Abstract 5653: Novel targets mediating resistance to EGFR/c-Met tyrosine kinase inhibitors in NSCLC.
Authors: Jacobs Ryan , Fong Jason , Moravec David , Botting Greg , Bomgarden Ryan , Rogers John , Viner Rosa , Blank Michael , Puri Neelu 
Published: August 2014

Abstract
Combination therapy of c-Met/EGFR TKIs against c-Met/EGFR, while initially effective, has been shown to be susceptible to acquired resistance. To establish a mechanism of TKI resistance, the NSCLC model cell lines H2170 and H358 were made resistant to the c-Met/EGFR TKIs SU11274, erlotinib, or a combination by exposing the cells to progressively increasing concentrations of inhibitors. Phospho-Met (Tyr1003) was found to be downregulated 4- and 1.5- fold in H2170 and H358 cells, respectively. Phospho-EGFR was found to be upregulated in H2170 cells and downregulated in H358 cells. Interestingly, no significant differences were observed in c-Met/EGFR downstream signaling proteins, indicating that alternative signaling pathways such as the canonical Wnt pathway may confer TKI resistance. To determine differences in Wnt signaling between parental and resistant cell lines, immunoblotting was performed. In the H2170 erlotinib resistant (H2170-ER) cell line, a 5- and 6-fold increase in phospho-LRP5/6 as well as a 2- and 3-fold increase in total-LRP5/6 was observed in the presence or absence of erlotinib, respectively. Furthermore, H2170-ER cells exhibited a 3- and 16-fold increase in NKD2 as well as a 2.5- and 6-fold increase in Wnt3A in the presence or absence of EGF, respectively. A 2- to 2.5-fold increase was also observed in DVL2/3 in the absence of EGF. However, the Wnt pathway did not seem to have a significant role in H358 cells. Since EGFR and Wnt signaling are known to exhibit crosstalk, activation of the Wnt pathway may stimulate activation of EGFR causing resistance. This is suggested as upregulation of proteins involved in the Wnt pathway was seen only in the H2170 cell line. These results were confirmed by treating H2170 cells with the Wnt inhibitor XAV939. MTT analysis indicated that parental cells showed no significant response to XAV939, however, resistant cell lines showed a 2-fold decrease in viability when compared to a c-Met/EGFR TKI combination treatment. We propose a new treatment modality with Wnt inhibitors which have not yet entered clinical testing.
To identify new targets for NSCLC, stable isotope labeling by heavy/light amino acids in cell culture (SILAC) coupled with mass spectroscopy was used. Analysis revealed more than 200 proteins modulated greater than 1.5-fold, six of which have significant potential to influence cell growth and survival based on their biological roles in NSCLC. These proteins include total-β-catenin, TPR, syntenin, HMGB2, TOM34 and AIF. We are currently using tandem mass tagging (TMT) to further investigate additional modulated proteins. Proteins of interest identified by this method include TPD52, erbB2, MESDC2, PIN1, PEA15 and NRAS. Proteins confirmed to be upregulated/activated in resistant cell lines will be further investigated as targets which induce drug resistance in NSCLC.
Citation Format: Ryan Jacobs, Jason Fong, David Moravec, Greg Botting, Ryan Bomgarden, John Rogers, Rosa Viner, Michael Blank, Neelu Puri. Novel targets mediating resistance to EGFR/c-Met tyrosine kinase inhibitors in NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5653. doi:10.1158/1538-7445.AM2013-5653
Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.