Cullin 3 targets the tumor suppressor gene ARMC5 for ubiquitination and degradation

in Endocrine-Related Cancer
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  • 1 Université de Paris, Institut Cochin, INSERM, CNRS, Paris, France
  • 2 Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
  • 3 Université de Paris, PARCC, INSERM, Paris, France
  • 4 Adrenal Unit, Hormone and Molecular Genetic Laboratory/LIM42, Hospital of Clinics, School of Medicine, University of São Paulo, São Paulo, Brazil
  • 5 Department of Endocrinology, APHP, Cochin Hospital, Paris, France

Correspondence should be addressed to I P Cavalcante or B Ragazzon: isadoracavalcante@gmail.com or bruno.ragazzon@inserm.fr

*(A Vaczlavik and L Drougat contributed equally to this work)

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ARMC5 (Armadillo repeat containing 5 gene) was identified as a new tumor suppressor gene responsible for hereditary adrenocortical tumors and meningiomas. ARMC5 is ubiquitously expressed and encodes a protein which contains a N-terminal Armadillo repeat domain and a C-terminal BTB (Bric-a-Brac, Tramtrack and Broad-complex) domain, both docking platforms for numerous proteins. At present, expression regulation and mechanisms of action of ARMC5 are almost unknown. In this study, we showed that ARMC5 interacts with CUL3 requiring its BTB domain. This interaction leads to ARMC5 ubiquitination and further degradation by the proteasome. ARMC5 alters cell cycle (G1/S phases and cyclin E accumulation) and this effect is blocked by CUL3. Moreover, missense mutants in the BTB domain of ARMC5, identified in patients with multiple adrenocortical tumors, are neither able to interact and be degraded by CUL3/proteasome nor alter cell cycle. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives in the understanding of its tumor suppressor activity.

Supplementary Materials

    • Figure S1 - ARMC5 regulates cell cycle and cyclin E turnover in HEK293 cells Propidium iodide was used to determine DNA content. (A) Flow cytometry analysis after ARMC5 depletion revealed a decrease in the percentage of cells in G1 phase and an increase in S phase. (B) Depletion of ARMC5 led to an increase in full length (FL) and low molecular weight (LMW) cyclin E. (C) Overexpression of WT ARMC5 increases the number of cells in G1 phase. However, co-expression of WT ARMC5 and CUL3, as well as overexpression of p.L754P mutated ARMC5 have no longer an effect in cell cycle. Images are representative of at least three independent experiments. Significance was assessed by using two-way ANOVA, followed by Bonferroni post-test.
    • Figure S2 - ARMC5 depletion increases CCNE1 mRNA transcription in both (A) H295R and (B) HEK293 cells. Significance was assessed by using student’s t-test.
    • Figure S3 - ARMC5 depletion favours cell cycle progression in H295R cells Propidium iodide was used to determine DNA content. (A) Flow cytometry analysis after ARMC5 depletion. (B) Synchronization of cells in late G1 phase with aphidicolin (10µM) for 24h. (C) Flow cytometry analysis 4h (C), 8h (D), 12h (E) and 24h (F) after release from aphidicolin. Significance was assessed by using two-way ANOVA, followed by Bonferroni post-test.
    • Supplemental table 1

 

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  • Alencar GA, Lerario AM, Nishi MY, Mariani BM, Almeida MQ, Tremblay J, Hamet P, Bourdeau I, Zerbini MC, Pereira MA, et al. 2014 ARMC5 mutations are a frequent cause of primary macronodular adrenal hyperplasia. Journal of Clinical Endocrinology and Metabolism 99 E1501E1509. (https://doi.org/10.1210/jc.2013-4237)

    • Search Google Scholar
    • Export Citation
  • Assie G, Libe R, Espiard S, Rizk-Rabin M, Guimier A, Luscap W, Barreau O, Lefevre L, Sibony M, Guignat L, et al. 2013 ARMC5 mutations in macronodular adrenal hyperplasia with Cushing’s syndrome. New England Journal of Medicine 369 21052114. (https://doi.org/10.1056/NEJMoa1304603)

    • Search Google Scholar
    • Export Citation
  • Assie G, Letouze E, Fassnacht M, Jouinot A, Luscap W, Barreau O, Omeiri H, Rodriguez S, Perlemoine K, Rene-Corail F, et al. 2014 Integrated genomic characterization of adrenocortical carcinoma. Nature Genetics 46 607612. (https://doi.org/10.1038/ng.2953)

    • Search Google Scholar
    • Export Citation
  • Bennett EJ, Rush J, Gygi SP & Harper JW 2010 Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell 143 951965. (https://doi.org/10.1016/j.cell.2010.11.017)

    • Search Google Scholar
    • Export Citation
  • Berthon A, Faucz F, Bertherat J & Stratakis CA 2017a Analysis of ARMC5 expression in human tissues. Molecular and Cellular Endocrinology 441 140145. (https://doi.org/10.1016/j.mce.2016.08.018)

    • Search Google Scholar
    • Export Citation
  • Berthon A, Faucz FR, Espiard S, Drougat L, Bertherat J & Stratakis CA 2017b Age-dependent effects of Armc5 haploinsufficiency on adrenocortical function. Human Molecular Genetics 26 34953507. (https://doi.org/10.1093/hmg/ddx235)

    • Search Google Scholar
    • Export Citation
  • Bourcigaux N, Gaston V, Logie A, Bertagna X, Le Bouc Y & Gicquel C 2000 High expression of cyclin E and G1 CDK and loss of function of p57KIP2 are involved in proliferation of malignant sporadic adrenocortical tumors. Journal of Clinical Endocrinology and Metabolism 85 322330. (https://doi.org/10.1210/jcem.85.1.6303)

    • Search Google Scholar
    • Export Citation
  • Bourdeau I, Oble S, Magne F, Levesque I, Caceres KY, Nolet S, Awadalla P, Tremblay J, Hamet P, Fragoso MC, et al. 2016 ARMC5 mutations in a large French-Canadian family with cortisol-secreting beta-adrenergic/vasopressin responsive bilateral macronodular adrenal hyperplasia. European Journal of Endocrinology 174 8596. (https://doi.org/10.1530/EJE-15-0642)

    • Search Google Scholar
    • Export Citation
  • Cavalcante IP, Nishi M, Zerbini MCN, Almeida MQ, Brondani VB, Botelho MLAA, Tanno FY, Srougi V, Chambo JL, Mendonca BB, et al. 2018 The role of ARMC5 in human cell cultures from nodules of primary macronodular adrenocortical hyperplasia (PMAH). Molecular and Cellular Endocrinology 460 3646. (https://doi.org/10.1016/j.mce.2017.06.027)

    • Search Google Scholar
    • Export Citation
  • Ciechanover A 2017 Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Best Practice and Research: Clinical Haematology 30 341355. (https://doi.org/10.1016/j.beha.2017.09.001)

    • Search Google Scholar
    • Export Citation
  • Davidge B, Rebola KGO, Agbor LN, Sigmund CD & Singer JD 2019 Cul3 regulates cyclin E1 protein abundance via a degron located within the N-terminal region of cyclin E. Journal of Cell Science 132 jcs.233049. (https://doi.org/10.1242/jcs.233049)

    • Search Google Scholar
    • Export Citation
  • Dubiel W, Dubiel D, Wolf DA & Naumann M 2018 Cullin 3-based ubiquitin ligases as master regulators of mammalian cell differentiation. Trends in Biochemical Sciences 43 95107. (https://doi.org/10.1016/j.tibs.2017.11.010)

    • Search Google Scholar
    • Export Citation
  • Elbelt U, Trovato A, Kloth M, Gentz E, Finke R, Spranger J, Galas D, Weber S, Wolf C, Konig K, et al. 2015 Molecular and clinical evidence for an ARMC5 tumor syndrome: concurrent inactivating germline and somatic mutations are associated with both primary macronodular adrenal hyperplasia and meningioma. Journal of Clinical Endocrinology and Metabolism 100 E119E128. (https://doi.org/10.1210/jc.2014-2648)

    • Search Google Scholar
    • Export Citation
  • Espiard S, Drougat L, Libe R, Assie G, Perlemoine K, Guignat L, Barrande G, Brucker-Davis F, Doullay F, Lopez S, et al. 2015 ARMC5 mutations in a large cohort of primary macronodular adrenal hyperplasia: clinical and functional consequences. Journal of Clinical Endocrinology and Metabolism 100 E926E935. (https://doi.org/10.1210/jc.2014-4204)

    • Search Google Scholar
    • Export Citation
  • Faucz FR, Zilbermint M, Lodish MB, Szarek E, Trivellin G, Sinaii N, Berthon A, Libe R, Assie G, Espiard S, et al. 2014 Macronodular adrenal hyperplasia due to mutations in an armadillo repeat containing 5 (ARMC5) gene: a clinical and genetic investigation. Journal of Clinical Endocrinology and Metabolism 99 E1113E1119. (https://doi.org/10.1210/jc.2013-4280)

    • Search Google Scholar
    • Export Citation
  • Gagliardi L, Schreiber AW, Hahn CN, Feng J, Cranston T, Boon H, Hotu C, Oftedal BE, Cutfield R, Adelson DL, et al. 2014 ARMC5 mutations are common in familial bilateral macronodular adrenal hyperplasia. Journal of Clinical Endocrinology and Metabolism 99 E1784E1792. (https://doi.org/10.1210/jc.2014-1265)

    • Search Google Scholar
    • Export Citation
  • Hu Y, Lao L, Mao J, Jin W, Luo H, Charpentier T, Qi S, Peng J, Hu B, Marcinkiewicz MM, et al. 2017 Armc5 deletion causes developmental defects and compromises T-cell immune responses. Nature Communications 8 13834. (https://doi.org/10.1038/ncomms13834)

    • Search Google Scholar
    • Export Citation
  • Huttlin EL, Bruckner RJ, Paulo JA, Cannon JR, Ting L, Baltier K, Colby G, Gebreab F, Gygi MP, Parzen H, et al. 2017 Architecture of the human interactome defines protein communities and disease networks. Nature 545 505509. (https://doi.org/10.1038/nature22366)

    • Search Google Scholar
    • Export Citation
  • Lu A & Pfeffer SR 2013 Golgi-associated RhoBTB3 targets cyclin E for ubiquitylation and promotes cell cycle progression. Journal of Cell Biology 203 233250. (https://doi.org/10.1083/jcb.201305158)

    • Search Google Scholar
    • Export Citation
  • Ma ZY, Song ZJ, Chen JH, Wang YF, Li SQ, Zhou LF, Mao Y, Li YM, Hu RG, Zhang ZY, et al. 2015 Recurrent gain-of-function USP8 mutations in Cushing’s disease. Cell Research 25 306317. (https://doi.org/10.1038/cr.2015.20)

    • Search Google Scholar
    • Export Citation
  • Morreale FE & Walden H 2016 Types of ubiquitin ligases. Cell 165 248248.e1. (https://doi.org/10.1016/j.cell.2016.03.003)

  • Ragazzon B, Cazabat L, Rizk-Rabin M, Assie G, Groussin L, Fierrard H, Perlemoine K, Martinez A & Bertherat J 2009 Inactivation of the Carney complex gene 1 (protein kinase A regulatory subunit 1A) inhibits SMAD3 expression and TGF beta-stimulated apoptosis in adrenocortical cells. Cancer Research 69 72787284. (https://doi.org/10.1158/0008-5472.CAN-09-1601)

    • Search Google Scholar
    • Export Citation
  • Reincke M, Sbiera S, Hayakawa A, Theodoropoulou M, Osswald A, Beuschlein F, Meitinger T, Mizuno-Yamasaki E, Kawaguchi K, Saeki Y, et al. 2015 Mutations in the deubiquitinase gene USP8 cause Cushing’s disease. Nature Genetics 47 3138. (https://doi.org/10.1038/ng.3166)

    • Search Google Scholar
    • Export Citation
  • Scortegagna M, Berthon A, Settas N, Giannakou A, Garcia G, Li JL, James B, Liddington RC, Vilches-Moure JG, Stratakis CA, et al. 2017 The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion. JCI Insight 2 97128. (https://doi.org/10.1172/jci.insight.97128)

    • Search Google Scholar
    • Export Citation
  • Singer JD, Gurian-West M, Clurman B & Roberts JM 1999 Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells. Genes and Development 13 23752387. (https://doi.org/10.1101/gad.13.18.2375)

    • Search Google Scholar
    • Export Citation
  • Tissier F, Louvel A, Grabar S, Hagnere AM, Bertherat J, Vacher-Lavenu MC, Dousset B, Chapuis Y, Bertagna X & Gicquel C 2004 Cyclin E correlates with malignancy and adverse prognosis in adrenocortical tumors. European Journal of Endocrinology 150 809817. (https://doi.org/10.1530/eje.0.1500809)

    • Search Google Scholar
    • Export Citation
  • Wilkins A, Ping Q & Carpenter CL 2004 RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex. Genes and Development 18 856861. (https://doi.org/10.1101/gad.1177904)

    • Search Google Scholar
    • Export Citation
  • Zhang DD, Lo SC, Cross JV, Templeton DJ & Hannink M 2004 Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Molecular and Cellular Biology 24 1094110953. (https://doi.org/10.1128/MCB.24.24.10941-10953.2004)

    • Search Google Scholar
    • Export Citation
  • Zheng S, Cherniack AD, Dewal N, Moffitt RA, Danilova L, Murray BA, Lerario AM, Else T, Knijnenburg TA, Ciriello G, et al. 2016 Comprehensive pan-genomic characterization of adrenocortical carcinoma. Cancer Cell 30 363. (https://doi.org/10.1016/j.ccell.2016.07.013)

    • Search Google Scholar
    • Export Citation
  • Zhou Z, Xu C, Chen P, Liu C, Pang S, Yao X & Zhang Q 2015 Stability of HIB-Cul3 E3 ligase adaptor HIB is regulated by self-degradation and availability of its substrates. Scientific Reports 5 12709. (https://doi.org/10.1038/srep12709)

    • Search Google Scholar
    • Export Citation