Abstract
Oxygen is an indispensable element for cell survival and maintenance. Eukaryotic cells are equipped with a series of signaling pathways that cope with hypoxia. The dioxygenase factor inhibiting HIF (FIH) is an oxygen sensor that regulates the transcriptional activity of hypoxia-inducible factor (HIF) through asparaginyl hydroxylation. Given that HACE1 was detected as an FIH-interacting protein in a previous proteomics study, we tested whether the E3 ubiquitin ligase HACE1 is a substrate for FIH. FIH interacted with HACE1, in cells and in vitro, and was determined to hydroxylate HACE1 at the N191 residue within the ankyrin repeat domain. Hydroxylation disrupted the physical association between HACE1 and its representative target, Rac1. Under hypoxic conditions, HACE1 is less hydroxylated due to the inactivation of FIH, and subsequently functions to ubiquitinate the active form of Rac1, leading to the proteasomal degradation of Rac1. Since Rac1 stimulates cell movement, HACE1 inhibits cell migration and invasion in breast cancer by removing active Rac1. Such an effect of HACE1 is reinforced under hypoxia because HACE1 escapes from FIH-mediated hydroxylation. In clinical datasets, HACE1 downregulation is associated with poor outcomes in patients with breast cancer. Taken together, FIH is likely to act as an oxygen sensor that determines oxygen-dependent cancer progression.
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References
Semenza GL. Hypoxia and human disease-and the Journal of Molecular Medicine. J Mol Med. 2007;85:1293–4.
Semenza GL, Wang GL. A nuclear factor induced by hypoxia via denovo protein-synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol. 1992;12:5447–54.
Mahon PC, Hirota K, Semenza GL. FIH-1: a novel protein that interacts with HIF-1 alpha and VHL to mediate repression of HIF-1 transcriptional activity. Gene Dev. 2001;15:2675–86.
Yu F, White SB, Zhao Q, Lee FS. HIF-1 alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci USA. 2001;98:9630–5.
Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Bio. 2004;5:343–54.
Pettersen EO, Juul NO, Ronning OW. Regulation of protein-metabolism of human-cells during and after acute-hypoxia. Cancer Res. 1986;46:4346–51.
Lee SD, Kim W, Jeong JW, Park JW, Kim JEAK-1. a SIRT2 inhibitor, destabilizes HIF-1alpha and diminishes its transcriptional activity during hypoxia. Cancer Lett. 2016;373:138–45.
Berndsen CE, Wolberger C. New insights into ubiquitin E3 ligase mechanism. Nat Struct Mol Biol. 2014;21:301–7.
Hewitson KS, McNeill LA, Riordan MV, Tian YM, Bullock AN, Welford RW, et al. Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J Biol Chem. 2002;277:26351–5.
Lando D, Peet DJ, Gorman JJ, Whelan DA, Whitelaw ML, Bruick RK. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev. 2002;16:1466–71.
Cockman ME, Lancaster DE, Stolze IP, Hewitson KS, McDonough MA, Coleman ML, et al. Posttranslational hydroxylation of ankyrin repeats in IkappaB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH). Proc Natl Acad Sci USA. 2006;103:14767–72.
Cockman ME, Webb JD, Kramer HB, Kessler BM, Ratcliffe PJ. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins. Mol Cell Proteom. 2009;8:535–46.
Anglesio MS, Evdokimova V, Melnyk N, Zhang L, Fernandez CV, Grundy PE, et al. Differential expression of a novel ankyrin containing E3 ubiquitin-protein ligase, Hace1, in sporadic Wilms’ tumor versus normal kidney. Hum Mol Genet. 2004;13:2061–74.
Torrino S, Visvikis O, Doye A, Boyer L, Stefani C, Munro P, et al. The E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of active Rac1. Dev Cell. 2011;21:959–65.
Liu Z, Chen P, Gao H, Gu Y, Yang J, Peng H, et al. Ubiquitylation of autophagy receptor Optineurin by HACE1 activates selective autophagy for tumor suppression. Cancer Cell. 2014;26:106–20.
Tortola L, Nitsch R, Bertrand MJM, Kogler M, Redouane Y, Kozieradzki I, et al. The tumor suppressor Hace1 Is a critical regulator of TNFR1-mediated cell fate. Cell Rep. 2016;16:3414.
Zhang L, Anglesio MS, O’Sullivan M, Zhang F, Yang G, Sarao R, et al. The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers. Nat Med. 2007;13:1060–9.
Castillo-Lluva S, Tan CT, Daugaard M, Sorensen PH, Malliri A. The tumour suppressor HACE1 controls cell migration by regulating Rac1 degradation. Oncogene. 2013;32:1735–42.
Goka ET, Lippman ME. Loss of the E3 ubiquitin ligase HACE1 results in enhanced Rac1 signaling contributing to breast cancer progression. Oncogene. 2015;34:5395–405.
Rodriguez J, Pilkington R, Garcia Munoz A, Nguyen LK, Rauch N, Kennedy S, et al. Substrate-trapped interactors of PHD3 and FIH cluster in distinct signaling pathways. Cell Rep. 2016;14:2745–60.
Cockman ME, Webb JD, Ratcliffe PJ. FIH-dependent asparaginyl hydroxylation of ankyrin repeat domain-containing proteins. Ann N Y Acad Sci. 2009;1177:9–18.
Elkins JM, Hewitson KS, McNeill LA, Seibel JF, Schlemminger I, Pugh CW, et al. Structure of factor-inhibiting hypoxia-inducible factor (HIF) reveals mechanism of oxidative modification of HIF-1 alpha. J Biol Chem. 2003;278:1802–6.
Linke S, Stojkoski C, Kewley RJ, Booker GW, Whitelaw ML, Peet DJ. Substrate requirements of the oxygen-sensing asparaginyl hydroxylase factor-inhibiting hypoxia-inducible factor. J Biol Chem. 2004;279:14391–7.
Hewitson KS, Holmes SL, Ehrismann D, Hardy AP, Chowdhury R, Schofield CJ, et al. Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH). J Biol Chem. 2008;283:25971–8.
Wilkins SE, Abboud MI, Hancock RL, Schofield CJ. Targeting protein-protein interactions in the HIF system. ChemMedChem. 2016;11:773–86.
Karttunen S, Duffield M, Scrimgeour NR, Squires L, Lim WL, Dallas ML, et al. Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel. J Cell Sci. 2015;128:225–31.
Yang M, Chowdhury R, Ge W, Hamed RB, McDonough MA, Claridge TD, et al. Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains. FEBS J. 2011;278:1086–97.
Kallberg M, Wang H, Wang S, Peng J, Wang Z, Lu H, et al. Template-based protein structure modeling using the RaptorX web server. Nat Protoc. 2012;7:1511–22.
Bosco EE, Mulloy JC, Zheng Y. Rac1 GTPase: a “Rac” of all trades. Cell Mol Life Sci. 2009;66:370–4.
Andrio E, Lotte R, Hamaoui D, Cherfils J, Doye A, Daugaard M, et al. Identification of cancer-associated missense mutations in hace1 that impair cell growth control and Rac1 ubiquitylation. Sci Rep. 2017;7:44779.
Tan EY, Campo L, Han C, Turley H, Pezzella F, Gatter KC, et al. Cytoplasmic location of factor-inhibiting hypoxia-inducible factor is associated with an enhanced hypoxic response and a shorter survival in invasive breast cancer. Breast Cancer Res. 2007;9:R89.
Yan M, Rayoo M, Takano EA, Investigators KC, Fox SB. BRCA1 tumours correlate with a HIF-1alpha phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression. Br J Cancer. 2009;101:1168–74.
Hyseni A, van der Groep P, van der Wall E, van Diest PJ. Subcellular FIH-1 expression patterns in invasive breast cancer in relation to HIF-1alpha expression. Cell Oncol. 2011;34:565–70.
Kretschmer C, Sterner-Kock A, Siedentopf F, Schoenegg W, Schlag PM, Kemmner W. Identification of early molecular markers for breast cancer. Mol Cancer. 2011;10:15.
Brunner AL, Li J, Guo X, Sweeney RT, Varma S, Zhu SX, et al. A shared transcriptional program in early breast neoplasias despite genetic and clinical distinctions. Genome Biol. 2014;15:R71.
Lesurf R, Aure MR, Mork HH, Vitelli V, Oslo Breast Cancer Research C, Lundgren S, et al. Molecular features of subtype-specific progression from ductal carcinoma in situ to invasive breast cancer. Cell Rep. 2016;16:1166–79.
van ‘t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002;415:530–6.
Zucchi I, Mento E, Kuznetsov VA, Scotti M, Valsecchi V, Simionati B, et al. Gene expression profiles of epithelial cells microscopically isolated from a breast-invasive ductal carcinoma and a nodal metastasis. Proc Natl Acad Sci USA. 2004;101:18147–52.
Loi S, Haibe-Kains B, Desmedt C, Lallemand F, Tutt AM, Gillet C, et al. Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol. 2007;25:1239–46.
Gyorffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat. 2010;123:725–31.
Coleman ML, McDonough MA, Hewitson KS, Coles C, Mecinovic J, Edelmann M, et al. Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor. J Biol Chem. 2007;282:24027–38.
Janke K, Brockmeier U, Kuhlmann K, Eisenacher M, Nolde J, Meyer HE, et al. Factor inhibiting HIF-1 (FIH-1) modulates protein interactions of apoptosis-stimulating p53 binding protein 2 (ASPP2). J Cell Sci. 2013;126:2629–40.
Lachance V, Degrandmaison J, Marois S, Robitaille M, Genier S, Nadeau S, et al. Ubiquitylation and activation of a Rab GTPase is promoted by a beta(2)AR-HACE1 complex. J Cell Sci. 2014;127:111–23.
Palicharla VR, Maddika S. HACE1 mediated K27 ubiquitin linkage leads to YB-1 protein secretion. Cell Signal. 2015;27:2355–62.
Giaccia A, Siim BG, Johnson RS. HIF-1 as a target for drug development. Nat Rev Drug Discov. 2003;2:803–11.
Lancaster DE, McDonough MA, Schofield CJ. Factor inhibiting hypoxia-inducible factor (FIH) and other asparaginyl hydroxylases. Biochem Soc Trans. 2004;32:943–5.
Scholz CC, Rodriguez J, Pickel C, Burr S, Fabrizio JA, Nolan KA, et al. FIH regulates cellular metabolism through hydroxylation of the deubiquitinase OTUB1. PLoS Biol. 2016;14:e1002347.
Ferguson JE 3rd, Wu Y, Smith K, Charles P, Powers K, Wang H, et al. ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism. Mol Cell Biol. 2007;27:6407–19.
So JH, Kim JD, Yoo KW, Kim HT, Jung SH, Choi JH, et al. FIH-1, a novel interactor of mindbomb, functions as an essential anti-angiogenic factor during zebrafish vascular development. PLoS ONE. 2014;9:e109517.
Rankin EB, Giaccia AJ. Hypoxic control of metastasis. Science. 2016;352:175–80.
Semenza GL. The hypoxic tumor microenvironment: a driving force for breast cancer progression. Biochim Biophys Acta. 2016;1863:382–91.
Hordijk PL. Regulation of NADPH oxidases: the role of Rac proteins. Circ Res. 2006;98:453–62.
Masson N, Singleton RS, Sekirnik R, Trudgian DC, Ambrose LJ, Miranda MX, et al. The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity. EMBO Rep. 2012;13:251–7.
Daugaard M, Nitsch R, Razaghi B, McDonald L, Jarrar A, Torrino S, et al. Hace1 controls ROS generation of vertebrate Rac1-dependent NADPH oxidase complexes. Nat Commun. 2013;4:2180.
Zhang L, Chen X, Sharma P, Moon M, Sheftel AD, Dawood F, et al. HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress. Nat Commun. 2014;5:3430.
Chun YS, Choi E, Kim GT, Lee MJ, Lee MJ, Lee SE, et al. Zinc induces the accumulation of hypoxia-inducible factor (HIF)-1alpha, but inhibits the nuclear translocation of HIF-1beta, causing HIF-1 inactivation. Biochem Biophys Res Commun. 2000;268:652–6.
Lee GY, Shin SH, Shin HW, Chun YS, Park JW. NDRG3 lowers the metastatic potential in prostate cancer as a feedback controller of hypoxia-inducible factors. Exp Mol Med. 2018;50:61.
Acknowledgements
This study was supported by a grant from the National Research Foundation of Korea (2017R1A4A1015015; to J.-W.P).
Author contributions
Conception and design: I.K., J.-W.P. Development of methodology: I.K. Acquisition of data: I.K., S.-H.S., J.E.L. Analysis and interpretation of data: I.K., S.-H.S. Writing, review, and/or revision of the manuscript: I.K., J.-W.P. Study supervision: J.-W.P.
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Kim, I., Shin, SH., Lee, J.E. et al. Oxygen sensor FIH inhibits HACE1-dependent ubiquitination of Rac1 to enhance metastatic potential in breast cancer cells. Oncogene 38, 3651–3666 (2019). https://doi.org/10.1038/s41388-019-0676-y
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DOI: https://doi.org/10.1038/s41388-019-0676-y
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