RÉSUMÉ
Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell-like pathways hijacked by tumor cells. SIGNIFICANCE: This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.
Sujet(s)
Marqueurs biologiques tumoraux/métabolisme , Résistance aux médicaments antinéoplasiques , Régulation de l'expression des gènes tumoraux , Mélanome/anatomopathologie , Crête neurale/anatomopathologie , Cellules souches neurales/anatomopathologie , Récepteurs à l'acide phosphatidique/métabolisme , Animaux , Antinéoplasiques/pharmacologie , Apoptose , Marqueurs biologiques tumoraux/génétique , Prolifération cellulaire , Humains , Mélanome/traitement médicamenteux , Mélanome/génétique , Mélanome/métabolisme , Souris , Souris de lignée NOD , Souris SCID , Crête neurale/effets des médicaments et des substances chimiques , Crête neurale/métabolisme , Cellules souches neurales/effets des médicaments et des substances chimiques , Cellules souches neurales/métabolisme , Pronostic , Récepteurs à l'acide phosphatidique/génétique , Transcriptome , Cellules cancéreuses en culture , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
In this Letter, two relevant references were omitted; see the accompanying Amendment for details. The original Letter has not been corrected.
RÉSUMÉ
Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.
Sujet(s)
Résistance aux médicaments antinéoplasiques/effets des médicaments et des substances chimiques , Mélanome/traitement médicamenteux , Mélanome/génétique , Mitogen-Activated Protein Kinases/antagonistes et inhibiteurs , Mutation , Inhibiteurs de protéines kinases/pharmacologie , Protéines proto-oncogènes B-raf/génétique , Transduction du signal/effets des médicaments et des substances chimiques , p21-Activated Kinases/métabolisme , Animaux , Apoptose/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Résistance aux médicaments antinéoplasiques/génétique , Activation enzymatique/effets des médicaments et des substances chimiques , Femelle , Humains , JNK Mitogen-Activated Protein Kinases/composition chimique , JNK Mitogen-Activated Protein Kinases/métabolisme , Système de signalisation des MAP kinases/effets des médicaments et des substances chimiques , Mélanome/enzymologie , Souris , Mitogen-Activated Protein Kinase Kinases/composition chimique , Mitogen-Activated Protein Kinase Kinases/métabolisme , Phosphorylation/effets des médicaments et des substances chimiques , Protéines proto-oncogènes c-raf/composition chimique , Protéines proto-oncogènes c-raf/métabolisme , Sérine-thréonine kinases TOR/métabolisme , bêta-Caténine/composition chimique , bêta-Caténine/métabolisme , p21-Activated Kinases/antagonistes et inhibiteurs , p21-Activated Kinases/génétiqueRÉSUMÉ
Cutaneous malignant melanomas originate primarily within epidermal melanocytic cells. Melanoma cells share many characteristics with melanocyte precursors, suggesting that melanoma cells utilize the developmental programs of their normal counterpart for their own progression. The pigmentation system provides an advantageous model to assess survival pathway interactions in the melanocytic lineage, as genetic alterations controlling melanocyte development can be easily detectable by coat color phenotype that do not affect the viability of an animal. By integrating combinatorial gene knockout approaches, cell-based assays and immunohistochemical observations, recent studies have illustrated several genes and pathways that play important roles both in melanocyte specification and maintenance and in melanoma formation and progression. We are reviewing those genes and pathways to understand the connection between normal and cancerous development and to reveal therapeutic potential of targeting developmental pathways for melanoma therapy.
Sujet(s)
Mélanocytes/métabolisme , Mélanome/métabolisme , Tumeurs cutanées/métabolisme , Animaux , Différenciation cellulaire , Évolution de la maladie , Endothélines/métabolisme , Humains , Mélanocytes/anatomopathologie , Mélanome/étiologie , Mélanome/anatomopathologie , Facteur de transcription associé à la microphtalmie/métabolisme , Phénotype , Récepteurs Notch/métabolisme , Facteurs de transcription SOX/métabolisme , Transduction du signal , Tumeurs cutanées/étiologie , Tumeurs cutanées/anatomopathologie , Voie de signalisation Wnt , bêta-Caténine/métabolismeRÉSUMÉ
Epithelial-mesenchymal transition (EMT) is an important developmental process hijacked by cancer cells for their dissemination. Here, we show that Exo70, a component of the exocyst complex, undergoes isoform switching mediated by ESRP1, a pre-mRNA splicing factor that regulates EMT. Expression of the epithelial isoform of Exo70 affects the levels of key EMT transcriptional regulators such as Snail and ZEB2 and is sufficient to drive the transition to epithelial phenotypes. Differential Exo70 isoform expression in human tumors correlates with cancer progression, and increased expression of the epithelial isoform of Exo70 inhibits tumor metastasis in mice. At the molecular level, the mesenchymal-but not the epithelial-isoform of Exo70 interacts with the Arp2/3 complex and stimulates actin polymerization for tumor invasion. Our findings provide a mechanism by which the exocyst function and actin dynamics are modulated for EMT and tumor invasion.
Sujet(s)
Épissage alternatif/physiologie , Tumeurs du sein/génétique , Tumeurs du sein/secondaire , Transition épithélio-mésenchymateuse/physiologie , Protéines du transport vésiculaire/génétique , Protéines du transport vésiculaire/métabolisme , Complexe Arp-2-3/métabolisme , Séquence d'acides aminés , Animaux , Évolution de la maladie , Femelle , Hétérogreffes , Humains , Cellules MCF-7 , Souris , Souris nude , Données de séquences moléculaires , Invasion tumorale , Transplantation tumorale , Protéines de liaison à l'ARN/génétique , Protéines de liaison à l'ARN/métabolismeRÉSUMÉ
Dynamic shape changes of the plasma membrane are fundamental to many processes, ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here, we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations toward the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. In cells, the membrane-deformation function of Exo70 is required for protrusion formation and directional cell migration. Exo70 thus represents a membrane-bending protein that may couple actin dynamics and plasma membrane remodeling for morphogenesis.
Sujet(s)
Mouvement cellulaire/physiologie , Simulation numérique , Morphogenèse/physiologie , Pseudopodes/physiologie , Protéines du transport vésiculaire/physiologie , Cytosquelette d'actine/physiologie , Cytosquelette d'actine/ultrastructure , Actines/physiologie , Animaux , Lignée cellulaire tumorale , Membrane cellulaire/physiologie , Dimérisation , Protéines à fluorescence verte , Cellules HEK293 , Humains , Mélanome , Protéines membranaires/physiologie , Souris , Microscopie électronique , Microscopie de fluorescence , Modèles biologiques , Pseudopodes/ultrastructure , Imagerie accélérée , Protéines du transport vésiculaire/composition chimiqueRÉSUMÉ
Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration. Using in vitro actin polymerization assay and time-lapse total internal reflection fluorescence microscopy, we found that Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of the Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors. At the cellular level, the stimulatory effect of Exo70 on the Arp2/3 complex is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.
Sujet(s)
Complexe Arp-2-3/métabolisme , Actines/métabolisme , Mouvement cellulaire , Pseudopodes/physiologie , Protéines du transport vésiculaire/métabolisme , Animaux , Lignée cellulaire , Humains , Polymérisation , Rats , Famille de protéines du syndrome de Wiskott-Aldrich/métabolismeRÉSUMÉ
Exocytosis is a fundamental membrane trafficking event in eukaryotic cells in which membrane proteins or lipids are incorporated into the plasma membrane and vesicle contents are secreted to the exterior of the cell. The exocyst, an evolutionarily conserved octameric protein complex, plays a crucial role in the targeting of secretory vesicles to the plasma membrane during exocytosis. The exocyst has been shown to be involved in diverse cellular processes requiring polarized exocytosis such as yeast budding, epithelial polarity establishment, and neurite outgrowth. Recently, the exocyst has also been implicated in cell migration through mechanisms independent of its role in exocytosis. In this review, we will first summarize our knowledge on the exocyst complex at a molecular and structural level. Then, we will discuss the specific functions of the exocyst in exocytosis in various cell types. Finally, we will review the emerging roles of the exocyst during cell migration and tumor cell invasion.
Sujet(s)
Mouvement cellulaire , Exocytose , Protéines du transport vésiculaire/métabolisme , Complexe Arp-2-3/métabolisme , Animaux , Humains , Structure quaternaire des protéines , Protéines du transport vésiculaire/physiologie , Protéines G ral/métabolismeRÉSUMÉ
Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, we report that the exocyst plays a pivotal role in invadopodial activity. With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. We have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, our results suggest that the exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.
Sujet(s)
Actines/métabolisme , Prolongements cytoplasmiques/métabolisme , Secreted matrix metalloproteinases/métabolisme , Tumeurs , Vésicules de sécrétion/métabolisme , Complexe Arp-2-3/génétique , Complexe Arp-2-3/métabolisme , Animaux , Invasion tumorale , Tumeurs/métabolisme , Tumeurs/anatomopathologie , Interférence par ARN , Rats , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/métabolisme , Protéines du transport vésiculaire/génétique , Protéines du transport vésiculaire/métabolismeRÉSUMÉ
The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P(2). Disrupting Exo70-PI(4,5)P(2) interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P(2)-binding-deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P(2) interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.
Sujet(s)
Membrane cellulaire/effets des médicaments et des substances chimiques , Membrane cellulaire/métabolisme , Exocytose/effets des médicaments et des substances chimiques , Phosphatidylinositol diphosphate-4,5/pharmacologie , Protéines du transport vésiculaire/métabolisme , Séquence d'acides aminés , Animaux , Cellules HeLa , Humains , Données de séquences moléculaires , Mutation/génétique , Liaison aux protéines , Petit ARN interférent/génétique , Rats , Protéines du transport vésiculaire/composition chimique , Protéines du transport vésiculaire/génétiqueRÉSUMÉ
ATP binding and hydrolysis are required for P-glycoprotein mediated multidrug resistance. To investigate the molecular mechanism involved in ATP binding and hydrolysis, a three-dimensional model of the carboxyl-terminal nucleotide binding domain (NBD2) was built by homology modeling. Modeling revealed the human P-glycoprotein ATP-binding site and the possible role of conserved Gln1118 residue. Recombinant NBD2 was overexpressed in Escherichia coli and the conserved Gln1118 residue was mutated to an alanine residue. The Vmax for ATP hydrolysis by the mutant NBD2 was approximately 56% of the Vmax of wild-type NBD2. But both proteins displayed similar affinity for ATP, with Km of 479 and 466 microM for mutant and wild-type NBD2, respectively. These results suggest that the possible role of Gln1118 is as an activating residue for ATP hydrolysis. The molecular model also provided structural information about the interactions between NBD2 and the chemosensitizer quercetin. The complex indicated that quercetin was tightly bound to the ATP-binding site and competed for binding. The three-dimensional model of NBD2 can be used to both guide enzymological studies and provide a theoretical basis for the design of potential multidrug resistance reversers.
Sujet(s)
Glycoprotéine P/composition chimique , Adenosine triphosphatases/composition chimique , Adénosine triphosphate/composition chimique , Modèles moléculaires , Glycoprotéine P/génétique , Glycoprotéine P/métabolisme , Adenosine triphosphatases/génétique , Adenosine triphosphatases/métabolisme , Adénosine triphosphate/métabolisme , Substitution d'acide aminé , Humains , Hydrolyse , Mutation ponctuelle , Structure tertiaire des protéines/génétiqueRÉSUMÉ
Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, there is no such universal system available up to now. A vector system was constructed based on pPIC9- Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of mouse IgG1 and His-tag were cloned into the Pichia expression vector pPIC9. A model ScFv was introduced into pPIC9-Fc, which can bind Glutathione-S-transferase (GST) from Schistosoma japonicum, to yield the expression cassette pPIC9-ScFv-Fc. Following fermentation in a 5-liter reactor, the fusion was expressed at high levels in the methylotrophic yeast Pichia Pastoris, secreted as a dimeric form in the culture, and purified by Ni2+-NTA column chromatography. The expression yield can reach 10-30 mg/liter of culture medium. The ScFv-Fc fusion retains the biological binding ability of the parent ScFv, and can be applied as anti-GST antibodies for the detection of GST and GST-fusion proteins. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-mouse antibodies.