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

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.

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.

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.