RESUMEN
MicroRNAs (miRNAs) are small, endogenous, non-protein-coding RNAs that are an important means of posttranscriptional gene regulation. Deletion of Dicer, a key miRNA processing enzyme, is embryonic lethal in mice, and tissue-specific Dicer deletion results in developmental defects. Using a conditional knockout model, we generated mice lacking Dicer in the adrenal cortex. These Dicer-knockout (KO) mice exhibited perinatal mortality and failure of the adrenal cortex during late gestation between embryonic day 16.5 (E16.5) and E18.5. Further study of Dicer-KO adrenals demonstrated a significant loss of steroidogenic factor 1-expressing cortical cells that was histologically evident as early as E16.5 coincident with an increase in p21 and cleaved-caspase 3 staining in the cortex. However, peripheral cortical proliferation persisted in KO adrenals as assessed by staining of proliferating cell nuclear antigen. To further characterize the embryonic adrenals from Dicer-KO mice, we performed microarray analyses for both gene and miRNA expression on purified RNA isolated from control and KO adrenals of E15.5 and E16.5 embryos. Consistent with the absence of Dicer and the associated loss of miRNA-mediated mRNA degradation, we observed an up-regulation of a small subset of adrenal transcripts in Dicer-KO mice, most notably the transcripts coded by the genes Nr6a1 and Acvr1c. Indeed, several miRNAs, including let-7, miR-34c, and miR-21, that are predicted to target these genes for degradation, were also markedly down-regulated in Dicer-KO adrenals. Together these data suggest a role for miRNA-mediated regulation of a subset of genes that are essential for normal adrenal growth and homeostasis.
Asunto(s)
Corteza Suprarrenal/embriología , Corteza Suprarrenal/metabolismo , ARN Helicasas DEAD-box/metabolismo , Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Ribonucleasa III/metabolismo , Corteza Suprarrenal/citología , Animales , Animales Recién Nacidos , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Secuencia de Bases , Complejo CD3/metabolismo , Ciclo Celular/genética , Muerte Celular/genética , Proliferación Celular , Secuencia Conservada/genética , ARN Helicasas DEAD-box/deficiencia , Daño del ADN/genética , Regulación hacia Abajo/genética , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Integrasas/metabolismo , Ratones , Ratones Noqueados , MicroARNs/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleasa III/deficiencia , Programas Informáticos , Análisis de SupervivenciaRESUMEN
Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal ß-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal ß-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized ß-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized ß-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/patología , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Factor II del Crecimiento Similar a la Insulina/metabolismo , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Corticoesteroides/metabolismo , Neoplasias de la Corteza Suprarrenal/genética , Animales , Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Impresión Genómica , Humanos , Hiperplasia , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Ratones Noqueados , Análisis Multivariante , Mutación/genética , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Modelos de Riesgos Proporcionales , Estabilidad Proteica , Transporte de Proteínas , Regulación hacia Arriba/genéticaRESUMEN
PURPOSE: The purpose of this study was to examine the expression and molecular significance of JAG1, a ligand for the Notch developmental signaling pathway, in adrenocortical carcinoma (ACC). EXPERIMENTAL DESIGN: Human microarray data were analyzed for genes expressing ligands for the Notch pathway and validated with quantitative real-time PCR (QPCR) and immunoblots of RNA and protein, respectively. ACC cells lines were assessed for Notch pathway member expression by immunoblot, QPCR, and immunofluorescence. Notch pathway activity was also determined using a reporter gene (luciferase) activation. Proliferation experiments using a Jag1 knockdown strategy (Jag1KD) and an inhibitor of Notch-dependent transcription (DNMaml) used a coculture system with fluorescence-activated cell-sorting (FACS) analysis. Tumor stage and mitotic rate of human ACC samples were correlated to JAG1 expression. RESULTS: The Notch ligand JAG1 mRNA and protein are upregulated in ACCs. JAG1 upregulation can be modeled in the Y1 mouse ACC cell line that expresses Jag1, Notch receptors, downstream signaling molecules, and exhibits density-dependent Notch activation. Jag1 enhances cell proliferation through activation of canonical Notch signaling as shown through Jag1KD and coculture experiments. Inhibition of Notch signaling at the level of postreceptor signaling (DNMaml), results in similar inhibition of cell proliferation. Analysis of clinical data indicates that Jag1 expression correlates with both grade and stage of ACCs, supporting a role of JAG1-dependent Notch activation in late-stage ACCs. CONCLUSIONS: JAG1 is the primary upregulated Notch ligand in ACCs and enhances ACC cell proliferation and tumor aggressiveness in a non-cell-autonomous manner through activation of Notch signaling in adjacent cells.
Asunto(s)
Carcinoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/patología , Proliferación Celular , Perfilación de la Expresión Génica , Janus Quinasa 1/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Carcinoma Corticosuprarrenal/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Janus Quinasa 1/genética , Luciferasas/metabolismo , Clasificación del Tumor , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The continuous centripetal repopulation of the adrenal cortex is consistent with a population of cells endowed with the stem/progenitor cell properties of self-renewal and pluripotency. The adrenocortical capsule and underlying undifferentiated cortical cells are emerging as critical components of the stem/progenitor cell niche. Recent genetic analysis has identified various signaling pathways including Sonic Hedgehog (Shh) and Wnt as crucial mediators of adrenocortical lineage and organ homeostasis. Shh expression is restricted to the peripheral cortical cells that express a paucity of steroidogenic genes but give rise to the underlying differentiated cells of the cortex. Wnt/ß-catenin signaling maintains the undifferentiated state and adrenal fate of adrenocortical stem/progenitor cells, in part through induction of its target genes Dax1 and inhibin-α, respectively. The pathogenesis of ACC, a rare yet highly aggressive cancer with an extremely poor prognosis, is slowly emerging from studies of the stem/progenitor cells of the adrenal cortex coupled with the genetics of familial syndromes in which ACC occurs. The frequent observation of constitutive activation of Wnt signaling due to loss-of-function mutations in the tumor suppressor gene APC or gain-of-function mutation in ß-catenin in both adenomas and carcinomas, suggests perhaps that the Wnt pathway serves an early or initiating insult in the oncogenic process. Loss of p53 might be predicted to cooperate with additional genetic insults such as IGF2 as both are the most common genetic abnormalities in malignant versus benign adrenocortical neoplasms. It is unclear whether other factors such as Pod1 and Pref1, which are implicated in stem/progenitor cell biology in the adrenal and/or other organs, are also implicated in the etiology of adrenocortical carcinoma. The rarity and heterogeneous presentation of ACC makes it difficult to identify the cellular origin and the molecular progression to cancer. A more complete understanding of adrenocortical stem/progenitor cell biology will invariably aid in characterization of the molecular details of ACC tumorigenesis and may offer new options for therapeutic intervention.
Asunto(s)
Neoplasias de la Corteza Suprarrenal , Corteza Suprarrenal , Carcinoma Corticosuprarrenal , Transformación Celular Neoplásica , Células Madre Neoplásicas , Células Madre Pluripotentes , Corteza Suprarrenal/metabolismo , Corteza Suprarrenal/patología , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/genética , Carcinoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/patología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Mutación , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Vía de Señalización Wnt/genéticaRESUMEN
Scientists have long hypothesized the existence of tissue-specific (somatic) stem cells and have searched for their location in different organs. The theory that adrenocortical organ homeostasis is maintained by undifferentiated stem or progenitor cells can be traced back nearly a century. Similar to other organ systems, it is widely believed that these rare cells of the adrenal cortex remain relatively undifferentiated and quiescent until needed to replenish the organ, at which time they undergo proliferation and terminal differentiation. Historical studies examining cell cycle activation by label retention assays and regenerative potential by organ transplantation experiments suggested that the adrenocortical progenitors reside in the outer periphery of the adrenal gland. Over the past decade, the Hammer laboratory, building on this hypothesis and these observations, has endeavored to understand the mechanisms of adrenocortical development and organ maintenance. In this review, we summarize the current knowledge of adrenal organogenesis. We present evidence for the existence and location of adrenocortical stem/progenitor cells and their potential contribution to adrenocortical carcinomas. Data described herein come primarily from studies conducted in the Hammer laboratory with incorporation of important related studies from other investigators. Together, the work provides a framework for the emerging somatic stem cell field as it relates to the adrenal gland.
Asunto(s)
Corteza Suprarrenal/citología , Células Madre/citología , Corteza Suprarrenal/embriología , Corteza Suprarrenal/fisiología , Animales , Diferenciación Celular/fisiología , Células Clonales/citología , Células Clonales/fisiología , Humanos , Organogénesis/fisiologíaRESUMEN
Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum factor(s) in cell proliferation. Using prostatic cancer cell (PCC) lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII), as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS). Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic fibroblast growth factor was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-15 expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer growth and metastasis.