Mabrouka Doghman

Inhibition of adrenocortical carcinoma cell proliferation by steroidogenic factor-1 inverse agonists.

CONTEXT Transcription factor steroidogenic factor-1 (SF-1) plays a pivotal role in the control of adrenocortical cell steroidogenesis and proliferation. SF-1 amplification and overexpression are found in most cases of childhood adrenocortical tumors (ACTs). OBJECTIVE Our objective was to investigate the effect of SF-1 inverse agonists of the alkyloxyphenol and isoquinolinone classes on the proliferation of human adrenocortical cell lines expressing SF-1 (H295R), in conditions of basal and increased SF-1 expression, or negative for SF-1 expression (SW-13). MAIN OUTCOME MEASURES Proliferation assays, immunoblots, flow cytometric analyses, steroid hormone assays, and reverse transcription quantitative PCR were used. RESULTS SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and -negative ACT cells, whereas SF-1 inverse agonists of the isoquinolinone class selectively inhibited cell proliferation elicited by SF-1 overexpression. These drugs also inhibited stimulated steroid hormone secretion and CYP21 and CYP17 mRNA expression. CONCLUSION SF-1 inhibitors may represent a useful tool in the chemotherapy of ACTs.

The T cell factor/β-Catenin Antagonist PKF115–584 Inhibits Proliferation of Adrenocortical Carcinoma Cells

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.

SNP array profiling of childhood adrenocortical tumors reveals distinct pathways of tumorigenesis and highlights candidate driver genes

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.

Efficacy of the novel dual PI3-kinase/mTOR inhibitor NVP-BEZ235 in a preclinical model of adrenocortical carcinoma

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.

Integrative analysis of SF-1 transcription factor dosage impact on genome-wide binding and gene expression regulation

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.

Beyond steroidogenesis: Novel target genes for SF-1 discovered by genomics

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.

High frequency of loss of heterozygosity at 11p15 and IGF2 overexpression are not related to clinical outcome in childhood adrenocortical tumors positive for the R337H TP53 mutation

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.

Genetics and genomics of childhood adrenocortical tumors

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.

A matter of dosage: SF-1 in adrenocortical development and cancer

Steroidogenic factor-1 (SF-1), a nuclear receptor transcripion factor, has a pivotal role in adrenal and gonadal development n humans and mice. It was previously shown that Sf-1 dosage ritically regulates adrenal size and function in mice, while in umans mutation of a single SF-1 allele leads to adrenal and/or onadal dysgenesis. Recent studies have elucidated the role of n increased SF-1 dosage as an important pathogenetic factor n childhood adrenocortical cancer and in experimental adrenoortical cancer in mice and identified genes that are regulated y SF-1 in a dosage-dependent fashion. Drugs targeting SF-1 ranscriptional activity may then represent potential therapeutic ools to be associated to current chemotherapeutic regimens for he treatment of adrenocortical cancer in children. The adrenal cortex is an essential component of the ypothalamic-pituitary-adrenal axis, synthesizing essential teroid hormones (mineralocorticoids, glucocorticoids and ndrogens) and regulating diverse biological processes, such as tress response, blood pressure, glucose metabolism and sexual evelopment. The structure of the fetal adrenal cortex is unique in human nd primates compared to other species. In fact, during intrauterne life, most of the inner part of the adrenal cortex (fetal zone) s occupied by large, steroid-secreting cells, which synthesize arge amounts of dehydroepiandrosterone and its sulfate derivaive (reviewed in [1]). Conversely, a few layers of cells lying in n outer, subcapsular position (definitive zone) are nonsteroidoenic and constitute a proliferative compartment in which most robably the precursors of fetal zone cells are generated. An dditional zone (transitional zone) develops between the definiive and the fetal zone late in pregnancy and synthesizes cortisol. teroid hormones produced by the fetal zone regulate intrauterne homeostasis, the maturation of the fetal organs necessary for

Role of Agouti-related protein in adrenal steroidogenesis.

The levels of Agouti-related protein (AgRP) mRNA in the adrenal are second only to those in the hypothalamus, raising questions regarding its target binding sites and its specific role in adrenal steroidogenesis. We and others demonstrated the presence of a population of melanocortin receptor-4 (MC4R) positively coupled to steroidogenesis in adrenal cells. Moreover, AgRP inhibited both the acute and long-term steroidogenic responses of these cells to NDP-alphaMSH through its antagonistic properties towards MC4R. Although AgRP had no antagonistic properties towards the MC2R and did not modify the acute steroidogenic response to ACTH, it exerted a biphasic sustained inhibitory effect on the long-term response to ACTH through an undefined alternate mechanism. Since adrenal cells release a relatively large amount of AgRP, this protein likely exerts a local paracrine/autocrine control on adrenal steroidogenesis.