Senji Shirasawa

Fukuoka University, Hukuoka, Fukuoka, Japan

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Publications (167)981.4 Total impact


  • No preview · Article · Jan 2016
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    Full-text · Article · Jan 2016
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    ABSTRACT: ANRIL is a long noncoding RNA transcribed from the INK4 locus that encodes three tumor suppressor genes, p15, p16, and ARF. Previous studies demonstrated that ANRIL represses p15 and p16, which positively regulate the pRB pathway, leading to repression of cellular senescence of human normal fibroblasts. However, the role of ANRIL in cancer cell proliferation is less well understood. Here we report that ANRIL is involved in the proliferation of colorectal cancer HCT116 cells in two- and three-dimensional culture. Silencing ANRIL by both transfection with small interfering RNA and retrovirally produced small hairpin RNA reduced HCT116 cell proliferation in both two- and three-dimensional culture. HCT116 cells depleted for ANRIL were arrested in the S phase of cell cycle. Notably, silencing ANRIL did not result in the activation of expression of the INK4 locus. These results suggest that ANRIL positively regulates the proliferation of HCT116 cells in two- and three-dimensional culture in a p15/p16-pRB pathway-independent manner.
    No preview · Article · Dec 2015 · Molecular and Cellular Biochemistry
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    ABSTRACT: To examine the cytotoxic activity of congenes of 3-amino-isoquinoline, we performed the phenotypic screening using panel of 60 cell lines and found that (N-(6,7-dimethoxy-1-methyl-isoquinolin-3-yl)-4-{[(1-ethyl-4-methyl-1H-pyrazol-3-yl)methyl]amino}benzamide (4d)) exhibited the significant effect against different tumor cell lines while showing the high activity toward human colorectal cancer HCT-116 cells (IC50 = 18 μM) and human breast cancer T-47D cells (GI50 = 1.9 μM). Virtual screening indicated that these compounds targets protein kinases and phosphodiesterases (PDE). However, wet screening among panel of protein kinases did not show any significant activity. By contrast, 50μM of 4c and 4d inhibited the growth of HKe3-mtKRAS spheroids in the 3D floating (3DF) culture suggesting that 4c and 4d targets PDE4B which is selectively upregulated by mtKRAS in 3DF culture. This article is protected by copyright. All rights reserved.
    No preview · Article · Nov 2015 · Chemical Biology & Drug Design
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    ABSTRACT: In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.
    Preview · Article · Sep 2015 · PLoS ONE
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    ABSTRACT: Purpose: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumour initiation, neo-vascularization and metastasis in a wide range of epithelial and mesenchymal cancers, however its role in colorectal cancer (CRC) recurrence and progression is unclear. Experimental Design: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumours (N=338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive CRC cell line models. Results: Colorectal tumours displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III CRC tissues, in both univariate and multivariate analyses. Pre-clinically, we found that EphA2 was highly expressed in KRASMT CRC cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines and down-regulation of EphA2 using RNAi or recombinant EFNA1, suppressed migration and invasion of KRASMT CRC cells. Conclusions: These data show that EpHA2 is a poor prognostic marker in stage II/III CRC, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target. http://clincancerres.aacrjournals.org/content/early/2015/08/15/1078-0432.CCR-15-0603.abstract
    Full-text · Article · Aug 2015 · Clinical Cancer Research
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    ABSTRACT: Apoptosis-inducing factor, mitochondrion-associated 2 (Aifm2), is a DNA-binding oxoreductase protein that promotes apoptosis. To assess its potential role in erythropoiesis we analyzed the effects of Aifm2 loss-of-function in the murine erythroleukemia line (MEL). MEL cells were transfected with siRNA targeting Aifm2 for 24 h and evaluated by cell counting, flow cytometry with annexin V and PI staining and gene expression analysis. Aifm2 knockdown did not affect the apoptotic status of MEL cells. However, Aifm2 knockdown significantly increased expression of the erythropoietic transcription factor Klf1 (2.9±0.2-fold, p<0.05) and decreased α- and β-globin expression (0.6±0.2-fold, p<0.05 and 0.5±0.2-fold, p<0.01). Aifm2 may function in differentiation of erythroid MEL cells in vitro. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
    No preview · Article · Aug 2015 · Anticancer research
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    ABSTRACT: Oncogenic mutations in the KRAS gene are critically involved in many human tumors but drugs targeting oncogenic KRAS have not yet been clinically developed. Herein, we established a three-dimensional floating (3DF) culture system for screening drugs that target KRAS-mediated signaling molecules. HKe3 cells, derived from colorectal cancer HCT116 cells and disrupted at mutated (mt) KRAS gene, were infected with a retrovirus expressing wild-type (wt) KRAS or mtKRAS to establish HKe3-derived cells expressing wtKRAS or mtKRAS. Established cells were cultured in 96-well plates with an ultra-low attachment surface and round bottom for 3DF culture. HKe3-wtKRAS and HKe3-mtKRAS cells in 3DF culture rapidly assembled into respective single spherical structures (spheroids). Furthermore, mtKRAS but not wtKRAS expression inhibited luminal apoptosis in spheroids indicating that the 3DF culture was compatible with the 3D matrigel culture. This 3DF culture system could be useful for screening drugs that target KRAS-mediated signaling molecules. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
    Full-text · Article · Aug 2015 · Anticancer research
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    ABSTRACT: Zfat is a nuclear protein that harbours putative DNA-binding domains. T-cell specific deletion of Zfat in Zfat(f/f)-CD4Cre mice yields a significant decrease in the number of peripheral T-cells with a lower surface expression of interleukin-7 receptor-α (IL-7Rα). However, the molecular mechanism by which Zfat controls IL-7Rα expression remains unknown. Expression levels of the molecules involved in IL-7Rα expression were determined by immunoblotting. Zfat-deficient peripheral T-cells showed a marked reduction in the FoxO1 protein that regulates IL-7Rα expression; however, the FoxO1 mRNA expression level was not affected by Zfat-deficiency. Furthermore, treatment of Zfat-deficient T-cells with a proteasome inhibitor, epoxomicin, restored FoxO1 expression levels, indicating that the loss of Zfat enhanced the proteasomal degradation of the FoxO1 protein. These results suggest that Zfat is required for peripheral T-cell homeostasis through IL-7Rα expression by controlling the FoxO1 protein. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
    No preview · Article · Aug 2015 · Anticancer research
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    ABSTRACT: Detachment of non-malignant intestinal epithelial cells from the extracellular matrix (ECM) triggers their growth arrest and, ultimately, apoptosis. In contrast, colorectal cancer cells can grow without attachment to the ECM. This ability is critical for their malignant potential. We found previously that detachment-induced growth arrest of non-malignant intestinal epithelial cells is driven by their detachment-triggered autophagy, and that RRAS, a major oncogene, promotes growth of detached cells by blocking such autophagy. In an effort to identify the mechanisms of detachment-induced autophagy and growth arrest of non-malignant cells we found here that detachment of these cells causes upregulation of ATG3 and that ATG3 upregulation contributes to autophagy and growth arrest of detached cells. We also observed that when ATG3 expression is artificially increased in the attached cells, ATG3 promotes neither autophagy nor growth arrest but triggers their apoptosis. ATG3 upregulation likely promotes autophagy of the detached but not that of the attached cells because detachment-dependent autophagy requires other detachment-induced events, such as the upregulation of ATG7. We further observed that those few adherent cells that do not die by apoptosis induced by ATG3 become resistant to apoptosis caused by cell detachment, a property that is critical for the ability of normal epithelial cells to become malignant. We conclude that cell-ECM adhesion can switch ATG3 functions: when upregulated in detached cells in the context of other autophagy-promoting events, ATG3 contributes to autophagy. However, when overexpressed in the adherent cells, in the circumstances not favoring autophagy, ATG3 triggers apoptosis.
    Full-text · Article · Jun 2015 · Autophagy
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    ABSTRACT: ZFAT is a transcriptional regulator, containing eighteen C2H2-type zinc-fingers and one AT-hook, involved in autoimmune thyroid disease, apoptosis, and immune-related cell survival. We determined the solution structures of the thirteen individual ZFAT zinc-fingers (ZF) and the tandemly arrayed zinc-fingers in the regions from ZF2 to ZF5, by NMR spectroscopy. ZFAT has eight uncommon bulged-out helix-containing zinc-fingers, and six of their structures (ZF4, ZF5, ZF6, ZF10, ZF11, and ZF13) were determined. The distribution patterns of the putative DNA-binding surface residues are different among the ZFAT zinc-fingers, suggesting the distinct DNA sequence preferences of the N-terminal and C-terminal zinc-fingers. Since ZFAT has three to five consecutive tandem zinc-fingers, which may cooperatively function as a unit, we also determined two tandemly arrayed zinc-finger structures, between ZF2 to ZF4 and ZF3 to ZF5. Our NMR spectroscopic analysis detected the interaction between ZF4 and ZF5, which are connected by an uncommon linker sequence, KKIK. The ZF4–ZF5 linker restrained the relative structural space between the two zinc-fingers in solution, unlike the other linker regions with determined structures, suggesting the involvement of the ZF4–ZF5 interfinger linker in the regulation of ZFAT function. Electronic supplementary material The online version of this article (doi:10.1007/s10969-015-9196-3) contains supplementary material, which is available to authorized users.
    Full-text · Article · Mar 2015 · Journal of Structural and Functional Genomics
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    ABSTRACT: Zfat, which is a nuclear protein harboring an AT-hook domain and 18-repeats of C2H2 zinc-finger motif, is highly expressed in immune-related tissues, including the thymus and spleen. T cell specific deletion of the Zfat gene by crossing Zfatf/f mice with LckCre mice yields a significant reduction in the number of CD4+CD8+double-positive (DP) thymocytes. However, physiological role for Zfat in T cell development in the thymus remains unknown. Here, we found that Zfat-deficient CD4+CD8+double-positive (DP) thymocytes in Zfatf/f-LckCre mice were susceptible to apoptosis both at an unstimulated state and in response to T cell receptor (TCR)-stimulation. The phosphorylation levels of p38 and JNK were elevated in Zfat-deficient thymocytes at an unstimulated state with an enhanced phosphorylation of ATF2 and with an over-expression of Gadd45α. On the other hand, the activation of JNK in the Zfat-deficient thymocytes, but not p38, was strengthened and prolonged in response to TCR-stimulation. All these results demonstrate that Zfat critically participates in the development of DP thymocytes through regulating the activities of p38 and JNK. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
    No preview · Article · Jan 2015 · Journal of Cellular Biochemistry
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    ABSTRACT: Recent ultrahigh-density tiling array and large-scale transcriptome analysis have revealed that large numbers of long non-coding RNAs (lncRNAs) are transcribed in mammals. Several lncRNAs have been implicated in transcriptional regulation, organization of nuclear structure, and post-transcriptional processing. However, the regulation of expression of lncRNAs is less well understood. Here, we show that the exogenous and endogenous expression of an oncogenic form of small GTPase Ras (called oncogenic Ras) decrease the expression of lncRNA ANRIL (antisense non-coding RNA in the INK4 locus), which is involved in the regulation of cellular senescence. We also show that forced expression of oncogenic Ras increases the expression of lncRNA PANDA (p21 associated ncRNA DNA damage activated), which is involved in the regulation of apoptosis. Microarray analysis demonstrated that expression of multiple lncRNAs fluctuated by forced expression of oncogenic Ras. These findings indicate that oncogenic Ras regulates the expression of a large number of lncRNAs including functional lncRNAs, such as ANRIL and PANDA.
    No preview · Article · Dec 2014 · Cytotechnology
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    ABSTRACT: KRAS gene mutations occur in approximately 40% of colorectal cancers (CRCs) and are associated with resistance to anti-epidermal growth factor receptor antibody therapy. We previously demonstrated that (18)F-FDG accumulation in PET was significantly higher in CRCs with mutated KRAS than in those with wild-type KRAS in a clinical setting. Here, we investigated the mechanisms by which mutated KRAS increased (18)F-FDG accumulation. Using paired isogenic human CRC cell lines that differ only in the mutational status of the KRAS gene, we measured (18)F-FDG accumulation in these cells in vitro and in vivo. We also investigated the roles of proteins that have a function in (18)F-FDG accumulation. Finally, we examined the relationship among mutated KRAS, hypoxia-inducible factor 1α (HIF-1α), and maximum standardized uptake value with 51 clinical CRC samples. In the in vitro experiments, (18)F-FDG accumulation was significantly higher in KRAS-mutant cells than in wild-type controls under normoxic conditions. The expression levels of glucose transporter 1 (GLUT1) and hexokinase type 2 (HK2) were higher in KRAS-mutant cells, and (18)F-FDG accumulation was decreased by knockdown of GLUT1. Hypoxic induction of HIF-1α was higher in KRAS-mutant cells than in wild-type controls; in turn, elevated HIF-1α resulted in higher GLUT1 expression and (18)F-FDG accumulation. In addition, HIF-1α knockdown decreased (18)F-FDG accumulation under hypoxic conditions only in the KRAS-mutant cells. Small-animal PET scans showed in vivo (18)F-FDG accumulation to be significantly higher in xenografts with mutated KRAS than in those with wild-type KRAS. The immunohistochemistry of these xenograft tumors showed that staining of GLUT1 was consistent with that of HIF-1α and pimonidazole. In a retrospective analysis of clinical samples, KRAS mutation exhibited a significantly positive correlation with expressions of GLUT1 and HIF-1α and with maximum standardized uptake value. Mutated KRAS caused higher (18)F-FDG accumulation possibly by upregulation of GLUT1; moreover, HIF-1α additively increased (18)F-FDG accumulation in hypoxic lesions. (18)F-FDG PET might be useful for predicting the KRAS status noninvasively. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.
    Full-text · Article · Dec 2014 · Journal of Nuclear Medicine
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    ABSTRACT: Human carcinomas are comprised of complex mixtures of tumor cells that are known to compete indirectly for nutrients and growth factors. Whether tumor cells could also compete directly, for example by elimination of rivals, is not known. Here we show that human cells can directly compete by a mechanism of engulfment called entosis. By entosis, cells are engulfed, or cannibalized while alive, and subsequently undergo cell death. We find that the identity of engulfing ("winner") and engulfed ("loser") cells is dictated by mechanical deformability controlled by RhoA and actomyosin, where tumor cells with high deformability preferentially engulf and outcompete neighboring cells with low deformability in heterogeneous populations. We further find that activated Kras and Rac signaling impart winner status to cells by downregulating contractile myosin, allowing for the internalization of neighboring cells that eventually undergo cell death. Finally, we compute the energy landscape of cell-in-cell formation, demonstrating that a mechanical differential between winner and loser cells is required for entosis to proceed. These data define a mechanism of competition in mammalian cells that occurs in human tumors.Cell Research advance online publication 24 October 2014; doi:10.1038/cr.2014.138.
    Full-text · Article · Nov 2014 · Cell Research

  • No preview · Article · Oct 2014 · Cancer Research
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    ABSTRACT: Background: We have previously reported the crucial roles of oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) in inhibiting apoptosis and disrupting cell polarity via the regulation of phosphodiesterase 4 (PDE4) expression in human colorectal cancer HCT116 cells in three-dimensional cultures (3DC). Herein we evaluated the effects of resveratrol, a PDE4 inhibitor, on the luminal cavity formation and the induction of apoptosis in HCT116 cells. Materials and methods: Apoptosis was detected by immunofluorescence using confocal laser scanning microscopy with an antibody against cleaved caspase-3 in HCT116 cells treated with or without resveratrol in a two-dimensional culture (2DC) or 3DC. Results: Resveratrol did not induce apoptosis of HCT116 cells in 2DC, whereas the number of apoptotic HCT116 cells increased after resveratrol treatment in 3DC, leading to formation of a luminal cavity. Conclusion: Resveratrol induced apoptosis of HCT116 cells in 3DC, resulting in the formation of a luminal cavity, probably by inhibiting PDE4 activity.
    Full-text · Article · Aug 2014 · Anticancer research
  • Keiko Doi · Shuhei Ishikura · Senji Shirasawa
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    ABSTRACT: ZFAT (zinc-finger gene in AITD susceptibility region), originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in various cellular processes and several common diseases including multiple sclerosis. Recent studies revealed that mouse Zfat is a novel critical regulator for both thymocyte differentiation and peripheral T-cell homeostasis. Zfat deficiency at early thymocyte developmental stages results in the inhibition of the development of CD4(+)CD8(+) thymocytes with an impaired positive selection. Zfat deficiency in peripheral T-cells results in a reduction in the number of T-cells with decreased expression of the interleukin-7 receptor-α (IL-7Rα) that is critical for T-cell homeostasis. In addition, T-cell antigen receptor stimulation-induced responses of Zfat-deficient T-cells are also impaired, with reduced IL-2Rα expression. This review highlights and discusses the roles of Zfat in thymocyte differentiation of T-cells and in the homeostasis of naive T-cells with recent work.
    No preview · Article · Aug 2014 · Anticancer research
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    ABSTRACT: Background/aim: Docking protein 2 (Dok2) is an adapter protein which is involved in hematopoiesis. However, it still remains unclear how Dok2 functions in regulation of transcription of hematopoietic genes. To address this issue, we knocked-down Dok2 mRNA in mouse erythroleukemia cells which highly express Dok2 intrinsically. Materials and methods: Mouse erythroleukemia cells were transfected with Dok2 siRNA for 24 h and gene expression of erythroid differentiation-related genes, such as GATA binding protein 1 (Gata1), Krüppel-like factor 1 (Klf1), α-globin and β-globin were assessed by real-time polymerase chain reaction. Results: Among the tested genes, expression of Klf1 exhibited a 1.94-fold increase when compared to the control 24 h after transfection. Immunocytochemistry and chromatin immunoprecipitation assays revealed that Dok2 protein localizes in the nucleus and binds to the promoter region of Klf1 gene. Conclusion: Dok2 is able to control Klf1 expression by transcriptional regulation through directly binding to its promoter region.
    No preview · Article · Aug 2014 · Anticancer research
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    ABSTRACT: There are currently no approved targeted therapies for advanced KRAS mutant (KRASMT) colorectal cancer (CRC). Using a unique systems biology approach, we identified JAK1/2-dependent activation of STAT3 as the key mediator of resistance to MEK inhibitors in KRASMT CRC in vitro and in vivo. Further analyses identified acute increases in c-MET activity following treatment with MEK inhibitors in KRASMT CRC models, which was demonstrated to promote JAK1/2-STAT3-mediated resistance. Furthermore, activation of c-MET following MEK inhibition was found to be due to inhibition of the ERK-dependent metalloprotease ADAM17, which normally inhibits c-MET signaling by promoting shedding of its endogenous antagonist, soluble "decoy" MET. Most importantly, pharmacological blockade of this resistance pathway with either c-MET or JAK1/2 inhibitors synergistically increased MEK-inhibitor-induced apoptosis and growth inhibition in vitro and in vivo in KRASMT models, providing clear rationales for the clinical assessment of these combinations in KRASMT CRC patients.
    Full-text · Article · Jun 2014 · Cell Reports

Publication Stats

6k Citations
981.40 Total Impact Points

Institutions

  • 2007-2015
    • Fukuoka University
      • Faculty of Medicine
      Hukuoka, Fukuoka, Japan
  • 2002
    • Harvard Medical School
      • Department of Neurobiology
      Boston, Massachusetts, United States
  • 1993-2001
    • Kyushu University
      • Division of Cancer Genetics
      Hukuoka, Fukuoka, Japan
  • 2000
    • Howard Hughes Medical Institute
      Ашбърн, Virginia, United States
  • 1998
    • Sunnybrook Health Sciences Centre
      Toronto, Ontario, Canada