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Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability.

Martín-Villar E, Borda-d'Agua B, Carrasco-Ramirez P, Renart J, Parsons M, Quintanilla M, Jones GE - Oncogene (2014)

Bottom Line: Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation.Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly.Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC-UAM), Madrid, Spain.

ABSTRACT
Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.

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Podoplanin defines adhesion rings at invadopodia of SCC cellsConfocal images showing specific localization of podoplanin fused to GFP (PDPN-GFP) at invadopodia in HN5 cells cultured on glass coverslips covered with cross-linked gelatin (A-B) or TRITC-gelatin (C). Note that podoplanin is not present in all invadopodia (A; left lower panels) but clusters to active invadopodia forming a ring structure around the actin or cortactin core (A-B; lower panels and C). Graphs indicate fluorescent intensity (in arbitrary units) of PDPN-GFP with respect to F-actin or cortactin over the indicated line scan. Data shown are representative from 6 invadopodia analysed per condition from two independent experiments. Bars = 10 μm.
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Figure 2: Podoplanin defines adhesion rings at invadopodia of SCC cellsConfocal images showing specific localization of podoplanin fused to GFP (PDPN-GFP) at invadopodia in HN5 cells cultured on glass coverslips covered with cross-linked gelatin (A-B) or TRITC-gelatin (C). Note that podoplanin is not present in all invadopodia (A; left lower panels) but clusters to active invadopodia forming a ring structure around the actin or cortactin core (A-B; lower panels and C). Graphs indicate fluorescent intensity (in arbitrary units) of PDPN-GFP with respect to F-actin or cortactin over the indicated line scan. Data shown are representative from 6 invadopodia analysed per condition from two independent experiments. Bars = 10 μm.

Mentions: We next investigated whether podoplanin is a component of invadopodia in SCC cells. The presence of both endogenous and GFP-tagged podoplanin (PDPN-GFP) at invadopodia was confirmed by immunofluorescence co-staining with the invadopodium markers actin and cortactin, and co-localization with areas of matrix degradation. Confocal microscopy analysis revealed that podoplanin was mostly absent from the invadopodia actin core but accumulated to podosome-like adhesion rings of actively degrading invadopodia in HN5 and SCC13 cells (Fig 2, S1A and S2A). The presence of adhesion rings in these cells was confirmed by co-staining of vinculin, a component of invadopodia adhesion rings,7 and the adhesion molecule talin (Fig S1B-C). The localization of podoplanin at invadopodium rings was also confirmed in MDA-MB-231 breast adenocarcinoma cells upon podoplanin ectopic expression (Fig S3A-C). These results demonstrate that podoplanin is a novel component of invadopodium adhesion rings and that this specific localization is not cell-type dependent.


Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability.

Martín-Villar E, Borda-d'Agua B, Carrasco-Ramirez P, Renart J, Parsons M, Quintanilla M, Jones GE - Oncogene (2014)

Podoplanin defines adhesion rings at invadopodia of SCC cellsConfocal images showing specific localization of podoplanin fused to GFP (PDPN-GFP) at invadopodia in HN5 cells cultured on glass coverslips covered with cross-linked gelatin (A-B) or TRITC-gelatin (C). Note that podoplanin is not present in all invadopodia (A; left lower panels) but clusters to active invadopodia forming a ring structure around the actin or cortactin core (A-B; lower panels and C). Graphs indicate fluorescent intensity (in arbitrary units) of PDPN-GFP with respect to F-actin or cortactin over the indicated line scan. Data shown are representative from 6 invadopodia analysed per condition from two independent experiments. Bars = 10 μm.
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Related In: Results  -  Collection

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Figure 2: Podoplanin defines adhesion rings at invadopodia of SCC cellsConfocal images showing specific localization of podoplanin fused to GFP (PDPN-GFP) at invadopodia in HN5 cells cultured on glass coverslips covered with cross-linked gelatin (A-B) or TRITC-gelatin (C). Note that podoplanin is not present in all invadopodia (A; left lower panels) but clusters to active invadopodia forming a ring structure around the actin or cortactin core (A-B; lower panels and C). Graphs indicate fluorescent intensity (in arbitrary units) of PDPN-GFP with respect to F-actin or cortactin over the indicated line scan. Data shown are representative from 6 invadopodia analysed per condition from two independent experiments. Bars = 10 μm.
Mentions: We next investigated whether podoplanin is a component of invadopodia in SCC cells. The presence of both endogenous and GFP-tagged podoplanin (PDPN-GFP) at invadopodia was confirmed by immunofluorescence co-staining with the invadopodium markers actin and cortactin, and co-localization with areas of matrix degradation. Confocal microscopy analysis revealed that podoplanin was mostly absent from the invadopodia actin core but accumulated to podosome-like adhesion rings of actively degrading invadopodia in HN5 and SCC13 cells (Fig 2, S1A and S2A). The presence of adhesion rings in these cells was confirmed by co-staining of vinculin, a component of invadopodia adhesion rings,7 and the adhesion molecule talin (Fig S1B-C). The localization of podoplanin at invadopodium rings was also confirmed in MDA-MB-231 breast adenocarcinoma cells upon podoplanin ectopic expression (Fig S3A-C). These results demonstrate that podoplanin is a novel component of invadopodium adhesion rings and that this specific localization is not cell-type dependent.

Bottom Line: Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation.Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly.Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC-UAM), Madrid, Spain.

ABSTRACT
Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.

Show MeSH
Related in: MedlinePlus