Abstract
Activation of epidermal growth factor receptor (EGFR), which occurs in many types of tumour, promotes tumour progression1,2. However, no extracellular antagonist of human EGFR has been identified. We found that human macrophage migration inhibitory factor (MIF) is O-GlcNAcylated at Ser 112/Thr 113 at its carboxy terminus. The naturally secreted and O-GlcNAcylated MIF binds to EGFR, thereby inhibiting the binding of EGF to EGFR and EGF-induced EGFR activation, phosphorylation of ERK and c-Jun, cell invasion, proliferation and brain tumour formation. Activation of EGFR through mutation or its ligand binding enhances the secretion of MMP13, which degrades extracellular MIF, and results in abrogation of the negative regulation of MIF on EGFR. The finding that EGFR activation downregulates its antagonist in the tumour microenvironment represents an important feedforward mechanism for human tumour cells to enhance EGFR signalling and promote tumorigenesis.
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References
Moscatello, D. K. et al. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res. 55, 5536–5539 (1995).
Nicholson, R. I., Gee, J. M. & Harper, M. E. EGFR and cancer prognosis. Eur. J. Cancer 37 (suppl. 4), S9–S15 (2001).
Suzuki, H. et al. The relationship between tyrosine kinase inhibitor therapy and overall survival in patients with non-small cell lung cancer carrying EGFR mutations. Chin. J. Cancer 32, 136–140 (2013).
Lu, Z., Jiang, G., Blume-Jensen, P. & Hunter, T. Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase. Mol. Cell Biol. 21, 4016–4031 (2001).
Avraham, R. & Yarden, Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nat. Rev. Mol. Cell Biol. 12, 104–117 (2011).
Klein, D. E., Stayrook, S. E., Shi, F., Narayan, K. & Lemmon, M. A. Structural basis for EGFR ligand sequestration by Argos. Nature 453, 1271–1275 (2008).
Schweitzer, R., Howes, R., Smith, R., Shilo, B. Z. & Freeman, M. Inhibition of Drosophila EGF receptor activation by the secreted protein Argos. Nature 376, 699–702 (1995).
Lippitz, B. E. Cytokine patterns in patients with cancer: a systematic review. Lancet Oncol. 14, e218–e228 (2013).
Calandra, T. & Roger, T. Macrophage migration inhibitory factor: a regulator of innate immunity. Nat. Rev. Immunol. 3, 791–800 (2003).
Schwartz, V. et al. A functional heteromeric MIF receptor formed by CD74 and CXCR4. FEBS Lett. 583, 2749–2757 (2009).
Conroy, H., Mawhinney, L. & Donnelly, S. C. Inflammation and cancer: macrophage migration inhibitory factor (MIF)–the potential missing link. Q. J. Med. 103, 831–836 (2010).
Krockenberger, M. et al. Macrophage migration inhibitory factor expression in cervical cancer. J. Cancer Res. Clin. Oncol. 136, 651–657 (2010).
Xu, X. et al. Overexpression of macrophage migration inhibitory factor induces angiogenesis in human breast cancer. Cancer Lett. 261, 147–157 (2008).
Verjans, E. et al. Dual role of macrophage migration inhibitory factor (MIF) in human breast cancer. BMC Cancer 9, 230 (2009).
Kuan, C. T., Wikstrand, C. J. & Bigner, D. D. EGF mutant receptor vIII as a molecular target in cancer therapy. Endocr. Relat. Cancer 8, 83–96 (2001).
Brinckerhoff, C. E. & Matrisian, L. M. Matrix metalloproteinases: a tail of a frog that became a prince. Nat. Rev. Mol. Cell Biol. 3, 207–214 (2002).
Hadler-Olsen, E., Winberg, J. O. & Uhlin-Hansen, L. Matrix metalloproteinases in cancer: their value as diagnostic and prognostic markers and therapeutic targets. Tumour Biol. 34, 2041–2051 (2013).
Gilmour, A. M. et al. A novel epidermal growth factor receptor-signaling platform and its targeted translation in pancreatic cancer. Cell Signal. 25, 2587–2603 (2013).
Moy, F. J. et al. High-resolution solution structure of the catalytic fragment of human collagenase-3 (MMP-13) complexed with a hydroxamic acid inhibitor. J. Mol. Biol. 302, 671–689 (2000).
Slawson, C. & Hart, G. W. O-GlcNAc signalling: implications for cancer cell biology. Nat. Rev. Cancer 11, 678–684 (2011).
Yang, Y. R. & Suh, P. G. O-GlcNAcylation in cellular functions and human diseases. Adv. Biol. Regul. 54, 68–73 (2014).
Sugimoto, H., Suzuki, M., Nakagawa, A., Tanaka, I. & Nishihira, J. Crystal structure of macrophage migration inhibitory factor from human lymphocyte at 2.1 Å resolution. FEBS Lett. 389, 145–148 (1996).
Hofer, S., Rushing, E., Preusser, M. & Marosi, C. Molecular biology of high-grade gliomas: what should the clinician know? Chin. J. Cancer 33, 4–7 (2014).
Inda, M. M. et al. Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma. Genes Dev. 24, 1731–1745 (2010).
Klein, D. E., Nappi, V. M., Reeves, G. T., Shvartsman, S. Y. & Lemmon, M. A. Argos inhibits epidermal growth factor receptor signalling by ligand sequestration. Nature 430, 1040–1044 (2004).
Xia, Y. et al. c-Jun downregulation by HDAC3-dependent transcriptional repression promotes osmotic stress-induced cell apoptosis. Mol. Cell 25, 219–232 (2007).
Yi, W. et al. Phosphofructokinase 1 glycosylation regulates cell growth and metabolism. Science 337, 975–980 (2012).
Zheng, Y. et al. FAK phosphorylation by ERK primes ras-induced tyrosine dephosphorylation of FAK mediated by PIN1 and PTP-PEST. Mol. Cell 35, 11–25 (2009).
Yang, W. et al. ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat. Cell Biol. 14, 1295–1304 (2012).
Acknowledgements
We thank X. Yu (University of Michigan Medical School) for the pCMV-Myc-OGT plasmid, D. M. F. Van Aalten (University of Dundee, UK) for the pEBG-6P-hOGA plasmid, and M. Wade in the Department of Scientific Publications at The University of Texas MD Anderson Cancer Center for critically reading this manuscript. We thank J. Gumin in the Department of Neurosurgery at The University of Texas MD Anderson Cancer Center for her help in mice intracranial injection. This work was supported by National Cancer Institute grants 2R01 CA109035 (Z.L.) and 1R0 CA169603 (Z.L.), National Institute of Neurological Disorders and Stroke grant 1R01 NS089754 (Z.L.), MD Anderson Support Grant CA016672, the James S. McDonnell Foundation 21st Century Science Initiative in Brain Cancer Research Award 220020318 (Z.L.), 2P50 CA127001 (Brain Cancer SPORE), a Sister Institution Network Fund from MD Anderson (Z.L.), National Institutes of Health Grant 1S10 OD012304-01 (D.H.H.), and Cancer Prevention and Research Institute of Texas research grant RP130397 (D.H.H.). Z.L. is a Ruby E. Rutherford Distinguished Professor.
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This study was conceived by Z.L.; Y.Z. and Z.L. designed research, Y.Z., X.L., X.Q., Y.W., J.-H.L., Y.X., D.H.H., G.Z. and J.L. performed experiments; Z.L. wrote the paper with comments from all authors.
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Supplementary Figure 2 Purified EGF Binds to EGFR.
Immunoprecipitated EGFR from A431 cells was incubated with purified recombinant EGF (50 ng ml−1) for 10 min. The beads were washed three times with PBS, and Western blotting analyses were performed with the indicated antibodies. WB, Western blot. Data represent one out of 3 experiments.
Supplementary Figure 3 MIF S112/T113 O-GlcNAcylation Results in Inhibition of EGF-induced EGFR Activation.
Western blotting analyses were performed with the indicated antibodies. WB, Western blot. Data represent one out of 3 experiments. (a) Purified His-MIF from bacteria (Gelcode Blue stained gel, left panel) was incubated with A431 cells for 30 min before EGF (50 ng ml−1) treatment for 30 min (right panel). (b) U251 (left panel) and DU145 (right panel) cells were treated with or without PUGNAc (10 μM) for 24 h. The cell lysates were treated without (as a control) or with the permissive mutant β-1,4-galactosyltransferase (Gal-T1 Y289L), which modified O-GlcNAc residues of cellular proteins with azido-modified galactose. The azide-modified proteins were then labeled with biotin and incubated with streptavidin beads. (c) Purified WT Flag-MIF (500 ng ml−1) or Flag-MIF S112/T113A (500 ng ml−1) protein was incubated with A431 cells for 30 min before EGF (50 ng ml−1) treatment for 30 min.
Supplementary Figure 4 CIP Treatment Does Not Affect MIF-inhibited EGFR Activation.
(a,b,d) Western blotting analyses were performed with the indicated antibodies. WB, Western blot. Data represent one out of 3 experiments. (a) Immunoprecipitated and purified Flag-MIF or cell lysates of A431 cells were treated with or without CIP (10 units) for 30 min. (b) Immunoprecipitated full-length EGFR from A431 cells (left panel) or immobilized and purified extracellular domain of His-EGFR (right panel) was incubated with CIP (10 units, 30 min)-treated or untreated purified Flag-MIF proteins (500 ng ml−1). (c) A431 cells were incubated with CIP (10 units, 30 min)-treated or untreated purified Flag-MIF proteins (500 ng ml−1) or Flag peptide (500 ng ml−1) for 30 min before being treated with Texas red-labeled EGF (50 ng ml−1) for 10 min. Immunofluorescence analysis was performed with an anti-EGFR antibody. Scale bar, 10 μm. The relative fluorescence intensity of Texas-red labeled EGF was quantified. The data represent the mean ± SD (n = 30 cells, 3 independent experiments). A two-tailed Student’s t test was used. *, P < 0.05. (d) A431 cells were incubated with CIP (10 units, 30 min)-treated or untreated purified Flag-MIF proteins (500 ng ml−1) or Flag peptide (500 ng ml−1) for 30 min before being treated with EGF (50 ng ml−1) for 30 min.
Supplementary Figure 5 The O-GlcNAcylation of MIF Inhibits EGF-induced Tumor Cell Invasion.
The cells that migrated to the opposite side of the insert were stained with crystal violet. Representative photomicrographs were taken with a digital camera mounted on a microscope. The Matrigel membranes that contained invading cells were dissolved in 4% deoxycholic acid and read colorimetrically at 590 nm for quantification of invasion. Images represent one out of 3 experiments. Scale bar, 100 μm. Column data represent the mean ± SD (n = 3 independent experiments). A two-tailed Student’s t test was used. *, P < 0.05; NS, not significant. (a) A431 cells with or without WT Flag-MIF or Flag-MIF S112/T113A expression were plated on the top surface of Matrigel inserts and treated with or without EGF (50 ng ml−1) for 24 h. (b) A431 cells, which were incubated with purified WT Flag-MIF (500 ng ml−1) or Flag-MIF S112/T113A (500 ng ml−1) protein, were plated on the top surface of Matrigel inserts and treated with or without EGF (50 ng ml−1) for 24 h. (c) U87 or U87/EGFR VIII cells with or without expression WT Flag-MIF or Flag-MIF S112/T113A were plated on the top surface of Matrigel inserts for 24 h.
Supplementary Figure 6 The O-GlcNAcylation of MIF Inhibits EGF-induced Brain Tumorigenesis, But Has No Effect On EGFRvIII-induced Tumor Growth.
A total of 5 × 105 U87 cells with or without MIF shRNA and EGFR shRNA (a) or U87 cells with or without WT Flag-MIF or Flag-MIF S112/T113A expression (b) or U87/EGFRvIII cells with or without expression of MIF WT or MIF S112/T113A mutant (c) were intracranially injected into athymic nude mice (n = 8 mice per group). After 2 weeks, the mice were euthanized and examined for tumor growth. Hematoxylin and eosin-stained coronal brain sections show representative tumor xenografts. Tumor volumes were measured by using length (a) and width (b) and calculated using the following equation: V = ab2/2. Data represent the means ± SD (n = 8 mice per group). A two-tailed Student’s t test was used. *, P < 0.05; NS, not significant.
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Zheng, Y., Li, X., Qian, X. et al. Secreted and O-GlcNAcylated MIF binds to the human EGF receptor and inhibits its activation. Nat Cell Biol 17, 1348–1355 (2015). https://doi.org/10.1038/ncb3222
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DOI: https://doi.org/10.1038/ncb3222
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