Suppression of mTORC1 activity in senescent Ras-transformed cells neither restores autophagy nor abrogates apoptotic death caused by inhibition of MEK/ERK kinases

mTORC1 suppression does not increase viability of senescent cells treated by MEK/ERK inhibitor

The work was performed using rat embryo fibroblasts, transformed with E1A+cHa-Ras oncogenes (ERas cells). Senescence was induced with histone deacetylase inhibitor sodium butyrate (NaBut, 4 mM). Consistent with our previous data, senescent cells are very sensitive to MEK/ERK inhibition, so treatment with specific MEK1,2 kinase inhibitor PD0325901 leads to a significant decrease of cellular viability and apoptotic death [14]. Senescent cells were unable to complete cytoprotective autophagy in response to MEK/ERK suppression. Given that mTORC1 is a negative regulator of autophagy, we used a specific mTOR kinase inhibitor pp242 in 200 nM concentration to suppress mTORC1 activity. The effect of pp242 on cellular viability is concentration-dependent. While cells tolerate the 200 nm concentration, treatment with 1500 nM leads to a significant decrease of cellular viability (Fig. 1A). 200 nM concentration of pp242 decreases phosphorylation of 4E-BP1, a target of mTORC1, after treatment for 72 h (Fig. 1B). It was shown that mTOR inhibitors (pp242, rapamycin, Torin1,2) decelerate senescence [21]. Low concentration of pp242 (200 nM) was used to suppress mTORC1 but only partially decelerate senescence, as deceleration of senescence program leads to proliferation of cells. Our analysis of senescence markers shows that pp242 at 200 nM concentration causes only a partial decrease of senescence markers according to data on Senescence-Associated β-Galactosidase expression and evaluation of the cell size (Suppl. Fig. 1A,B). Senescent cells are characterized with suppression of proliferation. We analyzed cellular regrowth ability after 72 h of pp242 treatment and showed that senescent cells after mTORC1 suppression demonstrate higher ability to proliferate than untreated senescent cells (Suppl. Fig. 1C). Then we questioned whether mTORC1 suppression would rescue viability of senescent cells exposed to MEK/ERK inhibition. However, mTORC1 suppression does not restore cellular viability of senescent cells upon MEK/ERK suppression, as follows from MTT data (Fig. 1C, D, E).

Figure 1. mTORC1 suppression does not rescue viability of senescent ERas cells exposed to the MEK/ERK inhibitor. (A)Viability of control and senescent ERas cells exposed to mTOR inhibitor pp242 (200 nM, 500 nM, 750 nM, 1500 nM), as assayed by MTT test. (B) Suppression of 4E-BP1 phosphorylation by pp242 in senescent ERas cells monitored by Western-blotting. Numbers below represent densitometry of the bands. (C) Viability of senescent ERas cells exposed to pp242 (200 nM) and MEK/ERK inhibitor PD0325901 (PD, 1 µM) assayed by MTT test. (D) Senescent cells are unable to restore proliferation after MEK/ERK suppression. Cells were exposed to NaBut, PD0325901 and pp242 for 72h then supplemented with a medium without inhibitors for 48 h. Cells were stained with Crystal Violet. (E) Senescent ERas cells undergo apoptosis upon mTORC1 and MEK/ERK suppression. DNA fragmentation analysis in 1,5% agarose gel electrophoresis. Serum- starved ERas (LS) were used as positive control for apoptotic DNA fragmentation.

mTORC1 suppression with 200 nM of pp242 leads to mitochondria damage and increase of lysosomal activity as well as to a transient activation of autophagy

Recent reports data have shown that mitochondrial stress affects lysosomal activity [25,26]. In particular, acute mitochondria damage leads to an increase of lysosomal biogenesis [25]. Using in vivo staining with Mitotracker Orange (potential-dependent) and Lysotracker Green, we checked mitochondrial damage and lysosomal activity in senescent cells upon mTORC1 suppression. Data obtained show that mTORC1 suppression leads to mitochondria damage as manifested by a decrease of Mito-Orange signal and in the same time an increase in the lysosome activity (as follows from data acquired using Lysotracker Green) (Fig. 2).

Figure 2. mTORC1 suppression induces mitochondria damage and an increase of lysosome levels in senescent ERas cells. Mitochondria are stained with Mitotracker Orange, lysosomes are stained with Lysotracker Green and visualized at proper wavelengths. Nuclei were stained with Hoechst33342. Magnification 40x obj + zoom. Graphs below represent intensities of Mitotracker Orange and Lysotracker Green in control and treated cells, measured using ImageJ software.

Mitochondria damage results in induction of autophagy, as demonstrated by an increase of LC3 I to LC3 II conversion after treatment with pp242 for 4 h (Fig. 3A). To analyze the completeness of autophagy induced by mTORC1 inhibition, we transfected cells with a plasmid that encodes LC3 fused with GFP and mRFP (Addgene #21074). When autophagosomes fuse with lysosomes, GFP-LC3 fluorescence vanishes due to acidification [27], thus allowing to distinguish autophagosomes from autophagolysosomes and to estimate the completeness of autophagic process. In control cells, both GFP and RFP signals distribute diffusely in the cytoplasm (Fig. 3B). Given that GFP signal disappears, whereas the clamps of mRFP arise after mTORC1 suppression with pp242 for 4 h, this evidence that the autophagosomes are formed and fuse with the lysosomes. However, later (24 h - 72 h) the number of mRFP clamps decreases, implying that formation of autophagosomes is attenuated (Fig. 3B).

Figure 3. mTORC1 inhibition induces an autophagy flux, which then terminates 24 h thereafter. (A) Western-blot analysis of autophagy markers and its modulators: LC3 I and II forms, phospho-Ulk1 (Ser757, Ser555), phospho-AMPK (Thr172) as well as ERK1,2 phosphorylation. (B) Expression of GFP-mRFP-pft-LC3 vector in senescent cells exposed to pp242 for 4, 24, 72 h visualizing formation of autophagosomes and fusion of autophagosomes with lysosomes. The nuclei stained with DAPI. Histogram presents the average number mRFP foci (autophagosomes fused with lysosomes) per cell after 4, 24, 72 h of mTORC1 inhibition.

ERas cells treated with mTOR inhibitor cease autophagy and eliminate damaged mitochondria through an alternative pathway

Given that upon 24 h of mTORC1 suppression autophagy is disrupted, the cells fail to degrade the damaged mitochondria. To find out how the damaged mitochondria are degraded, we performed Transmission Electron Microscopy (TEM) as well as in vivo staining with Mito-tracker Green (binds to all mitochondria) and Mitotracker Orange (binds only non-damaged mitochondria that retain their membrane potential). For IF staining, antibodies against lysosomal protein (LAMP1) and transmembrane protein on the outer membranes of mitochondria (TOM20) were used. TEM data showed abundance of damaged mitochondria with severe disorders of cristae in senescent cells with suppressed mTORC1 (Fig. 4C, D, F). However, after 72 h the vacuoles containing numerous membrane structures can be seen, pointing that the damaged mitochondria concentrate in the vacuoles (Fig. 4C, D, F). As shown by Mitotracker Green staining data as well from data acquired in the combined staining with Mitotracker Orange and Lysotracker Green, these mitochondria are in fact damaged. This follows from the results that no Mitotracker Orange signal is revealed in the vacuoles, where LysoGreen clamps reside. Onwards, TEM images show that cells exhibit intense exfoliations at their edges, where some membrane structures, probably, mitochondria remnants can be seen (Fig. 4E, F). Nevertheless, in the cytoplasm outside of the clamps normal mitochondria are visualized, as Mitotracker Orange intensity, that was decreased after 24 h of treatment, returns almost to the control values after 72 h, implying the recovery of functioning mitochondria located outside of the vacuoles. Senescent cells exhibit an increase of the oxygen species (ROS) levels. Mitochondria damaged upon mTORC1 suppression are a source of ROS. However, after 72 h of mTORC1 inhibition the ROS levels become lower as compared with senescent cells retaining active mTORC1, thereby indicating that there is a rescue mechanism that counters accumulation of ROS through isolation of the damaged mitochondria (Fig. 4H).

Figure 4. Senescent cells segregate damaged mitochondria into LC3-negative vacuoles after mTORC1 suppression. (A) IF images showing segregation of damaged mitochondria in the specific LC3-negative vacuoles in senescent cells exposed to mTOR inhibitor for 72 h. Upper panel: in vivo staining with Mitotracker Green and Mitotracker Orange visualized with the Leica TSC Microscope, 40x obj + zoom. Nuclei stained with Hoechst 33342. White arrow points the damaged mitochondria (Mitotracker Green only) inside of the vacuoles. Bottom panel: IF pictures after staining with antibodies against TOM20 and LAMP1 taken on the Olympus Fluoview 3000, 60x obj. Nuclei stained with DAPI. Arrow points the mitochondria colocalized with the lysosomes. (B) TEM image of senescent ERas cell with active mTORC1 (72 h) exhibiting the non-damaged mitochondria (m) and the enriched Golgi complex (G). (C) TEM image of senescent ERas cell treated with mTOR inhibitor pp242 for 72 h exhibiting the damaged mitochondria (m). Inset presents normal non-damaged mitochondria of untreated, non-senescent ERas cell. (D) Accumulation of membranous structures (mitochondria remnants, m) in the vacuole of senescent cell upon mTORC1 suppression. (E) Accumulation of lysosomes (Lys) in the vacuole of senescent cell upon mTOR suppression. (F) The vacuole containing lysosomes and membrane structure close to the plasma membrane of senescent ERas cell treated with mTOR inhibitor. (G) Membrane and electron-dense structures excreted from the cell. Scale bars in TEM images: 1 µm; inset (B) – 0,5 µm. (H) The level of reactive oxygen species (ROS) in senescent cells after 72 h of mTOR suppression measured using DCF-DA at proper wavelength.

IF analysis with antibodies against a lysosome marker LAMP1 showed that these vacuoles contains LAMP1 positive clamps, implying that lysosomes also accumulate therein (Fig. 5A). The results of immunofluorescence are confirmed by data on morphological Giemza staining that show acidic inclusions within the vacuoles of the cells as well as outside of the cells (Fig. 4B). LAMP1 immunofluorescence shows that after mTORC1 suppression with pp242 for 72 h the LAMP1 and TOM20 fully colocalizes in the clamps that is a direct evidence for aggregation of mitochondria and lysosomes within the large vacuoles (Fig. 4A). Transmission electron microscopy also revealed big vacuoles filled with lysosome-like cargo, thereby confirming data acquired with Lyso-tracker Green and LAMP1 staining (Fig. 4E). However, no LC3 signal is observed inside of the vacuoles together with LAMP1. Moreover, the senescent ERas cells transfected with LC3-GFP/RFP plasmid (ERas-LC3 cells) and treated with pp242 demonstrate neither GFP-LC3, nor mRFP-LC3 aggregations (Fig. 3B). This proves that the vacuoles are devoid of autophagosomes and autophagolysosomes. Analysis of p62/SQSTM1 shows its presence is discovered in the vacuoles, but not in co-localization with the LC3 (Fig. 5C). In case of canonical autophagy, when autophagic process is successfully finished, p62/SQSTM1 degrades together with the autophagolysosomal cargo. In senescent cells with suppressed mTORC1 activity, autophagy is interrupted, therefore, p62/SQSTM1 does not degrade and redistributes to the vacuoles. Thus, mTORC1 suppression with low doses of pp242 induces mitochondrial damage in senescent cells, but the cells get rescued by a special mechanism when damaged mitochondria are placed in the vacuoles together with the lysosomes. However, upon increased concentration of pp242 (1500 nM) the cells fail to implement this mechanism and die by apoptosis (Fig. 6A, B, C). Data obtained are consistent with the fact that viability of Ras-expressing cells is dependent on mitochondria, and processes observed in cells treated with low concentrations of pp242 result in elimination of damaged mitochondria. We’ve applied mTORC1 inhibitor rapamycin that doesn’t cause mitochondria damage, according to our previous works. Senescent cells treated with rapamycin don’t reveal vacuole formation as cells treated with 200 nM pp242 (Fig. 6D). The absence of mitochondria damage in cells treated with rapamycin might be explaining the differences in responses to pp242 and rapamycin.

Figure 5. Senescent pp242-treated cells with disrupted autophagy form the LC3-negative cavities where lysosomes and p62/SQSTM1 accumulate. (A) IF analysis of LC3 and lysosome (LAMP1) localization in senescent cells and mTORC1- suppressed senescent cells. Arrows show LAMP1 clamps in the LC3-negative vacuole (left image) and outside of the cell (right image). Nuclei stained with DAPI. (B) Morphology of senescent cells after 72 h of mTORC1 inhibition. Arrow shows acidic clamps secreted from the cells. (C) P62/SQSTM1 accumulates in vacuoles of senescent cells exposed to pp242. Nuclei stained with DAPI.

Figure 6. Senescent cells exposed to high concentration of mTOR inhibitor pp242 (1500 nM) fail to rescue themselves by forming the LC3-negative vacuoles and undergo cell death. (A) IF pictures after staining with antibodies against TOM20 and LAMP1. The mitochondria and lysosomes are segregated in the vacuoles of cells exposed to 200 nM pp242, but not to 1500 nM pp242. Nuclei stained with DAPI. (B) IF with antibodies against LAMP1 and LC3. (C) Viability of senescent ERas cells treated with 1500 nM pp242 and assayed by MTT test. (D) IF with antibodies against LAMP1 and LC3 showing the absence of the mitochondria and lysosomes-containing vacuoles after mTORC1 suppression with 200 nM of rapamycin.

Several groups reported that mTORC1 is localized on the surface of lysosomes [7]. IF analysis of senescent cells shows that after pp242 treatment for 72 h a small part of mTOR is detected in the vacuoles together with the lysosomes (Fig. 7A). Besides, mTORC1-lysosome aggregates can be seen outside the cells, and the total amount of mTOR decreases, according to Western-blot data (Fig. 7B). However, the most part of mTOR remains around the nucleus outside of the LAMP1-positive vacuoles, and distribution of the mTOR signals are clearly uncoupled from the LAMP1 signals (Fig. 7A). Therefore, mTORC1-lysosome interaction is weakened. Main negative regulator of mTORC1 – TSC2 protein, was also found in the vacuoles. Thus, the formation of the large vacuoles, where damaged mitochondria and lysosomes are segregated and then removed from the cell, can be a novel rescue mechanism. According to MTT data, this mechanism provides nearby 70% survival after 120h of treatment.

Figure 7. mTOR inhibition with pp242 uncouples mTOR from the lysosomes. (A) IF images showing pTSC2 (upper panel), mTOR (bottom panel) and lysosome (LAMP1) localization in senescent cells treated with pp242. Arrows show segregation of mTOR and pTSC2 (upper) and mTOR together with lysosomes (lower) in vacuoles and outside of cells. Nuclei stained with DAPI. (B) Western-blot analysis of mTOR levels in senescent cells after 72 h of pp242 treatment. The numbers represent densitometry of the bands.

Senescent cells require active MEK/ERK to implement non-autophagous rescue pathway

Ras-transformed cells exhibit constitutive activity of downstream Raf/MEK/ERK pathway. According to the Western-blot data, MEK/ERK activity retains on a high level in senescent cells regardless of mTORC1 activity (Fig. 3A). IF data show that senescent cells with suppressed MEK/ERK (NaBut + PD0325901) do not reveal accumulation of mitochondria and lysosomes in the content of the large vacuoles (Fig. 8A). Suppression of mTORC1 in senescent cells with inhibited MEK/ERK activity does not lead neither to restoration of autophagy nor to the large vacuole formation. This result is supported by TEM that shows the damaged mitochondria are distributed within the cell, but no vacuoles do appear (Fig. 8B, C). As follows from the MTT, 120 h treatment triggers almost 90% of cells to undergo apoptosis, thereby pointing out that senescent cells are more sensitive to MEK/ERK inhibitor than to mTOR inhibitor. One may conclude that active MEK/ERK pathway in ERas cells is indispensable to implement the mechanism of the LC3-lacking vacuole formation.

Figure 8. Senescent cells require active MEK/ERK pathway to implement degradation of the damaged mitochondria through their segregation into the LC3-negative vacuoles. (A) Cells exposed to MEK/ERK inhibitor do not segregate of lysosomes in the LC3-negative vacuoles (upper panel). IF images after staining with antibodies against LC3 and LAMP1) or mitochondria (bottom panel), IF images after staining with antibodies against TOM20 and LAMP1) of the LC3-negative vacuoles. (B) TEM of a senescent cell after MEK/ERK suppression undergoing complete destruction of the cytoplasm without segregation and elimination of the damaged mitochondria. (C) TEM image of senescent cells exposed to pp242 + PD0325901 showing destruction of the cytoplasm and dispersed damaged mitochondria. Scale bars: 1 µm.

Cells

Rat embryonic fibroblasts were co-transformed with E1A region of Ad5 DNA and human cHa-Ras oncogene (Q12 and G61 mutated). Cells were cultured in DMEM with 10% FBS (HyClone) in 5% CO2 at 37 ^°C. Histone deacetylase inhibitor sodium butyrate (NaBut, 4 mM, Sigma) was used to induce senescence. For mTOR suppression, specific kinase inhibitor pp242 was used in 200nM concentration. PD0325901 (PD, 1 µM, Sigma) was used to inhibit MEK1,2 activity and ERK1,2 phosphorylation.

Cell viability

For MTT test cells were seeded at 12-well plate at initial density 2x10⁴ cells per well and treated with inhibitors for indicated time. Then medium was changed for 0,5 mg/ml MTT solution for 1h at 37C. Then formazan produced was dissolved in DMSO and measured at 572 nm wavelength. For regrowth analysis, cells were seeded on 12-well plates, treated with inhibitors for 72h, then supplemented with fresh medium without inhibitors. After 48h of cultivation cells were stained with Crystal Violet.

Cellular transfection

Cells were transfected with pft-LC3 plasmid (Addgene #21074) using Lipofectamine 2000 as prescribed by manufacturer (Thermo Fischer, 11668027).

Nucleosomal DNA fragmentation assay

Cells were seeded at 100mM dishes and treated with inhibitors, then lysed in TES buffer containing 10 mM Tris-HCl (pH 8,0), 0,1M EDTA (pH 8,0) and 0,5% SDS, supplemented with RNAse A and NaCl at 37C, then left overnight with Proteinase K and SDS. Next day DNA was deproteinized by a mixture of water-saturated phenol pH 8.0/chloroform/izoamyl alcohol. DNA was reprecipitated, washed with 70% of ethanol, dried and dissolved in 1 mM TE buffer pH 8.0. DNA was subjected to electrophoresis in 2% agarose gel (Sigma). DNA bands were stained with ethidium bromide. DNA isolated from ERas cells starved for 24 h in 0.5% FBS undergoing apoptosis [40] was used as a positive control.

Immunofluorescence

Cells were seeded at coverslips and treated for indicated time, that fixed, permeabilized with 0,2% TRITON X100, blocked in 3% BSA in TBS-T, and incubated in primary antibodies overnight at +4°C following the incubation with secondary antibodies for how long and what temperature. Coverslips were mounted with DAPI-containing ProLong Gold mounting medium (Molecular Probes, P36931). Cells were visualized using Olympus Fluoview 3000 microscope (Olympus).

Western blotting

Cells were lysed in RIPA buffer (1% IGEPAL, 0,5% Sodium deoxycholate; 0,1% SDS; 50 mM TRIS-HCl pH 8,0; 150 mM sodium chloride; 5 mM EDTA pH 8,0; 60 mM sodium fluoride) + the Roche Protease inhibitor cocktail. 50 µg of protein were separated by SDS-PAGE electrophoresis in 12,5%, 12% or 10% gels and transferred to PVDF membranes (0,2 and 0,45 µM, EMD Millipore). Membranes were blocked with 5% fat-free milk (Sigma) for 1 h following the incubation with primary antibodies in TBS solution containing 3% BSA and 0,1% Tween-20 overnight at 4°C. The membranes were washed and incubated with secondary antibodies conjugated with horseradish peroxidase. Blots were developed by enhanced chemiluminescence (ECL, ThermoFisher Scientific). Densitometry was performed using GelPro3 Software (Media Cybernetics).

Lysosome detection by Lysotracker Green

For lysosome detection, Lysotracker Green was used as prescribed by manufacturer (Thermo Fischer Scientific, L7526). Nuclei were stained with Hoechst33342 (Thermo Fischer Scientific, 62249). Images were obtained using Leica TSC5 microscope (Leica Microsystems) and analyzed using ImageJ software.

Mitotracker Orange and Mitotracker Green staining for detection of mitochondrial integrity

Cells were seeded on coverslips and treated with the inhibitors for indicated time, then stained with Mitotracker Orange and Mitotracker Green (Invitrogen, M7510 and M7514) as described by manufacturer. The intensity of fluorescence was visualized using Leica TCS SP5 microscope (Leica Microsystems) at following wavelengths: Ex/Em=577/599 nm for Mitotracker Orange, 490/516 nm for Mitotracker Green. Images were analyzed using ImageJ software.

ROS levels

Cell were treated with inhibitors for the indicated time points and then incubated with 10 μM 2′-7′-dichlorodihydrofluorescene diacetate (DCFDA, Invitrogen D399) for 30 min. DCF-DA fluorescence was measured using FLUOstar Omega (BMG Labtech) microplate reader and normalized to cell number.

Senescence-associated β-galactosidase detection

Sa-β-Gal expression was analyzed as previously described [41]. Images were acquired using LSM5 Pascal microscope (Carl Zeiss Microscopy).

Cell size

Cell were seeded on 60mm dishes and treated with inhibitors for 72h, then collected and forward scattering was measured using Odam (Brucker, France) flow cytometer.

Transmission electron microscopy

Cells adhered to the coverslips were mechanically detached and fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.2-7.4, for 1 h at 4°C, postfixed in 1% aqueous OsO4 for 1 h, dehydrated, and embedded in Epon and Araldit, and then sectioned with a diamond knife on a LKB-ultratome (Sweden). Ultrathin sections were collected on fine mesh copper grids, and stained with uranyl acetate and lead citrate for examination with a Zeiss Libra 120 electron microscope (Carl Zeiss, Germany) at an accelerating voltage of 80 kV.

Statistical analysis

Confocal images were analyzed using ImageJ software; densitometry of Western Blotting was performed using free distributed GelPro Analyzer software. Data are presented as means ± S.E.M of three independent experiments.

Antibodies

The following primary antibodies were used: pan-LC3 (MBL, #PM036); LAMP1 (Santa-Cruz, sc-17768); phospho-Ulk1 Ser757 (Cell Signaling, #6888S); phospho-Ulk1 Ser555 (Cell Signalling, #5869); Tom20 (Santa-Cruz, sc-17764); phospho-AMPK T172 (Cell Signaling, #2535S); phospho-4E-BP1 Thr37/46 (Cell Signaling, #2855S); phospho-42/44 MAPK Thr202/Tyr204 (Cell Signaling, #4377S); mTOR (Cell Signaling, #2983S); pTSC2 (Cell Signalling, #3617), GAPDH (Cell signaling, #2118), Tubulin (Sigma, T5168). The following secondary antibodies were used: Goat-anti-Rabbit IgG (H+L) Alexa Flour 488 (Invitrogen, A11088); Rabbit-anti-Mouse IgG (H+L) Alexa Fluor 568 (Invitrogen, A11031); Goat-anti-Rabbit IgG HRP Conjugated (Sigma-Aldrich, A0545); Rabbit-anti-Mouse IgG HRP Conjugated (Sigma-Aldrich, A9044).