Abstract LB-57: Paradoxical roles of IL-6 in modulating growth of stem vs. non-stem cells of non-small cell lung cancer
Authors: Lee Soo O. , Tsai Ying , Stronjy Laura , Hsu Jong-Wei , Duan Shan-Zhou , Keng Peter , Chen Yuhchyau 
Published: May 2015

Abstract
Background: Elevated serum levels of interleukin-6 (IL-6) in lung cancer patients have been observed and have been implicated in lung cancer progression. However, the exact role and the mechanism of IL-6 actions have not been clearly dissected. Especially, the role of IL-6 in lung cancer stem cells (CSCs) has never been addressed.
Methods: In this study, we isolated CD133+ cancer stem cells (CSCs) and CD133- non-CSCs from 3 non-small cell lung cancer (NSCLC) cell lines, A549, H1299, and H157, and investigated the roles of IL-6 in modulating the growth of these two cell populations separately. After isolation of CD133+ and CD133- cells by magnetic sorting, and after identification of CSC characteristics in the isolated CD133+ cells, cells were treated with IL-6 for 2 weeks and their growth analyzed by direct cell counting for non-CSCs and by sphere formation assay for CSCs. To investigate the role of IL-6 in the IL-6-expressing A549 and H157 cell lines further, we manipulated IL-6 expression by lentiviral infection prior to the isolation of the CD133+ and CD133- cells. As the 3rd approach, we long-term treated H1299 cells with IL-6 and then isolated CD133+ and CD133- cells from this culture, as well as from the passage-matched non-treated parental cells.
We further confirmed the in vitro results by performing in vivo xenografted mouse studies. We subcutaneously injected mice with CD133+ cells isolated from the IL-6 knocked-down A549 (A549IL-6si) or control (A549sc) cells and monitored tumor growth. Finally, we dissected the mechanism by which IL-6 promoted self-renewal of CSCs.
Results: Results of these 3 approaches clearly indicated that IL-6 played opposite roles in regulating the growth of the two different cell populations; an inhibitory role in the growth of the CD133- cells, but a promoting role in the self-renewal of CD133+ CSCs. In mouse studies, we observed higher tumor incidence and higher tumor growth in mice injected with the A549sc-CD133+ cells compared to mice injected with A549IL-6si-CD133+ cells, confirming the promoter role of IL-6 in CD133+ cell growth in vivo. Further mechanism dissection studies using Western blot analyses revealed that IL-6 modulated the activation of the Hedgehog (hhg) and MEK/Erk signaling pathways, and increased the expression of anti-apoptotic proteins, bcl-2. Inhibitor studies using the inhibitors of these signaling pathways confirmed that increased self-renewal of CD133+ cells were blocked by inhibitions of hhg and bcl-2.
Conclusions and Impacts: Together, these results, for the first time, showed differential effects of IL-6 in regulating non-CSCs vs. CSCs of NSCLC that are paradoxical, which may explain why previous researchers had reported inconsistent results of IL-6 effects. The target molecules revealed in the mechanistic studies could be further utilized for developing future therapeutic approaches to target CSCs of NSCLC.
Citation Format: Soo Ok Lee, Ying Tsai, Laura Stronjy, Jong-Wei Hsu, Shan-Zhou Duan, Peter Keng, Yuhchyau Chen. Paradoxical roles of IL-6 in modulating growth of stem vs. non-stem cells of 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 LB-57. doi:10.1158/1538-7445.AM2014-LB-57