The Mechanism Of TGF-β/SMAD Pathway And Related Gene Regulated EMT And Cell Invasion In Non-small Cell Lung Cancer

Part1 Ski prevents TGF-β/SMAD pathway induced EMT and cell invasion in non-small cell lung cancerBackground and Objectives:Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis, which confers cancer cells with increased motility and invasiveness, and EMT is characterized by loss of epithelial marker E-cadherin and gain of mesenchymal marker N-cadherin. Transforming growth factor-P (TGF-β) signaling is a crucial inducer of EMT in various cancers. Ski is an important negative regulator of TGF-β signaling, which may interact with SMADs to repress TGF-β signaling activity. Although there is accumulating evidence that Ski functions as a promoter or suppressor in human cancers, the molecular mechanisms by which Ski affects TGF-β-induced EMT and invasion in non-small cell lung cancer (NSCLC) are not largely elucidated.Methods:(1) qRT-PCR and western blot analysis of the expression of Ski in NSCLC cell lines and tissues. (2) Try to create stable Ski-silenced A549 cells (A549-sh-Ski) and Ski-overexpressed A549 cells (A549-Ski). (3) A549-sh-Ski and A549-Ski cells were treated with or without TGF-β1 for 24 h, respectively. Cell morphology was examined and photographed using a phase-contrast microscope. The protein levels of E-cadherin, N-cadherin, SMAD3 and p-SMAD3 were determined by western blot. (4) A549-sh-Ski and A549-Ski cells transfected with PAI-1 promoter luciferase constructs were incubated for 24 h in the absence or presence TGF-β1, and then subjected to luciferase assays. (5) Transwell assay was performed for the effect of Ski on TGF-β1-mediated cell invasion. And MMP-2 mRNA levels were determined by qRT-PCR.Results:Ski was significantly reduced in metastatic NSCLC cells or tissues when compared with non-metastatic NSCLC cells or tissues. Moreover, on TGF-β1 stimulation, Ski-silenced A549 cells had more significant features of EMT and higher invasive activity compared with A549 cells overexpressing Ski. Mechanistically, Ski-silenced and-overexpressed A549 cells showed an increase and a reduction in SMAD3 phosphorylation level, respectively. This was supported by PAI-1 promoter activity obtained in Ski-silenced and-overexpressed A549 cells. However, after treatment of SIS3 (inhibitor of SMAD3 phosphorylation) followed by TGF-β1 stimulation, we did not observe any effect of Ski on TGF-β-induced EMT and invasion in Ski-silenced and -overexpressed A549 cells.Conclusion:In conclusion, our findings suggest that Ski represses TGF-β-induced EMT and invasion mostly through inhibiting SMAD-dependent signaling in NSCLC.Part2 TGF-β-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancerBackground and Objectives:Epithelial-mesenchymal transition (EMT) is a key event during early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation.Methods:(1) Western blot and qRT-PCR analysis of CDH2 expression in NSCLC cells in the presence of TGF-β1i. (2) We try to create a series of truncated CDH2 5’-promoter luciferase constructs and identify the TGF-P-response region. (3) ChIP assay to certify the TFs recruited to the promoter of CDH2. (4) RNAi experiments to detect the effect of the TFs on the expression of N-cadherin. (5) Correlation assays in NSCLC tissues.Results:The amount of CDH2 mRNAs and proteins were significantly increased in A549, SPC-A1 and H1650 cells when TGF-β1 treated. Luciferase reporter assay revealed that a functional TGF-P-response element was located at position-1078 to-891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to-891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively.Conclusions:We found that TGF-P-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element which was in the promoter of CDH2 gene. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.