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Investigation of N-cadherin/ß-catenin expression in adrenocortical tumors
Beatrice Rubin1 . Daniela Regazzo1 . Marco Redaelli2 · Carla Mucignat2 .
Marilisa Citton3 . Maurizio Iacobone3 . Carla Scaroni1 . Corrado Betterle1 .
Franco Mantero1 . Ambrogio Fassina4 . Raffaele Pezzani1 . Marco Boscaro1
Received: 30 March 2016 / Accepted: 15 July 2016
C International Society of Oncology and BioMarkers (ISOBM) 2016 ☐
Abstract ß-catenin is a multifunctional protein; it is a key component of the Wnt signaling, and it plays a central role in cadherin-based adhesions. Cadherin loss promotes tumori- genesis by releasing membrane-bound ß-catenin, hence stimulating Wnt signaling. Cadherins seem to be involved in tumor development, but these findings are limited in adreno- cortical tumors (ACTs). The objective of this study was to evaluate alterations in key components of cadherin/catenin adhesion system and of Wnt pathway. This study included eight normal adrenal samples (NA) and 95 ACT: 24 adreno- cortical carcinomas (ACCs) and 71 adrenocortical adenomas (ACAs). 3-catenin mutations were evaluated by sequencing, and ß-catenin and cadherin (E-cadherin and N-cadherin) expression was analyzed by quantitative reverse transcription PCR (qRT-PCR) and by immunohistochemistry (IHC). We identified 18 genetic alterations in 3-catenin gene. qRT-PCR showed overexpression of ß-catenin in 50 % of ACC (12/24) and in 48 % of ACA (21/44). IHC data were in accordance
Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-5257-x) contains supplementary material, which is available to authorized users.
☒ Beatrice Rubin beatrice.rubin.1@unipd.it
1 Division of Endocrinology, Department of Medicine (DIMED), University of Padua, via Ospedale Civile, 105, 35128 Padua, Italy
2 Department of Molecular Medicine, University of Padova, via Marzolo 3, 35131 Padova, Italy
3 Division of Minimally Invasive Endocrine Surgery, Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy
4 Division of Pathology and Cytopathology, Department of Medicine (DIMED), University of Padua, Padua, Italy
with qRT-PCR results: 47 % of ACC (7/15) and 33 % of ACA (11/33) showed increased cytoplasmic or nuclear ß-catenin accumulation. N-cadherin downregulation has been found in 83 % of ACC (20/24) and in 59 % of ACA (26/44). Similar results were obtained by IHC: N-cadherin downregulation was observed in 100 % (15/15) of ACC and in 55 % (18/33) of ACA. ß-catenin overexpression together with the aberrant expression of N-cadherin may play important role in ACT tumorigenesis. The study of differentially expressed genes (such as N-cadherin and ß-catenin) may enhance our understanding of the biology of ACT and may contribute to the discovery of new diagnostic and prognostic tools.
Keywords Adrenocortical tumors · Adrenocortical carcinomas · Wnt/beta-catenin signaling pathway · N-cadherin
Introduction
Adrenocortical tumors (ACTs) are very common neoplasms, affecting 3-10 % of the human population, and most are small benign adrenocortical adenomas (ACAs) [1, 2]. In contrast, adrenocortical carcinomas (ACCs) are a very rare and aggressive tumors with an annual incidence of 0.7-2.0 cases per million. ACC can occur at any age, with a peak incidence between 40 and 50 years, and the long-term outcomes are poor with a very low 5-year survival rate [3, 4].
The differential diagnosis between ACA and localized ACC can be demanding, and accurate distinction between these two different tumor types is very important, since their treatment is radically different [5]. At present, the modified Weiss score (WS) is the most widely accepted pathological system for classifying ACT [6]. This histopathological scoring system involves nine criteria (high mitotic rate, atypical mitoses, high nuclear grade, low percentage of clear cells,
necrosis, diffuse architecture of the tumor, capsular invasion, sinusoidal invasion, and venous invasion), but its reliability is challenged in borderline cases, having only few criteria represented [7]. To this purpose, the discovery of new molecular targets is urgent to discriminate between adrenocortical adenomas and carcinomas.
Wnt/ß-catenin signaling has been identified as one of the key signaling pathways in both normal adrenal development (regulating cell growth, motility, and differentiation) and tumorigenesis [8]. It is essential for embryonic development and cell renewal, but its ectopic constitutive activation is associated with cancer development in a number of neoplastic tissues (such as ovarian, colon, melanoma, endometrial, pros- tate, and hepatocellular) [9]. Abnormal activation of ß-catenin plays an important role also in ACT development [10]. Indeed, ß-catenin is an essential regulator of Wnt pathway; it is normally maintained at low levels in the absence of Wnt stimulation, due to its association to cadherin/catenin complex and subsequent phosphorylation and degradation by ubiquitin/proteasome system. Therefore, the activity of the Wnt signaling pathway is crucial in controlling the cytoplasmic and nuclear accumulation of ß-catenin [11, 12].
Gene alterations leading to a constitutive activation of this pathway are common events in ACC [13]. Somatic activating mutations of the CTNNB 1 gene (ß-catenin gene) are present in approximately 15-36 % of both benign and malignant tumors, and abnormal cytoplasmic or nuclear accumulation of ß-catenin is seen in 24-40 % of adrenocortical adenomas and in 30-80 % of ACC [14, 15]. Other mutations have been detected in components of the Wnt pathway, such as APC and AXIN2 genes. Gaujoux and collaborators showed the presence of a single silent APC gene mutation in 20 ACC analyzed [16], and Chapman’s research group identified AXIN2 alterations in 2 of 20 adenomas (7 %), in 1 of 6 ACC (17 %), and in H295R adrenocortical cell line [17]. However, despite the lack of CTNNB1, APC, and AXIN2 mutations in some tumors, the majority of ACC was found positive for cytoplasmic or nuclear ß-catenin accumulation, which suggests an additional mechanism of Wnt activation involving stabilization of ß-catenin [18].
In addition to its role as a transcriptional regulator in the Wnt signaling pathway, ß-catenin is also a key regulator of cadherin-mediated adhesion; it is a fundamental component of adherens junctions (AJs) [19]. ß-catenin is a multifunctional protein, which can be found associated with cadherins at the plasma membrane or as a cytoplasmic/nuclear protein. When associated with cadherins, ß-catenin is involved in cell-cell adhesion processes and a disruption of this interaction is thought to participate in the malignant progression of epithelial tumors [10, 20, 21].
Cadherins comprise a large family of transmembrane adhesion molecules that mediate calcium-dependent cell-cell adhesion. In cadherins, the conserved cytoplasmic domain
forms a complex with the catenins, such as p120catenin, ß-catenin, and «-catenin, which are possible regulators of cadherin junction and can bind to the cytoskeleton [22]. In this superfamily of adhesion molecules, the most extensively studied are epithelial cadherin (E-cadherin (E-CAD)) and neural cadherin (N-cadherin (N-CAD)). Whereas E-cadherin is mainly found in epithelial cells, promoting tight cell-cell associations, N-cadherin is primarily found in neuronal tissues and fibroblasts [23]. The cadherin family is closely related to tumor invasion and metastasis, because downregulation of cadherins correlate with an increased metastatic potential that arises from the loss of their adhesive properties. Therefore, elevated nuclear accumulation of ß-catenin by its dissociations from cadherin junction is frequently observed during tumorigenesis and may reveal some promising therapeutic targets [24, 25].
The aims of this study were to analyze the genetic (3-catenin, AXIN2 genes) and molecular profiles (ß-catenin, E-cadherin, and N-cadherin expression) of adrenocortical tumors, with the purpose of identifying useful markers for differentiating ACA to ACC and to better understand adrenocortical tumorigenesis.
Materials and methods
Human adrenocortical samples and adrenocortical tumor cell lines
Patients included in the study were investigated for sporadic ACT in the Department of Medicine at the Endocrinology Unit of University of Padua. Ninety-five ACT tissues were obtained surgically from patients with adrenocortical carcinomas (ACC, n = 24) and adrenocorti- cal adenomas (ACA, n = 71). ACA were further subdivided into aldosterone-producing adenomas (APA, n = 37) and non-aldosterone-producing cortical adenomas (NAPACA, n = 34). Adrenocortical normal samples (NA, n = 8) were obtained from renal transplantation surgical procedures. The type of adrenocortical mass was preoperatively characterized based on biochemical parameters and by magnetic resonance imaging or computed tomography and checked by adrenal histology. Malignancy was diagnosed on the histopathological criteria proposed by Weiss [6]. Staging was based on imaging studies and confirmed by the findings at surgery, by using the European Network for the Study of Adrenal Tumors (ENSAT) staging system [26]. Of 24 ACC patients, five were stage II and the others stage IV. All patients gave written informed consent to the collection and use of adrenal tissue for research purposes; the study was approved by the local ethics committee (conforming to the Declaration of Helsinki, revised in Tokyo, 2004).
H295R tumor cell line was obtained from the American Type Culture Collection (ATCC, Rockwille, MD) and used as described elsewhere [27].
DNA extraction and PCR amplification analysis
Tissue fragments obtained at surgery were immediately frozen in liquid nitrogen and stored at -80 ℃ until use. Genomic DNA was isolated from frozen tissues with a QIAmp DNA Tissue Mini Kit (Qiagen GmbH, Hilden, Germany). DNA quantity and quality were evaluated by spectrophotometry (UV-Visible NanoDrop® ND-1000) and PCR amplification performed as described elsewhere [28]. 3-catenin exon 3 was amplified with the primer pairs used by Taniguchi [29], and AXIN2 exon 7 was amplified with the primer sequences used by Hayes [30]. The 3-catenin primers (forward, 5’-AGTAACATTTCCAATCTACTAATGC-3’, and reverse, 5’-CTGACTTTCAGTAAGGCAATG-3’) and AXIN2 primers (forward, 5’-CAAAGCACAAAAAAGGCCTAC-3’, and reverse, 5’-AGGGTCCTGGGTGAACAGGT-3’) were purchased by the Invitrogen Life Technologies Company (Monza MB, Italy).
High-resolution melting curve analysis and mutation analysis
Mutation analysis from tissues and cell lines was performed using high-resolution melting (HRM) screening and direct sequencing as reported elsewhere [31]. For each sample, nor- malized melting curves were evaluated, and the analyzed sam- ples were compared to wild-type controls. Only samples with altered melting curves were undergone direct sequencing. ß-catenin and AXIN2 somatic mutation analysis was per- formed using an ABI PRISM3130 genetic analyzer (Applied Biosystems) according to the manufacturer’s protocol. Resulting sequences were aligned to each other and to a published wild-type sequence: exon 3 of 3-catenin gene (NM_001904) and exon 7 of AXIN2 gene (NM_004655.3).
B-Catenin and cadherin expression by real time-PCR (qRT-PCR)
Quantitative reverse transcription PCR (qRT-PCR) method was followed as described elsewhere [28]. Briefly, total RNA was extracted from 68 ACT samples (24 ACC and 44 ACA), 8 NA, and from H295R cells with an RNeasy Mini Kit (SABioscience) in accordance with the manufacturer’s proto- col. E-cadherin (CDH1 gene) (NM_004360.3), N-cadherin (CDH2 gene) (NM_001792.3), 3-catenin (CTNNB1 gene) (NM_001904), and 3-actin (housekeeping gene) (ACTB gene) (NM_001101) primer sequences were performed as de- scribed elsewhere [28, 32, 33, 34]. The E-cadherin primers (forward, 5’-CATTGCCACATACACTCTCTTCT-3’,
and reverse, 5’-CGGTTACCGTGATCAAAATCTC-3’), N- cadherin primers (forward, 5’ - TCCAGACCCCAATTCAATTAATATTAC-3’, and reverse, 5’- AAAATCACCATTAAGCCGAGTGA-3’), 3-catenin primers (forward, 5’-CTTGCTCAGGACAAGGAAGC-3’, and reverse, 5’-CATATGTCGCCACACCTTCA-3’), and 3-actin primers (forward, 5’-GGGACGACATGGAGAAAATCTG-3’, and re- verse, 5’-CACGCAGCTCATTGTAGAAGGT-3’) were pur- chased by the Invitrogen Life Technologies Company (Monza MB, Italy). Data were obtained as Ct values according to the MIQE guidelines and used to determine ACt values (ACt = Ct of the target gene minus Ct of the housekeeping gene) [35]. The equation 2-AACt was used to calculate the fold changes in gene expression between the categories of samples (e.g., ACC vs NA, ACA vs NA, ACC vs ACA).
Immunohistochemistry analysis
Immunohistochemistry (IHC) was performed on 5-um-thick paraffin-embedded sections of 48 available tumor samples (15 ACC, 33 ACA) and 10 NA. All IHC stainings were performed automatically (Bond-maX) with ß-catenin (Clone 17C2, Mouse, Novocastra, 1:100), E-cadherin (Clone NCH38, Mouse, DakoCytomation, 1:200), and N-cadherin (clone 6G11, Mouse, DakoCytomation, 1:100) antibodies. The section was counterstained with hematoxylin, running concurrently with appropriate positive and negative controls. The reactions were labeled relying on a simple scale: 0 (0-5 %), 1 (6-33 %), 2 (34-66 %), and 3 (67-100 %). Two independent pathologists (AF and RP) have examined and scored the samples.
Protein extraction and Western blot analysis
Proteins were extracted from two ACCs, two adrenocortical adenomas (one APA, one NAPACA), and four normal adjacent adrenocortical tissue (NA) samples, as described elsewhere [27]. Moreover, ACC-2 T and ACC-2 N were frac- tionated into cytoplasmic and nuclear fractions with NE-PER Nuclear and Citoplasmic Extraction Reagent Kit (Thermo Scientific, Pierce Biotecnology, Rockford, USA) according to the manufacturer’s protocol. Briefly, proteins were extracted, loaded onto SDS/PAGE-GEL, electro-blotted onto nitrocellulose membranes, blocked, and incubated overnight with different primary antibodies: anti-ß-actin (catalog num- ber A5441) (Sigma-Aldrich, St. Louis, USA), anti-N-cadherin (EPR1792Y) (catalog number GTX61612) (Genetex, Irvine, USA), anti-ß-catenin (catalog number 610154) (BD Transduction Laboratories, England, UK). After anti-mouse (catalog number 315-035-003) or anti-rabbit (catalog number 111-035-003) HRP-labeled secondary antibody (Jackson Immunoresearch Labs, West Grove, USA) was added, immu- noreactivity was detected with LiteAblot Extend Long Lasting
Chemiluminescent Substrate (EuroClone, Milan, Italy). Films were scanned and band intensity quantified with ImageJ software 1.48 s. Each experiment was performed in triplicate.
Transfection
H295R cells were plated in 6-well plates, 80-90 % confluent, cultured in DMEM/F12 antibiotic free medium for 24 h, and then transfected with empty vector (pcDNA3.1) or N-cadherin plasmid. Incorporation of the N-cadherin plasmid was achieved using Lipofectamine 2000 (Invitrogen Life Technologies, Dublin, Ireland). Plasmid was kindly provided by Dr. Phan and Prof. Scott Filler (Division of Infectious Diseases, Los Angeles Biomedical Research Institute, UCLA Medical Center, CA). The primers for the N-cadherin plasmid were 5’-TCCATGTGCCGGATAGCG-3’ and 5’- AGTTCAGTCATCACCTCCACCATACA-3’, and it was cloned into the pcDNA3.1 expression vector by the donating authors as described elsewhere [36]. The expression of N-cadherin and ß-catenin in H295R cells, at 24 and 72 h after transfection with empty vector or N-cadherin plasmid, was confirmed by Western blotting, real-time PCR, and confocal microscopy analysis.
Confocal microscopy analysis
H295R cells were fixed with 4 % paraformaldehyde and then permeabilized with 0.1 % Triton X-100 in PBS. After incubation at 4 ℃ overnight with the primary antibodies, anti-N-cadherin (EPR1792Y) (Genetex, Irvine, USA) and anti-ß-catenin (BD Transduction Laboratories, England, UK), the cells were washed with PBS. Secondary antibodies, donkey anti-rabbit Alexa Fluor 488 (Life Technologies A21206; 1:100), and goat anti-mouse Alexa Fluor 594 (Life Technologies A11005; 1:1000) were then added to the cells for 60 min. Cell nuclei were counterstained with DRAQ5
(Cell Signaling Technology). The samples were analyzed by a confocal microscope (Leica TCS SP8, Leica Microsystems) with a z-interval of 1 um using a 63×/1.4 oil immersion lens. Images were acquired with a digital system (DFC365FX camera, Leica Microsystems) and processed using the Leica Application Suite (LAS-AF) 3.1.1 software (Leica Microsystems). Sections were analyzed independently by three experts (AF, RP, and MR).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA) and Microsoft Excel software. Data comparisons in Western blot analysis were performed using Kruskal-Wallis analysis followed by Dunn’s test (*P < 0.05). For clinicopathologic studies, a non-parametric test (Kolmogorov-Smirnov test) was used. P < 0.01 was considered as statistically significant. Spearman’s correlation coefficient analysis was used to evaluate the correlations between ß-catenin and N-cadherin expression.
Results
Clinicopathologic characteristics
The clinicopathologic characteristics of the patients are summarized in Table 1. As expected, ACC median tumor size (median 83 mm) is greater than ACA median tumor size (median 27 mm) (P < 0.0001). Adenomas were subdivided in aldosterone secreting and non-aldosterone forms; non-aldosterone-producing cortical adenomas (NAPACA) are larger (median 38 mm) than APA (median 20 mm) (P< 0.0001).
| ACC (n =24) | ACA (n =71) | |
|---|---|---|
| Male | 10 | 26 |
| Female | 14 | 45 |
| Age (years) | 53 years (1-80 years) | 49 years (21-80 years) |
| Tumor size (mm) | 83 mm (40-150 mm) | 27 mm (5-60 mm) |
| 3-catenin mutations (% of total mutations) | 5/24 (20 %) | 13/71 (18.3 %) |
| Functional status | ||
| · Cortisol-producing | 9 | 25 |
| · Cortisol-producing and androgen-producing | 8 | 3 |
| · Aldosterone-producing | – | 36 |
| · Aldosterone-producing and cortisol-producing | – | 1 |
| · Non-functioning | 7 | 6 |
B-Catenin genetic alterations occur frequently in human ACT
HRM analysis in 95 ACT and 8 NA samples was used to evaluate the presence of alterations in key components of the Wnt/ß-catenin signaling pathway. Samples with an irregular melting curve (compared to wild type) were further investigated by direct Sanger sequencing (Fig. 1a-c).
We identified 18 genetic alterations in 3-catenin gene in 13 ACA (18.3 %) and 5 in ACC (20 %) (Fig. 1d) and only a single mutation in AXIN2 gene in H295R cells (c.2013_2024del12, p. T672_R675del) (Fig. 1e).
Mutations were found in secreting and non-secreting tumors. The most frequent 3-catenin mutations were found at the amino acid serine 45 (14/18), fundamental key-point for GSK3ß-mediated phosphorylation, and subsequent ß-catenin degradation. Furthermore, we observed two deletions, p. S45del (c.133_135del TCT) and p. A43_E53del (c.127del33bp), in a cortisol and androgen- producing ACC and in one APA, respectively, and two other combined mutations, p. P44A + p. S45P (c.133 T>C + c.136 C>G), were found in one NAPACA and in one APA (Table 2).
Moreover, we found that H295R cell line carried an activating 3-catenin mutation (c.133 T>C, p. S45P) in the
exon 3, thus confirming this cell line model as an excellent line of reference (Table 2).
ß-Catenin and cadherin expression analysis
ß-catenin and cadherin (E-cadherin and N-cadherin) expression in sporadic ACT was analyzed by qRT-PCR, IHC, and Western blot (WB). qRT-PCR showed overexpres- sion of ß-catenin in 50 % of ACC (12/24) (P = 0.0461) and in 48 % of ACA (21/44) (P = 0.0378) if compared to normal adrenocortical tissues (NA) (Fig. 2a).
IHC data were in accordance with the qRT-PCR results. In 47 % of ACC (7/15) and in 33 % of ACA (11/33), a cytoplas- mic and/or nuclear ß-catenin accumulation was found. NA showed ß-catenin presence only at plasma membrane level. Differently, ß-catenin staining was essentially present in the cytoplasm and nucleus of ACC and ACA (Fig. 2b-g).
E-cadherin was not expressed at messenger RNA (mRNA) level. We did not find expression of E-cadherin in any NA or ACC (0 %) by IHC (Fig. 3b-c). Only two ACA (6 %) were positive for E-cadherin.
Conversely, N-cadherin downregulation was found in 83 % of ACC (20/24) (P = 0.0006) and in 59 % of ACA (26/44)
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| Histological diagnosis | Patient sex/age (year) | Functional status | ß-Catenin mutation | |
|---|---|---|---|---|
| 1 | ACC | 53/F | Cortisol-producing and androgen-producing | c.107 A>C p. H36P |
| 2 | ACC | 63/F | Cortisol-producing | c.101 G>C p. G34A |
| 3 | ACC | 66/F | Cortisol-producing | c.107 A>C p. H36P |
| 4 | ACC | 34/F | Cortisol-producing and androgen-producing | c.134 C>T p. S45P |
| 5 | ACC | 69/F | Cortisol-producing and androgen-producing | c.133_135del TCT p. S45del |
| 6 | ACA | 60/M | Non-functioning | c.134 C>T p. S45F |
| 7 | ACA | 61/F | Non-functioning | c.133 T>C p. S45P |
| 8 | ACA | 66/F | Aldosterone-producing | c.127del 33 bp p. A43_E53del |
| 9 | ACA | 48/M | Aldosterone-producing | c.133 T>C + c.136 C>G p. P44A + S45P |
| 10 | ACA | 53/M | Aldosterone-producing | c.121 A>G p. T41A |
| 11 | ACA | 77/F | Aldosterone-producing | c.133 T>C p. S45P |
| 12 | ACA | 22/F | Aldosterone-producing and cortisol-producing | c. 134 C>G p. S45C |
| 13 | ACA | 50/M | Cortisol-producing | c.134 C>T p. S45F |
| 14 | ACA | 34/F | Cortisol-producing | c.133 T>C p. S45P |
| 15 | ACA | 68/M | Cortisol-producing | c.134 C>T p. S45F |
| 16 | ACA | 49/M | Cortisol-producing | c.134 C>G p. S45C |
| 17 | ACA | 42/F | Cortisol-producing | c.133 T>C + c.136 C>G p. P44A + S45P |
| 18 | ACA | 73/F | Cortisol-producing | c.134 C>T p. S45F |
| 19 | H295R cells | Derived from a 48-year-old female patient with ACC | Mineralocorticoids, glucocorticoids, adrenal androgens | c.133 T>C p. S45P (c.2013_2024del12 p. T672_R675del in AXIN2 gene)ª |
a
AXIN2 genetic alteration found in H295R cell line
(P=0.0075). N-cadherin mRNA expression was significantly lower in ACC samples compared to NA (Fig. 3a).
Similar results were obtained by IHC: N-cadherin downregulation was observed in 100 % (15/15) of ACC and in 55 % (18/33) of ACA if compared to NA (Fig. 3d-g).
By WB, we investigated two fundamental key factors of adherens junctions: ß-catenin and N-cadherin. We analyzed two ACC, one NAPACA, one APA-1, and four normal adjacent adrenocortical tissues (NA). A deletion (c.133_135del TCT, p. S45del) and a missense mutation (c.134 C>T, p. S45F) of 3-catenin gene were present in ACC-1 and NAPACA-1. Conversely, no mutation of the AXIN2 gene was detected in these eight samples. WB data confirmed previous qRT-PCR and IHC data. N-cadherin expression was observed in NA tissues, but it was very low or absent in ACT; conversely, ß-catenin was overexpressed in all ACT tissues (Fig. 4a), as it is perceivable by band quantification (Supplementary Figure S1). Furthermore, to evaluate the presence of ß-catenin and N-cadherin in nuclear and cytoplasmic fractions, we have analyzed one carcinoma
(ACC-2). N-cadherin was present in normal sample (ACC-2 N) and in the cytoplasm of this normal tissue. Conversely, ß-catenin was present in carcinoma (ACC-2 T), in nucleus, and in the cytoplasm but is low in the cytoplasm of normal tissue (Fig. 4b).
ß-Catenin and N-cadherin correlation analysis
The correlation analysis between the expression levels of ß-catenin and N-cadherin was represented in Spearman’s analysis. Correlation analysis showed significant positive correlation between high ß-catenin and low N-cadherin expression in 68 ACT analyzed (Spearman r = 0.6023; P < 0.0001) (Fig. 4c).
N-cadherin plasmid transfection
H295R cell transfection with N-cadherin plasmid resulted in an upregulation of N-cadherin expression and a
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downregulation of ß-catenin (Fig. 4d). Similar results were obtained by qRT-PCR (data not showed).
An increase of membranous N-cadherin expression, a decrease of ß-catenin cytoplasmic expression, and a shift of ß-catenin in the membrane with N-cadherin was observed in N-cadherin transfected cells (N-CAD) by confocal microsco- py analysis. In H295R cells transfected with the empty vector (EV), ß-catenin remains cytoplasmic while N-cadherin was not detectable (Supplementary Figure S2).
Discussion
The present study investigated the expression and mutational status of fundamental genes encoding key effectors of the Wnt/ß-catenin signaling pathway, such as B-catenin (CTNNB1 gene) and AXIN2, in a large number of ACT patients. Activation of this pathway has been already
demonstrated, suggesting that Wnt/ß-catenin pathway may be involved in ACT pathogenesis [14, 37]. Moreover, we evaluated the cadherin-catenin expressions, because ß-catenin is also a key regulator of cadherin-mediated adhesion. Nevertheless, no large, comprehensive and single-center study that evaluated catenin-cadherin association has been performed up to date.
We detected ß-catenin alterations in a large portion of ACT, and we have shown ß-catenin overexpression in about 50 % of ACT with a ß-catenin cytoplasmic and/or nuclear localization. We found that about 21 % of ACC (5/24) and 18 % of ACA (13/71) have 3-catenin genetic mutations, these results are in agreement with previous reports in the literature [38, 39, 40]. The most frequent B-catenin mutations affect the amino acid serine 45 (14/ 18 ACT), important point for GSK3ß-mediated phosphor- ylation and subsequent ß-catenin degradation by the proteasome complex; therefore in these mutated samples,
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the Wnt/ß-catenin signaling pathway resulted constitutive- ly activated. ß-catenin overexpression was detected in the majority of samples, but 3-catenin somatic mutations were not present in all samples. It is possible that other components of the Wnt signaling may be involved in ß-catenin accumulation. This is the reason why we decided to investigate AXIN2 gene, a key component of the B-catenin complex degradation. We identified only one alteration in H295R cell line, a homozygous deletion of 12 bp (c.2013_2024del12, p. T672_R675del), but we did not find any alteration in adrenocortical tissues. These results, in accordance with Chapman and collaborators [17], confirm that other genes or other mechanisms may be involved in ß-catenin cytoplasmic and nuclear accumu- lation observed in most ACC and ACA.
Indeed, a part of the ß-catenin accumulation may also be a result of N-cadherin absence. Therefore, if N-cadherin is not expressed in cellular membrane, ß-catenin is not complexed with N-cadherin and it accumulates in the cytoplasm or in the
nucleus. This is why we have analyzed cadherin expression in adrenocortical tumors.
We observed a drastic N-cadherin decrease in ACT tis- sues compared to NA samples; N-cadherin downregulation was present in 84-100 % of ACC. These results are in accordance with Tsuchiya and colleagues, who showed N-cadherin but not E-cadherin expressions in cortical and medullary adrenal glands [41]. Moreover, our results are further supported by Khorram-Manesh and collaborators who did not find E-cadherin expression in normal and pathological adrenal tissues, but N-cadherin overexpres- sion was demonstrated in malignant pheochromocytomas and N-cadherin downexpression in adrenocortical carcino- mas [42]. Other additional studies confirmed the absence of E-cadherin expression [43, 44] and the N-cadherin downregulation in ACC [45]. N-cadherin overexpression in ACC has been reported only by Yarom’s research group where they used an adjuvant treatment with ADH-1 (inhibitor of N-cadherin) with poor results [46].
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The acquisition of aggressive, invasive, and metastatic properties during tumor progression is related to changes in the expression of adhesion molecules regulating the interac- tions of cancer cells with the extracellular matrix (ECM) and neighboring cells. This study has demonstrated that ß-catenin was overexpressed and N-cadherin was down-expressed in ACT, both at the protein and mRNA level. Conversely, E- cadherin expression was not demonstrated in the normal ad- renal cortex and in the adrenal tumors analyzed. E-cadherin does not seem to be involved in normal adrenal gland function or tumorigenesis, but this point needs to be further elucidated.
Nowadays, high ß-catenin accumulation due to its dissociation from the cadherins is a frequently observed event during tumorigenesis of some cancers, such as colon or breast epithelial tumors [47]. In this study, we have demonstrated that ß-catenin and N-cadherin expression are correlated in ACT. We have obtained a statistically significant correlation between the expression of ß-catenin and N-cadherin represented in Spearman’s analysis; then, we may hypothesize that both molecules could participate to adrenal tumorigene- sis. Therefore, elevated nuclear accumulation of ß-catenin by
its dissociations from cadherin junction is frequently observed during tumorigenesis and may reveal some promising therapeutic target in adrenocortical tumors.
Finally, we transfected H295R cells with N-cadherin to increase its expression to further demonstrate the link between ß-catenin and N-cadherin in vitro. We observed a downregu- lation of ß-catenin expression when N-cadherin is overexpressed. Indeed, when N-cadherin is overexpressed in H295R cells by transfection, we could appreciate an increase of N-cadherin in the plasma membrane accompanied by a decrease of ß-catenin cytoplasmic expression and by an increased localization of ß-catenin in cellular membrane. This may propose a real connection between the expressions of the two proteins.
Our data suggest that ß-catenin overexpression together with the aberrant expression of N-cadherin may play a very important role in ACT tumorigenesis. This study of differen- tially expressed genes (such as N-cadherin and ß-catenin) may enhance our understanding of the molecular biology of ACT and may contribute to the discovery of new diagnostic and prognostic tools.
Acknowledgments This work was partially supported by European Network for the Study of Adrenal Tumors (ENS@T-CANCER grant agreement no. 259735), Futuro in Ricerca (FIRB grant agreement no. RBAP1153LS), Fondazione Guido Berlucchi per la Ricerca sul Cancro, and by Associazione Italiana per la Ricerca Oncologica di Base (AIROB, Padua, Italy).
Compliance with ethical standards
Conflicts of interest None
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