Insights into the Molecular Biology of Adrenocortical Tumors
Authors
Affiliation
L. Groussin, J. Bertherat, C. Gicquel, Y. le Bouc, X. Bertagna
Clinique des Maladies Endocriniennes et Métaboliques, and Endocrinology Department, Institut Cochin Faculté de Médecine René and Laboratoire d’Explorations Fonctionnelles Trousseau, and the COMETE network, Paris, France
received 15.8.2005 first decision 27.11.2006
accepted 22.1.2007
Bibiliography DOI 10.1055/s-2007-970411 Exp Clin Endocrinol Diabetes 2007; 115: 175-178
@ J. A. Barth Verlag in Georg Thieme Verlag KG . Stuttgart . New York . ISSN 0947-7349
Correspondence X. Bertagna
Clinique des Maladies Endocriniennes et Métaboliques Hôpital Cochin 27 rue du Fg-St-Jacques 75014 Paris France
Tel .: +33/1/58 41 17 90
Fax: +33/1/46 33 80 60 xavier.bertagna@cch.aphp.fr
Introduction
& Adrenocortical tumors are rare tumors which can cause morbidity secondary to hypersecretion (i. e. Cushing’s syndrome in adrenocortical adenoma or carcinoma, mineralocorticoid excess in Conn’s adenoma) or to their oncogenic growth and metastases.
In the recent years progress has been made in our understanding of the pathophysiology of these endocrine tumors (Latronico and Chrousos, 1997; Bornstein et al., 1999; Reincke et al., 2000; Bertherat et al., 2002). Some molecular mecha- nisms of hormone hypersecretion or growth of adrenocortical tumors have been unraveled this last decade and offer new support to our under- standing of adrenocortical tumor development. In particular, studies of familial forms of adreno- cortical tumors have given us new insight in the genetics of these tumors. Some of these advances have already lead to new therapeutical or diag- nostic approaches.
We will not provide here an extensive review of all studies that have been performed in adreno- cortical tumors; we will rather focus on some aspects that are thought to be of major interest.
Clonal Analysis of Adrenocortical Tumors
&
Determination of the clonal composition of neo- plastic tissues has been instrumental in estab- lishing the cellular origins of many human tumors. To elucidate the pathophysiological mechanism that generates sporadic steroid-pro- ducing adrenocortical tumors in man, and even- tually distinguish between different processes, it was necessary first to study their clonal composi- tion. The method used to identify the clonal ori- gin of tumors has been extensively described (Gicquel et al., 1994).
We showed that whereas all adrenocortical car- cinomas are monoclonal, adrenocortical adeno- mas can be either monoclonal or polyclonal with various intermediate forms that probably corre- spond to cell-mosaicism with a monoclonal sub- population (Gicquel et al., 1994). Others have obtained similar results (Beuschlein et al., 1994).
Activation of the cAMP Pathway in Benign Adrenocortical Tumors Causing Cushing’s Syndrome &
Multiple examples of alterations at the level of membrane receptors and the proteins regulating intracellular transduction signals have been described this last decade in endocrine tumors. Most involve the cyclic AMP (cAMP) pathway. Constitutive activation (without the need of ligand) of this pathway has been described in endo- crine tumors: somatic activating point mutations of the alpha subunit of Gs protein (termed onco- gene Gsp) have been observed in about 40% of somatotroph adenomas and in toxic thyroid ade- nomas. Similarly, activating mutations of the TSH receptor have been found in the majority of hyperfunctioning thyroid adenomas.
The pituitary peptide hormone adrenocortico- tropin (ACTH) is required for adrenocortical cortex activity. It stimulates adrenocortical steroid production through its action on steroidogenic enzyme expression. ACTH also activates the cAMP pathway and may stimulate adrenocortical cortex hyperplasia in animals. In humans, chronic ACTH stimulation, as observed for instance in Cushing’s disease, leads to adrenocortical cortex hyperplasia and cortisol hypersecretion.
Illegitimate expression of transmembrane receptors in adrenocortical tumors Recently abnormal expression of transmembrane receptors has been observed in cases of Cushing’s
syndrome with ACTH-independent macronodular bilateral adreno- cortical hyperplasia (AIMAH) or adrenocortical tumors. Most of these alterations involve seven-transmembrane domain recep- tors that positively regulate the cAMP cascade. The first case of food dependent hypercortisolism was reported more than 18 years ago. The explanation of this unusual rhythm of cortisol secretion in a case of Cushing’s syndrome was given simultane- ously by two groups in 1992 (Lacroix et al., 1992; Reznik et al., 1992). Cortisol response to GIP (an insulinotropic peptide secreted from the duodenum after food ingestion) is observed in vivo in these patients. Binding or RT-PCR studies have shown the abnor- mal expression of the GIP receptor in adrenocortical tumor tissue from patients with food-dependent hypercortisolism, while no or very low expression is observed in adjacent adrenocortical tissue, in adenomas from patients who do not have food dependent Cushing’s syndrome, or in normal adrenocortical tissue.
Since these initial observations, the concept of “illegitimate” receptor expression in AIMAH and - rarely - in unilateral tumors, has extended to many other receptors besides the GIP receptor (Lacroix et al., 2001). In a recent study (Bertherat et al., 2005) we have shown the simultaneous occurrence of several “illegiti- mate” receptors in the same patient.
The McCune-Albright Syndrome (MCAS)
Constitutive activation of the cAMP pathway by mutation of the a subunit of Gs protein (termed Gsp) has been also observed in ACTH-independent hypercortisolism associated with the McCune-Albright Syndrome (MCAS) (Weinstein et al., 1991). In MCAS, Cushing’s syndrome is secondary to AIMAH. The a subu- nit Gs mutant is present in the adrenocortical nodes from these patients, as well as in several other tissues. Acromegaly and thy- roid nodes could also be observed in MCAS, in keeping with the observation of somatic Gsp mutation in sporadic somatotroph adenomas and thyroid tumors.
The Carney Complex
The Carney Complex (CNC) is a dominantly inherited syndrome responsible for a variety of manifestations. The main characteris- tics of CNC are spotty skin pigmentation (lentiginosis), endocrine overactivity and cardiac myxomas (Carney et al., 1985). Among the tumors observed in CNC patients are growth hormone (GH)- secreting pituitary adenomas (acromegaly), thyroid adenomas or carcinomas, testicular tumors (large cell calcifying Sertoli cell tumors), ovarian cysts, melanocytic schwannomas, and breast ductal adenomas. ACTH-independent Cushing’s syndrome due to primary pigmented nodular adrenocortical disease (PPNAD) is observed in 25 to 30 % of patients with CNC. PPNAD is a very rare cause of Cushing’s syndrome due to a primary bilateral adrenal defect that can be also observed in some patients without other CNC manifestations or familial history.
CNC seems to be a genetically heterogeneous disease and at least two loci have been described: 2p16 and 17q22-24 (Kirschner et al., 2000a; Kirschner et al., 2000b). The gene located on 17q22-24 has been identified as the regulatory subunit R1A of the protein kinase A (PRKAR1A) (Kirschner et al., 2000a). PRKAR1A is a key compo- nent of the cAMP signaling pathway that has been implicated in endocrine tumorigenesis (Bertherat, 2001). Heterozygous inacti- vating mutations of PRKAR1A have been reported initially in about 45 % of CNC families. In the tumors of CNC patients LOH at 17q22- 24 are observed, suggesting that PRKAR1A is a tumor suppressor gene. In CNC patients with Cushing’s syndrome the frequency of PRKAR1A mutations is about 80 %, suggesting that families with
PPNAD are more likely to be associated with a 17q22-24 defect (Groussin et al., 2002a). Interestingly, patients with isolated PPNAD and no familial history of CNC can also present a germline de novo mutation of PRKAR1A (Groussin et al., 2002a). Somatic mutation of PRKAR1A can occur in PPNAD as a mechanism of inactivation of the wild type allele in a patient already presenting a germline mutation (Groussin et al., 2002b).
In sporadic adrenocortical tumors somatic PRKAR1A mutations have been found in secreting adrenocortical adenomas (Bertherat et al., 2003). Adrenocortical adenomas harboring somatic PRKAR1A mutations present with clinical, hormonal and patho- logical features quite similar to PPNAD. LOH at 17q22-24 have also been observed in sporadic adrenocortical tumors. In adreno- cortical adenomas these LOH seem quite restricted to the PRKAR1A locus, suggesting that this tumor suppressor gene might be involved. By contrast in AC, LOH seem to affect a large part of 17q and PRKAR1A alteration might play a minor (or no) role in the growth of these malignant tumors.
Adrenocortical Carcinoma
&
Adrenocortical carcinoma (AC) is a rare tumor with an annual incidence of approximately two per million and a dismal prog- nosis with less than 20% survival at five years after diagnosis (Luton et al., 1990; Wajchenberg et al., 2000; Allolio et al., 2004). Our current therapeutic means are limited for patients not cured by a complete surgery, usually achieved when the tumor is small. New chemotherapeutic regimens are needed for metastastic disease or as adjuvant therapy after apparently complete surgery. Markers to predict the natural course of adrenocortical tumors are needed and elucidating the patho- genesis of AC should hopefully provide us with new diagnostic tools. Several factors are possibly involved in the malignant transformation of cells in patients with AC. A simple approach is to examine various candidate genes in a comparative fashion between benign and malignant tumors. Genetic syndromes predisposing to adrenocortical tumors (Table 1) have also pro- vided interesting insights into the pathogenesis of AC.
Comparative genomic hybridization and genotyping by LOH analysis, analyzing the whole genome, showed that genetic alterations were frequent in AC and involved chromosomes 2, 4, 11, 17 and 18.
We focus here on the genetic abnormalities most often involved in AC.
The IGF system and the 11p15 locus in AC
Study of the IGF system showed that strong overexpression of the IGF-II gene, which encodes an important fetal growth factor,
| Syndrome | Genetics |
|---|---|
| Li-Fraumeni | Mutation of p53 (17p13) |
| Wiedemann-Beckwith | Genetic and epigenetic abnor- malities of the 11p15.5 locus/IGFII imprinting |
| McCune-Albright | Activating mutation of GNAS 1 (mosaicism) |
| Carney Complex | 2p16, 17q22-24 (PRKARIA: the cAMP pathway) |
| MEN 1 | Mutation of menin (11q13) |
is a frequent feature of the malignant state, occuring in about 90% of malignant tumors but none of benign tumors. Two stud- ies assessing the gene profiles of adrenocortical tumors have independently confirmed the specific and high prevalence of IGF-II overexpression in ACs (Giordano et al., 2003; de Fraipont et al., 2005). The IGF-II gene maps to the 11p15 region that is submitted to parental imprinting. Mechanism of IGF-II gene overexpression is, at least partly, related to pathological imprint- ing. Indeed, most tumors with overexpression of the IGF-II gene also exhibit paternal isodisomy (loss of maternal allele and duplication of the paternal active IGF-II allele) or, less frequently, loss of imprinting (with maintenance of the maternal allele but a paternal-like IGF-II gene expression pattern) (Gicquel et al., 1997). The altered expression of IGF-II has also been implicated in Beckwith-Wiedemann syndrome (BWS), a fetal overgrowth syndrome predisposing to childhood tumors, including adreno- cortical carcinomas (Table 1). IGF-II mRNA is efficiently trans- lated and malignant tumors contain large amounts of IGF-II protein, partly as a prohormone form of IGF-II. The mitogenic effect of IGF-II is dependent on the presence of the IGF-I recep- tor and we showed that IGF-II is directly involved in prolifera- tion of the human adrenocortical carcinoma NCI H295R cell line and acts through the IGF-I receptor (Logie et al., 1999). IGF-II effects are restricted to tumors and systemic plasma levels of IGF-II always are in the normal range. IGF-binding proteins (IGFBPs) modulate the biological effects of IGFs depending on their abundance and affinity for the growth factors. Analysis of IGFBP expression profile in H295R cells and adrenocortical tumors showed an enhanced IGFBP-2 content in H295R cells and adrenocortical tumors overexpressing IGF-II gene, suggest- ing that IGFBP-2 may regulate IGF-II effects in malignant adren- ocortical tumors.
The 11p15 imprinted region includes other candidate genes for adrenocortical tumorigenesis. The H19 gene, encoding an untranslated RNA with a putative growth suppressor function, and the p57KIP2 gene, encoding a G1 cyclin dependent kinase inhibitor (CKI), are highly expressed in adrenocortical tissues. Their maternal expression and their function suggest that they are good tumor suppressor gene candidates for adrenocortical tumorigenesis. Indeed, their expression was reported to be abro- gated in malignant adrenocortical tumors.
All these data concerning the IGF system and the 11p15 genes are consistent with a major role of dysregulation of the imprinted 11p15 region in transition from benign to malignant adrenocor- tical tumors. Whatever the exact mechanisms for this dysregu- lation are, these molecular markers could permit a more accurate diagnosis of malignancy and probably also a better assessment of prognosis of adrenocortical tumors.
Chromosome 17p13 and the p53 gene in AC
The prevalence of loss of heterozygosity (LOH) at 17p13 is high in AC, occurring in about 85% of such tumors (Gicquel et al., 2001). It has been shown to be specifically associated with the malignant phenotype, and it is an independent prognostic marker of recurrence after complete surgery (Gicquel et al., 2001).
The 17p13 region includes the p53 gene that is the suppressor gene most often involved in almost all types of carcinomas. Germline mutations in the p53 gene are responsible for the autosomal dominant Li-Fraumeni syndrome, characterized by an elevated risk of early onset cancers (Table 1). In comparison with 17p13 LOH, p53 mutations only occur in about 20% of AC.
This discrepancy between p53 mutations and 17p13 LOH sug- gests that another tumor suppressor gene in this region is important for adrenocortical tumorigenesis. The HIC-1 gene (Hypermethylated In Cancer) is such a candidate; it encodes a transcription factor triggered by p53 and is inactivated by hyper- methylation or allelic losses in different cancers.
Cell cycle control genes in adrenocortical carcinomas
Cell cycle dysregulation is tightly linked to tumorigenesis. Among the genes involved in cell cycle control, cyclin dependent kinase inhibitors (CKI) negatively control kinase activities of cyclin-CDK complexes and thus progression through the cell cycle. We have already focused on the p57KIP2 gene that maps to the 11p15.5 region and encodes a CKI from the CIP/KIP family. P16, a CKI from the INK4 family, is also involved in malignant adrenocortical tumors. LOH within 9p21 associated with absence of p16 immunostaining is found in a large proportion of malig- nant tumors (Pilon et al., 1999).
Type 1 Multiple Endocrine Neoplasia (MEN 1) &
MEN1 is an autosomal dominant disease associated with pituitary, enteropancreatic and parathyroid tumors. Adrenocortical hyper- plasia and benign tumors are present in about 40% of MEN 1 cases. Malignant adrenocortical tumors have also rarely been observed in MEN 1. The MEN1 gene is located on chromosome 11q13 and encodes the tumor suppressor, Menin. In sporadic adrenocortical tumors, LOH in 11q13 occurs frequently in carcinomas but rarely in adenomas (10%). These 11q13 LOH are not associated with a MEN1 mutation, suggesting the involvement of a different tumor sup- pressor gene on this chromosome (Kjellman et al., 1999).
References
1 Allolio B, Hahner S, Weismann D, Fassnacht M: Management of adren- ocortical carcinoma. Clin Endocrinol 2004; 60: 273-287
2 Bertherat J: Protein kinase A in Carney Complex: a new example of cAMP pathway alteration in endocrine tumors. Eur J Endocrinol 2001; 144: 209-211
3 Bertherat J, Gicquel C, Le Bouc Y, Bertagna X: Molecular genetics of adrenal tumours. In: John AH Wass and Stephen M Shalet, (eds). Oxford textbook of Endocrinology and Diabetes. Oxford: Oxford Uni- versity Press, 2002; 831-842
4 Bertherat J, Groussin L, Sandrini F, Matyakhina L, Bei T, Stergiopoulos S, Papageorgiou T, Bourdeau I, Kirschner LS, Vincent-Dejean C, Perlemoine K, Gicquel C, Bertagna X, Stratakis CA: Molecular and functional anal- ysis of PRKAR1A and its locus (17q22-24) in sporadic adrenocortical tumors: 17q losses, somatic mutations, and protein kinase A expres- sion and activity. Cancer Res 2003; 63: 5308-5319
5 Bertherat J, Contesse V, Louiset E, Barrande G, Duparc C, Groussin L, Emy P, Bertagna X, Kuhn JM, Vaudry H, Lefebvre H: In vivo and in vitro screening for illegitimate receptors in AIMAH causing Cushing’s syn- drome: identification of two cases of gonadotropin/GIP-dependent hypercortisolism. J Clin Endocrinol Metab 2005; 90: 1302-1310
6 Beuschlein F, Reincke M, Karl M, Travis WD, Jaursch-Hancke C, Abdel- hamid S, Chrousos GP, Allolio B: Clonal composition of human adren- ocortical neoplasms. Cancer Res 1994; 54: 4927-4932
7 Bornstein SR, Stratakis CA, Chrousos GP: Adrenocortical tumors: recent advances in basic concepts and clinical management. Ann Intern Med 1999; 130: 759-771
8 Carney JA, Gordon H, Carpenter PC, Shenoy BV, Go VL: The complex of myxomas, spotty pigmentation, and endocrine overactivity. Medicine (Baltimore) 1985; 64: 270-283
9 De Fraipont F, El Atifi M, Le Moigne G, Defaye G, Houlgatte R, Bertherat J, Bertagna X, Plouin PF, Baudin E, Berger F, Gicquel C, Chabre O, Feige JJ: Gene expression profiling of human adrenocortical tumors using complementary cDNA micro-arrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 2005; 90: 1819-1829
10 Gicquel C, Leblond-Francillard M, Bertagna X, Louvel A, Chapuis Y, Luton JP, Girard F, Le Bouc Y: Clonal analysis of human adrenocortical carci- nomas and secreting-adenomas. Clin Endocrinol (Oxf) 1994; 40: 465-477
11 Gicquel C, Raffin-Sanson ML, Gaston V, Bertagna X, Plouin PF, Schlum- berger M, Louvel A, Luton JP, Le Bouc Y: Structural and functional abnor- malities at 11p15 are associated with the malignant phenotype in sporadic adrenocortical tumors. Study on a series of 82 tumors. J Clin Endocrinol Metab 1997; 87: 2559-2565
12 Gicquel C, Bertagna X, Gaston V, Coste J, Louvel A, Baudin E, Bertherat J, Chapuis Y, Duclos JM, Schlumberger M, Plouin PF, Luton JP, Le Bouc Y: Molecular markers and long-term recurrences in a large cohort of patients with sporadic adrenocortical tumors. Cancer Res 2001; 61: 6762-6767
13 Giordano TJ, Thomas DG, Kuick R, Lizyness M, Misek DE, Smith AL, Sand- ers D, Aljundi RT, Gauger PG, Thompson NW, Taylor JM, Hanash SM: Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. Am J Pathol 2003; 162: 521-531
14 Groussin L, Kirschner LS, Vincent-Dejean C, Perlemoine K, Jullian E, Dele- mer B, Zacharieva S, Pignatelli D, Carney JA, Luton JP, Bertagna X, Stra- takis CA, Bertherat J: Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adren- ocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD. Am J Hum Genet 2002; 71: 1433-1442
15 Groussin L, Jullian E, Perlemoine K, Louvel A, Leheup B, Luton JP, Bertagna X, Bertherat J: Mutations of the PRKAR1A gene in Cushing’s syndrome due to sporadic primary pigmented nodular adrenocortical disease. J Clin Endocrinol Metab 2002; 87: 4324-4329
16 Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS, Cho- Chung YS, Stratakis CA: Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex. Nat Genet 2000; 26: 89-92
17 Kirschner LS, Sandrini F, Monbo J, Lin JP, Carney JA, Stratakis CA: Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in
patients with the Carney complex. Hum Mol Genet 2000; 9: 3037-3046
18 Kjellman M, Roshani L, Teh BT, Kallioniemi OP, Hoog A, Gray S, Farnebo LO, Holst M, Backdahl M, Larsson C: Genotyping of adrenocortical tumors: very frequent deletions of the MEN1 locus in 11q13 and of a 1-centimorgan region in 2p16. J Clin Endocrinol Metab 1999; 84: 730-735
19 Lacroix A, Bolte E, Tremblay J, Dupre J, Poitras P, Fournier H, Garon J, Garrel D, Bayard F, Taillefer R et al: Gastric inhibitory polypeptide- dependent cortisol hypersecretion — a new cause of Cushing’s syn- drome. N Engl J Med 1992; 327: 974-980
20 Lacroix A, Ndiaye N, Tremblay J, Hamet P: Ectopic and abnormal hor- mone receptors in adrenal Cushing’s syndrome. Endocrinol Rev 2001; 22: 75-110
21 Latronico AC, Chrousos G: Extensive personal experience: adrenocorti- cal tumors. J Clin Endocrinol Metab 1997; 85: 1317-1324
22 Logié A, Boulle N, Gaston V, Perin L, Boudou P, Le Bouc Y, Gicquel C: Autocrine role of IGF-II in proliferation of human adrenocortical car- cinoma NCI H295R cell line. J Mol Endocrinol 1999; 23: 23-32
23 Luton JP, Cerdas S, Billaud L, Thomas G, Guilhaume B, Bertagna X, Laudat MH, Louvel A, Chapuis Y, Blondeau P, Bricaire H: Clinical features of adrenocortical carcinoma, prognostic factors, and the effect of mito- tane therapy. N Engl J Med 1990; 322: 1195-1201
24 Pilon C, Pistorello M, Moscon A, Altavilla G, Pagotto U, Boscaro M, Fallo F: Inactivation of the p16 tumor suppressor gene in adrenocortical tumors. J Clin Endocrinol Metab 1999; 84: 2776-2779
25 Reincke M, Beuschlein F, Slawik M, Borm K: Molecular adrenocortical tumourigenesis. Eur J Clin Invest 2000; 30: 63-68
26 Reznik Y, Allali-Zerah V, Chayvialle A, Leroyer R, Leymarie P, Travert G, Lebrethon MC, Budi I, Balliere AM, Mahoudeau J: Food- dependent Cushing’s syndrome mediated by aberrant adrenocortical sensitivity to gastric inhibitory polypeptide. N Engl J Med 1992; 327: 981-986
27 Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM: Activating mutations of the stimulatory G protein in the McCune- Albright syndrome. N Engl J Med 1991; 325: 1688-1716
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