Carney triad (CTr) describes the association of paragangliomas (PGL), pulmonary chondromas, and gastrointestinal (GI) stromal tumors (GISTs) with a variety of other lesions, including pheochromocytomas and adrenocortical tumors. The gene(s) that cause CTr remain(s) unknown. PGL and GISTs may be caused by loss-of-function mutations in succinate dehydrogenase (SDH) (a condition known as Carney–Stratakis syndrome (CSS)). Mitochondrial structure and function are abnormal in tissues that carry SDH defects, but they have not been studied in CTr. For the present study, we examined mitochondrial structure in human tumors and GI tissue (GIT) of mice with SDH deficiency. Tissues from 16 CTr tumors (n=12), those with isolated GIST (n=1), and those with CSS caused by SDHC (n=1) and SDHD (n=2) mutations were studied by electron microscopy (EM). Samples of GIT from mice with a heterozygous deletion in Sdhb (Sdhb + /−, n=4) were also studied by EM. CTr patients presented with mostly epithelioid GISTs that were characterized by plump cells containing a centrally located, round nucleus and prominent nucleoli; these changes were almost identical to those seen in the GISTs of patients with SDH. In tumor cells from patients, regardless of diagnosis or tumor type, cytoplasm contained an increased number of mitochondria with a ‘hypoxic’ phenotype: mitochondria were devoid of cristae, exhibited structural abnormalities, and were of variable size. Occasionally, mitochondria were small and round; rarely, they were thin and elongated with tubular cristae. Many mitochondria exhibited amorphous fluffy material with membranous whorls or cystic structures. A similar mitochondrial hypoxic phenotype was seen in Sdhb + /− mice. We concluded that tissues from SDH-deficient tumors, those from mouse GIT, and those from CTr tumors shared identical abnormalities in mitochondrial structure and other features. Thus, the still-elusive CTr defect(s) is(are) likely to affect mitochondrial function, just like germline SDH-deficiency does.
Eva Szarek, Evan R Ball, Alessio Imperiale, Maria Tsokos, Fabio R Faucz, Alessio Giubellino, François-Marie Moussallieh, Izzie-Jacques Namer, Mones S Abu-Asab, Karel Pacak, David Taïeb, J Aidan Carney, and Constantine A Stratakis
Bruno Ragazzon, Guillaume Assié, and Jérôme Bertherat
Transcriptome analysis has been successfully used to study the gene profile expression of adrenocortical tumors (ACT) for 7 years. The various studies reported to date have produced an abundance of new information on adrenocortical cancer (ACC), underlying the validity of this approach to study the molecular genetics and pathogenesis of these tumors. The gene expression profile of ACC clearly differs from that of benign adrenocortical adenomas (ACA). Interestingly, transcriptome analysis has the ability to establish a subclassification of ACC based on the gene expression profile. In particular, it is able to identify two groups of tumors with different outcomes (i.e. good prognosis and poor prognosis). This approach has been used to develop molecular markers for ACC diagnosis and prognostication. An IGF2 cluster of genes up-regulated in ACC has been identified. Transcriptome analysis has shown that, in comparison with ACA, IGF2 is indeed the gene most overexpressed in ACC. By contrast, genes associated with steroidogenesis are down-regulated in ACC. Genes controlling the cell cycle are dysregulated in ACC, and several are dramatically overexpressed. Analysis regarding the level of expression of Wnt/β-catenin and p53 signaling has shown alterations, in keeping with the known molecular somatic genetic defects of these pathways that are observed in ACC. This review summarizes the main findings of studies reporting ACC transcriptome analysis, demonstrating its power for ACT classification, and examines the resulting progress in understanding the pathogenesis of ACC. The potential for both ACC diagnosis and the identification of new therapeutic targets will be discussed.
Kiran Nadella, Fabio R Faucz, and Constantine A Stratakis
Protein kinase A (PKA) regulatory subunit type 1A (PRKAR1A) defects lead to primary pigmented nodular adrenocortical disease (PPNAD). The KIT protooncogene (c-KIT) is not known to be expressed in the normal adrenal cortex (AC). In this study, we investigated the expression of c-KIT and its ligand, stem cell factor (SCF), in PPNAD and other cortisol-producing tumors of the adrenal cortex. mRNA and protein expression, by qRT-PCR, immunohistochemistry (IHC) and immunoblotting (IB), respectively, were studied. We then tested c-KIT and SCF responses to PRKAR1A introduction and PKA stimulation in adrenocortical cell lines CAR47 and H295R, which were also treated with the KIT inhibitor, imatinib mesylate (IM). Mice xenografted with H295R cells were treated with IM. There was increased c-KIT mRNA expression in PPNAD; IHC showed KIT and SCF immunoreactivity within certain nodular areas in PPNAD. IB data was consistent with IHC and mRNA data. PRKAR1A-deficient CAR47 cells expressed c-KIT; this was enhanced by forskolin and lowered by PRKAR1A reintroduction. Knockdown of PKA’s catalytic subunit (PRKACA) by siRNA reduced c-KIT levels. Treatment of the CAR47 cells with IM resulted in reduced cell viability, growth arrest, and apoptosis. Treatment with IM of mice xenografted with H295 cells inhibited further tumor growth. We conclude that c-KIT is expressed in PPNAD, an expression that appears to be dependent on PRKAR1A and/or PKA activity. In a human adrenocortical cell line and its xenografts in mice, c-KIT inhibition decreased growth, suggesting that c-KIT inhibitors may be a reasonable alternative therapy to be tested in PPNAD, when other treatments are not optimal.
Zsófia Tömböl, Peter M Szabó, Viktor Molnár, Zoltán Wiener, Gergely Tölgyesi, János Horányi, Peter Riesz, Peter Reismann, Attila Patócs, István Likó, Rolf-Christian Gaillard, András Falus, Károly Rácz, and Peter Igaz
MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR–mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCTmiR-511 and dCTmiR-503 (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.
Peter M van Koetsveld, Giovanni Vitale, Richard A Feelders, Marlijn Waaijers, Diana M Sprij-Mooij, Ronald R de Krijger, Ernst-Jan M Speel, Johannes Hofland, Steven W J Lamberts, Wouter W de Herder, and Leo J Hofland
Adrenocortical carcinoma (ACC) is an aggressive tumor with very poor prognosis. Novel medical treatment opportunities are required. We investigated the effects of interferon-β (IFN-β), alone or in combination with mitotane, on cell growth and cortisol secretion in primary cultures of 13 human ACCs, three adrenal hyperplasias, three adrenal adenomas, and in two ACC cell lines. Moreover, the interrelationship between the effects of IGF2 and IFN-β was evaluated. Mitotane inhibited cell total DNA content/well (representing cell number) in 7/11 (IC50: 38±9.2 μM) and cortisol secretion in 5/5 ACC cultures (IC50: 4.5±0.1 μM). IFN-β reduced cell number in 10/11 (IC50: 83±18 IU/ml) and cortisol secretion in 5/5 ACC cultures (IC50: 7.3±1.5 IU/ml). The effect of IFN-β on cell number included the induction of apoptosis. IFN-β strongly inhibited mRNA expression of STAR, CYP11A1, CYP17A1, and CYP11B1. Mitotane and IFN-β induced an additive inhibitory effect on cell number and cortisol secretion. IGF2 (10 nM) inhibited apoptosis and increased cell number and cortisol secretion. These effects were counteracted by IFN-β treatment. Finally, IFN-β inhibited IGF2 secretion and mRNA expression. In conclusion, IFN-β is a potent inhibitor of ACC cell growth in human primary ACC cultures, partially mediated by an inhibition of the effects of IGF2, as well as its production. The increased sensitivity of ACC cells to mitotane induced by treatment with IFN-β may open the opportunity for combined treatment regimens with lower mitotane doses. The inhibition of the expression of steroidogenic enzymes by IFN-β is a novel mechanism that may explain its inhibitory effect on cortisol production.
Milena Doroszko, Marcin Chrusciel, Joanna Stelmaszewska, Tomasz Slezak, Slawomir Anisimowicz, Ursula Plöckinger, Marcus Quinkler, Marco Bonomi, Slawomir Wolczynski, Ilpo Huhtaniemi, Jorma Toppari, and Nafis A Rahman
Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11–13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.
S G Creemers, P M van Koetsveld, W W De Herder, F Dogan, G J H Franssen, R A Feelders, and L J Hofland
Chemotherapy for adrenocortical carcinoma (ACC) has limited efficacy and is accompanied by severe toxicity. This lack of effectiveness has been associated with high tumoral levels of the multidrug resistance (MDR) pump P-glycoprotein (P-gp), encoded by the MDR1 gene. In this study, effects of P-gp inhibition on the sensitivity of ACC cells to cytotoxic drugs were evaluated. MDR1 mRNA and P-gp expression were determined in human adrenal tissues and cell lines. H295R, HAC15 and SW13 cells were treated with mitotane, doxorubicin, etoposide, cisplatin and streptozotocin, with or without the P-gp inhibitors verapamil and tariquidar. Cell growth and surviving fraction of colonies were assessed. MDR1 mRNA and P-gp protein expression were lower in ACCs than in adrenocortical adenomas (P < 0.0001; P < 0.01, respectively). MDR1 and P-gp expression were positively correlated in ACC (P < 0.0001, ρ = 0.723). Mitotane, doxorubicin, cisplatin and etoposide dose dependently inhibited cell growth in H295R, HAC15 and SW13. Tariquidar, and in H295R also verapamil, increased the response of HAC15 and H295R to doxorubicin (6.3- and 7.5-fold EC50 decrease in H295R, respectively; all P < 0.0001). Sensitivity to etoposide was increased in H295R and HAC15 by verapamil and tariquidar (all P < 0.0001). Findings were confirmed when assessing colony formation. We show that cytotoxic drugs, except streptozotocin, used for ACC treatment, inhibit ACC cell growth and colony formation at clinically achievable concentrations. P-gp inhibition increases sensitivity to doxorubicin and etoposide, suggesting that MDR1 is involved in sensitivity to these drugs and could be a potential target for cytotoxic treatment improvement in ACC.
Fabio L Forti and Hugo A Armelin
Arginine vasopressin (AVP), a vasoactive peptide hormone that binds to three G-protein coupled receptors (V1R, V2R, and V3R), has long been known to activate V1R and elicit mitogenesis in several cell types, including adrenal glomerulosa cells. However, in the mouse Y1 adrenocortical malignant cell line, AVP triggers not only a canonical mitogenic response but also novel RhoA-GTP-dependent mechanisms which downregulate cyclin D1, irreversibly inhibiting K-ras oncogene-driven proliferation. In Y1 cells, AVP blocks cyclin D1 expression, induces senescence-associated β-galactosidase (SAβ-Gal) and inhibits proliferation. However, ectopic expression of cyclin D1 renders Y1 cells resistant to both SAβ-Gal induction and proliferation inhibition by AVP. In addition, ectopic expression of the dominant negative RhoAN19 mutant blocks RhoA activation, yielding Y1 cell sub-lines which are no longer susceptible to cyclin D1 downregulation, SAβ-Gal induction, or proliferation inhibition by AVP. Furthermore, inhibiting RhoA with C3 exoenzyme protects Y1 cells from AVP proliferation inhibition and SAβ-Gal induction. On the other hand, AVP treatment does not activate caspases 3 and 7, and the caspase inhibitor Ac-DEVD-CMK does not protect Y1 cells from proliferation inhibition by AVP, implying that AVP does not trigger apoptosis. These results underline a pivotal survival activity of cyclin D1 that protects K-ras oncogene-dependent malignant cells from senescence.
Michaela Luconi, Monica Mangoni, Stefania Gelmini, Giada Poli, Gabriella Nesi, Michela Francalanci, Nicola Pratesi, Giulia Cantini, Adriana Lombardi, Monica Pepi, Tonino Ercolino, Mario Serio, Claudio Orlando, and Massimo Mannelli
Adrenocortical carcinoma (ACC) is a rare aggressive tumor with a poor prognosis. The lack of a specific and effective medical treatment is due to the poor knowledge of the mechanisms underlying tumor growth. Research on potential drugs able to specifically interfere with tumor proliferation is essential to develop more efficacious therapies. We evaluated for the first time the in vivo effect of rosiglitazone (RGZ), an anti-diabetic drug with in vitro anti-tumor properties, on ACC proliferation in a xenograft model obtained by s.c. injection of human ACC H295R cells in athymic mice. When the tumor size reached 5 mm, animals were allocated to 5 mg/kg RGZ- or water-treated groups. Tumor volume was measured twice a week. A significant reduction of tumor growth in RGZ versus control (control) group was observed and was already maximal following 17 day treatment (1−T/C=75.4% (43.7–93.8%)). After 31 days of treatment, mice were killed and tumor analyzed. Tumor histological evaluation revealed characteristics of invasiveness, richness in small vessels and mitotic figures in control group, while RGZ group tumors presented non infiltrating borders, few vessels, and many apoptotic bodies. Tumor immunohistochemistry showed that Ki-67 was reduced in RGZ versus control group. Quantitative real-time RT-PCR demonstrated a significant reduction in the expression of angiogenic (VEGF), vascular (CD31), proliferation (BMI-1), and anti-apoptotic (Bcl-2) genes in RGZ versus control group tumors. The same inhibitory effects were confirmed in in vitro RGZ-treated H295R. Our findings support and expand the role of RGZ in controlling ACC proliferation and angiogenesis in vivo and in vitro.
O Chabre, R Libé, G Assie, O Barreau, J Bertherat, X Bertagna, J-J Feige, and N Cherradi
Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Local and distant recurrences occur in a subset of tumors classified as ‘aggressive’ ACC (aACC), as opposed to ‘non-aggressive’ ACC (naACC). In this study, we investigated whether tissue and serum microRNAs (miRNAs) are predictive of ACC prognosis. Tissue miRNA expression profiles were determined using microarrays in a test series of six adrenocortical adenomas (ACAs), six naACCs, and six aACCs. Eight miRNAs were selected for further validation by quantitative RT-PCR (ten ACAs, nine naACCs, nine aACCs, and three normal adrenals). Serum levels of five miRNAs were measured in samples from 56 subjects (19 healthy controls (HC), 14 ACA, nine naACC, and 14 aACC patients). MiR-195 and miR-335 levels were significantly decreased in both tumor and serum samples of ACC patients relative to ACA patients or HC. MiR-139-5p and miR-376a levels were significantly increased in aACC compared with naACC patients in tumor samples only. Tissue miR-483-5p was markedly upregulated in a majority of ACC compared with ACA patients or HC, but most importantly, serum miR-483-5p was detected only in aACC patients. High circulating levels of miR-483-5p or low circulating levels of miR-195 were associated with both shorter recurrence-free survival (P=0.0004 and P=0.0014 respectively) and shorter overall survival (P=0.0005 and P=0.0086 respectively). In conclusion, this study reports for the first time that circulating miR-483-5p and miR-195 are promising noninvasive biomarkers with a highly specific prognostic value for the clinical outcome of ACC patients.