mTOR inhibitors blunt the p53 response to nucleolar stress by regulating RPL11 and MDM2 levels

Abstract

target of rapamycin; MTT, (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide); PI, propidium iodide Mechanistic target of rapamycin (mTOR) is a master regulator of cell growth through its ability to stimulate ribosome biogenesis and mRNA translation. In contrast, the p53 tumor suppressor negatively controls cell growth and is activated by a wide range of insults to the cell. The mTOR and p53 signaling pathways are connected by a number of different mechanisms. Chemotherapeutics that inhibit ribosome biogenesis often induce nucleolar stress and activation of p53. Here we have investigated how the p53 response to nucleolar stress is affected by simultaneous mTOR inhibition in osteosarcoma and glioma cell lines. We found that inhibitors of the mTOR pathway including rapamycin, wortmannin, and caffeine blunted the p53 response to nucleolar stress induced by actinomycin D. Synthetic inhibitors of mTOR (temsirolimus, LY294.002 and PP242) also impaired actinomycin D triggered p53 stabilization and induction of p21. Ribosomal protein (RPL11) is known to be required for p53 protein stabilization following nucleolar stress. Treatment of cells with mTOR inhibitors may lead to reduced synthesis of RPL11 and thereby destabilize p53. We found that rapamycin mimicked the effect of RPL11 depletion in terms of blunting the p53 response to nucleolar stress. However, the extent to which the levels of p53 and RPL11 were reduced by rapamycin varied between cell lines. Additional mechanisms whereby rapamycin blunts the p53 response to nucleolar stress are likely to be involved. Indeed, rapamycin increased the levels of endogenous MDM2 despite inhibition of its phosphorylation at Ser-166. Our findings may have implications for the design of combinatorial cancer treatments with mTOR pathway inhibitors.

EBF1-Mediated Upregulation of Ribosome Assembly Factor PNO1 Contributes to Cancer Progression by Negatively Regulating the p53 Signaling Pathway

“…Therefore, further study is needed to explore precisely how PNO1 knockdown activates p53. The ribosome protein RPL11 binds to MDM2 and inhibits its ubiquitin ligase activity toward p53, resulting in p53 accumulation (21,39,45). We therefore detected the binding of RPL11 and MDM2 in HCT116 after PNO1 knockdown and found that this knockdown significantly increased the binding of RPL11 to MDM2, suggesting that ribosomal stress increases RPL11 binding to MDM2 and decreases ubiquitination and degradation of p53; this may contribute to the accumulation of p53.…”

mTOR: An attractive therapeutic target for osteosarcoma?

“…The different conditions of cell living in between pre-clincal test and clinical study and the distrinct type of drug combined with mTOR inhibitor may lead to dissatisfied result. Moreover, nucleolar stress, induced by chemotherapeutic drugs, stimulates p53-dependent signaling pathways which contribute to cell cycle arrest, apoptosis, and mTOR inhibitor can alleviate this p53 response to nucleolar stress [79-85]. The cross-linking of p53-dependent signaling pathways and mTOR pathway may explain this inconsistent result.…”

Reduced Expression of PROX1 Transitions Glioblastoma Cells into a Mesenchymal Gene Expression Subtype

“…For analysis of detergent-soluble proteins, we used 0.5% Nonidet P-40 lysis buffer with complete protease and phosphatase inhibitors (PhosSTOP, cOmplete ULTRA; Roche). Analysis of detergent-soluble proteins, coimmunoprecipitation (co-IP), and IB were conducted as described (33). For co-IP ("large-scale") followed by mass spectrometry, the cells were harvested and prepared according to the nuclear complex co-IP kit instructions (Nuclear Complex Co-IP Kit; Nordic Biolabs), using the high stringency buffer option (Active Motif).…”