Adrenocortical Tumor Cell Line Research Articles

New insights for screening etomidate analogues in the human H295R cell model

Etomidate is a sedative-hypnotic with excellent pharmacological effects, including rapid onset and hemodynamic stability. However, etomidate causes adrenocortical toxicity via binding to 11β-hydroxylase. Therefore, developing an approach to screen new etomidate analogues without endocrine-disrupting effects is urgently warranted. In this study, we employed the adrenocortical tumour cell line, NCI-H295R, as an in vitro system for etomidate analogues screening and detected the hormone levels in these cells using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. After obtaining the concentration-response curves of hormone release, the “Adrenocortical Inhibitory Index” was used to evaluate the adrenocortical inhibitory potency of each compound. In summary, we demonstrate the benefits of our methods for screening of etomidate analogues that lack adrenocortical suppression, especially when this in vitro system is combined with in vivo testing.

MON-185 ACTH-Derived Peptide Antagonists as an Alternative Treatment Strategy for Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by different enzyme deficiencies in the steroid hormone synthesis leading to a disturbed cortisol biosynthesis. The medical treatment of CAH includes suboptimal ACTH-suppressing high glucocorticoid doses to reduce adrenal hyperplasia and overproduction of androgens. These inappropriate corticoid substitutions are often associated with undesirable side effects such as arterial hypertension, growth failure and obesity. Since the current therapy of patients with CAH is often unsatisfactory, innovative treatment options are required.The aim of our study was to specifically block the melanocortin 2 receptor (MC2R) signaling pathway as an alternative treatment strategy for CAH. We tested ACTH-derived selective peptides with incorporation of various synthetic non-natural amino acids in the activation motif of ACTH. To study the antagonistic activity of the peptides, cAMP production of MC2R/MRAP stably transfected human embryonic kidney (HEK) 293 cells were measured. All new synthesized peptide antagonists reduced ACTH-stimulated MC2R activity as competitive inhibitors indicated by a reduced in vitro cAMP response. Cells pre-incubated with peptide LNP009 showed the most efficient blockade of the MC2R and the highest shift of EC50 of ACTH (33.8 nM ± 0.08 vs. 7.3 nM ± 0.09). LNP009 was additionally tested for specificity concerning the other known melanocortin receptors and showed no antagonistic effect up to 1 μM on MC3, MC4 or MC5 receptor transiently transfected HEK 293 cells. To further investigate the inhibitory effect of our most potent antagonist peptide LNP009 on the steroid hormone response, we assessed steroidogenic enzyme expression of the human adrenocortical tumor cell line NCI-H295RA and performed mass spectrometry analyzes of steroids in the cell culture supernatant. Pre-incubation with LNP009 reduced the expression of the genes CYP21A2, CYP11B1 and HSD3B2 in NCI-H295RA cells and significantly reduced the synthesis of aldosterone (P=0.046; n=3), cortisol (P=0.020; n=3) and corticosterone (P=0.035; n=3).With the successful blocking of the ACTH binding and signal transduction by our antagonistic peptides, we anticipate an alternative approach for optimizing the treatment of CAH patients lacking the side effects of the currently used ACTH-suppressing corticoid therapy.

Biological Effects of EF24, a Curcumin Derivative, Alone or Combined with Mitotane in Adrenocortical Tumor Cell Lines.

Background: Curcumin has numerous properties and is used in many preclinical conditions, including cancer. It has low bioavailability, while its derivative EF24 shows enhanced solubility. However, its effects have never been explored in adrenocortical tumor cell models. The efficacy of EF24 alone or combined with mitotane (reference drug for adrenocortical cancer) was evaluated in two adrenocortical tumor cell lines, SW13 and H295R. Method and Results: EF24 reduced cell viability with an IC50 (half maximal inhibitory concentration) of 6.5 ± 2.4 μM and 4.9 ± 2.8 μM for SW13 and H295R cells, respectively. Combination index (EF24 associated with mitotane) suggested an additivity effect in both cell lines. Cell cycle analysis revealed an increase in subG0/G1 phase, while motility assay showed a decrease in migratory cell capacity, and similarly, clonogenic assay indicated that EF24 could reduce colony numbers. Furthermore, Wnt/β-catenin, NF-κB, MAPK, and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with EF24 alone or combined with mitotane. In addition, intracellular reactive oxygen species levels increased in both cell lines. Conclusion: This work analyzed EF24 in adrenocortical tumor cell lines for the first time. These results suggest that EF24 could potentially impact on adrenocortical tumors, laying the foundation for further research in animal models.

Identification of a novel putative interaction partner of the nucleoporin ALADIN

ABSTRACTIt has been shown that the nucleoporin ALADIN plays a significant role in the redox homeostasis of the cell, but its function in steroidogenesis contributing to adrenal atrophy in triple A syndrome remains largely unknown. In an attempt to identify new interaction partners of ALADIN, co-immunoprecipitation followed by proteome analysis was conducted in different expression models using the human adrenocortical tumour cell line NCI-H295R. Our results suggest an interaction of ALADIN with the microsomal protein PGRMC2. PGRMC2 is shown to be activity regulator of CYP P450 enzymes and, therefore, to be a possible target for adrenal dysregulation in triple A syndrome. We show that there is a sexual dimorphism regarding the expression of Pgrmc2 in adrenals and gonads of wild-type (WT) and Aaas knock-out (KO) mice. Female Aaas KO mice are sterile due to delayed oocyte maturation and meiotic spindle assembly. A participation in meiotic spindle assembly confirms the recently investigated involvement of ALADIN in mitosis and emphasises an interaction with PGRMC2 which is a regulator of the cell cycle. By identification of a novel interaction partner of ALADIN, we provide novel aspects for future research of the function of ALADIN during cell cycle and for new insights into the pathogenesis of triple A syndrome.

Effects of Type 1 Insulin-Like Growth Factor Receptor Silencing in a Human Adrenocortical Cell Line.

Type 1 insulin-like growth factor receptor (IGF-1R) is overexpressed in a variety of human cancers, including adrenocortical tumors. The aim of the work was to investigate the effects of IGF-1R downregulation in a human adrenocortical cell line by small interfering RNA (siRNA). The human adrenocortical tumor cell line NCI H295R was transfected with 2 specific IGF1R siRNAs (# 1 and # 2) and compared with untreated cells and a negative control siRNA. IGF1R expression was determined by quantitative reverse-transcription PCR (qRTPCR) and Western blot. The effects of IGF-1R downregulation on cell proliferation and apoptosis were assessed. IGF-1R levels were significantly decreased in cells treated with IGF-1R siRNA # 1 or # 2. Relative expression of IGF1R mRNA decreased approximately 50% and Western blot analysis revealed a 30% of reduction in IGF-1R protein. Downregulation of this gene resulted in 40% reduction in cell growth in vitro and 45% increase in apoptosis using siRNA # 2. These findings demonstrate that decreasing IGF-1R mRNA and protein expression in NCI H295R cells can partially inhibit adrenal tumor cell growth in vitro. Targeting IGF1R is a promising therapy for pediatric malignant adrenocortical tumor and can still be an option for adult adrenocortical cancer based on personalized genomic tumor profiling.

PK11195 effect on steroidogenesis is not mediated through the translocator protein (TSPO).

Translocator protein (TSPO) is a mitochondrial outer membrane protein of unknown function with high physiological expression in steroidogenic cells. Using TSPO gene-deleted mice, we recently demonstrated that TSPO function is not essential for steroidogenesis. The first link between TSPO and steroidogenesis was established in studies showing modest increases in progesterone production by adrenocortical and Leydig tumor cell lines after treatment with PK11195. To reconcile discrepancies between physiological and pharmacological interpretations of TSPO function, we generated TSPO-knockout MA-10 mouse Leydig tumor cells (MA-10:TspoΔ/Δ) and examined their steroidogenic potential after exposure to either dibutyryl-cAMP or PK11195. Progesterone production in MA-10:TspoΔ/Δ after dibutyryl-cAMP was not different from control MA-10:Tspo+/+ cells, confirming that TSPO function is not essential for steroidogenesis. Interestingly, when treated with increasing concentrations of PK11195, both control MA-10:Tspo+/+ cells and MA-10:TspoΔ/Δ cells responded in a similar dose-dependent manner showing increases in progesterone production. These results show that the pharmacological effect of PK11195 on steroidogenesis is not mediated through TSPO.

Effects of everolimus, nilotinib, imatinib and ZOL in combination with mitotane in monolayer and spheroids human adrenocortical tumor cell line

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Sonic Hedgehog Signaling Is Active in Human Adrenal Cortex Development and Deregulated in Adrenocortical Tumors

The sonic hedgehog (SHH) pathway plays a key role in rodent adrenal cortex development and is involved in tumorigenesis in several human tissues, but data in human adrenal glands are limited. The objectives of the study were to analyze the involvement of the SHH pathway in human adrenal development and tumorigenesis and the effects of SHH inhibition on an adrenocortical tumor (ACT) cell line. Expression of SHH pathway components was evaluated by immunohistochemistry in 51 normal adrenals (33 fetal) and 34 ACTs (23 pediatric) and by quantitative PCR in 81 ACTs (61 pediatric) and 19 controls (10 pediatric). The effects of SHH pathway inhibition on gene expression and cell viability in the NCI-H295A adrenocortical tumor cell line after cyclopamine treatment were analyzed. SHH pathway proteins were present in fetal and postnatal normal adrenals and showed distinct patterns of spatiotemporal expression throughout development. Adult adrenocortical carcinomas presented with higher expression of PTCH1, SMO, GLI3, and SUFU compared with normal adult adrenal cortices. Conversely, pediatric ACTs showed lower mRNA expression of SHH, PTCH1, SMO, GLI1, and GLI3 compared with normal pediatric adrenal cortices. In vitro treatment with cyclopamine resulted in decreased GLI3, SFRP1, and CTNNB1 mRNA expression and β-catenin staining as well as decreased cell viability. The SHH pathway is active in human fetal and postnatal adrenals, up-regulated in adult adrenocortical carcinomas, and down-regulated in pediatric ACTs. SHH pathway antagonism impaired cell viability. The SHH pathway is deregulated in ACTs and might provide a new target therapy to be explored.

Abstract DD01-03: TAK-700, an inhibitor of 17,20-lyase

Abstract Background: Recent studies suggest that residual adrenal androgen after castration could be responsible for the progression of castration-resistant prostate cancer (CRPC). To develop an effective therapy for CRPC that inhibits the production of adrenal and testicular androgens, we searched for a novel, non-steroidal, selective, and potent inhibitor of 17,20-lyase, a key enzyme in androgen synthesis, and identified TAK-700 as a clinical candidate. Methods: Enzyme assay was conducted using radiolabeled substrates. The enzyme reaction products were separated by thin layer chromatography and the radioactivity was measured by a bio-image analyzer. We assessed the activity and specificity of TAK-700 on androgen production in vitro in monkey adrenal cells and human adrenocortical tumor cell line, using a radioimmunoassay. Using a single dose of [14C]TAK-700 1 mg/kg, we investigated the pharmacokinetics and bioavailability of TAK-700 in cynomolgus monkeys, and assessed the effects of TAK-700 7.5 and 15 mg/kg twice daily (BID) for 7 days on serum androgen levels in castrated and intact cynomolgus monkeys. Results: TAK-700 potently inhibited the activities of human and monkey 17, 20-lyase (CYP17A1) with IC50 values of 140 and 27nmol/L, respectively. The inhibitory activity of TAK-700 on 17-hydroxylase was lower than that on 17, 20-lyase in all the enzyme preparations. IC50 values of ketoconazole and abiraterone for human 17, 20-lyase were 110 and 27nmol/L, respectively. TAK-700 did not inhibit monkey 11-hydroxylase up to 10μmol/L. In addition, IC50 values of TAK-700 for all CYP-specific activities (drug-metabolizing enzymes) examined were higher than 10 μmol/L. TAK-700 suppressed the production of DHEA, androstenedione, cortisol, corticosterone and aldosterone by monkey adrenal cells with IC50 values of 110, 130, 310, >100,000 and 4,400nmol/L, respectively. The IC50 value for cortisol production was only 3-fold higher than that for DHEA production in monkeys, reflecting low specificity of inhibition between monkey 17, 20-lyase and 17-hydroxylase activity (IC50 values: 27 vs 38nmol/L). Ketoconazole suppressed the production of DHEA, androstenedione, cortisol, corticosterone and aldosterone by monkey adrenal cells with IC50 values of 340, 580, 340, 1,500 and 350nmol/L, respectively. TAK-700 inhibited the production of dehydroepiandrosterone (DHEA) and cortisol in human adrenocortical tumor line NCI-H295R cells with IC50 values of 37 and 990nmol/L, respectively, showing about 27-fold selectivity for DHEA production. Ketoconazole inhibited the production of DHEA and cortisol with IC50 values of 330 and 480nmol/L (selectivity 1.5-fold), respectively, and abiraterone inhibited the production of DHEA and cortisol with IC50 values of 2.7 and 23nmol/L (selectivity: 8.5-fold), respectively. TAK-700 showed the highest specificity of inhibition for DHEA production among these 3 compounds. After oral administration of [14C]TAK-700 1 mg/kg, maximum plasma concentration (Cmax) was 0.147 μg/mL, time to Cmax (Tmax) was 1.7 hours, elimination half-life (t1/2) was 3.8 hours, and exposure (area under the plasma concentration-time curve in 24 hours, AUC0-24h) was 0.727 μg·h/mL. Oral bioavailability of TAK-700 in monkeys was 70.5%. Oral dosing of TAK-700 7.5 and 15 mg/kg BID rapidly (within 1 day) suppressed serum levels of the androgens dehydroepiandrosterone (DHEA) and testosterone and of cortisol in both castrated and gonadally intact monkeys. While suppression of DHEA and cortisol levels persisted during the 7-day treatment period in castrated monkeys, suppression of testosterone was attenuated from the second day of treatment, possibly due to activation of the hypothalamus-pituitary-gonad axis. TAK-700 had a milder suppressive effect on cortisol levels than on DHEA levels. During the treatment period, mean DHEA levels decreased to 9.0% and 6.9% of baseline levels in castrated monkeys treated with TAK-700 7.5 and 15 mg/kg, respectively. The mean cortisol levels decreased to 26.0% and 17.3% of baseline levels with TAK-700 7.5 and 15 mg/kg, respectively. In vitro results have shown that the specificity of TAK-700 suppression of DHEA production over cortisol production is 10-fold greater in human adrenal-origin cells than in monkey adrenal cells. Therefore, the impact of TAK-700 on human serum cortisol levels may be smaller than that in monkeys. Conclusion: As TAK-700 potently and selectively inhibited 17, 20-lyase activity and DHEA production by H295R cells and suppressed serum androgen levels in castrated monkeys, TAK-700 may be effective for at least some types of CRPC, and clinical evaluation of TAK-700 is now underway in patients with CRPC. Citation Format: Masuo Yamaoka, Takahito Hara, Hiroshi Miki, Takenori Hitaka, Tomohiro Kaku, Naohiro Kawaguchi, Hitomi Yamasaki, Toshiyuki Takeuchi, Akihiro Tasaka, Masami Kusaka. TAK-700, an inhibitor of 17,20-lyase [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr DD01-03

Bone Morphogenetic Proteins 2 and 5 Are Down-regulated in Adrenocortical Carcinoma and Modulate Adrenal Cell Proliferation and Steroidogenesis

Bone morphogenetic proteins (BMP) have been shown to affect tumorigenesis in a variety of tumors. Quantitative PCR analysis revealed down-regulation of BMP2 and BMP5 in tissue samples from adrenocortical carcinoma and adrenocortical tumor cell lines compared with normal adrenal glands. Integrity of BMP-dependent pathways in these cell lines could be shown by activation of the Smad1/5/8 pathway with subsequent increase of ID protein expression upon incubation with BMP2 or BMP5. On a functional level, BMP treatment resulted in inhibition of cell proliferation and viability in a dose- and time-dependent manner. This growth inhibitory effect was associated with BMP-dependent reduction of AKT phosphorylation under baseline conditions and under insulin-like growth factor costimulation. Furthermore, steroidogenic function, including melanocortin-2 receptor and steroidogenic enzyme expressions, was profoundly reduced. In vitro demethylation treatment and overexpression of GATA6 resulted in reactivation of BMP-dependent pathways with concomitant modulation of steroidogenesis. Taken together, we show that loss of expression of members of the BMP family of ligands is a common finding in adrenocortical tumors and we provide evidence that BMP-dependent pathways are likely to be involved in the modulation of the malignant and functional phenotype of adrenocortical cancer cells.

ACTH Induces ERK 1/2 Activation in Rat Adrenal Primary Cultures

Adrenocorticotropic hormone (ACTH) is the most potent stimulator of adrenal cortex, acting through the Melanocortin-2 receptor (MC2R). ACTH induces secretion of steroid hormones, critical for the normal stress response and plays also an important role on cell proliferation and differentiation. MC2R is a classical G-Protein coupled receptor (GPCR), thus activating Protein Kinase A (PKA). However, many studies suggested a cross-talk between different signalling pathways and a more complex intracellular network. In fact, in adrenocortical Y1 tumour cell line, ACTH may activate Extracellular Regulated Kinases 1/2 (ERK 1/2), which belong to the Mitogen-Activated Protein Kinases (MAPKs) family. In addition, this pathway was implicated in in vivo proliferation and steroidogenesis as shown by our group. In order to further explore and clarify ACTH signalling mechanisms, we made the present work to establish a model of primary cultures of rat adrenal cells.

Expression of Insulin-Like Growth Factor-II and Its Receptor in Pediatric and Adult Adrenocortical Tumors

Adrenocortical tumors are heterogeneous neoplasms with incompletely understood pathogenesis. IGF-II overexpression has been consistently demonstrated in adult adrenocortical carcinomas. The objective of the study was to analyze expression of IGF-II and its receptor (IGF-IR) in pediatric and adult adrenocortical tumors and the effects of a selective IGF-IR kinase inhibitor (NVP-AEW541) on adrenocortical tumor cells. Fifty-seven adrenocortical tumors (37 adenomas and 20 carcinomas) from 23 children and 34 adults were studied. Gene expression was determined by quantitative real-time PCR. Cell proliferation and apoptosis were analyzed in NCI H295 cells and a new cell line established from a pediatric adrenocortical adenoma. IGF-II transcripts were overexpressed in both pediatric adrenocortical carcinomas and adenomas. Otherwise, IGF-II was mainly overexpressed in adult adrenocortical carcinomas (270.5 +/- 130.2 vs. 16.1 +/- 13.3; P = 0.0001). IGF-IR expression was significantly higher in pediatric adrenocortical carcinomas than adenomas (9.1 +/- 3.1 vs. 2.6 +/- 0.3; P = 0.0001), whereas its expression was similar in adult adrenocortical carcinomas and adenomas. IGF-IR expression was a predictor of metastases in pediatric adrenocortical tumors in univariate analysis (hazard ratio 1.84; 95% confidence interval 1.28-2.66; P = 0.01). Furthermore, NVP-AEW541 blocked cell proliferation in a dose- and time-dependent manner in both cell lines through a significant increase of apoptosis. IGF-IR overexpression was a biomarker of pediatric adrenocortical carcinomas. Additionally, a selective IGF-IR kinase inhibitor had antitumor effects in adult and pediatric adrenocortical tumor cell lines, suggesting that IGF-IR inhibitors represent a promising therapy for human adrenocortical carcinoma.

Leukemia inhibitory factor protein and receptors are expressed in the bovine adrenal cortex and increase cortisol and decrease adrenal androgen release

The release of adrenal steroids during acute stress is primarily regulated by adrenocorticotropic hormone (ACTH). In contrast, during chronic inflammatory stress additional factors are involved in regulating adrenal function. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that increases ACTH release from the pituitary. In addition, LIF and LIF receptors (LIFR) are expressed in the human adrenal cortex and the human adrenocortical tumor cell line H295R. Furthermore, LIF increases basal and ACTH-stimulated cortisol release from H295R cells. However, the expression of LIF and LIFR in non-human adrenal glands and the effects of LIF on the release of cortisol from adrenal cells of non-human species have not been determined. Furthermore, the effects of LIF on adrenal androgen release from all species are unknown. In this study, immunohistochemistry, Western blots, RT-PCR, and nucleotide sequencing was utilized to demonstrate that LIF and its receptor are expressed throughout the bovine adrenal cortex. Although LIF did not modify basal cortisol release from dispersed cells isolated from the bovine adrenal zona fasciculate, this cytokine increased ACTH-stimulated release of cortisol from these cells in a manner dependent on the LIF concentration and exposure interval. In contrast, LIF in a concentration-dependent and time-dependent manner decreased basal and ACTH-stimulated adrenal androgen release from dispersed cells isolated from the bovine adrenal zona reticularis. Because LIF release increases during inflammatory stress and this cytokine stimulates adrenal cortisol release and inhibits adrenal androgen release, this cytokine may play an important role in regulating the release of adrenal steroids during inflammatory stress.

Side Population Does Not Define Stem Cell-Like Cancer Cells in the Adrenocortical Carcinoma Cell Line NCI h295R

Recent evidence suggests the existence of a stem cell-like subpopulation of cells in hematological and solid tumor entities, which determine the malignant phenotype of a given tumor through their proliferative potential and chemotherapy resistance. A recently used technique for the isolation of this cell population is through exclusion of the vital dye Hoechst 33342, which defines the so-called side population (SP). Herein we demonstrate the presence of SP cells in a variety of adrenal specimens, including primary cultures of human adrenocortical tumors and normal adrenal glands as well as established human and murine adrenocortical cancer cell lines by fluorescence-activated cell sorter analysis and confocal microscopy. On a functional level, SP cells from the human adrenocortical tumor cell line NCI h295R revealed an expression pattern consistent with a less differentiated phenotype, including lower expression of steroidogenic enzymes such as steroid acute regulatory protein (StAR) and side-chain cleavage enzyme (P450scc) in comparison with non-SP cells. However, proliferation between SP and non-SP cells did not differ (105.6 +/- 18.1 vs. 100.0 +/- 3.5%). Furthermore, re-sorting and tracing experiments revealed the capacity for both cell types to give rise to the original SP- and non-SP-containing cell population. Similarly to the baseline growth kinetics, no survival benefit was evident in SP cells after treatment with cytotoxic agents commonly used in adrenocortical carcinomas. Taken together, these findings provide evidence that Hoechst dye exclusion, in contrast to what has been reported for other tumor entities, is not a major tumor stem cell defining marker in adrenocortical NCI h295R tumor cells.

Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells

Angiotensin-II (Ang-II) regulates adrenal steroid production and gene transcription through several signaling pathways. Changes in gene transcription occur within minutes after Ang-II stimulation, causing an increase in aldosterone production and subsequent increase in the overall capacity to produce aldosterone. Our goal was to compare the Ang-II regulation of early gene expression and confirm the up-regulation of selected genes using quantitative real-time RT-PCR (qPCR) across three species, such as, human, bovine, and rat. Microarray analyses were performed using samples from control and Ang-II (10 nM)-treated (1 h) cells from human adrenocortical tumor cell line H295R, and primary adrenal glomerulosa cells from bovine and rat, applied respectively to human, bovine, and rat chips. qPCR was performed to confirm up-regulation of selected genes using mRNA. The microarray comparison revealed 18% similarity among the top 50 up-regulated genes, with human/rat, 20%; human/bovine, 36%; and rat/bovine, 26% similarity. The gene list generated by this comparison included: activating transcription factor 3, B-cell translocation gene (BTG2), Nuclear receptor subfamily 4, group A, member 1 (NR4A1), NR4A2, NR4A3, early growth response 1, v-fos FBJ murine osteosarcoma viral oncogene homolog (c-FOS), FOSB, and Jun family member B (JUNB). Pretreatment of H295R cells with cycloheximide had no effect on Ang-II induction of these genes, suggesting that they are direct targets of Ang-II signaling. The Ang-II gene targets have been defined in three different adrenocortical model systems. Several of the listed genes have previously been described as being key regulators of adrenocortical function. The presence of adrenal cell common genes in such distinct cell models strengthens the hypothesis that these genes are regulators of aldosterone production.

Adrenocortical Tumorigenesis

Through the widespread use of imaging techniques with great sensitivity adrenal tumors are often diagnosed as an incidental finding. Although the majority of these adrenal lesions are benign and without evidence of endocrine activity or malignancy, hormone hypersecretion needs to be ruled out by specific tests. In addition to the classical forms of overt adrenocortical hypersecretion, it has become evident over the recent years that modest adrenocortical steroid autonomy as present in normokalemic primary aldosteronism and subclinical Cushing's syndrome is also associated with a significant morbidity. However, detection and differential diagnosis of these subtle changes in adrenal steroidogenesis can pose a diagnostic challenge to the clinician and is dependent on tests with reliable sensitivity and specificity. Regulation of adrenocortical development and growth, which results in clinical symptoms if disrupted, is dependent upon the distinct spatiotemporal expression of a variety of transcription factors as well as stimulation by extra-adrenal peptide hormones. Contributions to the elucidation of growth regulation of the adrenal cortex come from rare familiar syndromes associated with adrenocortical tumors, expression studies of adrenal tumor samples, in vitro studies on adrenocortical tumor cell lines, and mouse models displaying adrenal growth defects. In this review, we will summarize the important molecular aspects of adrenal tumorigenesis and highlight some prospects for clinical applications.

C-Ki-ras oncogene amplification and FGF2 signaling pathways in the mouse Y1 adrenocortical cell line

The mouse Y1 adrenocortical tumor cell line is highly responsive to FGF2-(Fibroblast Growth Factor 2) and possesses amplified and over-expressed c-Ki-ras proto-oncogene. We previously reported that this genetic lesion leads to high constitutive levels of activation of the c-Ki-Ras-GTP-->PI3K-->Akt signaling pathway (Forti et al. 2002). On the other hand, activation levels of another important pathway downstream of c-Ki-Ras-GTP, namely, Raf-->MEK-->ERK, remain strictly dependent on FGF2 stimulation (Rocha et al. 2003). Here we show that, first, FGF2 transiently up-regulates the c-Ki-Ras-GTP-->PI3K-->Akt pathway, in spite of its high basal levels. Second, c-Ki-Ras-GTP transient up-regulation likely underlies activation of the ERK1/2 pathway by FGF2. Third, c-Ki-Ras-GTP high basal levels suppress activation of the c-H-Ras onco-protein. But, Y1 cells, expressing dominant negative mutant RasN17, display a rapid and transient up-regulation of c-H-Ras-GTP upon FGF2 treatment. Elucidation of FGF2-signaling pathways in Y1 tumor cells can uncover new targets for drug development of interest in cancer therapy.

Leptin Interferes with Adrenocorticotropin/3′,5′-Cyclic Adenosine Monophosphate (cAMP) Signaling, Possibly through a Janus Kinase 2-Phosphatidylinositol 3-Kinase/Akt-Phosphodiesterase 3-cAMP Pathway, to Down-Regulate Cholesterol Side-Chain Cleavage Cytochrome P450 Enzyme in Human Adrenocortical NCI-H295 Cell Line

Obesity has adverse effects on adrenocortical functions. Adipocyte-derived leptin, a biomarker molecule of obesity, may directly control adrenal steroidogenesis via an unclear mechanism. We studied the mechanism underlying leptin action on adrenal steroidogenesis in human adrenocortical NCI-H295 tumor cell line. Levels of progesterone, cortisol, and cAMP were determined by ELISA. Western blotting was used to detect protein amounts of P450 side-chain cleavage (P450scc), Janus kinase 2 (JAK2), Akt, and their phosphorylated forms. The mRNA expressions of P450scc and leptin receptors were measured by RT-PCR and real-time PCR. P450scc promoter activity was analyzed with a luciferase reporter system. Cholera toxin mimicked ACTH action by increasing adrenal cAMP levels and steroid secretion. Leptin did not affect basal release but significantly inhibited ACTH/cholera toxin-induced steroid secretion. The concomitant inhibitions by leptin on cholera toxin-induced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/cholera toxin-induced CYP11A1 promoter activity via a known cAMP-responsive region located between -1.7 and -1.5 kb. Leptin activated phosphorylations of JAK2 and Akt. Inhibitory effects of leptin on ACTH/cholera toxin-induced cAMP levels, CYP11A1 promoter activity, and steroid secretion were blunted by either inhibitor of JAK2 (AG490) or phosphatidylinositol 3-kinase/Akt (wortmannin) as well as inhibitors of cAMP-degrading phosphodiesterases (PDEs), including nonspecific 3-isobutyl-1-methylxanthine and PDE3-specific SKF94836. Leptin failed to affect the inductions of CYP11A1 promoter activity and steroid secretion by PDE-nonhydrolyzable N(6)-monobutyryl-cAMP. Leptin interferes with ACTH/cAMP signaling, possibly through a cAMP-degrading mechanism involving activation of JAK2, phosphatidylinositol 3-kinase, and PDE3, to down-regulate P450scc expression and consequent adrenal steroidogenesis.

Adrenocorticotropin-Dependent Changes in SF-1/DAX-1 Ratio Influence Steroidogenic Genes Expression in a Novel Model of Glucocorticoid-Producing Adrenocortical Cell Lines Derived from Targeted Tumorigenesis

We established the first adrenocortical tumor cell lines with complete zona fasciculata (ZF) cell phenotype from tumors induced in transgenic mice by large T-antigen of simian virus 40 under the control of the aldose reductase-like akr1b7 gene promoter. Adrenocortical tumor cell lines produced high amounts of corticosterone and were responsive to ACTH. All genes that are supportive for glucocorticoid synthesis including cyp21a1 and cyp11b1 were expressed, and most of them were transiently up-regulated by ACTH at transcriptional level: stimulation culminated after 3-6 h and returned to basal levels after 24 h. Taking advantage of these cells, we have examined the effect of ACTH on DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on X-chromosome, gene 1) and SF-1 (steroidogenic factor 1), two transcription factors known to respectively repress and activate adrenocortical steroidogenesis by acting on common target genes. According to their antagonistic activities, DAX-1 mRNA and protein levels were transiently down-regulated by ACTH, whereas those of SF-1 were stimulated, with kinetics paralleling those of steroidogenic genes expression, notably of two known SF-1 target genes, star and akr1b7. This suggests an essential role of SF-1/DAX-1 proteins ratio to achieve proper ACTH control of steroidogenic gene expression in cells derived from ZF. This was confirmed in mice adrenals, where repression of dax-1 gene and concomitant up-regulation of sf-1, star, and akr1b7 genes were observed in response to ACTH stimulation. In conclusion, using both unique differentiated cell lines and in vivo approaches, we provide the first evidence that hormonally induced changes in SF-1/DAX-1 ratio are part of the molecular arsenal of ZF cells to fine tune ACTH responsiveness.

The adrenocortical tumor cell line NCI-H295R as an in vitro screening system for the evaluation of CYP11B2 (aldosterone synthase) and CYP11B1 (steroid-11β-hydroxylase) inhibitors

Aldosterone plays a key role in salt and water homeostasis but is also involved in the development and progression of congestive heart failure and myocardial fibrosis. As a new pharmacological strategy for the treatment of these diseases, we propose the inhibition of the key enzyme of mineralcorticoid formation, CYP11B2 (aldosterone synthase). For studies of the effects of CYP11B2 inhibitors on the adrenal cortex, we selected the NCI-H295R cell line which expresses most of the key enzymes necessary for steroidogenesis. To evaluate this cell line as a test system for effects and side effects of CYP inhibitors, we established assays using radiolabeled substrates of CYP11B2 and CYP11B1 and subsequently tested a series of CYP11B2 inhibitors including the CYP19 inhibitor fadrozole. Fadrozole and compounds 6, 9 and 10 were more potent towards CYP11B2 compared to CYP11B1 with IC 50 values in the nanomolar range. To analyze their overall effect, the formation of steroids in the cell culture supernatant was monitored. All compounds led to a concentration-dependent reduction of the aldosterone secretion but also reduced the formation of cortisol and androgens. In conclusion, the H295R cell line is a suitable tool for the prediction of overall side effects of CYP11B2 inhibitors on steroidogenesis.