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ADAMTS-1 disrupts HGF/c-MET signaling and HGF-stimulated cellular processes in fibrosarcoma. Exp Cell Res 2018; 363:271-282. [PMID: 29355494 DOI: 10.1016/j.yexcr.2018.01.017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2017] [Revised: 12/21/2017] [Accepted: 01/12/2018] [Indexed: 01/10/2023]
Abstract
Extracellular matrix (ECM) serves as a reservoir for biologically active factors, such as growth factors and proteases that influence the tumor cell behavior. ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a secreted protease that has the ability to modify the ECM during physiological and pathological processes. Here, we analyzed the role played by ADAMTS-1 regulating HGF and TGF-β1 activities in the high-grade fibrosarcoma cell line (HT1080). We generated HT1080 and HEK293T cells overexpressing ADAMTS-1. HT1080 cells overexpressing ADAMTS-1 (HT1080-MPA) exhibited a significant decrease in cell proliferation and migration velocity, both in presence of HGF. We obtained similar results with ADAMTS-1-enriched conditioned medium from other cell type. However, ADAMTS-1 overexpression failed to affect TGF-β1 activity associated with HT1080 cell proliferation and migration velocity. Immunoblotting showed that ADAMTS-1 overexpression disturbs c-Met activation upon HGF stimulation. Downstream ERK1/2 and FAK signaling pathways are also influenced by this protease. Additionally, ADAMTS-1 decreased the size of the fibrosarcospheres, both under normal conditions and in the presence of HGF. Likewise, in presence of HGF, ADAMTS-1 overexpression in HT1080 disrupted microtumors formation in vivo. These microtumors, including individual cells, presented characteristics of non-invasive lesions (rounded morphology). Our results suggest that ADAMTS-1 is involved in regulating HGF-related functions on fibrosarcoma cells. This protease may then represent an endogenous mechanism in controlling the bioavailability of different growth factors that have a direct influence on tumor cell behavior.
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Parikh PK, Ghate MD. Recent advances in the discovery of small molecule c-Met Kinase inhibitors. Eur J Med Chem 2018; 143:1103-1138. [DOI: 10.1016/j.ejmech.2017.08.044] [Citation(s) in RCA: 76] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2017] [Revised: 08/03/2017] [Accepted: 08/21/2017] [Indexed: 12/17/2022]
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Nishimura Y, Hyuga S, Takiguchi S, Hyuga M, Itoh K, Hanawa T. Ephedrae herba stimulates hepatocyte growth factor-induced MET endocytosis and downregulation via early/late endocytic pathways in gefitinib-resistant human lung cancer cells. Int J Oncol 2016; 48:1895-906. [PMID: 26983447 DOI: 10.3892/ijo.2016.3426] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2015] [Accepted: 12/23/2015] [Indexed: 11/06/2022] Open
Abstract
The MET tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), are known to be overexpressed in a variety of malignant tumor cells, and are implicated in the development of gefitinib-resistance in human non-small cell lung cancer (NSCLC) cells. Ephedrae herba was previously reported to prevent HGF-induced cancer cell motility by directly suppressing HGF/MET signaling through the inhibition of MET tyrosine kinase, and treatment with its extract also considerably reduced MET protein levels. To further investigate the mechanism underlying the Ephedrae herba-induced inhibition of MET phosphorylation as well as its degradation and subsequent disappearance, we examined the effect of Ephedrae herba on HGF-stimulated MET endocytosis and downregulation via early/late endocytic pathways in an NSCLC cell line. Using immunofluorescence microscopy, we found that pretreatment of cells with Ephedrae herba extract dramatically changed the intracellular distribution of plasma membrane-associated MET, and that the resultant MET staining was distributed throughout the cytoplasm. Pretreatment of the cells with Ephedrae herba extract also led to the rapid loss of MET and phosphorylated (p)-MET in HGF-stimulated cells. In contrast, inefficient endocytic delivery of MET and p-MET from early to late endosomes was observed in the absence of Ephedrae herba extract, since considerable amounts of the internalized MET accumulated in the early endosomes and were not delivered to lysosomes up to 1 h after HGF-stimulation. Furthermore, large amounts of MET and p-MET that had accumulated in late endosomes of Ephedrae herba-pretreated cells after HGF stimulation were observed along with bafilomycin A1. Therefore, we inferred that degradation of MET occurred in the late endosome/lysosome pathway. Moreover, western blot analysis revealed the accelerated degradation of MET and p-MET proceeds in cells pretreated with Ephedrae herba extract. Collectively, our results suggest that some components of Ephedrae herba have a novel role in promoting HGF-stimulated MET and p-MET endocytosis followed by its downregulation, likely mediated by the early/late endocytic pathways.
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Affiliation(s)
- Yukio Nishimura
- Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
| | - Sumiko Hyuga
- Department of Clinical Research, Oriental Medicine Research Center of Kitasato University, Tokyo 108-8642, Japan
| | - Soichi Takiguchi
- Institute for Clinical Research, National Kyushu Cancer Center, Fukuoka 811-1395, Japan
| | - Masashi Hyuga
- Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo 158-8501, Japan
| | - Kazuyuki Itoh
- Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan
| | - Toshihiko Hanawa
- Department of Clinical Research, Oriental Medicine Research Center of Kitasato University, Tokyo 108-8642, Japan
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Aptamers Binding to c-Met Inhibiting Tumor Cell Migration. PLoS One 2015; 10:e0142412. [PMID: 26658271 PMCID: PMC4676636 DOI: 10.1371/journal.pone.0142412] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2015] [Accepted: 10/21/2015] [Indexed: 01/04/2023] Open
Abstract
The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.
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Pérez-Ramírez C, Cañadas-Garre M, Jiménez-Varo E, Faus-Dáder MJ, Calleja-Hernández MÁ. MET: a new promising biomarker in non-small-cell lung carcinoma. Pharmacogenomics 2015; 16:631-47. [PMID: 25893986 DOI: 10.2217/pgs.15.11] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Non-small-cell lung cancer (NSCLC) leads cancer-related deaths worldwide. Mutations in the kinase domain of the EGFR gene provide sensitivity to tyrosine kinase inhibitors (TKI) drugs. TKI show initial response rates over 75% in mutant EGFR-NSCLC patients, although most of these patients acquire resistance to EGFR inhibitors after therapy. EGFR-TKI resistance mechanisms include amplification in MET and its ligand, and also MET mutations. MET signaling dysregulation has been involved in tumor cell growth, survival, migration and invasion, angiogenesis and activation of several pathways, therefore representing an attractive target for anticancer drug development. In this review, we will discuss MET-related mechanisms of EGFR-TKI resistance in NSCLC, as well as the main drugs targeted to inhibit MET pathway.
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Affiliation(s)
- Cristina Pérez-Ramírez
- Pharmacogenetics Unit, UGC Provincial de Farmacia de Granada, Instituto de Investigación Biosanitaria de Granada, Complejo Hospitalario Universitario de Granada, Avda Fuerzas Armadas, 2, 18014 Granada, Spain
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Nishimura Y, Takiguchi S, Ito S, Itoh K. Evidence that depletion of the sorting nexin 1 by siRNA promotes HGF-induced MET endocytosis and MET phosphorylation in a gefitinib-resistant human lung cancer cell line. Int J Oncol 2013; 44:412-26. [PMID: 24297483 DOI: 10.3892/ijo.2013.2194] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2013] [Accepted: 10/22/2013] [Indexed: 11/05/2022] Open
Abstract
The receptor tyrosine kinase MET and its ligand HGF are known to be overexpressed in malignant tumor cells, and they have been implicated in gefitinib resistance in lung cancer cells. We recently found that sorting nexin 1 (SNX1), a protein that interacts with EGFR, exhibited negative regulation of EGFR trafficking out of early to late endosomes in gefitinib-resistant NSCLC cell lines. To investigate the role of SNX1 on HGF-stimulated MET endocytosis and its downregulation via the early/late endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in NSCLC cells. Using immunofluorescence, we found that the silencing of SNX1 by siRNA caused a dramatic change in the intracellular distribution of plasma membrane-associated MET and that the resultant MET staining was spread throughout the cytoplasm, and it co-localized well with the endocytosed Texas red-labeled transferrin in the siRNA-SNX1-transfected cells. We also found efficient MET phosphorylation and rapid endocytic delivery of phosphorylated MET from early endosomes to late endosomes in the siRNA-SNX1-transfected cells. By contrast, the siRNA-control transfected cells showed inefficient endocytic delivery of phosphorylated MET from early endosomes to late endosomes. Furthermore, large amounts of phosphorylated MET that had accumulated in late endosomes were seen even after 60 min of HGF-stimulation in the presence of bafilomycin A1, indicating that degradation of phosphorylated MET proceeds in a late endosome/lysosome pathway. Western blot analysis revealed that depletion of SNX1 by siRNA induced a maximal and dramatic increase in phosphorylated MET at 60 min, followed by an accelerated degradation of phosphorylated MET after HGF stimulation in the cells. Taken together, we suggest that SNX1 plays a suppressive role in the regulation of HGF-stimulated MET/phosphorylated MET endocytosis and downregulation via the early/late endocytic pathway in the gefitinib-resistant NSCLC cells.
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Affiliation(s)
- Yukio Nishimura
- Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
| | - Soichi Takiguchi
- Institute for Clinical Research, National Kyushu Cancer Center, Fukuoka 811-1395, Japan
| | - Shigeru Ito
- Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan
| | - Kazuyuki Itoh
- Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan
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Knockdown of RON receptor kinase delays but does not prevent tumor progression while enhancing HGF/MET signaling in pancreatic cancer cell lines. Oncogenesis 2013; 2:e76. [PMID: 24100611 PMCID: PMC3816215 DOI: 10.1038/oncsis.2013.36] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2013] [Revised: 06/07/2013] [Accepted: 08/13/2013] [Indexed: 12/14/2022] Open
Abstract
In this study, the role of RON (receptor originated from nantes) in tumor progression was further investigated in context with MET expression and activity. RON and MET expressions were not detected in an immortalized normal human pancreas cell line (HPNE), but were co-expressed in five of seven pancreatic ductal adenocarcinoma (PDAC) cell lines (PANC-1, BxPC-3, Capan-2, CFPAC-1 and AsPC-1). RON expression was knocked down by an shRNA approach in two PDAC cell lines (BxPC-3 and CFPAC-1) that co-express MET. Knockdown of RON significantly inhibited cell growth, clonogenicity and macrophage stimulating protein (MSP), RON ligand induced invasion by in vitro assays and significantly inhibited tumor growth (P<0.001) and metastasis (P<0.009) in an orthotopic pancreatic cancer mouse model at week 7. However, by week 9, the mice implanted with RON knockdown cells had developed similar size primary tumors and metastases compared with that seen in the control group at week 7. Western blotting and immunohistochemistry analyses showed that MET remains highly expressed in cells and tumor tissues where RON was knocked down. Moreover, knockdown of RON did not prevent hepatocyte growth factor (HGF) stimulated invasion in in vitro Matrigel assays. Treating cells with MSP induced the transphosphorylation of MET, suggesting that signaling may be modulated by relative levels of RON and MET receptors and their corresponding ligands. To this point, HGF treatment of RON knockdown cells caused an increase in intensity and duration of MET signaling, suggesting that MET signaling may compensate for loss of RON signaling. Treatment of cells with an MET inhibitor, PHA-665752, had minimal effects on inhibiting cell growth but significantly inhibited cell invasion induce by ligands for either MET or RON. These results suggest that HGF/MET signaling may have a more important role in tumor cell invasion and metastasis rather than in tumor cell proliferation. This study indicates that specific inhibition of RON delays but does not prevent progression of PDAC. Moreover, specific signaling may be modulated by the interaction of RON and MET receptors. This dynamic interaction of RON and MET in pancreatic cancer cells suggests that dual targeting of both RON and MET will be preferable to inhibition of either target alone.
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Okoshi R, Shu CL, Ihara S, Fukui Y. Scattering of MCF7 cells by heregulin ß-1 depends on the MEK and p38 MAP kinase pathway. PLoS One 2013; 8:e53298. [PMID: 23308187 PMCID: PMC3538754 DOI: 10.1371/journal.pone.0053298] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2012] [Accepted: 11/30/2012] [Indexed: 12/29/2022] Open
Abstract
Heregulin (HRG) β1 signaling promotes scattering of MCF7 cells by inducing breakdown of adherens and tight junctions. Here, we show that stimulation with HRG-β1 causes the F-actin backbone of junctions to destabilize prior to the loss of adherent proteins and scattering of the cells. The adherent proteins dissociate and translocate from cell–cell junctions to the cytosol. Moreover, using inhibitors we show that the MEK1 pathway is required for the disappearance of F-actin from junctions and p38 MAP kinase activity is essential for scattering of the cells. Upon treatment with a p38 MAP kinase inhibitor, adherens junction complexes immediately reassemble, most likely in the cytoplasm, and move to the plasma membrane in cells dissociated by HRG-β1 stimulation. Subsequently, tight junction complexes form, most likely in the cytoplasm, and move to the plasma membrane. Thus, the p38 MAP kinase inhibitor causes a re-aggregation of scattered cells, even in the presence of HRG-β1. These results suggest that p38 MAP kinase signaling to adherens junction proteins regulates cell aggregation, providing a novel understanding of the regulation of cell–cell adhesion.
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Affiliation(s)
- Rintaro Okoshi
- Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan, Republic of China
| | - Chung-Li Shu
- Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan, Republic of China
| | - Sayoko Ihara
- Division of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan
| | - Yasuhisa Fukui
- Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan, Republic of China
- Laboratory of Signal Transduction, Hoshi University, Tokyo, Japan
- * E-mail:
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McCleese JK, Bear MD, Kulp SK, Mazcko C, Khanna C, London CA. Met interacts with EGFR and Ron in canine osteosarcoma. Vet Comp Oncol 2011; 11:124-39. [PMID: 22235915 DOI: 10.1111/j.1476-5829.2011.00309.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2011] [Revised: 10/26/2011] [Accepted: 11/05/2011] [Indexed: 12/24/2022]
Abstract
The receptor tyrosine kinase (RTK) Met is known to be over-expressed in canine osteosarcoma (OSA). In human cancers, the RTKs Met, epidermal growth factor receptor (EGFR) and Ron are frequently co-expressed and engage in heterodimerization, altering signal transduction and promoting resistance to targeted therapeutics. We found that EGFR and Ron are expressed in canine OSA cell lines and primary tissues, EGFR and Ron are frequently phosphorylated in OSA tumour samples, and Met is co-associated with EGFR and Ron in canine OSA cell lines. Transforming growth factor alpha (TGFα) and hepatocyte growth factor (HGF) stimulation induced amplification of ERK1/2 and STAT3 phosphorylation in OSA cells and Met was phosphorylated following TGFα stimulation providing evidence for receptor cross-talk. Lastly, treatment of OSA cells with combined gefitinib and crizotinib inhibited cell proliferation in an additive manner. Together, these data support the notion that Met, EGFR and Ron interact in OSA cells and as such, may represent viable targets for therapeutic intervention.
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Affiliation(s)
- J K McCleese
- Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA
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Kenyon C. The first long-lived mutants: discovery of the insulin/IGF-1 pathway for ageing. Philos Trans R Soc Lond B Biol Sci 2011; 366:9-16. [PMID: 21115525 PMCID: PMC3001308 DOI: 10.1098/rstb.2010.0276] [Citation(s) in RCA: 228] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Inhibiting insulin/IGF-1 signalling extends lifespan and delays age-related disease in species throughout the animal kingdom. This life-extension pathway, the first to be defined, was discovered through genetic studies in the small roundworm Caenorhabditis elegans. This discovery is described here.
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Cai YR, Zhang HQ, Zhang ZDE, Mu J, Li ZH. Detection of MET and SOX2 amplification by quantitative real-time PCR in non-small cell lung carcinoma. Oncol Lett 2010; 2:257-264. [PMID: 22866074 DOI: 10.3892/ol.2010.229] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2010] [Accepted: 12/09/2010] [Indexed: 01/15/2023] Open
Abstract
Non-small cell lung carcinoma is a leading cause of cancer-related death. Amplification of the two oncogenes MET and SOX2 is frequently encountered in non-small-cell lung carcinoma. This study aimed to use real-time quantitative PCR to assess the correlation of MET and SOX2 amplification with clinicopathological factors. This study was conducted using 115 tissue samples including 57 squamous cell carcinomas (SCCs), 50 adenocarcinomas (ADCs) and 8 adenosquamous carcinomas (ADSCs). A total of 67 patients (58.3%) had a history of smoking. Our results showed that the frequency of MET amplification in SCCs was significantly higher compared to ADCs (χ(2)=8.0, P=0.005). SOX2 showed a markedly preferential amplification in SCCs compared to ADCs in the smoking group cases (P=0.014). Lymph node invasion correlated with MET amplification in SCCs marginally more significantly compared to ADCs (P=0.02). The amplified MET occurred more frequently in SCCs compared to ADCs correlated to tumor dimension at a small scale (<5 cm) (P=0.01). No significant difference in SOX2 amplification was found with regards to lymph node metastasis or tumor dimension. SOX2 and MET amplifications were not associated with gender or age. However, MET amplification in SCCs among patients younger than 64 years of age was higher compared to ADCs and ADSCs (P=0.03). Among ADSCs, MET was not amplified among patients who had never been smokers or were younger than 64 years of age. Neither MET nor SOX2 were amplified in tumors with dimensions <5 cm and without lymph node invasion. Findings of this study showed that MET and SOX2 amplifications are more common in the SCCs of smokers. Moreover, MET amplification is intrinsic in SCCs particularly among smokers, with regards to tumor growth, lymph node invasion and negative correlation to SOX2 amplification. The incidence of discrepancy in the amplifications of MET and SOX2 in SCCs and ADCs suggests that the MET and SOX2 genes play different roles in SCC and ADC tumorigenesis, respectively, particularly among smokers.
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Affiliation(s)
- Yi-Ran Cai
- Department of Pathology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Beijing 101149, P.R. China
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Naing A, Kurzrock R, Adams LM, Kleha JF, Laubscher KH, Bonate PL, Weller S, Fitzgerald C, Xu Y, LoRusso PM. A comparison of the pharmacokinetics of the anticancer MET inhibitor foretinib free base tablet formulation to bisphosphate salt capsule formulation in patients with solid tumors. Invest New Drugs 2010; 30:327-34. [PMID: 20842406 DOI: 10.1007/s10637-010-9536-x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2010] [Accepted: 08/18/2010] [Indexed: 11/26/2022]
Abstract
PURPOSE This phase I, open-label, randomized, 2-part crossover study assessed the safety, pharmacokinetics and relative bioavailability of single doses of the anticancer MET inhibitor foretinib (formerly known as GSK1363089, EXEL-2880 and XL-880) free base tablet formulation compared to a bisphosphate salt capsule formulation (Part 1), and assessed the safety, efficacy, and pharmacokinetics of the bisphosphate salt capsule administered 3 times a week in cancer patients (Part 2). PATIENTS AND METHODS In Part 1, patients were randomized in a crossover manner to receive a single oral dose of foretinib formulated as a bisphosphate salt capsule (240 mg; 183 mg free base equivalent) followed one week later by a single dose of a free base tablet (180 mg), or vice versa where the treatment sequence was reversed. In Part 2, patients self-administered oral doses of bisphosphate salt capsules (200 mg) 3 times a week until disease progression. RESULTS Twelve patients with solid tumors were enrolled and completed Part 1, and 10 patients continued into Part 2. Most AEs were mild or moderate in severity. The most common drug-related AEs were fatigue, diarrhea, and nausea. The least-squares (LS) mean total area under the curve was 3144 and 3514 ng*h/mL for the free base tablet and bisphosphate salt capsule, respectively, with a ratio of 0.89 (90% confidence interval, CI: 0.69, 1.16). The LS mean maximal concentration (Cmax) was 81.6 and 98.5 ng/mL for the free base and bisphosphate salt, respectively, with a ratio of 0.83 (90% confidence interval, CI: 0.67, 1.02). The time to reach Cmax was ∼4 h for both formulations. The pharmacokinetics of foretinib were not clinically different between the 2 formulations. Of the 10 patients assessed for efficacy, 3 patients achieved stable disease. CONCLUSIONS Foretinib was well tolerated as single doses of both the free base and bisphosphate salt formulations. The pharmacokinetics and relative bioavailability of the 2 formulations were not clinically different. The bisphosphate salt formulation was well tolerated on a 3-times a week dosing schedule, and reached steady-state plasma concentration after 2 weeks.
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Affiliation(s)
- Aung Naing
- Division of Cancer Medicine, Department of Investigational Cancer Therapeutics, Unit 455, University of Texas MD Anderson Cancer Center, Houston, TX 77030-1402, USA.
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Belli C, Anand S, Tassi G, Fennell D, Mutti L. Translational therapies for malignant pleural mesothelioma. Expert Rev Respir Med 2010; 4:249-60. [PMID: 20406091 DOI: 10.1586/ers.10.17] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Malignant pleural mesothelioma is a highly invasive tumor arising from the mesothelial cells of serosal surfaces. Several chemotherapeutic agents have been tested for the treatment of this disease and doublet cisplatin with antifolates has been demonstrated to have significant efficacy in Phase III studies. However, the benefit of these treatments remains poor and the median survival time of patients is low, ranging between 9 and 17 months. Targeted therapies are being developed in oncology and emerging evidence suggests that they offer disease control in several tumors. This article reviews the knowledge on the malignant pleural mesothelioma molecular pathway and focuses on results of clinical trials conducted on this devastating disease.
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Affiliation(s)
- Carmen Belli
- Oncology Department, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy.
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The Ron receptor tyrosine kinase negatively regulates mammary gland branching morphogenesis. Dev Biol 2009; 333:173-85. [PMID: 19576199 DOI: 10.1016/j.ydbio.2009.06.028] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2008] [Revised: 06/05/2009] [Accepted: 06/24/2009] [Indexed: 11/23/2022]
Abstract
The Ron receptor tyrosine kinase is expressed in normal breast tissue and is overexpressed in approximately 50% of human breast cancers. Despite the recent studies on Ron in breast cancer, nothing is known about the importance of this protein during breast development. To investigate the functional significance of Ron in the normal mammary gland, we compared mammary gland development in wild-type mice to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of Ron (TK-/-). Mammary glands from RonTK-/- mice exhibited accelerated pubertal development including significantly increased ductal extension and branching morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK, which predominantly localized to the epithelium, were associated with increased branching morphogenesis. Additionally, purified RonTK-/- epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK-/- glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development.
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Abstract
The MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) have been implicated in transformation of a variety of malignancies. Chronic or dysregulated activation of the MET/HGF pathway may lead to increased cell growth, invasion, angiogenesis, and metastasis, reduced apoptosis, altered cytoskeletal functions and other biological changes. It has been suggested that ligand activated MET stimulation can be sufficient for a transforming phenotype. In addition, amplification and activation mutations (germline and/or somatic) within the tyrosine kinase domain, juxtamembrane domain, or semaphorin domain have been identified for MET. MET gain-of-function mutations lead to either deregulated or prolonged tyrosine kinase activity, which are instrumental to its transforming activity. A number of therapeutic strategies targeting ligand-dependent activation or the kinase domain have been employed to inhibit MET. The different structural requirements for activation of signaling events and biological functions regulated by MET will be summarized. Therapeutic targets and current pre-clinical and clinical approaches will be described. Targeting the HGF/MET pathway, alone or in combination with standard therapies, is likely to improve present therapies in MET-dependent malignancies.
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Scott A, Salgia R. Biomarkers in lung cancer: from early detection to novel therapeutics and decision making. Biomark Med 2008; 2:577-586. [PMID: 19802373 DOI: 10.2217/17520363.2.6.577] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Lung cancer remains a significant cause of mortality worldwide. While advances in therapy continue to be made, the overall prognosis for patients diagnosed with lung cancer remains poor. Historically, markers such as age, performance status and disease stage have been used to risk-stratify patients and guide therapeutic decisions. These parameters provide some useful information, but more sensitive markers are clearly needed. Molecular and genetic studies have identified several such markers, which appear to play critical roles in carcinogenesis and affect patient outcomes. This article reviews a number of biomarkers that have been identified in lung cancer, and their prognostic and predictive roles.
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Affiliation(s)
- April Scott
- University of Chicago, Department of Medicine, Section of Hematology/Oncology, and University of Chicago Cancer Research Center, 5841 S Maryland Avenue, Chicago, IL 60637, USA
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Ariztia EV, Lee CJ, Gogoi R, Fishman DA. The Tumor Microenvironment: Key to Early Detection. Crit Rev Clin Lab Sci 2008; 43:393-425. [PMID: 17050079 DOI: 10.1080/10408360600778836] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The tumor microenvironment plays an important role equal to the tumor cell population in the progression of cancer. Consisting of stromal fibroblasts, inflammatory cells, components of the vasculature, normal epithelia, and extracellular matrix, the surrounding environment interacts or "cross-talks" with tumor cells through the release of growth factors, cytokines, proteases, and other bioactive molecules. Tumor growth, formation of new vascular networks, evasion of the host immune system, and invasion and metastasis are processes that co-evolve and become finely optimized and regulated within the tumor microenvironment. However, relatively recent reports on three areas of study have come together to add new levels of complexity to the tumor microenvironment. These include ectodomain shedding of proteins, shedding of membrane-derived vesicles, and novel roles for phospholipids. These dynamic changes that take place in the tumor microenvironment provide new avenues for study and for the early detection of cancer, whereas proteomic technologies provide the means to detect these unique proteins and lipids. Here we review the evolving concepts of the tumor microenvironment that, together with advances in proteomic technologies, hold the promise to facilitate the detection of early-stage cancer.
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Affiliation(s)
- Edgardo V Ariztia
- Department of Obstetrics and Gynecology, New York University School of Medicine, New York, NY 10016, USA
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19
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Cipriani NA, Abidoye OO, Vokes E, Salgia R. MET as a target for treatment of chest tumors. Lung Cancer 2008; 63:169-79. [PMID: 18672314 DOI: 10.1016/j.lungcan.2008.06.011] [Citation(s) in RCA: 96] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2008] [Accepted: 06/15/2008] [Indexed: 12/11/2022]
Abstract
The receptor tyrosine kinase MET has been studied of a large variety of human cancers, including lung and mesothelioma. The MET receptor and its ligand HGF (hepatocyte growth factor) play important roles in cell growth, survival and migration, and dysregulation of the HGF-MET pathway leads to oncogenic changes including tumor proliferation, angiogenesis and metastasis. In small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and malignant pleural mesothelioma (MPM), MET is dysregulated via overexpression, constitutive activation, gene amplification, ligand-dependent activation, mutation or epigenetic mechanisms. New drugs targeted against MET and HGF are currently being investigated in vitro and in vivo, with promising results. These drugs function at a variety of steps within the HGF-MET pathway, including MET expression at the RNA or protein level, the ligand-receptor interaction, and tyrosine kinase function. This paper will review the structure, function, mechanisms of tumorigenesis, and potential for therapeutic inhibition of the MET receptor in lung cancer and mesothelioma.
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Affiliation(s)
- Nicole A Cipriani
- Department of Medicine, University of Chicago Medical Center, Chicago, IL 60637, USA
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20
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Sun Q, Nawabi-Ghasimi F, Basile JR. Semaphorins in vascular development and head and neck squamous cell carcinoma-induced angiogenesis. Oral Oncol 2008; 44:523-31. [DOI: 10.1016/j.oraloncology.2007.10.005] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2007] [Revised: 10/28/2007] [Accepted: 10/10/2007] [Indexed: 01/01/2023]
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21
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Sattler M, Salgia R. c-Met and hepatocyte growth factor: Potential as novel targets in cancer therapy. Curr Oncol Rep 2007; 9:102-8. [PMID: 17288874 DOI: 10.1007/s11912-007-0005-4] [Citation(s) in RCA: 95] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Receptor tyrosine kinases have come to fruition as therapeutic targets in a variety of malignancies. In this group of targets, the c-Met receptor tyrosine kinase plays an important role in increased cell growth, reduced apoptosis, altered cytoskeletal function, increased metastasis, and other biologic changes. The ligand for c-Met is hepatocyte growth factor (HGF), also known as scatter factor. Met is overexpressed and mutated in a variety of malignancies, among which germline mutations are of particular interest. Most mutations of Met have been found in the juxtamembrane, the tyrosine kinase, and the semaphorin domain. Met gain-of-function mutations lead to deregulated or prolonged tyrosine kinase activity, which is instrumental to its transforming activity. This review summarizes the biologic functions regulated by Met and its structural requirements as well as related developments in targeted therapy. Treatment approaches, including antagonism of HGF binding to Met, targeting of RNA and the Met protein, and inhibition of the tyrosine kinase domain of Met, are highlighted. Targeting of the HGF/Met pathway, alone or in combination with standard therapies, is likely to improve current therapies in Met-dependent malignancies.
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Affiliation(s)
- Martin Sattler
- Department of Medicine, Pritzker School of Medicine, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA
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22
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Abstract
Various cytokines and soluble growth factors upon interaction with their membrane receptors are responsible for inducing cellular proliferation, differentiation, movement, and protection from anoikis (a planned suicide activated by normal cells in absence of attachment to neighboring cells or extracellular matrix (EMC)). Among those soluble factors a major position is exerted by hepatocyte growth factor (HGF) together with its receptor MET and macrophage-stimulating protein (MSP) in cooperation with its receptor RON.
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Affiliation(s)
- Silvia Benvenuti
- Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, Candiolo (Torino), Italy
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23
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Abstract
Retroviruses have played profound roles in our understanding of the genetic and molecular basis of cancer. Jaagsiekte sheep retrovirus (JSRV) is a simple retrovirus that causes contagious lung tumors in sheep, known as ovine pulmonary adenocarcinoma (OPA). Intriguingly, OPA resembles pulmonary adenocarcinoma in humans, and may provide a model for this frequent human cancer. Distinct from the classical mechanisms of retroviral oncogenesis by insertional activation of or virus capture of host oncogenes, the native envelope (Env) structural protein of JSRV is itself the active oncogene. A major pathway for Env transformation involves interaction of the Env cytoplasmic tail with as yet unidentified cellular adaptor(s), leading to the activation of PI3K/Akt and MAPK signaling cascades. Another potential mechanism involves the cell-entry receptor for JSRV, Hyaluronidase 2 (Hyal2), and the RON receptor tyrosine kinase, but the exact roles of these proteins in JSRV Env transformation remain to be better understood. Recently, a mouse model of lung cancer induced by JSRV Env has been developed, and the tumors in mice resemble those seen in sheep infected with JSRV and in humans. In this review, we summarize recent progress in our understanding the molecular mechanisms of oncogenic transformation by JSRV Env protein, and discuss the relevance to human lung cancer.
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Affiliation(s)
- S-L Liu
- Department of Microbiology and Immunology, McGill University, Montreal, Canada.
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24
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Abstract
Malignant mesothelioma (MM) is an uncommon tumor with high mortality and morbidity rates. It arises from mesothelial cells that line the pleural, pericardial, peritoneal, and testicular cavities. This is a disease with an indolent course because tumors arise 20 to 40 years after exposure to an inciting agent. Extensive research has shown that mesothelial cells are transformed into MM cells through various chromosomal and cellular pathway defects. These changes alter the normal cells' ability to survive, proliferate, and metastasize. This article discusses the alterations that occur in transforming normal mesothelial cells into MM. It also details some of the signal transduction pathways that seem to be important in MM with the potential for novel targeted therapeutics.
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Affiliation(s)
- Evan Pisick
- Department of Medicine, Section of Hematology/Oncology, Tufts-New England Medical Center, Boston, MA, USA
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25
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Sattler M, Ma PC, Salgia R. Therapeutic targeting of the receptor tyrosine kinase Met. Cancer Treat Res 2006; 119:121-38. [PMID: 15164876 DOI: 10.1007/1-4020-7847-1_7] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Affiliation(s)
- Martin Sattler
- Department of Medical Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
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26
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Pongchairerk U, Guan JL, Leardkamolkarn V. Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1. World J Gastroenterol 2005; 11:5845-52. [PMID: 16270396 PMCID: PMC4479687 DOI: 10.3748/wjg.v11.i37.5845] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.
METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunop-recipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.
RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations. FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.
CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.
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Affiliation(s)
- Urai Pongchairerk
- Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
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27
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Pillich RT, Scarsella G, Risuleo G. Reduction of apoptosis through the mitochondrial pathway by the administration of acetyl-l-carnitine to mouse fibroblasts in culture. Exp Cell Res 2005; 306:1-8. [PMID: 15878327 DOI: 10.1016/j.yexcr.2005.01.019] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2004] [Revised: 12/22/2004] [Accepted: 01/03/2005] [Indexed: 10/25/2022]
Abstract
It is shown in literature that stress, such as deprivation of trophic factors and hypoxia, induces apoptosis in cultured cells and in tissues. In light of these results, we explored the possibility of protecting cells from programmed death by improving the metabolism of the mitochondrion. To this end, acetyl-L-carnitine was administered at various concentrations under conditions of serum deprivation. The choice of this drug was based on the accepted notion that acetyl-L-carnitine is able to stabilize mitochondrial membranes and to increase the supply of energy to the organelle. The results presented here indicate that the drug protects cells from apoptotic death: this is demonstrated by a lower positivity to the TUNEL reaction and by a strong reduction of the apoptotic DNA ladder in serum-deprived cells. The involvement of the mitochondrial apoptotic pathway was assessed by cytochrome C release and immunoreactivity to caspase 3. Moreover, acetyl-L-carnitine stimulates cell proliferation.
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Affiliation(s)
- Rudolf Tito Pillich
- Dipartimento di Biologia Cellulare e dello Sviluppo, Italy; Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, P.le A. Moro, 5-00185 Roma, Italy
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28
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Yokoyama N, Ischenko I, Hayman MJ, Miller WT. The C terminus of RON tyrosine kinase plays an autoinhibitory role. J Biol Chem 2005; 280:8893-900. [PMID: 15632155 DOI: 10.1074/jbc.m412623200] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
RON is a receptor tyrosine kinase in the MET family. We have expressed and purified active RON using the Sf9/baculovirus system. The constructs used in this study comprise the kinase domain alone and the kinase domain plus the C-terminal region. The construct containing the kinase domain alone has a higher specific activity than the construct containing the kinase and C-terminal domains. Purified RON undergoes autophosphorylation, and the exogenous RON C terminus serves as a substrate. Peptides containing a dityrosine motif derived from the C-terminal tail inhibit RON in vitro or when delivered into intact cells, consistent with an autoinhibitory mechanism. Phenylalanine substitutions within these peptides increase the inhibitory potency. Moreover, introduction of these Phe residues into the dityrosine motif of the RON kinase leads to a decrease in kinase activity. Taken together, our data suggest a model in which the C-terminal tail of RON regulates kinase activity via an interaction with the kinase catalytic domain.
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Affiliation(s)
- Noriko Yokoyama
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, USA
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29
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Kalnina Z, Zayakin P, Silina K, Linē A. Alterations of pre-mRNA splicing in cancer. Genes Chromosomes Cancer 2005; 42:342-57. [PMID: 15648050 DOI: 10.1002/gcc.20156] [Citation(s) in RCA: 141] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Recent genomewide analyses of alternative splicing (AS) indicate that up to 70% of human genes may have alternative splice forms, suggesting that AS together with various posttranslational modifications plays a major role in the production of proteome complexity. Splice-site selection under normal physiological conditions is regulated in the developmental stage in a tissue type-specific manner by changing the concentrations and the activity of splicing regulatory proteins. Whereas spliceosomal errors resulting in the production of aberrant transcripts rarely occur in normal cells, they seem to be an intrinsic property of cancer cells. Changes in splice-site selection have been observed in various types of cancer and may affect genes implicated in tumor progression (for example, CD44, MDM2, and FHIT) and in susceptibility to cancer (for example, BRCA1 and APC). Splicing defects can arise from inherited or somatic mutations in cis-acting regulatory elements (splice donor, acceptor and branch sites, and exonic and intronic splicing enhancers and silencers) or variations in the composition, concentration, localization, and activity of regulatory proteins. This may lead to altered efficiency of splice-site recognition, resulting in overexpression or down-regulation of certain splice variants, a switch in splice-site usage, or failure to recognize splice sites correctly, resulting in cancer-specific splice forms. At least in some cases, changes in splicing have been shown to play a functionally significant role in tumorigenesis, either by inactivating tumor suppressors or by gain of function of proteins promoting tumor development. Moreover, cancer-specific splicing events may generate novel epitopes that can be recognized by the host's immune system as cancer specific and may serve as targets for immunotherapy. Thus, the identification of cancer-specific splice forms provides a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention.
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Affiliation(s)
- Zane Kalnina
- Biomedical Research and Study Centre, University of Latvia, Ratsupites St 1, LV-1067 Riga, Latvia
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30
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Higuchi T, Orita T, Katsuya K, Yamasaki Y, Akiyama K, Li H, Yamamoto T, Saito Y, Nakamura M. MUC20 suppresses the hepatocyte growth factor-induced Grb2-Ras pathway by binding to a multifunctional docking site of met. Mol Cell Biol 2004; 24:7456-68. [PMID: 15314156 PMCID: PMC506992 DOI: 10.1128/mcb.24.17.7456-7468.2004] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
A cDNA encoding a novel mucin protein, MUC20, was isolated as a gene that is up-regulated in the renal tissues of patients with immunoglobulin A nephropathy. We demonstrate here that the C terminus of MUC20 associates with the multifunctional docking site of Met without ligand activation, preventing Grb2 recruitment to Met and thus attenuating hepatocyte growth factor (HGF)-induced transient extracellular signal-regulated kinase-1 and -2 activation. Production of MUC20 reduced HGF-induced matrix metalloproteinase expression and proliferation, which require the Grb2-Ras pathway, whereas cell scattering, branching morphogenesis, and survival via the Gab1/phosphatidylinositol 3-kinase (PI3K) pathways was not affected. Thus, MUC20 reduces HGF-induced activation of the Grb2-Ras pathway but not the Gab1/PI3K pathways. We further demonstrate that the cytoplasmic domain of MUC20 has the ability to oligomerize and that the oligomerization augments its affinity for Met. Taken together, these results suggest that MUC20 is a novel regulator of the Met signaling cascade which has a role in suppression of the Grb2-Ras pathway.
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Affiliation(s)
- Toshio Higuchi
- Central Pharmaceutical Research Institute, Pharmaceutical Frontier Research Laboratories, Japan Tobacco Inc., Yokohama, 236-0004, Japan
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31
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Basile JR, Barac A, Zhu T, Guan KL, Gutkind JS. Class IV semaphorins promote angiogenesis by stimulating Rho-initiated pathways through plexin-B. Cancer Res 2004; 64:5212-24. [PMID: 15289326 DOI: 10.1158/0008-5472.can-04-0126] [Citation(s) in RCA: 190] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
The semaphorins are a large family of secreted and cell surface proteins that provide attractive and repulsive cues for axon guidance during neuronal development. Semaphorins share a conserved NH(2)-terminal Sema domain with their receptors, the plexins, which mediate neuronal cell adhesion, axon guidance, and maintenance of established neuronal pathways in the adult. Both semaphorins and plexins share structural homology with the extracellular domain of c-Met, a member of the scatter factor family of receptors. However, the highly conserved cytoplasmic region of plexins has no homology with the c-Met tyrosine kinase or with any other known protein. Using a recently developed antibody and RNA analysis, we found that high levels of plexin-B1 are expressed in endothelial cells. Whereas c-Met, with which plexin-B1 can interact, is known to be a potent promoter of angiogenesis, the effects of semaphorin-mediated plexin activation in endothelial cells are still poorly understood. Here, we examined the role of plexin-B1 activation in angiogenesis using a purified, secreted form of its ligand, Semaphorin 4D (Sema4D). Sema4D potently induced chemotaxis and tubulogenesis in endothelial cells and enhanced blood vessel formation in an in vivo mouse model. Interestingly, responses to Sema4D did not require c-Met activation. Instead, the use of chimeric plexin-B1 receptors, Rho inhibitors, and lentiviral gene delivery of interfering molecules revealed that these proangiogenic effects are dependent on a COOH-terminal PDZ-binding motif of plexin-B1, which binds two guanine nucleotide exchange factors for the small GTPase Rho, PDZ-RhoGEF and LARG, and are mediated by the activation of Rho-initiated pathways.
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Affiliation(s)
- John R Basile
- Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research/NIH, 30 Convent Drive, Bethesda, MD 20892, USA
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32
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Miyata Y, Ashida S, Nakamura T, Mochizuki Y, Koga S, Kanetake H, Shuin T, Kanda S. Overexpression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo. Biochem Biophys Res Commun 2003; 302:892-7. [PMID: 12646256 DOI: 10.1016/s0006-291x(03)00281-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
We examined the role of increased expression of HGFR kinase in in vivo growth of renal carcinoma. Human renal carcinoma cell line, ACHN cells, was transfected with plasmid encoding wild-type HGFR gene to generate cell lines with increased HGFR protein. ACHN cells with elevated HGFR expression, denoted clones 8 and 10, respectively, showed higher basal kinase activities of HGFR and PI3-kinase than those of empty-vector (mock)-transfected cells. Clone 8 and 10 cells grew similar to mock cells in culture. In mice, tumors of these clones grew more rapidly than those of mock cells. Microvessel density of clone 8 or 10 tumors was higher than that of mock tumors. Clone 8 and 10 cells secreted vascular endothelial growth factor-A (VEGF-A) more than mock cells and the secretion was PI3-kinase inhibitor, LY294002-sensitive. Anti-VEGF-A neutralizing antibody significantly inhibited tumor growth of clones 8 and 10 in mice. These results indicate for the first time that overexpression of HGFR tyrosine kinase in renal carcinoma cells participates in rapid tumor growth in vivo.
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Affiliation(s)
- Yasuyoshi Miyata
- Department of Urology, Nagasaki University Graduate School of Medical Science, Nagasaki, Japan
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33
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Tanimura S, Nomura K, Ozaki KI, Tsujimoto M, Kondo T, Kohno M. Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility. J Biol Chem 2002; 277:28256-64. [PMID: 12032150 DOI: 10.1074/jbc.m202866200] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We examined the signaling pathway by which hepatocyte growth factor (HGF) induces cell motility, with special focus on the role of extracellular signal-regulated kinase (ERK) in the nucleus. We used Madin-Darby canine kidney cells overexpressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked, and sustained activation and rapid nuclear accumulation of ERK1/2, but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059 treatment of the cells 30 min after HGF stimulation abolishes the HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by the expression of an inactive form of MKP-3 cytoplasmic phosphatase inhibits the cell motility response. Although epidermal growth factor stimulation of the cells induces the activation and nuclear accumulation of ERK1/2, it does not induce the prolonged nuclear retention of the activated ERK1/2, and fails to induce cell motility. In the nucleus, activated ERK1/2 continuously phosphorylate Elk-1, leading to the prolonged expression of c-fos, which results in the expression of several genes such as matrix metalloproteinase (mmp)-9; MMP-9 activity is required for the induction of the cell motility response. Our results indicate that the sustained activity of ERK1/2 in the nucleus is required for the induction of HGF-induced cell motility.
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Affiliation(s)
- Susumu Tanimura
- Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan
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Maulik G, Shrikhande A, Kijima T, Ma PC, Morrison PT, Salgia R. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev 2002; 13:41-59. [PMID: 11750879 DOI: 10.1016/s1359-6101(01)00029-6] [Citation(s) in RCA: 299] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Receptor tyrosine kinases have become important therapeutic targets for anti-neoplastic molecularly targeted therapies. c-Met is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. Stimulation of c-Met via its ligand hepatocyte growth factor also known as scatter factor (HGF/SF), leads to a plethora of biological and biochemical effects in the cell. There has been considerable knowledge gained on the role of c-Met-HGF/SF axis in normal and malignant cells. This review summarizes the structure of c-Met and HGF/SF and their family members. Since there are known mutations of c-Met in solid tumors, particularly in papillary renal cell carcinoma, we have summarized the various mutations and over-expression of c-Met known thus far. Stimulation of c-Met can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. The biological functions altered by c-Met are quite unique and described in detail. Along with biological functions, various signal transduction pathways, including the cytoskeleton are altered with the activation of c-Met-HGF/SF loop. We have recently shown the phosphorylation of focal adhesion proteins, such as paxillin and p125FAK in response to c-Met stimulation in lung cancer cells, and this is detailed here. Finally, c-Met when mutated or over-expressed in malignant cells serves as an important therapeutic target and the most recent data in terms of inhibition of c-Met and downstream signal transduction pathways is summarized.
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Affiliation(s)
- Gautam Maulik
- Department of Medicine, Division of Adult Oncology, Lowe Center for Thoracic Oncology, Binney Street, Boston, MA 02115, USA
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35
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Nakamura T, Kanda S, Yamamoto K, Kohno T, Maeda K, Matsuyama T, Kanetake H. Increase in hepatocyte growth factor receptor tyrosine kinase activity in renal carcinoma cells is associated with increased motility partly through phosphoinositide 3-kinase activation. Oncogene 2001; 20:7610-23. [PMID: 11753639 DOI: 10.1038/sj.onc.1204975] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2001] [Revised: 08/28/2001] [Accepted: 09/13/2001] [Indexed: 11/08/2022]
Abstract
Dysregulated cell motility is one of the major characteristics of invasion and metastatic potentials of malignant tumor cells. Here, we examined the hepatocyte growth factor (HGF)-induced cell motility of two human renal carcinoma cell lines, ACHN and VMRC-RCW. Scattering and migration was induced in ACHN in an HGF-dependent manner, whereas they were maintained in VMRC-RCW even in the absence of HGF. In VMRC-RCW, HGF receptor (HGFR) tyrosine kinase was constitutively active, and sequence analysis showed N375S, A1209G and V1290L mutations. However, transfection experiments using porcine aortic endothelial (PAE) cells demonstrated that no single mutation or combination of two or three mutations caused HGF-independent constitutive activation. Conversely, the expressed amount of receptor protein had a pivotal role in the basal kinase activity. With respect to downstream signaling molecules of HGFR in ACHN or VMRC-RCW, the Ras-MAPK pathway was downregulated, whereas phosphoinositide 3-kinase (PI3-kinase) was not further activated by HGF-treatment in VMRC-RCW cells. The PI3-kinase inhibitors, wortmannin and LY294002 strongly inhibited spontaneous migration of VMRC-RCW. One transfected PAE cell line with massive overexpression of HGFR demonstrated scattered morphology and increased PI3-kinase activity in association with increased motility, which was partially inhibited by LY294002. Taken together, our results indicate that the overexpression of HGFR causes increase in cellular motility and PI3-kinase shows the important contribution on the increased motility of renal carcinoma cells.
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Affiliation(s)
- T Nakamura
- Department of Urology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
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36
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Helou K, Walentinsson A, Kost-Alimova M, Levan G. Hgfr/Met oncogene acts as target for gene amplification in DMBA-induced rat sarcomas: free chromatin fluorescence in situ hybridization analysis of amplicon arrays in homogeneously staining regions. Genes Chromosomes Cancer 2001; 30:416-20. [PMID: 11241796 DOI: 10.1002/1098-2264(2001)9999:9999<::aid-gcc1109>3.0.co;2-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
Analysis of chromosome rearrangements in tumors is an important means for revealing genetic pathways underlying tumorigenesis and tumor progression. In five of 17 DMBA-induced rat sarcomas, cytogenetic analysis had disclosed homogeneously staining regions (hsrs), which are generally accepted to be cytogenetic signs of gene amplification. Using comparative genomic hybridization (CGH), regional increases in DNA copy number of the proximal part of rat chromosome (RNO) 4 were detected in four of the tumors harboring hsrs. Amplification of the Hgfr/Met oncogene, located at RNO4q21.2, was detected by fluorescence in situ hybridization (FISH) in five tumors. In four of them, a number of flanking genes located in the close vicinity of Hgfr/Met, including Cav1 (q21.1), Wnt2 (q21.2-q21.3), and Cftr (q21.3), also were amplified, though amplification was seen in a lower fraction of the cells than was Hgfr/Met amplification. In the fifth tumor (BL150T), Hgfr/Met was amplified in all cells and was the sole amplified gene of those tested. In addition, the Hgfr/Met FISH signals in BL150T were tightly clustered and formed compact and intense spots compared with the signals seen in the other four tumors. Application of the free chromatin FISH technique to BL150T showed that the genomic Hgfr/Met probe stained the extended chromatin fibers of up to 1.5 Mb with an almost uninterrupted signal, indicating that the BL150T amplicon was build up solely of Hgfr/Met gene sequences. Our results suggest that the Hgfr/Met oncogene is the primary target for amplification in a subset of rat DMBA sarcomas.
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Affiliation(s)
- K Helou
- Department of Cell and Molecular Biology-Genetics, Lundberg Laboratory, Göteborg University, Sweden.
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Abstract
As the sequencing of the human genome is completed by the Human Genome Project, the analysis of this rich source of information will illuminate many areas in medicine and biology. The protein tyrosine kinases are a large multigene family with particular relevance to many human diseases, including cancer. A search of the human genome for tyrosine kinase coding elements identified several novel genes and enabled the creation of a nonredundant catalog of tyrosine kinase genes. Ninety unique kinase genes can be identified in the human genome, along with five pseudogenes. Of the 90 tyrosine kinases, 58 are receptor type, distributed into 20 subfamilies. The 32 nonreceptor tyrosine kinases can be placed in 10 subfamilies. Additionally, mouse orthologs can be identified for nearly all the human tyrosine kinases. The completion of the human tyrosine kinase family tree provides a framework for further advances in biomedical science.
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Affiliation(s)
- D R Robinson
- Department of Biological Chemistry, UC Davis School of Medicine, UC Davis Cancer Center, Sacramento, California, CA 95817, USA
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Martoglio AM, Tom BDM, Starkey M, Corps AN, Charnock-Jones DS, Smith SK. Changes in Tumorigenesis- and Angiogenesis-related Gene Transcript Abundance Profiles in Ovarian Cancer Detected by Tailored High Density cDNA Arrays. Mol Med 2000. [DOI: 10.1007/bf03402191] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
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Follenzi A, Bakovic S, Gual P, Stella MC, Longati P, Comoglio PM. Cross-talk between the proto-oncogenes Met and Ron. Oncogene 2000; 19:3041-9. [PMID: 10871856 DOI: 10.1038/sj.onc.1203620] [Citation(s) in RCA: 150] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Scatter Factors control a complex genetic program known as 'invasive growth'. HGF (Scatter factor 1) and MSP (Scatter Factor 2) bind to tyrosine kinase receptors encoded by the proto-oncogenes MET and RON. Using the appropriate 'kinase inactive' mutant receptors, we show that ligand-induced activation of Met results in transphosphorylation of Ron, and vice versa. Transphosphorylation is direct, as it occurs in Met or Ron receptors lacking the docking sites for signal transducers. Phosphate groups are transferred to the tyrosine phosphorylation sites responsible both for kinase up-regulation (Met: Y1234/Y1235 and Ron: Y1238/Y1239) and for generation of signal transducer docking sites (Met: Y1349/Y1356 and Ron Y1353/Y1360). The transphosphorylation specifically takes place for the receptor subfamily, as it is not observed between Met or Ron and ErbB1, ErbB2 or TrkA. Cross-linking experiments show that non-covalent Met-Ron complexes are present on the cell surface, before ligand-induced dimerization. Co-expression of a kinase inactive Ron receptor with naturally-occurring oncogenic Met mutants suppresses the transforming phenotype, suggesting a dominant negative role for the inefficient kinase partner. These data show that, while specific for their ligands, scatter factor receptors cross-talk and cooperate in intracellular signaling.
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Affiliation(s)
- A Follenzi
- Institute for Cancer Research and Treatment (IRCC), University of Torino, School of Medicine 10060, Candiolo, Italy
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Wallenius V, Hisaoka M, Helou K, Levan G, Mandahl N, Meis-Kindblom JM, Kindblom LG, Jansson JO. Overexpression of the hepatocyte growth factor (HGF) receptor (Met) and presence of a truncated and activated intracellular HGF receptor fragment in locally aggressive/malignant human musculoskeletal tumors. THE AMERICAN JOURNAL OF PATHOLOGY 2000; 156:821-9. [PMID: 10702398 PMCID: PMC1876854 DOI: 10.1016/s0002-9440(10)64950-4] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 11/29/1999] [Indexed: 01/08/2023]
Abstract
Enhanced hepatocyte growth factor (HGF) receptor (Met) signaling has been suggested to play an important role in the development and progression of various epithelial and nonepithelial tumors. N-terminally truncated forms of the HGF receptor have been shown to be constitutively activated and tumorigenic in animal experiments. In the present study, 102 benign and malignant human musculoskeletal tumors were examined for expression of the HGF receptor by Western blotting and/or immunohistochemistry. A clear predominance of HGF receptor expression was seen in malignant as compared to benign tumors (Western blotting, P < 0.001; immunohistochemistry, P < 0.02). For the first time we show HGF receptor expression in the following four tumor types: dermatofibrosarcoma protuberans, clear cell sarcoma of tendons, malignant primitive neuroectodermal tumor, and benign fibrous histiocytoma. In three cases of sarcoma with high HGF receptor expression by Western blotting, we found indications of a short 85-kd N-terminally truncated HGF receptor that was tyrosine phosphorylated and located in the cytoplasm. Although fragments of this length were seen in 18 of 65 tumors, most were not tyrosine-phosphorylated. Northern blotting revealed only the 7.5-kb full-length HGF receptor transcript, suggesting that the 85-kd fragment is generated by an alternative initiation of translation or by proteolytic cleavage. Southern blotting detected no amplification of the Hgfr/Met gene in the 35 tumors examined, in contrast to our recent report of Hgfr/Met gene amplification in 7, 12-dimethylbenz(a)anthracene (DMBA)-induced rat sarcomas. The present data suggest that the locally aggressive and malignant properties of human mesenchymal tumors maybe related, in part, to high levels of full-length HGF receptors, and in some cases to the occurrence of N-terminally truncated HGF receptors, activated independently of HGF.
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MESH Headings
- Blotting, Western
- Bone Neoplasms/chemistry
- Bone Neoplasms/metabolism
- Bone Neoplasms/pathology
- Dermatofibrosarcoma/chemistry
- Dermatofibrosarcoma/metabolism
- Dermatofibrosarcoma/pathology
- Hepatocyte Growth Factor/biosynthesis
- Histiocytoma, Benign Fibrous/chemistry
- Histiocytoma, Benign Fibrous/metabolism
- Histiocytoma, Benign Fibrous/pathology
- Humans
- Neuroectodermal Tumors, Primitive/chemistry
- Neuroectodermal Tumors, Primitive/metabolism
- Neuroectodermal Tumors, Primitive/pathology
- Peptide Fragments/analysis
- Proto-Oncogene Proteins c-met/biosynthesis
- Receptor Protein-Tyrosine Kinases/biosynthesis
- Receptors, Cell Surface/biosynthesis
- Sarcoma, Clear Cell/chemistry
- Sarcoma, Clear Cell/metabolism
- Sarcoma, Clear Cell/pathology
- Soft Tissue Neoplasms/chemistry
- Soft Tissue Neoplasms/metabolism
- Soft Tissue Neoplasms/pathology
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Affiliation(s)
- V Wallenius
- Research Center for Endocrinology and Metabolism (RCEM), Department of Internal Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden.
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Xiao ZQ, Chen YQ, Wang MH. Requirement of both tyrosine residues 1330 and 1337 in the C-terminal tail of the RON receptor tyrosine kinase for epithelial cell scattering and migration. Biochem Biophys Res Commun 2000; 267:669-75. [PMID: 10631120 DOI: 10.1006/bbrc.1999.2011] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
RON is a receptor tyrosine kinase that mediates cell scattering, migration, and tubular formation. This study focused on the function of two tyrosines, Y1330 and Y1337, in the C-terminus of RON in regulating epithelial cell scattering and migration. Substitution of both tyrosine residues with phenylalanine causes complete loss of cell scattering and migration in kidney 293 cells. In contrast, single mutation of either tyrosine residue has no effect. We found that mutation at Y1330 or Y1337 alone does not significantly affect the association of RON with PI-3 kinase, whereas a double mutation abolishes the recruitment of substrates. RON-mediated cell migration was inhibited by PI-3 kinase inhibitor wortmannin. This effect was also achieved by a dominant inhibitory p85 of PI-3 kinase. We conclude that Y1330 and Y1337 are required for RON-mediated cell motility. By associating with PI-3 kinase, the Y1330-Y1337 docking site plays a critical role in transducing motile signals of RON.
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Affiliation(s)
- Z Q Xiao
- Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine and Denver Health Medical Center, Denver, Colorado 80204, USA
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Williams TA, Longati P, Pugliese L, Gual P, Bardelli A, Michieli P. MET(PRC) mutations in the Ron receptor result in upregulation of tyrosine kinase activity and acquisition of oncogenic potential. J Cell Physiol 1999; 181:507-14. [PMID: 10528237 DOI: 10.1002/(sici)1097-4652(199912)181:3<507::aid-jcp15>3.0.co;2-q] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Ron and Met are structurally related receptor tyrosine kinases that elicit a complex biological response leading to invasive growth. Naturally occurring point mutations activate the Met kinase in papillary renal carcinomas (MET(PRC) mutations). By site-directed mutagenesis, we generated homologous amino acid substitutions in the Ron kinase domain and analyzed the biochemical and biological properties of the mutant receptors. Among the mutations studied, D(1232)H and M(1254)T displayed transforming activity in NIH3T3 cells, inducing focus formation and anchorage-independent growth. The D(1232)H and M(1254)T substitutions resulted in increased Ron autophosphorylation both in vivo and in vitro and constitutive binding to intracellular signal transducers. Both mutations yielded a dramatic increase in catalytic efficiency, indicating a direct correlation between kinase activity and oncogenic potential. Molecular modeling of the Ron D(1232)H mutation suggests that this single amino acid substitution favors the transition of the kinase from the inactive to the active state. These data demonstrate that point mutations can confer transforming activity to the Ron receptor and show that RON is a potential oncogene.
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Affiliation(s)
- T A Williams
- Department of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), University of Torino School of Medicine, Torino, Italy
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Helou K, Wallenius V, Qiu Y, Ohman F, Ståhl F, Klinga-Levan K, Kindblom LG, Mandahl N, Jansson JO, Levan G. Amplification and overexpression of the hepatocyte growth factor receptor (HGFR/MET) in rat DMBA sarcomas. Oncogene 1999; 18:3226-34. [PMID: 10359528 DOI: 10.1038/sj.onc.1202658] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
In the present study subcutaneous fibrosarcomas were induced by the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) in rats from F1 generation cross breedings of two different inbred strains. Comparative genomic hybridization (CGH) analysis, which allows detection of DNA sequence copy changes, was applied to one of the tumors and it was found that there were increased copy numbers of sequences at chromosome 4q12-q21 in this tumor. We have previously determined that the loci for the hepatocyte growth factor (Hgf) and hepatocyte growth factor receptor (Hgfr/Met), a protooncogene, are situated in this particular chromosome region. Using probes for the two genes in FISH (fluorescence in situ hybridization) and in Southern blots we found that the Hgfr/Met gene was amplified in five of the 19 sarcomas studied, and that the Hgf gene was coamplified in two of them. Northern and Western blots and tyrosine phosphorylation analysis showed that the HGF receptor was overexpressed and functional in all five tumors, as well as in two additional tumors. In summary, both amplification and overexpression of the Hgfr/Met gene was found in about 25% of DMBA-induced experimental rat sarcomas, and HGF receptor overexpression alone was seen in two additional tumors. Possibly this reflects an involvement in paracrine or autocrine stimulation of growth and invasiveness by HGF. Our finding could provide a rodent model system to increased knowledge about causality and therapy, which may be applicable to the sizeable fraction of human musculoskeletal tumors displaying MET overexpression.
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Affiliation(s)
- K Helou
- Department of Cell and Molecular Biology-Genetics, Göteborg University, Gothenburg, Sweden
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Price M, Fivaz J, Jotterand A, Mirkovitch J. Tissue-specific chromatin structure at the hepatocyte growth factor/scatter factor gene promoter. Gene 1998; 211:141-50. [PMID: 9573349 DOI: 10.1016/s0378-1119(98)00089-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatocyte growth factor/scatter factor (HGF/SF) is a recently characterised molecule with many remarkable functions. Its involvement in important processes such as cell proliferation, cell migration, morphogenesis and organ development implies that its activity should be tightly regulated. To understand the molecular mechanisms controlling HGF/SF transcription, we have analysed DNaseI hypersensitive sites (DHS) along rat and human HGF/SF genes in various tissues and cell types. We identified five DHS along the rat gene, two in the 5'-flanking region and three in the first intron. These sites are only found in rat tissues and rat cell lines, which express HGF/SF. The strongest hypersensitive site map to a region that corresponds to the promoter by start site analysis. A single tissue-specific DHS is present in human cell lines that express HGF/SF and corresponds to the promoter region. Our results suggest that chromatin accessibility plays a major role in the regulation of HGF/SF transcription regulation.
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Affiliation(s)
- M Price
- Swiss Institute for Experimental Cancer Research (ISREC), Chemin des Boveresses 155, CH-1066, Epalinges, Switzerland
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Giordano S, Bardelli A, Zhen Z, Menard S, Ponzetto C, Comoglio PM. A point mutation in the MET oncogene abrogates metastasis without affecting transformation. Proc Natl Acad Sci U S A 1997; 94:13868-72. [PMID: 9391119 PMCID: PMC28399 DOI: 10.1073/pnas.94.25.13868] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/1997] [Accepted: 08/18/1997] [Indexed: 02/05/2023] Open
Abstract
The MET oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF), known to stimulate invasive growth of epithelial cells. MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. To investigate whether this oncogene is directly responsible for the acquisition of the metastatic phenotype, we exploited a single-hit oncogenic version of MET, able to transform and to confer invasive and metastatic properties to nontumorigenic cells, both in vitro and in nude mice. We mutagenized the signal transducer docking site of Met (Y1349VHVX3Y1356VNV), which has the uncommon property of binding and activating multiple src homology region 2 (SH2)-containing intracellular effectors. Notably, a point mutation (H1351 --> N) increased the transforming ability of the oncogene but abolished its metastatic potential. This mutation duplicates the Grb2 binding site, super-activating the Ras pathway and preventing the binding of the other intracellular transducers. Complementation in trans with another nonmetastatic mutant (N1358 --> H), recruiting all the transducers downstream to Met except Grb2, rescued the invasive-metastatic phenotype. It is concluded that the metastatic potential of the MET oncogene relies on the properties of its multifunctional docking site, and that a single point mutation affecting signal transduction can dissociate neoplastic transformation from metastasis.
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Affiliation(s)
- S Giordano
- Institute for Cancer Research, University of Torino Medical School, Strada Provinciale 142, Km 3.95, 10060 Candiolo, Italy.
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Holland SJ, Gale NW, Gish GD, Roth RA, Songyang Z, Cantley LC, Henkemeyer M, Yancopoulos GD, Pawson T. Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells. EMBO J 1997; 16:3877-88. [PMID: 9233798 PMCID: PMC1170012 DOI: 10.1093/emboj/16.13.3877] [Citation(s) in RCA: 227] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, we have expressed the EphB2 receptor (formerly Nuk/Cek5/Sek3) in neuronal NG108-15 cells, and have observed the tyrosine phosphorylation of multiple cellular proteins upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induced the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn formed a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also bound through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexed with activated EphB2 in vivo. We have localized an in vitro RasGAP-binding site to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2, and demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signalling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the + 3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signalling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture.
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Affiliation(s)
- S J Holland
- Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada
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Anastasi S, Giordano S, Sthandier O, Gambarotta G, Maione R, Comoglio P, Amati P. A natural hepatocyte growth factor/scatter factor autocrine loop in myoblast cells and the effect of the constitutive Met kinase activation on myogenic differentiation. J Cell Biol 1997; 137:1057-68. [PMID: 9166406 PMCID: PMC2136220 DOI: 10.1083/jcb.137.5.1057] [Citation(s) in RCA: 145] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/1996] [Revised: 03/10/1997] [Indexed: 02/04/2023] Open
Abstract
As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way. To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65(tpr-met)) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65(tpr-met) highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand-receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65(tpr-met) kinase. These data indicate that a met kinase signal released from differentiation-dependent control provides a negative stimulus for the onset of myogenic differentiation.
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Affiliation(s)
- S Anastasi
- Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Università di Roma La Sapienza, 00161 Roma, Italy
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Santoro MM, Collesi C, Grisendi S, Gaudino G, Comoglio PM. Constitutive activation of the RON gene promotes invasive growth but not transformation. Mol Cell Biol 1996; 16:7072-83. [PMID: 8943362 PMCID: PMC231710 DOI: 10.1128/mcb.16.12.7072] [Citation(s) in RCA: 79] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
MET, RON, and SEA are members of a gene family encoding tyrosine kinase receptors with distinctive properties. Besides mediating growth, they control cell dissociation, motility ("scattering"), and formation of branching tubules. While there are transforming counterparts of MET and SEA, no oncogenic forms of RON have yet been identified. A chimeric Tpr-Ron, mimicking the oncogenic form of Met (Tpr-Met) was generated to investigate its transforming potential. For comparison, a chimeric Tpr-Sea was also constructed. Fusion with Tpr induced constitutive activation of the Ron and Sea kinases. While Tpr-Sea was more efficient than Tpr-Met in transformation, Tpr-Ron did not transform NIH 3T3 cells. The differences in the transforming abilities of Tpr-Met and Tpr-Ron were linked to the functional features of the respective tyrosine kinases using the approach of swapping subdomains. Kinetic analysis showed that the catalytic efficiency of Tpr-Ron is five times lower than that of Tpr-Met. Moreover, constitutive activation of Ron resulted in activation of the MAP kinase signaling cascade approximately three times lower than that attained by Tpr-Met. However, constitutive activation of Ron did induce a mitogenic-invasive response, causing cell dissociation, motility, and invasion of extracellular matrices. Tpr-Ron also induced formation of long, unbranched tubules in tridimensional collagen gels. These data show that RON has the potential to elicit a motile-invasive rather than a transformed phenotype.
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Affiliation(s)
- M M Santoro
- Institute for Cancer Research, University of Turin Medical School, Italy
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