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1
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Uribe PM, Hudson AM, Lockard G, Jiang M, Harding J, Steyger PS, Coffin AB. Hepatocyte growth factor mimetic confers protection from aminoglycoside-induced hair cell death in vitro. Hear Res 2023; 434:108786. [PMID: 37192594 DOI: 10.1016/j.heares.2023.108786] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Revised: 04/18/2023] [Accepted: 05/05/2023] [Indexed: 05/18/2023]
Abstract
Loss of sensory hair cells from exposure to certain licit drugs, such as aminoglycoside antibiotics, can result in permanent hearing damage. Exogenous application of the neurotrophic molecule hepatocyte growth factor (HGF) promotes neuronal cell survival in a variety of contexts, including protecting hair cells from aminoglycoside ototoxicity. HGF itself is not an ideal therapeutic due to a short half-life and limited blood-brain barrier permeability. MM-201 is a chemically stable, blood-brain barrier permeable, synthetic HGF mimetic that serves as a functional ligand to activate the HGF receptor and its downstream signaling cascade. We previously demonstrated that MM-201 robustly protects zebrafish lateral line hair cells from aminoglycoside ototoxicity. Here, we examined the ability of MM-201 to protect mammalian sensory hair cells from aminoglycoside damage to further evaluate MM-201's clinical potential. We found that MM-201 exhibited dose-dependent protection from neomycin and gentamicin ototoxicity in mature mouse utricular explants. MM-201's protection was reduced following inhibition of mTOR, a downstream target of HGF receptor activation, implicating the activation of endogenous intracellular substrates by MM-201 as critical for the observed protection. We then asked if MM-201 altered the bactericidal properties of aminoglycosides. Using either plate or liquid growth assays we found that MM-201 did not alter the bactericidal efficacy of aminoglycoside antibiotics at therapeutically relevant concentrations. We therefore assessed the protective capacity of MM-201 in an in vivo mouse model of kanamycin ototoxicity. In contrast to our in vitro data, MM-201 did not attenuate kanamycin ototoxicity in vivo. Further, we found that MM-201 was ototoxic to mice across the dose range tested here. These data suggest species- and tissue-specific differences in otoprotective capacity. Next generation HGF mimetics are in clinical trials for neurodegenerative diseases and show excellent safety profiles, but neither preclinical studies nor clinical trials have examined hearing loss as a potential consequence of pharmaceutical HGF activation. Further research is needed to determine the consequences of systemic MM-201 application on the auditory system.
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Affiliation(s)
- Phillip M Uribe
- Department of Integrative Physiology and Neuroscience, Washington State University, 14204 NE Salmon Creek Ave, Vancouver, WA 98686 USA
| | - Alexandria M Hudson
- Department of Integrative Physiology and Neuroscience, Washington State University, 14204 NE Salmon Creek Ave, Vancouver, WA 98686 USA
| | - Gavin Lockard
- Department of Integrative Physiology and Neuroscience, Washington State University, 14204 NE Salmon Creek Ave, Vancouver, WA 98686 USA
| | - Meiyan Jiang
- Oregon Hearing Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA
| | - Joseph Harding
- Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA 99164 USA
| | - Peter S Steyger
- Translational Hearing Center, Creighton University, Omaha, NE, 68178, USA
| | - Allison B Coffin
- Department of Integrative Physiology and Neuroscience, Washington State University, 14204 NE Salmon Creek Ave, Vancouver, WA 98686 USA.
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2
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Kalinin EV, Chalenko YM, Sysolyatina EV, Midiber KY, Gusarov AM, Kechko OI, Kulikova AA, Mikhaleva LM, Mukhachev AY, Stanishevskyi YM, Mitkevich VA, Sobyanin KA, Ermolaeva SA. Bacterial hepatocyte growth factor receptor agonist stimulates hepatocyte proliferation and accelerates liver regeneration in a partial hepatectomy rat model. Drug Dev Res 2021; 82:123-132. [PMID: 32830369 DOI: 10.1002/ddr.21737] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Revised: 08/06/2020] [Accepted: 08/07/2020] [Indexed: 12/20/2022]
Abstract
Hepatocyte growth factor (HGF) is central to liver regeneration. The Internalin B (InlB) protein is a virulence factor produced by the pathogenic bacterium Listeria monocytogenes. InlB is known to mimic HGF activity by interacting with the HGF receptor (HGFR) and activating HGFR-controlled signaling pathways. We expressed and purified the HGFR-binding InlB domain, InlB321/15, cloned from the fully virulent clinical L. monocytogenes strain. HGFR and Erk1/2 phosphorylation was determined using Western blotting. The capacity of InlB321/15 to bind HGFR was measured using microscale thermophoresis. Liver regeneration was studied in a model of 70% partial hepatectomy (70%PHx) in male Wistar rats. The nuclear grade parameters were quantified using manual (percentage of binuclear hepatocytes), automated (nuclear diameters), or combined (Ki67 proliferation index) scoring methods. Purified InlB321/15 stimulated HGFR and Erk1/2 phosphorylation and accelerated the proliferation of HepG2 cells. InlB321/15 bound HGFR with Kd = 7.4 ± 1.3 nM. InlB321/15 injected intravenously on the second, fourth, and sixth days after surgery recovered the liver mass and improved the nuclear grade parameters. Seven days post 70% PHx, the liver weight indexes were 2.9 and 2.0%, the hepatocyte proliferation indexes were 19.8 and 0.6%, and the percentages of binucleated hepatocytes were 6.7 and 4.0%, in the InlB321/15-treated and control animals, respectively. Obtained data demonstrated that InlB321/15 improved hepatocyte proliferation and stimulated liver regeneration in animals with 70% hepatectomy.
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Affiliation(s)
- Egor V Kalinin
- Gamaleya Research Center of Epidemiology and Microbiology, Moscow, Russia
- Peoples' Friendship University of Russia (RUDN University), Moscow, Russia
| | - Yaroslava M Chalenko
- Gamaleya Research Center of Epidemiology and Microbiology, Moscow, Russia
- Federal Research Center for Virology and Microbiology (FRCVM), Russian Academy of Sciences, Moscow, Russia
| | | | | | - Alexey M Gusarov
- Gamaleya Research Center of Epidemiology and Microbiology, Moscow, Russia
| | - Olga I Kechko
- Engelhardt Institute of Molecular Biology, Moscow, Russia
| | | | | | | | | | | | | | - Svetlana A Ermolaeva
- Gamaleya Research Center of Epidemiology and Microbiology, Moscow, Russia
- Federal Research Center for Virology and Microbiology (FRCVM), Russian Academy of Sciences, Moscow, Russia
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3
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Garcia C, Buffet C, El Khattabi L, Rizk-Rabin M, Perlemoine K, Ragazzon B, Bertherat J, Cormier F, Groussin L. MET overexpression and activation favors invasiveness in a model of anaplastic thyroid cancer. Oncotarget 2019; 10:2320-2334. [PMID: 31040922 PMCID: PMC6481343 DOI: 10.18632/oncotarget.26798] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2018] [Accepted: 03/04/2019] [Indexed: 11/25/2022] Open
Abstract
In thyroid cancers, MET receptor overexpression has been associated with higher risk of metastatic progression. In this study, it was shown that the anaplastic thyroid cancer (ATC)-derived TTA1 cell line overexpressed MET. By using FISH and relative quantification by qPCR, it was demonstrated that this overexpression resulted from a MET amplification with more than 20 copies. As expected, MET overexpression led to its constitutive activation and upregulated signaling towards the MAPK, PI3K/AKT, STAT3 and NF-κB pathways. Since the usual feature of MET-amplified cell lines is the "MET addiction" for their cell proliferation, the effect of the highly selective ATP competitive MET inhibitor PHA665752 was analyzed. While PHA665752 strongly inhibited the MAPK pathway, it did not reduce cell proliferation in TTA1 cells (IC50 = 4100 nM). This resistance to PHA665752 of the TTA1 cell line was demonstrated to be related to EGFR-MET functional cross-talk and PI3K/AKT and NF-κB signaling. Nevertheless, PHA665752 suppressed the anchorage-independent growth capacity of the TTA1 cell line and reduced cell migration and invasion in a transwell assay. The role of activated MET in these neoplastic properties of the TTA1 cells was also proved with si-MET-RNA targeting. Thus, this work highlights the TTA1 cell line as the first model of MET amplification in an ATC cell line, which leads to MET constitutive activation and underlies its neoplastic properties. Besides being a useful model for MET inhibitors screening, the TTA1 cell line also supports the argument for searching for MET amplification in ATC, as it could have therapeutic implications.
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Affiliation(s)
- Cyril Garcia
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France.,Hôpital d'Instruction des Armées BEGIN, Saint-Mandé, France
| | - Camille Buffet
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Laila El Khattabi
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France.,Cytogenetics Laboratory, APHP, Cochin Hospital, Paris, France
| | - Marthe Rizk-Rabin
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Karine Perlemoine
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Bruno Ragazzon
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Jérôme Bertherat
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France.,Department of Endocrinology, APHP, Cochin Hospital, Paris, France
| | - Françoise Cormier
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Lionel Groussin
- INSERM Unité 1016, Institut Cochin, Paris, France.,Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Institut Cochin, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France.,Department of Endocrinology, APHP, Cochin Hospital, Paris, France
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4
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MET Activation by a Macrocyclic Peptide Agonist that Couples to Biological Responses Differently from HGF in a Context-Dependent Manner. Int J Mol Sci 2018; 19:ijms19103141. [PMID: 30322054 PMCID: PMC6213957 DOI: 10.3390/ijms19103141] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2018] [Revised: 10/10/2018] [Accepted: 10/10/2018] [Indexed: 12/11/2022] Open
Abstract
Non-native ligands for growth factor receptors with distinct chemical properties and different biological activities have the potential to become therapeutic applications. We previously generated MET/hepatocyte growth factor (HGF) receptor agonists using bivalent macrocyclic peptides. The highest MET-activating agonists exhibited biological activity that was indistinguishable from the effects of HGF. In this study, we investigated MET activation, signal characteristics, and biological responses induced by a macrocyclic peptide partial agonist known as aML5-PEG11. aML5-PEG11 induced weak tyrosine phosphorylation of MET while enhancing cell migration with potency comparable to HGF. aML5-PEG11 induced marked AKT (protein kinase B) and ERK (extracellular signal-regulated kinase) activation at a comparable potency and time-dependency to HGF, which suggests that enhancement of cell motility is attributable to activation of these molecules. In a 3-D culture of bile duct cancer cells in collagen gel, HGF induced robust activation of MET, ERK, and AKT, which was associated with enhanced expression of genes involved in bile duct development and subsequent branching of tubulogenesis. In contrast, aML5-PEG11 induced marginal activation of MET, ERK, and AKT (levels near the detection limits), which was associated with failure to enhance the expression of genes involved in bile duct development and a lack of tubulogenic response. Thus, MET activation by aML5-PEG11 couples to biological responses differently from HGF in an extracellular context-dependent manner.
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5
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Zhang H, Wang Y, Bai M, Wang J, Zhu K, Liu R, Ge S, Li J, Ning T, Deng T, Fan Q, Li H, Sun W, Ying G, Ba Y. Exosomes serve as nanoparticles to suppress tumor growth and angiogenesis in gastric cancer by delivering hepatocyte growth factor siRNA. Cancer Sci 2018; 109:629-641. [PMID: 29285843 PMCID: PMC5834801 DOI: 10.1111/cas.13488] [Citation(s) in RCA: 112] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2017] [Revised: 12/05/2017] [Accepted: 12/19/2017] [Indexed: 12/20/2022] Open
Abstract
Exosomes derived from cells have been found to mediate signal transduction between cells and to act as efficient carriers to deliver drugs and small RNA. Hepatocyte growth factor (HGF) is known to promote the growth of both cancer cells and vascular cells, and the HGF‐cMET pathway is a potential clinical target. Here, we characterized the inhibitory effect of HGF siRNA on tumor growth and angiogenesis in gastric cancer. In addition, we showed that HGF siRNA packed in exosomes can be transported into cancer cells, where it dramatically downregulates HGF expression. A cell co‐culture model was used to show that exosomes loaded with HGF siRNA suppress proliferation and migration of both cancer cells and vascular cells. Moreover, exosomes were able to transfer HGF siRNA in vivo, decreasing the growth rates of tumors and blood vessels. The results of our study demonstrate that exosomes have potential for use in targeted cancer therapy by delivering siRNA.
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Affiliation(s)
- Haiyang Zhang
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Yi Wang
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Ming Bai
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Junyi Wang
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Kegan Zhu
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Rui Liu
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Shaohua Ge
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - JiaLu Li
- Department of Gastroenterology, Tianjin First Center Hospital, Tianjin, China
| | - Tao Ning
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Ting Deng
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Qian Fan
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Hongli Li
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Wu Sun
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Guoguang Ying
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
| | - Yi Ba
- Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China
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6
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Dally J, Khan JS, Voisey A, Charalambous C, John HL, Woods EL, Steadman R, Moseley R, Midgley AC. Hepatocyte Growth Factor Mediates Enhanced Wound Healing Responses and Resistance to Transforming Growth Factor-β₁-Driven Myofibroblast Differentiation in Oral Mucosal Fibroblasts. Int J Mol Sci 2017; 18:ijms18091843. [PMID: 28837064 PMCID: PMC5618492 DOI: 10.3390/ijms18091843] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2017] [Revised: 08/21/2017] [Accepted: 08/23/2017] [Indexed: 01/07/2023] Open
Abstract
Oral mucosal wounds are characterized by rapid healing with minimal scarring, partly attributable to the "enhanced" wound healing properties of oral mucosal fibroblasts (OMFs). Hepatocyte growth factor (HGF) is a pleiotropic growth factor, with potential key roles in accelerating healing and preventing fibrosis. HGF can exist as full-length or truncated (HGF-NK), NK1 and NK2 isoforms. As OMFs display elevated HGF expression compared to dermal fibroblasts (DFs), this study investigated the extent to which HGF mediates the preferential cellular functions of OMFs, and the influence of pro-fibrotic, transforming growth factor-β₁ (TGF-β₁) on these responses. Knockdown of HGF expression in OMFs by short-interfering RNA (siHGF) significantly inhibited OMF proliferative and migratory responses. Supplementation with exogenous TGF-β₁ also significantly inhibited proliferation and migration, concomitant with significantly down-regulated HGF expression. In addition, knockdown abrogated OMF resistance to TGF-β₁-driven myofibroblast differentiation, as evidenced by increased α-smooth muscle actin (α-SMA) expression, F-actin reorganisation, and stress fibre formation. Responses were unaffected in siHGF-transfected DFs. OMFs expressed significantly higher full-length HGF and NK1 levels compared to patient-matched DFs, whilst NK2 expression was similar in both OMFs and DFs. Furthermore, NK2 was preferentially expressed over NK1 in DFs. TGF-β₁ supplementation significantly down-regulated full-length HGF and NK1 expression by OMFs, while NK2 was less affected. This study demonstrates the importance of HGF in mediating "enhanced" OMF cellular function. We also propose that full-length HGF and HGF-NK1 convey desirable wound healing properties, whilst fibroblasts preferentially expressing more HGF-NK2 readily undergo TGF-β₁-driven differentiation into myofibroblasts.
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Affiliation(s)
- Jordanna Dally
- Stem Cells, Wound Repair & Regeneration, Oral & Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff CF14 4XY, UK.
- Cardiff Institute of Tissue Engineering & Repair (CITER), Cardiff University, Cardiff CF10 3AX, UK.
| | - Jabur S Khan
- Stem Cells, Wound Repair & Regeneration, Oral & Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff CF14 4XY, UK.
- Cardiff Institute of Tissue Engineering & Repair (CITER), Cardiff University, Cardiff CF10 3AX, UK.
| | - Alex Voisey
- Stem Cells, Wound Repair & Regeneration, Oral & Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff CF14 4XY, UK.
- Wales Kidney Research Unit (WKRU), Systems Immunity Research Institute, Division of Infection and Immunity, College of Biomedical & Life Sciences, Cardiff University, Cardiff CF14 4XN, UK.
| | - Chrisandrea Charalambous
- Stem Cells, Wound Repair & Regeneration, Oral & Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff CF14 4XY, UK.
- Wales Kidney Research Unit (WKRU), Systems Immunity Research Institute, Division of Infection and Immunity, College of Biomedical & Life Sciences, Cardiff University, Cardiff CF14 4XN, UK.
| | - Hannah L John
- Stem Cells, Wound Repair & Regeneration, Oral & Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff CF14 4XY, UK.
- Wales Kidney Research Unit (WKRU), Systems Immunity Research Institute, Division of Infection and Immunity, College of Biomedical & Life Sciences, Cardiff University, Cardiff CF14 4XN, UK.
| | - Emma L Woods
- Stem Cells, Wound Repair & Regeneration, Oral & Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff CF14 4XY, UK.
- Cardiff Institute of Tissue Engineering & Repair (CITER), Cardiff University, Cardiff CF10 3AX, UK.
| | - Robert Steadman
- Cardiff Institute of Tissue Engineering & Repair (CITER), Cardiff University, Cardiff CF10 3AX, UK.
- Wales Kidney Research Unit (WKRU), Systems Immunity Research Institute, Division of Infection and Immunity, College of Biomedical & Life Sciences, Cardiff University, Cardiff CF14 4XN, UK.
| | - Ryan Moseley
- Stem Cells, Wound Repair & Regeneration, Oral & Biomedical Sciences, School of Dentistry, Cardiff University, Cardiff CF14 4XY, UK.
- Cardiff Institute of Tissue Engineering & Repair (CITER), Cardiff University, Cardiff CF10 3AX, UK.
| | - Adam C Midgley
- Cardiff Institute of Tissue Engineering & Repair (CITER), Cardiff University, Cardiff CF10 3AX, UK.
- Wales Kidney Research Unit (WKRU), Systems Immunity Research Institute, Division of Infection and Immunity, College of Biomedical & Life Sciences, Cardiff University, Cardiff CF14 4XN, UK.
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7
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Abstract
A fibroinflammatory stromal reaction cooperates with oncogenic signaling to influence pancreatic ductal adenocarcinoma (PDAC) initiation, progression, and therapeutic outcome, yet the mechanistic underpinning of this crosstalk remains poorly understood. Here we show that stromal cues elicit an adaptive response in the cancer cell including the rapid mobilization of a transcriptional network implicated in accelerated growth, along with anabolic changes of an altered metabolome. The close overlap of stroma-induced changes in vitro with those previously shown to be regulated by oncogenic Kras in vivo suggests that oncogenic Kras signaling-a hallmark and key driver of PDAC-is contingent on stromal inputs. Mechanistically, stroma-activated cancer cells show widespread increases in histone acetylation at transcriptionally enhanced genes, implicating the PDAC epigenome as a presumptive point of convergence between these pathways and a potential therapeutic target. Notably, inhibition of the bromodomain and extraterminal (BET) family of epigenetic readers, and of Bromodomain-containing protein 2 (BRD2) in particular, blocks stroma-inducible transcriptional regulation in vitro and tumor progression in vivo. Our work suggests the existence of a molecular "AND-gate" such that tumor activation is the consequence of mutant Kras and stromal cues, providing insight into the role of the tumor microenvironment in the origin and treatment of Ras-driven tumors.
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8
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Mungunsukh O, Lee YH, Bottaro DP, Day RM. The hepatocyte growth factor isoform NK2 activates motogenesis and survival but not proliferation due to lack of Akt activation. Cell Signal 2016; 28:1114-23. [PMID: 27224506 DOI: 10.1016/j.cellsig.2016.05.012] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2016] [Accepted: 05/16/2016] [Indexed: 12/14/2022]
Abstract
Hepatocyte growth factor (HGF) is a pleiotrophic factor involved in cellular proliferation, migration and morphogenesis. HGF is required for normal tissue and organ development during embryogenesis, but in the adult HGF has been demonstrated to drive normal tissue repair and inhibit fibrotic remodeling. HGF has two naturally occurring human isoforms as a result of alternative splicing, NK1 and NK2. While NK1 has been defined as an agonist for HGF receptor, Met, NK2 is defined as a partial Met antagonist. Furthermore, under conditions of fibrotic remodeling, NK2 is still expressed while full length HGF is suppressed. Furthermore, the mechanism by which NK2 partially signals through Met is not completely understood. Here, we investigated the mitogenic, motogenic, and anti-apoptotic activities of NK2 compared with full length HGF in primary human bronchial epithelial cells (BEpC) and bovine pulmonary artery endothelial cells (PAEC). In human BEpC, NK2 partial activated Met, inducing Met phosphorylation at Y1234/1235 in the tyrosine-kinase domain but not at Y1349 site in the multifunctional docking domain. Partial phosphorylation of Met by NK2 resulted in activation of MAPK and STAT3, but not AKT. This correlated with motogenesis and survival in a MAPK-dependent manner, but not cell proliferation. Overexpression of a constitutively active AKT complemented NK2 signaling, allowing NK2 to induce cell proliferation. These data indicate that NK2 and HGF drive motogenic and anti-apoptotic signaling but only HGF drives cell proliferation by activating AKT-pathway signaling. These results have implications for the biological consequences of differential regulation of the two isoforms under pro-fibrotic conditions.
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Affiliation(s)
- Ognoon Mungunsukh
- The Uniformed Services University of the Health Sciences, Department of Pharmacology, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Young H Lee
- Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Donald P Bottaro
- Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Regina M Day
- The Uniformed Services University of the Health Sciences, Department of Pharmacology, 4301 Jones Bridge Road, Bethesda, MD 20814, USA.
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9
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Pérez-Ramírez C, Cañadas-Garre M, Molina MÁ, Faus-Dáder MJ, Calleja-Hernández MÁ. MET/HGF targeted drugs as potential therapeutic strategies in non-small cell lung cancer. Pharmacol Res 2015; 102:90-106. [PMID: 26413995 DOI: 10.1016/j.phrs.2015.09.016] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/06/2015] [Revised: 09/22/2015] [Accepted: 09/22/2015] [Indexed: 12/15/2022]
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, Granada, Spain; Department of Biochemistry, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, s/n, 18071 Granada, Spain.
| | - Marisa Cañadas-Garre
- Pharmacogenetics Unit, UGC Provincial de Farmacia de Granada, Instituto de Investigación Biosanitaria de Granada, Complejo Hospitalario Universitario de Granada, Avda. Fuerzas Armadas 2, Granada, Spain.
| | - Miguel Ángel Molina
- Pangaea Biotech, S.L., Hospital Universitario Quirón Dexeus, C/Sabino Arana, 5-19, 08028 Barcelona, Spain.
| | - María José Faus-Dáder
- Department of Biochemistry, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, s/n, 18071 Granada, Spain.
| | - Miguel Ángel Calleja-Hernández
- Pharmacogenetics Unit, UGC Provincial de Farmacia de Granada, Instituto de Investigación Biosanitaria de Granada, Complejo Hospitalario Universitario de Granada, Avda. Fuerzas Armadas 2, Granada, Spain; Department of Pharmacology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, s/n, 18071 Granada, Spain.
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10
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Cecchi F, Lih CJ, Lee YH, Walsh W, Rabe DC, Williams PM, Bottaro DP. Expression array analysis of the hepatocyte growth factor invasive program. Clin Exp Metastasis 2015; 32:659-76. [PMID: 26231668 DOI: 10.1007/s10585-015-9735-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2014] [Accepted: 07/13/2015] [Indexed: 02/17/2023]
Abstract
Signaling by human hepatocyte growth factor (hHGF) via its cell surface receptor (MET) drives mitogenesis, motogenesis and morphogenesis in a wide spectrum of target cell types and embryologic, developmental and homeostatic contexts. Oncogenic pathway activation also contributes to tumorigenesis and cancer progression, including tumor angiogenesis and metastasis, in several prevalent malignancies. The HGF gene encodes full-length hHGF and two truncated isoforms known as NK1 and NK2. NK1 induces all three HGF activities at modestly reduced potency, whereas NK2 stimulates only motogenesis and enhances HGF-driven tumor metastasis in transgenic mice. Prior studies have shown that mouse HGF (mHGF) also binds with high affinity to human MET. Here we show that, like NK2, mHGF stimulates cell motility, invasion and spontaneous metastasis of PC3M human prostate adenocarcinoma cells in mice through human MET. To identify target genes and signaling pathways associated with motogenic and metastatic HGF signaling, i.e., the HGF invasive program, gene expression profiling was performed using PC3M cells treated with hHGF, NK2 or mHGF. Results obtained using Ingenuity Pathway Analysis software showed significant overlap with networks and pathways involved in cell movement and metastasis. Interrogating The Cancer Genome Atlas project also identified a subset of 23 gene expression changes in PC3M with a strong tendency for co-occurrence in prostate cancer patients that were associated with significantly decreased disease-free survival.
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Affiliation(s)
- Fabiola Cecchi
- Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892-1501, USA
| | - Chih-Jian Lih
- Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research, Inc. and Frederick National Laboratory for Cancer Research, Frederick, MD, 21702-1201, USA
| | - Young H Lee
- Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892-1501, USA
| | - William Walsh
- Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research, Inc. and Frederick National Laboratory for Cancer Research, Frederick, MD, 21702-1201, USA
| | - Daniel C Rabe
- Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892-1501, USA
| | - Paul M Williams
- Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research, Inc. and Frederick National Laboratory for Cancer Research, Frederick, MD, 21702-1201, USA
| | - Donald P Bottaro
- Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892-1501, USA. .,Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Bldg 10 CRC Rm 2-3952, 10 Center Drive MSC 1107, Bethesda, MD, 20892-1107, USA.
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11
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Tanaka S, Kawahara E, Nakagawa T. Myogenic cell response to muscle contraction with short electrical stimulation. J Phys Ther Sci 2015; 27:2349-52. [PMID: 26311981 PMCID: PMC4540879 DOI: 10.1589/jpts.27.2349] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Accepted: 04/16/2015] [Indexed: 01/16/2023] Open
Abstract
[Purpose] The present study aimed to determine the effects of short muscle strength exercise on hepatocyte growth factor expression and satellite cell activation. [Subjects] The study included 72 2-12-week-old male Sprague-Dawley rats. [Methods] The rat plantaris muscle was contracted with a 5-min electrical stimulation of the sciatic nerve, and then, the mRNA expressions of hepatocyte growth factor and myogenic regulatory factors in the plantaris muscle were determined, and the phosphorylation of the hepatocyte growth factor receptor (c-Met) was examined. [Results] The mRNA expressions of hepatocyte growth factor and myogenic regulatory factors increased after a short muscle contraction compared to that un-contraction. Immunofluorescence analysis showed the expression of hepatocyte growth factor protein and the possibility that downstream biological changes occurred in the hepatocyte growth factor-bound c-Met. [Conclusion] Our results demonstrated that activation of satellite cells induced hepatocyte growth factor expression during muscle contraction with a short 5-min electrical stimulation, which simulates short muscle strength exercise in physical therapy. The present study provides evidence for the use of short muscle strength exercise in physical therapy.
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Affiliation(s)
- Shoji Tanaka
- Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Japan
| | - Ei Kawahara
- Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Japan
| | - Takao Nakagawa
- Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Japan
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12
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Li F, Qu H, Cao HC, Li MH, Chen C, Chen XF, Yu B, Yu L, Zheng LM, Zhang W. Both FOXO3a and FOXO1 are involved in the HGF-protective pathway against apoptosis in endothelial cells. Cell Biol Int 2015; 39:1131-7. [PMID: 25952685 DOI: 10.1002/cbin.10486] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2015] [Accepted: 04/29/2015] [Indexed: 12/11/2022]
Affiliation(s)
- Fang Li
- Biomedical Research Institute; Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center; Shenzhen Guangdong China
| | - Huan Qu
- Department of Cardiovascularology; Peking University Shenzhen Hospital; Shenzhen Guangdong China
| | - Heng-Chang Cao
- Department of Emergency Surgery; Peking University Shenzhen Hospital; Shenzhen Guangdong China
| | - Mei-Hong Li
- Department of Cardiovascularology; Peking University Shenzhen Hospital; Shenzhen Guangdong China
| | - Chen Chen
- Department of Cardiovascularology; Peking University Shenzhen Hospital; Shenzhen Guangdong China
| | - Xiao-Fan Chen
- Biomedical Research Institute; Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center; Shenzhen Guangdong China
| | - Bo Yu
- Department of Dermatology; Peking University Shenzhen Hospital; Shenzhen Guangdong China
| | - Lin Yu
- Department of obstetrics and gynaecology; Peking University Shenzhen Hospital; Shenzhen Guangdong China
| | - Le-Min Zheng
- Department of Cardiovascular Sciences; Peking University Health Science Center; Peking University; Peking China
| | - Wei Zhang
- Biomedical Research Institute; Shenzhen Peking University-the Hong Kong University of Science and Technology Medical Center; Shenzhen Guangdong China
- Department of Dermatology; Peking University Shenzhen Hospital; Shenzhen Guangdong China
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13
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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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14
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Abstract
Skeletal muscles in vertebrates have a phenomenal regenerative capacity. A muscle that has been crushed can regenerate fully both structurally and functionally within a month. Remarkably, efficient regeneration continues to occur following repeated injuries. Thousands of muscle precursor cells are needed to accomplish regeneration following acute injury. The differentiated muscle cells, the multinucleated contractile myofibers, are terminally withdrawn from mitosis. The source of the regenerative precursors is the skeletal muscle stem cells-the mononucleated cells closely associated with myofibers, which are known as satellite cells. Satellite cells are mitotically quiescent or slow-cycling, committed to myogenesis, but undifferentiated. Disruption of the niche after muscle damage results in their exit from quiescence and progression towards commitment. They eventually arrest proliferation, differentiate, and fuse to damaged myofibers or make de novo myofibers. Satellite cells are one of the well-studied adult tissue-specific stem cells and have served as an excellent model for investigating adult stem cells. They have also emerged as an important standard in the field of ageing and stem cells. Several recent reviews have highlighted the importance of these cells as a model to understand stem cell biology. This chapter begins with the discovery of satellite cells as skeletal muscle stem cells and their developmental origin. We discuss transcription factors and signalling cues governing stem cell function of satellite cells and heterogeneity in the satellite cell pool. Apart from satellite cells, a number of other stem cells have been shown to make muscle and are being considered as candidate stem cells for amelioration of muscle degenerative diseases. We discuss these "offbeat" muscle stem cells and their status as adult skeletal muscle stem cells vis-a-vis satellite cells. The ageing context is highlighted in the concluding section.
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Affiliation(s)
- Ramkumar Sambasivan
- Institute for Stem Cell Biology and Regenerative Medicine, GKVK, Bellary Road, Bangalore, 560065, India,
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15
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Mungunsukh O, McCart EA, Day RM. Hepatocyte Growth Factor Isoforms in Tissue Repair, Cancer, and Fibrotic Remodeling. Biomedicines 2014; 2:301-326. [PMID: 28548073 PMCID: PMC5344272 DOI: 10.3390/biomedicines2040301] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2014] [Revised: 10/21/2014] [Accepted: 10/27/2014] [Indexed: 01/18/2023] Open
Abstract
Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a pleotropic factor required for normal organ development during embryogenesis. In the adult, basal expression of HGF maintains tissue homeostasis and is up-regulated in response to tissue injury. HGF expression is necessary for the proliferation, migration, and survival of epithelial and endothelial cells involved in tissue repair in a variety of organs, including heart, lung, kidney, liver, brain, and skin. The administration of full length HGF, either as a protein or using exogenous expression methodologies, increases tissue repair in animal models of tissue injury and increases angiogenesis. Full length HGF is comprised of an N-terminal hairpin turn, four kringle domains, and a serine protease-like domain. Several naturally occurring alternatively spliced isoforms of HGF were also identified. The NK1 variant contains the N-terminal hairpin and the first kringle domain, and the NK2 variant extends through the second kringle domain. These alternatively spliced forms of HGF activate the same receptor, MET, but they differ from the full length protein in their cellular activities and their biological functions. Here, we review the species-specific expression of the HGF isoforms, their regulation, the signal transduction pathways they activate, and their biological activities.
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Affiliation(s)
- Ognoon Mungunsukh
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
| | - Elizabeth A McCart
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
| | - Regina M Day
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
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16
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Dang Q, Song W, Xu D, Ma Y, Li F, Zeng J, Zhu G, Wang X, Chang LS, He D, Li L. Kaempferol suppresses bladder cancer tumor growth by inhibiting cell proliferation and inducing apoptosis. Mol Carcinog 2014; 54:831-40. [PMID: 24700700 DOI: 10.1002/mc.22154] [Citation(s) in RCA: 71] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2013] [Revised: 02/26/2014] [Accepted: 03/10/2014] [Indexed: 12/25/2022]
Abstract
The effects of the flavonoid compound, kaempferol, which is an inhibitor of cancer cell proliferation and an inducer of cell apoptosis have been shown in various cancers, including lung, pancreatic, and ovarian, but its effect has never been studied in bladder cancer. Here, we investigated the effects of kaempferol on bladder cancer using multiple in vitro cell lines and in vivo mice studies. The MTT assay results on various bladder cancer cell lines showed that kaempferol enhanced bladder cancer cell cytotoxicity. In contrast, when analyzed by the flow cytometric analysis, DNA ladder experiment, and TUNEL assay, kaempferol significantly was shown to induce apoptosis and cell cycle arrest. These in vitro results were confirmed in in vivo mice studies using subcutaneous xenografted mouse models. Consistent with the in vitro results, we found that treating mice with kaempferol significant suppression in tumor growth compared to the control group mice. Tumor tissue staining results showed decreased expressions of the growth related markers, yet increased expressions in apoptosis markers in the kaempferol treated group mice tissues compared to the control group mice. In addition, our in vitro and in vivo data showed kaempferol can also inhibit bladder cancer invasion and metastasis. Further mechanism dissection studies showed that significant down-regulation of the c-Met/p38 signaling pathway is responsible for the kaempferol mediated cell proliferation inhibition. All these findings suggest kaempferol might be an effective and novel chemotherapeutic drug to apply for the future therapeutic agent to combat bladder cancer.
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Affiliation(s)
- Qiang Dang
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Wenbin Song
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Defeng Xu
- School of Pharmaceutical and Life Sciences, Changzhou University, Changzhou, Jiangsu, PR, China
| | - Yanmin Ma
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Feng Li
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Jin Zeng
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Guodong Zhu
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Xinyang Wang
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Luke S Chang
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Dalin He
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
| | - Lei Li
- Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR, China
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17
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Surriga O, Rajasekhar VK, Ambrosini G, Dogan Y, Huang R, Schwartz GK. Crizotinib, a c-Met inhibitor, prevents metastasis in a metastatic uveal melanoma model. Mol Cancer Ther 2013; 12:2817-26. [PMID: 24140933 DOI: 10.1158/1535-7163.mct-13-0499] [Citation(s) in RCA: 80] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Uveal melanoma is the most common primary intraocular malignant tumor in adults and half of the primary tumors will develop fatal metastatic disease to the liver and the lung. Crizotinib, an inhibitor of c-Met, anaplastic lymphoma kinase (ALK), and ROS1, inhibited the phosphorylation of the c-Met receptor but not of ALK or ROS1 in uveal melanoma cells and tumor tissue. Consequently, migration of uveal melanoma cells was suppressed in vitro at a concentration associated with the specific inhibition of c-Met phosphorylation. This effect on cell migration could be recapitulated with siRNA specific to c-Met but not to ALK or ROS1. Therefore, we developed a uveal melanoma metastatic mouse model with EGFP-luciferase-labeled uveal melanoma cells transplanted by retro-orbital injections to test the effect of crizotinib on metastasis. In this model, there was development of melanoma within the eye and also metastases to the liver and lung at 7 weeks after the initial transplantation. When mice were treated with crizotinib starting 1 week after the transplantation, we observed a significant reduction in the development of metastases as compared with untreated control sets. These results indicate that the inhibition of c-Met activity alone may be sufficient to strongly inhibit metastasis of uveal melanoma from forming, suggesting crizotinib as a potential adjuvant therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease.
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Affiliation(s)
- Oliver Surriga
- Corresponding Author: Gary K. Schwartz, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065.
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18
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Mungunsukh O, Day RM. Transforming growth factor-β1 selectively inhibits hepatocyte growth factor expression via a micro-RNA-199-dependent posttranscriptional mechanism. Mol Biol Cell 2013; 24:2088-97. [PMID: 23657814 PMCID: PMC3694793 DOI: 10.1091/mbc.e13-01-0017] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Hepatocyte growth factor (HGF) is a multipotent endogenous repair factor. The profibrotic cytokine transforming growth factor (TGF)-β1 inhibits HGF expression by a micro-RNA-199 (miR-199)-dependent posttranscriptional mechanism. In contrast, NK2, a truncated isoform of HGF that inhibits normal repair, is protected from TGF-β1–induced downregulation by miR-199. Hepatocyte growth factor (HGF) is a multipotent endogenous repair factor secreted primarily by mesenchymal cells with effects on cells expressing its receptor, Met. HGF promotes normal tissue regeneration and inhibits fibrotic remodeling in part by promoting proliferation and migration of endothelial and epithelial cells and protecting these cells from apoptosis. HGF also inhibits myofibroblast proliferation. The profibrotic cytokine transforming growth factor beta 1 (TGF-β1) suppresses HGF expression but not the expression of NK2, an HGF splice variant that antagonizes HGF-induced proliferation. We investigated the mechanism for differential regulation of HGF and NK2 by TGF-β1. TGF-β1 down-regulated HGF in primary human adult pulmonary fibroblasts (HLFb) and increased the expression of miR-199a-3p, a microRNA (miRNA) associated with fibrotic remodeling. HGF and NK2 contain completely different 3′ untranslated regions (UTRs), and we determined that miR-199a-3p targeted HGF mRNA for suppression but not NK2. A pre–miR-199 mimic inhibited the expression of a luciferase reporter harboring the HGF 3′ UTR but not a pmirGLO reporter containing the NK2 3′ UTR. In contrast, an anti-miRNA inhibitor specific for miR-199a-3p prevented TGF-β1–induced reduction of both HGF mRNA and HGF protein secretion. Taken together, these findings demonstrate that HGF is distinctly regulated at the posttranscriptional level from its antagonist NK2.
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Affiliation(s)
- Ognoon Mungunsukh
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA
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19
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Grzelakowska-Sztabert B, Dudkowska M. Paradoxical action of growth factors: antiproliferative and proapoptotic signaling by HGF/c-MET. Growth Factors 2011; 29:105-18. [PMID: 21631393 DOI: 10.3109/08977194.2011.585609] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-MET) signaling is usually associated with the promotion of cellular growth and often with progression of tumors. Nevertheless, under certain conditions HGF can also act as an antiproliferative and proapoptotic factor and can sensitize various cancer cells, treated with anticancer drugs, to apoptosis. Not only HGF but also its various truncated forms as well as intracellular fragments of its membrane receptor, c-MET, may act as antiproliferative and proapoptotic factors toward various cells. This review focuses on different mechanisms responsible for such paradoxical action of the known typical growth factor. It also points toward the possibilities of usage of this information in anticancer therapy.
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20
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Felici A, Giubellino A, Bottaro DP. Gab1 mediates hepatocyte growth factor-stimulated mitogenicity and morphogenesis in multipotent myeloid cells. J Cell Biochem 2011; 111:310-21. [PMID: 20506405 DOI: 10.1002/jcb.22695] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Hepatocyte growth factor (HGF)-stimulated mitogenesis, motogenesis and morphogenesis in various cell types begins with activation of the Met receptor tyrosine kinase and the recruitment of intracellular adaptors and kinase substrates. The adapter protein Gab1 is a critical effector and substrate of activated Met, mediating morphogenesis, among other activities, in epithelial cells. To define its role downstream of Met in hematopoietic cells, Gab1 was expressed in the HGF-responsive, Gab1-negative murine myeloid cell line 32D. Interestingly, the adhesion and motility of Gab1-expressing cells were significantly greater than parental cells, independent of growth factor treatment. Downstream of activated Met, Gab1 expression was specifically associated with rapid Shp-2 recruitment and activation, increased mitogenic potency, suppression of GATA-1 expression and concomitant upregulation of GATA-2 transcription. In addition to enhanced proliferation, continuous culture of Gab1-expressing 32D cells in HGF resulted in cell attachment, filopodia extension and phenotypic changes suggestive of monocytic differentiation. Our results suggest that in myeloid cells, Gab1 is likely to enhance HGF mitogenicity by coupling Met to Shp-2 and GATA-2 expression, thereby potentially contributing to normal myeloid differentiation as well as oncogenic transformation.
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Affiliation(s)
- Angelina Felici
- Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National, Institutes of Health, Bethesda, Maryland 20892-1501, USA
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Abstract
Pulmonary remodeling is characterized by the permanent and progressive loss of the normal alveolar architecture, especially the loss of alveolar epithelial and endothelial cells, persistent proliferation of activated fibroblasts, or myofibroblasts, and alteration of extracellular matrix. Hepatocyte growth factor (HGF) is a pleiotropic factor, which induces cellular motility, survival, proliferation, and morphogenesis, depending upon the cell type. In the adult, HGF has been demonstrated to play a critical role in tissue repair, including in the lung. Administration of HGF protein or ectopic expression of HGF has been demonstrated in animal models of pulmonary fibrosis to induce normal tissue repair and to prevent fibrotic remodeling. HGF-induced inhibition of fibrotic remodeling may occur via multiple direct and indirect mechanisms including the induction of cell survival and proliferation of pulmonary epithelial and endothelial cells, and the reduction of myofibroblast accumulation.
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22
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Panganiban RAM, Day RM. Hepatocyte growth factor in lung repair and pulmonary fibrosis. Int J Radiat Biol 2010; 89:656-67. [PMID: 21131996 DOI: 10.3109/09553002.2012.711502] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Pulmonary remodeling is characterized by the permanent and progressive loss of the normal alveolar architecture, especially the loss of alveolar epithelial and endothelial cells, persistent proliferation of activated fibroblasts, or myofibroblasts, and alteration of extracellular matrix. Hepatocyte growth factor (HGF) is a pleiotropic factor, which induces cellular motility, survival, proliferation, and morphogenesis, depending upon the cell type. In the adult, HGF has been demonstrated to play a critical role in tissue repair, including in the lung. Administration of HGF protein or ectopic expression of HGF has been demonstrated in animal models of pulmonary fibrosis to induce normal tissue repair and to prevent fibrotic remodeling. HGF-induced inhibition of fibrotic remodeling may occur via multiple direct and indirect mechanisms including the induction of cell survival and proliferation of pulmonary epithelial and endothelial cells, and the reduction of myofibroblast accumulation.
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Affiliation(s)
- Ronald Allan M Panganiban
- Department of Pharmacology, Uniformed Services University of Health Sciences, Bethesda, MD 20852, USA
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23
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Lee YH, Marquez AP, Mungunsukh O, Day RM. Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants. Mol Biol Cell 2010; 21:4240-50. [PMID: 20926686 PMCID: PMC2993751 DOI: 10.1091/mbc.e10-04-0341] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2010] [Revised: 09/03/2010] [Accepted: 09/23/2010] [Indexed: 01/07/2023] Open
Abstract
Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner.
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Affiliation(s)
- Young H. Lee
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
| | - Ana P. Marquez
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
| | - Ognoon Mungunsukh
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
| | - Regina M. Day
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
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24
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Pao W, Iafrate AJ, Su Z. Genetically informed lung cancer medicine. J Pathol 2010; 223:230-40. [PMID: 21125677 DOI: 10.1002/path.2788] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2010] [Revised: 09/09/2010] [Accepted: 09/16/2010] [Indexed: 02/06/2023]
Abstract
Knowledge of tumour mutation status has become increasingly important for the prioritization of targeted therapies for cancer. We review here the major known 'driver' mutations with therapeutic relevance in non-small cell lung cancer. We also discuss a variety of methods now being employed in molecular diagnostic laboratories to detect genetic alterations in lung tumours. A genetically-informed approach to lung cancer medicine is rapidly becoming the standard of care worldwide and should lead to improved outcomes for patients.
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Affiliation(s)
- William Pao
- Vanderbilt-Ingram Cancer Center, Department of Medicine/Division of Hematology-Oncology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
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25
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Mungunsukh O, Lee YH, Marquez AP, Cecchi F, Bottaro DP, Day RM. A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation. Am J Physiol Lung Cell Mol Physiol 2010; 299:L905-14. [PMID: 20889677 DOI: 10.1152/ajplung.00094.2010] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB₃₆₋₃₂₁) was demonstrated to bind to and partially activate the HGF receptor Met. InlB₃₆₋₃₂₁ has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB₃₆₋₃₂₁ (1×InlB₃₆₋₃₂₁) and engineered a head-to-tail repeat of InlB₃₆₋₃₂₁ with a linker peptide (2×InlB₃₆₋₃₂₁); 1×InlB₃₆₋₃₂₁ and 2×InlB₃₆₋₃₂₁ were purified from E. coli. Both 1× and 2×InlB₃₆₋₃₂₁ activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB₃₆₋₃₂₁ activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB₃₆₋₃₂₁ activated only STAT3 and ERK1/2. The 2×InlB₃₆₋₃₂₁ promoted improved motility compared with 1×InlB₃₆₋₃₂₁ and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB₃₆₋₃₂₁ prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB₃₆₋₃₂₁ to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.
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Affiliation(s)
- Ognoon Mungunsukh
- Dept. of Pharmacology, Uniformed Services University of the Health Sciences, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
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Abstract
Dysregulation of mesenchymal-epithelial transition factor receptor tyrosine kinase pathway leads to cell proliferation, protection from apoptosis, angiogenesis, invasion, and metastasis. It can be dysregulated through overexpression, constitutive activation, gene amplification, ligand-dependent activation or mutation. New drugs targeting various mesenchymal-epithelial transition factor pathways are being investigated with promising results.
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Agarwal S, Zerillo C, Kolmakova J, Christensen JG, Harris LN, Rimm DL, Digiovanna MP, Stern DF. Association of constitutively activated hepatocyte growth factor receptor (Met) with resistance to a dual EGFR/Her2 inhibitor in non-small-cell lung cancer cells. Br J Cancer 2009; 100:941-9. [PMID: 19240716 PMCID: PMC2661782 DOI: 10.1038/sj.bjc.6604937] [Citation(s) in RCA: 81] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
There is a pressing need to identify new drug targets and novel approaches for treatment of non-small-cell lung carcinoma (NSCLC). Members of the epidermal growth factor receptor (EGFR) and Met receptor families have been identified as important molecular targets for NSCLC. Two EGFR tyrosine kinase inhibitors (TKIs; erlotinib and gefitinib) are in current clinical use, but a majority of patients do not respond to these targeted therapies. We used receptor TK (RTK) capture arrays to identify receptors active in NSCLC cell lines. As Met and ErbBs were active, we explored the potential therapeutic advantage of combined targeting of Met with ErbB receptor family inhibitors for treatment of NSCLC. We found that Met physically interacts with both EGFR and Her2 in a NSCLC cell line with overexpression/overactivation of Met. Combined use of a dual EGFR/Her2 inhibitor with a Met inhibitor yields maximal growth inhibition compared with the use of EGFR and/or Met inhibitors. This suggests that simultaneous inhibition of multiple RTKs may be needed to effectively abrogate tumour cell growth. Phosphoproteomic analysis by RTK capture arrays may be a valuable tool for identifying the subset of tumours with functional receptor activation, regardless of mechanism.
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Affiliation(s)
- S Agarwal
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.
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28
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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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29
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Lee YH, Suzuki YJ, Griffin AJ, Day RM. Hepatocyte growth factor regulates cyclooxygenase-2 expression via beta-catenin, Akt, and p42/p44 MAPK in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2008; 294:L778-86. [PMID: 18245266 DOI: 10.1152/ajplung.00410.2007] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Hepatocyte growth factor (HGF) is upregulated in response to lung injury and has been implicated in tissue repair through its antiapoptotic and proliferative activities. Cyclooxygenase-2 (COX-2) is an inducible enzyme in the biosynthetic pathway of prostaglandins, and its activation has been shown to play a role in cell growth. Here, we report that HGF induces gene transcription of COX-2 in human bronchial epithelial cells (HBEpC). Treatment of HBEpC with HGF resulted in phosphorylation of the HGF receptor (c-Met), activation of Akt, and upregulation of COX-2 mRNA. Adenovirus-mediated gene transfer of a dominant negative (DN) Akt mutant revealed that HGF increased COX-2 mRNA in an Akt-dependent manner. COX-2 promoter analysis in luciferase reporter constructs showed that HGF regulation required the beta-catenin-responsive T cell factor-4 binding element (TBE). The HGF activation of the COX-2 gene transcription was blocked by DN mutant of beta-catenin or by inhibitors that blocked activation of Akt. Inhibition of p42/p44 MAPK pathway blocked HGF-mediated activation of beta-catenin gene transcription but not Akt activation, suggesting that p42/p44 MAPK acts in a parallel mechanism for beta-catenin activation. We also found that inhibition of COX-2 with NS-398 blocked HGF-induced growth in HBEpC. Together, the results show that the HGF increases COX-2 gene expression via an Akt-, MAPK-, and beta-catenin-dependent pathway in HBEpC.
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Affiliation(s)
- Young H Lee
- Department of Pharmacology, The Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA
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30
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Bonine-Summers AR, Aakre ME, Brown KA, Arteaga CL, Pietenpol JA, Moses HL, Cheng N. Epidermal growth factor receptor plays a significant role in hepatocyte growth factor mediated biological responses in mammary epithelial cells. Cancer Biol Ther 2007; 6:561-70. [PMID: 17495520 PMCID: PMC3395216 DOI: 10.4161/cbt.6.4.3851] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Breast cancers often have deregulated hepatocyte growth factor (HGF) and c-Met signaling that results in increased tumor growth and invasion. Elucidating the mechanism responsible for HGF/c-Met action in breast cancer progression has been difficult as c-Met communicates with a number of secondary receptors that can lead to various pathological outcomes. Understanding how these secondary receptors facilitate HGF/c-Met cellular responses will aid in the development of better therapeutic treatment options for breast cancer patients with elevated HGF signaling. In the present study it was shown that the epidermal growth factor receptor (EGFR) plays a significant role in HGF/c-Met mediated biological activities indicative of advanced tumor pathology, including enhanced proliferation and invasion. The clinically relevant EGFR inhibitor gefitinib was used to determine the role of EGFR in HGF-induced proliferation and motility in several mammary carcinoma cells including PyVmT, MDA-MB-231 and 4T1. Our analyses indicated that EGFR inhibition significantly blocked HGF activation of c-Met and EGFR and that inhibition of these pathways mitigated HGF induced proliferation and motility. The data indicate that this inhibition was not through a direct effect of gefitinib on c-Met, but that EGFR is necessary for c-Met activation in the assays performed. These results provide a novel mechanism of action for EGFR as a mediator of HGF signaling thereby linking EGFR to the oncogenic potential of c-Met in mammary carcinomas cells.
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Affiliation(s)
| | | | | | | | | | | | - Nikki Cheng
- Correspondence to: Nikki Cheng; Department of Cancer Biology, Vanderbilt-Ingram Cancer Center; Vanderbilt University; 2220 Pierce Ave; Nashville, Tennessee 37232-6838 USA; Tel.: 615.936.1507; Fax: 615.938.1790;
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31
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Esaki M, Takemura G, Kosai KI, Takahashi T, Miyata S, Li L, Goto K, Maruyama R, Okada H, Kanamori H, Ogino A, Ushikoshi H, Minatoguchi S, Fujiwara T, Fujiwara H. Treatment with an adenoviral vector encoding hepatocyte growth factor mitigates established cardiac dysfunction in doxorubicin-induced cardiomyopathy. Am J Physiol Heart Circ Physiol 2007; 294:H1048-57. [PMID: 18083897 DOI: 10.1152/ajpheart.01102.2007] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Hepatocyte growth factor (HGF) reportedly exerts beneficial effects on the heart following myocardial infarction and during nonischemic cardiomyopathy, but the precise mechanisms underlying the latter have not been well elucidated. We generated nonischemic cardiomyopathy in mice by injecting them with doxorubicin (15 mg/kg ip). Two weeks later, when cardiac dysfunction was apparent, an adenoviral vector encoding human HGF gene (Ad.CAG-HGF, 1x10(11) particles/mouse) was injected into the hindlimb muscles; LacZ gene served as the control. Left ventricular dilatation and dysfunction normally seen 4 wk after doxorubicin administration were significantly mitigated in HGF-treated mice, as were the associated cardiomyocyte atrophy/degeneration and myocardial fibrosis. Myocardial expression of GATA-4 and a sarcomeric protein, myosin heavy chain, was downregulated by doxorubicin, but the expression of both was restored by HGF treatment. The protective effect of HGF against doxorubicin-induced cardiomyocyte atrophy was confirmed in an in vitro experiment, which also showed that neither cardiomyocyte apoptosis nor proliferation plays significant roles in the present model. Upregulation of c-Met/HGF receptor was noted in HGF-treated hearts. Among the mediators downstream of c-Met, the activation of extracellular signal-regulated kinase (ERK) was reduced by doxorubicin, but the activity was restored by HGF. Levels of transforming growth factor-beta1 and cyclooxygenase-2 did not differ between the groups. Our findings suggest the HGF gene delivery exerts therapeutic antiatrophic/degenerative and antifibrotic effects on myocardium in cases of established cardiac dysfunction caused by doxorubicin. These beneficial effects appear to be related to HGF-induced ERK activation and upregulation of c-Met, GATA-4, and sarcomeric proteins.
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Affiliation(s)
- Masayasu Esaki
- Division of Cardiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan
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Pozner-Moulis S, Pappas DJ, Rimm DL. Met, the hepatocyte growth factor receptor, localizes to the nucleus in cells at low density. Cancer Res 2007; 66:7976-82. [PMID: 16912172 DOI: 10.1158/0008-5472.can-05-4335] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Some breast cancer cases in our previous immunohistochemical studies show Met expression in the nucleus. Given nuclear localization of other receptor tyrosine kinases, we proceeded to investigate Met. Nuclear Met is seen in numerous cell lines and in germinal regions of many tissues using four unique antibodies. Cell fractionation reveals a 60-kDa band recognized by COOH-terminal Met antibodies that is present independent of hepatocyte growth factor treatment. Green fluorescent protein (GFP) fusion proteins of the cytoplasmic domain of Met transfected into HEK293 cells are found in the nucleus whereas the full-length Met-GFP fusion is membranous. Further deletions of the Met-GFP fusions identify a region of the juxtamembrane domain required for nuclear translocation. In a CaCo2 cell line model for epithelial maturation, we find that Met is initially nuclear, and then becomes membranous, after confluence. This work suggests processing of the Met receptor, analogous to ErbB4, resulting in the release of the cytoplasmic domain and its translocation to the nucleus in cells at low density.
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Affiliation(s)
- Sharon Pozner-Moulis
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
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Lee YH, Kayyali US, Sousa AM, Rajan T, Lechleider RJ, Day RM. Transforming growth factor-beta1 effects on endothelial monolayer permeability involve focal adhesion kinase/Src. Am J Respir Cell Mol Biol 2007; 37:485-93. [PMID: 17585111 PMCID: PMC2176121 DOI: 10.1165/rcmb.2006-0439oc] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Transforming growth factor (TGF)-beta1 activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathologic conditions, including pulmonary edema and vessel inflammation. In endothelial monolayers, TGF-beta1 increases permeability, and a number of studies have demonstrated the alteration of cell-cell contacts by TGF-beta1. We hypothesized that focal adhesion complexes also likely contribute to alterations in endothelial permeability. We examined early signal transduction events associated with rapid changes in monolayer permeability and the focal adhesion complex of bovine pulmonary artery endothelial cells. Western blotting revealed rapid tyrosine phosphorylation of focal adhesion kinase (FAK) and Src kinase in response to TGF-beta1; inhibition of both of these kinases using pp2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), ameliorates TGF-beta1-induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor downstream of the TGF-beta receptors, and is blocked by the epidermal growth factor receptor inhibitor AG1478. Immunohistochemistry showed that actin and the focal adhesion proteins paxillin, vinculin, and hydrogen peroxide-inducible clone-5 (Hic-5) are rearranged in response to TGF-beta1; these proteins are released from focal adhesion complexes. Rearrangement of paxillin and vinculin by TGF-beta1 is not blocked by the FAK/Src inhibitor, pp2, or by SB431542 inhibition of the TGF-beta type I receptor, anaplastic lymphoma kinase 5; however, pp1 (4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which inhibits both type I and type II TGF-beta receptors, does block paxillin and vinculin rearrangement. Hic-5 protein rearrangement requires FAK/Src activity. Together, these results suggest that TGF-beta1-induced monolayer permeability involves focal adhesion and cytoskeletal rearrangement through both FAK/Src-dependent and -independent pathways.
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Affiliation(s)
- Young H Lee
- Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA
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34
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Inducing effects of hepatocyte growth factor on the expression of vascular endothelial growth factor in human colorectal carcinoma cells through MEK and PI3K signaling pathways. Chin Med J (Engl) 2007. [DOI: 10.1097/00029330-200705010-00002] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
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35
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Day RM, Agyeman AS, Segel MJ, Chévere RD, Angelosanto JM, Suzuki YJ, Fanburg BL. Serotonin induces pulmonary artery smooth muscle cell migration. Biochem Pharmacol 2006; 71:386-97. [PMID: 16316635 PMCID: PMC1831537 DOI: 10.1016/j.bcp.2005.10.035] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2005] [Revised: 10/14/2005] [Accepted: 10/20/2005] [Indexed: 10/25/2022]
Abstract
The chronic phase of pulmonary arterial hypertension (PAH) is associated with vascular remodeling, especially thickening of the smooth muscle layer of large pulmonary arteries and muscularization of small pulmonary vessels, which normally have no associated smooth muscle. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMC), and may be important for in vivo pulmonary vascular remodeling. Here, we show that 5-HT stimulates migration of pulmonary artery PASMC. Treatment with 5-HT for 16h increased migration of PASMC up to four-fold as monitored in a modified Boyden chamber assay. Increased migratory responses were associated with cellular morphological changes and reorganization of the actin cytoskeleton. 5-HT-induced alterations in morphology were previously shown in our laboratory to require cAMP [Lee SL, Fanburg BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Phys 1992;150(2):396-405], and the 5-HT4 receptor was pharmacologically determined to be the primary activator of cAMP in bovine PASMC [Becker BN, Gettys TW, Middleton JP, Olsen CL, Albers FJ, Lee SL, et al. 8-Hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992;42(5):817-25]. We examined the role of the 5-HT4 receptor and cAMP in 5-HT-induced bovine PASMC migration. PASMC express 5-HT4 receptor mRNA, and a 5-HT4 receptor antagonist and a cAMP antagonist completely blocked 5-HT-induced cellular migration. Consistent with our previous report that a cAMP-dependent Cl(-) channel is required for 5-HT-induced morphological changes in PASMC, phenylanthranilic acid, a Cl(-) channel blocker, inhibited actin cytoskeletal reorganization and migration produced by 5-HT. We conclude that 5-HT stimulates PASMC migration and associated cytoskeletal reorganization through the 5-HT4 receptor and cAMP activation of a chloride channel.
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MESH Headings
- Animals
- Blotting, Western
- Cattle
- Cell Movement/drug effects
- Cells, Cultured
- Cyclic AMP/antagonists & inhibitors
- Cyclic AMP/biosynthesis
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/metabolism
- Pulmonary Artery/cytology
- Pulmonary Artery/drug effects
- Pulmonary Artery/metabolism
- Receptors, Serotonin, 5-HT4/biosynthesis
- Serotonin/pharmacology
- Serotonin 5-HT4 Receptor Antagonists
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Affiliation(s)
- Regina M Day
- Department of Pharmacology, The Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
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Day RM, Lee YH, Park AM, Suzuki YJ. Retinoic acid inhibits airway smooth muscle cell migration. Am J Respir Cell Mol Biol 2006; 34:695-703. [PMID: 16456186 PMCID: PMC1831536 DOI: 10.1165/rcmb.2005-0306oc] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-alpha, -beta, -gamma, and retinoid X receptor (RXR)-alpha, -beta, but not RXR-gamma. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2-2 microM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein-protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.
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Affiliation(s)
- Regina M Day
- Department of Pharmacology, Georgetown University Medical Center, NW403 Medical-Dental Building, Washington, DC 20057, USA
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37
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Castillo J, Ammendrup-Johnsen I, Codina M, Navarro I, Gutiérrez J. IGF-I and insulin receptor signal transduction in trout muscle cells. Am J Physiol Regul Integr Comp Physiol 2006; 290:R1683-90. [PMID: 16439672 DOI: 10.1152/ajpregu.00294.2005] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
In this study, primary cultures of trout skeletal muscle cells were used to investigate the main signal transduction pathways of insulin and IGF-I receptors in rainbow trout muscle. At different stages of in vitro development (myoblasts on day 1, myocytes on day 4, and fully developed myotubes on day 11), we detected in these cells the presence of immunoreactivity against ERK 1/2 MAPK and Akt/PKB proteins, components of the MAPK and the phosphatidylinositol 3-kinase-Akt pathways, respectively, two of the main intracellular transduction pathways for insulin and IGF-I receptors. Both insulin and IGF-I activated both pathways, although the latter provoked higher immunoreactivity of phosphorylated MAPKs and Akt proteins. At every stage, increases in total MAPK immunoreactivity levels were observed when cells were stimulated with IGF-I or insulin, while total Akt immunoreactivity levels changed little under stimulation of peptides. Total Akt and total MAPK levels increased as skeletal muscle cells differentiated in culture. Moreover, when cells were incubated with IGF-I or insulin, MAPK-P immunoreactivity levels showed greater increases over the basal levels on days 1 and 4, with no effect observed on day 11. Although Akt-P immunoreactivity displayed improved responses on days 1 and 4 as well, a stimulatory effect was still observed on day 11. In addition, the present study demonstrates that purified trout insulin receptors possess higher phosphorylative activity per unit of receptor than IGF-I receptors. In conclusion, these results indicate that trout skeletal muscle culture is a suitable model to study the insulin and IGF-I signal transduction molecules and that there is a different regulation of MAPK and Akt pathways depending on the developmental stage of the muscle cells.
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Affiliation(s)
- Juan Castillo
- Department de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain
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38
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Bardelli C, Sala M, Cavallazzi U, Prat M. Agonist Met antibodies define the signalling threshold required for a full mitogenic and invasive program of Kaposi's Sarcoma cells. Biochem Biophys Res Commun 2005; 334:1172-9. [PMID: 16039997 DOI: 10.1016/j.bbrc.2005.07.020] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2005] [Accepted: 07/11/2005] [Indexed: 01/08/2023]
Abstract
We previously showed that the Kaposi Sarcoma line KS-IMM express a functional Met tyrosine kinase receptor, which, upon HGF stimulation, activates motogenic, proliferative, and invasive responses. In this study, we investigated the signalling pathways activated by HGF, as well as by Met monoclonal antibodies (Mabs), acting as full or partial agonists. The full agonist Mab mimics HGF in all biological and biochemical aspects. It elicits the whole spectrum of responses, while the partial agonist Mab induces only wound healing. These differences correlated with a more prolonged and sustained tyrosine phosphorylation of the receptor and MAPK evoked by HGF and by the full agonist Mab, relative to the partial agonist Mab. Since Gab1, JNK and PI 3-kinase are activated with same intensity and kinetics by HGF and by the two agonist antibodies, it is concluded that level and duration of MAPK activation by Met receptor are crucial for the induction of a full HGF-dependent mitogenic and invasive program in KS cells.
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Affiliation(s)
- Claudio Bardelli
- Dipt. Scienze Mediche, Università del Piemonte Orientale A. Avogadro Via Solaroli 17, 28100 Novara, Italy
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Abstract
Remarkable phenotype plasticity of epithelial cells underlies morphogenesis, epithelial repair and tumor invasiveness. Detailed understanding of the contextual cues and molecular mediators that control epithelial plasticity will be required in order to develop viable therapeutic approaches targeting epithelial-to-mesenchymal transition (EMT), an advanced manifestation of epithelial plasticity. Members of the transforming growth factor (TGF-beta) family of growth factors can initiate and maintain EMT in a variety of biological systems and pathophysiological context by activating major signaling pathways and transcriptional regulators integrated in extensive signaling networks. Here we will review the distinct physiological contexts of EMT and the underlying molecular signaling networks controlled by TGF-beta.
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Affiliation(s)
- Jiri Zavadil
- Department of Pathology, NYU Cancer Institute, New York University School of Medicine, New York, NY 10016, USA
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40
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Jia Z, Barbier L, Stuart H, Amraei M, Pelech S, Dennis JW, Metalnikov P, O'Donnell P, Nabi IR. Tumor cell pseudopodial protrusions. Localized signaling domains coordinating cytoskeleton remodeling, cell adhesion, glycolysis, RNA translocation, and protein translation. J Biol Chem 2005; 280:30564-73. [PMID: 15985431 DOI: 10.1074/jbc.m501754200] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The pseudopodial protrusions of Moloney sarcoma virus (MSV)-Madin-Darby canine kidney (MDCK)-invasive (INV) variant cells were purified on 1-microm pore polycarbonate filters that selectively allow passage of the pseudopodial domains but not the cell body. The purified pseudopodial fraction contains phosphotyrosinated proteins, including Met and FAK, and various signaling proteins, including Raf1, MEK1, ERK2, PKBalpha (Akt1), GSK3alpha, GSK3beta, Rb, and Stat3. Pseudopodial proteins identified by liquid chromatography tandem mass spectrometry included actin and actin-regulatory proteins (ERM, calpain, filamin, myosin, Sra-1, and IQGAP1), tubulin, vimentin, adhesion proteins (vinculin, talin, and beta1 integrin), glycolytic enzymes, proteins associated with protein translation, RNA translocation, and ubiquitin-mediated protein degradation, as well as protein chaperones (HSP90 and HSC70) and signaling proteins (RhoGDI and ROCK). Inhibitors of MEK1 (U0126) and HSP90 (geldanamycin) significantly reduced MSV-MDCK-INV cell motility and pseudopod expression, and geldanamycin treatment inhibited Met phosphorylation and induced the expression of actin stress fibers. ROCK inhibition did not inhibit cell motility but transformed the pseudopodial protrusions of MSV-MDCK-INV cells into extended lamellipodia. Dominant negative Rho disrupted pseudopod expression and, in serum-starved cells, L-alpha-lysophosphatidic acid (oleoyl) activation of Rho induced pseudopodial protrusions or, in the presence of the ROCK inhibitor, extended lamellipodia. RNA was localized to the actin-rich pseudopodial domains of MSV-MDCK-INV cells, but the extent of colocalization with dense actin ruffles was reduced in the extended lamellipodia formed upon ROCK inhibition. Rho/ROCK activation in epithelial tumor cells therefore regulates RNA translocation to a pseudopodial domain that contains proteins involved in signaling, cytoskeleton remodeling, cell adhesion, glycolysis, and protein translation and degradation.
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Affiliation(s)
- Zongjian Jia
- Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3
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41
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Christensen JG, Burrows J, Salgia R. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention. Cancer Lett 2004; 225:1-26. [PMID: 15922853 DOI: 10.1016/j.canlet.2004.09.044] [Citation(s) in RCA: 421] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2004] [Accepted: 09/29/2004] [Indexed: 10/26/2022]
Abstract
Receptor tyrosine kinase (RTK) targeted agents such as trastuzumab, imatinib, bevacizumab, and gefitinib inhibitors have illustrated the utility of targeting this protein class for treatment of selected cancers. A unique member of the RTK family, c-Met, also represents an intriguing target for cancer therapy that is yet to be explored in a clinical setting. The proto-oncogene, c-Met, encodes the high-affinity receptor for hepatocyte growth factor (HGF) or scatter factor (SF). c-Met and HGF are each required for normal mammalian development and have been shown to be particularly important in cell migration, morphogenic differentiation, and organization of three-dimensional tubular structures (e.g. renal tubular cells, gland formation, etc.) as well as cell growth and angiogenesis. Both c-Met and HGF have been shown to be deregulated in and to correlate with poor prognosis in a number of major human cancers. New data describing the constitutive phosphorylation of c-Met in a number of human tumors is presented here along with a variety of mechanisms by which c-Met can become activated, including mutation and gene amplification. In support of the clinical data implicating c-Met activation in the pathogenesis of human cancers, introduction of c-Met and HGF (or mutant c-Met) into cells conferred the properties of motility, invasiveness, and tumorgenicity to the transformed cells. Conversely, the inhibition of c-Met with a variety of receptor antagonists inhibited the motility, invasiveness, and tumorgenicity of human tumor cell lines. Consistent with this observation, small-molecule inhibitors of c-Met were developed that antagonized c-Met/HGF-dependent phenotypes and tumor growth in mouse models. This review will address the potential for development of c-Met inhibitors for treatment of human cancers with particular emphasis on recent findings with small-molecule inhibitors.
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Halevy O, Cantley LC. Differential regulation of the phosphoinositide 3-kinase and MAP kinase pathways by hepatocyte growth factor vs. insulin-like growth factor-I in myogenic cells. Exp Cell Res 2004; 297:224-34. [PMID: 15194438 DOI: 10.1016/j.yexcr.2004.03.024] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2003] [Revised: 03/01/2004] [Indexed: 12/20/2022]
Abstract
Hepatocyte growth factor (HGF) promotes the proliferation of adult myoblasts and inhibits their differentiation, whereas insulin-like growth factor I (IGF-I) enhances both processes. Recent studies indicate that activation of the phosphoinositide 3'-kinase (PI3K) pathway promotes myoblast differentiation, whereas activation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) promotes proliferation and inhibits their differentiation. This simple model is confounded by the fact that both HGF and IGF-I have been shown to activate both pathways. In this study, we have compared the ability of HGF and IGF-I to activate PI3K and MAPK/ERK in i28 myogenic cells. We find that, although the two stimuli result in comparable recruitment of the p85alpha subunit of PI3K into complexes with tyrosine-phosphorylated proteins, the p85beta regulatory subunit and p110alpha catalytic subunit of PI3K are preferentially recruited into these complexes in response to IGF-I. In agreement with this observation, IGF-I is much more potent than HGF in stimulating phosphorylation of Akt/PKB, a protein kinase downstream of PI3K. In contrast, MAPK/ERK phosphorylation was higher in response to HGF and lasted longer, relative to IGF-I. Moreover, the specific PI3K inhibitor, Wortmannin, abolished MAPK/ERK and Elk-1 phosphorylation in HGF-treated cells, suggesting the requirement of PI3K in mediating the HGF-induced MAPK pathway. UO126, a specific MAPK pathway inhibitor, had no effect on PI3K activity or Akt phosphorylation, implying that at least in muscle cells, the MAPK/ERK pathway is not required for HGF-induced PI3K activation. These results provide a biochemical rationale for the previous observations that HGF and IGF-I have opposite effects on myogenic cells, consistent with studies linking PI3K activation to differentiation and MAPK/ERK activation to proliferation in these cells. Moreover, the finding that PI3K activity is required for HGF-induced MAPK activation suggests its additional role in proliferation, rather than exclusively in the differentiation of adult myoblasts.
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Affiliation(s)
- Orna Halevy
- Division of Signal Transduction, Beth Israel Deaconess Medical Center, Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
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43
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Taniguchi F, Harada T, Deura I, Iwabe T, Tsukihara S, Terakawa N. Hepatocyte growth factor promotes cell proliferation and inhibits progesterone secretion via PKA and MAPK pathways in a human granulosa cell line. Mol Reprod Dev 2004; 68:335-44. [PMID: 15112327 DOI: 10.1002/mrd.20076] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Hepatocyte growth factor (HGF) is a mesenchymal-derived paracrine factor that acts through a c-met receptor. The activated c-met receptor recruits various signal proteins. We used a steroidogenic human granulosa-like tumor cell line (KGN cells) to analyze the biological function of HGF in human ovary cells. First, we designed a method to analyze local production and action of HGF in the human ovary. Although c-met mRNA is expressed in KGN cells, granulosa lutein, theca, and ovarian stroma cells, we observed HGF mRNA only in theca and stroma cells. Adding HGF to the medium enhanced mitogenic activity in KGN cells. We next examined the activation of intracellular signal transduction molecules induced by HGF in KGN cells. Here, we showed that HGF activated the distinct phosphorylation of Raf-1, MEK1/2, and ERK1/2, but did not induce phosphorylation of Akt. HGF enhanced the phosphorylation of Elk-1 and c-Jun as nuclear transcription factors. U0126, a MEK1/2 inhibitor, completely abrogated the phosphorylation of ERK1/2 and the cell proliferation in response to HGF. In contrast, H-89, a protein kinase A inhibitor, further enhanced the HGF-induced phosphorylation of ERK1/2 and cell proliferation. In addition, we revealed that HGF suppressed progesterone synthesis in KGN cells. Adding HGF suppressed the forskolin-induced steroidogenic acute regulatory protein (StAR) expression, which is a key regulator in progesterone synthesis. Crosstalk signals between PKA and the mitogen-activated protein kinase (MAPK) pathway were mutually inhibitory. These results demonstrated for the first time that theca cell-derived HGF may be capable of stimulating the proliferation of granulosa cells and suppressing progesterone synthesis via an activating MAPK pathway.
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Affiliation(s)
- Fuminori Taniguchi
- Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago, Japan.
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Li XN, Ding YQ, Liu GB. Transcriptional gene expression profiles of HGF/SF-met signaling pathway in colorectal carcinoma. World J Gastroenterol 2003; 9:1734-8. [PMID: 12918110 PMCID: PMC4611533 DOI: 10.3748/wjg.v9.i8.1734] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the transcriptional gene expression profiles of HGF/SF-met signaling pathway in colorectal carcinoma to understand mechanisms of the signaling pathway at so gene level.
METHODS: Total RNA was isolated from human colorectal carcinoma cell line LoVo treated with HGF/SF (80 ng/L) for 48 h. Fluorescent probes were prepared from RNA labeled with cy3-dUTP for the control groups and with cy5-dUTP for the HGF/SF-treated groups through reverse-transcription. The probes were mixed and hybridized on the microarray at 60 °C for 15-20 h, then the microarray was scanned by laser scanner (GenePix 4000B). The intensity of each spot and ratios of Cy5/Cy3 were analyzed and finally the differentially expressed genes were selected by GenePix Pro 3.0 software. 6 differential expression genes (3 up-regulated genes and 3 down-regulated genes) were selected randomly and analyzed by β-actin semi-quantitative RT-PCR.
RESULTS: The fluorescent intensities of built-in negative control spots were less than 200, and the fluorescent intensities of positive control spots were more than 5000. Of the 4004 human genes analyzed by microarray, 129 genes (holding 3.22% of the investigated genes) revealed differential expression in HGF/SF-treated groups compared with the control groups, of which 61 genes were up-regulated (holding 1.52% of the investigated genes) and 68 genes were down-regulated (holding 1.70% of the investigated genes), which supplied abundant information about target genes of HGF/SF-met signaling.
CONCLUSION: HGF/SF-met signaling may up-regulate oncogenes, signal transduction genes, apoptosis-related genes, metastasis related genes, and down-regulate a number of genes. The complexity of HGF/SF-met signaling to control the gene expression is revealed as a whole by the gene chip technology.
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Affiliation(s)
- Xue-Nong Li
- Department of Pathology, First Military Medical University, Guangzhou 510515, Guangdong Province, China.
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Martin TA, Parr C, Davies G, Watkins G, Lane J, Matsumoto K, Nakamura T, Mansel RE, Jiang WG. Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist. Carcinogenesis 2003; 24:1317-23. [PMID: 12807719 DOI: 10.1093/carcin/bgg072] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Hepatocyte growth factor/scatter factor (HGF/SF) is a cytokine primarily produced by stromal fibroblasts and is a known angiogenic and invasion-inducing factor. It is increased in patients with breast cancer. This study examined the effect of NK4, a newly described HGF/SF antagonist, on HGF/SF-promoted growth of a human breast cancer. Both in vitro (invasion and migration assays) and in vivo (murine tumour model) methods were used to ascertain the effect of NK4 on HGF/SF from two sources: human fibroblast-derived HGF/SF and recombinant HGF/SF. In the in vitro invasion assay and migration assay, both HGF/SF and human fibroblasts, which secrete bioactive HGF/SF, increased the invasiveness and migration of the breast cancer cells (MDA MB 231). NK4 significantly reduced this invasiveness and motility. In the animal model, tumour volume and weight was significantly reduced with addition of NK4. It also suppressed HGF/SF-induced growth and markedly retarded tumour growth induced by fibroblasts (MRC5), secreting bioactive HGF/SF. Tumour angiogenesis was assessed by immunohistochemical staining of primary tissue sections using VE-cadherin (an endothelial cell specific cell-cell adhesion molecule). Again, NK4 reduced the effects of both HGF/SF and fibroblasts. We conclude that NK4 has a significant effect on the growth of human breast tumours in nude mice, particularly when stimulated by HGF/SF or fibroblasts. This may occur by decreasing angiogenesis. This gives a clear indication of the therapeutic worth of NK4.
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Affiliation(s)
- Tracey A Martin
- Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
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Derksen PWB, de Gorter DJJ, Meijer HP, Bende RJ, van Dijk M, Lokhorst HM, Bloem AC, Spaargaren M, Pals ST. The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma. Leukemia 2003; 17:764-74. [PMID: 12682635 DOI: 10.1038/sj.leu.2402875] [Citation(s) in RCA: 122] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
The evolution of multiple myeloma (MM) depends on complex signals from the bone marrow (BM) microenvironment, supporting the proliferation and survival of malignant plasma cells. An interesting candidate signal is hepatocyte growth factor/scatter factor (HGF), since its receptor Met is expressed on MM cells, while HGF is produced by BM stromal cells and by some MM cell lines, enabling para- or autocrine interaction. To explore this hypothesis, we studied the biological effects of HGF stimulation on MM cell lines and on primary MMs. We observed that Met is expressed by the majority of MM cell lines and by approximately half of the primary plasma cell neoplasms tested. Stimulation of MM cells with HGF led to the activation of the RAS/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathways, signaling routes that have been implicated in the regulation of cell proliferation and survival. Indeed, functional studies demonstrated that HGF has strong proliferative and anti-apoptotic effects on both MM cell lines and primary MM cells. Furthermore, by applying specific signal-transduction inhibitors, we demonstrated that MEK is required for HGF-induced proliferation, whereas activation of PI3K is required for both HGF-induced proliferation and for rescue of MM cells from apoptosis. Taken together, our data indicate that HGF is a potent myeloma growth and survival factor and suggest that the HGF/Met pathway is a potential therapeutic target in MM.
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Affiliation(s)
- P W B Derksen
- Department of Pathology, Academic Medical Center, University of Amsterdam, The Netherlands
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47
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Kitta K, Day RM, Kim Y, Torregroza I, Evans T, Suzuki YJ. Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells. J Biol Chem 2003; 278:4705-12. [PMID: 12468531 DOI: 10.1074/jbc.m211616200] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Hepatocyte growth factor (HGF) is released in response to myocardial infarction and may play a role in regulating cardiac remodeling. Recently, HGF was found to inhibit the apoptosis of cardiac muscle cells. Because GATA-4 can induce cell survival, the effects of HGF on GATA-4 activity were investigated. Treatment of HL-1 cells or primary adult rat cardiac myocytes with HGF, at concentrations that can be detected in the human serum after myocardial infarction, rapidly enhances GATA-4 DNA-binding activity. The enhanced DNA-binding activity is associated with the phosphorylation of GATA-4. HGF-induced phosphorylation and activation of GATA-4 is abolished by MEK inhibitors or the mutation of the ERK phosphorylation site (S105A), suggesting that HGF activates GATA-4 via MEK-ERK pathway-dependent phosphorylation. HGF enhances the expression of anti-apoptotic Bcl-x(L), and this is blocked by dominant negative mutants of MEK or GATA-4. Forced expression of wild-type GATA-4, but not the GATA-4 mutant (S105A) increases the expression of Bcl-x(L). Furthermore, expression of the GATA-4 mutant (S105A) suppresses HGF-mediated protection of cells against daunorubicin-induced apoptosis. These results demonstrate that HGF protects cardiac muscle cells against apoptosis via a signaling pathway involving MEK/ERK-dependent phosphorylation of GATA-4.
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Affiliation(s)
- Kazumi Kitta
- Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111, USA
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48
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Price JA, Kovach SJ, Johnson T, Koniaris LG, Cahill PA, Sitzmann JV, McKillop IH. Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma. Hepatology 2002; 36:1089-97. [PMID: 12395318 DOI: 10.1053/jhep.2002.36158] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Hepatocyte growth factor-scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P <.05). Despite activation of a MAPK pathway and increased proto-oncogene expression, IGF-I failed to significantly affect cell mitogenesis. In contrast, HGF significantly inhibited cell mitogenesis in HCC lines (68.4% +/- 9.4% vs. control, n = 4, P <.05). Pretreatment of HCC cells with IGF-I (60 minutes) led to significant HGF-SF stimulation of total cell mitogenesis dependent on both IGF-I and HGF-SF dose (194% +/- 8% increase vs. control, n = 4, P <.05). In conclusion, tumor burden is important in altering intrahepatic growth factor synthesis. Signal cooperation between multiple cytokine pathways is an important factor in the progression of HCC.
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Affiliation(s)
- Julie A Price
- Department of Surgery, University of Rochester Medical Center, NY, USA
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49
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Liu F, Schaphorst KL, Verin AD, Jacobs K, Birukova A, Day RM, Bogatcheva N, Bottaro DP, Garcia JGN. Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3beta. FASEB J 2002; 16:950-62. [PMID: 12087056 DOI: 10.1096/fj.01-0870com] [Citation(s) in RCA: 131] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The stabilization of endothelial cell (EC) barrier function within newly formed capillaries is a critical feature of angiogenesis. We examined human lung EC barrier regulation elicited by hepatocyte growth factor (HGF), a recognized angiogenic factor and EC chemoattractant. HGF rapidly and dose-dependently elevated transendothelial electrical resistance (TER) of EC monolayers (>50% increase at 100 ng/ml), with immunofluorescence microscopic evidence of both cytoplasmic actin stress fiber dissolution and strong augmentation of the cortical actin ring. HGF rapidly stimulated phosphatidylinositol 3'-kinase, ERK, p38 mitogen-activated protein kinase, and protein kinase C activities. Pharmacological inhibitor studies demonstrated each pathway to be intimately involved in HGF-induced increases in TER, cortical actin thickening, and phosphorylation of the Ser/Thr glycogen synthase kinase-3beta (GSK-3beta), a potential target for the HGF barrier-promoting response. GSK-3beta phosphorylation was strongly correlated with reductions in both HGF-induced TER and enhanced beta-catenin immunoreactivity observed at cell-cell junctions. Our data suggest a model in which HGF-mediated EC cytoskeletal rearrangement and barrier enhancement depend critically on the activation of a complex kinase cascade that converges at GSK-3beta to increase the availability of beta-catenin, thereby enhancing endothelial junctional integrity and vascular barrier function.
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Affiliation(s)
- Feng Liu
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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50
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Edme N, Downward J, Thiery JP, Boyer B. Ras induces NBT-II epithelial cell scattering through the coordinate activities of Rac and MAPK pathways. J Cell Sci 2002; 115:2591-601. [PMID: 12045229 DOI: 10.1242/jcs.115.12.2591] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Cell dissociation and cell migration are the two main components of epithelium-mesenchyme transitions (EMT). We previously demonstrated that Ras is required for the accomplishment of both of these processes during the EGF-induced EMT of the NBT-II rat carcinoma cell line in vitro. In this study,we examined the downstream targets of Ras that are responsible for the dissociation and motility of NBT-II cells. Overexpression of activated forms of c-Raf and MEK1 (a component of the mitogen-activated protein kinase pathway, MAPK) led to cell dissociation, as inferred by the loss of desmosomes from the cell periphery. By contrast, active PI3K, RalA and RalB did not induce desmosome breakdown. The MEK1 inhibitor PD098059 inhibited EGF- and Ras-induced cell dispersion, whereas the PI3K inhibitor LY294002 had no effect. Accordingly, among the partial loss-of-function mutants of Ras(RasV12) that were used to distinguish between downstream targets of Ras, we found that the Raf-specific Ras mutants RasV12S35 and RasV12E38 induced cell dissociation. The PI3K- and RalGDS-activating Ras mutants had, in contrast, no effect on cell dispersion. However, MEK1 was unable to promote cell motility,whereas RasV12S35 and RasV12E38 induced cell migration, suggesting that another Ras effector was responsible for cell motility. We found that the small GTPase Rac is necessary for EGF-mediated cell dispersion since overexpression of a dominant-negative mutant of Rac1 (Rac1N17) inhibited EGF-induced NBT-II cell migration. All stimuli that promoted cell migration also induced Rac activation. Finally, coexpression of active Rac1 and active MEK1 induced the motility of NBT-II cells, suggesting that Ras mediates NBT-II cell scattering through the coordinate activation of Rac and the Raf/MAPK pathway.
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Affiliation(s)
- Natacha Edme
- Laboratoire de Régulations Cellulaires et Oncogénése UMR146, Institut Curie Section de Recherche, Centre Universitaire Paris-Sud, 91405 Orsay, France
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