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

Tang Z, Du R, Jiang S, Wu C, Barkauskas DS, Richey J, Molter J, Lam M, Flask C, Gerson S, Dowlati A, Liu L, Lee Z, Halmos B, Wang Y, Kern JA, Ma PC - Br. J. Cancer (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, University Hospitals Case Medical Center, Cleveland, OH 44106, USA.

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.

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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).
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fig2: 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).

Mentions: As we found that many NSCLC cell lines co-express EGFR and MET, including H1975 cells, we first investigated if MET inhibition using the small-molecule inhibitor SU11274 can be effective in the erlotinib-resistant H1975 cells. H1975 cell line was chosen because it expresses the ‘gatekeeper'-resistant T790M-EGFR mutation (in-cis with L858R) in the receptor kinase domain hydrophobic pocket, representing a major mechanism of resistance to reversible EGFR-TKI (erlotinib/gefitinib) (Kobayashi et al, 2005a; Pao et al, 2005). SU11274 was previously characterised as a reversible inhibitor of MET, inhibiting specific tyrosine phosphorylation of the juxtamembrane CBL-binding phosphosite (pY1003), the major kinase autophosphorylation sites (pY1234/1235), as well as downstream signalling (Sattler et al, 2003; Ma et al, 2005a). It exhibits >60-fold selectivity for MET over FLK and >400-fold selectivity over RON, FGFR-1, SRC, CDK2, PDGFR-β, EGFR, and Tie-2 (Ma et al, 2005a). Here, we tested the pro-apoptotic effect of SU11274 treatment, in comparison to erlotinib, in the EGFR-TKI-resistant cell line H1975 (L858R/T790M-EGFR, wild-type KRAS). SU11274 at 2 μM induced apoptosis (Annexin V- and propidium iodide-stained positive cells) in 14.8±2.4% of T790M-EGFR expressing H1975 cells, which is 5.5-fold (P<0.001) higher than diluent control and 3.9-fold (P=0.0015) higher than erlotinib. To further demonstrate that the pro-apoptotic effect of SU11274 seen above was not a result of off-target effects, we also tested the EGFR-negative and MET-negative H520 cell line as negative control. SU11274 at 5 μM did not result in any significant apoptosis in H520 cells (0.44±0.30%, P=0.22). Similarly, H520 cells were also insensitive to erlotinib without any significant apoptosis induced by the drug (0.3±0.1%, P=0.35) (Figure 2).


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

Tang Z, Du R, Jiang S, Wu C, Barkauskas DS, Richey J, Molter J, Lam M, Flask C, Gerson S, Dowlati A, Liu L, Lee Z, Halmos B, Wang Y, Kern JA, Ma PC - Br. J. Cancer (2008)

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).
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Related In: Results  -  Collection

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fig2: 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).
Mentions: As we found that many NSCLC cell lines co-express EGFR and MET, including H1975 cells, we first investigated if MET inhibition using the small-molecule inhibitor SU11274 can be effective in the erlotinib-resistant H1975 cells. H1975 cell line was chosen because it expresses the ‘gatekeeper'-resistant T790M-EGFR mutation (in-cis with L858R) in the receptor kinase domain hydrophobic pocket, representing a major mechanism of resistance to reversible EGFR-TKI (erlotinib/gefitinib) (Kobayashi et al, 2005a; Pao et al, 2005). SU11274 was previously characterised as a reversible inhibitor of MET, inhibiting specific tyrosine phosphorylation of the juxtamembrane CBL-binding phosphosite (pY1003), the major kinase autophosphorylation sites (pY1234/1235), as well as downstream signalling (Sattler et al, 2003; Ma et al, 2005a). It exhibits >60-fold selectivity for MET over FLK and >400-fold selectivity over RON, FGFR-1, SRC, CDK2, PDGFR-β, EGFR, and Tie-2 (Ma et al, 2005a). Here, we tested the pro-apoptotic effect of SU11274 treatment, in comparison to erlotinib, in the EGFR-TKI-resistant cell line H1975 (L858R/T790M-EGFR, wild-type KRAS). SU11274 at 2 μM induced apoptosis (Annexin V- and propidium iodide-stained positive cells) in 14.8±2.4% of T790M-EGFR expressing H1975 cells, which is 5.5-fold (P<0.001) higher than diluent control and 3.9-fold (P=0.0015) higher than erlotinib. To further demonstrate that the pro-apoptotic effect of SU11274 seen above was not a result of off-target effects, we also tested the EGFR-negative and MET-negative H520 cell line as negative control. SU11274 at 5 μM did not result in any significant apoptosis in H520 cells (0.44±0.30%, P=0.22). Similarly, H520 cells were also insensitive to erlotinib without any significant apoptosis induced by the drug (0.3±0.1%, P=0.35) (Figure 2).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, University Hospitals Case Medical Center, Cleveland, OH 44106, USA.

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.

Show MeSH
Related in: MedlinePlus