- Email: [email protected]
The molecular basis of adrenocortical cancer
Cancer Genetics 205 (2012) 131e137
REVIEW
The molecular basis of adrenocortical cancer Tomasz Lehmann*, Tomasz Wrzesinski Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, Poznan, Poland Adrenocortical tumors (ACTs) are common, and most are benign adrenocortical adenomas (ACAs). Malignant adrenocortical carcinoma (ACC) is a rare tumor type and is observed at the rate of one or two cases per million annually. ACTs are classified as either ACAs or ACCs by histopathologic methods that are based on nine Weiss scoring criteria, including the nuclear grade, mitotic rate, presence of necrosis, and others. In this review, we describe the findings of studies that have examined the molecular basis of ACTs, and we compare transcriptome analysis with other diagnostic approaches. ACTs are occasionally difficult to classify. Therefore, molecular techniques, such as microarray analysis, have recently been applied to overcome some of these diagnostic problems. We also discuss the likelihood of the diagnosis and discernment between ACAs and ACCs based on the molecular tests. To show the recent progress in understanding the etiology of ACTs, we highlight the relationship between genetic analysis and transcriptome analysis. We attempt to understand the role of abnormal cell growth and steroid hormone secretion. Genetic and transcriptome analyses have improved our understanding of ACTs considerably, yet many unanswered questions remain. Keywords Adrenal, cancer, gene expression, microarray ª 2012 Elsevier Inc. All rights reserved.
Symptoms, clinical diagnosis, classification, staging, and prognosis Diagnosis Most frequently, malignant tumors in the adrenal glands are formed from the metastasis of cancers that have initiated growth in other organs, largely the lungs and breast. Most nonmetastatic adrenal masses are asymptomatic, benign tumors called adrenocortical adenomas (ACAs). The adrenocortical tumors (ACTs) revealed during diagnostic procedures for unrelated disorders are called incidentaloma (1). Most ACAs are likely undiagnosed, and among all diagnosed ACAs, 52% are subsequently removed through surgical operation (2). ACAs are frequent in the population (found in >3% of people older than 50 y) (3). Conversely, the prevalence of malignant adrenocortical carcinoma (ACC) is estimated to be very infrequent, with only 1e12 cases per million adults (3). It remains a matter of debate whether ACA is a separate disease or is part of the process of tumor progression. ACCs possess metastatic potential with secreting or nonsecreting
Received August 28, 2011; received in revised form February 4, 2012; accepted February 21, 2012. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergen.2012.02.009
characteristics. The 5-year survival rate of patients with metastatic ACC is 16e38% (4e6). ACC may be detected during a routine visualization test or diagnosed using a steroid hormone assay, or based on the symptoms resulting from tumor pressure on neighboring organs.
Imaging methods Proper imaging and diagnosis of ACTs are indispensable for effective patient care. In 2006, the European Network for the Study of Adrenal Tumors (ENSAT; www.ensat.org) proposed standards for the diagnostic procedures in patients with suspected or established ACC. In addition to hormonal tests, imaging by computer tomography (CT) or magnetic resonance (MRI) is recommended. Positron emission tomography with 18F-2-fluoro-2-deoxy-D-glucose (FDG-PET) is an optional method for the diagnosis of an adrenocortical tumor (7).
Test for adrenal hormones Approximately half of all diagnosed adrenal masses are hormonally active (8). For incidentaloma alone, the percentage of hormonally active adrenal tumors falls to 15%, because most of these masses are benign and asymptomatic. Among the hypersecreting incidentalomas, 9.2% were classified as subclinical Cushing’s syndrome, 4.2% as
132 pheochromocytomas, and 1.6% as aldosterone-producing adrenal masses (9). Mantero et al. showed that, in the Italian population, of the patients who underwent adrenalectomy, 52% were diagnosed as having ACA, 12% ACC, and 36% other histological diagnoses. Most of the removed ACAs were nonfunctioning (69%), whereas 25% secreted slightly excessive levels of cortisol, and 6% secreted aldosterone (9). In addition, other investigators have shown that 10e75% of ACCs are hypersecreting. Simultaneous abnormalities in the secretion of cortisol and adrenal androgens occur in 47% of diagnosed ACCs, abnormalities in cortisol secretion occur in 27%, and abnormalities in androgen secretion occur in 6% (9,10). ENSAT has proposed standards for the diagnostic procedures pertaining to patients with ACC. Because at least 10% of ACCs are hormonally active, ENSAT standards have been developed based on blood and urinary hormonal assays. In addition, ENSAT proposes to include an analysis of urine catecholamine excretion to exclude pheochromocytoma (7).
Histological and molecular tests Adrenal masses diagnosed by imaging and hormonal tests may require surgery. Material removed during surgery (or, rarely, from biopsies) is further analyzed using histological and molecular methods. The Tumor, Node, Metastasis (TNM) system, based on the McFarlane classification scale, provides the most reliable approach for estimating tumor stage and malignancy. The TNM scale was published in 2004 by the International Union Against Cancer and the American Joint Committee on Cancer with the World Health Organization (7). Four stages of disease are defined based on the tumor size (<5 cm stage I, >5 cm stage II), the presence of positive lymph nodes (stage III), or metastasis (stage IV). However, accurate ACC patientesurvival predictions are limited when the disease is classified between stages II and III. This observation prompted ENSAT to propose an alternative classification that takes advantage of McFarlane’s scale but presents a more predictive prognostic value (7). To evaluate the tumor malignancy, nine microscopic parameters are used in the Weiss score test, which is based on three structural traits, three cytological traits, and three determinants of the invasion tendency (11). The Weiss score reliably distinguishes between ACAs and ACCs. However, some tumors are classified in the middle of the four-step scale (between steps II and III), presenting difficulties in categorizing a tumor as ACA or ACC. These problems prompted researchers to use genetic methods to create new diagnostic and prognostic markers that confirm the Weiss score (12,13). One example is the assessment of Ki-67 antigen levels by immunostaining or the examination of insulin-like growth factor 2 (IGF2) protein levels. IGF2 has been shown to be overexpressed in 90% of ACCs (14,15). In addition to creating a more precise method to distinguish ACA from ACC, investigators have attempted to design a unified molecular method to allow the early diagnosis and prognosis of ACTs. The focus of these studies has been on selecting several genetic markers for investigation, which may provide expression profiles that could lead to highly accurate diagnoses. This goal has been achieved by
T. Lehmann, T. Wrzesinski comparing gene expression in tumors and in histologically normal adrenocortical tissue using DNA microarray technology and reverse transcriptionepolymerase chain reaction (RT-PCR) analyses. Most microarray and RT-PCR analyses of gene expression levels in ACCs or ACAs are consistent with preceding histopathological classifications (Supplementary Table 1) (16e19). Moreover, transcriptome analyses help to establish markers for favorable and unfavorable prognosis groups (17). The comparison of a massive number of genes ultimately permitted the determination of a smaller set of marker genes for further investigation. The analysis of DLG7 and PINK1 expression improved the diagnosis of ACC and ACA relative to the Weiss scale, particularly for score II and III tumors (17).
Steroid-secreting tumors Monitoring the serum and urine for steroids is usually sufficient for diagnosing steroid-secreting tumors. It has been established that the secretion profile correlates with a specific gene expression signature in steroid-secreting tumors. Cortisol-producing adenomas (CPAs) overexpress specific genes involved in glucocorticoid secretion, especially CYP11A1, CYP17A1, HSD3B1, and CYP11B1. In the same samples, the expression of CYP21A2 and CYP11B2 was not distinct from histologically normal adrenocortical tissue (20). The transcriptome was also investigated in aldosteroneproducing adenomas (APAs) from patients with primary aldosteronism (17,20,21). The RT-PCR array revealed that the CYP11B2 transcript level is frequently elevated in APAs in comparison with that of normal zona glomerulosa (ZG) cells (17,20,22). Investigations of other steroidogenic genes involved in cortisol and androgen synthesis revealed slight changes in CYP17A1, CYB5, CYP11B1, and CYP21A2 expression (17,20,22). Some APAs did not overexpress gene encoding aldosterone synthase (CYP11B2). This effect may be explained by an increase in the ZG mass or by the impact of factors increasing aldosterone synthase activity (21,23). Therefore, the CYP11B2 gene cannot be used in diagnosing APAs because its expression is heterogeneous. Gene-array experiments have also shown that the high density lipoprotein (HDL) receptor and the angiotensin receptor 1 (AT1) G proteinecoupled receptor were overexpressed in APAs (17,24,25). One-third of patients who underwent adrenalectomy were diagnosed with neither ACA nor ACC. Sometimes, these non-ACA, non-ACC patients show macronodular adrenal hyperplasia (26). An expression profile of macronodular adrenal hyperplasia has been analyzed using cDNA microarrays and revealed abnormal levels of gene transcripts encoding Wnt pathway proteins. In addition, investigators observed an upregulation of PRKAR1A, which encodes the regulatory subunit type I of protein kinase A (PKA), the main mediator of cyclic adenosine monophosphate (cAMP) signaling in mammals (26). Genes encoding the CYP11A1, CYP17A1, and CYP21A2 proteins, which are enzymes involved in steroidogenesis, were downregulated. Moreover, contrary to other histological types of adrenocortical benign tumors, IGF2 has also been shown to be downregulated in macronodular adrenal hyperplasia (26).
Molecular basis of adrenocortical cancer In conclusion, numerous studies have shown that the expression of genes responsible for cortisol and aldosterone synthesis is elevated in CPAs and APAs, respectively; however, the basis of these abnormalities has not been established.
Survival, metastasis and prognosis During diagnosis, approximately 17e36% of ACC patients already showed oncogenic invasion of other organs, largely the liver, lymph nodes, lungs, bones, and brain (27e29). A highly accurate method to predict the prognosis and recurrence in patients with ACT has been achieved using transcriptome analysis. The precision of expression analysis is similar to the precision of the TNM scale and the mitotic index (18,30). De Reynies et al. have focused on two genes, BUB1B and PINK1. The comparison of the expression levels of the two genes in different patients resulted in a more accurate prognosis of recurrence than the use of McFarlane’s scale (17).
Etiology Genetics of adrenocortical tumors Various analyses were applied to characterize the genetic background of ACA and ACC and to distinguish between both types of tumors. Comparative genomic hybridization (CGH) analysis has shown that the accumulation of chromosomal aberrations correlates with the size of a tumor (31). The loss or gain of chromosomes 17 and 9 was observed in 28e75% of ACA cases (31e35). The most frequent aberrations (39e47%) in ACC cases were the amplification of chromosomes 5 and 12 and the loss of chromosome 1 (Supplementary Table 2) (31e35). Recently, a genetic test based on quantitative PCR that discriminates between ACAs and ACCs has been designed (13). The greater stability of DNA may be the advantage of such DNA markers in comparison with cDNA markers.
Gene expression Microchip methods have been used widely for several years to investigate ACTs (Supplementary Table 1). Numerous studies have attempted to distinguish between ACA and ACC based on transcriptome analysis. However, the synthesis of a compiled set of data obtained in various laboratories is a challenging task. First, tumor heterogeneity is frequently observed in ACC. Second, variations in experimental protocols result in inconsistent observations. In addition, to determine the causes of aberrant gene expression, complex analyses are required to correlate the transcription signature with abnormalities in the genome and epigenome. Giordano and colleagues attempted to correlate the deregulated primary transcription found in ACC samples with chromosomal amplifications (12q and 5q) and losses (11q, 1p, and 17p) (18). Using gene microchips, they demonstrated that genes encoding proteins involved in the cell cycle, replication, and steroidogenesis as well as genes encoding growth factors were differentially expressed in ACA
133 and ACC. Based on this analysis, these same authors were able to divide ACC samples into two groups, depending on the prognosis for survival.
Cell cycle A comparison of the transcriptomes of ACC and ACA has shown that the deregulation of the cell cycle genes is largely responsible for alterations to the G1/S and G2/M transitions of the cell cycle (36). Overexpression of the G1 cyclin genes CCND1, CCNE1, and CCNE2 and the kinase genes CDK2 and CDK4 as well as a decrease in CDKN1C (p57, Kip2) were observed in ACC (37,38). The genes encoding the cell cycle activators in the G2/M transition, CDK1, CDC25B, and TOP2A, were overexpressed in ACC (36). These changes promote cell division and an uncontrolled increase in cell number.
Steroidogenesis A number of genes encoding proteins involved in steroidogenesis are variably expressed in ACC and ACA. Lower transcriptional activity of particular cytochrome P450 genes (CYP), monooxygenase component ferredoxin (FDX1), and a dehydrogenase gene (HSD3B1) has been observed in malignant adrenal tumors. Receptor-encoding genes are also abnormally expressed in ACC, including an ACTH receptor gene (MC2R) and an HDL receptor (SCARB1) (18,39e41). Steroidogenic factor 1 (SF-1, encoded by NR5A1) regulates the initiation of the transcription of most of these adrenocortical genes. Nevertheless, the data concerning expression levels of NR5A1 are inconsistent. It has been described earlier that the expression of NR5A1 is similar in normal adrenal cortex, ACA, and ACC samples (18,42). In a recent study, it has been shown that in adenomas, the 9q34 region, which includes the NR5A1 locus, is commonly gained and associated with an overexpression of NR5A1 (13). Therefore, the inconsistency of the data concerning NR5A1 expression makes this gene a poor marker for distinguishing between ACAs and ACCs. The transcription of HSD3B and CYP11B2 is controlled by the transcription factor NR4A2, and similarly, to SF-1, it is a member of the nuclear receptor family. The expression of NR4A2 was decreased in ACCs (18) and increased in APAs. CYP11B2 as well as other components of the aldosterone synthesis pathway, including HSD3B, were overexpressed (43). These data confirm the hypothesis that the deregulation of steroidogenic enzymes is at least in part a result of the deregulation of their transcription factors. CYP and HSD3B gene expression abnormalities could not be the result of chromosomal aberrations in loci encoding CYP and HSD3B, but rather the upstream regulators of these genes may be responsible.
Retinoic acid pathway Retinoids are involved in the pathogenesis of several tumors and are used in cancer therapy and prevention. Both retinoic acid synthesis and function may be defective in ACC cells. The rate-limiting step of retinoic acid synthesis requires
134 aldehyde dehydrogenase 1A (ALDH1A1e3) (44). Several microarray studies of ACT samples revealed that the ALDH1A1 and ALDH1A3 messenger RNA (mRNA) levels were lower in ACC than in ACA (16e19,45). A significant reduction in the expression of the retinoic acid receptor RXR alpha (RXRA) was observed in ACC (36). A direct comparison of the results of the CGH analysis and the Gene Set Enrichment Analysis (GSEA; http://www.broadinstitute.org/ gsea/index.jsp) revealed that the decreased expression of RXRB is correlated with the loss of chr 6p21, where RXRB is located (38). This observation suggests that there is a genetic basis for the abnormal retinoic acid production in ACC, but this hypothesis remains to be confirmed.
MicroRNA Small RNAs regulate target genes by accelerating the damage of their transcribed mRNAs and inhibiting their translation. By this method, microRNAs (miRNAs) control numerous processes in a cell, and the abnormal expression of miRNAs has been observed in several types of cancers, including adrenocortical cancer. Levels of miR-210, miR-184, miR-4835p, and miR-503 are increased and miR-375, miR-511, miR335, miR-195, and miR-214 are decreased in ACC cases compared with levels in normal adrenal cases (36,45). It has been postulated that miR-511 and miR-503 could be used as markers to distinguish between ACA and ACC (36). Decreased miR-195 and increased miR-483-5p levels were observed in ACC patients with the worst prognoses (45). Nevertheless, detailed studies revealing the origin of abnormal expression of miRNA and mRNA are required.
IGF2, CTNNB1, and TP53 genes in ACC Numerous studies have shown abnormalities in the expression of IGF2, the b-catenin oncogene (CTNNB1) and the p53 suppressor gene (TP53) in most ACCs. Chromosomal aberrations leading to abnormal mRNA levels of these genes could promote tumorigenesis. A milestone in the investigation of adrenocortical cancer would be in understanding the link between mutations (IGF2, CTNNB1, and TP53) and several key pathways in adrenocortical cells (cell cycle deregulation as well as retinoic acid and steroidogenesis pathways). A gene expression profile of ACC generated using transcriptome analysis led to the classification of two groups of ACCs: tumors with better and poorer outcomes. In the latter group which showed poor prognoses, 50% of the tumors contained mutations in or the inactivation of the tumor suppressor gene TP53, or activating mutations of CTNNB1. All TP53 and CTNNB1 mutations appeared to be mutually exclusive. Approximately 50% of the tumors indicating a poor prognosis displayed neither a TP53 nor a CTNNB1 alteration, suggesting that other unidentified molecular defects may be present (46).
IGF2 The IGF2 gene is located at 11p15 and is a potent mitogen involved in the differentiation and development of adrenals (47e52). IGF2 is indispensable for normal embryonic growth,
T. Lehmann, T. Wrzesinski and the IGF2 gene is subject to tissue-specific maternal imprinting. Therefore, only the paternal allele is expressed (53). IGF2 regulates growth and apoptosis through an interaction with the IGF-1 receptor, and the overexpression of the human IGF-1 receptor promotes a ligand-dependent neoplastic transformation in the adrenal glands (49). The interaction of IGF2 with the IGF-1 receptor activates two pathways involved in carcinogenesis (PI-3/AKT/mTOR and Raf/MEK/MAPK), thereby triggering cell proliferation and migration. In many cell types, the activation of the IGF-1 receptor correlates with tumor progression and poor prognosis. Several genetic alterations, such as the loss of maternal imprinting or the loss of heterozygosity (two paternal alleles) of the 11p15 gene locus, lead to considerable overexpression of IGF2 in ACCs and have been found in the majority of ACAs (90%). The loss of heterozygosity at the 11p15 region correlates with a higher risk of recurrence and occurs more frequently in ACCs (78.5%) than in ACAs (9.5%) (52). In the adrenal cortex of an ACC patient, both the mRNA and protein levels of IGF2 are increased, though they remain unchanged in serum (26,30,54). In H295R cells, IGF2 stimulates expression of the ACTH receptor gene (MC2R), CYP17A1, and HSD3B1, as well as steroid secretion (55). IGF2 may be one of the candidate proteins involved in the pathogenesis of ACC, because the loss of maternal imprinting or the loss of heterozygosity leads to abnormal IGF2 overexpression, which promotes adrenocortical cell proliferation and an increase in steroid secretion.
Wnt pathway Canonical Wnt signals function by regulating the translocation of b-catenin to the nucleus, where b-catenin controls key gene expression programs through interactions with Tcf/Lef and other families of transcription factors. Wnt can also act through noncanonical pathways that do not involve b-catenin activation but instead involve small GTPases/JNK kinase and intracellular calcium (56). Wnt stimulates steroidogenesis via the SF-1 transcription factor (57). An accumulation of bcatenin, detected by immunohistochemical staining, occurs in more than 50% of ACAs and ACCs (12). Nevertheless, CTNNB1 mutations are mostly observed in larger, nonsecreting ACAs, suggesting that the Wnt/b-catenin pathway activation is associated with the development of nonsecreting ACAs and ACCs (12,56). CTNNB1 mutations are involved in the tumorigenesis of primary pigmented nodular adrenocortical disease (PPNAD) (26). The Wnt/b-catenin pathway is also activated in PPNAD and ACA with PRKAR1A mutations, suggesting that there is crosstalk between the cAMP and Wnt/b-catenin pathways during ACT development (58).
Tumor suppressor gene TP53 The suppressor gene TP53, which functions in cell proliferation control, is mutated in many types of tumor cells (59). Mutations in TP53 were observed in 50e80% of children with sporadic ACC in North America and Europe (60,61). In 25e70% of adults with sporadic adrenal cancers, TP53 mutations were observed (62,63). The loss of heterozygosity at locus 17p13 has been identified as typical in ACC (52,64). These changes have been observed in 85% of ACCs and in
Molecular basis of adrenocortical cancer 15% of ACAs. It has been postulated that the homozygosity of 17p13 may be used as a molecular marker of adrenal tumor malignancy. An investigation of a large group of patients with adrenal masses has shown the loss of heterozygosity at 17p13 to be the marker of recurrence after resection (52).
Treatment The treatment strategies for ACCs include resection with or without adjuvant therapy or radiotherapy. Tumors diagnosed in stages IeIII by imagining techniques are removed surgically. Surgery may also be considered for stage IV tumors, yet the therapy depends on the metastasis of the cancer. The overall 5-year survival rate after tumor resection has been estimated at 35% (65,66). The elimination of an adrenocortical tumor in stage IV could diminish steroid hypersecretion and improve the effectiveness of other therapies. The use of laparoscopic therapy of adrenal cancer is currently under debate because of possible tumor cell peritoneal dissemination (67). Radiotherapy is usually ineffective for tumor elimination. However, it has been observed that radiotherapy of the postoperative region may prevent tumor recurrence (66). Several chemical agents, such as antibodies, antisense RNA, and small molecules, have been tested in the third phase of clinical trials on patients with adrenal cancer (68). Most patients benefit from adjuvant mitotane treatment. New targeted therapies, such as IGF-1 receptor inhibitors, are under investigation and may soon improve current treatment options (7,69). It has been shown that isoquinolinone compounds (SF-1 inverse agonists) inhibit the increase in proliferation triggered by an augmented SF-1 dosage in adrenocortical tumor cells and reduce their steroid production. This latter property may also reveal benefits for drugs used in the therapy of ACTs to alleviate the symptoms of virilization and Cushing’s syndrome often associated with the tumor burden (70).
Future considerations ACA and ACC tumors are highly heterogeneous. It remains a goal to design a reliable method to distinguish between benign ACA and malignant ACC based on differences among the transcriptomes. However, transcriptome analysis has revealed many abnormalities in gene expression. Additionally, the primary cause of adrenocortical cancer is still unclear. The prognostic value of the mRNA-based test is also limited. These types of cancers, such as ACC, require international coordinated cooperation to increase the number of analyzed samples. To better understand carcinogenesis in the adrenal cortex and to reveal the mechanisms governing the cancer progression, parallel studies of the genome, proteome, and epigenetics are required.
Acknowledgments We thank Ms. Beata Raczak and M.Sc. Bogumiła Ratajczak for their indispensable help during the preparation of this manuscript.
135
Supplementary data Supplementary data related to this article can be found online at doi:10.1016/j.cancergen.2012.02.009.
References 1. Mannelli M, Colagrande S, Valeri A, et al. Incidental and metastatic adrenal masses. Semin Oncol 2010;37:649e661. 2. Angeli A, Osella G, Ali A, et al. Adrenal incidentaloma: an overview of clinical and epidemiological data from the National Italian Study Group. Horm Res 1997;47:279e283. 3. Grumbach MM, Biller BM, Braunstein GD, et al. Management of the clinically inapparent adrenal mass ("incidentaloma"). Ann Intern Med 2003;138:424e429. 4. Luton JP, Martinez M, Coste J, et al. Outcome in patients with adrenal incidentaloma selected for surgery: an analysis of 88 cases investigated in a single clinical center. Eur J Endocrinol 2000;143:111e117. 5. Allolio B, Fassnacht M. Clinical review: adrenocortical carcinoma: clinical update. J Clin Endocrinol Metab 2006;91:2027e2037. 6. Kirschner LS. Emerging treatment strategies for adrenocortical carcinoma: a new hope. J Clin Endocrinol Metab 2006;91:14e21. 7. Fassnacht M, Allolio B. Clinical management of adrenocortical carcinoma. Best Pract Res Clin Endocrinol Metab 2009;23: 273e289. 8. Icard P, Chapuis Y, Andreassian B, et al. Adrenocortical carcinoma in surgically treated patients: a retrospective study on 156 cases by the French Association of Endocrine Surgery. Surgery 1992;112:972e979. discussion 979e980. 9. Mantero F, Terzolo M, Arnaldi G, et al; Study Group on Adrenal Tumors of the Italian Society of Endocrinology. A survey on adrenal incidentaloma in Italy. J Clin Endocrinol Metab 2000;85: 637e644. 10. Abiven G, Coste J, Groussin L, et al. Clinical and biological features in the prognosis of adrenocortical cancer: poor outcome of cortisol-secreting tumors in a series of 202 consecutive patients. J Clin Endocrinol Metab 2006;91:2650e2655. 11. Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol 1984;8:163e169. 12. Tissier F, Cavard C, Groussin L, et al. Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Res 2005;65:7622e7627. 13. Barreau O, de Reynies A, Wilmot-Roussel H, et al. Clinical and pathophysiological implications of chromosomal alterations in adrenocortical tumors: an integrated genomic approach. J Clin Endocrinol Metab 2012;97:E301eE311. 14. Nakazumi H, Sasano H, Iino K, et al. Expression of cell cycle inhibitor p27 and Ki-67 in human adrenocortical neoplasms. Mod Pathol 1998;11:1165e1170. 15. Schmitt A, Saremaslani P, Schmid S, et al. IGFII and MIB1 immunohistochemistry is helpful for the differentiation of benign from malignant adrenocortical tumours. Histopathology 2006;49: 298e307. 16. Velazquez-Fernandez D, Laurell C, Geli J, et al. Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy. Surgery 2005;138:1087e1094. 17. de Reynies A, Assie G, Rickman DS, et al. Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival. J Clin Oncol 2009;27:1108e1115. 18. Giordano TJ, Kuick R, Else T, et al. Molecular classification and prognostication of adrenocortical tumors by transcriptome profiling. Clin Cancer Res 2009;15:668e676.
136 19. Laurell C, Velazquez-Fernandez D, Lindsten K, et al. Transcriptional profiling enables molecular classification of adrenocortical tumours. Eur J Endocrinol 2009;161:141e152. 20. Bassett MH, Mayhew B, Rehman K, et al. Expression profiles for steroidogenic enzymes in adrenocortical disease. J Clin Endocrinol Metab 2005;90:5446e5455. 21. Lenzini L, Seccia TM, Aldighieri E, et al. Heterogeneity of aldosterone-producing adenomas revealed by a whole transcriptome analysis. Hypertension 2007;50:1106e1113. 22. Wang T, Satoh F, Morimoto R, et al. Gene expression profiles in aldosterone-producing adenomas and adjacent adrenal glands. Eur J Endocrinol 2011;164:613e619. 23. Fallo F, Pezzi V, Barzon L, et al. Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas. Eur J Endocrinol 2002;147:795e802. 24. Saner-Amigh K, Mayhew BA, Mantero F, et al. Elevated expression of luteinizing hormone receptor in aldosteroneproducing adenomas. J Clin Endocrinol Metab 2006;91: 1136e1142. 25. Ye P, Mariniello B, Mantero F, et al. G-protein-coupled receptors in aldosterone-producing adenomas: a potential cause of hyperaldosteronism. J Endocrinol 2007;195:39e48. 26. Tadjine M, Lampron A, Ouadi L, et al. Detection of somatic betacatenin mutations in primary pigmented nodular adrenocortical disease (PPNAD). Clin Endocrinol (Oxf) 2008;69:367e373. 27. Søreide JA, Brabrand K, Thoresen SO. Adrenal cortical carcinoma in Norway, 1970-1984. World J Surg 1992;16:663e667. discussion 668. 28. Kasperlik-Zaluska AA, Migdalska BM, Zgliczynski S, et al. Adrenocortical carcinoma. A clinical study and treatment results of 52 patients. Cancer 1995;75:2587e2591. 29. Hanna NN, Kenady DE. Advances in the management of adrenal tumors. Curr Opin Oncol 2000;12:49e53. 30. de Fraipont F, El Atifi M, Cherradi N, et al. Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 2005; 90:1819e1829. 31. Sidhu S, Marsh DJ, Theodosopoulos G, et al. Comparative genomic hybridization analysis of adrenocortical tumors. J Clin Endocrinol Metab 2002;87:3467e3474. 32. Kjellman M, Kallioniemi OP, Karhu R, et al. Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy. Cancer Res 1996;56:4219e4223. 33. Russell AJ, Sibbald J, Haak H, et al. Increasing genome instability in adrenocortical carcinoma progression with involvement of chromosomes 3, 9 and X at the adenoma stage. Br J Cancer 1999;81:684e689. 34. Zhao J, Speel EJ, Muletta-Feurer S, et al. Analysis of genomic alterations in sporadic adrenocortical lesions. Gain of chromosome 17 is an early event in adrenocortical tumorigenesis. Am J Pathol 1999;155:1039e1045. 35. Dohna M, Reincke M, Mincheva A, et al. Adrenocortical carcinoma is characterized by a high frequency of chromosomal gains and high-level amplifications. Genes Chromosomes Cancer 2000;28:145e152. 36. Tombol Z, Szabo PM, Molnar V, et al. Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis. Endocr Relat Cancer 2009;16:895e906. 37. Lombardi CP, Raffaelli M, Pani G, et al. Gene expression profiling of adrenal cortical tumors by cDNA macroarray analysis. Results of a preliminary study. Biomed Pharmacother 2006;60:186e190. 38. Szabo PM, Tamasi V, Molnar V, et al. Meta-analysis of adrenocortical tumour genomics data: novel pathogenic pathways revealed. Oncogene 2010;29:3163e3172.
T. Lehmann, T. Wrzesinski 39. Reincke M, Mora P, Beuschlein F, et al. Deletion of the adrenocorticotropin receptor gene in human adrenocortical tumors: implications for tumorigenesis. J Clin Endocrinol Metab 1997;82: 3054e3058. 40. Reincke M, Beuschlein F, Menig G, et al. Localization and expression of adrenocorticotropic hormone receptor mRNA in normal and neoplastic human adrenal cortex. J Endocrinol 1998; 156:415e423. 41. Slater EP, Diehl SM, Langer P, et al. Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors. Eur J Endocrinol 2006;154:587e598. 42. West AN, Neale GA, Pounds S, et al. Gene expression profiling of childhood adrenocortical tumors. Cancer Res 2007;67: 600e608. 43. Lu L, Suzuki T, Yoshikawa Y, et al. Nur-related factor 1 and nerve growth factor-induced clone B in human adrenal cortex and its disorders. J Clin Endocrinol Metab 2004;89: 4113e4118. 44. el Akawi Z, Napoli JL. Rat liver cytosolic retinal dehydrogenase: comparison of 13-cis-, 9-cis-, and all-trans-retinal as substrates and effects of cellular retinoid-binding proteins and retinoic acid on activity. Biochemistry 1994;33:1938e1943. 45. Soon PS, Gill AJ, Benn DE, et al. Microarray gene expression and immunohistochemistry analyses of adrenocortical tumors identify IGF2 and Ki-67 as useful in differentiating carcinomas from adenomas. Endocr Relat Cancer 2009;16: 573e583. 46. Ragazzon B, Libe R, Gaujoux S, et al. Transcriptome analysis reveals that p53 and {beta}-catenin alterations occur in a group of aggressive adrenocortical cancers. Cancer Res 2010;70: 8276e8281. 47. Hermsen IG, Fassnacht M, Terzolo M, et al. Plasma concentrations of o, p’DDD, o, p’DDA, and o, p’DDE as predictors of tumor response to mitotane in adrenocortical carcinoma: results of a retrospective ENS@T multicenter study. J Clin Endocrinol Metab 2011;96:1844e1851. 48. Ilvesmaki V, Kahri AI, Miettinen PJ, et al. Insulin-like growth factors (IGFs) and their receptors in adrenal tumors: high IGF-II expression in functional adrenocortical carcinomas. J Clin Endocrinol Metab 1993;77:852e858. 49. Mesiano S, Mellon SH, Jaffe RB. Mitogenic action, regulation, and localization of insulin-like growth factors in the human fetal adrenal gland. J Clin Endocrinol Metab 1993;76:968e976. 50. Gicquel C, Bertagna X, Le Bouc Y. Recent advances in the pathogenesis of adrenocortical tumours. Eur J Endocrinol 1995; 133:133e144. 51. Gicquel C, Raffin-Sanson ML, Gaston V, et al. Structural and functional abnormalities at 11p15 are associated with the malignant phenotype in sporadic adrenocortical tumors: study on a series of 82 tumors. J Clin Endocrinol Metab 1997;82: 2559e2565. 52. Gicquel C, Bertagna X, Gaston V, et al. Molecular markers and long-term recurrences in a large cohort of patients with sporadic adrenocortical tumors. Cancer Res 2001;61:6762e6767. 53. DeChiara TM, Robertson EJ, Efstratiadis A. Parental imprinting of the mouse insulin-like growth factor II gene. Cell 1991;64: 849e859. 54. Giordano TJ, Thomas DG, Kuick R, et al. Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. Am J Pathol 2003;162:521e531. 55. l’Allemand D, Penhoat A, Lebrethon MC, et al. Insulin-like growth factors enhance steroidogenic enzyme and corticotropin receptor messenger ribonucleic acid levels and corticotropin steroidogenic responsiveness in cultured human adrenocortical cells. J Clin Endocrinol Metab 1996;81:3892e3897. 56. El Wakil A, Lalli E. The Wnt/beta-catenin pathway in adrenocortical development and cancer. Mol Cell Endocrinol 2011;332: 32e37.
Molecular basis of adrenocortical cancer 57. Schinner S, Willenberg HS, Krause D, et al. Adipocyte-derived products induce the transcription of the StAR promoter and stimulate aldosterone and cortisol secretion from adrenocortical cells through the Wnt-signaling pathway. Int J Obes (Lond) 2007;31:864e870. 58. Gaujoux S, Tissier F, Groussin L, et al. Wnt/beta-catenin and 30 ,50 -cyclic adenosine 50 -monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors. J Clin Endocrinol Metab 2008;93:4135e4140. 59. Caron de Fromentel C, Soussi T. TP53 tumor suppressor gene: a model for investigating human mutagenesis. Genes Chromosomes Cancer 1992;4:1e15. 60. Wagner J, Portwine C, Rabin K, et al. High frequency of germline p53 mutations in childhood adrenocortical cancer. J Natl Cancer Inst 1994;86:1707e1710. 61. Varley JM, McGown G, Thorncroft M, et al. Are there lowpenetrance TP53 Alleles? evidence from childhood adrenocortical tumors. Am J Hum Genet 1999;65:995e1006. 62. Lin SR, Lee YJ, Tsai JH. Mutations of the p53 gene in human functional adrenal neoplasms. J Clin Endocrinol Metab 1994;78: 483e491.
137 63. Barzon L, Chilosi M, Fallo F, et al. Molecular analysis of CDKN1C and TP53 in sporadic adrenal tumors. Eur J Endocrinol 2001;145:207e212. 64. Yano T, Linehan M, Anglard P, et al. Genetic changes in human adrenocortical carcinomas. J Natl Cancer Inst 1989;81:518e523. 65. Schteingart DE, Doherty GM, Gauger PG, et al. Management of patients with adrenal cancer: recommendations of an international consensus conference. Endocr Relat Cancer 2005;12: 667e680. 66. Libe R, Fratticci A, Bertherat J. Adrenocortical cancer: pathophysiology and clinical management. Endocr Relat Cancer 2007;14:13e28. 67. Cobb WS, Kercher KW, Sing RF, et al. Laparoscopic adrenalectomy for malignancy. Am J Surg 2005;189:405e411. 68. Baehner FL, Lee M, Demeure MJ, et al. Genomic signatures of cancer: basis for individualized risk assessment, selective staging and therapy. J Surg Oncol 2011;103:563e573. 69. Fassnacht M, Libe R, Kroiss M, et al. Adrenocortical carcinoma: a clinician’s update. Nat Rev Endocrinol 2011;7:323e335. 70. Doghman M, Madoux F, Hodder P, et al. Identification and characterization of steroidogenic factor-1 inverse agonists. Methods Enzymol 2010;485:3e23.
REVIEW
The molecular basis of adrenocortical cancer Tomasz Lehmann*, Tomasz Wrzesinski Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, Poznan, Poland Adrenocortical tumors (ACTs) are common, and most are benign adrenocortical adenomas (ACAs). Malignant adrenocortical carcinoma (ACC) is a rare tumor type and is observed at the rate of one or two cases per million annually. ACTs are classified as either ACAs or ACCs by histopathologic methods that are based on nine Weiss scoring criteria, including the nuclear grade, mitotic rate, presence of necrosis, and others. In this review, we describe the findings of studies that have examined the molecular basis of ACTs, and we compare transcriptome analysis with other diagnostic approaches. ACTs are occasionally difficult to classify. Therefore, molecular techniques, such as microarray analysis, have recently been applied to overcome some of these diagnostic problems. We also discuss the likelihood of the diagnosis and discernment between ACAs and ACCs based on the molecular tests. To show the recent progress in understanding the etiology of ACTs, we highlight the relationship between genetic analysis and transcriptome analysis. We attempt to understand the role of abnormal cell growth and steroid hormone secretion. Genetic and transcriptome analyses have improved our understanding of ACTs considerably, yet many unanswered questions remain. Keywords Adrenal, cancer, gene expression, microarray ª 2012 Elsevier Inc. All rights reserved.
Symptoms, clinical diagnosis, classification, staging, and prognosis Diagnosis Most frequently, malignant tumors in the adrenal glands are formed from the metastasis of cancers that have initiated growth in other organs, largely the lungs and breast. Most nonmetastatic adrenal masses are asymptomatic, benign tumors called adrenocortical adenomas (ACAs). The adrenocortical tumors (ACTs) revealed during diagnostic procedures for unrelated disorders are called incidentaloma (1). Most ACAs are likely undiagnosed, and among all diagnosed ACAs, 52% are subsequently removed through surgical operation (2). ACAs are frequent in the population (found in >3% of people older than 50 y) (3). Conversely, the prevalence of malignant adrenocortical carcinoma (ACC) is estimated to be very infrequent, with only 1e12 cases per million adults (3). It remains a matter of debate whether ACA is a separate disease or is part of the process of tumor progression. ACCs possess metastatic potential with secreting or nonsecreting
Received August 28, 2011; received in revised form February 4, 2012; accepted February 21, 2012. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergen.2012.02.009
characteristics. The 5-year survival rate of patients with metastatic ACC is 16e38% (4e6). ACC may be detected during a routine visualization test or diagnosed using a steroid hormone assay, or based on the symptoms resulting from tumor pressure on neighboring organs.
Imaging methods Proper imaging and diagnosis of ACTs are indispensable for effective patient care. In 2006, the European Network for the Study of Adrenal Tumors (ENSAT; www.ensat.org) proposed standards for the diagnostic procedures in patients with suspected or established ACC. In addition to hormonal tests, imaging by computer tomography (CT) or magnetic resonance (MRI) is recommended. Positron emission tomography with 18F-2-fluoro-2-deoxy-D-glucose (FDG-PET) is an optional method for the diagnosis of an adrenocortical tumor (7).
Test for adrenal hormones Approximately half of all diagnosed adrenal masses are hormonally active (8). For incidentaloma alone, the percentage of hormonally active adrenal tumors falls to 15%, because most of these masses are benign and asymptomatic. Among the hypersecreting incidentalomas, 9.2% were classified as subclinical Cushing’s syndrome, 4.2% as
132 pheochromocytomas, and 1.6% as aldosterone-producing adrenal masses (9). Mantero et al. showed that, in the Italian population, of the patients who underwent adrenalectomy, 52% were diagnosed as having ACA, 12% ACC, and 36% other histological diagnoses. Most of the removed ACAs were nonfunctioning (69%), whereas 25% secreted slightly excessive levels of cortisol, and 6% secreted aldosterone (9). In addition, other investigators have shown that 10e75% of ACCs are hypersecreting. Simultaneous abnormalities in the secretion of cortisol and adrenal androgens occur in 47% of diagnosed ACCs, abnormalities in cortisol secretion occur in 27%, and abnormalities in androgen secretion occur in 6% (9,10). ENSAT has proposed standards for the diagnostic procedures pertaining to patients with ACC. Because at least 10% of ACCs are hormonally active, ENSAT standards have been developed based on blood and urinary hormonal assays. In addition, ENSAT proposes to include an analysis of urine catecholamine excretion to exclude pheochromocytoma (7).
Histological and molecular tests Adrenal masses diagnosed by imaging and hormonal tests may require surgery. Material removed during surgery (or, rarely, from biopsies) is further analyzed using histological and molecular methods. The Tumor, Node, Metastasis (TNM) system, based on the McFarlane classification scale, provides the most reliable approach for estimating tumor stage and malignancy. The TNM scale was published in 2004 by the International Union Against Cancer and the American Joint Committee on Cancer with the World Health Organization (7). Four stages of disease are defined based on the tumor size (<5 cm stage I, >5 cm stage II), the presence of positive lymph nodes (stage III), or metastasis (stage IV). However, accurate ACC patientesurvival predictions are limited when the disease is classified between stages II and III. This observation prompted ENSAT to propose an alternative classification that takes advantage of McFarlane’s scale but presents a more predictive prognostic value (7). To evaluate the tumor malignancy, nine microscopic parameters are used in the Weiss score test, which is based on three structural traits, three cytological traits, and three determinants of the invasion tendency (11). The Weiss score reliably distinguishes between ACAs and ACCs. However, some tumors are classified in the middle of the four-step scale (between steps II and III), presenting difficulties in categorizing a tumor as ACA or ACC. These problems prompted researchers to use genetic methods to create new diagnostic and prognostic markers that confirm the Weiss score (12,13). One example is the assessment of Ki-67 antigen levels by immunostaining or the examination of insulin-like growth factor 2 (IGF2) protein levels. IGF2 has been shown to be overexpressed in 90% of ACCs (14,15). In addition to creating a more precise method to distinguish ACA from ACC, investigators have attempted to design a unified molecular method to allow the early diagnosis and prognosis of ACTs. The focus of these studies has been on selecting several genetic markers for investigation, which may provide expression profiles that could lead to highly accurate diagnoses. This goal has been achieved by
T. Lehmann, T. Wrzesinski comparing gene expression in tumors and in histologically normal adrenocortical tissue using DNA microarray technology and reverse transcriptionepolymerase chain reaction (RT-PCR) analyses. Most microarray and RT-PCR analyses of gene expression levels in ACCs or ACAs are consistent with preceding histopathological classifications (Supplementary Table 1) (16e19). Moreover, transcriptome analyses help to establish markers for favorable and unfavorable prognosis groups (17). The comparison of a massive number of genes ultimately permitted the determination of a smaller set of marker genes for further investigation. The analysis of DLG7 and PINK1 expression improved the diagnosis of ACC and ACA relative to the Weiss scale, particularly for score II and III tumors (17).
Steroid-secreting tumors Monitoring the serum and urine for steroids is usually sufficient for diagnosing steroid-secreting tumors. It has been established that the secretion profile correlates with a specific gene expression signature in steroid-secreting tumors. Cortisol-producing adenomas (CPAs) overexpress specific genes involved in glucocorticoid secretion, especially CYP11A1, CYP17A1, HSD3B1, and CYP11B1. In the same samples, the expression of CYP21A2 and CYP11B2 was not distinct from histologically normal adrenocortical tissue (20). The transcriptome was also investigated in aldosteroneproducing adenomas (APAs) from patients with primary aldosteronism (17,20,21). The RT-PCR array revealed that the CYP11B2 transcript level is frequently elevated in APAs in comparison with that of normal zona glomerulosa (ZG) cells (17,20,22). Investigations of other steroidogenic genes involved in cortisol and androgen synthesis revealed slight changes in CYP17A1, CYB5, CYP11B1, and CYP21A2 expression (17,20,22). Some APAs did not overexpress gene encoding aldosterone synthase (CYP11B2). This effect may be explained by an increase in the ZG mass or by the impact of factors increasing aldosterone synthase activity (21,23). Therefore, the CYP11B2 gene cannot be used in diagnosing APAs because its expression is heterogeneous. Gene-array experiments have also shown that the high density lipoprotein (HDL) receptor and the angiotensin receptor 1 (AT1) G proteinecoupled receptor were overexpressed in APAs (17,24,25). One-third of patients who underwent adrenalectomy were diagnosed with neither ACA nor ACC. Sometimes, these non-ACA, non-ACC patients show macronodular adrenal hyperplasia (26). An expression profile of macronodular adrenal hyperplasia has been analyzed using cDNA microarrays and revealed abnormal levels of gene transcripts encoding Wnt pathway proteins. In addition, investigators observed an upregulation of PRKAR1A, which encodes the regulatory subunit type I of protein kinase A (PKA), the main mediator of cyclic adenosine monophosphate (cAMP) signaling in mammals (26). Genes encoding the CYP11A1, CYP17A1, and CYP21A2 proteins, which are enzymes involved in steroidogenesis, were downregulated. Moreover, contrary to other histological types of adrenocortical benign tumors, IGF2 has also been shown to be downregulated in macronodular adrenal hyperplasia (26).
Molecular basis of adrenocortical cancer In conclusion, numerous studies have shown that the expression of genes responsible for cortisol and aldosterone synthesis is elevated in CPAs and APAs, respectively; however, the basis of these abnormalities has not been established.
Survival, metastasis and prognosis During diagnosis, approximately 17e36% of ACC patients already showed oncogenic invasion of other organs, largely the liver, lymph nodes, lungs, bones, and brain (27e29). A highly accurate method to predict the prognosis and recurrence in patients with ACT has been achieved using transcriptome analysis. The precision of expression analysis is similar to the precision of the TNM scale and the mitotic index (18,30). De Reynies et al. have focused on two genes, BUB1B and PINK1. The comparison of the expression levels of the two genes in different patients resulted in a more accurate prognosis of recurrence than the use of McFarlane’s scale (17).
Etiology Genetics of adrenocortical tumors Various analyses were applied to characterize the genetic background of ACA and ACC and to distinguish between both types of tumors. Comparative genomic hybridization (CGH) analysis has shown that the accumulation of chromosomal aberrations correlates with the size of a tumor (31). The loss or gain of chromosomes 17 and 9 was observed in 28e75% of ACA cases (31e35). The most frequent aberrations (39e47%) in ACC cases were the amplification of chromosomes 5 and 12 and the loss of chromosome 1 (Supplementary Table 2) (31e35). Recently, a genetic test based on quantitative PCR that discriminates between ACAs and ACCs has been designed (13). The greater stability of DNA may be the advantage of such DNA markers in comparison with cDNA markers.
Gene expression Microchip methods have been used widely for several years to investigate ACTs (Supplementary Table 1). Numerous studies have attempted to distinguish between ACA and ACC based on transcriptome analysis. However, the synthesis of a compiled set of data obtained in various laboratories is a challenging task. First, tumor heterogeneity is frequently observed in ACC. Second, variations in experimental protocols result in inconsistent observations. In addition, to determine the causes of aberrant gene expression, complex analyses are required to correlate the transcription signature with abnormalities in the genome and epigenome. Giordano and colleagues attempted to correlate the deregulated primary transcription found in ACC samples with chromosomal amplifications (12q and 5q) and losses (11q, 1p, and 17p) (18). Using gene microchips, they demonstrated that genes encoding proteins involved in the cell cycle, replication, and steroidogenesis as well as genes encoding growth factors were differentially expressed in ACA
133 and ACC. Based on this analysis, these same authors were able to divide ACC samples into two groups, depending on the prognosis for survival.
Cell cycle A comparison of the transcriptomes of ACC and ACA has shown that the deregulation of the cell cycle genes is largely responsible for alterations to the G1/S and G2/M transitions of the cell cycle (36). Overexpression of the G1 cyclin genes CCND1, CCNE1, and CCNE2 and the kinase genes CDK2 and CDK4 as well as a decrease in CDKN1C (p57, Kip2) were observed in ACC (37,38). The genes encoding the cell cycle activators in the G2/M transition, CDK1, CDC25B, and TOP2A, were overexpressed in ACC (36). These changes promote cell division and an uncontrolled increase in cell number.
Steroidogenesis A number of genes encoding proteins involved in steroidogenesis are variably expressed in ACC and ACA. Lower transcriptional activity of particular cytochrome P450 genes (CYP), monooxygenase component ferredoxin (FDX1), and a dehydrogenase gene (HSD3B1) has been observed in malignant adrenal tumors. Receptor-encoding genes are also abnormally expressed in ACC, including an ACTH receptor gene (MC2R) and an HDL receptor (SCARB1) (18,39e41). Steroidogenic factor 1 (SF-1, encoded by NR5A1) regulates the initiation of the transcription of most of these adrenocortical genes. Nevertheless, the data concerning expression levels of NR5A1 are inconsistent. It has been described earlier that the expression of NR5A1 is similar in normal adrenal cortex, ACA, and ACC samples (18,42). In a recent study, it has been shown that in adenomas, the 9q34 region, which includes the NR5A1 locus, is commonly gained and associated with an overexpression of NR5A1 (13). Therefore, the inconsistency of the data concerning NR5A1 expression makes this gene a poor marker for distinguishing between ACAs and ACCs. The transcription of HSD3B and CYP11B2 is controlled by the transcription factor NR4A2, and similarly, to SF-1, it is a member of the nuclear receptor family. The expression of NR4A2 was decreased in ACCs (18) and increased in APAs. CYP11B2 as well as other components of the aldosterone synthesis pathway, including HSD3B, were overexpressed (43). These data confirm the hypothesis that the deregulation of steroidogenic enzymes is at least in part a result of the deregulation of their transcription factors. CYP and HSD3B gene expression abnormalities could not be the result of chromosomal aberrations in loci encoding CYP and HSD3B, but rather the upstream regulators of these genes may be responsible.
Retinoic acid pathway Retinoids are involved in the pathogenesis of several tumors and are used in cancer therapy and prevention. Both retinoic acid synthesis and function may be defective in ACC cells. The rate-limiting step of retinoic acid synthesis requires
134 aldehyde dehydrogenase 1A (ALDH1A1e3) (44). Several microarray studies of ACT samples revealed that the ALDH1A1 and ALDH1A3 messenger RNA (mRNA) levels were lower in ACC than in ACA (16e19,45). A significant reduction in the expression of the retinoic acid receptor RXR alpha (RXRA) was observed in ACC (36). A direct comparison of the results of the CGH analysis and the Gene Set Enrichment Analysis (GSEA; http://www.broadinstitute.org/ gsea/index.jsp) revealed that the decreased expression of RXRB is correlated with the loss of chr 6p21, where RXRB is located (38). This observation suggests that there is a genetic basis for the abnormal retinoic acid production in ACC, but this hypothesis remains to be confirmed.
MicroRNA Small RNAs regulate target genes by accelerating the damage of their transcribed mRNAs and inhibiting their translation. By this method, microRNAs (miRNAs) control numerous processes in a cell, and the abnormal expression of miRNAs has been observed in several types of cancers, including adrenocortical cancer. Levels of miR-210, miR-184, miR-4835p, and miR-503 are increased and miR-375, miR-511, miR335, miR-195, and miR-214 are decreased in ACC cases compared with levels in normal adrenal cases (36,45). It has been postulated that miR-511 and miR-503 could be used as markers to distinguish between ACA and ACC (36). Decreased miR-195 and increased miR-483-5p levels were observed in ACC patients with the worst prognoses (45). Nevertheless, detailed studies revealing the origin of abnormal expression of miRNA and mRNA are required.
IGF2, CTNNB1, and TP53 genes in ACC Numerous studies have shown abnormalities in the expression of IGF2, the b-catenin oncogene (CTNNB1) and the p53 suppressor gene (TP53) in most ACCs. Chromosomal aberrations leading to abnormal mRNA levels of these genes could promote tumorigenesis. A milestone in the investigation of adrenocortical cancer would be in understanding the link between mutations (IGF2, CTNNB1, and TP53) and several key pathways in adrenocortical cells (cell cycle deregulation as well as retinoic acid and steroidogenesis pathways). A gene expression profile of ACC generated using transcriptome analysis led to the classification of two groups of ACCs: tumors with better and poorer outcomes. In the latter group which showed poor prognoses, 50% of the tumors contained mutations in or the inactivation of the tumor suppressor gene TP53, or activating mutations of CTNNB1. All TP53 and CTNNB1 mutations appeared to be mutually exclusive. Approximately 50% of the tumors indicating a poor prognosis displayed neither a TP53 nor a CTNNB1 alteration, suggesting that other unidentified molecular defects may be present (46).
IGF2 The IGF2 gene is located at 11p15 and is a potent mitogen involved in the differentiation and development of adrenals (47e52). IGF2 is indispensable for normal embryonic growth,
T. Lehmann, T. Wrzesinski and the IGF2 gene is subject to tissue-specific maternal imprinting. Therefore, only the paternal allele is expressed (53). IGF2 regulates growth and apoptosis through an interaction with the IGF-1 receptor, and the overexpression of the human IGF-1 receptor promotes a ligand-dependent neoplastic transformation in the adrenal glands (49). The interaction of IGF2 with the IGF-1 receptor activates two pathways involved in carcinogenesis (PI-3/AKT/mTOR and Raf/MEK/MAPK), thereby triggering cell proliferation and migration. In many cell types, the activation of the IGF-1 receptor correlates with tumor progression and poor prognosis. Several genetic alterations, such as the loss of maternal imprinting or the loss of heterozygosity (two paternal alleles) of the 11p15 gene locus, lead to considerable overexpression of IGF2 in ACCs and have been found in the majority of ACAs (90%). The loss of heterozygosity at the 11p15 region correlates with a higher risk of recurrence and occurs more frequently in ACCs (78.5%) than in ACAs (9.5%) (52). In the adrenal cortex of an ACC patient, both the mRNA and protein levels of IGF2 are increased, though they remain unchanged in serum (26,30,54). In H295R cells, IGF2 stimulates expression of the ACTH receptor gene (MC2R), CYP17A1, and HSD3B1, as well as steroid secretion (55). IGF2 may be one of the candidate proteins involved in the pathogenesis of ACC, because the loss of maternal imprinting or the loss of heterozygosity leads to abnormal IGF2 overexpression, which promotes adrenocortical cell proliferation and an increase in steroid secretion.
Wnt pathway Canonical Wnt signals function by regulating the translocation of b-catenin to the nucleus, where b-catenin controls key gene expression programs through interactions with Tcf/Lef and other families of transcription factors. Wnt can also act through noncanonical pathways that do not involve b-catenin activation but instead involve small GTPases/JNK kinase and intracellular calcium (56). Wnt stimulates steroidogenesis via the SF-1 transcription factor (57). An accumulation of bcatenin, detected by immunohistochemical staining, occurs in more than 50% of ACAs and ACCs (12). Nevertheless, CTNNB1 mutations are mostly observed in larger, nonsecreting ACAs, suggesting that the Wnt/b-catenin pathway activation is associated with the development of nonsecreting ACAs and ACCs (12,56). CTNNB1 mutations are involved in the tumorigenesis of primary pigmented nodular adrenocortical disease (PPNAD) (26). The Wnt/b-catenin pathway is also activated in PPNAD and ACA with PRKAR1A mutations, suggesting that there is crosstalk between the cAMP and Wnt/b-catenin pathways during ACT development (58).
Tumor suppressor gene TP53 The suppressor gene TP53, which functions in cell proliferation control, is mutated in many types of tumor cells (59). Mutations in TP53 were observed in 50e80% of children with sporadic ACC in North America and Europe (60,61). In 25e70% of adults with sporadic adrenal cancers, TP53 mutations were observed (62,63). The loss of heterozygosity at locus 17p13 has been identified as typical in ACC (52,64). These changes have been observed in 85% of ACCs and in
Molecular basis of adrenocortical cancer 15% of ACAs. It has been postulated that the homozygosity of 17p13 may be used as a molecular marker of adrenal tumor malignancy. An investigation of a large group of patients with adrenal masses has shown the loss of heterozygosity at 17p13 to be the marker of recurrence after resection (52).
Treatment The treatment strategies for ACCs include resection with or without adjuvant therapy or radiotherapy. Tumors diagnosed in stages IeIII by imagining techniques are removed surgically. Surgery may also be considered for stage IV tumors, yet the therapy depends on the metastasis of the cancer. The overall 5-year survival rate after tumor resection has been estimated at 35% (65,66). The elimination of an adrenocortical tumor in stage IV could diminish steroid hypersecretion and improve the effectiveness of other therapies. The use of laparoscopic therapy of adrenal cancer is currently under debate because of possible tumor cell peritoneal dissemination (67). Radiotherapy is usually ineffective for tumor elimination. However, it has been observed that radiotherapy of the postoperative region may prevent tumor recurrence (66). Several chemical agents, such as antibodies, antisense RNA, and small molecules, have been tested in the third phase of clinical trials on patients with adrenal cancer (68). Most patients benefit from adjuvant mitotane treatment. New targeted therapies, such as IGF-1 receptor inhibitors, are under investigation and may soon improve current treatment options (7,69). It has been shown that isoquinolinone compounds (SF-1 inverse agonists) inhibit the increase in proliferation triggered by an augmented SF-1 dosage in adrenocortical tumor cells and reduce their steroid production. This latter property may also reveal benefits for drugs used in the therapy of ACTs to alleviate the symptoms of virilization and Cushing’s syndrome often associated with the tumor burden (70).
Future considerations ACA and ACC tumors are highly heterogeneous. It remains a goal to design a reliable method to distinguish between benign ACA and malignant ACC based on differences among the transcriptomes. However, transcriptome analysis has revealed many abnormalities in gene expression. Additionally, the primary cause of adrenocortical cancer is still unclear. The prognostic value of the mRNA-based test is also limited. These types of cancers, such as ACC, require international coordinated cooperation to increase the number of analyzed samples. To better understand carcinogenesis in the adrenal cortex and to reveal the mechanisms governing the cancer progression, parallel studies of the genome, proteome, and epigenetics are required.
Acknowledgments We thank Ms. Beata Raczak and M.Sc. Bogumiła Ratajczak for their indispensable help during the preparation of this manuscript.
135
Supplementary data Supplementary data related to this article can be found online at doi:10.1016/j.cancergen.2012.02.009.
References 1. Mannelli M, Colagrande S, Valeri A, et al. Incidental and metastatic adrenal masses. Semin Oncol 2010;37:649e661. 2. Angeli A, Osella G, Ali A, et al. Adrenal incidentaloma: an overview of clinical and epidemiological data from the National Italian Study Group. Horm Res 1997;47:279e283. 3. Grumbach MM, Biller BM, Braunstein GD, et al. Management of the clinically inapparent adrenal mass ("incidentaloma"). Ann Intern Med 2003;138:424e429. 4. Luton JP, Martinez M, Coste J, et al. Outcome in patients with adrenal incidentaloma selected for surgery: an analysis of 88 cases investigated in a single clinical center. Eur J Endocrinol 2000;143:111e117. 5. Allolio B, Fassnacht M. Clinical review: adrenocortical carcinoma: clinical update. J Clin Endocrinol Metab 2006;91:2027e2037. 6. Kirschner LS. Emerging treatment strategies for adrenocortical carcinoma: a new hope. J Clin Endocrinol Metab 2006;91:14e21. 7. Fassnacht M, Allolio B. Clinical management of adrenocortical carcinoma. Best Pract Res Clin Endocrinol Metab 2009;23: 273e289. 8. Icard P, Chapuis Y, Andreassian B, et al. Adrenocortical carcinoma in surgically treated patients: a retrospective study on 156 cases by the French Association of Endocrine Surgery. Surgery 1992;112:972e979. discussion 979e980. 9. Mantero F, Terzolo M, Arnaldi G, et al; Study Group on Adrenal Tumors of the Italian Society of Endocrinology. A survey on adrenal incidentaloma in Italy. J Clin Endocrinol Metab 2000;85: 637e644. 10. Abiven G, Coste J, Groussin L, et al. Clinical and biological features in the prognosis of adrenocortical cancer: poor outcome of cortisol-secreting tumors in a series of 202 consecutive patients. J Clin Endocrinol Metab 2006;91:2650e2655. 11. Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol 1984;8:163e169. 12. Tissier F, Cavard C, Groussin L, et al. Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Res 2005;65:7622e7627. 13. Barreau O, de Reynies A, Wilmot-Roussel H, et al. Clinical and pathophysiological implications of chromosomal alterations in adrenocortical tumors: an integrated genomic approach. J Clin Endocrinol Metab 2012;97:E301eE311. 14. Nakazumi H, Sasano H, Iino K, et al. Expression of cell cycle inhibitor p27 and Ki-67 in human adrenocortical neoplasms. Mod Pathol 1998;11:1165e1170. 15. Schmitt A, Saremaslani P, Schmid S, et al. IGFII and MIB1 immunohistochemistry is helpful for the differentiation of benign from malignant adrenocortical tumours. Histopathology 2006;49: 298e307. 16. Velazquez-Fernandez D, Laurell C, Geli J, et al. Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy. Surgery 2005;138:1087e1094. 17. de Reynies A, Assie G, Rickman DS, et al. Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival. J Clin Oncol 2009;27:1108e1115. 18. Giordano TJ, Kuick R, Else T, et al. Molecular classification and prognostication of adrenocortical tumors by transcriptome profiling. Clin Cancer Res 2009;15:668e676.
136 19. Laurell C, Velazquez-Fernandez D, Lindsten K, et al. Transcriptional profiling enables molecular classification of adrenocortical tumours. Eur J Endocrinol 2009;161:141e152. 20. Bassett MH, Mayhew B, Rehman K, et al. Expression profiles for steroidogenic enzymes in adrenocortical disease. J Clin Endocrinol Metab 2005;90:5446e5455. 21. Lenzini L, Seccia TM, Aldighieri E, et al. Heterogeneity of aldosterone-producing adenomas revealed by a whole transcriptome analysis. Hypertension 2007;50:1106e1113. 22. Wang T, Satoh F, Morimoto R, et al. Gene expression profiles in aldosterone-producing adenomas and adjacent adrenal glands. Eur J Endocrinol 2011;164:613e619. 23. Fallo F, Pezzi V, Barzon L, et al. Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas. Eur J Endocrinol 2002;147:795e802. 24. Saner-Amigh K, Mayhew BA, Mantero F, et al. Elevated expression of luteinizing hormone receptor in aldosteroneproducing adenomas. J Clin Endocrinol Metab 2006;91: 1136e1142. 25. Ye P, Mariniello B, Mantero F, et al. G-protein-coupled receptors in aldosterone-producing adenomas: a potential cause of hyperaldosteronism. J Endocrinol 2007;195:39e48. 26. Tadjine M, Lampron A, Ouadi L, et al. Detection of somatic betacatenin mutations in primary pigmented nodular adrenocortical disease (PPNAD). Clin Endocrinol (Oxf) 2008;69:367e373. 27. Søreide JA, Brabrand K, Thoresen SO. Adrenal cortical carcinoma in Norway, 1970-1984. World J Surg 1992;16:663e667. discussion 668. 28. Kasperlik-Zaluska AA, Migdalska BM, Zgliczynski S, et al. Adrenocortical carcinoma. A clinical study and treatment results of 52 patients. Cancer 1995;75:2587e2591. 29. Hanna NN, Kenady DE. Advances in the management of adrenal tumors. Curr Opin Oncol 2000;12:49e53. 30. de Fraipont F, El Atifi M, Cherradi N, et al. Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 2005; 90:1819e1829. 31. Sidhu S, Marsh DJ, Theodosopoulos G, et al. Comparative genomic hybridization analysis of adrenocortical tumors. J Clin Endocrinol Metab 2002;87:3467e3474. 32. Kjellman M, Kallioniemi OP, Karhu R, et al. Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy. Cancer Res 1996;56:4219e4223. 33. Russell AJ, Sibbald J, Haak H, et al. Increasing genome instability in adrenocortical carcinoma progression with involvement of chromosomes 3, 9 and X at the adenoma stage. Br J Cancer 1999;81:684e689. 34. Zhao J, Speel EJ, Muletta-Feurer S, et al. Analysis of genomic alterations in sporadic adrenocortical lesions. Gain of chromosome 17 is an early event in adrenocortical tumorigenesis. Am J Pathol 1999;155:1039e1045. 35. Dohna M, Reincke M, Mincheva A, et al. Adrenocortical carcinoma is characterized by a high frequency of chromosomal gains and high-level amplifications. Genes Chromosomes Cancer 2000;28:145e152. 36. Tombol Z, Szabo PM, Molnar V, et al. Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis. Endocr Relat Cancer 2009;16:895e906. 37. Lombardi CP, Raffaelli M, Pani G, et al. Gene expression profiling of adrenal cortical tumors by cDNA macroarray analysis. Results of a preliminary study. Biomed Pharmacother 2006;60:186e190. 38. Szabo PM, Tamasi V, Molnar V, et al. Meta-analysis of adrenocortical tumour genomics data: novel pathogenic pathways revealed. Oncogene 2010;29:3163e3172.
T. Lehmann, T. Wrzesinski 39. Reincke M, Mora P, Beuschlein F, et al. Deletion of the adrenocorticotropin receptor gene in human adrenocortical tumors: implications for tumorigenesis. J Clin Endocrinol Metab 1997;82: 3054e3058. 40. Reincke M, Beuschlein F, Menig G, et al. Localization and expression of adrenocorticotropic hormone receptor mRNA in normal and neoplastic human adrenal cortex. J Endocrinol 1998; 156:415e423. 41. Slater EP, Diehl SM, Langer P, et al. Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors. Eur J Endocrinol 2006;154:587e598. 42. West AN, Neale GA, Pounds S, et al. Gene expression profiling of childhood adrenocortical tumors. Cancer Res 2007;67: 600e608. 43. Lu L, Suzuki T, Yoshikawa Y, et al. Nur-related factor 1 and nerve growth factor-induced clone B in human adrenal cortex and its disorders. J Clin Endocrinol Metab 2004;89: 4113e4118. 44. el Akawi Z, Napoli JL. Rat liver cytosolic retinal dehydrogenase: comparison of 13-cis-, 9-cis-, and all-trans-retinal as substrates and effects of cellular retinoid-binding proteins and retinoic acid on activity. Biochemistry 1994;33:1938e1943. 45. Soon PS, Gill AJ, Benn DE, et al. Microarray gene expression and immunohistochemistry analyses of adrenocortical tumors identify IGF2 and Ki-67 as useful in differentiating carcinomas from adenomas. Endocr Relat Cancer 2009;16: 573e583. 46. Ragazzon B, Libe R, Gaujoux S, et al. Transcriptome analysis reveals that p53 and {beta}-catenin alterations occur in a group of aggressive adrenocortical cancers. Cancer Res 2010;70: 8276e8281. 47. Hermsen IG, Fassnacht M, Terzolo M, et al. Plasma concentrations of o, p’DDD, o, p’DDA, and o, p’DDE as predictors of tumor response to mitotane in adrenocortical carcinoma: results of a retrospective ENS@T multicenter study. J Clin Endocrinol Metab 2011;96:1844e1851. 48. Ilvesmaki V, Kahri AI, Miettinen PJ, et al. Insulin-like growth factors (IGFs) and their receptors in adrenal tumors: high IGF-II expression in functional adrenocortical carcinomas. J Clin Endocrinol Metab 1993;77:852e858. 49. Mesiano S, Mellon SH, Jaffe RB. Mitogenic action, regulation, and localization of insulin-like growth factors in the human fetal adrenal gland. J Clin Endocrinol Metab 1993;76:968e976. 50. Gicquel C, Bertagna X, Le Bouc Y. Recent advances in the pathogenesis of adrenocortical tumours. Eur J Endocrinol 1995; 133:133e144. 51. Gicquel C, Raffin-Sanson ML, Gaston V, et al. Structural and functional abnormalities at 11p15 are associated with the malignant phenotype in sporadic adrenocortical tumors: study on a series of 82 tumors. J Clin Endocrinol Metab 1997;82: 2559e2565. 52. Gicquel C, Bertagna X, Gaston V, et al. Molecular markers and long-term recurrences in a large cohort of patients with sporadic adrenocortical tumors. Cancer Res 2001;61:6762e6767. 53. DeChiara TM, Robertson EJ, Efstratiadis A. Parental imprinting of the mouse insulin-like growth factor II gene. Cell 1991;64: 849e859. 54. Giordano TJ, Thomas DG, Kuick R, et al. Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. Am J Pathol 2003;162:521e531. 55. l’Allemand D, Penhoat A, Lebrethon MC, et al. Insulin-like growth factors enhance steroidogenic enzyme and corticotropin receptor messenger ribonucleic acid levels and corticotropin steroidogenic responsiveness in cultured human adrenocortical cells. J Clin Endocrinol Metab 1996;81:3892e3897. 56. El Wakil A, Lalli E. The Wnt/beta-catenin pathway in adrenocortical development and cancer. Mol Cell Endocrinol 2011;332: 32e37.
Molecular basis of adrenocortical cancer 57. Schinner S, Willenberg HS, Krause D, et al. Adipocyte-derived products induce the transcription of the StAR promoter and stimulate aldosterone and cortisol secretion from adrenocortical cells through the Wnt-signaling pathway. Int J Obes (Lond) 2007;31:864e870. 58. Gaujoux S, Tissier F, Groussin L, et al. Wnt/beta-catenin and 30 ,50 -cyclic adenosine 50 -monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors. J Clin Endocrinol Metab 2008;93:4135e4140. 59. Caron de Fromentel C, Soussi T. TP53 tumor suppressor gene: a model for investigating human mutagenesis. Genes Chromosomes Cancer 1992;4:1e15. 60. Wagner J, Portwine C, Rabin K, et al. High frequency of germline p53 mutations in childhood adrenocortical cancer. J Natl Cancer Inst 1994;86:1707e1710. 61. Varley JM, McGown G, Thorncroft M, et al. Are there lowpenetrance TP53 Alleles? evidence from childhood adrenocortical tumors. Am J Hum Genet 1999;65:995e1006. 62. Lin SR, Lee YJ, Tsai JH. Mutations of the p53 gene in human functional adrenal neoplasms. J Clin Endocrinol Metab 1994;78: 483e491.
137 63. Barzon L, Chilosi M, Fallo F, et al. Molecular analysis of CDKN1C and TP53 in sporadic adrenal tumors. Eur J Endocrinol 2001;145:207e212. 64. Yano T, Linehan M, Anglard P, et al. Genetic changes in human adrenocortical carcinomas. J Natl Cancer Inst 1989;81:518e523. 65. Schteingart DE, Doherty GM, Gauger PG, et al. Management of patients with adrenal cancer: recommendations of an international consensus conference. Endocr Relat Cancer 2005;12: 667e680. 66. Libe R, Fratticci A, Bertherat J. Adrenocortical cancer: pathophysiology and clinical management. Endocr Relat Cancer 2007;14:13e28. 67. Cobb WS, Kercher KW, Sing RF, et al. Laparoscopic adrenalectomy for malignancy. Am J Surg 2005;189:405e411. 68. Baehner FL, Lee M, Demeure MJ, et al. Genomic signatures of cancer: basis for individualized risk assessment, selective staging and therapy. J Surg Oncol 2011;103:563e573. 69. Fassnacht M, Libe R, Kroiss M, et al. Adrenocortical carcinoma: a clinician’s update. Nat Rev Endocrinol 2011;7:323e335. 70. Doghman M, Madoux F, Hodder P, et al. Identification and characterization of steroidogenic factor-1 inverse agonists. Methods Enzymol 2010;485:3e23.