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Jahangiri L. The link between the trans-Golgi network and tumour progression. Mol Biol Rep 2025; 52:435. [PMID: 40293576 PMCID: PMC12037649 DOI: 10.1007/s11033-025-10548-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2025] [Accepted: 04/25/2025] [Indexed: 04/30/2025]
Abstract
The trans-Golgi network is a major sorting organelle consisting of a tubular membrane originating from the trans-Golgi cisternae. Proteins and lipids synthesised in the endoplasmic reticulum are transported through the Golgi apparatus and sorted in the trans-Golgi network into pleomorphic transport carriers targeted for various destinations. These destinations include the apical and basolateral membranes, early and late, recycling endosomes, and secretory granules. The trans-Golgi network also accepts retrograde endosome traffic, contributing to the recycling of proteins and lipids, and, therefore, sits at the crossroads of secretory and endosomal systems. Cancer is a somatic evolutionary process that comprises the accumulation of mutations that contribute to tumourigenesis, growth, progression, immune evasion, and resistance to therapy. This study aims to catalogue how multiple components and players of the trans-Golgi network affect tumour progression. Further, the link between the tumour microenvironment, the trans-Golgi network, and tumour progression will be dissected. A more profound understanding of these mechanisms will inform better treatment options.
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Affiliation(s)
- Leila Jahangiri
- School of Science and Technology, Nottingham Trent University, Clifton Site, Nottingham, NG11 8NS, UK.
- Division of Cellular and Molecular Pathology, Department of Pathology, Addenbrookes Hospital, University of Cambridge, Cambridge, CB0 2QQ, UK.
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2
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Godinez-Macias KP, Chen D, Wallis JL, Siegel MG, Adam A, Bopp S, Carolino K, Coulson LB, Durst G, Thathy V, Esherick L, Farringer MA, Flannery EL, Forte B, Liu T, Godoy Magalhaes L, Gupta AK, Istvan ES, Jiang T, Kumpornsin K, Lobb K, McLean KJ, Moura IMR, Okombo J, Payne NC, Plater A, Rao SPS, Siqueira-Neto JL, Somsen BA, Summers RL, Zhang R, Gilson MK, Gamo FJ, Campo B, Baragaña B, Duffy J, Gilbert IH, Lukens AK, Dechering KJ, Niles JC, McNamara CW, Cheng X, Birkholtz LM, Bronkhorst AW, Fidock DA, Wirth DF, Goldberg DE, Lee MCS, Winzeler EA. Revisiting the Plasmodium falciparum druggable genome using predicted structures and data mining. NPJ DRUG DISCOVERY 2025; 2:3. [PMID: 40066064 PMCID: PMC11892419 DOI: 10.1038/s44386-025-00006-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/07/2024] [Accepted: 01/22/2025] [Indexed: 03/19/2025]
Abstract
Identification of novel drug targets is a key component of modern drug discovery. While antimalarial targets are often identified through the mechanism of action studies on phenotypically derived inhibitors, this method tends to be time- and resource-consuming. The discoverable target space is also constrained by existing compound libraries and phenotypic assay conditions. Leveraging recent advances in protein structure prediction, we systematically assessed the Plasmodium falciparum genome and identified 867 candidate protein targets with evidence of small-molecule binding and blood-stage essentiality. Of these, 540 proteins showed strong essentiality evidence and lack inhibitors that have progressed to clinical trials. Expert review and rubric-based scoring of this subset based on additional criteria such as selectivity, structural information, and assay developability yielded 27 high-priority antimalarial target candidates. This study also provides a genome-wide data resource for P. falciparum and implements a generalizable framework for systematically evaluating and prioritizing novel pathogenic disease targets.
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Affiliation(s)
| | - Daisy Chen
- Department of Pediatrics, University of California, San Diego, La Jolla, CA USA
| | | | | | - Anna Adam
- MMV Medicines for Malaria Venture, 1215, Geneva, Switzerland
| | - Selina Bopp
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA USA
| | - Krypton Carolino
- Department of Pediatrics, University of California, San Diego, La Jolla, CA USA
| | - Lauren B. Coulson
- Holistic Drug Discovery and Development (H3D) Centre, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa
| | - Greg Durst
- Lgenia, Inc., 412 S Maple St, Fortville, IN USA
| | - Vandana Thathy
- Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY USA
- Center for Malaria Therapeutics and Antimicrobial Resistance, Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY USA
| | - Lisl Esherick
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA USA
| | - Madeline A. Farringer
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA USA
- Division of Infectious Diseases, Boston Children’s Hospital, Boston, MA USA
| | | | - Barbara Forte
- Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Science, University of Dundee, Dundee, UK
| | - Tiqing Liu
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA USA
| | - Luma Godoy Magalhaes
- Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Science, University of Dundee, Dundee, UK
| | - Anil K. Gupta
- Calibr-Skaggs Institute for Innovative Medicines, a division of The Scripps Research Institute, La Jolla, CA USA
| | - Eva S. Istvan
- Division of Infectious Diseases, Washington University School of Medicine, Saint Louis, MO USA
| | - Tiantian Jiang
- Department of Pediatrics, University of California, San Diego, La Jolla, CA USA
| | - Krittikorn Kumpornsin
- Calibr-Skaggs Institute for Innovative Medicines, a division of The Scripps Research Institute, La Jolla, CA USA
| | - Karen Lobb
- Lgenia, Inc., 412 S Maple St, Fortville, IN USA
| | - Kyle J. McLean
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA USA
| | - Igor M. R. Moura
- Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY USA
- São Carlos Institute of Physics, University of São Paulo, São Carlos, São Paulo, Brazil
| | - John Okombo
- Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY USA
- Center for Malaria Therapeutics and Antimicrobial Resistance, Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY USA
| | - N. Connor Payne
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA USA
- Center for Systems Biology, Massachusetts General Hospital, Boston, MA USA
| | - Andrew Plater
- Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Science, University of Dundee, Dundee, UK
| | | | - Jair L. Siqueira-Neto
- Department of Pediatrics, University of California, San Diego, La Jolla, CA USA
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA USA
| | | | - Robert L. Summers
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA USA
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, MA USA
| | - Rumin Zhang
- Global Health Drug Discovery Institute, Beijing, China
| | - Michael K. Gilson
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA USA
| | | | - Brice Campo
- MMV Medicines for Malaria Venture, 1215, Geneva, Switzerland
| | - Beatriz Baragaña
- Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Science, University of Dundee, Dundee, UK
| | - James Duffy
- MMV Medicines for Malaria Venture, 1215, Geneva, Switzerland
| | - Ian H. Gilbert
- Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Science, University of Dundee, Dundee, UK
| | - Amanda K. Lukens
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA USA
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, MA USA
| | | | - Jacquin C. Niles
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA USA
| | - Case W. McNamara
- Calibr-Skaggs Institute for Innovative Medicines, a division of The Scripps Research Institute, La Jolla, CA USA
| | - Xiu Cheng
- Global Health Drug Discovery Institute, Beijing, China
| | - Lyn-Marie Birkholtz
- Department of Biochemistry, Genetics & Microbiology, Institute for Sustainable Malaria Control, University of Pretoria, Private Bag X20, Hatfield, Pretoria, South Africa
| | | | - David A. Fidock
- Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY USA
- Center for Malaria Therapeutics and Antimicrobial Resistance, Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY USA
| | - Dyann F. Wirth
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA USA
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, MA USA
| | - Daniel E. Goldberg
- Division of Infectious Diseases, Washington University School of Medicine, Saint Louis, MO USA
- Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO USA
| | - Marcus C. S. Lee
- Division of Biological Chemistry and Drug Discovery, Wellcome Centre for Anti-Infectives Research, University of Dundee, Dundee, UK
| | - Elizabeth A. Winzeler
- Department of Pediatrics, University of California, San Diego, La Jolla, CA USA
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA USA
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Godinez-Macias KP, Chen D, Wallis JL, Siegel MG, Adam A, Bopp S, Carolino K, Coulson LB, Durst G, Thathy V, Esherick L, Farringer MA, Flannery EL, Forte B, Liu T, Magalhaes LG, Gupta AK, Istvan ES, Jiang T, Kumpornsin K, Lobb K, McLean K, Moura IMR, Okombo J, Payne NC, Plater A, Rao SPS, Siqueira-Neto JL, Somsen BA, Summers RL, Zhang R, Gilson MK, Gamo FJ, Campo B, Baragaña B, Duffy J, Gilbert IH, Lukens AK, Dechering KJ, Niles JC, McNamara CW, Cheng X, Birkholtz LM, Bronkhorst AW, Fidock DA, Wirth DF, Goldberg DE, Lee MCS, Winzeler EA. Revisiting the Plasmodium falciparum druggable genome using predicted structures and data mining. RESEARCH SQUARE 2024:rs.3.rs-5412515. [PMID: 39649165 PMCID: PMC11623766 DOI: 10.21203/rs.3.rs-5412515/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/10/2024]
Abstract
The identification of novel drug targets for the purpose of designing small molecule inhibitors is key component to modern drug discovery. In malaria parasites, discoveries of antimalarial targets have primarily occurred retroactively by investigating the mode of action of compounds found through phenotypic screens. Although this method has yielded many promising candidates, it is time- and resource-consuming and misses targets not captured by existing antimalarial compound libraries and phenotypic assay conditions. Leveraging recent advances in protein structure prediction and data mining, we systematically assessed the Plasmodium falciparum genome for proteins amenable to target-based drug discovery, identifying 867 candidate targets with evidence of small molecule binding and blood stage essentiality. Of these, 540 proteins showed strong essentiality evidence and lack inhibitors that have progressed to clinical trials. Expert review and rubric-based scoring of this subset based on additional criteria such as selectivity, structural information, and assay developability yielded 67 high priority candidates. This study also provides a genome-wide data resource and implements a generalizable framework for systematically evaluating and prioritizing novel pathogenic disease targets.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | - Anil K Gupta
- Calibr-Skaggs Institute for Innovative Medicines
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | - Xiu Cheng
- Global Health Drug Discovery Institute
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Chen Y, Wu Y, Tian X, Shao G, Lin Q, Sun A. Golgiphagy: a novel selective autophagy to the fore. Cell Biosci 2024; 14:130. [PMID: 39438975 PMCID: PMC11495120 DOI: 10.1186/s13578-024-01311-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2024] [Accepted: 10/08/2024] [Indexed: 10/25/2024] Open
Abstract
The Golgi apparatus is the central hub of the cellular endocrine pathway and plays a crucial role in processing, transporting, and sorting proteins and lipids. Simultaneously, it is a highly dynamic organelle susceptible to degradation or fragmentation under various physiological or pathological conditions, potentially contributing to the development of numerous human diseases. Autophagy serves as a vital pathway for eukaryotes to manage intracellular and extracellular stress and maintain homeostasis by targeting damaged or redundant organelles for removal. Recent research has revealed that autophagy mechanisms can specifically degrade Golgi components, known as Golgiphagy. This review summarizes recent findings on Golgiphagy while also addressing unanswered questions regarding its mechanisms and regulation, aiming to advance our understanding of the role of Golgiphagy in human disease.
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Affiliation(s)
- Yifei Chen
- Institute of Urinary System Diseases, The Affiliated People's Hospital, Jiangsu University, 8 Dianli Road, Zhenjiang, 212002, China
- Department of Basic Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, 212013, China
| | - Yihui Wu
- Department of Basic Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, 212013, China
| | - Xianyan Tian
- Department of Basic Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, 212013, China
| | - Genbao Shao
- Institute of Urinary System Diseases, The Affiliated People's Hospital, Jiangsu University, 8 Dianli Road, Zhenjiang, 212002, China
- Department of Basic Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, 212013, China
| | - Qiong Lin
- Institute of Urinary System Diseases, The Affiliated People's Hospital, Jiangsu University, 8 Dianli Road, Zhenjiang, 212002, China.
- Department of Basic Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
| | - Aiqin Sun
- Institute of Urinary System Diseases, The Affiliated People's Hospital, Jiangsu University, 8 Dianli Road, Zhenjiang, 212002, China.
- Department of Basic Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
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5
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Wang Y, Li Q, Ding Y, Luo C, Yang J, Wang N, Jiang N, Yao T, Wang G, Shi G, Hou SX. Novel Arf1 Inhibitors Drive Cancer Stem Cell Aging and Potentiate Anti-Tumor Immunity. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2404442. [PMID: 39225354 PMCID: PMC11497069 DOI: 10.1002/advs.202404442] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Revised: 07/01/2024] [Indexed: 09/04/2024]
Abstract
The small G protein Arf1 has been identified as playing a selective role in supporting cancer stem cells (CSCs), making it an attractive target for cancer therapy. However, the current Arf1 inhibitors have limited translational potential due to their high toxicity and low specificity. In this study, two new potent small-molecule inhibitors of Arf1, identified as DU101 and DU102, for cancer therapy are introduced. Preclinical tumor models demonstrate that these inhibitors triggered a cascade of aging in CSCs and enhance anti-tumor immunity in mouse cancer and PDX models. Through single-cell sequencing, the remodeling of the tumor immune microenvironment induced by these new Arf1 inhibitors is analyzed and an increase in tumor-associated CD8+ CD4+ double-positive T (DPT) cells is identified. These DPT cells exhibit superior features of active CD8 single-positive T cells and a higher percentage of TCF1+PD-1+, characteristic of stem-like T cells. The frequency of tumor-infiltrating stem-like DPT cells correlates with better disease-free survival (DFS) in cancer patients, indicating that these inhibitors may offer a novel cancer immunotherapy strategy by converting the cold tumor immune microenvironment into a hot one, thus expanding the potential for immunotherapy in cancer patients.
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Affiliation(s)
- Yuetong Wang
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Qiaoming Li
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Yahui Ding
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Chenfei Luo
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Jun Yang
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Na Wang
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Ning Jiang
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Tiange Yao
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Guohao Wang
- The Basic Research LaboratoryCenter for Cancer ResearchNational Cancer Institute at FrederickNational Institutes of HealthFrederickMD21702USA
| | - Guoming Shi
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
| | - Steven X. Hou
- Department of Cell and Developmental Biology at School of Life SciencesState Key Laboratory of Genetic EngineeringInstitute of Metabolism and Integrative BiologyHuman Phenome InstituteDepartment of Liver Surgery and Transplantation of Liver Cancer Institute at Zhongshan HospitalFudan UniversityShanghai200438China
- Leading Contact
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Lee ZY, Lee WH, Lim JS, Ali AAA, Loo JSE, Wibowo A, Mohammat MF, Foo JB. Golgi apparatus targeted therapy in cancer: Are we there yet? Life Sci 2024; 352:122868. [PMID: 38936604 DOI: 10.1016/j.lfs.2024.122868] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2024] [Revised: 06/14/2024] [Accepted: 06/20/2024] [Indexed: 06/29/2024]
Abstract
Membrane trafficking within the Golgi apparatus plays a pivotal role in the intracellular transportation of lipids and proteins. Dysregulation of this process can give rise to various pathological manifestations, including cancer. Exploiting Golgi defects, cancer cells capitalise on aberrant membrane trafficking to facilitate signal transduction, proliferation, invasion, immune modulation, angiogenesis, and metastasis. Despite the identification of several molecular signalling pathways associated with Golgi abnormalities, there remains a lack of approved drugs specifically targeting cancer cells through the manipulation of the Golgi apparatus. In the initial section of this comprehensive review, the focus is directed towards delineating the abnormal Golgi genes and proteins implicated in carcinogenesis. Subsequently, a thorough examination is conducted on the impact of these variations on Golgi function, encompassing aspects such as vesicular trafficking, glycosylation, autophagy, oxidative mechanisms, and pH alterations. Lastly, the review provides a current update on promising Golgi apparatus-targeted inhibitors undergoing preclinical and/or clinical trials, offering insights into their potential as therapeutic interventions. Significantly more effort is required to advance these potential inhibitors to benefit patients in clinical settings.
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Affiliation(s)
- Zheng Yang Lee
- School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, 47500 Subang Jaya, Selangor, Malaysia
| | - Wen Hwei Lee
- School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, 47500 Subang Jaya, Selangor, Malaysia
| | - Jing Sheng Lim
- School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, 47500 Subang Jaya, Selangor, Malaysia
| | - Afiqah Ali Ajmel Ali
- School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, 47500 Subang Jaya, Selangor, Malaysia
| | - Jason Siau Ee Loo
- School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, 47500 Subang Jaya, Selangor, Malaysia; Digital Health and Medical Advancements Impact Lab, Taylor's University, Subang Jaya 47500, Selangor, Malaysia
| | - Agustono Wibowo
- Faculty of Applied Science, Universiti Teknologi MARA (UiTM) Pahang, Jengka Campus, 26400 Bandar Tun Abdul Razak Jengka, Pahang, Malaysia
| | - Mohd Fazli Mohammat
- Organic Synthesis Laboratory, Institute of Science, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia
| | - Jhi Biau Foo
- School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, 47500 Subang Jaya, Selangor, Malaysia; Digital Health and Medical Advancements Impact Lab, Taylor's University, Subang Jaya 47500, Selangor, Malaysia
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Ferreira A, Castanheira P, Escrevente C, Barral DC, Barona T. Membrane trafficking alterations in breast cancer progression. Front Cell Dev Biol 2024; 12:1350097. [PMID: 38533085 PMCID: PMC10963426 DOI: 10.3389/fcell.2024.1350097] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Accepted: 02/12/2024] [Indexed: 03/28/2024] Open
Abstract
Breast cancer (BC) is the most common type of cancer in women, and remains one of the major causes of death in women worldwide. It is now well established that alterations in membrane trafficking are implicated in BC progression. Indeed, membrane trafficking pathways regulate BC cell proliferation, migration, invasion, and metastasis. The 22 members of the ADP-ribosylation factor (ARF) and the >60 members of the rat sarcoma (RAS)-related in brain (RAB) families of small GTP-binding proteins (GTPases), which belong to the RAS superfamily, are master regulators of membrane trafficking pathways. ARF-like (ARL) subfamily members are involved in various processes, including vesicle budding and cargo selection. Moreover, ARFs regulate cytoskeleton organization and signal transduction. RABs are key regulators of all steps of membrane trafficking. Interestingly, the activity and/or expression of some of these proteins is found dysregulated in BC. Here, we review how the processes regulated by ARFs and RABs are subverted in BC, including secretion/exocytosis, endocytosis/recycling, autophagy/lysosome trafficking, cytoskeleton dynamics, integrin-mediated signaling, among others. Thus, we provide a comprehensive overview of the roles played by ARF and RAB family members, as well as their regulators in BC progression, aiming to lay the foundation for future research in this field. This research should focus on further dissecting the molecular mechanisms regulated by ARFs and RABs that are subverted in BC, and exploring their use as therapeutic targets or prognostic markers.
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Bingham R, McCarthy H, Buckley N. Exploring Retrograde Trafficking: Mechanisms and Consequences in Cancer and Disease. Traffic 2024; 25:e12931. [PMID: 38415291 DOI: 10.1111/tra.12931] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2023] [Revised: 01/25/2024] [Accepted: 01/31/2024] [Indexed: 02/29/2024]
Abstract
Retrograde trafficking (RT) orchestrates the intracellular movement of cargo from the plasma membrane, endosomes, Golgi or endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) in an inward/ER-directed manner. RT works as the opposing movement to anterograde trafficking (outward secretion), and the two work together to maintain cellular homeostasis. This is achieved through maintaining cell polarity, retrieving proteins responsible for anterograde trafficking and redirecting proteins that become mis-localised. However, aberrant RT can alter the correct location of key proteins, and thus inhibit or indeed change their canonical function, potentially causing disease. This review highlights the recent advances in the understanding of how upregulation, downregulation or hijacking of RT impacts the localisation of key proteins in cancer and disease to drive progression. Cargoes impacted by aberrant RT are varied amongst maladies including neurodegenerative diseases, autoimmune diseases, bacterial and viral infections (including SARS-CoV-2), and cancer. As we explore the intricacies of RT, it becomes increasingly apparent that it holds significant potential as a target for future therapies to offer more effective interventions in a wide range of pathological conditions.
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Affiliation(s)
- Rachel Bingham
- School of Pharmacy, Queen's University Belfast, Belfast, UK
| | - Helen McCarthy
- School of Pharmacy, Queen's University Belfast, Belfast, UK
| | - Niamh Buckley
- School of Pharmacy, Queen's University Belfast, Belfast, UK
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9
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Martins M, Vieira J, Pereira-Leite C, Saraiva N, Fernandes AS. The Golgi Apparatus as an Anticancer Therapeutic Target. BIOLOGY 2023; 13:1. [PMID: 38275722 PMCID: PMC10813373 DOI: 10.3390/biology13010001] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/16/2023] [Revised: 12/12/2023] [Accepted: 12/15/2023] [Indexed: 01/27/2024]
Abstract
Although the discovery of the Golgi apparatus (GA) was made over 125 years ago, only a very limited number of therapeutic approaches have been developed to target this complex organelle. The GA serves as a modification and transport center for proteins and lipids and also has more recently emerged as an important store for some ions. The dysregulation of GA functions is implicated in many cellular processes associated with cancer and some GA proteins are indeed described as cancer biomarkers. This dysregulation can affect protein modification, localization, and secretion, but also cellular metabolism, redox status, extracellular pH, and the extracellular matrix structure. Consequently, it can directly or indirectly affect cancer progression. For these reasons, the GA is an appealing anticancer pharmacological target. Despite this, no anticancer drug specifically targeting the GA has reached the clinic and few have entered the clinical trial stage. Advances in nanodelivery approaches may help change this scenario by specifically targeting tumor cells and/or the GA through passive, active, or physical strategies. This article aims to examine the currently available anticancer GA-targeted drugs and the nanodelivery strategies explored for their administration. The potential benefits and challenges of modulating and specifically targeting the GA function in the context of cancer therapy are discussed.
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Affiliation(s)
- Marta Martins
- CBIOS—Universidade Lusófona’s Research Center for Biosciences & Health Technologies, Campo Grande 376, 1749-024 Lisboa, Portugal; (M.M.); (J.V.); (C.P.-L.)
- Department of Biomedical Sciences, University of Alcalá, Ctra. Madrid-Barcelona Km. 33.600, Alcalá de Henares, 28871 Madrid, Spain
| | - João Vieira
- CBIOS—Universidade Lusófona’s Research Center for Biosciences & Health Technologies, Campo Grande 376, 1749-024 Lisboa, Portugal; (M.M.); (J.V.); (C.P.-L.)
- Department of Biomedical Sciences, University of Alcalá, Ctra. Madrid-Barcelona Km. 33.600, Alcalá de Henares, 28871 Madrid, Spain
| | - Catarina Pereira-Leite
- CBIOS—Universidade Lusófona’s Research Center for Biosciences & Health Technologies, Campo Grande 376, 1749-024 Lisboa, Portugal; (M.M.); (J.V.); (C.P.-L.)
- LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
| | - Nuno Saraiva
- CBIOS—Universidade Lusófona’s Research Center for Biosciences & Health Technologies, Campo Grande 376, 1749-024 Lisboa, Portugal; (M.M.); (J.V.); (C.P.-L.)
| | - Ana Sofia Fernandes
- CBIOS—Universidade Lusófona’s Research Center for Biosciences & Health Technologies, Campo Grande 376, 1749-024 Lisboa, Portugal; (M.M.); (J.V.); (C.P.-L.)
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10
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Sawada S, Yoshikawa M, Tsutsui K, Miyazaki T, Kano K, Mishiro-Sato E, Tsukiji S. Palmitoylation-Dependent Small-Molecule Fluorescent Probes for Live-Cell Golgi Imaging. ACS Chem Biol 2023; 18:1047-1053. [PMID: 37098188 DOI: 10.1021/acschembio.3c00046] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/27/2023]
Abstract
Small-molecule fluorescent probes enabling visualization of the Golgi apparatus in living cells are essential tools for studying Golgi-associated biological processes and diseases. So far, several fluorescent Golgi stains have been developed by linking ceramide lipids to fluorophores. However, ceramide-based probes suffer from cumbersome staining procedures and low Golgi specificity. Here, we introduce fluorescent Golgi-staining probes based on the tri-N-methylated myristoyl-Gly-Cys (myrGC3Me) motif. The cell-permeable myrGC3Me motif localizes to the Golgi membrane upon S-palmitoylation. By modularly conjugating the myrGC3Me motif to fluorophores, we developed blue, green, and red fluorescent Golgi probes, all of which allowed simple and rapid staining of the Golgi in living cells with high specificity and no cytotoxicity. The probe was also applicable to the visualization of dynamic changes of the Golgi morphology induced by drug treatments and during cell division. The present work provides an entirely new series of live-cell Golgi probes useful for cell biological and diagnostic applications.
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Affiliation(s)
- Shunsuke Sawada
- Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Nagoya 466-8555, Japan
| | - Masaru Yoshikawa
- Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Nagoya 466-8555, Japan
| | - Keita Tsutsui
- Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Nagoya 466-8555, Japan
| | - Tomoki Miyazaki
- Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Nagoya 466-8555, Japan
| | - Keiko Kano
- Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya 464-8602, Japan
| | - Emi Mishiro-Sato
- Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya 464-8602, Japan
| | - Shinya Tsukiji
- Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Nagoya 466-8555, Japan
- Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Nagoya 466-8555, Japan
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11
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Qiao L, Sinha S, Abd El‐Hafeez AA, Lo I, Midde KK, Ngo T, Aznar N, Lopez‐Sanchez I, Gupta V, Farquhar MG, Rangamani P, Ghosh P. A circuit for secretion-coupled cellular autonomy in multicellular eukaryotic cells. Mol Syst Biol 2023; 19:e11127. [PMID: 36856068 PMCID: PMC10090951 DOI: 10.15252/msb.202211127] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2022] [Revised: 02/06/2023] [Accepted: 02/07/2023] [Indexed: 03/02/2023] Open
Abstract
Cancers represent complex autonomous systems, displaying self-sufficiency in growth signaling. Autonomous growth is fueled by a cancer cell's ability to "secrete-and-sense" growth factors (GFs): a poorly understood phenomenon. Using an integrated computational and experimental approach, here we dissect the impact of a feedback-coupled GTPase circuit within the secretory pathway that imparts secretion-coupled autonomy. The circuit is assembled when the Ras-superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Giαβγ and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed-loop control, allow the two GTPases to coregulate each other, and convert the expected switch-like behavior of Arf1-dependent secretion into an unexpected dose-response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self-sustained by stimulus-proportionate secretion. Proteomic studies and protein-protein interaction network analyses pinpoint GFs (e.g., the epidermal GF) as key stimuli for such self-sustenance. Findings highlight how the enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion-coupled autonomy of growth factors.
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Affiliation(s)
- Lingxia Qiao
- Department of Mechanical and Aerospace Engineering, Jacob's School of EngineeringUniversity of California San DiegoLa JollaCAUSA
| | - Saptarshi Sinha
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
| | - Amer Ali Abd El‐Hafeez
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
- Present address:
Pharmacology and Experimental Oncology Unit, Cancer Biology Department, National Cancer InstituteCairo UniversityCairoEgypt
| | - I‐Chung Lo
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
| | - Krishna K Midde
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
| | - Tony Ngo
- Skaggs School of Pharmacy and Pharmaceutical ScienceUniversity of California San DiegoLa JollaCAUSA
| | - Nicolas Aznar
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
| | - Inmaculada Lopez‐Sanchez
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
| | - Vijay Gupta
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
| | - Marilyn G Farquhar
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
| | - Padmini Rangamani
- Department of Mechanical and Aerospace Engineering, Jacob's School of EngineeringUniversity of California San DiegoLa JollaCAUSA
| | - Pradipta Ghosh
- Department of Cellular and Molecular Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
- Moores Comprehensive Cancer CenterUniversity of California San DiegoLa JollaCAUSA
- Department of Medicine, School of MedicineUniversity of California San DiegoLa JollaCAUSA
- Veterans Affairs Medical CenterLa JollaCAUSA
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12
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Khine MN, Sakurai K. Golgi-Targeting Anticancer Natural Products. Cancers (Basel) 2023; 15:cancers15072086. [PMID: 37046746 PMCID: PMC10093635 DOI: 10.3390/cancers15072086] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2023] [Revised: 02/12/2023] [Accepted: 02/15/2023] [Indexed: 04/03/2023] Open
Abstract
The Golgi apparatus plays an important role in maintaining cell homeostasis by serving as a biosynthetic center for glycans, lipids and post-translationally modified proteins and as a sorting center for vesicular transport of proteins to specific destinations. Moreover, it provides a signaling hub that facilitates not only membrane trafficking processes but also cellular response pathways to various types of stresses. Altered signaling at the Golgi apparatus has emerged as a key regulator of tumor growth and survival. Among the small molecules that can specifically perturb or modulate Golgi proteins and organization, natural products with anticancer property have been identified as powerful chemical probes in deciphering Golgi-related pathways and, in particular, recently described Golgi stress response pathways. In this review, we highlight a set of Golgi-targeting natural products that enabled the characterization of the Golgi-mediated signaling events leading to cancer cell death and discuss the potential for selectively exploiting these pathways for the development of novel chemotherapeutic agents.
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13
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Selvapandiyan A, Puri N, Kumar P, Alam A, Ehtesham NZ, Griffin G, Hasnain SE. Zooming in on common immune evasion mechanisms of pathogens in phagolysosomes: potential broad-spectrum therapeutic targets against infectious diseases. FEMS Microbiol Rev 2023; 47:6780197. [PMID: 36309472 DOI: 10.1093/femsre/fuac041] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2022] [Revised: 10/06/2022] [Accepted: 10/18/2022] [Indexed: 01/19/2023] Open
Abstract
The intracellular viral, bacterial, or parasitic pathogens evade the host immune challenges to propagate and cause fatal diseases. The microbes overpower host immunity at various levels including during entry into host cells, phagosome formation, phagosome maturation, phagosome-lysosome fusion forming phagolysosomes, acidification of phagolysosomes, and at times after escape into the cytosol. Phagolysosome is the final organelle in the phagocyte with sophisticated mechanisms to degrade the pathogens. The immune evasion strategies by the pathogens include the arrest of host cell apoptosis, decrease in reactive oxygen species, the elevation of Th2 anti-inflammatory response, avoidance of autophagy and antigen cross-presentation pathways, and escape from phagolysosomal killing. Since the phagolysosome organelle in relation to infection/cure is seldom discussed in the literature, we summarize here the common host as well as pathogen targets manipulated or utilized by the pathogens established in phagosomes and phagolysosomes, to hijack the host immune system for their benefit. These common molecules or pathways can be broad-spectrum therapeutic targets for drug development for intervention against infectious diseases caused by different intracellular pathogens.
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Affiliation(s)
| | - Niti Puri
- Cellular and Molecular Immunology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India
| | - Pankaj Kumar
- Department of Biochemistry, Jamia Hamdard, New Delhi, 110062, India.,Centre for Tuberculosis Research, Department of Medicine, Johns Hopkins University, Baltimore, MD, 21218, United States
| | - Anwar Alam
- ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, 110029, India.,Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, New Delhi, 110016, India
| | - Nasreen Zafar Ehtesham
- ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, 110029, India
| | - George Griffin
- Department of Cellular and Molecular Medicine, St. George's University of London, London, SW17 0RE, United Kingdom
| | - Seyed Ehtesham Hasnain
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, New Delhi, 110016, India.,Department of Life Science, School of Basic Sciences and Research, Sharda University, Knowledge Park III, Greater Noida, 201310, India
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14
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Li RS, Wen C, Huang CZ, Li N. Functional molecules and nano-materials for the Golgi apparatus-targeted imaging and therapy. Trends Analyt Chem 2022. [DOI: 10.1016/j.trac.2022.116714] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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15
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Zhang J, Li P, Lu R, Ouyang S, Chang MX. Structural and functional analysis of the small GTPase ARF1 reveals a pivotal role of its GTP-binding domain in controlling of the generation of viral inclusion bodies and replication of grass carp reovirus. Front Immunol 2022; 13:956587. [PMID: 36091067 PMCID: PMC9459132 DOI: 10.3389/fimmu.2022.956587] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Accepted: 08/12/2022] [Indexed: 11/13/2022] Open
Abstract
Grass carp reovirus (GCRV) is the most pathogenic double-stranded (ds) RNA virus among the isolated aquareoviruses. The molecular mechanisms by which GCRV utilizes host factors to generate its infectious compartments beneficial for viral replication and infection are poorly understood. Here, we discovered that the grass carp ADP ribosylation factor 1 (gcARF1) was required for GCRV replication since the knockdown of gcARF1 by siRNA or inhibiting its GTPase activity by treatment with brefeldin A (BFA) significantly impaired the yield of infectious viral progeny. GCRV infection recruited gcARF1 into viral inclusion bodies (VIBs) by its nonstructural proteins NS80 and NS38. The small_GTP domain of gcARF1 was confirmed to be crucial for promoting GCRV replication and infection, and the number of VIBs reduced significantly by the inhibition of gcARF1 GTPase activity. The analysis of gcARF1-GDP complex crystal structure revealed that the 27AAGKTT32 motif and eight amino acid residues (A27, G29, K30, T31, T32, N126, D129 and A160), which were located mainly within the GTP-binding domain of gcARF1, were crucial for the binding of gcARF1 with GDP. Furthermore, the 27AAGKTT32 motif and the amino acid residue T31 of gcARF1 were indispensable for the function of gcARF1 in promoting GCRV replication and infection. Taken together, it is demonstrated that the GTPase activity of gcARF1 is required for efficient replication of GCRV and that host GTPase ARF1 is closely related with the generation of VIBs.
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Affiliation(s)
- Jie Zhang
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
| | - Pengwei Li
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
- College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
| | - Riye Lu
- Innovation Academy for Seed Design, Chinese Academy of Sciences, Wuhan, China
| | - Songying Ouyang
- Innovation Academy for Seed Design, Chinese Academy of Sciences, Wuhan, China
- *Correspondence: Ming Xian Chang, ; Songying Ouyang,
| | - Ming Xian Chang
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
- College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
- Innovation Academy for Seed Design, Chinese Academy of Sciences, Wuhan, China
- *Correspondence: Ming Xian Chang, ; Songying Ouyang,
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16
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Kunimasa K, Ikeda-Ishikawa C, Tani Y, Tsukahara S, Sakurai J, Okamoto Y, Koido M, Dan S, Tomida A. Spautin-1 inhibits mitochondrial complex I and leads to suppression of the unfolded protein response and cell survival during glucose starvation. Sci Rep 2022; 12:11533. [PMID: 35798783 PMCID: PMC9262966 DOI: 10.1038/s41598-022-15673-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 06/28/2022] [Indexed: 11/09/2022] Open
Abstract
The unfolded protein response (UPR) is an adaptive stress response pathway that is essential for cancer cell survival under endoplasmic reticulum stress such as during glucose starvation. In this study, we identified spautin-1, an autophagy inhibitor that suppresses ubiquitin-specific peptidase 10 (USP10) and USP13, as a novel UPR inhibitor under glucose starvation conditions. Spautin-1 prevented the induction of UPR-associated proteins, including glucose-regulated protein 78, activating transcription factor 4, and a splicing variant of x-box-binding protein-1, and showed preferential cytotoxicity in glucose-starved cancer cells. However, USP10 and USP13 silencing and treatment with other autophagy inhibitors failed to result in UPR inhibition and preferential cytotoxicity during glucose starvation. Using transcriptome and chemosensitivity-based COMPARE analyses, we identified a similarity between spautin-1 and mitochondrial complex I inhibitors and found that spautin-1 suppressed the activity of complex I extracted from isolated mitochondria. Our results indicated that spautin-1 may represent an attractive mitochondria-targeted seed compound that inhibits the UPR and cancer cell survival during glucose starvation.
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Affiliation(s)
- Kazuhiro Kunimasa
- Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan
| | - Chika Ikeda-Ishikawa
- Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan
| | - Yuri Tani
- Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan
| | - Satomi Tsukahara
- Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan
| | - Junko Sakurai
- Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan
| | - Yuka Okamoto
- Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan
| | - Masaru Koido
- Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan.,Division of Molecular Pathology, Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan
| | - Shingo Dan
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan
| | - Akihiro Tomida
- Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan.
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17
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Martins M, Fernandes AS, Saraiva N. GOLGI: Cancer cell fate control. Int J Biochem Cell Biol 2022; 145:106174. [PMID: 35182766 DOI: 10.1016/j.biocel.2022.106174] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2021] [Revised: 02/11/2022] [Accepted: 02/12/2022] [Indexed: 11/16/2022]
Abstract
Growing evidence connects many of the Golgi known functions with cellular events related to cancer initiation and progression, including regulation of cell survival/death, proliferation, motility, metabolism and immune evasion. However, a broad and integrated understanding of the impact of the Golgi on cancer cell phenotype has not yet been achieved. Multiple cellular events involving the Golgi are associated with protein and lipid modification and trafficking. However, less explored aspects of this enigmatic organelle also contribute to cell fate decision-making by impacting signal transduction, redox and ion homeostasis. This article focuses on the molecular mechanisms and Golgi proteins underlying the impact of the Golgi on cancer cell phenotype. Special emphasis is given to emerging knowledge on redox and ion homeostasis. Current and potential cancer progression therapeutic strategies associated with this organelle will also be addressed.
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Affiliation(s)
- Marta Martins
- CBIOS - Universidade Lusófona's Research Center for Biosciences & Health Technologies, Campo Grande 376, 1749-024 Lisboa, Portugal
| | - Ana Sofia Fernandes
- CBIOS - Universidade Lusófona's Research Center for Biosciences & Health Technologies, Campo Grande 376, 1749-024 Lisboa, Portugal
| | - Nuno Saraiva
- CBIOS - Universidade Lusófona's Research Center for Biosciences & Health Technologies, Campo Grande 376, 1749-024 Lisboa, Portugal.
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18
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Chang HY, Yang WY. Golgi quality control and autophagy. IUBMB Life 2022; 74:361-370. [PMID: 35274438 DOI: 10.1002/iub.2611] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2021] [Accepted: 02/12/2022] [Indexed: 11/09/2022]
Abstract
Organelles can easily be disrupted by intracellular and extracellular factors. Studies on ER and mitochondria indicate that a wide range of responses are elicited upon organelle disruption. One response thought to be of particular importance is autophagy. Cells can target entire organelles into autophagosomes for removal. This wholesale nature makes autophagy a robust means for eliminating compromised organelles. Recently, it was demonstrated that the Golgi apparatus is a substrate of autophagy. On the other hand, various reports have shown that components traffic away from the Golgi for elimination in an autophagosome-independent manner when the Golgi apparatus is stressed. Future studies will reveal how these different pieces of machinery coordinate to drive Golgi degradation. Quantitative measurements will be needed to determine how much autophagy contributes to the maintenance of the Golgi apparatus.
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Affiliation(s)
- Hsiang-Yi Chang
- Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
| | - Wei Yuan Yang
- Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.,Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan
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19
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Saraon P, Snider J, Schormann W, Rai A, Radulovich N, Sánchez-Osuna M, Coulombe-Huntington J, Huard C, Mohammed M, Lima-Fernandes E, Thériault B, Halabelian L, Chan M, Joshi D, Drecun L, Yao Z, Pathmanathan S, Wong V, Lyakisheva A, Aboualizadeh F, Niu L, Li F, Kiyota T, Subramanian R, Joseph B, Aman A, Prakesch M, Isaac M, Mamai A, Poda G, Vedadi M, Marcellus R, Uehling D, Leighl N, Sacher A, Samaržija M, Jakopović M, Arrowsmith C, Tyers M, Tsao MS, Andrews D, Al-Awar R, Stagljar I. Chemical Genetics Screen Identifies COPB2 Tool Compounds That Alters ER Stress Response and Induces RTK Dysregulation in Lung Cancer Cells. J Mol Biol 2021; 433:167294. [PMID: 34662547 DOI: 10.1016/j.jmb.2021.167294] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Revised: 09/27/2021] [Accepted: 09/27/2021] [Indexed: 12/12/2022]
Abstract
Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.
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Affiliation(s)
- Punit Saraon
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada.
| | - Jamie Snider
- Donnelly Centre, University of Toronto, Ontario, Canada
| | - Wiebke Schormann
- Biological Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada
| | - Ankit Rai
- Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3548CH Utrecht, the Netherlands
| | - Nikolina Radulovich
- Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada
| | - Maria Sánchez-Osuna
- Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Downtown Station, Montreal, QC H3C 3J7, Canada
| | - Jasmin Coulombe-Huntington
- Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Downtown Station, Montreal, QC H3C 3J7, Canada
| | - Caroline Huard
- Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Downtown Station, Montreal, QC H3C 3J7, Canada
| | - Mohammed Mohammed
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | | | - Brigitte Thériault
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Levon Halabelian
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada
| | - Manuel Chan
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Dhananjay Joshi
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Luka Drecun
- Donnelly Centre, University of Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Ontario, Canada
| | - Zhong Yao
- Donnelly Centre, University of Toronto, Ontario, Canada
| | - Shivanthy Pathmanathan
- Donnelly Centre, University of Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Ontario, Canada
| | - Victoria Wong
- Donnelly Centre, University of Toronto, Ontario, Canada
| | | | | | - Li Niu
- Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada
| | - Fengling Li
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada
| | - Taira Kiyota
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | | | - Babu Joseph
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Ahmed Aman
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Michael Prakesch
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Methvin Isaac
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Ahmed Mamai
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Gennady Poda
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada; University of Toronto, Leslie Dan Faculty of Pharmacy, Toronto, Ontario, Canada
| | - Masoud Vedadi
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada; Department of Pharmacology and Toxicology, University of Toronto, Ontario, Canada
| | - Richard Marcellus
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - David Uehling
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada
| | - Natasha Leighl
- Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada
| | - Adrian Sacher
- Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada
| | - Miroslav Samaržija
- Department for Lung Diseases Jordanovac, Clinical Hospital Centre Zagreb, University of Zagreb, Zagreb, Croatia
| | - Marko Jakopović
- Department for Lung Diseases Jordanovac, Clinical Hospital Centre Zagreb, University of Zagreb, Zagreb, Croatia
| | - Cheryl Arrowsmith
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
| | - Mike Tyers
- Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Downtown Station, Montreal, QC H3C 3J7, Canada
| | - Ming-Sound Tsao
- Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - David Andrews
- Biological Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada
| | - Rima Al-Awar
- Drug Discovery Program, Ontario Institute for Cancer Research, Ontario, Canada; Department of Pharmacology and Toxicology, University of Toronto, Ontario, Canada.
| | - Igor Stagljar
- Donnelly Centre, University of Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Ontario, Canada; Department of Biochemistry, University of Toronto, Ontario, Canada; Mediterranean Institute for Life Sciences, Split, Croatia; School of Medicine, University of Split, Split, Croatia.
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20
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Autophagy Induction by Trichodermic Acid Attenuates Endoplasmic Reticulum Stress-Mediated Apoptosis in Colon Cancer Cells. Int J Mol Sci 2021; 22:ijms22115566. [PMID: 34070303 PMCID: PMC8197497 DOI: 10.3390/ijms22115566] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2021] [Revised: 05/14/2021] [Accepted: 05/19/2021] [Indexed: 12/26/2022] Open
Abstract
Colorectal cancer (CRC) is the third leading malignant tumor in the world, which has high morbidity and mortality. In this study we found that trichodermic acid (TDA), a secondary metabolite isolated from the plant endophytic fungus Penicillium ochrochloronthe with a variety of biological and pharmacological activities, exhibited the antitumor effects on colorectal cancer cells in vitro and in vivo. Our results showed that TDA inhibited the proliferation of colon cancer cells in a dose-dependent manner. TDA induces sustained endoplasmic reticulum stress, which triggers apoptosis through IRE1α/XBP1 and PERK/ATF4/CHOP pathways. In addition, we found that TDA mediated endoplasmic reticulum stress also induces autophagy as a protective mechanism. Moreover, combined treatment of TDA with autophagy inhibitors significantly enhanced its anticancer effect. In conclusion, our results indicated that TDA can induce ER stress and autophagy mediated apoptosis, suggesting that targeting ER stress and autophagy may be an effective strategy for the treatment of CRC.
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21
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Qiao L, Sinha S, El-hafeez AAA, Lo I, Midde KK, Ngo T, Aznar N, Lopez-sanchez I, Gupta V, Farquhar MG, Rangamani P, Ghosh P. A Circuit for Secretion-coupled Cellular Autonomy in Multicellular Eukaryotes.. [DOI: 10.1101/2021.03.18.436048] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/02/2023]
Abstract
ABSTRACTCancers represent complex autonomous systems, displaying self-sufficiency in growth signaling. Autonomous growth is fueled by a cancer cell’s ability to ‘secrete-and-sense’ growth factors: a poorly understood phenomenon. Using an integrated systems and experimental approach, here we dissect the impact of a feedback-coupled GTPase circuit within the secretory pathway that imparts secretion-coupled autonomy. The circuit is assembled when the Ras-superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Giαβγ and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed-loop control (CLC), allow the two GTPases to coregulate each other, and convert the expected switch-like behavior of Arf1-dependent secretion into an unexpected dose response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self-sustained by stimulus-proportionate secretion. Proteomic studies and protein-protein interaction network analyses pinpoint growth factors (e.g., the epidermal growth factor; EGF) as a key stimuli for such self-sustenance. Findings highlight how enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion-coupled autonomy of growth factors.SYNOPSIS IMAGESTANDFIRST TEXTThis work defines the inner workings of a Golgi-localized molecular circuitry comprised of coupled GTPases, which empowers cells to achieve self-sufficiency in growth factor signaling by creating a secrete-and-sense autocrine loop.HIGHLIGHTS/MAIN FINDINGSModeling and experimental approaches were used to dissect a coupled GTPase circuit.Coupling enables closed loop feedback and mutual control of GTPases.Coupling generates dose response alignment behavior of sensing and secretion of growth factors.Coupling is critical for multiscale feedback control to achieve secretion-coupled autonomy.
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Gray JL, von Delft F, Brennan PE. Targeting the Small GTPase Superfamily through Their Regulatory Proteins. Angew Chem Int Ed Engl 2020; 59:6342-6366. [PMID: 30869179 PMCID: PMC7204875 DOI: 10.1002/anie.201900585] [Citation(s) in RCA: 99] [Impact Index Per Article: 19.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Revised: 03/11/2019] [Indexed: 12/11/2022]
Abstract
The Ras superfamily of small GTPases are guanine-nucleotide-dependent switches essential for numerous cellular processes. Mutations or dysregulation of these proteins are associated with many diseases, but unsuccessful attempts to target the small GTPases directly have resulted in them being classed as "undruggable". The GTP-dependent signaling of these proteins is controlled by their regulators; guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in the Rho and Rab subfamilies, guanine nucleotide dissociation inhibitors (GDIs). This review covers the recent small molecule and biologics strategies to target the small GTPases through their regulators. It seeks to critically re-evaluate recent chemical biology practice, such as the presence of PAINs motifs and the cell-based readout using compounds that are weakly potent or of unknown specificity. It highlights the vast scope of potential approaches for targeting the small GTPases in the future through their regulatory proteins.
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Affiliation(s)
- Janine L. Gray
- Structural Genomics ConsortiumUniversity of Oxford, NDMRBOld Road CampusOxfordOX3 7DQUK
- Target Discovery InstituteNuffield Department of MedicineUniversity of OxfordOld Road CampusOxfordOX3 7FZUK
- Diamond Light SourceHarwell Science and Innovation CampusDidcotOX11 0QXUK
| | - Frank von Delft
- Structural Genomics ConsortiumUniversity of Oxford, NDMRBOld Road CampusOxfordOX3 7DQUK
- Diamond Light SourceHarwell Science and Innovation CampusDidcotOX11 0QXUK
- Department of BiochemistryUniversity of JohannesburgAuckland Park2006South Africa
| | - Paul E. Brennan
- Structural Genomics ConsortiumUniversity of Oxford, NDMRBOld Road CampusOxfordOX3 7DQUK
- Target Discovery InstituteNuffield Department of MedicineUniversity of OxfordOld Road CampusOxfordOX3 7FZUK
- Alzheimer's Research (UK) Oxford Drug Discovery InstituteNuffield Department of MedicineUniversity of OxfordOxfordOX3 7FZUK
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Detection of the in vitro modulation of Plasmodium falciparum Arf1 by Sec7 and ArfGAP domains using a colorimetric plate-based assay. Sci Rep 2020; 10:4193. [PMID: 32144363 PMCID: PMC7061341 DOI: 10.1038/s41598-020-61101-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2019] [Accepted: 12/09/2019] [Indexed: 11/18/2022] Open
Abstract
The regulation of human Arf1 GTPase activity by ArfGEFs that stimulate GDP/GTP exchange and ArfGAPs that mediate GTP hydrolysis has attracted attention for the discovery of Arf1 inhibitors as potential anti-cancer agents. The malaria parasite Plasmodium falciparum encodes a Sec7 domain-containing protein - presumably an ArfGEF - and two putative ArfGAPs, as well as an Arf1 homologue (PfArf1) that is essential for blood-stage parasite viability. However, ArfGEF and ArfGAP-mediated activation/deactivation of PfArf1 has not been demonstrated. In this study, we established an in vitro colorimetric microtiter plate-based assay to detect the activation status of truncated human and P. falciparum Arf1 and used it to demonstrate the activation of both proteins by the Sec7 domain of ARNO, their deactivation by the GAP domain of human ArfGAP1 and the inhibition of the respective reactions by the compounds SecinH3 and QS11. In addition, we found that the GAP domains of both P. falciparum ArfGAPs have activities equivalent to that of human ArfGAP1, but are insensitive to QS11. Library screening identified a novel inhibitor which selectively inhibits one of the P. falciparum GAP domains (IC50 4.7 µM), suggesting that the assay format is suitable for screening compound collections for inhibitors of Arf1 regulatory proteins.
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Gray JL, Delft F, Brennan PE. Targeting der kleinen GTPasen über ihre regulatorischen Proteine. Angew Chem Int Ed Engl 2020. [DOI: 10.1002/ange.201900585] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Affiliation(s)
- Janine L. Gray
- Structural Genomics ConsortiumUniversity of Oxford, NDMRB Old Road Campus Oxford OX3 7DQ Großbritannien
- Target Discovery InstituteNuffield Department of MedicineUniversity of Oxford Old Road Campus Oxford OX3 7FZ Großbritannien
- Diamond Light Source Harwell Science and Innovation Campus Didcot OX11 0QX Großbritannien
| | - Frank Delft
- Structural Genomics ConsortiumUniversity of Oxford, NDMRB Old Road Campus Oxford OX3 7DQ Großbritannien
- Diamond Light Source Harwell Science and Innovation Campus Didcot OX11 0QX Großbritannien
- Department of BiochemistryUniversity of Johannesburg Auckland Park 2006 Südafrika
| | - Paul E. Brennan
- Structural Genomics ConsortiumUniversity of Oxford, NDMRB Old Road Campus Oxford OX3 7DQ Großbritannien
- Target Discovery InstituteNuffield Department of MedicineUniversity of Oxford Old Road Campus Oxford OX3 7FZ Großbritannien
- Alzheimer's Research (UK) Oxford Drug Discovery InstituteNuffield Department of MedicineUniversity of Oxford Oxford OX3 7FZ Großbritannien
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25
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Design and Synthesis of Arf1-Targeting γ-Dipeptides as Potential Agents against Head and Neck Squamous Cell Carcinoma. Cells 2020; 9:cells9020286. [PMID: 31991585 PMCID: PMC7072570 DOI: 10.3390/cells9020286] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Revised: 01/20/2020] [Accepted: 01/21/2020] [Indexed: 12/24/2022] Open
Abstract
Background: Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related deaths and calls for new druggable targets. We have previously highlighted the critical role of ADP-ribosylation factor-1 (Arf1) activation in HNSCC. In the present study, we address the question whether targeting Arf1 could be proposed as a valuable strategy against HNSCC. Methods: We rationally designed and synthesized constrained ATC-based (4-amino-(methyl)-1,3-thiazole-5-carboxylic acid) γ-dipeptides to block Arf1 activation. We evaluated the effects of these γ-dipeptides in HNSCC cells: The cell viability was determined in 2D and 3D cell cultures after 72 h treatment and Arf1 protein levels and activity were assessed by GGA3 pull-down and Western blotting assays. Results: Targeting Arf1 offers a valuable strategy to counter HNSCC. Our new Arf1-targeting compounds revealed a strong in vitro cytotoxicity against HNSCC cells, through inhibiting Arf1 activation and its downstream pathways. Conclusions: Arf1-targeting γ-dipeptides developed in this study may represent a promising targeted therapeutic to improve managing the HNSCC disease.
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26
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Boncompain G, Gareil N, Tessier S, Lescure A, Jones TR, Kepp O, Kroemer G, Del Nery E, Perez F. BML-265 and Tyrphostin AG1478 Disperse the Golgi Apparatus and Abolish Protein Transport in Human Cells. Front Cell Dev Biol 2019; 7:232. [PMID: 31681765 PMCID: PMC6797785 DOI: 10.3389/fcell.2019.00232] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2019] [Accepted: 09/27/2019] [Indexed: 11/28/2022] Open
Abstract
The steady-state localization of Golgi-resident glycosylation enzymes in the Golgi apparatus depends on a balance between anterograde and retrograde transport. Using the Retention Using Selective Hooks (RUSH) assay and high-content screening, we identified small molecules that perturb the localization of Mannosidase II (ManII) used as a model cargo for Golgi resident enzymes. In particular, we found that two compounds known as EGFR tyrosine kinase inhibitors, namely BML-265 and Tyrphostin AG1478 disrupt Golgi integrity and abolish secretory protein transport of diverse cargos, thus inducing brefeldin A-like effects. Interestingly, BML-265 and Tyrphostin AG1478 affect Golgi integrity and transport in human cells but not in rodent cells. The effects of BML-265 are reversible since Golgi integrity and protein transport are quickly restored upon washout of the compounds. BML-265 and Tyrphostin AG1478 do not lead to endosomal tubulation suggesting that, contrary to brefeldin A, they do not target the trans-Golgi ARF GEF BIG1 and BIG2. They quickly induce COPI dissociation from Golgi membranes suggesting that, in addition to EGFR kinase, the cis-Golgi ARF GEF GBF1 might also be a target of these molecules. Accordingly, overexpression of GBF1 prevents the effects of BML-265 and Tyrphostin AG1478 on Golgi integrity.
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Affiliation(s)
- Gaelle Boncompain
- Dynamics of Intracellular Organization Laboratory, Institut Curie, PSL Research University, Sorbonne Université, Centre National de la Recherche Scientifique, UMR 144, Paris, France
| | - Nelly Gareil
- Dynamics of Intracellular Organization Laboratory, Institut Curie, PSL Research University, Sorbonne Université, Centre National de la Recherche Scientifique, UMR 144, Paris, France
| | - Sarah Tessier
- BioPhenics High-Content Screening Laboratory, Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, PSL Research University, Translational Research Department, Paris, France
| | - Aurianne Lescure
- BioPhenics High-Content Screening Laboratory, Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, PSL Research University, Translational Research Department, Paris, France
| | - Thouis R. Jones
- BioPhenics High-Content Screening Laboratory, Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, PSL Research University, Translational Research Department, Paris, France
| | - Oliver Kepp
- Equipe Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, INSERM U1138, Centre de Recherche des Cordeliers, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy, Villejuif, France
| | - Guido Kroemer
- Equipe Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, INSERM U1138, Centre de Recherche des Cordeliers, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy, Villejuif, France
- Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China
- Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France
- Department of Women’s and Children’s Health, Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden
| | - Elaine Del Nery
- BioPhenics High-Content Screening Laboratory, Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, PSL Research University, Translational Research Department, Paris, France
| | - Franck Perez
- Dynamics of Intracellular Organization Laboratory, Institut Curie, PSL Research University, Sorbonne Université, Centre National de la Recherche Scientifique, UMR 144, Paris, France
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Gangopadhyay A, Chakraborty HJ, Datta A. Employing virtual screening and molecular dynamics simulations for identifying hits against the active cholera toxin. Toxicon 2019; 170:1-9. [PMID: 31494206 DOI: 10.1016/j.toxicon.2019.09.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2019] [Revised: 08/22/2019] [Accepted: 09/01/2019] [Indexed: 12/24/2022]
Abstract
Cholera is a major global threat, affecting millions each year. The ADP ribosyltransferase activity of the active cholera toxin catalyses the massive loss of water and electrolytes during cholera infections. The active toxin heterodimer comprises the A1 subunit from Vibrio cholerae and ARF (ADP Ribosylation Factor) from the human host. Although the active toxin is a potential target for drug discovery against cholera, it has been scarcely targeted to date. The A1-ARF interface contains a potential druggable site for small molecule inhibitors. By combining a sequential docking and scoring strategy with molecular dynamics (MD) simulations, this study identified hits against the protein-protein interface (PPI) of the active cholera toxin from an in-house library of 9,175 ADMET-screened alkaloids. The docking algorithms and scoring functions of Glide SP, Glide XP, and AutoDock were employed for initial library screening. Three alkaloids were initially selected by docking-based virtual screening. The stability of the hit-toxin complexes was validated by MD simulations. Two of the three hits, namely, A6225 (7-formyldehydrothalicsimidine) and A16503 (1,2,7,8-tetrahydroxy dibenz[cd,f]indol-4(5H)-one), formed stable complexes with the toxin. Analyses of the hydrogen bond occupancies revealed that the hits formed stable hydrogen bonds with the toxin PPI. The hits identified herein can serve as reference compounds for drug discovery against cholera in the future.
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Affiliation(s)
- Aditi Gangopadhyay
- Department of Chemical Technology, University of Calcutta, 92, APC Road, Kolkata 700009, West Bengal, India; DBT Centre for Bioinformatics, Presidency University, Kolkata 700073, West Bengal, India.
| | - Hirak Jyoti Chakraborty
- DBT Centre for Bioinformatics, Presidency University, Kolkata 700073, West Bengal, India; Central Inland Fisheries Research Institute, Barrackpore, Kolkata 700120, West Bengal, India
| | - Abhijit Datta
- DBT Centre for Bioinformatics, Presidency University, Kolkata 700073, West Bengal, India; Department of Botany, Jhargram Raj College, Jhargram 721507, Paschim Medinipur, India
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28
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Kamata H, Sadahiro S, Yamori T. Discovery of Inhibitors of Membrane Traffic from a Panel of Clinically Effective Anticancer Drugs. Biol Pharm Bull 2019; 42:814-818. [DOI: 10.1248/bpb.b18-01026] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Affiliation(s)
- Hiroko Kamata
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research
- Graduate School of Medicine, Tokai University
| | | | - Takao Yamori
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research
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29
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Prieto-Dominguez N, Parnell C, Teng Y. Drugging the Small GTPase Pathways in Cancer Treatment: Promises and Challenges. Cells 2019; 8:E255. [PMID: 30884855 PMCID: PMC6468615 DOI: 10.3390/cells8030255] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2019] [Revised: 03/08/2019] [Accepted: 03/13/2019] [Indexed: 02/07/2023] Open
Abstract
Small GTPases are a family of low molecular weight GTP-hydrolyzing enzymes that cycle between an inactive state when bound to GDP and an active state when associated to GTP. Small GTPases regulate key cellular processes (e.g., cell differentiation, proliferation, and motility) as well as subcellular events (e.g., vesicle trafficking), making them key participants in a great array of pathophysiological processes. Indeed, the dysfunction and deregulation of certain small GTPases, such as the members of the Ras and Arf subfamilies, have been related with the promotion and progression of cancer. Therefore, the development of inhibitors that target dysfunctional small GTPases could represent a potential therapeutic strategy for cancer treatment. This review covers the basic biochemical mechanisms and the diverse functions of small GTPases in cancer. We also discuss the strategies and challenges of inhibiting the activity of these enzymes and delve into new approaches that offer opportunities to target them in cancer therapy.
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Affiliation(s)
- Néstor Prieto-Dominguez
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA 30912, USA.
- Institute of Biomedicine (IBIOMED), University of León, León 24010, Spain.
| | | | - Yong Teng
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA 30912, USA.
- Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.
- Department of Medical laboratory, Imaging and Radiologic Sciences, College of Allied Health, Augusta University, Augusta, GA 30912, USA.
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30
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Singh SR, Aggarwal P, Hou SX. Cancer Stem Cells and Stem Cell Tumors in Drosophila. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1167:175-190. [DOI: 10.1007/978-3-030-23629-8_10] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
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31
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Portela M, Segura-Collar B, Argudo I, Sáiz A, Gargini R, Sánchez-Gómez P, Casas-Tintó S. Oncogenic dependence of glioma cells on kish/TMEM167A regulation of vesicular trafficking. Glia 2018; 67:404-417. [PMID: 30506943 DOI: 10.1002/glia.23551] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2018] [Revised: 09/03/2018] [Accepted: 09/04/2018] [Indexed: 12/17/2022]
Abstract
Genetic lesions in glioblastoma (GB) include constitutive activation of PI3K and EGFR pathways to drive cellular proliferation and tumor malignancy. An RNAi genetic screen, performed in Drosophila melanogaster to discover new modulators of GB development, identified a member of the secretory pathway: kish/TMEM167A. Downregulation of kish/TMEM167A impaired fly and human glioma formation and growth, with no effect on normal glia. Glioma cells increased the number of recycling endosomes, and reduced the number of lysosomes. In addition, EGFR vesicular localization was primed toward recycling in glioma cells. kish/TMEM167A downregulation in gliomas restored endosomal system to a physiological state and altered lysosomal function, fueling EGFR toward degradation by the proteasome. These endosomal effects mirrored the endo/lysosomal response of glioma cells to Brefeldin A (BFA), but not the Golgi disruption and the ER collapse, which are associated with the undesirable toxicity of BFA in other cancers. Our results suggest that glioma growth depends on modifications of the vesicle transport system, reliant on kish/TMEM167A. Noncanonical genes in GB could be a key for future therapeutic strategies targeting EGFR-dependent gliomas.
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32
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Alborzinia H, Ignashkova TI, Dejure FR, Gendarme M, Theobald J, Wölfl S, Lindemann RK, Reiling JH. Golgi stress mediates redox imbalance and ferroptosis in human cells. Commun Biol 2018; 1:210. [PMID: 30511023 PMCID: PMC6262011 DOI: 10.1038/s42003-018-0212-6] [Citation(s) in RCA: 115] [Impact Index Per Article: 16.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2017] [Accepted: 11/05/2018] [Indexed: 12/15/2022] Open
Abstract
Cytotoxic activities of several Golgi-dispersing compounds including AMF-26/M-COPA, brefeldin A and golgicide A have previously been shown to induce autophagy or apoptosis. Here, we demonstrate that these Golgi disruptors also trigger ferroptosis, a non-apoptotic form of cell death characterized by iron-dependent oxidative degradation of lipids. Inhibitors of ferroptosis not only counteract cell death, but they also protect from Golgi dispersal and inhibition of protein secretion in response to several Golgi stress agents. Furthermore, the application of sublethal doses of ferroptosis-inducers such as erastin and sorafenib, low cystine growth conditions, or genetic knockdown of SLC7A11 and GPX4 all similarly protect cells from Golgi stress and lead to modulation of ACSL4, SLC7A5, SLC7A11 or GPX4 levels. Collectively, this study suggests a previously unrecognized function of the Golgi apparatus, which involves cellular redox control and prevents ferroptotic cell death. Hamed Alborzinia et al. show that Golgi-dispersing compounds trigger iron-dependent oxidative degradation of lipids, inducing a non-apoptotic cell death called ferroptosis. This study provides insight into the role of Golgi apparatus for preventing ferroptotic cell death through its cellular redox control.
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Affiliation(s)
- Hamed Alborzinia
- BioMed X Innovation Center, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany.,4Present Address: Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ) and Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | | | - Francesca R Dejure
- BioMed X Innovation Center, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany
| | - Mathieu Gendarme
- BioMed X Innovation Center, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany
| | - Jannick Theobald
- 2Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
| | - Stefan Wölfl
- 2Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
| | - Ralph K Lindemann
- 3Translational Innovation Platform Oncology, Merck Biopharma, Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany
| | - Jan H Reiling
- BioMed X Innovation Center, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany.,5Present Address: Institute for Applied Cancer Science and Center for Co-Clinical Trials, University of Texas MD Anderson Cancer Center, Houston, TX USA
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Almiron Bonnin DA, Havrda MC, Israel MA. Glioma Cell Secretion: A Driver of Tumor Progression and a Potential Therapeutic Target. Cancer Res 2018; 78:6031-6039. [PMID: 30333116 DOI: 10.1158/0008-5472.can-18-0345] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2018] [Revised: 05/30/2018] [Accepted: 08/14/2018] [Indexed: 11/16/2022]
Abstract
Cellular secretion is an important mediator of cancer progression. Secreted molecules in glioma are key components of complex autocrine and paracrine pathways that mediate multiple oncogenic pathologies. In this review, we describe tumor cell secretion in high-grade glioma and highlight potential novel therapeutic opportunities. Cancer Res; 78(21); 6031-9. ©2018 AACR.
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Affiliation(s)
- Damian A Almiron Bonnin
- Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire.,Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire
| | - Matthew C Havrda
- Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire.,Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire
| | - Mark A Israel
- Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire. .,Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire.,Departments of Medicine and Pediatrics, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire
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34
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Mishev K, Lu Q, Denoo B, Peurois F, Dejonghe W, Hullaert J, De Rycke R, Boeren S, Bretou M, De Munck S, Sharma I, Goodman K, Kalinowska K, Storme V, Nguyen LSL, Drozdzecki A, Martins S, Nerinckx W, Audenaert D, Vert G, Madder A, Otegui MS, Isono E, Savvides SN, Annaert W, De Vries S, Cherfils J, Winne J, Russinova E. Nonselective Chemical Inhibition of Sec7 Domain-Containing ARF GTPase Exchange Factors. THE PLANT CELL 2018; 30:2573-2593. [PMID: 30018157 PMCID: PMC6241273 DOI: 10.1105/tpc.18.00145] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/14/2018] [Revised: 06/25/2018] [Accepted: 07/17/2018] [Indexed: 05/12/2023]
Abstract
Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.
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Affiliation(s)
- Kiril Mishev
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium
- Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium
- Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
| | - Qing Lu
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium
- Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium
| | - Bram Denoo
- Department of Organic and Macromolecular Chemistry, Ghent University, 9000 Ghent, Belgium
| | - François Peurois
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique, Ecole Normale Supérieure Paris-Saclay, 94235 Cachan, France
| | - Wim Dejonghe
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium
- Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium
| | - Jan Hullaert
- Department of Organic and Macromolecular Chemistry, Ghent University, 9000 Ghent, Belgium
| | - Riet De Rycke
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium
- Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium
- VIB BioImaging Core, 9052 Ghent, Belgium
| | - Sjef Boeren
- Laboratory of Biochemistry, Wageningen University, 6708 Wageningen, The Netherlands
| | - Marine Bretou
- Laboratory for Membrane Trafficking, VIB Center for Brain and Disease Research, KU Leuven, Department of Neurosciences, 3000 Leuven, Belgium
| | - Steven De Munck
- Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium
- Center for Inflammation Research, VIB, 9052 Ghent, Belgium
| | - Isha Sharma
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium
- Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium
| | - Kaija Goodman
- Laboratory of Cell and Molecular Biology and Departments of Botany and Genetics, University of Wisconsin-Madison, Wisconsin 53706
| | - Kamila Kalinowska
- School of Life Sciences Weihenstephan, Technical University of Munich, 85354 Freising, Germany
| | - Veronique Storme
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium
- Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium
| | - Le Son Long Nguyen
- VIB Screening Core, 9052 Ghent, Belgium
- Expertise Centre for Bioassay Development and Screening (C-BIOS), Ghent University, 9052 Ghent, Belgium
| | - Andrzej Drozdzecki
- VIB Screening Core, 9052 Ghent, Belgium
- Expertise Centre for Bioassay Development and Screening (C-BIOS), Ghent University, 9052 Ghent, Belgium
| | - Sara Martins
- Institute for Integrative Biology of the Cell (I2BC), CNRS/CEA/Université Paris Sud, Université Paris-Saclay, Gif-sur-Yvette 91198, France
| | - Wim Nerinckx
- Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium
- Center for Medical Biotechnology, VIB, 9052 Ghent, Belgium
| | - Dominique Audenaert
- VIB Screening Core, 9052 Ghent, Belgium
- Expertise Centre for Bioassay Development and Screening (C-BIOS), Ghent University, 9052 Ghent, Belgium
| | - Grégory Vert
- Institute for Integrative Biology of the Cell (I2BC), CNRS/CEA/Université Paris Sud, Université Paris-Saclay, Gif-sur-Yvette 91198, France
| | - Annemieke Madder
- Department of Organic and Macromolecular Chemistry, Ghent University, 9000 Ghent, Belgium
| | - Marisa S Otegui
- Laboratory of Cell and Molecular Biology and Departments of Botany and Genetics, University of Wisconsin-Madison, Wisconsin 53706
| | - Erika Isono
- School of Life Sciences Weihenstephan, Technical University of Munich, 85354 Freising, Germany
- Department of Biology, University of Konstanz, 78457 Konstanz, Germany
| | - Savvas N Savvides
- Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium
- Center for Inflammation Research, VIB, 9052 Ghent, Belgium
| | - Wim Annaert
- Laboratory for Membrane Trafficking, VIB Center for Brain and Disease Research, KU Leuven, Department of Neurosciences, 3000 Leuven, Belgium
| | - Sacco De Vries
- Laboratory of Biochemistry, Wageningen University, 6708 Wageningen, The Netherlands
| | - Jacqueline Cherfils
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique, Ecole Normale Supérieure Paris-Saclay, 94235 Cachan, France
| | - Johan Winne
- Department of Organic and Macromolecular Chemistry, Ghent University, 9000 Ghent, Belgium
| | - Eugenia Russinova
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium
- Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium
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35
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Luchsinger C, Aguilar M, Burgos PV, Ehrenfeld P, Mardones GA. Functional disruption of the Golgi apparatus protein ARF1 sensitizes MDA-MB-231 breast cancer cells to the antitumor drugs Actinomycin D and Vinblastine through ERK and AKT signaling. PLoS One 2018; 13:e0195401. [PMID: 29614107 PMCID: PMC5882166 DOI: 10.1371/journal.pone.0195401] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2017] [Accepted: 03/21/2018] [Indexed: 12/26/2022] Open
Abstract
Increasing evidence indicates that the Golgi apparatus plays active roles in cancer, but a comprehensive understanding of its functions in the oncogenic transformation has not yet emerged. At the same time, the Golgi is becoming well recognized as a hub that integrates its functions of protein and lipid biosynthesis to signal transduction for cell proliferation and migration in cancer cells. Nevertheless, the active function of the Golgi apparatus in cancer cells has not been fully evaluated as a target for combined treatment. Here, we analyzed the effect of perturbing the Golgi apparatus on the sensitivity of the MDA-MB-231 breast cancer cell line to the drugs Actinomycin D and Vinblastine. We disrupted the function of ARF1, a protein necessary for the homeostasis of the Golgi apparatus. We found that the expression of the ARF1-Q71L mutant increased the sensitivity of MDA-MB-231 cells to both Actinomycin D and Vinblastine, resulting in decreased cell proliferation and cell migration, as well as in increased apoptosis. Likewise, the combined treatment of cells with Actinomycin D or Vinblastine and Brefeldin A or Golgicide A, two disrupting agents of the ARF1 function, resulted in similar effects on cell proliferation, cell migration and apoptosis. Interestingly, each combined treatment had distinct effects on ERK1/2 and AKT signaling, as indicated by the decreased levels of either phospho-ERK1/2 or phospho-AKT. Our results suggest that disruption of Golgi function could be used as a strategy for the sensitization of cancer cells to chemotherapy.
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Affiliation(s)
- Charlotte Luchsinger
- Department of Physiology, School of Medicine, Universidad Austral de Chile, Valdivia, Chile
- Center for Interdisciplinary Studies of the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
| | - Marcelo Aguilar
- Department of Physiology, School of Medicine, Universidad Austral de Chile, Valdivia, Chile
- Center for Interdisciplinary Studies of the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
| | - Patricia V. Burgos
- Department of Physiology, School of Medicine, Universidad Austral de Chile, Valdivia, Chile
- Center for Interdisciplinary Studies of the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
- Center for Cell Biology and Biomedicine (CEBICEM), School of Medicine and Science, Universidad San Sebastián, Santiago, Chile
- Center for Aging and Regeneration (CARE), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Pamela Ehrenfeld
- Center for Interdisciplinary Studies of the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
- Department of Anatomy, Histology and Pathology, School of Medicine, Universidad Austral de Chile, Valdivia, Chile
| | - Gonzalo A. Mardones
- Department of Physiology, School of Medicine, Universidad Austral de Chile, Valdivia, Chile
- Center for Interdisciplinary Studies of the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
- Center for Cell Biology and Biomedicine (CEBICEM), School of Medicine and Science, Universidad San Sebastián, Santiago, Chile
- * E-mail:
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36
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Zappa F, Failli M, De Matteis MA. The Golgi complex in disease and therapy. Curr Opin Cell Biol 2018; 50:102-116. [PMID: 29614425 DOI: 10.1016/j.ceb.2018.03.005] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2018] [Revised: 03/02/2018] [Accepted: 03/11/2018] [Indexed: 10/17/2022]
Abstract
The Golgi complex occupies a strategic position in the endomembrane system and acts not only as a key trafficking and sorting station and a vital biosynthetic center for glycoproteins and lipids, but also as an active signaling hub. As such, the Golgi complex participates in the establishment and maintenance of cell compartmentalization and in general, cell processes such as cell growth and apoptosis. The different functions of the Golgi complex are executed by composite molecular machineries that have been exhaustively dissected over the last three decades. These machineries can become dysfunctional as a result of mutations in the respective encoding genes or may be hijacked by infectious agents or misregulated in the course of multifactorial diseases such as neurodegeneration and cancer. Small molecules targeting components of these machineries have been instrumental in dissecting their functions in in vitro studies and some of them have been developed or are currently under development for clinical use.
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Affiliation(s)
- Francesca Zappa
- Telethon Institute of Genetics and Medicine, Pozzuoli (Naples), Italy
| | - Mario Failli
- Telethon Institute of Genetics and Medicine, Pozzuoli (Naples), Italy
| | - Maria Antonietta De Matteis
- Telethon Institute of Genetics and Medicine, Pozzuoli (Naples), Italy; Department of Molecular Medicine and Medical Biotechnology, Federico II University, Naples, Italy.
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37
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Xie X, Tang SC, Cai Y, Pi W, Deng L, Wu G, Chavanieu A, Teng Y. Suppression of breast cancer metastasis through the inactivation of ADP-ribosylation factor 1. Oncotarget 2018; 7:58111-58120. [PMID: 27517156 PMCID: PMC5295416 DOI: 10.18632/oncotarget.11185] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2016] [Accepted: 08/05/2016] [Indexed: 12/21/2022] Open
Abstract
Metastasis is the major cause of cancer-related death in breast cancer patients, which is controlled by specific sets of genes. Targeting these genes may provide a means to delay cancer progression and allow local treatment to be more effective. We report for the first time that ADP-ribosylation factor 1 (ARF1) is the most amplified gene in ARF gene family in breast cancer, and high-level amplification of ARF1 is associated with increased mRNA expression and poor outcomes of patients with breast cancer. Knockdown of ARF1 leads to significant suppression of migration and invasion in breast cancer cells. Using the orthotopic xenograft model in NSG mice, we demonstrate that loss of ARF1 expression in breast cancer cells inhibits pulmonary metastasis. The zebrafish-metastasis model confirms that the ARF1 gene depletion suppresses breast cancer cells to metastatic disseminate throughout fish body, indicating that ARF1 is a very compelling target to limit metastasis. ARF1 function largely dependents on its activation and LM11, a cell-active inhibitor that specifically inhibits ARF1 activation through targeting the ARF1-GDP/ARNO complex at the Golgi, significantly impairs metastatic capability of breast cancer cell in zebrafish. These findings underline the importance of ARF1 in promoting metastasis and suggest that LM11 that inhibits ARF1 activation may represent a potential therapeutic approach to prevent or treat breast cancer metastasis.
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Affiliation(s)
- Xiayang Xie
- Department of Oral Biology, Augusta University, Augusta, GA, USA.,Department of Pediatrics, Emory Children's Center, Emory University, Atlanta, GA, USA
| | - Shou-Ching Tang
- Georgia Cancer Center, Augusta University, Augusta, GA, USA.,Tianjin Medical University Cancer Institute and Hospital, Tianjin, P.R. China
| | - Yafei Cai
- Georgia Cancer Center, Augusta University, Augusta, GA, USA
| | - Wenhu Pi
- Georgia Cancer Center, Augusta University, Augusta, GA, USA
| | - Libin Deng
- Georgia Cancer Center, Augusta University, Augusta, GA, USA
| | - Guangyu Wu
- Department of Pharmacology and Toxicology, Augusta University, Augusta, GA, USA
| | - Alain Chavanieu
- Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université de Montpellier, CNRS, ENSCM, France
| | - Yong Teng
- Department of Oral Biology, Augusta University, Augusta, GA, USA.,Georgia Cancer Center, Augusta University, Augusta, GA, USA.,Department of Biochemistry and Molecular Biology, Augusta University, Augusta, GA, USA
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38
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ARF1 promotes prostate tumorigenesis via targeting oncogenic MAPK signaling. Oncotarget 2018; 7:39834-39845. [PMID: 27213581 PMCID: PMC5129974 DOI: 10.18632/oncotarget.9405] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2014] [Accepted: 03/11/2016] [Indexed: 11/25/2022] Open
Abstract
ADP-ribosylation factor 1 (ARF1) is a crucial regulator in vesicle-mediated membrane trafficking and involved in the activation of signaling molecules. However, virtually nothing is known about its function in prostate cancer. Here we have demonstrated that ARF1 expression is significantly elevated in prostate cancer cells and human tissues and that the expression levels of ARF1 correlate with the activation of mitogen-activated protein kinases (MAPK) ERK1/2. Furthermore, we have shown that overexpression and knockdown of ARF1 produce opposing effects on prostate cancer cell proliferation, anchorage-independent growth and tumor growth in mouse xenograft models and that ARF1-mediated cell proliferation can be abolished by the Raf1 inhibitor GW5074 and the MEK inhibitors U0126 and PD98059. Moreover, inhibition of ARF1 activation achieved by mutating Thr48 abolishes ARF1's abilities to activate the ERK1/2 and to promote cell proliferation. These data demonstrate that the aberrant MAPK signaling in prostate cancer is, at least in part, under the control of ARF1 and that, similar to Ras, ARF1 is a critical regulator in prostate cancer progression. These data also suggest that ARF1 may represent a key molecular target for prostate cancer therapeutics and diagnosis.
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39
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Ohashi Y, Okamura M, Katayama R, Fang S, Tsutsui S, Akatsuka A, Shan M, Choi HW, Fujita N, Yoshimatsu K, Shiina I, Yamori T, Dan S. Targeting the Golgi apparatus to overcome acquired resistance of non-small cell lung cancer cells to EGFR tyrosine kinase inhibitors. Oncotarget 2017; 9:1641-1655. [PMID: 29416720 PMCID: PMC5788588 DOI: 10.18632/oncotarget.22895] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2017] [Accepted: 11/17/2017] [Indexed: 11/25/2022] Open
Abstract
Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKIs) were demonstrated to provide survival benefit in patients with non-small cell lung cancer (NSCLC) harboring activating mutations of EGFR; however, emergence of acquired resistance to EGFR-TKIs has been shown to cause poor outcome. To overcome the TKI resistance, drugs with different mode of action are required. We previously reported that M-COPA (2-methylcoprophilinamide), a Golgi disruptor, suppressed the growth of gastric cancers overexpressing receptor tyrosine kinases (RTKs) such as hepatocyte growth factor receptor (MET) via downregulating their cell surface expression. In this study, we examined the antitumor effect of M-COPA on NSCLC cells with TKI resistance. As a result, M-COPA effectively downregulated cell surface EGFR and its downstream signals, and finally exerted in vivo antitumor effect in NSCLC cells harboring secondary (T790M/del19) and tertiary (C797S/T790M/del19) mutated EGFR, which exhibit acquired resistance to first- and third generation EGFR-TKIs, respectively. M-COPA also downregulated MET expression potentially involved in the acquired resistance to EGFR-TKIs via bypassing the EGFR pathway blockade. These results provide the first evidence that targeting the Golgi apparatus might be a promising therapeutic strategy to overcome the vicious cycle of TKI resistance in EGFR-mutated NSCLC cells via downregulating cell surface RTK expression.
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Affiliation(s)
- Yoshimi Ohashi
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Mutsumi Okamura
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Ryohei Katayama
- Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Siyang Fang
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Saki Tsutsui
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Akinobu Akatsuka
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Mingde Shan
- Eisai AiM Institute, Eisai Inc., Andover, MA, USA
| | | | - Naoya Fujita
- Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | | | - Isamu Shiina
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan
| | - Takao Yamori
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.,Present address: Center for Product Evaluation, Pharmaceuticals and Medical Devices Agency, Tokyo, Japan
| | - Shingo Dan
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
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40
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Obata Y, Horikawa K, Shiina I, Takahashi T, Murata T, Tasaki Y, Suzuki K, Yonekura K, Esumi H, Nishida T, Abe R. Oncogenic Kit signalling on the Golgi is suppressed by blocking secretory trafficking with M-COPA in gastrointestinal stromal tumours. Cancer Lett 2017; 415:1-10. [PMID: 29196126 DOI: 10.1016/j.canlet.2017.11.032] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2017] [Revised: 11/14/2017] [Accepted: 11/23/2017] [Indexed: 02/08/2023]
Abstract
Most gastrointestinal stromal tumours (GISTs) are caused by constitutively active mutations in Kit tyrosine kinase. The drug imatinib, a specific Kit inhibitor, improves the prognosis of metastatic GIST patients, but these patients become resistant to the drug by acquiring secondary mutations in the Kit kinase domain. We recently reported that a Kit mutant causes oncogenic signals only on the Golgi apparatus in GISTs. In this study, we show that in GIST, 2-methylcoprophilinamide (M-COPA, also known as "AMF-26"), an inhibitor of biosynthetic protein trafficking from the endoplasmic reticulum (ER) to the Golgi, suppresses Kit autophosphorylation at Y703/Y721/Y730/Y936, resulting in blockade of oncogenic signalling. Results of our M-COPA treatment assay show that Kit Y703/Y730/Y936 in the ER are dephosphorylated by protein tyrosine phosphatases (PTPs), thus the ER-retained Kit is unable to activate downstream molecules. ER-localized Kit Y721 is not phosphorylated, but not due to PTPs. Importantly, M-COPA can inhibit the activation of the Kit kinase domain mutant, resulting in suppression of imatinib-resistant GIST proliferation. Our study demonstrates that Kit autophosphorylation is spatio-temporally regulated and may offer a new strategy for treating imatinib-resistant GISTs.
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Affiliation(s)
- Yuuki Obata
- Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda 278-0022, Chiba, Japan
| | - Keita Horikawa
- Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda 278-0022, Chiba, Japan
| | - Isamu Shiina
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku 162-8601, Tokyo, Japan
| | - Tsuyoshi Takahashi
- Department of Surgery, Graduate School of Medicine, Osaka University, Suita 565-0871, Osaka, Japan
| | - Takatsugu Murata
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku 162-8601, Tokyo, Japan
| | - Yasutaka Tasaki
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku 162-8601, Tokyo, Japan
| | - Kyohei Suzuki
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku 162-8601, Tokyo, Japan
| | - Keita Yonekura
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku 162-8601, Tokyo, Japan
| | - Hiroyasu Esumi
- Division of Clinical Research, Research Institute for Biomedical Sciences, Tokyo, University of Science, Japan
| | - Toshirou Nishida
- National Cancer Center Hospital, Chuo-ku, 104-0045, Tokyo, Japan
| | - Ryo Abe
- Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda 278-0022, Chiba, Japan.
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Benabdi S, Peurois F, Nawrotek A, Chikireddy J, Cañeque T, Yamori T, Shiina I, Ohashi Y, Dan S, Rodriguez R, Cherfils J, Zeghouf M. Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles. Biochemistry 2017; 56:5125-5133. [PMID: 28858527 DOI: 10.1021/acs.biochem.7b00706] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.
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Affiliation(s)
- Sarah Benabdi
- Laboratoire de Biologie et Pharmacologie Appliquée CNRS, Ecole Normale Supérieure Paris-Saclay , 61 avenue du président Wilson, 94235 Cachan, France
| | - François Peurois
- Laboratoire de Biologie et Pharmacologie Appliquée CNRS, Ecole Normale Supérieure Paris-Saclay , 61 avenue du président Wilson, 94235 Cachan, France
| | - Agata Nawrotek
- Laboratoire de Biologie et Pharmacologie Appliquée CNRS, Ecole Normale Supérieure Paris-Saclay , 61 avenue du président Wilson, 94235 Cachan, France
| | - Jahnavi Chikireddy
- Laboratoire de Biologie et Pharmacologie Appliquée CNRS, Ecole Normale Supérieure Paris-Saclay , 61 avenue du président Wilson, 94235 Cachan, France
| | - Tatiana Cañeque
- Institut Curie, PSL Research University , Chemical Cell Biology group, 26 rue d'Ulm, 75248 Paris Cedex 05, France.,CNRS UMR3666 , 75005 Paris, France.,INSERM U1143 , 75005 Paris, France
| | - Takao Yamori
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research , Tokyo 135-8550, Japan
| | - Isamu Shiina
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science , Tokyo 162-8601, Japan
| | - Yoshimi Ohashi
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research , Tokyo 135-8550, Japan
| | - Shingo Dan
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research , Tokyo 135-8550, Japan
| | - Raphaël Rodriguez
- Institut Curie, PSL Research University , Chemical Cell Biology group, 26 rue d'Ulm, 75248 Paris Cedex 05, France.,CNRS UMR3666 , 75005 Paris, France.,INSERM U1143 , 75005 Paris, France
| | - Jacqueline Cherfils
- Laboratoire de Biologie et Pharmacologie Appliquée CNRS, Ecole Normale Supérieure Paris-Saclay , 61 avenue du président Wilson, 94235 Cachan, France
| | - Mahel Zeghouf
- Laboratoire de Biologie et Pharmacologie Appliquée CNRS, Ecole Normale Supérieure Paris-Saclay , 61 avenue du président Wilson, 94235 Cachan, France
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42
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Lang L, Shay C, Zhao X, Teng Y. Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2017; 36:112. [PMID: 28830537 PMCID: PMC5568197 DOI: 10.1186/s13046-017-0583-4] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/19/2017] [Accepted: 08/16/2017] [Indexed: 02/07/2023]
Abstract
Background Although major improvements have been made in surgical management, chemotherapeutic, and radiotherapeutic of prostate cancer, many prostate cancers remain refractory to treatment with standard agents. Therefore, the identification of new molecular targets in cancer progression and development of novel therapeutic strategies to target them are very necessary for achieving better survival for patients with prostate cancer. Activation of small GTPases such as Ras and Arf1 is a critical component of the signaling pathways for most of the receptors shown to be upregulated in advanced prostate cancer. Methods The drug effects on cell proliferation were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay. The drug effects on cell migration and invasion were determined by Radius™ 24-well and Matrigel-coated Boyden chambers. The drug effects on apoptosis were assessed by FITC Annexin V Apoptosis Detection Kit with 7-AAD and Western blot with antibodies against cleaved PARP and Caspase 3. A NOD/SCID mouse model generated by subcutaneous injection was used to assess the in vivo drug efficacy in tumor growth. ERK activation and tumor cell proliferation in xenografts were examined by immunohistochemistry. Results We show that Exo2, a small-molecule inhibitor that reduces Arf1 activation, effectively suppresses prostate cancer cell proliferation by blocking ERK1/2 activation. Exo2 also has other effects, inhibiting migration and invasion of PCa cells and inducing apoptosis. The Ras inhibitor salirasib augments Exo2-induced cytotoxicity in prostate cancer cells partially by enhancing the suppression of ERK1/2 phosphorylation. In a xenograft mouse model of prostate cancer, Exo2 reduces prostate tumor burden and inhibits ERK1/2 activation at a dose of 20 mg/kg. Synergistic treatment of salirasib and Exo2 exhibits a superior inhibitory effect on prostate tumor growth compared with either drug alone, which may be attributed to the more efficient inhibition of ERK1/2 phosphorylation. Conclusion This study suggests that simultaneous blockade of Arf1 and Ras activation in prostate cancer cells is a potential targeted therapeutic strategy for preventing prostate cancer development. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0583-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Liwei Lang
- Department of Oral Biology, Augusta University, Augusta, GA, 30912, USA
| | - Chloe Shay
- Department of Pediatrics, Emory Children's Center, Emory University, Atlanta, GA, 30322, USA
| | - Xiangdong Zhao
- Department of Oral Biology, Augusta University, Augusta, GA, 30912, USA
| | - Yong Teng
- Department of Oral Biology, Augusta University, Augusta, GA, 30912, USA. .,Georgia Cancer Center, Augusta University, 1120 15th Street, Augusta, GA, 30912, USA. .,Department of Biochemistry and Molecular Biology, Augusta University, Augusta, GA, 30912, USA.
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43
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Ignashkova TI, Gendarme M, Peschk K, Eggenweiler HM, Lindemann RK, Reiling JH. Cell survival and protein secretion associated with Golgi integrity in response to Golgi stress-inducing agents. Traffic 2017; 18:530-544. [PMID: 28485883 DOI: 10.1111/tra.12493] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2017] [Revised: 05/05/2017] [Accepted: 05/05/2017] [Indexed: 12/29/2022]
Abstract
The Golgi apparatus is part of the secretory pathway and of central importance for modification, transport and sorting of proteins and lipids. ADP-ribosylation factors, whose activation can be blocked by brefeldin A (BFA), play a major role in functioning of the Golgi network and regulation of membrane traffic and are also involved in proliferation and migration of cancer cells. Due to high cytotoxicity and poor bioavailability, BFA has not passed the preclinical stage of drug development. Recently, AMF-26 and golgicide A have been described as novel inhibitors of the Golgi system with antitumor or bactericidal properties. We provide here further evidence that AMF-26 closely mirrors the mode of action of BFA but is less potent. Using several human cancer cell lines, we studied the effects of AMF-26, BFA and golgicide A on cell homeostasis including Golgi structure, endoplasmic reticulum (ER) stress markers, secretion and viability, and found overall a significant correlation between these parameters. Furthermore, modulation of ADP-ribosylation factor expression has a profound impact on Golgi organization and survival in response to Golgi stress inducers.
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Affiliation(s)
- Tatiana I Ignashkova
- Metabolism and Signaling in Cancer, BioMed X Innovation Center, Heidelberg, Germany
| | - Mathieu Gendarme
- Metabolism and Signaling in Cancer, BioMed X Innovation Center, Heidelberg, Germany
| | - Katrin Peschk
- Medicinal Chemistry, Merck Biopharma, Merck KGaA, Darmstadt, Germany
| | | | - Ralph K Lindemann
- Translational Innovation Platform Oncology, Merck Biopharma, Merck KGaA, Darmstadt, Germany
| | - Jan H Reiling
- Metabolism and Signaling in Cancer, BioMed X Innovation Center, Heidelberg, Germany
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Hara Y, Obata Y, Horikawa K, Tasaki Y, Suzuki K, Murata T, Shiina I, Abe R. M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells. PLoS One 2017; 12:e0175514. [PMID: 28403213 PMCID: PMC5389679 DOI: 10.1371/journal.pone.0175514] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2016] [Accepted: 03/27/2017] [Indexed: 01/28/2023] Open
Abstract
Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.
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Affiliation(s)
- Yasushi Hara
- Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan
| | - Yuuki Obata
- Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan
- * E-mail:
| | - Keita Horikawa
- Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan
| | - Yasutaka Tasaki
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan
| | - Kyohei Suzuki
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan
| | - Takatsugu Murata
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan
| | - Isamu Shiina
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan
| | - Ryo Abe
- Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan
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45
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The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila. Nature 2016; 538:109-113. [PMID: 27680705 DOI: 10.1038/nature19788] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2015] [Accepted: 08/18/2016] [Indexed: 01/18/2023]
Abstract
Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. Here we investigated stem-cell death in the digestive system of adult Drosophila melanogaster. We found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively killed normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spared differentiated cells. The dying stem cells were engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduced CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers.
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Abstract
Members of the ADP-ribosylation factor (Arf) family of small GTP-binding (G) proteins regulate several aspects of membrane trafficking, such as vesicle budding, tethering and cytoskeleton organization. Arf family members, including Arf-like (Arl) proteins have been implicated in several essential cellular functions, like cell spreading and migration. These functions are used by cancer cells to disseminate and invade the tissues surrounding the primary tumor, leading to the formation of metastases. Indeed, Arf and Arl proteins, as well as their guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) have been found to be abnormally expressed in different cancer cell types and human cancers. Here, we review the current evidence supporting the involvement of Arf family proteins and their GEFs and GAPs in cancer progression, focusing on 3 different mechanisms: cell-cell adhesion, integrin internalization and recycling, and actin cytoskeleton remodeling.
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Affiliation(s)
- Cristina Casalou
- a CEDOC, NOVA Medical School - Faculdade de Ciências Médicas, Universidade NOVA de Lisboa , Lisbon , Portugal
| | - Alexandra Faustino
- a CEDOC, NOVA Medical School - Faculdade de Ciências Médicas, Universidade NOVA de Lisboa , Lisbon , Portugal.,b ProRegeM PhD Program, NOVA Medical School - Faculdade de Ciências Médicas, Universidade NOVA de Lisboa , Lisbon , Portugal
| | - Duarte C Barral
- a CEDOC, NOVA Medical School - Faculdade de Ciências Médicas, Universidade NOVA de Lisboa , Lisbon , Portugal
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Thinon E, Morales-Sanfrutos J, Mann DJ, Tate EW. N-Myristoyltransferase Inhibition Induces ER-Stress, Cell Cycle Arrest, and Apoptosis in Cancer Cells. ACS Chem Biol 2016; 11:2165-76. [PMID: 27267252 PMCID: PMC5077176 DOI: 10.1021/acschembio.6b00371] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2016] [Accepted: 05/24/2016] [Indexed: 12/16/2022]
Abstract
N-Myristoyltransferase (NMT) covalently attaches a C14 fatty acid to the N-terminal glycine of proteins and has been proposed as a therapeutic target in cancer. We have recently shown that selective NMT inhibition leads to dose-responsive loss of N-myristoylation on more than 100 protein targets in cells, and cytotoxicity in cancer cells. N-myristoylation lies upstream of multiple pro-proliferative and oncogenic pathways, but to date the complex substrate specificity of NMT has limited determination of which diseases are most likely to respond to a selective NMT inhibitor. We describe here the phenotype of NMT inhibition in HeLa cells and show that cells die through apoptosis following or concurrent with accumulation in the G1 phase. We used quantitative proteomics to map protein expression changes for more than 2700 proteins in response to treatment with an NMT inhibitor in HeLa cells and observed down-regulation of proteins involved in cell cycle regulation and up-regulation of proteins involved in the endoplasmic reticulum stress and unfolded protein response, with similar results in breast (MCF-7, MDA-MB-231) and colon (HCT116) cancer cell lines. This study describes the cellular response to NMT inhibition at the proteome level and provides a starting point for selective targeting of specific diseases with NMT inhibitors, potentially in combination with other targeted agents.
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Affiliation(s)
- Emmanuelle Thinon
- Department
of Chemistry, Imperial College London, Exhibition Road, London SW72AZ, United Kingdom
- Department
of Life Sciences, Imperial College London, Exhibition Road, London SW72AZ, United Kingdom
| | - Julia Morales-Sanfrutos
- Department
of Chemistry, Imperial College London, Exhibition Road, London SW72AZ, United Kingdom
| | - David J. Mann
- Department
of Life Sciences, Imperial College London, Exhibition Road, London SW72AZ, United Kingdom
- Institute
of Chemical Biology, Department of Chemistry, Imperial College London, Exhibition Road, London SW72AZ, United Kingdom
| | - Edward W. Tate
- Department
of Chemistry, Imperial College London, Exhibition Road, London SW72AZ, United Kingdom
- Institute
of Chemical Biology, Department of Chemistry, Imperial College London, Exhibition Road, London SW72AZ, United Kingdom
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48
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Akatsuka A, Kojima N, Okamura M, Dan S, Yamori T. A novel thiophene-3-carboxamide analog of annonaceous acetogenin exhibits antitumor activity via inhibition of mitochondrial complex I. Pharmacol Res Perspect 2016; 4:e00246. [PMID: 28116099 PMCID: PMC5242172 DOI: 10.1002/prp2.246] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2015] [Revised: 06/15/2016] [Accepted: 06/17/2016] [Indexed: 01/07/2023] Open
Abstract
Previously we synthesized JCI‐20679, a novel thiophene‐3‐carboxamide analog of annonaceous acetogenins which have shown potent antitumor activity, with no serious side effects, in mouse xenograft models. In this study, we investigated the antitumor mechanism of JCI‐20679. The growth inhibition profile (termed “fingerprint”) of this agent across a panel of 39 human cancer cell lines (termed “JFCR39”) was measured; this fingerprint was analyzed by the COMPARE algorithm utilizing the entire drug sensitivity database for the JFCR39 panel. The JCI‐20679‐specific fingerprint exhibited a high similarity to those of two antidiabetic biguanides and a natural rotenoid deguelin which were already known to be mitochondrial complex I inhibitors. In addition, the fingerprint exhibited by JCI‐20679 was not similar to that displayed by any typical anticancer drugs within the database, suggesting that it has a unique mode of action. In vitro experiments using bovine heart‐derived mitochondria showed direct inhibition of mitochondrial complex I by JCI‐20679 and associated derivatives. This inhibition of enzymatic activity positively correlated with tumor cell growth inhibition. Furthermore, a fluorescently labeled derivative of JCI‐20679 localized to the mitochondria of live cancer cells in vitro. These results suggest that JCI‐20679 can inhibit cancer cell growth by inhibiting mitochondrial complex I. Our results show that JCI‐20679 is a novel anticancer drug lead with a unique mode of action.
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Affiliation(s)
- Akinobu Akatsuka
- Molecular Pharmacology Cancer Chemotherapy Center Japanese Foundation for Cancer Research Tokyo Japan
| | - Naoto Kojima
- Pharmaceutical Manufacturing Chemistry Kyoto Pharmaceutical University Kyoto Japan
| | - Mutsumi Okamura
- Molecular Pharmacology Cancer Chemotherapy Center Japanese Foundation for Cancer Research Tokyo Japan
| | - Shingo Dan
- Molecular Pharmacology Cancer Chemotherapy Center Japanese Foundation for Cancer Research Tokyo Japan
| | - Takao Yamori
- Molecular Pharmacology Cancer Chemotherapy Center Japanese Foundation for Cancer Research Tokyo Japan; Present address: Pharmaceutical and Medical Devices Agency Tokyo Japan
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Hattori T, Watanabe-Takahashi M, Shiina I, Ohashi Y, Dan S, Nishikawa K, Yamori T, Naito M. M-COPA, a novel Golgi system disruptor, suppresses apoptosis induced by Shiga toxin. Genes Cells 2016; 21:901-6. [PMID: 27302278 DOI: 10.1111/gtc.12386] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2016] [Accepted: 05/12/2016] [Indexed: 11/29/2022]
Abstract
Shiga toxin (Stx) is a main virulence factor of Stx-producing Escherichia coli (STEC) that contributes to diarrhea and hemorrhagic colitis and occasionally to fatal systemic complications. Therefore, the development of an antidote to neutralize Stx toxicity is urgently needed. After internalization into cells, Stx is transferred to the Golgi apparatus via a retrograde vesicular transport system. We report here that 2-methylcoprophilinamide (M-COPA), a compound that induces disassembly of the Golgi apparatus by inactivating ADP-ribosylation factor 1 (Arf1), suppresses Stx-induced apoptosis. M-COPA inhibited transport of Stx from the plasma membrane to the Golgi apparatus and suppressed degradation of anti-apoptotic proteins and the activation of caspases. These findings suggest that inhibition of Stx retrograde transport by M-COPA could be a novel approach to suppress Stx toxicity.
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Affiliation(s)
- Takayuki Hattori
- Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501, Japan
| | - Miho Watanabe-Takahashi
- Faculty of Life and Medical Sciences, Doshisha University, 1-3 Miyakotani, Tatara, Kyotanabe, Kyoto, 610-0394, Japan
| | - Isamu Shiina
- Department of Applied Chemistry, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo, 162-8601, Japan
| | - Yoshimi Ohashi
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo, 135-8550, Japan
| | - Shingo Dan
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo, 135-8550, Japan
| | - Kiyotaka Nishikawa
- Faculty of Life and Medical Sciences, Doshisha University, 1-3 Miyakotani, Tatara, Kyotanabe, Kyoto, 610-0394, Japan
| | - Takao Yamori
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo, 135-8550, Japan.,Center for Product Evaluation, Pharmaceuticals and Medical Device Agency, Shin-Kasumigaseki Building 3-3-2 Kasumigaseki, Tokyo, 100-0013, Japan
| | - Mikihiko Naito
- Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501, Japan
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50
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Ohashi Y, Okamura M, Hirosawa A, Tamaki N, Akatsuka A, Wu KM, Choi HW, Yoshimatsu K, Shiina I, Yamori T, Dan S. M-COPA, a Golgi Disruptor, Inhibits Cell Surface Expression of MET Protein and Exhibits Antitumor Activity against MET-Addicted Gastric Cancers. Cancer Res 2016; 76:3895-903. [PMID: 27197184 DOI: 10.1158/0008-5472.can-15-2220] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2015] [Accepted: 03/28/2016] [Indexed: 12/31/2022]
Abstract
The Golgi apparatus is responsible for transporting, processing, and sorting numerous proteins in the cell, including cell surface-expressed receptor tyrosine kinases (RTK). The small-molecule compound M-COPA [2-methylcoprophilinamide (AMF-26)] disrupts the Golgi apparatus by inhibiting the activation of Arf1, resulting in suppression of tumor growth. Here, we report an evaluation of M-COPA activity against RTK-addicted cancers, focusing specifically on human gastric cancer (GC) cells with or without MET amplification. As expected, the MET-addicted cell line MKN45 exhibited a better response to M-COPA than cell lines without MET amplification. Upon M-COPA treatment, cell surface expression of MET was downregulated with a concurrent accumulation of its precursor form. M-COPA also reduced levels of the phosphorylated form of MET along with the downstream signaling molecules Akt and S6. Similar results were obtained in additional GC cell lines with amplification of MET or the FGF receptor FGFR2 MKN45 murine xenograft experiments demonstrated the antitumor activity of M-COPA in vivo Taken together, our results offer an initial preclinical proof of concept for the use of M-COPA as a candidate treatment option for MET-addicted GC, with broader implications for targeting the Golgi apparatus as a novel cancer therapeutic approach. Cancer Res; 76(13); 3895-903. ©2016 AACR.
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MESH Headings
- Animals
- Anti-Inflammatory Agents, Non-Steroidal/pharmacology
- Apoptosis/drug effects
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Case-Control Studies
- Cell Proliferation/drug effects
- Female
- Follow-Up Studies
- Golgi Apparatus/drug effects
- Golgi Apparatus/metabolism
- Golgi Apparatus/pathology
- Humans
- Immunoenzyme Techniques
- Mice
- Mice, Inbred C57BL
- Mice, Nude
- Naphthols/pharmacology
- Neoplasm Staging
- Phosphorylation/drug effects
- Prognosis
- Proto-Oncogene Proteins c-met/genetics
- Proto-Oncogene Proteins c-met/metabolism
- Pyridines/pharmacology
- Receptor, Fibroblast Growth Factor, Type 2/genetics
- Receptor, Fibroblast Growth Factor, Type 2/metabolism
- Signal Transduction/drug effects
- Stomach Neoplasms/drug therapy
- Stomach Neoplasms/metabolism
- Stomach Neoplasms/pathology
- Tumor Cells, Cultured
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Yoshimi Ohashi
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Mutsumi Okamura
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Asaka Hirosawa
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Naomi Tamaki
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Akinobu Akatsuka
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Kuo-Ming Wu
- Next Generation Systems, Eisai Inc., Andover, Massachusetts
| | | | | | - Isamu Shiina
- Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan
| | - Takao Yamori
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Shingo Dan
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.
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