6 Advances in understanding the molecular URRENT PINION underpinnings of adrenocortical tumors
Norman G. Nicolson, Jianling Man, and Tobias Carling
Purpose of review
Adrenocortical tumors are divided into benign adenomas and malignant carcinomas. The former is relatively common and carries a favorable prognosis, whereas the latter is rare and frequently presents at an advanced stage, with poor outcomes. Advances in next-generation sequencing, genome analysis, and bioinformatics have allowed for high-throughput molecular characterization of adrenal tumorigenesis.
Recent findings
Although recent genomic, epigenomic, and transcriptomic studies in large tumor cohorts have confirmed the central roles of aberrant Wnt/B-catenin signaling, constitutive protein kinase A pathway activation, cell cycle dysregulation, and ion channelopathies in adrenal tumorigenesis, these studies also revealed novel signature events underlying malignant differentiation of adrenocortical carcinomas.
Summary
Recent advances in understanding of the molecular mechanisms underlying adrenocortical tumorigenesis provide new molecular diagnostic and prognostic tools and opportunities for novel therapeutic approaches. These findings are particularly important in adrenocortical carcinoma, for which current treatment options are limited.
Keywords
adrenocortical adenoma, adrenocortical carcinoma, epigenetics, genomics, molecular mechanism
INTRODUCTION
Adrenocortical tumors (ACTs) are divided into a vari- ety of clinical, pathological, and molecular catego- ries, with different natural histories and different approaches to diagnosis and treatment. Broadly, the adrenocortical lesions include adrenal hyperpla- sias, adrenocortical adenomas (ACAs), and adreno- cortical carcinomas (ACCs). ACTs may be further segmented into functioning hormone-producing tumors and nonfunctioning (nonproducing) tumors. ACA and ACC pose unique challenges in their diag- nosis, prognosis, and treatment options, and there- fore will be the focus of this review.
Patients present with symptoms related to hor- mone overproduction, mass effect, or an incidentally discovered adrenal mass (the ‘adrenal incidenta- loma’). ACA is common, at 2-3% prevalence among the general population, whereas ACC is rare, with an estimated incidence of 0.7-2 cases per 1 million patients per year [1,2]. Despite their common tissue origin, there is limited molecular evidence of an ade- noma-carcinoma sequence in ACT progression, unlike the well-described adenoma-carcinoma sequence in other cancer types.
The mainstay of treatment for ACAs focuses on ameliorating the effects of hormone excess, often requiring surgical resection in medically refractory cases. Large nonfunctional ACAs may be removed if they are compressing other structures, or to rule out carcinoma. ACCs, on the other hand, are extremely aggressive malignancies for which resection offers the only chance at cure. Even with a margin- negative resection, most patients with ACC will succumb to the disease within 5 years, owing in large part to the lack of available salvage therapy for patients who recur after surgery or who present at advanced stages [3]. Both traditional cytotoxic
Section of Endocrine Surgery, Yale Endocrine Neoplasia Laboratory, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, USA
Correspondence to Tobias Carling, MD, PHD, FACS, Section of Endo- crine Surgery, Yale Endocrine Neoplasia Laboratory, Department of Surgery, Yale University School of Medicine, 333 Cedar Street, FMB130A, New Haven, CT 06520, USA. Tel: +1 203 737 2036; fax: +1 203 737 4067; e-mail: tobias.carling@yale.edu
Curr Opin Oncol 2017, 29:000-000
DOI:10.1097/CCO.0000000000000415
KEY POINTS
· Whole-exome analyses confirmed prior findings in the Wnt signaling pathway and cell cycle regulation in ACC, and revealed new genes of interest in APA (ion channel mutations) and CPA (protein kinase A pathway genes).
· A hypermethylated phenotype in a subset of ACCs is strongly correlated with worse prognosis, in accordance with similar analyses in other cancers.
. Noncoding miRNA shows promise as a biomarker for adrenal tumors, and delivery of targeted miRNAs to tumors is a possible approach for development of novel therapeutics.
· Transcriptome analysis reveals signature gene expression profiles in ACTs, useful both in diagnosing histologically indeterminate tumors and for prognosticating in ACC.
chemotherapy and the adrenolytic mitotane have been used for ACC patients in the preop, postop, and metastatic setting, with limited success. In large part, the impetus for ongoing research in ACC is to identify candidates for the development of targeted molecular therapies, which have been successful in the treatment of some other malignancies. Further- more, molecular tools may be necessary for prog- nostication in ACC, as there are currently little data to guide clinicians and patients in what to expect.
Of further interest is the question of distinguish- ing ACA from ACC, as the two are not always easily distinguished, even by experienced pathologists at
high-volume centers. The Weiss scoring system has been the gold standard for discriminating between the two entities histopathologically, but leads to some cases with intermediate scores for which addi- tional evidence is needed [4].
Several novel molecular and computational tools have emerged in recent years to identify and characterize aberrant pathways that likely play a role in adrenocortical tumorigenesis. Genomics as a broad category may be interpreted to include a variety of molecular techniques, including analyses of individual gene mutations (discovered in many cases by whole-exome/genome sequencing), DNA methylation, microRNA expression, and gene tran- scription. At present, it is unclear if the identified pathways represent truly distinct mechanisms of pathogenesis or simply different compensatory dys- regulations following from the cellular evolutionary processes inherent to tumorigenesis.
UPDATES FROM WHOLE-EXOME SEQUENCING ANALYSES
Several recent studies have performed whole-exome sequencing of aldosterone-producing adenomas (APAs) [5-8], cortisol-producing adenomas (CPAs) [9-13], nonfunctional adenomas (NFAs) [13], and ACCs [14,15,16""]. These have identified several recurrent mutations in each tumor category, and further demonstrated the prevalence of private mutations in each individual tumor, revealing a surprisingly diverse and complex molecular back- ground underlying each subtype of ACT (Table 1) [5-15,16”].
| Study | Tumor type | WES cohort size | Recurrently mutated, amplified, or deleted genes (>5% of samples, most to least frequent) |
|---|---|---|---|
| Juhlin et al. [14] | ACC | 41 | TP53, CTNNB1, ZNRF3, KREMEN1, TERT |
| Assié et al. [15] | ACC | 45 | ZNRF3, CTNNB1, TP53, CDKN2A, RB1, MEN1, DAXX, MED12, TERT |
| Zheng et al. [16""] | ACC | 91 | TP53, ZNRF3, CDKN2A, CTNNB1, TERT, PRKAR1A, CDK4, TERF2, RB1, MEN1, CCNE1, MLL4 |
| Choi et al. [5] | APA | 4 | KCNJ5 |
| Scholl et al. [8] | APA | 14 | KCNJ5, CACNA1D, CTNNB1 |
| Azizan et al. [7] | APA | 10 | ATP1A1, CACNA1D, CTNNB 1 |
| Beuschlein et al. [6] | APA | 9 | ATP1A1, ATP2B3 |
| Goh et al. [10] | CPA/ACC | 25 | PRKACA, CTNNB1, TP53, RB1, USP6NL, CUL2, GNAS |
| Beuschlein et al. [9] | CPA | 10 | PRKACA |
| Sato et al. [11] | CPA | 8 | PRKACA, GNAS |
| Cao et al. [12] | CPA | 39 | PRKACA |
| Ronchi et al. [13] | CPA/NFA | 99 | CTNNB1, GNAS, PRKACA |
ACC, adrenocortical carcinoma; APA, aldosterone-producing adenoma; CPA, cortisol-producing adenoma; NFA, nonfunctional adenoma.
Aldosterone-producing adenomas
The signature genetic events driving most APAs are mutations in ion channel genes which regulate cell membrane potential and ultimately intracellular calcium concentration (Fig. 1). Aldosterone is a steroid hormone involved in the regulation of blood pressure and sodium/potassium homeostasis, and the production of aldosterone is tied to flow of potassium ions into cells, ultimately leading to cal- cium influx and hormone production [5]. Mutations in the potassium channel KCNJ5, calcium channel CACNA1, and ATP-driven pumps ATP1A1 (sodium- potassium transporter) and ATP2B3 (calcium trans- porter) have all been identified in APAs [5-8]. Inter- estingly, a germline mutation in CACNA1H, another calcium channel, has been associated with a syn- drome of premature onset of hypertension in child- hood but not typically an adenoma-development phenotype [17]. Other researchers have shown that adrenal glands without macroscopic adenomas may nonetheless contain islands of intensive aldosterone production, which may carry mutations in the above membrane potential modulators [18]. The mecha- nism underlying the relationship between hormone overproduction and tumorigenesis has yet to be elu- cidated.
Previous work had shown Wnt/ß-catenin sig- naling to be constitutively active in up to 70% of APAs [19]. However, only 5% of APAs seem to have activating CTNNB1 mutations, and these are
apparently mutually exclusive with the ion channel mutations listed above [20]. Interestingly, although these CTNNB1 mutant tumors tended to be larger than their ion-channel-mutant analogs, and poten- tially represent a distinct pathway toward tumori- genesis, the two groups were not distinguishable histologically [20].
Cortisol-producing adenomas
CPAs are characterized in large part by dysregula- tion of the protein kinase A pathway, which is involved in cortisol production in response to ACTH stimulation (Fig. 2). Previously described mutations along this pathway included gain of function mutations in GNAS (an upstream G-pro- tein coupled receptor subunit) or inactivating mutations in PRKAR1A (an inhibitory component of the protein kinase A complex); both result in constitutive activation of cortisol production [21,22]. Several exome analyses demonstrated that an activating mutation in the catalytic subunit of protein kinase A (PRKACA) could result in a similar phenotype of overproduction of cortisol [9-12]. A more recent exome sequencing study in CPAs without the most common PRKACA mutation dem- onstrated alternative activating mutations in GNAS, PRKACA, and CTNNB1, though CTNNB1 mutations were more common in hormonally inac- tive tumors [13].
Na+
Na+
AT II
K+
K+
Ca2+
Ca2+
Ca2+
Ca2+
1
T
KCNJ5*
KCNJ5
AT1R
ATP1Α1
ATP1A1*
CACNA1D
CACNA1D*
ATP2B3
ATP2B3*
ATP
ADP
ATP
ADP
K+
K+
Na+
Na+
Depolarization
Ca2+
Ca2+
K+
Ca2+
Ca2+ Ca2+
Ca2+
Na+
CYP11B2
Aldosterone synthesis
ACTH
MC2R
Adenylyl
UUV
cyclase
CAMP
ATP
Gs
PRKAR1A
PRKACA
PRKAR1A
PRKACA
PRKAR1A
PRKACA
PRKAR1A
PRKACA
CREB
P
CREB
Cortisol synthesis
Nonproducing adenomas
Despite being the most common form of ACT in the population, NFAs tend to be under-represented in research, as they are infrequently removed from patients in light of their slow growth and lack of hormone-excess symptomatology [13]. Nonfunc- tioning adenomas are therefore less well character- ized than the other ACTs described above, but show some overlap in mutational profiles. A prior study and recent whole exome analysis demonstrated that among all adenomas, those harboring CTNNB1 mutations tended to be larger and were less likely to produce hormones overall [13,23].
Adrenocortical carcinomas
Multiple whole-exome analyses of ACC have con- firmed the prevalence of frequent Wnt/ß-catenin pathway aberrations, most commonly in the CTNNB1 gene itself or in upstream/downstream regulators such as ZNRF3 (Fig. 3A). Activating CTNNB1 mutations and inactivating ZNRF3 mutations or deletions (a negative Wnt pathway regulator) both result in constitutively active Wnt signaling; these mutations were mutually exclusive across all three whole exome studies, likely indicating their function via a final common pathway in Wnt signaling [14,15,16""].
Cell cycle regulatory genes were also found to be recurrently mutated or deleted in ACC, including TP53 and RB1 [14,15,16""]. TP53 is known to be mutated in many other cancers; dysregulation of
this gene leads to unchecked cell proliferation and failure of expected apoptosis in response to accumu- lation of DNA damage (Fig. 3B). As in many other cancer types, TP53 mutation is a clear marker of malignancy, as it is infrequently mutated in benign ACAs.
A subset of ACCs demonstrated mutations in genes controlling chromatin remodeling, including MEN1 and ATRX. One recent analysis found whole genome doubling in a subset of ACCs, and this was associated with worse prognosis [16""]. Copy num- ber amplifications in the TERT gene, which encodes a telomerase gene responsible for lengthening telo- meres and likely preventing cell senescence, were also identified in several ACC samples in each of the studies [14,15,16""]. Dysregulated TERT activity has been described in numerous other human cancers.
Functional validation of additional genes iden- tified through next-generation sequencing studies has confirmed their predicted roles in ACC, includ- ing FOXO1, DKK3, NR5A1, VAV2, BIRC7, SLC12A7, CYP2A6, and RARRES2 [24”-27”,28,29”,30”].
UPDATES FROM MICRORNA ANALYSES
MicroRNAs are small, noncoding RNAs which typi- cally function in a targeted regulatory role within cells, modulating translation of mRNA. A given miRNA (typically a few dozen base pairs in length) may target more than one mRNA transcript, making a precise 1 : 1 mapping of each miRNA to its target
(a)
(b)
Wnt
Frizzled
LRP5/ LRP6
ZNRF3
DVL2
MDM2
TP53
Apoptosis
CK1a
AXIN
T
APC
GSK3ß
CTNNB1
CDKN2A
CDK4
RB1
Target transcription
T
Cell cycle
Phosphorylation
CTNNB1
DNA damage
Degradation
gene suppressed difficult. That said, specific patterns of miRNA expression have been described in ACTs, and may be useful for diagnosis, prognostication, and therapy; for example, significant downregula- tion of miR-375 has been shown in APAs compared to normal adrenals, and treatment of an established aldosterone-producing ACC cell line with this miRNA resulted in reduced cell growth [31]. Several analyses have found miRNAs to be potentially sig- nificant players in ACC tumorigenesis [15,16"",32”]. The most promising marker for ACC in these studies seems to be upregulation of miR-483-5p, which appears to correlate with overexpression of IGF2, a growth factor which is frequently overexpressed in ACC; in fact, this miRNA is found within the IGF2 gene, so it is not clear if its overexpression in ACC is the cause of, or simply a marker for, the malignant phenotype [33”]. These analyses suggest that the miRNA profile can be used to distinguish ACC from APA or normal adrenal, and even to classify ACCs into discrete prognostic groups. The use of miRNA in the serum for a ‘liquid biopsy’ may allow for preci- sion biomarker-directed treatment without any invasive procedures to obtain a specimen from the tumor itself [34]. Furthermore, systems to deliver targeted miRNAs to cells are in development, though miRNA has not made its way into human trials in ACC to date [35].
UPDATES FROM METHYLOME ANALYSES
Methylation of CpG islands within the promoter regions of genes has been previously described as an epigenetic modulator across numerous tissue types, with CpG island methylator phenotype (CIMP) tumors in several different cancer types expressing distinct behaviors in vivo [36-38]. In recent pan-geno- mic analyses of ACC, CIMP tumors were more aggres- sive and associated with worse survival [15,16""]. Moreover, a recent methylation study using a smaller subset of genes demonstrated targeted methylome analysis as an independent predictor of overall and disease-specific survival, comparable within multivar- iate analysis to tumor stage and Ki-67 index in prog- nostic power [39”]. The molecular mechanism of this striking finding has not been described in detail in ACC but likely results from methylation and resultant silencing of target tumor suppressor genes.
UPDATES FROM TRANSCRIPTOME ANALYSES
Using bioinformatics approaches, mRNA transcrip- tion profiles can distinguish subgroups within the larger cohort, useful for development of molecular diagnostic tools as well as characterization of novel dysregulated signaling pathways. In ACC, transcriptome analysis has previously been shown
to sort tumors into the C1A and C1B subtypes, which correlate with worse and better prognosis, respec- tively, a strategy which was confirmed in the two recent genomic studies on ACC [15,16"",40]. These analyses have all relied on expression analyses of large numbers of genes, but subsequent studies have been able to identify smaller subsets with comparable dis- criminatory power, which would be more practical for personalized clinical applications. Overexpression of IGF2 has been previously shown to be a key marker of malignancy in ACTs; notably, patients with Beck- with-Wiedemann syndrome have dysregulated IGF2 production and develop ACCs at high rates [41]. Previous work has demonstrated transcriptome signatures for APA (with overexpression of CYP11B2, the enzyme responsible for the key step in aldosterone synthesis) and CPA, providing another potential tool for molecular diagnostics in these tumors [42-46,47”].
CONCLUSION
Recent advances in the molecular understanding of ACTs provide an exciting opportunity to improve diagnostics and develop personalized therapeutics for this family of diseases. In addition to validating previously identified drivers of adrenocortical tumorigenesis, genomic analyses of ACTs have iden- tified a new set of candidate genes that may prove to be effective therapeutic targets, greatly needed espe- cially in ACC. In combination with the recent advances in ACT epigenomics, the strides in ACT genome analysis will provide a solid foundation for the much needed translational studies and ulti- mately clinical use to improve the diagnosis and treatment of this heterogeneous family of tumors.
Acknowledgements
The authors would like to thank Reju Korah, PhD, for helpful content review of this article.
Financial support and sponsorship
None.
Conflicts of interest
The authors report no conflicts of interest relevant to the current review.
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