Integrated genomic characterization of Growth Hormone-secreting Pituitary Adenomas – INTE-GRAL

Somatotroph pituitary adenomas are responsible for chronic Growth Hormone (GH) excess leading to gigantism during childhood and acromegaly in adulthood, both of which are associated with significant morbidity and, if untreated, increased mortality. The first line treatment of somatotroph adenomas, which are unequivocally benign, is pituitary surgery, that aims at removing the adenoma, but which is frequently incomplete, as proximity to adjacent brain structures often limits the surgical resection. Multimodal medical treatment, which is burdensome, extremely expensive and has variable therapeutic efficacy, is currently required in more than 60% of the patients. New therapeutic approaches are therefore necessary for controlling GH secretion and adenoma growth. This research project addresses two unsolved and clinically relevant issues regarding 1) the molecular mechanisms underlying somatotroph adenoma development and 2) the molecular basis of their invasive behavior.
Besides activating GNAS gene mutations, promoting adenoma development in ~30% of cases, the molecular pathogenesis of somatotroph adenomas is poorly understood. We have identified among 41 somatotroph adenomas a group of GNAS mutation-negative adenomas from patients with paradoxical increase of GH to oral glucose load, presenting with ectopic pituitary Glucose-dependent Insulinotropic Polypeptide Receptor (GIPR) expression and displaying an overall hypermethylator phenotype. Our first objective is to perform an integrated genomic characterization of somatotroph adenomas without GNAS mutation. Our pilot methylome analysis did not include samples without GNAS mutation and non-expressing GIPR; we will thus extend this analysis on the entire sample collection. In an original mouse model of specific biallelic Gnas inactivation in somatotroph lineage (Gnasflox/flox Ghrh-r Cre+/0 mice) that we have generated, we will analyze how down tuning of cAMP tonus due to Gnas disruption in somatotroph cells impacts DNA methylation profile. We will further examine the relationship between DNA methylation and gene expression changes using RNA sequencing and perform exome sequencing to establish the mutational signature in GNAS mutation negative somatotropinomas.
Our second objective is to investigate the molecular determinants of extrasellar growth of somatotroph adenomas, which significantly limits the treatment outcomes of these benign lesions. We have obtained a unique collection of invasive somatotroph adenomas including their non-invasive intrasellar portions and portions invading the cavernous sinus. RNA sequencing performed on 19 sample pairs pointed out to an up-regulation of the epithelial to mesenchymal transition pathway and of the stem cell pathway in invasive versus non-invasive paired samples. We will consolidate these results by analyzing supplemental samples. We will characterize the molecular functions of selected genes using in vitro methods of cell migration and invasion. For this purpose, expression level of selected genes will be modulated (overexpression or downexpression) in rat pituitary GH3 cells by using transient transfection assays or siRNA. Further, stably transfected GH3 cells will be injected into the pituitary gland of the rats (or alternatively subcutaneously in the flank region) to study in vivo the acquisition of an invasive phenotype. Finally, we will perform exome sequencing of the invasive and non-invasive part of the adenomas to seek somatic mutations that could account for the differential gene expression profile.
This ambitious project will be supervised by a young researcher whose input over the last years allowed studies on pituitary tumorigenesis to be established, and will permit to consolidate this research axis in his Inserm Unit. This project will provide scientific basis for a deeper insight into pathophysiological mechanisms of somatotroph pituitary adenoma development and invasiveness and may rapidly open new therapeutic avenues.