Abstract 1939: Calcineurin/NFAT signaling regulates tumor initiating cell properties in non-small cell lung cancer

Abstract

Abstract Background: Targeting specific pathways responsible for Tumor Initiating Cells (TIC) maintenance is postulated to be an important therapeutic strategy for more effective control of tumors. Nuclear Factors of Activated T-cells (NFATs), consisting of 4 homologues (NFATc1-c4), are calcium-dependent transcription factors that are activated upon dephosphorylation by the protein phosphatase calcineurin. NFAT is known to be involved in T cell activation and differentiation. Recently, its roles in regulating embryonic stem cell proliferation and cancer survival have been reported. However, the involvement of Calcineurin/NFAT signaling in TIC maintenance remains unknown. Objective: To study the role of Calcineurin/NFAT in regulating TIC properties in non-small cell lung cancer (NSCLC). Methods and results: Analysis of NFAT mRNA expression in a panel of established and early passage patient derived NSCLC cell lines showed NFATc2 and NFATc4 were up-regulated in 92.6% (25 of 27) and 72% (20 of 27) of cancer cells compared to normal bronchial cell line (BEAS-2B), respectively. To study its role in TIC, tumor spheres or flow cytometry-isolated ALDHhigh/CD44high cells were obtained and NFAT expressions and activities were compared between TIC and non-TIC. The expression of NFAT and downstream targets were significantly higher in tumor spheres and ALDHhigh/CD44high cells compared to the corresponding monolayers and ALDHlow/CD44low cells. Correspondingly, luciferase reporter assay also showed higher NFAT activities in tumor spheres compared to monolayers. To study its functional importance, calcineurin and NFAT were inhibited and effects on TIC properties were analyzed. Inhibition of calcineurin by pharmacological inhibitors cyclosporin A (CSA) and FK506 resulted in reduced sphere formation ability, down-regulation of pluripotent genes (SOX2, OCT4, NANOG) and TIC markers (ALDH1A1, CD133, CD166) expression, as well as decreased TIC proportions (ALDHhigh/CD44high and CD133+) by flow cytometry. These effects were verified using siRNA knockdown of PPP3R1 which encodes one of the calcineurin regulatory subunits. Apart from expression changes, stable knockdown of NFATc2 using lentiviral shRNA inhibited in vitro and in vivo tumorigenecity as shown in soft agar colony formation and mice xenografts compared to shRNA control. To explore the clinical relevance of NFAT, NFAT expression levels were analyzed in human resected lung cancers by quantitative PCR and immunohistochemsitry. Results showed that NFATs were significantly up-regulated in clinical NSCLC compared to corresponding normal lung, and higher NFAT expression predicted shorter recurrence free survival. Conclusions: Calcineurin/NFAT signaling is active in both NSCLC and lung TIC. It plays an important role in TIC maintenance. Inhibition of this pathway is a promising approach for long term lung cancer control. Citation Format: Zhi-Jie Xiao, Jing Liu, Vicky Pui-Chi Tin, Maria Pik Wong. Calcineurin/NFAT signaling regulates tumor initiating cell properties in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2014-1939