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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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Expression of surface integrin in A5RT3CTRL and A5RT3TAK1. (a and b) FACS analysis of A5RT3CTRL and A5RT3TAK1 cells immunostained with antibodies against indicated surface integrin β subunits (a) and with integrin α5β1 (b). The distribution of tumor cells with regard to their extent of integrin staining was presented in histogram plots with the identity of the stained integrin subunit indicated (top left). Cells stained with only control IgG served as the negative controls. Each image is representative of at least three different experiments. Values shown indicate mean fluorescence intensity. (c) PLA assay of α5β1 integrin expression (upper panel) and active integrin β1:active Rac1 (lower panel) n A5RT3CTRL and A5RT3TAK1 cells. Representative images were shown with mean number of PLA spots per nucleus±S.D. indicated; n=3
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fig3: Expression of surface integrin in A5RT3CTRL and A5RT3TAK1. (a and b) FACS analysis of A5RT3CTRL and A5RT3TAK1 cells immunostained with antibodies against indicated surface integrin β subunits (a) and with integrin α5β1 (b). The distribution of tumor cells with regard to their extent of integrin staining was presented in histogram plots with the identity of the stained integrin subunit indicated (top left). Cells stained with only control IgG served as the negative controls. Each image is representative of at least three different experiments. Values shown indicate mean fluorescence intensity. (c) PLA assay of α5β1 integrin expression (upper panel) and active integrin β1:active Rac1 (lower panel) n A5RT3CTRL and A5RT3TAK1 cells. Representative images were shown with mean number of PLA spots per nucleus±S.D. indicated; n=3

Mentions: Our above observations suggest that TAK1 deficiency in A5RT3 facilitated TGFβ1-induced EMT. EMT involves the transition of the epithelial cancer cells from predominantly cell–cell adhesion to a cell–ECM interaction. Integrins are major cell surface receptors that enable bidirectional communication between the cells with the ECM are thus well apt to influence cellular behavior involved in EMT including migration and adhesion.17, 18 Thus, we examined the surface expression of integrins β1, β3, β4, β5 and β7 on A5RT3TAK1 (Figure 3a). We found that A5RT3TAK1 had significantly increased surface levels of β1 and β3 integrins (Figure 3a). We further conducted staining of α5β1 integrin and found that it is also significantly elevated in a manner reminiscent of integrin β1 (Figure 3b). Proximity ligation assay (PLA) also confirmed higher level of surface α5β1 integrin in A5RT3TAK1 than A5RT3CTRL (Figure 3c, upper panel). PLA assay was conducted without Triton-X permeabilization to detect only to surface integrin levels. PLA signals for integrin α5β1 were predominantly situated along the cell–cell boundary (Figure 3c, upper panel). PLA assay performed using antibody against active integrin β1 and configuration-specific monoclonal anti-Rac1-GTP antibody further confirmed that the increase expression of surface integrin was associated with an elevated Rac1 activation, suggesting a remodeling of the cytoskeleton (Figure 3c, lower panel).


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)

Expression of surface integrin in A5RT3CTRL and A5RT3TAK1. (a and b) FACS analysis of A5RT3CTRL and A5RT3TAK1 cells immunostained with antibodies against indicated surface integrin β subunits (a) and with integrin α5β1 (b). The distribution of tumor cells with regard to their extent of integrin staining was presented in histogram plots with the identity of the stained integrin subunit indicated (top left). Cells stained with only control IgG served as the negative controls. Each image is representative of at least three different experiments. Values shown indicate mean fluorescence intensity. (c) PLA assay of α5β1 integrin expression (upper panel) and active integrin β1:active Rac1 (lower panel) n A5RT3CTRL and A5RT3TAK1 cells. Representative images were shown with mean number of PLA spots per nucleus±S.D. indicated; n=3
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3824649&req=5

fig3: Expression of surface integrin in A5RT3CTRL and A5RT3TAK1. (a and b) FACS analysis of A5RT3CTRL and A5RT3TAK1 cells immunostained with antibodies against indicated surface integrin β subunits (a) and with integrin α5β1 (b). The distribution of tumor cells with regard to their extent of integrin staining was presented in histogram plots with the identity of the stained integrin subunit indicated (top left). Cells stained with only control IgG served as the negative controls. Each image is representative of at least three different experiments. Values shown indicate mean fluorescence intensity. (c) PLA assay of α5β1 integrin expression (upper panel) and active integrin β1:active Rac1 (lower panel) n A5RT3CTRL and A5RT3TAK1 cells. Representative images were shown with mean number of PLA spots per nucleus±S.D. indicated; n=3
Mentions: Our above observations suggest that TAK1 deficiency in A5RT3 facilitated TGFβ1-induced EMT. EMT involves the transition of the epithelial cancer cells from predominantly cell–cell adhesion to a cell–ECM interaction. Integrins are major cell surface receptors that enable bidirectional communication between the cells with the ECM are thus well apt to influence cellular behavior involved in EMT including migration and adhesion.17, 18 Thus, we examined the surface expression of integrins β1, β3, β4, β5 and β7 on A5RT3TAK1 (Figure 3a). We found that A5RT3TAK1 had significantly increased surface levels of β1 and β3 integrins (Figure 3a). We further conducted staining of α5β1 integrin and found that it is also significantly elevated in a manner reminiscent of integrin β1 (Figure 3b). Proximity ligation assay (PLA) also confirmed higher level of surface α5β1 integrin in A5RT3TAK1 than A5RT3CTRL (Figure 3c, upper panel). PLA assay was conducted without Triton-X permeabilization to detect only to surface integrin levels. PLA signals for integrin α5β1 were predominantly situated along the cell–cell boundary (Figure 3c, upper panel). PLA assay performed using antibody against active integrin β1 and configuration-specific monoclonal anti-Rac1-GTP antibody further confirmed that the increase expression of surface integrin was associated with an elevated Rac1 activation, suggesting a remodeling of the cytoskeleton (Figure 3c, lower panel).

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