Abstract
Cushing's disease is caused by corticotroph adenomas of the pituitary. To explore the molecular mechanisms of endocrine autonomy in these tumors, we performed exome sequencing of 10 corticotroph adenomas. We found somatic mutations in the USP8 deubiquitinase gene in 4 of 10 adenomas. The mutations clustered in the 14-3-3 protein binding motif and enhanced the proteolytic cleavage and catalytic activity of USP8. Cleavage of USP8 led to increased deubiqutination of the EGF receptor, impairing its downregulation and sustaining EGF signaling. USP8 mutants enhanced promoter activity of the gene encoding proopiomelanocortin. In summary, our data show that dominant mutations in USP8 cause Cushing's disease via activation of EGF receptor signaling.
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Acknowledgements
We thank Minoru Fukuda and Mitsunori Fukuda for the LAMP2 antibody and EGFP-Rab11AS25A expression vector, respectively. The study was supported in by the Else Kröner-Fresenius-Stiftung (grant 2012_A103 to M.R.), Bundesministerium für Bildung und Forschung (grant BMBF 01EO1004-D2 to M.F. and B.A.), the Wilhelm Sander-Stiftung (grant 2012.095.1 to B.A.), and Grants-in-aid from the Ministry of Education, Culture, Science and Technology of Japan (grant 24112003 to M.K. and 24112008 to Y.S.). W.S. is supported by funds from Novartis AG, Pfizer and NovoNordisk and Ipsen for the Hypophysenregister der Arbeitsgemeinschaft Hypophyse of the German Society of Endocrinology. M.T. is supported by a grant from the German Federal Ministry of Education and Research (01EX1021B, Spitzencluster M4, Verbund Personalisierte Medizin, Teilprojekt NeoExNET (PM1)). We thank B. Mauracher, P. Rank, J. Stalla and J.L. Monteserin-Garcia for excellent technical assistance.
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M.R., M.F. and M.K. planned the study, conceived and designed the experiments, analyzed the data and wrote the paper. S.S., A.H., M.T., F.B., T.M., E.M.-Y., K.K., T.W., E.G. W.S., B.A. and T.M.S. designed and performed experiments and analyzed data. A.O., Y. S., K.T. and C.L.R. performed experiments. M.B. provided tumor tissue and clinical data.
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Integrated supplementary information
Supplementary Figure 1 Identification of somatic USP8 mutations in corticotroph adenomas.
Shown are somatic heterozygous mutations in USP8 (a, 2153C->CG and b, 2159C->CG) which were identified in two corticotroph pituitary adenomas resulting in Ser718Cys and Pro720Arg substitution, respectively. The mutations were not present in corresponding normal leukocyte DNA (c).
Supplementary Figure 2 USP8 immunofluorescence score in normal human pituitary and human corticotroph adenomas.
USP8 immunofluorescence staining in normal human pituitary tissue from autopsies (n=3) and in patients with corticotroph adenomas (n=14). wt = adenoma with wild-type USP8 sequence; mut = adenoma with mutant USP8. A semi-quantitative H-score was calculated by multiplying the staining intensity grading score (0-3) with the proportion score (0 if 0% were positive, 0.1 if 1–9%, 0.5 if 10–49%, and 1 if ≥50%) as described38.
Supplementary Figure 3 Biochemical characterization of USP8-C40.
a, Flag-tagged full-length and C40 forms of USP8 were expressed in COS-7 cells, immunoprecipitated from their lysates with anti-Flag antibody, and eluted with a Flag peptide. Eluted proteins were detected by anti-Flag immunoblotting. USP8-C40 was loaded at serial dilutions. Comparison of their band intensity lead to an estimation that ~9% of USP8-C40 preparation contains roughly equal molar numbers of USP8 as full-length USP8 preparation. b, Flag-tagged C40 forms derived from indicated USP8 mutants were immunopurified from transfected COS-7 cells, detected by Coomassie staining (bottom), and incubated with Lys63 Ub oligomers for 1 hour at 37°C. The reaction products were immunoblotted with anti-Ub antibody (top). c, Immunopurified C40 forms prepared in b, together with immunopurified full-length USP8, were immunoblotted with indicated antibodies.
Supplementary Figure 4 USP8 mutants do not affect the steady-state cell surface level of EGFR.
HeLa cells were transfected with indicated Flag-tagged USP8 proteins, untreated with EGF and stained with anti-Flag antibody (magenta), anti-EGFR antibody (green), and DAPI (blue). Asterisks indicate cells not expressing Flag-USP8 proteins. Bars, 10 µm.
Supplementary Figure 5 Subcellular localization of EGFR in the absence or presence of EGF stimulation.
HeLa cells were untreated (a-c”) or treated with EGF for 1 hour (d-f”), and stained with anti-EGFR antibody (a-f) together with anti-Hrs (a’ and d’), anti-LAMP2 (b’ and e’), or both anti-Hrs and anti-LAMP2 (c’ and f’) antibodies. a”-f” are merged images in which nuclei were stained in blue. Arrowheads indicate co-localization of EGFR with Hrs or LAMP2. Bars, 10 µm.
Supplementary Figure 6 USP8 mutants do not induce ERK phosphorylation in the absence of EGF stimulation.
HeLa cells were transfected with indicated Flag-tagged USP8 proteins, untreated with EGF and stained with anti-Flag (magenta) and anti-phospho-Erk1/2 (green) antibodies. Asterisks indicate cells not expressing Flag-USP8 proteins. Bars, 10 µm.
Supplementary Figure 7 Overexpression of USP8 mutants does not significantly elevate the level of ERK phosphorylation compared to that in untransfected cells after 30 minutes of EGF stimulation.
HeLa cells were transfected with indicated Flag-USP8 proteins, treated with EGF for 30 minutes and stained with anti-Flag (magenta) and anti-phospho-Erk1/2 (green) antibodies. Asterisks indicate cells not expressing Flag-USP8 proteins. Bars, 10 µm.
Supplementary Figure 8 Effect of the single mutations of USP8 p.[Leu713Arg;Tyr717Cys] on Pomc promoter activity.
AtT-20 cells were transfected with EGFR and wild-type USP8 (black), USP8 p.Leu713Arg;Tyr717Cys (grey) or the single mutant forms p.Leu713Arg and p.Tyr717Cys (white). Data are means ± standard deviation of 3 measurements, calculated as luciferase to β-galactosidase ratio and presented as percentage of wild-type USP8. RLA: relative luciferase activity. *P<0.01 to wild-type USP8. Transfected cells were left for 24 hours in medium supplemented with 2% fetal calf serum.
Supplementary Figure 9 Effect of USP8 on Pomc gene transcription.
a, Real time RT-PCR on RNA from cells co-transfected with EGFR plus wild-type USP8, USP8CA, C40 or C40CA. The empty pME-Flag vector was used as control. b, Real time RT-PCR on RNA from cells co-transfected with EGFR plus wild-type USP8, p.Ser718Pro, p.Ser718Cys, p.Pro720Arg, p.[Leu713Arg;Tyr717Cys] or p.Ser718del. Data are means ± standard deviation of 2 measurements and presented as Pomc/TfIIb. Transfected cells were left for 24 hours in medium supplemented with 2% fetal calf serum.
Supplementary Figure 10 Effect of Erk1/2 inhibition on USP8 action on Pomc.
a, Real time RT-PCR on RNA from AtT-20 cells co-transfected with EGFR plus wild-type USP8, USP8CA, C40 or C40CA. Cell were treated with 100 ng/ml EGF alone (black columns) or after 1 hour pretreatment with the Erk1/2 kinase inhibitor PD098059 (1 µM; white). The empty pME-Flag and pRC/CMV vectors were used as control. Data are means ± standard deviation of 2 measurements and presented as Pomc/TfIIb percentage of empty plasmid control. b, Effect of PD098059 pretreatment on EGF action on Pomc promoter in AtT-20 cells overexpressing EGFR plus wild-type USP8, USP8CA, C40 or C40CA. Data are means ± standard deviation of 3 measurements, calculated as luciferase to β-galactosidase ratio and presented as percentage of EGFR + pME-Flag (EGFR). RLA: relative luciferase activity. *P<0.001 to EGF-treated EGFR, #P<0.05 to EGF+PD098059-treated EGFR. c, Effect of PD098059 pretreatment on EGF action on Pomc promoter in AtT-20 cells overexpressing EGFR plus wild-type USP8 or its mutants. Data are means ± standard deviation of 3 measurements, calculated as luciferase to β-galactosidase ratio and presented as percentage of EGFR + wild-type USP8. *P<0.05 to wild-type USP8. In all experiments, transfected cells were left for 24 hours in medium supplemented with 2% fetal calf serum.
Supplementary Figure 11 Effect of USP8 on Erk1/2 and Akt phosphorylation.
a, Effect of EGFR plus wild-type USP8, USP8CA, C40 or C40CA on Erk1/2 and Akt phosphorylation. Transfected cells were left for 24 hours in medium supplemented with 2% fetal calf serum, serum deprived overnight and treated with 100 ng/ml EGF for 30 minutes. Phosphorylated pErk1/2-Thr202/Tyr204 and pAkt-Ser473 were normalized with total Erk1/2 and Akt values. Immunoblots for total Erk1/2 and total Akt were performed on the same blot after stripping with Tris pH2.0 buffer. Bands were quantified by densitometry (ChemiDoc imaging system, BioRad). Data are means ± standard error of 2 experiments and presented as percentage of EGFR control. a.u.: arbitrary units. b, Effect of EGFR plus USP8 mutants. Data are presented as percentage of wild-type USP8. c, Representative immunoblots of AtT-20 cells overexpressing EGFR plus wild-type USP8 or mutants for pErk1/2-Thr202/Tyr204 and pAkt-Ser473. Each membrane was stripped and blotted for total Erk1/2 and Akt, respectively, and then for β-actin.
Supplementary Figure 12 Effect of USP8 on Pomc promoter elements.
a, AtT-20 cells overexpressing EGFR plus wild-type USP8, USP8CA, C40 or C40CA were transfected with the luciferase reporter constructs bearing NurRE, NBRE and AP1 responsive elements. Data are means ± standard deviation of 3 measurements, calculated as luciferase to β-galactosidase ratio and presented as percentage of EGFR plus pME-Flag (EGFR). *P<0.01 to EGFR. b, Effect of the USP8 mutants on Pomc promoter elements. *P<0.05 and #P<0.01 to wild-type USP8. In all experiments, transfected cells were left for 24 hours in medium supplemented with 2% fetal calf serum. Data are means ± standard deviation of 3 measurements, calculated as luciferase to β-galactosidase ratio and presented as percentage of EGFR + wild-type USP8. RLA: relative luciferase activity.
Supplementary Figure 13 Cell growth promoting effect of USP8.
AtT-20 cells were stably transfected with EGFR and wild-type or mutant USP8s. a, Effect of wild-type USP8, USP8CA, C40 or C40CA on cell viability. Cells were serum deprived for 20 hours and treated with vehicle or 100 ng/ml EGF for 48 hours. Data are means ± standard deviation of 3 measurements and presented as percentage of EGFR-overexpressing cells. *P<0.05 and **P<0.01 to each EGFR, #P<0.05 to vehicle-treated EGFR. b, Effect of USP8 mutants on cell viability. Data are means ± standard deviation of 3 measurements and presented as percentage of EGFR + wild-type USP8. *P<0.05, **P<0.01 and ***P<0.001 to each EGFR + wild-type USP8, #P<0.05 to vehicle-treated EGFR.
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Reincke, M., Sbiera, S., Hayakawa, A. et al. Mutations in the deubiquitinase gene USP8 cause Cushing's disease. Nat Genet 47, 31–38 (2015). https://doi.org/10.1038/ng.3166
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DOI: https://doi.org/10.1038/ng.3166
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