ERK1/2 activation mediated by the nutlin‑3‑induced mitochondrial translocation of p53

Lee,Sun-Young; Shin,Seok  Joon; Kim,Ho-Shik

doi:10.3892/ijo.2013.1764

ERK1/2 activation mediated by the nutlin‑3‑induced mitochondrial translocation of p53

Authors:
- Sun-Young Lee
- Seok Joon Shin
- Ho-Shik Kim
View Affiliations

Affiliations: Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea
Published online on: January 10, 2013 https://doi.org/10.3892/ijo.2013.1764
Pages: 1027-1035

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Nutlin-3 is a small-molecule antagonist of murine double minute 2 (MDM2) that blocks its binding to p53, leading to an increase in p53 protein levels. The tumor suppressor p53 induces growth arrest or apoptosis in response to genotoxic stress. Along with its growth‑suppressive effect, it has been reported that p53 stimulates the mitogen-activated protein kinase (MAPK) pathway via the upregulation of heparin- binding epidermal growth factor-like growth factor (HB-EGF), an epidermal growth factor receptor (EGFR) ligand, and discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, at the transcription level. In this study, we propose a novel mechanism involved in the p53-induced MAPK activation. Nutlin-3 induced the phosphorylation of EGFR, MAPK/ERK kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 in U2OS human osteosarcoma cells harboring wild-type p53. This phosphorylation was completely inhibited by p53 siRNA, but not by pifithrin (PFT)-α, an inhibitor of the transcriptional activity of p53. While the nutlin-3-induced EGFR phosphorylation was prevented by the inactivation of ERK1/2, the nutlin-3-induced MEK1/2-ERK1/2 phosphorylation was still observed in the cells in which EGFR phosphorylation was inhibited using EGFR siRNA and AG1478, an inhibitor of EGFR tyrosine kinase. Of note, nutlin-3 caused the accumulation of mitochondrial reactive oxygen species (ROS) and this correlated with the mitochondrial translocation of p53. 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), a ROS scavenger, prevented the phosphorylation of ERK1/2. PFT-μ, which prevented the mitochondrial localization of p53, suppressed ERK1/2 phosphorylation, as well as ROS accumulation. Finally, we analyzed the effect of ERK1/2 activation on nutlin-3-induced apoptosis. The knockdown of MEK1/2 and ERK1/2 activity using U0126, a MEK inhibitor, or siRNAs, resulted in the enhancement of nutlin-3-induced apoptosis. In addition, TEMPO and PFT-μ also promoted nutlin-3-induced apoptosis. Taking the above findings into account, it can be concluded that nutlin-3 activates ERK1/2 prior to EGFR phosphorylation via ROS generation following the mitochondrial translocation of p53 and that nutlin-3-induced ERK1/2 activation may play a role in protecting U2OS cells from p53-dependent apoptosis, constituting a negative feedback loop for p53-induced apoptosis.

View Figures

View References

1	Menendez D, Inga A and Resnick MA: The expanding universe of p53 targets. Nat Rev Cancer. 9:724–737. 2009. View Article : Google Scholar : PubMed/NCBI
2	Riley T, Sontag E, Chen P and Levine A: Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol. 9:402–412. 2008. View Article : Google Scholar : PubMed/NCBI
3	Honda R, Tanaka H and Yasuda H: Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 420:25–27. 1997. View Article : Google Scholar : PubMed/NCBI
4	Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW and Vogelstein B: Oncoprotein MDM2 conceals the activation domain of tumor suppressor p53. Nature. 362:857–860. 1993. View Article : Google Scholar : PubMed/NCBI
5	Reinhardt HC and Schumacher B: The p53 network: cellular and systemic DNA damage responses in aging and cancer. Trends Genet. 28:128–136. 2012. View Article : Google Scholar : PubMed/NCBI
6	Sill H, Olipitz W, Zebisch A, Schulz E and Wölfler A: Therapy-related myeloid neoplasms: pathobiology and clinical characteristics. Br J Pharmacol. 162:792–805. 2011. View Article : Google Scholar : PubMed/NCBI
7	Travis LB, Ng AK, Allan JM, et al: Second malignant neoplasms and cardiovascular disease following radiotherapy. J Natl Cancer Inst. 104:357–370. 2012. View Article : Google Scholar : PubMed/NCBI
8	Shangary S and Wang S: Targeting the MDM2-p53 interaction for cancer therapy. Clin Cancer Res. 14:5318–5324. 2008. View Article : Google Scholar : PubMed/NCBI
9	Dickens MP, Fitzgerald R and Fischer PM: Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Semin Cancer Biol. 20:10–18. 2010. View Article : Google Scholar : PubMed/NCBI
10	Vassilev LT, Vu BT, Graves B, et al: In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science. 303:844–848. 2004. View Article : Google Scholar : PubMed/NCBI
11	Secchiero P, Bosco R, Celeghini C and Zauli G: Recent advances in the therapeutic perspectives of nutlin-3. Curr Pharm Des. 17:569–577. 2011. View Article : Google Scholar : PubMed/NCBI
12	Tovar C, Rosinski J, Filipovic Z, et al: Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy. Proc Natl Acad Sci USA. 103:1888–1893. 2006. View Article : Google Scholar : PubMed/NCBI
13	Mendrysa SM, O’Leary KA, McElwee MK, et al: Tumor suppression and normal aging in mice with constitutively high p53 activity. Genes Dev. 20:16–21. 2006. View Article : Google Scholar : PubMed/NCBI
14	Mebratu Y and Tesfaigzi Y: How ERK1/2 activation controls cell proliferation and cell death: Is subcellular localization the answer? Cell Cycle. 8:1168–1175. 2009. View Article : Google Scholar : PubMed/NCBI
15	McCubrey JA, Steelman LS, Chappell WH, et al: Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta. 1773:1263–1284. 2007. View Article : Google Scholar : PubMed/NCBI
16	Persons DL, Yazlovitskaya EM and Pelling JC: Effect of extracellular signal-regulated kinase on p53 accumulation in response to cisplatin. J Biol Chem. 275:35778–35785. 2000. View Article : Google Scholar : PubMed/NCBI
17	Sablina AA, Chumakov PM, Levine AJ and Kopnin BP: p53 activation in response to microtubule disruption is mediated by integrin-Erk signaling. Oncogene. 20:899–909. 2001. View Article : Google Scholar : PubMed/NCBI
18	She QB, Bode AM, Ma WY, Chen NY and Dong Z: Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase. Cancer Res. 61:1604–1610. 2001.PubMed/NCBI
19	Lin T, Mak NK and Yang MS: MAPK regulate p53-dependent cell death induced by benzo[a]pyrene: involvement of p53 phosphorylation and acetylation. Toxicology. 247:145–153. 2008.PubMed/NCBI
20	Lee SW, Fang L, Igarashi M, Ouchi T, Lu KP and Aaronson SA: Sustained activation of Ras/Raf/mitogen-activated protein kinase cascade by the tumor suppressor p53. Proc Natl Acad Sci USA. 97:8302–8305. 2000. View Article : Google Scholar : PubMed/NCBI
21	Fang L, Li G, Liu G, Lee SW and Aaronson SA: p53 induction of heparin-binding EGF-like growth factor counteracts p53 growth suppression through activation of MAPK and PI3K/Akt signaling cascades. EMBO J. 20:1931–1939. 2001. View Article : Google Scholar
22	Ongusaha PP, Kim JI, Fang L, et al: p53 induction and activation of DDR1 kinase counteracts p53-mediated apoptosis and influence p53 regulation through a positive feedback loop. EMBO J. 22:1289–1301. 2003. View Article : Google Scholar : PubMed/NCBI
23	Yin Y, Liu YX, Jin YJ, Hall EJ and Barrett JC: PAC1 phosphatase is a transcription target of p53 in signaling apoptosis and growth suppression. Nature. 422:527–531. 2003. View Article : Google Scholar : PubMed/NCBI
24	Ueda K, Arakawa H and Nakamura Y: Dual-specificity phosphatase 5 (DUSP5) as a direct transcriptional target of tumor suppressor p53. Oncogene. 22:5586–5591. 2003. View Article : Google Scholar : PubMed/NCBI
25	Li M, Zhou JY, Ge Y, Matherly LH and Wu GS: The phosphatase MKP1 is a transcriptional target of p53 involved in cell cycle regulation. J Biol Chem. 278:41059–41068. 2003. View Article : Google Scholar : PubMed/NCBI
26	Kojima K, Konopleva M, Samudio IJ, Ruvolo V and Andreeff M: Mitogen-activated protein kinase kinase inhibition enhances nuclear proapoptotic function of p53 in acute myelogenous leukemia cells. Cancer Res. 67:3210–3219. 2007. View Article : Google Scholar
27	Zhang W, Konopleva M, Burks JK, et al: Blockade of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase and murine double minute synergistically induces apoptosis in acute myeloid leukemia via BH3-only proteins Puma and Bim. Cancer Res. 70:2424–2434. 2010. View Article : Google Scholar
28	Schmittgen TD and Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 3:1101–1108. 2008. View Article : Google Scholar : PubMed/NCBI
29	Jang JY, Kim MK, Jeon YK, et al: Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells. Exp Mol Med. 44:251–259. 2012. View Article : Google Scholar
30	Sauer L, Gitenay D, Vo C and Baron VT: Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells. Oncogene. 29:2628–2637. 2010. View Article : Google Scholar : PubMed/NCBI
31	McCubrey JA, Lahair MM and Franklin RA: Reactive oxygen species-induced activation of the MAP kinase signaling pathways. Antioxid Redox Signal. 8:1775–1789. 2006. View Article : Google Scholar : PubMed/NCBI
32	Liu CM, Sun YZ, Sun JM, Ma JQ and Cheng C: Protective role of quercetin against lead-induced inflammatory response in rat kidney through the ROS-mediated MAPKs and NF-κB pathway. Biochim Biophys Acta. 1820:1693–1703. 2012.PubMed/NCBI
33	Liu B, Chen Y and St Clair DK: ROS and p53: a versatile partnership. Free Radic Biol Med. 44:1529–1535. 2008. View Article : Google Scholar : PubMed/NCBI
34	Sansome C, Zaika A, Marchenko ND and Moll UM: Hypoxia death stimulus induces translocation of p53 protein to mitochondria. Detection by immunofluorescence on whole cells. FEBS Lett. 488:110–115. 2001. View Article : Google Scholar : PubMed/NCBI
35	Mihara M, Erster S, Zaika A, et al: p53 has a direct apoptogenic role at the mitochondria. Mol Cell. 11:577–590. 2003. View Article : Google Scholar : PubMed/NCBI
36	Palacios G and Moll UM: Mitochondrially targeted wild-type p53 suppresses growth of mutant p53 lymphomas in vivo. Oncogene. 25:6133–6139. 2006. View Article : Google Scholar : PubMed/NCBI
37	Talos F, Petrenko O, Mena P and Moll UM: Mitochondrially targeted p53 has tumor suppressor activities in vivo. Cancer Res. 65:9971–9981. 2005. View Article : Google Scholar : PubMed/NCBI
38	Vaseva AV, Marchenko ND and Moll UM: The transcription-independent mitochondrial p53 program is a major contributor to nutlin-induced apoptosis in tumor cells. Cell Cycle. 8:1711–1719. 2009. View Article : Google Scholar : PubMed/NCBI
39	Zhao Y, Chaiswing L, Velez JM, et al: p53 translocation to mitochondria precedes its nuclear translocation and targets mitochondrial oxidative defense protein-manganese superoxide dismutase. Cancer Res. 65:3745–3750. 2005. View Article : Google Scholar
40	Strom E, Sathe S, Komarov PG, et al: Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation. Nat Chem Biol. 2:474–479. 2006. View Article : Google Scholar : PubMed/NCBI