Abstract
Recent studies have demonstrated that acquisition of cancer stem-like properties plays an essential role in promoting epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) resistance in non-small cell lung cancer (NSCLC); however, how to regulate cancer stem-like properties and EGFR-TKI resistance is largely unclear. In this study, we discovered that increased iroquois-class homeodomain protein 4 (IRX4) was related to gefitinib resistance in NSCLC cells. Knockdown of IRX4 inhibited cell proliferation, sphere formation, and the expression of CD133, ALDH1A1, NANOG, Sox2 and Notch1, and the transcriptional activity of NANOG promoter. IRX4 overexpression increased the protein level of NANOG and CD133 in PC-9 cells. Combination of knocking-down IRX4 with gefitinib increased cell apoptosis and decreased cell viability and the expression of p-EGFR and NANOG in PC-9/GR cells. IRX4 knockdown in a PC-9/GR xenograft tumor model inhibited tumor progression and the expression of NANOG and CD133 more effectively than single treatment alone. Knockdown of NANOG inhibited the expression of CD133 and restored gefitinib cytotoxicity, and NANOG overexpression-induced cancer stem-like properties and gefitinib resistance could be obviously reversed by knocking-down IRX4. Further, we found that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) reduced obviously the expression of IRX4 and NANOG by inhibiting the activation of TGF-β1/Smad3 signaling pathway; moreover, combination of 1,25(OH)2D3 and gefitinib decreased cell viability and proliferation or tumor progression and the expression of IRX4 and NANOG compared with single treatment alone both in PC-9/GR cells and in a PC-9/GR xenograft tumor model. These results reveal that inhibition of IRX4-mediated cancer stem-like properties by regulating 1,25(OH)2D3 signaling may increase gefitinib cytotoxicity. Combination therapy of gefitinib and 1,25(OH)2D3 by targeting IRX4 and NANOG, could provide a promising strategy to improve gefitinib cytotoxicity.
Highlights
Lung cancer has been noted due to the increasing rate of morbidity and mortality worldwide[1]
IRX4 expression is upregulated by gefitinib exposure We found that IRX4 was widely expressed in lung adenocarcinoma (LUAD)
The IRX4 was mainly expressed in the nucleus and the nuclear expression of IRX4 was higher in PC-9/GR cells than that in PC-9 cells (Fig. 1h), indicating IRX4 functions in the nucleus
Summary
Lung cancer has been noted due to the increasing rate of morbidity and mortality worldwide[1]. Many patients with lung adenocarcinoma (LUAD), a major subtype of lung cancer[2], harbor mutations in the epidermal growth factor receptor (EGFR) in their cancer tissues and initially react well to molecular targeted drugs such as gefitinib, which inhibits the EGFR-tyrosine kinase[3,4]. Official journal of the Cell Death Differentiation Association. Jia et al Cell Death and Disease (2020)11:670. Several mechanisms of acquired resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have been elucidated, mainly including the mutation of EGFR T790M, MET and HER2 amplification[7]. Whereas, underlying resistance mechanism remains undefined in a significant percentage of patients. It is of great significance to investigate potential mechanisms and alternative strategies for reversing gefitinib resistance or enhancing its efficacy
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