Blockade of EGFR has been proved useful in enhancing the effect of radiotherapy, but the advantages of new-generation EGFR tyrosine kinase inhibitors (TKI) in radiosensitization are not well known. We used two human bladder cancer cells with wild-type EGFR to study the synergism between irradiation and afatinib (an EGFR/HER2 dual kinase inhibitor) or erlotinib (an EGFR kinase inhibitor). Here, we showed that afatinib has better radiosensitizing effect than erlotinib in increasing cancer cell killing, the percentage of apoptotic cells, and DNA damage. Afatinib is also superior to erlotinib in combining radiation to decrease tumor size, inhibit glucose metabolism, and enhance apoptotic proteins in vivo. Finally, erlotinib suppressed cell growth and induced more DNA damage in bladder cancer cells transfected with HER2 shRNA, but not in control vector-treated cells. In conclusion, concomitant blockade of radiation-activated EGFR and HER2 signaling by a new-generation EGFR TKI better inhibits the growth of bladder cancer cells both in vitro and in vivo. The absence of radiosensitization by EGFR inhibition alone and the greater radiosensitizing effect of EGFR inhibitor in HER2 knocked down cells suggest the synergism between HER2 and EGFR in determining radiosensitivity. The regained radiosensitizing activity of erlotinib implies that with proper HER2 inhibition, EGFR tyrosine kinase is still a potential target to enhance radiotherapy effect in these seemingly unresponsive bladder cancer cells.

No 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

<p>Western blotting of T24 cell lysate precipitated with EGFR antibody</p>

<p>The radiosensitizing effect of erlotinib in HER2 knocked-down NTUB1 cells also emphasizes the synergism between EGFR and HER2 in determining radiosensitivity</p>