ASK1 inhibits proliferation and migration of lung cancer cells via inactivating TAZ

Abstract

ASK1 (Apoptosis Signal-regulating Kinase 1, also MEKK5) is known to mediate cellular stress signaling pathways through activating p38 kinase. We here observed that ectopically expression of ASK1, but not its kinase-dead mutant, impaired cell proliferation and migration in lung cancer A549 and NCI-H1975 cells. To our surprise, this inhibitory effect of ASK1 is independent on activation of p38 kinase. We further discovered that ASK1 interacts with the WW domain of YAP and TAZ (also WWTR1) that are transcriptional co-activators and the Hippo signaling effectors. Overexpression of wild type ASK1, but not the kinase-dead mutant, in the lung cancer cells down-regulated the expression of the YAP/TAZ target genes CYR61 and CTGF. It seems that ASK1 specifically inactivates TAZ, not YAP, as ASK1 blocked nuclear translocation of TAZ only, while had no effect on YAP. Furthermore, knockdown of TAZ in the lung cancer cells caused the same inhibitory effect on cell proliferation and migration as that of overexpression of ASK1. Thus, our studies have defined a new signaling pathway of ASK1 for regulation of lung cancer cell proliferation and migration via interacting with and inactivating TAZ.

Keywords: ASK1; TAZ; lung cancer; migration; proliferation.

Figure 1

Overexpression of ASK1 inhibits lung cancer cell proliferation. A. The expression level of endogenous ASK1 in various cancer cell lines. B and C. The effect of overexpression of ASK1 on cell proliferation in the non-small cell lung cancer (NSCLC) cell lines A549 and NCI-H1975. The ASK1 (ASK1-WT) or its kinase-dead mutant ASK1-K709A (ASK1-KD) was stably expressed in the lung cancer cells using a lentiviral mammalian expression system. The expressed protein level of ASK1 or its kinase-dead mutant was detected by immunoblotting of the cell lysates with an anti-ASK1. The p38 and phosphor-p38 were also detected by immunoblotting. Treatment with TNFα (50 ng/ml) was employed for activation of ASK1 in A549 cells. The lung cancer cell proliferation was quantified by counting the cell number under a phase microscope with a hemocytometer. The data used for quantification were from three independent experiments.

Figure 2

Overexpression of ASK1 inhibits lung cancer cell migration. The effect of overexpression of ASK1 (ASK1-WT) or the kinase-dead mutant (ASK1-KD) on cell migration determined by the wound-healing assay (A and B) and by the transwell assay (C and D). EGF (50 ng/ml) was used for stimulation of cell migration. The cell line is labeled in the Figure. For quantification in the wound-healing assay, the area covered by the migrated cells was quantified with Image J software (from NIH). The percentage of the covered area by migrated cells is used as the migration rate. The data used for quantification are from three independent experiments. For quantification in the transwell assay, the migrated cells were fixed, stained with crystal violet, and counted under a microscope from three randomly selected fields. **P < 0.01; ***P < 0.001.

Figure 3

ASK1 inhibits cell proliferation and migration independent of the p38 pathway. A. The p38 kinase inhibitor SB203380 has a minor inhibitory effect on proliferation of lung cancer A549 cells and does not affect the inhibitory effect of ASK1 on cell proliferation. B and C. The p38 kinase inhibitor SB203380 inhibits migration of lung cancer A549 cells, but does not change the inhibitory effect of ASK1 on cell migration determined by both the wound-healing and the transwell assays. EGF (50 ng/ml) was used for stimulation of cell migration. SB203380 (10 μM) was used for inhibition of the p38 kinase. The data quantification was the same as described in Figure 2. **P < 0.01; ***P < 0.001.

Figure 4

ASK1 binds to the WW domain of YAP and TAZ. (A) A structural sketch of the YAP and TAZ and their WW domains. Tead-BD, Tead binding domains; WW, WW domain; WW1, the first WW domain; WW2, the second WW domain; TAD, Tead activating domain; PDZ-BD, PDZ binding domain. The numbers labeled in YAP and TAZ constructs indicate the amino acid residue positions. (B) ASK1 binds to the WW domain of YAP and TAZ. The GST fused WW domain was expressed in bacteria, and purified with the glutathione-conjugated agarose beads. The bead-bound GST-WW domain was incubated with the ASK1-expressed HEK293T cell lysates. The co-precipitated ASK1 was detected by immunoblotting with an anti-ASK1 antibody. (C and D) The amino acid residues 799-PPFY-882 in ASK1 is not the YAP- or TAZ-binding site. The GST-WW domain pulldown assay was performed the same as in (B) except the bead-bound GST-WW domain was also incubated with the HEK293T cell lysates containing the ASK1 mutant ASK1-Y882A in which the 799-PPFY-882 is mutated into 799-PPFA-882. (C) The GST-YAP-WW domain pulldown assay; (D) The GST-TAZ-WW domain pulldown assay.

Figure 5

Overexpression of ASK1 inhibits the expression of the YAP/TAZ target genes CYR61 and CTGF. (A and B) The mRNA level of the YAP/TAZ target genes CYR61 and CTGF was dramatically reduced upon overexpression of ASK1, but not the kinase-dead mutant. The cells were serum-starved for 24 hrs followed by stimulation with 10% FBS for 30 min. Total RNA was purified and the mRNA level of CYR61 and CTGF was determined by the quantitative RT-PCR (qRT-PCR) assay. (A) In A549 cells; (B) In NCI-H1975 cells. (C) The protein level of CYR61 was reduced upon overexpression of ASK1, but not the kinase-dead mutant, in A549 cells. The serum starvation and stimulation were performed the same as in (A and B). The cells were lysed and CYR61 was detected by immunoblotting with an anti-CYR61 antibody. (D) Overexpression of ASK1 or the kinase-dead mutant in A549 cells had no significant effect on phosphorylation of the Hippo signaling kinase LATS1.

Figure 6

Overexpression of ASK1 inhibits nuclear localization of TAZ, but not YAP. Lung cancer A549 cells stably expressed ASK1 or its kinase-dead mutant were cultured on glass cover-slips, serum-starved 24 hrs followed by stimulation with 10% serum for 30 min. A. The cells were fixed and immuno-stained with the anti-YAP or the anti-TAZ antibody, and the nuclei were stained with fluorescent dye DAPI. B. The expression of ASK1 and its kinase-dead mutant was detected by immunoblotting of the cell lysates with an anti-ASK1 antibody. Bar, 20 mm.

Figure 7

Depletion of TAZ in lung cancer inhibits cell proliferation and migration. A. Knockdown of TAZ by shTAZ-1 and shTAZ-2 in the NSCLC cell line A549. The effect of shTAZs on the protein level of TAZ and the target gene product CYR61 was determined by immunoblotting of the cell lysates with anti-TAZ and anti-CYR61 antibodies. B. The knockdown of TAZ by shTAZs inhibits A549 cell proliferation. The data used for quantitation were from three independent experiments. C and D. Knockdown of TAZ by shTAZs inhibits A549 cell migration. C. The wound-healing cell migration assay; D. The transwell cell migration assay. EGF (50 ng/ml) was used for stimulation of cell migration. The data quantification was the same as described in Figure 2. **P < 0.01; ***P < 0.001.