Bcl-2-dependent synthetic lethal interaction of the IDF-11774 with the V0 subunit C of vacuolar ATPase (ATP6V0C) in colorectal cancer

Abstract

Background: The IDF-11774, a novel clinical candidate for cancer therapy, targets HSP70 and inhibits mitochondrial respiration, resulting in the activation of AMPK and reduction in HIF-1α accumulation.

Methods: To identify genes that have synthetic lethality to IDF-11774, RNA interference screening was conducted, using pooled lentiviruses expressing a short hairpin RNA library.

Results: We identified ATP6V0C, encoding the V0 subunit C of lysosomal V-ATPase, knockdown of which induced a synergistic growth-inhibitory effect in HCT116 cells in the presence of IDF-11774. The synthetic lethality of IDF-11774 with ATP6V0C possibly correlates with IDF-11774-mediated autolysosome formation. Notably, the synergistic effect of IDF-11774 and the ATP6V0C inhibitor, bafilomycin A1, depended on the PIK3CA genetic status and Bcl-2 expression, which regulates autolysosome formation and apoptosis. Similarly, in an experiment using conditionally reprogramed cells derived from colorectal cancer patients, synergistic growth inhibition was observed in cells with low Bcl-2 expression.

Conclusions: Bcl-2 is a biomarker for the synthetic lethal interaction of IDF-11774 with ATP6V0C, which is clinically applicable for the treatment of cancer patients with IDF-11774 or autophagy-inducing anti-cancer drugs.

Fig. 1

RNA interference (RNAi) screening to identify candidate genes sensitizing HCT116 cells to IDF-11774. a Knockdown effect of candidate genes on cell growth of HCT116 cells treated with 10 µM IDF-11774. b Effect of ATP6V0C knockdown on the growth of HCT116 cells treated with different concentrations of IDF-11774 for 72 h. Cell viability was analyzed by the sulforhodamine B (SRB) assay. Results are shown as the mean ± S.D. of the three independent experiments. c, d Effect of ATP6V0C knockdown on the growth of HCT116 cells treated with 5 μM IDF-11774 or 5 μM 2-phenylethynesulfonamide (PES). e–g Effect of bafilomycin A1 (BM, 1 nM) on the growth of HCT116 cells treated with 5 μM IDF-11774, 5 μM PES or 5 μM fluorouracil (5-FU). Cell growth was evaluated by live-cell imaging (IncuCyte ZOOM system). Data are presented as the single representative result of three experiments as the mean ± S.D. In each experiment, each treatment was repeated in triplet wells, and each well was analyzed in 4 frames

Fig. 2

Synthetic lethal interaction of IDF-11774 with ATP6V0C in HCT116 cells. a, b Effect of autophagy inhibitor on LC3B accumulation in cells treated with IDF-11774 as analyzed by western blot analysis (a) and immunofluorescence (b). HCT116 cells were pre-incubated with 5 mM 3-methyladenine (3-MA) or 1 nM bafilomycin A1 (BM) for 3 h and then treated with IDF-11774 for 18 h. Scale bar, 10 µm. Western blot band intensities were quantified using ImageJ. The signal intensity of LC3B was normalized to that of GAPDH. c, d Autolysosome formation of cells treated with IDF-11774 and 1 nM bafilomycin A1 (BM). Representative images of confocal fluorescence microscopy (c) and quantification of positive cells for mRFP (red) and mRFP-GFP (yellow) in HCT116 cells transfected with ptfLC3 (d). Positive cells were counted using fluorescence microscopy (n = ~70). Scale bar, 5 µm. e Effect of autophagy inhibitor on autolysosome formation in cells treated with 5 μM IDF-11774 and/or 1 nM bafilomycine A1 for 18 h. Autolysosome formation was assessed by LysoTracker staining. Arrows indicate autolysosomes. Scale bar, 10 µm. f Effect of ATP6V0C knockdown using siATP6V0C on LC3B accumulation in cells treated with 5 μM IDF-11774, as analyzed by western blotting. g, h Effect of ATP6V0C knockdown on autolysosome formation, as analyzed by LysoTracker staining (g) and the ratio of LysoTracker-positive to total HCT116 cells (h). Scale bar, 10 µm

Fig. 3

Synergistic effect of ATP6V0C and IDF-11774 on the growth of various colorectal cancer cells. a Western blot analysis of ATP6V0C levels in human colorectal cancer cells. The signal intensity of ATP6V0C was normalized to that of GAPDH. b Effect of ATP6V0C knockdown on the sensitivity of human colorectal cancer cells to IDF-11774. Cell viability was analyzed using the sulforhodamine B (SRB) assay. Results are shown as the mean ± S.D. of three independent experiments. c LysoTracker staining for autolysosomes in various colorectal cancer cells. LysoTracker-positive cells as observed by fluorescence microscopy. Ratios of LysoTracker-positive to total cells are shown in the graph. Scale bar, 100 µm. d Correlation of mutation status or expression of genes and combination index of two compounds. BM (bafilomycin A1), CCM (concanamycin A), and CQ (chloroquine) were used

Fig. 4

Combined treatment of bafilomycin A1 and IDF-11774 induces apoptosis in colorectal cancer cells. a FACScan analysis of cells treated with IDF-11774 and 1 nM bafilomycin A1 for 72 h by propidium iodide (PI) staining. b Western blot analysis of cells treated with IDF-11774 and 1 nM bafilomycin A1 for 72 h. The signal intensity of each protein was normalized to that of GAPDH. c Scatter-grams of fluorescein isothiocyanate (FITC)-Annexin V/propidium iodide (PI) staining of WiDr and HCT116 cells treated with IDF-11774 and 1 nM bafilomycin A1 for 72 h. d, e Measurement of caspase-3/7 activity using the CellPlayer kinetic caspase-3/7 reagent. Caspase-3/7-positive cells appeared green under a fluorescence microscope. WiDr (d) and HCT116 cells (e) treated with IDF-11774 and 1 nM bafilomycin A1 for 72 h using the IncuCyte ZOOM system. Scale bar, 200 µm. The concentration of IDF-11774 was 5 μM in WiDr and 10 μM in HCT116 cells

Fig. 5

Bcl-2 expression level is crucial for the synthetic lethality of IDF-11774 and bafilomycin A1. a Western blot analysis of Bcl-2 family protein levels in human colorectal cancer cells. The signal intensity of each protein was normalized to that of GAPDH. b–f HCT116 cells transfected with pCMV-SPORT6- BCL2 were treated with 10 µM IDF-11774 and 1 nM bafilomycin A1. b Immunofluorescence of LC3B. Scale bar, 10 µm. c Autolysosome formation. Caspase-3/7 activity (d, e) and cell growth (f) were measured by IncuCyte ZOOM system. Graphs are shown as the mean ± S.D. in different regions (n = 4) of the well. g Evaluation of the change in the interaction between Bcl-2 and Bax induced by bafilomycin A1 using immunoprecipitation assay. HCT116 cells transfected with pCMV3-HA-BCL2 and pCMV3-Myc-BAX were treated with 10 µM IDF-11774 and 1 nM bafilomycin A1. h Combination index of IDF-11774 with bafilomycin A1 in WiDr cells transfected with pCMV-SPORT6-BCL2 and in LoVo cells transfected with siBCL2. i Model depicting Bcl-2-dependent synthetic lethal interaction of IDF-11774 and ATP6V0C

Fig. 6

Regulation of IDF-1174 sensitivity to conditionally reprogramed cells (CRCs) derived from patients with colon cancer. a Western blot analysis of Bcl-2 expression in the CRCs. The signal intensity of each protein was normalized to that of GAPDH. b, c Autolysosome formation in CRCs treated with 10 µM IDF-11774. Scale bar, 200 µm. Bars indicate the ratio of the average of LysoTracker-positive cells in different regions (n = 9) of the well. d, e Growth inhibition of CRCs expressing different levels of Bcl-2 in the presence of 1 nM bafilomycin A1 and 5 µM IDF-11774 or 5 µM chloroquine, as measured using IncuCyte ZOOM system. Data are presented as the single representative result of three experiments and as the mean ± S.D. In each experiment, each treatment was repeated in triplet wells, and each well was analyzed in 4 frames. f Combination index of IDF-11774 with bafilomycin A1 (BM) or chloroquine (CQ) in CRCs