Targeting epidermal growth factor receptor/human epidermal growth factor receptor 2 signalling pathway by a dual receptor tyrosine kinase inhibitor afatinib for radiosensitisation in murine bladder carcinoma

Abstract

Given the promising control of bladder cancer achieved by combined chemotherapy/radiotherapy with selective transurethral resection, obstacles remain to the treatment of unresectable bladder cancer. The aim of this study was to determine whether targeting epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) can radiosensitise a murine bladder tumour (MBT-2) cell line. Cell survival, expression of signal proteins and cell cycle changes in MBT-2 cells treated in vitro and in vivo with afatinib, an irreversible EGFR/HER2 inhibitor, plus radiotherapy were investigated by colony formation assay, Western blot assay and flow cytometry, respectively. Ectopic xenografts were established by subcutaneous injection of MBT-2 cells in C3H/HeN mice. Mice were randomised into 4 groups to receive afatinib (10mg/kg/day on day 1-7) and/or radiotherapy (15Gy on day 4). Positron emission tomography (PET) on day 8 was used to evaluate the early treatment response. Afatinib (200-1000nM) increased cell killing by radiation (0-10Gy). Pre-treatment of irradiated cells with afatinib inhibited radiation-activated HER2 and EGFR phosphorylation. As compared to either treatment alone, the combination increased the level of the cleavage form of poly (ADP-ribose) polymerase, the expression of phospho-γH2AX and the percentage of cells in subG1 phase (indicating enhanced induction of apoptosis), and decreased tumour metabolism and inhibited tumour growth by 64%. Afatinib has therapeutic value as a radiosensitiser of murine bladder cancer cells. The synergism between afatinib and radiation likely enhances DNA damage, leading to increased cell apoptosis.