In the present study, we established HGF-overexpressing sublines of HCC827 cells and showed that these sublines are resistant to gefitinib both in vitro
and in vivo
. To investigate whether the resistance of HCC827-HGF cells to gefitinib is attributable to HGF-MET signaling, we examined the effects of the MET-TKI PHA-665752 and of TAK-701, a humanized monoclonal antibody to HGF, on signal transduction and cell growth. In both HCC827-HGF1 and -HGF2 cells as well as HCC827 GR5 cells, which are positive for MET
amplification, gefitinib alone did not inhibit AKT or ERK phosphorylation, whereas gefitinib in combination with PHA-665752 markedly suppressed the phosphorylation of these signaling molecules. Consistent with these results, PHA-665752 restored the sensitivity of cell growth to inhibition by gefitinib in HCC827-HGF cells as well as in HCC827 GR5 cells. These results indicate that the gefitinib resistance of these cell lines is mediated by MET signaling. TAK-701 has been shown to potently inhibit HGF binding to MET in cancer cells and xenograft models dependent on autocrine HGF-MET signaling.6
TAK-701 did not inhibit the phosphorylation of MET in HCC827 GR5 cells, suggesting that the activation of MET in these cells is not dependent on HGF. Indeed, we were not able to detect the secretion of HGF from HCC827 GR5 cells. In contrast, TAK-701 suppressed MET phosphorylation, and thus the combination of TAK-701 and gefitinib markedly inhibited both AKT and ERK signaling, in HCC827-HGF cells, resulting in their growth inhibition. These results indicate that autocrine HGF-MET signaling contributes to gefitinib resistance in HCC827-HGF cells. Similar ligand-mediated gefitinib resistance has been described previously, with insulin-like growth factor (IGF) having been found to rescue NSCLC cells expressing wild-type EGFR
from gefitinib-induced inhibition of cell growth (21
). These observations suggest that ligand-dependent receptor tyrosine kinase (RTK) activation (by HGF or IGF), as well as ligand-independent RTK activation (by MET
amplification), plays a pivotal role in the development of resistance to gefitinib. Further studies should reveal whether other ligand-RTK combinations contribute to gefitinib resistance.
We found that the baseline levels of both MET expression and MET phosphorylation in HCC827-HGF cells were lower than those in HCC827 GR5 cells (), whereas HCC827-HGF cells were resistant to gefitinib to the same extent as were HCC827 GR5 cells in vitro
(). These results suggest that phosphorylated MET activates downstream signaling through different pathways in HCC827 GR5 and HCC827-HGF cells. MET was recently shown to signal through ERBB3 in MET
amplification–positive NSCLC cells (6
) or through Grb2-associated binder 1 (Gab1) in NSCLC cells with HGF-induced gefitinib resistance (22
). Further studies are required to investigate whether the biological properties or the abilities of drugs to overcome gefitinib resistance are affected by differences in RTK downstream signaling.
In our HCC827-HGF xenograft model, we showed that HGF secreted from EGFR
mutation–positive NSCLC cells drives tumor growth even in the presence of gefitinib, and that combination therapy with TAK-701 and gefitinib was able to greatly inhibit the growth of HCC827-HGF tumors. These results indicate that interruption of HGF-MET signaling with TAK-701 represents a powerful strategy to abrogate gefitinib resistance induced by HGF derived from tumor cells. HGF was previously shown to be expressed predominantly by adenocarcinoma cells in NSCLC specimens, although a low level of HGF staining was also apparent in stromal cells (23
). Furthermore, marked expression of HGF has been detected in most lung cancers with intrinsic or acquired resistance to gefitinib (10
). These data suggest that our autocrine model systems based on stable overexpression of HGF are clinically relevant and should prove useful for the establishment of strategies to overcome gefitinib resistance. HGF is also produced by stromal cells of various tumor types (13
). Indeed, HGF derived from fibroblasts injected into nude mice together with EGFR
mutation–positive NSCLC cells induced gefitinib resistance in the NSCLC cells in vivo
). Further studies are required to clarify the major source of HGF that contributes to gefitinib resistance in patients with EGFR
mutation–positive lung cancer. Given that TAK-701 inhibits HGF binding to MET, TAK-701 may reverse gefitinib resistance induced by HGF derived not only from tumor cells but also from stromal cells.
In conclusion, we have shown that autocrine activation of MET by HGF confers resistance to gefitinib, and that TAK-701, a humanized monoclonal antibody to HGF, restored sensitivity to gefitinib in tumors with HGF-induced gefitinib resistance. TAK-701 is currently undergoing phase I trials as a single agent in patients with advanced solid tumors. Our results now indicate that the addition of TAK-701 to gefitinib is a potential strategy to overcome EGFR-TKI resistance induced by HGF and warrants clinical evaluation.