RESUMEN
The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.
Asunto(s)
Membrana Celular , Proteínas del Citoesqueleto , Citoesqueleto , Miosinas , Uniones Adherentes/metabolismo , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Miosinas/metabolismo , Uniones Estrechas/metabolismoRESUMEN
The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell-cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7-PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.
Asunto(s)
Calcio , Proteínas Portadoras , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Uniones Adherentes/metabolismo , Animales , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Células Cultivadas , Células HeLa , Humanos , Ratones , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
Zonula occludens-1 (ZO-1), the major scaffolding protein of tight junctions (TJs), recruits the cytoskeleton-associated proteins cingulin (CGN) and paracingulin (CGNL1) to TJs by binding to their N-terminal ZO-1 interaction motif. The conformation of ZO-1 can be either folded or extended, depending on cytoskeletal tension and intramolecular and intermolecular interactions, and only ZO-1 in the extended conformation recruits the transcription factor DbpA to TJs. However, the sequences of ZO-1 that interact with CGN and CGNL1 and the role of TJ proteins in ZO-1 TJ assembly are not known. Here, we used glutathione-S-transferase pulldowns and immunofluorescence microscopy to show that CGN and CGNL1 bind to the C-terminal ZU5 domain of ZO-1 and that this domain is required for CGN and CGNL1 recruitment to TJs and to phase-separated ZO-1 condensates in cells. We show that KO of CGN, but not CGNL1, results in decreased accumulation of ZO-1 at TJs. Furthermore, ZO-1 lacking the ZU5 domain showed decreased accumulation at TJs, was detectable along lateral contacts, had a higher mobile fraction than full-length ZO-1 by fluorescence recovery after photobleaching analysis, and had a folded conformation, as determined by structured illumination microscopy of its N-terminal and C-terminal ends. The CGN-ZU5 interaction promotes the extended conformation of ZO-1, since binding of the CGN-ZO-1 interaction motif region to ZO-1 resulted in its interaction with DbpA in cells and in vitro. Together, these results show that binding of CGN to the ZU5 domain of ZO-1 promotes ZO-1 stabilization and accumulation at TJs by promoting its extended conformation.
Asunto(s)
Proteínas del Citoesqueleto , Uniones Estrechas , Proteína de la Zonula Occludens-1 , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Técnicas de Silenciamiento del Gen , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Dominios Proteicos , Pliegue de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/química , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Transmembrane proteins of adherens and tight junctions are known targets for viruses and bacterial toxins. The coronavirus receptor ACE2 has been localized at the apical surface of epithelial cells, but it is not clear whether ACE2 is localized at apical Cell-Cell junctions and whether it associates with junctional proteins. Here we explored the expression and localization of ACE2 and its association with transmembrane and tight junction proteins in epithelial tissues and cultured cells by data mining, immunoblotting, immunofluorescence microscopy, and co-immunoprecipitation experiments. ACE2 mRNA is abundant in epithelial tissues, where its expression correlates with the expression of the tight junction proteins cingulin and occludin. In cultured epithelial cells ACE2 mRNA is upregulated upon differentiation and ACE2 protein is widely expressed and co-immunoprecipitates with the transmembrane proteins ADAM17 and CD9. We show by immunofluorescence microscopy that ACE2 colocalizes with ADAM17 and CD9 and the tight junction protein cingulin at apical junctions of intestinal (Caco-2), mammary (Eph4) and kidney (mCCD) epithelial cells. These observations identify ACE2, ADAM17 and CD9 as new epithelial junctional transmembrane proteins and suggest that the cytokine-enhanced endocytic internalization of junction-associated protein complexes comprising ACE2 may promote coronavirus entry.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Uniones Intercelulares/metabolismo , Uniones Intercelulares/virología , Proteína ADAM17/metabolismo , Uniones Adherentes/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular , Coronavirus/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/virología , Expresión Génica/genética , Tetraspanina 29/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo , Transcriptoma/genéticaRESUMEN
Tight and adherens junctions are specialized sites of cell-cell interaction in epithelia and endothelia, and are involved in barrier, adhesion, and signaling functions. These functions are orchestrated by a highly organized meshwork of macromolecules in the membrane and cytoplasmic compartments. In this review, we discuss the structural organization and functions of the major cytoplasmic scaffolding and adaptor proteins of vertebrate apical junctions (ZO proteins, afadin, PLEKHA7, cingulin, paracingulin, polarity complex proteins, and a few others), focusing on their interactions with cytoskeletal and signaling proteins. Furthermore, we discuss recent results highlighting how mechanical tension, protein-protein interactions and post-translational modifications regulate the conformation and function of scaffolding proteins, and how spontaneous phase separation into biomolecular condensates contributes to apical junction assembly. Using a sequence-based algorithm, a large fraction of cytoplasmic proteins of apical junctions are predicted to be phase separating proteins (PSPs), suggesting that formation of biomolecular condensates is a general mechanism to organize cell-cell contacts by clustering proteins.
Asunto(s)
Uniones Intercelulares/metabolismo , Vertebrados/metabolismo , Animales , Fenómenos Biofísicos , Citoplasma/metabolismo , LigandosRESUMEN
Pleckstrin homology domain-containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to Staphylococcus aureus α-toxin. Complex formation between PLEKHA7, PDZ domain-containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin-mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione S-transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a "hydrophobic hot spot" region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7-PDZD11 interaction modulate the ligand-binding properties of PLEKHA7.
Asunto(s)
Proteínas Portadoras/química , Tetraspaninas/química , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Humanos , Enlace de Hidrógeno , Ratones , Ratones Noqueados , Simulación del Acoplamiento Molecular , Dominios Proteicos , Estructura Cuaternaria de Proteína , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMEN
The barrier function of epithelia and endothelia depends on tight junctions, which are formed by the polymerization of claudins on a scaffold of ZO proteins. Two differentially spliced isoforms of ZO-1 have been described, depending on the presence of the α domain, but the function of this domain is unclear. ZO-1 also contains a C-terminal ZU5 domain, which is involved in a mechano-sensitive intramolecular interaction with the central (ZPSG) region of ZO-1. Here we use immunoblotting and immunofluorescence to map the binding sites for commercially available monoclonal and polyclonal antibodies against ZO-1, and for a new polyclonal antibody (R3) that we developed against the ZO-1 C-terminus. We demonstrate that antibody R40.76 binds to the α domain, and the R3 antibody binds to the ZU5 domain. The (α+) isoform of ZO-1 shows higher expression in epithelial versus endothelial cells, and in differentiated versus undifferentiated primary keratinocytes, suggesting a link to epithelial differentiation and a potential molecular adaptation to junctions subjected to stronger mechanical forces. These results provide new tools and hypotheses to investigate the role of the α and ZU5 domains in ZO-1 mechano-sensing and dynamic interactions with the cytoskeleton and junctional ligands.
Asunto(s)
Epitelio/metabolismo , Queratinocitos/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/fisiología , Animales , Diferenciación Celular , HumanosRESUMEN
We previously identified PLEKHA7 and other junctional proteins as host factors mediating death by S. aureus α-toxin, but the mechanism through which junctions promote toxicity was unclear. Using cell biological and biochemical methods, we now show that ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Instead, disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. These results uncover a dock-and-lock molecular mechanism to target ADAM10 to junctions and provide a paradigm for how junctions regulate transmembrane receptors through their clustering.
Asunto(s)
Proteína ADAM10/metabolismo , Toxinas Bacterianas/toxicidad , Proteínas Hemolisinas/toxicidad , Uniones Intercelulares/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Humanos , Uniones Intercelulares/efectos de los fármacos , Proteínas de Microfilamentos/metabolismo , Pinocitosis/efectos de los fármacos , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Tetraspaninas/metabolismoRESUMEN
Melanoma is one of the most lethal cancers when it reaches a metastatic stage. Despite advancements in targeted therapies (BRAF inhibitors) or immunotherapies (anti-CTLA-4 or anti-PD1), most patients with melanoma will need additional treatment. Thus, there is an urgent need to develop new therapeutical approaches to bypass resistance and achieve more prolonged responses. In this context, we were interested in E2F1, a transcription factor that plays a major role in the control of cell cycle under physiological and pathological conditions. Here we confirmed that E2F1 is highly expressed in melanoma cells. Inhibition of E2F1 activity further increased melanoma cell death and senescence, both in vitro and in vivo. Moreover, blocking E2F1 also induced death of melanoma cells resistant to BRAF inhibitors. In conclusion, our studies suggest that targeting the E2F1 signaling pathway may be therapeutically relevant for melanoma.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Muerte Celular/efectos de los fármacos , Factor de Transcripción E2F1 , Melanoma Experimental , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Factor de Transcripción E2F1/antagonistas & inhibidores , Factor de Transcripción E2F1/metabolismo , Femenino , Humanos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones Desnudos , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
HACE1 is an E3 ubiquitin ligase described as a tumour suppressor because HACE1-knockout mice develop multi-organ, late-onset cancers and because HACE1 expression is lost in several neoplasms, such as Wilms' tumours and colorectal cancer. However, a search of public databases indicated that HACE1 expression is maintained in melanomas. We demonstrated that HACE1 promoted melanoma cell migration and adhesion in vitro and was required for mouse lung colonisation by melanoma cells in vivo. Transcriptomic analysis of HACE1-depleted melanoma cells revealed an inhibition of ITGAV and ITGB1 as well changes in other genes involved in cell migration. We revealed that HACE1 promoted the K27 ubiquitination of fibronectin and regulated its secretion. Secreted fibronectin regulated ITGAV and ITGB1 expression, as well as melanoma cell adhesion and migration. Our findings disclose a novel molecular cascade involved in the regulation of fibronectin secretion, integrin expression and melanoma cell adhesion. By controlling this cascade, HACE1 displays pro-tumoural properties and is an important regulator of melanoma cell invasive properties.
Asunto(s)
Ubiquitina-Proteína Ligasas/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Fibronectinas/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Tissues of multicellular organisms are characterised by several types of specialised cell-cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton-associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ.
Asunto(s)
Citoesqueleto/metabolismo , Mecanotransducción Celular/fisiología , Animales , Cadherinas/metabolismo , Polaridad Celular , Humanos , Uniones Intercelulares/metabolismoRESUMEN
Extracellular vesicles (EVs) have been shown to play an important role in intercellular communication as carriers of DNA, RNA and proteins. While the intercellular transfer of miRNA through EVs has been extensively studied, the stability of extracellular miRNA (ex-miRNA) once engulfed by a recipient cell remains to be determined. Here, we identify the ex-miRNA-directed phenotype to be transient due to the rapid decay of ex-miRNA. We demonstrate that the ex-miR-223-3p transferred from polymorphonuclear leukocytes to cancer cells were functional, as demonstrated by the decreased expression of its target FOXO1 and the occurrence of epithelial-mesenchymal transition reprogramming. We showed that the engulfed ex-miRNA, unlike endogenous miRNA, was unstable, enabling dynamic regulation and a return to a non-invasive phenotype within 8 h. This transient phenotype could be modulated by targeting XRN1/PACMAN exonuclease. Indeed, its silencing was associated with slower decay of ex-miR-223-3p and subsequently prolonged the invasive properties. In conclusion, we showed that the 'steady step' level of engulfed miRNA and its subsequent activity was dependent on the presence of a donor cell in the surroundings to constantly fuel the recipient cell with ex-miRNAs and of XRN1 exonuclease, which is involved in the decay of these imported miRNA.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Exorribonucleasas/metabolismo , MicroARNs/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias/genética , Estabilidad del ARN , Línea Celular Tumoral , Exosomas/metabolismo , Humanos , Invasividad Neoplásica , Neoplasias/enzimología , Neoplasias/metabolismo , Neoplasias/patología , Neutrófilos/metabolismoRESUMEN
Melanoma is one of the most lethal cancers when it reaches a metastatic stage. Despite the spectacular achievements of targeted therapies (BRAF inhibitors) or immuno-therapies (anti-CTLA4 or anti-PD1), most patients with melanoma will need additional treatments. Here we used a photoactive NADPH analogue called NS1 to induce cell death by inhibition of NADPH oxidases NOX in melanoma cells, including melanoma cells isolated from patients. In contrast, healthy melanocytes growth was unaffected by NS1 treatment.NS1 established an early Endoplasmic Reticulum stress by the early release of calcium mediated by (a) calcium-dependent redox-sensitive ion channel(s). These events initiated autophagy and apoptosis in all tested melanoma cells independently of their mutational status. The autophagy promoted by NS1 was incomplete. The autophagic flux was blocked at late stage events, consistent with the accumulation of p62, and a close localization of LC3 with NS1 associated with NS1 inhibition of NOX1 in autophagosomes. This hypothesis of a specific incomplete autophagy and apoptosis driven by NS1 was comforted by the use of siRNAs and pharmacological inhibitors blocking different processes. This study highlights the potential therapeutic interest of NS1 inducing cell death by triggering a selective ER stress and incomplete autophagy in melanoma cells harbouring wt and BRAF mutation.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Melanoma/tratamiento farmacológico , NADPH Oxidasas/antagonistas & inhibidores , NADP/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Autofagia/efectos de los fármacos , Calcio/metabolismo , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , NADP/análogos & derivados , NADPH Oxidasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Células RAW 264.7 , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Proteína Sequestosoma-1/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factores de Tiempo , TransfecciónRESUMEN
We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.
Asunto(s)
Antineoplásicos/administración & dosificación , Resistencia a Antineoplásicos/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Melanoma/tratamiento farmacológico , Sulfonamidas/administración & dosificación , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Melanoma/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Specific BRAFV600E inhibitors (BRAFi) are highly effective in the treatment of melanoma. However, acquired drug resistances invariably develop after the initial response. Therefore, the identification of new mechanisms of acquired resistance gives important clues towards the development of therapies that could elicit long lasting responses. Here we report that CD271 confers resistance to BRAFi in melanoma cells. The expression of CD271 is increased by BRAFi through a stimulation of tumor necrosis factor-alpha (TNFα) secretion that leads to NF-κB signaling pathway activation. CD271 is upregulated in a subset of BRAFi-resistant melanoma cells. The inhibition of TNFα/NF-κB pathway and CD271 silencing restore the BRAFi sensitivity of resistant melanoma cells. Finally, increase of CD271 expression is validated in BRAFi-resistant xenografts tumors and also in tumors from the patients who relapsed under BRAFi. In summary, these results reveal a novel TNFα/NF-κB/CD271 axis whose activation contributes to the acquisition of resistance to BRAFi and therefore may represent a novel therapeutic target to improve the efficacy of therapy in melanoma.
RESUMEN
Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX(4) and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX(4) -associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Luz , Melanoma/metabolismo , NADP/farmacología , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Apoptosis , Western Blotting , Ciclo Celular , Proliferación Celular , Espectroscopía de Resonancia por Spin del Electrón , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , NADP/análogos & derivados , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Neovascularización Patológica , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Several reports have demonstrated the inhibitory effect of metformin, a widely used drug in the treatment of type 2 diabetes, on the proliferation of many cancers including melanoma. Recently, it has been shown that metformin is able to modulate the cAMP level in the liver. As cAMP has a crucial role in melanin synthesis and skin pigmentation, we investigated the effect of metformin on melanogenesis both in vitro and in vivo. We showed that metformin led to reduced melanin content in melanoma cells and in normal human melanocytes by decreasing cAMP accumulation and cAMP-responsive element-binding protein phosphorylation. This inhibitory effect is correlated with decreased expression of master genes of melanogenesis, microphthalmia-associated transcription factor, tyrosinase, dopachrome tautomerase, and tyrosinase-related protein 1. Furthermore, we demonstrated that the antimelanogenic effect of metformin is independent of the AMPK pathway. Interestingly, topical application of metformin induced tail whitening in mice. Finally, we confirmed the antimelanogenic effect of metformin on reconstituted human epidermis and on human skin biopsies. These data emphasize the depigmenting effect of metformin and suggest a clinical strategy for using metformin in the topical treatment of hyperpigmentation disorders.
Asunto(s)
Hipoglucemiantes/farmacología , Melaninas/metabolismo , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Metformina/farmacología , Piel/efectos de los fármacos , Piel/metabolismo , Animales , Biopsia , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Melanocitos/patología , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción Asociado a Microftalmía/metabolismo , Piel/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Pigmentación de la Piel/efectos de los fármacos , Factores de TiempoRESUMEN
Metformin was reported to inhibit the proliferation of many cancer cells, including melanoma cells. In this report, we investigated the effect of metformin on melanoma invasion and metastasis development. Using different in vitro approaches, we found that metformin inhibits cell invasion without affecting cell migration and independently of antiproliferation action. This inhibition is correlated with modulation of expression of proteins involved in epithelial-mesenchymal transition such as Slug, Snail, SPARC, fibronectin, and N-cadherin and with inhibition of MMP-2 and MMP-9 activation. Furthermore, our data indicate that this process is dependent on activation of AMPK and tumor suppressor protein p53. Finally, we showed that metformin inhibits melanoma metastasis development in mice using extravasation and metastasis models. The presented data reinforce the fact that metformin might be a good candidate for clinical trial in melanoma treatment.