Aim of the present study was to assess the effects of HSP-90 inhibition using the novel synthetic HSP-90 inhibitor SNX-2112 in tumor cells with MET amplification. We have found that HSP-90 inhibition induces degradation of MET, inhibition of MET and downstream PI3K/AKT and Ras/Raf/MEK/MAPK signaling as well as cell cycle arrest in 3 different MET-amplified tumor cell lines (EBC-1, GTL-16 and MKN-45). We were also able to demonstrate that SNX-5542, an orally bioavailable prodrug of SNX-2112, has significant antitumor efficacy in vivo in nude mice bearing MET-amplified tumor xenografts. Notably, HSP-90 inhibition maintained its in vitro and in vivo efficacy against MET-amplified tumor cells resistant to the highly specific MET kinase inhibitor PHA-665752, suggesting that HSP-90 inhibition could be a particularly valuable strategy in MET-amplified tumors with acquired resistance to selective MET kinase inhibition.
Tumor cells harboring amplification of the MET oncogene are largely dependent upon constitutive activation of the MET kinase for proliferation and survival, which renders them highly sensitive towards targeted MET inhibition (6
). Several small molecule MET inhibitors have been developed and have recently entered early-stage clinical trials (31
). However, as with other highly selective kinase inhibitors (e.g. gefitinib, imatinib) development of acquired resistance is likely. Here, we show that de novo
FGF-3 expression is a possible mechanism of resistance to the selective MET kinase inhibitor PHA-665752 in MET-amplified GTL-16 tumor cells. In the resistant cell line (PR-GTL-16), FGF-3, which displays tumorigenic potential in murine breast and colorectal cancer models (33
), maintains AKT and MAPK phosphorylation even when MET is completely inhibited, while knockdown of FGF-3 or selective inhibition of FGFR signaling using either of two selective small molecule FGFR inhibitors (PD173074 or PD330361) largely restores PHA-665752-sensitivity. Our data add to the growing body of evidence that primary or secondary activation of parallel growth factor receptor signaling resulting in sustained PI3K/AKT and/or Ras/Raf/MEK/MAPK pathway activation is a mechanism of resistance to selective tyrosine kinase inhibition in oncogene-addicted cancer cells: Secondary amplification of MET confers resistance to the EGFR inhibitor gefitinib in EGFR-mutant non small-cell lung cancer cells (38
), loss of IGF binding protein-3 expression with consecutive activation of the IGF receptor pathway mediates gefitinib resistance in A431 cells with EGFR amplification (39
), and an autocrine FGF-2 signaling loop is associated with resistance to targeted EGFR inhibition in a subset of non small-cell lung cancer cells (40
). Furthermore, it has recently been demonstrated by our group that HER-kinase activation confers resistance to targeted MET inhibition in MET-amplified gastric cancer cells (19
Since oncogene-addicted tumor cells may readily overcome the effects of highly selective kinase inhibition through activation of redundant survival pathways, solitary inhibition of the oncogenically activated kinase is unlikely to exhibit long-term antitumor efficacy in these malignancies. Instead, the therapeutic strategy should take into account both oncogenic kinase activation as well as potential signaling pathway redundancy, i.e. by simultaneously inhibiting multiple key signaling molecules. Conceivably, such an approach would decrease the likelihood of resistance as well as maintain antitumor efficacy in tumors that have become resistant to selective kinase inhibitor treatment.
A large number of oncogenic receptor tyrosine kinases and downstream signaling molecules require HSP-90 for conformational stability, including HER-2, EGFR, v-Src, Raf1, cyclin-dependent kinase-4 and AKT (10
). Given the important roles played by these HSP-90 clients in signal transduction, proliferation and survival, inhibition of HSP-90 has emerged as a potential antitumor treatment strategy (18
). Its underlying mechanism involves proteasomal degradation of HSP-90 client proteins leading to disruption of the tumor cell signaling network – both at the receptor tyrosine kinase and downstream signaling level. HSP-90 inhibition therefore exemplifies the above-mentioned concept of multi-targeted signaling pathway inhibition, making it a highly attractive strategy for the treatment of oncogene-addicted malignancies (41
). Indeed, it could be demonstrated that oncogene-addicted tumors – e.g. HER-2-amplified breast cancers or tumors with mutational activation of the EGFR – are particularly sensitive to HSP-90 inhibition both in vitro
and in vivo
In MET-amplified cells, a large number of HSP-90 clients (e.g. Raf-1, AKT, EGFR) are activated by MET either through MET-dependent downstream signaling or receptor cross-talk (5
). Moreover, recent studies have indicated that MET itself is an HSP-90 client protein (20
). We therefore hypothesized that MET oncogene-addicted tumors may be equally amenable to the effects of HSP-90 inhibition. Indeed, our study demonstrates that MET-amplified tumor cells are highly susceptible towards the antitumor effects of HSP-90 inhibition, with a sensitivity that is largely identical to that of HER-2-amplified breast cancer cells.
Of note, HSP-90 inhibition maintained its in vitro and in vivo antitumor efficacy in PR-GTL-16 cells with acquired resistance to the selective MET kinase inhibitor PHA-665752. In these cells, FGF-3 (through its receptors FGFR-1 and -2) induces sustained activation of the PI3K/AKT and Ras/Raf/MEK/MAPK pathways even when MET is completely inhibited, suggesting that combinatorial signal transduction blockade is required for antitumor activity. Due to its multi-targeted effect including up- (e.g. MET) and downstream (e.g. PI3K/AKT and Ras/Raf/MEK/MAPK) signaling molecules which are also targeted by FGF-3, HSP-90 inhibition was able to simultaneously abrogate MET, PI3K/AKT and Ras/Raf/MEK/MAPK signaling in the resistant cells, inducing growth inhibition both in vitro and in vivo at levels largely comparable to parental cells.
Recently, it could be demonstrated that secondary activation of EGFR signaling is another mechanism of resistance to targeted MET inhibition in MET-amplified tumor cells (30
). To assess whether HSP-90 inhibition using SNX-2112 would also be effective in such tumor cells which display both MET amplification as well as secondary activation of EGFR signaling, MET-amplified NCI-H820 cells which also harbor an activating T790M EGFR mutation were used as an in vitro
): As shown in Supplementary Figure 2
, these cells are resistant against both EGFR and MET inhibition, while a >50% inhibition of growth is observed upon tretament with SNX-2112. Importantly, these data not only suggest that HSP-90 inhibition could be a promising therapeutic strategy to overcome different mechanisms of MET kinase inhibitor resistance (e.g. secondary activation of FGFR or EGFR signaling) but also to treat EGFR-mutant non small-cell lung cancers which have become resistant to EGFR inhibition through secondary amplification of MET (38
In conclusion, our study provides a strong rationale for the use of HSP-90 inhibition in tumors that have become resistant to selective tyrosine kinase inhibition and further illustrates that combinatorial signal transduction blockade offers significant advantages over highly selective kinase inhibition in oncogene-addicted malignancies.