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

垂体腺瘤是垂体的良性肿瘤，垂体是位于大脑底部的内分泌腺。如果腺瘤在这里发展，它可能会导致问题，因为它的大小，它可能会损害周围的关键结构（例如视神经），或由于过量的激素释放。最近，一种名为AIP的基因突变被发现，它易患儿童或成年后发病的腺瘤，通常会导致一种称为乳腺癌的毁灭性疾病。尽管AIP蛋白存在于身体的每一个细胞中，但具有生殖系AIP突变的患者的独特特征是他们仅发展垂体腺瘤，最常见的是来自垂体的生长激素（GH）分泌细胞。我们研究的总体目标是确定AIP相关肿瘤发展的机制。基于我们的初步数据，该提议旨在阐明RET蛋白（细胞生长和死亡的重要调节因子）及其通路成员在AIP相关肿瘤发生机制中的作用。在过去的几年里，已经发现干细胞在人的一生中不断提供新的分化的内分泌细胞，并且有一种控制机制，细胞凋亡，去除细胞以维持正常脑垂体中的细胞数量。在脑垂体中，RET主要在生长激素细胞中表达，并促进该凋亡过程。凋亡RET途径，负责控制细胞死亡，提供了第一个生理途径，促进垂体细胞营业额在GH细胞。该项目将探索凋亡RET通路的破坏有助于人类AIP相关肿瘤发生的可能性。我们将使用促生长素细胞系来研究这些途径，以及从大鼠和小鼠脑垂体的细胞，因为他们更像生理生长激素分泌细胞比细胞系。我们将用突变的AIP转染细胞，或者我们将敲低细胞自身的AIP（因此使细胞缺乏AIP），并将研究RET途径成员如RET、半胱天冬酶3活性、PKC δ、CREB、JNK、p53和Pit-1蛋白的变化。我们还将探讨AIP作为热休克蛋白90的伴侣，是否在RET的生物合成、运输或降解中发挥“伴侣”作用。此外，我们将研究在手术过程中从AIP突变患者中取出的垂体瘤样本，并将数据与无AIP突变的人腺瘤样本进行比较。我们已经建立了一个小鼠模型，它在脑垂体中不表达AIP，我们将用它来研究出生前后脑垂体的发育，包括青春期，当AIP突变的人经常发展腺瘤时。在项目的最后部分，我们将把含有突变型或野生型、天然存在的AIP（wt-AIP）的病毒颗粒显微注射到新生和青春期动物的脑垂体中，并研究其对脑垂体和身体生长的影响。我们希望，确定AIP在RET调节的增殖和细胞死亡途径中的作用将有助于为患者确定新的治疗靶点。

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.