Adrenocortical carcinoma (ACC) is an aggressive tumor showing frequent metastatic spread and poor survival. Although recent genome-wide studies of ACC have contributed to our understanding of the disease, major challenges remain for both diagnostic and prognostic assessments. The aim of this study was to identify specific microRNAs (miRNAs) associated with malignancy and survival of ACC patients. miRNA expression profiles were determined in a series of ACC, adenoma, and normal cortices using microarray. A subset of miRNAs showed distinct expression patterns in the ACC compared with adrenal cortices and adenomas. Among others, miR-483-3p, miR-483-5p, miR-210, and miR-21 were found overexpressed, while miR-195, miR-497, and miR-1974 were underexpressed in ACC. Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195 or miR-497 reduced cell proliferation in human NCI-H295R ACC cells. In addition, downregulation of miR-483-3p, but not miR-483-5p, and increased expression of miR-195 or miR-497 led to significant induction of cell death. Protein expression of p53 upregulated modulator of apoptosis (PUMA), a potential target of miR-483-3p, was significantly decreased in ACC, and inversely correlated with miR-483-3p expression. In addition, high expression of miR-503, miR-1202, and miR-1275 were found significantly associated with shorter overall survival among patients with ACC (P values: 0.006, 0.005, and 0.042 respectively). In summary, we identified additional miRNAs associated with ACC, elucidated the functional role of four miRNAs in the pathogenesis of ACC cells, demonstrated the potential involvement of the pro-apoptotic factor PUMA (a miR-483-3p target) in adrenocortical tumors, and found novel miRNAs associated with survival in ACC.
Deniz M Özata, Stefano Caramuta, David Velázquez-Fernández, Pinar Akçakaya, Hong Xie, Anders Höög, Jan Zedenius, Martin Bäckdahl, Catharina Larsson, and Weng-Onn Lui
S G Creemers, P M van Koetsveld, F J van Kemenade, T G Papathomas, G J H Franssen, F Dogan, E M W Eekhoff, P van der Valk, W W de Herder, J A M J L Janssen, R A Feelders, and L J Hofland
Adrenocortical carcinoma (ACC) is a rare malignancy with a poor prognosis. Discrimination of ACCs from adrenocortical adenomas (ACAs) is challenging on both imaging and histopathological grounds. High IGF2 expression is associated with malignancy, but shows large variability. In this study, we investigate whether specific methylation patterns of IGF2 regulatory regions could serve as a valuable biomarker in distinguishing ACCs from ACAs. Pyrosequencing was used to analyse methylation percentages in DMR0, DMR2, imprinting control region (ICR) (consisting of CTCF3 and CTCF6) and the H19 promoter. Expression of IGF2 and H19 mRNA was assessed by real-time quantitative PCR. Analyses were performed in 24 ACCs, 14 ACAs and 11 normal adrenals. Using receiver operating characteristic (ROC) analysis, we evaluated which regions showed the best predictive value for diagnosis of ACC and determined the diagnostic accuracy of these regions. In ACCs, the DMR0, CTCF3, CTCF6 and the H19 promoter were positively correlated with IGF2 mRNA expression (P<0.05). Methylation in the most discriminating regions distinguished ACCs from ACAs with a sensitivity of 96%, specificity of 100% and an area under the curve (AUC) of 0.997±0.005. Our findings were validated in an independent cohort of 9 ACCs and 13 ACAs, resulting in a sensitivity of 89% and a specificity of 92%. Thus, methylation patterns of IGF2 regulatory regions can discriminate ACCs from ACAs with high diagnostic accuracy. This proposed test may become the first objective diagnostic tool to assess malignancy in adrenal tumours and facilitate the choice of therapeutic strategies in this group of patients.
Susanna Vuorenoja, Adolfo Rivero-Müller, Adam J Ziecik, Ilpo Huhtaniemi, Jorma Toppari, and Nafis A Rahman
Novel strategies are needed for the treatment of adrenocortical tumors that are usually resistant to chemotherapy. Hecate, a 23-amino acid lytic peptide, was conjugated to the 15-amino acid (81–95) fragment of the human chorionic gonadotropin β (CGβ) chain, which would selectively kill cancer cells expressing the LH receptor (LHR) sparing the normal ones with LHR. To prove the principle that Hecate-CGβ conjugate may eradicate tumors ectopically expressing plasma membrane receptors, transgenic (TG) inhibin α-subunit promoter (inhα)/Simian Virus 40 T-antigen mice, expressing LHR in their adrenal gland tumors, were used as the experimental model. Wild-type control littermates and TG mice with adrenal tumors were treated with either Hecate or Hecate-CGβ conjugate at the age of 6.5 months for 3 weeks and killed 7 days after the last treatment. The Hecate-CGβ conjugate reduced the adrenal tumor burden significantly in TG male but not in female mice, in comparison with Hecate-treated mice. Hecate-CGβ conjugate treatment did not affect normal adrenocortical function as the serum corticosterone level between Hecate and Hecate-CGβ conjugate groups were similar. The mRNA and protein expressions of GATA-4 and LHR colocalized only in tumor area, and a significant downregulation of gene expression was found after the Hecate-CGβ conjugate in comparison with Hecate- and/or non-treated adrenal tumors by western blotting. This finding provides evidence for a selective destruction of the tumor cells by the Hecate-CGβ conjugate. Hereby, our findings support the principle that Hecate-CGβ conjugate is able to specifically destroy tumor cells that ectopically express LHR.
Susanna Vuorenoja, Bidut Prava Mohanty, Johanna Arola, Ilpo Huhtaniemi, Jorma Toppari, and Nafis A Rahman
Lytic peptide Hecate (23-amino acid (AA)) fused with a 15-AA fragment of human chorionic gonadotropin-β (CG-β), Hecate-CGβ conjugate (H-CGβ-c) selectively binds to and destroys tumor cells expressing LH/chorionic gonadotropin receptor (Lhcgr). Transgenic mice (6.5 month old) expressing SV40 T-antigen under the inhibin-α promoter (inhα/Tag) presenting with Lhcgr expressing adrenal tumors were treated either with H-CGβ-c, GnRH antagonist (GnRH-a), estradiol (E2; only females) or their combinations for 1 month. We expected that GnRH-a or E2 in combination with H-CGβ-c could improve the treatment efficacy especially in females by decreasing circulating LH and eliminating the potential competition of serum LH with the H-CGβ-c. GnRH-a and H-CGβ-c treatments were successful in males (adrenal weights 14±2.8 mg and 60±26 vs 237±59 mg in controls; P<0.05). Histopathologically, GnRH-a apparently destroyed the adrenal parenchyma leaving only the fibrotic capsule with few necrotic foci. In females, H-CGβ-c was totally ineffective, whereas GnRH-a (19±5 mg) or E2 (77±50 mg) significantly reduced the adrenal weights compared with controls (330±70 mg). Adrenal morphometry, cell proliferation markers, post-treatment suppression of serum progesterone, and quantitative RT-PCR of GATA-4, Lhcgr, and GATA-6 further supported the positive outcome. H-CGβ-c selectively killed the Lhcgr expressing tumor cells, whereas GnRH-a blocked tumor progression through gonadotropin suppression, emphasizing the gonadotropin dependency of these adrenocortical tumors. If extrapolated to humans, H-CGβ-c could be considered for the treatment of gonadotropin-dependent adrenal tumors in males, whereas in females gonadotropin suppression, but not H-CGβ-c, would work better.
Tiantian Liu, Taylor C Brown, C Christofer Juhlin, Adam Andreasson, Na Wang, Martin Bäckdahl, James M Healy, Manju L Prasad, Reju Korah, Tobias Carling, Dawei Xu, and Catharina Larsson
The telomerase reverse transcriptase gene (TERT) encodes the reverse transcriptase component of the telomerase complex, which is essential for telomere stabilization and cell immortalization. Recent studies have demonstrated a transcriptional activation role for the TERT promoter mutations C228T and C250T in many human cancers, as well as a role in aggressive disease with potential clinical applications. Although telomerase activation is known in adrenal tumors, the underlying mechanisms are not established. We assessed C228T and C250T TERT mutations by direct Sanger sequencing in tumors of the adrenal gland, and further evaluated potential associations with clinical parameters and telomerase activation. A total of 199 tumors were evaluated, including 34 adrenocortical carcinomas (ACC), 47 adrenocortical adenomas (ACA), 105 pheochromocytomas (PCC; ten malignant and 95 benign), and 13 abdominal paragangliomas (PGL; nine malignant and four benign). TERT expression levels were determined by quantitative RT-PCR. The C228T mutation was detected in 4/34 ACCs (12%), but not in any ACA (P=0.028). C228T was also observed in one benign PCC and in one metastatic PGL. The C250T mutation was not observed in any case. In the ACC and PGL groups, TERT mutation-positive cases exhibited TERT expression, indicating telomerase activation; however, since expression was also revealed in TERT WT cases, this could denote additional mechanisms of TERT activation. To conclude, the TERT promoter mutation C228T is a recurrent event associated with TERT expression in ACCs, but rarely occurs in PGL and PCC. The involvement of the TERT gene in ACC represents a novel mutated gene in this entity.
Elisa Roca, Alfredo Berruti, Silviu Sbiera, Ida Rapa, Ester Oneda, Paola Sperone, Cristina L Ronchi, Laura Ferrari, Salvatore Grisanti, Antonina Germano, Barbara Zaggia, Giorgio Vittorio Scagliotti, Martin Fassnacht, Marco Volante, Massimo Terzolo, and Mauro Papotti
Topoisomerase II alpha (TOP2A) and thymidylate synthase (TS) are known prognostic parameters in several tumors and also predictors of efficacy of anthracyclines, topoisomerase inhibitors and fluoropirimidines, respectively. Expression of TOP2A and TS mRNA was assessed in 98 patients with adrenocortical carcinoma (ACC) and protein expression was assessed by immunohistochemistry in a subset of 39 tumors. Ninety-two patients were radically resected for stage II–III disease and 38 of them received adjuvant mitotane. Twenty-six patients with metastatic disease received the EDP-M (etoposide, doxorubicin, Adriamycin, cisplatin plus mitotane). TOP2A and TS expression in ACC tissue was directly correlated with the clinical data. Both markers were not associated with either disease free survival (DFS) or overall survival (OS) in multivariate analyses and failed to be associated to mitotane efficacy. Disease response or stabilization to EDP-M treatment was observed in 12/17 (71%) and 1/9 (11%) patients with high and low TOP2A expressing tumors (P = 0.0039) and 9/13 (69%) and 4/13 (31%) patients with high and low TS expressing ACC, respectively (P = 0.049). High TOP2A expression was significantly associated with longer time to progression (TTP) after EDP-M. TOP2A and TS proteins assessed by immunohistochemistry significantly correlated with mRNA expression. Immunohistochemical TOP2A expression was associated with a non-significant better response and longer TTP after EDP-M. TOP2A and TS were neither prognostic nor predictive of mitotane efficacy in ACC patients. The predictive role of TOP2A expression of EDP-M activity suggests a significant contribution of Adriamycin and etoposide for the efficacy of the EDP scheme.
Brian Harding, Manuel C Lemos, Anita A C Reed, Gerard V Walls, Jeshmi Jeyabalan, Michael R Bowl, Hilda Tateossian, Nicky Sullivan, Tertius Hough, William D Fraser, Olaf Ansorge, Michael T Cheeseman, and Rajesh V Thakker
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized in man by parathyroid, pancreatic, pituitary and adrenal tumours. The MEN1 gene encodes a 610-amino acid protein (menin) which is a tumour suppressor. To investigate the in vivo role of menin, we developed a mouse model, by deleting Men1 exons 1 and 2 and investigated this for MEN1-associated tumours and serum abnormalities. Men1 +/− mice were viable and fertile, and 220 Men1 +/− and 94 Men1 +/+ mice were studied between the ages of 3 and 21 months. Survival in Men1 +/− mice was significantly lower than in Men1 +/+ mice (<68% vs >85%, P<0.01). Men1 +/− mice developed, by 9 months of age, parathyroid hyperplasia, pancreatic tumours which were mostly insulinomas, by 12 months of age, pituitary tumours which were mostly prolactinomas, and by 15 months parathyroid adenomas and adrenal cortical tumours. Loss of heterozygosity and menin expression was demonstrated in the tumours, consistent with a tumour suppressor role for the Men1 gene. Men1 +/− mice with parathyroid neoplasms were hypercalcaemic and hypophosphataemic, with inappropriately normal serum parathyroid hormone concentrations. Pancreatic and pituitary tumours expressed chromogranin A (CgA), somatostatin receptor type 2 and vascular endothelial growth factor-A. Serum CgA concentrations in Men1 +/− mice were not elevated. Adrenocortical tumours, which immunostained for 3-β-hydroxysteroid dehydrogenase, developed in seven Men1 +/− mice, but resulted in hypercorticosteronaemia in one out of the four mice that were investigated. Thus, these Men1 +/− mice are representative of MEN1 in man, and will help in investigating molecular mechanisms and treatments for endocrine tumours.
M Seki, K Nomura, D Hirohara, M Kanazawa, T Sawada, K Takasaki, and H Demura
A 58-year-old man had adrenocortical carcinoma in the right adrenal gland. The tumour secreted excessive cortisol and dehydroepiandrosterone-sulphate (DHEA-S), and had invaded the right hepatic lobe and vena cava. Eleven months after surgical tumour resection, the serum DHEA-S levels again increased. Local tumour recurrence and a metastasis was found in the lung. Eleven months after surgery chemotherapy with mitotane (o,p'-DDD) was initiated. Twelve weeks of mitotane reduced serum DHEA-S levels and caused these tumours to disappear. The patient was then treated with low-dose mitotane (1.5-2.0 g/day) for 2 years. Serum levels of mitotane remained at less than 10 microg/ml. Although such low serum levels of mitotane and delayed initiation of mitotane after surgery have been proposed to weaken the antineoplastic effect of mitotane, the patient had a remission for 2 years. However, there was then local re-recurrence with an increase in serum DHEA-S and death 4 months later. The histological features of neoplastic cells were quite different comparing tumour resected at surgery and tumour at autopsy. The latter had more frequent mitotic nuclei. This tumour was initially sensitive to mitotane, but later became insensitive.
Debbie L Hay, Christopher S Walker, and David R Poyner
Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane protein, calcitonin receptor-like receptor and one of the two accessory proteins, receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the ‘ADMR’), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible.
Helene Myrtue Nielsen, Alexandre How-Kit, Carole Guerin, Frederic Castinetti, Hans Kristian Moen Vollan, Catherine De Micco, Antoine Daunay, David Taieb, Peter Van Loo, Celine Besse, Vessela N Kristensen, Lise Lotte Hansen, Anne Barlier, Frederic Sebag, and Jörg Tost
Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof.