Dual mTORC1/C2 inhibitors suppress cellular geroconversion (a senescence program)

Abstract

In proliferating cells, mTOR is active and promotes cell growth. When the cell cycle is arrested, then mTOR converts reversible arrest to senescence (geroconversion). Rapamycin and other rapalogs suppress geroconversion, maintaining quiescence instead. Here we showed that ATP-competitive kinase inhibitors (Torin1 and PP242), which inhibit both mTORC1 and TORC2, also suppressed geroconversion. Despite inhibition of proliferation (in proliferating cells), mTOR inhibitors preserved re-proliferative potential (RP) in arrested cells. In p21-arrested cells, Torin 1 and PP242 detectably suppressed geroconversion at concentrations as low as 1-3 nM and 10-30 nM, reaching maximal gerosuppression at 30 nM and 300 nM, respectively. Near-maximal gerosuppression coincided with inhibition of p-S6K(T389) and p-S6(S235/236). Dual mTOR inhibitors prevented senescent morphology and hypertrophy. Our study warrants investigation into whether low doses of dual mTOR inhibitors will prolong animal life span and delay age-related diseases. A new class of potential anti-aging drugs can be envisioned.

Keywords: Gerotarget; Pathology Section; aging; pan-mTOR inhibitors; rapalogs; rapamycin; senescence.

Figure 1. Cytostatic and gerosuppressive effects

A. Schema of experiment; B. Cytostatic and gerosupressive effects of deforolimus, Torin 1 and PP242 in HT-p21 cells. Cells were treated with serial dilutions of deforolimus, PP242 or Torin 1 as depicted in schema in (A). To determine cytostatic activity, cells were treated with drugs for 3 days, then drugs were washed out and cells were allowed to recover in drug-free medium. Colonies were stained after 5 days of regrowth. To investigate drug effects on geroconversion, HT-p21 cells were treated with IPTG in the presence of serial dilutions of the drugs. After 3-day treatment, drugs were washed out and colonies were allowed to form for 9 days, then stained and counted in triplicates. Data are mean ± SD.

Figure 2. Gerosuppression and mTOR inhibition

A. Geroconversion. Torin 1 and PP242 were added to IPTG-treated HT-p21 cells, as shown in Figure 1A. Cells plated at low density were treated with IPTG in the presence of serial dilutions of inhibitors. After 3 days, IPTG and inhibitors were washed out and cells were incubated in drug-free medium. Then colonies were stained with Crystal Violet and counted. Data are mean number of colonies per a sector of a well ± SD. B. Immunoblot analysis. HT-p21 cells were treated with IPTG in combination with Torin 1 or PP242 for 24 h and lysed. Rapamycin (R) at 500 nM was included as additional control. Immunoblotting was performed with the indicated antibodies. Note: IPTG-induced p21 is highly expressed in all samples, confirming that the inhibitors do not interfere with p21 induction by IPTG and cell cycle arrest.

Figure 3. Torin 1 and PP242 prevent senescent morphology and hypertrophy in HT-p21 cells induced to senesce by IPTG

A. Cells were treated with IPTG (I) in the presence of Torin 1, PP242 or Rapamycin (R) for 4 days and stained for beta-Gal. Bar – 100 μm. B. Cells were treated with IPTG in combination with Torin 1 (100 nM), PP242 (1000 nM) or rapamycin (500 nM). After 3 days, cells were counted and lysed. Protein concentrations were measured. Data present mean ± SD of ng of protein per cell.

Figure 4. Torin 1 and PP242 suppress geroconversion in human WI38t fibroblasts and SkBr3 cells

A. WI38t cells were treated with either 1 μg/ml etoposide or 50 ng/ml doxorubicin in the absence or presence of 100 nM Torin 1 or 1 μM PP242 for 4 days and stained for beta-Gal. Bar – 100 μm. B. Cells were treated as in (A). After 4-day treatment, cells were extensively washed to remove the drugs and allowed to regrow in drug-free medium and counted after 3 weeks in culture. RP (re-proliferative potential) was calculated by dividing final cell numbers by initially-plated number of cells. Data present mean ±SD from triplicate wells. C. Effect of Torin 1 on senescent morphology of SKBR3 cells undergoing senescence induced by PMA. Cells were pre-treated with Torin 1 (100 nM) for 24 h before addition of 100 nM PMA. After 3-day treatment with PMA, drugs were washed out, cells were incubated in drug-free medium for another 3 days and stained for beta-gal. Bar – 100 μm.