Publications by authors named "Oltea Sampetrean"

41 Publications

Lactate Reprograms Energy and Lipid Metabolism in Glucose-Deprived Oxidative Glioma Stem Cells.

Metabolites 2021 May 18;11(5). Epub 2021 May 18.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

Fast-growing tumors satisfy their bioenergetic needs by supplementing glucose with alternative carbon sources. Cancer stem cells are the most versatile and robust cells within malignant tumors. They avoid potentially lethal metabolic and other types of stress through flexible reprogramming of relevant pathways, but it has remained unclear whether alternative carbon sources are important for the maintenance of their tumor-propagating ability. Here we assessed the ability of glycolytic and oxidative murine glioma stem cells (GSCs) to grow in an ultralow glucose medium. Sphere formation assays revealed that exogenous lactate and acetate reversed the growth impairment of oxidative GSCs in such medium. Extracellular flux analysis showed that lactate supported oxygen consumption in these cells, whereas metabolomics analysis revealed that it increased the intracellular levels of tricarboxylic acid cycle intermediates, ATP, and GTP as well as increased adenylate and guanylate charge. Lactate also reversed the depletion of choline apparent in the glucose-deprived cells as well as reprogrammed phospholipid and fatty acid biosynthesis. This metabolic reprogramming was associated with a more aggressive phenotype of intracranial tumors formed by lactate-treated GSCs. Our results thus suggest that lactate is an important alternative energetic and biosynthetic substrate for oxidative GSCs, and that it sustains their growth under conditions of glucose deprivation.
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http://dx.doi.org/10.3390/metabo11050325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158503PMC
May 2021

MEK inhibition preferentially suppresses anchorage-independent growth in osteosarcoma cells and decreases tumors in vivo.

J Orthop Res 2021 Mar 10. Epub 2021 Mar 10.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Osteosarcoma is the most common high-grade malignancy of bone, and novel therapeutic options are urgently required. Previously, we developed mouse osteosarcoma AXT cells that can proliferate both under adherent and nonadherent conditions. Based on metabolite levels, nonadherent conditions were more similar to the in vivo environment than adherent conditions. A drug screen identified MEK inhibitors, including trametinib, that preferentially decreased the viability of nonadherent AXT cells. Trametinib inhibited the cell cycle and induced apoptosis in AXT cells, and both effects were stronger under nonadherent conditions. Trametinib also potently decreased viability in U2OS cells, but its effects were less prominent in MG63 or Saos2 cells. By contrast, MG63 and Saos2 cells were more sensitive to PI3K inhibition than AXT or U2OS cells. Notably, the combination of MAPK/ERK kinase (MEK) and PI3K inhibition synergistically decreased viability in U2OS and AXT cells, but this effect was less pronounced in MG63 or Saos2 cells. Therefore, signal dependence for cell survival and crosstalk between MEK-ERK and PI3K-AKT pathways in osteosarcoma are cell context-dependent. The activation status of other kinases including CREB varied in a cell context-dependent manner, which might determine the response to MEK inhibition. A single dose of trametinib was sufficient to decrease the size of the primary tumor and circulating tumor cells in vivo. Moreover, combined administration of trametinib and rapamycin or conventional anticancer drugs further increased antitumor activity. Thus, given optimal biomarkers for predicting its effects, trametinib holds therapeutic potential for the treatment of osteosarcoma.
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http://dx.doi.org/10.1002/jor.25023DOI Listing
March 2021

N-cadherin upregulation mediates adaptive radioresistance in glioblastoma.

J Clin Invest 2021 Mar;131(6)

Department of Neurosurgery, School of Medicine and O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Glioblastoma (GBM) is composed of heterogeneous tumor cell populations, including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance in GBM, we generated radioresistant human and mouse GSCs by exposing them to repeat cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by a reduction in cell proliferation and an increase in cell-cell adhesion and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of β-catenin at the cell surface, which suppressed Wnt/β-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin, a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation.
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http://dx.doi.org/10.1172/JCI136098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954595PMC
March 2021

Heterogeneous Glioma Cell Invasion Under Interstitial Flow Depending on Their Differentiation Status.

Tissue Eng Part A 2021 Apr 15;27(7-8):467-478. Epub 2021 Feb 15.

Department of System Design Engineering, Keio University, Yokohama, Japan.

Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor. A deeper mechanistic understanding of the invasion of heterogeneous GBM cell populations is crucial to develop therapeutic strategies. A key regulator of GBM cell invasion is interstitial flow. However, the effect of an interstitial flow on the invasion of heterogeneous GBM cell populations composed of glioma initiating cells (GICs) and relatively differentiated progeny cells remains unclear. In the present study, we investigated how GICs invade three-dimensional (3D) hydrogels in response to an interstitial flow with respect to their differentiation status. Microfluidic culture systems were used to apply an interstitial flow to the cells migrating from the cell aggregates into the 3D hydrogel. Phase-contrast microscopy revealed that the invasion and protrusion formation of the GICs in differentiated cell conditions were significantly enhanced by a forward interstitial flow, whose direction was the same as that of the cell invasion, whereas those in stem cell conditions were not enhanced by the interstitial flow. The mechanism of flow-induced invasion was further investigated by focusing on differentiated cell conditions. Immunofluorescence images revealed that the expression of cell-extracellular matrix adhesion-associated molecules, such as integrin β1, focal adhesion kinase, and phosphorylated Src, was upregulated in forward interstitial flow conditions. We then confirmed that cell invasion and protrusion formation were significantly inhibited by PP2, a Src inhibitor. Finally, we observed that the flow-induced cell invasion was preceded by nestin-positive immature GICs at the invasion front and followed by tubulin β3-positive differentiated cells. Our findings provide insights into the development of novel therapeutic strategies to inhibit flow-induced glioma invasion. Impact statement A mechanistic understanding of heterogeneous glioblastoma cell invasion is crucial for developing therapeutic strategies. We observed that the invasion and protrusion formation of glioma initiating cells (GICs) were significantly enhanced by forward interstitial flow in differentiated cell conditions. The expression of integrin β1, focal adhesion kinase, and phosphorylated Src was upregulated, and the flow-induced invasion was significantly inhibited by a Src inhibitor. The flow-induced heterogeneous cell invasion was preceded by nestin-positive GICs at the invasion front and followed by tubulin β3-positive differentiated cells. Our findings provide insights into the development of novel therapeutic strategies to inhibit flow-induced glioma invasion.
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http://dx.doi.org/10.1089/ten.TEA.2020.0280DOI Listing
April 2021

2-Nitroimidazoles induce mitochondrial stress and ferroptosis in glioma stem cells residing in a hypoxic niche.

Commun Biol 2020 08 17;3(1):450. Epub 2020 Aug 17.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.

Under hypoxic conditions, nitroimidazoles can replace oxygen as electron acceptors, thereby enhancing the effects of radiation on malignant cells. These compounds also accumulate in hypoxic cells, where they can act as cytotoxins or imaging agents. However, whether these effects apply to cancer stem cells has not been sufficiently explored. Here we show that the 2-nitroimidazole doranidazole potentiates radiation-induced DNA damage in hypoxic glioma stem cells (GSCs) and confers a significant survival benefit in mice harboring GSC-derived tumors in radiotherapy settings. Furthermore, doranidazole and misonidazole, but not metronidazole, manifested radiation-independent cytotoxicity for hypoxic GSCs that was mediated by ferroptosis induced partially through blockade of mitochondrial complexes I and II and resultant metabolic alterations in oxidative stress responses. Doranidazole also limited the growth of GSC-derived subcutaneous tumors and that of tumors in orthotopic brain slices. Our results thus reveal the theranostic potential of 2-nitroimidazoles as ferroptosis inducers that enable targeting GSCs in their hypoxic niche.
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http://dx.doi.org/10.1038/s42003-020-01165-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431527PMC
August 2020

Antifungal Agent Luliconazole Inhibits the Growth of Mouse Glioma-initiating Cells in Brain Explants.

Keio J Med 2020 Dec 31;69(4):97-104. Epub 2020 Jul 31.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.

Imidazole antifungal compounds exert their antipathogenic effects through inhibition of sterol biosynthesis. These drugs have also recently been identified as candidate anticancer agents for several solid tumors including glioblastoma. However, their effects on glioma-initiating cells (GICs), i.e., glioma cells with stemlike properties that are able to initiate tumors, remain unclear. Consequently, we examined the effects of the optically active imidazole compound luliconazole on mouse GICs and GIC-based tumors. Luliconazole impaired in a concentration-dependent manner the growth of spheres formed by GICs in vitro. In contrast to the inhibitory effects of ionizing radiation and temozolomide on sphere growth, that of luliconazole was attenuated by the addition of exogenous cholesterol. Exposure to luliconazole of brain slices derived from mice with orthotopic GIC implants for 4 days in culture resulted in a marked increase in the number of tumor cells positive for cleaved caspase-3, but without a similar effect on normal cells. Furthermore, in brain slices, luliconazole inhibited the expansion of GIC-based tumors and the parenchymal infiltration of tumor cells. Our findings therefore indicate that luliconazole effectively targets GICs, thereby providing further support for the antitumorigenic effects of imidazole antifungal compounds.
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http://dx.doi.org/10.2302/kjm.2020-0001-OADOI Listing
December 2020

Downregulation of PD-L1 via FKBP5 by celecoxib augments antitumor effects of PD-1 blockade in a malignant glioma model.

Neurooncol Adv 2020 Jan-Dec;2(1):vdz058. Epub 2019 Dec 26.

Department of Neurosurgery, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan.

Background: Antitumor therapies targeting programmed cell death-1 (PD-1) or its ligand-1 (PD-L1) are used in various cancers. However, in glioblastoma (GBM), the expression of PD-L1 varies between patients, and the relationship between this variation and the efficacy of anti-PD-1 antibody therapy remains unclear. High expression levels of PD-L1 affect the proliferation and invasiveness of GBM cells. As COX-2 modulates PD-L1 expression in cancer cells, we tested the hypothesis that the COX-2 inhibitor celecoxib potentiates anti-PD-1 antibody treatment via the downregulation of PD-L1.

Methods: Six-week-old male C57BL/6 mice injected with murine glioma stem cells (GSCs) were randomly divided into four groups treated with vehicle, celecoxib, anti-PD-1 antibody, or celecoxib plus anti-PD-1 antibody and the antitumor effects of these treatments were assessed. To verify the mechanisms underlying these effects, murine GSCs and human GBM cells were studied in vitro.

Results: Compared with that with each single treatment, the combination of celecoxib and anti-PD-1 antibody treatment significantly decreased tumor volume and prolonged survival. The high expression of PD-L1 was decreased by celecoxib in the glioma model injected with murine GSCs, cultured murine GSCs, and cultured human GBM cells. This reduction was associated with post-transcriptional regulation of the co-chaperone FK506-binding protein 5 (FKBP5).

Conclusions: Combination therapy with anti-PD-1 antibody plus celecoxib might be a promising therapeutic strategy to target PD-L1 in glioblastoma. The downregulation of highly-expressed PD-L1 via FKBP5, induced by celecoxib, could play a role in its antitumor effects.
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http://dx.doi.org/10.1093/noajnl/vdz058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212915PMC
December 2019

Blood-Brain Barrier Permeability and Cytotoxicity of an Atorvastatin-Loaded Nanoformulation Against Glioblastoma in 2D and 3D Models.

Mol Pharm 2020 06 7;17(6):1835-1847. Epub 2020 May 7.

Functional Polymer Materials, Chair for Advanced Materials Synthesis, Department of Chemistry and Pharmacy and Bavarian Polymer Institute, University of Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b01117DOI Listing
June 2020

Author Correction: CD44 variant inhibits insulin secretion in pancreatic β cells by attenuating LAT1-mediated amino acid uptake.

Sci Rep 2020 Apr 3;10(1):6084. Epub 2020 Apr 3.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, 160-8582, 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/s41598-020-63111-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118093PMC
April 2020

Author Correction: CD44 variant inhibits insulin secretion in pancreatic β cells by attenuating LAT1-mediated amino acid uptake.

Sci Rep 2020 Apr 3;10(1):6084. Epub 2020 Apr 3.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, 160-8582, 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/s41598-020-63111-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118093PMC
April 2020

A Potent Blood-Brain Barrier-Permeable Mutant IDH1 Inhibitor Suppresses the Growth of Glioblastoma with IDH1 Mutation in a Patient-Derived Orthotopic Xenograft Model.

Mol Cancer Ther 2020 02 14;19(2):375-383. Epub 2019 Nov 14.

Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan.

Gliomas are the second most common primary brain tumors in adults. They are treated with combination therapies, including surgery, radiotherapy, and chemotherapy. There are currently limited treatment options for recurrent gliomas, and new targeted therapies need to be identified, especially in glioblastomas, which have poor prognosis. Isocitrate dehydrogenase (IDH) mutations are detected in various tumors, including gliomas. Most patients with IDH mutant glioma harbor the IDH1R132H subtype. Mutant IDH catalyzes the conversion of α-ketoglutarate to the oncometabolite 2-hydroxyglutarate (2-HG), which induces aberrant epigenetic status and contributes to malignant progression, and is therefore a potential therapeutic target for IDH mutant tumors. The present study describes a novel, orally bioavailable selective mutant IDH1 inhibitor, DS-1001b. The drug has high blood-brain barrier (BBB) permeability and inhibits IDH1R132H. Continuous administration of DS-1001b impaired tumor growth and decreased 2-HG levels in subcutaneous and intracranial xenograft models derived from a patient with glioblastoma with IDH1 mutation. Moreover, the expression of glial fibrillary acidic protein was strongly induced by DS-1001b, suggesting that inhibition of mutant IDH1 promotes glial differentiation. These results reveal the efficacy of BBB-permeable DS-1001b in orthotopic patient-derived xenograft models and provide a preclinical rationale for the clinical testing of DS-1001b in recurrent gliomas.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1349DOI Listing
February 2020

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma.

Nat Cell Biol 2019 08 1;21(8):1003-1014. Epub 2019 Aug 1.

Division of Human Biology, Solid Tumor and Translational Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.
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http://dx.doi.org/10.1038/s41556-019-0363-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686884PMC
August 2019

Visualization of spatiotemporal dynamics of human glioma stem cell invasion.

Mol Brain 2019 05 6;12(1):45. Epub 2019 May 6.

Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.
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http://dx.doi.org/10.1186/s13041-019-0462-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503361PMC
May 2019

Epidermal growth factor receptor promotes glioma progression by regulating xCT and GluN2B-containing N-methyl-d-aspartate-sensitive glutamate receptor signaling.

Cancer Sci 2018 Dec 5;109(12):3874-3882. Epub 2018 Nov 5.

Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan.

Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(-) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(-) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate-sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(-) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.
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http://dx.doi.org/10.1111/cas.13826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272110PMC
December 2018

HER2 Heterogeneity Is Associated with Poor Survival in HER2-Positive Breast Cancer.

Int J Mol Sci 2018 Jul 24;19(8). Epub 2018 Jul 24.

Department of Breast Surgery and Oncology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan.

Intratumoral human epidermal growth factor receptor 2 (HER2) heterogeneity has been reported in 16⁻36% of HER2-positive breast cancer and its clinical impact is under discussion. We examined the biological effects of HER2-heterogeneity on mouse models and analyzed metastatic brains by RNA sequence analysis. A metastatic mouse model was developed using 231-Luc (triple negative cells) and 2 HER2-positive cell lines, namely, HER2-60 and HER2-90 which showed heterogeneous and monotonous HER2 expressions, respectively. Metastatic lesions developed in 3 weeks in all the mice injected with HER2-60 cells, and in 69% of the mice injected with HER2-90 and 87.5% of the mice injected with 231-Luc. The median survival days of mice injected with 231-Luc, HER2-60, and HER2-90 cells were 29 ( = 24), 24 ( = 22) and 30 ( = 13) days, respectively. RNA sequence analysis showed that and its related genes were significantly downregulated in metastatic brain tumors with HER2-60 cells. The low expression of caspase-1 could be a new prognostic biomarker for early relapse in HER2-positive breast cancer.
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http://dx.doi.org/10.3390/ijms19082158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121890PMC
July 2018

Gold-nanofève surface-enhanced Raman spectroscopy visualizes hypotaurine as a robust anti-oxidant consumed in cancer survival.

Nat Commun 2018 04 19;9(1):1561. Epub 2018 Apr 19.

Department of Biochemistry, Keio University School of Medicine, Tokyo, 160-8582, Japan.

Gold deposition with diagonal angle towards boehmite-based nanostructure creates random arrays of horse-bean-shaped nanostructures named gold-nanofève (GNF). GNF generates many electromagnetic hotspots as surface-enhanced Raman spectroscopy (SERS) excitation sources, and enables large-area visualization of molecular vibration fingerprints of metabolites in human cancer xenografts in livers of immunodeficient mice with sufficient sensitivity and uniformity. Differential screening of GNF-SERS signals in tumours and those in parenchyma demarcated tumour boundaries in liver tissues. Furthermore, GNF-SERS combined with quantum chemical calculation identified cysteine-derived glutathione and hypotaurine (HT) as tumour-dominant and parenchyma-dominant metabolites, respectively. CD44 knockdown in cancer diminished glutathione, but not HT in tumours. Mechanisms whereby tumours sustained HT under CD44-knockdown conditions include upregulation of PHGDH, PSAT1 and PSPH that drove glycolysis-dependent activation of serine/glycine-cleavage systems to provide one-methyl group for HT synthesis. HT was rapidly converted into taurine in cancer cells, suggesting that HT is a robust anti-oxidant for their survival under glutathione-suppressed conditions.
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http://dx.doi.org/10.1038/s41467-018-03899-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908798PMC
April 2018

Tranilast inhibits the expression of genes related to epithelial-mesenchymal transition and angiogenesis in neurofibromin-deficient cells.

Sci Rep 2018 04 17;8(1):6069. Epub 2018 Apr 17.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, 160-8582, Japan.

Neurofibromatosis type 1 (NF1) is caused by germline mutations in the NF1 gene and is characterized by café au lait spots and benign tumours known as neurofibromas. NF1 encodes the tumour suppressor protein neurofibromin, which negatively regulates the small GTPase Ras, with the constitutive activation of Ras signalling resulting from NF1 mutations being thought to underlie neurofibroma development. We previously showed that knockdown of neurofibromin triggers epithelial-mesenchymal transition (EMT) signalling and that such signalling is activated in NF1-associated neurofibromas. With the use of a cell-based drug screening assay, we have now identified the antiallergy drug tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) as an inhibitor of EMT and found that it attenuated the expression of mesenchymal markers and angiogenesis-related genes in NF1-mutated sNF96.2 cells and in neurofibroma cells from NF1 patients. Tranilast also suppressed the proliferation of neurofibromin-deficient cells in vitro more effectively than it did that of intact cells. In addition, tranilast inhibited sNF96.2 cell migration and proliferation in vivo. Knockdown of type III collagen (COL3A1) also suppressed the proliferation of neurofibroma cells, whereas expression of COL3A1 and SOX2 was increased in tranilast-resistant cells, suggesting that COL3A1 and the transcription factor SOX2 might contribute to the development of tranilast resistance.
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http://dx.doi.org/10.1038/s41598-018-24484-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904101PMC
April 2018

CD44 variant inhibits insulin secretion in pancreatic β cells by attenuating LAT1-mediated amino acid uptake.

Sci Rep 2018 02 12;8(1):2785. Epub 2018 Feb 12.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, 160-8582, Japan.

CD44 variant (CD44v) contributes to cancer stemness by stabilizing the xCT subunit of system xc(-) and thereby promoting its glutamate-cystine antiporter activity. CD44 has also been implicated in autoimmune insulitis and inflammation in diabetic islets, but whether CD44v regulates insulin secretion has remained unclear. Here we show that CD44v inhibits insulin secretion by attenuating amino acid transport mediated by the L-type amino acid transporter LAT1. CD44v expression level was inversely related to insulin content in islets of normal and diabetic model mice. Knockdown of CD44 increased insulin secretion, the intracellular insulin level, and the transport of neutral amino acids mediated by LAT1 in Min6 cells. Attenuation of the uptake of neutral amino acids with a LAT inhibitor reduced insulin secretion and insulin content in Min6 cells, whereas overexpression of LAT1 increased insulin secretion. Moreover, inhibition of LAT1 prevented the increase in insulin secretion and content induced by CD44 depletion in Min6 cells. Our results thus implicate CD44v in the regulation of insulin secretion and reveal that amino acid transport is rate limiting for such secretion. They further suggest that amino acid transport mediated by LAT1 is a potential therapeutic target for diabetes.
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http://dx.doi.org/10.1038/s41598-018-20973-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809395PMC
February 2018

Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality.

Nat Commun 2017 12 19;8(1):2200. Epub 2017 Dec 19.

Program in Cancer & Stem Cell Biology, Duke-NUS Medical School, Singapore, 169857, Singapore.

Achieving robust cancer-specific lethality is the ultimate clinical goal. Here, we identify a compound with dual-inhibitory properties, named a131, that selectively kills cancer cells, while protecting normal cells. Through an unbiased CETSA screen, we identify the PIP4K lipid kinases as the target of a131. Ablation of the PIP4Ks generates a phenocopy of the pharmacological effects of PIP4K inhibition by a131. Notably, PIP4Ks inhibition by a131 causes reversible growth arrest in normal cells by transcriptionally upregulating PIK3IP1, a suppressor of the PI3K/Akt/mTOR pathway. Strikingly, Ras activation overrides a131-induced PIK3IP1 upregulation and activates the PI3K/Akt/mTOR pathway. Consequently, Ras-transformed cells override a131-induced growth arrest and enter mitosis where a131's ability to de-cluster supernumerary centrosomes in cancer cells eliminates Ras-activated cells through mitotic catastrophe. Our discovery of drugs with a dual-inhibitory mechanism provides a unique pharmacological strategy against cancer and evidence of cross-activation between the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways via a Ras˧PIK3IP1˧PI3K signaling network.
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http://dx.doi.org/10.1038/s41467-017-02287-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736559PMC
December 2017

Metabolic heterogeneity and plasticity of glioma stem cells in a mouse glioblastoma model.

Neuro Oncol 2018 02;20(3):343-354

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.

Background: Glioblastomas have been shown to rely on glycolysis as an energy source. However, recent evidence suggests that at least a subset of glioma cells with stem cell-like properties can thrive on oxidative phosphorylation. It remains unclear whether both metabolic phenotypes support tumor propagation, if they are independent, and how stable they are. The present study investigated these questions with the use of isogenic murine glioma stem cells (GSCs).

Methods: GSCs were established from tumors formed by Ink4a/Arf-null, H-RasV12-expressing glioma-initiating cells that differed in extracellular acidification potential. Metabolic characteristics of GSCs were determined by measurement of glucose, oxygen, and glutamine uptake, ATP content, and lactate production. Effects of metabolic inhibitors and changes in oxygen or nutrient availability on lactate production and tumorsphere growth were also determined.

Results: GSCs were found either to consume more glucose and produce more lactate or to consume more oxygen and maintain a higher ATP content depending on the metabolic characteristics of the tumor cells of origin. The latter, mitochondrial-type GSCs increased lactate production after treatment with the oxidative phosphorylation inhibitor oligomycin or phenformin. Exposure to hypoxia also increased lactate production and expression of glycolysis-related enzymes and metabolites in mitochondrial-type GSCs in a reversible manner.

Conclusions: Both glycolytic and mitochondrial-type energy production can sustain tumor propagation by isogenic GSCs. Whereas both phenotypes can be independent and stable, cells that rely on oxidative phosphorylation can also switch to a more glycolytic phenotype in response to metabolic stress, suggesting that plasticity is a further characteristic of GSC metabolism.
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http://dx.doi.org/10.1093/neuonc/nox170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817950PMC
February 2018

Organotypic brain explant culture as a drug evaluation system for malignant brain tumors.

Cancer Med 2017 Nov 4;6(11):2635-2645. Epub 2017 Oct 4.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.

Therapeutic options for malignant brain tumors are limited, with new drugs being continuously evaluated. Organotypic brain slice culture has been adopted for neuroscience studies as a system that preserves brain architecture, cellular function, and the vascular network. However, the suitability of brain explants for anticancer drug evaluation has been unclear. We here adopted a mouse model of malignant glioma based on expression of H-Ras in Ink4a/Arf neural stem/progenitor cells to establish tumor-bearing brain explants from adult mice. We treated the slices with cisplatin, temozolomide, paclitaxel, or tranilast and investigated the minimal assays required to assess drug effects. Serial fluorescence-based tumor imaging was sufficient for evaluation of cisplatin, a drug with a pronounced cytotoxic action, whereas immunostaining of cleaved caspase 3 (a marker of apoptosis) and of Ki67 (a marker of cell proliferation) was necessary for the assessment of temozolomide action and immunostaining for phosphorylated histone H3 (a marker of mitosis) allowed visualization of paclitaxel-specific effects. Staining for cleaved caspase 3 was also informative in the assessment of drug toxicity for normal brain tissue. Incubation of explants with fluorescently labeled antibodies to CD31 allowed real-time imaging of the microvascular network and complemented time-lapse imaging of tumor cell invasion into surrounding tissue. Our results suggest that a combination of fluorescence imaging and immunohistological staining allows a unified assessment of the effects of various classes of drug on the survival, proliferation, and invasion of glioma cells, and that organotypic brain slice culture is therefore a useful tool for evaluation of antiglioma drugs.
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http://dx.doi.org/10.1002/cam4.1174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673912PMC
November 2017

Convection-enhanced delivery of sulfasalazine prolongs survival in a glioma stem cell brain tumor model.

J Neurooncol 2018 Jan 19;136(1):23-31. Epub 2017 Sep 19.

Department of Neurosurgery, Tohoku University Graduate School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan.

Expression of CD44 in glioma cells was previously correlated with tumor grade and is considered a stem cell marker. CD44 stabilizes the cystine-glutamate transporter (xCT) and inhibits apoptosis in cancer stem cells (CSCs). Recently it was found that Sulfasalazine (SSZ), an anti-inflammatory drug, acts as an inhibitor of xCT and therefore has potential as a targeted therapy for CSCs. In this study, we tested an efficacy of SSZ against glioma stem cell model developed in rats. As poor penetration of blood-brain barrier resulted in insufficient efficacy of systemic SSZ treatment, SSZ was delivered locally with convection-enhanced delivery (CED). In vitro, expression of CD44 in glioma cells and efficacy of SSZ against glioma cells and glioma stem cells were confirmed. SSZ demonstrated anti-proliferative activity in a dose dependent manner against these cells. This activity was partially reversible with the addition of antioxidant, N-acetyl-L-cysteine, to the medium. In vivo, CED successfully delivered SSZ into the rat brain parenchyma. When delivered at 5 mM concentration, which was the highest possible concentration when SSZ was dissolved in water, CED of SSZ resulted in almost no tissue damage. Against highly malignant bRiTs-G3 brain tumor xenografted rat model; the glioma stem cell model, CED of SSZ at 5 mM concentration induced apoptosis and prolonged survival. Consequently, CED of SSZ induced glioma stem cell death without evidence of tissue damage to normal brain parenchyma. This strategy may be a promising targeted treatment against glioma stem cells.
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http://dx.doi.org/10.1007/s11060-017-2621-7DOI Listing
January 2018

Modeling phenotypes of malignant gliomas.

Cancer Sci 2018 Jan 15;109(1):6-14. Epub 2017 Nov 15.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Malignant gliomas are primary tumors of the central nervous system characterized by diffuse infiltration into the brain and a high recurrence rate. Advances in comprehensive genomic studies have provided unprecedented insight into the genetic and molecular heterogeneity of these tumors and refined our understanding of their evolution from low to high grade. However, similar levels of phenotypic characterization are indispensable to understanding the complexity of malignant gliomas. Experimental glioma models have also achieved great progress in recent years. Advances in transgenic technologies and cell culture have allowed the establishment of mouse models that mirror the human disease with increasing fidelity and which support single-cell resolution for phenotypic analyses. Here we review the major types of preclinical glioma models, with an emphasis on how recent developments in experimental modeling have shed new light on two fundamental aspects of glioma phenotype, their cell of origin and their invasive potential.
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http://dx.doi.org/10.1111/cas.13351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765309PMC
January 2018

Endothelium-induced three-dimensional invasion of heterogeneous glioma initiating cells in a microfluidic coculture platform.

Integr Biol (Camb) 2017 09;9(9):762-773

Department of System Design Engineering, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan.

Glioblastoma (GBM) is a highly invasive primary brain tumor that displays cellular heterogeneity, which is composed of glioma initiating cells (GICs) and their differentiated progeny. GICs play an important role in driving aggressive invasion. In particular, the interaction between GICs and blood vessels is critical because blood vessels are known to serve as routes for the invasion of GICs. However, the effect of endothelial cells on the three-dimensional (3D) invasion process of GICs as well as the spatial relationship between GICs and their differentiated progeny remains unclear. Here, we utilized a microfluidic device to recapitulate the 3D brain tumor microenvironments constituted by human umbilical vein endothelial cells (HUVECs) and type I collagen. Using the device, we found that HUVECs promoted the 3D invasion of heterogeneous glioma cell populations into type I collagen gel. The invasion induced by HUVECs was predominantly preceded by cells positive for nestin, a neural stem cell marker. In contrast, cells positive for tubulin β3 (TUBB3), a differentiated cell marker, rarely preceded invasion. In addition, HUVECs induced the upregulation of TUBB3 in GICs. Finally, we found that the genes associated with invasion, such as integrins α2 and β3, were significantly upregulated in the presence of HUVECs. These results as well as the experimental approach provide valuable knowledge for the development of effective therapeutic strategies targeting the aggressive invasion of GBM.
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http://dx.doi.org/10.1039/c7ib00091jDOI Listing
September 2017

Functional analysis of the DEPDC1 oncoantigen in malignant glioma and brain tumor initiating cells.

J Neurooncol 2017 Jun 29;133(2):297-307. Epub 2017 May 29.

Department of Neurosurgery, Keio University School of Medicine, Tokyo, Japan.

DEP domain containing 1 (DEPDC1) is a novel oncoantigen expressed in cancer cells, which presents oncogenic activity and high immunogenicity. Although DEPDC1 has been predicted to be a useful antigen for the development of a cancer vaccine, its pathophysiological roles in glioma have not been investigated. Here, we analyzed the expression and function of DEPDC1 in malignant glioma. DEPDC1 expression in glioma cell lines, glioma tissues, and brain tumor initiating cells (BTICs) was assessed by western blot and quantitative polymerase chain reaction (PCR). The effect of DEPDC1 downregulation on cell growth and nuclear factor kappa B (NFκB) signaling in glioma cells was investigated. Overall survival was assessed in mouse glioma models using human glioma cells and induced mouse brain tumor stem cells (imBTSCs) to determine the effect of DEPDC1 suppression in vivo. DEPDC1 expression was increased in glioma cell lines, tissues, and BTICs. Suppression of endogenous DEPDC1 expression by small interfering RNA (siRNA) inhibited glioma cell viability and induced apoptosis through NFκB signaling. In mouse glioma models using human glioma cells and imBTSCs, downregulation of DEPDC1 expression prolonged overall survival. These results suggest that DEPDC1 represents a target molecule for the treatment of glioma.
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http://dx.doi.org/10.1007/s11060-017-2457-1DOI Listing
June 2017

Blocking COX-2 induces apoptosis and inhibits cell proliferation via the Akt/survivin- and Akt/ID3 pathway in low-grade-glioma.

J Neurooncol 2017 04 10;132(2):231-238. Epub 2017 Mar 10.

Department of Neurosurgery, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima, 770-8503, Japan.

Approximately half of surgically-treated patients with low-grade-glioma (LGG) suffer recurrence or metastasis. Currently there is no effective drug treatment. While the selective COX-2 inhibitor celecoxib showed anti-neoplastic activity against several malignant tumors, its effects against LGG remain to be elucidated. Ours is the first report that the expression level of COX-2 in brain tissue samples from patients with LGG and in LGG cell lines is higher than in the non-neoplastic region and in normal brain cells. We found that celecoxib attenuated LGG cell proliferation in a dose-dependent manner. It inhibited the generation of prostaglandin E2 and induced apoptosis and cell-cycle arrest. We also show that celecoxib hampered the activation of the Akt/survivin- and the Akt/ID3 pathway in LGGs. These findings suggest that celecoxib may have a promising therapeutic potential and that the early treatment of LGG patients with the drug may be beneficial.
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http://dx.doi.org/10.1007/s11060-017-2380-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763415PMC
April 2017

RNA Sequencing Analysis Reveals Interactions between Breast Cancer or Melanoma Cells and the Tissue Microenvironment during Brain Metastasis.

Biomed Res Int 2017 22;2017:8032910. Epub 2017 Jan 22.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.

Metastasis is the main cause of treatment failure and death in cancer patients. Metastasis of tumor cells to the brain occurs frequently in individuals with breast cancer, non-small cell lung cancer, or melanoma. Despite recent advances in our understanding of the causes and in the treatment of primary tumors, the biological and molecular mechanisms underlying the metastasis of cancer cells to the brain have remained unclear. Metastasizing cancer cells interact with their microenvironment in the brain to establish metastases. We have now developed mouse models of brain metastasis based on intracardiac injection of human breast cancer or melanoma cell lines, and we have performed RNA sequencing analysis to identify genes in mouse brain tissue and the human cancer cells whose expression is associated specifically with metastasis. We found that the expressions of the mouse genes , , , and as well as those of the human genes , , , , , and were upregulated in brain tissue harboring metastases. Further characterization of such genes that contribute to the establishment of brain metastases may provide a basis for the development of new therapeutic strategies and consequent improvement in the prognosis of cancer patients.
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http://dx.doi.org/10.1155/2017/8032910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5292181PMC
February 2017

[p53 mutations in brain tumors].

Nihon Rinsho 2016 09;74 Suppl 7:112-116

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September 2016

Histopathological investigation of glioblastomas resected under bevacizumab treatment.

Oncotarget 2016 Aug;7(32):52423-52435

Department of Neurosurgery, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, Japan.

To date, no clinical observations have been reported for histopathological changes in human gliomas under antiangiogenic treatment.We collected six glioblastomas resected under bevacizumab treatment. Histopathological investigation was performed by hematoxilyn-eosin staining and immunohistochemistry for CD34, VEGF, VEGFR1/2, HIF-1α, CA9, and nestin as compared to eleven control glioblastomas to assess the differences in histological features, microvessel density, expression of VEGF and its receptors, tumor oxygenation, and status of glioma stem-like cells.In the six tumors resected under bevacizumab, microvascular proliferation was absent, and microvessel density had significantly decreased compared with that of the controls. The expressions of VEGF and its receptors were downregulated in two cases of partial response. HIF-1α or CA9 expression was decreased in five of the six tumors, whereas the decreased expression of these markers was noted in only one of the 11 control glioblastomas. The expression of nestin significantly decreased in the six tumors compared with that of the controls, with the remaining nestin-positive cells being relatively concentrated around vessels.We provide the first clinicopathological evidence that antiangiogenic therapy induces the apparent normalization of vascular structure, decrease of microvessel density, and improvement of tumor oxygenation in glioblastomas. These in situ observations will help to optimize therapy.
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http://dx.doi.org/10.18632/oncotarget.9387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5239563PMC
August 2016

Generation of heterozygous fibrillin-1 mutant cloned pigs from genome-edited foetal fibroblasts.

Sci Rep 2016 Apr 14;6:24413. Epub 2016 Apr 14.

Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, 160-8582, Japan.

Marfan syndrome (MFS) is an autosomal dominant genetic disease caused by abnormal formation of the extracellular matrix with an incidence of 1 in 3, 000 to 5, 000. Patients with Marfan syndrome experience poor quality of life caused by skeletal disorders such as scoliosis, and they are at high risk of sudden death from cardiovascular impairment. Suitable animal models of MFS are essential for conquering this intractable disease. In particular, studies employing pig models will likely provide valuable information that can be extrapolated to humans because of the physiological and anatomical similarities between the two species. Here we describe the generation of heterozygous fibrillin-1 (FBN1) mutant cloned pigs (+/Glu433AsnfsX98) using genome editing and somatic cell nuclear transfer technologies. The FBN1 mutant pigs exhibited phenotypes resembling those of humans with MFS, such as scoliosis, pectus excavatum, delayed mineralization of the epiphysis and disrupted structure of elastic fibres of the aortic medial tissue. These findings indicate the value of FBN1 mutant pigs as a model for understanding the pathogenesis of MFS and for developing treatments.
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http://dx.doi.org/10.1038/srep24413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830947PMC
April 2016