+ T cells. The frequencies of central and effector memory CD4+ T cell subsets and the secretion of interferon-γ after stimulation with thyroglobulin were increased in the cervical lymph nodes of mice with thyroid-irAE compared with controls. Histopathological analysis revealed infiltration of CD4+ T cells expressing granzyme B in thyroid glands and major histocompatibility complex class II expression on thyrocytes in mice with thyroid-irAE. Adoptive transfer of CD4+ T cells from cervical lymph nodes in mice with thyroid-irAE caused destruction of thyroid follicular architecture in the irradiated recipient mice. Flow cytometric analyses showed that the frequencies of central and effector memory CD4+ T cells expressing the cytotoxic marker CD27 were higher in peripheral blood mononuclear cells collected from patients with thyroid-irAE induced by PD-1-Ab versus those without. These data suggest a critical role for cytotoxic memory CD4+ T cells activated by PD-1-Ab in the pathogenesis of thyroid-irAE.

DOI： 10.1126/scitranslmed.abb7495

Oncolytic activity of naturally attenuated herpes-simplex virus HF10 against an immunocompetent model of oral carcinoma. Reviewed International journal

Gaku Takano, Shinichi Esaki, Fumi Goshima, Atsushi Enomoto, Yoshimi Hatano, Haruka Ozaki, Takahiro Watanabe, Yoshitaka Sato, Daisuke Kawakita, Shingo Murakami, Takayuki Murata, Yukihiro Nishiyama, Shinichi Iwasaki, Hiroshi Kimura

Molecular therapy oncolytics Vol. 20 page： 220 - 227 2021.3

Language：English Publishing type：Research paper (scientific journal)

Prognosis for advanced oral carcinoma remains poor. Oncolytic virotherapy uses replication-competent viruses to infect and kill only the tumor cells. However, it has been difficult to investigate the oncolytic activity of viruses against oral carcinomas in mouse models. This study established a mouse model of oral cancer and investigated the in vitro and in vivo anti-tumor effects of HF10, a highly attenuated, replication-competent herpes simplex virus (HSV)-1. Mouse tongue cancer was induced by injecting 4-nitroquinoline 1-oxide into the mouse tongue. The murine oral cancer cell line isolated from this tumor, named NMOC1, formed invasive carcinoma within a week when injected into mouse tongue. HF10 successfully infected, replicated, and spread in the cancer cells in vitro. HF10 was able to kill cancer cells isolated from human or mouse tongue tumor. HF10 injection into tongue carcinomas prolonged mouse survival without any side effects or weight loss. Intertumoral injection of GFP-expressing HF10 confirmed that viral spread was confined within the tumors. Immunohistochemical staining showed that HF10 induced infiltration of CD8-positive T cells around HSV-infected cells in the tumor mass, implying increased anti-tumor immunity. We successfully established an oral cancer cell line and showed that HF10 is a promising therapeutic agent for oral cancer.

DOI： 10.1016/j.omto.2020.12.007

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis. Reviewed International journal

Hiroki Kobayashi, Krystyna A Gieniec, Josephine A Wright, Tongtong Wang, Naoya Asai, Yasuyuki Mizutani, Tadashi Lida, Ryota Ando, Nobumi Suzuki, Tamsin R M Lannagan, Jia Q Ng, Akitoshi Hara, Yukihiro Shiraki, Shinji Mii, Mari Ichinose, Laura Vrbanac, Matthew J Lawrence, Tarik Sammour, Kay Uehara, Gareth Davies, Leszek Lisowski, Ian E Alexander, Yoku Hayakawa, Lisa M Butler, Andrew C W Zannettino, M Omar Din, Jeff Hasty, Alastair D Burt, Simon J Leedham, Anil K Rustgi, Siddhartha Mukherjee, Timothy C Wang, Atsushi Enomoto, Masahide Takahashi, Daniel L Worthley, Susan L Woods

Gastroenterology Vol. 160 ( 4 ) page： 1224 - + 2021.3

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. METHODS: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. RESULTS: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. CONCLUSIONS: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

DOI： 10.1053/j.gastro.2020.11.011

Vascular PDGFR-alpha protects against BBB dysfunction after stroke in mice. Reviewed International journal

Quang Linh Nguyen, Noriko Okuno, Takeru Hamashima, Son Tung Dang, Miwa Fujikawa, Yoko Ishii, Atsushi Enomoto, Takakuni Maki, Hoang Ngoc Nguyen, Van Tuyen Nguyen, Toshihiko Fujimori, Hisashi Mori, Johanna Andrae, Christer Betsholtz, Keizo Takao, Seiji Yamamoto, Masakiyo Sasahara

Angiogenesis Vol. 24 ( 1 ) page： 35 - 46 2021.2

Language：English Publishing type：Research paper (scientific journal)

Blood-brain barrier (BBB) dysfunction underlies the pathogenesis of many neurological diseases. Platelet-derived growth factor receptor-alpha (PDGFRα) induces hemorrhagic transformation (HT) downstream of tissue plasminogen activator in thrombolytic therapy of acute stroke. Thus, PDGFs are attractive therapeutic targets for BBB dysfunction. In the present study, we examined the role of PDGF signaling in the process of tissue remodeling after middle cerebral arterial occlusion (MCAO) in mice. Firstly, we found that imatinib increased lesion size after permanent MCAO in wild-type mice. Moreover, imatinib-induced HT only when administrated in the subacute phase of MCAO, but not in the acute phase. Secondly, we generated genetically mutated mice (C-KO mice) that showed decreased expression of perivascular PDGFRα. Additionally, transient MCAO experiments were performed in these mice. We found that the ischemic lesion size was not affected; however, the recruitment of PDGFRα/type I collagen-expressing perivascular cells was significantly downregulated, and HT and IgG leakage was augmented only in the subacute phase of stroke in C-KO mice. In both experiments, we found that the expression of tight junction proteins and PDGFRβ-expressing pericyte coverage was not significantly affected in imatinib-treated mice and in C-KO mice. The specific implication of PDGFRα signaling was suggestive of protective effects against BBB dysfunction during the subacute phase of stroke. Vascular TGF-β1 expression was downregulated in both imatinib-treated and C-KO mice, along with sustained levels of MMP9. Therefore, PDGFRα effects may be mediated by TGF-β1 which exerts potent protective effects in the BBB.

DOI： 10.1007/s10456-020-09742-w

Tumor microenvironment stimulates induction of cancer associated fibroblasts

Nakahara, R; Kato, M; Jiang, JR; Nishida, M; Tsuchida, R; Shimamura, T; Enomoto, A; Osawa, T

CANCER SCIENCE Vol. 112 page： 723 - 723 2021.2

CD109 regulates in vivo tumor invasion in lung adenocarcinoma through TGF-β signaling

Mii, S; Taki, T; Shiraki, Y; Enomoto, A; Takahashi, M

CANCER SCIENCE Vol. 112 page： 815 - 815 2021.2

CD109 regulates in vivo tumor invasion in lung adenocarcinoma through TGF-β signaling. Reviewed International journal

Tetsuro Taki, Yukihiro Shiraki, Atsushi Enomoto, Liang Weng, Chen Chen, Naoya Asai, Yoshiki Murakumo, Kohei Yokoi, Masahide Takahashi, Shinji Mii

Cancer science Vol. 111 ( 12 ) page： 4616 - 4628 2020.12

Language：English Publishing type：Research paper (scientific journal)

Stromal invasion is considered an important prognostic factor in patients with lung adenocarcinoma. The mechanisms underlying the formation of tumor stroma and stromal invasion have been studied in the lung; however, they are still unclear. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein highly expressed in several types of human malignant tumors including lung cancers. In this study, we investigated the in vivo functions of CD109 protein in malignant lung tumors. Initially, we identified an association between higher expression of CD109 protein in human lung adenocarcinoma and a significantly worse prognosis, according to immunohistochemical analysis. We also showed that CD109 deficiency significantly reduced the area of stromal invasive lesions in a genetically engineered CD109-deficient lung adenocarcinoma mouse model, which correlated with the results observed in human lung adenocarcinoma. Furthermore, we identified latent TGF-β binding protein-1 (LTBP1) as a CD109-interacting protein using mass spectrometry and confirmed their interaction by co-immunoprecipitation. Importantly, increased CD109 expression enhanced stromal TGF-β activation in the presence of LTBP1. Therefore, these data suggest the significance of the regulation of TGF-β signaling through CD109 and LTBP1 interaction in tumor stroma and also reveal the importance of CD109 expression levels in promoting lung cancer cell proliferation, migration, and invasion, and thus predicting the outcome of patients suffering from lung adenocarcinoma. Therefore, CD109 protein could be a potential therapeutic target for this disease.

DOI： 10.1111/cas.14673

The Daple-CK1ε complex regulates Dvl2 phosphorylation and canonical Wnt signaling. Reviewed International journal

Nobutoshi Esaki, Atsushi Enomoto, Maki Takagishi, Yasuyuki Mizutani, Tadashi Iida, Kaori Ushida, Yukihiro Shiraki, Shinji Mii, Masahide Takahashi

Biochemical and biophysical research communications Vol. 532 ( 3 ) page： 406 - 413 2020.11

Language：English Publishing type：Research paper (scientific journal)

The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of β-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1ε-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple ΔGCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1ε, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1ε trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of β-catenin transcriptional activity. Consistent with this, wild-type Daple promoted β-catenin transcriptional activity, following dissociation of β-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated β-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1ε-mediated activation of Dvl in the canonical Wnt signaling pathway.

DOI： 10.1016/j.bbrc.2020.08.066

Stromal imbalance of bone morphogenetic protein signaling drives colorectal carcinogenesis

Kobayashi, H; Gieniec, K; Wright, J; Wang, T; Asai, N; Mizutani, Y; Ida, T; Ando, R; Suzuki, N; Lannagan, T; Ng, J; Hara, A; Shiraki, Y; Mii, S; Ichinose, M; Vrbanac, L; Lawrence, M; Sammour, T; Uehara, K; Davies, G; Lisowski, L; Alexander, I; Hayakawa, Y; Butler, L; Zannettino, A; Din, MO; Hasty, J; Burt, A; Leedham, S; Rustgi, A; Wang, ST; Wang, TC; Enomoto, A; Takahashi, M; Worthley, D; Woods, S

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY Vol. 35 page： 160 - 160 2020.11

Complex roles of the actin-binding protein Girdin/GIV in DNA damage-induced apoptosis of cancer cells. Reviewed International journal

Chen Chen, Atsushi Enomoto, Liang Weng, Tetsuro Taki, Yukihiro Shiraki, Shinji Mii, Ryosuke Ichihara, Mitsuro Kanda, Masahiko Koike, Yasuhiro Kodera, Masahide Takahashi

Cancer science Vol. 111 ( 11 ) page： 4303 - 4317 2020.11

Language：English Publishing type：Research paper (scientific journal)

The actin-binding protein Girdin is a hub protein that interacts with multiple proteins to regulate motility and Akt and trimeric G protein signaling in cancer cells. Girdin expression correlates with poor outcomes in multiple human cancers. However, those findings are not universal, as they depend on study conditions. Those data suggest that multiple aspects of Girdin function and its role in tumor cell responses to anticancer therapeutics must be reconsidered. In the present study, we found that Girdin is involved in DNA damage-induced cancer cell apoptosis. An esophageal cancer cell line that exhibited high Girdin expression showed a marked sensitivity to UV-mediated DNA damage compared to a line with low Girdin expression. When transcriptional activation of endogenous Girdin was mediated by an engineered CRISPR/Cas9 activation system, sensitivity to DNA damage increased in both stationary and migrating HeLa cancer cells. High Girdin expression was associated with dysregulated cell cycle progression and prolonged G1 and M phases. These features were accompanied by p53 activation, which conceivably increases cancer cell vulnerability to UV exposure. These data highlight the importance of understanding complex Girdin functions that influence cancer cell sensitivity to therapeutics.

DOI： 10.1111/cas.14637

Generation of a mouse model of gastric adenocarcinoma and proximal polyposis of the stomach that causes stomach hyperplasia

Suzuki, N; Wright, J; Kobayashi, H; Wang, T; Ichinose, M; Vrbanac, L; Ng, J; Gieniec, K; Lannagan, T; Fujiwara, H; Enomoto, A; Chenevix-Trench, G; Hayakawa, Y; Koike, K; Woods, S; Worthley, D

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY Vol. 35 page： 165 - 165 2020.11

Melanoma cell adhesion molecule defines a proliferative subset of leptin receptor-lineage cells that substantially contribute to the colorectal cancer stroma

Gieniec, K; Kobayashi, H; Lannagan, T; Wang, T; Asai, N; Mizutani, Y; Ida, T; Ando, R; Suzuki, N; Ichinose, M; Wright, J; Vrbanac, L; Ng, J; Lawrence, M; Sammour, T; Hayakawa, Y; Klebe, S; Shin, AE; Asfaha, S; Arpaia, N; Butler, L; Burt, A; Leedham, S; Rustgi, A; Mukherjee, S; Takahashi, M; Wang, T; Enomoto, A; Woods, S; Worthley, D

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY Vol. 35 page： 163 - 163 2020.11

Connective tissue growth factor produced by cancer‑associated fibroblasts correlates with poor prognosis in epithelioid malignant pleural mesothelioma. Reviewed International journal

Yuuki Ohara, Atsushi Enomoto, Yuta Tsuyuki, Kotaro Sato, Tadashi Iida, Hiroki Kobayashi, Yasuyuki Mizutani, Yuki Miyai, Akitoshi Hara, Shinji Mii, Jun Suzuki, Kyoko Yamashita, Fumiya Ito, Yashiro Motooka, Nobuaki Misawa, Takayuki Fukui, Koji Kawaguchi, Kohei Yokoi, Shinya Toyokuni

Oncology reports Vol. 44 ( 3 ) page： 838 - 848 2020.9

Language：English Publishing type：Research paper (scientific journal)

Malignant mesothelioma is an aggressive neoplasm for which effective treatments are lacking. We often encounter mesothelioma cases with a profound desmoplastic reaction, suggesting the involvement of cancer‑associated fibroblasts (CAFs) in mesothelioma progression. While the roles of CAFs have been extensively studied in other tumors and have led to the view that the cancer stroma contains heterogeneous populations of CAFs, their roles in mesothelioma remain unknown. We previously showed that connective tissue growth factor (CTGF), a secreted protein, is produced by both mesothelioma cells and fibroblasts and promotes the invasion of mesothelioma cells in vitro. In this study, we examined the clinical relevance of CAFs in mesothelioma. Using surgical specimens of epithelioid malignant pleural mesothelioma, we evaluated the clinicopathological significance of the expression of α‑smooth muscle actin (αSMA), the most widely used marker of CAFs, the expression of CTGF, and the extent of fibrosis by immunohistochemistry and Elastica‑Masson staining. We also analyzed the expression of mesenchymal stromal cell‑ and fibroblast‑expressing Linx paralogue (Meflin; ISLR), a recently reported CAF marker that labels cancer‑restraining CAFs and differ from αSMA‑positive CAFs, by in situ hybridization. The extent of fibrosis and CTGF expression in mesothelioma cells did not correlate with patient prognosis. However, the expression of αSMA and CTGF, but not Meflin, in CAFs correlated with poor prognosis. The data suggest that CTGF+ CAFs are involved in mesothelioma progression and represent a potential molecular target for mesothelioma therapy.

DOI： 10.3892/or.2020.7669

Significance of meflin-positive cancer-associated fibroblasts in predicting response to immune checkpoint inhibitors in non-small cell lung cancer.

Miyai, Y; Enomoto, A; Ando, Y; Takahashi, M

JOURNAL OF CLINICAL ONCOLOGY Vol. 38 ( 15 ) 2020.5

Significance of meflin-positive cancer-associated fibroblasts in predicting response to immune checkpoint inhibitors in non-small cell lung cancer.

Yuki Miyai, Atsushi Enomoto, Yuichi Ando, Masahide Takahashi

Journal of Clinical Oncology Vol. 38 ( 15_suppl ) page： 3118 - 3118 2020.5

Publishing type：Research paper (scientific journal) Publisher：American Society of Clinical Oncology (ASCO)

3118

Background: Tumor immunity is regulated by complex interactions between cancer and immune cells, which also involves other components of the tumor microenvironment (TME). Recently, cancer-associated fibroblasts (CAFs), a major constituent of the TME, have emerged as important regulators of tumor immunity. Specifically, for example, α-smooth muscle actin or leucine-rich repeat containing 15-positive CAFs have been shown to be crucial for the suppression of tumor immunity. However, a comprehensive picture of how other CAF subset(s) are involved in tumor immunity is still lacking. Here, we show the involvement of a CAF subset highly expressing Meflin, which was recently identified as a marker of cancer-restraining CAFs in pancreatic cancer (Mizutani et al., Cancer Res, 2019), in the response of non-small cell lung cancer (NSCLC) patients to immune checkpoint inhibitors (ICIs). Methods: A sample cohort of 122 subjects with NSCLC who had received ICI monotherapy with nivolumab, pembrolizumab, or atezolizumab was identified at the Department of Respiratory Medicine at Nagoya University Hospital. We selected 92 eligible patients, collected formalin-fixed paraffin-embedded tumor tissues, and prepared 4–µm-thick slides for the analysis of Meflin expression by RNA-in situ hybridization assay, followed by the evaluation of treatment response of 88 patients using the iRECIST criteria. We assessed the number of Meflin-positive CAFs and divided the patients into Meflin-High (20% and more CAFs express Meflin) and -Low groups. The cut-off value was obtained by the ROC analysis. Primarily, objective response rate (ORR) was compared between Meflin-High and –Low groups. Overall survival (OS), and progression free survival (PFS) were also assessed. Results: Patients who started to receive ICIs till the end of March 2019 were enrolled and followed-up until the end of 2019. Analysis of the tumor tissues revealed that 24 (40.7%) of 59 Meflin-High patients responded to the ICI monotherapy. In contrast, none (0%) of 29 Meflin-Low patients showed any significant response (p-value: 0.0000174). Meflin-High groups showed statistically significant prolongations in both OS and PFS with the hazard ratios of 0.3114 [0.1591-0.6094] and 0.3997 [0.2290-0.6976], respectively. Conclusions: This retrospective observation indicated that the high infiltration of Meflin-positive CAFs may shape tumor-suppressive immune response and increase the sensitivity to ICIs, which differs from those of other CAF subsets.

DOI： 10.1200/jco.2020.38.15_suppl.3118

Cancer-associated fibroblasts that restrain cancer progression: Hypotheses and perspectives. Reviewed International journal

Yuki Miyai, Nobutoshi Esaki, Masahide Takahashi, Atsushi Enomoto

Cancer science Vol. 111 ( 4 ) page： 1047 - 1057 2020.4

Language：English Publishing type：Research paper (scientific journal)

The roles of cancer-associated fibroblasts (CAF) in the progression of various types of cancers are well established. CAF promote cancer progression through pleiotropic mechanisms, including the secretion of soluble factors and extracellular matrix, physical interactions with cancer cells, and the regulation of angiogenesis, immunity and metabolism. Their contribution to therapeutic resistance is also well appreciated. Therefore, CAF have been considered as a therapeutic target in cancer. However, recent studies in autochthonous pancreatic cancer models suggest that specific subset(s) of CAF exhibit cancer-restraining roles, indicating that CAF are functionally and molecularly heterogeneous, which is supported by recent single-cell transcriptome analyses. While cancer-promoting CAF (pCAF) have been extensively studied, the nature and specific marker(s) of cancer-restraining CAF (rCAF) have remained uncharacterized. Interestingly, a recent study provided insight into the nature of rCAF and suggested that they may share molecular properties with pancreatic stellate cells (PSC) and mesenchymal stem/stromal cells (MSC). Complicating this finding is that PSC and MSC have been shown to promote the formation of a tumor-permissive and tumor-promoting environment in xenograft tumor models. However, these cells undergo significant transcriptional and epigenetic changes during ex vivo culture, which confounds the interpretation of experimental results based on the use of cultured cells. In this short review, we describe recent studies and hypotheses on the identity of rCAF and discuss their analogy to fibroblasts that suppress fibrosis in fibrotic diseases. Finally, we discuss how these findings can be exploited to develop novel anticancer therapies in the future.

DOI： 10.1111/cas.14346

The Role of Meflin Expression in Fibroblasts During Development of Pulmonary Fibrosis

Hashimoto, N; Nakahara, Y; Sakamoto, K; Enomoto, A; Yokoi, T; Suzuki, A; Inoue, M; Wakahara, K; Hasegawa, Y

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Vol. 201 2020

Meflin-positive cancer-associated fibroblasts inhibit pancreatic carcinogenesis

Mizutani, Y; Kobayashi, H; Enomoto, A; Takahashi, M

CANCER RESEARCH Vol. 79 ( 24 ) 2019.12

Protein Kinase N Promotes Stress-Induced Cardiac Dysfunction Through Phosphorylation of Myocardin-Related Transcription Factor A and Disruption of Its Interaction With Actin. Reviewed International journal

Teruhiro Sakaguchi, Mikito Takefuji, Nina Wettschureck, Tomonari Hamaguchi, Mutsuki Amano, Katsuhiro Kato, Takuma Tsuda, Shunsuke Eguchi, Sohta Ishihama, Yu Mori, Yoshimitsu Yura, Tatsuya Yoshida, Kazumasa Unno, Takahiro Okumura, Hideki Ishii, Yuuki Shimizu, Yasuko K Bando, Koji Ohashi, Noriyuki Ouchi, Atsushi Enomoto, Stefan Offermanns, Kozo Kaibuchi, Toyoaki Murohara

Circulation Vol. 140 ( 21 ) page： 1737 - 1752 2019.11

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. METHODS: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1flox/flox and Pkn2flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. RESULTS: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. CONCLUSIONS: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.

DOI： 10.1161/CIRCULATIONAHA.119.041019

Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis. Reviewed International journal

Yasuyuki Mizutani, Hiroki Kobayashi, Tadashi Iida, Naoya Asai, Atsushi Masamune, Akitoshi Hara, Nobutoshi Esaki, Kaori Ushida, Shinji Mii, Yukihiro Shiraki, Kenju Ando, Liang Weng, Seiichiro Ishihara, Suzanne M Ponik, Matthew W Conklin, Hisashi Haga, Arata Nagasaka, Takaki Miyata, Makoto Matsuyama, Tomoe Kobayashi, Tsutomu Fujii, Suguru Yamada, Junpei Yamaguchi, Tongtong Wang, Susan L Woods, Daniel Worthley, Teppei Shimamura, Mitsuhiro Fujishiro, Yoshiki Hirooka, Atsushi Enomoto, Masahide Takahashi

Cancer research Vol. 79 ( 20 ) page： 5367 - 5381 2019.10

Language：English Publishing type：Research paper (scientific journal)

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. SIGNIFICANCE: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5367/F1.large.jpg.

DOI： 10.1158/0008-5472.CAN-19-0454

The new-defined mesenchymal stromal/stem cell marker has a protective role in the development of acute lung injury

Nakahara, Y; Hashimoto, N; Sakamoto, K; Ando, A; Inoue, M; Suzuki, A; Enomoto, A; Hasegawa, Y

EUROPEAN RESPIRATORY JOURNAL Vol. 54 2019.9

TDP-43 regulates early-phase insulin secretion via CaV1.2-mediated exocytosis in islets. Reviewed International journal

Kunihiko Araki, Amane Araki, Daiyu Honda, Takako Izumoto, Atsushi Hashizume, Yasuhiro Hijikata, Shinichiro Yamada, Yohei Iguchi, Akitoshi Hara, Kazuhiro Ikumi, Kaori Kawai, Shinsuke Ishigaki, Yoko Nakamichi, Shin Tsunekawa, Yusuke Seino, Akiko Yamamoto, Yasunori Takayama, Shihomi Hidaka, Makoto Tominaga, Mica Ohara-Imaizumi, Atsushi Suzuki, Hiroshi Ishiguro, Atsushi Enomoto, Mari Yoshida, Hiroshi Arima, Shin-Ichi Muramatsu, Gen Sobue, Masahisa Katsuno

The Journal of clinical investigation Vol. 129 ( 9 ) page： 3578 - 3593 2019.9

Language：English Publishing type：Research paper (scientific journal)

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured β cell line (MIN6) and β cell-specific Tardbp knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage-dependent calcium channels and thus plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.

DOI： 10.1172/JCI124481

Roles of the Mesenchymal Stromal/Stem Cell Marker Meflin in Cardiac Tissue Repair and the Development of Diastolic Dysfunction. Reviewed International journal

Akitoshi Hara, Hiroki Kobayashi, Naoya Asai, Shigeyoshi Saito, Takahiro Higuchi, Katsuhiro Kato, Takahiro Okumura, Yasuko K Bando, Mikito Takefuji, Yasuyuki Mizutani, Yuki Miyai, Shoji Saito, Shoichi Maruyama, Keiko Maeda, Noriyuki Ouchi, Arata Nagasaka, Takaki Miyata, Shinji Mii, Noriyuki Kioka, Daniel L Worthley, Toyoaki Murohara, Masahide Takahashi, Atsushi Enomoto

Circulation research Vol. 125 ( 4 ) page： 414 - 430 2019.8

Language：English Publishing type：Research paper (scientific journal)

RATIONALE: Myofibroblasts have roles in tissue repair following damage associated with ischemia, aging, and inflammation and also promote fibrosis and tissue stiffening, causing organ dysfunction. One source of myofibroblasts is mesenchymal stromal/stem cells that exist as resident fibroblasts in multiple tissues. We previously identified meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue), a glycosylphosphatidylinositol-anchored membrane protein, as a specific marker of mesenchymal stromal/stem cells and a regulator of their undifferentiated state. The roles of meflin in the development of heart disease, however, have not been investigated. OBJECTIVE: We examined the expression of meflin in the heart and its involvement in cardiac repair after ischemia, fibrosis, and the development of heart failure. METHODS AND RESULTS: We found that meflin has an inhibitory role in myofibroblast differentiation of cultured mesenchymal stromal/stem cells. Meflin expression was downregulated by stimulation with TGF (transforming growth factor)-β, substrate stiffness, hypoxia, and aging. Histological analysis revealed that meflin-positive fibroblastic cells and their lineage cells proliferated in the hearts after acute myocardial infarction and pressure-overload heart failure mouse models. Analysis of meflin knockout mice revealed that meflin is essential for the increase in the number of cells that highly express type I collagen in the heart walls after myocardial infarction induction. When subjected to pressure overload by transverse aortic constriction, meflin knockout mice developed marked cardiac interstitial fibrosis with defective compensation mechanisms. Analysis with atomic force microscopy and hemodynamic catheterization revealed that meflin knockout mice developed stiff failing hearts with diastolic dysfunction. Mechanistically, we found that meflin interacts with bone morphogenetic protein 7, an antifibrotic cytokine that counteracts the action of TGF-β and augments its intracellular signaling. CONCLUSIONS: These data suggested that meflin is involved in cardiac tissue repair after injury and has an inhibitory role in myofibroblast differentiation of cardiac fibroblastic cells and the development of cardiac fibrosis.

DOI： 10.1161/CIRCRESAHA.119.314806

The intercellular expression of type-XVII collagen, laminin-332, and integrin-β1 promote contact following during the collective invasion of a cancer cell population. Reviewed International journal

Kumagai Y, Nio-Kobayashi J, Ishida-Ishihara S, Tachibana H, Omori R, Enomoto A, Ishihara S, Haga H

Biochemical and biophysical research communications Vol. 514 ( 4 ) page： 1115 - 1121 2019.7

Language：English Publishing type：Research paper (scientific journal)

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-β1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-β1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-β1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-β1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

DOI： 10.1016/j.bbrc.2019.05.058

Identification of cancer-associated fibroblasts that suppress pancreatic cancer progression

Enomoto, A; Mizutani, Y; Kobayashi, H; Iida, T; Woods, SL; Worthley, DL; Takahashi, M

CANCER RESEARCH Vol. 79 ( 13 ) 2019.7

Dephosphorylation of Girdin by PP2A inhibits breast cancer metastasis. Reviewed

Li J, Enomoto A, Weng L, Sun L, Takahashi M

Biochemical and biophysical research communications Vol. 513 ( 1 ) page： 28 - 34 2019.5

Cancer-associated fibroblasts in gastrointestinal cancer. Reviewed

Kobayashi H, Enomoto A, Woods SL, Burt AD, Takahashi M, Worthley DL

Nature reviews. Gastroenterology & hepatology Vol. 16 ( 5 ) page： 282 - 295 2019.5

CD109: a multifunctional GPI-anchored protein with key roles in tumor progression and physiological homeostasis. Reviewed International journal

Shinji Mii, Atsushi Enomoto, Yukihiro Shiraki, Tetsuro Taki, Yoshiki Murakumo, Masahide Takahashi

Pathology international Vol. 69 ( 5 ) page： 249 - 259 2019.5

Language：English Publishing type：Research paper (scientific journal)

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein and a member of the α2 -macroglobulin/C3,C4,C5 family of thioester-containing proteins first identified as being expressed on blood cells, including activated T cells and platelets, and a subset of CD34 + bone marrow cells containing megakaryocyte progenitors. Although CD109 carries the biallelic platelet-specific alloantigen Gov, the physiological functions or roles of CD109 in human disease remain largely unknown. It was recently demonstrated that CD109 is expressed in many malignant tumors, including various squamous cell carcinomas and adenocarcinomas, and plays a role as a multifunctional coreceptor. CD109 reportedly associates with transforming growth factor (TGF)-β receptors and negatively regulates TGF-β signaling in keratinocytes. Additionally, CD109 is potentially related to signal transducer and activator of transcription-3 signaling and aberrant cell proliferation. In this review, we describe recent evidence of CD109-specific significance in malignant tumors shown in mouse models and human tissues. Furthermore, we discuss the physiological functions of CD109 in vitro and in vivo, including results of phenotype analyses of CD109-deficient mice exhibiting epidermal hyperplasia and osteopenia.

DOI： 10.1111/pin.12798

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma. Reviewed International journal

Ranjit M, Hirano M, Aoki K, Okuno Y, Ohka F, Yamamichi A, Kato A, Maeda S, Motomura K, Matsuo K, Enomoto A, Ino Y, Todo T, Takahashi M, Wakabayashi T, Kato T, Natsume A

Cell reports Vol. 26 ( 9 ) page： 2274 - + 2019.2

Language：English Publishing type：Research paper (scientific journal)

RET finger protein (RFP) forms a complex with histone deacetylase 1, resulting in aberrant deacetylation of H3K27ac and dysregulation of cis-regulatory elements. We evaluated the modulatory effects of RFP knockdown on cis-regulatory elements, gene expression, and chemosensitivity to temozolomide both in glioblastoma cells and in an intracranial glioblastoma model. The combination of RFP knockdown and temozolomide treatment markedly suppressed the glioblastoma cell growth due to oxidative stress and aberrant cell cycle and increased survival time in mice with glioblastoma. ChIP-seq and RNA-seq revealed that RFP knockdown increased or decreased activity of numerous cis-regulatory elements that lie adjacent to genes that control functions such as apoptosis, mitosis, DNA replication, and cell cycle: FOXO1, TBP2, and PARPBP. This study suggests that RFP contributes to chemoresistance via aberrant deacetylation of histone H3 at K27, whereas dysregulation of RFP-associated cis-regulatory elements in glioma and RFP knockdown combined with temozolomide is an effective treatment strategy for lethal glioma.

DOI： 10.1016/j.celrep.2019.01.109

Force-dependent arrest of keratinocyte proliferation and its potential defect in skin cancer.

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma Reviewed

Ranjit M, Hirano M, Aoki K, Okuno Y, Ohka F, Yamamichi A, Kato A, Maeda S, Motomura K, Matsuo K, Enomoto A, Ino Y, Todo T, Takahashi M, Wakabayashi T, Kato T, Natsume A.

Cell Rep Vol. 26 ( 9 ) page： 2274-2281.e5. 2019.2

Cancer-associated fibroblasts in gastrointestinal cancer Reviewed

Kobayashi H, Enomoto A, Woods SL, Burt AD, Takahashi M, Worthley DL.

Nat Rev Gastroenterol Hepatol. 2019.2

A rare pseudo tumour in the extraembryonic coelom in first trimester of pregnancy: ultrasound and pathology. Reviewed International journal

Sumigama S, Enomoto A, Matsukawa S, Ushida T, Imai K, Nakano T, Kotani T, Kikkawa F

Journal of obstetrics and gynaecology : the journal of the Institute of Obstetrics and Gynaecology Vol. 39 ( 4 ) page： 545 - 546 2019

Hirata, H; Enomoto, A; Sunagawa, M; Dobrokhotov, O; Takahashi, M; Samsonov, M; Sokabe, M

MOLECULAR BIOLOGY OF THE CELL Vol. 29 ( 26 ) 2018.12

Aberrant active-enhancers associated with downregulation of HDAC1-RFP complex overcome chemoresistance in glioblastoma

Hirano, M; Melissa, R; Ohka, F; Aoki, K; Yamamichi, A; Kato, T; Matsuo, K; Enomoto, A; Takahashi, M; Wakabayashi, T; Natsume, A

CANCER SCIENCE Vol. 109 page： 207 - 207 2018.12

A role of undifferentiated mesenchymal stem cells in cancer progression

Enomoto, A; Takahashi, M

CANCER SCIENCE Vol. 109 page： 182 - 182 2018.12

Analysis of the involvement of cancer-associated fibroblasts in the progression of malignant mesothelioma

Ohara, Y; Enomoto, A; Takahashi, M; Toyokuni, S

CANCER SCIENCE Vol. 109 page： 454 - 454 2018.12

Chemerin promotes angiogenesis in vivo. Reviewed International journal

Nobuhisa Nakamura, Keiko Naruse, Yasuko Kobayashi, Megumi Miyabe, Tomokazu Saiki, Atsushi Enomoto, Masahide Takahashi, Tatsuaki Matsubara

Physiological reports Vol. 6 ( 24 ) page： e13962 2018.12

Language：English Publishing type：Research paper (scientific journal)

Chemerin acts as a chemotactic factor for leukocyte populations expressing the G protein-coupled receptor CMKLR1 (ChemR23). It is also an adipocytokine involved in obesity and metabolic syndromes. Previous studies have demonstrated that chemerin promotes angiogenesis in vitro, although the precise mechanism has not been elucidated. In this study, we have investigated whether chemerin regulates angiogenic processes and validated the associated mechanisms. In this study, chemerin stimulated angiogenesis in mice, which was demonstrated using Matrigel plug implantation assay, mouse corneal models of angiogenesis, and ex vivo rat aortic ring assay. To explore the mechanisms by which chemerin induced angiogenesis, we examined the effects of chemerin in human umbilical vein endothelium cells (HUVECs). Chemerin stimulated the differentiation of HUVECs into capillary-like structures, promoted the proliferation of HUVECs, and functioned as a chemoattractant in migration assays. Chemerin induced the phosphorylation of Akt and p42/44 extracellular signal-regulated kinase (ERK) in HUVECs and chemerin promotes angiogenesis via Akt and ERK. SiRNA against the chemerin receptor CMKLR1 but not that against another chemerin receptor, CCRL2, completely inhibited the chemerin-induced migration and angiogenesis of HUVECs, which indicates that chemerin promotes the migration and angiogenic activities of HUVECs mainly through CMKLR1.

DOI： 10.14814/phy2.13962

Dissection of the function of CD109 in lung adenocarcinoma

Tetsuro Taki, Shinji Mii, Yukihiro Shiraki, Atsushi Enomoto, Masahide Takahashi

CANCER SCIENCE Vol. 109 page： 1407 - 1407 2018.12

Identification of cancer restraining CAF

Mizutani, Y; Enomoto, A; Takahashi, M

CANCER SCIENCE Vol. 109 page： 1396 - 1396 2018.12

Maintenance of Undifferentiated Mesenchymal Stromal Cells-Like Fibroblast by Meflin is Crucial for Heart Repair and Homeostasis

Hara, A; Enomoto, A; Murohara, T

CIRCULATION Vol. 138 2018.11

ABERRANT ACTIVE-ENHANCERS ASSOCIATED WITH DOWNREGULATION OF HDAC1-RET FINGER PROTEIN COMPLEX OVERCOME CHEMORESISTANCE IN GLIOBLASTOMA

Hirano, M; Ranjit, M; Ohka, F; Aoki, K; Yamamichi, A; Kato, T; Matsuo, K; Enomoto, A; Takahashi, M; Wakabayashi, T; Natsume, A

NEURO-ONCOLOGY Vol. 20 page： 111 - 111 2018.11

Girdin/GIV Regulates Collective Cancer Cell Migration by Controlling Cell Adhesion and Cytoskeletal Organization. Reviewed International journal

Wang X, Enomoto A, Weng L, Mizutani Y, Abudureyimu S, Esaki N, Tsuyuki Y, Chen C, Mii S, Asai N, Haga H, Ishida S, Yokota K, Akiyama M, Takahashi M

Cancer science Vol. 109 ( 11 ) page： 3643 - 3656 2018.11

Language：English Publishing type：Research paper (scientific journal)

Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. Here, we studied the role of the actin-binding protein Girdin, a specific regulator of collective migration of neuroblasts in the brain, in collective cancer cell migration. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to β-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the β-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells. Finally, we showed the correlation of the expression of Girdin with that of the components of the E-cadherin complex and the differentiation of human skin cancer. Collectively, our results suggest that Girdin is an important modulator of the collective behavior of cancer cells.

DOI： 10.1111/cas.13795

Trefoil factor 1 inhibits epithelial-mesenchymal transition of pancreatic intraepithelial neoplasm. Reviewed

Large cell neuroendocrine carcinoma of the breast showing triple negative, basal-like subtype Reviewed

Kawasaki T, Hasebe T, Ichihara S, Oiwa M, Sato Y, Morita T, Hayashi T, Kato A, Sugiyama K, Nozawa K, Enomoto A, Takahashi M, Horibe K, Naoe T, Osaki A, Saeki T

VIRCHOWS ARCHIV Vol. 473 page： S208 2018.9

Yamaguchi J, Yokoyama Y, Kokuryo T, Ebata T, Enomoto A, Nagino M

The Journal of clinical investigation Vol. 128 ( 8 ) page： 3619 - 3629 2018.8

The actin-binding protein Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization

Wang, XZ; Enomoto, A; Weng, L; Haga, H; Ishida, S; Takahashi, M

CANCER RESEARCH Vol. 78 ( 13 ) 2018.7

CD109 deficiency induces osteopenia with an osteoporosis-like phenotype in vivo. Reviewed International journal

Shinji Mii, Akiyoshi Hoshino, Atsushi Enomoto, Yoshiki Murakumo, Masako Ito, Akira Yamaguchi, Masahide Takahashi

Genes to cells : devoted to molecular & cellular mechanisms Vol. 23 ( 7 ) page： 590 - 598 2018.7

Language：English Publishing type：Research paper (scientific journal)

Osteoporosis is a global public health problem that is increasing along with an aging population. A major determinant of osteoporosis is high bone turnover, which results from osteoclast activation. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, a deficiency that leads to a psoriasis-like skin inflammation in mice. Although the expression of CD109 has been reported in mouse pre-osteoclast cells, its function in osteoclasts in vivo remains largely unknown. To investigate the physiological role of CD109 in bone metabolism, we analyzed bones from wild-type and CD109-deficient adult mice. Micro-computed tomography analysis of the femur (thigh bone) showed that bone volume was lower in CD109-deficient mice than in wild-type mice. Bone histomorphometric analysis showed not only a reduction in bone volume but also an increase in bone turnover in CD109-deficient mice as compared with wild-type mice. Additionally, we measured serum levels of several markers of bone turnover and found a significant increase in the N-terminal telopeptide of type I collagen, a bone resorption marker, as well as alkaline phosphatase, a bone formation marker, in CD109-deficient mice. These results indicate that CD109 deficiency induces a high-turnover, osteoporosis-like phenotype, which suggests that CD109 plays a role in bone metabolism in vivo.

DOI： 10.1111/gtc.12593

INHIBITION OF MTORC1 THROUGH AMINO ACID TRANSPORTER CD98/LAT1 MAINTAINS THE STEMNESS OF GLIOMA STEM CELL

Han, YP; Enomoto, A; Takahashi, M; Ma, J

NEURO-ONCOLOGY Vol. 20 page： 180 - 180 2018.6

Essential Role of Linx/Islr2 in the Development of the Forebrain Anterior Commissure Reviewed

Shaniya Abudureyimu, Naoya Asai, Atsushi Enomoto, Liang Weng, Hiroki Kobayashi, Xiaoze Wang, Chen Chen, Shinji Mii, Masahide Takahashi

Scientific Reports Vol. 8 ( 1 ) page： 7292 2018.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

Linx is a member of the leucine-rich repeat and immunoglobulin family of membrane proteins which has critical roles in the development of the peripheral nervous system and forebrain connectivity. A previous study showed that Linx is expressed in projection neurons in the cortex and in cells that comprise the passage to the prethalamus that form the internal capsule, indicating the involvement of Linx in axon guidance and cell-cell communication. In this study, we found that Linx-deficient mice develop severe hydrocephalus and die perinatally by unknown mechanisms. Importantly, mice heterozygous for the linx gene exhibited defects in the development of the anterior commissure in addition to hydrocephalus, indicating haploinsufficiency of the linx gene in forebrain development. In N1E-115 neuroblastoma cells and primary cultured hippocampal neurons, Linx depletion led to impaired neurite extension and an increase in cell body size. Consistent with this, but of unknown significance, we found that Linx interacts with and upregulates the activity of Rho-kinase, a modulator of many cellular processes including cytoskeletal organization. These data suggest a role for Linx in the regulation of complex forebrain connectivity, and future identification of its extracellular ligand(s) will help clarify this function.

DOI： 10.1038/s41598-018-24064-0

Regulation of keratin 5/14 intermediate filaments by CDK1, Aurora-B, and Rho-kinase Reviewed

金属接着凍結法による脂肪を含む凍結標本作製の試み

牛田かおり, 内山孝蔵, 浅井直也, 榎本篤, 高橋雅英

病理と臨床 Vol. 36 ( 5 ) page： 493 - 497 2018.5

Hironori Inaba, Daishi Yamakawa, Yasuko Tomono, Atsushi Enomoto, Shinji Mii, Kousuke Kasahara, Hidemasa Goto, Masaki Inagaki

Biochemical and Biophysical Research Communications Vol. 498 ( 3 ) page： 544 - 550 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier B.V.

We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.

DOI： 10.1016/j.bbrc.2018.03.016

Critical role of rabphilin-3A in the pathophysiology of experimental lymphocytic neurohypophysitis Reviewed

Yoshinori Yasuda, Shintaro Iwama, Atsushi Kiyota, Hisakazu Izumida, Kohtaro Nakashima, Naoko Iwata, Yoshihiro Ito, Yoshiaki Morishita, Motomitsu Goto, Hidetaka Suga, Ryoichi Banno, Atsushi Enomoto, Masahide Takahashi, Hiroshi Arima, Yoshihisa Sugimura

Journal of Pathology Vol. 244 ( 4 ) page： 469 - 478 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：John Wiley and Sons Ltd

Autoimmune hypophysitis (AH) is thought to be an autoimmune disease characterized by lymphocytic infiltration of the pituitary gland. Among AH pathologies, lymphocytic infundibulo-neurohypophysitis (LINH) involves infiltration of the neurohypophysis and/or the hypothalamic infundibulum, causing central diabetes insipidus resulting from insufficiency of arginine vasopressin secretion. The pathophysiological and pathogenetic mechanisms underlying LINH are largely unknown. Clinically, differentiating LINH from other pituitary diseases accompanied by mass lesions, including tumours, has often been difficult, because of similar clinical manifestations. We recently reported that rabphilin-3A is an autoantigen and that anti-rabphilin-3A antibodies constitute a possible diagnostic marker for LINH. However, the involvement of rabphilin-3A in the pathogenesis of LINH remains to be elucidated. This study was undertaken to explore the role of rabphilin-3A in lymphocytic neurohypophysitis and to investigate the mechanism. We found that immunization of mice with rabphilin-3A led to neurohypophysitis. Lymphocytic infiltration was observed in the neurohypophysis and supraoptic nucleus 1 month after the first immunization. Mice immunized with rabphilin-3A showed an increase in the volume of urine that was hypotonic as compared with control mice. Administration of a cocktail of monoclonal anti-rabphilin-3A antibodies did not induce neurohypophysitis. However, abatacept, which is a chimeric protein that suppresses T-cell activation, decreased the number of T cells specific for rabphilin-3A in peripheral blood mononuclear cells (PBMCs). It ameliorated lymphocytic infiltration of CD3+ T cells in the neurohypophysis of mice that had been immunized with rabphilin-3A. Additionally, there was a linear association between the number of T cells specific for rabphilin-3A in PBMCs and the number of CD3+ T cells infiltrating the neurohypophysis. In conclusion, we suggest that rabphilin-3A is a pathogenic antigen, and that T cells specific for rabphilin-3A are involved in the pathogenesis of neurohypophysitis in mice. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp
Sons, Ltd.

DOI： 10.1002/path.5046

Development of a method to preliminarily embed tissue samples using low melting temperature fish gelatin before sectioning: A technical note Reviewed

Kaori Ushida, Naoya Asai, Kozo Uchiyama, Atsushi Enomoto, Masahide Takahashi

Pathology International Vol. 68 ( 4 ) page： 241 - 245 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing

Embedding of tissue samples that maintains a desired orientation is critical for preparing sections suitable for diagnosis and study objectives. Methods to prepare tissue sections include: (i) paraffin embedding or snap-freezing followed by microtome or cryostat sectioning
and (ii) agarose embedding followed by cutting on a vibrating microslicer. Although these methods are useful for routine laboratory work, preparation of small and fragile tissues such as mouse organs, small human biopsy samples, and cultured floating spheres is difficult and requires special skills. In particular, tissue specimen orientation can be lost during embedding in molds and subsequent sectioning. Here, we developed a method using low melting temperature (LM) gelatin either alone or mixed with agarose to preliminarily embed collected tissues that are either prefixed or unfixed, followed by conventional fixation, paraffin embedding, freezing, and sectioning. The advantage of the method is that the LM gelatin and its mixture with agarose can be handled at room temperature but quickly hardens at 4°C, which allows embedding, trimming, and arranging of small and fragile tissues in a desired orientation and are compatible with traditional stainings. Thus, this method can have various laboratory applications and can be modified according to the needs of each laboratory.

DOI： 10.1111/pin.12652

Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex. Reviewed International journal

Liang Weng, Yi-Peng Han, Atsushi Enomoto, Yasuyuki Kitaura, Shushi Nagamori, Yoshikatsu Kanai, Naoya Asai, Jian An, Maki Takagishi, Masato Asai, Shinji Mii, Takashi Masuko, Yoshiharu Shimomura, Masahide Takahashi

PLoS biology Vol. 16 ( 3 ) page： e2005090 2018.3

Language：English Publishing type：Research paper (scientific journal)

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.

DOI： 10.1371/journal.pbio.2005090

The role of CD109 in PDGFB-induced glioma mouse model for sensitivity to Temozolomide

Shiraki Yukihiro, Mii Shinji, Asai Naoya, Enomoto Atsushi, Momota Hiroyuki, Natsume Atsushi, Wakabayashi Toshihiko, Takahashi Masahide

CANCER SCIENCE Vol. 109 page： 1130 - 1130 2018.1

ASC amino acid transporter 2, defined by enzyme-mediated activation of radical sources, enhances malignancy of GD2-positive small-cell lung cancer. Reviewed International journal

Nobutoshi Esaki, Yuki Ohkawa, Noboru Hashimoto, Yuhsuke Tsuda, Yuhsuke Ohmi, Robiul H Bhuiyan, Norihiro Kotani, Koichi Honke, Atsushi Enomoto, Masahide Takahashi, Keiko Furukawa, Koichi Furukawa

Cancer science Vol. 109 ( 1 ) page： 141 - 153 2018.1

Language：English Publishing type：Research paper (scientific journal)

Ganglioside GD2 is specifically expressed in small-cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme-mediated activation of radical sources combined with mass spectrometry in GD2+ SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2+ SCLC cells were enhanced by glutamine uptake, and were suppressed by L-γ-glutamyl-p-nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.

DOI： 10.1111/cas.13448

Aberrant super-enhancers associated with downregulation of RET finger protein overcomes chemoresistance in glioblastoma

Hirano, M; Ranjit, M; Yamamichi, A; Aoki, K; Ohka, F; Kato, T; Enomoto, A; Takahashi, M; Wakabayashi, T; Natsume, A

CANCER SCIENCE Vol. 109 page： 461 - 461 2018.1

Ganglioside GD2 coordinately works with ASCT2, which enhances malignant phenotypes in small cell lung cancer

Esaki, N; Ohkawa, Y; Hashimoto, N; Tuna, Y; Ohmi, Y; Bhuiya, R; Kotani, N; Honk, C; Enomoto, A; Takahashi, M; Furukawa, K; Furukawa, K

CANCER SCIENCE Vol. 109 page： 94 - 94 2018.1

Mechanisms for the control of amino acid signaling and drug resistance in cancer stem cells

Enomoto, A; Takahashi, M

CANCER SCIENCE Vol. 109 page： 813 - 813 2018.1

Aberrant super-enhancers associated with downregulation of RET finger protein overcomes chemoresistance in glioblastoma

Hirano Masaki, Ranjit Melissa, Yamamichi Akane, Aoki Kosuke, Ohka Fumiharu, Kato Takuya, Enomoto Atsushi, Takahashi Masahide, Wakabayashi Toshihiko, Natsume Atsushi

CANCER SCIENCE Vol. 109 page： 461-461 2018.1

Cullin-associated NEDD8-dissociated protein 1, a novel interactor of rabphilin-3A, deubiquitylates rabphilin-3A and regulates arginine vasopressin secretion in PC12 cells Reviewed

Kohtaro Nakashima, Seiji Takeuchi, Shintaro Iwama, Atsushi Kiyota, Yoshinori Yasuda, Naoko Iwata, Atsushi Enomoto, Hiroshi Arima, Yoshihisa Sugimura

Endocrine Journal Vol. 65 ( 3 ) page： 325 - 334 2018

Language：English Publishing type：Research paper (scientific journal) Publisher：Japan Endocrine Society

The molecular mechanism involved in the exocytosis of arginine vasopressin (AVP) is not fully known. Rabphilin-3A has been suggested as a novel autoantigen in infundibulo-neurohypophysitis (LINH), which leads to central diabetes insipidus through insufficient secretion of AVP. However, the role of rabphilin-3A in the pathogenesis of LINH remains unclear. Thus, the aim of the present study was to identify proteins binding rabphilin-3A in the posterior pituitary. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, cullin-associated NEDD8-dissociated protein 1 (CAND1) was identified as a rabphilin-3A-binding protein in the posterior pituitary. Co-immunoprecipitation assays indicated that CAND1 interacted endogenously with rabphilin-3A. In addition, immunohistochemistry experiments showed that CAND1 immunoreactivity was detected mainly in the posterior pituitary, intermediate lobe, and the supraoptic nucleus in the hypothalamus, and less in the anterior lobe, partially co-localizing with rabphilin-3A. Overexpression of CAND1 resulted in deubiquitylation of rabphilin-3A in PC12 cells. Moreover, overexpression of CAND1 in PC12 cells co-transfected with AVP enhanced both basal and KCl-stimulated AVP secretion. The findings indicate that CAND1 inhibits the ubiquitylation of rabphilin-3A and positively regulates AVP secretion. These data shed light on a novel potential mechanism involving rabphilin-3A in AVP secretion, and suggest a new role of CAND1 as a regulator of hormone or neurotransmitter secretion.

DOI： 10.1507/endocrj.EJ17-0399

Significance of perivascular tumour cells defined by CD109 expression in progression of glioma Reviewed

Yukihiro Shiraki, Shinji Mii, Atsushi Enomoto, Hiroyuki Momota, Yi-Peng Han, Takuya Kato, Kaori Ushida, Akira Kato, Naoya Asai, Yoshiki Murakumo, Kosuke Aoki, Hiromichi Suzuki, Fumiharu Ohka, Toshihiko Wakabayashi, Tomoki Todo, Seishi Ogawa, Atsushi Natsume, Masahide Takahashi

JOURNAL OF PATHOLOGY Vol. 243 ( 4 ) page： 468 - 480 2017.12

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY

In the progression of glioma, tumour cells often exploit the perivascular microenvironment to promote their survival and resistance to conventional therapies. Some of these cells are considered to be brain tumour stem cells (BTSCs); however, the molecular nature of perivascular tumour cells has not been specifically clarified because of the complexity of glioma. Here, we identified CD109, a glycosylphosphatidylinositol-anchored protein and regulator of multiple signalling pathways, as a critical regulator of the progression of lower-grade glioma (World Health Organization grade II/III) by clinicopathological and whole-genome sequencing analysis of tissues from human glioma. The importance of CD109-positive perivascular tumour cells was confirmed not only in human lower-grade glioma tissues but also in a mouse model that recapitulated human glioma. Intriguingly, BTSCs isolated from mouse glioma expressed high levels of CD109. CD109-positive BTSCs exerted a proliferative effect on differentiated glioma cells treated with temozolomide. These data reveal the significance of tumour cells that populate perivascular regions during glioma progression, and indicate that CD109 is a potential therapeutic target for the disease. Copyright (C) 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DOI： 10.1002/path.4981

ABERRANT SUPER-ENHANCERS ASSOCIATED WITH DOWNREGULATION OF RET FINGER PROTEIN OVERCOMES CHEMORESISTANCE IN GLIOBLASTOMA

Masaki Hirano, Melissa Ranjit, Akane Yamamichi, Kosuke Aoki, Fumiharu Ohka, Takuya Kato, Atsushi Enomoto, Masahide Takahashi, Toshihiko Wakabayashi, Atsushi Natsume

NEURO-ONCOLOGY Vol. 19 page： 103 - 103 2017.11

Correlation between SPARC expression and efficacy of nab-paclitaxel for advanced gastric cancer refractory to fluoropyrimidine: An exploratory analysis of a phase II trial, CCOG1303

Kobayashi, D; Enomoto, A; Mochizuki, Y; Matsui, T; Nakayama, H; Kawase, Y; Ishigure, K; Shikano, T; Torii, K; Kodera, Y

ANNALS OF ONCOLOGY Vol. 28 2017.9

脳腫瘍マウスモデルにおけるテモゾロミド感受性に対するCD109の機能解析

白木之浩, 三井伸二, 浅井直也, 榎本篤, 百田洋之, 夏目敦至, 若林俊彦, 高橋雅英

日本癌学会総会記事 Vol. 76回 page： P - 3324 2017.9

Daple Coordinates Planar Polarized Microtubule Dynamics in Ependymal Cells and Contributes to Hydrocephalus Reviewed

Maki Takagishi, Masato Sawada, Shinya Ohata, Naoya Asai, Atsushi Enomoto, Kunihiko Takahashi, Liang Weng, Kaori Ushida, Hosne Ara, Shigeyuki Matsui, Kozo Kaibuchi, Kazunobu Sawamoto, Masahide Takahashi

CELL REPORTS Vol. 20 ( 4 ) page： 960 - 972 2017.7

Language：English Publishing type：Research paper (scientific journal) Publisher：CELL PRESS

Motile cilia in ependymal cells, which line the cerebral ventricles, exhibit a coordinated beating motion that drives directional cerebrospinal fluid (CSF) flow and guides neuroblast migration. At the apical cortex of these multi-ciliated cells, asymmetric localization of planar cell polarity (PCP) proteins is required for the planar polarization of microtubule dynamics, which coordinates cilia orientation. Daple is a disheveledassociating protein that controls the non-canonical Wnt signaling pathway and cell motility. Here, we show that Daple-deficient mice present hydrocephalus and their ependymal cilia lack coordinated orientation. Daple regulates microtubule dynamics at the anterior side of ependymal cells, which in turn orients the cilial basal bodies required for the directional cerebrospinal fluid flow. These results demonstrate an important role for Daple in planar polarity in motile cilia and provide a framework for understanding the mechanisms and functions of planar polarization in the ependymal cells.

DOI： 10.1016/j.celrep.2017.06.089

Corrigendum: Girdin maintains the stemness of glioblastoma stem cells (Oncogene (2012) 31 (2715-2724) DOI: 10.1038/onc.2011.466) Reviewed

A. Natsume, T. Kato, S. Kinjo, A. Enomoto, H. Toda, S. Shimato, F. Ohka, K. Motomura, Y. Kondo, T. Miyata, M. Takahashi, T. Wakabayashi

Oncogene Vol. 31 ( 22 ) page： 2715 - 2724 2017.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

Glioblastomas (GBMs) are the most common and aggressive type of brain tumor. GBMs usually show hyperactivation of the PI3K-Akt pathway, a pro-tumorigenic signaling cascade that contributes to pathogenesis. Girdin, an actin-binding protein identified as a novel substrate of Akt, regulates the sprouting of axons and the migration of neural progenitor cells during early postnatal-stage neurogenesis in the hippocampus. Here, we show that Girdin is highly expressed in human glioblastoma (GBM). Stable Girdin knockdown in isolated GBM stem cells resulted in decreased expression of stem cell markers, including CD133, induced multilineage neural differentiation, and inhibited in vitro cell motility, ex vivo invasion, sphere-forming capacity and in vivo tumor formation. Furthermore, exogenous expression of the Akt-binding domain of Girdin, which competitively inhibits its Akt-mediated phosphorylation, diminished the expression of stem cell markers, SOX2 and nestin, and migration on the brain slice and induced the expression of neural differentiation markers glial fibrillary acidic protein/ΒIII Tubulin. Our results reveal that Girdin is required for GBM-initiating stem cells to sustain the stemness and invasive properties. © 2012 Macmillan Publishers Limited All rights reserved.

DOI： 10.1038/onc.2011.466

Tyrosine Phosphorylation of an Actin-Binding Protein Girdin Specifically Marks Tuft Cells in Human and Mouse Gut Reviewed

Daisuke Kuga, Kaori Ushida, Shinji Mii, Atsushi Enomoto, Naoya Asai, Masato Nagino, Masahide Takahashi, Masato Asai

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY Vol. 65 ( 6 ) page： 347 - 366 2017.6

Language：English Publishing type：Research paper (scientific journal) Publisher：SAGE PUBLICATIONS LTD

Tuft cells (TCs) are minor components of gastrointestinal epithelia, characterized by apical tufts and spool-shaped somas. The lack of reliable TC-markers has hindered the elucidation of its role. We developed site-specific and phosphorylation-status-specific antibodies against Girdin at tyrosine-1798 (pY1798) and found pY1798 immunostaining of mouse jejunum clearly depicted epithelial cells closely resembling TCs. This study aimed to validate pY1798 as a TC-marker. Double-fluorescence staining of intestines was performed with pY1798 and known TC-markers, for example, hematopoietic-prostaglandin-D-synthase (HPGDS), or doublecortin-like kinase 1 (DCLK1). Odds ratios (ORs) were calculated from cell counts to determine whether two markers were attracting (OR<1) or repelling (OR>1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs.

DOI： 10.1369/0022155417702586

Significance of low mTORC1 activity in defining the characteristics of brain tumor stem cells Reviewed

Girdin maintains the stemness of glioblastoma stem cells (vol 31, pg 2715, 2012) Reviewed

A. Natsume, T. Kato, S. Kinjo, A. Enomoto, H. Toda, S. Shimato, F. Ohka, K. Motomura, Y. Kondo, T. Miyata, M. Takahashi, T. Wakabayashi

ONCOGENE Vol. 36 ( 26 ) page： 3796 - 3796 2017.6

Yi-Peng Han, Atsushi Enomoto, Yukihiro Shiraki, Shen-Qi Wang, Xiaoze Wang, Shinya Toyokuni, Naoya Asai, Kaori Ushida, Hosne Ara, Fumiharu Ohka, Toshihiko Wakabayashi, Jie Ma, Atsushi Natsume, Masahide Takahashi

NEURO-ONCOLOGY Vol. 19 ( 5 ) page： 636 - 647 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS INC

The significance of mammalian target of rapamycin complex 1 (mTORC1) activity in the maintenance of cancer stem cells (CSCs) remains controversial. Previous findings showed that mTORC1 activation depleted the population of leukemia stem cells in leukemia, while maintaining the stemness in pancreatic CSCs. The purpose of this study was to examine the currently unknown role and significance of mTORC1 activity in brain tumor stem cells (BTSCs).
Basal mTORC1 activity and its kinetics were investigated in BTSC clones isolated from patients with glioblastoma and their differentiated progenies (DIFFs). The effects of nutrient deprivation and the mTORC1 inhibitors on cell proliferation were compared between the BTSCs and DIFFs. Tissue sections from patients with brain gliomas were examined for expression of BTSC markers and mTORC1 activity by immunohistochemistry.
BTSCs presented lower basal mTORC1 activity under each culture condition tested and a more rapid decline of mTORC1 activity after nutrient deprivation than observed in DIFFs. The self-renewal capacity of BTSCs was unaffected by mTORC1 inhibition, whereas it effectively suppressed DIFF proliferation. In agreement, immunohistochemical staining of glioma tissues revealed low mTORC1 activity in tumor cells positive for BTSC markers. In in vitro culture, BTSCs exhibited resistance to the antitumor agent temozolomide.
Our findings indicated the importance of low mTORC1 activity in maintaining the undifferentiated state of BTSCs, implicating the relevance of manipulating mTORC1 activity when developing future strategies that target BTSCs.

DOI： 10.1093/neuonc/now237