Tumor promoter-induced cellular senescence: cell cycle arrest followed by geroconversion.

Olga V Leontieva,Mikhail V Blagosklonny

doi:10.18632/oncotarget.3011

Abstract

Phorbol ester (PMA or TPA), a tumor promoter, can cause either proliferation or cell cycle arrest, depending on cellular context. For example, in SKBr3 breast cancer cells, PMA hyper-activates the MEK/MAPK pathway, thus inducing p21 and cell cycle arrest. Here we showed that PMA-induced arrest was followed by conversion to cellular senescence (geroconversion). Geroconversion was associated with active mTOR and S6 kinase (S6K). Rapamycin suppressed geroconversion, maintaining quiescence instead. In this model, PMA induced arrest (step one of a senescence program), whereas constitutively active mTOR drove geroconversion (step two). Without affecting Akt phosphorylation, PMA increased phosphorylation of S6K (T389) and S6 (S240/244), and that was completely prevented by rapamycin. Yet, T421/S424 and S235/236 (p-S6K and p-S6, respectively) phosphorylation became rapamycin-insensitive in the presence of PMA. Either MEK or mTOR was sufficient to phosphorylate these PMA-induced rapamycin-resistant sites because co-treatment with U0126 and rapamycin was required to abrogate them. We next tested whether activation of rapamycin-insensitive pathways would shift quiescence towards senescence. In HT-p21 cells, cell cycle arrest was caused by IPTG-inducible p21 and was spontaneously followed by mTOR-dependent geroconversion. Rapamycin suppressed geroconversion, whereas PMA partially counteracted the effect of rapamycin, revealing the involvement of rapamycin-insensitive gerogenic pathways. In normal RPE cells arrested by serum withdrawal, the mTOR/pS6 pathway was inhibited and cells remained quiescent. PMA transiently activated mTOR, enabling partial geroconversion. We conclude that PMA can initiate a senescent program by either inducing arrest or fostering geroconversion or both. Rapamycin can decrease gero-conversion by PMA, without preventing PMA-induced arrest. The tumor promoter PMA is a gero-promoter, which may be useful to study aging in mammals.

Highlights

IntroductionThe mTOR (Target of Rapamycin) signaling pathway is activated by nutrients (glucose, amino and fatty acids), growth factors, cytokines, oxygen, hormones and many other signals [1-4]
The mTOR (Target of Rapamycin) signaling pathway is activated by nutrients, growth factors, cytokines, oxygen, hormones and many other signals [1-4]
We found that PMA caused a large flat morphology with nucleoli enlargement and beta-Gal positivity (Fig. 1 A)

Summary

Introduction

The mTOR (Target of Rapamycin) signaling pathway is activated by nutrients (glucose, amino and fatty acids), growth factors, cytokines, oxygen, hormones and many other signals [1-4]. If the cell cycle is arrested, mTOR drives “futile growth” or geroconversion, converting reversible arrest to irreversible senescence [5, 20-22]. Serum withdrawal deactivates mTOR and MEK/MAPK pathways, causing reversible quiescence in normal cells [20, 26, 34-36]. Rapamycin (and other rapalogs), certain tumor suppressors, including p53, serum-withdrawal, hypoxia and contact inhibition all suppress geroconversion by deactivating mTOR [19, 28, 59-71], maintaining quiescence instead. Growth factor receptors, Ras, Raf, MEK, PI3K and Akt, which all activate the mTOR/S6K/S6 pathway, are involved in cellular senescence and cancer [72-76]. They are gerogenes, driving gerogenic conversion and oncogenic transformation [21, 64]. Phorbol ester is the most well known tumor promoter, which activates MEK/ERK and mTOR/S6K signaling pathways [77-85]

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Results

Conclusion