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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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Redox regulation of RhoA by Rac1 facilitates TGFβ1 induced EMT. (a) RhoA and Rac1 activities and LMW-PTP oxidation were compared with immunoblotting as respectively indicated. 10 ng/ml TGFβ1 and 100 μM of antioxidant NAC were used respectively for treatment duration of 48 h. (b) FACS analysis of tumor cells transfected with or without constitutive-active (G12V) and dominant-negative Rac1 (T17N). Antioxidant NAC-treated cells (100 μM) served as a negative control. Values shown indicate mean fluorescence intensity. (c) Representative images of E-cadherin (green/488 nm) immunostaining of tumor cells with respectively indicated treatments and transfection. Cell nucleus is stained with DAPI (blue). Scale bar, 100 μm. (d) Phase-contrast images of A5RT3TAK1 cells with and without TGFβ1 induction were inhibited with Y27632 (10 μM) for 24 h or transfected with siRNA for RhoA. Representative phase-contrast images of treated cells. Scale bar, 100 μm. (e) Representative blots of EMT markers in TGFβ1-induced A5RT3CTRL and A5RT3TAK1 treated with ROCK Y27632 or transfected with pooled siRNA for RhoA. Values shown with densitometry values indicated below respective lanes. Samples were normalized with tubulin as a loading control. Data represent means±S.D.; n=3
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fig6: Redox regulation of RhoA by Rac1 facilitates TGFβ1 induced EMT. (a) RhoA and Rac1 activities and LMW-PTP oxidation were compared with immunoblotting as respectively indicated. 10 ng/ml TGFβ1 and 100 μM of antioxidant NAC were used respectively for treatment duration of 48 h. (b) FACS analysis of tumor cells transfected with or without constitutive-active (G12V) and dominant-negative Rac1 (T17N). Antioxidant NAC-treated cells (100 μM) served as a negative control. Values shown indicate mean fluorescence intensity. (c) Representative images of E-cadherin (green/488 nm) immunostaining of tumor cells with respectively indicated treatments and transfection. Cell nucleus is stained with DAPI (blue). Scale bar, 100 μm. (d) Phase-contrast images of A5RT3TAK1 cells with and without TGFβ1 induction were inhibited with Y27632 (10 μM) for 24 h or transfected with siRNA for RhoA. Representative phase-contrast images of treated cells. Scale bar, 100 μm. (e) Representative blots of EMT markers in TGFβ1-induced A5RT3CTRL and A5RT3TAK1 treated with ROCK Y27632 or transfected with pooled siRNA for RhoA. Values shown with densitometry values indicated below respective lanes. Samples were normalized with tubulin as a loading control. Data represent means±S.D.; n=3

Mentions: Our above observations on increased CTF of untreated A5RT3TAK1 corresponded with the increased surface integrin expression, focal adhesion and stress fiber formation. RhoA and Rac1 have key roles in the formation of stress fiber, focal adhesion, cytoskeleton organization and cellular migration. We first examined their activities in A5RT3CTRL and A5RT3TAK1 with and without TGFβ1 induction. In both TGFβ1-treated and -untreated groups, the activities of Rac1 and RhoA were elevated and reduced, respectively, in A5RT3TAK1 compared with A5RT3CTRL (Figure 6a). We observed that NAC treatment increased RhoA activity, but has not significantly affected the level of active Rac1 (Figure 6a). This is consistent with our earlier observation that increased surface expression of integrin β1 activated Rac1, which subsequently engaged Nox1 for ROS generation. It also suggested that the activity of RhoA was redox-dependent. To further understand the relationship between Rac1 and ROS, we transfected A5RT3CTRL and A5RT3TAK1 with expression vectors containing constitutively active (G12V) and dominant-negative Rac mutant (T17N), respectively. FACS analysis revealed that A5RT3CTRL(G12V) cells have increased ROS content compared with control vector transfected A5RT3CTRL (Figure 6b). Conversely, the oxidative stress level of A5RT3TAK1(T17N) was significantly decreased compared with A5RT3TAK1 (Figure 6b). In contrast to their cognate parent cell lines, A5RT3CTRL(G12V) and A5RT3TAK1(T17N) showed increased and reduced TGFβ1-induced EMT, respectively (Figure 6c).


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)

Redox regulation of RhoA by Rac1 facilitates TGFβ1 induced EMT. (a) RhoA and Rac1 activities and LMW-PTP oxidation were compared with immunoblotting as respectively indicated. 10 ng/ml TGFβ1 and 100 μM of antioxidant NAC were used respectively for treatment duration of 48 h. (b) FACS analysis of tumor cells transfected with or without constitutive-active (G12V) and dominant-negative Rac1 (T17N). Antioxidant NAC-treated cells (100 μM) served as a negative control. Values shown indicate mean fluorescence intensity. (c) Representative images of E-cadherin (green/488 nm) immunostaining of tumor cells with respectively indicated treatments and transfection. Cell nucleus is stained with DAPI (blue). Scale bar, 100 μm. (d) Phase-contrast images of A5RT3TAK1 cells with and without TGFβ1 induction were inhibited with Y27632 (10 μM) for 24 h or transfected with siRNA for RhoA. Representative phase-contrast images of treated cells. Scale bar, 100 μm. (e) Representative blots of EMT markers in TGFβ1-induced A5RT3CTRL and A5RT3TAK1 treated with ROCK Y27632 or transfected with pooled siRNA for RhoA. Values shown with densitometry values indicated below respective lanes. Samples were normalized with tubulin as a loading control. Data represent means±S.D.; n=3
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fig6: Redox regulation of RhoA by Rac1 facilitates TGFβ1 induced EMT. (a) RhoA and Rac1 activities and LMW-PTP oxidation were compared with immunoblotting as respectively indicated. 10 ng/ml TGFβ1 and 100 μM of antioxidant NAC were used respectively for treatment duration of 48 h. (b) FACS analysis of tumor cells transfected with or without constitutive-active (G12V) and dominant-negative Rac1 (T17N). Antioxidant NAC-treated cells (100 μM) served as a negative control. Values shown indicate mean fluorescence intensity. (c) Representative images of E-cadherin (green/488 nm) immunostaining of tumor cells with respectively indicated treatments and transfection. Cell nucleus is stained with DAPI (blue). Scale bar, 100 μm. (d) Phase-contrast images of A5RT3TAK1 cells with and without TGFβ1 induction were inhibited with Y27632 (10 μM) for 24 h or transfected with siRNA for RhoA. Representative phase-contrast images of treated cells. Scale bar, 100 μm. (e) Representative blots of EMT markers in TGFβ1-induced A5RT3CTRL and A5RT3TAK1 treated with ROCK Y27632 or transfected with pooled siRNA for RhoA. Values shown with densitometry values indicated below respective lanes. Samples were normalized with tubulin as a loading control. Data represent means±S.D.; n=3
Mentions: Our above observations on increased CTF of untreated A5RT3TAK1 corresponded with the increased surface integrin expression, focal adhesion and stress fiber formation. RhoA and Rac1 have key roles in the formation of stress fiber, focal adhesion, cytoskeleton organization and cellular migration. We first examined their activities in A5RT3CTRL and A5RT3TAK1 with and without TGFβ1 induction. In both TGFβ1-treated and -untreated groups, the activities of Rac1 and RhoA were elevated and reduced, respectively, in A5RT3TAK1 compared with A5RT3CTRL (Figure 6a). We observed that NAC treatment increased RhoA activity, but has not significantly affected the level of active Rac1 (Figure 6a). This is consistent with our earlier observation that increased surface expression of integrin β1 activated Rac1, which subsequently engaged Nox1 for ROS generation. It also suggested that the activity of RhoA was redox-dependent. To further understand the relationship between Rac1 and ROS, we transfected A5RT3CTRL and A5RT3TAK1 with expression vectors containing constitutively active (G12V) and dominant-negative Rac mutant (T17N), respectively. FACS analysis revealed that A5RT3CTRL(G12V) cells have increased ROS content compared with control vector transfected A5RT3CTRL (Figure 6b). Conversely, the oxidative stress level of A5RT3TAK1(T17N) was significantly decreased compared with A5RT3TAK1 (Figure 6b). In contrast to their cognate parent cell lines, A5RT3CTRL(G12V) and A5RT3TAK1(T17N) showed increased and reduced TGFβ1-induced EMT, respectively (Figure 6c).

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