RESUMEN
Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.
Asunto(s)
Carcinogénesis/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Células A549 , Animales , Humanos , Espectrometría de Masas/métodos , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Proteómica , Ratas , Ratas Sprague-Dawley , Pez CebraRESUMEN
PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. Only a limited number of PP2A-regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Strikingly, we find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A-B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.
Asunto(s)
Proteína ADAM17/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteína ADAM17/genética , Secuencias de Aminoácidos , Animales , Sitios de Unión , Células HeLa , Humanos , Ratones , FosforilaciónRESUMEN
Desmoplasia is a common feature of aggressive cancers, driven by a complex interplay of protein production and degradation. Basigin is a type 1 integral membrane receptor secreted in exosomes or released by ectodomain shedding from the cell surface. Given that soluble basigin is increased in the circulation of patients with a poor cancer prognosis, we explored the putative role of the ADAM12-generated basigin ectodomain in cancer progression. We show that recombinant basigin ectodomain binds ß1 integrin and stimulates gelatin degradation and the migration of cancer cells in a matrix metalloproteinase (MMP)- and ß1-integrin-dependent manner. Subsequent in vitro and in vivo experiments demonstrated the altered expression of extracellular matrix proteins, including fibronectin and collagen type 5. Thus, we found increased deposits of collagen type 5 in the stroma of nude mice tumors of the human tumor cell line MCF7 expressing ADAM12-mimicking the desmoplastic response seen in human cancer. Our findings indicate a feedback loop between ADAM12 expression, basigin shedding, TGFß signaling, and extracellular matrix (ECM) remodeling, which could be a mechanism by which ADAM12-generated basigin ectodomain contributes to the regulation of desmoplasia, a key feature in human cancer progression.
Asunto(s)
Proteína ADAM12 , Basigina , Proteínas de la Matriz Extracelular , Animales , Femenino , Humanos , Ratones , Proteína ADAM12/metabolismo , Proteína ADAM12/genética , Basigina/metabolismo , Basigina/genética , Línea Celular Tumoral , Movimiento Celular , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Células MCF-7 , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/genética , Unión Proteica , Dominios Proteicos , Integrina beta1/metabolismoRESUMEN
Tumor progression and response to treatment are highly affected by interactions between cancer cells and the tumor microenvironment (TME). Many of the soluble factors and signaling receptors involved in this crosstalk are shed by a disintegrin and metalloproteinases (ADAMs). Upregulation of ADAM15 has been linked to worse survival in cancer patients and a tumor-promoting function both in vitro and in murine cancer models. Although ADAM15 has been involved in cell-cell and cell-extracellular matrix interactions, its role in the crosstalk between cancer cells and the TME in vivo remains unexplored. Therefore, we aimed to understand how ADAM15 regulates the cell composition of the TME and how it affects tumor progression. Here, we showed an upregulation of ADAM15 in tumor tissues from rectal cancer patients. Subcutaneous injection of wildtype and ADAM15-knockout CT26 colon cancer cells in syngeneic mice confirmed the protumorigenic role of ADAM15. Profiling of tumors revealed higher immune cell infiltration and cancer cell apoptosis in the ADAM15-deficient tumors. Specifically, loss of ADAM15 led to a reduced number of granulocytes and higher infiltration of antigen-presenting cells, including dendritic cells and macrophages, as well as more T cells. Using in vitro assays, we confirmed the regulatory effect of ADAM15 on macrophage migration and identified ADAM15-derived CYR61 as a potential molecular mediator of this effect. Based on these findings, we speculate that targeting ADAM15 could increase the infiltration of immune cells in colorectal tumors, which is a prerequisite for effective immunotherapy.
Asunto(s)
Neoplasias Colorrectales , Microambiente Tumoral , Humanos , Ratones , Animales , Transducción de Señal , Movimiento Celular , Neoplasias Colorrectales/genética , Proteínas de la Membrana , Proteínas ADAM/genéticaRESUMEN
BACKGROUND: The lactate receptor GPR81 contributes to cancer development through unclear mechanisms. Here, we investigate the roles of GPR81 in three-dimensional (3D) and in vivo growth of breast cancer cells and study the molecular mechanisms involved. METHODS: GPR81 was stably knocked down (KD) in MCF-7 human breast cancer cells which were subjected to RNA-seq analysis, 3D growth, in situ- and immunofluorescence analyses, and cell viability- and motility assays, combined with KD of key GPR81-regulated genes. Key findings were additionally studied in other breast cancer cell lines and in mammary epithelial cells. RESULTS: GPR81 was upregulated in multiple human cancer types and further upregulated by extracellular lactate and 3D growth in breast cancer spheroids. GPR81 KD increased spheroid necrosis, reduced invasion and in vivo tumor growth, and altered expression of genes related to GO/KEGG terms extracellular matrix, cell adhesion, and Notch signaling. Single cell in situ analysis of MCF-7 cells revealed that several GPR81-regulated genes were upregulated in the same cell clusters. Notch signaling, particularly the Notch ligand Delta-like-4 (DLL4), was strikingly downregulated upon GPR81 KD, and DLL4 KD elicited spheroid necrosis and inhibited invasion in a manner similar to GPR81 KD. CONCLUSIONS: GPR81 supports breast cancer aggressiveness, and in MCF-7 cells, this occurs at least in part via DLL4. Our findings reveal a new GPR81-driven mechanism in breast cancer and substantiate GPR81 as a promising treatment target.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Ácido Láctico/metabolismo , Ligandos , Transducción de Señal , Necrosis , Receptor Notch1/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
The transmembrane protease ADAM9 is frequently upregulated in human cancers, and it promotes tumour progression in mice. In vitro, ADAM9 regulates cancer cell adhesion and migration by interacting with integrins. However, how ADAM9 modulates integrin functions is not known. We here show that ADAM9 knockdown increases ß1 integrin levels through mechanisms that are independent of its protease activity. In ADAM9-silenced cells, adhesion to collagen and fibronectin is reduced, suggesting an altered function of the accumulated integrins. Mechanistically, ADAM9 co-immunoprecipitates with ß1 integrin, and both internalization and subsequent degradation of ß1 integrin are significantly decreased in ADAM9-silenced cells, with no effect on ß1 integrin recycling. Accordingly, the formation of focal adhesions and actin stress fibres in ADAM9-silenced cells is altered, possibly explaining the reduction in cell adhesion and migration in these cells. Taken together, our data provide mechanistic insight into the ADAM9-integrin interaction, demonstrating that ADAM9 regulates ß1 integrin endocytosis. Moreover, our findings indicate that the reduced migration of ADAM9-silenced cells is, at least in part, caused by the accumulation and altered activity of ß1 integrin at the cell surface.
Asunto(s)
Proteínas ADAM/metabolismo , Movimiento Celular , Endocitosis , Adhesiones Focales/metabolismo , Integrina beta1/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Proteínas ADAM/genética , Actinas/metabolismo , Adhesión Celular , Membrana Celular/metabolismo , Colágeno/metabolismo , Fibronectinas/metabolismo , Humanos , Proteínas de la Membrana/genética , Células PC-3RESUMEN
ADAM9 is an active member of the family of transmembrane ADAMs (a disintegrin and metalloproteases). It plays a role in processes such as bone formation and retinal neovascularization, and importantly, its expression in human cancers correlates with disease stage and poor prognosis. Functionally, ADAM9 can cleave several transmembrane proteins, thereby shedding their ectodomains from the cell surface. Moreover, ADAM9 regulates cell behavior by binding cell-surface receptors such as integrin and membrane-type matrix metalloproteases. Because these functions are mainly restricted to the cell surface, understanding the mechanisms regulating ADAM9 localization and activity at this site is highly important. To this end, we here investigated how intracellular trafficking regulates ADAM9 availability at the cell surface. We found that ADAM9 undergoes constitutive clathrin-dependent internalization and subsequent degradation or recycling to the plasma membrane. We confirmed previous findings of an interaction between ADAM9 and the intracellular sorting protein, sorting nexin 9 (SNX9), as well as its close homolog SNX18. Knockdown of either SNX9 or SNX18 had no apparent effects on ADAM9 internalization or recycling. However, double knockdown of SNX9 and SNX18 decreased ADAM9 internalization significantly, demonstrating a redundant role in this process. Moreover, SNX9 knockdown revealed a nonredundant effect on overall ADAM9 protein levels, resulting in increased ADAM9 levels at the cell surface, and a corresponding increase in the shedding of Ephrin receptor B4, a well-known ADAM9 substrate. Together, our findings demonstrate that intracellular SNX9-mediated trafficking constitutes an important ADAM9 regulatory pathway.
Asunto(s)
Proteínas ADAM/genética , Neoplasias de la Mama/metabolismo , Membrana Celular/metabolismo , Endocitosis , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Nexinas de Clasificación/metabolismo , Proteínas ADAM/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica , Transporte de Proteínas , Nexinas de Clasificación/genética , Células Tumorales CultivadasRESUMEN
The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.
Asunto(s)
Proteína ADAM12/metabolismo , Basigina/metabolismo , Proteína ADAM12/química , Proteína ADAM12/genética , Secuencia de Aminoácidos , Basigina/química , Basigina/genética , Sistemas CRISPR-Cas , Línea Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Reporteros , Humanos , Mutación , Especificidad por SustratoRESUMEN
High metabolic and proliferative rates in cancer cells lead to production of large amounts of H+ and CO2 , and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na+ /H+ exchanger NHE1, the Na+ - HCO3- cotransporter NBCn1, and the lactate-H+ cotransporters MCT1 and -4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone receptor status, NHE1 correlated negatively and NBCn1 positively with HER2 status, whereas MCT4 expression correlated with lymph node status. Stable shRNA-mediated knockdown (KD) of either NHE1 or NBCn1 in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line significantly reduced steady-state intracellular pH (pHi ) and capacity for pHi recovery after an acid load. Importantly, KD of any of the three transporters reduced in vivo primary tumor growth of MDA-MB-231 xenografts. However, whereas KD of NBCn1 or MCT4 increased tumor-free survival and decreased in vitro proliferation rate and colony growth in soft agar, KD of NHE1 did not have these effects. Moreover, only MCT4 KD reduced Akt kinase activity, PARP and CD147 expression and cell motility. This work reveals that different types of net acid extruding transporters, NHE1, NBCn1 and MCT4, are frequently expressed in patient mammary tumor tissue and demonstrates for the first time that they promote growth of TNBC human mammary tumors in vivo via distinct but overlapping mechanisms.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Supervivencia sin Enfermedad , Femenino , Xenoinjertos , Humanos , Estimación de Kaplan-Meier , RatonesRESUMEN
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
ADAM (a disintegrin and metalloproteinase) 12 is a metalloprotease implicated in cancer progression. ADAM12 can activate membrane-anchored proteins, such as sonic hedgehog, Delta-like 1 and certain epidermal growth factor receptor ligands, through a process called ectodomain shedding. We screened several membrane-anchored proteins to further dissect the substrate profile of ADAM12-mediated ectodomain shedding, and found shedding of five previously unreported substrates [Kitl1, VE-cadherin (vascular endothelial cadherin), Flk-1 (fetal liver kinase 1), Tie-2, and VCAM-1 (vascular cell adhesion molecule 1)], of which the latter four are specifically expressed by endothelial cells. We also observed that ADAM12 expression was increased in the tumour vasculature of infiltrating ductal carcinoma of the human breast as compared with little to no expression in normal breast tissue vasculature, suggesting a role for ADAM12 in tumour vessels. These results prompted us to further evaluate ADAM12-mediated shedding of two endothelial cell proteins, VE-cadherin and Tie-2. Endogenous ADAM12 expression was very low in cultured endothelial cells, but was significantly increased by cytokine stimulation. In parallel, the shed form of VE-cadherin was elevated in such cytokine-stimulated endothelial cells, and ADAM12 siRNA (small interfering RNA) knockdown reduced cytokine-induced shedding of VE-cadherin. In conclusion, the results of the present study demonstrate a role for ADAM12 in ectodomain shedding of several membrane-anchored endothelial proteins. We speculate that this process may have importance in tumour neovascularization or/and tumour cell extravasation.
Asunto(s)
Proteínas ADAM/biosíntesis , Proteínas ADAM/química , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/química , Células Endoteliales de la Vena Umbilical Humana/química , Proteínas de la Membrana/química , Proteínas ADAM/deficiencia , Proteína ADAM12 , Animales , Neoplasias de la Mama/genética , Línea Celular Transformada , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/patologíaRESUMEN
Radiotherapy is one of the most common cancer treatments, yet, some patients require high doses to respond. Therefore, the development of new strategies leans toward personalizing therapy to avoid unnecessary burden on cancer patients. This approach prevents the administration of ineffective treatments or uses combination strategies to increase the sensitivity of cancer cells. ADAM12 has been shown to be upregulated in many cancers and correlate with poor survival and chemoresistance, thus making it a potential candidate responsible for radioresistance. Here, we show that ADAM12 expression is upregulated in response to irradiation in both mouse and human cancer cells in vitro, as well as in tumor tissues from rectal cancer patients. Interestingly, the expression of ADAM12 following radiotherapy correlates with the initial disease stage and predicts the response of rectal cancer patients to the treatment. While we found no cell-autonomous effects of ADAM12 on the response of colon cancer cells to irradiation in vitro, depletion of ADAM12 expression markedly reduced the tumor growth of irradiated cancer cells when subcutaneously transplanted in syngeneic mice. Interestingly, loss of cancer cell-derived ADAM12 expression increased the number of CD31+FAP- cells in murine tumors. Moreover, conditioned medium from ADAM12-/- colon cancer cells led to increased tube formation when added to endothelial cell cultures. Thus, it is tempting to speculate that altered tumor vascularity may be implicated in the observed effect of ADAM12 on response to radiotherapy in rectal cancer. We conclude that ADAM12 represents a promising prognostic factor for stratification of rectal cancer patients receiving radiotherapy and suggest that targeting ADAM12 in combination with radiotherapy could potentially improve the treatment response.
Asunto(s)
Neoplasias del Colon , Neoplasias del Recto , Animales , Humanos , Ratones , Proteína ADAM12/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/radioterapia , Regulación Neoplásica de la Expresión Génica , Pronóstico , Neoplasias del Recto/genética , Neoplasias del Recto/radioterapiaRESUMEN
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
Macrophages in the tumor microenvironment have a substantial impact on tumor progression. Depending on the signaling environment in the tumor, macrophages can either support or constrain tumor progression. It is therefore of therapeutic interest to identify the tumor-derived factors that control macrophage education. With this aim, we correlated the expression of A Disintegrin and Metalloproteinase (ADAM) proteases, which are key mediators of cell-cell signaling, to the expression of protumorigenic macrophage markers in human cancer cohorts. We identified ADAM17, a sheddase upregulated in many cancer types, as a protein of interest. Depletion of ADAM17 in cancer cell lines reduced the expression of several protumorigenic markers in neighboring macrophages in vitro as well as in mouse models. Moreover, ADAM17-/- educated macrophages demonstrated a reduced ability to induce cancer cell invasion. Using mass spectrometry-based proteomics and ELISA, we identified heparin-binding EGF (HB-EGF) and amphiregulin, shed by ADAM17 in the cancer cells, as the implicated molecular mediators of macrophage education. Additionally, RNA-Seq and ELISA experiments revealed that ADAM17-dependent HB-EGF ligand release induced the expression and secretion of CXCL chemokines in macrophages, which in turn stimulated cancer cell invasion. In conclusion, we provide evidence that ADAM17 mediates a paracrine EGFR-ligand-chemokine feedback loop, whereby cancer cells hijack macrophages to promote tumor progression.
Asunto(s)
Proteína ADAM17 , Desintegrinas , Macrófagos , Invasividad Neoplásica , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Anfirregulina , Animales , Factor de Crecimiento Epidérmico , Receptores ErbB/metabolismo , Heparina , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Humanos , Ligandos , Macrófagos/metabolismo , Ratones , Microambiente TumoralRESUMEN
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
The disintegrin and metalloprotease ADAM12 has important functions in normal physiology as well as in diseases, such as cancer. Little is known about how ADAM12 confers its pro-tumorigenic effect; however, its proteolytic capacity is probably a key component. Thus selective inhibition of ADAM12 activity may be of great value therapeutically and as an investigative tool to elucidate its mechanisms of action. We have previously reported the inhibitory profile of TIMPs (tissue inhibitor of metalloproteinases) against ADAM12, demonstrating in addition to TIMP-3, a unique ADAM-inhibitory activity of TIMP-2. These findings strongly suggest that it is feasible to design a TIMP mutant selectively inhibiting ADAM12. With this purpose, we characterized the molecular determinants of the ADAM12-TIMP complex formation as compared with known molecular requirements for TIMP-mediated inhibition of ADAM17/TACE (tumour necrosis factor alpha-converting enzyme). Kinetic analysis using a fluorescent peptide substrate demonstrated that the molecular interactions of N-TIMPs (N-terminal domains of TIMPs) with ADAM12 and TACE are for the most part comparable, yet revealed strikingly unique features of TIMP-mediated ADAM12 inhibition. Intriguingly, we found that removal of the AB-loop in N-TIMP-2, which is known to impair its interaction with TACE, resulted in increased affinity to ADAM12. Importantly, using a cell-based epidermal growth factor-shedding assay, we demonstrated for the first time an inhibitory activity of TIMPs against the transmembrane ADAM12-L (full-length ADAM12), verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Taken together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic potential can be designed.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM12 , Catálisis , Línea Celular , Membrana Celular/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cinética , Proteínas de la Membrana/metabolismo , Mutación , Unión Proteica , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/farmacología , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
A disintegrin and metalloproteinase (ADAM)12 was previously found to be expressed in T cells in the inflamed brain. However, the function of ADAM12 in T-cell responses in general and in tissue inflammation has not been examined. Here, we studied the role of ADAM12 in T-cell responses, fate determination on activation, and its functions in T cells to mediate tissue inflammation. We identified ADAM12 as a costimulatory molecule that is expressed on naive T cells and downregulated on stimulation. ADAM12 mimics CD28 costimulatory signaling to activate and induce the proliferation of T-helper 1 (Th1) cells. Monoclonal ADAM12 Fab antibodies trigger T-cell activation by amplifying TCR signaling to stimulate T-bet-mediated IFNγ production. Lack of genomic ADAM12 and its knockdown in T cells diminished T-bet and IFNγ production in Th1 cells, whereas other T cells, including Th17 cells, were unaffected. ADAM12 had similar functions in vivo on myelin antigen (MOG35-55)-induced T-cell activation. We found that genetic loss of ADAM12 profoundly alleviated Th1-mediated neuroinflammation and thus disease severity in experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Transcriptomic profiling of MOG35-55-specific ADAM12-/- T cells revealed differentially expressed genes that are important for T-cell activation, proliferation, and costimulatory signaling and Th1 pathogenicity, consistent with their inability to cause T-cell-mediated skin inflammation in a model of adoptive delayed-type hypersensitivity. We conclude that ADAM12 is a T-cell costimulatory molecule that contributes to the pathogenesis of tissue inflammation and a potential target for the treatment of Th1-mediated diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Células TH1 , Animales , Inflamación/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Células Th17RESUMEN
ADAM12 belongs to the large family of ADAMs (a disintegrin and metalloproteases) and possesses extracellular metalloprotease and cell-binding functions, as well as intracellular signaling capacities. Interest in ADAM12 has increased recently because its expression is related to tumor progression and it is a potential biomarker for breast cancer. It is therefore important to understand ADAM12's functions. Many cellular roles for ADAM12 have been suggested. It is an active metalloprotease, and has been implicated in insulin-like growth factor (IGF) receptor signaling, through cleavage of IGF-binding proteins, and in epidermal growth factor receptor (EGFR) pathways, via ectodomain shedding of membrane-tethered EGFR ligands. These proteolytic events may regulate diverse cellular responses, such as altered cell differentiation, proliferation, migration, and invasion. ADAM12 may also regulate cell-cell and cell-extracellular matrix contacts through interactions with cell surface receptors - integrins and syndecans - potentially influencing the actin cytoskeleton. Moreover, ADAM12 interacts with several cytoplasmic signaling and adaptor molecules through its intracellular domain, thereby directly transmitting signals to or from the cell interior. These ADAM12-mediated cellular effects appear to be critical events in both biological and pathological processes. This review presents current knowledge on ADAM12 functions gained from in vitro and in vivo observations, describes ADAM12's role in both normal physiology and pathology, particularly in cancer, and discusses important areas for future investigation.
Asunto(s)
Proteínas ADAM/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM12 , Animales , Cardiomegalia/enzimología , Cardiomegalia/metabolismo , Regulación Enzimológica de la Expresión Génica , Salud , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Enfermedades Musculoesqueléticas/enzimología , Enfermedades Musculoesqueléticas/metabolismo , Neoplasias/enzimología , Neoplasias/metabolismo , Enfermedades del Sistema Nervioso/enzimología , Enfermedades del Sistema Nervioso/metabolismoRESUMEN
Osteoblasts and adipocytes may develop from common bone marrow mesenchymal precursors. Transgenic mice overexpressing DeltaFosB, an AP-1 transcription factor, under the control of the neuron-specific enolase (NSE) promoter show both markedly increased bone formation and decreased adipogenesis. To determine whether the two phenotypes were linked, we targeted overexpression of DeltaFosB in mice to the osteoblast by using the osteocalcin (OG2) promoter. OG2-DeltaFosB mice demonstrated increased osteoblast numbers and an osteosclerotic phenotype but normal adipocyte differentiation. This result firmly establishes that the skeletal phenotype is cell autonomous to the osteoblast lineage and independent of adipocyte formation. It also strongly suggests that the decreased fat phenotype of NSE-DeltaFosB mice is independent of the changes in the osteoblast lineage. In vitro, overexpression of DeltaFosB in the preadipocytic 3T3-L1 cell line had little effect on adipocyte differentiation, whereas it prevented the induction of adipogenic transcription factors in the multipotential stromal cell line ST2. Also, DeltaFosB isoforms bound to and altered the DNA-binding capacity of C/EBPbeta. Thus, the inhibitory effect of DeltaFosB on adipocyte differentiation appears to occur at early stages of stem cell commitment, affecting C/EBPbeta functions. It is concluded that the changes in osteoblast and adipocyte differentiation in DeltaFosB transgenic mice result from independent cell-autonomous mechanisms.