Mechanism Underlying ASK1-Mediated Inhibition Of Lung Cancer Cell Proliferation And Migration

Objective: This study is to explore the role of ASK1 in lung cancer cell proliferation and migration signaling,and to identify new molecular targets and provide novel strategies for diagnosis and treatment of lung cancer.Methods: 1、The lentiviral expression system was used to stably overexpress ASK1(ASK1-WT)or its kinase death mutant(ASK1-KD)in lung cancer lines A549 and H1975.The expression level of ASK1-WT or ASK1-KD was detected by western blotting.The effect of the overexpression on lung cancer cell proliferation was determined using the cell counting assay.2、 The effect of overexpression of ASK1-WT or ASK1-KD on lung cancer cell migration was examined by the transwell and the wound healing assays.3、The p38 kinase in lung cancer A549 cells was inhibited by treatment with the p38 kinase inhibitor SB203380.The effect of inhibition of p38 kinase by SB203380 on the ASK1-mediated lung cancer cell proliferation and migration was evaluated by the cell counting,the transwell and the wound healing assays.4、The GST-WW pulldown assay was used to determine the interaction of YAP/TAZ with ASK1.5、Lung cancer A549 and H1975 cells were treated with serum to inhibit the Hippo pathway,and the effect of ASK1 on expression of the m RNA and the protein level of the YAP/TAZ target genes CYR61 and CTGF was detected by the real-time quantitative PCR assay and western blotting,respectively.The effect of ASK1 on the nuclear localization of YAP and TAZ was detected by immunofluorescent staining.6、Lentivirus vector-loaded TAZ sh RNAs were used to deplete endogenous TAZ in lung cancer A549 cells,and the efficiency of the knockdown were examined by detecting the TAZ protein in the lysates with western blotting.The effect of TAZ knockdown on proliferation and migration of lung cancer A549 cells was determined using the cell counting,the transwell and the wound healing assays.Results: 1、Overexpression of ASK1-WT,but not the ASK1-KD,in either A549 or H1975 cells significantly inhibited cell proliferation and migration.2、Inhibition of p38 kinase with SB203380 did not cause siginificant changes in the inhibitory effect of ASK1 on A549 cells proliferation and migration.3、ASK1 interacted with the WW domains of YAP and TAZ.4、Overexpression of ASK1-WT in either A549 or H1975 cells significantly inhibited the m RNA expression of the YAP/TAZ target genes CYR61 and CTGF,while overexpression of ASK1-KD had no inhibitory effect on the m RNA expression of CYR61 and CTGF.5、Overexpression of ASK1-WT in A549,but not ASK1-KD,significantly reduced the protein level of CYR61.6、Immunofluorescent staining showed that YAP and TAZ were mainly localized on the nucleus of lung cancer A549 and H1975 cells.Overexpression of ASK1-WT,but not ASK1-KD,enhanced amount of cytoplasmic TAZ and reduced amount of nuclear TAZ.However,overexpression of either ASK1-WT or ASK1-KD did not significantly affect the nuclear localization of YAP.7、Knockdown of TAZ in A549 cells significantly inhibited cell proliferation and migration.Conclusions: 1、ASK1 mediates inhibitory signaling in lung cancer cell proliferation and migration,and this process depends on its kinase activity.2、The ASK1-mediated inhibition of proliferation and migration of lung cancer cell is independent on the p38 signaling pathway.3、Although ASK1 interacts with both YAP and TAZ,it seems to inhibit the transcriptional activity of TAZ only.4、Knockdown of TAZ in lung cancer A549 cells significantly inhibits the cell proliferation and migration,which is consistent with the effect of overexpression of ASK1.