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Loss of TAK1 increases cell traction force in a ROS-dependent manner to drive epithelial-mesenchymal transition of cancer cells.

Lam CR, Tan C, Teo Z, Tay CY, Phua T, Wu YL, Cai PQ, Tan LP, Chen X, Zhu P, Tan NS - Cell Death Dis (2013)

Bottom Line: We further show that TAK1 modulates Rac1 and RhoA GTPases activities via a redox-dependent downregulation of RhoA by Rac1, which involves the oxidative modification of low-molecular weight protein tyrosine phosphatase.Our findings suggest that a dysregulated balance in the activation of TGFβ-TAK1 and TGFβ-SMAD pathways is pivotal for TGFβ1-induced EMT.Thus, TAK1 deficiency in metastatic cancer cells increases integrin:Rac-induced ROS, which negatively regulated Rho by LMW-PTP to accelerate EMT.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is a crucial step in tumor progression, and the TGFβ-SMAD signaling pathway as an inductor of EMT in many tumor types is well recognized. However, the role of non-canonical TGFβ-TAK1 signaling in EMT remains unclear. Herein, we show that TAK1 deficiency drives metastatic skin squamous cell carcinoma earlier into EMT that is conditional on the elevated cellular ROS level. The expression of TAK1 is consistently reduced in invasive squamous cell carcinoma biopsies. Tumors derived from TAK1-deficient cells also exhibited pronounced invasive morphology. TAK1-deficient cancer cells adopt a more mesenchymal morphology characterized by higher number of focal adhesions, increase surface expression of integrin α5β1 and active Rac1. Notably, these mutant cells exert an increased cell traction force, an early cellular response during TGFβ1-induced EMT. The mRNA level of ZEB1 and SNAIL, transcription factors associated with mesenchymal phenotype is also upregulated in TAK1-deficient cancer cells compared with control cancer cells. We further show that TAK1 modulates Rac1 and RhoA GTPases activities via a redox-dependent downregulation of RhoA by Rac1, which involves the oxidative modification of low-molecular weight protein tyrosine phosphatase. Importantly, the treatment of TAK1-deficient cancer cells with Y27632, a selective inhibitor of Rho-associated protein kinase and antioxidant N-acetylcysteine augment and hinders EMT, respectively. Our findings suggest that a dysregulated balance in the activation of TGFβ-TAK1 and TGFβ-SMAD pathways is pivotal for TGFβ1-induced EMT. Thus, TAK1 deficiency in metastatic cancer cells increases integrin:Rac-induced ROS, which negatively regulated Rho by LMW-PTP to accelerate EMT.

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TAK1 deficiency increases cell traction force in A-5RT3 facilitating EMT. (a) Vinculin immunostaining (green) and phalloidin F-actin (red) staining were conducted of A5RT3CTRL and A5RT3TAK1 cell colonies with and without TGFβ1 treatment for 48 h with representative images shown. Scale bar, 100 μm. (b and c) Cell traction force profiling (b) and mean measured cell traction stress (c) of A5RT3CTRL and A5RT3TAK1 with and without TGFβ1 (10 ng/ml) treatment for 24 h. Color scale bar denotes traction stress (kPa). Scale bar, 10 μm. Values (mean+S.D.) of three independent measurements. (d) A5RT3CTRL and A5RT3TAK1 cells were seeded on a transwell membrane of pore size and evaluated for their migration through the pore with and without TGFβ1 treatment for 48 h. Migrated cells were stained with crystal violet and destained with a fixed volume of 0.5% Triton-X solution. Absorbance at 595 nm of the solutions was measured to quantify transwell migration. Graph displays mean absorbance values±S.D.; n=3
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fig4: TAK1 deficiency increases cell traction force in A-5RT3 facilitating EMT. (a) Vinculin immunostaining (green) and phalloidin F-actin (red) staining were conducted of A5RT3CTRL and A5RT3TAK1 cell colonies with and without TGFβ1 treatment for 48 h with representative images shown. Scale bar, 100 μm. (b and c) Cell traction force profiling (b) and mean measured cell traction stress (c) of A5RT3CTRL and A5RT3TAK1 with and without TGFβ1 (10 ng/ml) treatment for 24 h. Color scale bar denotes traction stress (kPa). Scale bar, 10 μm. Values (mean+S.D.) of three independent measurements. (d) A5RT3CTRL and A5RT3TAK1 cells were seeded on a transwell membrane of pore size and evaluated for their migration through the pore with and without TGFβ1 treatment for 48 h. Migrated cells were stained with crystal violet and destained with a fixed volume of 0.5% Triton-X solution. Absorbance at 595 nm of the solutions was measured to quantify transwell migration. Graph displays mean absorbance values±S.D.; n=3

Mentions: During EMT, integrins are recruited to distinct contact points to form focal adhesions accompanied by cytoskeletal remodeling of cortical actin network to filamentous stress fibers. Interestingly, we already observed more focal adhesions and better developed stress fiber formation in A5RT3TAK1 than A5RT3CTRL, before TGFβ1 treatment (Figure 4a). These differences in stress fiber formation correlated with vinculin-positive focal adhesion staining at their cell junctions of A5RT3TAK1 (Figure 4a). The vinculin staining also displayed differences in spatial distribution observed in PLA staining for integrin α5β1. As expected, the treatment with TGFβ1 increased stress fiber in both cell lines despite their different cell–cell dispersion. Differences in stress fibers and focal adhesion contact formation in cells are known to effect differential cell traction force (CTF) generation. These force differences represent the early detectable changes in cell responses, preceding the appearance of visible morphological changes. Thus, we measured the CTF generated in A5RT3TAK1 and A5RT3CTRL using CTF microscopy. Cells were cultured on fibronectin-treated compliant PDMS substrate (10 kPa) coated with a monolayer of fluorescence beads. Traction force is determined by measuring the displacement of the fluorescence beads in the x-y plane because of the contraction exerted by the cell. Notably, A5RT3TAK1 generated a considerably different CTF profile from that of control A5RT3CTRL cells. Although traction field was observed to be concentrated along the periphery of the A5RT3CTRL cell colony, intra-colony traction can only be detected in the A5RT3TAK1 group (Figure 4b). As the CTF exerted is also relative to the cell colony size and shape, measured CTF values are divided over the area covered by the cell colonies to derive traction stress values for comparison between cell colonies of differing sizes. Untreated A5RT3CTRL cells displayed a mean traction stress value of 93.4±14.8 Pa, whereas that of A5RT3TAK1 displayed a mean CTF of 170.7±20.2 Pa (Figure 4c), indicating higher contractility in TAK1-deficient cancer cells. TGFβ1 induction resulted in significant CTF increments for both A5RT3CTRL and A5RT3TAK1 as reflected in their elevated traction stress values of 143.96±31.7 Pa and 461.4±113.22 Pa, respectively (Figure 4c). Notably, TGFβ1-induced A5RT3CTRL cell colonies also adopted a CTF profile pattern comparable with that of untreated A5RT3TAK1. In transwell invasion assay, we found that A5RT3TAK1 significantly migrated faster through an 8-μm pore transwell with TGFβ1 as the chemotactic factor (Figure 4d). Our results indicate that TAK1 deficiency in A5RT3 promoted the expression of β1 and β3 integrins, and altered stress fiber formation. This cytoskeletal remodeling corresponded with an increased CTF profile and metastatic potential in A5RT3TAK1 cells, suggesting an inhibitory role of endogenous TAK1 during TGFβ1-induced EMT.


Loss of TAK1 increases cell traction force in a ROS-dependent manner to drive epithelial-mesenchymal transition of cancer cells.

Lam CR, Tan C, Teo Z, Tay CY, Phua T, Wu YL, Cai PQ, Tan LP, Chen X, Zhu P, Tan NS - Cell Death Dis (2013)

TAK1 deficiency increases cell traction force in A-5RT3 facilitating EMT. (a) Vinculin immunostaining (green) and phalloidin F-actin (red) staining were conducted of A5RT3CTRL and A5RT3TAK1 cell colonies with and without TGFβ1 treatment for 48 h with representative images shown. Scale bar, 100 μm. (b and c) Cell traction force profiling (b) and mean measured cell traction stress (c) of A5RT3CTRL and A5RT3TAK1 with and without TGFβ1 (10 ng/ml) treatment for 24 h. Color scale bar denotes traction stress (kPa). Scale bar, 10 μm. Values (mean+S.D.) of three independent measurements. (d) A5RT3CTRL and A5RT3TAK1 cells were seeded on a transwell membrane of pore size and evaluated for their migration through the pore with and without TGFβ1 treatment for 48 h. Migrated cells were stained with crystal violet and destained with a fixed volume of 0.5% Triton-X solution. Absorbance at 595 nm of the solutions was measured to quantify transwell migration. Graph displays mean absorbance values±S.D.; n=3
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: TAK1 deficiency increases cell traction force in A-5RT3 facilitating EMT. (a) Vinculin immunostaining (green) and phalloidin F-actin (red) staining were conducted of A5RT3CTRL and A5RT3TAK1 cell colonies with and without TGFβ1 treatment for 48 h with representative images shown. Scale bar, 100 μm. (b and c) Cell traction force profiling (b) and mean measured cell traction stress (c) of A5RT3CTRL and A5RT3TAK1 with and without TGFβ1 (10 ng/ml) treatment for 24 h. Color scale bar denotes traction stress (kPa). Scale bar, 10 μm. Values (mean+S.D.) of three independent measurements. (d) A5RT3CTRL and A5RT3TAK1 cells were seeded on a transwell membrane of pore size and evaluated for their migration through the pore with and without TGFβ1 treatment for 48 h. Migrated cells were stained with crystal violet and destained with a fixed volume of 0.5% Triton-X solution. Absorbance at 595 nm of the solutions was measured to quantify transwell migration. Graph displays mean absorbance values±S.D.; n=3
Mentions: During EMT, integrins are recruited to distinct contact points to form focal adhesions accompanied by cytoskeletal remodeling of cortical actin network to filamentous stress fibers. Interestingly, we already observed more focal adhesions and better developed stress fiber formation in A5RT3TAK1 than A5RT3CTRL, before TGFβ1 treatment (Figure 4a). These differences in stress fiber formation correlated with vinculin-positive focal adhesion staining at their cell junctions of A5RT3TAK1 (Figure 4a). The vinculin staining also displayed differences in spatial distribution observed in PLA staining for integrin α5β1. As expected, the treatment with TGFβ1 increased stress fiber in both cell lines despite their different cell–cell dispersion. Differences in stress fibers and focal adhesion contact formation in cells are known to effect differential cell traction force (CTF) generation. These force differences represent the early detectable changes in cell responses, preceding the appearance of visible morphological changes. Thus, we measured the CTF generated in A5RT3TAK1 and A5RT3CTRL using CTF microscopy. Cells were cultured on fibronectin-treated compliant PDMS substrate (10 kPa) coated with a monolayer of fluorescence beads. Traction force is determined by measuring the displacement of the fluorescence beads in the x-y plane because of the contraction exerted by the cell. Notably, A5RT3TAK1 generated a considerably different CTF profile from that of control A5RT3CTRL cells. Although traction field was observed to be concentrated along the periphery of the A5RT3CTRL cell colony, intra-colony traction can only be detected in the A5RT3TAK1 group (Figure 4b). As the CTF exerted is also relative to the cell colony size and shape, measured CTF values are divided over the area covered by the cell colonies to derive traction stress values for comparison between cell colonies of differing sizes. Untreated A5RT3CTRL cells displayed a mean traction stress value of 93.4±14.8 Pa, whereas that of A5RT3TAK1 displayed a mean CTF of 170.7±20.2 Pa (Figure 4c), indicating higher contractility in TAK1-deficient cancer cells. TGFβ1 induction resulted in significant CTF increments for both A5RT3CTRL and A5RT3TAK1 as reflected in their elevated traction stress values of 143.96±31.7 Pa and 461.4±113.22 Pa, respectively (Figure 4c). Notably, TGFβ1-induced A5RT3CTRL cell colonies also adopted a CTF profile pattern comparable with that of untreated A5RT3TAK1. In transwell invasion assay, we found that A5RT3TAK1 significantly migrated faster through an 8-μm pore transwell with TGFβ1 as the chemotactic factor (Figure 4d). Our results indicate that TAK1 deficiency in A5RT3 promoted the expression of β1 and β3 integrins, and altered stress fiber formation. This cytoskeletal remodeling corresponded with an increased CTF profile and metastatic potential in A5RT3TAK1 cells, suggesting an inhibitory role of endogenous TAK1 during TGFβ1-induced EMT.

Bottom Line: We further show that TAK1 modulates Rac1 and RhoA GTPases activities via a redox-dependent downregulation of RhoA by Rac1, which involves the oxidative modification of low-molecular weight protein tyrosine phosphatase.Our findings suggest that a dysregulated balance in the activation of TGFβ-TAK1 and TGFβ-SMAD pathways is pivotal for TGFβ1-induced EMT.Thus, TAK1 deficiency in metastatic cancer cells increases integrin:Rac-induced ROS, which negatively regulated Rho by LMW-PTP to accelerate EMT.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is a crucial step in tumor progression, and the TGFβ-SMAD signaling pathway as an inductor of EMT in many tumor types is well recognized. However, the role of non-canonical TGFβ-TAK1 signaling in EMT remains unclear. Herein, we show that TAK1 deficiency drives metastatic skin squamous cell carcinoma earlier into EMT that is conditional on the elevated cellular ROS level. The expression of TAK1 is consistently reduced in invasive squamous cell carcinoma biopsies. Tumors derived from TAK1-deficient cells also exhibited pronounced invasive morphology. TAK1-deficient cancer cells adopt a more mesenchymal morphology characterized by higher number of focal adhesions, increase surface expression of integrin α5β1 and active Rac1. Notably, these mutant cells exert an increased cell traction force, an early cellular response during TGFβ1-induced EMT. The mRNA level of ZEB1 and SNAIL, transcription factors associated with mesenchymal phenotype is also upregulated in TAK1-deficient cancer cells compared with control cancer cells. We further show that TAK1 modulates Rac1 and RhoA GTPases activities via a redox-dependent downregulation of RhoA by Rac1, which involves the oxidative modification of low-molecular weight protein tyrosine phosphatase. Importantly, the treatment of TAK1-deficient cancer cells with Y27632, a selective inhibitor of Rho-associated protein kinase and antioxidant N-acetylcysteine augment and hinders EMT, respectively. Our findings suggest that a dysregulated balance in the activation of TGFβ-TAK1 and TGFβ-SMAD pathways is pivotal for TGFβ1-induced EMT. Thus, TAK1 deficiency in metastatic cancer cells increases integrin:Rac-induced ROS, which negatively regulated Rho by LMW-PTP to accelerate EMT.

Show MeSH
Related in: MedlinePlus