Differential expression of protein kinase A catalytic and regulatory subunits in adrenocortical adenomas

Isabel Weigand; Cristina Ronchi; Marthe Rizk-Rabin; Dalmazi Guido Di; Davide Calebiro; Vanessa Wild; Felix Beuschlein; Bruno Allolio; Jerome Bertherat; Martin Fassnacht; Silviu Sbiera

doi:10.1530/endoabs.41.GP116

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Endocrine Abstracts (2016) 41 GP116 | DOI: 10.1530/endoabs.41.GP116

ECE2016 Guided Posters Endocrine Tumours (10 abstracts)

Differential expression of protein kinase A catalytic and regulatory subunits in adrenocortical adenomas

Isabel Weigand ¹ , Cristina Ronchi ^1, , Marthe Rizk-Rabin ² , Guido Di Dalmazi ³ , Davide Calebiro ⁴ , Vanessa Wild ⁵ , Felix Beuschlein ³ , Bruno Allolio ¹ , Jérôme Bertherat ² , Martin Fassnacht ^1, & Silviu Sbiera ^1,

Err

Author affiliations

¹Departement of Internal Medicine 1, Endocrine and Diabetes Unit, University Hospital Würzburg, Würzburg, Germany; ²Institut Cochin Department of Endocrinology, Paris, France; ³Medizinische Klinik and Poliklinik IV, Ludwig-Maximilians University, München, Germany; ⁴University of Wuerzburg, Institute of Pharmacology and Toxicology and Bioimaging Center, Würzburg, Germany; ⁵Institute of Pathology, University of Wuerzburg, Würzburg, Germany; ⁶Comprehensive Cancer Center Mainfranken, University of Wuerzburg, Würzburg, Germany.

Background: Heterozygous activating somatic mutations in the catalytic subunit α (Cα) of Protein Kinase A (PKA) underlie 30–40% of cortisol producing adrenocortical adenomas (CPA). The activity of the catalytic subunits α,β,γ is controlled by the regulatory subunits (Iα,Iβ, IIα, IIβ). Previous reports found uncommonly reduced levels of RIIβ in CPA compared to other adrenocortical tumors.

Aim: Investigating a possible correlation between Cα mutational status and RIIβ expression levels.

Methods: We performed immunohistochemistry staining of all PKA regulatory subunits and Cα on FFPE tissue from normal adrenal glands (n=12), adrenocortical adenomas (n=87 including 40 CPA, of which 18 Cα-mutated) and adrenocortical carcinomas (ACC, n=35). mRNA (n=19) and proteins (n=24) from CPAs were isolated to perform qPCR and immunoblotting. Functional experiments were performed in the ACC cell line NCI-H295R.

Results: RIIβ expression was strongly reduced in Cα mutated CPAs, especially in tumors harboring the frequent L206R mutation (n=13, mean expression: 0.4±0.5 vs 1.5±0.7, p<0.05). Similar results were observed for RIα (1.8±0.9 vs 2.6±0.6, p<0.05) but not for the other regulatory subunits. Cα levels were not influenced by the mutations. mRNA expression of all subunits was unchanged in CPA^Cα-wt compared to CPA^Cα-mut. Since no significant changes in mRNA expression were observed, downregulation of RIIβ and RIα seems a post-transcriptional event. However, in NCI-H295R cells, proteasome inhibition did not save RIIβ from degradation, rendering other degradation pathways more likely. Furthermore, we found different expression patterns of the PKA subunits in normal adrenal glands, overlapping zoning, with low RIIβ in the cortisol producing Z. fasciculata. The expression pattern of PKA subunits in normal adrenal glands indicates possible particular roles of these subunits on differentially regulating hormone secretion.

Conclusion: We demonstrate for the first time, that mutations in PKA Cα lead to post-transcriptional downregulation of the main regulatory subunits in adrenocortical tissues enhancing Cα catalytic activity leading to increased cortisol production.