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Liu Y, Tian W, Ge C, Zhang C, Huang Z, Zhang C, Yang Y, Tian H. SNX17 mediates STAT3 activation to promote hepatocellular carcinoma progression via a retromer dependent mechanism. Int J Biol Sci 2025; 21:2762-2779. [PMID: 40303303 PMCID: PMC12035908 DOI: 10.7150/ijbs.110506] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2025] [Accepted: 03/17/2025] [Indexed: 05/02/2025] Open
Abstract
Endocytosis has emerged as a key regulator of malignant behavior in cancer. Members of the sorting nexin (SNX) family have been found to be dysregulated in various cancers and play significant roles in regulating tumor metastasis. However, the role and mechanism of SNX17 in hepatocellular carcinoma (HCC) progression remain largely unknown. Here, we found that upregulation of SNX17 in HCC was associated with poor prognosis. Overexpression of SNX17 promoted HCC cell proliferation, migration, invasion, and metastasis, whereas silencing SNX17 expression resulted in opposite effects. Knockdown of SNX17 induced G1/S phase arrest and apoptosis. We discovered that SNX17 directly interacted with STAT3 and increased its phosphorylation in a retromer-dependent manner. SNX17-retromer complex acted as a platform for IL-6-induced STAT3 activation. Activated STAT3 then increased c-Myc expression and promoted mitochondrial oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis. SNX17 overexpression-induced OXPHOS was reversed by c-Myc inhibitor. Knockdown of STAT3 expression or treatment with a STAT3 inhibitor significantly attenuated SNX17-enhanced proliferation and invasion. Taken together, our results indicate that SNX17 promotes HCC cell proliferation and metastasis through direct interaction with STAT3 in a retromer-dependent manner, thereby activating the STAT3/c-Myc signaling pathway and enhancing OXPHOS. These findings suggest that SNX17 is a potential therapeutic target for HCC.
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Affiliation(s)
- Yuqi Liu
- State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Wei Tian
- State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Chao Ge
- State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Canxue Zhang
- State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Zhihong Huang
- State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Chi Zhang
- State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Yue Yang
- State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Hua Tian
- State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Department of Pathology, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise 533000, China
- The Key Laboratory of Molecular Pathology (Hepatobiliary Diseases) of Guangxi, Baise 533000, China
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Seymour L, Nuru N, Johnson KR, Gutierrez JMV, Njoku VT, Darie CC, Neagu AN. Roles of Post-Translational Modifications of Transcription Factors Involved in Breast Cancer Hypoxia. Molecules 2025; 30:645. [PMID: 39942749 PMCID: PMC11820228 DOI: 10.3390/molecules30030645] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2024] [Revised: 01/17/2025] [Accepted: 01/28/2025] [Indexed: 02/16/2025] Open
Abstract
BC is the most commonly diagnosed cancer and the second leading cause of cancer death among women worldwide. Cellular stress is a condition that leads to disrupted homeostasis by extrinsic and intrinsic factors. Among other stressors, hypoxia is a driving force for breast cancer (BC) progression and a general hallmark of solid tumors. Thus, intratumoral hypoxia is an important determinant of invasion, metastasis, treatment failure, prognosis, and patient mortality. Acquisition of the epithelial-mesenchymal transition (EMT) phenotype is also a consequence of tumor hypoxia. The cellular response to hypoxia is mainly regulated by the hypoxia signaling pathway, governed by hypoxia-inducible factors (HIFs), mainly HIF1α. HIFs are a family of transcription factors (TFs), which induce the expression of target genes involved in cell survival and proliferation, metabolic reprogramming, angiogenesis, resisting apoptosis, invasion, and metastasis. HIF1α cooperates with a large number of other TFs. In this review, we focused on the crosstalk and cooperation between HIF1α and other TFs involved in the cellular response to hypoxia in BC. We identified a cluster of TFs, proposed as the HIF1α-TF interactome, that orchestrates the transcription of target genes involved in hypoxia, due to their post-translational modifications (PTMs), including phosphorylation/dephosphorylation, ubiquitination/deubiquitination, SUMOylation, hydroxylation, acetylation, S-nitrosylation, and palmitoylation. PTMs of these HIF1α-related TFs drive their stability and activity, degradation and turnover, and the bidirectional translocation between the cytoplasm or plasma membrane and nucleus of BC cells, as well as the transcription/activation of proteins encoded by oncogenes or inactivation of tumor suppressor target genes. Consequently, PTMs of TFs in the HIF1α interactome are crucial regulatory mechanisms that drive the cellular response to oxygen deprivation in BC cells.
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Affiliation(s)
- Logan Seymour
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810, USA; (L.S.); (N.N.); (K.R.J.); (J.M.V.G.); (V.T.N.)
| | - Niyogushima Nuru
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810, USA; (L.S.); (N.N.); (K.R.J.); (J.M.V.G.); (V.T.N.)
| | - Kaya R. Johnson
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810, USA; (L.S.); (N.N.); (K.R.J.); (J.M.V.G.); (V.T.N.)
| | - Jennifer Michel Villalpando Gutierrez
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810, USA; (L.S.); (N.N.); (K.R.J.); (J.M.V.G.); (V.T.N.)
| | - Victor Tochukwu Njoku
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810, USA; (L.S.); (N.N.); (K.R.J.); (J.M.V.G.); (V.T.N.)
| | - Costel C. Darie
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810, USA; (L.S.); (N.N.); (K.R.J.); (J.M.V.G.); (V.T.N.)
| | - Anca-Narcisa Neagu
- Laboratory of Animal Histology, Faculty of Biology, “Alexandru Ioan Cuza” University of Iași, Carol I bvd. 20A, 700505 Iasi, Romania
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Bruno PS, Arshad A, Gogu MR, Waterman N, Flack R, Dunn K, Darie CC, Neagu AN. Post-Translational Modifications of Proteins Orchestrate All Hallmarks of Cancer. Life (Basel) 2025; 15:126. [PMID: 39860065 PMCID: PMC11766951 DOI: 10.3390/life15010126] [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: 12/23/2024] [Revised: 01/14/2025] [Accepted: 01/16/2025] [Indexed: 01/27/2025] Open
Abstract
Post-translational modifications (PTMs) of proteins dynamically build the buffering and adapting interface between oncogenic mutations and environmental stressors, on the one hand, and cancer cell structure, functioning, and behavior. Aberrant PTMs can be considered as enabling characteristics of cancer as long as they orchestrate all malignant modifications and variability in the proteome of cancer cells, cancer-associated cells, and tumor microenvironment (TME). On the other hand, PTMs of proteins can enhance anticancer mechanisms in the tumoral ecosystem or sustain the beneficial effects of oncologic therapies through degradation or inactivation of carcinogenic proteins or/and activation of tumor-suppressor proteins. In this review, we summarized and analyzed a wide spectrum of PTMs of proteins involved in all regulatory mechanisms that drive tumorigenesis, genetic instability, epigenetic reprogramming, all events of the metastatic cascade, cytoskeleton and extracellular matrix (ECM) remodeling, angiogenesis, immune response, tumor-associated microbiome, and metabolism rewiring as the most important hallmarks of cancer. All cancer hallmarks develop due to PTMs of proteins, which modulate gene transcription, intracellular and extracellular signaling, protein size, activity, stability and localization, trafficking, secretion, intracellular protein degradation or half-life, and protein-protein interactions (PPIs). PTMs associated with cancer can be exploited to better understand the underlying molecular mechanisms of this heterogeneous and chameleonic disease, find new biomarkers of cancer progression and prognosis, personalize oncotherapies, and discover new targets for drug development.
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Affiliation(s)
- Pathea Shawnae Bruno
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biochemistry, Clarkson University, Potsdam, NY 13699-5810, USA; (P.S.B.); (A.A.); (N.W.); (R.F.); (K.D.)
| | - Aneeta Arshad
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biochemistry, Clarkson University, Potsdam, NY 13699-5810, USA; (P.S.B.); (A.A.); (N.W.); (R.F.); (K.D.)
| | - Maria-Raluca Gogu
- Advanced Research and Development Center for Experimental Medicine (CEMEX), “Grigore T. Popa” University of Medicine and Pharmacy, University Street No. 16, 700115 Iasi, Romania;
| | - Natalie Waterman
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biochemistry, Clarkson University, Potsdam, NY 13699-5810, USA; (P.S.B.); (A.A.); (N.W.); (R.F.); (K.D.)
| | - Rylie Flack
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biochemistry, Clarkson University, Potsdam, NY 13699-5810, USA; (P.S.B.); (A.A.); (N.W.); (R.F.); (K.D.)
| | - Kimberly Dunn
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biochemistry, Clarkson University, Potsdam, NY 13699-5810, USA; (P.S.B.); (A.A.); (N.W.); (R.F.); (K.D.)
| | - Costel C. Darie
- Biochemistry & Proteomics Laboratories, Department of Chemistry and Biochemistry, Clarkson University, Potsdam, NY 13699-5810, USA; (P.S.B.); (A.A.); (N.W.); (R.F.); (K.D.)
| | - Anca-Narcisa Neagu
- Laboratory of Animal Histology, Faculty of Biology, “Alexandru Ioan Cuza” University of Iași, Carol I bvd. 20A, 700505 Iasi, Romania
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Bandaru M, Sultana OF, Islam MA, Rainier A, Reddy PH. Rlip76 in ageing and Alzheimer's disease: Focus on oxidative stress and mitochondrial mechanisms. Ageing Res Rev 2025; 103:102600. [PMID: 39617058 DOI: 10.1016/j.arr.2024.102600] [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: 08/14/2024] [Revised: 11/12/2024] [Accepted: 11/25/2024] [Indexed: 12/13/2024]
Abstract
RLIP76 (Rlip), a stress-responsive protein, plays a multifaceted role in cellular function. This protein acts primarily as a glutathione-electrophile conjugate (GS-E) transporter, crucial for detoxifying hazardous compounds and converting them into mercapturic acids. RLIP76 also modulates cytoskeletal motility and membrane plasticity through its role in the Ral-signaling pathway, interacting with RalA and RalB, key small GTPases involved in growth and metastasis. Beyond its ATP-dependent transport functions in various tissues, RLIP76 also demonstrates GTPase Activating Protein (GAP) activity towards Rac1 and Cdc42, with a preference for Ral-GTP over Ral-GDP. Its functions span critical physiological processes including membrane dynamics, oxidative stress response, and mitochondrial dynamics. The protein's widespread expression and evolutionary conservation underscore its significance. Our lab discovered that Rlip interacts with Alzheimer's disease (AD) proteins, amyloid beta and phosphorylated and induce oxidative stress, mitochondrial dysfnction and synaptic damage in AD. Our in vitro studies revealed that overexpression of Rlip reduces mitochondrial abnormalities. Further, our in vivo studies (Rlip+/- mice) revealed that a partial reduction of Rlip in mice (Rlip+/-), leads to mitochondrial abnormalities, elevated oxidative stress, and cognitive deficits resembling late-onset AD, emphasizing the protein's crucial role in neuronal health and disease. Finally, we discuss the experimental cross-breedings of overexpression of mice Rlip TG/TG or Rlip + /- mice with Alzheimer's disease models - earlyonset 5XFAD, late-onset APPKI and Tau transgenic mice, providing new insights into RLIP76's role in AD progression and development. This review summarizes RLIP76's structure, function, and cellular pathways, highlighting its implications in AD and its potential as a therapeutic target.
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Affiliation(s)
- Madhuri Bandaru
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Omme Fatema Sultana
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Md Ariful Islam
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Alvir Rainier
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - P Hemachandra Reddy
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Nutritional Sciences Department, College of Human Sciences, Texas Tech University, Lubbock, TX 79409, United States; Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Department of Neurology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA 5. Department of Public Health, Graduate School of Biomedical Sciences, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Department of Speech, Language, and Hearing Sciences, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
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Jiao F, Zhao Y, Zhou G, Meng C, Wang L, Wu S, Li J, Cao L, Zhou B, Luo Y, Jiao H. Multiple Functions of Hepatitis E Virus ORF3. Microorganisms 2024; 12:1405. [PMID: 39065173 PMCID: PMC11278674 DOI: 10.3390/microorganisms12071405] [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: 06/16/2024] [Revised: 07/05/2024] [Accepted: 07/08/2024] [Indexed: 07/28/2024] Open
Abstract
Hepatitis E (Hepatitis E, HE) is an acute and chronic infectious hepatitis caused by hepatitis E virus (Hepatitis E Virus, HEV) infection, which is responsible for most acute hepatitis in the world and is a significant public health problem. The pathogen, HEV, has three Open Reading Frames (ORFs) ORF1, ORF2, and ORF3, each of which has a different function. Most of the current research is focused on ORF1 and ORF2, while the research on ORF3 is still relatively small. To provide more ideas for the study of HEV pathogenesis and the prevention and treatment of HE, this paper reviews the effects of ORF3 on the ERK pathway, growth factors, immune response, and virus release.
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Affiliation(s)
- Fengyuan Jiao
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Yu Zhao
- Ministry of Agriculture and Rural Affairs Key Laboratory of Crop Genitic Resources and Germplasm Innovation in Karst Region, Institute of Animal Husbandry and Veterinary Medicine of Guizhou Academy of Agricultural Science, Guiyang 550005, China;
| | - Gengxu Zhou
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Chi Meng
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Lingjie Wang
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Shengping Wu
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Jixiang Li
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Liting Cao
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Bo Zhou
- Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Yujinxiang Street 573, Changchun 130102, China;
| | - Yichen Luo
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
| | - Hanwei Jiao
- The College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (F.J.); (G.Z.); (C.M.); (L.W.); (S.W.); (J.L.); (L.C.)
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Zhang H, Kong L, Cao Z, Zhu Y, Jiang Y, Wang X, Jiang R, Liu Y, Zhou J, Kang Y, Zhen X, Kong N, Wu M, Yan G, Sun H. EHD1 impaired decidualization of endometrial stromal cells in recurrent implantation failure: role of SENP1 in modulating progesterone receptor signalling†. Biol Reprod 2024; 110:536-547. [PMID: 38011671 DOI: 10.1093/biolre/ioad161] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2023] [Revised: 10/13/2023] [Accepted: 11/25/2023] [Indexed: 11/29/2023] Open
Abstract
Recurrent implantation failure (RIF) patients exhibit poor endometrial receptivity and abnormal decidualization with reduced effectiveness and exposure to progesterone, which is an intractable clinical problem. However, the associated molecular mechanisms remain elusive. We found that EH domain containing 1 (EHD1) expression was abnormally elevated in RIF and linked to aberrant endometrial decidualization. Here we show that EHD1 overexpressed in human endometrial stromal cells significantly inhibited progesterone receptor (PGR) transcriptional activity and the responsiveness to progesterone. No significant changes were observed in PGR mRNA levels, while a significant decrease in progesterone receptor B (PRB) protein level. Indeed, EHD1 binds to the PRB protein, with the K388 site crucial for this interaction. Overexpression of EHD1 promotes the SUMOylation and ubiquitination of PRB, leading to the degradation of the PRB protein. Supplementation with the de-SUMOylated protease SENP1 ameliorated EHD1-repressed PRB transcriptional activity. To establish a functional link between EHD1 and the PGR signalling pathway, sg-EHD1 were utilized to suppress EHD1 expression in HESCs from RIF patients. A significant increase in the expression of prolactin and insulin-like growth factor-binding protein 1 was detected by interfering with the EHD1. In conclusion, we demonstrated that abnormally high expression of EHD1 in endometrial stromal cells attenuated the activity of PRB associated with progesterone resistance in a subset of women with RIF.
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Affiliation(s)
- Hui Zhang
- Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Liping Kong
- Nanjing Vocational Health College, Nanjing, China
| | - Zhiwen Cao
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Yinchun Zhu
- Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
| | - Yue Jiang
- Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
| | - Xiaoying Wang
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Ruiwei Jiang
- Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Yang Liu
- Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Jidong Zhou
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Yu Kang
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Xin Zhen
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Na Kong
- Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
| | - Min Wu
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
| | - Guijun Yan
- Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
- State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China
| | - Haixiang Sun
- Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
- Center for Molecular Reproductive Medicine, Nanjing University, Nanjing, China
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Sun Z, Li Y, Tan X, Liu W, He X, Pan D, Li E, Xu L, Long L. Friend or Foe: Regulation, Downstream Effectors of RRAD in Cancer. Biomolecules 2023; 13:biom13030477. [PMID: 36979412 PMCID: PMC10046484 DOI: 10.3390/biom13030477] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2023] [Revised: 02/28/2023] [Accepted: 03/01/2023] [Indexed: 03/08/2023] Open
Abstract
Ras-related associated with diabetes (RRAD), a member of the Ras-related GTPase superfamily, is primarily a cytosolic protein that actives in the plasma membrane. RRAD is highly expressed in type 2 diabetes patients and as a biomarker of congestive heart failure. Mounting evidence showed that RRAD is important for the progression and metastasis of tumor cells, which play opposite roles as an oncogene or tumor suppressor gene depending on cancer and cell type. These findings are of great significance, especially given that relevant molecular mechanisms are being discovered. Being regulated in various pathways, RRAD plays wide spectrum cellular activity including tumor cell division, motility, apoptosis, and energy metabolism by modulating tumor-related gene expression and interacting with multiple downstream effectors. Additionally, RRAD in senescence may contribute to its role in cancer. Despite the twofold characters of RRAD, targeted therapies are becoming a potential therapeutic strategy to combat cancers. This review will discuss the dual identity of RRAD in specific cancer type, provides an overview of the regulation and downstream effectors of RRAD to offer valuable insights for readers, explore the intracellular role of RRAD in cancer, and give a reference for future mechanistic studies.
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Affiliation(s)
- Zhangyue Sun
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
| | - Yongkang Li
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
| | - Xiaolu Tan
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
| | - Wanyi Liu
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
| | - Xinglin He
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
| | - Deyuan Pan
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou 515041, China
- The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Shantou University Medical College, Shantou 515041, China
- Institute of Oncologic Pathology, Shantou University Medical College, Shantou 515041, China
| | - Enmin Li
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou 515041, China
- The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Shantou University Medical College, Shantou 515041, China
- Institute of Oncologic Pathology, Shantou University Medical College, Shantou 515041, China
| | - Liyan Xu
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou 515041, China
- The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Shantou University Medical College, Shantou 515041, China
- Institute of Oncologic Pathology, Shantou University Medical College, Shantou 515041, China
| | - Lin Long
- Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- Cancer Research Center, Institute of Basic Medical Science, Shantou University Medical College, Shantou 515041, China
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou 515041, China
- The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Shantou University Medical College, Shantou 515041, China
- Institute of Oncologic Pathology, Shantou University Medical College, Shantou 515041, China
- Correspondence: ; Tel.: +86-754-88900460; Fax: +86-754-88900847
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Castillo-Sanchez R, Cortes-Reynosa P, Lopez-Perez M, Garcia-Hernandez A, Salazar EP. Caveolae Microdomains Mediate STAT5 Signaling Induced by Insulin in MCF-7 Breast Cancer Cells. J Membr Biol 2023; 256:79-90. [PMID: 35751654 DOI: 10.1007/s00232-022-00253-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2022] [Accepted: 06/06/2022] [Indexed: 02/07/2023]
Abstract
Caveolae are small plasma membrane invaginations constituted for membrane proteins namely caveolins and cytosolic proteins termed cavins, which can occupy up to 50% of the surface of mammalian cells. The caveolae have been involved with a variety of cellular processes including regulation of cellular signaling. Insulin is a hormone that mediates a variety of physiological processes through activation of insulin receptor (IR), which is a tyrosine kinase receptor expressed in all mammalian tissues. Insulin induces activation of signal transducers and activators of transcription (STAT) family members including STAT5. In this study, we demonstrate, for the first time, that insulin induces phosphorylation of STAT5 at tyrosine-694 (STAT5-Tyr(P)694), STAT5 nuclear accumulation and an increase in STAT5-DNA complex formation in MCF-7 breast cancer cells. Insulin also induces nuclear accumulation of STAT5-Tyr(P)694, caveolin-1, and IR in MCF-7 cells. STAT5 nuclear accumulation and the increase of STAT5-DNA complex formation require the integrity of caveolae and microtubule network. Moreover, insulin induces an increase and nuclear accumulation of STAT5-Tyr(P)694 in MDA-MB-231 breast cancer cells. In conclusion, results demonstrate that caveolae and microtubule network play an important role in STAT5-Tyr(P)694, STAT5 nuclear accumulation and STAT5-DNA complex formation induced by insulin in breast cancer cells.
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Affiliation(s)
- Rocio Castillo-Sanchez
- Departamento de Biologia Celular, Cinvestav-IPN, Av. IPN # 2508, 07360, Mexico City, Mexico
| | - Pedro Cortes-Reynosa
- Departamento de Biologia Celular, Cinvestav-IPN, Av. IPN # 2508, 07360, Mexico City, Mexico
| | - Mario Lopez-Perez
- Departamento de Biologia Celular, Cinvestav-IPN, Av. IPN # 2508, 07360, Mexico City, Mexico
| | | | - Eduardo Perez Salazar
- Departamento de Biologia Celular, Cinvestav-IPN, Av. IPN # 2508, 07360, Mexico City, Mexico.
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9
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Pandita P, Bhalla R, Saini A, Mani I. Emerging tools for studying receptor endocytosis and signaling. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2023; 194:19-48. [PMID: 36631193 DOI: 10.1016/bs.pmbts.2022.10.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Ligands, agonists, or antagonists use receptor-mediated endocytosis (RME) to reach their intracellular targets. After the internalization of ligand-receptor complexes, it traffics through different subcellular organelles such as early endosome, recycling endosome, lysosome, etc. Further, after the ligand binding to the receptor, different second messengers are generated, such as cGMP, cAMP, IP3, etc. Several methods have been used, such as radioligand binding assay, western blotting, co-immunoprecipitation (co-IP), qRT-PCR, immunofluorescence and confocal microscopy, microRNA/siRNA, and bioassays to understand the various events, such as internalization, subcellular trafficking, signaling, metabolic degradation, etc. This chapter briefly discusses the key principles and methods used to study internalization, subcellular trafficking, signaling, and metabolic degradation of numerous receptors.
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Affiliation(s)
- Pratiksha Pandita
- Faculty of Medicine, Department of Infectious Disease, Imperial College London, London, United Kingdom
| | - Rhea Bhalla
- ICMR-National Institute of Virology, Pune, Maharashtra, India
| | - Ashok Saini
- Department of Microbiology, Institute of Home Economics, University of Delhi, New Delhi, India
| | - Indra Mani
- Department of Microbiology, Gargi College, University of Delhi, New Delhi, India.
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10
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Cusenza VY, Bonora E, Amodio N, Frazzi R. Spartin: At the crossroad between ubiquitination and metabolism in cancer. Biochim Biophys Acta Rev Cancer 2022; 1877:188813. [PMID: 36195276 DOI: 10.1016/j.bbcan.2022.188813] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2022] [Revised: 09/28/2022] [Accepted: 09/28/2022] [Indexed: 12/01/2022]
Abstract
SPART is a gene coding for a multifunctional protein called spartin, localized in various organelles of human cells. Mutations in the coding region are responsible for a hereditary form of spastic paraplegia called Troyer syndrome while the epigenetic silencing has been demonstrated for some types of tumors. The main functions of this gene are associated to endosomic trafficking and receptor degradation, microtubule interaction, cytokinesis, fatty acids and oxidative metabolism. Spartin has been shown to be a target regulated by STAT3 and localizes also at the level of the mitochondrial outer membrane, where it forms part of a complex maintaining the integrity of the membrane potential. The most recent evidences report a downregulation of spartin in tumor tissues when compared to adjacent normal samples. This intriguing evidence supports further research aimed at clarifying the role of this protein in cancer development and metabolism.
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Affiliation(s)
- Vincenza Ylenia Cusenza
- Laboratory of Translational Research, Azienda Unità Sanitaria Locale - IRCCS di Reggio Emilia, Reggio Emilia, Italy
| | - Elena Bonora
- Medical Genetics Unit, Department of Medical and Surgical Sciences, University of Bologna, Italy; IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
| | - Nicola Amodio
- Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Catanzaro, Italy
| | - Raffaele Frazzi
- Laboratory of Translational Research, Azienda Unità Sanitaria Locale - IRCCS di Reggio Emilia, Reggio Emilia, Italy.
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11
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Liang XH, Nichols JG, Tejera D, Crooke ST. Perinuclear positioning of endosomes can affect PS-ASO activities. Nucleic Acids Res 2021; 49:12970-12985. [PMID: 34878127 PMCID: PMC8682747 DOI: 10.1093/nar/gkab1198] [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: 08/24/2021] [Revised: 11/16/2021] [Accepted: 11/22/2021] [Indexed: 11/18/2022] Open
Abstract
Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs that act on cellular RNAs must enter cells and be released from endocytic organelles to elicit antisense activity. It has been shown that PS-ASOs are mainly released by late endosomes. However, it is unclear how endosome movement in cells contributes to PS-ASO activity. Here, we show that PS-ASOs in early endosomes display Brownian type motion and migrate only short distances, whereas PS-ASOs in late endosomes (LEs) move linearly along microtubules with substantial distances. In cells with normal microtubules and LE movement, PS-ASO-loaded LEs tend to congregate perinuclearly. Disruption of perinuclear positioning of LEs by reduction of dynein 1 decreased PS-ASO activity, without affecting PS-ASO cellular uptake. Similarly, disruption of perinuclear positioning of PS-ASO-LE foci by reduction of ER tethering proteins RNF26, SQSTM1 and UBE2J1, or by overexpression of P50 all decreased PS-ASO activity. However, enhancing perinuclear positioning through reduction of USP15 or over-expression of RNF26 modestly increased PS-ASO activity, indicating that LE perinuclear positioning is required for ensuring efficient PS-ASO release. Together, these observations suggest that LE movement along microtubules and perinuclear positioning affect PS-ASO productive release.
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Affiliation(s)
- Xue-Hai Liang
- Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, CA 92010, USA
| | - Joshua G Nichols
- Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, CA 92010, USA
| | - Dario Tejera
- Neurology, Ionis Pharmaceuticals, Inc., Carlsbad, CA 92010, USA
| | - Stanley T Crooke
- Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, CA 92010, USA
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12
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Mysore V, Cullere X, Mears J, Rosetti F, Okubo K, Liew PX, Zhang F, Madera-Salcedo I, Rosenbauer F, Stone RM, Aster JC, von Andrian UH, Lichtman AH, Raychaudhuri S, Mayadas TN. FcγR engagement reprograms neutrophils into antigen cross-presenting cells that elicit acquired anti-tumor immunity. Nat Commun 2021; 12:4791. [PMID: 34373452 PMCID: PMC8352912 DOI: 10.1038/s41467-021-24591-x] [Citation(s) in RCA: 71] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Accepted: 06/17/2021] [Indexed: 12/12/2022] Open
Abstract
Classical dendritic cells (cDC) are professional antigen-presenting cells (APC) that regulate immunity and tolerance. Neutrophil-derived cells with properties of DCs (nAPC) are observed in human diseases and after culture of neutrophils with cytokines. Here we show that FcγR-mediated endocytosis of antibody-antigen complexes or an anti-FcγRIIIB-antigen conjugate converts neutrophils into nAPCs that, in contrast to those generated with cytokines alone, activate T cells to levels observed with cDCs and elicit CD8+ T cell-dependent anti-tumor immunity in mice. Single cell transcript analyses and validation studies implicate the transcription factor PU.1 in neutrophil to nAPC conversion. In humans, blood nAPC frequency in lupus patients correlates with disease. Moreover, anti-FcγRIIIB-antigen conjugate treatment induces nAPCs that can activate autologous T cells when using neutrophils from individuals with myeloid neoplasms that harbor neoantigens or those vaccinated against bacterial toxins. Thus, anti-FcγRIIIB-antigen conjugate-induced conversion of neutrophils to immunogenic nAPCs may represent a possible immunotherapy for cancer and infectious diseases.
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Affiliation(s)
- Vijayashree Mysore
- Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Xavier Cullere
- Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Joseph Mears
- Center for Data Sciences, Brigham and Women's Hospital, Boston, MA, USA
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
- Division of Rheumatology, Immunology, Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Florencia Rosetti
- Departamento de Inmunología y Reumatología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico
| | - Koshu Okubo
- Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Pei X Liew
- Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Fan Zhang
- Center for Data Sciences, Brigham and Women's Hospital, Boston, MA, USA
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
- Division of Rheumatology, Immunology, Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Iris Madera-Salcedo
- Departamento de Inmunología y Reumatología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico
| | - Frank Rosenbauer
- Institute of Molecular Tumor Biology, University of Muenster, Muenster, Germany
| | - Richard M Stone
- Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA
| | - Jon C Aster
- Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Ulrich H von Andrian
- Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA
| | - Andrew H Lichtman
- Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Soumya Raychaudhuri
- Center for Data Sciences, Brigham and Women's Hospital, Boston, MA, USA
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
- Division of Rheumatology, Immunology, Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
- Arthritis Research UK Centre for Genetics and Genomics, Centre for Musculoskeletal Research, The University of Manchester, Manchester, UK
| | - Tanya N Mayadas
- Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
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13
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Singhal SS, Srivastava S, Mirzapoiazova T, Horne D, Awasthi S, Salgia R. Targeting the mercapturic acid pathway for the treatment of melanoma. Cancer Lett 2021; 518:10-22. [PMID: 34126193 DOI: 10.1016/j.canlet.2021.06.004] [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: 02/26/2021] [Revised: 06/03/2021] [Accepted: 06/07/2021] [Indexed: 02/07/2023]
Abstract
The treatment of metastatic melanoma is greatly hampered by the simultaneous dysregulation of several major signaling pathways that suppress apoptosis and promote its growth and invasion. The global resistance of melanomas to therapeutics is also supported by a highly active mercapturic acid pathway (MAP), which is responsible for the metabolism and excretion of numerous chemotherapy agents. The relative importance of the MAP in melanoma survival was not recognized until demonstrated that B16 melanoma undergoes dramatic apoptosis and regression upon the depletion or inhibition of the MAP transporter protein RLIP. RLIP is a multi-functional protein that couples ATP hydrolysis with the movement of substances. As the rate-limiting step of the MAP, the primary function of RLIP in the plasma membrane is to catalyze the ATP-dependent efflux of unmetabolized drugs and toxins, including glutathione (GSH) conjugates of electrophilic toxins (GS-Es), which are the precursors of mercapturic acids. Clathrin-dependent endocytosis (CDE) is an essential mechanism for internalizing ligand-receptor complexes that promote tumor cell proliferation through autocrine stimulation (Wnt5a, PDGF, βFGF, TNFα) or paracrine stimulation by hormones produced by fibroblasts (IGF1, HGF) or inflammatory cells (IL8). Aberrant functioning of these pathways appears critical for melanoma cell invasion, metastasis, and evasion of apoptosis. This review focuses on the selective depletion or inhibition of RLIP as a highly effective targeted therapy for melanoma that could cause the simultaneous disruption of the MAP and critical peptide hormone signaling that relies on CDE.
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Affiliation(s)
- Sharad S Singhal
- Department of Medical Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, 91010, USA.
| | - Saumya Srivastava
- Department of Medical Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, 91010, USA
| | - Tamara Mirzapoiazova
- Department of Medical Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, 91010, USA
| | - David Horne
- Department of Molecular Medicine, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, 91010, USA
| | - Sanjay Awasthi
- Department of Internal Medicine, Division of Hematology & Oncology, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA
| | - Ravi Salgia
- Department of Medical Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, 91010, USA
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14
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Schmidt-Arras D, Rose-John S. Endosomes as Signaling Platforms for IL-6 Family Cytokine Receptors. Front Cell Dev Biol 2021; 9:688314. [PMID: 34141712 PMCID: PMC8204807 DOI: 10.3389/fcell.2021.688314] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Accepted: 04/28/2021] [Indexed: 12/12/2022] Open
Abstract
Interleukin-6 (IL-6) is the name-giving cytokine of a family of eleven members, including IL-6, CNTF, LIF, and IL-27. IL-6 was first recognized as a B-cell stimulating factor but we now know that the cytokine plays a pivotal role in the orchestration of inflammatory processes as well as in inflammation associated cancer. Moreover, IL-6 is involved in metabolic regulation and it has been shown to be involved in major neural activities such as neuroprotection, which can help to repair and to reduce brain damage. Receptor complexes of all members formed at the plasma membrane contain one or two molecules of the signaling receptor subunit GP130 and the mechanisms of signal transduction are well understood. IL-6 type cytokines can also signal from endomembranes, in particular the endosome, and situations have been reported in which endocytosis of receptor complexes are a prerequisite of intracellular signaling. Moreover, pathogenic GP130 variants were shown to interfere with spatial activation of downstream signals. We here summarize the molecular mechanisms underlying spatial regulation of IL-6 family cytokine signaling and discuss its relevance for pathogenic processes.
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Affiliation(s)
- Dirk Schmidt-Arras
- Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany
| | - Stefan Rose-John
- Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany
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15
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Application of the antitussive agents oxelaidin and butamirate as anti-glioma agents. Sci Rep 2021; 11:10145. [PMID: 33980886 PMCID: PMC8115262 DOI: 10.1038/s41598-021-89238-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Accepted: 03/10/2021] [Indexed: 11/09/2022] Open
Abstract
Glioblastoma (GBM) is an aggressive brain tumor with a strong tendency of relapse and resistance to chemotherapy, but we currently lack non-toxic agents that effectively treat GBM. In this study, high-throughput screening of FDA-approved drugs was performed to identify safe and effective molecules and test their effect on GBM cell lines, LN229, U87 and T98G. Cough suppressants, oxelaidin and butamirate inhibited GBM growth. A Ras family GTPase, Ras-related associated with diabetes (RRAD), contributes to activation of STAT3, which is essential for survival and growth of many cancer types. Interestingly, oxelaidin and butamirate did not affect proliferation in RRAD negative GBM cells. Docking simulation analyses revealed selective interactions between oxelaidin and RRAD. The mechanism by which butamirate and oxelaidin inhibits GBM cell growth involves the suppression of STAT3 transcriptional activity, leading to down-regulation of cyclin D1 and survivin. In addition, components of RRAD-associated signaling cascades, including p-EGFR, p-Akt, and p-STAT3, were inhibited upon oxelaidin treatment. Intraperitoneal administration of oxelaidin or butamirate markedly suppressed tumor growth in a glioblastoma xenograft mouse model without significant adverse effects. Our collective findings indicate that oxelaidin and butamirate exert anti-tumor effects in glioblastoma, supporting its utility as a novel therapeutic candidate for glioblastoma.
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16
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Lu J, Xu S, Huo Y, Sun D, Hu Y, Wang J, Zhang X, Wang P, Li Z, Liang M, Wu Z, Liu P. Sorting nexin 3 induces heart failure via promoting retromer-dependent nuclear trafficking of STAT3. Cell Death Differ 2021; 28:2871-2887. [PMID: 33947971 DOI: 10.1038/s41418-021-00789-w] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 04/08/2021] [Accepted: 04/15/2021] [Indexed: 12/18/2022] Open
Abstract
Sorting nexins (SNXs), the retromer-associated cargo binding proteins, have emerged as critical regulators of the trafficking of proteins involved in the pathogenesis of diverse diseases. However, studies of SNXs in the development of cardiovascular diseases, especially cardiac hypertrophy and heart failure, are lacking. Here, we ask whether SNX3, the simplest structured isoform in the SNXs family, may act as a key inducer of myocardial injury. An increased level of SNX3 was observed in failing hearts from human patients and mice. Cardiac-specific Snx3 knockout (Snx3-cKO) mice and Snx3 transgenic (Snx3-cTg) mice were generated to evaluate the role of Snx3 in myocardial hypertrophy, fibrosis, and heart function by morphology, echocardiography, histological staining, and hypertrophic biomarkers. We report that Snx3-cKO in mice significantly protected against isoproterenol (ISO)-induced cardiac hypertrophy at 12 weeks. Conversely, Snx3-cTg mice were more susceptible to ISO-induced cardiac hypertrophy at 12 weeks and showed aggravated cardiac injury even heart failure at 24 weeks. Immunoprecipitation-based mass spectrometry, immunofluorescent staining, co-immunoprecipitation, localized surface plasmon resonance, and proximity ligation assay were performed to examine the direct interaction of SNX3-retromer with signal transducer and activator of transcription 3 (STAT3). We discovered that STAT3 was a new interacting partner of SNX3-retromer, and SNX3-retromer served as an essential platform for assembling gp130/JAK2/STAT3 complexes and subsequent phosphorylation of STAT3 by direct combination at EE. SNX3-retromer and STAT3 complexes were transiently imported into the nucleus after hypertrophic stimuli. The pharmacological inhibition or knockdown of STAT3 reversed SNX3 overexpression-induced myocardial injury. STAT3 overexpression blunts the beneficial function of SNX3 knockdown on hypertrophic cardiomyocytes. We show that SNX3-retromer promoted importin α3-mediated STAT3 nuclear trafficking and ultimately leading to cardiac injury. Taken together, our study reveals that SNX3 plays a key role in cardiac function and implicates SNX3 as a potential therapeutic target for cardiac hypertrophy and heart failure.
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Affiliation(s)
- Jing Lu
- School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-sen University, Guangzhou, P. R. China
| | - Suowen Xu
- Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.,Department of Endocrinology, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, P. R. China
| | - Yuqing Huo
- Department of Cellular Biology and Anatomy, Medical College of Georgia, Vascular Biology Center, Augusta University, Augusta, GA, USA
| | - Duanping Sun
- Center for Drug Research and Development, Guangdong Pharmaceutical University, Guangzhou, P.R. China
| | - Yuehuai Hu
- School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-sen University, Guangzhou, P. R. China
| | - Junjian Wang
- School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-sen University, Guangzhou, P. R. China
| | - Xiaolei Zhang
- School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-sen University, Guangzhou, P. R. China
| | - Panxia Wang
- School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-sen University, Guangzhou, P. R. China
| | - Zhuoming Li
- School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-sen University, Guangzhou, P. R. China
| | - Mengya Liang
- Department of Cardiac Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, P.R. China
| | - Zhongkai Wu
- Department of Cardiac Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, P.R. China.
| | - Peiqing Liu
- School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-sen University, Guangzhou, P. R. China.
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17
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Singhal SS, Mohanty A, Kulkarni P, Horne D, Awasthi S, Salgia R. RLIP depletion induces apoptosis associated with inhibition of JAK2/STAT3 signaling in melanoma cells. Carcinogenesis 2021; 42:742-752. [PMID: 33623991 DOI: 10.1093/carcin/bgab016] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 02/14/2021] [Accepted: 02/19/2021] [Indexed: 01/03/2023] Open
Abstract
The incidence of malignant melanoma, a neoplasm of melanocytic cells, is increasing rapidly. The lymph nodes are often the first site of metastasis and can herald systemic dissemination, which is almost uniformly fatal. RLIP, a multi-specific ATP-dependent transporter that is over-expressed in several types of cancers, plays a central role in cancer cell resistance to radiation and chemotherapy. RLIP appears to be necessary for cancer cell survival because both in vitro cell culture and in vivo animal tumor studies show that the depletion or inhibition of RLIP causes selective toxicity to malignant cells. RLIP depletion/inhibition triggers apoptosis in cancer cells by inducing the accumulation of endogenously formed glutathione-conjugates. In our in vivo studies, we administered RLIP antibodies or antisense oligonucleotides to mice bearing subcutaneous xenografts of SKMEL2 and SKMEL5 melanoma cells and demonstrated that both treatments caused significant xenograft regression with no apparent toxic effects. Anti-RLIP antibodies and antisense, which respectively inhibit RLIP-mediated transport and deplete RLIP expression, showed similar tumor regressing activities, indicating that the inhibition of RLIP transport activity at the cell surface is sufficient to achieve anti-tumor activity. Furthermore, RLIP antisense treatment reduced levels of RLIP, pSTAT3, pJAK2, pSrc, Mcl-1 and Bcl2, as well as CDK4 and cyclin B1, and increased levels of Bax and phospho 5' AMP-activated protein kinase (pAMPK). These studies indicate that RLIP serves as a key effector in the survival of melanoma cells and is a valid target for cancer therapy. Overall, compounds that inhibit, deplete or downregulate RLIP will function as wide-spectrum agents to treat melanoma, independent of common signaling pathway mutations.
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Affiliation(s)
- Sharad S Singhal
- Department of Medical Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, USA
| | - Atish Mohanty
- Department of Medical Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, USA
| | - Prakash Kulkarni
- Department of Medical Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, USA
| | - David Horne
- Department of Molecular Medicine, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, USA
| | - Sanjay Awasthi
- Department of Internal Medicine, Division of Hematology and Oncology, Texas Tech University Health Sciences Center, Lubbock, TX, USA
| | - Ravi Salgia
- Department of Medical Oncology, Beckman Research Institute, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, USA
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18
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Hanley SE, Cooper KF. Sorting Nexins in Protein Homeostasis. Cells 2020; 10:cells10010017. [PMID: 33374212 PMCID: PMC7823608 DOI: 10.3390/cells10010017] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2020] [Revised: 12/15/2020] [Accepted: 12/18/2020] [Indexed: 12/11/2022] Open
Abstract
Protein homeostasis is maintained by removing misfolded, damaged, or excess proteins and damaged organelles from the cell by three major pathways; the ubiquitin-proteasome system, the autophagy-lysosomal pathway, and the endo-lysosomal pathway. The requirement for ubiquitin provides a link between all three pathways. Sorting nexins are a highly conserved and diverse family of membrane-associated proteins that not only traffic proteins throughout the cells but also provide a second common thread between protein homeostasis pathways. In this review, we will discuss the connections between sorting nexins, ubiquitin, and the interconnected roles they play in maintaining protein quality control mechanisms. Underlying their importance, genetic defects in sorting nexins are linked with a variety of human diseases including neurodegenerative, cardiovascular diseases, viral infections, and cancer. This serves to emphasize the critical roles sorting nexins play in many aspects of cellular function.
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19
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Liu Y, Calmel C, Desbois-Mouthon C, Sobczak-Thépot J, Karaiskou A, Praz F. Regulation of the EGFR/ErbB signalling by clathrin in response to various ligands in hepatocellular carcinoma cell lines. J Cell Mol Med 2020; 24:8091-8102. [PMID: 32515546 PMCID: PMC7348188 DOI: 10.1111/jcmm.15440] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2019] [Revised: 05/10/2020] [Accepted: 05/12/2020] [Indexed: 12/20/2022] Open
Abstract
Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin‐dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin‐binding EGF‐like growth factor (HB‐EGF) and heregulin‐1β (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin‐mediated endocytosis (CME), by down‐regulating clathrin heavy chain expression, resulted in a cell‐ and ligand‐specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down‐regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB‐EGF or HRG, except in HRG‐stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down‐regulated Hep3B cells stimulated with any of the ligands, and in HRG‐stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down‐regulated cell lines upon stimulation with EGF or HB‐EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR‐stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.
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Affiliation(s)
- Yuanhui Liu
- INSERM UMR_S 938, Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Paris, France
| | - Claire Calmel
- INSERM UMR_S 938, Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Paris, France
| | | | - Joëlle Sobczak-Thépot
- INSERM UMR_S 938, Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Paris, France
| | - Anthi Karaiskou
- INSERM UMR_S 938, Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Paris, France
| | - Françoise Praz
- INSERM UMR_S 938, Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Paris, France.,Centre National de la Recherche Scientifique (CNRS), Paris, France
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20
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Zhang HX, Xu ZS, Lin H, Li M, Xia T, Cui K, Wang SY, Li Y, Shu HB, Wang YY. TRIM27 mediates STAT3 activation at retromer-positive structures to promote colitis and colitis-associated carcinogenesis. Nat Commun 2018; 9:3441. [PMID: 30143645 PMCID: PMC6109048 DOI: 10.1038/s41467-018-05796-z] [Citation(s) in RCA: 49] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2017] [Accepted: 07/24/2018] [Indexed: 12/26/2022] Open
Abstract
STAT3 is a transcription factor that plays central roles in various physiological processes and its deregulation results in serious diseases including cancer. The mechanisms on how STAT3 activity is regulated remains enigmatic. Here we identify TRIM27 as a positive regulator of II-6-induced STAT3 activation and downstream gene expression. TRIM27 localizes to retromer-positive punctate structures and serves as a critical link for recruiting gp130, JAK1, and STAT3 to and subsequent phosphorylation of STAT3 at the retromer-positive structures. Overexpression of TRIM27 promotes cancer cell growth in vitro and tumor growth in nude mice, whereas knockdown of TRIM27 has opposite effects. Deficiency of TRIM27 significantly impairs dextran sulfate sodium (DSS)-induced STAT3 activation, inflammatory cytokine expression and colitis as well as azoxymethane (AOM)/DSS-induced colitis-associated cancer in mice. These findings reveal a retromer-dependent mechanism for regulation of STAT3 activation, inflammation, and inflammation-associated cancer development. Aberrant and persistent activation of the transcription factor STAT3 has been found in various types of cancers. Here the authors identify TRIM27 as a positive regulator of IL-6-induced STAT3 activation through the formation of JAK1-STAT3 complex, thus impacting inflammation-induced colon cancer development.
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Affiliation(s)
- Hong-Xia Zhang
- College of Life Sciences, Wuhan University, 430072, Wuhan, China.,Medical Research Institute, School of Medicine, Wuhan University, 430071, Wuhan, China
| | - Zhi-Sheng Xu
- The Joint Center of Translational Precision Medicine, Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, 510623, Guangzhou, China.,Wuhan Institute of Virology, Chinese Academy of Sciences, 430071, Wuhan, China
| | - Hen Lin
- College of Life Sciences, Wuhan University, 430072, Wuhan, China
| | - Mi Li
- College of Life Sciences, Wuhan University, 430072, Wuhan, China
| | - Tian Xia
- College of Life Sciences, Wuhan University, 430072, Wuhan, China
| | - Kaisa Cui
- College of Life Sciences, Wuhan University, 430072, Wuhan, China
| | - Su-Yun Wang
- Wuhan Institute of Virology, Chinese Academy of Sciences, 430071, Wuhan, China
| | - Youjun Li
- College of Life Sciences, Wuhan University, 430072, Wuhan, China
| | - Hong-Bing Shu
- College of Life Sciences, Wuhan University, 430072, Wuhan, China. .,Medical Research Institute, School of Medicine, Wuhan University, 430071, Wuhan, China.
| | - Yan-Yi Wang
- Wuhan Institute of Virology, Chinese Academy of Sciences, 430071, Wuhan, China.
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21
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STAT3 associates with vacuolar H +-ATPase and regulates cytosolic and lysosomal pH. Cell Res 2018; 28:996-1012. [PMID: 30127373 PMCID: PMC6170402 DOI: 10.1038/s41422-018-0080-0] [Citation(s) in RCA: 68] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2018] [Revised: 06/28/2018] [Accepted: 07/16/2018] [Indexed: 12/02/2022] Open
Abstract
Dysregulated intracellular pH is emerging as a hallmark of cancer. In spite of their acidic environment and increased acid production, cancer cells maintain alkaline intracellular pH that promotes cancer progression by inhibiting apoptosis and increasing glycolysis, cell growth, migration, and invasion. Here we identify signal transducer and activator of transcription-3 (STAT3) as a key factor in the preservation of alkaline cytosol. STAT3 associates with the vacuolar H+-ATPase in a coiled-coil domain-dependent manner and increases its activity in living cells and in vitro. Accordingly, STAT3 depletion disrupts intracellular proton equilibrium by decreasing cytosolic pH and increasing lysosomal pH, respectively. This dysregulation can be reverted by reconstitution with wild-type STAT3 or STAT3 mutants unable to activate target genes (Tyr705Phe and DNA-binding mutant) or to regulate mitochondrial respiration (Ser727Ala). Upon cytosolic acidification, STAT3 is transcriptionally inactivated and further recruited to lysosomal membranes to reestablish intracellular proton equilibrium. These data reveal STAT3 as a regulator of intracellular pH and, vice versa, intracellular pH as a regulator of STAT3 localization and activity.
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22
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Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells. Oncotarget 2018; 7:66491-66511. [PMID: 27613843 PMCID: PMC5341816 DOI: 10.18632/oncotarget.11582] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2016] [Accepted: 08/01/2016] [Indexed: 12/12/2022] Open
Abstract
In prior work we reported that advanced stage, drug-resistant pancreatic cancer cells (the SW1990 line) can be sensitized to the EGFR-targeting tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib by treatment with 1,3,4-O-Bu3ManNAc (Bioorg. Med. Chem. Lett. (2015) 25(6):1223-7). Here we provide mechanistic insights into how this compound inhibits EGFR activity and provides synergy with TKI drugs. First, we showed that the sialylation of the EGFR receptor was at most only modestly enhanced (by ∼20 to 30%) compared to overall ∼2-fold increase in cell surface levels of this sugar. Second, flux-driven sialylation did not alter EGFR dimerization as has been reported for cancer cell lines that experience increased sialylation due to spontaneous mutations. Instead, we present evidence that 1,3,4-O-Bu3ManNAc treatment weakens the galectin lattice, increases the internalization of EGFR, and shifts endosomal trafficking towards non-clathrin mediated (NCM) endocytosis. Finally, by evaluating downstream targets of EGFR signaling, we linked synergy between 1,3,4-O-Bu3ManNAc and existing TKI drugs to a shift from clathrin-coated endocytosis (which allows EGFR signaling to continue after internalization) towards NCM endocytosis, which targets internalized moieties for degradation and thereby rapidly diminishes signaling.
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23
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Khanna P, Chua PJ, Wong BSE, Yin C, Thike AA, Wan WK, Tan PH, Baeg GH. GRAM domain-containing protein 1B (GRAMD1B), a novel component of the JAK/STAT signaling pathway, functions in gastric carcinogenesis. Oncotarget 2017; 8:115370-115383. [PMID: 29383166 PMCID: PMC5777778 DOI: 10.18632/oncotarget.23265] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2017] [Accepted: 12/03/2017] [Indexed: 12/31/2022] Open
Abstract
Dysregulated JAK/STAT signaling has been implicated in the molecular pathogenesis of gastric cancer. However, downstream effectors of STAT signaling that facilitate gastric carcinogenesis remain to be explored. We previously identified the Drosophila ortholog of human GRAMD1B in our genome-wide RNAi screen to identify novel components of the JAK/STAT signaling pathway in Drosophila. Here, we examined the involvement of GRAMD1B in JAK/STAT-associated gastric carcinogenesis. We found that GRAMD1B expression is positively regulated by JAK/STAT signaling and GRAMD1B inhibition decreases STAT3 levels, suggesting the existence of a positive feedback loop. Consistently, GRAMD1B and JAK/STAT signaling acted synergistically to promote gastric cancer cell survival by upregulating the expression of the anti-apoptotic molecule Bcl-xL. Interestingly, our immunohistochemical analysis for GRAMD1B revealed a gradual loss of cytoplasmic staining but an increase in the nuclear accumulation of GRAMD1B, as gastric tissue becomes malignant. GRAMD1B expression levels were also found to be significantly associated with clinicopathological features of the gastric cancer patients, particularly the tumor grades and lymph node status. Moreover, GRAMD1B and pSTAT3 (Tyr705) showed a positive correlation in gastric tissues, thereby confirming the existence of a close link between these two signaling molecules in vivo. This new knowledge about JAK/STAT-GRAMD1B regulation deepens our understanding of JAK/STAT signaling in gastric carcinogenesis and provides a foundation for the development of novel biomarkers in gastric cancer.
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Affiliation(s)
- Puja Khanna
- Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117594, Singapore
| | - Pei Jou Chua
- Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117594, Singapore
| | - Belinda Shu Ee Wong
- Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117594, Singapore
| | - Changhong Yin
- Department of Pediatrics, New York Medical College, Valhalla, NY 10595, USA
| | - Aye Aye Thike
- Division of Pathology, Singapore General Hospital, Singapore 169856, Singapore
| | - Wei Keat Wan
- Division of Pathology, Singapore General Hospital, Singapore 169856, Singapore.,Academic Clinical Program for Pathology, Duke-NUS Graduate Medical School, Singapore 169857, Singapore
| | - Puay Hoon Tan
- Division of Pathology, Singapore General Hospital, Singapore 169856, Singapore
| | - Gyeong Hun Baeg
- Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117594, Singapore
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24
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EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer. Nat Commun 2017; 8:2035. [PMID: 29229958 PMCID: PMC5725448 DOI: 10.1038/s41467-017-02185-w] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2017] [Accepted: 11/09/2017] [Indexed: 12/15/2022] Open
Abstract
Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.
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25
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Bridgewater RE, Streuli CH, Caswell PT. Extracellular matrix promotes clathrin-dependent endocytosis of prolactin and STAT5 activation in differentiating mammary epithelial cells. Sci Rep 2017; 7:4572. [PMID: 28676702 PMCID: PMC5496899 DOI: 10.1038/s41598-017-04783-6] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2016] [Accepted: 04/25/2017] [Indexed: 12/24/2022] Open
Abstract
The hormone prolactin promotes lactational differentiation of mammary epithelial cells (MECs) via its cognate receptor and the downstream JAK2-STAT5a signalling pathway. In turn this regulates transcription of milk protein genes. Prolactin signalling depends on a cross-talk with basement membrane extracellular matrix (ECM) via β1 integrins which activate both ILK and Rac1 and are required for STAT5a activation and lactational differentiation. Endocytosis is an important regulator of signalling. It can both enhance and suppress cytokine signalling, although the role of endocytosis for prolactin signalling is not known. Here we show that clathrin-mediated endocytosis is required for ECM-dependent STAT5 activation. In the presence of ECM, prolactin is internalised via a clathrin-dependent, but caveolin-independent, route. This occurs independently from JAK2 and Rac signalling, but is required for full phosphorylation and activation of STAT5. Prolactin is internalised into early endosomes, where the master early endosome regulator Rab5b promotes STAT5 phosphorylation. These data reveal a novel role for ECM-driven endocytosis in the positive regulation of cytokine signalling.
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Affiliation(s)
- Rebecca E Bridgewater
- Wellcome Trust Centre for Cell-Matrix Research, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK
| | - Charles H Streuli
- Wellcome Trust Centre for Cell-Matrix Research, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK
| | - Patrick T Caswell
- Wellcome Trust Centre for Cell-Matrix Research, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.
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26
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CMTM3 decreases EGFR expression and EGF-mediated tumorigenicity by promoting Rab5 activity in gastric cancer. Cancer Lett 2017; 386:77-86. [DOI: 10.1016/j.canlet.2016.11.015] [Citation(s) in RCA: 50] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2016] [Revised: 10/27/2016] [Accepted: 11/10/2016] [Indexed: 02/06/2023]
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27
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Krolopp JE, Thornton SM, Abbott MJ. IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells. Front Physiol 2016; 7:626. [PMID: 28066259 PMCID: PMC5167732 DOI: 10.3389/fphys.2016.00626] [Citation(s) in RCA: 67] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Accepted: 11/30/2016] [Indexed: 12/19/2022] Open
Abstract
Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism.
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Affiliation(s)
- James E Krolopp
- Department of Health Sciences and Kinesiology, Crean College of Health and Behavioral Sciences, Chapman University Orange, CA, USA
| | - Shantaé M Thornton
- Department of Health Sciences and Kinesiology, Crean College of Health and Behavioral Sciences, Chapman University Orange, CA, USA
| | - Marcia J Abbott
- Department of Health Sciences and Kinesiology, Crean College of Health and Behavioral Sciences, Chapman UniversityOrange, CA, USA; Department of Biological Sciences, Human and Evolutionary Biology Section, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern CaliforniaLos Angeles, CA, USA
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28
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Mikula M, Skrzypczak M, Goryca K, Paczkowska K, Ledwon JK, Statkiewicz M, Kulecka M, Grzelak M, Dabrowska M, Kuklinska U, Karczmarski J, Rumienczyk I, Jastrzebski K, Miaczynska M, Ginalski K, Bomsztyk K, Ostrowski J. Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy. Nucleic Acids Res 2016; 44:10150-10164. [PMID: 27587583 PMCID: PMC5137434 DOI: 10.1093/nar/gkw763] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2015] [Revised: 08/17/2016] [Accepted: 08/23/2016] [Indexed: 01/20/2023] Open
Abstract
Genome-wide mechanisms that coordinate expression of subsets of functionally related genes are largely unknown. Recent studies show that receptor tyrosine kinases and components of signal transduction cascades including the extracellular signal-regulated protein kinase (ERK), once thought to act predominantly in the vicinity of plasma membrane and in the cytoplasm, can be recruited to chromatin encompassing transcribed genes. Genome-wide distribution of these transducers and their relationship to transcribing RNA polymerase II (Pol2) could provide new insights about co-regulation of functionally related gene subsets. Chromatin immunoprecipitations (ChIP) followed by deep sequencing, ChIP-Seq, revealed that genome-wide binding of epidermal growth factor receptor, EGFR and ERK pathway components at EGF-responsive genes was highly correlated with characteristic mitogen-induced Pol2-profile. Endosomes play a role in intracellular trafficking of proteins including their nuclear import. Immunofluorescence revealed that EGF-activated EGFR, MEK1/2 and ERK1/2 co-localize on endosomes. Perturbation of endosome internalization process, through the depletion of AP2M1 protein, resulted in decreased number of the EGFR containing endosomes and inhibition of Pol2, EGFR/ERK recruitment to EGR1 gene. Thus, mitogen-induced co-recruitment of EGFR/ERK components to subsets of genes, a kinase module possibly pre-assembled on endosome to synchronize their nuclear import, could coordinate genome-wide transcriptional events to ensure effective cell proliferation.
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Affiliation(s)
- M Mikula
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland
| | - M Skrzypczak
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089, Poland
| | - K Goryca
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland
| | - K Paczkowska
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland
| | - J K Ledwon
- Medical Center for Postgraduate Education, Department of Gastroenterology, Hepatology and Clinical Oncology, Roentgena 5, 02-781 Warsaw, Poland
| | - M Statkiewicz
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland
| | - M Kulecka
- Medical Center for Postgraduate Education, Department of Gastroenterology, Hepatology and Clinical Oncology, Roentgena 5, 02-781 Warsaw, Poland
| | - M Grzelak
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089, Poland
| | - M Dabrowska
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland
| | - U Kuklinska
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland
| | - J Karczmarski
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland
| | - I Rumienczyk
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland
| | - K Jastrzebski
- International Institute of Molecular and Cell Biology, Trojdena 4, 02-109, Warsaw, Poland
| | - M Miaczynska
- International Institute of Molecular and Cell Biology, Trojdena 4, 02-109, Warsaw, Poland
| | - K Ginalski
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089, Poland
| | - K Bomsztyk
- University of Washington, Department of Medicine, 850 Republican Street, Seattle, WA, USA
| | - J Ostrowski
- Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Roentgena 5, 02-781 Warsaw, Poland.,Medical Center for Postgraduate Education, Department of Gastroenterology, Hepatology and Clinical Oncology, Roentgena 5, 02-781 Warsaw, Poland
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29
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Gao J, Wang F, Chen J, Wang J, Cai M, Xu H, Jiang J, Wang H. Super-resolution imaging of STAT3 cellular clustering during nuclear transport. RSC Adv 2016. [DOI: 10.1039/c6ra09591g] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
STAT3 cellular clustering revealed by super-resolution fluorescence microscopy.
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Affiliation(s)
- Jing Gao
- State Key Laboratory of Electroanalytical Chemistry
- Changchun Institute of Applied Chemistry
- Chinese Academy of Sciences
- Changchun, China
| | - Feng Wang
- Institute of Immunology
- The First Bethune Hospital Academy of Translational Medicine
- Jilin University
- Changchun, China
| | - Junling Chen
- State Key Laboratory of Electroanalytical Chemistry
- Changchun Institute of Applied Chemistry
- Chinese Academy of Sciences
- Changchun, China
- University of Chinese Academy of Sciences
| | - Jianzhong Wang
- School of Computer Science and Information Technology
- Northeast Normal University
- Changchun, China
| | - Mingjun Cai
- State Key Laboratory of Electroanalytical Chemistry
- Changchun Institute of Applied Chemistry
- Chinese Academy of Sciences
- Changchun, China
| | - Haijiao Xu
- State Key Laboratory of Electroanalytical Chemistry
- Changchun Institute of Applied Chemistry
- Chinese Academy of Sciences
- Changchun, China
| | - Junguang Jiang
- State Key Laboratory of Electroanalytical Chemistry
- Changchun Institute of Applied Chemistry
- Chinese Academy of Sciences
- Changchun, China
| | - Hongda Wang
- State Key Laboratory of Electroanalytical Chemistry
- Changchun Institute of Applied Chemistry
- Chinese Academy of Sciences
- Changchun, China
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30
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Jastrzębski K, Zdżalik-Bielecka D, Mamińska A, Kalaidzidis Y, Hellberg C, Miaczynska M. Multiple routes of endocytic internalization of PDGFRβ contribute to PDGF-induced STAT3 signaling. J Cell Sci 2016; 130:577-589. [DOI: 10.1242/jcs.191213] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2016] [Accepted: 12/05/2016] [Indexed: 12/31/2022] Open
Abstract
Platelet-derived growth factor receptor β (PDGFRβ) is a receptor tyrosine kinase which upon activation by PDGF-BB stimulates cell proliferation, migration and angiogenesis. Ligand binding induces intracellular signaling cascades but also internalization of the receptor, eventually resulting in its lysosomal degradation. However, endocytic trafficking of receptors often modulates their downstream signaling. We previously reported that internalization of PDGFRβ occurs via dynamin-dependent and -independent pathways but their further molecular determinants remained unknown. Here we show that in human fibroblasts expressing endogenous PDGFRβ and stimulated with 50 ng/ml PDGF-BB, ligand-receptor uptake proceeds via parallel routes of clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE). CME involves the canonical AP2 complex as a clathrin adaptor, while CIE requires RhoA-ROCK, Cdc42 and galectin-3, the latter indicating lectin-mediated internalization via clathrin-independent carriers (CLICs). Although different uptake routes appear to be partly interdependent, they cannot fully substitute for each other. Strikingly, inhibition of any internalization mechanism impaired activation of STAT3 but not of other downstream effectors of PDGFRβ. Our data indicate that multiple routes of internalization of PDGFRβ contribute to a transcriptional and mitogenic response of cells to PDGF.
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Affiliation(s)
- Kamil Jastrzębski
- Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland
| | - Daria Zdżalik-Bielecka
- Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland
| | - Agnieszka Mamińska
- Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland
| | - Yannis Kalaidzidis
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Carina Hellberg
- School of Biosciences, University of Birmingham, Birmingham, United Kingdom
| | - Marta Miaczynska
- Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland
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31
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Macrophage migration inhibitory factor has a permissive role in concanavalin A-induced cell death of human hepatoma cells through autophagy. Cell Death Dis 2015; 6:e2008. [PMID: 26633714 PMCID: PMC4720884 DOI: 10.1038/cddis.2015.349] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2015] [Revised: 10/29/2015] [Accepted: 11/03/2015] [Indexed: 02/06/2023]
Abstract
Concanavalin A (ConA) is a lectin and T-cell mitogen that can activate immune responses. In recent times, ConA-induced cell death of hepatoma cells through autophagy has been reported and its therapeutic effect was confirmed in a murine in situ hepatoma model. However, the molecular mechanism of ConA-induced autophagy is still unclear. As macrophage migration inhibitory factor (MIF), which is a proinflammatory cytokine, can trigger autophagy in human hepatoma cells, the possible involvement of MIF in ConA-induced autophagy was investigated in this study. We demonstrated that cell death is followed by an increment in MIF expression and secretion in the ConA-stimulated human hepatoma cell lines, HuH-7 and Hep G2. In addition, ConA-induced autophagy and cell death of hepatoma cells were blocked in the presence of an MIF inhibitor. Knockdown of endogenous MIF by small hairpin RNA confirmed that MIF is required for both ConA-induced autophagy and death of hepatoma cells. Furthermore, signal pathway studies demonstrated that ConA induces signal transducer and activator of transcription 3 (STAT3) phosphorylation to trigger MIF upregulation, which in turn promotes Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3)-dependent autophagy. By using a murine in situ hepatoma model, we further demonstrated that MIF contributes to anti-hepatoma activity of ConA by regulating STAT3-MIF-BNIP3-dependent autophagy. In summary, our findings uncover a novel role of MIF in lectin-mediated anti-hepatoma activities by regulating autophagy.
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Stasyk T, Huber LA. Spatio-Temporal Parameters of Endosomal Signaling in Cancer: Implications for New Treatment Options. J Cell Biochem 2015; 117:836-43. [PMID: 26506511 PMCID: PMC4949996 DOI: 10.1002/jcb.25418] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2015] [Accepted: 10/26/2015] [Indexed: 02/03/2023]
Abstract
The endo/lysosomal system in cells provides membranous platforms to assemble specific signaling complexes and to terminate signal transduction, thus, is essential for physiological signaling. Endocytic organelles can significantly extend signaling of activated cell surface receptors, and may additionally provide distinct locations for the generation of specific signaling outputs. Failures of regulation at different levels of endocytosis, recycling, degradation as well as aberrations in specific endo/lysosomal signaling pathways, such as mTORC1, might lead to different diseases including cancer. Therefore, a better understanding of spatio‐temporal compartmentalization of sub‐cellular signaling might provide an opportunity to interfere with aberrant signal transduction in pathological processes by novel combinatorial therapeutic approaches. J. Cell. Biochem. 117: 836–843, 2016. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.
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Affiliation(s)
- Taras Stasyk
- Biocenter, Division of Cell Biology, Innsbruck Medical University, Austria
| | - Lukas A Huber
- Biocenter, Division of Cell Biology, Innsbruck Medical University, Austria.,ADSI - Austrian Drug Screening Institute, Innsbruck, Austria
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Lau ST, Zhou T, Liu JAJ, Fung EYM, Che CM, Lang BHH, Ngan ESW. Dysregulation of clathrin promotes thyroid cell growth and contributes to multinodular goiter pathogenesis. Biochim Biophys Acta Mol Basis Dis 2015; 1852:1676-86. [DOI: 10.1016/j.bbadis.2015.05.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2015] [Revised: 05/06/2015] [Accepted: 05/07/2015] [Indexed: 11/15/2022]
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Yoo YH, Kim YR, Kim MS, Lee KJ, Park KH, Hahn JH. YAC tripeptide of epidermal growth factor promotes the proliferation of HaCaT keratinocytes through activation of EGFR. BMB Rep 2015; 47:581-6. [PMID: 25179402 PMCID: PMC4261517 DOI: 10.5483/bmbrep.2014.47.10.151] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2014] [Indexed: 01/22/2023] Open
Abstract
Epidermal growth factor (EGF) is known to play key roles in skin regeneration and wound-healing. Here, we demonstrate that Pep2-YAC, a tripeptide covering residues 29-31 in the B loop of EGF, promotes the proliferation of HaCaT keratinocytes with activity comparable to EGF. The treatment of HaCaT cells with Pep2-YAC induced phosphorylation, internalization, and degradation of EGFR and organization of signaling complexes, which consist of Grb2, Gab1, SHP2, and PI3K. In addition, it stimulated the phosphorylation of ERK1/2 at Thr 202/Tyr 204 and of Akt1 at Ser 473 and the nuclear translocation of EGFR, STAT3, c-Jun, and c-Fos. These results suggest that Pep2-YAC may be useful as a therapeutic agent for skin regeneration and wound-healing as an EGFR agonist.
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Affiliation(s)
- Yeon Ho Yoo
- Department of Anatomy and Cell Biology, School of Medicine, Kangwon National University, Chuncheon 200-701, Korea
| | - Yu Ri Kim
- Department of Anatomy and Cell Biology, School of Medicine, Kangwon National University, Chuncheon 200-701, Korea
| | - Min Seo Kim
- Department of Anatomy and Cell Biology, School of Medicine, Kangwon National University, Chuncheon 200-701, Korea
| | - Kyoung-Jin Lee
- Department of Anatomy and Cell Biology, School of Medicine, Kangwon National University, Chuncheon 200-701, Korea
| | - Kyeong Han Park
- Department of Anatomy and Cell Biology, School of Medicine, Kangwon National University, Chuncheon 200-701, Korea
| | - Jang-Hee Hahn
- Department of Anatomy and Cell Biology, School of Medicine, Kangwon National University, Chuncheon 200-701, Korea
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The molecular basis for functional plasticity in type I interferon signaling. Trends Immunol 2015; 36:139-49. [DOI: 10.1016/j.it.2015.01.002] [Citation(s) in RCA: 136] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2014] [Revised: 01/13/2015] [Accepted: 01/13/2015] [Indexed: 01/16/2023]
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Chung BM, Tom E, Zutshi N, Bielecki TA, Band V, Band H. Nexus of signaling and endocytosis in oncogenesis driven by non-small cell lung cancer-associated epidermal growth factor receptor mutants. World J Clin Oncol 2014; 5:806-823. [PMID: 25493220 PMCID: PMC4259944 DOI: 10.5306/wjco.v5.i5.806] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/22/2014] [Revised: 07/19/2014] [Accepted: 09/10/2014] [Indexed: 02/06/2023] Open
Abstract
Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling as a result of receptor overexpression and/or mutation occurs in many types of cancer. Tumor cells in non-small cell lung cancer (NSCLC) patients that harbor EGFR kinase domain mutations exhibit oncogene addiction to mutant EGFR, which confers high sensitivity to tyrosine kinase inhibitors (TKIs). As patients invariably develop resistance to TKIs, it is important to delineate the cell biological basis of mutant EGFR-induced cellular transformation since components of these pathways can serve as alternate therapeutic targets to preempt or overcome resistance. NSCLC-associated EGFR mutants are constitutively-active and induce ligand-independent transformation in nonmalignant cell lines. Emerging data suggest that a number of factors are critical for the mutant EGFR-dependent tumorigenicity, and bypassing the effects of TKIs on these pathways promotes drug resistance. For example, activation of downstream pathways such as Akt, Erk, STAT3 and Src is critical for mutant EGFR-mediated biological processes. It is now well-established that the potency and spatiotemporal features of cellular signaling by receptor tyrosine kinases such as EGFR, as well as the specific pathways activated, is determined by the nature of endocytic traffic pathways through which the active receptors traverse. Recent evidence indicates that NSCLC-associated mutant EGFRs exhibit altered endocytic trafficking and they exhibit reduced Cbl ubiquitin ligase-mediated lysosomal downregulation. More recent work has shown that mutant EGFRs undergo ligand-independent traffic into the endocytic recycling compartment, a behavior that plays a key role in Src pathway activation and oncogenesis. These studies are beginning to delineate the close nexus between signaling and endocytic traffic of EGFR mutants as a key driver of oncogenic processes. Therefore, in this review, we will discuss the links between mutant EGFR signaling and endocytic properties, and introduce potential mechanisms by which altered endocytic properties of mutant EGFRs may alter signaling and vice versa as well as their implications for NSCLC therapy.
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Yeom SY, Nam DH, Park C. RRAD promotes EGFR-mediated STAT3 activation and induces temozolomide resistance of malignant glioblastoma. Mol Cancer Ther 2014; 13:3049-61. [PMID: 25313011 DOI: 10.1158/1535-7163.mct-14-0244] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Glioblastoma multiforme (GBM) is an extremely aggressive brain cancer with a median survival of less than 2 years. GBM is characterized by abnormal activation of receptor tyrosine kinase and constitutively activated STAT3. Although EGFR phosphorylation and STAT3 activation are essential for the maintenance of GBM cancer stem cells, the molecular mechanism underlying endosome-mediated STAT3 activation is not fully understood. In the current study, we showed that GTP-binding protein RRAD (RAS associated with diabetes, RAD) physically associates with EGFR, and EEA1, enhancing the stability and endosome-associated nuclear translocation of EGFR. Functionally, RRAD contributes to the activation of STAT3 and expression of the stem cell factors OCT4, NANOG, and SOX2, thereby enhancing self-renewing ability, tumor sphere formation, EMT, and in vivo tumorigenesis. Most importantly, RRAD contributes to poor survival in patients with GBM. RRAD expression is correlated with temozolomide resistance, and, conversely, depletion of RRAD leads to sensitization of highly temozolomide-resistant GBM cells. Our data collectively support a novel function of RRAD in STAT3 activation and provide evidence that RRAD acts as a positive regulator in the EGFR signaling pathway. These results demonstrate a critical role for RRAD in GBM tumorigenesis and provide a rationale for the development of pharmacologic inhibitors of RRAD in GBM.
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Affiliation(s)
- Seon-Yong Yeom
- Research Institute for Future Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Do-Hyun Nam
- Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Chaehwa Park
- Research Institute for Future Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
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Ng IHW, Bogoyevitch MA, Jans DA. Cytokine-induced slowing of STAT3 nuclear import; faster basal trafficking of the STAT3β isoform. Traffic 2014; 15:946-60. [PMID: 24903907 DOI: 10.1111/tra.12181] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2014] [Revised: 06/02/2014] [Accepted: 06/03/2014] [Indexed: 01/01/2023]
Abstract
The STAT3 signal transducer and activator of transcription is a key mediator of gene transcription in response to cytokines such as oncostatin M (OSM). We performed direct live cell imaging of GFP-tagged STAT3 proteins for the first time, showing transient relocalization of STAT3α to the nucleus following OSM exposure, in contrast to sustained nuclear relocalization of the shorter STAT3β spliceform. To explore this further, we applied fluorescence recovery after photobleaching (FRAP) to determine the nuclear import kinetics of STAT3α and β, as well as of a C-terminal truncation derivative STAT3ΔC comprising only the sequence shared by the spliceforms, in the absence or presence of OSM. The rates of basal nuclear import for STAT3β and STAT3ΔC were significantly faster than those for STAT3α. Strikingly, OSM slowed the import rates of all the three STAT3 proteins, whereas the import rates of GFP alone or a classical importin-mediated cargo were unaffected, with analysis of Y705F mutant derivatives for all the three STAT3 constructs, or of a S727A mutant within the unique C-terminus of STAT3α, reinforcing the contribution of specific phosphorylation to the cytokine-stimulated changes. The results introduce a new paradigm where cytokine treatment prolongs nuclear retention simultaneous with decreasing rather than increasing the rate of nuclear import.
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Affiliation(s)
- Ivan H W Ng
- Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, 3800, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria, 3010, Australia
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Ng IHW, Yeap YYC, Ong LSR, Jans DA, Bogoyevitch MA. Oxidative stress impairs multiple regulatory events to drive persistent cytokine-stimulated STAT3 phosphorylation. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2014; 1843:483-94. [PMID: 24286865 DOI: 10.1016/j.bbamcr.2013.11.015] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/20/2013] [Revised: 10/31/2013] [Accepted: 11/19/2013] [Indexed: 12/30/2022]
Abstract
Although cytokine-driven STAT3 phosphorylation and activation are often transient, persistent activation of STAT3 is a hallmark of a range of pathologies and underpins altered transcriptional responses. As triggers in disease frequently include combined increases in inflammatory cytokine and reactive oxygen species levels, we report here how oxidative stress impacts on cytokine-driven STAT3 signal transduction events. In the model system of murine embryonic fibroblasts (MEFs), combined treatment with the interleukin-6 family cytokine Leukemia Inhibitory Factor (LIF) and hydrogen peroxide (H2O2) drove persistent STAT3 phosphorylation whereas STAT3 phosphorylation increased only transiently in response to LIF alone and was not increased by H2O2 alone. Surprisingly, increases in transcript levels of the direct STAT3 gene target SOCS3 were delayed during the combined LIF + H2O2 treatment, leading us to probe the impact of oxidative stress on STAT3 regulatory events. Indeed, LIF + H2O2 prolonged JAK activation, delayed STAT3 nuclear localisation, and caused relocalisation of nuclear STAT3 phosphatase TC-PTP (TC45) to the cytoplasm. In exploring the nuclear import/ export pathways, we observed disruption of nuclear/cytoplasmic distributions of Ran and importin-alpha3 in cells exposed to H2O2 and the resultant reduced nuclear trafficking of Classical importin-alpha/3-dependent protein cargoes. CRM1-mediated nuclear export persisted despite the oxidative stress insult, with sustained STAT3 Y705 phosphorylation enhancing STAT3 nuclear residency. Our studies thus reveal for the first time the striking impact of oxidative stress to sustain STAT3 phosphorylation and nuclear retention following disruption of multiple regulatory events, with significant implications for STAT3 function.
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Shaiken TE, Opekun AR. Dissecting the cell to nucleus, perinucleus and cytosol. Sci Rep 2014; 4:4923. [PMID: 24815916 PMCID: PMC4017230 DOI: 10.1038/srep04923] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2014] [Accepted: 04/22/2014] [Indexed: 12/25/2022] Open
Abstract
Cells have been described under the microscope as organelles containing cytoplasm and the nucleus. However, an unnoted structure exists between the cytoplasm and the nucleoplasm of eukaryotic cells. In addition to the nuclear envelope, there exists a perinuclear region (PNR or perinucleus) with unknown composition and function. Until now, an investigation of the role of the perinucleus has been restricted by the absence of a PNR isolation method. This manuscript describes a perinucleus isolation technique on the basis of its unique compact organization. The perinucleus was found to contain approximately 15 to 18% of the total proteins of the mammalian cell, almost half of the proteins of nuclei. Using four different normal and cancer cell lines, it was shown that the composition of PNR is highly dynamic. Application of the method showed that translocation of the p53 tumor-suppressor protein to the perinucleus in immortalized MEF cells is correlated with the translocation of p53-stabilizing protein, nucleophosmin (B23), to the PNR. Herein, the concept of the perinuclear region is advanced as a formal, identifiable structure. The roles of the perinucleus in maintaining genome integrity, regulation of gene expression and understanding of malignant transformation are discussed.
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Affiliation(s)
- Tattym E Shaiken
- Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
| | - Antone R Opekun
- Departments of Medicine & Pediatrics G.I. & S.A.H.S. Baylor College of Medicine-McNair Faculty Center A10.019 One Baylor Plaza (GI Medicine MS901), Houston, Texas 77030, USA
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Gaucci E, Altieri F, Turano C, Chichiarelli S. The protein ERp57 contributes to EGF receptor signaling and internalization in MDA-MB-468 breast cancer cells. J Cell Biochem 2014; 114:2461-70. [PMID: 23696074 DOI: 10.1002/jcb.24590] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2013] [Accepted: 05/03/2013] [Indexed: 12/27/2022]
Abstract
The disulfide isomerase ERp57 is a soluble protein mainly located in the endoplasmic reticulum, where it acts in the quality control of newly synthesized glycoproteins, in association with calreticulin and calnexin. It has been also detected in other cell compartments, such as the cytosol, the plasma membrane and the nucleus. In these locations it is implicated in various processes, participating in the rapid response to calcitriol, modulating the activity of STAT3 and being requested for the pre-apoptotic exposure of calreticulin on the plasma membrane. In the present work, the involvement of ERp57 in the activity of the EGF receptor was evaluated for the first time. EGFR is a tyrosine kinase receptor, which is able to activate numerous signaling cascades, leading to cell proliferation and inhibition of apoptosis. In the MDA-MB-468 breast adenocarcinoma cells, which overexpress EGFR, ERp57 expression has been knocked down by siRNA and the effects on EGFR have been studied. ERp57 silencing did not affect EGFR protein expression, cell membrane exposure or EGF binding, whereas the internalization and the phosphorylation of the receptor were impaired. The implication of ERp57 in the activity of EGFR, whose upregulation is known to be associated with tumors, could be relevant for cancer therapy.
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Affiliation(s)
- Elisa Gaucci
- Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, 00185, Italy; Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, 00185, Italy
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STAT3 Activation in Glioblastoma: Biochemical and Therapeutic Implications. Cancers (Basel) 2014; 6:376-95. [PMID: 24518612 PMCID: PMC3980601 DOI: 10.3390/cancers6010376] [Citation(s) in RCA: 101] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2013] [Revised: 01/19/2014] [Accepted: 01/29/2014] [Indexed: 02/04/2023] Open
Abstract
Signal transducer and activator of transcription 3 (STAT3) is a potent regulator of gliomagenesis through its induction of angiogenesis, host immunosuppression, and tumor invasion. Gain of function mutations result in constitutive activation of STAT3 in glioma cells, making STAT3 an attractive target for inhibition in cancer therapy. Nevertheless, some studies show that STAT3 also participates in terminal differentiation and apoptosis of various cell lines and in glioma with phosphatase and tensin homolog (PTEN)-deficient genetic backgrounds. In light of these findings, the utility of STAT3 as a prognostic indicator and as a target of drug therapies will be contingent on a more nuanced understanding of its pro- and anti-tumorigenic effects.
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The regulation of reactive changes around multiple sclerosis lesions by phosphorylated signal transducer and activator of transcription. J Neuropathol Exp Neurol 2014; 72:1135-44. [PMID: 24226263 DOI: 10.1097/nen.0000000000000011] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Activation of signal transducer and activator of transcription 3 (STAT3) by phosphorylation is thought to mediate anti-inflammatory responses to CNS injury. Several studies have reported an increase in phosphorylated STAT3 (pSTAT3) in peripheral T cells and monocytes from patients with multiple sclerosis (MS) during relapses, suggesting that pSTAT3 might represent an inflammatory marker. Here, we examined immunoreactivity for pSTAT3 in brain tissue samples from MS patients and controls. Phosphorylated STAT3 immunoreactivity was sparse within lesions, with no difference between active and inactive lesions. It was, however, significantly greater in white matter (WM) adjacent to active and inactive lesions; moreover, it was significantly greater in WM adjacent to active versus inactive lesions. Phosphorylated STAT3-positive cells were identified as astrocytes and macrophages/microglia. Phosphorylated STAT3 expression was also detected by Western blotting in WM of patients with MS. In comparison, pSTAT3 immunoreactivity was either rare or found focally in brain tissue samples from patients with other neurologic diseases. Our findings show that pSTAT3 does not correlate with inflammatory activity in MS lesions, but that it may play an important role in regulating reactive changes proximal to MS lesions.
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Ng IHW, Jans DA, Bogoyevitch MA. Hyperosmotic stress sustains cytokine-stimulated phosphorylation of STAT3, but slows its nuclear trafficking and impairs STAT3-dependent transcription. Cell Signal 2014; 26:815-24. [PMID: 24394455 DOI: 10.1016/j.cellsig.2013.12.012] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2013] [Accepted: 12/22/2013] [Indexed: 11/16/2022]
Abstract
Persistent STAT3 phosphorylation and nuclear retention are hallmarks of a range of pathologies suggesting the importance of STAT3 transcriptional responses in disease progression. Since hyperosmotic stress (HOS) is a hallmark of diseases such as diabetes and asthma, we analysed the impact of HOS on cytokine-stimulated STAT3 signalling. In contrast to transient STAT3 Y705 and S727 phosphorylation in murine embryonic fibroblasts (MEFs) stimulated by the interleukin-6 family cytokine, leukemia inhibitory factor (LIF), under non-stress conditions, HOS induced by sorbitol treatment increased STAT3 S727 but not Y705 phosphorylation. Strikingly, combined LIF+HOS treatment stimulated persistent STAT3 Y705 and S727 phosphorylation and nuclear localisation, but STAT3 nuclear accumulation was slowed during HOS, likely reflecting the mislocalisation of Ran and importin-α3 during HOS that also reduced the nuclear localisation of classical importin-α/β-recognised nuclear import cargoes. Strikingly, combined LIF+HOS exposure, even though stimulating STAT3 phosphorylation and nuclear accumulation did not elicit a transcriptional output, as demonstrated by qPCR analyses of its target genes SOCS3 and c-Fos. Our analysis thus shows for the first time that HOS can disconnect nuclear, phosphorylated STAT3 from transcriptional outcomes, and emphasizes the importance of assessing STAT3 target gene changes in addition to STAT3 phosphorylation status and localisation.
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Affiliation(s)
- Ivan H W Ng
- Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia; Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia
| | - David A Jans
- Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
| | - Marie A Bogoyevitch
- Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.
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Ai S, Jia T, Ai W, Duan J, Liu Y, Chen J, Liu X, Yang F, Tian Y, Huang Z. Targeted delivery of doxorubicin through conjugation with EGF receptor-binding peptide overcomes drug resistance in human colon cancer cells. Br J Pharmacol 2013; 168:1719-35. [PMID: 23146125 DOI: 10.1111/bph.12055] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2012] [Revised: 10/10/2012] [Accepted: 11/01/2012] [Indexed: 11/29/2022] Open
Abstract
BACKGROUND AND PURPOSE Induction of multidrug resistance by doxorubicin (DOX), together with non-specific toxicities, has restricted DOX-based chemotherapy. Recently, we demonstrated that DOX conjugated with an EGF receptor-binding peptide (DOX-EBP) had enhanced anticancer efficacy and reduced systemic toxicity when targeting EGF receptor-overexpressing tumours. Here we investigated whether DOX-EBP is able to overcome drug resistance and the underlying molecular mechanisms. EXPERIMENTAL APPROACH DOX-resistant SW480/DOX cells were derived from non-resistant SW480 cells by stepwise exposure to increasing concentrations of DOX, and P-glycoprotein overexpression induced by DOX was confirmed by Western blotting. Cytotoxicity and intracellular distribution of drugs were evaluated by MTT assay and fluorescence microscopy respectively. EGF receptor-mediated endocytosis was determined in EGF receptor and endocytosis inhibition assays. Drug accumulation in tumour cells and murine xenografts was determined by HPLC. KEY RESULTS The cytotoxicity and accumulation of DOX-EBP in SW480/DOX cells were almost the same as in SW480 cells, but those of free DOX were reduced. DOX-EBP accumulation was prevented by inhibitors of both EGF receptors and endocytosis, suggesting EGF receptors mediate endocytotic uptake. Tumour accumulation of DOX-EBP was significantly higher than free DOX in mice, and the levels of DOX-EBP were similar in DOX-resistant and non-resistant tumour tissues. Importantly, DOX-EBP, but not free DOX, was effective at inhibiting solid tumour growth and increased survival rate in both sensitive and resistant models. CONCLUSION AND IMPLICATIONS DOX-EBP can overcome DOX resistance of tumour cells and increase in vivo antitumour efficacy. Therefore, it has the potential to be a potent therapeutic agent for treating drug-resistant cancers.
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Affiliation(s)
- Shibin Ai
- Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan, China
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Harada H, Warabi E, Matsuki T, Yanagawa T, Okada K, Uwayama J, Ikeda A, Nakaso K, Kirii K, Noguchi N, Bukawa H, Siow RCM, Mann GE, Shoda J, Ishii T, Sakurai T. Deficiency of p62/Sequestosome 1 causes hyperphagia due to leptin resistance in the brain. J Neurosci 2013; 33:14767-77. [PMID: 24027277 PMCID: PMC6705174 DOI: 10.1523/jneurosci.2954-12.2013] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2012] [Revised: 08/05/2013] [Accepted: 08/07/2013] [Indexed: 01/08/2023] Open
Abstract
The cytoplasmic regulatory protein p62 (Sequestosome 1/A170) is known to modulate various receptor-mediated intracellular signaling pathways. p62 deficiency was shown to result in mature-onset obesity in mice, but the mechanisms underlying this abnormality remained unclear. Here we report that hyperphagia due to central leptin resistance is the cause of obesity in p62(-/-) mice. We found that these mice show hyperphagia. Restriction of food to the amount eaten by wild-type mice prevented excess body weight gain and fat accumulation, suggesting that overfeeding is the primary cause of obesity in p62(-/-) mice. Brain-specific p62 deficiency caused mature-onset obesity to the same extent as in p62(-/-) mice, further supporting a neuronal mechanism as the major cause of obesity in these mice. Immunohistochemical analysis revealed that p62 is highly expressed in hypothalamic neurons, including POMC neurons in the arcuate nucleus. Central leptin resistance was observed even in young preobese p62(-/-) mice. We found a defect in intracellular distribution of the transcription factor Stat3, which is essential for the action of leptin, in p62(-/-) mice. These results indicate that brain p62 plays an important role in bodyweight control by modulating the central leptin-signaling pathway and that lack of p62 in the brain causes leptin resistance, leading to hyperphagia. Thus, p62 could be a clinical target for treating obesity and metabolic syndrome.
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Affiliation(s)
- Harumi Harada
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Eiji Warabi
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Taizo Matsuki
- Department of Molecular Neuroscience and Integrative Physiology, Faculty of Medicine, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan
- Center for Behavioral Molecular Genetics, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Toru Yanagawa
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Kosuke Okada
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Junya Uwayama
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Akira Ikeda
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Kazuhiro Nakaso
- Department of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8504, Japan
| | - Kyoko Kirii
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Noriko Noguchi
- Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan, and
| | - Hiroki Bukawa
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Richard C. M. Siow
- Cardiovascular Division, British Heart Foundation Centre of Research Excellence, School of Medicine, King's College London, London SE1 9NH, United Kingdom
| | - Giovanni E. Mann
- Cardiovascular Division, British Heart Foundation Centre of Research Excellence, School of Medicine, King's College London, London SE1 9NH, United Kingdom
| | - Junichi Shoda
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Tetsuro Ishii
- Majors of Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
| | - Takeshi Sakurai
- Department of Molecular Neuroscience and Integrative Physiology, Faculty of Medicine, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan
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Gouttenoire EA, Lupo V, Calpena E, Bartesaghi L, Schüpfer F, Médard JJ, Maurer F, Beckmann JS, Senderek J, Palau F, Espinós C, Chrast R. Sh3tc2 deficiency affects neuregulin-1/ErbB signaling. Glia 2013; 61:1041-51. [PMID: 23553667 DOI: 10.1002/glia.22493] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2012] [Accepted: 02/01/2013] [Indexed: 12/19/2022]
Abstract
Mutations in SH3TC2 trigger autosomal recessive demyelinating Charcot-Marie-Tooth type 4C (CMT4C) neuropathy. Sh3tc2 is specifically expressed in Schwann cells and is necessary for proper myelination of peripheral axons. In line with the early onset of neuropathy observed in patients with CMT4C, our analyses of the murine model of CMT4C revealed that the myelinating properties of Sh3tc2-deficient Schwann cells are affected at an early stage. This early phenotype is associated with changes in the canonical Nrg1/ErbB pathway involved in control of myelination. We demonstrated that Sh3tc2 interacts with ErbB2 and plays a role in the regulation of ErbB2 intracellular trafficking from the plasma membrane upon Nrg1 activation. Interestingly, both the loss of Sh3tc2 function in mice and the pathological mutations present in CMT4C patients affect ErbB2 internalization, potentially altering its downstream intracellular signaling pathways. Altogether, our results indicate that the molecular mechanism for the axonal size sensing is disturbed in Sh3tc2-deficient myelinating Schwann cells, thus providing a novel insight into the pathophysiology of CMT4C neuropathy.
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48
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Mohapatra B, Ahmad G, Nadeau S, Zutshi N, An W, Scheffe S, Dong L, Feng D, Goetz B, Arya P, Bailey TA, Palermo N, Borgstahl GEO, Natarajan A, Raja SM, Naramura M, Band V, Band H. Protein tyrosine kinase regulation by ubiquitination: critical roles of Cbl-family ubiquitin ligases. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2012; 1833:122-39. [PMID: 23085373 DOI: 10.1016/j.bbamcr.2012.10.010] [Citation(s) in RCA: 171] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/19/2012] [Revised: 10/05/2012] [Accepted: 10/08/2012] [Indexed: 12/20/2022]
Abstract
Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell-cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant "activated PTK-selective" ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.
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Affiliation(s)
- Bhopal Mohapatra
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
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49
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Mohapatra B, Ahmad G, Nadeau S, Zutshi N, An W, Scheffe S, Dong L, Feng D, Goetz B, Arya P, Bailey TA, Palermo N, Borgstahl GEO, Natarajan A, Raja SM, Naramura M, Band V, Band H. Protein tyrosine kinase regulation by ubiquitination: critical roles of Cbl-family ubiquitin ligases. BIOCHIMICA ET BIOPHYSICA ACTA 2012. [PMID: 23085373 DOI: 10.1016/j.bbamcr] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell-cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant "activated PTK-selective" ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.
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Affiliation(s)
- Bhopal Mohapatra
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
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50
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Luwor RB, Chin X, McGeachie AB, Robinson PJ, Zhu HJ. Dynamin II function is required for EGF-mediated Stat3 activation but not Erk1/2 phosphorylation. Growth Factors 2012; 30:220-9. [PMID: 22574813 DOI: 10.3109/08977194.2012.683189] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Signalling from receptor tyrosine kinases is elicited by ligand binding which initiates the activation of many downstream signalling cascades. Endocytosis has been widely accepted as one mechanism in which cells inactivate signalling by internalising and subsequently degrading activated receptors. However, it is now evident that endocytosis of signalling receptors is important in initiation and sustaining downstream signalling. We and others have previously shown that epidermal growth factor receptor (EGFR) overexpression and activation of signal transducer and activator of transcription 3 (Stat3) are associated with tumourigenicity. Here, we examine the role of endocytosis in EGFR signal attenuation and differential signalling. Inhibition of dynamin II (Dyn II), a GTPase required for endocytosis, with a small molecular weight inhibitor, led to reduced EGF-mediated Stat3 phosphorylation and transcriptional activity in the A431 and HN5 human tumour cell lines. However, Dyn II inhibition had minimal effect on EGF-mediated EGFR and Erk1/2 phosphorylation, which is often regarded responsible for the tumourigenic function of the EGFR. Interestingly, this effect on Stat3 activation was not due to reduced EGFR/Stat3 association. Likewise, cells transfected with Dyn II siRNA or stably transfected with Dyn II shRNA had reduced EGF-mediated phospho-Stat3 levels but similar EGF-mediated phospho-EGFR and phospho-Erk1/2 levels compared with controls. Dyn II siRNA also reduced Stat3 transcriptional reporter activity and inhibits Stat3 accumulating into the nucleus. Taken together, our data suggest that the activation status of Stat3 and Erk1/2 and the sustainability of these signals are potentially due to the spatial and temporal control of the EGFR within the cell. This notion may have implications on therapeutic targeting and efficacy when using inhibitors to proteins either regulating endocytosis and/or signalling.
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Affiliation(s)
- Rodney B Luwor
- Department of Surgery (RMH/WH), University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria, Australia
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