Lung cancer is the leading cause of cancer-related deaths in the world. Non-small cell lung cancer (NSCLC) accounts for ~85% of the cases. NSCLC patients frequently harbor causal gene mutations. Epidermal Growth Factor Receptor (EGFR), an NSCLC causal gene, is mutated in 10-35% of NSCLC patients. Patients with EGFR activating mutation are treated with a tyrosine-kinase inhibitor, Erlotinib, specifically targeting EGFR. However, most patients develop Erlotinib resistance within a year. Although multiple mechanisms are involved in the development of Erlotinib resistance, the role of microRNAs in mediating Erlotinib resistance is largely unexplored. MicroRNAs (miRNAs) are small non-coding RNAs that regulate normal cellular physiology. In cancers, miRNAs are severely dysregulated, contributing to multiple cancer processes, including drug response. Despite the involvement of miRNAs in cancer, their direct role as drivers of drug resistance remain understudied, therefore, there is a critical need to understand the role of miRNAs in inducing Erlotinib resistance.
To this end, we propose an unbiased two-prong approach to identify the miRNAs that drive the development of Erlotinib resistance. Therefore, miRNAs are either being (i) overexpressed, or (ii) silenced in Erlotinib sensitive cells with the hypothesis that perturbed miRNA levels will drive Erlotinib resistance. Erlotinib sensitive NSCLC cell lines, EKVX and H322M were identified from the NCI-60 Developmental Therapeutics Project and their Erlotinib dose response curves were established. To perform the miRNA overexpression screen, EKVX and H322M cells stably expressing renilla and firefly luciferase genes were generated, which will be used to monitor cell number and transfection efficiency, respectively. The luciferase-expressing cells will be transfected with a library of 2,019 individually arrayed human miRNAs and cell growth in the presence of Erlotinib will be monitored. The second prong of the study will identify miRNAs, that when lost, confer Erlotinib resistance. 400-fold coverage of small guide RNA (sgRNA) library of the CRISPR-Cas9 system was transduced in Cas9 overexpressing EKVX cells, to knockout ~21,000 human encoded genes (~1800 miRNA genes). Cells are being cultured in the presence of 75% or 90% growth inhibitory concentrations (GI75 or GI90) of Erlotinib so that only cells with sgRNAs against genes critical for Erlotinib response, survive and grow due to acquired resistance. DNA from these cells will be harvested, the sgRNAs sequenced, and compared to the sgRNAs present in Erlotinib-untreated cells.
Successful completion of this project will identify miRNAs that drive Erlotinib resistance and may contribute towards development of miRNA therapeutics to enhance Erlotinib sensitivity of NSCLC tumors.
Citation Format: Arpita S. Pal, Alejandra Agredo, Andrea L. Kasinski. Aberrantly expressed microRNAs drive the development of acquired Erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2017-3142