5.4.1 Planned experiments
We have proved that both T24 and NTUB1 human bladder cancer cells have wild-type
EGFR. On the other hand, non-small cell lung cancer with the L858R or the ΔE746-E750
mutations in the tyrosine kinase domain of EGFR was reported to exhibit enhanced
sensitivity to radiation, and this strongly correlated with dramatically diminished capacity to
resolve radiation-induced DNA double strand breaks (Das et al., 2006). Moreover, non-small
cell lung cancer patients with mutant EGFR patients treated with radiotherapy had a better
clinical outcome than patients with wild-type EGFR in terms of locoregional control (Mak et
al., 2011) or response to whole brain radiotherapy (Gow et al., 2008). It is also reported that
the mutant EGFR in these non-small cell lung carcinomas failed to exhibit radiation-induced
nuclear translocation or binding to DNA-PKcs (Das et al., 2007). Therefore it is plausible to
hypothesize that EGFR mutations may confer sensitivity to radiation.
To introduce different forms of EGFRs into T24 cells, we used the pLenti6/directional
TOPO cloning kit. Full-length fragment of wild-type EGFR was amplified from
pcDNA3.1-EGFR-wt and cloned into pLenti6/directional TOPO vector according to the instructions of
the manufacturer. The L858R mutation was introduced by using site-directed mutagenesis kit
(Stratagene). We also planned to introduce the full length of E746-A750 del mutation from
cDNA of HCC827 cell line and cloned into pLenti6/directional TOPO vector. Correct
sequences were confirmed by sequencing for all vectors. However, although we successfully
introduced the plasmid with mutated EGFR to T24 bladder cancer cell, the majority of EGFR
in T24 cells was the wild-type. After evaluation we abandoned the rest of the experiments.
5.4.2 Lessons learned from recent literature
Interestingly, Zhang et al. recently showed that afatinib increases radiosensitivity of
non-small cell lung cancer cells with acquired EGFR T790M mutation (Zhang et al., 2015). Since
the experimental design is similar to ours, it deserves description and comparison. In Fig. 5-1
the authors showed the change of EGFR/Akt/ERK pathways in lung cancer cells after
irradiation and afatinib. The phosphorylation levels of EGFR, Akt and ERK increased after
irradiation in PC-9 (EGFR del E746-A750), PC-9-GR (PC-9 with gefitinib resistance) and
H1975 cells (EGFR L858R and T790M mutation). Pretreatment with afatinib remarkable
blocked basal level of the phosphorylations of EGFR and ERK proteins, and caused delays of
irradiation-induced phosphorylation of Akt in these cells.
Figure 5-1. Effects of afatinib on protein phosphorylation after irradiation or afatinib
pretreatment+irradiation in PC-9 (A), PC-9-GR (B) and H1975 (C) cells (Zhang et al., 2015)
But afatinib did not cause changes of the basal levels for phosphorated EGFR, Akt and
ERK proteins in H460 (wild-type EGFR) cells which has a low baseline of these proteins
(Fig. 5-2). Although we don’t have the data about EGFR-mutated bladder cancer cells, the
result confirmed our previous observation in Fig. 3-2, Fig 3-3 and Fig 4-1 that afatinib
inhibits certain radiation-activated signaling pathways.
Figure 5-2. The effects of afatinib on protein phosphorylations in lung cancer cells (Zhang et
al., 2015)
The authors also demonstrated the clonogenic assay in PC-9, PC-9-GR, H1975 and
H460 cells after the treatment of radiation and/or afatinib (Fig. 5-3). PC-9-GR cells which
acquired T790M mutation in addition to the original deletion in exon 19 (del E746-A750)
demonstrated the radiosensitizing effect of afatinib. Similar to our experimental design, the
authors showed that afatinib treatment lead to increased apoptosis (by flow cytometry) and
suppressed DNA damage repair (by γ-H2AX foci) in irradiated PC-9-GR cells, and enhanced
tumor growth inhibition when combined with irradiation in PC-9-GR xenografts. However,
the exact mechanism why lung cancer cells with different EGFR mutation have different
susceptibility to afatinib-induced radiosensitization was not addressed
Figure 5-3. Effect of afatinib on clonogenic survival in irradiated lung cancer cells
(Zhang et al., 2015)
It is also a pity that the expression of baseline and radiation-induced HER2
phosphorylation was not compared in this study. Although H460 cell as well as T24 and
NTUB1 bladder cancer cells in our study has wild-type EGFR, the extent of HER2 activation
after irradiation may explain the difference in radiosensitizing effect of afatinib. More
mechanistic studies are still awaited.
CHAPTER SIX: PROSPECT
6.1 Radiosensitizing Activity of Afatinib and Microenvironment
Till now our study of the radiosensitizing effects of new-generation EGFR inhibitor
focus on the factors influence cell growth, like DNA damage and apoptosis. On the other
hand, the interaction between cancer cells and microenvironment also determine the potential
to invasion and metastasis, therefore it is very important to the radiation effect. Rapidly
accumulating evidence suggests that radiation exposure also promotes cancer metastatic
ability through epithelial-mesenchymal transition (EMT), which has a central role in cancer
metastasis and has become the subject of intense investigation (Kawamoto et al., 2012; Liu et
al., 2014; S. Yan et al., 2013). However, the signaling molecular mechanisms underlying
radiation-induced EMT remain obscure (Cui et al., 2015), and there is virtually no report
regarding this field in bladder cancer research. Since the failure pattern of radiotherapy in
bladder cancer patients includes local recurrence and distant metastasis, the influence of how
new-generation EGFR TKIs like afatinib on these phenomena will be of great significance.
Till now we have some preliminary result. As shown in Fig. 6-1, Boyden chamber
invasion assay revealed that after 2.5Gy irradiation, there is an increase of cell invasion in
T24 and 5637 bladder cancer cells. After pretreatment with afatinib, the radiation-enhanced
invasion was decreased, but the pretreatment with erlotinib did not show this effect.
Figure 6-1. Invasion assay of T24 and 5637 bladder cancer cells treated with irradiation +/-
afatinib (BIBW) or erlotinib (Tar)
Then we tested the expression of phosphorylated EGFR and HER2 as well as MMP-9
and MMP-2 in T24 and 5637 bladder cancer cells. As shown in Fig. 6-2, after irradiation the
expression of p-EGFR, p-HER2 and MMP-9 was increased, and the effect was decreased
after the pretreatment with afatinib. However the pretreatment with erlotinib did not show
this effect.
Figure 6-2. Western blot of T24 and 5637 bladder cancer cells treated with irradiation +/-
afatinib (BIBW) or erlotinib (Tarceva)
In addition, enzyme activity in culture media using zymography was also tested. The
result showed that after irradiation the activity of MMP-9 was increased, and the effect was
decreased after the pretreatment with afatinib. The influence of erlotinib pretreatment is
more variable in T24 and 5637 cells. In contrary, there was no obvious change in MMP-2
activity (Fig. 6-3).
Figure 6-3. Gelatin zymography of culture media from T24 and 5637 bladder cancer cells
treated with irradiation +/- afatinib (BIBW) or erlotinib (Tarceva)
Currently mechanistic and animal studies are ongoing to find out more ways to improve
the treatment outcome of bladder cancer radiotherapy.