ARMC5 Role in Human Cell Cultures Obtained from Adrenal Nodules of Primary Macronodular Adrenocortical Hyperplasia (PMAH)

Presentation Number: OR01-1
Date of Presentation: April 3rd, 2017

Isadora Pontes Cavalcante1, Maria Claudia N Zerbini2, Mirian Y Nishi3, Jose Luiz Chambo3, Madson Q. Almeida4, Claudimara Ferini Pacicco Lotfi5 and Maria Candida Barisson Villares Fragoso*6
1Institute of Biomedical Sciences, University of Sao Paulo, São Paulo, Brazil, 2Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 3Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 4Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulol, 5Institute of Biomedical Sciences - University of Sao Paulo, Sao Paulo SP, Brazil, 6Hospital das Clinicas, University of Sao Paulo, School of Medicine, Sao Paulo, BRAZIL

Abstract

Background: The mechanisms causing hypercortisolism in PMAH are not fully clarified. The participation of ectopic receptors and regulation of intra-adrenal ACTH in clusters have been considered. Additionally, ARMC5 mutations have been described as main cause of PMAH. So far, the functional study to analyze the role of ARMC5 used the adrenocortical carcinoma cell line, H295R. Therefore, we propose a more suitable model in cell cultures obtained from nodules of PMAH. Aim: Characterization of PMAH cell cultures through morphological, functional and molecular analyses, as well as ARMC5 functional studies. Methods: Cell cultures obtained from adrenal nodules of 10 unrelated patients with PMAH with or without ARMC5 mutations were analyzed for: 1) ARMC5 direct sequencing; 2) Oil Red O staining; 3) ectopic receptors, steroidogenic enzymes, POMC and ARMC5 mRNA expression by qPCR; 4) ARMC5 and ACTH detection by respectively immunofluorescence/immunoblotting and immunocytochemistry; 5) ARMC5 silencing through lentiviral shRNA in non-mutated PMAH cells, followed by steroidogenic and proliferative functional studies. Results: 1) ARMC5 mutations were located mostly in exons 1, 2 and 3; ARMC5 germline mutations were identified in 7 cell cultures. In 5 of those, the germline mutations were associated with somatic mutations or LOH, and in 2 of them, it was not identified somatic mutations. In 3, no mutations were identified; 2) All of cell cultures presented an accumulation of lipid droplets in the cytoplasm; 3) ARMC5 expression was higher in the cell cultures without ARMC5 mutations, whereas the expression of HSD3B2, CYP17A1, MC2R, POMC and ectopic receptors were heterogeneous among cell cultures; 4) ARMC5 protein expression is prevalent in the cytoplasm, whereas ACTH expression is also cytoplasmatic but sparse and situated in cell clusters; 5) ARMC5 silencing in PMAH cell cultures (without ARMC5 mutation) caused a decrease in StAR (p=0.0276), CYP17A1 (p=0.0398) and MC2R (p=0.0036) mRNA expression, as well as an increase in CCNE1 (p=0.0120) and in its proliferative capacity after 72h (p<0.001). Conclusions: ARMC5 mutations in exons 1, 2 and 3 are located in the region responsible for protein-protein interactions of ARM repeat family, whereas the detection of lipid droplets, MC2R, HSD3B2 and CYP17A1 mRNA expression imply the presence of steroidogenic cells in the cultures obtained. Also, the presence of intraadrenal ACTH and ectopic receptors seem to be independent of ARMC5 mutations, given that both mutated and non-mutated cell cultures express them. Finally, we characterized for the first time PMAH cell cultures and confirm the importance of ARMC5 in the steroidogenesis related to PMAH previously described in H295R cells. In addition, we report the potential importance of ARMC5 in proliferation and cell cycle regulation of PMAH cell cultures, which needs to be further explored.

 

Nothing to Disclose: IPC, MCNZ, MYN, JLC, MQA, CFPL, MCBVF