Publications by authors named "Hiroto Katoh"

37 Publications

Focal adhesion ribonucleoprotein complex proteins are major humoral cancer antigens and targets in autoimmune diseases.

Commun Biol 2020 10 16;3(1):588. Epub 2020 Oct 16.

Department of Preventive Medicine, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.

Despite the accumulating evidences of the significance of humoral cancer immunity, its molecular mechanisms have largely remained elusive. Here we show that B-cell repertoire sequencing of 102 clinical gastric cancers and molecular biological analyses unexpectedly reveal that the major humoral cancer antigens are not case-specific neo-antigens but are rather commonly identified as ribonucleoproteins (RNPs) in the focal adhesion complex. These common antigens are shared as autoantigens with multiple autoimmune diseases, suggesting a direct molecular link between cancer- and auto-immunity on the focal adhesion RNP complex. This complex is partially exposed to the outside of cancer cell surfaces, which directly evokes humoral immunity and enables functional bindings of antibodies to cancer cell surfaces in physiological conditions. These findings shed light on humoral cancer immunity in that it commonly targets cellular components fundamental for cytoskeletal integrity and cell movement, pointing to a novel modality of immunotherapy using humoral immunological reactions to cancers.
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http://dx.doi.org/10.1038/s42003-020-01305-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567837PMC
October 2020

Clinicopathological and molecular analyses of linearly expanded epithelial cells with β-catenin alterations, "β-catenin signature", in the normal fallopian tube.

Histopathology 2020 Dec 17;77(6):880-889. Epub 2020 Oct 17.

Department of Cellular and Organ Pathology, Graduate School of Medicine, Akita University, Akita, Japan.

Aims: Recent advances in next-generation sequencing have made it clear that clonal expansion of cells harbouring driver gene mutations occurs in physiologically normal epithelium. Molecular analysis of tubal epithelium has been almost exclusively confined to the TP53 pathway, which is involved in serous carcinogenesis. Other oncogenic events have not been explored in detail. Here, we report the linear expansion of fallopian tubal epithelial cells exhibiting an altered β-catenin profile (β-catenin signature). Through molecular analyses, we determined the incidence and clinicopathological significance of β-catenin signatures.

Methods And Results: We evaluated 64 specimens of surgically removed bilateral fallopian tubes. Thirty-three β-catenin signatures were identified in 13 cases (20.3%); these patients were significantly younger than those without β-catenin signatures (median ages of 44 and 57 years, respectively, P = 0.0317). No correlation between β-catenin signature and any clinical factor was observed. CTNNB1 mutations were detected in three of eight β-catenin signatures when tissues were microdissected and subjected to Sanger sequencing in two representative cases.

Conclusions: This is the first report of the CTNNB1 mutation in clusters of morphologically bland tubal epithelial cells. The results of this study indicate that β-catenin signatures are common, and they may be a part of diverse molecular alterations occurring in normal tubal epithelium.
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http://dx.doi.org/10.1111/his.14227DOI Listing
December 2020

Accumulation of Molecular Aberrations Distinctive to Hepatocellular Carcinoma Progression.

Cancer Res 2020 09 8;80(18):3810-3819. Epub 2020 Jul 8.

Genome Science Division, RCAST, University of Tokyo, Tokyo, Japan.

Cancer develops through the accumulation of genetic and epigenetic aberrations. To identify sequential molecular alterations that occur during the development of hepatocellular carcinoma (HCC), we compared 52 early and 108 overt HCC samples by genome sequencing. Gene mutations in the p53/RB1 pathway, WNT pathway, MLL protein family, SWI/SNF complexes, and AKT/PI3K pathway were common in HCC. In the early phase of all entities, was the most frequently upregulated gene owing to diverse mechanisms. Despite frequent somatic mutations in driver genes, including and , early HCC was a separate molecular entity from overt HCC, as each had a distinct expression profile. Notably, WNT target genes were not activated in early HCC regardless of mutation status because β-catenin did not translocate into the nucleus due to the E-cadherin/β-catenin complex at the membrane. Conversely, WNT targets were definitively upregulated in overt HCC, with mutation associated with downregulation of and hypomethylation of CpG islands in target genes. Similarly, cell-cycle genes downstream of the p53/RB pathway were upregulated only in overt HCC, with or gene mutations associated with chromosomal deletion of 4q or 16q. HCC was epigenetically distinguished into four subclasses: normal-like methylation, global-hypomethylation (favorable prognosis), stem-like methylation (poor prognosis), and CpG island methylation. These methylation statuses were globally maintained through HCC progression. Collectively, these data show that as HCC progresses, additional molecular events exclusive of driver gene mutations cooperatively contribute to transcriptional activation of downstream targets according to methylation status. SIGNIFICANCE: In addition to driver gene mutations in the WNT and p53 pathways, further molecular events are required for aberrant transcriptional activation of these pathways as HCC progresses.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0225DOI Listing
September 2020

Defined lifestyle and germline factors predispose Asian populations to gastric cancer.

Sci Adv 2020 May 6;6(19):eaav9778. Epub 2020 May 6.

Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan.

Germline and environmental effects on the development of gastric cancers (GC) and their ethnic differences have been poorly understood. Here, we performed genomic-scale trans-ethnic analysis of 531 GCs (319 Asian and 212 non-Asians). There was one distinct GC subclass with clear alcohol-associated mutation signature and strong Asian specificity, almost all of which were attributable to alcohol intake behavior, smoking habit, and Asian-specific defective allele. Alcohol-related GCs have low mutation burden and characteristic immunological profiles. In addition, we found frequent (7.4%) germline variants among Japanese GCs, most of which were attributed to a few recurrent single-nucleotide variants shared by Japanese and Koreans, suggesting the existence of common ancestral events among East Asians. Specifically, approximately one-fifth of diffuse-type GCs were attributable to the combination of alcohol intake and defective allele or to variants. These results revealed uncharacterized impacts of germline variants and lifestyles in the high incidence areas.
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http://dx.doi.org/10.1126/sciadv.aav9778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202881PMC
May 2020

Post-mortem Plasma Cell-Free DNA Sequencing: Proof-of-Concept Study for the "Liquid Autopsy".

Sci Rep 2020 02 7;10(1):2120. Epub 2020 Feb 7.

Department of Cellular and Organ Pathology, Graduate School of Medicine, Akita University, Akita, Japan.

Recent genomic studies on cancer tissues obtained during rapid autopsy have provided insights into the clonal evolution and heterogeneity of cancer. However, post-mortem blood has not been subjected to genetic analyses in relation to cancer. We first confirmed that substantial quantities of cell-free DNA were present in the post-mortem plasma of 12 autopsy cases. Then, we focused on a pilot case of prostate cancer with multiple metastases for genetic analyses. Whole-exome sequencing of post-mortem plasma-derived cell-free DNA and eight frozen metastatic cancer tissues collected during rapid autopsy was performed, and compared their mutational statuses. The post-mortem plasma cell-free DNA was successfully sequenced and 344 mutations were identified. Of these, 160 were detected in at least one of the metastases. Further, 99% of the mutations shared by all metastases were present in the plasma. Sanger sequencing of 30 additional formalin-fixed metastases enabled us to map the clones harboring mutations initially detected only in the plasma. In conclusion, post-mortem blood, which is usually disposed of during conventional autopsies, can provide valuable data if sequenced in detail, especially regarding cancer heterogeneity. Furthermore, post-mortem plasma cell-free DNA sequencing (liquid autopsy) can be a novel platform for cancer research and a tool for genomic pathology.
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http://dx.doi.org/10.1038/s41598-020-59193-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005783PMC
February 2020

Capturing the differences between humoral immunity in the normal and tumor environments from repertoire-seq of B-cell receptors using supervised machine learning.

BMC Bioinformatics 2019 May 28;20(1):267. Epub 2019 May 28.

Department of Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan.

Background: The recent success of immunotherapy in treating tumors has attracted increasing interest in research related to the adaptive immune system in the tumor microenvironment. Recent advances in next-generation sequencing technology enabled the sequencing of whole T-cell receptors (TCRs) and B-cell receptors (BCRs)/immunoglobulins (Igs) in the tumor microenvironment. Since BCRs/Igs in tumor tissues have high affinities for tumor-specific antigens, the patterns of their amino acid sequences and other sequence-independent features such as the number of somatic hypermutations (SHMs) may differ between the normal and tumor microenvironments. However, given the high diversity of BCRs/Igs and the rarity of recurrent sequences among individuals, it is far more difficult to capture such differences in BCR/Ig sequences than in TCR sequences. The aim of this study was to explore the possibility of discriminating BCRs/Igs in tumor and in normal tissues, by capturing these differences using supervised machine learning methods applied to RNA sequences of BCRs/Igs.

Results: RNA sequences of BCRs/Igs were obtained from matched normal and tumor specimens from 90 gastric cancer patients. BCR/Ig-features obtained in Rep-Seq were used to classify individual BCR/Ig sequences into normal or tumor classes. Different machine learning models using various features were constructed as well as gradient boosting machine (GBM) classifier combining these models. The results demonstrated that BCR/Ig sequences between normal and tumor microenvironments exhibit their differences. Next, by using a GBM trained to classify individual BCR/Ig sequences, we tried to classify sets of BCR/Ig sequences into normal or tumor classes. As a result, an area under the curve (AUC) value of 0.826 was achieved, suggesting that BCR/Ig repertoires have distinct sequence-level features in normal and tumor tissues.

Conclusions: To the best of our knowledge, this is the first study to show that BCR/Ig sequences derived from tumor and normal tissues have globally distinct patterns, and that these tissues can be effectively differentiated using BCR/Ig repertoires.
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http://dx.doi.org/10.1186/s12859-019-2853-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6537402PMC
May 2019

Molecular Taxonomy of Interstitial Cystitis/Bladder Pain Syndrome Based on Whole Transcriptome Profiling by Next-Generation RNA Sequencing of Bladder Mucosal Biopsies.

J Urol 2019 08 8;202(2):290-300. Epub 2019 Jul 8.

Japanese Red Cross Medical Center , Tokyo.

Purpose: We systematically characterized gene expression, inflammation and neovascularization in patients with interstitial cystitis/bladder pain syndrome to obtain biological evidence supporting diagnosis and classification.

Materials And Methods: We sequenced RNA obtained from bladder mucosal biopsies of 33 patients with 3 subtypes of interstitial cystitis/bladder pain syndrome, including Hunner lesions in 12, no Hunner lesions in 11 but with glomerulations and neither Hunner lesions nor glomerulations in 10, and 9 controls. Differentially expressed genes of each subtype were searched to identify subtype specific biological pathways and candidate genes important for pathogenesis. Candidate genes were validated by quantitative polymerase chain reaction and immunohistochemistry. Digital immunohistochemical quantification was performed to assess subepithelial lymphoplasmacytic cell and microvessel density. Relationships between candidate gene over expression and symptom severity were explored.

Results: Patients with Hunner lesions showed a distinct gene expression profile associated with significant up-regulation of biological processes involving immune responses and infection, and an increase in subepithelial lymphoplasmacytic cell and microvessel density. Over expression of 2 candidate genes, VEGF and BAFF, correlated with symptom severity. Meanwhile, the gene expression profiles of patients with the 2 subtypes without Hunner lesions were similar to those of controls. No difference in biological pathways or subepithelial lymphoplasmacytic cell and microvessel density were detected between these 2 subtypes and controls.

Conclusions: Interstitial cystitis/bladder pain syndrome with Hunner lesions shows distinct genomic and histological features associated with immune responses and infection. In addition, VEGF and BAFF are potential disease biomarkers and therapeutic targets. This subtype should be considered separate from the syndrome.
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http://dx.doi.org/10.1097/JU.0000000000000234DOI Listing
August 2019

Functional genomics screen with pooled shRNA library and gene expression profiling with extracts of identify potential pathways for therapeutic targets in head and neck squamous cell carcinoma.

PeerJ 2019 1;7:e6464. Epub 2019 Mar 1.

Ganit Labs, Bio-IT Centre, Institute of Bioinformatics and Applied Biotechnology, Bangalore, India.

Tumor suppression by the extracts of (neem) works via anti-proliferation, cell cycle arrest, and apoptosis, demonstrated previously using cancer cell lines and live animal models. However, very little is known about the molecular targets and pathways that neem extracts and their associated compounds act through. Here, we address this using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell lines treated with crude neem leaf extracts, known for their anti-tumorigenic activity. We analyzed differences in global clonal sizes of the shRNA-infected cells cultured under no treatment and treatment with neem leaf extract conditions, assayed using next-generation sequencing. We found 225 genes affected the cancer cell growth in the shRNA-infected cells treated with neem extract. Pathway enrichment analyses of whole-genome gene expression data from cells temporally treated with neem extract revealed important roles played by the TGF-β pathway and -related gene network. Our results indicate that neem extract affects various important molecular signaling pathways in head and neck cancer cells, some of which may be therapeutic targets for this devastating tumor.
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http://dx.doi.org/10.7717/peerj.6464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398373PMC
March 2019

EVI1 expression is associated with aggressive behavior in intrahepatic cholangiocarcinoma.

Virchows Arch 2019 Jan 23;474(1):39-46. Epub 2018 Oct 23.

Department of Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Ecotropic virus integration site 1 protein homolog (EVI1), a well-known oncogenic transcriptional factor of hematopoietic cells, contributes to pancreatic cancer oncogenicity through increased expression of KRAS. Because EVI1 was upregulated in cholangiocarcinoma by referring The Cancer Genome Atlas, we investigated the importance of EVI1 in intrahepatic cholangiocarcinoma (ICC) which has been regarded as a heterogeneous group of cancers. Immunohistochemical analysis results demonstrated that EVI1 was overexpressed in about half of ICC (53/101, 52.5%). Moreover, all intraductal papillary neoplasms of the bile duct cases expressed EVI1 regardless of histological grading and subtypes such as gastric, intestinal, pancreatobiliary, or oncocytic (20/20, 100%). EVI1-positive ICC showed higher frequencies of aggressive pathological indicators such as periductal infiltrative growth (p = 0.022), hilar invasion (p = 0.041), advanced UICC stage (p = 0.026), major vascular invasion (p = 0.026), and perineural invasion (p = 0.007) than EVI1-negative ICC. Patients with EVI1-positive ICC showed worse overall survival and recurrence-free survival in all resected cases and in curative resected cases. Recently, we proposed type 1/2 (large/small duct types) classification of ICC based on mucin productivity and immunophenotypes (S100P, N-cadherin, and NCAM). Type 1 predominantly consisted of EVI1-positive ICC (33/42 cases, 79%), and the frequency was significantly higher than type 2 (18/55 cases, 32.7%) (p < 0.0001). EVI1-positive ICC was likely to express stomach-specific claudin CLDN18 (correlation coefficient r = 0.55373) and mucin MUC5AC (r = 0.42718). EVI1-positive ICC is an aggressive ICC showing both large-duct and/or gastric phenotypes. Consequently, a transcriptional factor EVI1 is associated with aggressive behavior in ICC and can be a therapeutic target molecule, while EVI1 might be a key molecule for the development of intraductal papillary neoplasms of the bile duct.
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http://dx.doi.org/10.1007/s00428-018-2476-0DOI Listing
January 2019

Frequent fusion in highly metastatic diffuse-type gastric cancer with relatively early onset.

Oncotarget 2018 Jun 29;9(50):29336-29350. Epub 2018 Jun 29.

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

CLDN18-ARHGAP26/6 fusions have been identified in gastric cancers, with a predominance in diffuse-type gastric cancers (DGCs). Although experiments have suggested an oncogenic role for fusions, the exact frequencies and clinicopathological characteristics of the fusion-positive cases are poorly understood. We analyzed 254 cases of gastric cancer (172 diffuse-type and 82 intestinal-type) using RT-PCR and FISH, and also analyzed TCGA transcriptome datasets to identify genes that are related to the aggressive behaviors of fusion-positive cancers. Our assays identified 26 fusion-positive cases, 22 of which were DGCs (22/172, 12.8%). Unlike fusion-negative DGCs, almost all fusion-positive DGCs retained E-cadherin expression (P = 0.036). Fusion-positive DGCs also showed a higher prevalence of lymphatic and distant organ metastases, and these trends were only significant in the younger age group (< 60 years). In this group, the majority of cases with distant organ metastases (4 of 6 cases) were fusion-positive, and the multivariate regression analysis revealed that fusion status was an independent predictive marker for distant organ metastases (P = 0.002). In the TCGA dataset analysis, carbonic anhydrase 9 was postulated to be a potential modulator of the age-specific effects of the fusion protein, compatible with the immunohistochemical analysis of our cohort. Therefore, CLDN18-ARHGAP26/6 fusion-positive DGCs are considered biologically distinct entities that will require more advanced therapeutic options.
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http://dx.doi.org/10.18632/oncotarget.25464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047683PMC
June 2018

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses.

Pathol Int 2018 Feb;68(2):63-90

Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine.
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http://dx.doi.org/10.1111/pin.12631DOI Listing
February 2018

EVI1 modulates oncogenic role of GPC1 in pancreatic carcinogenesis.

Oncotarget 2017 Nov 1;8(59):99552-99566. Epub 2017 Sep 1.

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

Glypican-1 (GPC1) protein in exosomes was recently identified as a biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analyses and assays were conducted to assess the usefulness of GPC1 as a PDAC biomarker, to reveal the biological role of GPC1 in pancreatic carcinogenesis, and to ascertain the regulation mechanism of GPC1. An aberrant overexpression of GPC1 protein which is usually absent in normal pancreatic duct, was a widespread marker across the full spectrum of human PDAC precursors, PDAC, and pancreatic cancerous stroma. In intraductal papillary-mucinous neoplasms (IPMNs), GPC1 tended to be positive in gastric-type IPMN. mutations were found in all GPC1-positive IPMN cases and in one-third of GPC1-negative IPMN cases. In pancreatic cell lines, GPC1 depletion caused remarkable inhibition of cell growth and migration, suggesting its oncogenic roles. GPC1 depletion upregulated the molecules associated with cell cycle arrest in pancreatic cell lines. Furthermore, KRAS and ecotropic viral integration site 1 (EVI1) oncoprotein upregulated GPC1 expression. In a clinical cohort, GPC1 overexpression was not correlated with pancreatic cancer prognosis. Taken together, these findings suggest the necessity of establishing a threshold of GPC1 value for detecting pancreatic malignancy because GPC1 is overexpressed even in low-grade PDAC precursors which do not always become malignant. Our study also reveals a new aspect of pancreatic carcinogenesis: KRAS and EVI1, two important molecules in early phases of pancreatic carcinogenesis, positively regulate GPC1 expression and likely promote pancreatic carcinogenesis.
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http://dx.doi.org/10.18632/oncotarget.20601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725114PMC
November 2017

Immunogenetic Profiling for Gastric Cancers Identifies Sulfated Glycosaminoglycans as Major and Functional B Cell Antigens in Human Malignancies.

Cell Rep 2017 08;20(5):1073-1087

Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, 113-8510 Tokyo, Japan. Electronic address:

Recent successes in tumor immunotherapies have highlighted the importance of tumor immunity. However, most of the work conducted to date has been on T cell immunity, while the role of B cell immunity in cancer remains more elusive. In this study, immunogenetic repertoire profiling for tumor-infiltrating B and T cells in gastric cancers was carried out to help reveal the architecture of B cell immunity in cancer. Humoral immunity in cancer was shown to involve oligoclonal expansions of tumor-specific and private B cell repertoires. We find that B cell repertoires in cancer are shaped by somatic hypermutation (SHM) either with or without positive selection biases, the latter of which tended to be auto-reactive. Importantly, we identified sulfated glycosaminoglycans (GAGs) as major functional B cell antigens among gastric tumors. Furthermore, natural anti-sulfated GAG antibodies discovered in gastric cancer tissues showed robust growth-suppressive functions against a wide variety of human malignancies of various organs.
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http://dx.doi.org/10.1016/j.celrep.2017.07.016DOI Listing
August 2017

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes.

Nat Cell Biol 2017 May 17;19(5):530-541. Epub 2017 Apr 17.

Department of Molecular Biology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan.

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.
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http://dx.doi.org/10.1038/ncb3509DOI Listing
May 2017

A new missense mutation in the paired domain of the mouse Pax3 gene.

Exp Anim 2017 Aug 6;66(3):245-250. Epub 2017 Apr 6.

Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.

Mice with dominant white spotting occurred spontaneously in the C3.NSY-(D11Mit74-D11Mit229) strain. Linkage analysis indicated that the locus for white spotting was located in the vicinity of the Pax3 gene on chromosome 1. Crosses of white-spotted mice showed that homozygosity for the mutation caused tail and limb abnormalities and embryonic lethality as a result of exencephaly; these phenotypes were analogous to those found in other Pax3 mutants. Sequence analysis identified a missense point mutation (c.101G>A) in exon 2 of Pax3 that resulted in a methionine to isoleucine conversion at amino acid 62 of the PAX3 protein. This mutation site was located in the N-terminal HTH (helix-turn-helix) motif of the paired domain of Pax3, which is necessary for binding to DNA and is highly conserved in vertebrate species. Alteration of DNA binding affinity was responsible for embryonic lethality in homozygotes and white spotting in heterozygotes. We named the mutant allele as Pax3. The C3H/HeN-Pax3 strain may be useful for analyzing the function of Pax3 as a new model of the human disease, Waardenburg Syndrome.
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http://dx.doi.org/10.1538/expanim.17-0013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543245PMC
August 2017

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor.

Cancer Sci 2017 Apr 20;108(4):785-794. Epub 2017 Apr 20.

Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama, Japan.

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.
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http://dx.doi.org/10.1111/cas.13175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406609PMC
April 2017

Genomic pathobiology of gastric carcinoma.

Pathol Int 2017 Feb 22;67(2):63-71. Epub 2016 Dec 22.

Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

With the recent advances in genome sequencing technologies, comprehensive cancer genomic profiling has revealed the in-depth molecular mechanisms of gastric malignancies. New insights into the carcinogenesis pathways of gastric cancers have been acquired, not only by DNA sequencing, but also by the expression profiling of the transcriptome, the identification of chimeric genes, and epi-genetic profiling (such as DNA hypermethylation). Global genomic profiling of gastric cancers, in combination with histopathology, etiology, and cancer biology, has clarified that gastric cancers can be categorized into four subtypes with specific genomic characteristics. Here, we summarize recent knowledge concerning the clinically relevant genomic classifications of gastric cancers and discuss the therapeutic implications for such genomic subtypes, including future perspectives for immune-checkpoint blockade therapies against gastric malignancies.
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http://dx.doi.org/10.1111/pin.12493DOI Listing
February 2017

Receptor tyrosine kinase amplification is predictive of distant metastasis in patients with oral squamous cell carcinoma.

Cancer Sci 2017 Feb;108(2):256-266

Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

This study aimed to clarify the genomic factors associated with the diagnosis and prognosis of oral squamous cell carcinoma via next-generation sequencing. We evaluated data from 220 cases of oral squamous cell carcinoma. Genomic DNA was eluted using formalin-fixed, paraffin-embedded samples, and targeted resequencing of 50 cancer-related genes was performed. In total, 311 somatic mutations were detected in 220 patients, consisting of 68 synonymous mutations and 243 non-synonymous mutations. Genes carrying mutations included TP53, CDKN2A, and PIK3CA in 79 (35.9%), 35 (15.9%), and 19 patients (8.6%), respectively. Copy number analysis detected amplification of PIK3CA and AKT1 in 38 (17.3%) and 11 patients (5.0%), respectively. Amplification of receptor tyrosine kinases was found in 37 patients (16.8%). Distant metastasis was noted in nine of 37 patients (24%) with receptor tyrosine kinase amplification, accounting for 43% of the 21 cases of distant metastasis. The cumulative 5-year survival rate was 64.6% in the receptor tyrosine kinase amplification group vs 85.2% in the no receptor tyrosine kinase amplification group. Moreover, we identified significantly poorer prognosis in the TP53 mutation/receptor tyrosine kinase amplification group, for which the cumulative 5-year survival rate was 41.6%. In conclusion, the results of this study demonstrated that receptor tyrosine kinase amplification is a prognostic factor for distant metastasis of oral squamous cell carcinoma, indicating the necessity of using next-generation sequencing in clinical sequencing.
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http://dx.doi.org/10.1111/cas.13126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5329163PMC
February 2017

CASTIN: a system for comprehensive analysis of cancer-stromal interactome.

BMC Genomics 2016 11 9;17(1):899. Epub 2016 Nov 9.

Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

Background: Cancer microenvironment plays a vital role in cancer development and progression, and cancer-stromal interactions have been recognized as important targets for cancer therapy. However, identifying relevant and druggable cancer-stromal interactions is challenging due to the lack of quantitative methods to analyze whole cancer-stromal interactome.

Results: We present CASTIN (CAncer-STromal INteractome analysis), a novel framework for the evaluation of cancer-stromal interactome from RNA-Seq data using cancer xenograft models. For each ligand-receptor interaction which is derived from curated protein-protein interaction database, CASTIN summarizes gene expression profiles of cancer and stroma into three evaluation indices. These indices provide quantitative evaluation and comprehensive visualization of interactome, and thus enable to identify critical cancer-microenvironment interactions, which would be potential drug targets. We applied CASTIN to the dataset of pancreas ductal adenocarcinoma, and successfully characterized the individual cancer in terms of cancer-stromal relationships, and identified both well-known and less-characterized druggable interactions.

Conclusions: CASTIN provides comprehensive view of cancer-stromal interactome and is useful to identify critical interactions which may serve as potential drug targets in cancer-microenvironment. CASTIN is available at: http://github.com/tmd-gpat/CASTIN .
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http://dx.doi.org/10.1186/s12864-016-3207-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103609PMC
November 2016

Hunner-Type (Classic) Interstitial Cystitis: A Distinct Inflammatory Disorder Characterized by Pancystitis, with Frequent Expansion of Clonal B-Cells and Epithelial Denudation.

PLoS One 2015 20;10(11):e0143316. Epub 2015 Nov 20.

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

Interstitial cystitis (IC) is a chronic bladder disease with urinary frequency, bladder discomfort or bladder pain of unknown etiology. Based on cystoscopic findings, patients with IC are classified as either Hunner-type/classic IC (HIC), presenting with a specific Hunner lesion, or non-Hunner-type IC (NHIC), presenting with no Hunner lesion, but post-hydrodistension mucosal bleeding. Inflammatory cell infiltration, composed predominantly of lymphocytes, plasma cells and epithelial denudation, has in the past been documented as a major pathological IC finding. However, the significance of the pathological evaluation of IC, especially with regard to the difference between HIC and NHIC, has been downplayed in recent years. In this study, we performed immunohistochemical quantification of infiltrating T-lymphocytes, B-lymphocytes and plasma cells, and measured the amount of residual epithelium in urinary bladder biopsy specimens taken from patients with HIC and NHIC, and those with no IC, using image analysis software. In addition, in situ hybridization of the light chains was performed to examine clonal B-cell expansion. Lymphoplasmacytic infiltration was significantly more severe in HIC specimens than in NHIC specimens (P <0.0001). Substantial lymphoplasmacytic inflammation (≥200 cells/mm2) was observed in 93% of HIC specimens, whereas only 8% of NHIC specimens were inflamed. Plasmacytic infiltration was more prominent in HIC specimens compared with NHIC and non-IC cystitis specimens (P <0.005). Furthermore, expansion of light-chain-restricted B-cells was observed in 31% of cases of HIC. The amount of residual epithelium was decreased in HIC specimens compared with NHIC specimens and non-IC cystitis specimens (P <0.0001). These results suggest that NHIC and HIC are distinct pathological entities, with the latter characterized by pancystitis, frequent clonal B-cell expansion and epithelial denudation. An abnormality in the B-cell population may be involved in the pathogenesis of HIC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143316PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654580PMC
June 2016

RHOA mutation in diffuse-type gastric cancer: a comparative clinicopathology analysis of 87 cases.

Gastric Cancer 2016 Apr 1;19(2):403-411. Epub 2015 Apr 1.

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

Background: Recent studies have discovered recurrent RHOA mutations in diffuse-type gastric cancers. These reports show mutant RhoA is an important cancer driver and is a potential therapeutic target. This study aims to investigate the clinicopathological features of diffuse-type gastric cancers with RHOA mutation.

Methods: We performed a thorough review of 87 diffuse-type gastric cancers, including 22 RHOA-mutated and 65 RHOA wild-type gastric cancers.

Results: Most advanced tumors with RHOA mutation appeared as Borrmann type 3 lesions (81 %) developing in the middle (50 %) or distal (32 %) third of the stomach. Histologically, although all of the tumors were predominantly or exclusively composed of poorly cohesive carcinoma, limited tubular differentiation was also observed in 73 % of the RHOA-mutated tumors. Notably, RHOA-mutated tumors more frequently showed a permeative growth pattern at the edge of the mucosal area (59 %) compared with RHOA wild-type tumors (29 %, P = 0.0202). Additionally, the size ratios of the deeply invasive components to the mucosal components were significantly lower in RHOA-mutated tumors [less than 1.45 (median) in 68 % of cases] than in RHOA wild-type tumors (less than 1.45 in 42 % of cases, P = 0.0482). RHOA mutation did not significantly impact survival in this study.

Conclusions: These observations suggest that RHOA mutation may be associated with the growth patterns of diffuse-type gastric cancer but have a limited prognostic impact in isolation. Further studies, including analyses of the other alterations involving the RhoA pathways, such as CLDN18-ARHGAP fusion, as well as functional studies of mutant RhoA, are necessary to clarify the significance of alterations in the RhoA-signaling pathway in diffuse-type gastric cancers.
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http://dx.doi.org/10.1007/s10120-015-0493-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824805PMC
April 2016

Recurrent gain-of-function mutations of RHOA in diffuse-type gastric carcinoma.

Nat Genet 2014 Jun 11;46(6):583-7. Epub 2014 May 11.

1] Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. [2] Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. [3] Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

Diffuse-type gastric carcinoma (DGC) is characterized by a highly malignant phenotype with prominent infiltration and stromal induction. We performed whole-exome sequencing on 30 DGC cases and found recurrent RHOA nonsynonymous mutations. With validation sequencing of an additional 57 cases, RHOA mutation was observed in 25.3% (22/87) of DGCs, with mutational hotspots affecting the Tyr42, Arg5 and Gly17 residues in RHOA protein. These positions are highly conserved among RHO family members, and Tyr42 and Arg5 are located outside the guanine nucleotide-binding pocket. Several lines of functional evidence indicated that mutant RHOA works in a gain-of-function manner. Comparison of mutational profiles for the major gastric cancer subtypes showed that RHOA mutations occur specifically in DGCs, the majority of which were histopathologically characterized by the presence of poorly differentiated adenocarcinomas together with more differentiated components in the gastric mucosa. Our findings identify a potential therapeutic target for this poor-prognosis subtype of gastric cancer with no available molecularly targeted drugs.
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http://dx.doi.org/10.1038/ng.2984DOI Listing
June 2014

FOXP3 regulates sensitivity of cancer cells to irradiation by transcriptional repression of BRCA1.

Cancer Res 2013 Apr 14;73(7):2170-80. Epub 2013 Jan 14.

Department of Pathology, University of Michigan, Ann Arbor, MI, USA.

FOXP3 is an X-linked tumor suppressor gene and a master regulator in T regulatory cell function. This gene has been found to be mutated frequently in breast and prostate cancers and to inhibit tumor cell growth, but its functional significance in DNA repair has not been studied. We found that FOXP3 silencing stimulates homologous recombination-mediated DNA repair and also repair of γ-irradiation-induced DNA damage. Expression profiling and chromatin-immunoprecipitation analyses revealed that FOXP3 regulated the BRCA1-mediated DNA repair program. Among 48 FOXP3-regulated DNA repair genes, BRCA1 and 12 others were direct targets of FOXP3 transcriptional control. Site-specific interaction of FOXP3 with the BRCA1 promoter repressed its transcription. Somatic FOXP3 mutants identified in breast cancer samples had reduced BRCA1 repressor activity, whereas FOXP3 silencing and knock-in of a prostate cancer-derived somatic FOXP3 mutant increased the radioresistance of cancer cells. Together our findings provide a missing link between FOXP3 function and DNA repair programs.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-2481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815443PMC
April 2013

FOXP3: genetic and epigenetic implications for autoimmunity.

J Autoimmun 2013 Mar 11;41:72-8. Epub 2013 Jan 11.

Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Hokkaido, Japan.

FOXP3 plays an essential role in the maintenance of self-tolerance and, thus, in preventing autoimmune diseases. Inactivating mutations of FOXP3 cause immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. FOXP3-expressing regulatory T cells attenuate autoimmunity as well as immunity against cancer and infection. More recent studies demonstrated that FOXP3 is an epithelial cell-intrinsic tumor suppressor for breast, prostate, ovary and other cancers. Corresponding to its broad function, FOXP3 regulates a broad spectrum of target genes. While it is now well established that FOXP3 binds to and regulates thousands of target genes in mouse and human genomes, the fundamental mechanisms of its broad impact on gene expression remain to be established. FOXP3 is known to both activate and repress target genes by epigenetically regulating histone modifications of target promoters. In this review, we first focus on germline mutations found in the FOXP3 gene among IPEX patients, then outline possible molecular mechanisms by which FOXP3 epigenetically regulates its targets. Finally, we discuss clinical implications of the function of FOXP3 as an epigenetic modifier. Accumulating results reveal an intriguing functional convergence between FOXP3 and inhibitors of histone deacetylases. The essential epigenetic function of FOXP3 provides a foundation for experimental therapies against autoimmune diseases.
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http://dx.doi.org/10.1016/j.jaut.2012.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622774PMC
March 2013

Epithelial homeostasis: elimination by live cell extrusion.

Curr Biol 2012 Jun;22(11):R453-5

Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.

To maintain a functional and harmonious epithelial society, the number and quality of cells need to be tightly controlled. Two recent studies reveal a novel cellular process for epithelial homeostasis: crowding-mediated live cell extrusion.
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http://dx.doi.org/10.1016/j.cub.2012.04.036DOI Listing
June 2012

FOXP3 orchestrates H4K16 acetylation and H3K4 trimethylation for activation of multiple genes by recruiting MOF and causing displacement of PLU-1.

Mol Cell 2011 Dec;44(5):770-84

Division of Immunotherapy, Department of Surgery, University of Michigan School of Medicine and Cancer Center, Ann Arbor, MI 48109, USA.

Both H4K16 acetylation and H3K4 trimethylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here, we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 trimethylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial cells disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells.
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http://dx.doi.org/10.1016/j.molcel.2011.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243051PMC
December 2011

Identification of a tumor suppressor relay between the FOXP3 and the Hippo pathways in breast and prostate cancers.

Cancer Res 2011 Mar 28;71(6):2162-71. Epub 2011 Jan 28.

Departments of Surgery, Pathology, and Internal Medicine, Division of Immunotherapy, University of Michigan School of Medicine and Cancer Center, Ann Arbor, Michigan 48109, USA.

Defective expression of LATS2, a negative regulator of YAP oncoprotein, has been reported in cancer of prostate, breast, liver, brain, and blood origins. However, no transcriptional regulators for the LATS2 gene have been identified. Here we report that spontaneous mutation of the transcription factor FOXP3 reduces expression of the LATS2 gene in mammary epithelial cells. shRNA-mediated silencing of FOXP3 in normal or malignant mammary epithelial cells of mouse and human origin repressed LATS2 expression and increased YAP protein levels. LATS2 induction required binding of FOXP3 to a specific sequence in the LATS2 promoter, and this interaction contributed to FOXP3-mediated growth inhibition of tumor cells. In support of these results, reduced expression and somatic mutations of FOXP3 correlated strongly with defective LATS2 expression in microdissected prostate cancer tissues. Thus, defective expression of LATS2 is attributable to FOXP3 defects and may be a major independent determinant of YAP protein elevation in cancer. Our findings identify a novel mechanism of LATS2 downregulation in cancer and reveal an important tumor suppressor relay between the FOXP3 and HIPPO pathways which are widely implicated in human cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-3268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070402PMC
March 2011

Signalling through FOXP3 as an X-linked tumor suppressor.

Int J Biochem Cell Biol 2010 Nov 1;42(11):1784-7. Epub 2010 Aug 1.

Division of Immunotherapy, Department of Surgery, University of Michigan School of Medicine and Cancer Center, Ann Arbor, MI 48109, USA.

The FOXP3 (forkhead box P3) gene is a member of forkhead winged helix family transcription factors and functions as both a transcriptional activator and a repressor. FOXP3 dysfunction is responsible for an X-linked autoimmune syndrome: immune dysregulation, polyendopathy, enterophathy, X-linked syndrome. In addition to its role as an essential transcription factor in regulatory T cells, the FOXP3 gene is an epithelial cell-intrinsic tumor suppressor for breast and prostate cancers. We will focus on the FOXP3 signalling pathway in epithelial cells and discuss how genetic and/or epigenetic inactivation of the FOXP3 contributes to the malignant transformation of cells.
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http://dx.doi.org/10.1016/j.biocel.2010.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2950213PMC
November 2010

Somatic single hits inactivate the X-linked tumor suppressor FOXP3 in the prostate.

Cancer Cell 2009 Oct;16(4):336-46

Division of Immunotherapy, Department of Surgery, University of Michigan School of Medicine and Cancer Center, Ann Arbor, MI 48109, USA.

Despite clear epidemiological and genetic evidence for X-linked prostate cancer risk, all prostate cancer genes identified are autosomal. Here, we report somatic inactivating mutations and deletion of the X-linked FOXP3 gene residing at Xp11.23 in human prostate cancer. Lineage-specific ablation of FoxP3 in the mouse prostate epithelial cells leads to prostate hyperplasia and prostate intraepithelial neoplasia. In both normal and malignant prostate tissues, FOXP3 is both necessary and sufficient to transcriptionally repress cMYC, the most commonly overexpressed oncogene in prostate cancer as well as among the aggregates of other cancers. FOXP3 is an X-linked prostate tumor suppressor in the male. Because the male has only one X chromosome, our data represent a paradigm of "single genetic hit" inactivation-mediated carcinogenesis.
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http://dx.doi.org/10.1016/j.ccr.2009.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758294PMC
October 2009