The role of the aryl hydrocarbon receptor interacting protein (AIP) in pituitary tumorigenesis: A novel animal model for investigating the role of AIP during embryogenesis and pituitary tumour formation

Pituitary adenomas (PAs) have a 1:1000 prevalence and carry significant morbidity despite their benign nature. Mutations in the AIP (aryl hydrocarbon receptor interacting protein) gene have been unambiguously associated with higher predisposition for PAs. These tumours pose a challenge in treatment due to delayed diagnosis, increased size, earlier onset, aggressive nature and considerable resistance to therapy. The exact mechanism of the tumour formation due to loss of AIP and the role of AIP in pituitary organogenesis is still unknown. A large-cohort retrospective (225 patients) and prospective (876 patients) clinical study was carried out on the pituitary adenoma patients of a tertiary referral centre in London assessing the prevalence of FIPA (familial isolated pituitary adenomas). 20% of FIPA patients are reported to harbour an AIP mutation, but half of the AIP mutation-positive probands are not aware of a positive family history. This study has shown that active inquiry of family history increases detection of previously unknown family history with nearly 3-fold, enabling the genetic screening of these families for early diagnosis and better customised therapy. The novel, pituitary- specific, biallelic Aip-knockout murine model (AipFlox/Flox; Hesx1Cre/+) generated within this study allowed for the first time to investigate the role of AIP during the embryonic stages. There is phenotypical difference (enlarged anterior lobe, incomplete fusion of the sphenoid bone) in the pituitaries 17.5 dpc AipFlox/Flox; Hesx1Cre/+ embryos when compared with the wildtype of the same embryonic stage, with no difference in cell lineage determination (dpc 15.5). A decrease in growth hormone and prolactin producing cells is described at terminal differentiation (dpc 17.5), which is intriguing because patients harbouring AIP mutations most commonly present with growth hormone and/or prolactin secreting 7 PAs. Further research has since been done efficiently using this model focusing on postnatal tumour formation, which is not part of this thesis. Deregulation of the Hippo signalling components has been increasingly investigated in relation to pituitary tumorigenesis, especially in human hormone secreting PAs, but no study had investigated the role of Hippo signalling in AIP-mediated tumour formation. A bioinformatic-based analysis was performed for the expression of 41 Hippo pathway associated genes in AIP-silenced rat pituitary somatomammotroph GH3 cells, pituitary specific AIP-knockout mice, humans with AIP mutation-positive and AIP mutation-negative familial and sporadic PAs. Both up- and downstream members of the Hippo signalling show significantly altered expression in the different subgroups, which warrants more targeted future studies. Additionally, in the analysed exome and whole genome sequencing of PA patients, variants of several key Hippo pathway components show segregation within families. These data (along with a global phosphorylation analysis) suggest that further studies are needed to investigate the role of Hippo signalling in AIP-mediated tumorigenesis

Tumor microenvironment defines the invasive phenotype of AIP-mutation-positive pituitary tumors

The molecular mechanisms leading to aryl hydrocarbon receptor interacting protein (AIP) mutation-induced aggressive, young-onset growth hormone-secreting pituitary tumors are not fully understood. In this study, we have identified that AIP-mutation-positive tumors are infiltrated by a large number of macrophages compared to sporadic tumors. Tissue from pituitary-specific Aip-knockout (AipFlox/Flox;Hesx1Cre/+) mice recapitulated this phenotype. Our human pituitary tumor transcriptome data revealed the "epithelial-to-mesenchymal transition (EMT) pathway" as one of the most significantly altered pathways in AIPpos tumors. Our in vitro data suggest that bone marrow-derived macrophage-conditioned media induces more prominent EMT-like phenotype and enhanced migratory and invasive properties in Aip-knockdown somatomammotroph cells compared to non-targeting controls. We identified that tumor-derived cytokine CCL5 is upregulated in AIP-mutation-positive human adenomas. Aip-knockdown GH3 cell-conditioned media increases macrophage migration, which is inhibited by the CCL5/CCR5 antagonist maraviroc. Our results suggest that a crosstalk between the tumor and its microenvironment plays a key role in the invasive nature of AIP-mutation-positive tumors and the CCL5/CCR5 pathway is a novel potential therapeutic target

122 Cardiac Abnormalities are Common in Patients Diagnosed with Phaeochromocytoma as Detected by Cardiovascular Magnetic Resonance Imaging.

INTRODUCTION: Sudden and/or chronic exposure to catecholamines may predispose patients with phaeochromocytoma to cardiac pathology, including left ventricular (LV) hypertrophy, myocardial infarction, stress-induced cardiomyopathy and heart failure. This is a prospective, multicentre study using cardiovascular magnetic resonance (CMR) imaging to describe the variety and incidence of cardiac abnormalities in patients diagnosed with phaeochromocytoma. METHODS: Patients diagnosed with phaeochromocytoma (n = 50) were included. We prospectively recruited patients newly-diagnosed with confirmed phaeochromocytoma (n = 20, age 51 ± 14 yrs) who underwent CMR before and after curative surgical resection of the phaeochromocytoma (median follow-up 1 year). Previously-diagnosed patients who had curative surgery (n = 30, age 52 ± 14 yrs) were also recruited. Patients with known cardiac conditions were excluded. CMR included cine imaging for LV function, T2-weighted imaging for oedema and late gadolinium enhancement imaging to detect scarring. RESULTS: In newly-diagnosed patients, the mean LV ejection fraction was 67 ± 10% (range 47-88%; normal range 57-81%); Of these patients, 20% (n = 4/20) had mild global LV dysfunction (EF 47-56%). A significant proportion (65%, n = 13/20) demonstrated scarring, all with a non-ischaemic pattern (midwall/subepicardial/patchy), but the areas were small (<10% myocardium); no patient had myocardial infarction (subendocardial scarring). One patient demonstrated global myocardial oedema with normal EF. All LV dysfunction or oedema were reversible and normalised at postoperative follow-up. Previously-diagnosed patients had a slightly higher EF of 73 ± 7% (56-86%) compared to newly-diagnosed patients (P < 0.03); only one (3%) had mild global LV dysfunction (EF = 56%). Compared to newly-diagnosed patients, a significantly smaller proportion of previously-diagnosed patients (17% vs. 65%; P < 0.001) demonstrated areas of scarring, which again were small in areas with a non-ischaemic pattern, except for one patient who suffered a small myocardial infarction. CONCLUSIONS: Cardiac abnormalities are common in patients newly-diagnosed with phaeochromocytoma, and include mild LV dysfunction, myocardial oedema and small areas of non-ischaemic scarring, with the former two demonstrating full reversibility and normalisation post surgical resection of the phaeochromocytoma. In patients who had previously undergone curative surgical resection of their phaeochromocytoma, the incidence of cardiac abnormalities is lower (17%), predominantly consisting of small areas of non-ischaemic fibrosis

Pheochromocytoma is characterized by catecholamine-mediated myocarditis, focal and diffuse myocardial fibrosis, and myocardial dysfunction

© 2016 American College of Cardiology Foundation.Background Pheochromocytoma is associated with catecholamine-induced cardiac toxicity, but the extent and nature of cardiac involvement in clinical cohorts is not well-characterized. Objectives This study characterized the cardiac phenotype in patients with pheochromocytoma using cardiac magnetic resonance (CMR). Methods A total of 125 subjects were studied, including patients with newly diagnosed pheochromocytoma (n = 29), patients with previously surgically cured pheochromocytoma (n = 31), healthy control subjects (n = 51), and hypertensive control subjects (HTN) (n = 14), using CMR (1.5-T) cine, strain imaging by myocardial tagging, late gadolinium enhancement, and native T1 mapping (Shortened Modified Look-Locker Inversion recovery [ShMOLLI]). Results Patients who were newly diagnosed with pheochromocytoma, compared with healthy and HTN control subjects, had impaired left ventricular (LV) ejection fraction (<56% in 38% of patients), peak systolic circumferential strain (p < 0.05), and diastolic strain rate (p < 0.05). They had higher myocardial T1 (974 ± 25 ms, as compared with 954 ± 16 ms in healthy and 958 ± 23 ms in HTN subjects; p < 0.05), areas of myocarditis (median 22% LV with T1 >990 ms, as compared with 1% in healthy and 2% in HTN subjects; p < 0.05), and focal fibrosis (59% had nonischemic late gadolinium enhancement, as compared with 14% in HTN subjects). Post-operatively, impaired LV ejection fraction typically normalized, but systolic and diastolic strain impairment persisted. Focal fibrosis (median 5% LV) and T1 abnormalities (median 12% LV) remained, the latter of which may suggest some diffuse fibrosis. Previously cured patients demonstrated abnormal diastolic strain rate (p < 0.001), myocardial T1 (median 12% LV), and small areas of focal fibrosis (median 1% LV). LV mass index was increased in HTN compared with healthy control subjects (p < 0.05), but not in the 2 pheochromocytoma groups. Conclusions This first systematic CMR study characterizing the cardiac phenotype in pheochromocytoma showed that cardiac involvement was frequent and, for some variables, persisted after curative surgery. These effects surpass those of hypertensive heart disease alone, supporting a direct role of catecholamine toxicity that may produce subtle but long-lasting myocardial alterations

Increased Population Risk of AIP-related Acromegaly and Gigantism in Ireland.

The aryl hydrocarbon receptor interacting protein (AIP) founder mutation R304* (or p.R304*; NM_003977.3:c.910C>T, p.Arg304Ter) identified in Northern Ireland (NI) predisposes to acromegaly/gigantism; its population health impact remains unexplored. We measured R304* carrier frequency in 936 Mid Ulster, 1000 Greater Belfast (both in NI) and 2094 Republic of Ireland (ROI) volunteers and in 116 acromegaly/gigantism patients. Carrier frequencies were 0.0064 in Mid Ulster (95%CI = 0.0027-0.013; P = 0.0005 vs. ROI), 0.001 in Greater Belfast (0.00011-0.0047) and zero in ROI (0-0.0014). R304* prevalence was elevated in acromegaly/gigantism patients in NI (11/87, 12.6%, P < 0.05), but not in ROI (2/29, 6.8%) vs. non-Irish patients (0-2.41%). Haploblock conservation supported a common ancestor for all the 18 identified Irish pedigrees (81 carriers, 30 affected). Time to most recent common ancestor (tMRCA) was 2550 (1275-5000) years. tMRCA-based simulations predicted 432 (90-5175) current carriers, including 86 affected (18-1035) for 20% penetrance. In conclusion, R304* is frequent in Mid Ulster, resulting in numerous acromegaly/gigantism cases. tMRCA is consistent with historical/folklore accounts of Irish giants. Forward simulations predict many undetected carriers; geographically-targeted population screening improves asymptomatic carrier identification, complementing clinical testing of patients/relatives. We generated disease awareness locally, necessary for early diagnosis and improved outcomes of AIP-related disease. This article is protected by copyright. All rights reserved.This article is freely available via Open Access. Click on the Additional Link above to access the full-text via the publisher's site.Accepted manuscript, 12 month embargo

Palmitoylation is required for efficient Fas cell death signaling

Localization of the death receptor Fas to specialized membrane microdomains is crucial to Fas-mediated cell death signaling. Here, we report that the post-translational modification of Fas by palmitoylation at the membrane proximal cysteine residue in the cytoplasmic region is the targeting signal for Fas localization to lipid rafts, as demonstrated in both cell-free and living cell systems. Palmitoylation is required for the redistribution of Fas to actin cytoskeleton-linked rafts upon Fas stimulation and for the raft-dependent, ezrin-mediated cytoskeleton association, which is necessary for the efficient Fas receptor internalization, death-inducing signaling complex assembly and subsequent caspase cascade leading to cell death