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ORIGINAL ARTICLE
Is there a role for epithelial-mesenchymal transition in adrenocortical tumors?
Daniel Bulzico 1,2 . Paulo Antônio Silvestre de Faria3 . Camila Bravo Maia3 .
Marcela Pessoa de Paula2 . Davi Coe Torres4 . Gerson Moura Ferreira5 .
Bruno Ricardo Barreto Pires5 . Rocio Hassan4 . Eliana Abdelhay5 . Mario Vaisman6 .
Leonardo Vieira Neto2,6
Received: 26 April 2017 / Accepted: 24 August 2017 @ Springer Science+Business Media, LLC 2017
Abstract
Purpose Epithelial-mesenchymal transition (EMT) is a biological dynamic process by which epithelial cells lose their epithelial phenotype and acquire mesenchymal inva- sive and migratory characteristics. This has been postulated as an essential step during cancer progression and metas- tasis. Although this is well described in other tumors, the role of EMT in adrenocortical tumors (ACT) has yet to be addressed.
Methods The aim of this study was to evaluate the expression of EMT markers e-cadherin, vimentin, and fibronectin, along with EMT-transcription factors (EMT- TFs), TWIST1, SIP1, and SNAIL in 24 adrenocortical carcinoma (ACC), 19 adrenocortical adenomas (ACA), 27
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12020-017-1409-z) contains supplementary material, which is available to authorized users.
☒ Daniel Bulzico danielbulzico@gmail.com
1 Endocrine Oncology Unit Brazilian National Cancer Institute- INCA, Rio de Janeiro, Brazil
2 Endocrinology Section Federal Hospital of Lagoa, Rio de Janeiro, Brazil
3 Division of Pathology Brazilian National Cancer Institute- INCA, Rio de Janeiro, Brazil
4 Laboratory of Oncovirology, Center for Bone Marrow Transplants Brazilian National Cancer Institute-INCA, Rio de Janeiro, Brazil
5 Stem cell Laboratory, Center for Bone Marrow Transplants Brazilian National Cancer Institute-INCA, Rio de Janeiro, Brazil
6 Department of Internal Medicine and Endocrinology Section, Medical School and Clementino Fraga Filho University Hospital, Rio de Janeiro Federal University, Rio de Janeiro, Brazil
childhood-onset adrenocortical tumors (CAT), and 12 nor- mal adrenal glands. The association of EMT and EMT-TFs with clinical outcomes and pathology features were also evaluated.
Results Cytoplasmic vimentin expression was increased among CAT samples when compared to ACC, ACA, and normal adrenal samples (p <0.001). There was no differ- ence in e-cadherin and fibronectin expression observed between groups. Nuclear and cytoplasmic expression of TWIST1 and SIP1 was stronger in CAT and ACC vs. ACA and normal tissue samples (all, p < 0.05). ACT, regardless of classification, exhibited increased SNAIL expression when compared to normal tissue (p <0.05). A significant correlation was observed between vimentin and TWIST1 (rs=0.44, p<0.001); SIP1 (rs=0.51, p<0.001); and SNAIL (r$=0.23, p<0.05). TWIST1 and SIP1 expres- sions demonstrated a significant correlation (rs=0.56, p < 0.001). High SIP1 expression was associated with a lower survival rate among ACC cases (p <0.05).
Conclusions Vimentin, TWIST1, and SIP1 expressions are increased in aggressive ACT. Therefore, EMT may play a relevant role in adrenal tumorigenesis.
Keywords Adrenal cortex . Carcinoma . Adenoma . Immunohistochemistry
Introduction
Adrenocortical tumors (ACT) are very common in the general population, with the majority of them represented by adulthood onset benign adenomas (ACA) [1]. Con- versely, childhood-onset adrenocortical tumors (CAT) and
adulthood carcinomas (ACC) are extremely rare with the former harboring a significant elevated incidence in south Brazil, due to the founder effect of an exon-10 (R337H) TP53 germline mutation [2-4]. While ACA generally har- bor an indolent clinical course, CAT and ACC can be very aggressive and are associated with a poor prognosis [5-7]. Despite significant advances to determine deregulated gene expression, the pathogenesis of ACT remains to be eluci- dated [7].
Epithelial-mesenchymal transition (EMT) is a physiolo- gical, molecular, and biologic dynamic process essential for embryonic development and first described by Elizabeth Hay in the early 1980s [8]. During the EMT process, epi- thelial cells lose their epithelial features, mainly the cell-cell adhesion property, and acquire a mesenchymal phenotype [9]. Recently, EMT has been described in oncology as a fundamental step by which in situ malignant cells acquire invasive and migratory properties, essential to cancer pro- gression and metastasis development [9-11]. This rever- sible phenotype switch is regulated predominantly by the transcription factors TWIST1, SNAIL, SLUG (SNAIL2), and SIP1 (ZEB2), which downregulate epithelial markers (e.g., e-cadherin, claudins, occludins) and upregulate mesenchymal markers (e.g., vimentin, n-cadherin, fibro- nectin) responsible for the migratory and invasiveness fea- tures [12-15]. Moreover, increased expression of theses transcription factors has been correlated with aggressive tumor features, unfavorable clinical outcomes, and failure to respond to conventional therapy in various types of solid cancers [16-25]. The role of EMT and EMT-transcription factors (EMT-TFs) in ACT has yet to be evaluated.
The aim of this study was to determine the role of the epithelial marker e-cadherin, mesenchymal markers vimentin and fibronectin, and EMT-TFs TWIST1, SIP1, and SNAIL in ACT (including CAT, ACA, and ACC) and normal adrenal tissue through immunohistochemistry ana- lysis. Additionally, a focus was to evaluate the association of these markers with clinical outcomes and the presence of a TP53 mutation.
Materials and methods
Study design and population
This observational and retrospective study was conducted by the Endocrine Oncology Unit in collaboration with the Division of Pathology (DIPAT), the Laboratory of Oncov- irology, and the Stem cell Laboratory from the Brazilian National Institute of Cancer-INCA, Rio de Janeiro, Brazil. Cases were identified using the DIPAT information system, which has been coding all cases since 1997. After filtering for adrenal cortex localization, all cases registered from
January 1st, 1997 to March 31st, 2015 were considered eligible. Exclusion criterion included evidence of histolo- gical diagnosis other than ACT. Cases were categorized in three groups:
A. CAT: all cases diagnosed under the age of 18 regardless of any other clinical and/or pathology feature.
B. ACC: all cases diagnosed ≥ the age of 18, with tumors possessing three or more pathology features according to the Weiss-score system [26]. Patients harboring tumors considered unresectable, but with a biopsy confirming adrenal origin, regardless of the presence of distant metastasis.
C. ACA: all cases diagnosed ≥ the age of 18, with tumors possessing a Weiss-score of 0-2.
A group of 12 normal adrenal tissues was also included for comparison to the three ACT groups in terms of immunohistochemical staining pattern analysis. These nor- mal adrenal tissues were retrieved from patients with renal carcinoma en-bloc resections.
Pathology diagnosis review
To confirm the ACT diagnosis, original hematoxylin-eosin (H&E) stained slides from resected or biopsied cases were initially reviewed by two investigators (D.B., P.A.S.F.). Investigators were blinded to the patient’s clinical outcomes. Following confirmation, the presence of each of the nine following histological features were evaluated and the Weiss-index [26] calculated: nuclear atypia [grade III/IV according to Fuhrman criteria [27]; increased mitotic rate [more than five mitotic figures in 50 high-power fields (40x objective)]; atypical mitotic figure; eosinophilic (“dark”) cytoplasm in at least 75% of tumor cells; diffuse pattern in tumor architecture; microscopic tumor necrosis; unequi- vocal venous invasion; sinusoidal invasion; and tumor capsule invasion. Cases submitted only to biopsy were excluded from the Weiss-score analysis. Additionally, ori- ginal H&E stained slides from normal adrenal tissue, derived from en-bloc renal carcinoma resections, were reviewed to assure no evidence of neoplastic invasion.
Clinical data
Medical records from CAT, ACA, and ACC patients were reviewed and a specific questionnaire was completed. The following variables were assessed: (a) gender; (b) age at presentation; (c) tumor size; (d) disease stage according to the European Network for the Study of Adrenal Tumors (ENSAT) for the adults and to Sandrini et al. [28] staging system for childhood-onset cases; (e) mortality; and (f) follow-up and survival time.
Immunohistochemistry
A representative formalin-fixed and paraffin-embedded tis- sue was selected for each case during the pathology diag- nosis review process. Four micron-thick serial sections were manually immunostained with previously reported technique [29] for Ki-67 (1:600, mouse monoclonal MIB-1-Dako, Glostrup, Denmark); e-cadherin (1:1200, mouse monoclonal SPM471-Santa Cruz, Dallas, USA); vimentin (1:100, goat polyclonal sc-7557-Santa Cruz, Dallas, USA); fibronectin (1:100, mouse monoclonal P1H11-Santa Cruz, Dallas, USA); TWIST1 (1:50, mouse monoclonal Twist2Cla- Santa Cruz, Dallas, USA), SIP1 (1:200, rabbit polyclonal sc- 48789-Santa Cruz, Dallas, USA), and SNAIL (1:400, rabbit monoclonal C15D3-Cell Signaling, Danvers, USA). Positive controls were performed using different human breast cancer sections (TWIST1, SIP1, SNAIL, and Ki-67), normal appendix (vimentin and fibronectin), and normal mammary ducts (e-cadherin). Negative controls were application of secondary antibody only (Fig. 1).
Immunohistochemistry scoring
Immunohistochemistry labeling was assessed only in neo- plastic cells (for CAT, ACC, and ACA groups) and normal adrenocortical cells (for the normal adrenal samples group). Scoring was independently performed by three observers (D.B., P.A.S.F., C.B.M.). Each was blinded to the patient’s clinical outcomes. Agreement between scorers was assessed by a correlation test for each antibody, with final significant accordance in most cases. Discordant scores were re- reviewed by the same initial observers to reach a consensus. Both the intensity and the extent of immunopositivity were considered. The mean staining intensity was scored as fol- lows: 0 = negative, 1 = weak, 2 = moderate, 3 = strong.
For e-cadherin, vimentin and fibronectin, the extent of positive staining tumor cells was scored as previously described by Zhou et al. [30]: < 10% scored as 1, 11-50% as 2, 51-75 as 3, and >75% as 4. The final score was deter- mined by multiplying the intensity and extent scores (0-12). For e-cadherin, only membranous staining pattern was considered, for vimentin only cytoplasmic, and for fibro- nectin, both cytoplasmic and membranous. The positive (+) expression was defined as a score of >3 for e-cadherin, and >1 for vimentin and fibronectin [30].
Due to unestablished labeling patterns [16, 19, 20, 31, 32] for TWIST1, SIP1, and SNAIL ana- lysis, both nuclear and cytoplasmic immunopositivity were assessed independently in each slide. The staining intensity was also scored as 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. However, the extent was defined semi- quantitatively from 0-100% of cells. The final score was calculated using a modified H score algorithm [33] as
Positive
Negative
E-cadherin
Vimentin
Fibronectin
TWIST1
SIP1
SNAIL
Ki-67
follows: (intensity of nuclear staining x % of stained nucleus) + (intensity of cytoplasmic staining × % of stained cytoplasm), leading to a score from 0-600, where 600 was equal to 100% of tumor cells nucleus and cytoplasm strongly labeled. High expression (+) of TWIST1, SIP1, and SNAIL was defined as a modified H score above the whole group median score.
The Ki-67 labeling index was defined as the percentage of positively stained neoplastic cells among the total num- ber of neoplastic cells.
TP53 sequencing
Genomic DNA was purified from 10 um thick sections of formalin-fixed paraffin embedded tissues from the patient’s adrenal tumor (groups A, B, and C) using the GeneRead DNA FFPE Kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. Mutation analysis of TP53 was performed as previously reported [29].
Outcomes
The principal outcome was to compare the immunohis- tochemistry expression of the epithelial marker e-cadherin, mesenchymal markers vimentin and fibronectin and EMT- TFs, TWIST1, SIP1, and SNAIL among CAT, ACA, ACC groups and normal adrenal tissues.
Secondary outcomes were to analyze the association of the same immunohistochemistry expressions described above with clinical and pathologic characteristics (sex, disease stage, presence of distant metastasis, microscopic capsular or vascular invasion, and death), Ki-67 labeling index, and the presence of a somatic TP53 mutation.
Statistical analysis
Analyses were performed using SPSS version 20.0 for Macintosh (IBM SPSS Statistics, Armonk, NY, USA) and GraphPad Prism 5 (Graphpad Software, Inc., La Jolla, CA, USA). In descriptive analysis, categorical variables were expressed as percentages, while numerical variables were expressed as mean ± (standard deviation) and/or median (minimum-maximum). The Kolmogorov-Smirnov test was used to evaluate whether numeric variables were normally distributed. The Kruskal-Wallis test was used to compare numerical variables among three or four groups and the Mann-Whitney test was performed for comparison between two groups. Chi-square or Fisher exact tests were used to evaluate categorical variables. A p-value of <0.05 was considered statistically significant, except for comparisons among more than two groups, and then p-values <0.013 (4 groups) were considered significant (Bonferroni post hoc analysis). Spearman rank correlation tests were used to
correlate numeric continuous variables among groups. Kaplan-Meier’s analysis was used to assess death risk according to TWIST1, SIP1, and SNAIL expression, cate- gorized as high or low according to median modified H score values. Peto-Prentice Wilcoxon test was used to compare survival distribution.
Ethical aspects
This study was approved by INCA’s independent institu- tional advisory committee on September 24th, 2014 (pro- tocol 33847514.4.0000.5274) and conducted according to the principles expressed in the Declaration of Helsinki. Informed consent was obtained from individuals partici- pants in the study.
Results
Population and tumor characteristics
A total of 70 cases of ACT with a mean follow up time of 50 ±47 months were included for analysis: 27 cases of CAT [median age, 3.6 years (1-15); 19 female, 8 male], 24 cases of ACC [median age, 47.3 years (22-76); 16 female, 8 male], and 19 cases of ACA [median age, 55 years (29-73); 14 female, 5 male]. Twelve cases of normal adrenal tissue were used for comparison [median age, 61.5 years (1-69)]. Previous detailed clinical and pathologic characteristics from CAT, ACA and ACC groups have been recently reported by our group [5, 6]. Basic demographic char- acteristics are described in Table 1.
Ki-67 immunohistochemistry
Ki-67 labeling index was significantly higher in CAT [median, 20 (0.5-90)] and ACC cases [median, 19 (0-90)], in comparison to ACA cases [median, 0.5 (0-8)] - p < 0.001 for both. No difference was observed between CAT and ACC samples.
Vimentin, e-cadherin and fibronectin immunohistochemistry
Figure 2 illustrates vimentin, e-cadherin and fibronectin immunohistochemistry patterns among the four groups. The cytoplasmic vimentin expression score was significantly higher among CAT samples [median, 7.5 (0.5-12)] when compared to ACA [median, 2.0 (0-7)] and normal adrenal tissue samples [median, 2.0 (0-3)], p <0.001 (Fig. 3d). No significant difference was observed in vimentin scores among ACC [median, 4.7 (0-12)], ACA and normal tissue, all p> 0.05. Similarly, no differences in e-cadherin and
| Clinical characteristic | CAT | ACC | ACA | Normal adrenal |
|---|---|---|---|---|
| N | 27 | 24 | 19 | 12 |
| Female, n (%) | 19 (70.4%) | 16 (66.7%) | 14 (73.7%) | 6 (50%) |
| Age, yr | ||||
| Mean (SD) | 5.1 (4.1) | 46.8 (16.4) | 52.5 (13.7) | 48.6 (26.8) |
| Median (min-max) | 3.7 (1-15) | 47.3 (22-77) | 55.0 (29-73) | 61.5 (1-69) |
| Follow up, mo | ||||
| Mean (SD) | 51 (49) | 41 (39) | 62 (52) | – |
| Tumor largest diameter, cm | ||||
| Mean (SD) | 8.2 (4.9) | 11.9 (4.9) | 3.6 (1.2) | – |
| Tumor stageª, n (%) | – | – | ||
| I | 11 (40.7%) | 1 (4.2%) | ||
| II | 5 (18.5%) | 11 (45.8%) | ||
| III | 6 (22.2%) | 5 (20.8%) | ||
| IV | 5 (18.5%) | 7 (29.2%) | ||
| Distant metastasis, n (%) | 5 (18.5%) | 7 (29.2%) | 0 | – |
| Death, n (%) | 10 (37%) | 11 (45.8%)b | 1 (5.3%) | – |
| Mean survival, months (SD) | 9.8 (7.8) | 24.8 (27.2) | 10.6 | – |
a Tumor stage according to Sandrini et al. (Sandrini et al. 1997) staging system for childhood-onset adrenocortical tumors, and the European Network for the Study of Adrenal Tumors (ENSAT) staging system for adulthood adrenocortical carcinomas
b Death could not be assessed in two cases of adulthood adrenocortical carcinoma because of lost of follow-up
fibronectin expression were observed between groups. Very low expression of e-cadherin and fibronectin scores were found, with median scores of 0 in all groups, except for the fibronectin score, which was 1 (0-6) only in the CAT group Data is illustrated in Table 2.
TWIST1, SIP1, and SNAIL immunohistochemistry
Nuclear and cytoplasmic expression of TWIST1, SIP1, and SNAIL were observed in all groups, with a median overall (n = 82) modified H score of 257.5 (10-531) for TWIST1, 162.2 (1-487.5) for SIP1, and 4.2 (0-290) for SNAIL. Stronger TWIST1 and SIP1 expression patterns were observed predominantly in CAT and ACC tumor cells samples. Figure 2 illustrates the immunohistochemistry pattern while Fig. 3 describes TWIST1, SIP1, and SNAIL modified H scores among groups. For TWIST1, crescent scores were observed in the following order: normal tissue [median, 155 (10-226)], ACA [median, 187.5 (20.5-379)], CAT [median, 267.5 (25-531)], and ACC [median, 314.7 (37-525)]-Fig. 3a. For SIP1: normal tissue [median, 14 (1-80)], ACA [median, 70 (37.5-250)], ACC [median, 185 (35-457)], and CAT [median, 216 (32.5-487.5)]-Fig. 3b. No difference on SNAIL (Fig. 3c) expression was observed among CAT, ACC, and ACA cases with median scores of 12.5 (0-290), 4.2 (0-240), and 5.0 (0-68), respectively.
Significant lower SNAIL expression was found amongst normal adrenal tissue in comparison to CAT (p<0.01), ACC and ACA (p=0.02, for both comparisons).
Significant correlations were observed between the expression levels of TWIST1 and vimentin (Fig. 4a); TWIST1 and SIP1 (Fig. 4b); SIP1 and vimentin (Fig. 4c); SIP1 and SNAIL (Fig. 4d); and vimentin and SNAIL (Fig. 4e). No significant correlation was observed between TWIST1 and SNAIL immunohistochemistry scores (Fig. 4f).
Association of the clinical and pathologic variables with vimentin, e-cadherin, fibronectin, TWIST1, SIP1, and SNAIL immunohistochemistry
Vimentin was positive in the majority of cases and both e- cadherin and fibronectin median scores were extremely low in all groups (Table 2) thus, the association of clinical and pathology features with the immunohistochemistry profile were assessed only for TWIST1, SIP1, and SNAIL. A detailed distribution of immunomarkers can be found in Supplemental Tables 1 and 2.
No differences were observed in the immunoexpression when focusing on gender or tumor size in any of studied groups. The number of adverse events was extremely low in the ACA group (only one death among 19 cases).
Normal
ACA
ACC
CAT
HE
Vimentin
E-cadherin
Fibronectin
TWIST1
SIP1
SNAIL
A
p < 0.001
B
p < 0.001
p < 0.05
p = 0.001
p < 0.001
600
600
p <0.001
p <0.01
p < 0.01
TWIST1 score
400
SIP1 score
400
p < 0.001
200
200
0
0
Normal
ACA
ACC
CAT
Normal
ACA
ACC
CAT
C
D
p <0.001
p < 0.001
p < 0.001
300
p < 0.05
15
200
1
p <0.05
T
T
100
SNAIL score
50
I
Vimentin score
15.0
10
12.5
10.0
7.5
5
5.0
2.5
0.0
0
Normal
ACA
ACC
CAT
Normal
ACA
ACC
CAT
Table 2 Vimentin, e-cadherin and fibronectin expression pattern
| CAT | ACC | ACA | Normal adrenal | P | |
|---|---|---|---|---|---|
| Vimentin | |||||
| N | 27 | 24 | 19 | 12 | |
| Positive (+) | 26 (96.3%) | 14 (58.3%) | 12 (63.2%) | 7 (58.3%) | 0.008* |
| Negative (-) | 1 (3.7%) | 10 (41.7%) | 7 (36.8%) | 5 (41.7%) | |
| Median score (min-max) | 7.5 (0.5-12) | 4.7 (0-12) | 2.0 (0-7) | 2.0 (0-3) | <0.001 ** |
| E-cadherin | |||||
| N | 27 | 24 | 19 | 9 | |
| Positive (+) | 3 (11.1%) | 2 (8.3%) | 0 (0%) | 2 (22.2%) | 0.26 |
| Negative (-) | 24 (88.9%) | 22 (91.7%) | 19 (100%) | 7 (77.8%) | |
| Median score (min-max) | 0 (0-12) | 0 (0-9) | 0 (0-0) | 0 (0-8) | 0.24 |
| Fibronectin | |||||
| N | 27 | 24 | 19 | 11 | |
| Positive (+) | 11 (40.7%) | 10 (41.7%) | 3 (15.8%) | 1 (9.1%) | 0.07 |
| Negative (-) | 16 (59.3%) | 14 (58.3%) | 16 (84.2%) | 10 (90.9%) | |
| Median score (min-max) | 1 (0-6) | 1 (0-9) | 0 (0-4) | 0 (0-4) | 0.08 |
*P 0.008 at ×2 test;
** P < 0.001 at Bonferroni post-hoc analysis when comparing childhood-onset tumors vs. adulthood carcinomas, adulthood adenomas and normal adrenals. P = NS for any other comparisons.
Therefore, the association of immunohistochemistry markers with poor prognosis and clinical and pathology characteristics were analyzed only for the CAT and ACC groups. In both the CAT and ACC cases, disease stage
(either Sandrini et al. [28] for CAT; and ENSAT for ACC cases), presence of distant metastasis, vascular invasion, and capsular invasion were not associated with any immunohistochemistry expression pattern
A
C
15
‘s = 0.44 , p < 0.001
B
600
r’s = 0.56 , p <0.001
15
r’s = 0.51 , p<0.001
Vimentin
10
400
10
SIP1
Vimentin
5
200
5
0
0
0
0
200
400
600
0
200
400
600
0
200
600
TWIST1
TWIST1
400
SIP1
D
E
F
400
r’s = 0.27 , p < 0.05
400
r’s = 0.23 , p < 0.05
400
‘s = 0.11 , p = 0.31
300
300
300
SNAIL
200
SNAIL
SNAIL
200
200
100
100
100
0
0
0
0
200
400
600
0
5
10
15
0
200
400
600
SIP1
Vimentin
TWIST1
(Supplemental Tables 1 and 2). Additionally, the median Weiss-score was similar among cases, regardless of high or low expression of TWIST1, SIP1, and SNAIL.
Survival curve analyses of CAT and ACC cases are detailed in Fig. 5. In the ACC group cases with high SIP1 expression had a significantly higher mortality risk (p= 0.04). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of high SIP1 expression for mortality among ACC cases were 72.7, 27.3, 66.7, 70, and 68%, respectively.
The expression of TWIST1 and SNAIL were not related to death risk in any of the studied groups.
TP53 sequencing analysis
Tumor DNA was extracted from all CAT, ACC, and ACA cases (n = 70). Exons 5-10 TP53 gene sequencing was successful in 22/27 (81.5%) of CAT cases; 15/19 (78.9%) of ACA cases; and 18/24 (75%) of the ACC cases. Non- synomimous TP53 mutations were found in 11/22 (50%) cases of CAT-four of them in homozygosis (diagnosed at age of two, three, four, and 12 years-old). Exon-10 R337H mutation was the most prevalent observed aberration (7/11). The remaining four TP53 mutations among children were three exon-5 mutations (R158H, two cases; and R175H, one case), and one E349G exon-10 mutations. Mutations were found in three ACC cases (16.7%): exon-10 R337H and R337C (both in heterozygosis, at age of 34 and 27 years- old, respectively); and an 8 base-pair deletion in exon-8
[29]. No mutations were observed among ACA cases. As expected, median age was significantly lower among patients harboring a TP53 mutation [4.0 (1-76) years vs. 36.4 (1-73) years, p = 0.002]. No association was detected between the presence of mutated TP53 and the following clinical variables: tumor size, disease stage, gender, or death. Moreover, no difference was observed on the expression of vimentin, e-cadherin, fibronectin, TWIST1, SIP1, and SNAIL among individuals with mutated vs. wild- type TP53 (all, p> 0.05) (data not shown).
Discussion
The present study originally describes the differences in the protein expression of EMT markers e-cadherin, vimentin, and fibronectin; and EMT-TFs TWIST1, SIP1, and SNAIL among CAT, ACA, ACC and normal adrenal samples. TWIST1 and SIP1 expression were significantly higher among the most aggressive tumors (CAT and ACC) in comparison to the benign tumor (ACA) and normal tissue. SNAIL expression was increased in CAT, ACC, and ACA tumor tissues in comparison to normal tissue. Vimentin expression was significantly higher among CAT samples, with lower expression in the remaining ACT and normal tissue samples. Lastly, high expression of SIP1 was related to poorer survival in ACC patients.
EMT is an essential biological process during embryonic development, wound healing and organ fibrosis [10, 11].
TWIST1
SIP1
SNAIL
1,0
Wilcoxon p = 0.70
1,0
Wilcoxon p = 0.64
1,0
Wilcoxon p = 0.72
0,8
0,8
0,8
Cumulative Survival
Cumulative Survival
Cumulative Survival
0,6
0,6
0,6
CAT
0,4
0,4
0,4
0,2
High TWIST1
0,2
High SIP1
0,2
High SNAIL
ANO
YES
-ANO
ANO .. ” YES
NO-censored
.”YES
NO-censored
NO-censored
YES-censored
YES-censored
YES-censored
0,0
0,0
0,0
,0
50,0
100,0
150,0
200,0
,0
50,0
100,0
150,0
200,0
,0
50,0
100,0
150,0
200,0
months
months
months
1,0
Wilcoxon p = 0.31
1,0
Wilcoxon p = 0.04
1,0
Wilcoxon p = 0.65
0,8
0,8
0,8
Cumulative Survival
Cumulative Survival
Cumulative Survival
0,6
0,6
0,6
ACC
0,4
0,4
0,4
+
0,2
High TWIST1
0,2
High SIP1
0,2
High SNAIL
ANO
YES
ANO
MNO
NO-censored
YES
YES
YES-censored
NO-censored
YES-censored
NO-censored
YES-censored
0,0
0,0
0,0
,0
25,0
50,0
75,0
100,0
125,0
,0
25,0
50,0
75,0
100,0
125,0
.0
25,0
50,0
75,0
100,0
125,0
months
months
months
Additionally, the role of EMT and EMT-TFs as pivotal for cancer progression has been reported [9, 11- 15, 22, 25, 34, 35]. In this context, EMT has been postu- lated as one of the major mechanisms for metastasis development [36]. TWIST1, a member of the basic helix- loop-helix transcription factor family; Smad-Interactin- Protein-1 (SIP1), (also known as zinc-finger E-box-bind- ing-ZEB2); and SNAIL, a zinc-finger containing tran- scription factor are responsible for interacting with the e- cadherin promoter region, leading to downregulation and consequent impairment in the cell-cell adhesion mechanism [37]. In the present study, the expression of e-cadherin was extremely low in all groups including normal adrenal samples. However, due to its origin in the embryonic mesoderm, low levels of e-cadherin are expected despite its glandular epithelial pattern. For the same reason, vimentin expression is expected in normal adrenal tissue, as was observed in our evaluation. However, the increased vimentin expression observed in malignant tumors as CAT and ACC can be attributed to the activation of EMT related genes. TWIST1, SIP1, and SNAIL are able to suppress e- cadherin, as well as to increase mesenchymal proteins
expression [38]. As reported by Mendes et al. [39], these EMT-related changes in intermediate filament composition occur to facilitate cell motility though the interaction of vimentin with motor proteins. In the present study, our data indicate that the malignant and invasive behavior of CAT and ACC might be related to EMT related interaction between vimentin and TWIST1, SIP1, and SNAIL.
Although this data has provided significant progress in the knowledge of molecular mechanisms involved in adre- nal development and homeostasis, the full understanding of adrenal tumorigenesis has yet to be elucidated [40]. Among the studied signaling pathways, the insulin-like growth factors (IGF) [41], Wnt/ß-catenin [42], Sonic-hedgehog (SHH) [43], and Notch [44] have been described both in the development/maintenance of the adrenal cortex and its tumorigenesis. Interestingly, the aberrant overexpression of these same pathways is able to independently activate EMT in different types of cancer [45-48]. In fact, the IGF path- way is a major contributor to ACC, as Gicquel et al. [49] have demonstrated that most malignant (but not benign) adrenocortical tumors accumulate high amounts of IGF2. These data was recently confirmed by The Cancer Genome
Atlas (TCGA) study [50]. In line with previous studies [51, 52], TCGA study also described that CTNNB1 muta- tions, which lead to cytoplasmic and nucleus accumulation of ß-catenin, are common events in both ACA and ACC [50]. In turn, Leal et al. reported that CTNNB1 mutations are less frequent in CAT [42]. Interestingly, Salomon et al. [53] reported that increased ß-catenin in H295R adrenocortical carcinoma cell lines is positively associated with growth and aggressiveness and with the acquisition of the expres- sion of mesenchymal markers vimentin and n-cadherin. In conclusion, although indispensable to the normal adrenal cortex development and homeostasis, the impairment and overexpression of IGF2, Wnt/ß-catenin, SHH, and Notch pathways are related to both malignant and benign adrenal tumorigensis, as well as to EMT and EMT-TFs activation in other cancers. Therefore, the observed significant pro- gressive increase in the expression of vimentin, TWIST1, SIP1, and SNAIL from normal adrenal tissue to ACC and CAT neoplastic cells are potentially related to the presence of eventual different tumoral pathways in each particular case. Another explanation for this observation may be related to an eventual malignant transformation of an existing adrenal mass, as proposed by Ozsari et al. [54].
TP53 mutations are the foremost common genetic aber- ration observed in CAT patients [3, 4]. We observed the peculiar exon-10 R337H missense mutation in 50% of our pediatric cases. Although previous studies reported up to a 15-fold higher incidence among children in southern Brazil, it is noteworthy that none of our TP53 mutated cases were from that region. Similarly to others, we observed no association with the presence of a TP53 mutation and worse clinical outcomes, suggesting that in this case mutTP53 acts only as a driver mutation in childhood onset tumors [42], and therefore may not be associated with later cancer pro- gression events such as EMT, as demonstrated. However, also in accordance to previous reports, the incidence of TP53 mutations among ACC was very low with no muta- tion being observed among ACA, suggesting that TP53 mutations may be a late event in the tumorigenesis of adult malignant tumors [55, 56]. Unfortunately, because of the low number of mutTP53 within adults in the present study, the assessment of its association with EMT and EMT-TFs factors was not feasible. The role of TP53 in EMT has been previously published. Kim et al. [57] demonstrated that wtTP53 could suppress EMT by directly activating the expression of miR-200 and miR-192 leading to SIP1 downregulation, and consequently maintaining the typical epithelial phenotype. Interestingly, miR200 and miR-192 have been proven to be downregulated in ACC patients and the H295R ACC cell line [58, 59].
EMT and the overexpression of EMT-TFs have been related to poor clinical outcomes, including higher histolo- gical grades, local invasiveness, nodal and distant
metastasis, recurrence, and failure to conventional therapy in different types of solid cancers regardless of their embryonic layer origin [16-25]. Giordano et al. [60] ori- ginally described higher TWIST1 expression in ACC sam- ples using microarray gene expression methodology. However, the present study is the first attempt to investigate the TWIST1 and SIP1 protein expression in ACT. Wald- mann et al. [61] found significantly higher immunohisto- chemical expression of SNAIL in 26 cases of ACC (mean
age of 47 years) in comparison to a control group of 12 ACA. Similar to our findings, e-cadherin expression was low in both benign and malignant tumors; however no analysis was performed in normal adrenal tissue. SNAIL expression was stronger among high-grade tumors and survival rates were decreased among immunopositive cases. Alternatively to Waldmann’s study, SNAIL analysis was performed with a monoclonal antibody in our study, which may be responsible for the lack of immunopositivity dif- ferences observed between malignant and benign tumors, but significantly higher in comparison to normal adrenal tissue. Moreover, staining score methodologies are not comparable between studies. To date, and the best of our knowledge, there has not been a study to previously com- pare the protein expression of EMT epithelial and mesenchymal markers, and EMT-TFs among pediatric, adulthood tumors, and normal adrenal tissue. In opposition to ACA, which harbor very good prognosis with low mortality rates, ACC and CAT are frequently associated to recurrence and unfavorable prognosis [7, 40, 62, 63]. Therefore, precocious prognostic factors are needed in order to identity those cases who require more intense surveil- lance. In this context, SIP1 higher expression was related to decreased survival among ACC cases, raising the possibility of a future marker of poor outcome. Validation is obviously warranted and will require larger studies. Other associations were not observed between clinical/pathology features and the expression of EMT phenotype markers expression and EMT-TFs.
Although the present study highlights interesting new findings into the context of ACT tumorigenesis, it also has limitations, which we believe do not compromise our results. The main potential limitation in immunohis- tochemistry analysis is related to its intrinsic subjectivity. To reduce this potential bias, independent multiple observer methodology was applied with a mean score used. Sec- ondly, as immunohistochemistry observers were blinded to the patients’ clinical outcomes, no bias on the association of studied scores and poor prognosis was possible. The small number of subjects in each ACT group potentially may have impaired the analysis of the association of clinical and pathology variables with EMT markers and EMT-TFs. However, ACC and CAT are extremely rare, and the fact that all patients treated in our Institution during an almost
20-year period were included in analysis must be acknowledged. Lastly, we must acknowledge that the EMT signature is extremely complex, involving not only the studied markers [9, 11]. Additional EMT markers (such as claudins, occludins, and n-cadherin among others), and EMT-TFs (such as ZEB1 and SLUG) were not assessed in this study. The role of these additional EMT markers and EMT-TFs definitely deserves to be investigated in future work in order to better characterize the EMT signature in ACT.
In summary, EMT is potentially related to the aggres- siveness of ACC and CAT, not through the classical pattern of suppressed e-cadherin expression, but with the increased expression of the mesenchymal marker vimentin. TWIST1 and SIP1 are overexpressed in ACC and CAT samples in comparison to ACA and normal adrenal tissue samples. Moreover, CAT, ACC, and ACA have comparable SNAIL expression levels, all of which are higher than normal tissue samples. Additionally, the data indicated that patients with high SIP1 expression have lower survival rates. Larger studies are required to validate our results and address the mechanisms underlying already known ACT tumorigenesis pathways and EMT/EMT-TFs.
Acknowledgements The authors are grateful to Dr. Marisa Dreyer Breitenbach for Institutional support. We are also grateful to Priscila Valverde and Maria Theresa Accioly for technical support with immunohistochemistry reactions.
Funding This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing interests.
Ethical approval This study was approved by INCA’s independent institutional advisory committee in September 24th, 2014 (protocol 33847514.4.0000.5274). All procedures performed in studies invol- ving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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