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Mossenta M, Busato D, Dal Bo M, Macor P, Toffoli G. Novel Nanotechnology Approaches to Overcome Drug Resistance in the Treatment of Hepatocellular Carcinoma: Glypican 3 as a Useful Target for Innovative Therapies. Int J Mol Sci 2022; 23:10038. [PMID: 36077433 PMCID: PMC9456072 DOI: 10.3390/ijms231710038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Revised: 08/30/2022] [Accepted: 08/30/2022] [Indexed: 11/23/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is the second most lethal tumor, with a 5-year survival rate of 18%. Early stage HCC is potentially treatable by therapies with curative intent, whereas chemoembolization/radioembolization and systemic therapies are the only therapeutic options for intermediate or advanced HCC. Drug resistance is a critical obstacle in the treatment of HCC that could be overcome by the use of targeted nanoparticle-based therapies directed towards specific tumor-associated antigens (TAAs) to improve drug delivery. Glypican 3 (GPC3) is a member of the glypican family, heparan sulfate proteoglycans bound to the cell surface via a glycosylphosphatidylinositol anchor. The high levels of GPC3 detected in HCC and the absence or very low levels in normal and non-malignant liver make GPC3 a promising TAA candidate for targeted nanoparticle-based therapies. The use of nanoparticles conjugated with anti-GPC3 agents may improve drug delivery, leading to a reduction in severe side effects caused by chemotherapy and increased drug release at the tumor site. In this review, we describe the main clinical features of HCC and the common treatment approaches. We propose the proteoglycan GPC3 as a useful TAA for targeted therapies. Finally, we describe nanotechnology approaches for anti-GPC3 drug delivery systems based on NPs for HCC treatment.
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Affiliation(s)
- Monica Mossenta
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, 33081 Aviano, Italy
- Department of Life Sciences, University of Trieste, 34127 Trieste, Italy
| | - Davide Busato
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, 33081 Aviano, Italy
- Department of Life Sciences, University of Trieste, 34127 Trieste, Italy
| | - Michele Dal Bo
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, 33081 Aviano, Italy
| | - Paolo Macor
- Department of Life Sciences, University of Trieste, 34127 Trieste, Italy
| | - Giuseppe Toffoli
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, 33081 Aviano, Italy
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2
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Kamimura K, Maeda N. Glypicans and Heparan Sulfate in Synaptic Development, Neural Plasticity, and Neurological Disorders. Front Neural Circuits 2021; 15:595596. [PMID: 33679334 PMCID: PMC7928303 DOI: 10.3389/fncir.2021.595596] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Accepted: 01/11/2021] [Indexed: 12/16/2022] Open
Abstract
Heparan sulfate proteoglycans (HSPGs) are components of the cell surface and extracellular matrix, which bear long polysaccharides called heparan sulfate (HS) attached to the core proteins. HSPGs interact with a variety of ligand proteins through the HS chains, and mutations in HSPG-related genes influence many biological processes and cause various diseases. In particular, recent findings from vertebrate and invertebrate studies have raised the importance of glycosylphosphatidylinositol-anchored HSPGs, glypicans, as central players in the development and functions of synapses. Glypicans are important components of the synapse-organizing protein complexes and serve as ligands for leucine-rich repeat transmembrane neuronal proteins (LRRTMs), leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (RPTPs), and G-protein-coupled receptor 158 (GPR158), regulating synapse formation. Many of these interactions are mediated by the HS chains of glypicans. Neurexins (Nrxs) are also synthesized as HSPGs and bind to some ligands in common with glypicans through HS chains. Therefore, glypicans and Nrxs may act competitively at the synapses. Furthermore, glypicans regulate the postsynaptic expression levels of ionotropic glutamate receptors, controlling the electrophysiological properties and non-canonical BMP signaling of synapses. Dysfunctions of glypicans lead to failures in neuronal network formation, malfunction of synapses, and abnormal behaviors that are characteristic of neurodevelopmental disorders. Recent human genetics revealed that glypicans and HS are associated with autism spectrum disorder, neuroticism, and schizophrenia. In this review, we introduce the studies showing the roles of glypicans and HS in synapse formation, neural plasticity, and neurological disorders, especially focusing on the mouse and Drosophila as potential models for human diseases.
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Affiliation(s)
- Keisuke Kamimura
- Developmental Neuroscience Project, Department of Brain and Neurosciences, Tokyo Metropolitan Institute of Medical Science, Setagaya, Japan
| | - Nobuaki Maeda
- Developmental Neuroscience Project, Department of Brain and Neurosciences, Tokyo Metropolitan Institute of Medical Science, Setagaya, Japan
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3
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Gu D, Xie Y, Wei J, Li W, Ye Z, Zhu Z, Tian J, Li X. MRI-Based Radiomics Signature: A Potential Biomarker for Identifying Glypican 3-Positive Hepatocellular Carcinoma. J Magn Reson Imaging 2020; 52:1679-1687. [PMID: 32491239 DOI: 10.1002/jmri.27199] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2020] [Revised: 05/03/2020] [Accepted: 05/05/2020] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Glypican 3 (GPC3) expression has proved to be a critical risk factor related to prognosis in hepatocellular carcinoma (HCC) patients. PURPOSE To investigate the performance of MRI-based radiomics signature in identifying GPC3-positive HCC. STUDY TYPE Retrospective. POPULATION An initial cohort of 293 patients with pathologically confirmed HCC was involved in this study, and patients were randomly divided into training (195) and validation (98) cohorts. FIELD STRENGTH/SEQUENCES Contrast-enhanced T1 -weight MRI was performed with a 1.5T scanner. ASSESSMENT A total of 853 radiomic features were extracted from the volume imaging. Univariate analysis and Fisher scoring were utilized for feature reduction. Subsequently, forward stepwise feature selection and radiomics signature building were performed based on a support vector machine (SVM). Incorporating independent risk factors, a combined nomogram was developed by multivariable logistic regression modeling. STATISTICAL TESTS The predictive performance of the nomogram was calculated using the area under the receive operating characteristic curve (AUC). Decision curve analysis (DCA) was applied to estimate the clinical usefulness. RESULTS The radiomics signature consisting of 10 selected features achieved good prediction efficacy (training cohort: AUC = 0.879, validation cohort: AUC = 0.871). Additionally, the combined nomogram integrating independent clinical risk factor α-fetoprotein (AFP) and radiomics signature showed improved calibration and prominent predictive performance with AUCs of 0.926 and 0.914 in the training and validation cohorts, respectively. DATA CONCLUSION The proposed MR-based radiomics signature is strongly related to GPC3-positive. The combined nomogram incorporating AFP and radiomics signature may provide an effective tool for noninvasive and individualized prediction of GPC3-positive in patients with HCC. J. MAGN. RESON. IMAGING 2020;52:1679-1687.
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Affiliation(s)
- Dongsheng Gu
- Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing, China
| | - Yongsheng Xie
- Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
| | - Jingwei Wei
- Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing, China
| | - Wencui Li
- Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
| | - Zhaoxiang Ye
- Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
| | - Zhongyuan Zhu
- Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
| | - Jie Tian
- Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China.,Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, School of Medicine, Beihang University, Beijing, China.,Engineering Research Center of Molecular and Neuro Imaging of Ministry of Education, School of Life Science and Technology, Xidian University, Xi'an, China
| | - Xubin Li
- Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
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Miyoshi E, Kamada Y, Suzuki T. Functional glycomics: Application to medical science and hepatology. Hepatol Res 2020; 50:153-164. [PMID: 31750967 DOI: 10.1111/hepr.13459] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/18/2019] [Revised: 10/20/2019] [Accepted: 10/29/2019] [Indexed: 02/08/2023]
Abstract
Glycomics refers to the comprehensive analysis of glycans. Recent progress in glycotechnology enables the determination of a variety of biological functions of glycans. Among different glycosylation patterns, certain types of aberrant glycosylation are linked to cancer and/or inflammation, and thus have biological importance. Glycotechnology has been applied to many fields of medical science, including hepatology. In particular, dramatic changes in glycosylation are observed in the progression of liver diseases. As the liver produces so many serum glycoproteins, changes in glycosylation of these proteins might provide useful disease biomarkers. Furthermore, many patients with genetic diseases of glycosylation who have liver dysfunction have been found as a result from whole genome sequencing, and various kinds of glycotherapy have been developed, especially in immunotherapy. In this review, we describe our basic knowledge of glycobiology and discuss the application of these data to medical science, especially hepatology.
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Affiliation(s)
- Eiji Miyoshi
- Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
| | - Yoshihiro Kamada
- Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
| | - Tadashi Suzuki
- Glycometabolic Biochemistry Laboratory, RIKEN Cluster for Pioneering Research (CPR), Wako, Saitama, Japan
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Nishida T, Kataoka H. Glypican 3-Targeted Therapy in Hepatocellular Carcinoma. Cancers (Basel) 2019; 11:E1339. [PMID: 31510063 PMCID: PMC6770328 DOI: 10.3390/cancers11091339] [Citation(s) in RCA: 79] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Revised: 09/03/2019] [Accepted: 09/07/2019] [Indexed: 02/08/2023] Open
Abstract
Glypican-3 (GPC3) is an oncofetal glycoprotein attached to the cell membrane by a glycophosphatidylinositol anchor. GPC3 is overexpressed in some kinds of tumors, particularly hepatocellular carcinoma (HCC). The prognostic significance of serum GPC3 levels and GPC3 immunoreactivity in tumor cells has been defined in patients with HCC. In addition to its usefulness as a biomarker, GPC3 has attracted attention as a novel therapeutic target molecule, and clinical trials targeting GPC3 are in progress. The major mechanism of anti-GPC3 antibody (GPC3Ab) against cancer cells is antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity. Since GPC3Ab is associated with immune responses, a combination of protocols with immune checkpoint inhibitors has also been investigated. Moreover, some innovative approaches for GPC3-targeting therapy have emerged in recent years. This review introduces the results of recent clinical trials targeting GPC3 in HCC and summarizes the latest knowledge regarding the role of GPC3 in HCC progression and clinical application targeting GPC3.
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Affiliation(s)
- Takahiro Nishida
- Section of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
- Division of Gastrointestinal, Endocrine and Pediatric Surgery, Department of Surgery, University of Miyazaki Faculty of Medicine, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
| | - Hiroaki Kataoka
- Section of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
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6
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Zhang Y, Qiu D, Li R, Liu Y, Shi S, Wang Y. Preparation of a monoclonal antibody against the carcinoembryonic antigen, glypican‑3. Mol Med Rep 2019; 19:3889-3895. [PMID: 30896845 DOI: 10.3892/mmr.2019.10019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2018] [Accepted: 01/17/2019] [Indexed: 11/05/2022] Open
Abstract
The carcinoembryonic antigen, glypican‑3 (GPC3), is a putative therapeutic target and diagnostic marker of hepatoma. In the present study, a monoclonal antibody (mAb) specifically against GPC3 was obtained via cloning the sequence of GPC3 via polymerase chain reaction and inserting it into a pET16b vector prior to transfection into Escherichia coli (E. coli) BL21. BALB/c mice were immunized with 20 µg purified antigen by intrasplenic embedding. Splenocytes and mouse myeloma cells SP2/0 were fused; then, the hybridoma cells were screened by an indirect ELISA. The properties of the mAb were examined by western blotting and immunofluorescence analysis against the purified protein. The results revealed that the prokaryotic expression vector of GPC3 had been successfully generated and GPC3 was stably expressed in E. coli BL21. A stable hybridoma cell line, 2F3, was generated in the present study, which produced mAbs against GPC3. The mAb 2F3 had a high antibody titer and the isotype was identified as IgG1/κ; 2F3 hybridomas had a median chromosome number of 98. Western blot and immunofluorescence analyses revealed that 2F3 specifically recognized recombinant and native GPC3. The 2F3 clone was proposed as a stable secretor of this mAb against GPC3. The results of present study indicated that the successful preparation of recombinant GPC3 protein and an anti‑human GPC3 mouse mAb may be provide a basis for developments in the diagnosis and treatment of liver cancer.
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Affiliation(s)
- Yongdong Zhang
- Department of Stomatology, Tianjin First Central Hospital, Tianjin 300192, P.R. China
| | - Dongri Qiu
- Department of Stomatology, Tianjin First Central Hospital, Tianjin 300192, P.R. China
| | - Ronghua Li
- Department of Stomatology, Tianjin First Central Hospital, Tianjin 300192, P.R. China
| | - Yawu Liu
- Department of Clinical Radiology, Kuopio University Hospital, FIN‑70210 Kuopio, Finland
| | - Shuainan Shi
- Department of Clinical Laboratory Medicine, The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin 300211, P.R. China
| | - Yuliang Wang
- Department of Clinical Laboratory Medicine, The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin 300211, P.R. China
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Wang S, Kalim M, Liang K, Zhan J. Polyclonal antibody production against rGPC3 and their application in diagnosis of hepatocellular carcinoma. Prep Biochem Biotechnol 2018; 48:435-445. [PMID: 29561231 DOI: 10.1080/10826068.2018.1452258] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Glypican-3 (GPC3) is an integral membrane proteoglycan, which contains a core protein anchored to the cytoplasmic membrane through a glycosylphosphatidylinositol linkage. The glypican-3 can regulate the signaling pathways, thereby enhances cell division, growth, and apoptosis in certain cell types. It is almost nonexistent on the surface of the human normal cell membrane and highly expresses on the membrane of hepatocellular carcinoma (HCC) cells. It has been well established that GPC3 provides a useful diagnostic marker. For generating the polyclonal antibody of GPC3, we expected that GPC3 N-terminal region (amino acid sequence 26-358) could be expressed in Escherichia coli system, however, no active expression was observed after IPTG induction. Interestingly, after deletion of six proline residues from position 26 to 31 in the N-terminus, expression of recombinant GPC3 was clearly detected. We further analyzed the expressed protein deprived of six prolines, to immunize the New Zealand male rabbits for production of active antibodies. The binding affinity of antibody was analyzed by immunofluorescence analysis, immunohistochemical detection, and western blotting. The functional GPC3 N-terminal protein recombinant development, expression, purification, and the polyclonal antibody have been generated provide the basis for the diagnosis of HCC in cancer therapy.
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Affiliation(s)
- Shenghao Wang
- a Department of Biochemistry and Genetics , Zhejiang University School of Medicine , Hangzhou , China
| | - Muhammad Kalim
- a Department of Biochemistry and Genetics , Zhejiang University School of Medicine , Hangzhou , China
| | | | - Jinbiao Zhan
- a Department of Biochemistry and Genetics , Zhejiang University School of Medicine , Hangzhou , China
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Kaseb AO, Hassan M, Lacin S, Abdel-Wahab R, Amin HM, Shalaby A, Wolff RA, Yao J, Rashid A, Vennapusa B, Feng J, Ohtomo T. Evaluating clinical and prognostic implications of Glypican-3 in hepatocellular carcinoma. Oncotarget 2018; 7:69916-69926. [PMID: 27655712 PMCID: PMC5342524 DOI: 10.18632/oncotarget.12066] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2016] [Accepted: 09/05/2016] [Indexed: 12/21/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide. In patients with HCC, histopathogical differentiation is an important indicator of prognosis; however, because determination of HCC differentiation is difficult, the recently described immunohistochemical (IHC) marker glypican3 (GPC3) might assist in HCC prognostication.The goal of our study was to investigate GPC3's IHC staining pattern and define the relationship between its expression and patients' clinicopathologic features and overall survival. We retrieved clinical parameters from 101 pathologically diagnosed HCC patients' medical records and classified these patients into 4 clinical score categories (0–3) based on increasing GPC3 staining intensity and the percentage of stained tumor cells in their resection and biopsy specimens. Histopathological samples were well, moderately, and poorly differentiated in 33, 22, and 12 patients, respectively, and the GPC3 expression rate was 63%, 86%, and 92%,respectively. The median overall survival was 49.9 months (confidence interval (CI): 35.3–64.6 months) for clinical scores 0–1 and 30.7 months (CI: 19.4–41.9 months) for clinical scores 2–3. This difference was not statistically significant (P = .06) but showed a strong trend. In conclusion, a greater GPC3 expression is associated with a worse HCC prognosis and may be a promising prognostic marker.
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Affiliation(s)
- Ahmed Omar Kaseb
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Manal Hassan
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Sahin Lacin
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.,Department of Medical Oncology, Hacettepe University, Medical Faculty, Ankara, Turkey
| | - Reham Abdel-Wahab
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.,Department of Clinical Oncology, Assiut University Hospital, Assiut, Egypt
| | - Hesham M Amin
- Division of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Ahmed Shalaby
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Robert A Wolff
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - James Yao
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Asif Rashid
- Division of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | | | - Janine Feng
- Ventana Medical Systems, Inc., Tucson, Arizona, USA
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Gastric Cancer With Primitive Enterocyte Phenotype: An Aggressive Subgroup of Intestinal-type Adenocarcinoma. Am J Surg Pathol 2017; 41:989-997. [PMID: 28505005 DOI: 10.1097/pas.0000000000000869] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
A primitive cell-like gene expression signature is associated with aggressive phenotypes of various cancers. We assessed the expression of phenotypic markers characterizing primitive cells and its correlation with clinicopathologic and molecular characteristics in gastric cancer. Immunohistochemical analysis of a panel of primitive phenotypic markers, including embryonic stem cell markers (OCT4, NANOG, SALL4, CLDN6, and LIN28) and known oncofetal proteins (AFP and GPC3), was performed using tissue microarray on 386 gastric cancers. On the basis of the expression profiles, the 386 tumors were clustered into 3 groups: group 1 (primitive phenotype, n=93): AFP, CLDN6, GPC3, or diffuse SALL4 positive; group 2 (SALL4-focal, n=56): only focal SALL4 positive; and group 3 (negative, n=237): all markers negative. Groups 1 and 2 predominantly consisted of intestinal-type adenocarcinoma, including 13 fetal gut-like adenocarcinomas exclusively in group 1. Group 1 was significantly associated with higher T-stage, presence of vascular invasion and nodal metastasis when compared with groups 2 and 3. Group 1 was associated with patients' poor prognosis and was an independent risk factor for disease-free survival. Group 1 showed frequent TP53 overexpression and little association with Epstein-Barr virus or mismatch repair deficiency. Further analysis of the Cancer Genome Atlas data set validated our observations and revealed that tumors with primitive phenotypes were mostly classified as "chromosomal instability" in the Cancer Genome Atlas' molecular classification. We identified gastric cancer with primitive enterocyte phenotypes as an aggressive subgroup of intestinal-type/chromosomal instability gastric cancer. Therapeutic strategies targeting primitive markers, such as GPC3, CLDN6, and SALL4, are highly promising.
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Chen C, Huang X, Ying Z, Wu D, Yu Y, Wang X, Chen C. Can glypican-3 be a disease-specific biomarker? Clin Transl Med 2017; 6:18. [PMID: 28510121 PMCID: PMC5433957 DOI: 10.1186/s40169-017-0146-5] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2016] [Accepted: 03/30/2017] [Indexed: 12/12/2022] Open
Abstract
Background Glypican-3 (GPC3) is a cell surface-bound proteoglycan which has been identified as a potential biomarker candidate in hepatocellular carcinoma, lung carcinoma, severe pneumonia, and acute respiratory distress syndrome (ARDS). The aim of our review is to evaluate whether GPC3 has utility as a disease-specific biomarker, to discuss the potential involvement of GPC3 in cell biology, and to consider the changes of GPC3 gene and protein expression and regulation in hepatocellular carcinoma, lung cancer, severe pneumonia, and ARDS. Results Immunohistochemical studies have suggested that over-expression of GPC3 is associated with a poorer prognosis for hepatocellular carcinoma patients. Expression of GPC3 leads to an increased apoptosis response in human lung carcinoma tumor cells, and is considered to be a candidate lung tumor suppressor gene. Increased serum levels of GPC3 have been demonstrated in ARDS patients with severe pneumonia. Conclusions Glypican-3 could be considered as a clinically useful biomarker in hepatocellular carcinoma, lung carcinoma, and ARDS, but further research is needed to confirm and expand on these findings.
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Affiliation(s)
- Chaolei Chen
- Department of Pulmonary Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Xiaomin Huang
- Department of Pulmonary Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Zhaojian Ying
- Department of Pulmonary Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Dengmin Wu
- Department of Pulmonary Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Yani Yu
- Department of Pulmonary Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Xiangdong Wang
- Department of Pulmonary Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
| | - Chengshui Chen
- Department of Pulmonary Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
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11
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Study on a new high affinity anti-glypicans-3 antibody in diagnosis of early hepatocellular carcinoma by differential pulse voltammetry. J Solid State Electrochem 2017. [DOI: 10.1007/s10008-017-3535-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
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12
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Choi D, Lee TH, Spinelli C, Chennakrishnaiah S, D'Asti E, Rak J. Extracellular vesicle communication pathways as regulatory targets of oncogenic transformation. Semin Cell Dev Biol 2017; 67:11-22. [PMID: 28077296 DOI: 10.1016/j.semcdb.2017.01.003] [Citation(s) in RCA: 92] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2016] [Revised: 12/23/2016] [Accepted: 01/06/2017] [Indexed: 12/15/2022]
Abstract
Pathogenesis of human cancers bridges intracellular oncogenic driver events and their impact on intercellular communication. Among multiple mediators of this 'pathological connectivity' the role of extracellular vesicles (EVs) and their subsets (exosomes, ectosomes, oncosomes) is of particular interest for several reasons. The release of EVs from cancer cells represents a unique mechanism of regulated expulsion of bioactive molecules, a process that also mediates cell-to-cell transfer of lipids, proteins, and nucleic acids. Biological effects of these processes have been implicated in several aspects of cancer-related pathology, including tumour growth, invasion, angiogenesis, metastasis, immunity and thrombosis. Notably, the emerging evidence suggests that oncogenic mutations may impact several aspects of EV-mediated cell-cell communication including: (i) EV release rate and protein content; (ii) molecular composition of cancer EVs; (iii) the inclusion of oncogenic and mutant macromolecules in the EV cargo; (iv) EV-mediated release of genomic DNA; (v) deregulation of mechanisms responsible for EV biogenesis (vesiculome) and (vi) mechanisms of EV uptake by cancer cells. Intriguingly, EV-mediated intercellular transfer of mutant and oncogenic molecules between subpopulations of cancer cells, their indolent counterparts and stroma may exert profound biological effects that often resemble (but are not tantamount to) oncogenic transformation, including changes in cell growth, clonogenicity and angiogenic phenotype, or cause cell stress and death. However, several biological barriers likely curtail a permanent horizontal transformation of normal cells through EV-mediated mechanisms. The ongoing analysis and targeting of EV-mediated intercellular communication pathways can be viewed as a new therapeutic paradigm in cancer, while the analysis of oncogenic cargo contained in EVs released from cancer cells into biofluids is being developed for clinical use as a biomarker and companion diagnostics. Indeed, studies are underway to further explore the multiple links between molecular causality in cancer and various aspects of cellular vesiculation.
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Affiliation(s)
- Dongsic Choi
- Research Institute of the McGill University Health Centre, Glen Site, McGill University, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada
| | - Tae Hoon Lee
- Research Institute of the McGill University Health Centre, Glen Site, McGill University, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada
| | - Cristiana Spinelli
- Research Institute of the McGill University Health Centre, Glen Site, McGill University, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada
| | - Shilpa Chennakrishnaiah
- Research Institute of the McGill University Health Centre, Glen Site, McGill University, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada
| | - Esterina D'Asti
- Research Institute of the McGill University Health Centre, Glen Site, McGill University, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada
| | - Janusz Rak
- Research Institute of the McGill University Health Centre, Glen Site, McGill University, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada.
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Haruyama Y, Kataoka H. Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma. World J Gastroenterol 2016; 22:275-283. [PMID: 26755876 PMCID: PMC4698492 DOI: 10.3748/wjg.v22.i1.275] [Citation(s) in RCA: 89] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2015] [Accepted: 11/19/2015] [Indexed: 02/06/2023] Open
Abstract
Glypican-3 (GPC3) is a cell surface oncofetal proteoglycan that is anchored by glycosylphosphatidylinositol. Whereas GPC3 is abundant in fetal liver, its expression is hardly detectable in adult liver. Importantly, GPC3 is overexpressed in hepatocellular carcinoma (HCC), and several immunohistochemical studies reported that overexpression predicts a poorer prognosis for HCC patients. Therefore, GPC3 would serve as a useful molecular marker for HCC diagnosis and also as a target for therapeutic intervention in HCC. Indeed, some immunotherapy protocols targeting GPC3 are under investigations; those include humanized anti-GPC3 cytotoxic antibody, peptide vaccine and immunotoxin therapies. When considering the clinical requirements for GPC3-targeting therapy, companion diagnostics to select the appropriate HCC patients are critical, and both immunohistochemical analysis of tissue sections and measurement of serum GPC3 level have been suggested for this purpose. This review summarizes current knowledge regarding the clinical implication of GPC3 detection and targeting in the management of patients with HCC.
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Wang L, Yao M, Pan LH, Qian Q, Yao DF. Glypican-3 is a biomarker and a therapeutic target of hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int 2015; 14:361-366. [PMID: 26256079 DOI: 10.1016/s1499-3872(15)60396-4] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
BACKGROUND The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multi-step and complex process. Early diagnosis and effective treatments are of utmost importance. This review summarized the recent studies of oncofetal glypican-3 (GPC-3), a membrane-associated heparan sulfate proteoglycan, in the diagnosis and treatment of HCC. DATA SOURCES English-language reports published from June 2001 to September 2014 were searched from MEDLINE. The key words searched included: GPC-3, biomarker, target and HCC. The sensitivity, specificity, positive and negative predictive values were extracted, and the effect of GPC-3 targeted therapy on HCC was also evaluated. RESULTS GPC-3 plays a crucial role in HCC cell proliferation and metastasis. It mediates oncogenesis involving signaling pathways during hepatocyte malignant transformation. GPC-3 expression is increased in atypical hyperplasia and cancerous tissues. GPC-3 levels in HCC patients are related to HBV infection, TNM stage, periportal cancerous embolus, and extrahepatic metastasis. The diagnostic accuracy of the combination of serum GPC-3 and alpha-fetoprotein in HCC is up to 94.3%. Down-regulation of GPC-3 with specific siRNA or anti-GPC-3 antibody alters cell migration, metastasis and invasion behaviors. The nude mice xenograft tumor growth is inhibited by silencing GPC-3 gene transcription. CONCLUSION Oncofetal GPC-3 is a highly specific biomarker for the diagnosis of HCC and a promising target molecule for HCC gene therapy.
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Affiliation(s)
- Li Wang
- Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, China.
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15
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Qi XH, Wu D, Cui HX, Ma N, Su J, Wang YT, Jiang YH. Silencing of the glypican-3 gene affects the biological behavior of human hepatocellular carcinoma cells. Mol Med Rep 2014; 10:3177-84. [PMID: 25270552 DOI: 10.3892/mmr.2014.2600] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2013] [Accepted: 06/05/2014] [Indexed: 11/05/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in the world. The gene glypican-3 (GPC3) is reported to be a potential therapeutic target for HCC. In this study, we use RNA interference with lentiviral vectors to explore the effect of GPC3 silencing on the biological behavior of HCC cells and the potential role of the GPC3 protein in the activation of epithelial-mesenchymal transition (EMT), which relates to HCC cell invasion and migration. Our data suggest that GPC3 silencing leads to a decrease in HCC cell proliferation and to an increase in apoptosis. We demonstrated that GPC3 silencing regulates cell invasion and migration, most probably through the activation of the EMT cellular program. In conclusion, GPC3 is associated with the HCC cell biological behavior, while the relationship between GPC3 and EMT in tumorigenesis of HCC deserves future investigation.
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Affiliation(s)
- Xin-Hui Qi
- Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Di Wu
- Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Hui-Xia Cui
- Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Nan Ma
- Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Jia Su
- Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - Yu-Tong Wang
- Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
| | - You-Hong Jiang
- Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
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16
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ZHAO ZHENGYUAN, HAN CHENGGUANG, LIU JUNTAO, WANG CHANGLEI, WANG YI, CHENG LIYA. GPC5, a tumor suppressor, is regulated by miR-620 in lung adenocarcinoma. Mol Med Rep 2014; 9:2540-6. [DOI: 10.3892/mmr.2014.2092] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2013] [Accepted: 02/27/2014] [Indexed: 11/05/2022] Open
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17
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Kim YH, Kang JS. Expression of Glypican-3 in Mouse Embryo Stem Cells and its Derived Hepatic Lineage Cells Treated with Diethylnitrosamine in vitro. Asian Pac J Cancer Prev 2013; 14:6341-5. [DOI: 10.7314/apjcp.2013.14.11.6341] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
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18
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Glypican-3-mediated inhibition of CD26 by TFPI: a novel mechanism in hematopoietic stem cell homing and maintenance. Blood 2013; 121:2587-95. [PMID: 23327927 DOI: 10.1182/blood-2012-09-456715] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Directional migration determines hematopoietic stem/progenitor cell (HSPC) homing, which depends upon the interaction between the chemokine CXCL12 and its receptor CXCR4. CD26 is a widely expressed membrane-bound ectopeptidase that cleaves CXCL12 thereby depleting its chemokine activity. We identified tissue-factor pathway inhibitor (TFPI) as a biological inhibitor of CD26 in murine and human HSPCs. We observed low-level TFPI expression in endothelial cells in the bone marrow (BM), which did not increase following radiation injury. Treatment of HSPCs with TFPI in vitro led to enhanced HSPC migration toward CXCL12, as well as homing and engraftment in the BM upon transplantation. We found that Glypican-3 (GPC3), a heparan sulfate proteoglycan expressed on murine as well as human HSPCs, mediated this effect. TFPI did not affect CD26 activity, migration, or homing of GPC3(-/-) HSPCs, while it affected GPC1(-/-) HSPCs similar to wild-type HSPCs. Moreover, proliferation of GPC3(-/-) but not GPC1(-/-) BM HSPCs was significantly increased, which was associated with a decrease in the primitive HSC pool in BM and an increase in proportion of the circulating HSPCs in the peripheral blood. Hence, we present a novel role for TFPI and GPC3 in regulating HSC homing as well as retention in the BM.
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19
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Dwivedi PP, Lam N, Powell BC. Boning up on glypicans-opportunities for new insights into bone biology. Cell Biochem Funct 2013; 31:91-114. [DOI: 10.1002/cbf.2939] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2012] [Revised: 11/09/2012] [Accepted: 11/16/2012] [Indexed: 01/01/2023]
Affiliation(s)
| | - N. Lam
- Craniofacial Research Group; Women's and Children's Health Research Institute; North Adelaide; South Australia; Australia
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20
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Shiota G, Miura N. Biomarkers for hepatocellular carcinoma. Clin J Gastroenterol 2012; 5:177-82. [PMID: 26182317 DOI: 10.1007/s12328-012-0301-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/12/2012] [Accepted: 04/12/2012] [Indexed: 12/17/2022]
Abstract
Hepatocellular carcinoma (HCC) ranks high among the most common and fatal cancers in the world. HCC develops from chronic liver diseases, especially from hepatitis C virus-related and hepatitis B virus (HBV)-related liver diseases. In this sense, useful biomarkers for HCC detection for the patients at risk of HCC are quite important. Recently, new therapies for HCC have been developed, and the prognosis of the patients has improved. However, considering the recurrence rate of HCC after treatment is very high, biomarkers that detect recurrence at an early stage are also required. In addition, since new drugs such as multikinase inhibitors have been introduced to the clinical scene, surrogate biomarkers to predict the effectiveness of treatment will be required in the near future. So far, many biomarkers for HCC have been developed, and their clinical usefulness has been assessed. As a result, several biomarkers for HCC are widely used. However, investigations to discover more useful biomarkers that fit in clinical settings are under way. In this review article, biomarkers for HCC are overviewed to examine their clinical usefulness.
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Affiliation(s)
- Goshi Shiota
- Division of Molecular and Genetic Medicine, Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medicine, Tottori University, Yonago, 683-8503, Japan.
| | - Norimasa Miura
- Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University, Yonago, 683-8503, Japan
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21
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Ho M. Advances in liver cancer antibody therapies: a focus on glypican-3 and mesothelin. BioDrugs 2012; 25:275-84. [PMID: 21942912 DOI: 10.2165/11595360-000000000-00000] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Liver cancer is one of the most common malignancies worldwide. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common primary liver cancers, yet there have been no significant advances in effective therapeutics. There is an urgent need to identify molecular targets for the development of novel therapeutic approaches. In this review, glypican-3 (GPC3) and mesothelin are discussed, with a focus on their potential as targets for antibody therapy in liver cancer. GPC3 and mesothelin are glycosylphosphatidylinositol-anchored proteins present on the cell surface. They are attractive candidates for liver cancer therapy given that GPC3 and mesothelin show high expression in HCC and CCA, respectively. Antibody drugs targeting GPC3 or mesothelin have shown anti-cancer activity in mice. Humanized or chimeric IgG molecules based on first-generation murine monoclonal antibodies against these antigens are being evaluated in clinical studies. Recently, fully human monoclonal antibodies against GPC3 and mesothelin have been isolated by antibody phage display technology that may provide opportunities for novel cancer therapy.
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Affiliation(s)
- Mitchell Ho
- Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
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22
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Masuda T, Miyoshi E. Cancer biomarkers for hepatocellular carcinomas: from traditional markers to recent topics. Clin Chem Lab Med 2011; 49:959-966. [PMID: 21428856 DOI: 10.1515/cclm.2011.152] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Hepatocellular carcinomas (HCC) are the fifth most common tumor type and the third most common cause of cancer-related death worldwide. Some tumor markers for HCC, such as α-fetoprotein and des-γ-carboxyprothrombin, are used clinically. Recent advances in proteomics and glyco-proteomics might provide various types of novel tumor markers for HCC. While the clinical availability of these tumor markers is important, the molecular mechanisms underlying the production of tumor markers requires further clarification. Our group has investigated the glycobiology of tumor markers. In this review, we describe the impact of novel HCC markers and their possible implications for clinical use.
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Affiliation(s)
- Tomomi Masuda
- Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, Suita, Japan
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23
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Ho M, Kim H. Glypican-3: a new target for cancer immunotherapy. Eur J Cancer 2010; 47:333-8. [PMID: 21112773 DOI: 10.1016/j.ejca.2010.10.024] [Citation(s) in RCA: 158] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2010] [Accepted: 10/27/2010] [Indexed: 12/12/2022]
Abstract
Hepatocellular carcinoma (HCC) remains a common malignant cancer worldwide. There is an urgent need to identify new molecular targets for the development of novel therapeutic approaches. Herein, we review the structure, function and biology of glypican-3 (GPC3) and its role in human cancer with a focus on its potential as a therapeutic target for immunotherapy. GPC3 is a cell-surface protein that is over-expressed in HCC. Loss-of-function mutations of GPC3 cause Simpson-Golabi-Behmel syndrome (SGBS), a rare X-linked overgrowth condition. GPC3 binds Wnt and Hedgehog (Hh) signalling proteins. GPC3 is also able to bind basic growth factors such as fibroblast growth factor 2 through its heparan sulphate glycan chains. GPC3 is a promising candidate for liver cancer therapy given that it shows high expression in HCC. An anti-GPC3 monoclonal antibody has shown anti-cancer activity in mice and its humanised IgG molecule is currently undergoing clinical evaluation in patients with HCC. There is also evidence that soluble GPC3 may be a useful serum biomarker for HCC.
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Affiliation(s)
- Mitchell Ho
- Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
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24
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Yasuda E, Kumada T, Toyoda H, Kaneoka Y, Maeda A, Okuda S, Yoshimi N, Kozawa O. Evaluation for clinical utility of GPC3, measured by a commercially available ELISA kit with Glypican-3 (GPC3) antibody, as a serological and histological marker for hepatocellular carcinoma. Hepatol Res 2010; 40:477-85. [PMID: 20374302 DOI: 10.1111/j.1872-034x.2010.00624.x] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
AIMS We evaluated the clinical utility of glypican-3 (GPC3), which has been proposed as a potential novel tumor marker for hepatocellular carcinoma (HCC), as a serological and histological marker for HCC. METHODS The serum GPC3 level was compared between 200 patients with HCC and 200 patients with chronic liver disease (CLD). In addition, the expression of GPC3 was examined with immunohistochemistry on 38 resected specimens from patients with HCC. A commercially available GPC3 antibody was used for these analyses. RESULTS The median values of serum GPC3 in patients with HCC and with CLD were 924.8 pg/mL and 1161.6 pg/mL, respectively. We found no elevation of serum GPC3 level in patients with HCC in comparison with those with CLD; rather the level was higher in patients with CLD (P < 0.0001). In immunohistochemical analysis, 14 of 38 (36.9%) HCC tissues were positive for GPC3, whereas no corresponding non-cancerous tissue was positive. The positivity for GPC3 tended to increase with pathologic decreased differentiation of HCC. CONCLUSIONS We did not find serum GPC3 level, measured by a commercially available ELISA kit with GPC3 antibody, to be useful in the diagnosis of HCC. However, we did observe increased GPC3 staining in HCC tissue with moderate or poor differentiation, suggesting that GPC3 is produced by HCC tumors. This lack of utility could have been due to the measuring procedure used in the present study. Further evaluation of GPC3 in HCC with other measuring procedures is needed.
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Affiliation(s)
- Eisuke Yasuda
- Department of Medical Technology, Ogaki Municipal Hospital, Ogaki, Japan
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25
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Ushiku T, Uozaki H, Shinozaki A, Ota S, Matsuzaka K, Nomura S, Kaminishi M, Aburatani H, Kodama T, Fukayama M. Glypican 3-expressing gastric carcinoma: distinct subgroup unifying hepatoid, clear-cell, and alpha-fetoprotein-producing gastric carcinomas. Cancer Sci 2009; 100:626-32. [PMID: 19243386 PMCID: PMC11158094 DOI: 10.1111/j.1349-7006.2009.01108.x] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2008] [Revised: 12/01/2008] [Accepted: 12/02/2008] [Indexed: 12/19/2022] Open
Abstract
Gypican-3 (GPC3) has been recognized as an oncofetal protein in hepatic neoplasms and yolk sac tumors. To characterize a distinct subgroup of gastric carcinoma (GC) expressing GPC3 (GPC3-GC), primary and metastatic GC tissues were evaluated by immunohistochemistry with special focus on their related entities: hepatoid, clear-cell, and alpha-fetoprotein-producing GC. GPC3-GC was defined as focal GPC3-GC when 10-49% of neoplastic cells were positive, and as diffuse GPC3-GC when more than 50% of cells were positive. Among 926 GC cases, 101 (11%) were GPC3-GC, of which 45 were diffuse and 56 were focal GPC3-GC. Specific histological patterns, such as the hepatoid and clear-cell patterns, were frequently observed in diffuse GPC3-GC (38 and 49%, respectively) and in focal GPC3-GC (4 and 25%, respectively), whereas these patterns were extremely rare in GPC3-negative GC. Immunoreactive alpha-fetoprotein was only identified in GPC3-GC (38% of diffuse and 14% of focal GPC3-GC). Both diffuse and focal GPC3-GC showed nodal metastasis more frequently (67 and 55%, respectively) than GPC3-negative GC (34%), and the diffuse GPC3-GC had significantly more T2-4 and M1 stage cases. GPC3 immunostaining was present in 57 out of 61 nodal metastases (93%) and in all four liver metastases examined. Importantly, diffuse GPC3 expression was observed in the liver metastasis, even if the primary tumor was focal GPC3-GC. GPC3-GC is a distinctive group of GC, which unifies hepatoid, clear-cell, and alpha-fetoprotein-producing GC. GPC3 is expected to be a target of forthcoming immunotherapy for a patient bearing this specific type of GC.
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Affiliation(s)
- Tetsuo Ushiku
- Department of Pathology and Diagnostic Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
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Aviel-Ronen S, Lau SK, Pintilie M, Lau D, Liu N, Tsao MS, Jothy S. Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma. Mod Pathol 2008; 21:817-25. [PMID: 18469798 DOI: 10.1038/modpathol.2008.37] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Glypican-3 is a membrane-bound proteoglycan whose expression has been linked to malignancies through the existence of both mutations and aberrant protein expression. Reports on glypican-3 expression in lung cancer were limited, with some evidence for loss of expression, which suggested a tumor-suppressor role. We sought to evaluate glypican-3 expression in lung cancer at the protein and mRNA levels and correlate it with clinical, histological and genomic characteristics such as RAS mutation status. We used immunohistochemistry on tissue microarray to study glypican-3 expression in 97 patients, evaluated glypican-3 mRNA levels by quantitative polymerase chain reaction in 143 patients and identified RAS mutations by allele-specific oligonucleotide hybridization. We correlated the results with clinical and histological data. Glypican-3 immunostaining was negative in all normal lung tissues, but positive in 23% of lung carcinoma samples. High protein and mRNA expression was associated with squamous histology (positive stain in 55% of squamous cell carcinoma vs 8% of adenocarcinoma, P<0.0001 for both immunostaining and mRNA). RAS mutations were highly associated with adenocarcinoma and low glypican-3 mRNA expression (P<0.0001 for both). Among smokers, glypican-3 mRNA expression was reduced in adenocarcinoma patients (P=0.013), and was elevated in those with squamous cell carcinoma (P=0.03, interaction P=0.0009). These opposing associations also correlated with the smoking burden. Patients with tumors staining positively for glypican-3 smoked significantly more than patients with tumors staining negatively (P=0.013). No association was found between glypican-3 expression and patient outcome. In conclusion, glypican-3 was overexpressed in cancerous compared with normal lung tissue. Adenocarcinoma and squamous cell carcinoma had differential expression of glypican-3, with predilection to squamous cell carcinoma patients who smoked. Glypican-3 expression in squamous cell carcinoma as an oncofetal protein renders it a potential candidate marker for early detection of lung squamous cell carcinoma.
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Affiliation(s)
- Sarit Aviel-Ronen
- Department of Pathology, University Health Network-Princess, Margaret Hospital, Ontario Cancer Institute, Toronto, ON, Canada
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27
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Identification of Glypican3 as a novel GLUT4-binding protein. Biochem Biophys Res Commun 2008; 369:1204-8. [DOI: 10.1016/j.bbrc.2008.03.027] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2008] [Accepted: 03/06/2008] [Indexed: 12/23/2022]
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28
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Ieta K, Ojima E, Tanaka F, Nakamura Y, Haraguchi N, Mimori K, Inoue H, Kuwano H, Mori M. Identification of overexpressed genes in hepatocellular carcinoma, with special reference toubiquitin-conjugating enzyme E2Cgene expression. Int J Cancer 2007; 121:33-8. [PMID: 17354233 DOI: 10.1002/ijc.22605] [Citation(s) in RCA: 82] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
This study consisted of 2 aims: (i) to determine genes associated with hepatocellular carcinoma (HCC) by microarray analysis; and (ii) to evaluate the clinicopathological significance of human ubiquitin-conjugating enzyme E2C (Ube2c) found to be overexpressed in HCC from microarray analysis. Laser microdissection and cDNA-microarray were performed to identify genes associated with HCC. We then focused on the Ube2c gene. Using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), Ube2c expression status and clinicopathological significance were studied in 65 clinical HCC samples. A number of genes upregulated in HCC cells compared to noncancerous liver cells were identified, one of which was the Ube2c gene. Ube2c gene expression in the cancer tissue was higher than in the corresponding noncancerous tissue in 62 of the 65 cases (95.4%, p < 0.01). Tumors with high Ube2c expression showed higher frequencies of tumor invasion to capsular formation (fc-inf), invasion to portal vein (vp) and tumor de-differentiation (p < 0.05). Patients with high Ube2c expression also showed significantly worse disease-free survival rates than those with low Ube2c expression (p < 0.01). In addition, Ube2c expression was found to be an independent prognostic factor for disease-free survival rate in multivariate analysis. We identified differentially expressed genes between HCC and normal liver tissues. Of those, the Ube2c gene appeared to be associated with HCC progression, and may be useful as a prognostic indicator for HCC patients.
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Affiliation(s)
- Keisuke Ieta
- Department of Surgery and Molecular Oncology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu 874-0838, Japan
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29
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Boily G, Ouellet S, Langlois S, Larivière M, Drouin R, Sinnett D. In vivo footprinting analysis of the Glypican 3 (GPC3) promoter region in neuroblastoma cells. ACTA ACUST UNITED AC 2007; 1769:182-93. [PMID: 17350117 DOI: 10.1016/j.bbaexp.2007.01.014] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2006] [Revised: 01/24/2007] [Accepted: 01/29/2007] [Indexed: 11/17/2022]
Abstract
Glypican 3 (GPC3) is an X-linked gene that has its peak expression during development and is down-regulated in all studied tissues after birth. We have shown that GPC3 was expressed in neuroblastoma and Wilms' tumor. To understand the mechanisms regulating the transcription of this gene in neuroblastoma cells, we have focused our study on the identification of putative transcription factors binding the promoter. In this report we performed in vivo dimethylsulfate, UV type C irradiation and DNaseI footprinting analyses coupled with ligation-mediated PCR on nearly 1000 bp of promoter in two neuroblastoma cell lines, SJNB-7 (expressing GPC3) and SK-N-FI (not expressing GPC3). Nucleosome signature footprints were observed in the most distal part of the studied region in both cell lines. We detected eight large differentially protected regions, suggesting the presence of binding proteins in both cell lines but more DNA-protein interactions in GPC3-expressing cells. Sp1 was previously shown to be able to bind some of these regions. Here by combining electromobility shift assays and chromatin immunoprecipitations we showed that the transcription factor NFY was part of the DNA-protein complex found in footprinted regions upstream of the described minimal promoter. These studies performed on chromatin in situ suggest that NFY and yet unknown cell type-specific factors may play an important role in the regulation of GPC3.
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Affiliation(s)
- Gino Boily
- Division of Hemato-Oncology, Charles-Bruneau Cancer Center, Research Center, CHU Sainte- Justine, Montreal, QC, Canada H3T 1C5
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Abstract
Serological markers for hepatocellular carcinoma (HCC) are important for early diagnosis, as well as monitoring of tumour aggressiveness, treatment responsiveness, recurrence and survival. The three most common markers are total alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive AFP (AFP-L3) and protein induced by vitamin K absence or antagonist-II (PIVKA-II). Total AFP has the sensitivity of 60% and specificity of 90% for the detection of HCC. Increase in the percentage of AFP-L3 over the total AFP (>10%) is very specific for small HCC. PIVKA-II is also more specific than total AFP in detecting HCC. AFP-L3 and PIVKA-II levels correlate with tumour aggressiveness and prognosis. All three markers are useful for monitoring treatment responsiveness and tumour recurrence. Since the levels of the three markers are independent of each other, combination of measurement of two or three markers will increase the sensitivity and diagnostic accuracy. Some novel markers including glypican-3 are being extensively studied.
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Affiliation(s)
- Man-Fung Yuen
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China.
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Li H, Melford K, Judson A, Bensadoun A. Murine glypican-4 gene structure and expression; Sp1 and Sp3 play a major role in glypican-4 expression in 3T3-F442A cells. ACTA ACUST UNITED AC 2004; 1679:141-55. [PMID: 15297147 DOI: 10.1016/j.bbaexp.2004.06.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2004] [Revised: 06/08/2004] [Accepted: 06/11/2004] [Indexed: 02/08/2023]
Abstract
In this report we describe the genomic organization of the mouse glypican-4 (Gpc4), an analysis of its promoter and its transcriptional regulation in the 3T3-F442A adipocyte cell line. The Gpc4 gene consists of nine exons separated by eight introns. A series of deletion mutants and 4391 bp of the 5'-flanking region were cloned into pGL3-BASIC upstream of the luciferase reporter gene and transfected into 3T3-F442A adipocytes. Analysis of a 4.3-kb DNA fragment at the 5'-flanking region of this gene revealed that the Gpc4 promoter is a TATA-less promoter with a large cluster of GC boxes. Competitive electrophoretic mobility shift and supershift assays identified a cluster of nine functional GC boxes binding Sp1 and Sp3 in this region. Transactivation experiments in insect cells showed that both Sp1 and Sp3 are major activators of the Gpc4 promoter. Gpc4 is expressed in adipocytes where its expression is highest in confluent 3T3-F442A adipoblasts and decreases dramatically as cells differentiate. Sp protein analyses demonstrated a major decrease in Sp3 protein in differentiated adipocytes as compared to undifferentiated adipoblasts. These experiments show that Gpc4 is developmentally regulated in 3T3-F442A adipocytes and suggest that Sp transcription factors play a significant role in the regulated expression of Gpc4.
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Affiliation(s)
- Huaixing Li
- Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA
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Abstract
We have previously shown that the glypican 3 (GPC3) gene was expressed in neuroblastoma (NB) and Wilms' tumour (WT), two embryonal tumours. GPC3 is an X-linked gene that has its peak expression during development and that is downregulated in all investigated tissues after birth. GPC3 expression could be involved in the aetiology of embryonal tumours such as NB and WT. Methylation is known to play a role in gene silencing, notably in chromosome X inactivation. Southern blot- and PCR-based methylation assays were used to assess the methylation status of the GPC3 promoter on genomic DNA from both normal and embryonal tumour cells. In normal cells, the promoter was not methylated in males and partially methylated in females. Our results suggest that DNA methylation of the promoter region is not essential for the transcriptional repression of the GPC3 gene and that the methylation observed in females is probably linked to the inactive X chromosome. In tumour samples, methylation abnormalities have been found exclusively in female NB samples (loss of methylation) and mainly in male WT samples (gain of methylation). Overall, methylation did not significantly correlate with the expression status of GPC3. Although promoter methylation is likely to affect the expression status of the gene, our results suggest that the deregulation of GPC3 transcriptional expression seen in NB and WT involves other regulatory levels.
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Affiliation(s)
- G Boily
- Division of Hematology-oncology, Charles-Bruneau Cancer Center, Research Center, Sainte-Justine Hospital, 3175 chemin de la Côte-Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5
- Department of Pediatrics, University of Montreal, 3175 chemin de la Côte-Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5
| | - Z Saikali
- Division of Hematology-oncology, Charles-Bruneau Cancer Center, Research Center, Sainte-Justine Hospital, 3175 chemin de la Côte-Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5
- Department of Pediatrics, University of Montreal, 3175 chemin de la Côte-Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5
| | - D Sinnett
- Division of Hematology-oncology, Charles-Bruneau Cancer Center, Research Center, Sainte-Justine Hospital, 3175 chemin de la Côte-Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5
- Department of Pediatrics, University of Montreal, 3175 chemin de la Côte-Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5
- Division of Hematology-oncology, Charles-Bruneau Cancer Center, Research Center, Sainte-Justine Hospital, 3175 chemin de la Côte-Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5. E-mail:
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Wichert A, Stege A, Midorikawa Y, Holm PS, Lage H. Glypican-3 is involved in cellular protection against mitoxantrone in gastric carcinoma cells. Oncogene 2004; 23:945-55. [PMID: 14661052 DOI: 10.1038/sj.onc.1207237] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Elevated expression of the heparan sulphate proteoglycan glypican-3 (GPC3) was found on mRNA and protein levels in the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV, which was established by in vitro selection against mitoxantrone. In order to elucidate a putative role of GPC3 in the drug-resistant phenotype, the mitoxantrone-resistant cell line EPG85-257RNOV was transfected with an expression vector construct carrying an anti-GPC3 hammerhead ribozyme. It could be demonstrated that in anti-GPC3 ribozyme-transfected cell clones, the GPC3-specific mRNA and corresponding protein expression levels were decreased to levels that are similar to those observed in nonresistant, parental cells. The anti-GPC3 ribozyme-containing clones reduced the mitoxantrone resistance level up to 21% of the original resistance and the crossresistance against etoposide to 33% of the original value. This reversal of drug resistance was accompanied by an increased cellular mitoxantrone accumulation in the anti-GPC3 ribozyme-expressing cells. In conclusion, it was verified that GPC3 is involved in the cellular protection against mitoxantrone in the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV.
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Affiliation(s)
- Anke Wichert
- Institute of Pathology, Charité, Campus Mitte, Humboldt University Berlin, Schumannstr 20/21, Berlin D-10117, Germany
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Yu W, Inoue J, Imoto I, Matsuo Y, Karpas A, Inazawa J. GPC5 is a possible target for the 13q31-q32 amplification detected in lymphoma cell lines. J Hum Genet 2004; 48:331-335. [PMID: 12721791 DOI: 10.1007/s10038-003-0026-2] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2003] [Accepted: 03/13/2003] [Indexed: 01/02/2023]
Abstract
Comparative genomic hybridization (CGH) analyses have detected gains of copy number on 13q, especially at 13q31-q32, in cell lines and primary cases of various types of lymphoma. Since amplification of chromosomal DNA is one of the mechanisms that can activate tumor-associated genes, and because 13q amplification had been reported in various other types of tumors as well, we attempted to define by fluorescence in situ hybridization (FISH) a common region at 13q31-q32 in which to explore genes that might be targets for the amplification events. Although the commonly amplified region we defined was relatively large (approximately 4 Mb), only one true gene, GPC5, was found there. GPC5 was over-expressed in lymphoma cell lines that had shown amplification, in comparison with those that had not. Our findings suggest that GPC5 is a likely target for amplification, and that over-expression of this gene may contribute to development and/or progression of lymphomas and other tumors.
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Affiliation(s)
- Wei Yu
- Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Jun Inoue
- Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
- Theranostics Research Center (TRC), Otsuka Pharmaceutical Co., 463-10 Kagasuno Kawauchi-cho, Tokushima 771-0192, Japan
| | - Issei Imoto
- Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
- Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), 4-1-8 Honmachi, Kawaguchi 332-0012, Japan
| | - Yoshinobu Matsuo
- Fujisaki Cell Center, Hayashibara Biochemical Laboratories, 675-1 Fujisaki, Okayama 702-8006, Japan
| | - Abraham Karpas
- Department of Hematology, University of Cambridge Clinical School, MRC Centre, Hills Road, Cambridge, CB2 2QH, United Kingdom
| | - Johji Inazawa
- Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
- Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), 4-1-8 Honmachi, Kawaguchi 332-0012, Japan.
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Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 2003; 125:89-97. [PMID: 12851874 DOI: 10.1016/s0016-5085(03)00689-9] [Citation(s) in RCA: 672] [Impact Index Per Article: 30.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
BACKGROUND & AIMS Early detection of hepatocellular carcinoma (HCC) is critical for successful treatment. However, the differential diagnosis between HCC and benign hepatic lesions is sometimes difficult and new biochemical markers for HCC are required. It has been reported that glypican-3 (GPC3) messenger RNA (mRNA) is significantly increased in most HCCs compared with benign liver lesions or normal liver. The goal of this study is to determine whether GPC3 is also overexpressed at the protein level and whether GPC3 is detectable in the serum of patients with HCC. METHODS GPC3 was assessed in liver tissue sections by immunohistochemistry and in serum by enzyme-linked immunosorbent assay. Serum alpha-fetoprotein (AFP) level was also measured in the same patients. RESULTS Immunohistochemical studies showed that GPC3 is expressed in 72% of HCCs (21 of 29), whereas it is not detectable in hepatocytes from normal liver and benign liver diseases. Consistent with this, GPC3 was undetectable in the serum of healthy donors and patients with hepatitis, but its levels were significantly increased in 18 of 34 patients (53%) with HCC. In addition, only 1 of 20 patients with hepatitis plus liver cirrhosis displayed elevated levels of serum GPC3. Interestingly, in most cases, there was no correlation between GPC3 and AFP values. Thus, at least 1 of the 2 markers was elevated in 82% of the patients with HCC. CONCLUSIONS GPC3 is specifically overexpressed in most HCCs and is elevated in the serum of a large proportion of patients with HCC. The simultaneous determination of GPC3 and AFP may significantly increase the sensitivity for diagnosis of HCC.
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Affiliation(s)
- Mariana Capurro
- Division of Molecualr and Cell Biology, Sunnybrook and Women's College Health Sciences Center and Deaprtment of Medical Biophysics, University of Toronto, Ontario, Canada
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Peters MG, Farías E, Colombo L, Filmus J, Puricelli L, Bal de Kier Joffé E. Inhibition of invasion and metastasis by glypican-3 in a syngeneic breast cancer model. Breast Cancer Res Treat 2003; 80:221-32. [PMID: 12908826 DOI: 10.1023/a:1024549729256] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Glypican-3 (GPC3), a proteoglycan bound to the cell membrane through a GPI anchor, is widely expressed in the embryo but down regulated in most adult tissues, with some exceptions as mammary cells. GPC3 is involved in the regulation of cell proliferation and survival in specific cell types. LM3, a murine mammary tumor cell line unable to express GPC3, was stably transfected with the rat GPC3 gene to analyze its role in tumor progression. Upon injection into syngeneic BALB/c mice LM3-GPC3 clones showed less local invasiveness and developed fewer spontaneous and experimental lung metastasis than controls. GPC3-expressing cells were more sensitive to apoptosis induced by serum depletion, exhibited a delay in the first steps of spreading and were less motile than controls. On the other hand, LM3-GPC3 cells were significantly more adherent to FN than control ones. We observed that GPC3 transfectants presented a higher expression of E-cadherin and beta-catenin, molecules whose down regulation has been associated with tumor progression. Exogenous TGF-beta increased MMP-9 activity in both control and GPC3-expressing cells, but did not modulate MMP-2. Contrarily, GPC3 expression prevented the increase of MMP-2 activity induced by IGF-II. Our results suggest that GPC3 has a protective role against mammary cancer progression.
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Affiliation(s)
- M G Peters
- Research Area, Cell Biology Department, Institute of Oncology Angel H. Roffo, University of Buenos Aires, Buenos Aires, Argentina
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37
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Abstract
Glypicans are a family of heparan sulfate proteoglycans that are bound to the cell surface by a glycosyl-phosphatidylinositol anchor. Six members of this family have been identified in mammals. In general, glypicans are highly expressed during development, and their expression pattern suggests that they are involved in morphogenesis. One member of this family, glypican-3, is mutated in the Simpson-Golabi-Behmel syndrome. This syndrome is characterized by overgrowth and various developmental abnormalities that indicate that glypican-3 inhibits proliferation and cell survival in the embryo. It has consequently been proposed that glypicans can regulate the activity of several growth factors that play a critical role in morphogenesis.
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Affiliation(s)
- Howard H Song
- Division of Molecular and Cell Biology, Sunnybrook and Women's College Health Science Centre, Toronto, ON, Canada
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Affiliation(s)
- J Filmus
- Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
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39
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Abstract
Glypicans are proteins with very characteristic structures that are substituted with heparan sulfate and that are linked to the cell surface via glycosylphosphatidylinositol. The modular structure of the glypicans has been highly conserved throughout evolution. Six glypicans have been identified so far in vertebrates. Mutations in Drosophila, humans and mice reveal a role for these cell surface molecules in the control of cell growth and differentiation. Their mechanism of action is not yet clear. Most likely, glypicans activate or determine the activity ranges of morphogens and growth factors such as FGFs, BMPs, Wnts, Hhs and IGFs.
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Affiliation(s)
- B De Cat
- Laboratory for Glycobiology and Developmental Genetics, Center for Human Genetics, Flanders Interuniversity Institute for Biotechnology, University of Leuven, B-3000 Leuven, Belgium
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Hagihara K, Watanabe K, Chun J, Yamaguchi Y. Glypican-4 is an FGF2-binding heparan sulfate proteoglycan expressed in neural precursor cells. Dev Dyn 2000; 219:353-67. [PMID: 11066092 DOI: 10.1002/1097-0177(2000)9999:9999<::aid-dvdy1059>3.0.co;2-#] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
FGF2 is a crucial mitogen for neural precursor cells in the developing cerebral cortex. Heparan sulfate proteoglycans (HSPGs) are thought to play a role in cortical neurogenesis by regulating the action of FGF2 on neural precursor cells. In this article, we present data indicating that glypican-4 (K-glypican), a GPI-anchored cell surface HSPG, is involved in these processes. In the developing mouse brain, glypican-4 mRNA is expressed predominantly in the ventricular zone of the telencephalon. Neither the outer layers of the telencephalic wall nor the ventricular zone of other parts of the developing brain express significant levels of glypican-4, with the exception of the ventricular zone of the tectum. In cultures of E13 rat cortical precursor cells, glypican-4 is expressed in cells immunoreactive for nestin and the D1.1 antigen, markers of neural precursor cells. Glypican-4 expression was not detected in early postmitotic or fully differentiated neurons. Recombinant glypican-4 produced in immortalized neural precursor cells binds FGF2 through its heparan sulfate chains and suppressed the mitogenic effect of FGF2 on E13 cortical precursor cells. The spatiotemporal expression pattern of glypican-4 in the developing cerebral wall significantly overlaps with that of FGF2. These results suggest that glypican-4 plays a critical role in the regulation of FGF2 action during cortical neurogenesis.
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Affiliation(s)
- K Hagihara
- The Burnham Institute, La Jolla, California 92037, USA
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Szuchet S, Watanabe K, Yamaguchi Y. Differentiation/regeneration of oligodendrocytes entails the assembly of a cell-associated matrix. Int J Dev Neurosci 2000; 18:705-20. [PMID: 10978849 DOI: 10.1016/s0736-5748(00)00034-4] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
Oligodendrocytes assemble and maintain CNS myelin. We have shown that adhesion of ovine oligodendrocytes to the substratum, GRASP - a novel, horse serum heparin-binding glycoprotein - initiates their myelinogenic phenotype. Synthesis and vectorial transport to the plasma membrane of heparan sulfate proteoglycans is one of the many events that ensue upon adhesion. Proteoglycans play key roles in defining the line of communication between cells and their microenvironment. The nature of their association with cells varies. Often, proteoglycans are part of a complex extracellular network that either surrounds cells or is restricted to smaller areas of their surface. Such extracellular matrices form an integral part of the machinery that regulates cell function. As part of an effort to delineate the events and identify the molecules involved in the adhesion-induced-regeneration and possibly in differentiation of OLGs, we have undertaken to define the full repertoire of OLG proteoglycans. Oligodendrocytes express surface-associated proteoglycans and also secrete them to the medium. However, we observed a clear distinction between secreted and surface-associated proteoglycans in terms of types, temporal regulation and spacial distribution. Oligodendrocytes secrete chondroitin sulfate proteoglycans and keratan sulfate proteoglycans but have only heparan sulfate proteoglycans associated with their surface. Secreted proteoglycans are temporally modulated but adhesion-independent, whereas surface-associated proteoglycans are adhesion-induced. Herein, we present the biochemical characterization of oligodendrocyte proteoglycans. We report that a significant fraction of the surface-associated heparan sulfate proteoglycans are assembled into a cell-associated matrix. This finding is important. First, it reveals a closer parallel than hitherto documented with events that signal Schwann cell myelination. Second, it implicates HSPGs in the establishment of OLG differentiated phenotype. Third, it brings OLGs in tune with other cell types where the ECM (broadly defined) is critical for the orchestration of cues that generate tissue-specific gene expression and phenotypes.
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Affiliation(s)
- S Szuchet
- Department of Neurology and The Brain Research Institute, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA.
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Bandtlow CE, Zimmermann DR. Proteoglycans in the developing brain: new conceptual insights for old proteins. Physiol Rev 2000; 80:1267-90. [PMID: 11015614 DOI: 10.1152/physrev.2000.80.4.1267] [Citation(s) in RCA: 490] [Impact Index Per Article: 19.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Proteoglycans are a heterogeneous class of proteins bearing sulfated glycosaminoglycans. Some of the proteoglycans have distinct core protein structures, and others display similarities and thus may be grouped into families such as the syndecans, the glypicans, or the hyalectans (or lecticans). Proteoglycans can be found in almost all tissues being present in the extracellular matrix, on cellular surfaces, or in intracellular granules. In recent years, brain proteoglycans have attracted growing interest due to their highly regulated spatiotemporal expression during nervous system development and maturation. There is increasing evidence that different proteoglycans act as regulators of cell migration, axonal pathfinding, synaptogenesis, and structural plasticity. This review summarizes the most recent data on structures and functions of brain proteoglycans and focuses on new physiological concepts for their potential roles in the developing central nervous system.
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Affiliation(s)
- C E Bandtlow
- Brain Research Institute, University of Zurich and Swiss Federal Institute of Technology Zurich, Switzerland.
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Murthy SS, Shen T, De Rienzo A, Lee WC, Ferriola PC, Jhanwar SC, Mossman BT, Filmus J, Testa JR. Expression of GPC3, an X-linked recessive overgrowth gene, is silenced in malignant mesothelioma. Oncogene 2000; 19:410-6. [PMID: 10656689 DOI: 10.1038/sj.onc.1203322] [Citation(s) in RCA: 111] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Gene expression changes in rat asbestos-induced malignant mesothelioma (MM) cells were investigated by differential mRNA display. A mRNA transcript identified by this approach was abundant in normal rat mesothelial cells but not expressed in rat MM cell lines. Northern blot analysis confirmed that this transcript is uniformly silenced in rat MM cell lines and primary tumors. Nucleotide sequence analysis revealed that this transcript is encoded by the rat glypican 3 gene (GPC3), whose human homolog is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. Allelic loss at the GPC3 locus was infrequent (6.9%) in MM cell lines, and no mutations were found. GPC3 transcript levels were markedly decreased in 16 of 18 primary tumors and 17 of 22 human MM cell lines. Most of the cell lines were shown to have aberrant methylation of the GPC3 promoter region. In two of four human MM cell lines tested, GPC3 expression was restored after 2-deoxy 5-azacytidine (DAC)-mediated demethylation of its promoter region. Ectopic expression of GPC3 inhibited in vitro colony formation of human MM cells. Collectively, these data suggest that down-regulation of GPC3 is a common occurrence in MM and that GPC3, an X-linked recessive overgrowth gene, may encode a negative regulator of mesothelial cell growth.
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Affiliation(s)
- S S Murthy
- Human Genetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA
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Hagihara K, Watanabe K, Chun J, Yamaguchi Y. Glypican-4 is an FGF2-binding heparan sulfate proteoglycan expressed in neural precursor cells. Dev Dyn 2000. [DOI: 10.1002/1097-0177(2000)9999:9999%3c::aid-dvdy1059%3e3.0.co;2-%23] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
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Siebertz B, Stöcker G, Drzeniek Z, Handt S, Just U, Haubeck HD. Expression of glypican-4 in haematopoietic-progenitor and bone-marrow-stromal cells. Biochem J 1999; 344 Pt 3:937-43. [PMID: 10585884 PMCID: PMC1220719] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/14/2023]
Abstract
Heparan sulphate proteoglycans and the extracellular matrix of bone-marrow-stromal cells are important components of the microenvironment of haematopoietic tissues and are involved in the interaction of haematopoietic stem and stromal cells. Previous studies have emphasized the role of heparan sulphate proteoglycan synthesis by bone-marrow-stromal cells. In the present study we describe the expression of glypican-4 (GPC-4), belonging to the glypican family, in bone-marrow-stromal cells and haematopoietic-progenitor cells of human and murine origin. Expression of GPC-4 was shown on the mRNA-level by reverse transcription-PCR and Northern blot analysis. Amplification products were cloned and sequenced, to confirm these results. To analyze the expression of GPC-4 on the protein level, polyclonal antibodies against selected peptides were raised in rabbits. Western blot analysis showed expression of GPC-4 as a heparan sulphate proteoglycan in the human haematopoietic-progenitor cell line TF-1 and normal human bone marrow. These results were confirmed by FACS analysis of TF-1 cells. Furthermore, GPC-4-positive progenitor cells and stromal cells were enriched from normal human bone marrow by magnetic-cell sorting and analysed by confocal laser-scanning microscopy.
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Affiliation(s)
- B Siebertz
- Institute for Clinical Chemistry, University of Technology, Pauwelsstrasse 30, D-52057 Aachen, Germany
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Wang A, Miralem T, Templeton DM. Heparan sulfate chains with antimitogenic properties arise from mesangial cell-surface proteoglycans. Metabolism 1999; 48:1220-9. [PMID: 10535382 DOI: 10.1016/s0026-0495(99)90259-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Heparan sulfate (HS) chains accumulate in both the medium and the cell layer of mesangial cell cultures. When given in fresh medium to quiescent cultures at naturally occurring concentrations, they suppress entry into the cell cycle and progression to DNA synthesis. We have attempted to identify the proteoglycan (PG) source of the antimitogenic HS chains from mesangial cell layers (HS(c)) and medium (HS(c)). When cells were labeled for 16 hours with [35S]sulfate, 25% of the label was found in intracellular HS chains and 5% in extracellular HSPGs. Cell-surface HSPGs accounted for the remaining 70% of the label associated with cell-layer HS and were released by either trypsin or 2% Triton X-100. About 20% of this cell-surface fraction was released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), and probably represents glypican-like PG; glypican mRNA was present in the cells. The remainder of this fraction could be incorporated into liposomes, indicating the presence of hydrophobic transmembrane regions suggestive of syndecans. Upon purification and deglycosylation, an antiserum to rat liver HSPGs that reacts primarily with syndecan-2 showed a strong signal corresponding to this protein and three weaker bands that may represent additional syndecans. mRNAs for syndecan-1, -2, and -4 were present in the cultures. Syndecan-1 and -2 mRNAs were increased 30 minutes after stimulation of quiescent rat mesangial cells (RMCs) with serum. Heparin, HS(c), and HS(m) all prevented this increase. Syndecan-4 mRNA was not affected by serum, heparin, or HS. In pulse-chase experiments, the amount of 35S appearing in the cellular protein-free HS fraction was accounted for almost entirely by cell-surface PGs, as matrix-associated label was a minor contribution at the end of the pulse-labeling. The appearance of [35S]HS in cell extracts was unaffected by phospholipase C treatment, indicating that turnover of the newly labeled syndecan fraction is the source of the antimitogenic HS chains.
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Affiliation(s)
- A Wang
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada
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Veugelers M, De Cat B, Ceulemans H, Bruystens AM, Coomans C, Dürr J, Vermeesch J, Marynen P, David G. Glypican-6, a new member of the glypican family of cell surface heparan sulfate proteoglycans. J Biol Chem 1999; 274:26968-77. [PMID: 10480909 DOI: 10.1074/jbc.274.38.26968] [Citation(s) in RCA: 91] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding a Drosophila glypican, and in GPC3, the gene for human glypican-3, implicate glypicans in the control of cell growth and division. So far, five members of the glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, we identified a sixth member of the glypican family. The glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to glypican-4 (identity of 63%). Expression of this protein in Namalwa cells shows a core protein of approximately 60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human glypican-6, contains nine exons. Like GPC5, the gene encoding glypican-5, GPC6 maps to chromosome 13q32. Clustering of the GPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, glypican-1, glypican-2, glypican-4, and glypican-6 appear to define a subfamily of glypicans, differing from the subfamily comprising so far glypican-3 and glypican-5. Northern blottings indicate that glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might in part be regulated by the presence of glypican-6 on the cell surface.
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Affiliation(s)
- M Veugelers
- Laboratory for Glycobiology, Center for Human Genetics, University of Leuven, B-3000, Belgium
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Gonzalez AD, Kaya M, Shi W, Song H, Testa JR, Penn LZ, Filmus J. OCI-5/GPC3, a glypican encoded by a gene that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome, induces apoptosis in a cell line-specific manner. J Cell Biol 1998; 141:1407-14. [PMID: 9628896 PMCID: PMC2132788 DOI: 10.1083/jcb.141.6.1407] [Citation(s) in RCA: 142] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/1997] [Revised: 05/06/1998] [Indexed: 02/07/2023] Open
Abstract
OCI-5/GPC3 is a member of the glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the cell surface through a glycosyl-phosphatidylinositol anchor. It has recently been shown that the OCI-5/GPC3 gene is mutated in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre- and postnatal overgrowth and various visceral and skeletal dysmorphisms. Some of these dysmorphisms could be the result of deficient growth inhibition or apoptosis in certain cell types during development. Here we present evidence indicating that OCI-5/GPC3 induces apoptosis in cell lines derived from mesothelioma (II14) and breast cancer (MCF-7). This induction, however, is cell line specific since it is not observed in NIH 3T3 fibroblasts or HT-29 colorectal tumor cells. We also show that the apoptosis-inducing activity in II14 and MCF-7 cells requires the anchoring of OCI-5/GPC3 to the cell membrane. The glycosaminoglycan chains, on the other hand, are not required. MCF-7 cells can be rescued from OCI-5/GPC3-induced cell death by insulin-like growth factor 2. This factor has been implicated in Beckwith-Wiedemann, an overgrowth syndrome that has many similarities with SGBS. The discovery that OCI-5/GPC3 is able to induce apoptosis in a cell line- specific manner provides an insight into the mechanism that, at least in part, is responsible for the phenotype of SGBS patients.
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Affiliation(s)
- A D Gonzalez
- Division of Cancer Biology Research, Sunnybrook Health Science Centre, Toronto, Ontario M4N 3M5, Canada
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Asundi VK, Keister BF, Carey DJ. Organization, 5'-flanking sequence and promoter activity of the rat GPC1 gene. Gene 1998; 206:255-61. [PMID: 9469940 DOI: 10.1016/s0378-1119(97)00594-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Glypicans are a member of a family of glycosylphosphatidylinositol anchored heparan sulfate proteoglycans that are expressed in cell and development specific patterns. Rat GPC1 cDNA probes were used to screen rat genomic libraries. Three overlapping genomic clones that contained the entire rat GPC1 gene were isolated. The rat GPC1 gene is approximately 15kb in length and consists of eight exons interrupted by introns of varying lengths. Two of the introns are quite short, with lengths of 41 and 43 base pairs. Each exon-intron splice junction exhibited the consensus splice site sequence. Exon 1 encodes the putative signal peptide and the serine residue of the first putative heparan sulfate attachment site. The last exon encodes the cluster of three potential COOH-terminal heparan sulfate attachment sites, the putative GPI anchor and polypeptide cleavage site, and the 3'-untranslated region including the polyadenylation signal. One of the genomic clones extended approximately 2.8 kb 5' of the exon 1 coding sequence, and is thus likely to contain sequences that regulate GPC1 gene expression. Sequence analysis of the 5'-flanking sequence revealed a lack of consensus TATA and CAAT boxes. A search for potential transcription factor binding sites revealed a number of such motifs, including Sp1 (GC box), NF-kappaB, and MyoD (E-box). This region of the rat GPC1 gene shows significant sequence homology to the 5'-flanking region of the human GPC3 gene. Functional promoter activity of the rat GPC1 sequence was demonstrated by its ability to drive the expression of a luciferase reporter gene in several cell types.
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Affiliation(s)
- V K Asundi
- Henry Hood, MD, Research Program, Sigfried and Janet Weis Center for Research, Penn State University College of Medicine, Danville, PA 17822, USA.
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