RESUMEN
CONTEXT: DNA methylation has been identified among putative regulatory mechanisms for CYP11B2 expression in primary aldosteronism. OBJECTIVE: The objective of this work is to investigate DNA methylation and expression of genes encoding steroidogenic enzymes in benign adrenocortical tumors. DESIGN AND SETTING: This cross-sectional study took place at university hospitals. PATIENTS: We collected fresh-frozen tissues from patients with benign adrenocortical adenomas (n = 48) (nonfunctioning n = 9, autonomous cortisol secretion n = 9, Cushing syndrome n = 17, aldosterone-producing [APA] n = 13) and adrenal cortex adjacent to APA (n = 12). We collected formalin-fixed, paraffin-embedded (FFPE) specimens of paired APA and concurrent aldosterone-producing cell clusters (APCCs) (n = 6). INTERVENTION: DNA methylation levels were evaluated by quantitative bisulfite next-generation sequencing in fresh-frozen tissues (CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, HSD3B1, HSD3B2, NR5A1, STAR, and TSPO) and FFPE APA/APCC paired samples (CYP11B2). CYP11B1, CYP11B2, CYP17, CYP21, and STAR gene expressions were examined by quantitative real-time polymerase chain reaction. MAIN OUTCOME MEASURE: The main outcome measure was DNA methylation. RESULTS: CYP11B2 methylation levels were significantly lower in APA than in other adrenal tissues (P < .001). Methylation levels of the remaining genes were comparable among groups. Overall, CYP11B2 expression and DNA methylation were negatively correlated (ρ = -0.379; P = .003). In FFPE-paired APA/APCC samples, CYP11B2 methylation level was significantly lower in APA than in concurrent APCCs (P = .028). CONCLUSIONS: DNA methylation plays a regulatory role for CYP11B2 expression and may contribute to aldosterone hypersecretion in APA. Lower CYP11B2 methylation levels in APA than in APCCs may suggest an APCC-to-APA switch via progressive CYP11B2 demethylation. Conversely, DNA methylation seems not to be relevant in regulating the expression of genes encoding steroidogenic enzymes other than CYP11B2.
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Neoplasias de la Corteza Suprarrenal/genética , Adenoma Corticosuprarrenal/genética , Metilación de ADN , Enzimas/genética , Hormonas Esteroides Gonadales/biosíntesis , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/enzimología , Adenoma Corticosuprarrenal/metabolismo , Adenoma Corticosuprarrenal/patología , Adulto , Anciano , Aldosterona/metabolismo , Estudios de Cohortes , Estudios Transversales , Enzimas/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Redes y Vías Metabólicas/genética , Persona de Mediana EdadRESUMEN
Adrenocortical carcinomas (ACC) are aggressive tumors with a heterogeneous prognosis and limited therapeutic options for advanced stages. This study aims to identify novel drug targets for a personalized treatment in ACC. RNA was isolated from 40 formalin-fixed paraffin-embedded ACC samples. We evaluated gene expression of 84 known cancer drug targets by reverse transcriptase quantitative real time-PCR and calculated fold change using 5 normal adrenal glands as reference (overexpression by fold change >2.0). The most promising candidate cyclin-dependent kinase 4 (CDK4) was investigated at protein level in 104 ACC samples and tested by in vitro experiments in two ACC cell lines (NCI-H295R and MUC1). The most frequently overexpressed genes were TOP2A (100% of cases, median fold change = 16.5), IGF2 (95%, fold change = 52.9), CDK1 (80%, fold change = 6.7), CDK4 (62%, fold change = 2.6), PLK4 (60%, fold change = 2.8), and PLK1 (52%, fold change = 2.3). CDK4 was chosen for functional validation, as it is actionable by approved CDK4/6-inhibitors (e.g., palbociclib). Nuclear immunostaining of CDK4 significantly correlated with mRNA expression (R = 0.52, P < 0.005). We exposed both NCI-H295R and MUC1 cell lines to palbociclib and found a concentration- and time-dependent reduction of cell viability, which was more pronounced in the NCI-H295R cells in line with higher CDK4 expression. Furthermore, we tested palbociclib in combination with insulin-like growth factor 1/insulin receptor inhibitor linsitinib showing an additive effect. In conclusion, we demonstrate that RNA profiling is useful to discover potential drug targets and that CDK4/6 inhibitors are promising candidates for treatment of selected patients with ACC.
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Carcinoma Corticosuprarrenal/patología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Adolescente , Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Proliferación Celular , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/genética , Femenino , Estudios de Seguimiento , Humanos , Imidazoles/administración & dosificación , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Piperazinas/administración & dosificación , Pronóstico , Pirazinas/administración & dosificación , Piridinas/administración & dosificación , Tasa de Supervivencia , Células Tumorales Cultivadas , Adulto JovenRESUMEN
PURPOSE: To assess the diagnostic performance of the H-score of 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the histopathological diagnosis of adrenocortical tumors (ACT). METHODS: We retrospectively evaluated 199 cases of ACT, of which 85 were diagnosed as aldosterone-producing adenoma (APA), 66 as cortisol-producing adenoma (CPA), 9 as aldosterone-cortisol co-secreting adenoma, 30 as nonhyperfunctioning adenoma, and 9 as adrenocortical carcinoma (ACC). Immunohistochemical staining was performed using anti-CYP11B1 and anti-CYP11B2 monoclonal antibodies. The staining was quantified by the McCarty's H-score system. The diagnostic performance was assessed by the receiver operating characteristic curve (ROC). RESULTS: The H-score of CYP11B1 is highest in the CPA group and lowest in the ACC group. The H-score of CYP11B2 in the APA group is significantly higher than other ACT groups. The area under ROC (AUC) of an increased H-score of CYP11B2 (>65) for the diagnosis of APA was 0.971 (95%CI 0.937-0.990). The AUC of an increased H-score of CYP11B1 (>204) for the diagnosis of CPA was 0.725 (95%CI 0.658-0.786). The AUC of a decreased H-score of CYP11B1 (<85) for the diagnosis of ACC was 0.960 (95%CI 0.923-0.983). CONCLUSIONS: H-score of CYP11B1 and CYP11B2 are reliable tools for the histopathological subtyping of functional benign ACT and may offer some value in the histopathological diagnosis of malignant ACT.
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Adenoma/diagnóstico , Adenoma/enzimología , Neoplasias de la Corteza Suprarrenal/diagnóstico , Neoplasias de la Corteza Suprarrenal/enzimología , Carcinoma Corticosuprarrenal/diagnóstico , Carcinoma Corticosuprarrenal/enzimología , Citocromo P-450 CYP11B2/metabolismo , Adulto , Anciano , Aldosterona/metabolismo , Núcleo Celular/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Receptores de Esteroides/metabolismo , Reproducibilidad de los Resultados , Estudios Retrospectivos , Esteroide 11-beta-Hidroxilasa/metabolismoRESUMEN
OBJECTIVE: Mitotane is used for the treatment of adrenocortical carcinoma. High oral daily doses of typically 1- 6 g are required to attain therapeutic concentrations. The drug has a narrow therapeutic index and patient management is difficult because of a high volume of distribution, very long elimination half-life, and drug interaction through induction of metabolizing enzymes. The present evaluation aimed at the development of a population pharmacokinetic model of mitotane to facilitate therapeutic drug monitoring. METHODS: Appropriate dosing information, plasma concentrations (1137 data points) and covariates were available from therapeutic drug monitoring (TDM) of 76 adrenocortical carcinoma patients treated with mitotane. Using nonlinear mixed effects modeling, a simple structural model was first developed, with subsequent introduction of metabolic autoinduction. Covariate data were analyzed to improve overall model predictability. Simulations were performed to assess the attainment of therapeutic concentrations with clinical dosing schedules. RESULTS: A one-compartment pharmacokinetic model with first order absorption was found suitable to describe the data, with an estimated central volume of distribution of 6086 L related to a high interindividual variability of 81.5%. Increase in clearance of mitotane during treatment could be modeled by a linear enzyme autoinduction process. Body mass index was found to have an influence upon disposition kinetics of mitotane. Model simulations favor a high dose regimen to rapidly attain therapeutic concentrations, with the first TDM suggested on day 16 of treatment to avoid systemic toxicity. CONCLUSION: The proposed model describes mitotane pharmacokinetics and can be used to facilitate therapy by predicting plasma concentrations.
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Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Antineoplásicos Hormonales/farmacocinética , Mitotano/farmacocinética , Modelos Biológicos , Adolescente , Neoplasias de la Corteza Suprarrenal/enzimología , Carcinoma Corticosuprarrenal/enzimología , Adulto , Anciano , Antineoplásicos Hormonales/uso terapéutico , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitotano/uso terapéutico , Adulto JovenRESUMEN
The mechanism of aldosterone-producing adrenocortical adenoma (APA) pathogenesis and the role of microRNAs (miRNAs) in APA pathogenesis have not been completely clarified. We examined the expression and function of miR-140-3p, miR-193a-3p and miR-22-3p, which have binding sites in CYP11B2. Expression of miRNAs and CYP11B2 mRNA was measured by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation was monitored by colorimetric analysis, and cell apoptosis and cell cycle progression were analysed by flow cytometry. ELISA was carried out to detect aldosterone levels in cell culture supernatants. Luciferase reporter assays, qRT-PCR and Western blotting were performed to identify CYP11B2 as a target of miR-193a-3p. Of the three miRNAs examined, miR-193a-3p exhibited a significant decrease and CYP11B2 mRNA exhibited a significant increase in expression in APA compared with adjacent normal adrenal gland tissue. Transfection of miR-193a-3p mimic into the human adrenocortical cell line H295R showed that elevated miR-193a-3p expression inhibits proliferation and aldosterone secretion, induces G1-phase arrest and promotes apoptosis in H295R cells. Furthermore, in luciferase reporter assays, overexpression of miR-193a-3p in H295R cells significantly reduced the luciferase activity of the wild-type CYP11B2 3'-UTR construct, which could be reversed by mutation of the miR-193a-3p-binding site. Moreover, miR-193a-3p overexpression downregulated CYP11B2 mRNA and protein expression. Finally, overexpression of CYP11B2 diminished the effects of miR-193a-3p on H295R cells. Taken together, our results suggest that CYP11B2 levels may be modulated by miR-193a-3p in APA, which could explain, at least partially, why downregulation of miR-193a-3p during APA formation may promote cell growth and suppress apoptosis.
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Neoplasias de la Corteza Suprarrenal/enzimología , Adenoma Corticosuprarrenal/enzimología , Aldosterona/metabolismo , Citocromo P-450 CYP11B2/metabolismo , MicroARNs/metabolismo , Regiones no Traducidas 3' , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/genética , Adenoma Corticosuprarrenal/patología , Apoptosis , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Citocromo P-450 CYP11B2/genética , Regulación hacia Abajo , Puntos de Control de la Fase G1 del Ciclo Celular , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Vías SecretorasRESUMEN
The adrenal cortex accumulates lipofuscin granules with age. Lipofuscin accumulation is also seen in adrenocortical tumors associated with Cushing syndrome (CS), particularly those with PRKAR1A mutations, such as in primary pigmented nodular adrenocortical disease (PPNAD). We investigated the presence of lipofuscin in cortisol-producing adenomas (CPAs) responsible for CS with and without the PRKACA (pLeu206Arg) somatic mutation. Ten paraffin-embedded sections of CPAs from cases with overt CS with (n=4) and without (n=6) a PRKACA mutation were microscopically examined through three detection methods, the hematoxylin-Eosin (H & E) staining, the Fontana Masson (FM) staining using light microscopy, and lipofuscin autofluorescence, using confocal laser scanning microscopy (CLSM). Sections were examined quantitatively according to the intensity of the pigmentation, as well as qualitatively based on the total number of granular pigments at all visual fields per tissue slide. Tissues from CPAs were compared to peritumoral adjacent tissues (n=5), to Conn adenomas (n=4), and PPNAD (n=3). CPAs had significantly higher number of lipofuscin-pigment granules compared to peritumoral adrenal tissue and Conn adenomas (46.9±9.5 vs. 3.8±4.8, p=0.0001). The presence of the PRKACA mutation did not increase the chances of pigmentation in the form of lipofuscin granules within CPAs associated with CS. Thus, all CPAs leading to CS accumulate lipofuscin, which presents like pigmentation sometimes seen macroscopically but always detected microscopically. PPNAD caused by PRKAR1A mutations is the best known adrenal lesion leading to CS associated with intense lipofuscin pigmentation and this was confirmed here; CPAs harboring PRKACA mutations did not have statistically significantly more pigmentation than CPAs without mutation, but a larger study might have shown a difference.
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Adenoma/genética , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Hidrocortisona/biosíntesis , Lipofuscina/metabolismo , Mutación/genética , Adenoma/enzimología , Adenoma/patología , Neoplasias de la Corteza Suprarrenal/patología , Adulto , Femenino , Fluorescencia , Humanos , PigmentaciónRESUMEN
Topoisomerase II alpha (TOP2A) and thymidylate synthase (TS) are known prognostic parameters in several tumors and also predictors of efficacy of anthracyclines, topoisomerase inhibitors and fluoropirimidines, respectively. Expression of TOP2A and TS mRNA was assessed in 98 patients with adrenocortical carcinoma (ACC) and protein expression was assessed by immunohistochemistry in a subset of 39 tumors. Ninety-two patients were radically resected for stage II-III disease and 38 of them received adjuvant mitotane. Twenty-six patients with metastatic disease received the EDP-M (etoposide, doxorubicin, Adriamycin, cisplatin plus mitotane). TOP2A and TS expression in ACC tissue was directly correlated with the clinical data. Both markers were not associated with either disease free survival (DFS) or overall survival (OS) in multivariate analyses and failed to be associated to mitotane efficacy. Disease response or stabilization to EDP-M treatment was observed in 12/17 (71%) and 1/9 (11%) patients with high and low TOP2A expressing tumors (P = 0.0039) and 9/13 (69%) and 4/13 (31%) patients with high and low TS expressing ACC, respectively (P = 0.049). High TOP2A expression was significantly associated with longer time to progression (TTP) after EDP-M. TOP2A and TS proteins assessed by immunohistochemistry significantly correlated with mRNA expression. Immunohistochemical TOP2A expression was associated with a non-significant better response and longer TTP after EDP-M. TOP2A and TS were neither prognostic nor predictive of mitotane efficacy in ACC patients. The predictive role of TOP2A expression of EDP-M activity suggests a significant contribution of Adriamycin and etoposide for the efficacy of the EDP scheme.
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Neoplasias de la Corteza Suprarrenal/enzimología , ADN-Topoisomerasas de Tipo II/biosíntesis , Timidilato Sintasa/biosíntesis , Adolescente , Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Neoplasias de la Corteza Suprarrenal/genética , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cisplatino/administración & dosificación , ADN-Topoisomerasas de Tipo II/genética , Doxorrubicina/administración & dosificación , Etopósido/administración & dosificación , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mitotano/administración & dosificación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Timidilato Sintasa/genética , Adulto JovenRESUMEN
Adrenocortical tumor (ACT) is a malignancy with a low incidence rate and the current therapy for advanced disease has a limited impact on overall patient survival. A previous study from our group suggested that elevated expression of aurora-A and aurora-B is associated with poor outcome in childhood ACT. Similar results were also reported for adult ACTs. The present in-vitro study shows that AMG 900 inhibits aurora kinases in adrenocortical carcinoma cells. AMG 900 inhibited cell proliferation in NCI-H295 cells as well as in the ACT primary cultures and caused apoptosis in the cell line NCI-H295. Furthermore, it potentialized the mitotane, doxorubicin, and etoposide effects on apoptosis induction and acted synergistically with mitotane and doxorubicin in the inhibition of proliferation. In addition, we found that AMG 900 activated Notch signaling and rendered the cells sensitive to the combination of AMG 900 and Notch signaling inhibition. Altogether, these data show that aurora kinases inhibition using AMG 900 may be an adjuvant therapy to treat patients with invasive or recurrent adrenocortical carcinomas.
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Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Ftalazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/enzimología , Carcinoma Corticosuprarrenal/patología , Aurora Quinasas/antagonistas & inhibidores , Línea Celular Tumoral , Cisplatino/administración & dosificación , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Sinergismo Farmacológico , Histonas/metabolismo , Humanos , Mitotano/administración & dosificación , Mitotano/farmacología , Fosforilación/efectos de los fármacos , Ftalazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificaciónRESUMEN
Somatic mutations in PRKACA, coding for the catalytic α subunit of protein kinase A (PKA), have been recently identified as the most frequent genetic alteration in cortisol-secreting adrenocortical adenomas, which are responsible for adrenal Cushing's syndrome. The mutations identified so far lie at the interface between the catalytic (C) and regulatory (R) subunit of PKA. Detailed functional studies of the most frequent of these mutations (L206R) as well as of another one in the same region of the C subunit (199_200insW) have revealed that these mutations cause constitutive activation of PKA and lack of regulation by cAMP. This is due to interference with the binding of the R subunit, which keeps the C subunit inactive in the absence of cyclic AMP. Here, we review these recent findings, with a particular focus on the mechanisms of action of PRKACA mutations.
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Neoplasias de la Corteza Suprarrenal , Adenoma Corticosuprarrenal , Síndrome de Cushing , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico , Mutación Missense , Proteínas de Neoplasias , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/enzimología , Adenoma Corticosuprarrenal/genética , Adenoma Corticosuprarrenal/patología , Sustitución de Aminoácidos , Animales , Síndrome de Cushing/epidemiología , Síndrome de Cushing/genética , Síndrome de Cushing/patología , AMP Cíclico/genética , AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Sistemas de Mensajero Secundario/genéticaRESUMEN
Untargeted, next generation sequencing approaches have provided deep insights into genetic events that result in unopposed steroidogenesis from the adrenal cortex. In particular, somatic mutations in the gene encoding the catalytic subunit α of protein kinase A (PKA) (PRKACA) were identified independently by several groups as the most frequently altered gene in cortisol-producing adenomas. Detailed functional studies could explore the molecular consequences of these hot-spot mutations and large international cohorts have provided the basis to explore the clinical characteristics associated with this mutation. Thereby, PRKACA mutations are highly specific for cortisol over-secretion, while they are absent or very rare in the context of other adrenal diseases. Patients carrying these somatic mutations are affected by a more severe phenotype and are identified at a younger age. Thus, these genotype/phenotype correlations provide further evidence for the importance of PKA-dependent pathways for adrenal physiology and disease.
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Adenoma Corticosuprarrenal , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico , Mutación , Proteínas de Neoplasias , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/enzimología , Adenoma Corticosuprarrenal/genética , Adenoma Corticosuprarrenal/patología , Animales , Humanos , Hidrocortisona/genética , Hidrocortisona/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
Aldosterone-producing adrenocortical adenoma (APA) is responsible for the majority of cases clinically diagnosed as primary aldosteronism. Aldosterone synthase (CYP11B2) is one of the enzymes that play essential roles in aldosterone synthesis and is involved in the pathogenesis of APA. Recent studies have demonstrated that various factors and regulators influence the expression and function of CYP11B2 in APA. In particular, somatic mutations, such as gain-of-function and loss-of-function mutations, have been identified in several genes, each of which encodes a pivotal protein that affects the calcium signaling pathway, the expression of CYP11B2, and aldosterone production. The gain-of-function mutations were reported in KCNJ5 that encodes G-protein activated inward rectifier K+ channel 4 (Kir3.4) and in CACNA1D, encoding calcium channel, voltage-dependent, L type, alpha subunit Cav1.3. The loss-of-function mutations were found in ATP1A1 that encodes Na+/K+ ATPase α subunit and in ATP2B3, encoding Ca2+ ATPase. Furthermore, the aberrant expression of gonadotropin-releasing hormone receptor is associated with the overexpression of CYP11B2 and overproduction of aldosterone in APA with activating mutations in CTNNB1 encoding ß-catenin. On the other hand, CYP11B2 also catalyzes the conversion of cortisol to 18-hydroxycortisol and subsequently converts 18-hydroxycortisol to 18-oxocortisol. The recent studies have identified 18-oxocortisol as an important and distinct biomarker to diagnose primary aldosteronism. In this review, we summarize the recent findings on CYP11B2 and discuss the molecular pathogenesis of APA and the clinical significance of CYP11B2.
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Neoplasias de la Corteza Suprarrenal/enzimología , Adenoma Corticosuprarrenal/enzimología , Aldosterona/biosíntesis , Citocromo P-450 CYP11B2/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/patología , Forma de la Célula , Citocromo P-450 CYP11B2/genética , Humanos , Mutación/genética , Factores de Transcripción/metabolismoRESUMEN
The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations.
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Neoplasias de la Corteza Suprarrenal/metabolismo , Adenoma Corticosuprarrenal/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfoproteínas/metabolismo , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/genética , Adenoma Corticosuprarrenal/enzimología , Adenoma Corticosuprarrenal/genética , Adulto , Cromograninas/genética , Cromograninas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Genotipo , Humanos , Hidrocortisona/metabolismo , Inmunohistoquímica , Masculino , Mutación/genéticaRESUMEN
Human cytochome P450 2W1 (CYP2W1) enzyme is expressed in fetal colon and in colon tumors. The level of expression is higher in colon metastases than in the parent tumors and the enzyme is a possible drug target for treatment of colorectal cancer, as demonstrated in mouse xenograft studies. A previous study published in this journal reported that CYP2W1 is highly expressed in normal and transformed adrenal tissue. However, adrenal expression of CYP2W1 protein was not seen in previous studies in our research group. To clarify this inconsistency, we have used qRT-PCR and Western blotting with CYP2W1-specific antibodies to probe a panel of 27 adrenocortical carcinomas and 35 normal adrenal cortex samples. CYP2W1 mRNA expression is seen in all samples. However, significant CYP2W1 protein expression was found in only one tumor sample (a testosterone-producing adrenocortical carcinoma) and not in any normal tissue. Differences in the specificity of the CYP2W1 antibodies used in the two studies may explain the apparent discrepancy. We conclude that normal adrenal tissue lacks P450 2W1 enzyme expression; also, adrenocortical carcinomas generally do not express the enzyme. This information thus underline the colon cancer specificity of CYP2W1 enzyme expression and has implications for the development of anti-colon cancer therapies based on CYP2W1 as a drug target, since 2W1-dependent bioactivation of prodrugs for CYP2W1 will not take place in normal adrenal tissue or other non-transformed tissues.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/genética , Corteza Suprarrenal/enzimología , Carcinoma Corticosuprarrenal/genética , Familia 2 del Citocromo P450/genética , Corteza Suprarrenal/citología , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/enzimología , Carcinoma Corticosuprarrenal/patología , Anticuerpos Monoclonales/química , Western Blotting , Familia 2 del Citocromo P450/metabolismo , Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Adrenal Cushing syndrome (CS) is caused by the overproduction of cortisol in adrenocortical tumors including adrenal cortisol-producing adenoma (CPA). In CS, steroidogenic enzymes such as 17α-hydroxylase/17, 20-lase (CYP17A1), 3ß-hydroxysteroid dehydrogenase (HSD3B), and 11ß-hydroxylase (CYP11B1) are abundantly expressed in tumor cells. In addition, several transcriptional factors have been reported to play pivotal roles in the regulation of these enzymes in CPA, but their correlations with those enzymes above have still remained largely unknown. Therefore, in this study, we examined the status of steroidogenic enzymes and their transcriptional factors in 78 and 15 CPA cases by using immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR), respectively. Immunoreactivity of HSD3B2, CYP11B1, CYP17A1, steroidogenic factor-1 (SF1[NR5A1]), GATA6, and nerve growth factor induced-B (NGFIB[NR4A1]) was detected in tumor cells. Results of qPCR analysis revealed that expression of HSD3B2 mRNA was significantly higher than that of HSD3B1, and CYP11B1 mRNA was significantly higher than CYP11B2. In addition, the expression of CYP11B1 mRNA was positively correlated with those of NR5A1, GATA6, and NR4A1. These results all indicated that HSD3B2 but not HSD3B1 was mainly involved in cortisol overproduction in CPA. In addition, NR5A1, GATA6, and NR4A1 were all considered to play important roles in cortisol overproduction through regulating CYP11B1 gene transcription.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/genética , Adenoma Corticosuprarrenal/enzimología , Adenoma Corticosuprarrenal/genética , Biomarcadores de Tumor/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Hidrocortisona/biosíntesis , Inmunohistoquímica , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/patología , Adulto , Biomarcadores de Tumor/análisis , Femenino , Factor de Transcripción GATA6/genética , Humanos , Masculino , Persona de Mediana Edad , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Progesterona Reductasa/genética , ARN Mensajero/genética , Esteroide 11-beta-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/genética , Factor Esteroidogénico 1/genética , Factores de Transcripción/análisisRESUMEN
CONTEXT: Oncocytic tumors of the adrenal cortex are rare, mostly nonfunctioning and benign. SETTING: Report virilizing oncocytic adrenocortical carcinoma in a 50-year-old woman. PATIENT: She presented a recent and progressive virilization syndrome, associated with high blood pressure. Hormonal evaluation showed elevated serum testosterone and delta-4-androstenedione levels, normal urinary free cortisol level and incomplete suppression of cortisol at the 1 mg dexamethasone suppression test. CT scan of the abdomen revealed a 35 mm left adrenal mass. INTERVENTION: The patient underwent a left adrenalectomy, and the histological study showed a 3 cm oncocytic adrenocortical carcinoma with signs of malignancy. RESULTS: Immunohistochemical study revealed that tumor cells expressed the steroidogenic enzymes involved into androgen synthesis (3ßHSD and P450c17α), P450 aromatase and luteinizing hormone (LH) receptors. Post-operatively, signs of virilization improved rapidly, serum testosterone and delta-4-androstenedione levels returned to normal, as did the dexamethasone suppression test. During follow-up CT-scan and 18-FDG PET/CT showed a right ovary mass, corresponding to a follicular cyst associated with hyperthecosis. The patient is alive with no recurrence 48 months after adrenal surgery. CONCLUSION: Oncocytic adrenocortical carcinomas, although extremely rare, should be considered in women with a virilization syndrome. In this woman immunohistochimical studies revealed the presence of steroidogenic enzymes involved into androgen synthesis and aromatization, and LH receptors could be implicated in this pathology.
Asunto(s)
Adenoma Oxifílico/complicaciones , Neoplasias de la Corteza Suprarrenal/complicaciones , Carcinoma Corticosuprarrenal/complicaciones , Virilismo/etiología , Adenoma Oxifílico/enzimología , Adenoma Oxifílico/cirugía , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/cirugía , Carcinoma Corticosuprarrenal/enzimología , Carcinoma Corticosuprarrenal/cirugía , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Virilismo/enzimología , Virilismo/cirugíaRESUMEN
We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/enzimología , Adenoma Corticosuprarrenal/enzimología , Síndrome de Cushing/enzimología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/genética , Adenoma Corticosuprarrenal/patología , Dominio Catalítico , Línea Celular Tumoral , Síndrome de Cushing/genética , Síndrome de Cushing/patología , AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/química , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Mutación , Unión Proteica , Estabilidad ProteicaRESUMEN
BACKGROUND: Adrenocortical tumors comprise frequent adenomas (ACA) and rare carcinomas (ACC). Human cytochrome P450 2W1 (CYP2W1) is highly expressed in some cancers holding the potential to activate certain drugs into tumor cytotoxins. OBJECTIVE: To investigate the CYP2W1 expression in adrenal samples and its relationship with clinical outcome in ACC. MATERIAL AND METHODS: CYP2W1 expression was investigated by qRT-PCR in 13 normal adrenal glands, 32 ACA, 25 ACC, and 9 different non-adrenal normal tissue samples and by immunohistochemistry in 352 specimens (23 normal adrenal glands, 33 ACA, 239 ACC, 67 non-adrenal normal or neoplastic samples). RESULTS: CYP2W1 mRNA expression was absent/low in normal non-adrenal tissues, but high in normal and neoplastic adrenal glands (all P<0.01 vs non-adrenal normal tissues). Accordingly, CYP2W1 immunoreactivity was absent/low (H-score 0-1) in 72% of non-adrenal normal tissues, but high (H-score 2-3) in 44% of non-adrenal cancers, in 65% of normal adrenal glands, in 62% of ACAs and in 50% of ACCs (all P<0.001 vs non-adrenal normal tissues), being significantly increased in steroid-secreting compared to non-secreting tumors. In ACC patients treated with mitotane only, high CYP2W1 immunoreactivity adjusted for ENSAT stage was associated with longer overall survival and time to progression (P<0.05 and P<0.01, respectively), and with a better response to therapy both as palliative (response/stable disease in 42% vs 6%, P<0.01) or adjuvant option (absence of disease recurrence in 69% vs 45%, P<0.01). CONCLUSION: CYP2W1 is highly expressed in both normal and neoplastic adrenal glands making it a promising tool for targeted therapy in ACC. Furthermore, CYP2W1 may represent a new predictive marker for the response to mitotane treatment.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/enzimología , Glándulas Suprarrenales/enzimología , Carcinoma Corticosuprarrenal/enzimología , Antineoplásicos Hormonales/administración & dosificación , Sistema Enzimático del Citocromo P-450/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mitotano/administración & dosificación , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Neoplasias de la Corteza Suprarrenal/patología , Glándulas Suprarrenales/patología , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/patología , Familia 2 del Citocromo P450 , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Stimulation of the cAMP pathway by adrenocorticotropin (ACTH) is essential for adrenal cortex maintenance, glucocorticoid and adrenal androgens synthesis, and secretion. Various molecular and cellular alterations of the cAMP pathway have been observed in endocrine tumors. Protein kinase A (PKA) is a central key component of the cAMP pathway. Molecular alterations of PKA subunits have been observed in adrenocortical tumors. PKA molecular defects can be germline in hereditary disorders or somatic in sporadic tumors. Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) can be observed in patients with ACTH-independent Cushing's syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic cortisol-secreting adrenocortical adenomas. Germline gene duplication of the catalytic subunits Cα (PRKACA) has been observed in patients with PPNAD. Furthermore, exome sequencing revealed recently activating somatic mutations of PRKACA in about 40% of cortisol-secreting adrenocortical adenomas. In vitro and in vivo functional studies help in the progress to understand the mechanisms of adrenocortical tumors development due to PKA regulatory subunits alterations. All these alterations are observed in benign oversecreting tumors and are mimicking in some way cAMP pathway constitutive activation. On the long term, unraveling these alterations will open new strategies of pharmacological treatment targeting the cAMP pathway in adrenal tumors and cortisol-secretion disorders.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Animales , Biocatálisis , Humanos , Modelos Biológicos , Subunidades de Proteína/metabolismoRESUMEN
Oncocytomas are tumours predominantly or exclusively composed of oncocytes, cells with granular and eosinophilic cytoplasm filled with mitochondria. Although they can occur in every organ, they are rare in adrenal glands, and in paediatric patients they are even rarer, with only three case reports previously published. We present a preschool child developing Cushing's syndrome due to an adrenocortical oncocytoma, which was confirmed immunohistochemically with antibodies to the mitochondrial electron complex 2. A 5.8-year-old girl presented with clinical features of Cushing's syndrome. ACTH-independent hypercortisolism was confirmed biochemically and a left adrenal mass was detected by imaging and removed by laparotomy. Histopathological analysis revealed a tumour composed of more than 95 % of oncocytes, confirmed immunohistochemically with antibodies to subunits A and B of the mitochondrial enzyme succinate dehydrogenase. Using the Lin-Weiss-Bisceglia score system and the reticulin algorithm, this tumour was categorized as a benign adrenocortical oncocytoma. The patient currently has 64 months of follow-up, without any evidence of relapse of symptoms. To our knowledge, we herein present the youngest patient developing an adrenocortical oncocytoma and the first manifestation of Cushing's syndrome due to this rare neoplasm in paediatric patients. We also emphasize the clinical usefulness of immunohistochemistry to the mitochondrial enzyme succinate dehydrogenase to confirm the oxyphilic nature of adrenocortical oncocytomas.
Asunto(s)
Adenoma Oxifílico/diagnóstico , Neoplasias de la Corteza Suprarrenal/diagnóstico , Síndrome de Cushing/diagnóstico , Adenoma Oxifílico/enzimología , Adenoma Oxifílico/patología , Adenoma Oxifílico/cirugía , Neoplasias de la Corteza Suprarrenal/enzimología , Neoplasias de la Corteza Suprarrenal/patología , Neoplasias de la Corteza Suprarrenal/cirugía , Niño , Diagnóstico Diferencial , Complejo II de Transporte de Electrones/análisis , Femenino , Humanos , Inmunohistoquímica , Succinato Deshidrogenasa/análisisRESUMEN
Dysregulation of aldosterone or cortisol production can predispose to hypertension, as seen in aldosterone-producing adenoma, a form of primary aldosteronism. We investigated the role of microRNA (miRNA) in their production, with particular emphasis on the CYP11B1 (11ß-hydroxylase) and CYP11B2 (aldosterone synthase) genes, which produce the enzymes responsible for the final stages of cortisol and aldosterone biosynthesis, respectively. Knockdown of Dicer1, a key enzyme in miRNA maturation, significantly altered CYP11B1 and CYP11B2 expression in a human adrenocortical cell line. Screening of nondiseased human adrenal and aldosterone-producing adenoma samples yielded reproducible but distinctive miRNA expression signatures for each tissue type, with levels of certain miRNA, including microRNA-24 (miR-24), differing significantly between the 2. Bioinformatic analysis identified putative binding sites for several miRNA, including miR-24, in the 3' untranslated region of CYP11B1 and CYP11B2 mRNAs. In vitro manipulation of miR-24 confirmed its ability to modulate CYP11B1 and CYP11B2 expression, as well as cortisol and aldosterone production. This study demonstrates that Dicer-dependent miRNA, including miR-24, can post-transcriptionally regulate expression of the CYP11B1 and CYP11B2 genes. Normal adrenal tissue and aldosterone-producing adenoma differ significantly and reproducibly in their miRNA expression profiles, with miR-24 significantly downregulated in the latter. Adrenal miRNA may, therefore, be a novel and valid target for the therapeutic manipulation of corticosteroid biosynthesis.