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Context Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas. Methods We conducted an international, multicenter, cohort study. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent PBMAH with Cushing's syndrome. Adrenal samples from patients with PBMAH and Cushing's syndrome without food-dependent cortisol production were used as controls. We further collected somatotropinoma specimens from acromegaly patients followed at two expert endocrine centers in France. Results 17 patients with familial or sporadic GIP-dependent PBMAH with Cushing's syndrome were studied. We identified germline heterozygous mutations in the lysine demethylase 1A (KDM1A) gene in all 17 patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. None of the 25 patients in the control group had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in the adrenal lesions. RNA-sequencing revealed the global impact of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes including those encoding for GIPR and other G-Protein-Coupled Receptors that may be involved in adrenal tumorigenesis and regulation of steroidogenesis. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical H295R cells. Somatotropinoma samples from 78 patients with acromegaly were studied. 24% of these patients presented with a paradoxical rise of GH after oral glucose load and expressed ectopically GIP-receptor in their somatotropinoma. None of the somatotropinomas harbored KDM1A pathogenic variants, but those from patients with paradoxical GH response displayed a recurrent chromosome 1p loss. Discussion We identified germline inactivating KDM1A mutations and loss of heterozygosity as a genetic predisposition to GIP-dependent PBMAH with Cushing's syndrome following a tumor suppressor gene model of tumorigenesis. We currently perform genetic screening in first-degree relatives of patients with GIP-dependent PBMAH with Cushing's syndrome and clinical examination with biochemical testing in asymptomatic KDM1A variant carriers. We did not identify somatic KDM1A mutations in somatotropinomas expressing GIPR ectopically, however their recurrent 1p chromosome loss suggests that KDM1A haploinsufficiency may contribute to GIPR expression in those tumors. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

International audience; Mitotane (o,p'DDD), the most effective drug in adrenocortical carcinoma, concentrates into the mitochondria and impacts mitochondrial functions. To address the molecular mechanisms of mitotane action and to identify its potential target, metabolomic and lipidomic approaches as well as imaging analyses were employed in human adrenocortical H295R cells allowing identification of Mitochondria-Associated Membranes dysfunction as a critical impact of mitotane. Study of intracellular energetic metabolites by NMR spectroscopy showed that mitotane significantly decreased aspartate while concomitantly increased glutamate content in a time-and concentration-dependent manner. Such alterations were very likely linked to the previously described, mitotane-induced respiratory chain defect. Lipidomic studies of intracellular and intramitochondrial phospholipids revealed that mitotane exposure markedly reduced the phosphatidylserine/phosphatidylethanolamine ratio, indicative of a dysfunction of phosphatidylserine decarboxylase located in Mitochondria-Associated Membranes. Expression levels of Mitochondria-Associated Membranes proteins phosphatidylserine decarboxylase, DRP1, ATAD3A or TSPO were greatly reduced by mitotane as assessed by western blot analyses. Mitotane exposure markedly altered endogenous Mitochondria-Associated Membranes integrity and reduced the magnitude of mitochondria and the endoplasmic reticulum interactions as demonstrated by high resolution deconvolution microscopy and quantification. Finally, we showed that PK11195, a pharmacological inhibitor of the cholesterol translocator TSPO, embedded in Mitochondria-Associated Membranes, exerts a synergetic effect with mitotane in inducing Mitochondria-Associated Membranes disruption, apoptosis and in inhibiting steroid secretion. Altogether, our results demonstrate Mitochondria-Associated Membranes dysfunction in H295R cells treated with mitotane and that TSPO inhibition significantly potentiates mitotane antitumoral and antisecretory actions in vitro. This constitutes a potential and promising pharmacological strategy for patients with adrenocortical carcinoma.

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (o,p'-DDD) is the most effective medical therapy for adrenocortical carcinoma but its molecular mechanism of action remains poorly understood. Although mitotane is known to have mitochondrial (mt) effects, a direct link to mitochondrial dysfunction has never been established. We examined the functional consequences on proliferation, steroidogenesis, and mitochondrial respiratory chain, biogenesis and morphology, of mitotane exposure in two human adrenocortical cell lines, the steroid-secreting H295R line and the non-secreting SW13 line. Mitotane inhibited cell proliferation in a dose- and a time-dependent manner. At the concentration 50μM (14 mg/L), which corresponds to the threshold for therapeutic efficacy, mitotane drastically reduced cortisol and 17-hydroxyprogesterone secretions by 70%. This was accompanied by significant decreases in the expression of genes encoding mitochondrial proteins involved in steroidogenesis (STAR, CYP11B1, CYP11B2). In both H295R and SW13 cells, 50μM mitotane significantly inhibited (50%) the maximum velocity of the activity of the respiratory chain complex IV (cytochrome c oxidase, COX). This effect was associated with a drastic reduction in steady-state levels of the whole COX complex as revealed by Blue Native PAGE and reduced mRNA expression of both mtDNA-encoded COX2 and nuclear DNA-encoded COX4 subunits. In contrast, the activity and expression of respiratory chain complexes II and III were unaffected by mitotane treatment. Lastly, mitotane exposure enhanced mitochondrial biogenesis (increase in mtDNA content and PGC1α expression) and triggered fragmentation of the mitochondrial network. Altogether, our results provide first evidence that mitotane induced a mitochondrial respiratory chain defect in human adrenocortical cells.

International audience; Progesterone receptor (PR) isoforms (PRA and PRB) are implicated in the progression of breast cancers frequently associated with imbalanced PRA/PRB expression ratio. Antiprogestins represent potential antitumorigenic agents for such hormone-dependent cancers. To investigate the mechanism(s) controlling PR isoforms degradation/stability in the context of agonist and antagonist ligands, we used endometrial and mammary cancer cells stably expressing PRA and/or PRB. We found that the antiprogestin RU486 inhibited the agonist-induced turnover of PR isoforms through active mechanism(s) involving distinct MAPK-dependent phosphorylations. p42/44 MAPK activity inhibited proteasome-mediated degradation of RU486-bound PRB but not PRA in both cell lines. Ligand-induced PRB turnover required neosynthesis of a mandatory down-regulating partner whose interaction/function is negatively controlled by p42/44 MAPK. Such regulation strongly influenced expression of various endogenous PRB target genes in a selective manner, supporting functional relevance of the mechanism. Interestingly, in contrast to PRB, PRA stability was specifically increased by MAPK kinase kinase 1-induced p38 MAPK activation. Selective inhibition of p42/p44 or p38 activity resulted in opposite variations of the PRA/PRB expression ratio. Moreover, MAPK-dependent PR isoforms stability was independent of PR serine-294 phosphorylation previously proposed as a major sensor of PR down-regulation. In sum, we demonstrate that MAPK-mediated cell signaling differentially controls PRA/PRB expression ratio at posttranslational level through ligand-sensitive processes. Imbalance in PRA/PRB ratio frequently associated with carcinogenesis might be a direct consequence of disorders in MAPK signaling that might switch cellular responses to hormonal stimuli and contribute towards pathogenesis.

International audience; Primary glucocorticoid resistance (OMIM 138040) is a rare hereditary disease that causes a generalized partial insensitivity to glucocorticoid action, due to genetic alterations of the glucocorticoid receptor (GR). Investigation of adrenal incidentalomas led to the discovery of a family (eight affected individuals spanning three generations), prone to cortisol resistance, bilateral adrenal hyperplasia, arterial hypertension and hypokalemia. This phenotype exacerbated over time, cosegregates with the first heterozygous nonsense mutation p.R469[R,X] reported to date for the GR, replacing an arginine (CGA) by a stop (TGA) at amino-acid 469 in the second zinc finger of the DNA-binding domain of the receptor. In vitro, this mutation leads to a truncated 50-kDa GR lacking hormone and DNA binding capacity, devoid of hormone-dependent nuclear translocation and transactivation properties. In the proband's fibroblasts, we provided evidence for the lack of expression of the defective allele in vivo. The absence of detectable mutated GR mRNA was accompanied by a 50% reduction in wild type GR transcript and protein. This reduced GR expression leads to a significantly below-normal induction of glucocorticoid-induced target genes, FKBP5 in fibroblasts. We demonstrated that the molecular mechanisms of glucocorticoid signaling dysfunction involved GR haploinsufficiency due to the selective degradation of the mutated GR transcript through a nonsense-mediated mRNA Decay that was experimentally validated on emetine-treated propositus' fibroblasts. GR haploinsufficiency leads to hypertension due to illicit occupation of renal mineralocorticoid receptor by elevated cortisol rather than to increased mineralocorticoid production reported in primary glucocorticoid resistance. Indeed, apparent mineralocorticoid excess was demonstrated by a decrease in urinary tetrahydrocortisone-tetrahydrocortisol ratio in affected patients, revealing reduced glucocorticoid degradation by renal activity of the 11β-hydroxysteroid dehydrogenase type 2, a GR regulated gene. We propose thus that GR haploinsufficiency compromises glucocorticoid sensitivity and may represent a novel genetic cause of subclinical hypercortisolism, incidentally revealed bilateral adrenal hyperplasia and mineralocorticoid-independent hypertension.