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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 regulates cofilin phosphorylation at Ser3 through the RhoC/ROCK/LIMK pathway(A) Determination of active RhoA and RhoC GTPase levels in control and podoplanin knockdown cells by GTP affinity pull-down assays. Quantification of RhoA- and RhoC-GTP expression levels is shown in Figure S7B. (B) The status of cofilin phosphorylation at Ser3 (pCofilinS3) was evaluated by Western blot in podoplanin knock-down HN5 cells and HaCaT cells expressing PDPN-GFP. Quantification of pCofilinS3 levels (right panels) was performed relative to total cofilin levels and GAPDH loading control by densitometric analysis. Values were normalised to HN5 or HaCaT cells to which an arbitrary value of 1 was given. (C-D) Determination of pCofilinS3 levels during the stages of invadopodia formation in control and podoplanin-depleted HN5 cells. Invadopodia formation in HN5 cell transfectants was synchronised in order to evaluate invadopodia activity and the levels of pCofilinS3 during the stages of invadopodia formation (see Material and Methods section). Invadopodia activity was analysed by gelatin degradation assay (C), and pCofilinS3 changes were monitored by Westen blot and quantified by densitometric analysis (D and Fig S7D). pCofilinS3 levels were normalised to total cofilin levels and GAPDH loading control. Graphs represent means ± SEM of two (C) or three (D) independent experiments. (E) Western blot analysis of RhoC and RhoA expression in HN5 cells upon specific siRNA treatment. Quantification of RhoA and C expression levels is shown in Figure S7C. (F) Analysis of pCofilinS3 levels in RhoA- and RhoC-depleted cells. (G) Effects of RhoA and RhoC knock-down in invadopodia-mediated degradation of HN5 cells (images are depicted in Fig S7C). (H) Effects of ROCK inhibitor H-1152 in invadopodia-mediated degradation of control Sc and podoplanin-depleted HN5 cells. (I) Western blot analysis of pCofilinS3 levels after H-1152 treatment in control Sc and podoplanin-depleted HN5 cells. Quantification of pCofilinS3 levels is shown in the right panel. (J) Invadopodia-mediated gelatin degradation upon expression of the indicated LIMK1/2 mutant constructs in control Sc and podoplanin-depleted HN5 cells. (K) Western blot analysis of pCofilinS3 and LIMK1/2 mutant expression (GFP) in control and podoplanin-depleted HN5 cells. Quantification of pCofilinS3 levels is shown in the right panel. All Graphs represent means ± SEM of three or four (D and E) independent experiments. *p<0.01; **p<0.001; ***p<0.0001; ns = not significant.
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Figure 9: Podoplanin regulates cofilin phosphorylation at Ser3 through the RhoC/ROCK/LIMK pathway(A) Determination of active RhoA and RhoC GTPase levels in control and podoplanin knockdown cells by GTP affinity pull-down assays. Quantification of RhoA- and RhoC-GTP expression levels is shown in Figure S7B. (B) The status of cofilin phosphorylation at Ser3 (pCofilinS3) was evaluated by Western blot in podoplanin knock-down HN5 cells and HaCaT cells expressing PDPN-GFP. Quantification of pCofilinS3 levels (right panels) was performed relative to total cofilin levels and GAPDH loading control by densitometric analysis. Values were normalised to HN5 or HaCaT cells to which an arbitrary value of 1 was given. (C-D) Determination of pCofilinS3 levels during the stages of invadopodia formation in control and podoplanin-depleted HN5 cells. Invadopodia formation in HN5 cell transfectants was synchronised in order to evaluate invadopodia activity and the levels of pCofilinS3 during the stages of invadopodia formation (see Material and Methods section). Invadopodia activity was analysed by gelatin degradation assay (C), and pCofilinS3 changes were monitored by Westen blot and quantified by densitometric analysis (D and Fig S7D). pCofilinS3 levels were normalised to total cofilin levels and GAPDH loading control. Graphs represent means ± SEM of two (C) or three (D) independent experiments. (E) Western blot analysis of RhoC and RhoA expression in HN5 cells upon specific siRNA treatment. Quantification of RhoA and C expression levels is shown in Figure S7C. (F) Analysis of pCofilinS3 levels in RhoA- and RhoC-depleted cells. (G) Effects of RhoA and RhoC knock-down in invadopodia-mediated degradation of HN5 cells (images are depicted in Fig S7C). (H) Effects of ROCK inhibitor H-1152 in invadopodia-mediated degradation of control Sc and podoplanin-depleted HN5 cells. (I) Western blot analysis of pCofilinS3 levels after H-1152 treatment in control Sc and podoplanin-depleted HN5 cells. Quantification of pCofilinS3 levels is shown in the right panel. (J) Invadopodia-mediated gelatin degradation upon expression of the indicated LIMK1/2 mutant constructs in control Sc and podoplanin-depleted HN5 cells. (K) Western blot analysis of pCofilinS3 and LIMK1/2 mutant expression (GFP) in control and podoplanin-depleted HN5 cells. Quantification of pCofilinS3 levels is shown in the right panel. All Graphs represent means ± SEM of three or four (D and E) independent experiments. *p<0.01; **p<0.001; ***p<0.0001; ns = not significant.

Mentions: Some of the major regulatory signals for invadopodia assembly and maturation originate from Rho GTPases.8 Cdc42 has been implicated in the actin nucleation necessary for invadopodia formation,33 whereas RhoA and RhoC are essential for the control of invadopodia-mediated degradation.10,34 To ascertain whether podoplanin knockdown in HN5 cells disrupts Rho GTPase activity, we used pull down assays to evaluate the activation status of RhoA, Rac1, Cdc42 and RhoC. In agreement with previous observations,28 RhoA but not Rac1 or Cdc42 activity was significantly reduced in podoplanin-depleted cells with respect to control cells (Fig 9A and S7A-B). Interestingly, RhoC activity was also reduced in podoplanin-depleted cells (Fig 9A and S7A-B), suggesting that not only RhoA but also RhoC could mediate the function of podoplanin in invadopodia. The Rho/ROCK (Rho-associated protein kinase) pathway has been involved in invadopodia maturation through control of cofilin phosphorylation.10 Podoplanin knockdown cells showed a very small but consistent reduction of cofilin phosphorylation at Ser3 (pCofilinS3; Fig 9B). Conversely, pCofilinS3 was slightly increased upon ectopic expression of podoplanin in HaCaT cells (Fig 9B). To further define the kinetics of pCofilinS3 during invadopodia assembly, control and podoplanin-silenced HN5 cells were serum starved overnight and stimulated with FBS to synchronously induce invadopodia formation and associated matrix degradation (Fig 9C). Formation of non-functional immature invadopodia was observed during the first 30 min after stimulation in control cells. Matrix degradation initiated after 1h, and ~90% of control cells showed fully mature invadopodia after 3-4h, by which point the cells were no longer synchronised. pCofilinS3 levels were slightly decreased in control cells at 0-1h, correlating with initiation of invadopodia formation and actin polymerization (Fig 9D and S7D). After 1h, pCofilinS3 levels were increased accordingly with the stabilisation and maturation steps (2-4h). Podoplanin Knockdown led to a global and steady decrease in pCofilinS3 compared to control cells. Restoration of podoplanin expression in PDPNsh cells (Fig 9A) rescued the observed defects in matrix degradation and pCofilinS3.


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 regulates cofilin phosphorylation at Ser3 through the RhoC/ROCK/LIMK pathway(A) Determination of active RhoA and RhoC GTPase levels in control and podoplanin knockdown cells by GTP affinity pull-down assays. Quantification of RhoA- and RhoC-GTP expression levels is shown in Figure S7B. (B) The status of cofilin phosphorylation at Ser3 (pCofilinS3) was evaluated by Western blot in podoplanin knock-down HN5 cells and HaCaT cells expressing PDPN-GFP. Quantification of pCofilinS3 levels (right panels) was performed relative to total cofilin levels and GAPDH loading control by densitometric analysis. Values were normalised to HN5 or HaCaT cells to which an arbitrary value of 1 was given. (C-D) Determination of pCofilinS3 levels during the stages of invadopodia formation in control and podoplanin-depleted HN5 cells. Invadopodia formation in HN5 cell transfectants was synchronised in order to evaluate invadopodia activity and the levels of pCofilinS3 during the stages of invadopodia formation (see Material and Methods section). Invadopodia activity was analysed by gelatin degradation assay (C), and pCofilinS3 changes were monitored by Westen blot and quantified by densitometric analysis (D and Fig S7D). pCofilinS3 levels were normalised to total cofilin levels and GAPDH loading control. Graphs represent means ± SEM of two (C) or three (D) independent experiments. (E) Western blot analysis of RhoC and RhoA expression in HN5 cells upon specific siRNA treatment. Quantification of RhoA and C expression levels is shown in Figure S7C. (F) Analysis of pCofilinS3 levels in RhoA- and RhoC-depleted cells. (G) Effects of RhoA and RhoC knock-down in invadopodia-mediated degradation of HN5 cells (images are depicted in Fig S7C). (H) Effects of ROCK inhibitor H-1152 in invadopodia-mediated degradation of control Sc and podoplanin-depleted HN5 cells. (I) Western blot analysis of pCofilinS3 levels after H-1152 treatment in control Sc and podoplanin-depleted HN5 cells. Quantification of pCofilinS3 levels is shown in the right panel. (J) Invadopodia-mediated gelatin degradation upon expression of the indicated LIMK1/2 mutant constructs in control Sc and podoplanin-depleted HN5 cells. (K) Western blot analysis of pCofilinS3 and LIMK1/2 mutant expression (GFP) in control and podoplanin-depleted HN5 cells. Quantification of pCofilinS3 levels is shown in the right panel. All Graphs represent means ± SEM of three or four (D and E) independent experiments. *p<0.01; **p<0.001; ***p<0.0001; ns = not significant.
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Figure 9: Podoplanin regulates cofilin phosphorylation at Ser3 through the RhoC/ROCK/LIMK pathway(A) Determination of active RhoA and RhoC GTPase levels in control and podoplanin knockdown cells by GTP affinity pull-down assays. Quantification of RhoA- and RhoC-GTP expression levels is shown in Figure S7B. (B) The status of cofilin phosphorylation at Ser3 (pCofilinS3) was evaluated by Western blot in podoplanin knock-down HN5 cells and HaCaT cells expressing PDPN-GFP. Quantification of pCofilinS3 levels (right panels) was performed relative to total cofilin levels and GAPDH loading control by densitometric analysis. Values were normalised to HN5 or HaCaT cells to which an arbitrary value of 1 was given. (C-D) Determination of pCofilinS3 levels during the stages of invadopodia formation in control and podoplanin-depleted HN5 cells. Invadopodia formation in HN5 cell transfectants was synchronised in order to evaluate invadopodia activity and the levels of pCofilinS3 during the stages of invadopodia formation (see Material and Methods section). Invadopodia activity was analysed by gelatin degradation assay (C), and pCofilinS3 changes were monitored by Westen blot and quantified by densitometric analysis (D and Fig S7D). pCofilinS3 levels were normalised to total cofilin levels and GAPDH loading control. Graphs represent means ± SEM of two (C) or three (D) independent experiments. (E) Western blot analysis of RhoC and RhoA expression in HN5 cells upon specific siRNA treatment. Quantification of RhoA and C expression levels is shown in Figure S7C. (F) Analysis of pCofilinS3 levels in RhoA- and RhoC-depleted cells. (G) Effects of RhoA and RhoC knock-down in invadopodia-mediated degradation of HN5 cells (images are depicted in Fig S7C). (H) Effects of ROCK inhibitor H-1152 in invadopodia-mediated degradation of control Sc and podoplanin-depleted HN5 cells. (I) Western blot analysis of pCofilinS3 levels after H-1152 treatment in control Sc and podoplanin-depleted HN5 cells. Quantification of pCofilinS3 levels is shown in the right panel. (J) Invadopodia-mediated gelatin degradation upon expression of the indicated LIMK1/2 mutant constructs in control Sc and podoplanin-depleted HN5 cells. (K) Western blot analysis of pCofilinS3 and LIMK1/2 mutant expression (GFP) in control and podoplanin-depleted HN5 cells. Quantification of pCofilinS3 levels is shown in the right panel. All Graphs represent means ± SEM of three or four (D and E) independent experiments. *p<0.01; **p<0.001; ***p<0.0001; ns = not significant.
Mentions: Some of the major regulatory signals for invadopodia assembly and maturation originate from Rho GTPases.8 Cdc42 has been implicated in the actin nucleation necessary for invadopodia formation,33 whereas RhoA and RhoC are essential for the control of invadopodia-mediated degradation.10,34 To ascertain whether podoplanin knockdown in HN5 cells disrupts Rho GTPase activity, we used pull down assays to evaluate the activation status of RhoA, Rac1, Cdc42 and RhoC. In agreement with previous observations,28 RhoA but not Rac1 or Cdc42 activity was significantly reduced in podoplanin-depleted cells with respect to control cells (Fig 9A and S7A-B). Interestingly, RhoC activity was also reduced in podoplanin-depleted cells (Fig 9A and S7A-B), suggesting that not only RhoA but also RhoC could mediate the function of podoplanin in invadopodia. The Rho/ROCK (Rho-associated protein kinase) pathway has been involved in invadopodia maturation through control of cofilin phosphorylation.10 Podoplanin knockdown cells showed a very small but consistent reduction of cofilin phosphorylation at Ser3 (pCofilinS3; Fig 9B). Conversely, pCofilinS3 was slightly increased upon ectopic expression of podoplanin in HaCaT cells (Fig 9B). To further define the kinetics of pCofilinS3 during invadopodia assembly, control and podoplanin-silenced HN5 cells were serum starved overnight and stimulated with FBS to synchronously induce invadopodia formation and associated matrix degradation (Fig 9C). Formation of non-functional immature invadopodia was observed during the first 30 min after stimulation in control cells. Matrix degradation initiated after 1h, and ~90% of control cells showed fully mature invadopodia after 3-4h, by which point the cells were no longer synchronised. pCofilinS3 levels were slightly decreased in control cells at 0-1h, correlating with initiation of invadopodia formation and actin polymerization (Fig 9D and S7D). After 1h, pCofilinS3 levels were increased accordingly with the stabilisation and maturation steps (2-4h). Podoplanin Knockdown led to a global and steady decrease in pCofilinS3 compared to control cells. Restoration of podoplanin expression in PDPNsh cells (Fig 9A) rescued the observed defects in matrix degradation and pCofilinS3.

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