RESUMEN
Matrix metalloproteinases (MMPs), in particular MMP-2, MMP-9 and MMP-14, play a key role in various aspects of cancer pathology. Likewise, ADAMs (a disintegrin and metalloproteinases), including ADAM12, are upregulated in malignant tumors and contribute to the pathology of cancers. Here, we show that there is a positive correlation between MMP-14 and ADAM12 expression in human breast cancer. We demonstrated that in 293-VnR and human breast cancer cells expressing ADAM12 at the cell surface, endogenous MMP-14 was recruited to the cell surface, resulting in its activation. Subsequent to this activation, gelatin degradation was stimulated and tumor cell apoptosis was decreased, with reduced expression of the pro-apoptotic proteins BCL2L11 and BIK. The effect on gelatin degradation was abrogated by inhibition of the MMP-14 activity and appeared to be dependent on cell surface αVß3 integrin localization, but neither the catalytic activity of ADAM12 nor the cytoplasmic tail of ADAM12 were required. The significance of ADAM12-induced activation of MMP-14 was underscored by a reduction in MMP-14-mediated gelatin degradation and abolition of apoptosis-protective effects by specific monoclonal antibodies against ADAM12. Furthermore, orthotopic implantation of ADAM12-expressing MCF7 cells in nude mice produced tumors with increased levels of activated MMP-14 and confirmed that ADAM12 protects tumor cells against apoptosis, leading to increased tumor progression. In conclusion, our data suggest that a ternary protein complex composed of ADAM12, αVß3 integrin and MMP-14 at the tumor cell surface regulates the function of MMP-14. This interaction might point to a novel concept for the development of MMP-14-targeting drugs in treating cancer.
Asunto(s)
Proteínas ADAM/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Gelatina/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/inmunología , Proteína ADAM12 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Femenino , Células HEK293 , Xenoinjertos , Humanos , Integrina alfaVbeta3/metabolismo , Células MCF-7 , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos NODRESUMEN
ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor-bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.
Asunto(s)
Proteínas ADAM/metabolismo , Clatrina/metabolismo , Endocitosis , Proteína Adaptadora GRB2/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/análisis , Proteínas ADAM/química , Proteína ADAM12 , Neoplasias de la Mama/química , Neoplasias de la Mama/enzimología , Carcinoma/química , Endosomas/metabolismo , Femenino , Proteína Adaptadora GRB2/análisis , Células HEK293 , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Dominios Proteicos Ricos en Prolina , Dominios y Motivos de Interacción de ProteínasRESUMEN
Invadopodia are dynamic actin structures at the cell surface that degrade extracellular matrix and act as sites of signal transduction. The biogenesis of invadopodia, including the mechanisms regulating their formation, composition, and turnover is not entirely understood. Here, we demonstrate that antibody ligation of ADAM12, a transmembrane disintegrin and metalloprotease, resulted in the rapid accumulation of invadopodia with extracellular matrix-degrading capacity in epithelial cells expressing the αvß3 integrin and active c-Src kinase. The induction of invadopodia clusters required an intact c-Src interaction site in the ADAM12 cytoplasmic domain, but was independent of the catalytic activity of ADAM12. Caveolin-1 and transmembrane protease MMP14/MT1-MMP were both present in the ADAM12-induced clusters of invadopodia, and cholesterol depletion prevented their formation, suggesting that lipid-raft microdomains are involved in the process. Importantly, our data demonstrate that ADAM12-mediated ectodomain shedding of epidermal growth factor receptor ligands can occur within these invadopodia. Such localized growth factor signalling offers an interesting novel biological concept highly relevant to the properties of carcinoma cells, which often show upregulated ADAM12 and ß3 integrin expression, together with high levels of c-Src kinase activity.
Asunto(s)
Proteínas ADAM/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/genética , Proteína ADAM12 , Actinas/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Línea Celular , Membrana Celular/genética , Matriz Extracelular/genética , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Riñón/citología , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de la Membrana/genética , Unión Proteica/genética , Receptores de Vitronectina/metabolismo , Transducción de Señal/genética , Transfección , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
ADAM12 is an active metalloprotease playing an important role in tumour progression. Human ADAM12 exists in two splice variants: a long transmembrane form, ADAM12-L, and a secreted form, ADAM12-S. The subcellular localization of ADAM12-L is tightly regulated and involves intracellular interaction partners and signalling proteins. We demonstrate here a c-Src-dependent redistribution of ADAM12-L from perinuclear areas to actin-rich Src-positive structures at the cell periphery, and identified two separate c-Src binding sites in the cytoplasmic tail of ADAM12-L that interact with the SH3 domain of c-Src with different binding affinities. The association between ADAM12-L and c-Src is transient, but greatly stabilized when the c-Src kinase activity is disrupted. In agreement with this observation, kinase-active forms of c-Src induce ADAM12-L tyrosine phosphorylation. Interestingly, ADAM12-L was also found to enhance Src kinase activity in response to external signals, such as integrin engagement. Thus, we suggest that activated c-Src binds, phosphorylates, and redistributes ADAM12-L to specific sites at the cell periphery, which may in turn promote signalling mechanisms regulating cellular processes with importance in cancer.
Asunto(s)
Proteínas ADAM/metabolismo , Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas ADAM/genética , Proteína ADAM12 , Sitios de Unión/fisiología , Unión Competitiva , Proteína Tirosina Quinasa CSK , Línea Celular , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Adhesiones Focales/metabolismo , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Proteínas de la Membrana/genética , Modelos Biológicos , Mutación/fisiología , Fragmentos de Péptidos/metabolismo , Fosforilación/fisiología , Unión Proteica/fisiología , Transporte de Proteínas , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Vitronectina/metabolismo , Dominios Homologos src/fisiología , Familia-src QuinasasRESUMEN
A recently identified breast cancer-associated mutation in the metalloprotease ADAM12 alters a potential dileucine trafficking signal, which could affect protein processing and cellular localization. ADAM12 belongs to the group of A Disintegrin And Metalloproteases (ADAMs), which are typically membrane-associated proteins involved in ectodomain shedding, cell-adhesion, and signaling. ADAM12 as well as several members of the ADAM family are over-expressed in various cancers, correlating with disease stage. Three breast cancer-associated somatic mutations were previously identified in ADAM12, and two of these, one in the metalloprotease domain and another in the disintegrin domain, were investigated and found to result in protein misfolding, retention in the secretory pathway, and failure of zymogen maturation. The third mutation, p.L792F in the ADAM12 cytoplasmic tail, was not investigated, but is potentially significant given its location within a di-leucine motif, which is recognized as a potential cellular trafficking signal. The present study was motivated both by the potential relevance of this documented mutation to cancer, as well as for determining the role of the di-leucine motif in ADAM12 trafficking. Expression of ADAM12 p.L792F in mammalian cells demonstrated quantitatively similar expression levels and zymogen maturation as wild-type (WT) ADAM12, as well as comparable cellular localizations. A cell surface biotinylation assay demonstrated that cell surface levels of ADAM12 WT and ADAM12 p.L792F were similar and that internalization of the mutant occurred at the same rate and extent as for ADAM12 WT. Moreover, functional analysis revealed no differences in cell proliferation or ectodomain shedding of epidermal growth factor (EGF), a known ADAM12 substrate between WT and mutant ADAM12. These data suggest that the ADAM12 p.L792F mutation is unlikely to be a driver (cancer causing)-mutation in breast cancer.