Abstract 748: The glucose metabolism targeting therapies and withaferin A eliminate epidermal growth factor tyrosine kinase inhibitor-induced drug-tolerant persisters in non-small lung cancer cells
Authors: Kunimasa Kei , Nagano Tatsuya , Shimono Yohei , Tokunaga Shuntaro , Tamura Daisuke , Tachihara Motoko , Kobayashi Kazuyuki , Nishimura Yoshihiro 
Published: August 2015

Abstract
In pathway-targeted cancer drug therapies, the relatively rapid emergence of drug-tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. The aim of this study is (1) to clarify the subpopulations of DTPs in EGFR mutated non-small lung cancer (NSCLC) cells that have different roles in the resistance to epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI) and (2) to develop effective treatments.
In this study, we used two EGFR mutated lung adenocarcinoma cell lines, PC9 and II-18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 month. We analyzed the mRNA expression of the stem cell related markers by qRT-PCR and the expression of the cellular senescence associated proteins by Western blotting. Then, we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by the glucose metabolism targeting therapies and a stem-like cell targeting drug, withaferin A.
The time-dependent upregulation of the stem cell-related markers was observed until day 24. Western blot analysis revealed that the cellular senescence associated proteins were upregulated in a similar time course. Senescent cells secrete various inflammatory cytokines and chemokines, a phenomenon known as SASP. Recently, SASP has been reported to contribute to the emergence and maintenance of cancer stem-like cells. Using FACS analysis, DTPs were divided into the two populations depending on the surface expression of CD133. CD133high DTPs were characterized by the higher expression of stem cell related markers. On the other hand, CD133low DTPs were characterized by higher expression of the cellular senescence associated proteins. Rapamycin is reported to suppress SASP. Gefitnib and rapamycin effectively suppressed the emergence of CD133high DTPs. We found CD133low DTPs showed higher glucose metabolic state, consistent with the previous reports that show senescence-associated metabolic reprogramming is induced by anticancer therapies. We targeted this metabolic change using a glucose transporter inhibitor (phloretin) and a glycolytic blocker (2DG). These agents successfully suppressed the emergence of CD133high DTPs. Finally, withaferin A could inhibit the emergence of CD133high DTPs.
In conclusion, we found that the EGFR-TKI-induced DTPs in EGFR mutated NSCLC cells were composed of at least two distinct cell populations: CD133high DTPs with stem-like characters and CD133low DTPs with cellular senescent characters. CD133low DTPs supported CD133high DTPs via SASP and blocking SASP effectively suppressed the emergence of CD133high DTPs. Furthermore, our results suggest that the glucose metabolism targeting therapies and withaferin A can be new therapeutic candidates to ablate DTPs.
Citation Format: Kei Kunimasa, Tatsuya Nagano, Yohei Shimono, Shuntaro Tokunaga, Daisuke Tamura, Motoko Tachihara, Kazuyuki Kobayashi, Yoshihiro Nishimura. The glucose metabolism targeting therapies and withaferin A eliminate epidermal growth factor tyrosine kinase inhibitor-induced drug-tolerant persisters in non-small lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 748. doi:10.1158/1538-7445.AM2015-748