Abstract
BackgroundInvadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development.MethodsCo-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis.ResultsThe data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas.ConclusionThe main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs.831JMZGwFCMXv4_eLWR3W6Video abstract
Highlights
Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion
The role of protein tyrosine phosphatases (PTPs) in the formation of invadopodia remains unclear. We have addressed this issue by testing the involvement of the Src-homology region 2 (SH2) domain-containing phosphatase 1 (Shp1) in invadopodia formation and extracellular matrix (ECM) degradation, as it has a well-known role in the regulation of Src kinase [6]
We first analyzed the intracellular localization of endogenous Src homology region domain-containing phosphatase-1 (Shp1) in human melanoma A375MM cells, a cell model system extensively studied for the invadopodia formation process [32]
Summary
Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is known on the role of protein tyrosine phosphatases in this process. The initial steps of the metastatic process include the local invasion of cancerous cells into the extracellular matrix (ECM) and connective tissue with the subsequent penetration into the vascular and lymphatic systems. This invasive process is driven by the reorganization of the actin cytoskeleton and formation of cell structures, the invadopodia [2]. Invadopodia develop through a series of maturation events, which include the formation of a central-filamentous actin core, the invadopodium precursor, followed by actin polymerization that pushes the plasma membrane outward, and the recruitment of secreted and membrane-bound matrix metalloproteases (MMPs) [3]
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