Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane is the most effective medical therapy for adrenocortical carcinoma, but its molecular mechanism of action remains poorly understood. Although mitotane is known to have mitochondrial (mt) effects, a direct link to mt dysfunction has never been established. We examined the functional consequences of mitotane exposure on proliferation, steroidogenesis, and mt respiratory chain, biogenesis and morphology, in two human adrenocortical cell lines, the steroid-secreting H295R line and the non-secreting SW13 line. Mitotane inhibited cell proliferation in a dose- and a time-dependent manner. At the concentration of 50 μM (14 mg/l), which corresponds to the threshold for therapeutic efficacy, mitotane drastically reduced cortisol and 17-hydroxyprogesterone secretions by 70%. This was accompanied by significant decreases in the expression of genes encoding mt proteins involved in steroidogenesis (STAR, CYP11B1, and CYP11B2). In both H295R and SW13 cells, 50 μM mitotane significantly inhibited (50%) the maximum velocity of the activity of the respiratory chain complex IV (cytochrome c oxidase (COX)). This effect was associated with a drastic reduction in steady-state levels of the whole COX complex as revealed by blue native PAGE and reduced mRNA expression of both mtDNA-encoded COX2 (MT-CO2) and nuclear DNA-encoded COX4 (COX4I1) subunits. In contrast, the activity and expression of respiratory chain complexes II and III were unaffected by mitotane treatment. Lastly, mitotane exposure enhanced mt biogenesis (increase in mtDNA content and PGC1 α (PPARGC1A) expression) and triggered fragmentation of the mt network. Altogether, our results provide first evidence that mitotane induced a mt respiratory chain defect in human adrenocortical cells.
Ségolène Hescot, Abdelhamid Slama, Anne Lombès, Angelo Paci, Hervé Remy, Sophie Leboulleux, Rita Chadarevian, Séverine Trabado, Larbi Amazit, Jacques Young, Eric Baudin, and Marc Lombès
Alfred King-yin Lam
Adrenal lipomatous tumour is a group of adrenal tumours with a significant component of adipose tissue. According to the current World Health Organization (WHO) classification of tumours of endocrine organs, adrenal myelolipoma is the only entity amongst the group of tumours being described. In the literature, other more recently documented adrenal lipomatous tumours included 24 lipomas, 32 teratomas and 16 angiomyolipomas. Rare fatty tumours of the adrenal gland comprised liposarcoma, hibernoma, adrenocortical tumours with fat component and rare adrenal tumours with fat component. Myelolipoma comprises approximately 3% of primary adrenal tumour. It is noted more commonly in females and in the right adrenal gland. Approximately 40 bilateral myelolipomas were reported. The tumour is most frequently recorded in patients between fifth and seventh decades of life. Adrenal lipomas are often seen in males and in the right adrenal gland. They were commonly noted in patients in the sixth decade of life. The diagnosis could only be possible on examination of the surgically removed specimen. Adrenal teratomas were more common in females and with a bimodal age distribution. Slightly over 60% of the patients with adrenal teratoma are symptomatic. Adrenal angiomyolipomas were often symptomatic, more common in females and in the fifth decades of life. To conclude, adrenal lipomatous tumour is uncommon. They are often benign and non-functional. It is important to recognize the features of this group of lipomatous tumours in the adrenal gland as they are being detected on increasing incidence as a result of the wide-spread use of modern imaging modalities.
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.
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.
T M A Kerkhofs, M H T Ettaieb, I G C Hermsen, and H R Haak
Cancer of the adrenal cortex (ACC) is a rare endocrine malignancy with limited treatment options. Patients typically present with autonomous hormonal overproduction and/or a large abdominal mass. Hormonal assays and medical imaging can be diagnostic, but urinary steroid profiling might be a more sensitive technique to assess malignancy in adrenal tumours. The stage of the disease at diagnosis is the most important prognostic factor. The current staging system needs refinement, especially to separate aggressive from indolent disease in stage IV patients and to select patients who need adjuvant treatment after complete surgical resection. Regarding the latter, assessing the proliferation index Ki-67 seems the best tool currently available. Genomic profiling is expected to become of clinical relevance in the future. Medical therapy is centred on the adrenolytic drug mitotane, which carries considerable toxicity and is not easy to manage. Its tolerability and long plasma level build-up phase may be improved by therapeutic drug monitoring based on pharmacokinetic modelling and intensive counselling of patients. Current chemotherapy regimens can offer disease stabilization in about 50% of patients, but an objective response should be expected in <25%. Research on targeted therapy and immunotherapy is difficult in this rare disease with often heavily pre-treated patients and has not yet been successful. Quality of care should be ensured by treating patients in centres with established experience in multidisciplinary oncologic care, who adhere to prevailing guidelines and state-of-the-art in diagnostic and treatment concepts. International collaboration in fundamental research and clinical trials is the key to further elucidate the pathogenesis and to improve patient care.
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.
A Falchetti and M L Brandi
Multiple Endocrine Neoplasias type 1 (MEN 1) and type 2 (MEN 2) represent complex inherited (autosomal dominant traits) syndromes characterized by occurrence of distinct proliferative disorders of endocrine tissues, varying from hyperplasia to adenoma and carcinoma.
MEN 1 syndrome is characterized by parathyroid gland, anterior pituitary and endocrine pancreas tumors. Other endocrine and non endocrine tumors, such as carcinoids, lipomas, pinealomas, adrenocortical and thyroid follicular tumors, have been also described in MEN 1 patients occurring at higher frequency than in general population (Brandi ML et al. 1987). Recently also a spinal ependymoma has been found in a patient with MEN 1 syndrome (Kato H et al 1997)
MEN 2 syndromes recognize three main clinical entities, MEN 2A, characterized by medullary thyroid carcinoma (MTC), primary hyperparathyroidism (PHPT) and pheochromocytoma (PHEO); MEN 2B that exhibits MTC, usually developing sooner than the MEN 2A- associated one, pheochromocytoma, multiple neuromas of gastroenteric mucosa, myelinated corneal nerves (Gorlin RJ et al. 1968) and a typical marphanoid habitus; and familial medullary thyroid carcinoma only (FMTC) featuring by families with at least four members with MTC and no objective evidence of pheochromocytoma and parathyroid disease on screening of affected and at-risk members, as stated by the International RET Mutation Consortium (Larsson C et al. 1994).
This work was supported by grants of the Associazione Italiana per la Ricerca sul Cancro (to MLB), from CNR/PF ACRO (INV. 95.00316 PF 39) and by MURST 60% (to MLB).
S G Creemers, L J Hofland, E Korpershoek, G J H Franssen, F J van Kemenade, W W de Herder, and R A Feelders
Adrenocortical carcinoma (ACC) is a rare disease with a poor prognosis. Discrimination between ACCs and adrenocortical adenomas (ACAs) remains challenging, with the current gold standard being the Weiss score, consisting of several histopathological characteristics. However, new markers like Ki67, a marker for proliferation, and the staining of reticulins are promising not only as it comes to identifying malignancy but also as prognostic markers in patients with ACC. Currently, surgery is still the only curative treatment for ACC. Mitotane, an adrenolytic drug, is used in the adjuvant setting and in case of metastatic or advanced disease. Patients with progressive disease are frequently treated with mitotane, alone or in combination with etoposide, doxorubicine and cisplatin. Radiotherapy is indicated in selected cases. The low response rates and high toxicity of the systemic therapies emphasize the need for markers that enable the identification of responders and non-responders. Consequently, research is focusing on predictive factors varying from the expression of DNA repair genes to clinical patient characteristics. Subgroups of ACC with different prognosis have been identified based on transcriptome characteristics. As a conclusion from large molecular studies, ACCs appear to harbor many abnormalities compared to ACAs. Altered pathways driving ACC pathogenesis include the IGF, TP53 and the Wnt signaling pathway, allowing these as new potential targets for medical therapy. However, despite efforts in preclinical and clinical studies investigating efficacy of targeting these pathways, most novel therapies appear to be effective in only a subset of patients with ACC. New treatment concepts are therefore urgently needed.
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.
Deniz M Özata, Stefano Caramuta, David Velázquez-Fernández, Pinar Akçakaya, Hong Xie, Anders Höög, Jan Zedenius, Martin Bäckdahl, Catharina Larsson, and Weng-Onn Lui
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.