Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors

Abstract

Malignant pleural mesothelioma (MPM) originates in most of the cases from chronic inflammation of the mesothelium due to exposure to asbestos fibers. Given the limited effect of chemotherapy, a big effort is being made to find new treatment options. The PI3K/mTOR pathway was reported to be upregulated in MPM. We tested the cell growth inhibition properties of two dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 on 19 MPM cell lines. We could identify resistant and sensitive lines; however, there was no correlation to the downregulation of PI3K/mTOR activity markers. As a result of mTOR inhibition, both drugs efficiently induced long-term autophagy but not cell death. Autophagy blockade by chloroquine in combination with the dual PI3K/mTOR inhibitors significantly induced caspase-independent cell death involving RIP1 in the sensitive cell line SPC212. Cell death in the resistant cell line Mero-82 was less pronounced, and it was not induced via RIP1-dependent mechanism, suggesting the involvement of RIP1 downstream effectors. Cell death induction was confirmed in 3D systems. Based on these results, we identify autophagy as one of the main mechanisms of cell death resistance against dual PI3K/mTOR inhibitors in MPM. As PI3K/mTOR inhibitors are under investigation in clinical trials, these results may help interpreting their outcome and suggest ways for intervention.

Figure 1

Identification of mesothelioma cell lines sensitive versus resistant to PI3K/mTOR inhibition. IC50 of 19 MPM cell lines: ACC-Meso-1, SPC212, MSTO-211H, ZL34, ZL55, NCI-H2452, ZL5, NCI-H226, SPC111, NCI-H2052, Mero-25, Mero-95, SDM103T2, ACC-Meso-4, Mero-82, ONE58, Mero-14, Mero-84 and Mero-83 and Q-Q Plot normality test at 250 nM after 72 h treated with (a) NVP-BEZ235 and (b) GDC-0980

Figure 2

Decrease of PI3K/mTOR signaling is accompanied by G1 arrest, which is more pronounced in sensitive cell lines. (a) Protein lysates of sensitive cell lines SPC212, ZL34 and Mero-25 and resistant cell lines Mero-83, Mero-82 and ONE58 were analyzed by western blotting against PI3K/mTOR activity markers: phospho-AKT (Thr308), phospho-AKT (Ser473), AKT, phospho-S6, S6, phospho-4E-BP1, 4E-BP1, and Actin. Each cell line was serum-starved for 16 h and treated as indicated for 4 h with 1000 μg/ml IGF-1, 0.5 μM NVP-BEZ235, 0.5 μM GDC-0980 or treated for 72 h with increasing concentrations of NVP-BEZ235 or GDC-0980 as indicated. (b) Cell cycle profile of sensitive cell line SPC212 and resistant line Mero-82 treated with 0.2 μM NVP-BEZ235 or 0.5 μM GDC-0980 for 72 h. Data are presented as means±S.D. from ≥3 independent experiments. Significance was determined by Student's t-test (***P<0.005, **P<0.01 and *P<0.05; NS, not significant)

Figure 3

Mesothelioma cells exhibit a high level of autophagy, which is increased upon inhibition of PI3K/mTOR signaling. (a) Anti-P62 and -LC3BI/II and -Actin western blots of protein lysates of sensitive cell line SPC212 and resistant cell line Mero-82 treated for 72 h with increasing concentrations of NVP-BEZ235 or GDC-0980 as indicated. (b) Anti-LC3BI/II and -Actin western blots of mesothelial cell line SDM104 and MPM cell lines SPC111, SPC212, NCI-H226, NCI-H2052, NCI-H2452, Mero-14, Mero-25, Mero-82, Mero-83, Mero-84, Mero-95, ACC-Meso-1, ACC-Meso-4, MSTO-211H and ONE58 left with serum or serum-starved for 16 h. In the lower panel, protein level quantification of LC3BII normalized against Actin is represented for the cell lines described above with standard culture conditions

Figure 4

Inhibition of autophagy with CQ sensitizes mesothelioma cell lines to PI3K/mTOR inhibitors. (a) Anti-P62, L-C3BI/II and Actin western blots of protein lysates of sensitive cell line SPC212 and resistant cell line Mero-82 treated with 20 μM CQ, 0.5 μM GDC-0980 or in combination for 24, 48 and 72 h. (b) Light micrographs of SPC212 and Mero-82 treated with 0.2 μM NVP-BEZ235, 0.5 μM GDC-0980 alone or in combination with 20 μM CQ for 96 h. Scale bar represents 25 μm. (c) Quantification of the percentage of ATP content of SPC212 cells and Mero-82 cell treated with 0.2 μM NVP-BEZ235, 0.5 μM GDC-0980 alone or in combination with 20 μM CQ for 96 h. Data are presented as means±S.D. from ≥3 independent experiments. Significance was determined by analysis of variance test (***P<0.005, **P<0.01 and *P<0.05; NS, not significant)

Figure 5

Autophagy is necessary for mesothelioma cell growth. SPC212 and Mero-82 cell lines were stable transduced with virus particles carrying Mir30- Sh-Atg5 or Mir30-Sh-Ctrl. (a) Atg5 gene silencing was confirmed by anti-ATG5 western blotting. (b) Anti-P62, -LC3BI/II and -Actin of SPC212 and Mero-82 Sh-Atg5 and Sh-Ctrl untreated or treated with 20 μM CQ for 4 h. (c) Fold cell growth of SPC212 and Mero-82 Sh-Atg5 and Sh-Ctrl for 3 days. Data are presented as means±S.D. from three independent experiments. Significance was determined by Mann–Whitney U-test (*P<0.05)

Figure 6

Inhibition of autophagy with CQ combined with inhibition of PI3K/mTOR signaling induces caspase-independent cell death. (a) SPC212 and Mero-82 cell lines were treated as indicated with 0.2 μM NVP-BEZ235, 0.5 μM GDC-0980, 20 μM CQ, 20 μM ZVAD-FMK or 50 μM Nec-1 for 96 h. Cell death was assessed by GFP-Annexin V/PI staining and flow cytometry. Data are presented as means±S.D. from three independent experiments. Significance was determined by analysis of variance test (***P<0.005, **P<0.01 and *P<0.05; NS, not significant). (b) Anti- PARP, -Caspase 3 and -Actin western blots of SPC212 and Mero-82 treated as indicated with 0.2 μM NVP-BEZ235, 0.5 μM GDC-0980 and 20 μM CQ. SV40 immortalized wt MEFs treated for 15 h with 3 μg/ml Etoposide were used as control for apoptosis. (c) Anti-XIAP (asterisk (*) represents unspecific band), -RIP1 and -Actin western blots of protein lysates of SPC212 and Mero-82 treated as indicated with 0.2 μM NVP-BEZ235, 0.5 μM GDC-0980 and 20 μM CQ for 72 and 96 h

Figure 7

Inhibition of autophagy with CQ combined with inhibition of PI3K/mTOR signaling induces spheroid cell death. (a) Representative light micrographs of SPC212 and Mero-82 spheroids treated as indicated with 1 μM NVP-BEZ235, 1 μM GDC-0980 and 20 μM CQ for 6 days. (b) Fold increase volume (growth) of spheroids shown in panel (a). (c) Viability is presented as the percentage of ATP content in SPC212 and Mero-82 spheroids treated as described in panel (a). Data are presented as means±S.D. from ≥3 independent experiments. Significance was determined by analysis of variance test (***P<0.005 and *P<0.05; NS, not significant)