Inhibition of TBK1 attenuates radiation-induced epithelial–mesenchymal transition of A549 human lung cancer cells via activation of GSK-3β and repression of ZEB1

Abstract

Radiotherapy is an effective treatment method for lung cancer, particularly when the disease is at an advanced stage. However, previous researchers have observed that the majority of patients with conventional radiation therapy develop distant metastases and succumb to the disease. Thus, identifying and understanding novel pathways for the development of new therapeutic targets is a major goal in research on pulmonary neoplasms. Recent studies suggest that epithelial-mesenchymal transition (EMT) is the most important contributor to cancer metastasis. Induction of this complex process requires endogenously produced microRNAs; specifically, downregulation of the miRNA-200c causes an induction of EMT. We recently identified the tank-binding kinase-1 (TBK1) as a downstream effector of the miR-200c-driven pathway, but the biological function of TBK1 in EMT remains unknown. In this study, we tested whether TBK1 has a role in radiation-induced EMT and identified associated potential mechanisms. Human alveolar type II epithelial carcinoma A549 cells were irradiated with 60 Co g-rays. Western blotting revealed a time-and dose-dependent decrease in E-cadherin with a concomitant increase in vimentin after radiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. TBK1 siRNA significantly inhibited radiationinduced suppression of the epithelial marker E-cadherin and upregulation of the mesenchymal marker vimentin. The invasion and migratory potential of lung cancer cells upon radiation treatment was also reduced by TBK1 knockdown. Furthermore, radiation-induced EMT attenuated by TBK1 depletion was partially dependent on transcriptional factor ZEB1 expression. Finally, we found glycogen synthase kinase-3b (GSK-3b) is involved in regulation of radiation-induced EMT by TBK1. Thus, our findings reveal that TBK1 signaling regulates radiation-induced EMT by controlling GSK-3b phosphorylation and ZEB1 expression. TBK1 may therefore constitute a useful target for treatment of radiotherapy-induced metastasis diseases.

ZEB1: At the crossroads of epithelial-mesenchymal transition, metastasis and therapy resistance

“…16 How ATMmediated phosphorylation of ZEB1 leads to ZEB1 stabilization remains to be determined. Similarly, others also reported that radiation causes upregulation of ZEB1 in the A549 lung cancer cell line 67 and in the CNE1 and CNE2 nasopharyngeal cancer cell lines. 68 Taken together, these findings by us and others suggest that radiation treatment can give rise to increased ZEB1 levels and therapy-induced radioresistance.…”

MiR-203 Determines Poor Outcome and Suppresses Tumor Growth by Targeting TBK1 in Osteosarcoma

“…For example, TBK1 was identifies as a downstream effector of the miR-200c in human lung cancer cells [21]. Inhibition of TBK1 attenuates radiation-induced epithelial-mesenchymal transition via activation of GSK-3β and repression of ZEB1 in lung cancer [22]. More recently, in pancreatic ductal adenocarcinoma (PDAC), miR-429 inhibited PDAC cell lines growth by targeting TBK1 [23].…”

Radiation Enhances the Epithelial– Mesenchymal Transition of A549 Cells via miR3591-5p/USP33/PPM1A

“…The TGF-β/Smad signaling pathway is an important regulator for radiation-induced EMT [10,11]. To explore the potential molecular mechanisms how miR-3591-5p affected radiationinduced EMT, we assessed the activation of TGF-β/Smad pathway by measuring TGF-β concentrations in cultured medium and the phosphorylation of Smad2/3.…”

“…In A549 lung cancer cells, radiation can induce the EMT via the activation of TGF-β signaling [10,11]. A crucial step in TGF-β signaling is the phosphorylation and activation of Smad2/3 by TGF-β receptor.…”