The phosphatase Shp1 interacts with and dephosphorylates cortactin to inhibit invadopodia function

Abstract

Background 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 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. Methods Co-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. Results The 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. Conclusion The 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.

Inhibition of carbonic anhydrases IX/XII by SLC-0111 boosts cisplatin effects in hampering head and neck squamous carcinoma cell growth and invasion

“…Next, we investigated whether the combined treatment Cis-Pt with SLC-0111 could have any effect on metastatic spread in vivo , a preliminary study was carried out to refine the intervention and evaluate its acceptability and feasibility on mice bearing the orthotopic HNSCC xenografts. Mice were subjected after the different drug treatments to in vivo imaging using the FRI with ProSense 750, a cathepsin-activatable fluorescent imaging agent, commonly used to detect the metastatic process [ 30 ].…”

Glycerophosphoinositol Promotes Apoptosis of Chronic Lymphocytic Leukemia Cells by Enhancing Bax Expression and Activation

“…The activity of the tyrosine phosphatase SHP-1, known to promote apoptosis ( 18 , 25 ), has been shown to be impaired in CLL cells ( 26 ). Since GroPIns is a well-known regulator of SHP-1 in melanoma cells ( 17 ), we asked whether it promotes apoptosis of CLL cells through a SHP-1-dependent mechanism. B cells purified from peripheral blood of CLL patients were cultured for 24 h in the presence of 100 μM GroPIns and the percentage of early apoptotic Annexin V + /PI - cells was quantified by flow cytometry.…”

“…The active form of SHP-1 is phosphorylated on tyrosine 564 ( 37 ). We hypothesized that, similar to melanoma cells ( 17 ), GroPIns interacts with and activates SHP-1 in CLL cells, thereby promoting their apoptosis. To test this hypothesis, B cells purified from peripheral blood of CLL patients and healthy controls were cultured in the presence of GroPIns and the active, phosphorylated form of SHP-1 was quantified by flow cytometry using a phospho-Y564-specific antibody ( 37 ).…”

“…In human blood monocytes GroPIns counteracts the LPS-induced proinflammatory and prothrombotic responses, inhibiting TLR4 signaling and leading to a decrease in the NF-κB-dependent transcription of inflammatory genes ( 15 ). GroPIns has also been recently found to reduce the invasive potential of melanoma cells through its ability to interact with and regulate the non-receptor tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) ( 16 , 17 ). GroPIns interaction with SHP-1 facilitates SHP-1 localization to invadopodia where it dephosphorylates cortactin, with subsequent impaired invadopodia function and hampered metastasis of melanoma cells both in vitro and in vivo ( 17 ).…”

LAMC2 marks a tumor-initiating cell population with an aggressive signature in pancreatic cancer

“…Gelatin degradation (invasion) assay. Fluorescent-coated coverslips were prepared as described previously (Varone et al, 2021). Cells were plated on gelatin-coated coverslips in a 24-well plate and fixed after 24 and 48 hours with 4% PFA (v/v) for 15' at room temperature.…”