Hypermethylator Phenotype and Ectopic GIP Receptor in GNAS Mutation-Negative Somatotropinomas.

Besides GNAS gene mutations, the molecular pathogenesis of somatotroph adenomas responsible for gigantism and acromegaly remains elusive. To investigate alternative driver events in somatotroph tumorigenesis, focusing on a subgroup of acromegalic patients with a paradoxical increase in growth hormone (GH) secretion after oral glucose, resulting from ectopic glucose-dependent insulinotropic polypeptide receptor (GIPR) expression in their somatotropinomas. We performed combined molecular analyses, including array-comparative genomic hybridization, RNA/DNA fluorescence in situ hybridization, and RRBS DNA methylation analysis on 41 somatotropinoma samples from 38 patients with acromegaly and three sporadic giants. Ten patients displayed paradoxical GH responses to oral glucose. GIPR expression was detected in 13 samples (32%), including all 10 samples from patients with paradoxical GH responses. All GIPR-expressing somatotropinomas were negative for GNAS mutations. GIPR expression occurred through transcriptional activation of a single allele of the GIPR gene in all GIPR-expressing samples, except in two tetraploid samples, where expression occurred from two alleles per nucleus. In addition to extensive 19q duplications, we detected in four samples GIPR locus microamplifications in a certain proportion of nuclei. We identified an overall hypermethylator phenotype in GIPR-expressing samples compared with GNAS-mutated adenomas. In particular, we observed hypermethylation in the GIPR gene body, likely driving its ectopic expression. We describe a distinct molecular subclass of somatotropinomas, clinically revealed by a paradoxical increase of GH to oral glucose related to pituitary GIPR expression. This ectopic GIPR expression occurred through hypomorphic transcriptional activation and is likely driven by GIPR gene microamplifications and DNA methylation abnormalities.

Hage M ，Chaligné R ，Viengchareun S ，Villa C ，Salenave S ，Bouligand J ，Letouzé E ，Tosca L ，Rouquette A ，Tachdjian G ，Parker F ，Lombès M ，Lacroix A ，Gaillard S ，Chanson P ，Kamenický P ... -  《-》

The glucose-dependent insulinotropic polypeptide receptor is overexpressed amongst GNAS1 mutation-negative somatotropinomas and drives growth hormone (GH)-promoter activity in GH3 cells.

Somatic mutations in the GNAS1 gene, encoding the α-subunit of the heterotrimeric stimulatory G protein (Gαs), occur in approximately 40% of growth hormone (GH)-secreting pituitary tumours. By altering the adenylate cyclase-cAMP-protein kinase A pathway, they unequivocally give somatotroph cells a growth advantage. Hence, the pathogenesis of somatotropinomas could be linked to anomalies in receptors coupled to the cAMP second-messenger cascade. Among them, the glucose-dependent insulinotropic polypeptide receptor (GIPR) is already known to play a primary role in the impaired cAMP-dependent cortisol secretion in patients affected by food-dependent Cushing's syndrome. In the present study, 43 somatotropinomas and 12 normal pituitary glands were investigated for GIPR expression by quantitative reverse transcriptase-polymerase chain reaction, western blotting and immunohistochemistry. Tumoural specimens were also evaluated for GNAS1 mutational status. The effect of GIPR overexpression on cAMP levels and GH transcription was evaluated in an in vitro model of somatotropinomas, the GH-secreting pituitary cell line GH3. GIPR was expressed at higher levels compared to normal pituitaries in 13 GNAS1 mutation-negative somatotropinomas. GIP stimulated adenylyl cyclase and GH-promoter activity in GIPR-transfected GH3 cells, confirming a correct coupling of GIPR to Gαs. In a proportion of acromegalic patients, GIPR overexpression appeared to be associated with a paradoxical increase in GH after an oral glucose tolerance test. Whether GIPR overexpression in acromegalic patients may be associated with this paradoxical response or more generally involved in the pathogenesis of acromegaly, as suggested by the mutually exclusive high GIPR levels and GNAS1 mutations, remains an open question.

Occhi G ，Losa M ，Albiger N ，Trivellin G ，Regazzo D ，Scanarini M ，Monteserin-Garcia JL ，Fröhlich B ，Ferasin S ，Terreni MR ，Fassina A ，Vitiello L ，Stalla G ，Mantero F ，Scaroni C ... -  《-》

The GIP/GIPR axis is functionally linked to GH-secretion increase in a significant proportion of gsp(-) somatotropinomas.

Glucose-dependent insulinotropic polypeptide receptor (GIPR) overexpression has been recently described in a proportion of gsp- somatotropinomas and suggested to be associated with the paradoxical increase of GH (GH-PI) during an oral glucose load. This study was aimed at linking the GIP/GIPR pathway to GH secretion in 25 somatotropinomas-derived primary cultures and correlating molecular with clinical features in acromegalic patients. Given the impairment of the GIP/GIPR axis in acromegaly, an additional aim was to assess the effect of GH/IGF-1 stimulation on GIP expression in the enteroendocrine cell line STC-1. Nearly 80% of GIPR-expressing somatotropinomas, all of them negative for gsp mutations, show increased GH secretion upon GIP stimulation, higher sensitivity to Forskolin but not to somatostatin analogs. Besides increased frequency of GH-PI, GIPR overexpression does not appear to affect acromegalic patients' clinical features. In STC-1 cells transfected with GIP promoter-driven luciferase vector, IGF-1 but not GH induced dose-dependent increase in luciferase activity. We demonstrate that GIPR mediates the GH-PI in a significant proportion of gsp- acromegalic patients. In these cases, the stimulatory effect of IGF-1 on GIP promoter support the hypothesis of a functional GH/IGF-1/GIP axis. Further studies based on larger cohorts and the development of a stable transgenic model with inducible GIPR overexpression targeted to pituitary somatotroph lineage will be mandatory to establish the real role of GIPR in the pathogenesis of somatotropinomas.

Regazzo D ，Losa M ，Albiger NM ，Terreni MR ，Vazza G ，Ceccato F ，Emanuelli E ，Denaro L ，Scaroni C ，Occhi G ... -  《-》

KDM1A genotyping and expression in 146 sporadic somatotroph pituitary adenomas.

A paradoxical increase of growth hormone (GH) following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants and somatic loss of heterozygosity of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism. We aimed to analyze KDM1A gene sequence and KDM1A and GIPR expressions in somatotroph pituitary adenomas. We conducted a cohort study at university hospitals in France and in Italy. We collected pituitary adenoma specimens from acromegalic patients who had undergone pituitary surgery. We performed targeted exome sequencing (gene panel analysis) and array-comparative genomic hybridization on somatic DNA derived from adenomas and performed droplet digital PCR on adenoma samples to quantify KDM1A and GIPR expressions. One hundred and forty-six patients with sporadic acromegaly were studied; 72.6% presented unsuppressed classical GH response, whereas 27.4% displayed a paradoxical rise in GH after oral glucose load. We did not identify any pathogenic variant in the KDM1A gene in the adenomas of these patients. However, we identified a recurrent 1p deletion encompassing the KDM1A locus in 29 adenomas and observed a higher prevalence of paradoxical GH rise (P = .0166), lower KDM1A expression (4.47 ± 2.49 vs 8.56 ± 5.62, P < .0001), and higher GIPR expression (1.09 ± 0.92 vs 0.43 ± 0.51, P = .0012) in adenomas from patients with KDM1A haploinsufficiency compared with those with 2 KDM1A copies. Unlike in GIP-dependent primary bilateral macronodular adrenal hyperplasia, KDM1A genetic variations are not the cause of GIPR expression in somatotroph pituitary adenomas. Recurrent KDM1A haploinsufficiency, more frequently observed in GIPR-expressing adenomas, could be responsible for decreased KDM1A function resulting in transcriptional derepression on the GIPR locus.

Chasseloup F ，Regazzo D ，Tosca L ，Proust A ，Kuhn E ，Hage M ，Jublanc C ，Mokhtari K ，Dalle Nogare M ，Avallone S ，Ceccato F ，Tachdjian G ，Salenave S ，Young J ，Gaillard S ，Parker F ，Boch AL ，Chanson P ，Bouligand J ，Occhi G ，Kamenický P ... -  《-》

The pathogenic role of the GIP/GIPR axis in human endocrine tumors: emerging clinical mechanisms beyond diabetes.

Regazzo D ，Barbot M ，Scaroni C ，Albiger N ，Occhi G ... -  《-》