RESUMO
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.
Assuntos
Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Melanoma/patologia , Crista Neural/patologia , Células-Tronco Neurais/patologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Prognóstico , Receptores de Ácidos Lisofosfatídicos/genética , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this Letter, two relevant references were omitted; see the accompanying Amendment for details. The original Letter has not been corrected.
RESUMO
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.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/efeitos dos fármacos , Quinases Ativadas por p21/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/enzimologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Serina-Treonina Quinases TOR/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genéticaRESUMO
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.
Assuntos
Melanócitos/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Diferenciação Celular , Progressão da Doença , Endotelinas/metabolismo , Humanos , Melanócitos/patologia , Melanoma/etiologia , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Fenótipo , Receptores Notch/metabolismo , Fatores de Transcrição SOX/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
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.
Assuntos
Processamento Alternativo/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/secundário , Transição Epitelial-Mesenquimal/fisiologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Sequência de Aminoácidos , Animais , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Invasividade Neoplásica , Transplante de Neoplasias , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
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.
Assuntos
Movimento Celular/fisiologia , Simulação por Computador , Morfogênese/fisiologia , Pseudópodes/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/fisiologia , Animais , Linhagem Celular Tumoral , Membrana Celular/fisiologia , Dimerização , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Melanoma , Proteínas de Membrana/fisiologia , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Biológicos , Pseudópodes/ultraestrutura , Imagem com Lapso de Tempo , Proteínas de Transporte Vesicular/químicaRESUMO
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.
Assuntos
Movimento Celular , Exocitose , Proteínas de Transporte Vesicular/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Humanos , Estrutura Quaternária de Proteína , Proteínas de Transporte Vesicular/fisiologia , Proteínas ral de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Actinas/metabolismo , Extensões da Superfície Celular/metabolismo , Metaloproteinases da Matriz Secretadas/metabolismo , Neoplasias , Vesículas Secretórias/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Invasividade Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
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.