Sensitivity to EphB TKI was also demonstrated
Sensitivity to EphB3 TKI was also demonstrated in a colon cancer cell line, COLO 205, with high expression of EphB3. We found not only exogenous but also endogenous EphB3 protein was expressed in the COLO 205 MAFP receptor after transient transfection of pCMV6-AC-GFP/EphB3, and both were demonstrated to be sensitive to EphB3 TKI with showing increased cleaved PARP levels (Supplementary Fig. S3). Simultaneously decreased phosphorylation of MEK1/2 by EphB3 TKI was also observed, suggesting suppressed signaling through Ras-ERK1/2 pathway might have contributed to inhibition of tumor cell growth and subsequent induction of apoptosis of the colon cancer cells. Given that results from many previous studies investigating effects of EphB3 signaling on various cancers are not consistent, several unclarified molecular regulatory processes are likely to be involved in the EphB3 signaling. As a matter of fact, a previous study reported microRNA-149-induced downregulation of EphB3 and PI3K/Akt led to suppression of proliferation and invasion of colon cancer cell lines (Wang et al., 2018). Therefore, the complicated signal transduction mechanisms are encouraged to be further clarified according to various cancer types to understand functional diversity of EphB3. EMT has been demonstrated as a mechanism of AZD4547 resistance in GC cells (Grygielewicz et al., 2016). Despite upregulated expression of transforming growth factor β (TGF-β), known to be involved in EMT (Brunen et al., 2013), it was reported that treatment of AZD4547-resistant SNU-16 cells with a TGF-β inhibitor did not reverse the EMT phenotype (Grygielewicz et al., 2016). Given this, we evaluated whether phospho-EphB3 was related to induction of EMT in SNU-16R cells in our study. Interestingly, expression of EMT markers in SNU-16R cells was abrogated by treatment with EphB3 TKI, suggesting that signaling through EphB3 may be an alternative pathway by which those cells obtain EMT characteristics. The downregulation of RTKs with simultaneous induction of EMT is not an uncommon mechanism in acquisition of resistance to targeted agents. Consistent to our results, a previous study also reported loss of expression of FGFR2 accompanied by induction of EMT in an AZD4547-resistant SNU-16 cell line (Grygielewicz et al., 2016). Another study that clarified resistance mechanism of gefitinib using a NSCLC cell line also showed reduced expression of RTKs, EGFR, HER3, and MET, alongside obtaining EMT characteristics (Rho et al., 2009). Differently from previous studies, which have reported the phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR pathway exerts tumorigenic effect by regulating cell survival, division, and EMT (Gulhati et al., 2011; Mendoza et al., 2011), Akt-independent activation of mTOR through the Ras-ERK1/2 pathway seems to be in charge with regulation of cell motility in EphB3 phosphorylated, AZD 4547-resistant GC cells in this study. We also showed EphB3-mediated activation of mTOR could be reversed by everolimus, verifying the mechanism of resistance to AZD4547 in GC cells and, at the same time, offering a possible alternative therapeutic strategy to overcome it. Synergistic effect was observed by treatment of AZD4547 in addition to everolimus in inhibiting cell growth of parental GC cells, while no difference of cell viability was observed between SNU-16R cells treated by everolimus alone and the resistant GC cells treated by both everolimus and AZD4547. For the lack of the target for AZD4547, no synergistic effect of AZD4547 added to everolimus in SNU-16R cells had been expected. On the other hand, in parental SNU-16 GC cells, treatment of AZD4547 or everolimus was observed to abrogate activity of mTOR or FGFR2, respectively, and this may be a clue to explain the synergistic effect (Fig. 5E). No synergistic effect of the combined treatment with AZD4547 and everolimus (Fig. 5D) or EphB3 TKI (Fig. 3C) on the resistant GC cells also suggests SNU-16R cells escaped sensitivity of the RTK inhibitor, AZD4547.