Genetics of adrenocortical tumors: gatekeepers, landscapers and conductors in symphony
Constantine A. Stratakis
Section on Endocrinology and Genetics, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1862, USA
The genetic and histopathological backgrounds of adre- nocortical tumorigenesis remain poorly characterized. In other tissues, there is conclusive evidence that hyper- plasia and adenomas precede cancer. In the adrenal, there are few clinical cases of either hyperplasia or adenoma associated with later development of cancer, and there are few biological studies that attempt to characterize this process molecularly. Current research focuses on the early lesions of the adrenal cortex because of their possible molecular link with carcino- genesis, and evidence of their frequent association with atypical forms of Cushing’s and Conn’s syndromes, obesity, hypertension and/or diabetes. These studies indicate a model for oncogenesis that is the same as that in other tissues. The rarity of adrenal cancer com- pared to benign lesions could be a clue to unique fea- tures of adrenocortical cells. It might also highlight the function of genes that are associated with endocrine tumors in the context of which the concept of gene ‘conductors’ is introduced here.
The possible roles of several genes in the formation of adrenocortical tumors are reviewed elsewhere [1,2]. Briefly, adrenocortical hyperplasia is a polyclonal process but adenomas and carcinomas are mostly monoclonal lesions [3]. This indicates that genetic changes at specific loci in the genome are needed for adrenal tumorigenesis. These include the genes that encode p53 (TP53), KIP2/p57, insulin-like growth factor type II (IGF2), angiotensin II, endothelin 1, diminuto/Dwarf-1, adrenomedullin, uroten- sin II, novH and cAMP-early repressor. Perhaps the best characterized gene is TP53. However, expression of TP53 does not always correlate with prognosis and it is rarely seen in tumors that are monoclonal but highly differen- tiated, which indicates that mutations in TP53 are a late event in the formation of sporadic adrenal cancer [1,2,4]. The role of IGF2 in adrenal tumors is reviewed elsewhere [1,2]. Although overexpression of IGF2 is observed widely in cancer, its role in hyperplasias and adenomas is less clear [2]. Here, I present a model of tumorigenesis in the adrenal cortex that stems from data collected over the past ten years of using molecular genomic, positional cloning and animal-model approaches.
Genomic and molecular cytogenetic studies
Comparative genomic hybridization (CGH) indicates genetic aberrations in adrenocortical tumors from adult [5,6] and pediatric [7] patients, with several differences that might correlate with the clinical differences associ- ated with tumors that form at a young age. The most common genetic aberrations in carcinomas are gains of chromosomes 4 and 5, and losses of chromosomes 11 and 17 [5]. The loss of 17q was observed frequently in a recent CGH analysis of adrenal tumors from several age groups, whereas 9q34 amplification occurs in eight out of nine tumors studied from Brazilian children [7]. In another study of 18 benign and 13 malignant adrenal tumors [8], the most common gains were seen on chromosomes 5, 12, 19 and 4, whereas losses occurred most frequently at 1p, 17p, 22, 2q and 11q. Of the benign adenomas, the most common change was gain of 4q. Similar to previous studies, this investigation confirms that the genetic changes associated with benign and malignant tumors are significantly different.
A comprehensive investigation using fluorescent in situ hybridization (FISH) showed frequent alterations and polyploidy in adrenocortical tumors [9]. Whereas no normal adrenal tissues had any numerical chromosomal aberrations, tetrasomy of chromosomes 3, 7, 8, 11 and 12 was detected in 12 of the 17 adenomas associated with primary aldosteronism. DNA flow cytometry also revealed tetraploidy in 11 of the 17 cases of primary aldosteronism. These results indicate that DNA polyploidy is not uncommon, even in benign adrenal tumors, and is even more common in tumors associated with hyperaldosteron- ism [9]. Indeed, polyploidy seems to be a relatively frequent event in benign adrenocortical lesions [7-9], although it is rarely encountered in hyperplasias and adenomas of other tissues. Genomic instability of benign adrenocortical tumors might be related to their lack of centrosomal stability [10].
Some of the genes with proven involvement in adreno- cortical tumor formation are associated with 11p (p57/KIP2 and IGF2) and 17p (TP53) chromosomal changes [11]. In addition, molecular genetic analysis by microsatellite markers shows alterations in 2p16 [12,13], 11q13 [13] and 17q [14] in sporadic adrenal tumors [13] and those associated with the Carney complex (CNC), a syndrome associated with multiple endocrine and other
Corresponding author: C.A. Stratakis (stratakc@mail.nichd.nih.gov).
tumors [12], multiple endocrine neoplasia type 1 (MEN1) [15,16] and neurofibromatosis type 1 (NF 1) [14]. However, mutational analyses of the menin [15,16] and neuro- fibromin [14] tumor-suppressor genes in sporadic adreno- cortical tumors fail to show frequent, if any, somatic mutations.
The case for inhibin
Of the factors studied in adrenocortical tumors, the involvement of inhibin-A in human tumorigenesis remains a mystery. In rodents, inhibin plays a role in tumor formation [17]. Homozygous knockout Inha-/- mice develop gonadal tumors at 4-5 weeks of age and die at 12 weeks. Gonadectomy postpones the wasting syndrome, the development of adrenal tumors (21 weeks) and death (33-36 weeks) [18,19]. In another transgenic-mouse model that expresses a 6 kb fragment of the Inha promoter fused with the simian virus 40T-antigen (SV40-Tag), gonadal tumors appear in all animals at age 5-8 months [20]. In these animals, gonadectomy at 4 weeks of age is followed by the development of adrenal tumors at the age that gonadal tumors would have appeared.
The results of studies of INHA expression in human adrenal tumors are less clear with regards to its contribution to human tumorigenesis. INHA immuno- reactivity is present in the zona reticularis, and in focal collections of cells in the zona fasciculata of the normal adrenal cortex but not in cells of the zona glomerulosa [21,22]. INHA appears to be overexpressed in cortico- tropin-induced cortical hyperplasia, adrenal adenomas and most carcinomas, but some carcinomas do demon- strate a decrease in INHA immunoreactivity, as would be expected from its presumed tumor-suppressor role [23,24]. A recent, small, preliminary investigation identified three heterozygous, germline point mutations in INHA from unrelated pediatric patients with adrenal adenomas and carcinomas (C. Longui, et al., unpublished). However, the functional consequences of these sequence changes remain unclear, and further studies of INHA and the pathway involved in its molecular signaling need to be undertaken in adrenocortical tumors in light of the findings in animal models.
Low-penetrance TP53 mutations
It has been suggested that low-penetrance mutations of established tumor-suppressor genes underlie adrenocortical tumors in at least some patients [25]. Indeed, a germline mutation in TP53 (R337H) was described in 35 out of 36 Brazilian children with either adenomas or carcinomas but no other identifiable clinical syndromes (i.e. Li-Fraumeni and Beckwith-Wiedemann syndrome) [26]. The same mutation was identified subsequently by Latronico et al. in other children and adult Brazilian patients with adrenal tumors [27] and additional, low-penetrance mutations of TP53 have also been found recently in other populations [25]. It appears likely that the tumorigenic effect of the R337H mutation is related to pH-induced conformational changes of p53 [28]. Thus, at least some mutations of established tumor-suppressor genes that were not previously associ- ated with disease might not be simple polymorphisms: they could predispose adrenocortical masses and this
tumorigenic function might depend on complex genetic or even environmental interactions [29].
PRKAR1A: a gene for inherited and sporadic Cushing’s syndrome
Various components of the cAMP-dependent protein kinase A (PKA) signaling pathway, including the genes that encode the corticotropin (ACTH) receptor (MC2R) and the Gsa subunit (GNAS1) are also implicated in adreno- cortical tumorigenesis [30]. Recently, the gene that encodes the PKA type I-a regulatory subunit (RIa), PRKAR1A, was found to be responsible for most cases of a relatively rare form of bilateral adrenocortical hyper- plasia called primary pigmented nodular adrenocortical disease (PPNAD), which is often associated with CNC [31-34]. Germline, inactivating mutations in PRKAR1A were found in both patients with isolated PPNAD and those with CNC [35]. In PPNAD, as in other CNC tumors, loss of heterozygosity of the 17q PRKAR1A locus and abnormal activity of PKA have been demonstrated. This might indicate that PRKAR1A acts as a tumor-suppressor gene [31], despite evidence to the contrary from studies of RIa and PKA in cancer cell lines [36].
PPNAD might cause either classic Cushing’s syndrome or an insidious, clinically atypical form of hypercortisolism that could be associated with cyclical Cushing’s, simple disrturbances of the normal circadian variation of cortisol secretion and other atypical features. Most of these patients present with osteoporosis and, occasionally, myopathy and cachexia, but tend to lack severe obesity, persistent hypertension, moon face and other Cushing’s manifestations [37,38]. A modified interpretation of the classic Liddle’s test takes advantage of the paradoxical responses of PPNAD to dexamethasone administration and can be used diagnostically [34,38].
In a recent study, Bertherat et al. examined whether somatic alterations of PRKAR1A are associated with the development of sporadic adrenal adenomas and carci- nomas in 44 patients, including 26 with ACTH-indepen- dent Cushing’s syndrome. Hitherto, somatic changes in the PRKAR1A locus and gene had only been described in thyroid tumors [39] and not in > 100 pituitary tumors that were screened [40,41]. The results indicate that 17q22-24 allelic losses are relatively frequent in sporadic adrenal tumors (J. Bertherat et al., unpublished). Sequencing PRKAR1A in all samples identified three novel, inactivat- ing mutations in adenomas from unrelated patients with paradoxical responses to dexamethasone, in addition, one had cyclical Cushing’s syndrome. For the first time this indicates that there is a correlation between PRKAR1A genotype and clinical phenotype in sporadic adrenal tumors [42]. In this study, genetic alterations of PRKAR1A and/or its 17q22-24 locus correlated well with decreased expression of PRKAR1A and alterations in PKA activity [42], as in germline mutations in PRKAR1A that cause PPNAD [31,43].
Macronodular hyperplasia: ectopic receptors and other genes
In 1964, Kirschner et al. [44] described a 40-year-old woman with long-standing Cushing’s syndrome. Although
her disease was not ACTH-dependent, testing showed hyper-responsiveness to ACTH and that glucocorticoid production was not suppressed by the administration of dexamethasone. The patient underwent bilateral adrena- lectomy; her adrenal glands had multiple nodules and a combined weight of 94 g (the weight of adrenal glands is usually 8-12 g). There have been >200 patients with macronodular hyperplasia described since 1964 under various names [ACTH-independent macronodular adre- nocortical hyperplasia (AIMAH), massive macronodular extremous disease (MMAD), autonomous macronodular adrenal hyperplasia, ACTH-independent massive bilat- eral adrenal disease and giant or huge macronodular adrenal disease] [44-46]. In addition, bilateral adrenal nodules or tumors are reported to be present in up to 10-15% of patients with adrenal masses that are discovered incidentally [47]. AIMAH, similar to PPNAD, appears to have a bimodal age distribution: a small number of patients present during the first years of life when this form of the disease might be confused with McCune-Albright syndrome (MAS), whereas most patients present in the fifth decade of life [45,46].
In AIMAH, the secretion of steroid hormones is ACTH- independent; plasma ACTH levels are usually low or undetectable and administration of a high-dose of dexa- methasone fails to suppress cortisol secretion [45]. MC2R is expressed in AIMAH tissue, and most patients with AIMAH respond to ACTH with increases in baseline cortisol levels that depend on the size of the adrenocortical mass. This is in contrast to PPNAD, large benign adenomas and adrenocortical carcinomas, which are mostly unresponsive to ACTH [44,46].
ACTH responsiveness is not the only difference between adrenocortical cancer and AIMAH. The latter is a benign process that has never been shown to either metastasize or transform to cancer. The histology of the nodules is also distinct: they comprise two types of cells, some with clear cytoplasm (lipid-rich) that form cordon, nest-like struc- tures, and others with a compact cytoplasm (lipid-poor) that form small nest or island-like structures [45,47].
AIMAH is rarely familial [47-50]. This form of the disease must be distinguished from infants with MAS, adrenocortical hypeplasia and adenomas, and somatic GNAS1 mutations [51,52]. GNAS1 genetic defects do not explain most cases of AIMAH, which occur sporadically (I. Bourdeau et al., unpublished). Recently, a Brazilian study identified somatic mutations of GNAS1 in adrenocortical adenomas in 3 out of 5 patients with AIMAH [53]. These mutations were detected by denaturing-gel electrophoresis followed by sequencing of the respective amplicons but were not confirmed in the tumor tissue by other methods. These data might reflect regional differences or come from patients with mild forms of MAS.
Ectopic expression of receptors for neuroendocrine hormones, such as gastrointestinal inhibitory peptide, vasopressin, ß-adrenoceptor and other receptors, has been the most common and clinically useful feature in patients with AIMAH [45,54,55]. Increasingly, similar abnormal- ities are found in sporadic, non-AIMAH-related adrenal adenomas [56,57].
Despite advances in identifying AIMAH and investi- gating its pathophysiology, there has been little progress towards the underlying genetic mechanisms that are responsible for this form of adrenal hyperplasia. As discussed above, GNAS1 mutations are not often present in adrenocortical tumors associated with AIMAH or ectopic receptor expression, outside of MAS or exceptional patients [53,58]. Although cAMP-dependent PKA signal- ing appears to be involved in the pathophysiology of AIMAH, to date no PRKAR1A mutations have been identified in this disease; other components of this path- way can be altered functionally but are not mutated somatically (I. Bourdeau et al., unpublished). Part of the problem in characterizing AIMAH is its apparent clinical and, hence, genetic heterogeneity; the other difficulty is its somatic nature. Microarray technology [59] and genetic- linkage studies in individual families with inherited AIMAH might reveal these defects.
Immunohistochemical and other molecular markers
Cytokines, growth factors, and their receptors, which can be expressed eutopically or ectopically in adrenocortical tissue, have been implicated recently in carcinogenesis [60]. Expression of major histocompatibility complex class- II antigens in adrenocortical tissue correlates with adrenocortical cell differentiation [61]. The expression of both transforming growth factor & (TGFa) and epidermal growth factor receptor [62] is elevated markedly in carcinomas (unlike adenomas), and synaptophysin and other neuroendocrine markers are expressed in adreno- cortical cancer [63]. The unexpected presence of proteins with neuroendocrine and other functions in adrenal cancer follows a pattern similar to that observed in benign adrenocortical hyperplasias, such as AIMAH and PPNAD [64], although in cancer it seems to occur on a wider scale. These immunohistochemical properties can be associated with unusual clinical behavior and paradoxical responses to dexamethasone, which again mimic those seen in PPNAD.
Other ‘syndromic’ genes
Could other genes that cause human genetic-tumor syndromes also be involved in the pathogenesis of adrenocortical tumors in addition to TP53 and IGF2? I have already mentioned menin, PRKAR1A, GNAS1 and neurofibromin. Patients with familial polyposis coli and germline mutations of the APC gene also get, mostly nonfunctional, adrenal tumors [65,66]; however, APC has not been studied in sporadic adrenal tumors. Patients with the Carney triad (which consists of gastric stromal tumors, pulmonary chordomas and extra-adrenal paragangliomas) frequently have adrenocortical adenomas [67,68] but the gene for this disorder has not been identified.
A model for adrenocortical tumorigenesis: ‘conductor’ genes
The studies reviewed here support the notion that adrenocortical tumorigenesis, like oncogenesis in other tissues [69], is a multi-step process (Fig. 1). Every step in this process is associated with an increasing number of genetic changes, as shown by CGH [5-8]. Inactivating
(a)
Bilateral adrenocortical hyperplasia
Single adrenocortical adenoma
Adrenal cancer
Polyclonal
Monoclonal
Monoclonal, associated with TP53 mutations, allelic losses of 17p and other genetic changes
(b)
Absolute number of genetic changes (by CGH)
30
25
20
15
10
5
0
4
5
6
7
10
15
20
Size of adrenal tumor (cm)
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mutations of TP53 and either chromosomal deletions or gains are frequent in cancers and large adenomas. Although these changes are either rare or nonexistent in hyperplasias and small adenomas, in some cases, genetic alterations might correlate with the clinical phenotype of the tumor. For example, chromosomal abnormalities are more frequent in aldosteronomas than cortisol-producing adenomas [9] and PPNAD frequently presents with microsatellite alterations and genomic instability [12]. Furthermore, monoclonal expansion is the most important genetic feature that differentiates adenomas from hyper- plasias [3]. The continuum presented in Fig. 1 has only recently been demonstrated convincingly in adrenocortical tissue [70], but is indicated repeatedly in patients with 21-hydroxylase deficiency and adrenal tumors [71]. Other data support the notion that cancer of the adrenal glands is the same as that in other organs, including endocrine glands such as the thyroid [72].
Other than confirming the general model of tumorigen- esis, what additional knowledge has been gained from genetic studies on adrenal tumors? We have identified some features that might be true for many cell types, and others that might apply uniquely to the adrenal cortex and, perhaps, other endocrine tissues. First, a concept under investigation is that heterozygous mutations that were thought to cause disease only in hemizygotes might act as a first ‘hit’, albeit with low penetrance [26] (Fig. 2). Haploin- sufficiency of genes such as STK11/LKB1 and neurofibro- min also appears to be sufficient for abnormalities to
The two-hit hypothesis
Somatic first hit
Normal cell
Loss of heterozygosity
Somatic second hit
Cell carrying a predisposing mutation
Germline first hit
Second mutation
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appear in other tissues [73,74]. Second, the high frequency of genomic abnormalities, including polyploidy, which is a striking feature of adrenocortical adenomas, has been confirmed by CGH [5-8], FISH [9], and microsatellite [10,11] and centrosomal [12] studies. This is uncommon in other tissues, where such genomic changes occur almost exclusively in cancer. It is tempting to speculate that genomic instability in early tumoral adrenocortical tissue might explain the discrepancy between the high frequency of benign nodules (as high as 20% of the general population in some autopsy series) and the low incidence of adrenocortical cancer (0.05-0.2% of all cancers and an approximate prevalence of two new cases per million of population per year) [1,2].
Cells with extensive genomic abnormalities are not expected to readily develop a selection advantage that would lead them to acquire the highly aggressive molecular and clinical phenotype of adrenocortical cancer. Therefore, whatever causes genomic instability in the early stages of adrenocortical tumorigenesis also protects against malignancy. We have to assume that one or more of the early hits discussed here (PRKAR1A, low-penetrance inactivating mutations of TP53 and mutations in genes that have yet to be identified) are responsible for this genomic instability in benign adrenocortical tumors. PRKAR1A is a particularly good candidate because of its central role in the regulation of PKA [75].
How do inactivating mutations in a tumor-suppressor gene protect against cancer? There is at least one example of this in vitro - the STK11 /LKB1 tumor suppressor gene [73]. There are two ways that protection can be mediated: one passive and one active. The passive is exemplified by
genomic instability that occurs as a direct effect of a mutation, as hypothesized above for PKA and PRKAR1A- related tumorigenesis. The active has been described for STK11/LKB1, that is steering a cell away from taking a ‘hit’ that is necessary for the development of a malignant phenotype [73]. This change of direction could involve the activation of alternative signaling pathways either by direct interactions or by unleashing ‘hidden’ properties of the molecule in a tissue-dependent or cell-dependent manner.
These novel properties of genes that have been associated with tumorigenesis blur the traditional bound- aries between oncogenes and tumor suppressors. It is no coincidence that both loss of the normal allele and mutations that lead to tumors and increased apoptosis (depending on the tissue and developmental stage) have been described for RET, an oncogene that is associated with endocrine tumors [76,77].
If mutations in these genes cause tumors and, at the same time, have the potential to impede carcinogenesis, these genes do not appear to fit the traditional definition of either tumor suppressors or ‘oncogenes’ [78]. Genes such as TP53, which control major events in cell cycle or apoptosis, have been called ‘gatekeepers’, and those such as PTEN, which are involved in major signaling pathways and seem to regulate gatekeepers, have been called ‘landscapers’ [78,79].
What best characterizes these new groups? Most participate in complex and highly interactive signaling pathways and they not only control several other genes, but also communicate with many pathways, both horizon- tally and vertically. This is reminiscent of the internet, as has been proposed elsewhere [80]. In this internet-like structure, the web of possibilities is huge: mutations and functional alterations in genes that are expressed almost everywhere and play the role of hubs in this net have unpredictable consequences in the organism as a whole because of their tissue-specificity, cell-specificity, develop- mental-stage-dependence and numerous connections to other pathways.
I suggest that these genes should be called ‘conductors’. They are expressed in almost all tissues, control numerous cellular functions and can assume multiple roles because they are ultrasensitive and adapt according to cellular needs. ‘Conductors’ are one step above ‘landscapers’, just as the latter are one step above ‘gatekeepers’ in the complex process of tumorigenesis. These terms do not replace ‘tumor suppressors’ and ‘oncogenes’, but comp- lement them. Therefore, we might refer to a particular gene as a ‘tumor suppressor conductor’ or an ‘oncogene landscaper’, depending on the tissue, cell and even particular function.
These concepts have developed from our studies of adrenocortical tumors because of the unique features of this tissue, as discussed above. They might also apply to other tissues, namely the thyroid gland and, perhaps, nonendocrine organs [72,80]. PRKAR1A, which was cloned because of its involvement in adrenocortical tumorigen- esis, is an example of an endocrine gene that was identified as a tumor suppressor and can be thought of as a ‘conductor’. PRKAR1A is expressed in most cells and
controls cAMP-dependent PKA signaling. As a con- sequence, interacts with numerous other pathways and functions. Another example of an endocrine conductor that defies the classical definitions of tumor suppressor and oncogene is TGF-ß, which has also been extensively investigated in adrenocortical tumors. Microarray studies are expected to unravel additional information about such genes in adrenocortical [81] and other endocrine tumors.
Concluding remarks
This is an exciting time for clinicians and research scientists interested in primary tumors of the adrenal gland. Microarray technology and the identification of genes and molecular pathways that are specific to adrenocortical tumorigenesis are the ways of the immedi- ate future. As the molecular basis of adrenocortical tumors becomes better understood, clinicians now faced with an increasing incidence of these lesions [82] will be able to offer better therapies to their patients. And, importantly, new concepts in oncogenesis might be developed by elucidating the omnipotent presence and versatile func- tion of ‘endocrine’ tumor genes.
Acknowledgements
I thank Caroline Sandrini (Santa Catarina, Brazil) for Fig. 2.
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Books Received
We thank the publishers for sending us the books listed here. Those of interest to the readership of Trends in Endocrinology & Metabolism will be reviewed. Readers are invited to write book reviews by informing the Book Review Editor of the title and publisher of books you wish to review. Please address all books and correspondence for book reviews to Lee A. Meserve, PhD, Book Reviews, Trends in Endocrinology & Metabolism, c/o Dept of Biological Sciences, Room 217 Life Sciences Building, Bowling Green State University, Bowling Green, OH 43403-0212, USA.
Exercise Endocrinology
By Katarina T. Borer. Human Kinetics, 2003. US129.95 (xiii + 273 pages) ISBN 0 88011 566 1
Prader-Willi Syndrome as a Model for Obesity Edited by Urs Eiholzer, Dagmar l’Allemand and William B. Zipf. Karger, 2003. CHF 138.00/EUR 98.50/US$120.00 (238 pages) ISBN 3 8055 7574 2
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