Abstract 1464: Afatinib, an EGFR/Her-2 dual inhibitor, effectively radiosensitizes bladder cancer cells

Abstract

Over-expression of epidermal growth factor receptor (EGFR) had been correlated with poor prognosis in bladder cancer, but clinical trials using EGFR inhibitors alone in the treatment of bladder cancer did not show definite improvement. On the other hand, radiotherapy is the key element of bladder-preserving protocol for patients with muscle-invasive bladder cancer, and several preclinical data reported the advantage of combing radiation and tyrosine kinase inhibitor against EGFR. It is known that aberrantly activated signal transduction pathways can influence radiosensitivity of cancer cells. Herein, we attempt to demonstrate whether radiation can activate signal transduction pathways in bladder cancer cells, and whether the combined inhibition of such pathways can further enhance radiosensitivity. We used a RTK signaling antibody array to investigate the relative levels of phosphorylated signaling proteins. We detected that not only EGFR but also HER-2 and Akt (Ser473) were activated in T24 human bladder cancer cells after irradiation, and the phenomenon was more prominent in 10Gy dose than in 2.5Gy dose. The increased phosphorylation of EGFR and Her-2 was confirmed by Western blot. Then we performed colony formation assay of T24 and NTUB1 human bladder cancer cell lines for 7 days after treatment combining irradiation and erlotinib (an EGFR inhibitor), lapatinib (a reversible EGFR/Her-2 dual inhibitor) or afatinib (an irreversible EGFR/Her-2 dual inhibitor) in different doses. The combination index (CI) analysis revealed synergy between radiation (2.5Gy and 10 Gy) and afatinib in various doses. For T24 cells with afatinib 100nM, the CI was 0.97 in 2.5Gy and 0.66 in 10Gy. For NTUB1 cells with afatinib 100nM, the CI was 0.92 in 2.5Gy and 0.80 in 10Gy. In contrast, erlotinib or lapatinib showed only minor influence to enhance radiation effect in human bladder cancer cells. Using flow cytometry, the relative distribution of cells among various cell cycle phases were determined in cell lines treated with either DMSO, radiation 2.5Gy, afatinib 200nM, or the combination of radiation and afatinib. When compared with radiation alone, treatment combining radiation and afatinib resulted in accumulation of cells in sub-G1 phase but decrease in G2-M phase. Western blot showed the expression of cleaved poly (ADP-ribose) polymerase (PARP) was increased in the combination group when compared with radiation or afatinib alone. The results implicated that the adding of afatinib might enhance radiation effect by increasing apoptosis in bladder cancer cells. Our data clearly show that both EGFR and Her-2 signaling can be activated after radiation in human bladder cancer cells. Afatinib, an EGFR/Her-2 dual inhibitor, effectively radiosensitizes the bladder cancer cells by enhancing apoptosis. The result provides a basis for future in vivo and human studies. 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 1464. doi:1538-7445.AM2012-1464

A comprehensive review of the preclinical efficacy profile of the ErbB family blocker afatinib in cancer

“…Radiation treatment of bladder cancer cells dose-dependently induced phosphorylation of EGFR, HER2 and Akt; in a colony formation assay, afatinib showed synergism with radiation in such cells, indicating possible radiosensitization (Tasi et al 2012). A minor radiosensitization by afatinib had also been observed in HNSCC cell lines (Aravindan et al 2011; Schütze et al 2007).…”

“…Growth inhibition by afatinib was also observed in a panel of pancreatic cancer cell lines (Ioannou et al 2011 , 2013 ). While being tested on a smaller number of cell lines, afatinib exhibited growth inhibition in lines representing bladder cancer (Greulich et al 2012 ; Quesnelle and Grandis 2011 ; Tasi et al 2012 ), endometrial cancer (Greulich et al 2012 ), basal cell carcinoma (Eberl et al 2012 ) and epidermal cell carcinoma (Cha et al 2012 ). In melanoma cells with RAS or BRAF mutations, afatinib was found to be ineffective but gained efficacy when used in combination with other drugs (see below) (Held et al 2013 ).…”

“…In HNSCC cell lines, inhibition of the transcription factor ESX (epithelial-restricted with serine box) by siRNA or by a low molecular weight inhibitor reduced EGFR and HER2 promoter activity; the growth-inhibiting effects of these approaches were enhanced by afatinib (Zhang et al 2013 ). Radiation treatment of bladder cancer cells dose-dependently induced phosphorylation of EGFR, HER2 and Akt; in a colony formation assay, afatinib showed synergism with radiation in such cells, indicating possible radiosensitization (Tasi et al 2012 ). A minor radiosensitization by afatinib had also been observed in HNSCC cell lines (Aravindan et al 2011 ; Schütze et al 2007 ).…”