Abstract 850: Activation of autophagy by erlotinib protects NSCLC cells with wild-type EGFR from erlotinib cytotoxicity
Authors: Schwartz Edward L. , Ling Yi-He , Zou Yiyu , Piperdi Bilal , Perez-Soler Roman 
Published: August 2012

Erlotinib is an orally available EGFR tyrosine kinase inhibitor which has significant clinical activity in previously treated patients with non-small cell lung cancer (NSCLC), as well as in maintenance therapy after frontline chemotherapy. Although it has shown efficacy in NSCLC with both wild-type and mutant EGFR, it is much more effective in tumors with activating EGFR mutations than in those tumors which have wild-type EGFR. This study identifies autophagy induction as a potential mechanism for the relative resistance of wild-type EGFR NSCLC cells to erlotinib. Autophagy is a tightly regulated lysosomal self-digestion process that may promote cell survival and/or be involved in type II cell death. The potential role of autophagy in erlotinib-induced cytotoxicity has not been well defined. We used four wild-type EGFR NSCLC cell lines, which are either erlotinib-sensitive (H322 and H358) or erlotinib-resistant (H460 and A549), to determine if treatment with erlotinib induces autophagy. We then assessed the effect of disrupting autophagy by chemical and genetic approaches on erlotinib-induced cytotoxicity. Erlotinib at a clinically relevant concentration (2 μM) induced autophagy, as shown by increased MDC staining, increased conversion of LC3-I (the cytosolic form) to LC3-II (the lapidated form associated with autophagosome), and the formation of autophagic vacuoles, in all tested cell lines. Autophagy induction by erlotinib was greater in resistant cells than in sensitive cells, suggesting that autophagy is a cytoprotective mechanism. This was confirmed with the use of autophagy inhibitors chloroquine (CQ), 3-methyladenine, and bafilomycin A1, which synergistically increased erlotinib cytotoxicity. Similar enhancement was seen with knockdown of Atg-5 and Beclin-1 gene expression. Cotreatment with CQ did not alter erlotinib-induced EGFR signaling inhibition or G1-phase arrest, but markedly activated mitochondrial-mediated apoptosis. Furthermore, disrupting autophagy by CQ or other inhibitors enhanced erlotinib-induced upregulation of BIM expression, and knockdown of BIM expression by siRNA attenuated the cytotoxicity induced by the combination. In addition, inhibition of autophagy by CQ enhanced erlotinib-induced ROS generation and activation of p38 but not JNK signaling. Inhibition of ROS generation by N-acetyl-cysteine, or inhibition of p38 by SB202190 attenuated the cytotoxic effect of the combination, suggesting that ROS generation and p38 activation are potential mediators of the synergism observed. The ability to adapt to anti-EGFR therapy by triggering autophagy may be a major determinant of resistance to erlotinib in NSCLC tumors with wild-type EGFR. Inhibition of autophagy by CQ or other inhibitors represents a novel strategy to potentiate erlotinib efficacy in NSCLC tumors with wild-type EGFR. Supported by NIH/NCI grants 113360, 132783, and 154755.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 850. doi:1538-7445.AM2012-850