Lack of Ubiquitin Specific Protease 8 (USP8) Mutations in Canine Corticotroph Pituitary Adenomas

Abstract

Purpose: Cushing's disease (CD), also known as pituitary-dependent hyperadrenocorticism, is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. Affected humans and dogs have similar clinical manifestations, however, the incidence of the canine disease is thousand-fold higher. This makes the dog an obvious model for studying the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite certain similarities identified at the molecular level, the question still remains whether the two species have a shared oncogenetic background. Recently, hotspot recurrent mutations in the gene encoding for ubiquitin specific protease 8 (USP8) have been identified as the main driver behind the formation of ACTH-secreting pituitary adenomas in humans. In this study, we aimed to verify whether USP8 mutations also play a role in the development of such tumours in dogs.

Methods: Presence of USP8 mutations was analysed by Sanger and PCR-cloning sequencing in 38 canine ACTH-secreting adenomas. Furthermore, the role of USP8 and EGFR protein expression was assessed by immunohistochemistry in a subset of 25 adenomas.

Results: None of the analysed canine ACTH-secreting adenomas presented mutations in the USP8 gene. In a subset of these adenomas, however, we observed an increased nuclear expression of USP8, a phenotype characteristic for the USP8 mutated human tumours, that correlated with smaller tumour size but elevated ACTH production in those tumours.

Conclusions: Canine ACTH-secreting pituitary adenomas lack mutations in the USP8 gene suggesting a different genetic background of pituitary tumourigenesis in dogs. However, elevated nuclear USP8 protein expression in a subset of tumours was associated with a similar phenotype as in their human counterparts, indicating a possible end-point convergence of the different genetic backgrounds in the two species. In order to establish the dog as a useful animal model for the study of CD, further comprehensive studies are needed.

Fig 1. Sanger sequencing results of the 14-3-3 binding site of canine ubiquitin specific protease 8 (USP8).

Representation of protein (A) and DNA (B) USP8 sequence consensus around the 14-3-3 binding motif (red rectangle) between Homo sapiens (region corresponding to the amino-acids 715–720) and Canis lupus familiaris (region corresponding to the amino-acids 713–718). Sanger sequencing of directly (C) and cloning PCR (D) amplified DNA showed no mutations present in the 14-3-3 binding motif (red rectangle).

Fig 2. Interspecies specificity of the antibodies used for immunohistochemistry.

(A) Western blot analysis of EGFR, USP8 and α-tubulin expression proteins extracted from dog testis, dog pituitary adenoma and normal pituitary gland and from the human adrenocortical cell-line NCI-H295. (B) Protein sequence conservation status between human (upper sequence) and dog (lower sequence) in the area of the anti human USP8 antibody epitope (amino-acids 239–377, blue colour).

Fig 3. Immunohistological characterization of canine pituitary tissues.

Normal canine pituitary gland (A-D) and canine ACTH-secreting pituitary adenomas (E-T) stained with Haematoxylin/Eosine (A, E, I, M and Q), ACTH (B, F, J, N and R), EGFR (C, G, K, O and S) and USP8 (D, H, L, P and T). The ACTH-secreting pituitary adenomas have been stratified according to their nuclear USP8 staining from H-score 3 (H) to H-score 0 (T). Scale bars = 20μm.

Fig 4. Staining intensity distribution in normal canine pituitary glands vs. canine ACTH-secreting pituitary adenomas.

H-scores distribution of EGFR expression in the membrane (A), cytoplasm (B) and nucleus (C) and USP8 in the cytoplasm (D) and nucleus (E) of normal dog pituitary cells (empty circles) and ACTH producing pituitary adenoma cells (full circles).

Fig 5. Influence of nuclear USP8 protein expression on the pathophysiology of canine ACTH-secreting pituitary adenomas.

Correlations between nuclear USP8 staining intensity (X-axis) and ACTH staining intensity (A), blood ACTH levels pre-surgery (B) and tumour size reported as P/B ratio (C).