Carroll, B. , Nelson, G., Rabanal-Ruiz, Y., Kucheryavenko, O., Dunhill- Turner, N. A., Chesterman, C. C., Zahari, Q., Zhang, T., Conduit, S. E., Mitchell, C. A., Maddocks, O. D. K., Lovat, P., von Zglinicki, T., & Korolchuk, V. I. (2017). Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing. Journal of Cell Biology, 216(7), 1949-1957. https://doi.org/10.1083/jcb.201610113 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1083/jcb.201610113 Link to publication record in Explore Bristol Research PDF-document This is the final published version of the article (version of record). It first appeared online via The Rockefeller University Press at DOI:10.1083/jcb.201610113. Please refer to any applicable terms of use of the publisher. University of Bristol - Explore Bristol Research General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/ebr-terms/ Published Online: 31 May, 2017 | Supp Info: http://doi.org/10.1083/jcb.201610113 JCB: Report Downloaded from jcb.rupress.org on March 20, 2019 Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing Bernadette Carroll,1 Glyn Nelson,1 Yoana Rabanal-Ruiz,1 Olena Kucheryavenko,1 Natasha A. Dunhill-Turner,1 Charlotte C. Chesterman,1 Qabil Zahari,1 Tong Zhang,3 Sarah E. Conduit,4 Christina A. Mitchell,4 Oliver D.K. Maddocks,3 Penny Lovat,2 Thomas von Zglinicki,1 and Viktor I. Korolchuk1 1Institute for Cell and Molecular Biosciences and 2Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK 3Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, Scotland, UK 4Cancer Program, Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia Mammalian target of rapamycin complex 1 (mTORC1) and cell senescence are intimately linked to each other and to organismal aging. Inhibition of mTORC1 is the best-known intervention to extend lifespan, and recent evidence suggests that clearance of senescent cells can also improve health and lifespan. Enhanced mTORC1 activity drives characteristic phenotypes of senescence, although the underlying mechanisms responsible for increased activity are not well under- stood. We have identified that in human fibroblasts rendered senescent by stress, replicative exhaustion, or oncogene activation, mTORC1 is constitutively active and resistant to serum and amino acid starvation. This is driven in part by depolarization of senescent cell plasma membrane, which leads to primary cilia defects and a resultant failure to inhibit growth factor signaling. Further, increased autophagy and high levels of intracellular amino acids may act to support mTORC1 activity in starvation conditions. Interventions to correct these phenotypes restore sensitivity to the mTORC1 signaling pathway and cause death, indicating that persistent signaling supports senescent cell survival. Introduction Cellular senescence is an irreversible cell cycle exit that is a key tor of mTORC1 (Dibble and Cantley, 2015). TSC2 localization tumor suppressor mechanism and also directly contributes to to the lysosome, and therefore Rheb activity, is controlled by aging (López-Otín et al., 2013). Indeed, clearance of senescent availability of growth factors and amino acids, specifically ar- cells can improve aging phenotypes (Baker et al., 2011, 2016). ginine, (Demetriades et al., 2014; Menon et al., 2014; Carroll Senescence is characterized by proliferation arrest, increase in et al., 2016). Amino acids further regulate mTORC1 activity cell size and mitochondrial mass together with mitochondrial by controlling its localization at the lysosome via the signal- dysfunction, and increased secretion of proinflammatory and ing cascade upstream of Ragulator complex and Rag GTPases pro-oxidant signals (Passos et al., 2007, 2010; Rodier et al., (Laplante and Sabatini, 2012). Starvation of growth factors or 2009; López-Otín et al., 2013). This increase in cell growth amino acids inhibits mTORC1 and activates autophagy. Au- and metabolism is supported in part by mTORC1 (Zhang et tophagy involves the engulfment of cytoplasmic contents into THE JOURNAL OF CELL BIOLOGY CELL OF JOURNAL THE al., 2000; Demidenko and Blagosklonny, 2008; Carroll et al., double membrane–bound vesicles called autophagosomes, 2013; Xu et al., 2013; Herranz et al., 2015; Correia-Melo et which fuse with lysosomes, degrading their contents, which are al., 2016), a conserved serine/threonine kinase that specifically subsequently released into the cytoplasm (Carroll et al., 2015). regulates protein translation and nucleotide and lipid biogenesis Starvation therefore shifts the cell from an anabolic to a cata- and inhibits the catabolic process of autophagy (Laplante and bolic program to liberate nutrients and ensure cell survival. Sabatini, 2012; Carroll et al., 2015). mTORC1 activity promotes senescence phenotypes; how- Amino acids are necessary and sufficient for mTORC1 ever, it is unclear how mTORC1 signaling differs in senescent activation, the magnitude of which is greatly enhanced in the versus young cells. Indeed, its activity appears to be only mod- presence of growth factors (Hara et al., 1998; Long et al., 2005; erately elevated in senescence (Demidenko and Blagosklonny, Carroll et al., 2016). Growth factors signal via phosphoinositide 2008; Dalle Pezze et al., 2014; Correia-Melo et al., 2016), al- 3-kinase (PI3K)/Akt and tuberous sclerosis complex (TSC1/2) though it has been reported to become insensitive to serum in to activate the small GTPase Rheb, which is the master activa- senescent cells (Zhang et al., 2000). To further understand the underlying mechanisms by which mTORC1 is dysregulated in Correspondence to Bernadette Carroll: [email protected]; Viktor senescence, we investigated the ability of mTORC1 and auto- I. Korolchuk: [email protected] O. Kucheryavenko’s present address is the Federal Institute for Risk Assessment, Berlin, Germany. Abbreviations used: PI3K, phosphoinositide 3-kinase; TSC, tuberous sclerosis © 2017 Carroll et al. This article is available under a Creative Commons License (Attribution complex. 4.0 International, as described at https ://creativecommons .org /licenses /by /4 .0 /). The Rockefeller University Press J. Cell Biol. Vol. 216 No. 7 1949–1957 https://doi.org/10.1083/jcb.201610113 JCB 1949 phagy to sense and appropriately respond to changes in extra- tive lysosomal turnover remains to be investigated. At the same cellular nutrient availability in young and senescent cells. time, we noted that upon starvation of HeLa cells, patches of phospho-S6–positive cells were still observed in the confluent monolayer, which correlates with a consistent failure of these Results and discussion cells to grow primary cilia (Fig. S1 f). Together, these data indi- cate that PI3K/mTORC1 activity is negatively regulated by pri- Upon removal of serum and amino acids, proliferating pri- mary cilia, and signaling cannot be efficiently inhibited in their mary human fibroblasts (control) show a significant decrease in absence. Therefore, we asked whether a lack of cilia forma- mTORC1 signaling (phospho S6 and 4EBP1) and a concomi- tion could represent the underlying mechanism of constitutive tant increase in LC3B-II levels, a marker for autophagy (Fig. 1, mTORC1 activity in senescence. Indeed, although serum star- a and b). In contrast, mTORC1 activity persists in the absence vation of confluent fibroblasts induced significant elongation of of these mitogenic signals in stress-induced senescent (20 Gy ir- primary cilia, cilia present in senescent cells failed to elongate radiation), oncogene-induced senescent (B-RAFV600E transduc- upon starvation (Fig. 2, c–f), even though the same number of tion), and replicative senescent cells (Fig. 1, a and b; and Fig. S1 short cilia were observed in resting conditions (Fig. 2, c and d). a). This is accompanied by a lack of increase in LC3-II levels, Defects in cilia formation could result from differences in although interestingly, the basal levels of LC3B-II are signifi- the properties of the plasma membrane in senescent cells. In- cantly higher in senescent cells than in control cells (Narita et deed, we found that the plasma membrane becomes depolarized al., 2011). We confirmed that this phenotype is specific to senes- upon senescence acquisition (Fig. 3 a). Interestingly, membrane cence and is not simply a result of cell cycle exit (quiescence; depolarization is clearly linked to the acquisition of senescence, Fig. S1 a; Demidenko and Blagosklonny, 2008). Different star- as long-term culture of fibroblasts in the presence of pinacidil vation methods indicate that signaling pathways dependent (which can partially prevent membrane depolarization in se- on both growth factors (Zhang et al., 2000) and amino acids nescent cells) increases the rate of proliferation and postpones are perturbed in senescent cells. Indeed, we observed higher the onset of growth arrest (Fig. 3 b and Fig. S2, a and b). Im- levels of phosphorylated Akt and TSC during starvation (but portantly, restoration of senescent fibroblast plasma membrane no changes in AMPK) in addition to the insensitivity