Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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Folliculin regulates osteoclastogenesis through metabolic regulation

Baba, M., Endoh, M., Ma, W., Toyama, H., Hirayama, A., Nishikawa, K., Takubo, K., Hano, H., Hasumi, H., Umemoto, T., Hashimoto, M., Irie, N., Esumi, C., Kataoka, M., Nakagata, N., Soga, T., Yao, M., Kamba, T., Minami, T., Ishii, M., Suda, T.

J. Bone Miner. Res. (Journal of Bone and Mineral Research) 33 ( 10 ) 1785 - 1798 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 08840431

© 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR) Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. Here, we demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn-deficient osteoclast precursors reveal cell autonomous accelerated osteoclastogenesis with increased sensitivity to receptor activator of NF-κB ligand (RANKL). We demonstrate that Flcn regulates oxidative phosphorylation and purine metabolism through suppression of nuclear localization of the transcription factor Tfe3, thereby inhibiting expression of its target gene Pgc1. Metabolome studies revealed that Flcn-deficient osteoclast precursors exhibit significant augmentation of oxidative phosphorylation and nucleotide production, resulting in an enhanced purinergic signaling loop that is composed of controlled ATP release and autocrine/paracrine purinergic receptor stimulation. Inhibition of this purinergic signaling loop efficiently blocks accelerated osteoclastogenesis in Flcn-deficient osteoclast precursors. Here, we demonstrate an essential and novel role of the Flcn-Tfe3-Pgc1 axis in osteoclastogenesis through the metabolic reprogramming of oxidative phosphorylation and purine metabolism. © 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

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Beta-galactosidase-responsive synthetic biomarker for targeted tumor detection.

Nishihara, T. Kuno, S., Nonaka, H., Tabata, S., Saito, N., Fukuda, S., Tomita, M., Sando, S., *Soga, T.

Chem.Comm.(Camb) 4 ( 83 ) 11745 - 11745 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 13597345

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Metabolic characterization of antifolate responsiveness and nonresponsiveness in malignant pleural mesothelioma cells

Sato, Y., Matsuda, S., Maruyama, A., Nakayama, J., Miyashita, T., Udagawa, H., Umemura, S., Yanagihara, K., Ochiai, A., Tomita, M., Soga, T., Tsuchihara, K., Makinoshima, H.

Front. Pharmacol. 9 1129 2018.10

Research paper (scientific journal), Joint Work, Accepted

© 2007 - 2018 Frontiers Media S.A. All Rights Reserved. Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM.

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SRSF3, a Splicer of the PKM Gene, Regulates Cell Growth and Maintenance of Cancer-Specific Energy Metabolism in Colon Cancer Cells

Kuranaga, Y., Sugito, N., Shinohara, H., Tsujino, T., Taniguchi, K., Komura, K., Ito, Y., Soga, T., Akao, Y.

Int. J. Mol. Sci. (International Journal of Molecular Sciences) 19 ( 10 ) E3012 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 16616596

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Serine and arginine rich splicing factor 3 (SRSF3), an SR-rich family protein, has an oncogenic function in various kinds of cancer. However, the detailed mechanism of the function had not been previously clarified. Here, we showed that the SRSF3 splicer regulated the expression profile of the pyruvate kinase, which is one of the rate-limiting enzymes in glycolysis. Most cancer cells express pyruvate kinase muscle 2 (PKM2) dominantly to maintain a glycolysis-dominant energy metabolism. Overexpression of SRSF3, as well as that of another splicer, polypyrimidine tract binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), in clinical cancer samples supported the notion that these proteins decreased the Pyruvate kinase muscle 1 (PKM1)/PKM2 ratio, which positively contributed to a glycolysis-dominant metabolism. The silencing of SRSF3 in human colon cancer cells induced a marked growth inhibition in both in vitro and in vivo experiments and caused an increase in the PKM1/PKM2 ratio, thus resulting in a metabolic shift from glycolysis to oxidative phosphorylation. At the same time, the silenced cells were induced to undergo autophagy. SRSF3 contributed to PKM mRNA splicing by co-operating with PTBP1 and hnRNPA1, which was validated by the results of RNP immunoprecipitation (RIP) and immunoprecipitation (IP) experiments. These findings altogether indicated that SRSF3 as a PKM splicer played a positive role in cancer-specific energy metabolism.

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Canagliflozin reduces plasma uremic toxins and alters the intestinal microbiota composition in a chronic kidney disease mouse model

Mishima E., Fukuda S., Kanemitsu Y., Saigusa D., Mukawa C., Asaji K., Matsumoto Y., Tsukamoto H., Tachikawa T., Tsukimi T., Fukuda N., Ho H., Kikuchi K., Suzuki C., Nanto F., Suzuki T., Ito S., Soga T., Tomioka Y., Abe T.

Am. J. Physiol. Renal. Physiol. (American Journal of Physiology - Renal Physiology) 315 ( 4 ) F824 - F833 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 03636127

© 2018 the American Physiological Society. Mishima E, Fukuda S, Kanemitsu Y, Saigusa D, Mukawa C, Asaji K, Matsumoto Y, Tsukamoto H, Tachikawa T, Tsukimi T, Fukuda NN, Ho HJ, Kikuchi K, Suzuki C, Nanto F, Suzuki T, Ito S, Soga T, Tomioka Y, Abe T. Canagliflozin reduces plasma uremic toxins and alters the intestinal microbiota composition in a chronic kidney disease mouse model. Am J Physiol Renal Physiol 315: F824–F833, 2018. First published November 22, 2017; doi:10.1152/ajprenal.00314.2017.—Accumulation of uremic toxins, which exert deleterious effects in chronic kidney disease, is influenced by the intestinal environment; the microbiota contributes to the production of representative uremic toxins, including p-cresyl sulfate and indoxyl sulfate. Canagliflozin is a sodium-glucose cotransporter (SGLT) 2 inhibitor, and it also exerts a modest inhibitory effect on SGLT1. The inhibition of intestinal SGLT1 can influence the gastrointestinal environment. We examined the effect of canagliflozin on the accumulation of uremic toxins in chronic kidney disease using adenine-induced renal failure mice. Two-week canagliflozin (10 mg/kg po) treatment did not influence the impaired renal function; however, it significantly reduced the plasma levels of p-cresyl sulfate and indoxyl sulfate in renal failure mice (a 75% and 26% reduction, respectively, compared with the vehicle group). Additionally, canagliflozin significantly increased cecal short-chain fatty acids in the mice, suggesting the promotion of bacterial carbohydrate fermentation in the intestine. Analysis of the cecal microbiota showed that canagliflozin significantly altered microbiota composition in the renal failure mice. These results indicate that canagliflozin exerts intestinal effects that reduce the accumulation of uremic toxins including p-cresyl sulfate. Reduction of accumulated uremic toxins by canagliflozin could provide a potential therapeutic option in chronic kidney disease.

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A Metabolomic-Based Evaluation of the Role of Commensal Microbiota throughout the Gastrointestinal Tract in Mice

Yamamoto, Y., Nakanishi, Y., Murakami, S., Aw, W., Tsukimi, T., Nozu, R., Ueno, M., Hioki, K., Nakahigashi, K., Hirayama, A., Sugimoto, M., Soga, T., Ito, M., Tomita, M., Fukuda, S.

Microorganisms 6 ( 4 ) 101 2018.09

Research paper (scientific journal), Joint Work, Accepted

Commensal microbiota colonize the surface of our bodies. The inside of the gastrointestinal tract is one such surface that provides a habitat for them. The gastrointestinal tract is a long organ system comprising of various parts, and each part possesses various functions. It has been reported that the composition of intestinal luminal metabolites between the small and large intestine are different; however, comprehensive metabolomic and commensal microbiota profiles specific to each part of the gastrointestinal lumen remain obscure. In this study, by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome and 16S rRNA gene-based microbiome analyses of specific pathogen-free (SPF) and germ-free (GF) murine gastrointestinal luminal profiles, we observed the different roles of commensal microbiota in each part of the gastrointestinal tract involved in carbohydrate metabolism and nutrient production. We found that the concentrations of most amino acids in the SPF small intestine were higher than those in the GF small intestine. Furthermore, sugar alcohols such as mannitol and sorbitol accumulated only in the GF large intestine, but not in the SPF large intestine. On the other hand, pentoses, such as arabinose and xylose, gradually accumulated from the cecum to the colon only in SPF mice, but were undetected in GF mice. Correlation network analysis between the gastrointestinal microbes and metabolites showed that niacin metabolism might be correlated to Methylobacteriaceae. Collectively, commensal microbiota partially affects the gastrointestinal luminal metabolite composition based on their metabolic dynamics, in cooperation with host digestion and absorption.

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Trans-omic Analysis Reveals Selective Responses to Induced and Basal Insulin across Signaling, Transcriptional, and Metabolic Networks

Kawata K., Hatano A., Yugi K., Kubota H., Sano T., Fujii M., Tomizawa Y., Kokaji T., Tanaka K., Uda S., Suzuki Y., Matsumoto M., Nakayama K., Saitoh K., Kato K., Ueno A., Ohishi M., Hirayama A., Soga T., Kuroda S.

iScience 7 ( - ) 212 - 229 2018.09

Research paper (scientific journal), Joint Work, Accepted

The concentrations of insulin selectively regulate multiple cellular functions. To understand how insulin concentrations are interpreted by cells, we constructed a trans-omic network of insulin action in FAO hepatoma cells using transcriptomic data, western blotting analysis of signaling proteins, and metabolomic data. By integrating sensitivity into the trans-omic network, we identified the selective trans-omic networks stimulated by high and low doses of insulin, denoted as induced and basal insulin signals, respectively. The induced insulin signal was selectively transmitted through the pathway involving Erk to an increase in the expression of immediate-early and upregulated genes, whereas the basal insulin signal was selectively transmitted through a pathway involving Akt and an increase of Foxo phosphorylation and a reduction of downregulated gene expression. We validated the selective trans-omic network in vivo by analysis of the insulin-clamped rat liver. This integrated analysis enabled molecular insight into how liver cells interpret physiological insulin signals to regulate cellular functions.

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Gamma-aminobutyric acid signaling in brown adipose tissue promotes systemic metabolic derangement in obesity

Ikegami, R., Shimizu, I., Sato, T., Yoshida, Y., Hayashi, Y., Suda, M., Katsuumi, G., Li, J., Wakasugi, T., Minokoshi, Y., Okamoto, S., Hinoi, E., Nielsen, S., Jespersen, N. Z., Scheele, C., Soga, T., Minamino, T.

Cell Rep. 24 ( 11 ) 2827 - 2837 2018.09

Research paper (scientific journal), Joint Work, Accepted

Brown adipose tissue (BAT) is a metabolically active organ that contributes to the maintenance of systemic metabolism. The sympathetic nervous system plays important roles in the homeostasis of BAT and promotes its browning and activation. However, the role of other neurotransmitters in BAT homeostasis remains largely unknown. Our metabolomic analyses reveal that gamma-aminobutyric acid (GABA) levels are increased in the interscapular BAT of mice with dietary obesity. We also found a significant increase in GABA-type B receptor subunit 1 (GABA-BR1) in the cell membranes of brown adipocytes of dietary obese mice. When administered to obese mice, GABA induces BAT dysfunction together with systemic metabolic disorder. Conversely, the genetic inactivation or inhibition of GABA-BR1 leads to the re-browning of BAT under conditions of metabolic stress and ameliorated systemic glucose intolerance. These results indicate that the constitutive activation of GABA/GABA-BR1 signaling in obesity promotes BAT dysfunction and systemic metabolic derangement.

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Effect of storage conditions on salivary polyamines quantified via liquid chromatography-mass spectrometry

Tomita, A., Mori, M., Hiwatari, K., Yamaguchi, E., Itoi, T., Sunamura, M., Soga, T., Tomita, M., Sugimoto, M.

Sci. Rep. 8 ( 1 ) 12075 2018.08

Research paper (scientific journal), Joint Work, Accepted

© 2018, The Author(s). Salivary polyamines are potential non-invasive tools for screening various types of cancers. For clinical use, the reproducibility of these metabolites should be evaluated under various storage conditions, including duration and temperature, to establish standard operating protocols. Polyamines and amino acids in unstimulated whole saliva were quantified via liquid chromatography-mass spectrometry. Concentrations of time course samples were analysed after short-term storage for up to 240 min and long-term storage for up to 8 days under various storage conditions. As expected, storage at the lowest temperature (−18 °C) exerted the least pronounced effects on the quantified values in both tests. At a higher temperature, polyamines were more stable than amino acids, as evident from polyamine profiling. Addition of ethanol significantly stabilized polyamine profiles even at a higher temperature. Comparative processing of saliva revealed a minor effect of the solvent, whereas drying had a more prominent effect on polyamine profiles. Computational analyses evaluated the ability of polyamines to discriminate pancreatic cancer from controls. Repeated noise added tests were designed on the basis of the results of the storage tests; these analyses confirmed that the discriminative abilities were robust. These data contribute to the standardization of salivary storage conditions, thereby highlighting the clinical utility of saliva.

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Lacking ketohexokinase-A exacerbates renal injury in streptozotocin-induced diabetic mice

Doke, T., Ishimoto, T., Hayasaki, T., Ikeda, S., Hasebe, M., Hirayama, A., Soga, T., Kato, N., Kosugi, T., Tsuboi, N., Lanaspa, MA., Johnson, RJ., Kadomatsu, K., Maruyama, S.

Metabolism (Metabolism: Clinical and Experimental) 85 161 - 170 2018.08

Research paper (scientific journal), Joint Work, ISSN 00260495

© 2018 Elsevier Inc. Objective: Ketohexokinase (KHK), a primary enzyme in fructose metabolism, has two isoforms, namely, KHK-A and KHK-C. Previously, we reported that renal injury was reduced in streptozotocin-induced diabetic mice which lacked both isoforms. Although both isoforms express in kidney, it has not been elucidated whether each isoform plays distinct roles in the development of diabetic kidney disease (DKD). The aim of the study is to elucidate the role of KHK-A for DKD progression. Materials and Methods: Diabetes was induced by five consecutive daily intraperitoneal injections of streptozotocin (50 mg/kg) in C57BL/6J wild-type mice, mice lacking KHK-A alone (KHK-A KO), and mice lacking both KHK-A and KHK-C (KHK-A/C KO). At 35 weeks, renal injury, inflammation, hypoxia, and oxidative stress were examined. Metabolomic analysis including polyol pathway, fructose metabolism, glycolysis, TCA (tricarboxylic acid) cycle, and NAD (nicotinamide adenine dinucleotide) metabolism in kidney and urine was done. Results: Diabetic KHK-A KO mice developed severe renal injury compared to diabetic wild-type mice, and this was associated with further increases of intrarenal fructose, dihydroxyacetone phosphate (DHAP), TCA cycle intermediate levels, and severe inflammation. In contrast, renal injury was prevented in diabetic KHK-A/C KO mice compared to both wild-type and KHK-A KO diabetic mice. Further, diabetic KHK-A KO mice contained decreased renal NAD + level with the increase of renal hypoxia-inducible factor 1-alpha expression despite having increased renal nicotinamide (NAM) level. Conclusion: These results suggest that KHK-C might play a deleterious role in DKD progression through endogenous fructose metabolism, and that KHK-A plays a unique protective role against the development of DKD.

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Low tumor glutathione level as a sensitivity marker for glutamate-cysteine ligase inhibitors

Nishizawa S., Araki H., Ishikawa Y., Kitazawa S., Hata A., Soga T., Hara T.

Oncol. Lett. 15 ( 6 ) 8735 - 8743 2018.06

Research paper (scientific journal), Joint Work, ISSN 17921074

© 2018, Spandidos Publications. All rights reserved. Previous metabolomic analyses of cancer have revealed elevated glutathione levels in tumors. An inhibitor of cystine uptake was identified to suppress glutathione biosynthesis, leading to ferroptosis, a novel iron-dependent form of cell death that differs from apoptosis and necrosis. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione biosynthesis pathway. Buthionine sulfoximine (BSO), a GCL inhibitor, has previously demonstrated limited clinical benefits. Therefore, selecting patients who respond well to the inhibitor is a key approach for successful future drug development. Ferroptosis induction by BSO has not been fully examined in prior studies. Therefore, the present study investigated the pharmacological effects of BSO and the association between basal intracellular glutathione levels and sensitivity to BSO in cultured cell lines derived from various types of cancer, including those of the kidney [769P, 786-O, A-498, A704, ACHN, Caki-1, Caki-2, G401, G402, RCC4 VHL(-/-), RCC4 VHL(+/+), SK-NEP-1 and SW156] and ovaries (A2780 and A2780/CDDP). BSO was demonstrated to suppress glutathione levels and induce lipid peroxidation, thereby inhibiting cell viability. The viability-reducing effects of BSO were attenuated by ferroptosis inhibition and enhanced by iron, indicating that BSO induced ferroptosis in cancer cells. The cell lines sensitive to BSO, including G402, tended to exhibit non-significantly lower levels of glutathione compared with the BSO-insensitive cell lines, including Caki-2 (P=0.08). Patient sample data indicated the existence of a population of colorectal tumors with lower glutathione levels compared with those of matched normal tissues that might be suitable for the clinical testing of sensitivity to GCLC inhibitors. Collectively, these data suggest that GCL inhibition leads to ferroptosis in cancer cells, and that low glutathione tumor levels may be a patient selection marker for the use of GCL inhibitors in the treatment of tumors.

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Development of a sheathless CE‐ESI‐MS interface

Hirayama, A., Abe, H., Yamaguchi, N., Tabata, S., Tomita, T., Soga, T.

Electrophoresis 39 ( 11 ) 1382 - 1389 2018.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 01730835

© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
A sheath-flow interface is the most common ionization technique in CE-ESI-MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE-MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath-flow CE-MS. Because the interface is easy to construct, it is cost-effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE-MS-based metabolomic analysis.

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Changes of Liver Metabolites following Hepatectomy with Ischemia Reperfusion towards Liver Regeneration

Saito, Y., Morine Y., Iwahashi, S., Ikemoto, T., Imura, S., Yamanaka-Okumura, H., Hirayama, A., Soga, T., Tomita, M., Shimada, M.

Ann. Gastroenterol. Surg. 2 ( 3 ) 204 - 211 2018.05

Research paper (scientific journal), Joint Work, Accepted

Background: Metabolome analysis is one of the omics which investigates the final product of a central dogma. Changes of liver metabolites during liver regeneration following hepatectomy (Hx) continue to remain unclear. The aim of the present study was to investigate the changes of liver metabolites following Hx with ischemia reperfusion (I/R) towards liver regeneration. Methods: Twenty-three patients who underwent Hx were enrolled in this study. Non-tumor tissues were sampled immediately before and after Hx and a comparison was made between the liver samples taken before and after Hx using capillary electrophoresis (CE)-time-of-flight mass spectrometry (TOFMS) as metabolome analysis. Results: The metabolic pathway showed that there was a significant increase in "lactate" following Hx. There was a significant decrease in metabolites only in the first half of the tricarboxylic acid cycle (TCA) cycle, and adenosine triphosphate (ATP) by anaerobiotic glycolysis did not occur in time for energy consumption of the Hx. Principal component analysis revealed remarkably different component profiles between the samples taken before and after Hx. One hundred and three metabolites were selected as critical metabolites for separating components. Valine and tryptophan increased significantly after Hx and they were regulated by resected liver volume, ischemic time and liver function. Conclusion: The liver metabolites changed remarkably between before and after Hx. Especially, liver valine and tryptophan were increased.

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Thymidine catabolism promotes NADPH oxidase-derived reactive oxygen species (ROS) signalling in KB and yumoto cells

Tabata, S., Yamamoto, M., Goto, H., Hirayama, A., Ohishi, M., Kuramoto, T., Mitsuhashi, A., Ikeda, R., Haraguchi, M., Kawahara, K., Shinsato, Y., Minami, K., Saijo, A., Toyoda, Y., Hanibuchi, M., Nishioka, Y., Sone, S., Esumi, H., Tomita, M., Soga, T., Furukawa, T., Akiyama, S.

Sci. Rep. 8 ( 1 ) 6760 2018.04

Research paper (scientific journal), Joint Work, Accepted

Thymidine phosphorylase (TP) is a rate-limiting enzyme in the thymidine catabolic pathway. TP is identical to platelet-derived endothelial cell growth factor and contributes to tumour angiogenesis. TP induces the generation of reactive oxygen species (ROS) and enhances the expression of oxidative stress-responsive genes, such as interleukin (IL)-8. However, the mechanism underlying ROS induction by TP remains unclear. In the present study, we demonstrated that TP promotes NADPH oxidase-derived ROS signalling in cancer cells. NADPH oxidase inhibition using apocynin or small interfering RNAs (siRNAs) abrogated the induction of IL-8 and ROS in TP-expressing cancer cells. Meanwhile, thymidine catabolism induced by TP increased the levels of NADPH and intermediates of the pentose phosphate pathway (PPP). Both siRNA knockdown of glucose 6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme in PPP, and a G6PD inhibitor, dihydroepiandrosterone, reduced TP-induced ROS production. siRNA downregulation of 2-deoxy-D-ribose 5-phosphate (DR5P) aldolase, which is needed for DR5P to enter glycolysis, also suppressed the induction of NADPH and IL-8 in TP-expressing cells. These results suggested that TP-mediated thymidine catabolism increases the intracellular NADPH level via the PPP, which enhances the production of ROS by NADPH oxidase and activates its downstream signalling.

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Purine nucleotide metabolism regulates expression of the human immune ligand MICA

McCarthy, MT., Moncayo, G., Hiron, TK., Jakobsen, NA., Valli, A., Soga, T., Adam, J., O'Callaghan, CA.

J. Biol. Chem. 293 ( 11 ) 3913-3924 - 3924 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219258

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the cell-surface glycoprotein MHC class I polypeptide- related sequence A (MICA) is induced in dangerous, abnormal, or "stressed" cells, including cancer cells, virus-infected cells, and rapidly proliferating cells. MICA is recognized by the activating immune cell receptor natural killer group 2D (NKG2D), providing a mechanism by which immune cells can identify and potentially eliminate pathological cells. Immune recognition through NKG2D is implicated in cancer, atherosclerosis, transplant rejection, and inflammatory diseases, such as rheumatoid arthritis. Despite the wide range of potential therapeutic applications ofMICAmanipulation, the factors that control MICA expression are unclear. Here we use metabolic interventions and metabolomic analyses to show that the transition from quiescent cellular metabolism to a "Warburg" or biosynthetic metabolic state induces MICA expression. Specifically, we show that glucose transport into the cell and active glycolytic metabolism are necessary to up-regulate MICA expression. Active purine synthesis is necessary to support this effect of glucose, and increases in purine nucleotide levels are sufficient to induce MICA expression. Metabolic induction of MICA expression directly influences NKG2D-dependent cytotoxicity by immune cells. These findings support a model of MICA regulation whereby the purine metabolic activity of individual cells is reflected by cell-surface MICA expression and is the subject of surveillance by NKG2D receptor-expressing immune cells.

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PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth

Morita, M., Sato, T., Nomura, M., Sakamoto, Y., Inoue, Y., Tanaka, R., Ito, S., Kurosawa, K., Yamaguchi, K., Sugiura, Y., Takizaki, H., Yamashita, Y., Katakura, R., Sato, I., Kawai, M., Okada, Y., Watanabe, H., Kondoh, G., Matsumoto, S., Kishimoto, A., Obata, M., Matsumoto, M., Fukuhara, T., Motohashi, H., Suematsu, M., Komatsu, M., Nakayama, KI., Watanabe, T., Soga, T., Shima, H., Maemondo, M., Tanuma. N.

Cancer Cell (Cancer Cell) 33 ( 3 ) 355 - 367.e7 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 15356108

Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including small-cell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs.

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Urinary Polyamine Biomarker Panels with Machine-Learning Differentiated Colorectal Cancers, Benign Disease, and Healthy Controls

Nakajima, T., Katsumata, K., Kuwabara, H., Soya, R., Enomoto, M., Ishizaki, T., Tsuchida, A., Mori, M., Hiwatari, K., Soga, T., Tomita, M., Sugimoto, M.

Int. J. Mol. Sci. (Int. J. Mol. Sci.) 19 ( 3 ) 756 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 16616596

Colorectal cancer (CRC) is one of the most daunting diseases due to its increasing worldwide prevalence, which requires imperative development of minimally or non-invasive screening tests. Urinary polyamines have been reported as potential markers to detect CRC, and an accurate pattern recognition to differentiate CRC with early stage cases from healthy controls are needed. Here, we utilized liquid chromatography triple quadrupole mass spectrometry to profile seven kinds of polyamines, such as spermine and spermidine with their acetylated forms. Urinary samples from 201 CRCs and 31 non-CRCs revealed the N₁,N12-diacetylspermine showing the highest area under the receiver operating characteristic curve (AUC), 0.794 (the 95% confidence interval (CI): 0.704-0.885, p < 0.0001), to differentiate CRC from the benign and healthy controls. Overall, 59 samples were analyzed to evaluate the reproducibility of quantified concentrations, acquired by collecting three times on three days each from each healthy control. We confirmed the stability of the observed quantified values. A machine learning method using combinations of polyamines showed a higher AUC value of 0.961 (95% CI: 0.937-0.984, p < 0.0001). Computational validations confirmed the generalization ability of the models. Taken together, polyamines and a machine-learning method showed potential as a screening tool of CRC.

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Microhomology-assisted scarless genome editing in human iPSCs

Kim, S., Matsumoto, T., Kagawa, H., Nakamura, M., Hirohata, R., Ueno, A., Ohishi, M., Sakuma, T., Soga, T., Yamamoto, T., Woltjen, K.

Nat. Commun. 9 ( 1 ) 939 2018.03

Research paper (scientific journal), Joint Work, Accepted

© 2018 The Author(s). Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.

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Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors

Oshima, K., Saiki, N., Tanaka, M., Imamura, H. Niwa, A., Tanimura, A., Nagahashi;, A., Hirayama, A., Okita, K., Hotta, A., Kitayama, S., Osawa, M., Kaneko, S., Watanabe, A., Asaka, I., Fujibuchi W., Imai, K., Yabe, H., Kamachi, Y., Hara J., Kojima, S., Tomita, M., Soga, T., Noma, T., Nonoyama, S., Nakahata, T., Saito, M.K.

Biochem. Biophys. Res. Commun. 497 ( 2 ) 719-725 - 725 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells.

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Elevated Polyamines in Saliva of Pancreatic Cancer

Asai, Y., Itoi, T., Sugimoto, M., Sofuni, A., Tsuchiya, T, Tanaka, R., Tonozuka, R., Honjo, M., Mukai, S., Fujita, M., Yamamoto, K., Matsunami, Y., Kurosawa, T., Nagakawa, Y., Kaneko, M., Ota, S., Kawachi, S., Shimazu, M., Soga, T., Tomita, M., Sunamura., M.

Cancers 10 ( 2 ) pii: E43 2018.02

Research paper (scientific journal), Joint Work, Accepted

Detection of pancreatic cancer (PC) at a resectable stage is still difficult because of the lack of accurate detection tests. The development of accurate biomarkers in low or non-invasive biofluids is essential to enable frequent tests, which would help increase the opportunity of PC detection in early stages. Polyamines have been reported as possible biomarkers in urine and saliva samples in various cancers. Here, we analyzed salivary metabolites, including polyamines, using capillary electrophoresis-mass spectrometry. Salivary samples were collected from patients with PC (n = 39), those with chronic pancreatitis (CP, n = 14), and controls (C, n = 26). Polyamines, such as spermine, N₁-acetylspermidine, and N₁-acetylspermine, showed a significant difference between patients with PC and those with C, and the combination of four metabolites including N₁-acetylspermidine showed high accuracy in discriminating PC from the other two groups. These data show the potential of saliva as a source for tests screening for PC.

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