Publications by authors named "Kiyoshi Yamaguchi"

74 Publications

Mutant ASXL1 induces age-related expansion of phenotypic hematopoietic stem cells through activation of Akt/mTOR pathway.

Nat Commun 2021 03 23;12(1):1826. Epub 2021 Mar 23.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.
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http://dx.doi.org/10.1038/s41467-021-22053-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988019PMC
March 2021

TP53/p53-FBXO22-TFEB controls basal autophagy to govern hormesis.

Autophagy 2021 Mar 11:1-18. Epub 2021 Mar 11.

Division of Cancer Cell Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy. Mild stress-activated TP53 transcriptionally induced FBXO22, which in turn ubiquitinated KDM4B (lysine-specific demethylase 4B) complexed with MYC-NCOR1 suppressors for degradation, leading to transcriptional induction of TFEB. Upregulation of autophagy-related genes by increased TFEB dramatically enhanced autophagic activity and cell survival upon following a severe stressor. Mitogen-induced AKT1 activation counteracted this process through the phosphorylation of KDM4B, which inhibited FBXO22-mediated ubiquitination. Additionally, mice died within 10 h of birth, and their mouse embryonic fibroblasts (MEFs) showed a lowered basal autophagy, whereas FBXO22-overexpressing mice were resistant to chemotherapy. Taken together, these results suggest that TP53 upregulates basal autophagy through the FBXO22-TFEB axis, which governs the hormetic effect in chemotherapy.: BBC3/PUMA: BCL2 binding component 3; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; ChIP-seq: chromatin immunoprecipitation followed by sequencing; DDB2: damage specific DNA binding protein 2; DRAM: DNA damage regulated autophagy modulator; ESR/ER: estrogen receptor 1; FMD: fasting mimicking diet; HCQ: hydroxychloroquine; KDM4B: lysine-specific demethylase 4B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; NCOR1: nuclear receptor corepressor 1; SCF: SKP1-CUL-F-box protein; SQSTM1: sequestosome 1; TFEB: transcription factor EB.
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http://dx.doi.org/10.1080/15548627.2021.1897961DOI Listing
March 2021

Functional Restoration of Bacteriomes and Viromes by Fecal Microbiota Transplantation.

Gastroenterology 2021 May 9;160(6):2089-2102.e12. Epub 2021 Feb 9.

Department of Immunology and Genomics, Osaka City University, Graduate School of Medicine, Abeno-ku, Osaka, Japan; Division of Metagenome Medicine, Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan; Division of Innate Immune Regulation, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan; Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. Electronic address:

Background & Aims: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT.

Methods: The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria-phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated.

Results: Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented.

Conclusions: The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.
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http://dx.doi.org/10.1053/j.gastro.2021.02.013DOI Listing
May 2021

Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders.

Science 2021 01;371(6526):265-270

Division of Cancer Cell Biology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Removal of senescent cells (senolysis) has been proposed to be beneficial for improving age-associated pathologies, but the molecular pathways for such senolytic activity have not yet emerged. Here, we identified glutaminase 1 () as an essential gene for the survival of human senescent cells. The intracellular pH in senescent cells was lowered by lysosomal membrane damage, and this lowered pH induced kidney-type glutaminase (KGA) expression. The resulting enhanced glutaminolysis induced ammonia production, which neutralized the lower pH and improved survival of the senescent cells. Inhibition of KGA-dependent glutaminolysis in aged mice eliminated senescent cells specifically and ameliorated age-associated organ dysfunction. Our results suggest that senescent cells rely on glutaminolysis, and its inhibition offers a promising strategy for inducing senolysis in vivo.
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http://dx.doi.org/10.1126/science.abb5916DOI Listing
January 2021

Robust parameter design of human induced pluripotent stem cell differentiation protocols defines lineage-specific induction of anterior-posterior gut tube endodermal cells.

Stem Cells 2021 Apr 15;39(4):429-442. Epub 2021 Jan 15.

Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan.

Tissues and cells derived from pluripotent stem cells (PSC) are likely to become widely used in disease modeling, drug screening, and regenerative medicine. For these applications, the in vitro PSC differentiation process must be elaborately investigated and controlled to reliably obtain the desired end products. However, because traditional experimental methods, such as one factor at a time or brute-force approaches, are impractical for detailed screening of complex PSC cultivation conditions, more strategic and effective screening based on statistical design of experiments (DOE) ought to be indispensable. Among various DOE approaches, we regard robust parameter design (RPD) as particularly suited for differentiation protocol optimization due to its suitability for multifactorial screening. We confirmed the adaptability of RPD for investigating human induced PSC lineage specification toward anterior-posterior gut tube endodermal cells and clarified both the contribution of each cell signaling pathway and the effect of cell signaling condition alteration on marker RNA expression levels, while increasing the efficiency of the screening in 243-fold (18 vs 4374) compared with that of a brute-force approach. Specific induction of anterior foregut, hepatic, pancreatic, or mid-hindgut cells was achieved using seven iPSC strains with the optimal culture protocols established on the basis of RPD analysis. RPD has the potential to enable efficient construction and optimization of PSC differentiation protocols, and its use is recommended from fundamental research to mass production of PSC-derived products.
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http://dx.doi.org/10.1002/stem.3326DOI Listing
April 2021

Halcyon: An Accurate Basecaller Exploiting An Encoder-Decoder Model With Monotonic Attention.

Bioinformatics 2020 Nov 9. Epub 2020 Nov 9.

Health Intelligence Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Motivation: In recent years, nanopore sequencing technology has enabled inexpensive long-read sequencing, which promises reads longer than a few thousand bases. Such long-read sequences contribute to the precise detection of structural variations and accurate haplotype phasing. However, deciphering precise DNA sequences from noisy and complicated nanopore raw signals remains a crucial demand for downstream analyses based on higher-quality nanopore sequencing, although various basecallers have been introduced to date.

Results: To address this need, we developed a novel basecaller, Halcyon, that incorporates neural-network techniques frequently used in the field of machine translation. Our model employs monotonic-attention mechanisms to learn semantic correspondences between nucleotides and signal levels without any pre-segmentation against input signals. We evaluated performance with a human whole-genome sequencing dataset and demonstrated that Halcyon outperformed existing third-party basecallers and achieved competitive performance against the latest Oxford Nanopore Technologies' basecallers.

Availability: The source code (halcyon) can be found at https://github.com/relastle/halcyon.
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http://dx.doi.org/10.1093/bioinformatics/btaa953DOI Listing
November 2020

Efficacy of the novel tubulin polymerization inhibitor PTC-028 for myelodysplastic syndrome.

Cancer Sci 2020 Dec 2;111(12):4336-4347. Epub 2020 Nov 2.

Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.
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http://dx.doi.org/10.1111/cas.14684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734154PMC
December 2020

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 Nov 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Metagenome Data on Intestinal Phage-Bacteria Associations Aids the Development of Phage Therapy against Pathobionts.

Cell Host Microbe 2020 09 10;28(3):380-389.e9. Epub 2020 Jul 10.

Department of Immunology and Genomics, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan; Division of Metagenome Medicine, Human Genome Center, the Institute of Medical Sciences, the University of Tokyo, Tokyo 108-8639, Japan; Division of Innate Immune Regulation, International Research and Development Center for Mucosal Vaccines, the Institute of Medical Sciences, the University of Tokyo, Tokyo 108-8639, Japan; Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo 113-8657, Japan. Electronic address:

The application of bacteriophages (phages) is proposed as a highly specific therapy for intestinal pathobiont elimination. However, the infectious associations between phages and bacteria in the human intestine, which is essential information for the development of phage therapies, have yet to be fully elucidated. Here, we report the intestinal viral microbiomes (viromes), together with bacterial microbiomes (bacteriomes), in 101 healthy Japanese individuals. Based on the genomic sequences of bacteriomes and viromes from the same fecal samples, the host bacteria-phage associations are illustrated for both temperate and virulent phages. To verify the usefulness of the comprehensive host bacteria-phage information, we screened Clostridioides difficile-specific phages and identified antibacterial enzymes whose activity is confirmed both in vitro and in vivo. These comprehensive metagenome analyses reveal not only host bacteria-phage associations in the human intestine but also provide vital information for the development of phage therapies against intestinal pathobionts.
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http://dx.doi.org/10.1016/j.chom.2020.06.005DOI Listing
September 2020

EXOSC9 depletion attenuates P-body formation, stress resistance, and tumorigenicity of cancer cells.

Sci Rep 2020 06 9;10(1):9275. Epub 2020 Jun 9.

Department of System Biology, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Takaramachi, Kanazawa, Ishikawa, Japan.

Cancer cells adapt to various stress conditions by optimizing gene expression profiles via transcriptional and translational regulation. However, whether and how EXOSC9, a component of the RNA exosome complex, regulates adaptation to stress conditions and tumorigenicity in cancer cells remain unclear. Here, we examined the effects of EXOSC9 depletion on cancer cell growth under various stress conditions. EXOSC9 depletion attenuated growth and survival under various stress conditions in cancer cells. Interestingly, this also decreased the number of P-bodies, which are messenger ribonucleoprotein particles (mRNPs) required for stress adaptation. Meanwhile, EXOSC2/EXOSC4 depletion also attenuated P-body formation and stress resistance with decreased EXOSC9 protein. EXOSC9-mediated stress resistance and P-body formation were found to depend on the intact RNA-binding motif of this protein. Further, RNA-seq analyses identified 343 EXOSC9-target genes, among which, APOBEC3G contributed to defects in stress resistance and P-body formation in MDA-MB-231 cells. Finally, EXOSC9 also promoted xenografted tumor growth of MDA-MB-231 cells in an intact RNA-binding motif-dependent manner. Database analyses further showed that higher EXOSC9 activity, estimated based on the expression of 343 target genes, was correlated with poorer prognosis in some cancer patients. Thus, drugs targeting activity of the RNA exosome complex or EXOSC9 might be useful for cancer treatment.
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http://dx.doi.org/10.1038/s41598-020-66455-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283315PMC
June 2020

Correction: Importance of gastric cancer for the diagnosis and surveillance of Japanese Lynch syndrome patients.

J Hum Genet 2020 07;65(7):633

Division of Clinical Genome Research, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s10038-020-0747-5DOI Listing
July 2020

Response to the correspondence referring to our article "Development of an MSI-positive colon tumor with aberrant DNA methylation in a PPAP patient" by Pilar Mur, Claire Palles, Ian Tomlinson, Laura Valle.

J Hum Genet 2020 06 3;65(6):515-516. Epub 2020 Apr 3.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

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http://dx.doi.org/10.1038/s10038-020-0752-8DOI Listing
June 2020

Discovery of chemical probes that suppress Wnt/β-catenin signaling through high-throughput screening.

Cancer Sci 2020 Mar 5;111(3):783-794. Epub 2020 Feb 5.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Aberrant activation of the Wnt/β-catenin signaling pathway has been observed in a wide range of human tumors. Deregulation of the pathway is closely linked to various aspects of human carcinogenesis such as cell viability, regulation of cell cycle, epithelial-mesenchymal transition, and maintenance of stemness. In addition, recent studies have disclosed the involvement of Wnt signaling in immune evasion of tumor cells. The accumulation of β-catenin in the nucleus is a common feature of cancer cells carrying defects in the pathway, which leads to the continuous activation of T-cell factor (TCF)/LEF transcription factors. Consequently, a genetic program is switched on, leading to the uncontrolled growth, prolonged survival, and acquisition of mesenchymal phenotype. As β-catenin/TCF serves as a signaling hub for the pathway, β-catenin/TCF-dependent transcriptional activity is a relevant readout of the pathway. To date, a wide variety of synthetic TCF/LEF reporters has been developed, and high-throughput screening (HTS) using these reporters has made significant contributions to the discovery of Wnt inhibitors. Indeed, HTS led to the identification of chemical probes targeting porcupine, a membrane bound O-acyltransferase, and CREB-binding protein, a transcriptional coactivator. This review focuses on various screening strategies for the discovery of Wnt inhibitors and their mode of action to help the creation of new concepts for assay/screening methods.
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http://dx.doi.org/10.1111/cas.14297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060471PMC
March 2020

Medication prescription in older people receiving home medical care services.

Geriatr Gerontol Int 2019 Dec;19(12):1292-1293

Department of Home Care Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

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http://dx.doi.org/10.1111/ggi.13793DOI Listing
December 2019

Importance of gastric cancer for the diagnosis and surveillance of Japanese Lynch syndrome patients.

J Hum Genet 2019 Dec 7;64(12):1187-1194. Epub 2019 Oct 7.

Division of Clinical Genome Research, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

Lynch syndrome (LS) is an autosomal dominantly inherited disease predisposed to not only colorectal cancer but also other LS-related tumors. Although the clinical and genetic characteristics of LS in Western countries have been well characterized, the information of Japanese LS is limited. As a collaborative study of Japanese Society for Cancer of the Colon and Rectum (JSCCR), we registered colorectal cancer (CRC) patients who fulfilled the modified Amsterdam II criteria including gastric cancer as an LS-related tumor. Among 4030 CRC patients initially registered in this project, 85 patients (2.1%) fulfilled the modified criteria. An additional 26 patients who met the same criteria were enrolled in the analysis. We analyzed three major responsible genes, MLH1, MSH2, and MSH6 by direct sequencing, and further performed multiplex ligation-dependent probe amplification for MLH1 and MSH2. Consequently, we identified pathogenic variants in 64 of the 111 patients comprising of 34 patients in MLH1, 28 in MSH2, and 2 in MSH6. It is of note that large structural alterations were found in 17 patients. Among the 64 patients, 11 patients would not have been enrolled in the analysis if gastric cancer were not included in the modified criteria. In addition, 10 of the 64 variant carriers (15.6%) had medical history of gastric cancer. Furthermore, the standardized incidence ratio of gastric cancer in the LS patients to the Japanese population is estimated to be as high as 20.2. These data underscore the importance of gastric cancer in the diagnosis and healthcare of Japanese LS patients.
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http://dx.doi.org/10.1038/s10038-019-0674-5DOI Listing
December 2019

Anti-apoptotic effect by the suppression of IRF1 as a downstream of Wnt/β-catenin signaling in colorectal cancer cells.

Oncogene 2019 08 10;38(32):6051-6064. Epub 2019 Jul 10.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, the University of Tokyo, Tokyo, 108-8639, Japan.

Impaired Wnt signaling pathway plays a crucial role in the development of colorectal cancer through activation of the β-catenin/TCF7L2 complex. Although genes upregulated by Wnt/β-catenin signaling have been intensively studied, the roles of downregulated genes are poorly understood. Previously, we reported that interferon-induced proteins with tetratricopeptide repeats 2 (IFIT2) was downregulated by the Wnt/β-catenin signaling, and that the suppressed expression of IFIT2 conferred antiapoptotic property to colorectal cancer (CRC) cells. However, the mechanisms underlying how Wnt/β-catenin signaling regulates IFIT2 remain to be elucidated. In this study, we have uncovered that the expression of IFIT2 is induced by IRF1, which is negatively regulated by the Wnt/β-catenin signaling. In addition, we found that downregulation of IRF1 is mediated by its degradation through the ubiquitination-proteasome pathway, and that decreased activity of a deubiquitinase complex containing USP1 and UAF1 is involved in the degradation of IRF1 by Wnt/β-catenin signaling. These data should provide better understanding of the Wnt signaling pathway and human carcinogenesis.
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http://dx.doi.org/10.1038/s41388-019-0856-9DOI Listing
August 2019

Development of an MSI-positive colon tumor with aberrant DNA methylation in a PPAP patient.

J Hum Genet 2019 Aug 14;64(8):729-740. Epub 2019 May 14.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

Polymerase proofreading-associated polyposis (PPAP) is a disease caused by germline variations in the POLE and POLD1 genes that encode catalytic subunits of DNA polymerases. Studies of cancer genomes have identified somatic mutations in these genes, suggesting the importance of polymerase proofreading of DNA replication in suppressing tumorigenesis. Here, we identified a germline frameshift variation in the POLE gene (c.4191_4192delCT, p.Tyr1398*) in a case with multiple adenomatous polyps and three synchronous colon cancers. Interestingly, one of the colon cancers showed microsatellite instability-high (MSI-H) and another microsatellite stable. Immunohistochemical staining revealed that the MSI-H tumor cells lost the expression of MLH1 protein. Whole genome sequencing of the MSI-H tumor did not find pathogenic somatic mutations in mismatch repair genes but found frameshift mutations in the TET genes that catalyze 5-methylcytosine hydroxylation. Bisulfite sequencing of the tumor corroborated an increase in the number of hypermethylated regions including the MLH1 promoter. These data indicate that PPAP patients might develop MSI-positive tumors through epigenetic silencing of MLH1. These findings will contribute to comprehensive understanding of the molecular basis of tumors that involve deficiency of proofreading activity of DNA polymerases.
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http://dx.doi.org/10.1038/s10038-019-0611-7DOI Listing
August 2019

Fbxo22-mediated KDM4B degradation determines selective estrogen receptor modulator activity in breast cancer.

J Clin Invest 2018 12 12;128(12):5603-5619. Epub 2018 Nov 12.

Division of Cancer Cell Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

The agonistic/antagonistic biocharacter of selective estrogen receptor modulators (SERMs) can have therapeutic advantages, particularly in the case of premenopausal breast cancers. Although the contradictory effects of these modulators have been studied in terms of crosstalk between the estrogen receptor α (ER) and coactivator dynamics and growth factor signaling, the molecular basis of these mechanisms is still obscure. We identify a series of regulatory mechanisms controlling cofactor dynamics on ER and SERM function, whose activities require F-box protein 22 (Fbxo22). Skp1, Cullin1, F-box-containing complex (SCFFbxo22) ubiquitylated lysine demethylase 4B (KDM4B) complexed with tamoxifen-bound (TAM-bound) ER, whose degradation released steroid receptor coactivator (SRC) from ER. Depletion of Fbxo22 resulted in ER-dependent transcriptional activation via transactivation function 1 (AF1) function, even in the presence of SERMs. In living cells, TAM released SRC and KDM4B from ER in a Fbxo22-dependent manner. SRC release by TAM required Fbxo22 on almost all ER-SRC-bound enhancers and promoters. TAM failed to prevent the growth of Fbxo22-depleted, ER-positive breast cancers both in vitro and in vivo. Clinically, a low level of Fbxo22 in tumor tissues predicted a poorer outcome in ER-positive/human epidermal growth factor receptor type 2-negative (HER2-negative) breast cancers with high hazard ratios, independently of other markers such as Ki-67 and node status. We propose that the level of Fbxo22 in tumor tissues defines a new subclass of ER-positive breast cancers for which SCFFbxo22-mediated KDM4B degradation in patients can be a therapeutic target for the next generation of SERMs.
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http://dx.doi.org/10.1172/JCI121679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264734PMC
December 2018

Efficacy of liquid-based genetic diagnosis of endometrial cancer.

Cancer Sci 2018 Dec 26;109(12):4025-4032. Epub 2018 Oct 26.

Department of Obstetrics and Gynecology, Sapporo Medical University, Sapporo, Japan.

Although liquid-based cytology (LBC) has increased the sensitivity of cytological diagnosis of endometrial cancer (EC) compared with conventional smear cytology, the sensitivity of LBC for the detection of EC is between 70% and 96% and remains unsatisfactory. In the present study, we compared the efficacy of LBC with liquid-based genetic diagnosis (LBGDx) by amplicon sequencing of five genes including PTEN, PIK3CA, CTNNB1, KRAS, and TP53 in 48 LBC subjects who underwent endometrial screening. Consequently, LBC classified 15 samples as "positive or suspicious for malignancy" and the 15 were later confirmed as EC. However, LBC failed to identify five cases who were diagnosed as EC by additional transvaginal ultrasound and endometrial curettage, indicating that the sensitivity of cytology alone was 75% (15/20). LBGDx identified 11 pathogenic PTEN variants in 10 subjects, six PIK3CA variants in nine, three CTNNB1 variants in five, two KRAS variants in four, and three TP53 variants in three. Collectively, at least one pathogenic variant was identified in 19 subjects, which included 17 EC (15 endometrioid carcinoma and 2 endometrial carcinosarcomas), and one cervical adenocarcinoma. However, LBGDx did not identify any pathogenic mutations in three of the 20 EC, indicating that the sensitivity of LBGDx alone was 85% (17/20). Although five EC were negative for malignancy by LBC and three were negative for pathogenic mutations by LBGDx, the combination of LBC and LBGDx would successfully diagnose all 20 EC. These data suggested that LBGDx is a useful strategy to improve the sensitivity of screening of EC by LBC.
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http://dx.doi.org/10.1111/cas.13819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272085PMC
December 2018

Regulation of tankyrase activity by a catalytic domain dimer interface.

Biochem Biophys Res Commun 2018 09 26;503(3):1780-1785. Epub 2018 Jul 26.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Tankyrases (TNKS and TNKS2) are enzymes that catalyze poly-ADP-ribosylation (PARsylation) of their target proteins. Tankyrase-mediated PARsylation plays critical regulatory roles in cell signaling, particularly in the Wnt/β-catenin pathway. The sterile alpha motif (SAM) domain in tankyrases mediates their oligomerization, which is essential for tankyrase function. The oligomerization regulates the catalytic activity of tankyrases, but the underlying mechanism is unclear. Our analyses of crystal structures of the tankyrase catalytic domain suggest that formation of a head-to-head dimer regulates the catalytic activity. Our activity assays show that residues in the catalytic domain dimer interface are important for the PARsylation activity of tankyrases both in solution and cells. The dimer is weak and may only form in the context of the SAM domain-mediated oligomers of tankyrases, consistent with the dependence of the tankyrase activity on the SAM domain.
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http://dx.doi.org/10.1016/j.bbrc.2018.07.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6132069PMC
September 2018

EGF Receptor and mTORC1 Are Novel Therapeutic Targets in Nonseminomatous Germ Cell Tumors.

Mol Cancer Ther 2018 05 26;17(5):1079-1089. Epub 2018 Feb 26.

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas.

Germ cell tumors (GCT) are malignant tumors that arise from pluripotent embryonic germ cells and occur in children and young adults. GCTs are treated with cisplatin-based regimens which, while overall effective, fail to cure all patients and cause significant adverse late effects. The seminoma and nonseminoma forms of GCT exhibit distinct differentiation states, clinical behavior, and response to treatment; however, the molecular mechanisms of GCT differentiation are not fully understood. We tested whether the activity of the mTORC1 and MAPK pathways were differentially active in the two classes of GCT. Here we show that nonseminomatous germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma) from both children and adults display activation of the mTORC1 pathway, while seminomas do not. In seminomas, high levels of REDD1 may negatively regulate mTORC1 activity. In NSGCTs, on the other hand, EGF and FGF2 ligands can stimulate mTORC1 and MAPK signaling, and members of the EGF and FGF receptor families are more highly expressed. Finally, proliferation of NSGCT cells and is significantly inhibited by combined treatment with the clinically available agents erlotinib and rapamycin, which target EGFR and mTORC1 signaling, respectively. These results provide an understanding of the signaling network that drives GCT growth and a rationale for therapeutic targeting of GCTs with agents that antagonize the EGFR and mTORC1 pathways. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932259PMC
May 2018

Establishment and analysis of a novel mouse line carrying a conditional knockin allele of a cancer-specific FBXW7 mutation.

Sci Rep 2018 01 31;8(1):2021. Epub 2018 Jan 31.

Division of Clinical Genome Research, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

F-box and WD40 domain protein 7 (FBXW7) is a component of the SKP1-CUL1-F-box protein (SCF) complex that mediates the ubiquitination of diverse oncogenic target proteins. The exploration of FBXW7 mutations in human primary cancer has revealed three mutation hotspots at conserved arginine residues (Arg, Arg, and Arg) in the WD40 domain, which are critical for substrate recognition. To study the function of human FBXW7 , the most frequent mutation in human malignancies, we generated a novel conditional knockin mouse line of murine Fbxw7 corresponding to human FBXW7 . Systemic heterozygous knockin of the Fbxw7 mutation resulted in perinatal lethality due to defects in lung development, and occasionally caused an eyes-open at birth phenotype and cleft palate. Furthermore, mice carrying liver-specific heterozygous and homozygous Fbxw7 alleles cooperated with an oncogenic Kras mutation to exhibit bile duct hyperplasia within 8 months of birth and cholangiocarcinoma-like lesions within 8 weeks of birth, respectively. In addition, the substrates affected by the mutant Fbxw7 differed between the embryos, embryonic fibroblasts, and adult liver. This novel conditional knockin Fbxw7 line should be useful to gain a more profound understanding of carcinogenesis associated with mutation of FBXW7.
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http://dx.doi.org/10.1038/s41598-018-19769-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792591PMC
January 2018

Longitudinal changes of elderly patients' wishes about artificial nutrition and hydration during end-of-life care: A pilot study in a single hospital.

Geriatr Gerontol Int 2017 12;17(12):2635-2637

Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

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http://dx.doi.org/10.1111/ggi.13140DOI Listing
December 2017

Decreased expression of interferon-induced protein 2 (IFIT2) by Wnt/β-catenin signaling confers anti-apoptotic properties to colorectal cancer cells.

Oncotarget 2017 Nov 26;8(59):100176-100186. Epub 2017 Oct 26.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

Impaired Wnt signaling pathway plays a crucial role in the development of colorectal cancer through activation of the β-catenin/TCF7L2 complex. Although genes up-regulated by Wnt/β-catenin signaling have been intensively studied, the roles of down-regulated genes are poorly understood. In this study, we explored a global gene expression of colorectal cancer cells transfected with β-catenin siRNAs or a dominant negative form of TCF7L2 (dnTCF7L2), and identified a set of genes down-regulated by Wnt/β-catenin signaling. Among the genes, we focused here on , a gene encoding interferon-induced protein with tetratricopeptide repeats. A reporter assay using plasmids containing a 5'-flanking region of the gene showed that the reporter activity was enhanced by either transduction of β-catenin siRNA or dnTCF7L2, suggesting that the region is involved in the transcriptional regulation as a downstream of the β-catenin/TCF7L2 complex. Consistent with this result, expression of was significantly lower in colorectal cancer tissues than that in normal tissues. Exogenous IFIT2 expression decreased cell proliferation and increased apoptosis of colorectal cancer cells. These data suggested that the down-regulation of IFIT2 by Wnt/β-catenin signaling may play a vital role in human colorectal carcinogenesis through the suppression of apoptosis.
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http://dx.doi.org/10.18632/oncotarget.22122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725011PMC
November 2017

Requirement of glycosylation machinery in TLR responses revealed by CRISPR/Cas9 screening.

Int Immunol 2017 08;29(8):347-355

Division of Innate Immunity, Department of Microbiology and Immunology.

The Toll family of receptors sense microbial products and activate a defense response. The molecular machinery required for the TLR response is not yet fully understood. In the present study, we used a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CAS9 screening system to study TLR responses. We employed a cell line expressing TLR with an NF-κB-driven GFP reporter. The cell line was transduced with a guide RNA (gRNA) library and stimulated with TLR ligands. The cells impaired in GFP induction were sorted, and gRNAs were sequenced. Identified genes were ranked according to the count of sequence reads and the number of gRNA target sites. The screening system worked correctly, as molecules that were already known to be required for the TLR response were identified by the screening. Furthermore, this system revealed that the oligosaccharide transferase complex (OSTC) mediating co-translational glycosylation was required for TLR5, 7 and 9 responses. Protein expression of TLR5, but not an irrelevant molecule (CD44), was abolished by the lack of OSTC, suggesting the essential role of glycosylation in TLR5 protein stability. These results demonstrate that the screening system established here is able to reveal molecular mechanisms underlying the TLR response.
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http://dx.doi.org/10.1093/intimm/dxx044DOI Listing
August 2017

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

Biotechnol Bioeng 2017 12 29;114(12):2868-2882. Epub 2017 Aug 29.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway.
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http://dx.doi.org/10.1002/bit.26394DOI Listing
December 2017

Genetic alterations in Japanese extrahepatic biliary tract cancer.

Oncol Lett 2017 Jul 22;14(1):877-884. Epub 2017 May 22.

Division of Clinical Genome Research, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Biliary tract cancer (BTC) is one of the most devastating types of malignant neoplasms worldwide. However, the mechanisms underlying the development and progression of BTC remain unresolved. BTC includes extrahepatic bile duct carcinoma (EBDC), gallbladder carcinoma (GBC) and ampulla of Vater carcinoma (AVC), named according to the location of the tumor. Although genetic alterations of intrahepatic cholangiocarcinoma have been investigated, those of EBDC, GBC and AVC have not yet been fully understood. The present study analyzed somatic mutations of 50 cancer-associated genes in 27 Japanese BTC cells, including: 11 EBDC, 14 GBC and 2 AVC. Next-generation sequencing using an Ion AmpliSeq Cancer Panel identified a total of 44 somatic mutations across 14 cancer-associated genes. Among the 44 mutations, 42 were judged as pathological mutations. Frequent mutations were identified in tumor protein 53 () (14/27), SMAD family member 4 () (6/27), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α () (6/27), and Kirsten rat sarcoma () (6/27); no significant differences were identified between EBDC and GBC tissues. Notably, the frequency of the mutation was higher when compared with previous reports. This result may suggest that the activation of the PIK3CA-protein kinase B signaling pathway, in addition to the abrogation of p53, SMAD4 and RAS mitogen-activated protein kinase may have a crucial role in the carcinogenesis of Japanese BTC. These findings may be useful for the development of personalized therapies for BTC.
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http://dx.doi.org/10.3892/ol.2017.6224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494721PMC
July 2017

Altered expression of microRNAs in patients with pouchitis after restorative proctocolectomy.

Surg Today 2017 Dec 9;47(12):1484-1491. Epub 2017 Jun 9.

Department of Surgery, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

Background And Purpose: Pouchitis is the most common long-term complication of restorative proctocolectomy with ileal pouch-anal anastomosis. We investigated alterations in the expression of microRNAs, noncoding RNAs that act as potent negative regulators of gene expression, in pouchitis.

Methods: The subjects of this study were 16 patients with diagnosed pouchitis and 48 patients without pouchitis after restorative proctocolectomy, performed for ulcerative colitis. Total RNA was extracted from biopsies and microRNAs were quantified using a real-time polymerase chain reaction.

Results: The expression of microRNA 21 and 223 was higher, whereas that of microRNA 192 and 196a was lower, in the inflamed mucosa from the pouchitis patients than in the mucosa from the non-pouchitis patients. The levels of 14 microRNAs were significantly lower in the mucosa from the pouchitis patients, than in the non-inflamed proximal ileal mucosal samples. The expression of microRNA 192 was remarkably reduced in pouchitis. A significant negative correlation was found between microRNA 192 and interleukin 17 receptor A mRNA levels.

Conclusions: Significant alteration in miRNA expression in line with inflammatory bowel disease was evident in the mucosa from the pouchitis patients. Interleukin 17 receptor A may be involved in the pathogenesis of pouchitis through the downregulation of microRNA 192.
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http://dx.doi.org/10.1007/s00595-017-1550-6DOI Listing
December 2017

Identification of FERM domain-containing protein 5 as a novel target of β-catenin/TCF7L2 complex.

Cancer Sci 2017 Apr 20;108(4):612-619. Epub 2017 Apr 20.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Deregulation of the canonical Wnt signaling pathway plays an important role in human tumorigenesis through the accumulation of β-catenin and subsequent transactivation of TCF7L2. Although some of the consequences associated with the accumulated β-catenin have been clarified, the comprehensive effect of activated β-catenin/TCF7L2 transcriptional complex on tumorigenesis remains to be elucidated. To understand the precise molecular mechanisms underlying colorectal cancer, we searched for genes regulated by the complex in colorectal tumors. We performed expression profile analysis of HCT116 and SW480 colon cancer cells treated with β-catenin siRNAs, and ChIP-sequencing using anti-TCF7L2 antibody. Combination of these data with public microarray data of LS174 cells with a dominant-negative form of TCF7L2 identified a total of 11 candidate genes. In this paper, we focused on FERM domain-containing protein 5 (FRMD5), and confirmed that it is regulated by both β-catenin and TCF7L2. An additional reporter assay disclosed that a region in intron1 transcriptionally regulated the expression of FRMD5. ChIP assay also corroborated that TCF7L2 associates with this region. These data suggested that FRMD5 is a novel direct target of the β-catenin/TCF7L2 complex.
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http://dx.doi.org/10.1111/cas.13174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406541PMC
April 2017