Rapid proteasomal degradation of mutant proteins is the primary mechanism leading to tumorigenesis in patients with missense AIP mutations

CONTEXT The pathogenic effect of AIP mutations (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. OBJECTIVE To analyze the mechanism/speed of protein turnover of wild-type (WT) and missense AIP variants, correlating protein half-life with clinical parameters. DESIGN Half-life and protein-protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed. SETTING Clinical academic research institution. PATIENTS Data was obtained from our cohort of pituitary adenoma patients and literature-reported cases. INTERVENTIONS Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. GST pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by co-immunoprecipitation and gene knockdown. Relevant clinical data was collected. MAIN OUTCOME MEASURES Half-life of WT and mutant AIP proteins and its correlation with clinical parameters. RESULTS Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7h). AIP variants were divided in stable proteins (median 77.7h [IQR 60.7-92.9]), and those with short (27h [21.6-28.7]) or very short (7.7h [5.6-10.5]) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r=0.411, P=0.002). The FBXO3-containing SCF complex was identified as the E3 ubiquitin-ligase recognizing AIP. CONCLUSIONS AIP is a stable protein, driven to ubiquitination by the SCF complex. Enhanced proteasomal degradation is a novel pathogenic mechanism for AIPmuts, with direct implications for the phenotype

Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing's disease.

The CABLES1 cell cycle regulator participates in the adrenal-pituitary negative feedback, and its expression is reduced in corticotropinomas, pituitary tumors with a largely unexplained genetic basis. We investigated the presence of CABLES1 mutations/copy number variations (CNVs) and their associated clinical, histopathological and molecular features in patients with Cushing's disease (CD). Samples from 146 pediatric (118 germline DNA only/28 germline and tumor DNA) and 35 adult (tumor DNA) CD patients were screened for CABLES1 mutations. CNVs were assessed in 116 pediatric CD patients (87 germline DNA only/29 germline and tumor DNA). Four potentially pathogenic missense variants in CABLES1 were identified, two in young adults (c.532G > A, p.E178K and c.718C > T, p.L240F) and two in children (c.935G > A, p.G312D and c.1388A > G, and p.D463G) with CD; no CNVs were found. The four variants affected residues within or close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein and impaired its ability to block cell growth in a mouse corticotropinoma cell line (AtT20/D16v-F2). The four patients had macroadenomas. We provide evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene. Its function might link two of the main molecular mechanisms altered in corticotropinomas: the cyclin-dependent kinase/cyclin group of cell cycle regulators and the epidermal growth factor receptor signaling pathway. Further studies are needed to assess the prevalence of CABLES1 mutations among patients with other types of pituitary adenomas and to elucidate the pituitary-specific functions of this gene

Duplications disrupt chromatin architecture and rewire GPR101-enhancer communication in X-linked acrogigantism

X-linked acrogigantism (X-LAG) is the most severe form of pituitary gigantism and is characterized by aggressive growth hormone (GH)-secreting pituitary tumors that occur in early childhood. X-LAG is associated with chromosome Xq26.3 duplications (the X-LAG locus typically includes VGLL1, CD40LG, ARHGEF6, RBMX, and GPR101) that lead to massive pituitary tumoral expression of GPR101, a novel regulator of GH secretion. The mechanism by which the duplications lead to marked pituitary misexpression of GPR101 alone was previously unclear. Using Hi-C and 4C-seq, we characterized the normal chromatin structure at the X-LAG locus. We showed that GPR101 is located within a topologically associating domain (TAD) delineated by a tissue-invariant border that separates it from centromeric genes and regulatory sequences. Next, using 4C-seq with GPR101, RBMX, and VGLL1 viewpoints, we showed that the duplications in multiple X-LAG-affected individuals led to ectopic interactions that crossed the invariant TAD border, indicating the existence of a similar and consistent mechanism of neo-TAD formation in X-LAG. We then identified several pituitary active cis-regulatory elements (CREs) within the neo-TAD and demonstrated in vitro that one of them significantly enhanced reporter gene expression. At the same time, we showed that the GPR101 promoter permits the incorporation of new regulatory information. Our results indicate that X-LAG is a TADopathy of the endocrine system in which Xq26.3 duplications disrupt the local chromatin architecture forming a neo-TAD. Rewiring GPR101-enhancer interaction within the new regulatory unit is likely to cause the high levels of aberrant expression of GPR101 in pituitary tumors caused by X-LAG.The work was supported by the following funding sources: Fondazione Telethon, Italy grant no. GGP20130 (to G.T.); Society for Endocrinology equipment grant (to G.T.); Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), National Institutes of Health (NIH) Research project Z01-HD008920 (to C.A.S., supporting G.T., F.R.F.); Fonds d’Investissement pour la Recherche Scientifique (FIRS) of the Centre Hospitalier Universitaire de Liège (to A.F.D. and A.B.); the JABBS Foundation, UK (to A.B.); and Novo Nordisk Belgium Educational Grant, Belgium (to A.F.D. and A.B.). M.F. was funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#800396) and a Juan de la Cierva-Formación fellowship from the Spanish Ministry of Science and Innovation (FJC2018-038233-I). G.T. was funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#843843). A.F.D. and D.A. were supported by Action de Recherche Concertée (ARC) Grant 17/21-01 from Liège University. D.A. was supported by grants from Télévie (7461117 F, 7454719 F) and the Léon Fredericq Foundation, Belgium

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal alpha-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PMCID: PMC3534021This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

PAM variants in patients with thyrotrophinomas, cyclical Cushing’s disease and prolactinomas

IntroductionGermline loss-of-function variants in PAM, encoding peptidylglycine α-amidating monooxygenase (PAM), were recently discovered to be enriched in conditions of pathological pituitary hypersecretion, specifically: somatotrophinoma, corticotrophinoma, and prolactinoma. PAM is the sole enzyme responsible for C-terminal amidation of peptides, and plays a role in the biosynthesis and regulation of multiple hormones, including proopiomelanocortin (POMC).MethodsWe performed exome sequencing of germline and tumour DNA from 29 individuals with functioning pituitary adenomas (12 prolactinomas, 10 thyrotrophinomas, 7 cyclical Cushing’s disease). An unfiltered analysis was undertaken of all PAM variants with population prevalence <5%.ResultsWe identified five coding, non-synonymous PAM variants of interest amongst seven individuals (six germline, one somatic). The five variants comprised four missense variants and one truncating variant, all heterozygous. Each variant had some evidence of pathogenicity based on population prevalence, conservation scores, in silico predictions and/or prior functional studies. The yield of predicted deleterious PAM variants was thus 7/29 (24%). The variants predominated in individuals with thyrotrophinomas (4/10, 40%) and cyclical Cushing’s disease (2/7, 29%), compared to prolactinomas (1/12, 8%).ConclusionThis is the second study to demonstrate a high yield of suspected loss-of-function, predominantly germline, PAM variants in individuals with pathological pituitary hypersecretion. We have extended the association with corticotrophinoma to include the specific clinical entity of cyclical Cushing’s disease and demonstrated a novel association between PAM variants and thyrotrophinoma. PAM variants might act as risk alleles for pituitary adenoma formation, with a possible genotype-phenotype relationship between truncating variants and altered temporal secretion of cortisol

Landscape of FIPA : AIP and Prospective Diagnosis

Context: Familial isolated pituitary adenoma (FIPA) due to aryl hydrocarbon receptor interacting protein (AIP) gene mutations is an autosomal dominant disease with incomplete penetrance. Clinical screening of apparently unaffected AIP mutation (AIPmut) carriers could identify previously unrecognized disease. Objective: To determine the AIP mutational status of FIPA and young pituitary adenoma patients, analyzing their clinical characteristics, and to perform clinical screening of apparently unaffected AIPmut carrier family members. Design: This was an observational, longitudinal study conducted over 7 years. Setting: International collaborative study conducted at referral centers for pituitary diseases. Participants: FIPA families (n = 216) and sporadic young-onset (≤30 y) pituitary adenoma patients (n = 404) participated in the study. Interventions: We performed genetic screening of patients for AIPmuts, clinical assessment of their family members, and genetic screening for somatic GNAS1 mutations and the germline FGFR4 p.G388R variant. Main Outcome Measure(s): We assessed clinical disease in mutation carriers, comparison of characteristics of AIPmut positive and negative patients, results of GNAS1, and FGFR4 analysis. Results: Thirty-seven FIPA families and 34 sporadic patients had AIPmuts. Patients with truncating AIPmuts had a younger age at disease onset and diagnosis, compared with patients with nontruncating AIPmuts. Somatic GNAS1 mutations were absent in tumors from AIPmut-positive patients, and the studied FGFR4 variant did not modify the disease behavior or penetrance in AIPmut-positive individuals.Atotal of 164 AIPmut-positive unaffected family members were identified; pituitary disease was detected in 18 of those who underwent clinical screening. Conclusions: A quarter of the AIPmut carriers screened were diagnosed with pituitary disease, justifying this screening and suggesting a variable clinical course for AIPmut-positive pituitary adenomas

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

PubMed ID: 23555276This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Evaluation of the role of AIP, CDKN1B, miR-107 and AHRR in the pathogenesis of sporadic and familial pituitary adenomas

Pituitary adenomas are common benign intracranial neoplasms, with the vast majority of such tumours being sporadic. The pathogenesis of sporadic pituitary adenomas remains unclear; however, abnormal microRNAs (miRNAs) expression profiles have been recently associated with these adenomas, suggesting that miRNAs can contribute to tumour formation. In about 5% of cases, pituitary adenomas occur in a familial setting, often as part of multiple endocrine tumours syndromes, such as multiple endocrine neoplasia type 1 (MEN1) or Carney complex (CNC). Recently, germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) and in the CDKN1B (encoding the p27Kip1 protein) genes have been identified as causing pituitary adenoma predisposition (PAP) or associated to the development of multiple endocrine tumours, respectively. Several studies revealed AIP mutations in about 15% of kindreds with familial isolated pituitary adenomas (FIPA), a clinical entity in which pituitary tumours are the sole phenotypic manifestation among family members, while sporadic cases are only rarely mutated. Regarding CDKN1B, so far point mutations have been reported only in few subjects with clinical features of MEN1, including pituitary adenomas, but without MEN1 mutations, suggesting that they rarely cause a MEN-like phenotype in both sporadic and familial MEN1 patients. The aim of the present investigation was to study some of the tumorigenic mechanisms involved in pituitary adenoma formation both in sporadic and familial settings. Genomic DNA of a homogeneous cohort of Italian patients with either sporadic acromegaly or FIPA was analysed for point mutations and gross rearrangements in the AIP and CDKN1B genes. I detected three novel, presumably pathogenic, AIP variants in four apparently sporadic cases, and one known mutation (p.R304X) in a FIPA family. Interestingly, I found that another Italian FIPA family shares the same mutation and haplotype associated with the mutated gene, strongly supporting the presence of a founder effect. No known mutations were found in the CDKN1B gene. The analysis of miR-107, a miRNA involved in the pathogenesis of different types of tumours and predicted to downregulate AIP expression, revealed that it is overexpressed in pituitary adenomas and may act as a tumour suppressor gene. I also provided experimental evidences that AIP-3’UTR is a functional and direct target of miR-107. The study of aryl hydrocarbon receptor repressor (AHRR), a molecule which participates in one of the AIP-related pathways potentially involved in pituitary tumorigenesis, demonstrated an increased expression in GH-secreting adenomas compared to normal pituitaries. In conclusion, the main contribution of this PhD thesis was to provide new knowledge about the pathogenesis of pituitary adenomas. In particular, I found that: - mutations in the AIP and CDKN1B genes seem relatively uncommon in the Italian sporadic acromegalic patients, confirming what already reported in the literature for other populations; - a founder effect for the AIP p.R304X mutation exists in a region of central Italy; - the miRNA miR-107 interacts with AIP and may play roles in pituitary adenoma pathogenesis; - AHRR is overexpressed in somatotropinomas.Gli adenomi ipofisari rappresentano delle comuni neoplasie intracraniche benigne, che nella maggior parte dei casi insorgono in maniera sporadica. La patogenesi degli adenomi ipofisari sporadici rimane tuttora poco chiara; recentemente, tuttavia, degli anormali livelli di espressione dei microRNA (miRNA) sono stati associati con questi adenomi, suggerendo che i miRNA possano contribuire al loro sviluppo. In circa il 5% dei casi, gli adenomi ipofisari insorgono invece in forma familiare, spesso come componenti di neoplasie endocrine multiple, quali la neoplasia endocrina multipla di tipo 1 (MEN1) o il complesso di Carney (CNC). Recentemente, mutazioni germinali nei geni aryl hydrocarbon receptor-interacting protein (AIP) e CDKN1B (codificante la proteina p27Kip1) sono state identificate, rispettivamente, come predisponenti l’insorgenza di adenomi ipofisari o associate allo sviluppo di tumori endocrini multipli. Diversi studi hanno dimostrato come mutazioni in AIP siano presenti in circa il 15% delle famiglie con adenoma ipofisario isolato (FIPA), un’entità clinica nella quale i tumori ipofisari sono l’unica manifestazione fenotipica tra i soggetti affetti, mentre siano solo raramente ritrovate nei casi sporadici. Relativamente a CDKN1B, ad oggi mutazioni puntiformi sono state descritte solo in alcuni pazienti con caratteristiche cliniche associabili al fenotipo MEN1, tra cui la presenza di adenomi ipofisari, ma privi di mutazioni nel gene MEN1. Ciò fa quindi ritenere che tali mutazioni causino solo raramente un fenotipo associabile a MEN sia nei casi sporadici che familiari. Lo scopo di questo lavoro è stato quello di studiare alcuni dei meccanismi tumorigenici coinvolti nello sviluppo degli adenomi ipofisari, sia nelle loro forme sporadiche che in quelle familiari. Il DNA genomico di una coorte omogenea di pazienti acromegalici Italiani sia sporadici che familiari è stato analizzato per ricercare la presenza di mutazioni puntiformi e grossi riarrangiamenti nei geni AIP e CDKN1B. Ho individuato tre nuove varianti, presumibilmente patogeniche, nel gene AIP in quattro pazienti apparentemente sporadici, e una mutazione nota (p.R304X) sempre in AIP in una famiglia FIPA. E’ interessante notare come un’altra famiglia FIPA Italiana condivida la stessa mutazione e lo stesso aplotipo associato con il gene mutato, dimostrando quindi la presenza di un effetto fondatore. Nessuna mutazione nota nel gene CDKN1B è stata invece riscontrata nella coorte analizzata. L’analisi di miR-107, un miRNA coinvolto nella patogenesi di diversi tipi di tumore e predetto regolare negativamente l’espressione di AIP, ha rivelato come tale miRNA sia sovraespresso negli adenomi ipofisari e svolga verosimilmente un ruolo da gene oncosopressore. Ho inoltre dimostrato sperimentalemente che il 3’UTR di AIP è un reale e diretto bersaglio di miR-107. Lo studio del repressore del recettore degli idrocarburi (AHRR), una molecola coinvolta in una delle vie correlate ad AIP e potenzialmemente coinvolta nella tumorigenesi ipofisaria, ha dimostrato una sua incrementata espressione nei tumori GH-secernenti rispetto alle ipofisi normali. In conclusione, il contributo principale di questa tesi di dottorato è stato il fornire nuova conoscenza sui meccanismi patogenetici negli adenomi ipofisari. Più specificatamente, ho dimostrato che: - mutazioni nei geni AIP e CDKN1B sono relativamente rare nei pazienti acromegalici sporadici Italiani, in accordo con quanto riportato in letteratura per altre popolazioni; - esiste un effetto fondatore in una regione del centro Italia per la mutazione p.R304X del gene AIP; - il miRNA miR-107 interagisce con AIP e potrebbe avere un ruolo nella patogenesi degli adenomi ipofisari; - AHRR è sovraespresso nei somatotropinomi