Abstract
Recently, inhibitor of apoptosis proteins (IAPs) and some IAP antagonists were found to regulate autophagy, but the underlying mechanisms remain unclear. WX20120108 is an analogue of GDC-0152 (a known IAP antagonist) and displays more potent anti-tumor and autophagy-regulating activity in tumor cells, we investigated the regulatory mechanisms underlying WX20120108-induced autophagy. Using molecular docking and fluorescence polarization anisotropy (FPA) competitive assay, we first demonstrated that WX20120108, acting as an IAP antagonist, bound to the XIAP-BIR3, XIAP BIR2-BIR3, cIAP1 BIR3, and cIAP2 BIR3 domains with high affinities. In six cancer cell lines, WX20120108 inhibited the cell proliferation with potencies two to ten-fold higher than that of GDC-0152. In HeLa and MDA-MB-231 cells, WX20120108 induced caspase-dependent apoptosis and activated TNFα-dependent extrinsic apoptosis. On the other hand, WX20120108 induced autophagy in HeLa and MDA-MB-231 cells in dose- and time-dependent manners. We revealed that WX20120108 selectively activated Foxo3, evidenced by Foxo3 nuclear translocation in both gene modified cell line and HeLa cells, as well as the upregulated expression of Foxo3-targeted genes (Bnip3, Pik3c3, Atg5, and Atg4b), which played a key role in autophagy initiation. WX20120108-induced autophagy was significantly suppressed when Foxo3 gene was silenced. WX20120108 dose-dependently increased the generation of reactive oxygen species (ROS) in HeLa cells, and WX20120108-induced Foxo3 activation was completely blocked in the presence of catalase, a known ROS scavenger. However, WX20120108-induced ROS generation was not affected by cIAP1/2 or XIAP gene silencing. In conclusion, WX20120108-induced autophagy relies on activating ROS-Foxo3 pathway, which is independent of IAPs. This finding provides a new insight into the mechanism of IAP antagonist-mediated regulation of autophagy.
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References
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70.
Hunter AM, LaCasse EC, Korneluk RG. The inhibitors of apoptosis (IAPs) as cancer targets. Apoptosis. 2007;12:1543–68.
Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S, et al. Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. Clin Cancer Res. 2000;6:1796–803.
Sun C, Cai M, Gunasekera AH, Meadows RP, Wang H, Chen J, et al. NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature. 1999;401:818–22.
Chai J, Shiozaki E, Srinivasula SM, Wu Q, Datta P, Alnemri ES, et al. Structural basis of caspase-7 inhibition by XIAP. Cell. 2001;104:769–80.
Riedl SJ, Renatus M, Schwarzenbacher R, Zhou Q, Sun C, Fesik SW, et al. Structural basis for the inhibition of caspase-3 by XIAP. Cell. 2001;104:791–800.
Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science. 1998;281:1680–3.
de Almagro MC, Vucic D. The inhibitor of apoptosis (IAP) proteins are critical regulators of signaling pathways and targets for anti-cancer therapy. Exp Oncol. 2012;34:200–11.
Fulda S. Smac mimetics as IAP antagonists. Semin Cell Dev Biol. 2015;39:132–8.
Brunckhorst MK, Lerner D, Wang S, Yu Q. AT-406, an orally active antagonist of multiple inhibitor of apoptosis proteins, inhibits progression of human ovarian cancer. Cancer Biol Ther. 2012;13:804–11.
Flygare JA, Beresini M, Budha N, Chan H, Chan IT, Cheeti S, et al. Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152). J Med Chem. 2012;55:4101–13.
Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, Chai J, et al. A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature. 2001;410:112–6.
Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU, et al. IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell. 2007;131:682–93.
Gyrd-Hansen M, Meier P. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation and cancer. Nat Rev Cancer. 2010;10:561–74.
Lee YJ, Park BS, Park HR, Yu SB, Kang HM, Kim IR. XIAP inhibitor embelin induces autophagic and apoptotic cell death in human oral squamous cell carcinoma cells. Environ Toxicol. 2017;32:2371–8.
Li BX, Wang HB, Qiu MZ, Luo QY, Yi HJ, Yan XL, et al. Novel smac mimetic APG-1387 elicits ovarian cancer cell killing through TNF-alpha, Ripoptosome and autophagy mediated cell death pathway. J Exp Clin Cancer Res. 2018;37:53.
Wang YF, Zhang W, He KF, Liu B, Zhang L, Zhang WF, et al. Induction of autophagy-dependent cell death by the survivin suppressant YM155 in salivary adenoid cystic carcinoma. Apoptosis. 2014;19:748–58.
Wang Q, Chen Z, Diao X, Huang S. Induction of autophagy-dependent apoptosis by the survivin suppressant YM155 in prostate cancer cells. Cancer Lett. 2011;302:29–36.
Ding YH, Fan XD, Wu JJ, Deng ZK, Wei B, Li YF. Effect of YM155 on apoptosis and autophagy of K562 Cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2015;23:375–80.
Zhang L, Zhang W, Wang YF, Liu B, Zhang WF, Zhao YF, et al. Dual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma. Cell Death Dis. 2015;6:e1771.
Cheng SM, Chang YC, Liu CY, Lee JY, Chan HH, Kuo CW, et al. YM155 down-regulates survivin and XIAP, modulates autophagy and induces autophagy-dependent DNA damage in breast cancer cells. Br J Pharmacol. 2015;172:214–34.
Vequaud E, Seveno C, Loussouarn D, Engelhart L, Campone M, Juin P, et al. YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-κB network. Oncotarget. 2015;6:13476–86.
Pennati M, Sbarra S, De Cesare M, Lopergolo A, Locatelli SL, Campi E, et al. YM155 sensitizes triple-negative breast cancer to membrane-bound TRAIL through p38 MAPK- and CHOP-mediated DR5 upregulation. Int J Cancer. 2015;136:299–309.
Jane EP, Premkumar DR, Sutera PA, Cavaleri JM, Pollack IF. Survivin inhibitor YM155 induces mitochondrial dysfunction, autophagy, DNA damage and apoptosis in Bcl-xL silenced glioma cell lines. Mol Carcinog. 2017;56:1251–65.
Peiqi L, Rong H, Hongming D, Zhuogang L, Wei J, Miao M. GDC-0152-induced autophagy promotes apoptosis in HL-60 cells. Mol Cell Biochem. 2018;445:135–43.
Huang X, Wu Z, Mei Y, Wu M. XIAP inhibits autophagy via XIAP-Mdm2-p53 signalling. EMBO J. 2013;32:2204–16.
Lin F, Ghislat G, Luo S, Renna M, Siddiqi F, Rubinsztein DC. XIAP and cIAP1 amplifications induce Beclin 1-dependent autophagy through NFkappaB activation. Hum Mol Genet. 2015;24:2899–913.
Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–77.
Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene. 2004;23:2891–906.
Gump JM, Thorburn A. Autophagy and apoptosis- what’s the connection? Trends Cell Biol. 2011;21:387–92.
Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A. Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 2009;16:966–75.
Cai Q, Sun H, Peng Y, Lu J, Nikolovska-Coleska Z, McEachern D, et al. A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment. J Med Chem. 2011;54:2714–26.
Wang S. Design of small-molecule Smac mimetics as IAP antagonists. Curr Top Microbiol Immunol. 2011;348:89–113.
Nikolovska-Coleska Z, Wang R, Fang X, Pan H, Tomita Y, Li P, et al. Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal Biochem. 2004;332:261–73.
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990;82:1107–12.
Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc. 2006;1:1112–6.
Xiao D, Vogel V, Singh SV. Benzyl isothiocyanate-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species and regulated by Bax and Bak. Mol Cancer Ther. 2006;5:2931–45.
Li F, Wei A, Bu L, Long L, Chen W, Wang C, et al. Procaspase-3-activating compound 1 stabilizes hypoxia-inducible factor 1alpha and induces DNA damage by sequestering ferrous iron. Cell Death Dis. 2018;9:1025.
Shao Y, Gao Z, Marks PA, Jiang X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci USA. 2004;101:18030–5.
Chen W, Fan S, Xie X, Xue N, Jin X, Wang L. Novel PPAR pan agonist, ZBH ameliorates hyperlipidemia and insulin resistance in high fat diet induced hyperlipidemic hamster. PLoS One. 2014;9:e96056.
Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X, et al. Structural basis of IAP recognition by Smac/DIABLO. Nature. 2000;408:1008–12.
Zhang Y, Zhu J, Tang Y, Li F, Zhou H, Peng B, et al. X-linked inhibitor of apoptosis positive nuclear labeling: a new independent prognostic biomarker of breast invasive ductal carcinoma. Diagn Pathol. 2011;6:49.
Liu SS, Tsang BK, Cheung AN, Xue WC, Cheng DK, Ng TY, et al. Anti-apoptotic proteins, apoptotic and proliferative parameters and their prognostic significance in cervical carcinoma. Eur J Cancer. 2001;37:1104–10.
Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. A small molecule Smac mimic potentiates TRAIL- and TNFalpha-mediated cell death. Science. 2004;305:1471–4.
Klionsky DJ. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008;25:151–75.
Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 2016;12:1–222.
Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140:313–26.
McGovern SL, Caselli E, Grigorieff N, Shoichet BK. A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem. 2002;45:1712–22.
Webb AE, Brunet A. FOXO transcription factors: key regulators of cellular quality control. Trends Biochem Sci. 2014;39:159–69.
Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, et al. FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab. 2007;6:472–83.
Ameziane El Hassani R, Dupuy C. Detection of intracellular reactive oxygen species (CM-H2DCFDA). Bio-Protocol. 2013;3:e313.
Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T, et al. Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature. 2000;408:1004–8.
Finlay D, Vamos M, Gonzalez-Lopez M, Ardecky RJ, Ganji SR, Yuan H, et al. Small-molecule IAP antagonists sensitize cancer cells to TRAIL-induced apoptosis: roles of XIAP and cIAPs. Mol Cancer Ther. 2014;13:5–15.
Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis. 2008;13:1–9.
Steinman RM, Mellman IS, Muller WA, Cohn ZA. Endocytosis and the recycling of plasma membrane. J Cell Biol. 1983;96:1–27.
Turk B, Stoka V, Rozman-Pungercar J, Cirman T, Droga-Mazovec G, Oresic K, et al. Apoptotic pathways: involvement of lysosomal proteases. Biol Chem. 2002;383:1035–44.
Galluzzi L, Baehrecke EH, Ballabio A, Boya P, Bravo-San Pedro JM, Cecconi F, et al. Molecular definitions of autophagy and related processes. EMBO J. 2017;36:1811–36.
Noda NN, Inagaki F. Mechanisms of autophagy. Annu Rev Biophys. 2015;44:101–22.
He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67–93.
Yang Z, Klionsky DJ. Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol. 2010;22:124–31.
Sengupta A, Molkentin JD, Yutzey KE. FoxO transcription factors promote autophagy in cardiomyocytes. J Biol Chem. 2009;284:28319–31.
Yang J, Carra S, Zhu WG, Kampinga HH. The regulation of the autophagic network and its implications for human disease. Int J Biol Sci. 2013;9:1121–33.
Lv P, Huang J, Yang J, Deng Y, Xu J, Zhang X, et al. Autophagy in muscle of glucose-infusion hyperglycemia rats and streptozotocin-induced hyperglycemia rats via selective activation of m-TOR or FoxO3. PLoS One. 2014;9:e87254.
Hay N. Interplay between FOXO, TOR, and Akt. Biochim Biophys Acta. 2011;1813:1965–70.
Ho KK, McGuire VA, Koo CY, Muir KW, de Olano N, Maifoshie E, et al. Phosphorylation of FOXO3a on Ser-7 by p38 promotes its nuclear localization in response to doxorubicin. J Biol Chem. 2012;287:1545–55.
Klotz LO, Sanchez-Ramos C, Prieto-Arroyo I, Urbanek P, Steinbrenner H, Monsalve M. Redox regulation of FoxO transcription factors. Redox Biol. 2015;6:51–72.
Moscat J, Diaz-Meco MT. p62 at the crossroads of autophagy, apoptosis, and cancer. Cell. 2009;137:1001–4.
Toepfer N, Childress C, Parikh A, Rukstalis D, Yang W. Atorvastatin induces autophagy in prostate cancer PC3 cells through activation of LC3 transcription. Cancer Biol Ther. 2011;12:691–9.
Colosetti P, Puissant A, Robert G, Luciano F, Jacquel A, Gounon P, et al. Autophagy is an important event for megakaryocytic differentiation of the chronic myelogenous leukemia K562 cell line. Autophagy. 2009;5:1092–8.
Zheng Q, Su H, Ranek MJ, Wang X. Autophagy and p62 in cardiac proteinopathy. Circ Res. 2011;109:296–308.
Fujita K, Maeda D, Xiao Q, Srinivasula SM. Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degradation. Proc Natl Acad Sci USA. 2011;108:1427–32.
Nakaso K, Yoshimoto Y, Nakano T, Takeshima T, Fukuhara Y, Yasui K, et al. Transcriptional activation of p62/A170/ZIP during the formation of the aggregates: possible mechanisms and the role in Lewy body formation in Parkinson’s disease. Brain Res. 2004;1012:42–51.
Trocoli A, Bensadoun P, Richard E, Labrunie G, Merhi F, Schlafli AM, et al. p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells. Cell Death Differ. 2014;21:1852–61.
B’Chir W, Maurin AC, Carraro V, Averous J, Jousse C, Muranishi Y, et al. The eIF2alpha/ATF4 pathway is essential for stress-induced autophagy gene expression. Nucleic Acids Res. 2013;41:7683–99.
Cnop M, Abdulkarim B, Bottu G, Cunha DA, Igoillo-Esteve M, Masini M, et al. RNA sequencing identifies dysregulation of the human pancreatic islet transcriptome by the saturated fatty acid palmitate. Diabetes. 2014;63:1978–93.
Puissant A, Fenouille N, Auberger P. When autophagy meets cancer through p62/SQSTM1. Am J Cancer Res. 2012;2:397–413.
Acknowledgements
We would like to thank Professor Shaomeng Wang at the University of Michigan in the U.S. for his assistance in the FPA competition assay. This work was supported by the National Natural Science Foundation of China (Grant nos. 81803567 and 81773790) and the National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant nos. 2012ZX09301-001 and 2012ZX09301-003) and partially supported by a project of the State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University (FAMP201708K).
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RD, AHN and LLW designed the research; RD, XW, WC, ZL, ALW and QBW performed the research; RD and LLW wrote the paper; XW and AHN provided the testing compound; LLW, WC, ZL and AHN reviewed and modified the paper.
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Ding, R., Wang, X., Chen, W. et al. WX20120108, a novel IAP antagonist, induces tumor cell autophagy via activating ROS-FOXO pathway. Acta Pharmacol Sin 40, 1466–1479 (2019). https://doi.org/10.1038/s41401-019-0253-5
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DOI: https://doi.org/10.1038/s41401-019-0253-5
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