A Reversible Shift of Driver Dependence from EGFR to Notch1 in Non-Small Cell Lung Cancer as a Cause of Resistance to Tyrosine Kinase Inhibitors

SIMPLE SUMMARY: Notch1 and EGFR are two surface receptors activating different cellular processes in cancer cells. EGFR, harboring activating mutations, drives unlimited cell proliferation in non-small cell lung cancer (NSCLC), and treatment of tumors with tyrosine kinase inhibitors (TKIs) results in growth arrest and cell death. On the other hand, Notch1 plays a key role in the loss of epithelial characteristics and the concomitant acquisition of mesenchymal traits and invasive potential of tumor cells. Interestingly, high levels of Notch1 are associated with the resistance to EGFR TKIs. Here, we evaluated the mechanisms by which Notch1 causes resistance of NSCLC to EGFR TKIs, and provided evidence that high levels of activated Notch1 induce a decrease of EGFR, by modulating the activity of the promoter of the EGFR gene. Therefore, blocking the Notch1 pathway in tumors treated with EGFR inhibitors would prevent EGFR downregulation, maintaining drug sensitivity. ABSTRACT: Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the EGFR promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.