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TCF21/POD-1, a Transcritional Regulator of SF-1/NR5A1, as a Potential Prognosis Marker in Adult and Pediatric Adrenocortical Tumors
Barbara dos Santos Passaia1, Matheus Henrique Dias2, Jean Lucas Kremer1, Sonir Roberto Rauber Antonini3, Madson Queiroz de Almeida4, Maria Candida Barisson Villares Fragoso4 and Claudimara Ferini Pacicco Lotfi1*
1 Department of Anatomy, Institute of Biomedical Science, University of São Paulo, São Paulo, Brazil, 2 Special Laboratory of Applied Toxicology (LETA), Butantan Institute, São Paulo, Brazil, 3 Department of Pediatrics and Puericulture, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil, 4 Adrenal Unit, Hormone and Molecular Genetic Laboratory/LIM42, Hospital of Clinics, School of Medicine, University of São Paulo, São Paulo, Brazil
OPEN ACCESS
Edited by: Vincenzo Pezzi, University of Calabria, Italy
Reviewed by: Michaela Luconi, University of Florence, Italy Matthias Kroiss, University of Würzburg, Germany
*Correspondence: Claudimara Ferini Pacicco Lotfi clotfi@usp.br
Specialty section:
This article was submitted to Cancer Endocrinology, a section of the journal Frontiers in Endocrinology Received: 28 September 2017 Accepted: 30 January 2018 Published: 22 February 2018
Citation:
Passaia BS, Dias MH, Kremer JL, Antonini SRR, Almeida MQ, Fragoso MCBV and Lotfi CFP (2018) TCF21/POD-1, a Transcritional Regulator of SF-1/NR5A1, as a Potential Prognosis Marker in Adult and Pediatric Adrenocortical Tumors. Front. Endocrinol. 9:38. doi: 10.3389/fendo.2018.00038
With recent progress in understanding the pathogenesis of adrenocortical tumors (ACTs), identification of molecular markers to predict their prognosis has become possible. Transcription factor 21 (TCF21)/podocyte-expressed 1 (POD1) is a transcriptional regula- tory protein expressed in mesenchymal cells at sites of epithelial-mesenchymal transition during the development of different systems. Adult carcinomas express less TCF21 than adenomas, in addition, the KEGG pathway analysis has shown that BUB1B, among others genes, is negatively correlated with TCF21 expression. The difference between BUB1B and PTEN-induced putative kinase 1 (PINK1) expression has been described previously to be associated with survival in adult but not in pediatric carcinomas. Here, we analyzed the gene expression of TCF21, BUB1B, PINK1, and NR5A1 in adult and pediatric ACTs. We found a negative correlation between the relative expression levels of TCF21 and BUB1B in adult ACTs, but the relative expression levels of TCF21, BUB1B, PINK1, and NR5A1 were similar in childhood ACTs. In addition, we propose using the subtracted expression levels of the TCF21/POD-1 genes as a predictor of overall survival (OS) in adult carcinomas and TCF21-NR5A1 as a predictor of malignancy for pediatric tumors in patients aged <5 years. These results require further validation in different cohorts of both adult and pediatric samples. Finally, we observed that the OS for patients aged <5 years was markedly favorable compared with that for patients >5 years as well as adult patients with carcinoma. In summary, we propose TCF21/POD-1 as a new prognostic marker in adult and pediatric ACTs.
Keywords: adrenocortical tumors, adult and pediatric tumors, transcription factor 21, podocyte-expressed 1, BUB1B, PTEN-induced putative kinase 1, nuclear receptor subfamily 5 group A member 1, CRISPR/dCas9
INTRODUCTION
The molecular pathogenesis of adrenocortical tumors (ACTs) remains poorly understood despite recent advances provided by comprehensive clinical and molecular investigations (1, 2). Some advances are related to the development of different tran- scriptomes during the last decade [for a review, see Ref. (3)]. Among these studies, a microarray analysis (4) showed that 91 genes are differentially expressed between adrenocortical carcinomas (ACCs) and adrenocortical adenomas (ACAs) of adult patients, including the TCF21 gene, which was two times lower in ACCs than in ACAs or in normal adrenal cortex samples. Transcription factor 21 (TCF21)/podocyte-expressed 1 (POD1) also known as capsulin/epicardin is a bHLH transcriptional regulatory protein expressed at sites of epithelial-mesenchymal interactions in the developing urogenital, cardiovascular, respiratory, and gastroin- testinal systems (5-9). TCF21 directly regulates the expression of steroidogenic factor 1 (NR5A1/SF1) in human ACT cells by binding to the E-box sequence in the NR5Al’s promoter region (10). Also, in this study, we showed that the viability of ACC cells transfected with TCF21 was not affected. However, StAR expres- sion was downregulated following transfection with TCF21, in accordance with a decrease in SF1-mediated StAR transcription. In addition, KEGG analysis showed a significant enrichment in cell cycle regulation pathways involving genes whose expression was negatively correlated with TCF21 expression in ACCs, such as CDK1 and BUB1B (10). Budding uninhibited by benzimida- zoles 1 homolog beta (BUB1B) encodes a kinase with important functions in the mitotic checkpoint (11-13). In a study using microarrays to identify genes that discriminated ACC and ACA, they identified two clusters of ACC with different outcomes (14). de Reyniès and colleagues identified that the difference between the expression values of DLGAP5 (disks large associated protein 7) and PINK1 (PTEN induced putative kinase 1), and the dif- ference between the expression of values of BUB1B and PINK were, respectively, predictors of malignancy, and overall survival. Indeed, ACTBUBIB - ACTPINK1 was considered a prognostic factor in ACCs in two different cohorts (14, 15).
PTEN-induced putative kinase 1 (PINK1) is a key mediator of mitochondria quality control induced by the tumor suppressor gene PTEN (16), whose expression levels decline in more aggres- sive ACCs and in ovarian cancer (17).
For pediatric patients, there are limited data to define prog- nostic molecular markers that distinguish benign from malignant ACTs, despite attempts of histological criteria and molecular clas- sifications (18, 19). In fact, the only study using transcriptome profiling analysis of pediatric ACT did not discriminate ACAs and ACCs using unsupervised clustering (20). Therefore, due to clinical, histological, and molecular heterogeneity in malignant ACTs, there is a need to validate and correlate the driver genes with possible prognostic value for both adult and pediatric ACTs.
Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; ACT, adrenocortical tumor; ACTB, beta-actin; BUB1B, budding uninhibited by benzimidazoles 1 homolog beta; GFP, green fluorescent protein; GUSB, glucuro- nidase beta; NR5A1, nuclear receptor subfamily 5 group A member 1; PINK1, PTEN-induced putative kinase 1; POD1, podocyte-expressed 1; SF1, steroidogenic factor 1; TCF21, transcription factor 21.
In this study, we aimed to analyze the expression of TCF21 and genes that may have TCF21-related expression, such as BUB1B, PINK1, and NR5A1 in adult and pediatric ACTs. Through the analysis of TCF21, BUB1B, PINK1, and NR5A1 gene expression, we tested the value of this analysis to predict the OS of adult ACC and to distinguish between benign and malignant pediatric ACTs. Therefore, the overall aim of this study was to verify if TCF21 has a diagnostic and prognostic role in adult and pediatric tumors.
MATERIALS AND METHODS
Patients
This study was approved by the Ethics Committees of Hospital das Clinicas, Institute of Biomedical Sciences (#822/2016) and
| Adults | Characteristics | n = 78 | Adrenocortical adenoma (ACA) (n = 44) | Adrenocortical carcinoma (ACC) (n = 34) |
|---|---|---|---|---|
| Mean age (years) | 40.59 ± 13.8 | 41.68 ± 16.45 | ||
| Sex | Female | 36 | 23 | |
| Male | 8 | 11 | ||
| Weiss scoreb | ≤3 | 42 | 7 | |
| >3 | 1 | 27 | ||
| ENSAT stagec | I/II | 1 | 11 | |
| III/IV | 0 | 10 | ||
| Metastasisb | Yes | 0 | 16 | |
| No | 32 | 14 | ||
| Cancer-related | 1 | 15 | ||
| death (CRD)a | ||||
| Median OS (mo) | 41 | |||
| Follow-up (mo) | 59.4 ± 62.41 | 52.2 ± 69.94 | ||
| Pediatric | <5 years | n = 35 | ACA (n = 27) | ACC (n = 8) |
| Mean age (years) | 1.84 ± 0.76 | 2.44 ± 0.87 | ||
| Sex | Female | 19 | 4 | |
| Male | 8 | 4 | ||
| Weiss Scoreb | ≤3 | 9 | 0 | |
| >3 | 17 | 8 | ||
| Metastasisd | Yes | 0 | 7 | |
| No | 26 | 1 | ||
| CRD | 0 | 5 | ||
| Median OS (mo) | 197.7 | |||
| Mean follow-up | 100.2 ± 61.9 | 57.67 ± 65.05 | ||
| (months) | ||||
| Pediatric | >5 years | n = 15 | ACA (n = 5) | ACC (n = 10) |
| Mean age (years) | 10.4 ± 4.03 | 13.44 ± 3.98 | ||
| Sex | Female | 4 | 8 | |
| Male | 1 | 2 | ||
| Weiss score | ≤3 | 5 | 0 | |
| >3 | 0 | 10 | ||
| Metastasis | Yes | 0 | 7 | |
| No | 5 | 3 | ||
| CRD | 0 | 7 | ||
| Median OS (mo) | 23.3 | |||
| Mean follow-up | 96.5 ±61.14 | 30.21 ± 34.7 | ||
| (months) |
ª11 cases not informed.
b1 case not informed.
“55 cases not informed. d16 cases not informed.
OS, overall survival.
Department of Pediatrics and School of Medicine of Ribeirão Preto (#7534/2010), São Paulo, Brazil. Written informed consent was obtained from all the patients or from their parents.
The clinical and histological features of patients with ACTs are summarized in Table 1. Further details of clinical charac- teristics and molecular data are shown in Tables S1 and S2 in Supplementary Material.
Samples of ACTs were obtained from 128 patients, 78 adult patients (range: 18-83 years), 35 pediatric patients aged <5 years (range: 0.43-3.8 years), and 15 pediatric patients aged >5 years (range: 5.5-17.7 years). The pediatric patients were separated into two groups because the fetal zone of the human adrenal cortex undergoes involution after birth, with the glomerulosa and fasciculata zones achieving complete differentiation approximately 4 years of age (21, 22), while the reticularis zone
is formed from 6 to 9 years of age (23, 24). Patients were evalu- ated at Hospital das Clinicas, by the School of Medicine from University of São Paulo and at the Department of Pediatrics from the School of Medicine of Ribeirao Preto, São Paulo, Brazil, between 1981 and 2014.
The mean follow-up periods were 56.2 ± 65.9, 90.5 ± 65.1, and 52.3 ± 55.0 months for adult patients, pediatric patients aged <5 years, and pediatric patients aged > 5 years, respectively. The final diagnosis of the patients was determined according to the histopathological characteristics, clinical manifestation, and biological behavior of the tumor, as proposed by Wieneke et al. (18), and it was used to classify the tumors as adult and pediatric adenomas or carcinomas in this study. As shown in Table 1, the Weiss score for adult ACTs was different from the correct diagnosis based on the final diagnosis. Accordingly,
A 210.
p= 0.0005
B 210.
p< 0.0001
TCF21 base-2 log expression
25.
.
BUB1B base-2 log expression
25.
20.
20.
2
-5.
..
2-5.
ACA n=44
ACC n=34
ACA n=42
ACC n=33
C 210.
p= 0.758
D 210.
p= 0.247
NR5A1 base-2 log expression
25.
PINK1 base-2 log expression
25.
20.
20%
2-5.
2-5.
.
ACA n=44
ACC n=34
ACA n=42
ACC n=33
in our study, we used the final diagnosis (44 ACA; 34 ACC) instead of the Weiss score (49 ACA; 28 ACC) to classify adenomas and carcinomas for adult tumors. For the pediatric group, the Weiss criteria are not useful to discriminate the histopathological diagnosis because pediatric tumors present a favorable outcome even with a Weiss score ≥3 (18). Among pediatric patients up to 5 years of age, the final diagnosis was 27 ACAs and 8 ACCs; however, among pediatric patients from 5 to 18 years of age, 5 ACAs and 10 ACCs were diagnosed (Table 1).
Cell Cultures
Human ACC cell lines NCI-H295R (25) and SW-13 (26) and human embryonic kidney cell line HEK-293 (27) were obtained
from ATCC (The ATCC Cell Biology Collection). NCI-H295R, SW-13, and HEK-293 were cultured, respectively, in RPMI medium with 2% fetal bovine serum (FBS) and 1% insulin- transferrin-selenium, L-15 medium with 10% FBS, and DMEM medium with 10% FBS (Gibco, Grand Island, NY, USA) at 37°℃ in a 95% air-5% CO2, in fully humidified environment. The culture used was authenticated by STR DNA profiling analysis.
Quantitative Real-time PCR
Total RNA was extracted from previously frozen tumor fragments (stored in liquid nitrogen) using Trizol (Invitrogen, Carlsbad, CA, USA) and an automatic homogenizer (model 985370, Biospec Products, Bartlesville, OK, USA). The RNA integrity and concentration were evaluated by agarose gel electrophoresis (2%)
Pediatric < 5 years
A
210.
p= 0.132
B 210.
p= 0.629
TCF21 base-2 log expression
25.
BUB1B base-2 log expression
25.
20.
20-
2-5.
2-10.
2-5.
ACA n=27
ACC n=6
ACA n=27
ACC n=8
C
210.
p= 0.359
D 210.
NR5A1 base-2 log expression
PINK1 base-2 log expression
p= 0.445
25.
25.
20.
20-
2-5.
2-5.
2-10.
ACA n=27
ACC n=8
ACA n=26
ACC n=8
and spectrometry (NanoDrop 2000c, Thermo Fisher Scientific, Waltham, MA, USA). cDNA was generated from 1 µg of RNA using the SuperScript III First-Strand Synthesis Supermix kit (Invitrogen). Quantitative real-time PCR was performed using the 7500 Real Time PCR System Sequencer (Applied Biosystems, Foster City, CA, USA) and the TaqMan gene expression assay for gene quantification according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The assays IDs were as follows: human ß-glucuronidase, glucuronidase beta (GUSB) (Hs00939627_m1 ID), beta-actin (ACTB) (Hs99999903_m1 ID), TCF21 (Hs00162646_m1 ID), nuclear receptor subfamily 5 group A member 1 (NR5A1) (ID Hs00610436_m1), BUB1B (ID Hs01084828_m1), and PINK1 (ID Hs00260868_m1). A cycle threshold (CT) value was selected in the linear range of
amplification for each sample in triplicate and was normalized to the GUSB and ACTB expression levels. The relative expression levels were calculated using the 2-44Ct method (28), where AACt is the difference between the selected ACt value of a given sample and the ACt for a pool of commercial normal adrenals (BioChain, USA). For the pediatric groups, the ACt mean of eight pediatric normal adrenal samples were used as normalizer. These samples were collected from patients up to 5 years of age undergoing nephrectomy due to kidney cancer and were kindly provided by Dr. Sonir R. R. Antonini from the Department of Pediatrics of FMRP-USP.
A mean expression value of 1.0 was attributed to the target genes in the pool of normal adrenals. A relative increase in the expression levels was determined for each tumor sample. The
Pediatric > 5 years
A 210.
p= 0.437
B 210,
p= 0.11
4
TCF21 base-2 log expression
BUB1B base-2 log expression
25.
25.
.
.
.
20.
20%
2-5.
2-5.
ACA n=5
ACC n=9
ACA n=5
ACC n=9
C 210.
p= 0.945
D 210.
p= 0.744
NR5A1 base-2 log expression
25.
PINK1 base-2 log expression
25.
.
20.
20-
2-5.
2-5.
ACA n=5
ACC n=10
ACA n=5
ACC n=9
subtraction gene level expression was calculated using the formula ACt target gene 1 - ACt target gene 2, as described by de Reyniès et al. (14).
Transfection Assay
NCI-H295R cells were transiently transfected with pcMVMyc- Pod1, which was kindly provided by Dr. Masataka Nakamura (Tokyo Medical University, Japan), as described earlier by Funato et al. (29). Next, 1.1 × 106 cells were plated and transfected with 4 µg of plasmid DNA and 12 ul of Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) for 5 h. After 24 h of transfec- tion, total RNA was extracted with Trizol (Invitrogen). Three independent experiments were performed. A high (71.4 ± 2.8%) efficiency of transfection was verified using the pmaxGFP vec- tor (Amaxa Biosystems, Gaithersburg, MD, USA), and the cells
were analyzed in a fluorescence-inverted microscope (data not shown).
Transduction Assay for CRISPR/dCas9 Activation System
The lentiviruses were produced in HEK-293FT cells using plasmids sgRNA (MS2) (#61427; Addgene, Cambridge, MA, USA), dCas9-VP64 (#61425; Addgene, Cambridge, MA, USA), or MS2-P65-HSF1 (#61426; Addgene, Cambridge, MA, USA). The sgRNA sramble (SCR) was constructed with the sequence GCACTACCAGAGCTAACTCA and the sgRNA T2 with the sequence ACATTACAAGTTGCAAATCA, according to proto- col established by Konermann et al. (30).
Transduction and cell selection were performed serially: dCas9-VP64 was selected with blasticidin; MS2-P65-HSF1
A 1.0×105
B
1.5
p = 0.36
TCF21 mRNA relative expression
p = 0.003
8.0×104
NR5A1 mRNA relative expression
6.0×104
1.0
4.0×104
2.0×104
0.5
3
2
1
0
empty
TCF21
0.0
empty
TCF21
C
1.5
D
1.5
BUB1B mRNA relative expression
p = 0.05
PINK1 mRNA relative expression
p = 0.39
1.0-
1.0-
0.5.
0.5-
0.0
0.0
empty
TCF21
empty
TCF21
was selected with hygromycin; and sgRNA-SCR or sgRNA-T2 was selected with zeocin. The concentration of antibiotics used was determined through a dose-response curve. The cells were plated to reach 50% of confluency 48 h before transduction and maintained for 24 h with a solution (1:1) of viral supernatant in culture medium, followed by of antibiotic selection until control cells died.
Statistical Analysis
As described in the Section “Materials and Methods” (patients), the groups of adenomas and carcinomas were discriminated considering the final diagnosis. The data are presented as medians for the Mann-Whitney test or mean ± SD for Student’s t test, as indicated in the figure legends. The ROC curve analysis method was used to test the combined gene expression as molecular
A
1020.
p= 0.0002
B
100
1010.
80-
ACtBUB1B-ACtPINK1
Sensitivity (%)
60-
100-
40-
20-
BUB1B-PINK1 < 0.24
10-10.
0
AUC = 0.77; P = 0.0003
0
20
40
60
80
100
Specificity (%)
10-20.
ACA n=34
ACC n=27
C 1020,
D
100
p< 0.0001
80-
1010.
ACtTCF21-ACtBUB1B
Sensitivity (%)
60-
100%
40-
20-
TCF21-BUB1B > 0.19
AUC = 0.84; P <0.0001
10-10.
0
0
20
40
60
80
100
Specificity (%)
10-20
ACA n=42
ACC n=33
E 1020.
F
100
p= 0.02
80-
1010.
ACtTCF21-ACtPINK1
Sensitivity (%)
60-
100%
40-
20-
TCF21-PINK1 >-1.38
AUC = 0.66; P =0.016
10-10.
0
0
20
40
60
80
100
Specificity (%)
10-20.
ACA n=42
ACC n=33
predictors. The cut-off obtained was selected considering the best sensitivity and specificity. Due to the limitation to obtain tumor samples, cut-off points were obtained and tested in the same cohort. The log-rank test was used to demonstrate the applicabil- ity of the cut-off in the groups analyzed. The event considered in the OS curve was cancer-related death. The critical value for significance of P < 0.05 was used throughout the study.
RESULTS
Relative Expression Levels of TCF21, BUB1B, PINK1, and NR5A1 in Samples of Adult and Pediatric ACTs
The relative expression of TCF21 transcripts was higher in adult ACA than in ACC (0.49 vs 0.18; P = 0.0005) (Figure 1A), in line with previous studies (4, 10). By contrast, the relative expression of BUB1B transcripts was higher in ACC than in ACA (1.17 vs 0.27; P < 0.0001) (Figure 1B), suggesting a negative correlation between TCF21 and BUB1B, as previously proposed (10). The relative expression of NR5A1 and PINK1 (Figures 1C,D) was similar in adult ACC and ACA (0.57 vs 0.86; P = 0.758; 0.42 vs 0.5; P = 0.247, respectively). In addi- tion, the relative expression levels of TCF21, BUB1B, PINK1, and NR5A1 were similar in childhood ACTs, regardless of the group studied (Figures 2 and 3).
To test whether the induction of TCF21 expression affected BUB1B or PINK1 expression in the NCI-H295R ACC cell line, we performed RT-qPCR of BUB1B or PINK1 in cells transiently transfected with the expression vector pCMVMycPOD1. NCI- H295R cells transfected with pcCMVMycPOD1 (Figure 4A) did not significantly affect NR5A1 expression (Figure 4B), in contrast to that observed previously (10), probably due to the experimental variation obtained. In addition, pcCMVMycPOD1 transfection showed a tendency of reduction in BUB1B expres- sion (Figure 4C), but did not significantly affect PINK1 expres- sion (Figure 4D) compared with the control levels. The same test with different biological approach was performed in HEK-293 and SW-13 cell lines transduced with CRISPR/Cas9/TCF21 activation system, and TCF21 showed not reduction of BUB1B expression (Figure 10).
Molecular Predictors of Malignancy in Adult ACTs
We tested whether the ACtBUB1B - ACtPINK1 could discrimi- nate between adenomas and carcinomas in our cohort. After removing from the analysis the patients whose data overlapped with the previous study of our group (15), we found significant differences (P = 0.0002; Mann-Whitney test) in the expression level of BUB1B-PINK1 (Figure 5A) between patients whose final diagnosis was adenoma (median = 2.67; n = 34) and those whose final diagnosis was carcinoma (median = - 0.176; n = 27). To select the cut-off for BUB1B-PINK1 in our cohort, we applied the ROC curve analysis method (Figure 5B). The area under the curve (AUC) was 0.77 [95% confidence interval (CI): 0.65 to 0.90; P = 0.0003]. The cut-off determined was <0.24, with 55.6% of sensitivity and 97.1% of specificity.
Applying the same reasoning, wetested whether subtracting the expression level of TCF21 and BUB1B (ACtTCF21 -ACtBUB1B) could also discriminate between benign and malignant adult ACTs in the total cohort of adult patients. We found significant differences (P < 0.001; Mann-Whitney test) in the
A
100
Overall survival (%)
BUB1B-PINK1 >0.24 (n=12)
50-
BUB1B-PINK1 <0.24 (n=15)
0
Log rank P = 0.008
0
50
100
150
200
Time (months)
B
100
TCF21-BUB1B <0.19 (n=10)
Overall survival (%)
50-
TCF21-BUB1B >0.19 (n=23)
0
Log rank P = 0.004
0
100
200
300
400
C
Time (months)
100-
Overall survival (%)
TCF21-PINK1 ← 1.38 (n=8)
50-
TCF21-PINK1 >-1.38 (n=25)
0
Log rank P = 0.59
0
100
200
300
400
Time (months)
expression level of TCF21-BUB1B (Figure 5C) between adeno- mas (median = - 3.41; n = 42) and carcinomas (median = 1.52; n = 33). The AUC obtained from the ROC curve test was 0.84 (95% CI: 0.74 to 0.94; P < 0.0001; Figure 5D), and the cut-off determined was >0.19, with 69.7% of sensitivity and 97.6% of specificity. The analysis of ACtTCF21 - ACtPINK1 (Figure 5E) showed significant differences (P = 0.02; Mann-Whitney test) between ACA (median =- 0.71; n=42) and ACC (median= 1.36; n = 33). The AUC obtained from the ROC curve test was 0.66
(95% CI: 0.54 to 0.79; P = 0.016; Figure 5F), and the cut-off determined was >-1.38 with 75.8% of sensitivity and 38.1% of specificity.
TCF21-BUB1B As a Predictor of OS in Adult Carcinomas
Among the ACCs, subtraction of the expression level of BUB1B and PINK1 (ACtBUB1B - ACtPINK1) was a good predictor
A 1020.
B 100
p= 0.13
1010.
80-
ACtTCF21-ACtBUB1B
Sensitivity (%)
60-
100%
40-
20-
TCF21-BUB1B
10-10.
AUC = 0.78; P = 0.11
0
0
20
40
60
80
100
Specificity (%)
10-20.
ACA n=5
ACC n=8
C
1020.
D
100
p= 0.52
1010.
80-
ACtTCF21-ACtPINK1
Sensitivity (%)
60-
100.
40-
20-
TCF21-PINK1
10-10.
AUC = 0.63; P = 0.46
0
0
20
40
60
80
100
Specificity (%)
10-20.
ACA n=5
ACC n=8
E
1020.
F
100
p= 0.15
1010.
80-
ACtTCF21-ACtNR5A1
Sensitivity (%)
60-
100%
40-
20-
TCF21-NR5A1
10-10.
AUC = 0.76; P = 0.13
0
0
20
40
60
80
100
Specificity (%)
10-20.
ACA n=5
ACC n=9
of OS (14, 15). Applying the cutoff value <0.24 obtained for BUB1B-PINK1 to 27 adult patients with a malignant final diag- nosis (Figure 6A), we found two groups with different survival times (log-rank test P = 0.008). Accordingly, we applied the cut-off value >0.19 obtained for TCF21-BUB1B to 33 adult patients with a malignant final diagnosis (Figure 6B), and we could discriminate two groups with distinct survival times (log-rank test P = 0.004). The cutoff value >-1.38 obtained for TCF21-PINK1 was not associated with a statistically significant difference in OS (Figure 6C).
TCF21-NR5A1 As a Molecular Predictor of Malignancy in Pediatric ACTs
To determine whether TCF21 could be used as a molecular predictor of malignancy in pediatric patients, we analyzed the subtraction expression level of TCF21-BUB1B, TCF21- PINK1, and TCF21-NR5A1 in the pediatric cohort. These analyses did not show significant differences in the tumors of patients aged >5 years (Figure 7). In the cohort of pediatric patients aged <5 years, the subtraction expression level of TCF21-BUB1B and TCF21-PINK1 did not present significant
A 1020.
B
100
p= 0.6
80-
ACtTCF21-ACtBUB1B
1010.
Sensitivity (%)
60-
..
100.
40-
20-
TCF21-BUB1B
10-10.
AUC = 0.57; P = 0.58
0
0
20
40
60
80
100
Specificity (%)
10-20.
ACA n=27
ACC n=6
C 1020.
D
100
p= 0.09
80-
1010.
ACtTCF21-ACtPINK1
Sensitivity (%)
60-
100.
40-
20-
TCF21-PINK1
10-10.
AUC = 0.73; P = 0.08
0
0
20
40
60
80
100
Specificity (%)
10-20.
ACA n=26
ACC n=6
E 1020.
F
100
p= 0.027
80-
1010.
ACtTCF21-ACtNR5A1
Sensitivity (%)
60-
100%
40-
20-
TCF21-NR5A1 < 1.52
10-10.
AUC = 0.79; P = 0.028
0
0
20
40
60
80
100
Specificity (%)
10-20
ACA n=27
ACC n=6
differences (Figures 8A-D). However, ACtTCF21 - ACtNR5A1 (Figures 8E,F) showed significant differences (P = 0.026; Mann- Whitney test) between adenomas (median = 4.06; n = 27) and carcinomas (median = 1.06; n = 6). The AUC value was 0.79 (95% CI: 0.59 to 0.99; P = 0.028), and the cut-off determined was <1.52 with 66.7% of sensitivity and 92.6% of specificity. A poor outcome occurs when ACtTCF21 - ACtNR5A1 is <1.52. Due to the small number of carcinomas studied, in this case, the cutoff value was not applied as a predictor of OS.
The OS for Patients Aged <5 Years Is Markedly Favorable
In our cohort, the median survival for adult patients with carci- noma was 41 months for adults (Figure 9). For pediatric patients aged >5 years with ACTs, the median survival was 23.3 months, while the median survival for patients aged <5 years was 197.7 months. However, it was limited to the maximum follow-up time that was 230.07 months for this group. Therefore, as seen in Figure 9, the OS curves were significantly different (log rank P = 0.007) among the patients studied. In addition, the OS for patients aged <5 years was markedly favorable compared with that for patients >5 years and adult patients with carcinoma.
DISCUSSION
Different authors have relied on RNA-based global gene expres- sion profiles to identify molecular markers that differentiate malignant and benign adrenal tumors. Among these studies, de Reyniès et al. (14) identified three molecular markers in adult ACTs, DLGAP5, BUB1B, and PINK1, and found that BUB1B is overexpressed in carcinomas. According to their data, the com- bined expression of BUB1B and PINK1 was the best predictor of OS among carcinomas. Thereafter, BUB1B-PINK1 expression was
validated in a different cohort of adult and pediatric patients (15). Giordano et al. (4) identified the downregulation of TCF21 in ACCs in a microarray assay. In Franca et al. (10), we showed that TCF21 was markedly downregulated in adult ACCs compared with adenomas and normal tissue.
Here, we found that TCF21 was downregulated while BUB1B was upregulated in adult ACCs, in agreement with previous studies (10, 14). A previous study proposed that, among other cell cycle genes, BUBIB is negatively correlated with TCF21 (10). We tested the causal relationship between TCF21 and BUB1B and could not confirm the negative correlation between these genes in different experimental approaches and cell lines overexpressing TCF21.
We also analyzed whether the subtraction expression level of TCF21-BUB1B and TCF21-PINK1 could distinguish between adenomas and carcinomas. The study of the subtraction expres- sion levels of BUB1B-PINK1 and TCF21-BUB1B discriminated between adult adenoma and carcinoma in a similar pattern. In addition, among adult malignant tumors, the combined expression of TCF21 and BUBIB was a good predictor of OS. Accordingly, both BUB1B-PINK1 and TCF21-BUB1B seem reli- able molecular markers to be used in the clinical evaluation of adult adrenal tumors.
We employed these new molecular markers and the combined expression of TCF21-NR5A1 to discriminate between benignant and malignant tumors in a cohort of pediatric ACTs. Increased NR5A1 copy number has been associated with childhood adreno- cortical tumorigenesis (31, 32), although this increase does not correlate with NR5A1 protein levels (33). The functional role of NR5A1 extends beyond steroidogenesis because NR5A1 regu- lates proliferation in adrenocortical cells, angiogenesis, extracel- lular matrix adhesion, cytoskeleton dynamics, and apoptosis in the adrenal cortex (34). Although there was no difference in the
100
Overall Survival (%)
Pediatric ACT < 5 years (n=35)
50
Pediatric ACT > 5 years (n=15)
Adult ACC (n=34)
Log rank P = 0.007
0
0
100
200
300
400
Time (months)
FIGURE 9 | Comparison of the overall survival of adults with adrenocortical carcinomas (ACCs) and pediatric patients with adrenocortical tumors (ACTs). Adult patients with carcinoma (n = 34), pediatric patients aged <5 years with ACTs (n = 35), and pediatric patients aged >5 years with ACTs (n = 15).
A
C
POD-1/TCF21 mRNA relative expression
POD-1/TCF21 mRNA relative expression
400
15-
1
*
300-
4
10-
200-
100
5-
2
1-
0
HEK293
SCR
T2
0
SW13
SCR
T2
BUB1B mRNA relative expression
2.0-
BUB1B mRNA relative expression
1.5-
1.5-
1.0-
1.0-
0.5-
0.5-
0.0
HEK293
SCR
T2
0.0
SW13
SCR
T2
NR5A1 expression of adenomas and carcinomas in the cohort of patients <5 years of age, the subtraction expression level of TCF21-NR5A1 discriminates between benign and malignant tumors and may provide relevant information in addition to pathology analysis.
It is largely accepted that children have a better outcome than adolescents. As observed in the study by Wieneke and collaborators (18) in 83 pediatric tumors, there appears to be a biphasic age distribution with a poor clinical outcome in the group aged >5 years. In fact, we showed that the OS for patients aged <5 years was markedly favorable compared with that for patients aged >5 years and adult patients with carcinoma. This observation was utilized in the more recent study by Cecchetto et al. (35), where patients were separated in three different groups: <4 years, 4-10 years, and >10 years. Patients aged ≤4 years had a better outcome than the older ones. Indeed, <5 years patients have specific features distinguishing them from adolescent and adult tumors considering their genomic profiles and pathological and genetic mechanisms (36). Therefore, we suggest that separa- tion between children and adolescents should be considered in future analysis.
In summary, we could not establish a consistent relationship between the analyzed genes for adult and pediatric tumors, although TCF21 transfection in the H295R cell line has shown a tendency of reduction in BUB1B expression. In addition, the sub- traction of gene expression of TCF21 and BUB1B can be a good predictor of OS in adult carcinomas, whereas the TCF21-NR5A1
can be a molecular predictor of malignancy in pediatric ACTs. Moreover, we confirm that patients aged <5 years showed more favorable OS than adolescent patients. Finally, our study sug- gested a role of TCF21 in ACTs that should be explored in future studies.
ETHICS STATEMENT
This study was approved by the Ethics Committees of Hospital das Clinicas, Institute of Biomedical Sciences and Department of Pediatrics and School of Medicine of Ribeirão Preto, São Paulo, Brazil. Written informed consent was obtained from all the patients or from their parents.
AUTHOR CONTRIBUTIONS
BP performed the experiments, analyzed the data, and wrote the manuscript; SA provided the pediatric samples; MA provided the adult samples; MF provided the adult and pediatric samples; CL idealized the study, analyzed the data, and wrote the manuscript.
ACKNOWLEDGMENTS
We are grateful to Antonio Marcondes Lerario, Laboratory of Hormones and Molecular Genetics (LIM42), School of Medicine, University of São Paulo, for assistance with the analysis of the results; Rosana D. Prisco, Institute of Biomedical Science,
University of São Paulo, for statistical analysis and Cintia Fridman, Department of Legal Medicine and Medical Ethics, School of Medicine, University of São Paulo, for STR DNA profil- ing analysis in cell cultures.
FUNDING
BP is a recipient of a scholarship from FAPESP (no. 2013/23548- 1), the São Paulo State Research Foundation; CL received funding
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Conflict of Interest Statement: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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