The effect of AIP mutations on the apoptotic RET pathway in pituitary adenomas

Lead Research Organisation: Queen Mary University of London
Department Name: William Harvey Research Institute

Abstract

Pituitary adenomas are benign tumours of the pituitary, an endocrine gland located at the base of the brain. If an adenoma develops here it can cause problems either because of its size, where it can damage the surrounding crucial structures (e.g. optic nerve), or due to excess hormone release. Recently mutations in a gene called AIP have been identified which predisposes to childhood or young-adult onset adenomas often leading to a devastating condition called gigantism. Despite the AIP protein being present in every cell of the body, a unique feature of patients with germline AIP mutations is that they only develop pituitary adenoma, most commonly from the growth hormone (GH)-secreting cells of the pituitary gland. The overall aim of our study is to identify the mechanism by which AIP-related tumours develop. Based on our preliminary data this proposal aims to clarify the role of the RET protein, an important regulator of cell growth and death, and its pathway members in the mechanism of the AIP-related tumorigenesis.
Over the last few years it has been revealed that stem cells constantly provide new differentiated endocrine cells throughout a person's life and there is a control mechanism, apoptosis, removing cells to maintain the number of cells in the normal pituitary gland. In the pituitary gland RET is predominantly expressed in the growth hormone cells and contributes to this apoptotic process. The apoptotic RET pathway, responsible for controlled cell death, provides the first physiological pathway contributing to pituitary cell turnover in GH cells. This project will explore the possibility that disruption of the apoptotic RET pathway contributes to AIP-related tumorigenesis in humans. We will use a somatotroph cell line to study these pathways as well as cells from rat and mouse pituitary glands as they resemble physiological growth hormone-secreting cells better than the cell line. We will transfect the cells with mutant AIP or we will knock down the cell's own AIP (therefore making the cell deficient in AIP) and will study resulting changes in RET pathway members such RET, Caspase 3 activity, PKCdelta, CREB, JNK, p53 and Pit-1 proteins. We will also explore if AIP, as a partner to the heat-shock protein 90, plays a 'chaperone' role in RET biogenesis, trafficking or degradation. In addition, we will study pituitary tumour samples removed during surgery from patients with AIP mutations and compare data to human adenoma samples without AIP mutation. We have created a mouse model which does not express AIP in the pituitary gland and we will use this to study the development of the pituitary gland both before and after birth including at puberty, when humans with AIP mutations often develop adenomas. In the final part of the project we will microinject virus particles containing mutant or wild type, naturally occurring AIP (wt-AIP), into the pituitary gland of newborn and pubescent animals and will study their impact on pituitary gland and body growth.
We hope that identifying the role of AIP in the RET-regulated proliferation and cell death pathways will help to identify new therapeutic targets for patients.

Technical Summary

AIP-related pituitary disease is a novel clinical entity; however, the mechanism by which AIP, a ubiquitously expressed putative tumour suppressor gene, causes adenomas primarily from a single cell type is currently unclear. RET is a dual function protein with both tyrosine-kinase receptor and, in the absence of ligand, apoptosis-inducing 'dependence receptor' activity. RET is predominantly expressed in growth hormone cells of the pituitary gland and previous data show an interaction between RET and AIP. The current project will study the impact of AIP dysfunction on RET pathway components.
Our preliminary data show that knockdown of AIP or overexpression of mutant AIP can inhibit RET-induced apoptosis. Building on this work, we propose to set out and dissect the impact of AIP dysfunction (knockdown and mutated AIP overexpression) on members of the RET pathway (RET, IC-RET, Caspase 3, PKCdelta, CREB, JNK, Pit-1) ) in rat pituitary cell line GH4C1, and dissect the exact point in the pathway affected by AIP dysfunction. We will also explore if AIP, as a partner to heat-shock protein 90, plays a 'chaperone' role in RET biogenesis, trafficking or degradation. In addition to cell line work, we will use primary rat and mouse pituitary cultures expressing endogenous RET to confirm the observed changes in a more physiological setting. Human pituitary adenoma samples from patients carrying AIP mutations will be studied for apoptosis and RET pathway member expression compared to sporadic pituitary adenoma samples. We have developed a pituitary-specific transgenic AIP knockout mouse model and we will study embryonic and postnatal pituitaries for RET pathway member expression and phosphorylation status. Finally viral constructs containing mutated AIP will be microinjected into neonatal and pubertal rat pituitary glands to see the effect on growth hormone cells, hyperplasia and tumour formation and body growth during puberty and adulthood.

Planned Impact

Patients:

The ultimate beneficiaries of this research are patients with pituitary adenomas, as it can provide a mechanism explaining their disease. Doctors and scientists, both academic as well as those within the pharmaceutical industry, who are interested in pituitary tumours, their pathomechanisms and their treatments would benefit from the knowledge generated here. RET and its pathway is an important factor in many tumours and understanding its dual role as a tyrosine kinase and as a dependence receptor is important to understand tumorigenesis and to be able to advise treatment strategies where it is involved. Patients carrying AIP mutations typically have childhood onset aggressive tumours, which in the past could only be fully controlled with radiotherapy. This has severe consequences on cerebrovascular disease and brain tumours (P. Burman, JCEM. 2013;98:1466-1475.), hence any novel pathway, and critically, any potential new treatment option needs to be explored.

Prof Korbonits has set up a consortium of 170 clinicians who are referring their FIPA cases to her and these medical professionals will benefit from the current data. The information gained on specific mutations will be added to the locus specific database for AIP mutations (set up by the Korbonits lab, http://ftp.ebi.ac.uk/pub/databases/lrgex/LRG_460.xml). Data presented at an appropriate level will be added to the website we have set up for patients with the disease (www.fipapatients.org). Prof Korbonits is ideally placed to transform any basic science data into direct patient benefit, having this large network of collaborators in addition to being the clinical academic head of one of the largest adult endocrine departments in the UK. Facilities available at WHRI and their close collaboration with UCL Partners with its large pituitary surgery unit (Queen Square) are ideal to support this project.
Science Community:
Our research will provide highly skilled scientists proficient in a wealth of cutting edge techniques applicable to both translational and basic research, who will have received training in public engagement and have a clear emphasis on keeping the public informed about the research discoveries.
Researchers employed on the project will directly benefit via learning a range of useful methods such as primary cultures of tissue maintaining the phenotype, retrovirus production, cloning, plasmid production, transfections, Immunohistochemistry, FiJi management, Stereotactic techniques and biostatistics.
Prof Alvarez is part of a network of 20 different hospitals in Spain collecting pituitaries from patients undergoing surgery (REMAH). This collaboration will allow to transfer any knowledge gained in this Project that could also apply to sporadic pituitary somatotroph adenomas, at a fast pace to the human clinical setting.
As Executive Committee members of learned societies (for example the Prof Alvarez at the European Neuroendocrine Association) the PIs will have the opportunity to follow and contribute to research and opinion statements and include results from the project.
Economy:
There is still a clear deficit in our ability to successfully treat tumours without detrimental side effects which impact on the remaining lifespan of patients. Tumours arising in childhood therefore present a serious economic burden on the NHS. Affected individuals may require frequent absences from both school and future places of employment and this impact on the UK workforce further contributes to the cost of ineffective treatments. The aim of our research is to understand the process of tumorigenesis and identify novel beneficial treatments - we believe this is the only way to reduce this expenditure.
Both PIs are currently collaborating with major pharmaceutical companies. Prof Alvarez has close link with the EC-funded drug discovery company INNOPHARMA providing ample opportunity to develop drug targets and benefiting both health care and economy.

Publications

10 25 50
 
Title Artistic summary of my talk and data 
Description I gave a talk and an artist has generated a picture based on the talk and my data 
Type Of Art Artwork 
Year Produced 2023 
Impact I will use this for other evenets 
 
Description Finalising Paediatric pituitary adenoma guidelines
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact better care for patients
 
Description Success Charity meeting
Geographic Reach National 
Policy Influence Type Contribution to a national consultation/review
Impact Comissioning of paediatric pituitary adenoma services.
URL https://successcharity.org.uk/wp-content/uploads/2023/02/Success-Draft-Outline-Conference-Programme....
 
Description Taking part in numerous postgraduate teaching activities to educate about FIPA
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact Our work is unique in the world in assessing patient s with this rare disease. Group leader is giving 15-25 talks per year in various countries on the project, both clinical and basic science
 
Description Crinetics grant
Amount £18,910 (GBP)
Organisation Crinetics Pharmaceuticals 
Sector Private
Country United States
Start 02/2019 
End 02/2022
 
Description Discovering the molecular pathology and treatment of a novel paediatric disease
Amount £250,000 (GBP)
Organisation Sparks Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2022 
End 08/2025
 
Description NIHR Academic Clinical Fellowship
Amount £180,000 (GBP)
Funding ID ACF-2021-18-009 
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 07/2021 
End 08/2024
 
Description Rostrees project grant
Amount £30,000 (GBP)
Funding ID M505 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 12/2018
 
Description St. Bartholomew's Hospital Medical College Trust Clinical Training Fellowship
Amount £213,454 (GBP)
Funding ID Clinical Training Fellowship for Dr Chung Thong Lim Role of the extracellular protein CRTAC1 in the invasive behaviour of AIP mutation positive pituitary tumours 
Organisation Queen Mary University of London 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 09/2020
 
Description WHRI Studentship
Amount £110,000 (GBP)
Funding ID AIP-related pituitary tumorigenesis - dissecting the mechanism 
Organisation Queen Mary University of London 
Department William Harvey Research Institute
Sector Academic/University
Country United Kingdom
Start 08/2018 
End 09/2021
 
Title FACS of YFP labelled pituitary cells 
Description Pituitaries from control and knockout mice tagged with YFP are extracted and dissociated in an enzyme mix. The single cell suspension is then incubated with DAPI and the live cells are selected for YFP fluorescence and collected in Trizol for RNA extraction. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2016 
Provided To Others? No  
Impact This method allows the collection of a pure population of pituitary cells in which AIP has been deleted for further applications such as qPCR and RNA sequencing. 
 
Title Gordon and Sweet's Staining Protocol for Reticulin 
Description Reticulin fibres have low affinity for silver solutions so, they must be treated with potassium permanganate to produce sensitised sites on the fibres where silver deposition can be initiated. The silver is in a form readily able to precipitate as metallic silver (diamine silver solution)The Optimal pH for maximum uptake of silver ions is pH 9.0. A reducing agent, formalin, causes deposition of silver in the form of metal. Any excess silver in the unprecipitated state is removed by treating with hypo. Gold chloride treatment renders the preparation permanent and produces a neutral black colour of high intensity. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The Gordon and Sweet's silver staining method is used to demonstrate reticular fibres, so we can evaluate the hyperplasia in wax embedded tissues 
 
Title Immunohistochemistry/Immunofluorescence 
Description The paraffin-embedded fixed tissues are cut and mounted in microscope slides. This are subsequently deparaffined and rehydratated with ethanol washes; then the slides are unmasked, washed, blocked and immunostained with different antibodies. 
Type Of Material Biological samples 
Provided To Others? No  
Impact This allows us to identify the presence, absence or distribution of proteins in tissue samples. 
 
Title Myocardial Perfusion of animals 
Description We perform a paraformaldehyde (PFA) cardiac perfusion of mouse to improve the fixation of the mouse tissues. We firstly weight the mouse to calculate the volume of anesthesia (pentobarbital), and the time of saline and PFA perfusion. We inject the anesthesia, and after 10-15 min, we check with tail or corneal reflex the mouse is completely anesthetized, clean the mouse with chlorhexidine or ethanol, section superficially the skin in the middle line of the abdomen and subcostal bilaterally (in T), section the peritoneum (vertically), then also open the abdomen subcostal in both sides (section like a T), section the diaphragm and laterally the rib cage (left and right side), and fix with a clamp above the heart, then we separate the pericardium carefully and with the pump running at 1% max speed, insert the needle in the apex of left ventricle (the heart is still pumping); finally we section the right atrium, to allow the perfusion volume has an entrance (left ventricle) and exit (right atrium) and start the perfusion at 7% max speed, first with saline and then with PFA 4%, and run the time that have been calculated before depending on the weight. Then we collect the pituitary, brain, heart, lung, liver, stomach, small intestine, spleen, kidneys and testicles in PFA 4% at 4ºC After overnight fixation in PFA we store the tissues in ethanol at 4ºC. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This method is important for high quality immunohystochemistry assays 
 
Title OpenPedigree 
Description Family tree drawing program called Open Pedigree 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? Yes  
Impact now good free program available, this needs a bit more development 
URL https://shermanlo77.github.io/qmul-open-pedigree/
 
Title RNA extraction, retrotranscription and qPCR 
Description Total RNA is extracted from mouse tissues and GH3 rat somatotrope cell line using RNeasy Mini kit (QIAGEN) or TRIzol Reagent (Life Technologies) following the manufacturer's protocol. In all cases, total RNA concentration and purity is assessed using Nanodrop spectrophotometer (Thermo Scientific), and subsequently retro-transcribed using random hexamer primers and the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific). qPCR is performed using Brilliant III SYBR Green Master Mix in the Stratagene Mx3000p instrument (Agilent). 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This would allow identification of changes in expression levels of genes following tumour formation 
 
Title RNAScope 
Description RNAScope is a new technique and we set it up in the lab for this project 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? Yes  
Impact paper is in preparation this is new type if in situ equivalent, our machine is available for others in the lab 
 
Title Stem cell culture 
Description The method is to perform a primary culture of mouse pituitary in a medium that promotes stem cell to attach and grow forming colonies. Firstly the pituitary has to be mechanically and enzymatically dissociated with trypsin, collagenase and DNase and then, the cell suspension is counted and plated on 6well plates or chambered culture slides. The medium has to be replaced every 2 days with fresh medium. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This method allows us to evaluate the proliferation and assess different markers in the stem cells derived from different mouse models. 
 
Title Wax Embedding Protocol 
Description 1) Fix mouse tissues or embryos in 4% (w/v) fresh, cold PFA in PBS (day 1) 2) Wash 2 times in PBS (30 min each one) in cold PBS (or put the samples in ice) (day 2) 3) Dehydrate through ethanol serial dilution washes: - 25, 50 and 75% EtOH overnight (day 2) After this, if you want to keep in EtOH, keep in 75%. - 80, 95 and 100% EtOH overnight (day 3) 4) In the morning to do another 100% EtOH wash at room temperature. Then transfer to a glass vial (day 4) 5) Proceed to wax embedding 6) Orientate in a plastic or glass mold. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The wax embedding is essential for immunohystochemical protocols 
 
Title AIP viral particles injection to mice pituitary through the ear 
Description This method has been setup in Co-applicant Calra Alvarez lab. 
Type Of Material Data analysis technique 
Year Produced 2020 
Provided To Others? Yes  
Impact it will be a useful tool 
 
Title AIP-Hesx1 mouse model and AIP sox 2 mouse model 
Description we have generated 2 mouse model of the disease 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact first as an abstract and now one paper in press and another in preparation 
 
Description Carles Gaston-Massuet - Rosetrees 
Organisation Queen Mary University of London
Department School of Engineering and Materials Science
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed mouse AIP KO models in collaboration with Carles Gaston-Massuet key for the study of AIP and its role on pituitary tumorigenesis.
Collaborator Contribution Carles Gaston-Massuet has provided inducible AIP KO mouse that allow us to study the role of AIP before and after puberty.
Impact To date we just have preliminary data. We have successfully generated three novel conditional KO mice by crossing AipFlox/Flox with a pituitary-specific Cre lines: (i) HesxCre, (ii) the tamoxifen-inducible SoxCreERT2, and (iii) the triple transgenic AipFlox/Flox;Sox2CreERT2/+;Rosa26loxP-STOP-loxP-YFP/+.
Start Year 2012
 
Description Clara Alvarez MRC grant 
Organisation University of Santiago de Compostela
Country Spain 
Sector Academic/University 
PI Contribution We are working on AIPfl/flHesx1Cre/+ mice to investigate the role of the RET pathway in pituitary adenomas.
Collaborator Contribution Our collaborators are working on rats using known AIP mutants to determine their role in pituitary adenoma formation
Impact Still ongoing, but we have one paper in press and another in preparation
Start Year 2015
 
Description Oliver Haworth collaboration 
Organisation Queen Mary University of London
Department William Harvey Research Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution we are discovering new aspects of homozygous AIP disease molecular mechanisms
Collaborator Contribution key aspects were basic science ideas related lysosomes and autophagy
Impact Dr Haworth is an immunologist so yes multidisciplinary
Start Year 2018
 
Description Paul Chapple collaboration 
Organisation Queen Mary University of London
Department William Harvey Research Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Working on AIP activities and partnerships
Collaborator Contribution expertese on mitochondrial function and HSPs
Impact common papers (PMID: 22659247, 20506337, 18381572).
Start Year 2008
 
Description neurosurgery collaboration 
Organisation National Hospital for Neurology and Neurosurgery
Country United Kingdom 
Sector Hospitals 
PI Contribution Analysing data
Collaborator Contribution providing samples and clinical data
Impact plenty of papers
Start Year 2010
 
Description Fipapatients webiste 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We have setup a website for patients, doctors and the general public to inform about FIPA as no resources were previously available on this recently identified disease.
We have updated this website in order to include additional resources and appeal to non-FIPA pituitary adenoma patients.
Year(s) Of Engagement Activity 2016,2017,2018,2022
URL http://www.fipapatients.org
 
Description Television interviews 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact we had several programs on television related to our work with AIP mutatio atients
Year(s) Of Engagement Activity 2017,2018