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Supplementary Tables 1-2, Figures 1-5 from Regulation of Insulin-like Growth Factor–Mammalian Target of Rapamycin Signaling by MicroRNA in Childhood Adrenocortical Tumors

Supplementary Table 1 from Regulation of Insulin-like Growth Factor–Mammalian Target of Rapamycin Signaling by MicroRNA in Childhood Adrenocortical Tumors

MicroRNAs (miRNAs) act at the posttranscriptional level to control gene expression in virtually every biological process, including oncogenesis. Here, we report the identification of a set of miRNAs that are differentially regulated in childhood adrenocortical tumors (ACT), including miR-99a and miR-100. Functional analysis of these miRNAs in ACT cell lines showed that they coordinately regulate expression of the insulin-like growth factor–mammalian target of rapamycin (mTOR)–raptor signaling pathway through binding sites in their 3′-untranslated regions. In these cells, the active Ser2448-phosphorylated form of mTOR is present only in mitotic cells in association with the mitotic spindle and midbody in the G2-M phases of the cell cycle. Pharmacologic inhibition of mTOR signaling by everolimus greatly reduces tumor cell growth in vitro and in vivo. Our results reveal a novel mechanism of regulation of mTOR signaling by miRNAs, and they lay the groundwork for clinical evaluation of drugs inhibiting the mTOR pathway for treatment of adrenocortical cancer. Cancer Res; 70(11); 4666–75. ©2010 AACR.

The SF-1 transcription factor target gene FATE1 encodes a cancer-testis antigen that has an important role in regulating apoptosis and response to chemotherapy in adrenocortical carcinoma (ACC) cells. Autoantibodies directed against FATE1 were previously detected in patients with hepatocellular carcinoma. In this study, we investigated the prevalence of circulating anti-FATE1 antibodies in pediatric and adult patients with adrenocortical tumors using three different methods (immunofluorescence, ELISA and Western blot). Our results show that a pervasive anti-FATE1 immune response is present in those patients. Furthermore, FATE1 expression is a robust prognostic indicator in adult patients with ACC and is associated with increased steroidogenic and decreased immune response gene expression. These data can open perspectives for novel strategies in ACC immunotherapy.

Many types of cancer cells present constitutively activated ER stress pathways because of their significant burden of misfolded proteins coded by mutated and rearranged genes. Further increase of ER stress by pharmacological intervention may shift the balance towards cell death and can be exploited therapeutically. Recent studies have shown that an important component in the mechanism of action of mitotane, the only approved drug for the medical treatment of adrenocortical carcinoma (ACC), is represented by activation of ER stress through inhibition of the SOAT1 enzyme and accumulation of toxic lipids. Here we show that HA15, a novel inhibitor of the essential ER chaperone GRP78/BiP, inhibits ACC H295R cell proliferation and steroidogenesis and is able to synergize with mitotane action. These results suggest that convergent activation of ER stress pathways by drugs acting via different mechanisms represents a valuable therapeutic option for ACC.

International audience; Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.

ER-mitochondria contact sites represent hubs for signaling that control mitochondrial biology related to several aspects of cellular survival, metabolism, cell death sensitivity and metastasis, which all contribute to tumorigenesis. Altered ER-mitochondria contacts can deregulate Ca2+ homeostasis, phospholipid metabolism, mitochondrial morphology and dynamics. MAM represent both a hot spot in cancer onset and progression and an Achilles' heel of cancer cells that can be exploited for therapeutic perspectives. Over the past years, an increasing number of cancer-related proteins, including oncogenes and tumor suppressors, have been localized in MAM and exert their pro- or antiapoptotic functions through the regulation of Ca2+ transfer and signaling between the two organelles. In this review, we highlight the central role of ER-mitochondria contact sites in tumorigenesis and focus on chemotherapeutic drugs or potential targets that act on MAM properties for new therapeutic approaches in cancer.

SF-1 and LRH-1 are transcription factors that belong to the NR5A family of nuclear receptors that both have an essential role during development. Recent studies at the genome-wide scale have enabled the characterization of the cistrome and transcriptome regulated by SF-1 and LRH-1 in different cell lines and tissues. Those studies have allowed us to make a significant leap forward in our understanding of the mechanisms of transcriptional regulation of NR5A nuclear receptors in stem cells and cancer.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. Genomic studies have enabled progress in our understanding of the molecular bases of ACC, but factors that influence its prognosis are lacking. Amplification of the gene encoding the transcription factor steroidogenic factor-1 (SF-1; also known as NR5A1) is one of the genetic alterations common in ACC. We identified a transcriptional regulatory mechanism involving increased abundance of VAV2, a guanine nucleotide exchange factor for small GTPases that control the cytoskeleton, driven by increased expression of the gene encoding SF-1 in ACC. Manipulating SF-1 and VAV2 abundance in cultured ACC cells revealed that VAV2 was a critical factor for SF-1-induced cytoskeletal remodeling and invasion in culture (Matrigel) and in vivo (chicken chorioallantoic membrane) models. Analysis of ACC patient cohorts indicated that greater VAV2 abundance robustly correlated with poor prognosis in ACC patients. Because VAV2 is a druggable target, our findings suggest that blocking VAV2 may be a new therapeutic approach to inhibit metastatic progression in ACC patients.

Context Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with overall poor prognosis. The Ki67 labeling index (LI) has a major prognostic role in localized ACC after complete resection, but its estimates may suffer from considerable intra- and interobserver variability. VAV2 overexpression induced by increased Steroidogenic Factor-1 dosage is an essential factor driving ACC tumor cell invasion. Objective To assess the prognostic role of VAV2 expression in ACC by investigation of a large cohort of patients. Design, Setting, and Participants A total of 171 ACC cases (157 primary tumors, six local recurrences, eight metastases) from seven European Network for the Study of Adrenal Tumors centers were studied. Outcome Measurements H-scores were generated to quantify VAV2 expression. VAV2 expression was divided into two categories: low (H-score, Results VAV2 expression and Ki67 LI were significantly correlated with each other and with PFS and OS. Heterogeneity of VAV2 expression inside the same tumor was very low. Combined assessment of VAV2 expression and Ki67 LI improved patient stratification to low-risk and high-risk groups. Conclusion Combined assessment of Ki67 LI and VAV2 expression improves prognostic prediction in ACC.

Steroidogenic Factor-1 (SF-1) is a nuclear receptor transcription factor that has an essential role in the development of adrenal glands and gonads and in the regulation of steroidogenic gene expression. Recent studies using genomic approaches have revealed that SF-1 also has an important role in regulating proliferation of adrenocortical cells and have revealed its role in the control of a variety of biological processes as diverse as angiogenesis, adhesion to the extracellular matrix, cytoskeleton dynamics, transcriptional and post-transcriptional regulation of gene expression and apoptosis in the adrenal cortex. The identification of the complete set of SF-1 target genes will be of great importance to open new avenues for therapeutic intervention in adrenal diseases.

Steroidogenesis is a multistep process where interorganelle communications between the endoplasmic reticulum and mitochondria are critical. These intimate interactions physically occur through the Mitochondria-Associated ER membranes called MAMs. MAMs play important roles in mitochondrial morphology and in many cellular functions ranging from lipid metabolism, to calcium signaling and apoptosis together with a critical effect on steroidogenesis. Moreover, our recent characterization of new MAM resident proteins in adrenocortical cells extends the function of MAM in the mechanism of resistance of cancer cells to apoptotic stimuli and offers new perspectives in targeted therapeutic approaches for adrenocortical tumorigenesis.

International audience; Several stimuli induce programmed cell death by increasing Ca(2+) transfer from the endoplasmic reticulum (ER) to mitochondria. Perturbation of this process has a special relevance in pathologies as cancer and neurodegenerative disorders. Mitochondrial Ca(2+) uptake mainly takes place in correspondence of mitochondria-associated ER membranes (MAM), specialized contact sites between the two organelles. Here, we show the important role of FATE1, a cancer-testis antigen, in the regulation of ER-mitochondria distance and Ca(2+) uptake by mitochondria. FATE1 is localized at the interface between ER and mitochondria, fractionating into MAM FATE1 expression in adrenocortical carcinoma (ACC) cells under the control of the transcription factor SF-1 decreases ER-mitochondria contact and mitochondrial Ca(2+) uptake, while its knockdown has an opposite effect. FATE1 also decreases sensitivity to mitochondrial Ca(2+)-dependent pro-apoptotic stimuli and to the chemotherapeutic drug mitotane. In patients with ACC, FATE1 expression in their tumor is inversely correlated with their overall survival. These results show that the ER-mitochondria uncoupling activity of FATE1 is harnessed by cancer cells to escape apoptotic death and resist the action of chemotherapeutic drugs.

Context: Childhood adrenocortical tumors (ACT) are rare malignancies, except in southern Brazil, where a higher incidence rate is associated to a high frequency of the founder R337H TP53 mutation. To date, copy number alterations in these tumors have only been analyzed by low-resolution comparative genomic hybridization. Objective: We analyzed an international series of 25 childhood ACT using high-resolution single nucleotide polymorphism arrays to: 1) detect focal copy number alterations highlighting candidate driver genes; and 2) compare genetic alterations between Brazilian patients carrying the R337H TP53 mutation and non-Brazilian patients. Results: We identified 16 significantly recurrent chromosomal alterations (q-value < 0.05), the most frequent being −4q34, +9q33-q34, +19p, loss of heterozygosity (LOH) of chromosome 17 and 11p15. Focal amplifications and homozygous deletions comprising well-known oncogenes (MYC, MDM2, PDGFRA, KIT, MCL1, BCL2L1) and tumor suppressors (TP53, RB1, RPH3AL) were identified. In addition, eight focal deletions were detected at 4q34, defining a sharp peak region around the noncoding RNA LINC00290 gene. Although non-Brazilian tumors with a mutated TP53 were similar to Brazilian tumors, those with a wild-type TP53 displayed distinct genomic profiles, with significantly fewer rearrangements (P = 0.019). In particular, three alterations (LOH of chromosome 17, +9q33-q34, and −4q34) were significantly more frequent in TP53-mutated samples. Finally, two of four TP53 wild-type tumors displayed as sole rearrangement a copy-neutral LOH of the imprinted region at 11p15, supporting a major role for this region in ACT development. Conclusions: Our findings highlight potential driver genes and cellular pathways implicated in childhood ACT and demonstrate the existence of different oncogenic routes in this pathology.

Adrenocortical cells of several species have been reported to express significant levels of Agouti-related protein (Agrp) as well as melanocortin 4-receptor (MC4-R). In this study, we used the mouse tumoral adrenal cell line ATC7- L that secretes corticosterone in basal conditions with a 2- fold increase in response to ACTH treatment. We reported that these cells expressed functional MC4-R. They also expressed Agrp mRNA and secreted immunoreactive Agrp in the culture medium. Long-term treatment of ATC7-L with (Nle4,D-Phe7)-alpha MSH (NDP-alpha MSH) or forskolin as well as Agrp strongly reduced MC4-R level by more than 30%. On the contrary, leptin treatment did not modify this level although it significantly reduced MC2-R level. These results could be correlated to some data obtained in vivo on adrenal glands removed from diet-induced obese mice exhibiting a hyperleptinemia, where the level of both MC2-R and MC4-R appeared to be reduced as Agrp mRNA expression level was increased compared to Control mice. All these data would suggest the existence of a link between the metabolic status and the activation of the adrenal melanocortinergic system.

CONTEXT: Childhood adrenocortical tumors (ACTs) have a fetal adrenal phenotype and overexpress steroidogenic factor-1 (SF-1). Nephroblastoma overexpressed (NOV)/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3 mRNA is significantly down-regulated in childhood ACTs. OBJECTIVE: The objective of the study was to measure NOV protein levels in childhood ACTs and characterize NOV expression regulation and biological function in human adrenocortical cells. DESIGN AND SETTING: Protein extracts from ACT and normal adrenal cortex samples, human adrenocortical carcinoma H295R, primary adrenocortical tumors and fetal adrenal cultures, tissue culture supernatants, and cell lysates from H295R cells overexpressing SF-1 in an inducible fashion were used. MAIN OUTCOME MEASURES: NOV protein levels were measured by enzyme-linked immunoassay and immunoblot. Transient transfection assays were used to study the activity of NOV promoter. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, caspase assays, and flow cytometry were used to assess the proapoptotic activity of NOV on cells in culture. RESULTS: NOV mRNA and protein expression is lower in childhood ACTs than in normal adrenal cortex. No significant difference was observed between adenomas and carcinomas. SF-1 overexpression down-regulates NOV at the transcriptional level. NOV has a selective proapoptotic activity toward human adrenocortical cells. The C-terminal domain of NOV is responsible for its proapoptotic effect. NOV protein is expressed in DAX-1-positive human fetal adrenal cells. CONCLUSIONS: NOV is a selective proapoptotic factor for human adrenocortical cells. Reduced expression of NOV in ACTs may play an important role in the process of childhood ACT tumorigenesis, accounting at least in part for the defect of apoptotic regression of the fetal adrenal that has been proposed to be responsible for tumor formation.

Adrenocortical carcinomas (ACC) are rare malignant tumors of the adrenal cortex whose prognosis remains ominous. Considerable progress has been made during the last few years in the identification of the molecular mechanisms implicated in their pathogenesis. Overexpression of the transcription factor Steroidogenic Factor-1 (SF-1/NR5A1), a pivotal regulator of adrenocortical development and steroidogenic function, has a stage-independent negative prognostic value in patients with ACC.1 Studies in cell lines and animal models have shown that an increased SF-1 dosage is able to increase adrenocortical cell proliferation and triggers tumorigenesis.2 Those effects are, at least in part, explained by differential regulation by overexpressed SF-1 of a set of genes that are largely distinct from those ones that are regulated by the same transcription factor in conditions of basal expression.3 Understanding of the cellular and molecular function of those SF-1 dosage-dependent target genes is important to s...

Mitotane is a widely used drug in the therapy of adrenocortical carcinoma (ACC). It is important to set up preclinical protocols to study the possible synergistic effects of its association with new drugs for ACC therapy. We assessed the efficacy of different routes of administration of mitotane (i.p. and oral) in inhibiting growth of H295R ACC cell xenografts in an adjuvant setting. Both formulations of mitotane could inhibit H295R xenografts growth only at short times after carcinoma cells inoculation, even though plasma mitotane levels approached or fell within the therapeutic range in humans. Our results show that mitotane adjuvant therapy is inadequate to antagonize long-term growth of H295R cancer cells xenografts and that care should then be taken in the design of preclinical protocols to evaluate the performance of new drugs in association with mitotane.

Steroidogenic Factor-1 (SF-1) is a nuclear receptor that has a pivotal role in the development of adrenal glands and gonads and in the control of steroid hormone production, being also implicated in the pathogenesis of adrenocortical tumors. We have analyzed the mechanisms how SF-1 controls gene expression in adrenocortical cells and showed that it regulates different categories of genes according to its dosage. Significant correlations exist between the localization of SF-1-binding sites in chromatin under different dosage conditions and dosage-dependent regulation of gene expression. Our study revealed unexpected functional interactions between SF-1 and Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription Factor (NRSF/REST), which was first characterized as a repressor of neuronal gene expression in non-neuronal tissues, in the regulation of gene expression in steroidogenic cells. When overexpressed, SF-1 reshapes the repertoire of NRSF/REST—regulated genes, relieving repression of key steroidogenic genes. These data show that NRSF/REST has a novel function in regulating gene expression in steroidogenic cells and suggest that it may have a broad role in regulating tissue-specific gene expression programs.

International audience; Adrenocortical cancer is a rare malignancy for which current pharmacological therapies are still insufficient. We tested the effect of a novel PI3 kinase - mammalian target of rapamycin dual inhibitor (NVP-BEZ235) on proliferation of the H295R adrenocortical cancer cell line in vitro and grown as xenografts in immunodeficient mice. NVP-BEZ235 was able to significantly inhibit phosporylation of Akt kinase and S6 ribosomal protein in H295R cells and to significantly reduce their proliferation in vitro and xenograft growth in vivo. The drug also induced activation of Erk phosphorylation, which could be inhibited by simultaneous treatment with the Erk inhibitor FR180204. This latter drug synergized with NVP-BEZ235 in the inhibition of H295R proliferation in vitro. Our data suggest that dual PI3K/mTOR inhibitors may represent a useful pharmacological tool in the therapy of advanced adrenocortical cancer and that simultaneous inhibition of both Erk and PI3K - mTOR pathways may be required to obtain a higher antiproliferative effect in this type of tumor.

International audience; Adrenocortical tumors in children are usually diagnosed because of signs of virilization and their prognosis is poor. They possess several distinct pathological features compared to adrenocortical tumors in adults and have an exceptional prevalence in southern Brazil, where they are nearly invariably linked to the presence of a germline specific TP53 (R337H) mutation. Other important factors in childhood adrenocortical tumor pathogenesis are overexpression of the Steroidogenic Factor-1 transcription factor and imprinting defects in the 11p15 genomic region, causing overexpression of Insulin-like Growth Factor-2. Genomic studies have revealed the prognostic relevance of the expression of some Major Histocompatibility Complex genes and the deregulation of the Insulin-like Growth Factor/mammalian Target Of Rapamycin pathway by microRNAs in these tumors. Our hope is that these findings will constitute the basis for the development of novel therapies that will be more active against these tumors and less toxic for the patients.

MicroRNAs (miRNAs) act at the posttranscriptional level to control gene expression in virtually every biological process, including oncogenesis. Here, we report the identification of a set of miRNAs that are differentially regulated in childhood adrenocortical tumors (ACT), including miR-99a and miR-100. Functional analysis of these miRNAs in ACT cell lines showed that they coordinately regulate expression of the insulin-like growth factor–mammalian target of rapamycin (mTOR)–raptor signaling pathway through binding sites in their 3′-untranslated regions. In these cells, the active Ser2448-phosphorylated form of mTOR is present only in mitotic cells in association with the mitotic spindle and midbody in the G2-M phases of the cell cycle. Pharmacologic inhibition of mTOR signaling by everolimus greatly reduces tumor cell growth in vitro and in vivo. Our results reveal a novel mechanism of regulation of mTOR signaling by miRNAs, and they lay the groundwork for clinical evaluation of drugs inhibiting the mTOR pathway for treatment of adrenocortical cancer. Cancer Res; 70(11); 4666–75. ©2010 AACR.

International audience; The Li-Fraumeni syndrome (LFS) is a rare autosomal dominant hereditary cancer syndrome, characterized by a wide spectrum of neoplasms, occurring in children and young adults. The identification of germline TP53 mutations in LFS has given rise to a number of in vitro studies using cultures of cancer cells and non-tumoral fibroblasts presenting germline TP53 mutations. In the present study, we performed a detailed documentation of the pedigree of an LFS family with a comprehensive analysis of genotype-phenotype correlations. We sequenced the TP53 gene and verified that the proband carries a germline nonsense mutation in codon 146 in one allele, the TP53Arg72Pro polymorphism in the second, and other intronic polymorphisms in the TP53 gene. In order to investigate the disruption of the p53 function in a patient presenting this mutation and the TP53Arg72Pro polymorphism who had so far suffered five malignant tumors and a benign meningioma, we tested her fibroblasts in response to DNA damage by evaluating the proliferation rate, apoptosis, and disruption of the TP53 pathway. The proband's heterozygous fibroblasts were not as efficient as control fibroblasts or those of her mother, who carried only the TP53Arg72Pro polymorphism, in causing cell arrest and cell death after DNA damage, which was correlated with diminished TP21 protein levels.

The transcription factor Steroidogenic Factor-1 (Ad4BP/SF-1; NR5A1 according to the standard nomenclature) has an essential role in adrenogonadal development. Furthermore, SF-1 is amplified and overexpressed in most cases of adrenocortical tumor occurring in children; studies performed in transgenic mice have shown that an increased SF-1 dosage triggers tumor formation in the adrenal cortex. For these reasons, drugs interfering with SF-1 action would represent a promising tool to be added to the current pharmacological protocols in the therapy of adrenocortical cancer. Here, we describe the methods how isoquinolinone compounds inhibiting the constitutive transcriptional activity of SF-1 (SF-1 inverse agonists) were identified and characterized. These compounds have the attributes to inhibit the increase in proliferation triggered by an augmented SF-1 dosage in adrenocortical tumor cells and to reduce their steroid production. This latter property may also reveal beneficial for drugs used in the therapy of adrenocortical tumors to alleviate symptoms of virilization and Cushing often associated with tumor burden.

International audience; The transcription factor Steroidogenic Factor-1 (Ad4BP/SF-1; NR5A1 according to the standard nomenclature) has an essential role in adrenogonadal development. Furthermore, SF-1 is amplified and overexpressed in most cases of adrenocortical tumor occurring in children; studies performed in transgenic mice have shown that an increased SF-1 dosage triggers tumor formation in the adrenal cortex. For these reasons, drugs interfering with SF-1 action would represent a promising tool to be added to the current pharmacological protocols in the therapy of adrenocortical cancer. Here, we describe the methods how isoquinolinone compounds inhibiting the constitutive transcriptional activity of SF-1 (SF-1 inverse agonists) were identified and characterized. These compounds have the attributes to inhibit the increase in proliferation triggered by an augmented SF-1 dosage in adrenocortical tumor cells and to reduce their steroid production. This latter property may also reveal beneficial for drugs used in the therapy of adrenocortical tumors to alleviate symptoms of virilization and Cushing often associated with tumor burden.

International audience

CONTEXT: Mutations of the beta-catenin (CTNNB1) gene are frequently found in adrenocortical tumors. This has important consequences to deregulate the expression of transcriptional targets of the Wnt pathway, which may contribute to tumorigenesis. OBJECTIVE: The objective of the study was to investigate the effect of the small-molecule inhibitor of the T cell factor (Tcf)/beta-catenin complex PKF115-584 on beta-catenin-dependent transcription and proliferation of H295R adrenocortical tumor cells, which harbor mutations in CTNNB1 as well as the TP53 tumor suppressor gene. MAIN OUTCOME MEASURES: Immunofluorescence, transient transfection, proliferation assays, and flow cytometric analyses were used. RESULTS: Nuclear localization of beta-catenin and constitutive activation of beta-catenin-dependent transcription was observed in H295R cells. PKF115-584 dose-dependently inhibited beta-catenin-dependent transcription and H295R proliferation, even in the presence of increased steroidogenic factor-1 levels, which augment proliferation in this cell line. The drug had no effect on HeLa cells, a cell line in which the beta-catenin pathway is not activated. PKF115-584 decreased the percentage of H295R cells in S-phase and increased the percentage of apoptotic cells. CONCLUSIONS: Inhibitors of the Tcf/beta-catenin complex may prove useful in the treatment of adrenocortical tumors in which multiple genetic alterations have accumulated.

A germline TP53 R337H mutation is present in childhood adrenocortical tumors (ACT) from southern Brazil. Other genetic alterations are also frequently found in these tumors. This study was designed to assess whether alterations of the 11p15 region exist in childhood ACT, accounting for IGF2 overexpression in these tumors, and how they are related to clinical outcome. Tumor DNA of 12 children with ACT (4 adenomas and 8 carcinomas) and from the blood of their parents was analyzed. All patients showed 11p15 loss of heterozygosity (LOH) in the tumor. In contrast to the single case of paternal LOH, IGF2 was overexpressed in tumors with maternal allele loss. Our data show that 11p15 LOH is a widespread finding in childhood ACT not related with malignancy, contrary to adult ACT. Alterations in the expression of other genes in the same region (e.g., CDKN1C) may contribute to ACT tumorigenesis. ©2008 Elsevier Inc. All rights reserved. © 2008 Elsevier Inc. All rights reserved.

Steroidogenic factor-1 (SF-1/Ad4BP; NR5A1), a nuclear receptor transcription factor, has a pivotal role in adrenal and gonadal development in humans and mice. A frequent feature of childhood adrenocortical tumors is SF-1 amplification and overexpression. Here we show that an increased SF-1 dosage can by itself augment human adrenocortical cell proliferation through concerted actions on the cell cycle and apoptosis. This effect is dependent on an intact SF-1 transcriptional activity. Gene expression profiling showed that an increased SF-1 dosage regulates transcripts involved in steroid metabolism, the cell cycle, apoptosis, and cell adhesion to the extracellular matrix. Consistent with these results, increased SF-1 levels selectively modulate the steroid secretion profile of adrenocortical cells, reducing cortisol and aldosterone production and maintaining dehydroepiandrosterone sulfate secretion. As a model to understand the mechanisms of transcriptional regulation by increased SF-1 dosage, we studied FATE1, coding for a cancer-testis antigen implicated in the control of cell proliferation. Increased SF-1 levels increase its binding to a consensus site in FATE1 promoter and stimulate its activity through modulation of the recruitment of specific cofactors. On the other hand, sphingosine, which can compete with phospholipids for binding to SF-1, had no effect on the SF-1 dosage-dependent increase of adrenocortical cell proliferation and expression of the FATE1 promoter. In mice, increased Sf-1 dosage produces adrenocortical hyperplasia and formation of tumors expressing gonadal markers (Amh, Gata-4), which originate from the subcapsular region of the adrenal cortex. Gene expression profiling revealed that genes involved in cell adhesion and the immune response and transcription factor signal transducer and activator of transcription-3 (Stat3) are differentially expressed in Sf-1 transgenic mouse adrenals compared with wild-type adrenals. Our studies reveal a critical role for SF-1 dosage in adrenocortical tumorigenesis and constitute a rationale for the development of drugs targeting SF-1 transcriptional activity for adrenocortical tumor therapy.

The steroidogenic factor 1 (SF-1) gene encodes a transcription factor playing a pivotal role in the regulation of adrenogenital development. We have recently shown that SF-1 is amplified in childhood adrenocortical tumours (ACT). This study was aimed to assess if an increase in SF-1 gene copy number was associated with increased protein levels and to study the correlation between SF-1 expression and ACT clinical parameters. An increased SF-1 copy number was detected in eight of the 10 ACT cases studied. Conversely, the SF-1 protein was found to be overexpressed in all cases, compared to normal age-matched adrenal glands. No significant correlation was found between SF-1 protein levels and its gene copy number. Furthermore, no significant correlation existed with histological grade or with the clinical manifestation or evolution of disease. This data show that SF-1 overexpression is widespread in childhood ACT and is likely to play a role in its pathogenesis.

The adrenal gland is the second tissue after hypothalamus exhibiting high expression level of Agouti Related Protein (Agrp) mRNA, which suggests that this peptide may control adrenal cell functions. However, its role in this tissue remained to be determined. In this report, we studied the effects of a long-term treatment (24 h) of cultured bovine adrenal cells by Agrp on the (Nle4, d -Phe7)-αMSH (NDP-αMSH)- or ACTH-induced cortisol release. We showed that Agrp inhibited, in a dose-dependent manner, the 10 −9 M NDP-αMSH-induced cortisol production through its antagonistic properties towards MSH at the level of MC4-R. Surprisingly, we found that Agrp in the same conditions of cell treatment also induced a strong inhibition of the ACTH-induced cortisol release. These effects were stronger using low concentrations of Agrp and disappeared for higher concentrations resulting in U-shaped curve data. There was no effect of SHU9119 in the same conditions of stimulation of the cells. Our data confirmed that Agrp is not an antagonist of ACTH at the level of MC2-R and that its sustained effect on ACTH-induced steroidogenesis did not involve its antagonistic properties at the level of MC4-R. The hypothesis would be that Agrp is acting on adrenal steroidogenesis through an alternate mechanism.

Agouti-related protein (Agrp), primarily expressed in the hypothalamus, is an endogenous antagonist of αMSH at the level of melanocortin 3 receptor (MC3-R) and MC4-R, but the adrenal gland represents the second major Agrp-expressing tissue. In adrenal fasciculata cells, the glucocorticoid secretion is under the control of ACTH, which binds specifically MC2-R, the only functional melanocortin receptor described in these cells to date. Nevertheless, using cultured bovine fasciculata adrenal cells, we report that Agrp has no antagonistic properties against ACTH at the level of MC2-R. In our studies, (Nle4, d-Phe7)-αMSH (NDP-αMSH) stimulated the production of cortisol in a dose-dependent manner, and these effects were abolished by Agrp or SHU9119, a synthetic antagonist of MC3-R and MC4-R. Using a more specific antagonist (JKC-363) and RT-PCR analysis, we can postulate that the effects of NDP-αMSH were mediated via MC4-R. These results are suggestive that adrenal glucocorticoid production could be regulated through MC4-R that may have some relevance in the physiology of adrenal cells. Moreover, Agrp might exert an autocrine control on adrenal cells because a protein with biological Agrp-like activity is secreted by these cells. This peptide could then modulate locally the functions of some peripheral tissues such as adrenals.