Abstract B47: Modeling epithelial plasticity-induced erlotinib resistance in non-small cell lung cancer
Authors: Hailun Wang, Jessica Cades, Zachary Yochum, Katriana Nugent, Charles Rudin, Timothy Burns, Phuoc Tran
Journal: Cancer Research (2018)

Abstract Background: Advanced non-small lung cancer (NSCLC) patients with EGFR mutations initially respond to treatment with the EGFR-targeted tyrosine kinase inhibitors (TKIs) such as erlotinib, but will invariably acquire resistance with progression of disease within 10–16 months. Mechanisms of EGFR TKIs resistance include second-site EGFR mutations (&amp;gt;50%), MET amplfication (5–10%), and mutations in PIK3CA (&amp;lt;5%). A much less well-defined mechanism of TKIs resistance are lung cancer cells that have appeared to undergone a morphologic change to a more mesenchymal phenotype (~5-30% of cases) or epithelial-mesenchymal transition (EMT). Whether epithelial plasticity programs like EMT are directly causative for erlotinib-acquired resistance has not been shown definitively in vivo. Methods: To examine the relationship between epithelial plasticity and erlotinib resistance in EGFR mutant lung cancers, we created an inducible CCSP-rtTA/tetO-EgfrL858R/Twist1 (CET) transgenic mice (Twist1 is a key regulator of EMT). We utilized the tetracycline-inducible gene expression system to control EgfrL858R/Twist1 gene expression in the lung by providing or withdrawing doxycycline to the mice. The mice were treated for 3 weeks with erlotinib and scanned by CT each week, and survival of the mice was also recorded. The tumor tissues were collected 1 week and 3 weeks after the start of treatment and used for immunohistochemical staining for H&amp;E, Ki67, and cleaved caspase 3 (CC3). To investigate the mechanisms of treatment resistance, mice lung tumor tissues were collected after 3 weeks' treatment with erlotinib, and tumor lysates were used for RTK signaling antibody array analysis. Results: The lung tumors in the CET mice were more anaplastic than those seen in CE (CCSP-rtTA/tetO-EgfrL858R) mice, with larger, more irregular nuclei. This is consistent with reports of cancers expressing Twist1 being more aggressive. Both genotypes display diffuse hyperplasia instead of discrete tumors, which is similar to the disease seen in EGFR mutant NSCLC in humans. After 3 weeks of erlotinib treatment, a majority of CE mice demonstrated complete and partial responses as well as stable disease. Conversely, over half of the CET mice had tumor progression over the three weeks of treatment. CET mice median overall survival time, from the beginning of treatment, was 6.8 weeks, while CE mice lived a median of 8.7 weeks (p&amp;lt;0.01, Mantel–Cox test). When erlotinib-treated tissue was stained for Ki-67, there was no significant difference between proliferation levels in CE and CET mice. Apoptosis assessed through staining for CC3 showed apoptosis levels were decreased in CET erlotinib-treated compared to the CE erlotinib-treated lung tumors. RTK signaling antibody array analysis showed several signaling pathways that were found to be dysregulated between CE and CET lung tumors, with the highest signal observed from the Src kinase pathway. Targeting the Src pathway with 30 mg/kg of dasatinib in combination with erlotinib in CET mice demonstrated significant reduction in tumor burden and increased survival in comparison to the other single-drug groups (p&amp;lt;0.05, Mantel–Cox test). Conclusions: Expression of Twist1 induces resistance to erlotinib in EGFR mutant NSCLC, partially through activation of the Src signaling pathway. Combining erlotinib treatment with the Src inhibitor, dasatinib, in the CET mice leads to tumor regression, prolonged overall survival, as well as decreased proliferation and downstream signaling. Therefore, this combined treatment can potentially overcome Twist1-induced erlotinib resistance in NSCLC. Citation Format: Hailun Wang, Jessica Cades, Zachary Yochum, Katriana Nugent, Charles Rudin, Timothy Burns, Phuoc Tran. Modeling epithelial plasticity-induced erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B47.