DAX1 Overexpression in Pediatric Adrenocortical Tumors: A Synergic Role with SF1 in Tumorigenesis
| Authors | G. R.V. de Sousa1, I. C. Soares2, A. M. Faria1, V. B. Domingues3, A. Wakamatsu3, A. M. Lerario1, 4, V. A. F. Alves3, M. C. N. Zerbini3, B. B. Mendonca1, M. C. B. V. Fragoso1, 4, A. C. Latronico1, M. Q. Almeida1, 4 |
| Affiliations | Affiliation addresses are listed at the end of the article |
Key words DAX1 SF1 expression adrenocortical tumors
Abstract
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DAX1 transcription factor is a key determinant of adrenogonadal development, acting as a repres- sor of SF1 targets in steroidogenesis. It was recently demonstrated that DAX1 regulates pluripotency and differentiation in murine embryonic stem cells. In this study, we investi- gated DAX1 expression in adrenocortical tumors (ACTs) and correlated it with SF1 expression and clinical parameters. DAX1 and SF1 protein expression were assessed in 104 ACTs from 34 children (25 clinically benign and 9 malignant) and 70 adults (40 adenomas and 30 carcinomas). DAX1 gene expression was studied in 49 ACTs by quantitative real-time PCR. A strong DAX1 pro- tein expression was demonstrated in 74% (25 out of 34) and 24% (17 out of 70) of pediatric and
adult ACTs, respectively (x2=10.1, p=0.002). In the pediatric group, ACTs with a strong DAX1 expression were diagnosed at earlier ages than ACTs with weak expression [median 1.2 (range, 0.5-4.5) vs. 2.2 (0.9-9.4), p=0.038]. DAX1 expres- sion was not associated with functional status in ACTs. Interestingly, a positive correlation was observed between DAX1 and SF1 protein expres- sion in both pediatric and adult ACTs (r=0.55 for each group separately; p<0.0001). In addition, DAX1 gene expression was significantly corre- lated with SF1 gene expression (p<0.0001, r=0.54). In conclusion, DAX1 strong protein expression was more frequent in pediatric than in adult ACTs. Additionally, DAX1 and SF1 expres- sion positively correlated in ACTs, suggesting that these transcription factors might cooperate in adrenocortical tumorigenesis.
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received 23.09.2014 accepted 18.12.2014
Bibliography
DOI http://dx.doi.org/ 10.1055/s-0034-1398560 Published online:
May 18, 2015 Horm Metab Res 2015; 47:656-661 @ Georg Thieme Verlag KG Stuttgart . New York ISSN 0018-5043
Correspondence
M. Q. Almeida, MD Unidade de Suprarrenal, Laboratório de Hormônios e Genética Molecular LIM-42, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo Av. Dr. Enéas de Carvalho Aguiar, 155, 2 andar, Bloco 6 05403-900 São Paulo SP Brazil
Tel .: +55/11/26617 512
Fax: +55/11 26617 519
Introduction
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DAX1 (dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on X chro- mosome gene 1; NR0B1) is an orphan member of the nuclear receptor superfamily and a key deter- minant of human adrenogonadal development and function [1,2]. In humans, inactivating mutations in the DAX1 gene cause X-linked adre- nal hypoplasia congenita associated with hypog- onadotropic hypogonadism [1,2]. Similarly, SF1 (steroidogenic factor 1 gene; NR5A1) inactivating mutations were later identified in patients with adrenal insufficiency caused also by congenital adrenal hypoplasia [3]. Although DAX1 and SF1 inactivating mutations produce a corresponding adrenal phenotype in humans, DAX1 inhibits SF1- mediated transactivation of target genes in both steroidogenic and nonsteroidogenic tissues [4]. Adrenocortical carcinoma is a rare malignancy with incompletely understood pathogenesis [5]. In Southern Brazil, the incidence of adrenocorti-
cal tumors (ACTs) in children is remarkably high, being estimated as 10-15 times greater than the worldwide incidence [6]. We and others demon- strated an important role of SF1 gene in pediatric and adult tumorigenesis [7-9]. SF1 gene amplifi- cation and overexpression are frequent molecular events in pediatric ACTs [9]. On the other side, SF1 gene amplification is less common in adult ACTs, but its protein overexpression is correlated with a reduced overall and disease-free survival [7,8]. A novel role for Dax1 in murine embryogenesis has recently been demonstrated [10,11]. Dax1 is expressed in high levels in murine embryonic stem cells and contributes to the maintenance of a relatively undifferentiated state [10, 11]. Khal- fallah et al. [12] showed that Dax1 knockdown induces upregulation of multilineage differentia- tion markers and decreases embryonic stem cells viability and proliferation. In addition, DAX1 directly regulates oncogenesis in Ewing’s Sar- coma, modulating gene expression and mediat- ing tumor phenotype progression [13].
Besides the essential role of DAX1 in the adrenal development, the involvement of this transcription factor in the adrenocortical tumorigenesis remains to be determined. DAX1 expression was previously studied in small cohorts of adult ACTs and correlated with nonfunctioning tumors [14-16]. In this study, we investi- gated the expression of DAX1, a stem cell fate regulator, in a large series of pediatric and adult ACTs, and correlated it with SF1 expression and clinical parameters.
Patients and Methods
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The study was approved by the Ethics Committee of Hospital das Clinicas, Sao Paulo, Brazil, and informed written consent was obtained from all patients and/or parents. DAX1 protein expres- sion was assessed in ACTs obtained from 34 children [25 clini- cally benign (CB) and 9 clinically malignant (CM)] and 70 adults (40 adenomas and 30 carcinomas) (· Table 1). Forty-nine spo- radic ACTs from 20 children (15 clinically benign and 5 clinically malignant) and 29 adults (14 adenomas and 15 carcinomas) were used to analyze DAX1 gene expression. This same cohort of patients was previously studied for SF1 protein and gene expres- sion [8]. In adults, the Weiss criteria were used to classify adeno- mas and carcinomas (Weiss score <3 and ≥3, respectively). Pediatric ACTs (age <15 years) were classified as CB or CM, as proposed by Wieneke et al. [17], according to advanced tumor stage (III or IV) and/or poor clinical outcome, since Weiss scores are a poor predictor of prognosis in the pediatric group [18]. Clinical parameters, such as sex, age at diagnosis, date of surgery, tumor size, pathological classification, and hormone analysis were collected from patient records. Tumor stage was classified according to the European Network for the Study of Adrenal Tumors (ENSAT) classification [19]. Presence of distant metasta- ses or recurrence was evaluated at the time of diagnosis and during follow-up visits by computerized tomography of chest and abdomen every 3-6 months.
Tissue microarray (TMA) and immunohistochemical analysis
Representative areas of the 104 adrenocortical tumors and 2 normal adrenal tissue samples were identified on hematoxylin and eosin stained slides, and marked on paraffin donor blocks. The procedures used for TMA construction and immunohisto-
chemical analysis were performed according to a previous description [8]. One set of 3 slides was selected (one slide from each TMA block of the triplicate) for staining with DAX1 anti- body (K-17, Santa Cruz Biotechnology, Santa Cruz, CA, USA) [20,21]. An additional TMA containing 13 normal adrenals obtained from autopsies (7 from children and 6 from adults ) was constructed.
Two investigators (ICS and VBD), who were unaware of clinical data, independently, evaluated DAX1 staining. The staining intensity and the percentage of positive tumor nuclei were cal- culated for each specimen to obtain a final semi-quantitative H score [22]. Only nuclear immunoreactivity was considered for quantification. The median value of all the H scores was a priori chosen as the cutoff point for separating adrenocortical tumors with strong DAX1 staining from tumors with weak or negative staining for DAX1, as previously described [22]. Then, the posi- tive samples were categorized as weak (H score from 0.0 to 0.49) or strong (H score≥0.5). The inter-observer agreement was 0.83. The mean of the 2 evaluations (each one in triplicate) was taken for statistical analysis.
Quantitative real-time PCR (qRT-PCR)
After surgical resection, tumor fragments were immediately fro- zen in liquid nitrogen and stored at -80℃ until total RNA extraction using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was generated using the High Capacity kit (Applied Biosystems). Quantitative real-time PCR was performed using TaqMan Gene Expression Assays according to the manufactur- er’s instructions. The assay IDs were: DAX1, Hs00230864_m1 and B-actin, 43263. The relative expression levels were analyzed using the 2-AACT method [23]. The mean expression of the target genes in a pool of 62 normal adrenals (Clontech, Palo Alto, CA, USA) was assigned an expression value of 1.0 and fold change in the DAX1 expression levels was determined for each tumor sample.
Statistical analysis
All statistical analyses were performed with the SPSS 16.0 (SPSS, Chicago, IL, USA). Weighted kappa coefficient of agreement was used to evaluate the agreement between the observers. Categor- ical variables were compared using chi-square (x2) test. Pear- son’s coefficient was used to correlate DAX1 and SF1 staining. Continuous data are expressed as mean±SEM. Differences in
| Children (n=34) Weak DAX-1 staining (n=9) | Strong DAX-1 staining (n=25) | Adults (n=70) Weak DAX-1 staining (n=43) | Strong DAX-1 staining (n=27) | |
|---|---|---|---|---|
| Age (years)£ | 1.6±1.3 | 3.0±2.3 | 34.1±13.8 | 40.7±15.9 |
| Sex (F/M) | 2:1 | 1.8:1 | 7.8:1 | 8:1 |
| Follow-up (m)£ | 171±95 | 125±71 | 101±86 | 98±71 |
| Clinical presentation | ||||
| Cushing | 0/9 (17%) | 1/25 (4%) | 20/43 (47%) | 11/27 (40%) |
| Virilizing only | 6/9 (67%) | 17/25 (68 %) | 2/43 (5%) | 4/27 (15%) |
| Mixed | 3/9 (33%) | 7/25 (28%) | 12/43 (28 %) | 4/27 (15%) |
| Nonfunctioning | 0/12 | 0/24 | 9/43 (20%) | 8/27 (30%) |
| Diagnosis | ||||
| CB * /Adenoma | 8/9 (89%) | 20/25 (80%) | 22/43 (51%) | 18/27 (67%) |
| CM * /Carcinoma | 1/9 (11 %) | 5/25 (20%) | 21/43 (49%) | 9/27 (33%) |
* Adrenocortical tumors in children were classified as clinically benign (CB) or clinically malignant (CM) £Average ± SD
expression levels were analyzed by the two-tailed Mann-Whit- ney U test. Survival analysis was performed using Kaplan-Meier curves and log rank test. All p-values less than 0.05 were consid- ered significant.
Results
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DAX1 nuclear expression was detected in the normal adrenal cortex from children and adults ( Fig. 1). A strong DAX1 protein expression was demonstrated in 74% (25 out of 34) and 24% (17 out of 70) of pediatric and adult ACTs, respectively (x2=10.1, p=0.002) ( Fig. 2). In the pediatric group, ACTs with a strong DAX1 expression were diagnosed at earlier ages than ACTs with weak expression [median 1.2 (range, 0.5 to 4.5) vs. 2.2 (0.9 to 9.4), p=0.038]. A strong DAX1 protein expression was detected in 71% (20 out of 28) of CB and in 83% (5 out of 6) of CM pediat- ric tumors. Additionally, DAX1 expression was not associated with reduced overall and disease-free survival in pediatric tumors (p=0.9 and p=0.7, respectively) ( Fig. 3a).
In adults, a strong expression for DAX1 was detected in 45% (18 out of 40) of adenomas and in 30% (9 out of 30) carcinomas (p=0.2). Similar to children, DAX1 protein expression did not cor- relate with overall and disease-free survival in adult adrenocorti- cal carcinomas (p=0.8 and p=0.1, respectively) ( Fig. 3b).
There was a trend for association between DAX1 protein and gene expression (r=0.4, p=0.05; n=49). Similar to protein data,
DAX1 gene expression was not associated with histopathological diagnosis and prognosis. Among adult ACTs, DAX1 overexpres- sion was demonstrated in 8 out 14 adenomas (57%) and in 7 out of 15 carcinomas (47%; p=0.57). In children, DAX1 overexpres- sion was evidenced in 9 out of 15 CB (60%) and in 2 out of 5 carcinomas (40%; p=0.43).
Endocrine syndromes were diagnosed in all children with ACTs (· Table 1). In the adult group, 24% of ACTs were nonfunction- ing. DAX1 expression did not correlate with the functional status of ACTs in adults. The frequency of nonfunctioning tumors was not significantly different between adrenocortical tumors with weak or strong DAX1 staining (20% vs. 30%, respectively; p=0.4). Similar to protein data, DAX1 gene expression was not associated with functional status.
To evaluate if DAX1 represses SF1 expression in ACTs, we analyzed the correlation between DAX1 and SF1 expression ( Fig. 4). Interestingly, a positive correlation was observed between DAX1 and SF1 protein expression in ACTs (p<0.0001, r=0.58) ( Fig. 4a). The positive DAX1/SF1 correlation was iden- tified in both pediatric and adult ACTs (r=0.55 for each group separately; p<0.0001). In addition, DAX1 gene expression was significantly correlated with SF1 gene expression (p<0.0001, r=0.54) ( Fig. 4b). Considering that DAX1 protein expression correlated with DAX1 gene expression and that a positive DAX1/ SF1 correlation was demonstrated at messenger and protein levels, we can rely on our findings using the K-17 antibody.
a
b
Fig. 1 Normal adrenals from a child a and an adult b displaying positive immunoreactivity for DAX1 (Color figure available online only).
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Pediatric ACTs (n= 34)
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Discussion ▼
DAX1 was initially cloned as the gene responsible for X-linked congenital adrenal hypoplasia [1,2], but it was recently found to be a stem cell fate regulator [10,11]. DAX1 down-regulation in murine embryonic stem cells increases the expression of genes involved in tissue differentiation and control of proliferation [12]. In the current study, a strong nuclear DAX1 expression was more frequent in ACTs diagnosed in children than in adults. Interestingly, pediatric ACTs with a strong immunoreactivity for DAX1 were diagnosed at earlier ages than those with a weak immunoreactivity for DAX1. Since pediatric ACTs are often diag- nosed before age 1-year-old and their hormonal profile resem- bles the fetal adrenal zone, it is reasonable to speculate that a
strong DAX1 protein expression could be a marker of an embry- onic origin of pediatric ACTs.
A higher frequency of SF1 gene amplification and protein over- expression in pediatric than in adult ACTs was recently demon- strated by our group [8]. In this new study, we reveal a significant and positive correlation between DAX1 and SF1 expression in ACTs at protein and messenger level. Indeed, DAX1 promoter contains a putative SF1 response element that specifically bound SF1 [24]. It was previously demonstrated that SF1 acts as posi- tive regulator of DAX1 transcription in the human adrenocorti- cal carcinoma cell line NCI-H295 [25]. In addition, SF1 and DAX1 cooperate to mediate somatic cell differentiation during mouse testis development [26]. Thus, although SF1 and DAX1 have opposite functions with regards to transcripional regulation of many target genes encoding steroidogenic enzymes, they may act cooperatively with regards to other cellular functions. Although specificity issues have been raised against the DAX1 K-17 antibody [27], we should emphasize that DAX1/SF1 corre- lation was demonstrated at both protein and messenger levels and DAX1 staining was significantly correlated with DAX1 gene expression.
Wnt signaling has also been implicated in the regulation of DAX1 transcription in murine embryonic stem cells [12]. Somatic acti- vating mutations of the CTNNB1 gene are the most frequent genetic defects identified both in adrenocortical adenomas and carcinomas in adult patients [28]. Mizusaki et al. [29] demon- strated that Dax1 gene transcription is activated by beta-catenin, a key signal-transducing protein in the Wnt pathway, acting in synergy with SF1. In turn, SF1 functions as a co-activator directly interacting with the beta-catenin-LEF/TCF complex to induce DAX1 transcription [29].
Low DAX1 expression was previously demonstrated in cortisol- and aldosterone-producing adenomas [14, 15]. In contrast, Bas- sett et al. [16] showed DAX1 upregulation in both cortisol- and aldosterone-producing adrenocortical adenomas. In adults, the hormonal status of ACTs with strong and weak DAX1 protein expression was similar. Moreover, DAX1 expression at protein and mRNA level was not different between functioning and non- functioning ACTs. In addition, DAX1 gene and protein expression was not associated with prognosis in both children and adults. When analyzing the publicly online database from the Michigan University, we could confirm that DAX1 gene expression was not a predictor of poor outcome [30]. It should be also emphasized that SF1 protein expression did not predict poor outcome in our cohort [8].
DAX1 has been recently considered a therapeutic target in can- cer cells [31]. Preclinical experiments showed that DAX1 silenc- ing induces growth arrest in the A673 Ewing’s cell line and severely impairs its capability to form tumors in immunodefi- cient mice [32]. Oda et al. [33] have also demonstrated that a high DAX1 expression in lung adenocarcinoma correlated with higher rates of lymph node metastasis and recurrence. In addi- tion, positive DAX1 immunoreactivity in breast cancer indicates a possible failure of endocrine therapies [21].
In conclusion, DAX1 strong protein expression was more fre- quent in pediatric ACTs than in adult tumors. Additionally, DAX1 expression positively correlated with SF1 expression in ACTs, suggesting that these transcription factors might cooperate in adrenocortical tumorigenesis.
Acknowledgements
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Financial support for this work was obtained from FAPESP Grant number 2011/09092-0 (to A.M.F. and M.Q.A.), 2012/21272-6 (to G.R.V.S. and M.Q.A.), 06/00244-3 (to M.Q.A.); CNPq Grant num- ber 470428/2013-9 (to M.Q.A.), CNPq grant 305743/2011-2 (to BBM), 470631/2012-0 (to M.Q.A.), and 302825/2011-8 (to A.C.L.).
Conflict of Interest
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The authors declare no conflict of interest ..
Affiliations
1 Unidade de Suprarrenal & Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular (Laboratório de Investigação Medica, LIM 42) da Disciplina de Endocrinologia do Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
2 Hospital do Câncer de Barretos, RO, Brazil
3 Divisão de Anatomia Patologia, Laboratório de Patologia Hepática/LIM14 do Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, SP, Brazil
1Serviço de Endocrinologia, Instituto do Câncer do Estado de São Paulo, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
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