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Fonseca Teixeira A, Wu S, Luwor R, Zhu HJ. A New Era of Integration between Multiomics and Spatio-Temporal Analysis for the Translation of EMT towards Clinical Applications in Cancer. Cells 2023; 12:2740. [PMID: 38067168 PMCID: PMC10706093 DOI: 10.3390/cells12232740] [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: 10/25/2023] [Revised: 11/28/2023] [Accepted: 11/28/2023] [Indexed: 12/18/2023] Open
Abstract
Epithelial-mesenchymal transition (EMT) is crucial to metastasis by increasing cancer cell migration and invasion. At the cellular level, EMT-related morphological and functional changes are well established. At the molecular level, critical signaling pathways able to drive EMT have been described. Yet, the translation of EMT into efficient diagnostic methods and anti-metastatic therapies is still missing. This highlights a gap in our understanding of the precise mechanisms governing EMT. Here, we discuss evidence suggesting that overcoming this limitation requires the integration of multiple omics, a hitherto neglected strategy in the EMT field. More specifically, this work summarizes results that were independently obtained through epigenomics/transcriptomics while comprehensively reviewing the achievements of proteomics in cancer research. Additionally, we prospect gains to be obtained by applying spatio-temporal multiomics in the investigation of EMT-driven metastasis. Along with the development of more sensitive technologies, the integration of currently available omics, and a look at dynamic alterations that regulate EMT at the subcellular level will lead to a deeper understanding of this process. Further, considering the significance of EMT to cancer progression, this integrative strategy may enable the development of new and improved biomarkers and therapeutics capable of increasing the survival and quality of life of cancer patients.
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Affiliation(s)
- Adilson Fonseca Teixeira
- Department of Surgery, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3050, Australia (S.W.); (R.L.)
- Huagene Institute, Kecheng Science and Technology Park, Pukou District, Nanjing 211800, China
| | - Siqi Wu
- Department of Surgery, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3050, Australia (S.W.); (R.L.)
- Huagene Institute, Kecheng Science and Technology Park, Pukou District, Nanjing 211800, China
| | - Rodney Luwor
- Department of Surgery, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3050, Australia (S.W.); (R.L.)
- Huagene Institute, Kecheng Science and Technology Park, Pukou District, Nanjing 211800, China
- Fiona Elsey Cancer Research Institute, Ballarat, VIC 3350, Australia
- Health, Innovation and Transformation Centre, Federation University, Ballarat, VIC 3350, Australia
| | - Hong-Jian Zhu
- Department of Surgery, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3050, Australia (S.W.); (R.L.)
- Huagene Institute, Kecheng Science and Technology Park, Pukou District, Nanjing 211800, China
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Interactions between 14-3-3 Proteins and Actin Cytoskeleton and Its Regulation by microRNAs and Long Non-Coding RNAs in Cancer. ENDOCRINES 2022. [DOI: 10.3390/endocrines3040057] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
14-3-3s are a family of structurally similar proteins that bind to phosphoserine or phosphothreonine residues, forming the central signaling hub that coordinates or integrates various cellular functions, thereby controlling many pathways important in cancer, cell motility, cell death, cytoskeletal remodeling, neuro-degenerative disorders and many more. Their targets are present in all cellular compartments, and when they bind to proteins they alter their subcellular localization, stability, and molecular interactions with other proteins. Changes in environmental conditions that result in altered homeostasis trigger the interaction between 14-3-3 and other proteins to retrieve or rescue homeostasis. In circumstances where these regulatory proteins are dysregulated, it leads to pathological conditions. Therefore, deeper understanding is needed on how 14-3-3 proteins bind, and how these proteins are regulated or modified. This will help to detect disease in early stages or design inhibitors to block certain pathways. Recently, more research has been devoted to identifying the role of MicroRNAs, and long non-coding RNAs, which play an important role in regulating gene expression. Although there are many reviews on the role of 14-3-3 proteins in cancer, they do not provide a holistic view of the changes in the cell, which is the focus of this review. The unique feature of the review is that it not only focuses on how the 14-3-3 subunits associate and dissociate with their binding and regulatory proteins, but also includes the role of micro-RNAs and long non-coding RNAs and how they regulate 14-3-3 isoforms. The highlight of the review is that it focuses on the role of 14-3-3, actin, actin binding proteins and Rho GTPases in cancer, and how this complex is important for cell migration and invasion. Finally, the reader is provided with super-resolution high-clarity images of each subunit of the 14-3-3 protein family, further depicting their distribution in HeLa cells to illustrate their interactions in a cancer cell.
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Quantitative Expression of SFN, lncRNA CCDC18-AS1, and lncRNA LINC01343 in Human Breast Cancer as the Regulator Biomarkers in a Novel ceRNA Network: Based on Bioinformatics and Experimental Analyses. Genet Res (Camb) 2022; 2022:6787791. [PMID: 36160032 PMCID: PMC9484965 DOI: 10.1155/2022/6787791] [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: 05/18/2022] [Revised: 07/15/2022] [Accepted: 08/05/2022] [Indexed: 11/21/2022] Open
Abstract
Breast cancer (BC) is one of the leading cancers in the world, which has become an increasing serious problem. In this context, reports demonstrate that some long noncoding RNAs (lncRNAs) play significant regulatory roles in breast tumorigenesis and BC progression via various pathways and act as endogenous RNAs. Finding their dysregulation in cancer and evaluating their interaction with other molecules, such as short noncoding RNAs “microRNA (miRNAs)” as well as various genes, are the most important parts in cancer diagnostics. In this study, after performing GSEA and microarray analysis on the GSE71053 dataset, a new ceRNA network of CCDC18-AS1, LINC01343, hsa-miR4462, and SFN in BC was detected by bioinformatics analysis. Therefore, the expression of SFN, CCDC18-AS1, and LINC01343 was quantitatively measured in 24 BC and normal paired tissues using qRT-PCR. CCDC18-AS1, LINC01343, and SFN were expressed higher in BC than in the control (normal paired) tissues based on qRT-PCR data. Furthermore, a significant positive correlation was observed between CCDC18-AS1 and LINC01343 expression in the samples investigated in this study. The investigation of clinicopathological parameters showed that SFN was highly expressed in tumor size of <5 cm and in nonmenopausal ages, while CCDC18-AS1 and LINC01343 indicated a high expression in stages II-III and III of BC, respectively. The overall survival analysis displayed high and low survival in patients with high expression of SFN and CCDC18-AS1, respectively. The ROC curve analysis disclosed that SFN, CCDC18-AS1, and LINC01343 might be suggested as potential biological markers in BC patients. The high expression of CCDC18-AS1, LINC01343, and SFN in BC samples suggests their potential role in BC tumorigenesis and could be considered hallmarks for the diagnosis and prognosis of BC, although this will require further clinical investigations.
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Tsamouri MM, Steele TM, Mudryj M, Kent MS, Ghosh PM. Comparative Cancer Cell Signaling in Muscle-Invasive Urothelial Carcinoma of the Bladder in Dogs and Humans. Biomedicines 2021; 9:1472. [PMID: 34680588 PMCID: PMC8533305 DOI: 10.3390/biomedicines9101472] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 10/12/2021] [Accepted: 10/12/2021] [Indexed: 12/15/2022] Open
Abstract
Muscle-invasive urothelial carcinoma (MIUC) is the most common type of bladder malignancy in humans, but also in dogs that represent a naturally occurring model for this disease. Dogs are immunocompetent animals that share risk factors, pathophysiological features, clinical signs and response to chemotherapeutics with human cancer patients. This review summarizes the fundamental pathways for canine MIUC initiation, progression, and metastasis, emerging therapeutic targets and mechanisms of drug resistance, and proposes new opportunities for potential prognostic and diagnostic biomarkers and therapeutics. Identifying similarities and differences between cancer signaling in dogs and humans is of utmost importance for the efficient translation of in vitro research to successful clinical trials for both species.
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Affiliation(s)
- Maria Malvina Tsamouri
- Veterans Affairs-Northern California Health System, Mather, CA 95655, USA; (T.M.S.); (M.M.)
- Department of Urologic Surgery, School of Medicine, University of California Davis, Sacramento, CA 95718, USA
- Graduate Group in Integrative Pathobiology, University of California Davis, Davis, CA 95616, USA
| | - Thomas M. Steele
- Veterans Affairs-Northern California Health System, Mather, CA 95655, USA; (T.M.S.); (M.M.)
- Department of Urologic Surgery, School of Medicine, University of California Davis, Sacramento, CA 95718, USA
| | - Maria Mudryj
- Veterans Affairs-Northern California Health System, Mather, CA 95655, USA; (T.M.S.); (M.M.)
- Department of Medical Microbiology and Immunology, School of Medicine, University of California Davis, Davis, CA 95616, USA
| | - Michael S. Kent
- Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, USA;
| | - Paramita M. Ghosh
- Veterans Affairs-Northern California Health System, Mather, CA 95655, USA; (T.M.S.); (M.M.)
- Department of Urologic Surgery, School of Medicine, University of California Davis, Sacramento, CA 95718, USA
- Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, Sacramento, CA 95718, USA
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Silvestrini VC, Lanfredi GP, Masson AP, Poersch A, Ferreira GA, Thomé CH, Faça VM. A proteomics outlook towards the elucidation of epithelial-mesenchymal transition molecular events. Mol Omics 2020; 15:316-330. [PMID: 31429845 DOI: 10.1039/c9mo00095j] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The main cause of death in cancer is the spread, or metastasis, of cancer cells to distant organs with consequent tumor formation. Additionally, metastasis is a process that demands special attention, as the cellular transformations make cancer at this stage very difficult or occasionally even impossible to be cured. The main process that converts epithelial tumor cells to mesenchymal-like metastatic cells is the Epithelial to Mesenchymal Transition (EMT). This process allows stationary and polarized epithelial cells, which are connected laterally to several types of junctions as well as the basement membrane, to undergo multiple biochemical changes that enable disruption of cell-cell adherence and apical-basal polarity. Moreover, the cells undergo important reprogramming to remodel the cytoskeleton and acquire mesenchymal characteristics such as enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and a large increase in the production of ECM components. As expected, the alterations of the protein complement are extensive and complex, and thus exploring this by proteomic approaches is of particular interest. Here we review the overall findings of proteome modifications during EMT, mainly focusing on molecular signatures observed in multiple proteomic studies as well as coordinated pathways, cellular processes and their clinical relevance for altered proteins. As a result, an interesting set of proteins is highlighted as potential targets to be further investigated in the context of EMT, metastasis and cancer progression.
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Affiliation(s)
- Virgínia Campos Silvestrini
- Department of Biochemistry and Immunology - FMRP - University of São Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil.
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Differential Subcellular Distribution and Translocation of Seven 14-3-3 Isoforms in Response to EGF and During the Cell Cycle. Int J Mol Sci 2020; 21:ijms21010318. [PMID: 31906564 PMCID: PMC6981507 DOI: 10.3390/ijms21010318] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Revised: 12/05/2019] [Accepted: 12/28/2019] [Indexed: 12/12/2022] Open
Abstract
Multiple isoforms of 14-3-3 proteins exist in different organisms. In mammalian cells, 14-3-3 protein has seven isoforms (α/β, ε, η, γ, σ, θ/τ, and δ/ζ), with α and δ representing the phosphorylated versions of β and ζ, respectively. While the existence of multiple isoforms may represent one more level of regulation in 14-3-3 signaling, our knowledge regarding the isoform-specific functions of 14-3-3 proteins is very limited. Determination of the subcellular localization of the different 14-3-3 isoforms could give us important clues of their specific functions. In this study, by using indirect immunofluorescence, subcellular fractionation, and immunoblotting, we studied the subcellular localization of the total 14-3-3 protein and each of the seven 14-3-3 isoforms; their redistribution throughout the cell cycle; and their translocation in response to EGF in Cos-7 cells. We showed that 14-3-3 proteins are broadly distributed throughout the cell and associated with many subcellular structures/organelles, including the plasma membrane (PM), mitochondria, ER, nucleus, microtubules, and actin fibers. This broad distribution underlines the multiple functions identified for 14-3-3 proteins. The different isoforms of 14-3-3 proteins have distinctive subcellular localizations, which suggest their distinctive cellular functions. Most notably, 14-3-3ƞ is almost exclusively localized to the mitochondria, 14-3-3γ is only localized to the nucleus, and 14-3-3σ strongly and specifically associated with the centrosome during mitosis. We also examined the subcellular localization of the seven 14-3-3 isoforms in other cells, including HEK-293, MDA-MB-231, and MCF-7 cells, which largely confirmed our findings with Cos-7 cells.
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Thorsen SF, Gromova I, Christensen IJ, Fredriksson S, Andersen CL, Nielsen HJ, Stenvang J, Moreira JM. Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer. Int J Mol Sci 2019; 20:ijms20236082. [PMID: 31810358 PMCID: PMC6929140 DOI: 10.3390/ijms20236082] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 11/18/2019] [Accepted: 11/18/2019] [Indexed: 12/15/2022] Open
Abstract
The burden of colorectal cancer (CRC) is considerable-approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level (p = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3.
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Affiliation(s)
- Stine F. Thorsen
- Institute of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark;
| | - Irina Gromova
- Danish Cancer Society Research Center, 2100 Copenhagen, Denmark;
| | - Ib J. Christensen
- Department of Surgical Gastroenterology, Hvidovre Hospital, University of Copenhagen, 2650 Hvidovre, Denmark; (I.J.C.); (H.J.N.)
| | | | - Claus L. Andersen
- Department of Molecular Medicine, Aarhus University Hospital, 8200 Aarhus, Denmark;
| | - Hans J. Nielsen
- Department of Surgical Gastroenterology, Hvidovre Hospital, University of Copenhagen, 2650 Hvidovre, Denmark; (I.J.C.); (H.J.N.)
| | - Jan Stenvang
- Institute of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark;
- Correspondence: (J.S.); (J.M.A.M.)
| | - José M.A. Moreira
- Institute of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark;
- Correspondence: (J.S.); (J.M.A.M.)
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8
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Interaction between Rho GTPases and 14-3-3 Proteins. Int J Mol Sci 2017; 18:ijms18102148. [PMID: 29036929 PMCID: PMC5666830 DOI: 10.3390/ijms18102148] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2017] [Revised: 10/11/2017] [Accepted: 10/13/2017] [Indexed: 01/25/2023] Open
Abstract
The Rho GTPase family accounts for as many as 20 members. Among them, the archetypes RhoA, Rac1, and Cdc42 have been the most well-characterized. Like all members of the small GTPases superfamily, Rho proteins act as molecular switches to control cellular processes by cycling between active, GTP-bound and inactive, GDP-bound states. The 14-3-3 family proteins comprise seven isoforms. They exist as dimers (homo- or hetero-dimer) in cells. They function by binding to Ser/Thr phosphorylated intracellular proteins, which alters the conformation, activity, and subcellular localization of their binding partners. Both 14-3-3 proteins and Rho GTPases regulate cell cytoskeleton remodeling and cell migration, which suggests a possible interaction between the signaling pathways regulated by these two groups of proteins. Indeed, more and more emerging evidence indicates the mutual regulation of these two signaling pathways. There have been many documented reviews of 14-3-3 protein and Rac1 separately, but there is no review regarding the interaction and mutual regulation of these two groups of proteins. Thus, in this article we thoroughly review all the reported interactions between the signaling pathways regulated by 14-3-3 proteins and Rho GTPases (mostly Rac1).
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Khorrami A, Sharif Bagheri M, Tavallaei M, Gharechahi J. The functional significance of 14-3-3 proteins in cancer: focus on lung cancer. Horm Mol Biol Clin Investig 2017; 32:/j/hmbci.ahead-of-print/hmbci-2017-0032/hmbci-2017-0032.xml. [PMID: 28779564 DOI: 10.1515/hmbci-2017-0032] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2017] [Accepted: 07/03/2017] [Indexed: 02/07/2023]
Abstract
The 14-3-3 family proteins are phosphoserine/phosphothreonine binding proteins constituting a conserved class of proteins which are detected in all eukaryotic cells. In mammalians, 14-3-3 proteins have seven distinct isoforms (β, γ, ε, η, ζ, σ and τ/θ) which are involved in various cellular processes including signal transduction, cell cycle, cell proliferation, apoptosis, differentiation and survival. 14-3-3 proteins do not have a distinct catalytic activity and often regulate the activity, stability, subcellular localization and interactions of other proteins. The 14-3-3 family proteins function through interacting with their client proteins or facilitating the interaction of other proteins likely as adaptor proteins. The versatile functions of these proteins in the regulation of cell growth, cell division, cell death and cell migration make them candidate proteins for which an important role in cancer development could be envisioned. Indeed, analysis of cancer cell lines and tumor-derived tissues have indicated the differential abundance or post-translational modification of some 14-3-3 isoforms. In this review, we aimed to show how deregulation of 14-3-3 proteins contributes to initiation, establishment and progression of cancers with a particular emphasis on lung cancer. The role of these proteins in cancer-relevant processes including cell cycle, cell migration, cell-cell communication and programmed cell death will be discussed in detail.
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Affiliation(s)
- Afshin Khorrami
- Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
| | - Mahyar Sharif Bagheri
- Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
| | - Mahmood Tavallaei
- Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
| | - Javad Gharechahi
- Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
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Huhe M, Liu S, Zhang Y, Zhang Z, Chen Z. Expression levels of transcription factors c-Fos and c-Jun and transmembrane protein HAb18G/CD147 in urothelial carcinoma of the bladder. Mol Med Rep 2017; 15:2991-3000. [PMID: 28358415 PMCID: PMC5428553 DOI: 10.3892/mmr.2017.6411] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2015] [Accepted: 02/06/2017] [Indexed: 01/24/2023] Open
Abstract
The aim of the present study was to investigate the prognostic significance of the expression of transcription factors, c-Fos, c-Jun and transmembrane protein CD147, in urothelial carcinoma of the bladder (UCB). The current study investigated the clinical significance of these factors in the development, progression and survival analysis of UCB. Immunohistochemistry was employed to analyze c-Fos, c-Jun and CD147 expression in 41 UCB cases and 34 non-cancerous human bladder tissues. These results were scored in a semi-quantitative manner based on the intensity and percentage of tumor cells that presented immunoreactivity. Protein levels of CD147, c-Fos and c-Jun expression were upregulated in 22 (53.7%), 10 (24.4%) and 9 (22.0%) UCB cases, respectively. High levels of c-Jun correlated with the AJCC cancer staging manual (7th edition; P=0.038). Univariate analysis revealed that upregulated CD147 (P=0.038) or c-Jun (P=0.008) was associated with poor overall survival (OS), respectively. Further analysis revealed that either CD147-c-Fos-c-Jun co-expression (P=0.004), or CD147-c-Jun co-expression (P=0.037) and c-Fos-c-Jun co-expression (P<0.001) were associated with poor OS. Multivariate analysis suggested that either upregulation of CD147, c-Jun or c-Fos were independent risk indicators for death in UCB patients. Increased expression of c-Jun or CD147, as well as co-expression of CD147-c-Jun, c-Jun-c-Fos or CD147-c-Jun-c-Fos has prognostic significance for UCB patients. Therefore, high CD147 and c-Jun expression may serve roles in tumor progression and may be diagnostic and therapeutic targets in UCB whether alone or in combination.
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Affiliation(s)
- Muren Huhe
- Cell Engineering Research Center and Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Shuangshuang Liu
- Cell Engineering Research Center and Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Yang Zhang
- Cell Engineering Research Center and Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Zheng Zhang
- Cell Engineering Research Center and Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
| | - Zhinan Chen
- Cell Engineering Research Center and Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
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11
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Integrated genomic and molecular characterization of cervical cancer. Nature 2017; 543:378-384. [PMID: 28112728 PMCID: PMC5354998 DOI: 10.1038/nature21386] [Citation(s) in RCA: 1107] [Impact Index Per Article: 138.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2015] [Accepted: 01/14/2017] [Indexed: 02/06/2023]
Abstract
Cervical cancer remains one of the leading causes of cancer-related deaths worldwide. Reported here is an extensive molecular characterization of 228 primary cervical cancers, the largest comprehensive genomic study of cervical cancer to date. We observed striking APOBEC mutagenesis patterns and identified SHKBP1, ERBB3, CASP8, HLA-A, and TGFBR2 as novel significantly mutated genes in cervical cancer. We also discovered novel amplifications in immune targets CD274/PD-L1 and PDCD1LG2/PD-L2, and the BCAR4 lncRNA that has been associated with response to lapatinib. HPV integration was observed in all HPV18-related cases and 76% of HPV16-related cases, and was associated with structural aberrations and increased target gene expression. We identified a unique set of endometrial-like cervical cancers, comprised predominantly of HPV-negative tumors with high frequencies of KRAS, ARID1A, and PTEN mutations. Integrative clustering of 178 samples identified Keratin-low Squamous, Keratin-high Squamous, and Adenocarcinoma-rich subgroups. These molecular analyses reveal new potential therapeutic targets for cervical cancers.
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12
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Suzuki A, Watanabe H, Mizutani T, Sato T, Ohta Y, Iguchi T. Global Gene Expression in Mouse Vaginae Exposed to Diethylstilbestrol at Different Ages. Exp Biol Med (Maywood) 2016; 231:632-40. [PMID: 16636312 DOI: 10.1177/153537020623100518] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Estrogens regulate proliferation and differentiation of cells in target organs such as the female reproductive tract. In mature mice, estrogens stimulate cell proliferation, whereas ovariectomy results in atrophy of the female reproductive tract. In contrast, perinatal exposure to estrogens, including diethylstilbestrol (DES), induces persistent, ovary-independent vaginal stratification and cervico-vaginal tumors later in life. These effects are due to altered cell fate following DES exposure during a critical developmental period. The detailed mechanisms underlying the reversible and irreversible cell proliferation in vaginae induced by DES at different ages has not been clarified. Therefore, we examined differences in gene expression pattern using DNA microarray analysis in mouse vaginae 6 hrs after a single injection of 2 μg DES per gram of body weight, and proliferation of vaginal epithelial and stromal cells 24 hrs after the injection at postnatal days (PNDs) 0, 5, 20, and 70. After DES stimulation, vaginal epithelial and stromal cells showed cell proliferation at PNDs 20 and 70, and at PNDs 0 and 5, respectively. DNA microarray analysis exhibited 54 DES-induced genes and 9 DES-repressed genes in vaginae at PND 0, whereas more than 200 DES-induced genes were found in vaginae at PNDs 5 and 20, and 350 genes at PND 70. Clustering analysis of DES-induced genes in the vaginae at different ages revealed that genes induced by DES at PND 5 were closer to the adult type than that of PND 0. Genes related to keratinocyte differentiation, such as Gadd45α, p21, 14–3–3 sigma, small proline-rich protein 2f (Sprr2f), and Krupple-like factor 4 (Klf4), were induced by DES. The number of DES-induced genes during the critical period, PND 0, was smaller than those found after the critical period. These results give insight toward understanding the molecular mechanisms underlying the critical period in mouse vaginae.
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Affiliation(s)
- Atsuko Suzuki
- The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 753-8515, Japan
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13
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Frantzi M, van Kessel KE, Zwarthoff EC, Marquez M, Rava M, Malats N, Merseburger AS, Katafigiotis I, Stravodimos K, Mullen W, Zoidakis J, Makridakis M, Pejchinovski M, Critselis E, Lichtinghagen R, Brand K, Dakna M, Roubelakis MG, Theodorescu D, Vlahou A, Mischak H, Anagnou NP. Development and Validation of Urine-based Peptide Biomarker Panels for Detecting Bladder Cancer in a Multi-center Study. Clin Cancer Res 2016; 22:4077-86. [PMID: 27026199 DOI: 10.1158/1078-0432.ccr-15-2715] [Citation(s) in RCA: 84] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2015] [Accepted: 03/11/2016] [Indexed: 11/16/2022]
Abstract
PURPOSE Urothelial bladder cancer presents high recurrence rates, mandating continuous monitoring via invasive cystoscopy. The development of noninvasive tests for disease diagnosis and surveillance remains an unmet clinical need. In this study, validation of two urine-based biomarker panels for detecting primary and recurrent urothelial bladder cancer was conducted. EXPERIMENTAL DESIGN Two studies (total n = 1,357) were performed for detecting primary (n = 721) and relapsed urothelial bladder cancer (n = 636). Cystoscopy was applied for detecting urothelial bladder cancer, while patients negative for recurrence had follow-up for at least one year to exclude presence of an undetected tumor at the time of sampling. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was employed for the identification of urinary peptide biomarkers. The candidate urine-based peptide biomarker panels were derived from nested cross-sectional studies in primary (n = 451) and recurrent (n = 425) urothelial bladder cancer. RESULTS Two biomarker panels were developed on the basis of 116 and 106 peptide biomarkers using support vector machine algorithms. Validation of the urine-based biomarker panels in independent validation sets, resulted in AUC values of 0.87 and 0.75 for detecting primary (n = 270) and recurrent urothelial bladder cancer (n = 211), respectively. At the optimal threshold, the classifier for detecting primary urothelial bladder cancer exhibited 91% sensitivity and 68% specificity, while the classifier for recurrence demonstrated 87% sensitivity and 51% specificity. Particularly for patients undergoing surveillance, improved performance was achieved when combining the urine-based panel with cytology (AUC = 0.87). CONCLUSIONS The developed urine-based peptide biomarker panel for detecting primary urothelial bladder cancer exhibits good performance. Combination of the urine-based panel and cytology resulted in improved performance for detecting disease recurrence. Clin Cancer Res; 22(16); 4077-86. ©2016 AACR.
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Affiliation(s)
- Maria Frantzi
- Mosaiques diagnostics GmbH, Hannover, Germany. Biotechnology Division, Biomedical Research Foundation Academy of Athens, Athens, Greece.
| | - Kim E van Kessel
- Department of Pathology, Erasmus MC Cancer Institute, Erasmus Medical Center, Rotterdam, the Netherlands
| | - Ellen C Zwarthoff
- Department of Pathology, Erasmus MC Cancer Institute, Erasmus Medical Center, Rotterdam, the Netherlands
| | - Mirari Marquez
- Genetic and Molecular Epidemiology Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain
| | - Marta Rava
- Genetic and Molecular Epidemiology Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain
| | - Núria Malats
- Genetic and Molecular Epidemiology Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain
| | | | - Ioannis Katafigiotis
- Department of Urology, Laikon Hospital, Medical School of Athens, Athens, Greece
| | | | - William Mullen
- Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Jerome Zoidakis
- Biotechnology Division, Biomedical Research Foundation Academy of Athens, Athens, Greece
| | - Manousos Makridakis
- Biotechnology Division, Biomedical Research Foundation Academy of Athens, Athens, Greece
| | | | - Elena Critselis
- Biotechnology Division, Biomedical Research Foundation Academy of Athens, Athens, Greece
| | | | - Korbinian Brand
- Institute of Clinical Chemistry, Hannover Medical School, Hannover, Germany
| | | | - Maria G Roubelakis
- Laboratory of Biology, Department of Basic Medical Sciences, University of Athens School of Medicine, Athens, Greece
| | - Dan Theodorescu
- University of Colorado, Department of Surgery and Pharmacology, Aurora, Colorado. University of Colorado Comprehensive Cancer Center, Aurora, Colorado
| | - Antonia Vlahou
- Biotechnology Division, Biomedical Research Foundation Academy of Athens, Athens, Greece
| | - Harald Mischak
- Mosaiques diagnostics GmbH, Hannover, Germany. Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Nicholas P Anagnou
- Laboratory of Biology, Department of Basic Medical Sciences, University of Athens School of Medicine, Athens, Greece. Laboratory of Cell and Gene Therapy, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
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Yang G, Xu Z, Lu W, Li X, Sun C, Guo J, Xue P, Guan F. Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method. PLoS One 2015; 10:e0134727. [PMID: 26230496 PMCID: PMC4521931 DOI: 10.1371/journal.pone.0134727] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2015] [Accepted: 07/13/2015] [Indexed: 12/26/2022] Open
Abstract
The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia), KK47 (low grade nonmuscle invasive bladder cancer, NMIBC), and YTS1 (metastatic bladder cancer) have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC) progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO) term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer.
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Affiliation(s)
- Ganglong Yang
- The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Zhipeng Xu
- Shaanxi Provincial People’s Hospital, Xi’an, China
| | - Wei Lu
- The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Xiang Li
- Wuxi Medical School, Jiangnan University, Wuxi, China
| | - Chengwen Sun
- Department of Urology, Affiliated Hospital of Jiangnan University, Wuxi, China
| | - Jia Guo
- The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Peng Xue
- Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- * E-mail: (PX); (FG)
| | - Feng Guan
- The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
- * E-mail: (PX); (FG)
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Suárez-Bonnet A, Herráez P, Aguirre M, Suárez-Bonnet E, Andrada M, Rodríguez F, Espinosa de Los Monteros A. Expression of cell cycle regulators, 14-3-3σ and p53 proteins, and vimentin in canine transitional cell carcinoma of the urinary bladder. Urol Oncol 2015; 33:332.e1-7. [PMID: 25979650 DOI: 10.1016/j.urolonc.2015.04.006] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2014] [Revised: 04/07/2015] [Accepted: 04/08/2015] [Indexed: 02/07/2023]
Abstract
OBJECTIVES The study of the expression of 14-3-3σ, p53, and vimentin proteins in canine transitional cell carcinoma (TCC) evaluating differences with normal bladder tissues, and the association with clinicopathological variables. METHODS We analyze by immunohistochemistry in 19 canine TCCs the expression of 14-3-3σ, p53, and vimentin using monoclonal antibodys. A semiquantitative scoring method was employed and statistical analysis was performed to display relationships between variables. RESULTS In contrast to normal urinary bladder epithelium, which showed high levels of 14-3-3σ, its expression was decreased in 53% of the studied tumors (P = 0.0344). The 14-3-3σ protein was expressed by neoplastic emboli and by highly infiltrative neoplastic cells. The p53 protein was expressed in 26% of TCCs, but no significant association between 14-3-3σ and p53 was detected. Neoplastic epithelial cells displayed vimentin immunoreactivity in 21% of TCCs, and a positive correlation with mitotic index was observed (P = 0.042). Coexpression of vimentin and 14-3-3σ by highly infiltrative neoplastic cells was also observed. CONCLUSIONS 14-3-3σ is deregulated in canine TCCs and its expression by highly infiltrative tumor cells may be related to the acquisition of aggressive behavior. Furthermore, this article reinforce the role of canine TCC as relevant model of human urothelial carcinoma and we suggest 14-3-3σ as a potential therapeutic target. Further studies are necessary to clarify the role of 14-3-3σ in canine TCC.
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Affiliation(s)
- Alejandro Suárez-Bonnet
- Unit of Histology and Animal Pathology, Institute for Animal Health, Veterinary School, University of Las Palmas de Gran Canaria, Arucas, Spain.
| | - Pedro Herráez
- Unit of Histology and Animal Pathology, Institute for Animal Health, Veterinary School, University of Las Palmas de Gran Canaria, Arucas, Spain
| | - Maria Aguirre
- Department of Surgery and Internal Medicine, Veterinary School, University of Las Palmas de Gran Canaria, Arucas, Spain
| | - Elena Suárez-Bonnet
- Unit of Histology and Animal Pathology, Institute for Animal Health, Veterinary School, University of Las Palmas de Gran Canaria, Arucas, Spain
| | - Marisa Andrada
- Unit of Histology and Animal Pathology, Institute for Animal Health, Veterinary School, University of Las Palmas de Gran Canaria, Arucas, Spain
| | - Francisco Rodríguez
- Unit of Histology and Animal Pathology, Institute for Animal Health, Veterinary School, University of Las Palmas de Gran Canaria, Arucas, Spain
| | - Antonio Espinosa de Los Monteros
- Unit of Histology and Animal Pathology, Institute for Animal Health, Veterinary School, University of Las Palmas de Gran Canaria, Arucas, Spain
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Kageyama S, Isono T, Iwaki H, Hanada E, Tomita K, Yoshida T, Yoshiki T, Kawauchi A. Proteome research in urothelial carcinoma. Int J Urol 2015; 22:621-8. [DOI: 10.1111/iju.12793] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Revised: 03/01/2015] [Accepted: 03/18/2015] [Indexed: 01/10/2023]
Affiliation(s)
| | - Takahiro Isono
- Central Research Laboratory; Shiga University of Medical Science; Otsu Shiga
| | - Hideaki Iwaki
- Department of Urology; Shiga University of Medical Science
| | - Eiki Hanada
- Department of Urology; Shiga University of Medical Science
| | - Keiji Tomita
- Department of Urology; Shiga University of Medical Science
| | | | - Tatsuhiro Yoshiki
- Department of Clinical Oncology; Kyoto Pharmaceutical University; Kyoto Japan
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Tsai MM, Wang CS, Tsai CY, Chi HC, Tseng YH, Lin KH. Potential prognostic, diagnostic and therapeutic markers for human gastric cancer. World J Gastroenterol 2014; 20:13791-13803. [PMID: 25320517 PMCID: PMC4194563 DOI: 10.3748/wjg.v20.i38.13791] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2014] [Revised: 04/18/2014] [Accepted: 05/26/2014] [Indexed: 02/06/2023] Open
Abstract
The high incidence of gastric cancer (GC) and its consequent mortality rate severely threaten human health. GC is frequently not diagnosed until a relatively advanced stage. Surgery is the only potentially curative treatment. Thus, early screening and diagnosis are critical for improving prognoses in patients with GC. Gastroscopy with biopsy is an appropriate method capable of aiding the diagnosis of specific early GC tumor types; however, the stress caused by this method together with it being excessively expensive makes it difficult to use it as a routine method for screening for GC on a population basis. The currently used tumor marker assays for detecting GC are simple and rapid, but their use is limited by their low sensitivity and specificity. In recent years, several markers have been identified and tested for their clinical relevance in the management of GC. Here, we review the serum-based tumor markers for GC and their clinical significance, focusing on discoveries from microarray/proteomics research. We also review tissue-based GC tumor markers and their clinical application, focusing on discoveries from immunohistochemical research. This review provides a brief description of various tumor markers for the purposes of diagnosis, prognosis and therapeutics, and we include markers already in clinical practice and various forthcoming biomarkers.
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18
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Zou J, Mi L, Yu XF, Dong J. Interaction of 14-3-3σ with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells. World J Gastroenterol 2013; 19:3770-3780. [PMID: 23840115 PMCID: PMC3703518 DOI: 10.3748/wjg.v19.i24.3770] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/16/2012] [Revised: 02/01/2013] [Accepted: 03/23/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells.
METHODS: Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells.
RESULTS: The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1, quinone oxidoreductase (NQO2), hydroxyisobutyrate dehydrogenase (HIBADH) and 14-3-3σ. For the subsequent coimmunoprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immunoprecipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc.
CONCLUSION: Genes of the proteins that interacted with 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.
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19
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Chung H, Kim B, Jung SH, Won KJ, Jiang X, Lee CK, Lim SD, Yang SK, Song KH, Kim HS. Does phosphorylation of cofilin affect the progression of human bladder cancer? BMC Cancer 2013; 13:45. [PMID: 23374291 PMCID: PMC3568060 DOI: 10.1186/1471-2407-13-45] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2012] [Accepted: 01/28/2013] [Indexed: 12/11/2022] Open
Abstract
Background We determined the differently expressed protein profiles and their functions in bladder cancer tissues with the aim of identifying possible target proteins and underlying molecular mechanisms for taking part in their progression. Methods We examined the expression of proteins by proteomic analysis and western blot in normal urothelium, non-muscle-invasive bladder cancers (NMIBCs), and muscle-invasive bladder cancers (MIBCs). The function of cofilin was analyzed using T24 human bladder cancer cells. Results The expression levels of 12 proteins were altered between bladder cancers and normal bladder tissues. Of these proteins, 14-3-3σ was upregulated in both NMIBCs and MIBCs compared with controls. On the other hand, myosin regulatory light chain 2, galectin-1, lipid-binding AI, annexin V, transthyretin, CARD-inhibitor of NF-κB-activating ligand, and actin prepeptide were downregulated in cancer samples. Cofilin, an actin-depolymerizing factor, was prominent in both NMIBCs and MIBCs compared with normal bladder tissues. Furthermore, we confirmed that cofilin phosphorylation was more prominent in MIBCs than in NMIBCs using immunoblotting and immunohistochemcal analyses. Epidermal growth factor (EGF) increased the phosphorylation of cofilin and elevated the migration in T24 cells. Knockdown of cofilin expression with small interfering RNA attenuated the T24 cell migration in response to EGF. Conclusions These results demonstrate that the increased expression and phosphorylation of cofilin might play a role in the occurrence and invasiveness of bladder cancer. We suspected that changes in cofilin expression may participate in the progression of the bladder cancer.
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Affiliation(s)
- Hong Chung
- Department of Urology, School of Medicine, Konkuk University, 82 Gugwon-daero, Chungju, Chungbuk 380-704, Republic of Korea
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Radhakrishnan VM, Putnam CW, Martinez JD. Activation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling and the consequent induction of transformation by overexpressed 14-3-3γ protein require specific amino acids within 14-3-3γ N-terminal variable region II. J Biol Chem 2012; 287:43300-11. [PMID: 23115241 PMCID: PMC3527917 DOI: 10.1074/jbc.m112.397877] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2012] [Revised: 10/10/2012] [Indexed: 11/06/2022] Open
Abstract
Members of the 14-3-3 superfamily regulate numerous cellular functions by binding phosphoproteins. The seven human isoforms (and the myriad of other eukaryotic 14-3-3 proteins) are highly conserved in amino acid sequence and secondary structure, yet there is abundant evidence that the various isoforms manifest disparate as well as common functions. Several of the human 14-3-3 isoforms are dysregulated in certain cancers and thus have been implicated in oncogenesis; experimentally, 14-3-3γ behaves as an oncogene, whereas 14-3-3σ acts as a tumor suppressor. In this study, we sought to localize these opposing phenotypes to specific regions of the two isoforms and then to individual amino acids therein. Using a bioinformatics approach, six variable regions (VRI-VRVI) were identified. Using this information, two sets of constructs were created in which N-terminal portions (including either VRI-IV or only VRI and VRII) of 14-3-3γ and 14-3-3σ were swapped; NIH3T3 cells overexpressing the four chimeric proteins were tested for transformation activity (focus formation, growth in soft agar) and activation of PI3K and MAPK signaling. We found that the specific phenotypes of 14-3-3γ are associated with the N-terminal 40 amino acids (VRI and VRII); in like fashion, VRI and VRII of 14-3-3σ dictated its tumor suppressor function. Using individual amino acid substitutions within the 14-3-3γ VRII, we identified two residues required for and two contributing to the γ-specific phenotypes. Our observations suggest that isoform-specific phenotypes are dictated by a relatively few amino acids within variable regions.
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Affiliation(s)
| | - Charles W. Putnam
- From the Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724
| | - Jesse D. Martinez
- From the Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724
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21
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Gromova I, Gromov P, Kroman N, Wielenga VT, Simon R, Sauter G, Moreira JMA. Immunoexpression analysis and prognostic value of BLCAP in breast cancer. PLoS One 2012; 7:e45967. [PMID: 23049907 PMCID: PMC3458104 DOI: 10.1371/journal.pone.0045967] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2012] [Accepted: 08/23/2012] [Indexed: 01/15/2023] Open
Abstract
Bladder Cancer Associated Protein (BLCAP, formerly Bc10), was identified by our laboratory as being down-regulated in bladder cancer with progression. BLCAP is ubiquitously expressed in different tissues, and several studies have found differential expression of BLCAP in various cancer types, such as cervical and renal cancer, as well as human tongue carcinoma and osteosarcoma. Here we report the first study of the expression patterns of BLCAP in breast tissue. We analyzed by immunohistochemistry tissue sections of normal and malignant specimens collected from 123 clinical high-risk breast cancer patients within the Danish Center for Translational Breast Cancer Research (DCTB) prospective study dataset. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. We observed weak immunoreactivity for BLCAP in mammary epithelial cells, almost exclusively localizing to the cytoplasm and found that levels of expression of BLCAP were generally higher in malignant cells as compared to normal cells. Quantitative IHC analysis of BLCAP expression in breast tissues confirmed this differential BLCAP expression in tumor cells, and we could establish, in a 62-patient sample matched cohort, that immunostaining intensity for BLCAP was increased in tumors relative to normal tissue, in more than 45% of the cases examined, indicating that BLCAP may be of value as a marker for breast cancer. We also analyzed BLCAP expression and prognostic value using a set of tissue microarrays comprising an independent cohort of 2,197 breast cancer patients for which we had follow-up clinical information.
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Affiliation(s)
- Irina Gromova
- Cancer Proteomics, Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark
- Danish Centre for Translational Breast Cancer Research (DCTB), Copenhagen, Denmark
- * E-mail: (JM); (IG)
| | - Pavel Gromov
- Cancer Proteomics, Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark
- Danish Centre for Translational Breast Cancer Research (DCTB), Copenhagen, Denmark
| | - Niels Kroman
- Department of Breast Surgery, Copenhagen University Hospital, Copenhagen, Denmark
| | - Vera Timmermans Wielenga
- Danish Centre for Translational Breast Cancer Research (DCTB), Copenhagen, Denmark
- Department of Pathology, the Centre of Diagnostic Investigations, Copenhagen University Hospital, Copenhagen, Denmark
| | - Ronald Simon
- Department of Pathology, Diagnostic Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Guido Sauter
- Department of Pathology, Diagnostic Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - José M. A. Moreira
- Cancer Proteomics, Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark
- Danish Centre for Translational Breast Cancer Research (DCTB), Copenhagen, Denmark
- Section of Pathobiology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- * E-mail: (JM); (IG)
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Wang L, Huang H, Liu D, Fang S, Xian Y, Zhou J, Zuo Y, Wang F, Huang O, He M. Evaluation of 14-3-3 protein family levels and associated receptor expression of estrogen and progesterone in human uterine leiomyomas. Gynecol Endocrinol 2012; 28:665-8. [PMID: 22329840 DOI: 10.3109/09513590.2012.650768] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
OBJECTIVE Uterine leiomyomas represents a major public health problem. Despite their prevalence, the causation and pathogenesis of leiomyomas are poorly understood. A broad range of organisms and tissues contain 14-3-3 proteins which have been associated with the pathogenesis of many diseases through participating in signal transduction pathways. This study was designed to evaluate which 14-3-3 isoforms might be optimal targets in leiomyomas, and to further explore their relationship with estrogen and progesterone receptor (ER and PR). METHODS Paired samples of leiomyoma and adjacent myometrium were obtained from 80 subjects who had surgical excision of uterine leiomyomas. The expression of 14-3-3 isoforms was detected by Western bolt and RT-PCR, and their relationship with ER and PR was analysed by immunohistochemistry. RESULTS The expressions of 14-3-3σ had decreased significantly in leiomyoma compared with that in normal myometrium and was negatively correlated with ER and PR by immunohistochemistry. CONCLUSION The down-regulation of 14-3-3σ in leiomyoma suggests that 14-3-3σ may play a role in tumorigenesis, and that its mechanism may be involved in the up-regulation of ER and PR.
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Affiliation(s)
- Liqun Wang
- Division of Obstetrics, Jiangxi Maternal & Child Health Hospital, Nanchang, PR China
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Proteome alterations in response to aristolochic acids in experimental animal model. J Proteomics 2012; 76 Spec No.:79-90. [PMID: 22796065 DOI: 10.1016/j.jprot.2012.06.026] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2012] [Revised: 06/26/2012] [Accepted: 06/29/2012] [Indexed: 11/23/2022]
Abstract
Strong indications have been presented that dietary poisoning with aristolochic acids (AA) is responsible for Endemic Nephropathy (EN) and AA associated cancer of the upper urinary tract (UUTC). Our recent investigation showed drastic urinary proteome changes in AA treated mice. This study was designed to identify proteome changes associated with AA nephrotoxicity in experimental animal model. The DBA and C57BL mice, which differ in AA sensitivity, were exposed to AA for 4 days. The strategy for urinary, plasma and kidney tissue proteome study of AA exposed and control mice integrated gel-based and in-solution tryptic digestion combined with LC-ESI-MS/MS. To maximize proteome coverage, plasma fractionation scheme was developed and MS compatible sequential tissue extraction procedure was established. Proteomic analyses of urinary, plasma and kidney tissue tryptic digests resulted in identification of several cytoskeletal proteins, as well as proteins involved in kidney development and inflammatory response, that are differentially expressed in both AA exposed and control mice. These proteins are consistent with renal pathogenesis of endotoxicity and cancer. This proteomic strategy could be effectively translated for unbiased discovery of potential biomarkers for EN and associated UUTC in humans. At the same time, these results highlight the significance of AA exposure with EN. This article is part of a Special Issue entitled: Integrated omics.
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Koh SS, Wei JPJ, Li X, Huang RR, Doan NB, Scolyer RA, Cochran AJ, Binder SW. Differential gene expression profiling of primary cutaneous melanoma and sentinel lymph node metastases. Mod Pathol 2012; 25:828-37. [PMID: 22411186 DOI: 10.1038/modpathol.2012.32] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Limited understanding of molecular mechanisms of metastasis in melanoma contributes to the absence of effective treatments. Increased knowledge of alterations in genes that underpin critical molecular events that lead to metastasis is essential. We have investigated the gene expression profiles of primary melanomas and melanoma metastases in sentinel lymph nodes. A total of 19 samples (10 primary melanomas and 9 sentinel lymph node metastases) were evaluated. Melanoma cells were dissected from tissue blocks. Total mRNA was isolated, amplified, and labeled using an Ambion Recover All Total Nucleic Acid Isolation kit, Nu-GEN WT-Ovation formalin-fixed, paraffin-embedded RNA Amplification System, and FL-Ovation cDNA Biotin Module V2, respectively. Samples were hybridized to the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data were analyzed using Partek Genomics Suite Version 6.4. Genes selected showed ≥2-fold difference in expression and P<5.00E-2. Validation studies used standard immunohistochemical assays. Hierarchical clustering disclosed two distinct groups: 10 primary melanomas and 9 sentinel lymph node metastases. Gene expression analysis identified 576 genes that showed significant differential expression. Most differences reflected decreased gene expression in metastases relative to primaries. Reduced gene expression in primaries was less frequent and less dramatic. Genes significantly increased or decreased in sentinel lymph node metastases were active in cell adhesion/structural integrity, tumor suppression, cell cycle regulation, and apoptosis. Validation studies indicate that MAGEC1 (melanoma antigen family C1) and FCRL1 (Fc receptor-like 1) are involved in melanoma progression. There are striking differential gene expression patterns between primary and nodally metastatic melanomas. Similar findings were seen with autologous paired primary melanomas and sentinel lymph node metastases, supporting involvement of these gene alterations in evolution of metastases. With further study, it may be possible to determine the exact sequence of molecular events that underlie melanoma metastases.
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Affiliation(s)
- Stephen S Koh
- Department of Pathology and Laboratory Medicine, University of California Los Angeles/David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1732, USA
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Deng J, Gao G, Wang L, Wang T, Yu J, Zhao Z. Stratifin expression is a novel prognostic factor in human gliomas. Pathol Res Pract 2011; 207:674-9. [PMID: 21940111 DOI: 10.1016/j.prp.2011.08.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/08/2011] [Revised: 07/31/2011] [Accepted: 08/01/2011] [Indexed: 11/19/2022]
Abstract
Stratifin (14-3-3σ or SFN) is a member of the 14-3-3 family of proteins which play critical roles in different cellular signaling processes. Stratifin as a potential tumor suppressor gene plays an important role in carcinogenesis and metastasis. The aim of this study was to investigate the expression of Stratifin in human gliomas and to analyze its expression profile with respect to tumor development. The expression pattern of Stratifin was analyzed by immunohistochemistry and/or Western blotting in tumor samples from 186 patients with different grades of gliomas. Prognostic significance was assessed using Kaplan-Meier survival estimates and Cox regression analyses. The expression pattern of Stratifin was correlated with the pathological and clinical characteristics of the patients with gliomas. Western blot analysis indicated that the average optical densitometry (OD) ratio of Stratifin in high-grade gliomas (World Health Organization [WHO] grade III/IV) was lower than in low-grade tumors (WHO grade I/II, p=0.026). In addition, statistical analysis showed that patients expressing a high level of Stratifin have favorable overall survival rates relative to those expressing a low level of this protein. Cox multi-factor analysis showed that Stratifin (p=0.02) was an independent prognosis factor for human gliomas. Our results provide convincing evidence that the expression of Stratifin is down-regulated in human gliomas. Its expression level is correlated with the clinicopathological parameters and prognosis in patients with gliomas. Pending validation targeting, Stratifin might also be a novel opportunity to improve the therapy of this tumor.
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Affiliation(s)
- Jianping Deng
- Department of Neurosurgery, Institute for Functional Neurosurgery P.L.A, TangDu Hospital, Fourth Military Medical University, #1, Xinsi Road, Baqiao District, Xi'an 710038, PR China
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Radhakrishnan VM, Jensen TJ, Cui H, Futscher BW, Martinez JD. Hypomethylation of the 14-3-3σ promoter leads to increased expression in non-small cell lung cancer. Genes Chromosomes Cancer 2011; 50:830-6. [PMID: 21755566 DOI: 10.1002/gcc.20904] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2011] [Revised: 06/08/2011] [Accepted: 06/14/2011] [Indexed: 11/12/2022] Open
Abstract
The 14-3-3 proteins are a set of seven highly conserved proteins that have recently been implicated in having a role in human tumorigenesis. However, the mechanism by which 14-3-3 proteins may act in this capacity is not well understood. In this study, we examined the expression of one of the 14-3-3 family members, 14-3-3σ, since it was shown previously to be aberrantly altered in human tumors. Using quantitative rtPCR and immunohistochemistry, we found that the expression levels of 14-3-3σ were elevated in the majority of human non-small cell lung cancers (NSCLC) we examined. Surprisingly, we found that the 14-3-3σ gene was hypomethylated in lung tumors relative to normal lung tissue suggesting that decreased DNA methylation resulted in increased expression of 14-3-3σ in NSCLC. We also determined the gene copy number for 14-3-3σ in tumor samples and found no significant correlation with elevated mRNA expression. And also no mutations were found in 14-3-3σ gene. Overall, our data suggest that misregulated expression of 14-3-3σ gene may be due to altered methylation status. © 2011 Wiley-Liss, Inc.
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Yao Y, Li J, Lu Z, Tong A, Wang W, Su X, Zhou Y, Mu B, Zhou S, Li X, Chen L, Gou L, Song H, Yang J, Wei Y. Proteomic analysis of the interleukin-4 (IL-4) response in hepatitis B virus-positive human hepatocelluar carcinoma cell line HepG2.2.15. Electrophoresis 2011; 32:2004-12. [PMID: 21739463 DOI: 10.1002/elps.201100147] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2011] [Revised: 04/19/2011] [Accepted: 04/25/2011] [Indexed: 02/05/2023]
Abstract
Hepatitis B virus (HBV) infection is the leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. In recent decades, significant progress toward understanding the molecular virology and pathogenesis of HBV infection has been made. In addition, multiple treatment modalities have been developed for persons with HBV infection. In the present study, we demonstrated that IL-4 inhibits the expression of hepatitis B surface antigen and hepatitis B e antigen in a HBV stably transfected hepatocellular carcinoma cell line (HepG2.2.15). To reveal the anti-HBV mechanism of IL-4 by proteomics, 2-DE and MS technology were utilized to profile global changes in protein expression in HepG2.2.15 cells after IL-4 treatment. A total of 56 differentially expressed proteins were identified in IL-4-treated HepG2.2.15 cells. To find out the interaction of these changed proteins by bioinformatics, signaling network analysis with the STRING tool showed that the identified proteins are primarily involved in transcription and proteolysis. Taken together, these results offer valuable clues for understanding the molecular mechanisms of the IL-4-mediated anti-HBV response.
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Affiliation(s)
- Yuqin Yao
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan, P R China
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14-3-3 protein beta/alpha as a urinary biomarker for renal cell carcinoma: proteomic analysis of cyst fluid. Anal Bioanal Chem 2011; 401:245-52. [PMID: 21553213 DOI: 10.1007/s00216-011-5057-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2011] [Revised: 04/06/2011] [Accepted: 04/22/2011] [Indexed: 01/22/2023]
Abstract
Although various samples, including tissue, cells, serum, and urine, from patients with renal cell carcinoma (RCC) have been analyzed, biomarkers with diagnostic value have yet to be identified. We used a proteomics approach to analyze cyst fluid in cases of cyst-associated RCC to identify accessible and abundant proteins that are overexpressed and/or secreted by RCC cells. Proteins in the cyst fluid were separated by reverse-phase high-performance liquid chromatography and agarose two-dimensional gel electrophoresis and were identified by tandem mass spectrometry. We conducted a National Center for Biotechnology Information search and a MEDLINE search to predict the function of these identified proteins and to select a tumor-marker candidate protein. Our search resulted in the identification and selection of the differentially regulated protein known as 14-3-3 protein beta/alpha, which was overexpressed in cyst fluid from cyst-associated RCC but has not been previously associated with RCC. We then measured its incidence through Western blotting of various normal and RCC samples (serum, urine, tissue, and cyst fluid). The expression levels of 14-3-3 protein beta/alpha were higher in urine samples from patients with RCC than in samples from healthy volunteers. Receiver operating characteristic (ROC) curve analyses were performed to assess this potential biomarker; these data (area under the ROC curve value was 0.8813) indicate a high degree of accuracy for this screening method. 14-3-3 Protein beta/alpha may be a diagnostically useful biomarker for early diagnosis of RCC.
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Boominathan L. The guardians of the genome (p53, TA-p73, and TA-p63) are regulators of tumor suppressor miRNAs network. Cancer Metastasis Rev 2011; 29:613-39. [PMID: 20922462 DOI: 10.1007/s10555-010-9257-9] [Citation(s) in RCA: 85] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The tumor suppressor p53 homologues, TA-p73, and p63 have been shown to function as tumor suppressors. However, how they function as tumor suppressors remains elusive. Here, I propose a number of tumor suppressor pathways that illustrate how the TA-p73 and p63 could function as negative regulators of invasion, metastasis, and cancer stem cells (CSCs) proliferation. Furthermore, I provide molecular insights into how TA-p73 and p63 could function as tumor suppressors. Remarkably, the guardians--p53, p73, and p63--of the genome are in control of most of the known tumor suppressor miRNAs, tumor suppressor genes, and metastasis suppressors by suppressing c-myc through miR-145/let-7/miR-34/TRIM32/PTEN/FBXW7. In particular, p53 and TA-p73/p63 appear to upregulate the expression of (1) tumor suppressor miRNAs, such as let-7, miR-34, miR-15/16a, miR-145, miR-29, miR-26, miR-30, and miR-146a; (2) tumor suppressor genes, such as PTEN, RBs, CDKN1a/b/c, and CDKN2a/b/c/d; (3) metastasis suppressors, such as Raf kinase inhibitory protein, CycG2, and DEC2, and thereby they enlarge their tumor suppressor network to inhibit tumorigenesis, invasion, angiogenesis, migration, metastasis, and CSCs proliferation.
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Expression of 14-3-3 σ protein in normal and neoplastic canine mammary gland. Vet J 2011; 190:345-51. [PMID: 21251859 DOI: 10.1016/j.tvjl.2010.12.015] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2010] [Revised: 12/09/2010] [Accepted: 12/12/2010] [Indexed: 11/22/2022]
Abstract
14-3-3 σ protein is a negative cell cycle regulator, with both reduced and elevated levels associated with cancer in humans. This study assessed the expression of this protein in canine mammary tissues using immunohistochemistry and Western blotting. 14-3-3 σ was detected in 97% of the mammary tissue samples examined and was found in both myoepithelial (MECs) and epithelial (ECs) cells. Expression levels were elevated and reduced in neoplastic ECs and MECs, respectively (P<0.001). Intense expression of 14-3-3 σ was detected in neoplastic ECs infiltrating blood vessels and lymph nodes and suggests a possible role for this protein in the malignant transformation of mammary neoplasms. Moreover, double immunostaining for 14-3-3 σ and the MEC-specific marker p63, confirmed that 14-3-3 σ is a highly sensitive marker of MECs since all p63-positive cells were also positive for 14-3-3 σ. However, this protein is not exclusive to MECs as ECs also labelled positively.
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Lu Y, Liu J, Lin C, Wang H, Jiang Y, Wang J, Yang P, He F. Peroxiredoxin 2: a potential biomarker for early diagnosis of hepatitis B virus related liver fibrosis identified by proteomic analysis of the plasma. BMC Gastroenterol 2010; 10:115. [PMID: 20939925 PMCID: PMC2959091 DOI: 10.1186/1471-230x-10-115] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/21/2009] [Accepted: 10/13/2010] [Indexed: 12/12/2022] Open
Abstract
Background Liver fibrosis is a middle stage in the course of chronic Hepatitis B virus (HBV) infection, which will develop into cirrhosis and eventually hepatocellular carcinoma (HCC) if not treated at the early stage. Considering the limitations and patients' reluctance to undergo liver biopsy, a reliable, noninvasive diagnostic system to predict and assess treatment and prognosis of liver fibrosis is needed. The aim of this study was to identify biomarkers for early diagnosis of HBV related liver fibrosis. Method Plasma samples from 7 healthy volunteers and 27 HBV infected patients with different stages of fibrosis were selected for 2-DIGE proteomic screening. One-way ANOVA analysis was used to assess differences in protein expression among all groups. The alteration was further confirmed by western blotting. Plasma levels of 25 serological variables in 42 healthy volunteers and 68 patients were measured to establish a decision tree for the detection of various stages fibrosis. Result The up-regulated proteins along with fibrosis progress included fibrinogen, collagen, macroglobulin, hemopexin, antitrypsin, prealbumin and thioredoxin peroxidase. The down-regulated proteins included haptoglobin, serotransferrin, CD5 antigen like protein, clusterin, apolipoprotein and leucine-rich alpha-2-glycoprotein. For the discrimination of milder stage fibrosis, the area under curve for Prx II was the highest. Four variables (PT, Pre, HA and Prx II) were selected from the 25 variables to construct the decision tree. In a training group, the correct prediction percentage for normal control, milder fibrosis, significant fibrosis and early cirrhosis was 100%, 88.9%, 95.2% and 100%, respectively, with an overall correct percent of 95.9%. Conclusion This study showed that 2-D DIGE-based proteomic analysis of the plasma was helpful in screening for new plasma biomarkers for liver disease. The significant up-expression of Prx II could be used in the early diagnosis of HBV related liver fibrosis.
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Affiliation(s)
- Ye Lu
- Department of Chemistry, Fudan University, Shanghai, China
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Ren HZ, Pan GQ, Wang JS, Wen JF, Wang KS, Luo GQ, Shan XZ. Reduced stratifin expression can serve as an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma. Dig Dis Sci 2010; 55:2552-60. [PMID: 20108042 DOI: 10.1007/s10620-009-1065-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/03/2009] [Accepted: 11/20/2009] [Indexed: 12/20/2022]
Abstract
UNLABELLED Stratifin plays an important role in cancer biology by interfering with intracellular signalling pathways and cell-cycle checkpoints. Decreased expression of stratifin gene has been reported to be a poor prognostic indicator in a variety of human malignant tumors. AIM To clarify the role and prognostic significance of stratifin in esophageal squamous cell carcinoma (ESCC). METHODS The alteration of stratifin messenger RNA (mRNA) and protein was analyzed by reverse-transcription and quantitative real-time polymerase chain reaction (QRT-PCR) and Western blotting in 20 paired ESCC and nonneoplastic esophageal mucosa tissues, respectively. Then, immunohistochemistry (IHC) was used to evaluate expression of stratifin in tissues of 148 ESCC patients (including the former 20 pairs of tissues) and correlate it with clinicopathological parameters and prognosis of ESCC patients. RESULTS The stratifin level of mRNA and protein was markedly downregulated in ESCC tissue compared with in corresponding nonneoplastic esophageal epithelium (P<0.05). Similarly, the positive rate of stratifin protein expression was lower in the esophageal cancer than in paired nonneoplastic esophageal epithelium as detected by IHC (P=0.007). Statistically, the downregulation of stratifin expression was correlated with tumor infiltration depth (P=0.003), lymph node metastasis (P=0.008), distant metastasis (P=0.013), and lymphovascular invasion (P=0.007) of ESCC. Furthermore, the reduced stratifin expression was associated with shorter 5-year survival rate of ESCC patients after curative surgery (P<0.0001). On the basis of univariate and multivariate Cox regression analysis, we found that reduced stratifin expression, T4 stage, lymph node metastasis, and distant metastasis were independent risk factors for worse prognosis in ESCC patients. CONCLUSION The present report indicates that stratifin could be a useful indicator for prognosis of this disease, as well as a potential target for more effective therapy.
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MESH Headings
- 14-3-3 Proteins/analysis
- 14-3-3 Proteins/genetics
- Adult
- Aged
- Biomarkers, Tumor/analysis
- Biomarkers, Tumor/genetics
- Blotting, Western
- Carcinoma, Squamous Cell/chemistry
- Carcinoma, Squamous Cell/genetics
- Carcinoma, Squamous Cell/mortality
- Carcinoma, Squamous Cell/secondary
- Carcinoma, Squamous Cell/surgery
- Chi-Square Distribution
- Down-Regulation
- Esophageal Neoplasms/chemistry
- Esophageal Neoplasms/genetics
- Esophageal Neoplasms/mortality
- Esophageal Neoplasms/pathology
- Esophageal Neoplasms/surgery
- Esophagectomy
- Exonucleases/analysis
- Exonucleases/genetics
- Exoribonucleases
- Female
- Gene Expression Regulation, Neoplastic
- Humans
- Immunohistochemistry
- Kaplan-Meier Estimate
- Lymphatic Metastasis
- Male
- Middle Aged
- Neoplasm Invasiveness
- Neoplasm Staging
- Predictive Value of Tests
- Proportional Hazards Models
- RNA, Messenger/analysis
- Reverse Transcriptase Polymerase Chain Reaction
- Risk Assessment
- Risk Factors
- Time Factors
- Treatment Outcome
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Affiliation(s)
- Hong-Zheng Ren
- Department of Pathology, Xiangya Basic Medical College, Central South University, Changsha, China
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Zhong WD, Chen QB, Ye YK, Han ZD, Bi XC, Dai QS, Liang YX, Zeng GH, Wang YS, Zhu G, Chen ZN, He HC. Extracellular matrix metalloproteinase inducer expression has an impact on survival in human bladder cancer. Cancer Epidemiol 2010; 34:478-82. [DOI: 10.1016/j.canep.2010.04.007] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2010] [Revised: 04/04/2010] [Accepted: 04/09/2010] [Indexed: 11/15/2022]
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Mathias RA, Chen YS, Wang B, Ji H, Kapp EA, Moritz RL, Zhu HJ, Simpson RJ. Extracellular remodelling during oncogenic Ras-induced epithelial-mesenchymal transition facilitates MDCK cell migration. J Proteome Res 2010; 9:1007-19. [PMID: 19954229 DOI: 10.1021/pr900907g] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Epithelial-mesenchymal transition (EMT) describes a process whereby immotile epithelial cells escape structural constraints imposed by cellular architecture and acquire a phenotype characteristic of migratory mesenchymal cells. Implicated in carcinoma progression and metastasis, EMT has been the focus of several recent proteomics-based studies aimed at identifying new molecular players. To gain insights into extracellular mediators associated with EMT, we conducted an extensive proteomic analysis of the secretome from MDCK cells following oncogenic Ras-induced EMT (21D1 cells). Using Orbitrap technology and a label-free quantitative approach, differential expression of several secreted modulators were revealed. Proteomic findings were further substantiated by mRNA transcript expression analysis with 71% concordance. MDCK cells undergoing Ras-induced EMT remodel the extracellular matrix (ECM) via diminished expression of basement membrane constituents (collagen type IV and laminin 5), up-regulation of extracellular proteases (MMP-1, kallikreins -6 and -7), and increased production and secretion of ECM constituents (SPARC, collagen type I, fibulins -1 and -3, biglycan, and decorin). Collectively, these findings suggest that hierarchical regulation of a subset of extracellular effectors may coordinate a biological response during EMT that enhances cell motility. Transient silencing of MMP-1 in 21D1 cells via siRNA-mediated knockdown attenuated cell migration. Many of the secretome proteins identified broaden our understanding of the EMT process.
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Affiliation(s)
- Rommel A Mathias
- Joint Proteomics Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia
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Novel snail1 target proteins in human colon cancer identified by proteomic analysis. PLoS One 2010; 5:e10221. [PMID: 20421926 PMCID: PMC2857666 DOI: 10.1371/journal.pone.0010221] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2010] [Accepted: 03/26/2010] [Indexed: 11/19/2022] Open
Abstract
Background The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT), a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT. Methodology/Principal Findings We identified by proteomic analysis using 2D-DIGE electrophoresis combined with MALDI-TOF-TOF and ESI-linear ion trap mass spectrometry a number of proteins with variable functions whose expression is modulated by Snail1 in SW480-ADH human colon cancer cells. Validation was performed by Western blot and immunofluorescence analyses. Snail1 repressed several members of the 14-3-3 family of phosphoserine/phosphothreonine binding proteins and also the expression of the Proliferation-associated protein 2G4 (PA2G4) that was mainly localized at the nuclear Cajal bodies. In contrast, the expression of two proteins involved in RNA processing, the Cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and the Splicing factor proline/glutamine-rich (SFPQ), was higher in Snail1-expressing cells than in controls. The regulation of 14-3-3ε, 14-3-3τ, 14-3-3ζ and PA2G4 by Snail1 was reproduced in HT29 colon cancer cells. In addition, we found an inverse correlation between 14-3-3σ and Snail1 expression in human colorectal tumors. Conclusions/Significance We have identified a set of novel Snail1 target proteins in colon cancer that expand the cellular processes affected by Snail1 and thus its relevance for cell function and phenotype.
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Moreira JMA, Ohlsson G, Gromov P, Simon R, Sauter G, Celis JE, Gromova I. Bladder cancer-associated protein, a potential prognostic biomarker in human bladder cancer. Mol Cell Proteomics 2009; 9:161-77. [PMID: 19783793 DOI: 10.1074/mcp.m900294-mcp200] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
It is becoming increasingly clear that no single marker will have the sensitivity and specificity necessary to be used on its own for diagnosis/prognosis of tumors. Interpatient and intratumor heterogeneity provides overwhelming odds against the existence of such an ideal marker. With this in mind, our laboratory has been applying a long term systematic approach to identify multiple biomarkers that can be used for clinical purposes. As a result of these studies, we have identified and reported several candidate biomarker proteins that are deregulated in bladder cancer. Following the conceptual biomarker development phases proposed by the Early Detection Research Network, we have taken some of the most promising candidate proteins into postdiscovery validation studies, and here we report on the characterization of one such biomarker, the bladder cancer-associated protein (BLCAP), formerly termed Bc10. To characterize BLCAP protein expression and cellular localization patterns in benign bladder urothelium and urothelial carcinomas (UCs), we used two independent sets of samples from different patient cohorts: a reference set consisting of 120 bladder specimens (formalin-fixed as well as frozen biopsies) and a validation set consisting of 2,108 retrospectively collected UCs with long term clinical follow-up. We could categorize the UCs examined into four groups based on levels of expression and subcellular localization of BLCAP protein and showed that loss of BLCAP expression is associated with tumor progression. The results indicated that increased expression of this protein confers an adverse patient outcome, suggesting that categorization of staining patterns for this protein may have prognostic value. Finally, we applied a combinatorial two-marker discriminator using BLCAP and adipocyte-type fatty acid-binding protein, another UC biomarker previously reported by us, and found that the combination of the two markers correlated more closely with grade and/or stage of disease than the individual markers. The implications of these results in biomarker discovery are discussed.
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Affiliation(s)
- José M A Moreira
- Department of Proteomics in Cancer, Institute of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen Ø, Denmark.
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Towards understanding epithelial–mesenchymal transition: A proteomics perspective. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2009; 1794:1325-31. [DOI: 10.1016/j.bbapap.2009.05.001] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/17/2009] [Accepted: 05/05/2009] [Indexed: 11/19/2022]
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Markers of epithelial-mesenchymal transition and epithelial differentiation in sarcomatoid carcinoma: utility in the differential diagnosis with sarcoma. Appl Immunohistochem Mol Morphol 2009; 16:251-62. [PMID: 18301244 DOI: 10.1097/pai.0b013e318156e9b4] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The distinction between sarcomatoid carcinoma (SC) and bona fide sarcoma can be difficult using conventional immunohistochemical markers. Epithelial-mesenchymal transition (EMT) has been proposed as a histogenetic mechanism for the development of SC. Expression of selected markers of EMT (Twist and Slug) was compared with other markers of epithelial differentiation in SC and spindle cell sarcoma to determine the utility of these antigens in this differential diagnosis. Twenty-seven cases of SC (excluding those of gynecologic origin) were stained by immunohistochemistry for cytokeratins (AE1/AE3, 5D3, CK5/6, and 34betaE12), p63, claudin-1, claudin-7, epithelial cadherin, placental cadherin, epithelial cell adhesion molecule/epithelial-specific antigen, 14-3-3sigma, Twist, and Slug. A comparison group of 21 spindle or pleomorphic spindle cell sarcomas was also studied. Immunohistochemical stains were scored in a semiquantitative manner and subsequent exploratory analyses were performed using logistic regression and chi2 tests. Only cytokeratin AE1/AE3 specifically labeled SC in a statistically significant manner. Other epithelial-specific markers tested did not distinguish SC from sarcoma primarily owing to low sensitivity. However, when positive, immunostains such as CK5/6, membranous epithelial cadherin, and nuclear p63 may aid in the distinction of SC from sarcoma. EMT markers were expressed in most cases of both SC and sarcoma, and were not useful in making a differential diagnosis between these neoplasms.
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Li J, Gromov P, Gromova I, Moreira JM, Timmermans-Wielenga V, Rank F, Wang K, Li S, Li H, Wiuf C, Yang H, Zhang X, Bolund L, Celis JE. Omics-based profiling of carcinoma of the breast and matched regional lymph node metastasis. Proteomics 2009; 8:5038-52. [PMID: 19003862 DOI: 10.1002/pmic.200800303] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Axillary lymph node (ALN) status is currently used as an important clinical indicator of breast cancer prognosis. However, the molecular mechanisms underlying lymph node metastasis are poorly understood and the relationship between ALN metastasis and the primary tumor remains unclear. In an effort to reveal structural changes in the genome and related protein responses that may drive regional metastatic progression we have analyzed matched pairs of primary breast tumors and ALN metastases both at the genomic and proteomic levels using comparative genomic hybridization (CGH) array, quantitative high-resolution 2-D PAGE in combination with MS, and immunohistochemistry (IHC). Array CGH revealed a remarkable similarity in genomic aberration profiles between the matched primary tumors and the ALN metastases. Quantitative profiling of 135 known proteins also revealed striking similarities in their overall expression patterns, although we observed distinct changes in the levels of individual proteins in some sample pairs. The remarkable similarities of the overall genomic and proteomic profiles between primary tumors and matched ALN metastases are taken to suggest that, in general, key biological characteristics of the primary breast tumor are maintained in the corresponding lymph node metastases. Given that the omics-based technologies are oblivious to changes that only occur in minor cellular subsets we validated the proteomic data using IHC, which provides protein expression information with a valuable topological component. Besides confirming the omics-derived data, the IHC analysis revealed that in two cases the ALN metastases may have been derived from a distinct minor cell subpopulation present in the primary tumor rather than from the bulk of it.
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Affiliation(s)
- Jian Li
- Institute of Human Genetics, University of Aarhus, Aarhus C, Denmark.
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Hill JJ, Tremblay TL, Cantin C, O'Connor-McCourt M, Kelly JF, Lenferink AEG. Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-beta induced epithelial to mesenchymal transition. Proteome Sci 2009; 7:2. [PMID: 19128513 PMCID: PMC2651118 DOI: 10.1186/1477-5956-7-2] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2008] [Accepted: 01/08/2009] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND TGF-beta acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-beta can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-beta in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01. RESULTS Preliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin). CONCLUSION Interestingly, despite the fact that TGF-beta induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.
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Affiliation(s)
- Jennifer J Hill
- Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada.
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Sun B, Zhang S, Zhang D, Li Y, Zhao X, Luo Y, Guo Y. Identification of Metastasis-Related Proteins and Their Clinical Relevance to Triple-Negative Human Breast Cancer. Clin Cancer Res 2008; 14:7050-9. [PMID: 18981002 DOI: 10.1158/1078-0432.ccr-08-0520] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Affiliation(s)
- Baocun Sun
- Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin, People's Republic of China
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42
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Lv J, Zhu X, Dong K, Lin Y, Hu Y, Zhu C. Reduced expression of 14-3-3 gamma in uterine leiomyoma as identified by proteomics. Fertil Steril 2008; 90:1892-8. [DOI: 10.1016/j.fertnstert.2007.08.039] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2007] [Revised: 08/14/2007] [Accepted: 08/14/2007] [Indexed: 11/28/2022]
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Wang Z, Tropè CG, Suo Z, Trøen G, Yang G, Nesland JM, Holm R. The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas. BMC Cancer 2008; 8:308. [PMID: 18950492 PMCID: PMC2577690 DOI: 10.1186/1471-2407-8-308] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2008] [Accepted: 10/24/2008] [Indexed: 11/13/2022] Open
Abstract
Background 14-3-3 sigma (σ) promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown. Methods We investigated the 14-3-3σ expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome. Results In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3σ protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3σ in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (p = 0.001, p = 0.002 and p = 0.001, respectively) and deep invasion (p = 0.01, p = 0.001 and p = 0.007, respectively). Variations of 14-3-3σ protein expression were not associated to disease-specific survival. Conclusion Our results indicate that 14-3-3σ may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3σ protein. Neither cytoplasmic nor nuclear level of 14-3-3σ expression was associated with prognosis.
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Affiliation(s)
- Zhihui Wang
- Division of Pathology, The Norwegian Radium Hospital, Rikshospitalet University Hospital, Oslo, Norway.
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Moreira JMA, Shen T, Ohlsson G, Gromov P, Gromova I, Celis JE. A combined proteome and ultrastructural localization analysis of 14-3-3 proteins in transformed human amnion (AMA) cells: definition of a framework to study isoform-specific differences. Mol Cell Proteomics 2008; 7:1225-1240. [PMID: 18378962 DOI: 10.1074/mcp.m700439-mcp200] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/26/2024] Open
Abstract
The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 beta, epsilon, sigma, tau, and zeta protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3 proteins.
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Affiliation(s)
- José M A Moreira
- Department of Proteomics in Cancer, Institute of Cancer Biology and Danish Centre for Translational Breast Cancer Research (DCTB), Danish Cancer Society, DK-2100 Copenhagen, Denmark
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Yuan C, Jiao L, Yang L, Ying W, Hu Z, Liu J, Cui F, Li L, Qian L, Teng Y, Hang H, Qian X, Yang X. The up-regulation of 14-3-3 proteins in Smad4 deficient epidermis and hair follicles at catagen. Proteomics 2008; 8:2230-43. [DOI: 10.1002/pmic.200700760] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
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Wu TF, Ku WL, Tsay YG. Proteome-based diagnostics and prognosis of bladder transitional cell carcinoma. Expert Rev Proteomics 2007; 4:639-47. [PMID: 17941819 DOI: 10.1586/14789450.4.5.639] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
More than 90% of bladder tumors are diagnosed as bladder transitional cell carcinoma and the majority of these lesions (70%) are diagnosed as superficial papillary lesions (stage pTa, T1). Recurrences are common to superficial tumors and few lesions will progress to a higher grade and/or stage and muscle invasion. Thus, diagnosing cancer at an early stage, predicting whether a tumor will recur and/or progress and identification of novel targets for cancer intervention, become the main focus of bladder cancer research. The purpose of this article is to briefly review what has been accomplished to date by using proteomic technology in order to develop a new strategy to resolve the problems of early detection, recurrence or therapeutic intervention.
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Affiliation(s)
- Ting-Feng Wu
- Southern Taiwan University, Department of Biotechnology, Tainan, Taiwan.
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Medina A, Ghaffari A, Kilani RT, Ghahary A. The role of stratifin in fibroblast-keratinocyte interaction. Mol Cell Biochem 2007; 305:255-64. [PMID: 17646930 DOI: 10.1007/s11010-007-9538-y] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2007] [Accepted: 06/21/2007] [Indexed: 10/23/2022]
Abstract
Stratifin is a member of 14-3-3 protein family, a highly conserved group of proteins constituted by seven isoforms. They are involved in numerous crucial intracellular functions such as cell cycle and apoptosis, regulation of signal transduction pathways, cellular trafficking, cell proliferation and differentiation, cell survival, and protein folding and processing, among others. At epidermal level, stratifin (also called 14-3-3 sigma) has been described as molecule with relevant functions. For instance, this isoform is a marker associated with keratinocyte differentiation. In this maturation process, the presence of dominant negative molecules of p53 induces a "stemness condition" of keratinocyte precursor cells and suppression of stratifin expression. In addition, the recently described keratinocyte-releasable form of stratifin is involved in dermal fibroblast MMP-1 over-expression through c-Fos and c-Jun activity. This effect is mediated, at least in part, by p38 mitogen-activated protein kinase (MAPK). Other MMP family members such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), neutrophil collagenase (MMP-8), and membrane-type MMP-24 (MT5-MMP) are also up-regulated by stratifin. Within fibroproliferative disorder of skin, hypertrophic scar and keloids exhibit a high content of collagen, proteoglycans, and fibronectin. Thus, the MMP profile induced by stratifin is an interesting starting point to establish new therapeutic tools to control the process of wound healing. In this review, we will focus on site of synthesis and mode of action of stratifin in skin and wound healing.
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Affiliation(s)
- Abelardo Medina
- BC Professional Fire Fighters' Burn and Wound Healing Laboratory, Division of Plastic Surgery, University of British Columbia, Vancouver, BC, Canada
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48
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Goc A, Abdalla M, Al-Azayzih A, Somanath PR. Tuning cell cycle regulation with an iron key. PLoS One 2007; 7:e40594. [PMID: 22808202 PMCID: PMC3396618 DOI: 10.1371/journal.pone.0040594] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2012] [Accepted: 06/11/2012] [Indexed: 01/20/2023] Open
Abstract
14-3-3 proteins are ubiquitously expressed dimeric adaptor proteins that have emerged as key mediators of many cell signaling pathways in multiple cell types. Its effects are mainly mediated by binding to selective phosphoserine/threonine proteins. The importance of 14-3-3 proteins in cancer have only started to become apparent and its exact role in cancer progression as well as the mechanisms by which 14-3-3 proteins mediate cancer cell function remain unknown. While protein 14-3-3σ is widely accepted as a tumor suppressor, 14-3-3ζ, β and γ isoforms have been shown to have tumor promoting effects. Despite the importance of 14-3-3 family in mediating various cell processes, the exact role and mechanism of 14-3-3ζ remain unexplored. In the current study, we investigated the role of protein 14-3-3ζ in prostate cancer cell motility and transendothelial migration using biochemical, molecular biology and electric cell-substrate impedance sensing approaches as well as cell based functional assays. Our study indicated that expression with wild-type protein 14-3-3ζ significantly enhanced Rac activity in PC3 cells. In contrast, expression of dimer-resistant mutant of protein 14-3-3ζ (DM-14-3-3) inhibited Rac activity and associated phosphorylation of p21 activated kinase-1 and 2. Expression with wild-type 14-3-3ζ or constitutively active Rac1 enhanced extracellular matrix recognition, lamellipodia formation, cell migration and trans-endothelial migration by PC3 cells. In contrast, expression with DM 14-3-3ζ or DN-Rac1 in PC3 cells significantly inhibited these cell functions. Our results demonstrate for the first time that 14-3-3ζ enhances prostate cancer cell-matrix interactions, motility and transendothelial migration in vitro via activation of Rac1-GTPase and is an important target for therapeutic interventions for prostate cancer.
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Affiliation(s)
- Anna Goc
- Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, Georgia, United States of America
- Charlie Norwood VA Medical Center, Augusta, Georgia, United States of America
| | - Maha Abdalla
- Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, Georgia, United States of America
- Charlie Norwood VA Medical Center, Augusta, Georgia, United States of America
| | - Ahmad Al-Azayzih
- Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, Georgia, United States of America
- Charlie Norwood VA Medical Center, Augusta, Georgia, United States of America
| | - Payaningal R. Somanath
- Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, Georgia, United States of America
- Charlie Norwood VA Medical Center, Augusta, Georgia, United States of America
- Department of Medicine, Georgia Health Sciences University, Augusta, Georgia, United States of America
- * E-mail:
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Saldanha RG, Molloy MP, Bdeir K, Cines DB, Song X, Uitto PM, Weinreb PH, Violette SM, Baker MS. Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy. J Proteome Res 2007; 6:1016-28. [PMID: 17330942 DOI: 10.1021/pr060518n] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Urokinase plasminogen activator (uPA) and its high affinity receptor (uPAR) play crucial proteolytic and non-proteolytic roles in cancer metastasis. In addition to promoting plasmin-mediated degradation of extracellular matrix barriers, cell surface engagement of uPA through uPAR binding results in the activation of a suite of diverse cellular signal transduction pathways. Because uPAR is bound to the plasma membrane through a glycosyl-phosphatidylinositol anchor, these signalling sequelae are thought to occur through the formation of multi-protein cell surface complexes involving uPAR. To further characterize uPAR-driven protein complexes, we co-immunoprecipitated uPAR from the human ovarian cancer cell line, OVCA 429, and employed sensitive proteomic methods to identify the uPAR-associated proteins. Using this strategy, we identified several known, as well as numerous novel, uPAR associating proteins, including the epithelial restricted integrin, alphavbeta6. Reverse immunoprecipitation using anti-beta6 integrin subunit monoclonal antibodies confirmed the co-purification of this protein with uPAR. Inhibition of uPAR and/or beta6 integrin subunit using neutralizing antibodies resulted in the inhibition of uPA-mediated ERK 1/2 phosphorylation and subsequent cell proliferation. These data suggest that the association of beta6 integrin (and possibly other lynchpin cancer regulatory proteins) with uPAR may be crucial in co-transmitting uPA signals that induce cell proliferation. Our findings support the notion that uPAR behaves as a lynchpin in promoting tumorigenesis by forming functionally active multiprotein complexes.
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Affiliation(s)
- Rohit G Saldanha
- Australian Proteome Analysis Facility Ltd and Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, 2109, NSW Australia
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Wilker EW, van Vugt MATM, Artim SA, Huang PH, Petersen CP, Reinhardt HC, Feng Y, Sharp PA, Sonenberg N, White FM, Yaffe MB. 14-3-3sigma controls mitotic translation to facilitate cytokinesis. Nature 2007; 446:329-32. [PMID: 17361185 DOI: 10.1038/nature05584] [Citation(s) in RCA: 168] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2006] [Accepted: 01/09/2007] [Indexed: 02/06/2023]
Abstract
14-3-3 proteins are crucial in a wide variety of cellular responses including cell cycle progression, DNA damage checkpoints and apoptosis. One particular 14-3-3 isoform, sigma, is a p53-responsive gene, the function of which is frequently lost in human tumours, including breast and prostate cancers as a result of either hypermethylation of the 14-3-3sigma promoter or induction of an oestrogen-responsive ubiquitin ligase that specifically targets 14-3-3sigma for proteasomal degradation. Loss of 14-3-3sigma protein occurs not only within the tumours themselves but also in the surrounding pre-dysplastic tissue (so-called field cancerization), indicating that 14-3-3sigma might have an important tumour suppressor function that becomes lost early in the process of tumour evolution. The molecular basis for the tumour suppressor function of 14-3-3sigma is unknown. Here we report a previously unknown function for 14-3-3sigma as a regulator of mitotic translation through its direct mitosis-specific binding to a variety of translation/initiation factors, including eukaryotic initiation factor 4B in a stoichiometric manner. Cells lacking 14-3-3sigma, in marked contrast to normal cells, cannot suppress cap-dependent translation and do not stimulate cap-independent translation during and immediately after mitosis. This defective switch in the mechanism of translation results in reduced mitotic-specific expression of the endogenous internal ribosomal entry site (IRES)-dependent form of the cyclin-dependent kinase Cdk11 (p58 PITSLRE), leading to impaired cytokinesis, loss of Polo-like kinase-1 at the midbody, and the accumulation of binucleate cells. The aberrant mitotic phenotype of 14-3-3sigma-depleted cells can be rescued by forced expression of p58 PITSLRE or by extinguishing cap-dependent translation and increasing cap-independent translation during mitosis by using rapamycin. Our findings show how aberrant mitotic translation in the absence of 14-3-3sigma impairs mitotic exit to generate binucleate cells and provides a potential explanation of how 14-3-3sigma-deficient cells may progress on the path to aneuploidy and tumorigenesis.
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Affiliation(s)
- Erik W Wilker
- Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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