Abstract 3872: Targeting EGFR/HER2 signaling pathway by a dual receptor tyrosine kinase inhibitor BIBW2992 for radiosensitization in murine urothelial carcinoma
Authors: Yeh Chih-Hsien , Cheng Jason Chia-Hsien , Tsai Yu-Chieh , Tuan Tsung Fan , Ho Pei-Yin , Pu Yeong-Shiau , Cheng Ann-Lii 
Published: August 2012

Abstract
Background: Combined chemotherapy and radiotherapy (CCRT) has been successful in phase II trials with selected patients for preserving bladder after transurethral resection of bladder tumor. However, the obstacles remain in the unsatisfactory effect of CCRT for unresectable disease of bladder or upper urinary tract. The purpose of this study was to determine whether the EGFR/HER2 targeting agent combined with radiotherapy has the potential of radiosensitization in urothelial carcinoma. Methods: BIBW2992, an irreversible EGFR/HER2 inhibitor, was selected as the target agent to combine with radiotherapy. Murine bladder tumor cell line, MBT-2, was used to evaluate the in vitro and in vivo synergism of combining BIBW2992 with irradiation. Colony formation assay was conducted to evaluate the synergistic effect of BIBW2992 and radiation on colony number analyzed using CompuSyn software. The related signaling protein expressions were investigated by Western blot assay. Flow cytometry analyzed the cell cycle changes. Ectopic xenografts were established by subcutaneous injection of MBT-2 cell (2× 106) into the right hind leg of C3H mouse. As the tumors became established (mean starting tumor volume= 200 mm3), the mice were randomized into 4 groups (n = 4) that received BIBW2992 and/or radiotherapy (10 mg/kg/day for 7 days and/or radiotherapy of 15 Gy on day 4). The tumor size was measured weekly using calipers and the volumes were calculated. Positron emission tomography (PET) was obtained on day 7 as the imaging modality to evaluate the early treatment response. Results: BIBW2992 (0-1000 nM) synergistically sensitized MBT-2 cells to radiation (0-10 Gy) in clonogenic cell survivals. Based on the rapid increase of phosphorylation of Her2 and EGFR shortly (2-6 hrs) after irradiation, pre-treatment of BIBW2992 on irradiated MBT-2 cells inhibited Her2 and EGFR phosphorylation dramatically. As compared to either treatment alone, combined BIBW2992 and irradiation increased the cleavage form of PARP. It was compatible with the flow cytometry finding of significantly increased cells in subG1 phase, indicating the synergistic induction of cell apoptosis. Besides, the combination of BIBW2992 and irradiation enhanced p-γH2AX (double-strand DNA damage) expression compared with either treatment alone. The inhibitory effect on ectopic tumor metabolism was early observed on PET after combined BIBW2992 and radiotherapy, as compared to either modality alone or sham treatment. The tumor growth delay between irradiation and combined treatment was 8.5 days. Conclusions: BIBW2992 is an orally bioavailable dual EGFR/HER2 inhibitor with therapeutic value as a radiosensitizer of murine bladder cancer. The synergism likely associates with the enhanced effect on radiation induced DNA damage, and leads to the increased cell apoptosis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3872. doi:1538-7445.AM2012-3872