Abstract 1608: Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells

Abstract

Abstract Tyrosine kinase inhibitors (TKIs) against c-MET and EGFR have positive therapeutic effects in a subset of Non-Small Cell Lung Cancer (NSCLC) patients; however, their clinical efficacies are limited due to the development of TKI resistance. To determine mechanisms of TKI resistance, two NSCLC model cell lines H2170 and H358 were made resistant to the c-Met/EGFR TKIs SU11274, erlotinib or a combination of both by exposing the cells to progressively increasing concentrations of inhibitor. Initial characterization of these cell lines using phospho-specific antibodies showed that phospho-c-Met was found to be downregulated 4- and 1.5- fold in SU11274-resistant (SR) H2170 and SR H358 cells, respectively. Phospho-EGFR was found to be constitutively autophosphorylated (upregulated 19-fold) in erlotinib-resistant (ER) H2170 cells, but downregulated 6-fold in ER H358 cells. Interestingly, c-Met/EGFR downstream signaling proteins p-mTOR, p-p70S6K and p-ERK were found to be 2-4-fold upregulated, 2-fold upregulated and 2-5-fold upregulated, respectively, in ER H2170 and H358 cells. Phospho-p70S6K was also found to be upregulated 20-fold in SR H2170 cells. These results indicate that alternative signaling pathways may confer TKI resistance in these cell lines. To identify which signaling pathways may be responsible for TKI resistance, H358 parental and resistant cell lines were analyzed by mass spectrometry (MS). Differences in protein expression in cell lines treated with and without EGF and/or erlotinib were measured using higher multiplexing Thermo Scientific™ Tandem Mass Tag™ (TMT™) reagents. More than 1500 protein groups were identified and quantified using a Thermo Scientific™ Orbitrap™ Fusion™ platform with HCD-MS2 and MS3 fragmentation methods. Protein expression in ER H358 cells showed a marked increase in expression of DNA replication proteins, transcription factors, apoptosis inhibitors and some Ras-associated signal transduction pathways. To further investigate the mechanism of TKI resistance, phospho-peptide enrichment was also used in combination with TMT quantitation to profile changes in protein phosphorylation state. Overall, the combination of phospho-peptide enrichment coupled with higher multiplexing TMT and high resolution MS analysis enabled deeper global profiling of TKI resistance signaling pathways compared to previous studies without enrichment. Citation Format: Ryan D. Bomgarden, Ryan Jacobs, Jason Fong, David Moravec, Gregory M. Botting, Michael Blank, Rosa I. Viner, John C. Rogers, Neelu Puri. Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1608. doi:10.1158/1538-7445.AM2014-1608