PML at Mitochondria-Associated Membranes Is Critical for the Repression of Autophagy and Cancer Development

Abstract

The precise molecular mechanisms that coordinate apoptosis and autophagy in cancer remain to be determined. Here, we provide evidence that the tumor suppressor promyelocytic leukemia protein (PML) controls autophagosome formation at mitochondria-associated membranes (MAMs) and, thus, autophagy induction. Our in vitro and in vivo results demonstrate how PML functions as a repressor of autophagy. PML loss promotes tumor development, providing a growth advantage to tumor cells that use autophagy as a cell survival strategy during stress conditions. These findings demonstrate that autophagy inhibition could be paired with a chemotherapeutic agent to develop anticancer strategies for tumors that present PML downregulation.

Graphical abstract

Figure 1

PML Represses Autophagic Processes (A) Percentages of GFP-LC3 puncta-positive cells in Pml WT and KO MEFs transfected with the GFP-LC3 plasmid under basal conditions (fed) and after starvation (starved). Bars, SEM. ^∗∗p < 0.01, n = 4. (B) Representative images of GFP-LC3 puncta in MEFs. Scale bar, 10 μm. (C) Conversion of endogenous LC3-I to LC3-II, monitored via immunoblotting in Pml^+/+ and Pml^−/− MEFs cultured in regular medium (fed) or without nutrients (starved). (D) Ultrastructural evidence of higher autophagy levels in MEFs in the absence of PML compared with WT conditions basal conditions (fed) or after serum deprivation (starved). Scale bar, 500 nm. Bars, SEM. ^∗p < 0.05, n = 3. (E) Immunoblotting of endogenous LC3 in the liver and skeletal muscle of fed or starved (24 hr) Pml^+/+ and Pml^−/− mice. (F) Immunoblot of subcellular fractions isolated from Pml^+/+ and Pml^−/− MEFs, where IP3R3 was used as an ER marker, Sigma 1-R as a MAM marker, Cyt c as a mitochondria marker and ATG14/STX17 as autophagosome formation markers. C, cytosol; ER, endoplasmic reticulum; MAMs, mitochondria-associated membranes; Mp, pure mitochondria.

Figure 2

PML Localization at ER/MAM Domains Controls the Levels of Autophagy (A) Percentages of GFP-LC3 puncta-positive cells induced by the transfection of erPML and nuPML chimeras into Pml^−/− MEFs under resting conditions (fed) and after serum deprivation (starved). Bars, SEM. ^∗∗p < 0.01, n = 4. (B) Representative images of GFP-LC3 puncta in Pml^−/− MEFs before and after re-introduction of the two chimeras, erPML and nuPML. Scale bar, 10 μm. (C) Immunoblotting to detect LC3 in PML KO MEFs after the re-introduction of erPML and nuPML chimeras both under basal conditions (fed) and after serum deprivation (starved). (D) Images of autophagic ultrastructures in Pml^−/− MEFs following the transfection of erPML and nuPML chimeras. Scale bar, 500 nm. Bars, SEM. ^∗∗∗p < 0.005, n = 3.

Figure 3

Delocalization of PML from the MAMs Is p53 Dependent (A) Immunoblot detection of PML in p53^−/− MEF fractions. C, cytosol; ER, endoplasmic reticulum; MAMs, mitochondria-associated membranes; Mp, pure mitochondria. (B) Pre-embedding immunogold labeling of PML in p53^+/+ and p53^−/− MEFs. In WT cells, PML localizes mainly at discrete sites in the nucleus (PML nuclear bodies) and on the ER, outer mitochondria membrane (OMM) and MAMs (arrows indicate PML-positive MAMs). In p53^−/− MEFs, while PML still localizes at PML nuclear bodies and on the ER membranes, the association with OMM and in particular the presence at MAMs is reduced (arrows indicate PML negative MAMs). Scale bar, left panel, 1 μm; upper-right panel, 500 nm; lower-right panel, 250 nm. (C and D) The co-localization of PML (red) and Sigma 1-R-EGFP (used as a MAM marker, green) in (C) p53^−/− MEFs and in (D) p53^−/− MEFs following re-introduction of the erPML chimera was analyzed based on immunofluorescence using confocal images. The lower-left panels display the merged image of the two stains. The lower-right panels display the PML signal overlaid with MAMs (MAM boundaries are highlighted in yellow) in a rainbow lookup table (LUT) (MAMsPML: Manders coefficient for PML staining was calculated according to Manders coefficient method as the proportion of PML signal overlapping with the Sigma 1-R marker). Scale bar, 10 μm. (E–G) Reduced levels of autophagy were observed in p53^−/− MEFs following erPML transfection as determined based on the percentage of LC3-GFP puncta (E and F) and on immunoblotting to detect LC3-I conversion into LC3-II (G). Representative images are shown. Bars, SEM. ^∗∗p < 0.01, n = 4. Scale bar, 10 μm. (H) PML and p53 localization following the re-introduction of p53^wt as analyzed via immunoblotting in H1299 p53^−/− cell fractions, where IP3R3 was used as an ER marker, Sigma 1-R as a MAM marker, Tubulin as a cytosolic marker and Cyt c as a mitochondria marker. C, cytosol; ER, endoplasmic reticulum; MAMs, mitochondria-associated membranes; Mp, pure mitochondria. (I and J) Immunofluorescence of Pml (red) and p53 (blue) in H1299 cells after the re-introduction of p53^wt (I) and mutant p53^K382R (J). The lower-left panels display the merged image of the two stains. The lower-right panels display the Pml signal overlaid with MAMs (MAM boundaries are highlighted in yellow) in a rainbow LUT. Scale bar, 10 μm.

Figure 4

PML Modulates Autophagy through the AMPK/mTOR/Ulk1 Pathway in a Ca²⁺-Dependent Manner (A) Immunoblot detection of the phosphorylation status of AMPK, ACCα, p70^S6K, mTOR, and Ulk1 in Pml^+/+ and Pml^−/− MEFs. (B) Detection of autophagy and AMPK-mTOR-Ulk1 phosphorylation levels in the liver and skeletal muscle of Pml^+/+ and Pml^−/− mice. (C) Representative traces of increased mitochondrial Ca²⁺ levels in Pml^−/− MEFs after MCU overexpression. Pml^−/−: [Ca²⁺]_m peak 33.7 ± 2.55; Pml^−/− + MCU: [Ca²⁺]_m peak 59.2 ± 6.22) SEM. ^∗∗p < 0.01, n = 3. (D–F) Quantification of autophagy in Pml^−/− MEFs following MCU overexpression via (D and E) analysis of GFP-LC3 puncta or (F) immunoblotting. Representative images are shown. Bars, SEM. ^∗∗p < 0.01, n = 3. Scale bar, 10 μm. (G) Schematic model of autophagy regulation by PML. In the absence of Pml, the release of Ca²⁺ from the ER into the mitochondria and the production of ATP are reduced. This low-energy status induces AMPK activation, mTOR inhibition, and Ulk1 phosphorylation, leading to increased autophagy.

Figure 5

PML Deletion Favors Cell Survival under Stress Conditions Due to Autophagy Activation (A) Cytosolic ATP levels in Pml^+/+ and Pml^−/− MEFs as measured by luciferase expression under starvation conditions (glucose deprivation for 15 min). ^∗∗p < 0.01, compared to WT conditions, n = 3. (B) Analysis of mitochondrial membrane potential (Ψ_m) as measured by TMRM intensity in Pml^+/+ and Pml^−/− MEFs. Where indicated, cells were deprived of glucose or exposed to 1 μM carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP). On the bottom-right side, representative images of the TMRM signal in the presence or absence of glucose are shown. Normalized TMRM intensity is displayed as a rainbow LUT (statistical analysis cross, average; line, median; box, 25 and 75 percentile; bars, maximum and minimum values; ^∗p < 0.05, n = 3). Scale bar, 10 μm. (C and D) Pml^−/− cells are more resistant to metabolic stress-induced cell death than Pml^+/+ cells as confirmed by (C) immunoblot for apoptotic markers and by (D) cell viability assay. Metabolic stress is induced by glucose deprivation (3 hr for western blot and the indicated hours for cell viability). Bars, SEM. ^∗p < 0.05, ^∗∗∗p < 0.005, n = 3. (E) Basal-dependent (i) and ATP synthase-dependent (ii, + Oligomycin) mitochondrial O₂ consumption rates (OCR) in Pml^−/− and Pml^+/+ MEFs under starvation conditions (glucose, pyruvate, and glutamine deprivation for 1 hr). Bars, SEM. ^∗∗p < 0.01, ^∗∗∗p < 0.005, n = 4.

Figure 6

Selective Degradation of the PML-RARα Oncogenic Fusion Protein in APL Human Cancer Restores a Correct PML Localization and the Sensitivity to Metabolic Stress (A) Quantification of LC3 dots in NB4 cells transfected with the GFP-LC3 plasmid under basal conditions (0 hr) and after ATO (1 μM) treatment. Bars, SEM. ^∗∗p < 0.01, n = 4. (B) Representative images of GFP-LC3 dots showing the effects of ATO on autophagic process in NB4 cells. (C) PML-RARα degradation and LC3-II and p62 accumulation after ATO treatment (1 μM) in NB4 cells. PML-RARα was detected monitoring the band at 110 kDa either with a PML antibody or with a RARα antibody. (D) Analysis of autophagic flux in NB4 cells after ATO treatment (1 μM). (E) Quantification of basal (i) and Oligomycin ATP synthase-dependent (ii) mitochondrial O₂ consumption rates (OCR) either in vehicle or ATO treated NB4 cells under starvation conditions (glucose, pyruvate, and glutamine deprivation for 1 hr). Bars, SEM. ^∗p < 0.05, n = 7. (F) Immunoblot of subcellular fractions isolated from NB4 cells and PML protein quantification at MAM regions and ER normalized to the amount of Sigma 1-R and IP3R3. Where indicated, cells were exposed to ATO treatment (1 μM, 12 hr). IP3R3, Sigma 1-R and Cyt c was used as ER, MAMs, and mitochondria marker, respectively.

Figure 7

Inhibition of Autophagy Augments the Cytotoxicity of Chemotherapy Treatment in Pml KO Tumors (A and B) Pml^+/+ MEFs are sensitive to 5-fluoracil-induced cell death (5-FU, 25 μM for 16 hr), whereas Pml^−/− cells are resistant. Inhibition of autophagy by treatment with 3-MA (2.5 mM for 16 hr) or CQ (5 μM for 16 hr) increases apoptosis induced via chemotherapy treatment with 5-FU (25 μM for 16 hr) in Pml^−/− MEFs. (C) Tumor growth of Pml^−/− and Pml^+/+ transformed MEF xenografts. (D) Representative images of mouse fibrosarcoma xenografts. (E) Increased levels of autophagy in tumors derived from Pml^−/−-transformed MEFs as analyzed by immunoblotting. Tumors were excised on day 19 after inoculation. (F) Analysis of apoptosis based on the intensity of fluorescence (SR-FLIVO) emitted in tumor tissue sections, accompanied by statistical analysis (cross, average; line, median; box, 25 and 75 percentile; bars, maximum and minimum values; ^∗∗∗p < 0.005, n = 3). Scale bar, 50 μm. (G) Increased cytotoxicity-chemotherapy effects on Pml^−/− tumors after autophagy inhibition. (H) Representative histological sections of human colon cancer immunostained for PML and LC3 accompanied by statistical analysis expressed as the percentage of staining intensity. Bars, SEM. ^∗p < 0.05, ^∗∗∗p < 0.005, n = 13. Magnification 20×.