5.2.1 Significance
We demonstrated in this study the in vitro and in vivo radiosensitising effect of afatinib
(an irreversible EGFR/HER2 dual inhibitor) on MBT-2 murine bladder cancer model. The
synergism with irradiation was likely mediated through enhancement of radiation-induced
DNA damage and apoptosis. We also showed that afatinib suppressed radiation-activated
EGFR and HER2 signaling. To our knowledge, the present study is the first to report that the
combined blockade of EGFR and other Erb-B family receptor tyrosine kinases enhances the
radiosensitivity of bladder cancer cells.
EGFR specific tyrosine kinase inhibitor like gefitinib (Colquhoun et al., 2007;
Dominguez-Escrig et al., 2004; Maddineni, Sangar, Hendry, Margison, & Clarke, 2005) and
erlotinib (Colquhoun & Mellon, 2004) has been reported to enhance the radiosensitivity of
bladder cancer cells. Compared to the gefitinib, the radiosensitizing effect of afatinib in our
study was superior, indicating the critical role of HER2 signaling pathway in determining the
radiosensitivity of bladder cancer cells. Similarly, monoclonal antibody to HER2/neu receptor
has been shown to modulate the repair of radiation-induced DNA damage and enhance the
radiosensitivity of breast and esophageal cancer cells overexpressing HER2 (Pietras et al.,
1999; Sato et al., 2005). Furthermore, the Radiation Therapy Oncology Group trial on
bladder preservation demonstrated a significant association of HER2 staining of bladder
tumor tissue with complete response after tri-modality treatment (Chakravarti et al., 2005).
Lapatinib, a reversible EGFR/HER2 dual inhibitor, had been shown to have a
radiosensitizing effect in certain breast cancer cells (Sambade, Camp, Kimple, Sartor, &
Shields, 2009; Sambade et al., 2010) and patients (Abboud, Saghir, Salame, & Geara, 2010;
Harrington et al., 2009). However, its effect was unsatisfactory in our pilot study. Compared
to lapatinib, afatinib (an irreversible inhibitor of EGFR and HER2) was the more potent
radiosensitizer (Li et al., 2008). One large randomized trial recently reported the clinical
benefit of afatinib for patients with non-small-cell lung cancer unresponsive to EGFR
inhibitors or chemotherapy (Miller et al., 2012). Furthermore, afatinib had been shown to
enhance responses to an EGFR specific monoclonal antibody in bladder cancer cells resistant
to this treatment (Quesnelle & Grandis, 2011).
Schütze et al. investigated radiosensitization by afatinib of human squamous cell
carcinoma cells only marginally affected by radiation (Abraham, Pagano, Gomella, & Baffa,
2007). The design of the present study differed from their design in the in vivo dosing
schedule of afatinib (10 mg/kg for 7 days in our study versus 20 mg/kg until reaching a tumor
diameter of 15 mm in their study) and radiotherapy (15 Gy on day 4 in this study versus 20
Gy after the last afatinib dose in their study). Moreover, we measured not only the volume of
the ectopic tumor but also its glucose metabolism by PET, as well as the corresponding
expression of EGFR and HER2 immunohistochemically. We additionally showed that
afatinib enhanced radiation-induced DNA damage and apoptosis. The molecular response to
irradiation combined with afatinib might vary between different malignant cells (Krause,
Gurtner, Deuse, & Baumann, 2009; Toulany, Dittmann, Baumann, & Rodemann, 2005) and
this variation might account for discrepancies between our study and the one by Schutze et al.
MBT-2 cells are poorly differentiated urothelial carcinoma and derived from a
carcinogen-induced bladder tumor in C3H/He mice. The tumor resembles its human
counterpart both grossly and histologically (Horiguchi et al., 2008). The advantage of the
MBT-2 murine bladder cancer model in this study is the immune-competent system for the in
vivo data with the integrated physiological response after irradiation (Blomgren, Edsmyr, von
Stedingk, & Wasserman, 1986) and inhibition of Erb-B family receptor tyrosine kinases
(Hamilton et al., 2012; Y. Yan et al., 2006).
In this study, radiosensitization by afatinib was mediated through enhanced
irradiation-induced DNA damage as indicated by the increase in γ-H2AX foci. γ-H2AX is a marker of
DNA double-strand breaks (Rogakou, Pilch, Orr, Ivanova, & Bonner, 1998). Increased numbers of γ-H2AX foci have been found in bronchial carcinoma and breast adenoma cells
treated with a EGFR inhibitor for radiosensitization (Dittmann, Mayer, & Rodemann, 2005),
and this increase has been correlated with dead or dying cells (Gonzalez, Barquinero, Lee,
Garcia, & Casaco, 2012). Double-strand breaks of DNA are the principal lesions responsible
for cell killing by ionizing radiation. The radiosensitivity and clonogenic survival of
irradiated bladder cancer cell lines have been previously assessed by alkaline comet assay, a
method to detect DNA strand break damage (Moneef et al., 2003). Our findings of reduced clonogenic survival and significantly increased numbers of γ-H2AX foci after combined
afatinib and irradiation imply that afatinib augments DNA damage. Given that DNA is the
most important cellular target of ionizing radiation, afatinib is an ideal radiosensitizer for
bladder cancer.
5.2.2 Limitation
Although we demonstrate that afatinib suppresses irradiation-activated EGFR and HER2
pathways, the full mechanism of enhanced radiosensitization of bladder cancer cells remains
elusive. Sambade et al. reported that lapatinib mediates radiosensitization in breast cancer
cells primarily by inhibiting the Raf > MEK > ERK mitogen-activated protein kinase cascade
(Sambade et al., 2009). We showed that afatinib inhibits post-radiation Akt phosphorylation
in response to EGFR and HER2 signaling. Nicolle et al. similarly found that growth and
invasiveness inhibition by gefitinib in urothelial carcinoma cell lines involves strong
phosphorylation of Akt/MAPK pathways in association with activation of EGFR (Nicolle et
al., 2006). Further investigation is required to identify the pathways through which DNA
damage and apoptosis signals are transduced.
5.2.3 Conclusion
In this murine bladder cancer model we demonstrated for the first time the in vitro and
in vivo radiosensitizing activity of afatinib, an orally bioavailable EGFR/HER2 dual inhibitor.
Afatinib likely mediates its effect on bladder cancer cells by suppressing radiation-activated
EGFR and HER2 signals and thereby causing enhanced DNA damage and cell apoptosis. The
greater potency of afatinib as a radiosensitizer compared with previously reported EGFR
inhibitors underscores the importance of other Erb-B family receptor tyrosine kinases such as
HER2, and indicates a new direction for future clinical trials in bladder cancer.