RESUMO
Cancer cells form actin-rich degradative protrusions (invasive pseudopods and invadopodia), which allows their efficient dispersal during metastasis. Using biochemical and advanced imaging approaches, we demonstrate that the N-WASP-interactors WIP and WICH/WIRE play non-redundant roles in cancer cell invasion. WIP interacts with N-WASP and cortactin and is essential for invadopodium assembly, whereas WICH/WIRE regulates N-WASP activation to control invadopodium maturation and degradative activity. Our data also show that Nck interaction with WIP and WICH/WIRE modulates invadopodium maturation; changes in WIP and WICH/WIRE levels induce differential distribution of Nck. We show that WIP can replace WICH/WIRE functions and that elevated WIP levels correlate with high invasiveness. These findings identify a role for WICH/WIRE in invasiveness and highlight WIP as a hub for signaling molecule recruitment during invadopodium generation and cancer progression, as well as a potential diagnostic biomarker and an optimal target for therapeutic approaches.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Podossomos/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cortactina/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas dos Microfilamentos , Invasividade Neoplásica , Transdução de Sinais , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.
Assuntos
Proteínas de Transporte/metabolismo , Estruturas da Membrana Celular/metabolismo , Movimento Celular , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Western Blotting , Proteínas de Transporte/genética , Células Cultivadas , Quimiocina CXCL13/farmacologia , Quimiotaxia/efeitos dos fármacos , Proteínas do Citoesqueleto , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Microscopia Confocal , Proteínas Oncogênicas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ligação Proteica , Pseudópodes/metabolismo , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
In immature dendritic cells (DCs) podosomes form and turn over behind the leading edge of migrating cells. The Arp2/3 complex activator Wiskott-Aldrich Syndrome Protein (WASP) localises to the actin core of forming podosomes together with WASP-Interacting Protein (WIP). A second weaker Arp2/3 activator, cortactin, is also found at podosomes where it has been proposed to participate in matrix metalloproteinase (MMP) secretion. We have previously shown that WIP(-/-) DCs are unable to make podosomes. WIP binds to cortactin and in this report we address whether WIP regulates cortactin-mediated MMP activity. Using DCs derived from splenic murine precursors, we found that wild-type cells were able to localise MMPs at podosomes where matrix degradation takes place. In contrast, WIP(-/-) DCs remain able to synthesise MMPs but do not degrade the extracellular matrix. Infection of WIP KO DCs with lentivirus expressing WIP restored both podosome formation and their ability to degrade the extracellular matrix, implicating WIP-induced podosomes as foci of functional MMP location. When WIP KO DCs were infected with a mutant form of WIP lacking the cortactin-binding domain (WIPΔ110-170) DCs were only able to elaborate disorganised podosomes that were unable to support MMP-mediated matrix degradation. Taken together, these results suggest a role for WIP not only in WASP-mediated actin polymerisation and podosome formation, but also in cortactin-mediated extracellular matrix degradation by MMPs.
