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.
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.
Patricia de Cremoux, Dan Rosenberg, Jacques Goussard, Catherine Brémont-Weil, Frédérique Tissier, Carine Tran-Perennou, Lionnel Groussin, Xavier Bertagna, Jérôme Bertherat, and Marie-Laure Raffin-Sanson
Adrenal tumors occur more frequently in women and are the leading cause of Cushing's syndrome during pregnancy. We aimed to evaluate the potential role of sex steroids in the susceptibility of women to adrenocortical tumors. We evaluated the presence of the progesterone receptor (PR), estradiol receptors (ERs), and aromatase in 5 patients with primary pigmented nodular adrenal disease (PPNAD), 15 adrenocortical adenomas (ACAs) and adjacent normal tissues, 12 adrenocortical carcinomas (ACCs), and 3 normal adrenal glands (NA). The expression of PR and ERα was evaluated by enzyme immunoassays, real-time RT-PCR, immunohistochemistry, and cytosol-based ligand-binding assays. ERβ and aromatase levels were evaluated by real-time RT-PCR. ERα concentrations were low in NA, in adrenal tissues adjacent to ACA (51±33), in ACC (53±78), and lower in ACA (11±11 fmol/mg DNA). Conversely, PR concentrations were high in NA and adrenal tissues adjacent to ACA, at 307±216 fmol/mg DNA, and were even higher in tumors – 726±706 fmol/mg DNA in ACA and 1154±1586 fmol/mg DNA in ACC – and in isolated PPNAD nodules. Binding study results in four tumors were compatible with binding to a steroid receptor. In patients with PPNAD, a strong positive immunohistochemical signal was associated with the sole isolated nodular regions. ERβ transcript levels were very high in all samples except those for two ACCs, whereas aromatase levels were low. PR and ERβ are clearly present in normal adrenal glands and adrenal tumors. Further studies may shed light on the possible pathogenic role of these receptors in adrenal proliferation.
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.
Sandra Rodríguez-Rodero, Elías Delgado-Álvarez, Agustín F Fernández, Juan L Fernández-Morera, Edelmiro Menéndez-Torre, and Mario F Fraga
Aberrant epigenetics is a hallmark of cancer, and endocrine-related tumors are no exception. Recent research has been identifying an ever-growing number of epigenetic alterations in both genomic DNA methylation and histone post-translational modification in tumors of the endocrine system. Novel microarray and ultra-deep sequencing technologies have allowed the identification of genome-wide epigenetic patterns in some tumor types such as adrenocortical, parathyroid, and breast carcinomas. However, in other cancer types, such as the multiple endocrine neoplasia syndromes and thyroid cancer, tumor information is limited to candidate genes alone. Future research should fill this gap and deepen our understanding of the functional role of these alterations in cancer, as well as defining their possible clinical uses.
P S H Soon, A J Gill, D E Benn, A Clarkson, B G Robinson, K L McDonald, and S B Sidhu
The management of adrenocortical tumors (ACTs) is complex. The Weiss score is the present most widely used system for ACT diagnosis. An ACT is scored from 0 to 9, with a higher score correlating with increased malignancy. However, ACTs with a score of 3 can be phenotypically benign or malignant. Our objective is to use microarray profiling of a cohort of adrenocortical carcinomas (ACCs) and adrenocortical adenomas (ACAs) to identify discriminatory genes that could be used as an adjunct to the Weiss score. A cohort of Weiss score defined ACCs and ACAs were profiled using Affymetrix HGU133plus2.0 genechips. Genes with high-discriminatory power were identified by univariate and multivariate analyses and confirmed by quantitative real-time reverse transcription PCR and immunohistochemistry (IHC). The expression of IGF2, MAD2L1, and CCNB1 were significantly higher in ACCs compared with ACAs while ABLIM1, NAV3, SEPT4, and RPRM were significantly lower. Several proteins, including IGF2, MAD2L1, CCNB1, and Ki-67 had high-diagnostic accuracy in differentiating ACCs from ACAs. The best results, however, were obtained with a combination of IGF2 and Ki-67, with 96% sensitivity and 100% specificity in diagnosing ACCs. Microarray gene expression profiling accurately differentiates ACCs from ACAs. The combination of IGF2 and Ki-67 IHC is also highly accurate in distinguishing between the two groups and is particularly helpful in ACTs with Weiss score of 3.