Dual MET-EGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer

Abstract

Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by T790M-EGFR mutation in half of the relapses. We show that MET is highly expressed in lung cancer, often concomitantly with epidermal growth factor receptor (EGFR), including H1975 cell line. The erlotinib-resistant lung cancer cell line H1975, which expresses L858R/T790M-EGFR in-cis, was used to test for the effect of MET inhibition using the small molecule inhibitor SU11274. H1975 cells express wild-type MET, without genomic amplification (CNV = 1.1). At 2 microM, SU 11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P = 0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells. In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay. Using small-animal microPET/MRI, SU11274 treatment was found to induce an early tumour metabolic response in H1975 tumour xenografts. MET and EGFR pathways were found to exhibit collaborative signalling with receptor cross-activation, which had different patterns between wild type (A549) and L858R/T790M-EGFR (H1975). SU11274 plus erlotinib/CL-387,785 potentiated MET inhibition of downstream cell proliferative survival signalling. Knockdown studies in H1975 cells using siRNA against MET alone, EGFR alone, or both, confirmed the enhanced downstream inhibition with dual MET-EGFR signal path inhibition. Finally, in our time-lapse video-microscopy and in vivo multimodal molecular imaging studies, dual SU11274-erlotinib concurrent treatment effectively inhibited H1975 cells with enhanced abrogation of cytoskeletal functions and complete regression of the xenograft growth. Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer. Furthermore, optimised inhibition may be further achieved with MET inhibition in combination with erlotinib or an irreversible EGFR-TKI.

Figures

Figure 1

Co-expression pattern of MET and EGFR in lung cancer. The expression pattern of MET (1st panel) and EGFR (2nd panel) was examined using standard immunoblotting of the whole cell lysates (WCLs) from the following lung cancer cell lines cultured under serum-containing conditions (10% FBS): A549, H1975, H226, H441, H520, H596, H661, H1437, H1838, H2122, and SW900. The downstream signalling effector STAT3 (3rd panel) was also included in the immunoblot analysis. β-Actin was included as loading control (bottom panel).

Figure 2

MET inhibition with SU11274 in EGFR-TKI-resistant H1975 lung cancer cells: induction of apoptosis in vitro and inhibition of cytoskeletal functions. The MET kinase inhibitor SU11274 was used to treat H1975 cells (L858R/T790M-EGFR, wild-type KRAS) and H520 cells (negative expression for both EGFR and MET) as control. The effect of SU11274 was examined using Annexin-V/propidium iodide (PI)-FITC cellular apoptosis assay. Untreated diluent control (U) and erlotinib were included in the experiment as treatment controls for comparison. Erlotinib (EGFR inhibition) was ineffective in promoting apoptosis in any of these above cell lines at 72 h. On the other hand, MET inhibition by SU11274 at 2 μM induced significant cellular apoptosis in the EGFR-TKI-resistant H1975 cells (14.8±2.4%, P=0.0015), when compared with erlotinib (3.8±0.7%). For the EGFR-negative and MET-negative H520 cells, neither SU11274 (0.44±0.30%, P=0.22) nor erlotinib (0.28±0.13%, P=0.35) at 5 μM induced any significant cellular apoptosis when compared with diluent control (0.18±0.10%). Mean values of percent cells in early apoptosis (Annexin-V plus PI staining) from three independent experiments for each of the treatment conditions were plotted in the graphs shown here. Error bar, s.e.m. (N=3).

Figure 3

MET inhibition with SU11274 successfully induced in vivo tumour response in EGFR-TKI-resistant H1975 cells in murine xenograft model assessed by multimodal molecular imaging. (A) In vivo tumour xenografts for H1975-luc cells were established as described in the Materials and Methods section in 6-week old nude mice. Daily SU11274 (100 μg per xenograft) treatment was administered to the H1975-luc lung cancer tumour xenografts in nude mice as described. DMSO diluent control was included for comparison. Imaging was performed using a Xenogen IVIS 200 System cooled CCD camera at indicated times. (a) Representative BLI digital pictures of nude mouse from each of the treatment conditions are illustrated. SU11274 significantly inhibited L858R/T790M-EGFR expressing H1975-luc in vivo tumorigenesis within the treatment durations (6 days). (b) Mean values of relative BLI flux of each group are plotted here (H1975-luc). N=4 per treatment group. Error bar, s.e.m. (*), P=0.025 for H1975-luc. Representative tumour xenograft micrographs from H1975-luc (c) cell line under haematoxylin and eosin (H&E) staining are also shown here for the control and SU11274 treatment animals. Magnification × 100 (inset, × 200). (d) SU11274 inhibited HGF-driven signalling activation in H1975 cells. H1975 cells were stimulated with HGF (50 ng ml−1, 15 min) and inhibited by MET inhibitor SU11274 (1 μM, 4 h) in vitro, and analysed with 7.5% SDS–PAGE and immunoblotting with the indicated antibodies as described in Materials and Methods. (B, C) Magnetic resonance imaging (MRI) and microPET molecular imaging studies of MET inhibition of H1975 in vivo xenograft. H1975 in vivo xenografts were established as above for treatment with either diluent control (N=2) or SU11274 (N=2). The nude mice with H1975 xenografts were subjected to MRI and microPET imaging as described in the Materials and Methods section at 0, and 24 h with the MET inhibitor SU11274 treatment or diluent control. (B) Examples of the transverse sections of high-resolution MRI images of the tumour xenografts at baseline between the two treatment groups were shown here for illustration (left). The MRI tumour volumes were analysed digitally with the calculated tumour volume changes at the indicated time intervals (0 and 24 h) plotted. Comparing with baseline, the control xenograft tumour volume increased by 105.1±8.3% at 24 h, whereas the SU11274-treated xenografts increased by 120.2±16.2% at 24 h. The MRI tumour volumes changes at 24 h post-treatment between the two groups were not statistically significant (*P=0.360). Error bar, s.e.m. Quantitative microPET radiotracer uptake of the H1975 tumour xenografts at 60 min of radiotracer tail-vein infusion in the animals' pretreatment baseline (0 h) and post-MET-TKI treatment at 24 h is shown graphically (right). N=2 in each treatment group: Control and SU11274. Error bar, s.e.m. Representative co-registered pictures of the microPET/MRI (low-resolution) images of each xenograft from the two treatment groups are shown in (C). SU11274 induced early tumour metabolic response, as early as 24-h post-TKI treatment, with statistically significant inhibition of glucose metabolism as evident in the decrease in microPET uptake signal intensity by 45% (P=0.0226) in SU11274-treated xenografts, when compared with diluent control. The degree of increase in the glucose uptake in the H1975 tumour xenograft in diluent control is also consistent with the average rate of the xenograft growth (increase of 50.8% BLI flux per day) as reflected in the bioluminescence imaging.

Figure 3

MET inhibition with SU11274 successfully induced in vivo tumour response in EGFR-TKI-resistant H1975 cells in murine xenograft model assessed by multimodal molecular imaging. (A) In vivo tumour xenografts for H1975-luc cells were established as described in the Materials and Methods section in 6-week old nude mice. Daily SU11274 (100 μg per xenograft) treatment was administered to the H1975-luc lung cancer tumour xenografts in nude mice as described. DMSO diluent control was included for comparison. Imaging was performed using a Xenogen IVIS 200 System cooled CCD camera at indicated times. (a) Representative BLI digital pictures of nude mouse from each of the treatment conditions are illustrated. SU11274 significantly inhibited L858R/T790M-EGFR expressing H1975-luc in vivo tumorigenesis within the treatment durations (6 days). (b) Mean values of relative BLI flux of each group are plotted here (H1975-luc). N=4 per treatment group. Error bar, s.e.m. (*), P=0.025 for H1975-luc. Representative tumour xenograft micrographs from H1975-luc (c) cell line under haematoxylin and eosin (H&E) staining are also shown here for the control and SU11274 treatment animals. Magnification × 100 (inset, × 200). (d) SU11274 inhibited HGF-driven signalling activation in H1975 cells. H1975 cells were stimulated with HGF (50 ng ml−1, 15 min) and inhibited by MET inhibitor SU11274 (1 μM, 4 h) in vitro, and analysed with 7.5% SDS–PAGE and immunoblotting with the indicated antibodies as described in Materials and Methods. (B, C) Magnetic resonance imaging (MRI) and microPET molecular imaging studies of MET inhibition of H1975 in vivo xenograft. H1975 in vivo xenografts were established as above for treatment with either diluent control (N=2) or SU11274 (N=2). The nude mice with H1975 xenografts were subjected to MRI and microPET imaging as described in the Materials and Methods section at 0, and 24 h with the MET inhibitor SU11274 treatment or diluent control. (B) Examples of the transverse sections of high-resolution MRI images of the tumour xenografts at baseline between the two treatment groups were shown here for illustration (left). The MRI tumour volumes were analysed digitally with the calculated tumour volume changes at the indicated time intervals (0 and 24 h) plotted. Comparing with baseline, the control xenograft tumour volume increased by 105.1±8.3% at 24 h, whereas the SU11274-treated xenografts increased by 120.2±16.2% at 24 h. The MRI tumour volumes changes at 24 h post-treatment between the two groups were not statistically significant (*P=0.360). Error bar, s.e.m. Quantitative microPET radiotracer uptake of the H1975 tumour xenografts at 60 min of radiotracer tail-vein infusion in the animals' pretreatment baseline (0 h) and post-MET-TKI treatment at 24 h is shown graphically (right). N=2 in each treatment group: Control and SU11274. Error bar, s.e.m. Representative co-registered pictures of the microPET/MRI (low-resolution) images of each xenograft from the two treatment groups are shown in (C). SU11274 induced early tumour metabolic response, as early as 24-h post-TKI treatment, with statistically significant inhibition of glucose metabolism as evident in the decrease in microPET uptake signal intensity by 45% (P=0.0226) in SU11274-treated xenografts, when compared with diluent control. The degree of increase in the glucose uptake in the H1975 tumour xenograft in diluent control is also consistent with the average rate of the xenograft growth (increase of 50.8% BLI flux per day) as reflected in the bioluminescence imaging.

Figure 4

Signalling cross-activation between MET and EGFR signalling pathways. (A) Cross-activation between MET and EGFR signalling in lung cancer cells, A549 and H1975. A549 or H1975 cells were cultured in serum-starved conditions with exogenous stimulation with RTK ligands: EGF alone, HGF alone, or both EGF and HGF. Cells without any ligand stimulation were included as control. Both MET and EGFR signalling pathways are functional and ligand-sensitive in A549 and H1975 cells. There was augmented downstream signalling with combined EGF-HGF co-stimulation, with also more durable signalling induction. In A549 cells, although HGF alone did not activate EGFR phosphorylation appreciably, under co-stimulation conditions with EGF together, HGF further enhanced the EGFR phosphorylation in A549 cells to a level higher than that with EGF alone. On the other hand, EGF stimulation of H1975 cells co-activated MET receptor to enhance the level of MET phosphorylation. (B) MET–EGFR cross-activation in lung cancer. Left panel (A549), HGF cross-activated p-EGFR in A549 cells in the presence of co-stimulation with EGF. A549 cells were cultured in serum-starved conditions overnight, then stimulated with EGF alone (100 ng ml−1, 15 min), HGF alone (50 ng/ml, 15 min), or both. Whole cell lysates were collected for immunoprecipitation with EGFR antibody, followed by immunoblotting (WB) with antibodies against p-EGFR[Y1068] (upper panel) and total EGFR (lower panel). Right panel (H1975), EGF cross-activates phospho-MET in H1975 cells. H1975 cells were cultured in starved media overnight, then stimulated with EGF alone (100 ng ml−1, 15 min), HGF alone (50 ng ml−1, 15 min), or both. Whole cell lysates were collected for immunoprecipitation with MET antibody (C-12), followed by immunoblotting with antibodies against p-MET[Y1234/1235] (upper panel) and total MET (lower panel). The MET and EGFR genotypes of the A549 and H1975 cells, as well as their MET genomic copy numbers, are shown in the bottom.

Figure 5

SU11274 inhibition of MET in combination with EGFR inhibitor in erlotinib-resistant NSCLC cell signalling. (A) Potentiated inhibition of cellular cytoskeletal functions by combined MET–EGFR inhibition (SU11274 plus erlotinib) in H1975 cells under video microscopy. H1975 cells had constitutively activated cytoskeletal functions with enhanced cell motility and migration under the serum-starved culture conditions. Comparing with the untreated control (left panel), drug treatment using SU11274 (right panel) substantially inhibited the constitutively activated cytoskeletal functions of H1975 cells (Supplementary Figure 3). H1975 cells were cultured in serum-stimulated conditions (10% FBS) and treated with the following for video microscopy digital video recording as described in the Materials and Methods: (a) DMSO diluent control, (b) Erlotinib alone (2 μM), (c) SU11275 alone (5 μM), and (d) combined concurrent SU11274 (5 μM)+erlotinib (2 μM). Complete abrogation of cytoskeletal functions with inhibition of cell motility and migration was only evident in the dual SU11274/erlotinib TKI-treated cells (d). (B) MET inhibition using SU11274, in combination with EGFR inhibition (erlotinib), induced cooperative downstream signalling inhibition in A549 (left panel) and H1975 (right panel) cells in vitro. EGFR-TKI-resistant A549 and H1975 cells were cultured in serum-starved conditions with EGF and HGF dual ligands stimulation as described in the Materials and Methods section. The cells were treated with SU11274 alone, erlotinib alone, or combination SU11274 plus erlotinib, then analysed in immunoblotting as indicated. (C) MET inhibition using specific siRNA-MET, in combination with EGFR inhibition (erlotinib) induced cooperative downstream signalling inhibition in A549 cells in vitro. Cells were transfected with control siRNA or siRNA-MET as described in Methods. Forty-eight hours after transfection, cells were cultured in starved media overnight, then treated with or without erlotinib and alone or in combination with siRNA-MET as indicated. After 4 h of inhibitor treatment, cells were then stimulated with both EGF (100 ng ml−1) and HGF (50 ng ml−1) ligands as indicated for 15 min. Whole cell lysates were then collected for immunoblotting analysis as in panel B above. (D) Rescue from alternative RTK ligand-stimulated signalling (MET–HGF vs EGFR–EGF) against TKI in A549 cells. A549 cells were cultured under serum-starved conditions, and then treated with either HGF or EGF, and in the presence or absence of the corresponding targeted inhibitor SU11274 or erlotinib as indicated (lanes 2, 4). Dual ligand stimulation (HGF and EGF) with single or dual TKIs treatment was included as indicated (lanes 1, 3, 5, 6). Although receptor-specific TKI was able to inhibit the downstream signalling driven by the corresponding ligand stimulation, alternative ligand stimulation in the form of dual ligand stimulation rescued the inhibited downstream signals. Dual TKI SU11274 plus erlotinib inhibition was required to fully knockdown the dual ligand-stimulated downstream signal activation of AKT, ERK1/2, and STAT3 (lane 6). (E) MET inhibition with SU11274, in combination with EGFR inhibition using CL-387,785 (irreversible EGFR-TKI), induced cooperative downstream signalling inhibition in H1975 cells in vitro. Cells were cultured in starved media overnight, then treated with or without CL-387,785 and alone or in combination with MET inhibitor SU11274 as indicated. After 4 h of inhibitor treatment, cells were then stimulated with both EGF (100 ng ml−1) and HGF (50 ng ml−1) ligands as indicated for 15 min. Whole cell lysates were then collected for immunoblotting analysis. Similar to erlotinib, CL-387,785 further sensitised H1975 cells to SU11274 inhibition with enhanced cooperative inhibition of signalling pathways downstream of the two RTKs.

Figure 6

Dual SU11274–erlotinib inhibition induced cooperative inhibition in H1975 cell viability in vitro and murine xenograft tumour growth in vivo. (A) In vitro inhibition using MET inhibitor SU11274 combined with erlotinib was more effective in H1975 cell viability inhibition under serum-stimulated conditions. Enhanced inhibition of cell viability was evident with dual SU11274-erlotinib treatment in combination (at 3 μM of each TKI as indicated). *P<0.05 (SU11274/erlotinib vs SU11274); **P<0.02 (SU11274/erlotinib vs erlotinib); and ***P=0.281 (SU11274 vs erlotinib). Error bar, s.d. (B) Combined knockdown of MET and EGFR signalling using short-interfering RNA (siRNA) in H1975 cells resulted in enhanced downstream signal transduction inhibition. H1975 cells cultured under serum-stimulated conditions were treated with siRNAs specifically targeted against mRNA of MET alone, EGFR alone, or both MET and EGFR as described in the Materials and Methods section. Cells with siRNA knockdown as indicated were harvested for immunoblotting using antibodies against phosphotyrosine (left panel) to survey the effects on global cellular phosphotyrosine phosphoproteomic profiles. Cells were also immunoblotted with antibodies against the MET and EGFR signal paths including the downstream pro-survival AKT and STAT3 pathways (right panel). Concurrent dual knockdown of MET and EGFR signalling by siRNA in H1975 cells led to optimally enhanced downregulation of global phosphorylated cellular proteome (left panel) including the pro-survival downstream p-AKT and p-STAT3 signal activation (right panel). (C) In vivo treatment using SU11274 combined with erlotinib induced cooperative complete regression of EGFR-TKI-resistant H1975 tumour xenograft growth. EGFR-TKI-resistant H1975-luc cells were used to establish nude mouse xenograft in vivo as described in the Materials and Methods section. The nude mice with H1975-luc xenografts were then treated with diluent control, EGFR inhibitor (erlotinib, 100 mg/kg/day) alone, MET inhibitor (SU11274, 50 μg per xenograft per day) alone, or both inhibitors concurrently (SU11274 plus erlotinib). Tumour xenograft growth was monitored by BLI at pretreatment baseline (day 0), and on post-treatment days 6 and 13. SU11274, in combination with erlotinib, induced complete tumour xenograft regression of H1975 cells in vivo. The mean relative BLI flux from each treatment group was plotted graphically (N=4 per treatment group). Error bar, s.e.m. (*), (SU11274/erlotinib vs erlotinib) P=0.0006. (**), (SU11274/erlotinib vs SU11274) P=0.0003. (***), (SU11274 vs erlotinib) P=0.0070. (D) H1975 tumour xenograft micrographs under H&E staining at × 100 magnification (and × 200, inset) showed substantial viable tumour cells in panel (a) DMSO control, and (b) erlotinib-treated animals, whereas there were necrotic and apoptotic tumour cells seen in panel (c) SU11274 (suboptimal dose: 50 μg per xenograft) and massively so in panel (d) in combined SU11274 plus erlotinib-treated animals.