LAMTOR1 depletion induces p53-dependent apoptosis via aberrant lysosomal activation

Abstract

Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.

Figure 1

LAMTOR-1 depletion induces an increase in both p53 and p21 expression. SHEP cells were transfected with scrambled or LAMTOR1 (B) siRNAs, and viable cells were counted (a) or stained with BrdU and PI for cell cycle analysis (b), 72 h after transfection. Values are means+S.E.M. of three independent experiments. **P<0.01 using Mann–Whitney test. (c) FACS analysis of Annexin V/PI staining of SHEP cells transfected with the indicated siRNAs as above and treated or not with zFA (20 μM) or zVAD (20 μM) for 72 h; n=3. **P<0.01 using Mann–Whitney test. (d) SHEP cells transfected with scrambled or LAMTOR1 (A or B) siRNAs were analyzed for expression levels of P53, P21, LAMTOR1 and actin proteins by western blotting; n=5

Figure 2

LAMTOR-1 depletion induces p53-mediated apoptosis. (a) Representative FACS analysis of Annexin V/PI staining of HCT116 cells transfected with scrambled or LAMTOR1 (B) siRNAs for 36 h; n=3. **P<0.01 using Mann–Whitney test. (b) HCT116 cells transfected with scrambled or LAMTOR1 (B) siRNAs for 36 h were analyzed for expression levels of P53, P21, LAMTOR1 and actin proteins by western blotting; n=4. (c) MCF10-A cells were transfected with scrambled shRNA plasmid (shCtrl) or shRNA plasmids targeted against Rictor or Raptor. The corresponding protein expression levels are shown in the left panel. These cells further transfected with scrambled or LAMTOR1 (A) siRNAs for 72 h were analyzed for expression levels of P53, P21, LAMTOR1 and actin proteins (right panel); n=3

Figure 3

LAMTOR1 depletion induces lysosomal expansion. (a) Electron microscopy of SHEP cells transfected with scrambled siRNA (top panels), or with LAMTOR1 siRNA B (middle and bottom panels). Scale bars are indicated. Autophagosomes (white arrows), autolysosomes (black arrows) and lysosomes (arrow heads). (b) Confocal images of representative cathepsin D immuno-staining in SHEP cells transfected with scrambled or LAMTOR1 (B) siRNAs. Scale bar= 10 μm. (c) SHEP cells were transfected with scrambled or LAMTOR1 (B) siRNAs for the indicated times and the expression levels of P53, P21, mature cathepsin D, LAMP-1 and LAMTOR1 proteins were analyzed by western blotting; n=3. (d) SHEP cells transfected with scrambled or LAMTOR1 (B) siRNAs for 72h, were further treated or not during the last 6 h with 20 μM CQ to inhibit lysosomal activity. Expression levels of LC3-I, LC3-II, P62, LAMTOR1 and actin proteins were analyzed by western blotting; n=4

Figure 4

The increased lysosomal activity generates ROS. (a) Representative FACS histograms of H₂DCF-DA analysis of scrambled or LAMTOR1 (B) siRNA-transfected SHEP cells for 72 h, treated or not with 2.5 μM of CQ; n=5. (b) LAMTOR1 (B) siRNA-treated SHEP cells were treated with indicated concentrations of CQ for 72 h. The maturation process of cathepsin D was analyzed by western blotting. Pro, pro-cathepsin D; Int, intermediate form of cathespsin D; Mat, mature form of cathespsin D; n=2 (c) Untransfected or transfected SHEP cells with the indicated siRNAs (LAMTOR1 siRNA B) for 72 h, were analyzed for expression levels of cathepsin D, LC3-I, LC3-II, p62, Beclin1, LAMTOR1, and actin by western blotting n=2. (d) Representative FACS histograms of H₂DCF-DA analysis of SHEP cells transfected with the indicated siRNAs for 72 h; n=5

Figure 5

ROS are causative in LAMTOR-1 depletion-induced cell death. (a) Representative FACS analysis of Annexin V/PI-stained cells. SHEP cells transfected with scrambled or LAMTOR1 (A) siRNAs were treated or not with 2.5 μM of CQ for 72 h; n=3. **P<0.01 using Mann–Whitney test. (b) SHEP cells transfected with the indicated scrambled or LAMTOR1 (B) siRNAs, were treated or not with anti-oxydant TROLOX at the indicated concentrations, for 72 h. The expression levels of P53, P21, cathepsin D, LAMTOR1 and actin were measured by western blotting; n=3. (c) SHEP cells transfected with scrambled or LAMTOR1 (B) siRNAs were treated or not with TROLOX (500 nM) for 72 h, and viable cells were counted. Values are mean+S.E.M. of three independent experiments. **P<0.01 using Mann–Whitney test