Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 2, Pages 481-503Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.011341
Keywords
cancer therapy; cancer biology; molecular pharmacology; receptor tyrosine kinase; hepatocyte growth factor (HGF); c-Met; hepatocyte growth factor receptor; MET proto-oncogene receptor tyrosine kinase (MET); N-myc downstream-regulated gene-1 (NDRG1); thiosemicarbazone
Categories
Funding
- National Health and Medical Research Council of Australia (NHMRC) [1060482]
- Priority-driven Collaborative Cancer Research Scheme Grant - Cure Cancer Australia Foundation of Australia [1086449]
- Cancer Australia
- NHMRC/PdCCRS Cancer Australia Project Grant - NBCF [NHMRC APP1146599]
- National Breast Cancer Foundation (NBCF) [IIRS-19-048]
- Research Training Program Scholarship from the University of Sydney
- Cancer Institute of New South Wales Career Development Fellowship [CDF171147]
- NHMRC Peter Doherty Early Career Fellowship [1037323]
- Cancer Institute New South Wales Early Career Fellowship [12-ECF2-17]
- NHMRC RD Wright Fellowship [APP1140447]
- Cancer Institute New South Wales Career Development Fellowship [CDF171126]
- NHMRC Senior Principal Research Fellowship [1062607]
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Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. NDRG1 silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after NDRG1 silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 (MIG6), decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. In fact, Dp44mT increased c-Met CTF while decreasing the ICD. Dp44mT and a ?-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors, EDTA and batimastat, partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3), had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage.
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