ECE2015 Oral Communications Adrenal 1 (5 abstracts)
Functional study of ARMC5 (armadillo repeat containing 5), a new tumour suppressor gene involved in primary bilateral macronodular adrenal hyperplasia
ludivine Drougat 1 , Stéphanie Espiard 1 , Stéphane Doly 1 , Stéphanie Rodriguez 1 , Marthe Rizk-Rabin 1 , Rossella Libé 1, , Guillaume Assié 1, , Stefano Marullo 1 , Bruno Ragazzon 1 & Jérôme Bertherat 1,
1Institut Cochin, Université Paris Descartes, CNRS (UMR 8104)- Inserm, U1016, PARIS, France; 2AP-HP, Hôpital Cochin, Department of Endocrinology, Center for rare adrenal diseases, PARIS, France.
Introduction: Primary bilateral macronodular adrenal hyperplasia (PBMAH) are adrenocortical tumors leading to adrenal Cushing’s syndrome. Recently, our laboratory has identified the first gene predisposing frequently to PBMAH in adults, named ARMC5 (Armadillo Repeat Containing 5)1. The ARMC5-inactivating mutations identified in leucocyte and tumour DNA in PBMAH patients suggest that ARMC5 is a tumour suppressor gene. However, the mechanisms of action of ARMC5 remain unknown. The aim of this work is to understand both the biological role of ARMC5 protein and how mutations alter this function.
Methods: The human adrenocortical cells H295R were transiently transfected with expression vectors of WT or mutants ARMC5. The eight mutations studied (F700del, C657R, I664S, L754P, Y736S, R362L, R315W, L331P, C139R) have been identified in leukocyte or tumour DNA of operated PBMAH patients. After 8 and 14 h of transfection, apoptosis was analysed by flow cytometry and immunofluorescence analysis (cleaved caspase 3 staining). In parallel, co-immunoprecipitation experiments were carried out to identify partners of the ARMC5 protein by mass spectrometry.
Results: Cell overexpressing WT ARMC5 promptly undergo apoptosis compared to cells transfected with mutants ARMC5 that all lose the ability to induce apoptosis. We identified, by mass spectrometry, 16 proteins specifically interacting with WT ARMC5. These proteins are involved in the degradation of proteins, the redox system and the cAMP/PKA pathway including subunits of PKA. The interaction between ARMC5 and subunits of PKA were then confirmed by co-immunoprecipitations and BRET (Bioluminescence Resonance Energy Transfert) experiments.
Conclusion: These results confirm the role of ARMC5 in the regulation of apoptosis and the loss of this function by the mutants identified in PBMAH. By its binding with actors involved in the cAMP/PKA pathway, known to play an essential role in the adrenocortical function, ARMC5 seems to be a major factor in the adrenal cortex physiology and pathophysiology. 1- Assié et al., NEJM, 2014.
Disclosure: This study was supported in part by the Agence Nationale de la Recherche (ANR-10-Blan-1136), the COMETE Network (Programme Hospitalier de Recherche Clinique Grant AOM95201), the Assistance Publique-Hôpitaux de Paris.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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