novel insights into the genetics and pathophysiology of adrenocortical tumors

International audienceAdrenocortical tumors (ACTs) are typically unilateral and can be classified as benign adrenocortical adenomas (ACAs) or malignant adrenocortical cancers (ACCs). In rare cases, tumors may occur in both adrenal glands as micronodular hyperplasia (primary pigmented nodular adrenal dysplasia) or as macronodular hyperplasia (primary bilateral macronodular adrenal hyperplasia, PBMAH). The study of certain tumor predisposition syndromes has improved our understanding of sporadic ACTs. Most ACAs are associated with abnormalities of the cAMP signaling pathway, whereas most ACCs are linked to alterations in IGF2, TP53, or the Wnt/βcatenin pathways. Over the past year, single-nucleotide polymorphism array technology and next-generation sequencing have identified novel genetic alterations in ACTs that shed new light on the molecular mechanisms of oncogenesis. Among these are somatic mutations of PKA catalytic subunit alpha gene (PRKACA) in ACA, germline, and somatic mutations of armadillo repeat containing 5 gene (ARMC5) in primary bilateral macronodular adrenal hyperplasia and somatic alterations of the E3 ubiquitin ligase gene ZNRF3 in ACC. This review focuses on the recent discoveries and their diagnostic, prognostic, and therapeutic implications

Silencing mutated β-catenin inhibits cell proliferation and stimulates apoptosis in the adrenocortical cancer cell line H295R

Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating β-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/β-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target. Similar to patient tumor specimen the H295 cell line derived from an ACC harbors a natural activating β-catenin mutation. We herein assess the in vitro and in vivo effect of β-catenin inactivation using a doxycyclin (dox) inducible shRNA plasmid in H295R adrenocortical cancer cells line (clone named shβ). Following dox treatment a profound reduction in β-catenin expression was detectable in shβ clones in comparison to control clones (Ctr). Accordingly, we observed a decrease in Wnt/βcatenin-dependent luciferase reporter activity as well as a decreased expression of AXIN2 representing an endogenous β-catenin target gene. Concomitantly, β-catenin silencing resulted in a decreased cell proliferation, cell cycle alterations with cell accumulation in the G1 phase and increased apoptosis in vitro. In vivo, on established tumor xenografts in athymic nude mice, 9 days of β-catenin silencing resulted in a significant reduction of CTNNB1 and AXIN2 expression. Moreover, continous β-catenin silencing, starting 3 days after tumor cell inoculation, was associated with a complete absence of tumor growth in the shβ group while tumors were present in all animals of the control group. In summary, these experiments provide evidences that Wnt/β-catenin pathway inhibition in ACC is a promising therapeutic target

Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of β-catenin in adrenocortical carcinoma

International audienceAdrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/β-catenin signaling pathway. However, the adrenal-specific targets of oncogenic β-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous β-catenin activating mutation was done to identify the Wnt/β-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that AFF3 mediates the oncogenic effects of β-catenin in ACC. The Wnt response element site located at nucleotide position − 1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/β-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/β-catenin pathway involved in ACC, acting on transcription and RNA splicing

Cushing's Syndrome and Fetal Features Resurgence in Adrenal Cortex–Specific Prkar1a Knockout Mice

Carney complex (CNC) is an inherited neoplasia syndrome with endocrine overactivity. Its most frequent endocrine manifestation is primary pigmented nodular adrenocortical disease (PPNAD), a bilateral adrenocortical hyperplasia causing pituitary-independent Cushing's syndrome. Inactivating mutations in PRKAR1A, a gene encoding the type 1 α-regulatory subunit (R1α) of the cAMP–dependent protein kinase (PKA) have been found in 80% of CNC patients with Cushing's syndrome. To demonstrate the implication of R1α loss in the initiation and development of PPNAD, we generated mice lacking Prkar1a specifically in the adrenal cortex (AdKO). AdKO mice develop pituitary-independent Cushing's syndrome with increased PKA activity. This leads to autonomous steroidogenic genes expression and deregulated adreno-cortical cells differentiation, increased proliferation and resistance to apoptosis. Unexpectedly, R1α loss results in improper maintenance and centrifugal expansion of cortisol-producing fetal adrenocortical cells with concomitant regression of adult cortex. Our data provide the first in vivo evidence that loss of R1α is sufficient to induce autonomous adrenal hyper-activity and bilateral hyperplasia, both observed in human PPNAD. Furthermore, this model demonstrates that deregulated PKA activity favors the emergence of a new cell population potentially arising from the fetal adrenal, giving new insight into the mechanisms leading to PPNAD

Aldo Keto Reductase 1B7 and Prostaglandin F2α Are Regulators of Adrenal Endocrine Functions

Prostaglandin F2α (PGF2α), represses ovarian steroidogenesis and initiates parturition in mammals but its impact on adrenal gland is unknown. Prostaglandins biosynthesis depends on the sequential action of upstream cyclooxygenases (COX) and terminal synthases but no PGF2α synthases (PGFS) were functionally identified in mammalian cells. In vitro, the most efficient mammalian PGFS belong to aldo-keto reductase 1B (AKR1B) family. The adrenal gland is a major site of AKR1B expression in both human (AKR1B1) and mouse (AKR1B3, AKR1B7). Thus, we examined the PGF2α biosynthetic pathway and its functional impact on both cortical and medullary zones. Both compartments produced PGF2α but expressed different biosynthetic isozymes. In chromaffin cells, PGF2α secretion appeared constitutive and correlated to continuous expression of COX1 and AKR1B3. In steroidogenic cells, PGF2α secretion was stimulated by adrenocorticotropic hormone (ACTH) and correlated to ACTH-responsiveness of both COX2 and AKR1B7/B1. The pivotal role of AKR1B7 in ACTH-induced PGF2α release and functional coupling with COX2 was demonstrated using over- and down-expression in cell lines. PGF2α receptor was only detected in chromaffin cells, making medulla the primary target of PGF2α action. By comparing PGF2α-responsiveness of isolated cells and whole adrenal cultures, we demonstrated that PGF2α repressed glucocorticoid secretion by an indirect mechanism involving a decrease in catecholamine release which in turn decreased adrenal steroidogenesis. PGF2α may be regarded as a negative autocrine/paracrine regulator within a novel intra-adrenal feedback loop. The coordinated cell-specific regulation of COX2 and AKR1B7 ensures the generation of this stress-induced corticostatic signal

Contrôle hormonal de la stéroïdogenèse et tumorigenèse cortico-surrénalienne : utilisation de la trangenèse chez la souris pour le développement de nouvelles lignées cellulaires et de modèles animaux de pathologies tumorales par oncogenèse ciblée

The use of the adrenocortical specific promoter in the mouse has enabled to establish new cell lines with zona fasciculata adrenocortical cell phenotype (ATC cell line) and to test the capacity of gene candidates in the development of adrenocortical tumors.L'utilisation de transgènes actifs dans le cortex surrénalien de souris a permis de dériver de nouvelles lignées cellulaires de la zone fasciculée de la corticosurrénale (lignées ATC) et de tester le pouvoir transformant de gènes candidats au développement de tumeurs corticosurrénaliennes. Les cellules ATC produisent de la corticostérone et expriment l'ensemble des gènes impliqués dans la stéroïdogenèse sous le contrôle de l'ACTH. Elles ont permis d'explorer les rôles antagonistes des facteurs de transcription SF1 et DAX1 dans le mécanisme d'action de l'ACTH. Les modèles cellulaires et murins surexprimant IGF2 ou ayant perdu en partie l'expression de p57kip2 ont conduit à rejeter leur implication individuelle dans le développement des corticosurrénalomes. Nous avons montré que l'expression dans la corticosurrénale de souris d'une sous unité régulatrice RIalpha tronquée de la PKA reproduit l'hyperactivité endocrine rencontrée dans les hyperplasies micronodulaires pigmentées des surrénale

Contrôle hormonal de la stéroïdogenèse et tumorigenèse cortico-surrénalienne (utilisation de la trangenèse chez la souris pour le développement de nouvelles lignées cellulaires et de modèles animaux de pathologies tumorales par oncogenèse ciblée)

L'utilisation de transgènes actifs dans le cortex surrénalien de souris a permis de dériver de nouvelles lignées cellulaires de la zone fasciculée de la corticosurrénale (lignées ATC) et de tester le pouvoir transformant de gènes candidats au développement de tumeurs corticosurrénaliennes. Les cellules ATC produisent de la corticostérone et expriment l'ensemble des gènes impliqués dans la stéroïdogenèse sous le contrôle de l'ACTH. Elles ont permis d'explorer les rôles antagonistes des facteurs de transcription SF1 et DAX1 dans le mécanisme d'action de l'ACTH. Les modèles cellulaires et murins surexprimant IGF2 ou ayant perdu en partie l'expression de p57kip2 ont conduit à rejeter leur implication individuelle dans le développement des corticosurrénalomes. Nous avons montré que l'expression dans la corticosurrénale de souris d'une sous unité régulatrice RIalpha tronquée de la PKA reproduit l'hyperactivité endocrine rencontrée dans les hyperplasies micronodulaires pigmentées des surrénalesCLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

8Cl-cAMP modifies the balance between PKAR1 and PKAR2 and modulates the cell cycle, growth and apoptosis in human adrenocortical H295R cells

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