The Calcineurin/Nfat Pathway Mediates Tumor Initiating Cell And Drug Resistance In Lung Cancer Through Sox2 Enhancer Binding And Aldh Upregulation

Tumor initiating cells (TIC) are a small population of cancer cells with enhanced tumorigenicity and drug resistance. Their stemness properties could be activated by stress-induced cell plasticity but the mechanisms remain to be studied. Calcium signaling is important for integrating cell responses to intra- and extracellular stresses. The role of its downstream pathway, calcineurin/NFAT, in lung TIC has not been explored. This study investigated the role and mechanisms of calcineurin/NFAT in regulating TIC and drug resistance by analyzing NFATc2 expression in multiple TIC populations and functional evaluation using in vitro and in vivo models. In tumor spheres and TIC isolated by marker-based flow cytometry, NFATc2 showed overexpression and higher luciferase reporter activities. Knockdown of NFATc2 reduced TIC marker proportions and suppressed TIC properties including pluripotency genes expression, sphere formation, cell mobility and drug resistance in vitro. Xenograft models and limiting dilution assay showed shNFATc2 effectively suppressed tumorigenesis and reduced TIC frequency. Stable NFATc2 knockdown sensitized tumors to cisplatin and gefitinib treatment. In contrast, TIC-supportive effects were observed in vitro and in vivo when NFATc2 was overexpressed. Further, NFATc2 was activated in stable gefitinib or cisplatin resistant cell lines, and knockdown of NFATc2 sensitized them to drug treatment. These data implicated NFATc2 is involved in lung TIC maintenance and drug resistance. To further investigate the molecular mechanisms of how NFATc2 mediate these roles, we analyzed pluripotency genes and observed a significant correlation between NFATc2 and SOX2 expression in lung cancer cell lines and human lung carcinomas. Computational analysis predicted NFATc2 binding sequences in SOX2 regulatory regions. Detailed studies including luciferase reporter assay, site-directed mutagenesis and CHIP-qRCR confirmed NFATc2 could regulate SOX2 expression through binding to its enhancer. Furthermore, immunohistochemistry on human lung cancers showed significant correlation between expressions of SOX2 and the anti-oxidative stem cell marker ALDH, suggesting SOX2 regulates ALDH expression. In vitro, NFATc2 knockdown suppressed ALDH1A1 expression but it was rescued by SOX2 overexpression. Vice versa, ALDH1A1 was upregulated in NFATc2 overexpressing cells but this was prevented by SOX2 knockdown. Functionally, reactive oxidative species (ROS) levels assessed by flow cytometry were suppressed by activated NFATc2/SOX2/ALDH1A1 axis, while treatment with the ROS inhibitor N-acetylcysteine reversed the sensitization of shNFATc2 on cisplatin treatment. Together, the data showed NFATc2 is involved in the regulation of TIC phenotypes in lung cancer. It increases drug resistance by ROS scavenging mediated by the SOX2/ALDH1A1 axis. Citation Format: Zhi-Jie Xiao, Jing Liu, Vicky PC Tin, Maria P. Wong. The Calcineurin/NFAT pathway mediates tumor initiating cell and drug resistance in lung cancer through SOX2 enhancer binding and ALDH upregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2482.