Link between steroidogenesis, the cell cycle, and PKA in adrenocortical tumor cells

International audienceAdrenocortical tumors (ACTs) frequently cause steroid excess and present cell-cycle dysregulation. cAMP/PKA signaling is involved in steroid synthesis and play a role in cell-cycle regulation. We investigated, by cell synchronization in the different phases of the cell-cycle, the control of steroidogenesis and the contribution of PKA in adrenocortical cells (H295R and culture of primary pigmented nodular adrenocortical disease cells). Cells showed increased steroidogenesis and a maximal PKA activity at G2 phase, and a reduction at G1 phase. PRKACA overexpression, or cAMP stimulation, enhanced PKA activity and induced steroidogenesis in all synchronized groups but is not sufficient to drive cell-cycle progression. PRKAR1A inactivation enhanced PKA activity and induced STAR gene expression, only in cells in G1, and triggered cell-cycle progression in all groups.These findings provide evidence for a tight association between steroidogenesis and cell-cycle in ACTs. Moreover, PRKAR1A is essential for mediating the function of PKA activity on both steroidogenesis and cell-cycle progression in adrenocortical cells

Cofilin is a cAMP effector in mediating actin cytoskeleton reorganization and steroidogenesis in mouse and human adrenocortical tumor cells

cAMP pathway plays a major role in the pathogenesis of cortisol-producing adrenocortical adenomas (CPA). cAMP-induced steroidogenesis is preceded by actin cytoskeleton reorganization, a process regulated by cofilin activity. In this study we investigated cofilin role in mediating cAMP effects on cell morphology and steroidogenesis in adrenocortical tumor cells. We demonstrated that forskolin induced cell rounding and strongly reduced phosphorylated (P)-cofilin/total cofilin ratio in Y1 (-52 ± 16%, p < 0.001) and human CPA cells (-53 ± 18%, p < 0.05). Cofilin silencing significantly reduced both forskolin-induced morphological changes and progesterone production (1.3-fold vs 1.8-fold in controls, p < 0.05), whereas transfection of wild-type or S3A (active), but not S3D (inactive) cofilin, potentiated forskolin effects on cell rounding and increased 3-fold progesterone synthesis with respect to control (p < 0.05). Furthermore, cofilin dephosphorylation by a ROCK inhibitor potentiated forskolin-induced cell rounding and steroidogenesis (2-fold increase vs forskolin alone). Finally, we found a reduced P-cofilin/total cofilin ratio and increased cofilin expression in CPA vs endocrine inactive adenomas by western blot and immunohistochemistry. Overall, these results identified cofilin as a mediator of cAMP effects on both morphological changes and steroidogenesis in mouse and human adrenocortical tumor cells

KDM1A inactivation causes hereditary food-dependent Cushing syndrome

International audiencePURPOSE: This study aimed to investigate the genetic cause of food-dependent Cushing syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the glucose-dependent insulinotropic polypeptide receptor. Germline ARMC5 alterations have been reported in about 25% of PBMAH index cases but are absent in patients with FDCS. METHODS: A multiomics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNA sequencing, single-nucleotide variant array, methylome, miRNome, exome sequencing). RESULTS: The integrative analysis revealed 3 molecular groups with different clinical features, namely G1, comprising 16 patients with ARMC5 inactivating variants; G2, comprising 6 patients with FDCS with glucose-dependent insulinotropic polypeptide receptor ectopic expression; and G3, comprising 14 patients with a less severe phenotype. Exome sequencing revealed germline truncating variants of KDM1A in 5 G2 patients, constantly associated with a somatic loss of the KDM1A wild-type allele on 1p, leading to a loss of KDM1A expression both at messenger RNA and protein levels (P = 1.2 × 10(-12) and P < .01, respectively). Subsequently, KDM1A pathogenic variants were identified in 4 of 4 additional index cases with FDCS. CONCLUSION: KDM1A inactivation explains about 90% of FDCS PBMAH. Genetic screening for ARMC5 and KDM1A can now be offered for most PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management

Identification of predictive criteria for pathogenic variants of primary bilateral macronodular adrenal hyperplasia (PBMAH) gene ARMC5 in 352 unselected patients

International audienceAbstract Objective Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a heterogeneous disease characterized by adrenal macronodules and variable levels of cortisol excess, with not clearly established clinical diagnostic criteria. It can be caused by ARMC5 germline pathogenic variants. In this study, we aimed to identify predictive criteria for ARMC5 variants. Methods We included 352 consecutive index patients from 12 European centers, sequenced for germline ARMC5 alteration. Clinical, biological and imaging data were collected retrospectively. Results 52 patients (14.8%) carried ARMC5 germline pathogenic variants and showed a more distinct phenotype than non-mutated patients for cortisol excess (24-h urinary free cortisol 2.32 vs 1.11-fold ULN, respectively, P &lt; 0.001) and adrenal morphology (maximal adrenal diameter 104 vs 83 mm, respectively, P &lt; 0.001) and were more often surgically or medically treated (67.9 vs 36.8%, respectively, P &lt; 0.001). ARMC5-mutated patients showed a constant, bilateral adrenal involvement and at least a possible autonomous cortisol secretion (defined by a plasma cortisol after 1 mg dexamethasone suppression above 50 nmol/L), while these criteria were not systematic in WT patients (78.3%). The association of these two criteria holds a 100% sensitivity and a 100% negative predictive value for ARMC5 pathogenic variant. Conclusion We report the largest series of index patients investigated for ARMC5 and confirm that ARMC5 pathogenic variants are associated with a more severe phenotype in most cases. To minimize negative ARMC5 screening, genotyping should be limited to clear bilateral adrenal involvement and autonomous cortisol secretion, with an optimum sensitivity for routine clinical practice. These findings will also help to better define PBMAH diagnostic criteria