Egfr/Dna Targeting Molecule Potentiates Radiation Response In A Murine Breast Cancer Model

Epidermal growth factor receptor EGFR is often overexpressed in human malignancies and it is associated with the activation of the AKT pathway leading to anti-apoptotic effects. This also includes the lack of sensitivity of some tumors to several existing cytotoxic therapies such as radio and chemotherapies. Therefore, it is of great importance to develop novel anti-cancer drugs effective against EGFR-overexpressing cancer cells. ZRBA1 is a binary targeting molecule, which not only blocks EGFR at the TK domain but also induces DNA breaks. However, the binary property of this molecule can be limited by DNA repair mechanisms in cancer cells. Therefore to further increase the efficacy of treatment we combined ZRBA1 with ionizing radiation. Using MDA-MB-468 breast cancer and 4T1 mouse mammary cancer cells, we performed colony forming assay to determine the radio-modulating effect of ZRBA1. The combined treatment was tested at multiple schedules (before radiation, concurrent and after radiation). Using Western blot analysis and flow cytometry, we have also evaluated the effect of each of the treatments alone and in combination on the level of apoptosis and cell cycle arrest. Finally, we have confirmed our in vitro data in a syngeneic breast cancer model by performing a tumor growth delay assay. Our colony forming assay showed a radiation dose enhancement factor (DEF) of 1.5 when ZRBA1 (22 uM) is combined with radiation. Moreover, our flow cytometry analysis showed a significant increase in G2/M cell cycle arrest when ZRBA1 and radiation are combined (p < 0.046). Western blot results demonstrated the highest level of cleaved PARP when radiation and ZRBA1 are combined as a late response. Importantly, our in vivo tumor growth delay assay strongly suggests that ZRBA1 potentiates the radiation response in our 4T1 breast cancer model. In addition, tumor growth delay was nearly doubled in combined treated group compared to irradiated only group (26 days in irradiated group vs 47 days in combined treatment group) with a statistical significance of 0.0456 on day 31 post-treatment (radiation+ ZRBA1 vs radiation alone). Our results have shown that ZRBA1 potentiates the radiation response in a breast cancer model In vitro and In vivo. The higher potency of this combination is due to increased cell cycle arrest, and induced apoptosis.