MET and PI3K/mTOR as a potential combinatorial therapeutic target in malignant pleural mesothelioma

Abstract

Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.

Figure 1. Expression of p-AKT and PTEN in archival mesothelioma tumor tissue samples.

Mesothelioma TMA containing 212 tumor samples and 192 normal lung tissue samples probed with p-AKT and PTEN representative images are shown in (A). (B) Comparative average scores of p-AKT and PTEN in TMA samples. (C) Representative immunoblot of mesothelioma cell lines for MET, p-110, p-85, Total AKT, p-AKT and loading control β Actin.

Figure 2. Effect of MET and PI3K/mTOR dual inhibitors on growth of human mesothelioma tumor cell lines.

Met-5A, H2596, H513, H2461 and H2052 were treated with ARQ 197, NVP-BEZ235, and GDC-0980 for 72 h at the indicated concentrations. Viability was measured by Alamar Blue assay. The data shown represent the mean ± SEM. (A) ARQ 197, (B) GDC-0980, (C) NVP-BEZ235, (D) Crizotinib.

Figure 3. Synergistic anti-tumor activity of combination of ARQ 197 with NVP-BEZ235 and GDC-0980 in mesothelioma cell lines.

Cells were treated with ARQ 197, NVP-BEZ235 and GDC-0980 alone or in combination at serial concentrations for 72 h. Cell viability was measured by Alamar Blue assay. Combination index (CI) plot analysis of ARQ 197/GDC-0980 and ARQ 197/NVP-BEZ235 combinations show that they interact synergistically in H2596 and H513 cells. CI = 1 shows additive effect, CI<1 is synergism and CI>1 is antagonism. Each experiment was carried out independently and repeated at least three times. (A) CI plot of ARQ 197/BEZ235 in H2596 cells. (B) CI plot of ARQ 197/BEZ235 in H513 cells. (C) CI plot of ARQ 197/GDC0980 in H2596 cells. (D) CI plot of ARQ 197/GDC0980 in H513 cells.

Figure 4. MET inhibition alone or in combination with PI3K/mTOR dual inhibitors suppresses cell motility.

Wound healing assay was performed in H513 and H2596 cells treated with ARQ 197(0.2 µM), GDC-0980 (0.2 µM), NVP-BEZ235 (60 nM) or combinations for 24 h as described in the methods. Representative pictures of the degree of wound closure in control and treated (A) H513 and (B) H2596 cells at 12 h respectively. The open wound at each time point was quantified and normalized to 0 h for (C) H513 and (D) H2596 cells. The experiments were repeated three times in triplicate and average closure ± SEM is shown.

Figure 5. MET inhibition alone or in combination with PI3K/mTOR dual inhibitors induces cell cycle arrest and apoptosis.

H2596 cells were treated with ARQ 197, GDC-0980, NVP-BEZ235 alone and in combination for 48 h. Cell cycle profile was determined using flow cytometry after staining with PI/RNase. Fig. A shows the percentage of cells in G1, S, and G2/M phases was quantified and the results expressed as the mean ± SEM of four independent experiments. H2596 (B) and H513 (C) cells, were treated with ARQ 197, GDC-0980, NVP-BEZ235 alone and in combination for 48 h. Cell lysates were prepared and immunoblotted for total PARP, cleaved PARP, cyclin D1 and actin as a loading control. H2596 cells treated with ARQ 197, GDC-0980, NVP-BEZ235 alone and in combination for 48 h, the cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. Results are expressed as mean percentage of apoptotic cells ± SEM of four independent experiments (D).

Figure 6. Effect of MET inhibitor ARQ 197 on kinase activation profile and downstream signaling pathways in MPM.

The protein tyrosine kinase activity profile obtained using H513 lysates treated with ARQ 197 as tested on PamChip microarrays. The color-coded response signature (A) is shown as heatmap in which treatment related upregulation of kinase activity is shown by red and downregulation by blue. Protein tyrosine kinase activity is down regulated in a dose dependent manner for most of the peptides. (B) H2596 and H513 cells were starved in 0.5% BSA media for 16 to 18 h and then treated with ARQ 197 at indicated concentrations for 24 h. The cells were then stimulated with human recombinant HGF (100 ng/ml) for 15 min before preparing the cell lysates. Immunobloting was then performed with the following antibodies Phospho-MET^Y1234/1235, Phospho-MET^Y1003, Phospho-MET^Y1349, Phospho-Paxillin, Phospho-AKT ^Ser473, Phospho -AKT ^thr308, Phospho-ERK, phospho-S6K and the corresponding total antibodies.

Figure 7. Effect of combined MET and PI3K/mTOR inhibition on kinase activation profile and downstream signaling pathways in MPM.

H2596 and H513 cells were plated in 10 cm tissue culture plates overnight and next day treated with indicated inhibitors for 4 h. Phospho-AKT (p-AKT ^Ser473, p-AKT ^thr308), total AKT, p-S6K and total S6K were assessed in (A) H2596 and (B) H513 by immunoblotting. (C) H2596 cells were plated in 10 cm tissue culture plates overnight and next day treated with indicated inhibitors for 24 h. PIP3 content was measured via dot blot and densitometric analysis for each blot is shown.

Figure 8. ARQ 197 and GDC-0980 inhibit the growth of tumor xenografts in nude mice and their combination has synergistic inhibition of tumor growth.

Results from tumor xenograft experiments testing the efficacy of ARQ 197 and GDC-0980 alone and/or combination in inhibiting the growth of H2596 cell line tumors in nude mice. Oral gavage treatment with ARQ 197 (200 mg/kg/day) and/or GDC-0980 (5 mg/kg/day) reduced H2596 tumor growth significantly relative to vehicle control (p<0.001). The combined use of ARQ 197/GDC-0980 was much more effective than any single agent alone, in inhibiting the tumor growth (p<0.01). (A) Tumor sizes were recorded every three days till the end of the experiment. (B) Representative images of mice and tumors treated with vehicle control, ARQ 197, GDC-0980 and their combination. (C) Immunohistochemical staining of tumor tissues with total CD31 and p-AKT and quantification. Bar graphs indicate average expression with standard error.