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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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Role of elevated ROS during TGFβ1-induced EMT in A5RT3TAK1. (a) PLA assay of active Rac1 and Nox1 in A5RT3CTRL and A5RT3TAK1 cell. Representative images were shown with mean number of PLA spots per nucleus±S.D. indicated; n=3. (b) A5RT3CTRL and A5RT3TAK1 with or without 48 h of TGFβ1 (10 ng/ml) treatment were stained with DCF and analyzed with flow cytometry. Antioxidant NAC-treated cells (100 μM) served as a negative control. Image shown is representative of three different experiments. Values shown indicate mean fluorescence intensity. (c) Phase-contrast images showing DCF staining of A5RT3CTRL and A5RT3TAK1 after 48 h of TGFβ1 (10 ng/ml) treatment. Scale bar, 100μm. (d) Phase-contrast images A5RT3TAK1 cells subjected to indicated treatements. NAC (100 μM) was used to quench ROS. Scale bar, 100 μm. (e) qPCR analysis of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment. Samples were normalized with reference gene, L27. (f) Representative blots of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment were 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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fig5: Role of elevated ROS during TGFβ1-induced EMT in A5RT3TAK1. (a) PLA assay of active Rac1 and Nox1 in A5RT3CTRL and A5RT3TAK1 cell. Representative images were shown with mean number of PLA spots per nucleus±S.D. indicated; n=3. (b) A5RT3CTRL and A5RT3TAK1 with or without 48 h of TGFβ1 (10 ng/ml) treatment were stained with DCF and analyzed with flow cytometry. Antioxidant NAC-treated cells (100 μM) served as a negative control. Image shown is representative of three different experiments. Values shown indicate mean fluorescence intensity. (c) Phase-contrast images showing DCF staining of A5RT3CTRL and A5RT3TAK1 after 48 h of TGFβ1 (10 ng/ml) treatment. Scale bar, 100μm. (d) Phase-contrast images A5RT3TAK1 cells subjected to indicated treatements. NAC (100 μM) was used to quench ROS. Scale bar, 100 μm. (e) qPCR analysis of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment. Samples were normalized with reference gene, L27. (f) Representative blots of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment were 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: TAK1 deficiency was reported to upregulate ROS level in human keratinocytes and in cancer cells.9, 19 Integrin-Rac1 signaling mediates multiple pathways that control actin cytoskeletal changes, transcriptional activity and ROS production.20, 21 First, we examine if the increased active Rac1 in A5RT3TAK1 (Figure 3c) was associated with a higher recruitment of Rac1-dependent Nox1 for ROS generation. PLA assay using antibodies against active Rac1 and Nox1 revealed higher number of PLA signals in A5RT3TAK1 than A5RT3CTRL, indicating an elevated Rac1-dependent activation of Nox1 (Figure 5a). Next, we evaluated intracellular ROS production in A5RT3TAK1 and A5RT3CTRL using fluorescence dye CM-H2DCFDA followed by FACS analysis. We found that A5RT3TAK1 cells have increased ROS level, which was further potentiated by TGFβ1 induction. In fact, the ROS level in untreated A5RT3TAK1 was comparable with TGF-β1-treated A5RT3CTRL (Figure 5b). The difference in ROS level between A5RT3CTRL and A5RT3TAK1 upon TGFβ1 treatment was also verified by fluorescence microscopy. Interestingly, we observed that A5RT3TAK1 cells that have separated from the tumor colony were more intensely stained with CM-H2DCFDA compared with A5RT3CTRL (Figure 5c). As ROS upregulation in A5RT3TAK1 correlated with the mesenchymal phenotype induced by TGFβ1, it was conceivable that the observed elevated oxidative stress may be mechanistically involved in EMT. Antioxidant N-acetylcysteine (NAC) addition was utilized to quench ROS. First, we determine the optimal concentration of NAC (30 μM–30 mM) for our experiments. At NAC concentrations of 3–30 mM, annexin V/propidium iodide staining of A5RT3TAK1 revealed increased percentage of apoptotic cells (Supplementary Figure S4A). A corresponding CM-H2DCFDA staining showed that the ROS level was increased in cells treated with high 30 mM NAC concentration, most likely due to cell death. The treatment with 300 μM–3 mM of NAC did not lower the ROS further than that obtained by 100 μM of NAC. At 30 μM of NAC, the decrease in ROS was less effective (Figure 4b). Thus, we determine the optimal NAC concentration to be 100 μM, which lowers the ROS level without substantially increasing cell death. We quenched the ROS in the cancer cells with NAC and observed that the treatment hindered the cell–cell separation characteristic of TGFβ1-induced EMT in A5RT3TAK1 (Figure 5d). The NAC-treated cells were significantly diminished of their EMT traits (Figures 5e and f). NAC treatment neither altered the phenotype of A5RT3CTRL cells (Supplementary Figure S4C) nor elicited any off-target effects as verified by immunoblotting of various EMT markers (Supplementary Figure S4D).


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)

Role of elevated ROS during TGFβ1-induced EMT in A5RT3TAK1. (a) PLA assay of active Rac1 and Nox1 in A5RT3CTRL and A5RT3TAK1 cell. Representative images were shown with mean number of PLA spots per nucleus±S.D. indicated; n=3. (b) A5RT3CTRL and A5RT3TAK1 with or without 48 h of TGFβ1 (10 ng/ml) treatment were stained with DCF and analyzed with flow cytometry. Antioxidant NAC-treated cells (100 μM) served as a negative control. Image shown is representative of three different experiments. Values shown indicate mean fluorescence intensity. (c) Phase-contrast images showing DCF staining of A5RT3CTRL and A5RT3TAK1 after 48 h of TGFβ1 (10 ng/ml) treatment. Scale bar, 100μm. (d) Phase-contrast images A5RT3TAK1 cells subjected to indicated treatements. NAC (100 μM) was used to quench ROS. Scale bar, 100 μm. (e) qPCR analysis of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment. Samples were normalized with reference gene, L27. (f) Representative blots of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment were 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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fig5: Role of elevated ROS during TGFβ1-induced EMT in A5RT3TAK1. (a) PLA assay of active Rac1 and Nox1 in A5RT3CTRL and A5RT3TAK1 cell. Representative images were shown with mean number of PLA spots per nucleus±S.D. indicated; n=3. (b) A5RT3CTRL and A5RT3TAK1 with or without 48 h of TGFβ1 (10 ng/ml) treatment were stained with DCF and analyzed with flow cytometry. Antioxidant NAC-treated cells (100 μM) served as a negative control. Image shown is representative of three different experiments. Values shown indicate mean fluorescence intensity. (c) Phase-contrast images showing DCF staining of A5RT3CTRL and A5RT3TAK1 after 48 h of TGFβ1 (10 ng/ml) treatment. Scale bar, 100μm. (d) Phase-contrast images A5RT3TAK1 cells subjected to indicated treatements. NAC (100 μM) was used to quench ROS. Scale bar, 100 μm. (e) qPCR analysis of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment. Samples were normalized with reference gene, L27. (f) Representative blots of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment were 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: TAK1 deficiency was reported to upregulate ROS level in human keratinocytes and in cancer cells.9, 19 Integrin-Rac1 signaling mediates multiple pathways that control actin cytoskeletal changes, transcriptional activity and ROS production.20, 21 First, we examine if the increased active Rac1 in A5RT3TAK1 (Figure 3c) was associated with a higher recruitment of Rac1-dependent Nox1 for ROS generation. PLA assay using antibodies against active Rac1 and Nox1 revealed higher number of PLA signals in A5RT3TAK1 than A5RT3CTRL, indicating an elevated Rac1-dependent activation of Nox1 (Figure 5a). Next, we evaluated intracellular ROS production in A5RT3TAK1 and A5RT3CTRL using fluorescence dye CM-H2DCFDA followed by FACS analysis. We found that A5RT3TAK1 cells have increased ROS level, which was further potentiated by TGFβ1 induction. In fact, the ROS level in untreated A5RT3TAK1 was comparable with TGF-β1-treated A5RT3CTRL (Figure 5b). The difference in ROS level between A5RT3CTRL and A5RT3TAK1 upon TGFβ1 treatment was also verified by fluorescence microscopy. Interestingly, we observed that A5RT3TAK1 cells that have separated from the tumor colony were more intensely stained with CM-H2DCFDA compared with A5RT3CTRL (Figure 5c). As ROS upregulation in A5RT3TAK1 correlated with the mesenchymal phenotype induced by TGFβ1, it was conceivable that the observed elevated oxidative stress may be mechanistically involved in EMT. Antioxidant N-acetylcysteine (NAC) addition was utilized to quench ROS. First, we determine the optimal concentration of NAC (30 μM–30 mM) for our experiments. At NAC concentrations of 3–30 mM, annexin V/propidium iodide staining of A5RT3TAK1 revealed increased percentage of apoptotic cells (Supplementary Figure S4A). A corresponding CM-H2DCFDA staining showed that the ROS level was increased in cells treated with high 30 mM NAC concentration, most likely due to cell death. The treatment with 300 μM–3 mM of NAC did not lower the ROS further than that obtained by 100 μM of NAC. At 30 μM of NAC, the decrease in ROS was less effective (Figure 4b). Thus, we determine the optimal NAC concentration to be 100 μM, which lowers the ROS level without substantially increasing cell death. We quenched the ROS in the cancer cells with NAC and observed that the treatment hindered the cell–cell separation characteristic of TGFβ1-induced EMT in A5RT3TAK1 (Figure 5d). The NAC-treated cells were significantly diminished of their EMT traits (Figures 5e and f). NAC treatment neither altered the phenotype of A5RT3CTRL cells (Supplementary Figure S4C) nor elicited any off-target effects as verified by immunoblotting of various EMT markers (Supplementary Figure S4D).

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