Glucose metabolism-targeted therapy and withaferin A are effective for epidermal growth factor receptor tyrosine kinase inhibitor-induced drug-tolerant persisters

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. In this study, we investigated the features of epidermal growth factor receptor–tyrosine kinase inhibitor-induced DTPs and explored a new treatment strategy to overcome the emergence of these DTPs. 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 months. We analyzed the mRNA expression of the stem cell-related markers by quantitative RT-PCR and the expression of the cellular senescence-associated proteins. 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 glucose metabolism targeting therapies and a stem-like cell targeting drug, withaferin A. Drug-tolerant persisters were composed of at least two types of cells, one with the properties of cancer stem-like cells (CSCs) and the other with the properties of therapy-induced senescent (TIS) cells. The CD133^high cell population had CSC properties and the CD133^low cell population had TIS properties. The CD133^low cell population containing TIS cells showed a senescence-associated secretory phenotype that supported the emergence of the CD133^high cell population containing CSCs. Glucose metabolism inhibitors effectively eliminated the CD133^low cell population. Withaferin A effectively eliminated the CD133^high cell population. The combination of phloretin and withaferin A effectively suppressed gefitinib-resistant tumor growth.