RESUMO
Intratumor morphological heterogeneity of pancreatic ductal adenocarcinoma (PDAC) predicts clinical outcomes but is only partially understood at the molecular level. To elucidate the gene expression programs underpinning intratumor morphological variation in PDAC, we investigated and deconvoluted at single cell level the molecular profiles of histologically distinct clusters of PDAC cells. We identified three major morphological and functional variants that co-exist in varying proportions in all PDACs, display limited genetic diversity, and are associated with a distinct organization of the extracellular matrix: a glandular variant with classical ductal features; a transitional variant displaying abortive ductal structures and mixed endodermal and myofibroblast-like gene expression; and a poorly differentiated variant lacking ductal features and basement membrane, and showing neuronal lineage priming. Ex vivo and in vitro evidence supports the occurrence of dynamic transitions among these variants in part influenced by extracellular matrix composition and stiffness and associated with local, specifically neural, invasion.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Membrana Basal/metabolismo , Sistema NervosoRESUMO
Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing, and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodium cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a MYO6-DOCK7 axis essential for spatially restricting RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative-mode motion in otherwise solid and static carcinoma cell collectives.
Assuntos
Mama , Pseudópodes , Cicatrização , Movimento (Física)RESUMO
Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodia cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a novel MYO6-DOCK7 axis that is critical for spatially restriction of RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative mode motion in otherwise solid and static carcinoma cell collectives. Highlights: Collective motion of jammed epithelia requires myosin VI activityThe MYO6-DOCK7 axis is critical to restrict the activity of RAC1 in a planar polarized fashionMYO6-DOCK7-RAC1 activation ensures long-range coordination of movements by promoting orientation and persistence of cryptic lamellipodiaMyosin VI overexpression is exploited by infiltrating breast cancer cells.
RESUMO
The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.
Assuntos
Actinas , Neoplasias , DNA , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Citosol/metabolismo , Transdução de SinaisRESUMO
Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Dano ao DNA , Exodesoxirribonucleases/metabolismo , Membrana Nuclear/metabolismo , Fosfoproteínas/metabolismo , Animais , Linhagem Celular , Senescência Celular , Colágeno/metabolismo , Progressão da Doença , Feminino , Humanos , Camundongos , Invasividade Neoplásica , Membrana Nuclear/ultraestrutura , Proteólise , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The accurate quantification of cellular motility and of the structural changes occurring in multicellular aggregates is critical in describing and understanding key biological processes, such as wound repair, embryogenesis and cancer invasion. Current methods based on cell tracking or velocimetry either suffer from limited spatial resolution or are challenging and time-consuming, especially for three-dimensional (3D) cell assemblies. Here we propose a conceptually simple, robust and tracking-free approach for the quantification of the dynamical activity of cells via a two-step procedure. We first characterise the global features of the collective cell migration by registering the temporal stack of the acquired images. As a second step, a map of the local cell motility is obtained by performing a mean squared amplitude analysis of the intensity fluctuations occurring when two registered image frames acquired at different times are subtracted. We successfully apply our approach to cell monolayers undergoing a jamming transition, as well as to monolayers and 3D aggregates that exhibit a cooperative unjamming-via-flocking transition. Our approach is capable of disentangling very efficiently and of assessing accurately the global and local contributions to cell motility.
Assuntos
Imageamento Tridimensional , Movimento Celular , Movimento (Física)RESUMO
ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Núcleo Celular/metabolismo , Estresse Mecânico , Citoesqueleto de Actina , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Encéfalo , Cromatina , Citoplasma , Citoesqueleto/metabolismo , Dano ao DNA , Camundongos Knockout , Metástase Neoplásica , Neurogênese , Membrana Nuclear/metabolismoRESUMO
Selective evolutionary pressure shapes the processes and genes that enable cancer survival and expansion in a tumour-suppressive environment. A distinguishing lethal feature of malignant cancer is its dissemination and seeding of metastatic foci. A key requirement for this process is the acquisition of a migratory/invasive ability. However, how the migratory phenotype is selected for during the natural evolution of cancer and what advantage, if any, it might provide to the growing malignant cells remain open issues. In this opinion piece, we discuss three possible answers to these issues. We will examine lines of evidence from mathematical modelling of cancer evolution that indicate that migration is an intrinsic selectable property of malignant cells that directly impacts on growth dynamics and cancer geometry. Second, we will argue that migratory phenotypes can emerge as an adaptive response to unfavourable growth conditions and endow cells not only with the ability to move/invade, but also with specific metastatic traits, including drug resistance, self-renewal and survival. Finally, we will discuss the possibility that migratory phenotypes are coincidental events that emerge by happenstance in the natural evolution of cancer. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.
Assuntos
Evolução Biológica , Carcinogênese/metabolismo , Movimento Celular/genética , Neoplasias/metabolismo , Seleção Genética , Humanos , FenótipoRESUMO
During wound repair, branching morphogenesis and carcinoma dissemination, cellular rearrangements are fostered by a solid-to-liquid transition, known as unjamming. The biomolecular machinery behind unjamming and its pathophysiological relevance remain, however, unclear. Here, we study unjamming in a variety of normal and tumorigenic epithelial two-dimensional (2D) and 3D collectives. Biologically, the increased level of the small GTPase RAB5A sparks unjamming by promoting non-clathrin-dependent internalization of epidermal growth factor receptor that leads to hyperactivation of the kinase ERK1/2 and phosphorylation of the actin nucleator WAVE2. This cascade triggers collective motility effects with striking biophysical consequences. Specifically, unjamming in tumour spheroids is accompanied by persistent and coordinated rotations that progressively remodel the extracellular matrix, while simultaneously fluidizing cells at the periphery. This concurrent action results in collective invasion, supporting the concept that the endo-ERK1/2 pathway is a physicochemical switch to initiate collective invasion and dissemination of otherwise jammed carcinoma.
Assuntos
Diferenciação Celular , Movimento Celular , Linhagem Celular Tumoral , Proliferação de Células , Receptores ErbB/metabolismo , Humanos , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Collagen prolyl hydroxylation (CPH), which is catalyzed by prolyl 4-hydroxylase (P4H), is the most prevalent posttranslational modification in humans and requires vitamin C (VitC). Here, we demonstrate that CPH acts as an epigenetic modulator of cell plasticity. Increased CPH induced global DNA/histone methylation in pluripotent stem and tumor cells and promoted cell state transition (CST). Interfering with CPH by either genetic ablation of P4H subunit alpha-2 (P4HA2) or pharmacologic treatment reverted epigenetic changes and antagonized CST. Mechanistically, we suggest that CPH modifies the epigenetic landscape by reducing VitC for DNA and histone demethylases. Repurposed drugs targeting CPH-mediated metabolic perturbation, such as the antiasthmatic budesonide, blocked metastatic dissemination of breast cancer cells in vivo by preventing mesenchymal transition. Our study provides mechanistic insights into how metabolic cues and epigenetic factors integrate to control CST and paves the way for the development of novel antimetastatic strategies. SIGNIFICANCE: A phenotype-based high-throughput screening reveals unforeseen metabolic control of cell plasticity and identifies budesonide as a drug candidate for metastatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3235/F1.large.jpg.
Assuntos
Neoplasias da Mama/patologia , Colágeno/metabolismo , Epigênese Genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células-Tronco Pluripotentes/patologia , Prolil Hidroxilases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Hidroxilação , Células-Tronco Pluripotentes/metabolismo , Prolil Hidroxilases/genéticaRESUMO
Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.
Assuntos
Sequências de Repetição em Tandem/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Adulto , Animais , Sequência de Bases , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Feminino , Expressão Gênica , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Mucosa Bucal/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Fatores de Transcrição/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/deficiência , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
How cells move chemotactically remains a major unmet challenge in cell biology. Emerging evidence indicates that for interpreting noisy, shallow gradients of soluble cues a system must behave as an excitable process. Here, through an RNAi-based, high-content screening approach, we identify RAB35 as necessary for the formation of growth factors (GFs)-induced waves of circular dorsal ruffles (CDRs), apically restricted actin-rich migratory protrusions. RAB35 is sufficient to induce recurrent and polarized CDRs that travel as propagating waves, thus behaving as an excitable system that can be biased to control cell steering. Consistently, RAB35 is essential for promoting directed chemotactic migration and chemoinvasion of various cells in response to gradients of motogenic GFs. Molecularly, RAB35 does so by directly regulating the activity of p85/PI3K polarity axis. We propose that RAB35 is a molecular determinant for the control of an excitable, oscillatory system that acts as a steering wheel for GF-mediated chemotaxis and chemoinvasion.
Assuntos
Quimiotaxia/genética , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Expressão Gênica , Células HeLa , Humanos , Camundongos , Imagem Molecular , Fator de Crescimento Derivado de Plaquetas/farmacologia , Cultura Primária de Células , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
KRAS is the key mutated gene in pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that KRas modulates endocytic uptake. The present study aimed to explore the fate of early endosomal trafficking under the control of KRas expression in PDAC. Surprisingly, PANC-1 cells lacking KRas exhibited significantly enlarged early and late endosomes containing internalized dextran and epidermal growth factor. Endosome enlargement was accompanied by reduced endosomal degradation. Both KRas silencing and lysosomal blockade caused an upregulation of the master regulator of early endosome biogenesis, RAB5A, which is likely responsible for the expansion of the early endosomal compartment, because simultaneous KRAS/RAB5A knockdown abolished endosome enlargement. In contrast, early endosome shrinkage was seen in MIA PaCa-2 cells despite RAB5A upregulation, indicating that distinct KRas-modulated responses operate in different metabolic subtypes of PDAC. In conclusion, mutant KRAS promotes endosomal degradation in PDAC cell lines, which is impaired by KRAS silencing. Moreover, KRAS silencing activates RAB5A upregulation and drives PDAC subtype-dependent modulation of endosome trafficking.
Assuntos
Endossomos/metabolismo , Neoplasias Pancreáticas/metabolismo , Pinocitose , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Endossomos/patologia , Humanos , Neoplasias Pancreáticas/patologia , Transporte ProteicoRESUMO
The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Complexo de Golgi/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Endossomos/metabolismo , Exocitose , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Inativação Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Invasividade Neoplásica , Prognóstico , Transporte Proteico , Proteólise , Recidiva , Proteínas Supressoras de Tumor/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.
Assuntos
Forma Celular/genética , DNA Ligases/genética , Replicação do DNA/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Western Blotting , Adesão Celular/genética , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Transformada , Movimento Celular/genética , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/deficiência , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Microscopia de Fluorescência , Mutação , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Imagem com Lapso de Tempo/métodosRESUMO
The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-ß1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.
Assuntos
Células Epiteliais/citologia , Regulação Neoplásica da Expressão Gênica , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Actomiosina/metabolismo , Animais , Caderinas/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Linhagem Celular Tumoral , Endocitose , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Antígenos de Histocompatibilidade Menor , Transplante de Neoplasias , Receptor ErbB-2/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5-dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and ß3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.
Assuntos
Neoplasias da Mama/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/fisiologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Matriz Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Invasividade Neoplásica/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Proteólise , Transplante Heterólogo , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination.