Role of c-Met and EGFR synergism and identification of targets causing resistance to their inhibitors
thesisposted on 13.12.2012, 00:00 authored by Jason T. Fong
While the use of specific Tyrosine Kinase Inhibitors (TKIs) against epidermal growth factor receptor (EGFR) and Hepatocyte Growth Factor Receptor (c-Met) in Non-Small Cell Lung Cancer (NSCLC) are very effective at increasing patient progression free survival (PFS), their efficacy is limited by the subsequent development of resistance and tumor recurrence. Therefore, to establish the mechanism of TKI resistance in NSCLC, two cell lines (H2170 and H358) were made resistant to c-Met (SU11274) and EGFR (erlotinib) inhibitors (4-6-fold and 11-22-fold increase in IC50 of SU11274 and erlotinib, respectively). Rather than focusing on secondary mutations, several of which are currently established, we have studied alternative signaling pathways that may be essential in the development of acquired resistance. We have developed two models of c-Met/EGFR TKI resistant NSCLC cell lines. In our resistant cell lines, one model displays constitutive phosphorylation of EGFR and increased phosphorylation of c-Met, while the other model exhibits decreased phosphorylation of both EGFR and c-Met. Interestingly, in both models we find a 2-4-fold increased activation of mTOR (mammalian target of rapamycin), its substrates 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) and S6 Kinase (Ribosomal protein S6 kinase beta-1) and increased activation of β-catenin (beta catenin), which plays a role in the Wnt signaling pathway. To confirm the role of mTOR and Wnt pathways in resistance and to explore options to break this resistance, parental and resistant cell lines were treated with an mTOR inhibitor, everolimus, and a Wnt inhibitor, XAV939, in addition to c-Met/EGFR TKIs SU11274 and erlotinib. When everolimus was added to the c-Met/EGFR TKI combination treatment, resistant cell lines were found to be significantly (p<.001) more susceptible in comparison to parental cells. However in response to XAV939, parental cell lines displayed no sensitivity, while resistant lines displayed a significant decrease in viability when used alone (p<.001) or when added to the c-Met/EGFR TKI combination treatment (p<.001). These results suggest that targeting the mTOR and Wnt pathways may be viable options for NSCLC patients with acquired resistance to EGFR/c-Met inhibitors. Furthermore, results from DNA sequencing of EGFR exons 18-21 (kinase domain and sites of activating EGFR mutations and secondary resistance mutations) in our resistant cells confirm no T790M or D761Y secondary mutations that commonly cause erlotinib-resistance. Hence, our models would establish a mechanism of inhibitor resistance that is separate from secondary resistance mutations. In addition to mTOR and the Wnt signaling pathways, we also identified several proteins exhibiting modulated expression in resistant cells. These include downregulation of pro-apoptotic proteins such as PDCD6 (Programmed cell death 6) and AIF1 (Apoptosis-inducing factor), downregulation of total β-catenin, which would increase tumorigenicity, and upregulation of translation proteins such as HMGB2 (High-mobility group protein B2) and TOM34 (Translocase of outer mitochondrial membrane 34). While unconfirmed, these proteins may play a role in EGFR/c-Met TKI resistance and warrant further study. Overall our studies find modulations in the Wnt and mTOR pathways after resistance is acquired to EGFR and c-Met TKIs in NSCLC, and these modulations could be used as targets to overcome c-Met/EGFR TKI resistance in NSCLC patients.