REVIEW ARTICLE
Molecular markers and targeted therapies for adrenocortical carcinoma
Yunze Xu ** t, Yicheng Qit, Yu Zhu*, Guang Ning# and Yiran Huangt
*Department of Urology, School of Medicine, Ruijin Hospital, Shanghai Jiao Tong University, Department of Urology, School of Medicine, Renji Hospital, Shanghai Jiao Tong University and ¿Department of Endocrinology, Clinical Center of Shanghai Endocrine and Metabolic Diseases, School of Medicine, Ruijin Hospital, Shanghai Jiaotong University, Shanghai, China
Summary
Adrenocortical carcinoma (ACC) is a lethal disease with poor prognosis and lack of effective therapeutic options. Systemic treatment is often employed to treat patients with advanced ACC, but outcomes are disappointing. During the last decade, some of the causative genetic mutations in sporadic ACCs have been identified. Molecular analysis has had a significant impact on the understanding of the pathogenetic mechanism of ACC development and the evaluation of prognostic and predictive markers. Preclinical investigations and clinical trials of tyrosine kinase inhibitors and anti-angiogenic compounds have been ini- tiated to seek target therapy of ACCs. This review summarizes the current view of molecular alterations involved in the patho- physiology of adrenocortical carcinogenesis. The rationale for testing targeted therapies of ACC is also presented.
(Received 5 August 2013; returned for revision 13 September 2013; finally revised 9 October 2013; accepted 28 October 2013)
Introduction
Adrenocortical carcinoma (ACC), derived from the adrenal cor- tex, is a rare but aggressive endocrine malignancy with an esti- mated annual incidence of 0-7-2-0 cases per million population, accounting for <5% of adrenal incidentalomas.14 ACC can develop at any age, but the tumour shows bimodal distribution peaks, the first occurring in children <5 years of age and the sec- ond in adults between age 40 and 50 years.3 There is an anoma- lously high incidence in southern Brazil with 3-4-4-2 cases per million children under 15 years, a rate that is 12-18 times
higher than other countries, which can be attributed to the endemic germline TP53 R337H mutation.5-8
The majority of patients with ACC (60%) present with signs and symptoms of adrenal steroid excess. Rapidly progressing Cushing syndrome with or without virilization is the most fre- quent presentation.9,10 In contrast, patients with a nonfunction- ing ACC may present with abdominal discomfort or back pain referable to the tumour mass, which is usually at least 10 cm in diameter.11 ACC can also be discovered incidentally by improved abdominal imaging in asymptomatic individuals. ACC is staged on the basis of a revised TNM classification, which was reported by the European Network for the Study of Adrenal Tumors (ENSAT). This classification scheme restricts stage IV ACC to patients with distant metastatic disease and defines stage III ACC as the presence of venous tumour thrombosis, tumour infiltration into surrounding tissue or positive lymph node(s). However, because of the rarity of ACC, the prognostic value of the revised TNM system remains controversial.
There is no effective treatment of patients with advanced ACC, and poor understanding of the molecular alterations in the tumourigenesis of ACC might be one of the most significant reasons for this situation. Complete surgical resection is virtually the sole hope of cure in ACC,12 but recurrence occurs in approximately 60-80% of patients after so-called complete resection.13,14 Those patients with apparently localized tumours at diagnosis frequently develop metastatic disease within 6-24 months of surgical resection.15 Another 30-40% of patients have clear evidence for metastatic disease (most frequent in lung or liver) at the time of primary diagnosis, and surgical removal then becomes unfeasible.16 In cases of advanced ACC, the com- bination of cytotoxic drugs and mitotane is recommended as first-line therapy, but the overall prognosis is still very poor.17 Patients with recurrent disease or stage IV disease at diagnosis have a 5-year survival <5%,15,18 and untreated patients with unresectable disease survive only 3-9 months. 13,17,19
Most ACCs are sporadic neoplasms of undetermined aetiol- ogy, but some cases occur as a component of hereditary tumour syndromes, including the Li-Fraumeni syndrome (LFS), Beckwith-Wiedemann syndrome (BWS), multiple endocrine
Correspondence: Yiran Huang, Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 200 Pujian Road, Shanghai, 200127, China; and Yu Zhu, Department of Urology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, No. 197 Ruijin Er Road, 200025, Shanghai, China. Tel .: +86 21 64370045; E-mails: hyrrenji2@yahoo.com.cn; zyyyhyq@163.com
Yunze Xu and Yicheng Qi contributed equally to this work.
neoplasia 1, Carney complex and Lynch syndrome.20,21 The causative genetic mutations in these syndromes have also been identified to be involved in the tumourigenesis of ACC. Recently, advances in the understanding of the molecular patho- genesis of ACC have been made based on studies of gene expres- sion profiling and of the above genetic syndromes associated with the development of ACC.22,23 Accordingly, a rapidly grow- ing number of clinical trials testing novel therapies for ACC have been initiated (Table 1). The goal of this review is to pro- vide insights into the pathophysiology of adrenocortical carcino- genesis and the rationale for testing targeted therapies.
Genetic alterations
Studies using loss of heterozygosity (LOH) analysis have shown that LOH of 17p13, 11p15, 11q13, 17q22-24 and 2p16 tends to occur more frequently in sporadic ACCs than in adrenocortical adenomas (ACAs),20,24 whereas comparative genomic hybridiza- tion (CGH) analyses have revealed that genetic abnormalities and chromosomal losses are more likely to occur in ACCs than in ACAs,25 suggesting that genetic disruption is associated with the malignant phenotype in sporadic adrenocortical tumours (ACTs). The molecular pathogenesis of ACC remains an area of uncertainty and controversy.20,26
TP53 gene
The tumour suppressor gene TP53 is the most frequently mutated gene in human cancers27: somatic TP53 mutations occur in about half of all human cancers and constitute a cor- nerstone in tumourigenesis.28,29 Germline mutations of the p53 gene located at the 17p13 locus are present in 70% of patients with LFS, in which the frequency of ACC is up to 4%.30,31 LFS is a rare familial cancer syndrome characterized by the early onset of tumours and multiple tumours in at least two-first- degree relatives. This syndrome confers a high susceptibility to breast carcinoma, soft tissue sarcoma, brain tumours, osteosar- coma, leukaemia and ACC.31,32
Mutations of TP53 in the highly conserved region of exons 5-8 have been observed in 20-27% of sporadic ACCs.33 Somatic mutations of TP53 have been observed in 20-35% of cases of sporadic ACC in adults,33,34 and 50-80% of children with spo- radic ACC have germline mutations of the p53 tumour suppres- sor gene.35 TP53 somatic mutations define a subgroup of ACC with different tumourigenesis and poor outcome.36
In children with ACC in southern Brazil, a recurrent TP53 germline mutation, p.R337H, leading to a pH-sensitive and tem- perature-dependent alteration in the function of the p53 protein, has been demonstrated.37 This p.R337H mutation was found in 78% of children with sporadic ACTs originating from Southern Brazil and was also found in 13% of adult patients with ACTs.38 More recently, Achatz et al.39 have screened for TP53 mutations in 45 unrelated Brazilian individuals with family histories fulfill- ing the clinical definitions of LFS or Li-Fraumeni-like (LFL) syndromes. The germline p.R337H mutation was found in 46% of patients harbouring TP53 mutations, and families with the R337H mutation were associated with a wide spectrum of tumours, including breast cancers (30%), brain cancers (11%), soft tissue sarcomas (10-7%) and ACTs (8.9%). The mutation is related to unfavourable prognosis in most of the adults, but not in the children with ACTs.38,40
Loss of heterozygosity at 17p13 has been demonstrated in 80% of ACCs, but it does not always correlate with the presence of TP53 mutation,34,41 suggesting that another tumour suppres- sor gene exists in this locus. 17p13 LOH is predictive of long- term outcome in adults with ACTs,41 but it does not correlate with aggressive tumour behaviour and poor prognosis in paedi- atric ACTs.40 Compared with ACAs, a minimal region of loss of 10-4-Mb on 17p13 and significantly down-regulated expression of ACADVL and ALOX15B genes within this region have been identified in ACCs.24
Insulin-like growth factor 2
Another hereditary syndrome associated with ACC is the BWS, caused by alterations at 11p15,42 a chromosomal locus including
| Agent | Rationale | Outcome | References |
|---|---|---|---|
| Bevacizumab + capecitabine | Inhibition of VEGF signalling + cytotoxic drug | No response in all 10 patients | Wortmann et al.110 |
| OSI-906 | A small-molecule tyrosine kinase IGF-1R inhibitor | A partial tumour response in 5 of 16 patients with advanced ACC | Carden et al.55 |
| Erlotinib+ gemcitabine | Inhibition of EGFR signalling + cytotoxic drug | A minor response in 10 patients with advanced ACC | Quinkler et al.91 |
| Figitumumab Gefitinib Thalidomide | An anti-IGF-1R monoclonal antibody Inhibition of EGFR signalling An anti-angiogenic compound that inhibits the activity of bFGF-2 | Stable disease in 8 of 14 patients with refractory ACC No response in all 19 patients A partial response in a patient with advanced chemoresistant ACC | Haluska et al.54 Samnotra et al.112 Chacon et al.96 |
| Imatinib | Inhibition of c-KIT and PDGF | No response in all four patients | Gross et al.111 |
ACC, adrenocortical carcinoma; bFGF-2, basic fibroblast growth factor; EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor.
the insulin-like growth factor 2 (IGF2), H19 and cyclin-dependent kinase inhibitor 1C (CDKN1C also known as p57kip2) genes. Rear- rangements, LOH and abnormal imprinting of the 11p15.5 locus, resulting in elevated IGF2 and low CDKN1C and H19 expression levels, have been observed in the majority of ACCs.43,44 The IGF system, which has growth-promoting as well as differentiation functions in the adrenal gland, comprises two peptide ligands (IGF1 and IGF2), two IGF receptors (IGF1R and IGF2R/man- nose-6-phosphate receptor) and six high-affinity binding proteins (IGF-binding proteins 1-6).45,46 Both IGF1 and IGF2 can induce steroidogenesis in adrenocortical cells in vitro and in vivo, but IGF1 has not been shown to be overexpressed in sporadic ACCs.47
Structural abnormalities at the 11p15 locus, including duplica- tions of the paternal 11p15 allele and loss of the maternal allele containing the H19 gene, can increase IGF2 expression.44 In fact, IGF2 is the single most up-regulated transcript in 80-90% of ACCs, compared with ACAs and normal adrenal cortices.22,48 Higher IGF2 expression levels are associated with a more malig- nant phenotype and a higher risk of ACC recurrence.41,47 IGF2 elicits its cellular effects through the IGF1 receptor (IGF1R), which is also overexpressed in most ACCs.49 More recently, Doghman and colleagues confirmed IGF1R overexpression in paediatric ACTs and showed that miR-100 down-regulation is one of the mechanisms to explain IGF1R overexpression in pae- diatric ACT.50
Furthermore, LOH of the 11p15 locus has been demonstrated to be more frequent in ACCs than in ACAs (78.5 vs 9.5%) and is associated with a higher risk of tumour recurrence.41 Thus, activation of the IGF pathway may be a critical mechanism employed by tumour cells and triggers a series of molecular events during adrenocortical tumourigenesis. Based on the reported data that antagonism of the IGF1R inhibits the growth of tumour cells in other solid tumours, several preclinical studies involving IGF-IR inhibition have been initiated in ACC. A highly selective IGF1R antagonist, NVP-AEW541, showed anti- proliferative and proapoptotic effects in a dose- and time-depen- dent manner on both the H295R ACC cell line and another cell line derived from a paediatric ACTs.51 Barlaskar et al. have also reported the preliminary results that IGF1R antagonists, includ- ing NVP-AEW541 and IMC-A12, caused significant dose-depen- dent inhibition of growth of ACC cell lines in vitro and in xenograft models and that mitotane enhanced growth suppres- sion in culture and in xenografts when used in combination with the IGF1R antagonists. Statistical analysis indicated that IGFIR antagonists as monotherapy were more potent than mito- tane to reduce tumour growth, and the association of mitotane and IGFIR antagonist produced synergistic effect.52,53
Treatment with an anti-IGF1R monoclonal antibody fig- itumumab resulted in stable disease in 8 of 14 patients with refractory ACC (57%) in another phase I study. The results of this study are somehow less encouraging, because no relevant clinical activity was demonstrated. However, four patients experi- enced tumour shrinkage without meeting the RECIST criteria of partial response. The side effect profile and pharmacokinetics of figitumumab in ACC were similar to patients with other solid tumours, but treatment with figitumumab increased serum
insulin and growth hormone levels.54 A phase I study of a small- molecule tyrosine kinase IGF1R inhibitor (OSI-906) demon- strated one patient had a partial response and four had stable disease by RECIST.55 Currently, an international phase 3 trial of OSI-906 has completed its accrual goal of 135 patients. The results of this trial are eagerly awaited. Early preclinical data suggested the activity of IGF-1R-targeted drugs in ACC, but the initial enthusiasm quickly encountered several challenges and disappointment.
ß-catenin
The Wnt signalling pathway plays an important role in adrenal cortex development, and ß-catenin is a key component of this signalling pathway.56 Wnt binds to its receptor complex and then inhibits the axin-adenomatous polyposis coli (APC)-glyco- gen synthase kinase 3ß (GSK-3) complex, leading to a block in B-catenin phosphorylation by GSK-3 and accumulation of B-catenin in the cytoplasm. After translocation into the nucleus, B-catenin is available to bind the TCF/LEF family of transcrip- tion factors and to induce Wnt target gene expression.
In both benign and malignant ACTs, ß-catenin accumulation has frequently been identified, indicating activation of the Wnt signalling pathway.59,60 Tissier et al.61 have analysed the Wnt pathway in 26 ACAs and 13 ACCs, and abnormal cytoplasmic and/or nuclear accumulation of ß-catenin was found in 10 of 26 (38%) ACAs and 11 of 13 (77%) ACCs. In a subset of these tumours, somatic mutations of the ß-catenin gene (CTNNB1) abolish or reduce GSK3ß phosphorylation of ß-catenin, which leads to its accumulation by preventing its degradation by the ubiquitin-proteasome system.20 Immunohistochemical studies have shown abnormal distribution of the ß-catenin protein in ACCs. There is a higher rate of abnormal immunohistochemis- try for ß-catenin in ACCs than in ACAs. However, similar fre- quencies of mutations of the ß-catenin gene are found in both ACAs and ACCs (27 vs 31%).61 Constitutive activation of B-catenin in transgenic mice has recently been shown to trigger benign aldosterone-secreting tumour development and promote adrenal cancer development.62 Ragazzon et al.36 also suggest that alterations to the Wnt/B-catenin pathway are a poor prognostic factor in ACC.
Preclinical studies have evaluated the effect of a small-mole- cule inhibitor of Wnt signalling (PKF115-584) on proliferation of the human ACC cell line H295R, which has a constitutively active ß-catenin due to a heterozygous mutation, S45P.63 Doghman et al.64 have demonstrated that PKF115-584 not only inhibited ß-catenin-dependent transcription and proliferation of an adre- nocortical cancer cell line but also overruled the effect of enhanced SF-1 levels on H295R proliferation. So far, ß-catenin antagonists remain in preliminary preclinical investigation and may prove useful in the treatment for ACC in the future.
Steroidogenic factor-1
Steroidogenic factor 1 (SF1) is a nuclear transcription factor, which has a pivotal role in the development and function of
steroidogenic tissues and in the regulation of cytochrome P450 steroidogenic enzyme expression in the adrenal cortex.65,66 SF-1 amplification and overexpression have been demonstrated widely in adult and paediatric ACCs, suggesting an association with adrenocortical tumourigenesis.67,68
Recently, Almeida et al.69 assessed SF-1 protein expression in a cohort of 103 ACTs from 36 children and 67 adults and analy- sed gene amplification in 38 tumours. Increased SF-1 copy num- ber was found in 47% of paediatric ACTs and in only 10% of adult tumours, confirming a higher frequency of SF-1 overex- pression and gene amplification in paediatric than in adult ACTs. SF-1 expression is not correlated with functional status of ACTs, indicating that SF-1 functions are more related to modu- lation of cell proliferation and apoptosis than to steroidogenesis in adrenal tumour cells.69,70 SF-1 overexpression might be caused by additional mechanisms other than gene amplification, because strong SF-1 staining without SF-1 amplification could be identified.69 In addition, increased SF-1 expression has been shown to promote the proliferation of human adrenocortical cells in vitro and to trigger tumourigenesis in mice.70,71 Further- more, as a stage-independent prognostic marker, SF-1 expression is significantly correlated with poor clinical outcome in a cohort of adult ACCs.72
SF-1 inverse agonists have recently been identified by Dogh- man to affect ACTs cell proliferation. Studies have demonstrated that SF-1 inverse agonists of the isoquinolinone class selectively down-regulated H295R proliferation elicited by SF-1 overexpres- sion and steroidogenesis in vitro, whereas they had no inhibitory effect on proliferation of the SW-13 cell line, which does not express SF-1.73 In addition, SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and SF-1-negative ACT cells.
Melanocortin 2 receptor
The binding of ACTH to the melanocortin 2 receptor (MC2R; also known as the ACTH receptor), a member of the G protein- coupled receptor superfamily, regulates the hypothalamus-pitui- tary-adrenal axis and stimulates steroidogenesis in the adrenal gland.74 LOH of MC2R has been found in one of 16 ACAs and two of four ACCs, which suggests that MC2R may play a role in cellular differentiation and adrenocortical tumourigenesis.75 Chida et al.76 have generated mice with an inactivating mutation of the MC2R gene to elucidate that disruption of MC2R leads to adrenocortical atrophy and reduced production of glucocortic- oids and aldosterone. MC2R mRNA expression is up-regulated in patients with functional ACAs but down-regulated in those with nonfunctional ACAs or carcinomas.77 However, no activat- ing mutations of the MC2R gene were identified in adrenal tumours, including ACAs, ACCs and hyperplasias.77
Growth factors
Many growth factors and cytokines have been shown to regulate tumour growth and function in the adrenal glands, including
vascular endothelial growth factor (VEGF), basic fibroblast growth factor 2 (bFGF-2), transforming growth factor-a (TGF- a), transforming growth factor-ß1 (TGF-ß1) and interleukins (Fig. 1).12,78
Vascular endothelial growth factor has been proposed to be an important and powerful stimulant of angiogenesis, and assessment of VEGF expression is a way to quantify the angio- genic status of a tumour. VEGF binding to the tyrosine kinase VEGF receptor, usually VEGF-R2, activates a signalling cascade, which induces endothelial cell (EC) proliferation and promotes EC migration.79 All these actions eventually lead to neovascular- ization.80 Zacharieva and de Fraipont et al. demonstrated that expression of VEGF is significantly elevated in tumour tissue and circulating blood in patients with ACC,81,82 and elevated levels of VEGF in circulating blood fall after successful tumour resection.83,84 Recently, we assessed VEGF and its receptor VEGF-R2 staining in a cohort of 44 adult ACTs; the results were consistent with previous studies showing increased VEGF expression in 71% of ACCs and in only 25% of adenomas. VEGFR-2 was strongly expressed in the majority of ACCs (79%, 19/24) and weakly expressed in the ACA group (25%, 5/20).85 However, ACC is characterized by VEGF overexpression and low vascularization, indicating a disassociation between angiogenic status and neoangiogenic capabilities of these tumours.86 Bev- acizumab is a humanized monoclonal antibody that prevents VEGF binding to its receptors on the surface of ECs by binding to VEGF, leading to an inhibitory effect on VEGF-induced increased vessel permeability and EC migration and proliferation.
Growth Factors (VEGF, IGF-1R etc.)
EGF
Antibodies
Kinase Inhibitors
IGF-IR
Cytoplasm
P
P
P
P
PI3K
RAS
S
AKT
T
RAF
A
T
mTOR
MEK
PP
Myc
qelin DI
DNA
Nucleus
Jun Fos
Myc
Proliferation
Apoptosis
Angiogenesis
Metastasis
A study of 10 patients with advanced ACC reported no response to a combination of the anti-VEGF antibody bevacizumab plus capecitabine given as salvage treatment, suggesting that this combination has no activity in patients with advanced ACC and cannot be recommended as a salvage therapy.87
The epidermal growth factor receptor (EGFR) is overexpressed in more than 90% of ACCs but hardly expressed in ACAs.88,89 However, no mutations of the EGFR gene have been found, and EGFR expression does not correlate with clinical outcome in patients with ACC.90 Fassnacht et al. have released preclinical data at the Ninth European Congress of Endocrinology, suggest- ing that inhibition of the EGFR signalling pathway leads to a significant inhibition of proliferation in the H295R ACC cell line. Nevertheless, Quinkler et al.91 have reported that the com- bination of the EGFR inhibitor erlotinib with gemcitabine exhib- ited only limited efficacy as salvage therapy in 10 patients with advanced ACC. Only one individual had a minor response in this study, whereas the remainder experienced progressive dis- ease or toxicities at the first staging. Similarly, no activity of the EGFR antagonist gefitinib was seen in 19 patients with advanced ACC previously treated with mitotane or chemotherapy. Fur- thermore, another EGFR inhibitor, BMS-690514, is currently being investigated in a multicentre phase I study enrolling patients with advanced/metastatic solid tumours (including ACC).92 In regard to synergy, there is well-documented func- tional crosstalk between the IGF1R and EGFR signalling path- ways.93 It has been demonstrated that tumours that are sensitive to IGF1R inhibitors rapidly up-regulate signalling through the EGFR system as a means to resistance. In addition, reciprocal EGFR compensation could mediate resistance to IGF1R inhibi- tion. For this reason, targeting both these pathways together would seem a rational approach likely to enhance the effective- ness of the agents.
Fibroblast growth factors (FGF) including FGF-1 and FGF-2 are expressed in normal adrenal cortex and bind to a family of four fibroblast growth factors receptor (FGFR) tyrosine kinases. Two members of this kinase family, FGFR1 and FGFR4, are overexpressed in ACTs,94 and expression of FGFR4 is much higher in ACC than in ACA in paediatric patients.95 Thalido- mide, an anti-angiogenic compound that inhibits the activity of bFGF-2, has an effect on ECs by interacting with heparan- sulphate proteoglycans and tyrosine kinase FGF receptors. In a single case report, a partial response to thalidomide has been detailed in a patient with advanced chemoresistant ACC.96 Part of the limitation to targeting FGF signalling has been the lack of agents influencing this pathway.
MicroRNA (miRNA) in ACC
The human genome is transcribed to produce protein coding and noncoding RNAs (ncRNAs). As protein coding genes have been the focus of most research, the functional role of ncRNAs has long been underestimated. Accumulating research indicates that ncRNAs, especially miRNAs, are increasingly being impli- cated in ACC.97 Both in vitro and in vivo studies have shown
that alterations of miRNAs could have profound effects on hun- dreds of target genes, thus possibly implicating multiple biologi- cal pathways.98 miRNAs are conserved small ncRNAs that are involved in the epigenetic regulation of cellular processes. One- third of coding genes are regulated by miRNAs and therefore changes in miRNA expression maybe associated with cancer development and progression.99 This emerging field is anticipated to profoundly affect clinical research, diagnosis and therapy.
The first study examining for deregulated miRNAs in ACCs was performed on seven ACCs, nine cortisol producing ACAs (CPAs), 10 hormonally inactive ACAs and 10 normal adreno- cortical tissue samples using quantitative PCR (qPCR). Low- density array revealed significant differences in the expression of six miRNAs: miR-184, miR-210, miR-214, miR-375, miR-503 and miR-511. Of these, miR-184, miR-210 and miR-503 were up-regulated in ACCs compared with ACAs or normal adrenal cortices. The difference between dCT mir-511 and dCT mir-503 (delta cycle threshold) can help to accurately distinguish between ACCs and ACAs.100 miR-375 is located at 2q35. It has been reported that underexpression of miR-375 might influence ACC pathogenesis through direct interaction with the ß-catenin pathway.101
Another miRNA analysis of 22 ACAs, 27 ACCs and six nor- mal adrenal cortices suggested that 23 miRNAs were significantly differentially expressed between ACCs and ACAs, 14 of which were up-regulated and the remaining nine down-regulated. This observation found that miR-195 and miR-335 were down-regu- lated, and miR-483-5p was up-regulated in ACCs compared with ACAs. Patients with ACC with up-regulated miR-483-5p and down-regulated miR-195 defined a subgroup of ACC with sig- nificantly poorer prognosis.102,103 Assessing miR-483-5p and miR-195 expression levels in serum may serve as promising non- invasive biomarkers with a highly specific prognostic value for the clinical outcome in patients with ACC.21,104,105 Down-regu- lation of miR-195 and miR-335 in ACCs compared with ACAs was confirmed by another study. In this study of six ACAs and 12 ACCs, the authors used miRXplore microarrays to evaluate the expression profiles of 728 human miRNAs. They found a total of 12 differentially expressed miRNAs between ACCs and ACAs, of which five were down-regulated and seven up-regu- lated in ACCs. Furthermore, 29 miRNAs were significantly dif- ferentially expressed between two subgroups of patients with ACC, consisting of six recurrent (subgroup A) and six nonrecur- rent tumours (subgroup B). The levels of miR-335 were further down-regulated in the subgroup of recurrent ACCs as compared to nonrecurrent ACCs.106
Another study of 26 ACAs, 10 ACCs and 21 normal adrenal cortices found a total of 23 differentially expressed miRNAs between ACCs and ACAs, of which five were up-regulated and 18 were down-regulated. Expression of miR-483-5p predicted malignant cases with high sensitivity and specificity. In addition, up-regulated miR-483-5p and down-regulated miR-195 signifi- cantly correlated with poorer disease-specific survival in patients with ACC, thus confirming the study by Soon et al. miR-483-5p
is located at 11p15.5 within the second intron of IGF2. The high expression of miR-483-5p was statistically significantly co-expressed with IGF2 in ACCs, suggesting that miR-483-5p may be indicative of the expression levels of IGF2.98
Schmitz et al.106 studied four ACCs, three ACCs with metasta- sis, nine ACAs and four normal adrenocortical tissue samples. miRNA profiling for 667 miRNAs was undertaken using a qPCR low-density array and revealed down-regulation of 159 and up-regulation of 89 miRNAs in ACCs compared with ACAs. Three key miRNAs, including miR-139-3p, miR-675 and miR-335, were found to be significantly down-regulated in ACCs as compared to ACAs. miR-139-5p was found to be significantly up-regulated in ACCs with very poor outcome. As discussed earlier, studies have shown that miRNAs may be useful biomar- kers in ACCs because of their stability and the sensitivity of the detection methods available.
DNA methylation in ACC
More recently, changes at the epigenetic level have been impli- cated in carcinogenesis and found to be diagnostic markers. The best-defined epigenetic modification is DNA methylation of cytosines, which affects a number of different cellular processes including apoptosis, cell cycle, DNA damage repair, growth fac- tor response, signal transduction and tumour architecture, all of which can result in tumourigenesis and its progression.107 Rech- ache et al.108 performed a comprehensive genome-wide study of the DNA methylome of normal (n = 19), benign (n = 48), pri- mary malignant (n = 8) and metastatic malignant (n = 12) adre- nocortical tissue samples using a platform with 485,421 cytosine probe sites. They found that ACCs have a global hypomethyla- tion pattern compared with normal and benign adrenocortical tissue samples. Methylation patterns could distinguish normal, benign, primary malignant and metastatic tissue samples. Deter- mination of the methylation profile difference in certain probe sites in ACTs may be able to classify primary ACC and benign ACTs. Several CpG sites were differentially methylated in ACC samples compared with benign tissue samples, including those associated with KCTD12, KIRREL, SYNGR1 and NTNG2 and those in the chromosome 11p15 imprinted region including IGF2 and H19.
Fonseca et al.109 investigated the DNA methylation levels of 27,578 CpG sites in bisulphite-modified DNA from six normal adrenocortical tissue samples, 27 ACAs and 15 ACCs. When comparing the methylome of normal adrenal cortex and ACAs, genes known to be involved in cell cycle regulation, apoptosis and transcriptional regulation of cancer such as CDKN2A, GATA4, SCGB3A1, PYCARD and DLEC1 were highly and sig- nificantly hypermethylated in ACC. Comparing benign vs malig- nant ACTs, a total of 212 CpG islands were identified as significantly hypermethylated in ACC. Furthermore, treatment of H295R cells with a demethylating agent increased expression of the hypermethylated genes CDKN2A, GATA4, DLEC1, HDAC10, PYCARD and SCGB3A1/HIN1. Methylation profile differences may accurately distinguish ACC from benign tumours with high sensitivity and specificity.
Conclusion
Adrenocortical carcinoma (ACC) remains a deadly disease. His- torically, the rarity and complexity of ACC have confounded multinational trials. Recent advances in our understanding of the molecular pathogenesis of ACC offer hope that targeted therapies can be developed and successfully implemented. It is acknowledged that improving the clinical care of patients with ACC will require a combination of basic science, translational research and clinical trials.
Acknowledgements
This study was supported by the grants from the National Natu- ral Science Foundation of China (No. 81272936; No. 81272841; No. 91129725) and Shanghai Municipal Natural Science Foun- dation (No. 134119a2700).
Conflict of interests
Nothing to declare.
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