Background
Human hemochorial placentation closely resembles processes otherwise seen in cancer. The capacity of trophoblasts to migrate and invade the maternal decidua, as well as their ability to form the syncytiotrophoblast (SCT) via cell-fusion, shares high similarities with tumor cells [
1]. As a consequence, trophoblasts are often characterized to be of pseudo-malignant nature. Not surprisingly, expression patterns of tumor suppressor genes in cancer are similar to those found in placenta [
2,
3]. Recently, retinoic acid receptor responder 1 (
RARRES1), also known as Tazarotene-induced gene 1 (
TIG1), was identified as an important tumor suppressor gene [
4,
5]. It was initially described as a novel retinoic acid (RA) receptor (
RARβ and
γ) regulated gene in skin graft cultures [
6].
RARRES1 is located on chromosome 3q25 and was reported to be one of the most commonly methylated loci in multiple cancers [
7‐
9]. Epigenetic silencing of
RARRES1 expression and its effect on tumor cell invasion, proliferation and survival further underscored its tumor suppressive properties [
10]. In prostate cancer an association between
RARRES1 hypermethylation and worse clinical outcome was reported [
8].
Recently, we identified RARRES1 in the human placenta and determined its regulation by RA derivates. In the placenta, uncontrolled trophoblast growth result in malignant transformation as observed in choriocarcinomas [
11].
Thus, in the light of previous reports regarding the role of epigenetic regulation of tumor suppressor genes in the human placenta [
12,
13], we investigated the epigenetic and transcriptional regulation of the tumor suppressor
RARRES1 in the human placenta, choriocarcinoma cell lines and biopsies, as well as its potential functional properties.
Methods
Patient and tissue collection
Human term placentas (third trimester) were obtained from healthy patients (
n = 74) after elective Caesarean section and processed within 1 h. The clinical data were summarized in Additional file
1: Table S1. After removal of the basal plate and chorionic membrane, a biopsy was obtained near the cord from every placenta. Placental tissues were snap frozen in liquid nitrogen and stored at −80 °C until further use. Formalin-fixed and paraffin-embedded (FFPE) sections were supplied by the Institute of Pathology, University Hospital Erlangen. Handling of patients and tissues was approved by the Ethics Committee at the University Erlangen-Nuremberg (No. 2180). A written informed consent was obtained from all participants. FFPE sections of 10 choriocarcinoma cases were provided by the Institute of Pathology of the University Hospital Leipzig. Five cases were intramolar and five pure choriocarcinomas.
Immunohistochemical (IHC) staining
Human FFPE placental sections were deparaffinized and rinsed in a series of descending ethanol concentrations. IHC stains were performed on tissue sections using the LSAB + HRP kit (Agilent, Hamburg, Germany) according to the manufacturer’s instructions. Anti-human RARRES1 (ab92884, Abcam, Cambridge, United Kingdom, 1:6000) antibody was used. Nuclei were counter-stained by hematoxylin.
Immunofluorescence (IF) staining
Human FFPE placental sections were deparaffinized and rehydrated as stated above. Slides were blocked using 1% bovine serum albumin in Tris-buffered saline (BSA/TBS) for 30 min at room temperature. Anti-human RARRES1 antibody was diluted in 1% BSA/TBS (1:400) and slides were incubated at 4 °C over-night. As secondary antibody Alexa Fluor 488 Donkey Anti-Mouse IgG (H + L) (Molecular Probes, Eugene, USA) was used at a dilution of 1:400 in 1% BSA/TBS and slides were incubated for 2 h at room temperature. Staining of the nuclei was performed using DAPI (Thermo Fischer, Darmstadt, Germany).
Villous cytotrophoblast isolation
Villous cytotrophoblasts (VT) from healthy placentas of 15 different individuals were isolated using the Trypsin-DNase-Dispase/Percoll method, as previously described [
14]. Before DNA and RNA extraction the fractionated trophoblasts were cryopreserved in liquid nitrogen. By fluorescence activated cell sorting (FACSCalibur, BD Biosciences) we could determine a percentage of 86.6–90% of the fractionated cells being trophoblastic [
14].
Cell culture
The choriocarcinoma cell lines BeWo, Jeg-3 and JAR were cultured under the following conditions: BeWo was grown in DMEM:F12 phenol-red free (high Glucose; Thermo Fischer, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS, Thermo Fischer, Darmstadt, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin (1% P/S, Sigma-Aldrich, Taufkirchen, Germany). Jeg-3 cells were cultured in phenol-red free DMEM:F12 (Thermo Fischer, Darmstadt, Germany) supplemented with 10% FCS, 1% P/S and JAR cells in RPMI 1640 media (Thermo Fischer, Darmstadt, Germany) with 10% FCS, 10 mM Hepes (Sigma-Aldrich, Taufkirchen, Germany), 2 mM L-Glutamin (Sigma-Aldrich, Taufkirchen, Germany) and 0.1 mM non-essential amino acids (NEAA, Sigma Aldrich, Taufkirchen, Germany). The first trimester cell line Swan71 was a kind gift of Prof. G. Mor, Department of Obstetrics, Gynaecology and Reproductive Sciences, Reproductive Immunology Unit, Yale University School of Medicine, USA [
15]. The cell line was grown in phenol red free DMEM:F12 with 10% FCS, 1%P/S and 0.1 mM NEAA.
For stimulation experiments cells were seeded at a density of 3x104/ml (Swan71, Jeg-3 and JAR) or 4x104/ml (BeWo) in a 12-well cell culture plate. Cell lines were cultivated in medium supplemented with 2.5% (Swan71) or 5% (Jeg-3, JAR, BeWo) charcoal treated fetal bovine serum (CTS, Thermo Fisher, Darmstadt, Germany) 24 h prior to and during stimulation. Cells were treated with 1.0 μM all-trans-retinoic acid (ATRA, Biomol, Hamburg, Germany), Tazarotene (TAZA, Sigma-Aldrich, Taufkirchen, Germany) or 200 nM Am580 (Tocris, Lille Cedex, France) for 24 to 72 h. In order to induce global DNA demethylation, the cell lines were additionally treated with 0.5 or 1.0 μM 2′-deoxy-5-azacytidine (AZA, Sigma-Aldrich, Taufkirchen, Germany) for 72 h.
For the analysis of RARRES1 expression in dependence of cell density, Swan71 and Jeg-3 cells were seeded at 0.75, 1.50, 3.0, 6.0, 12.0 and 18.0x10
4 cells/ml in 12-well dishes. After 48 h cells were harvested for RNA isolation (Additional file
2: Figure S1). Jeg-3 cells were additionally treated with AZA for 72 h before harvesting.
Genomic DNA was isolated from cultivated cell lines or 50–100 mg placental tissues as previously described in detail [
16]. DNA was dissolved in 0.01% DEPC water.
Bisulfite treatment and PCR amplification
Bisulfite treatment of 0.5–2.0 μg genomic DNA of placental tissues and trophoblast-like cell lines was performed using the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Converted DNA (100 ng) was amplified using a thermal cycler (Thermo Fischer, Darmstadt, Germany) and the HRM-Mastermix (Qiagen, Hilden, Germany). Bisulfite treated DNA was used in a 25 μl reaction volume with 10 μM of both the forward and the reverse primer (Additional file
3: Table S2). PCR conditions were 95 °C for 15 min, 50 cycles of denaturation (30s at 95 °C), annealing (30s at 50 °C for the CpG region 1 and 55 °C for region 2) and extension (30s at 72 °C), followed by a final extension period of 7 min at 72 °C. Successful conversion and amplification were controlled using capillary electrophoresis (QIAxcel, Qiagen, Hilden, Germany).
Pyrosequencing
Pyrosequencing (PyroMark Q24, Qiagen, Hilden, Germany) was performed according to the manufacturer’s instructions using PyroMark Gold Q24-reagents (Qiagen, Hilden, Germany). PCR and sequencing primer were designed using the PSW Assay design software 1.0.6 (Biotage, Sweden) as well as the online program MethPrimer [
17]. 75 pmol of the respective CpG region specific sequencing primer was used (Additional file
3: Table S2). Each CpG region of interest was split into two sections and analyzed separately to obtain reproducible results. The second section was sequenced using serial pyrosequencing according to Tost et al. (2006) [
18]. The methylation pattern was quantified by the PyroMark Q24-CpQ-Assay (Qiagen, Hilden, Germany). Correct sequencing was monitored using the EpiTect-PCR-Control-DNA set including methylated, unmethylated and unconverted control DNA. Data were analyzed by the PyroMark Q24 software.
RNA extraction and cDNA synthesis
RNA was isolated from third trimester placental tissues as well as cultivated cell lines using peqGOLD TriFast (PEQLAB, Erlangen, Germany), as previously described [
16]. Isolated RNA was treated with DNase I (Roche, Mannheim, Germany) to avoid DNA contamination during expression analysis. The High-Capacity-cDNA-Reverse-Transcription kit (Thermo Fischer, Darmstadt, Germany) was used to generate cDNA in a thermal cycler (ABI2720) for 2 h at 37 °C.
Quantitative Realtime PCR (qRT-PCR)
Quantification of
RARRES1 mRNA expression in placental tissues and cell lines was achieved by qRT-PCR analysis as previously described [
14]. In short, 10 nM forward and reverse primers and 40 ng cDNA were used to analyze the amplification of
RARRES1 via SYBR-green based technology (SYBR Select Master Mix, Thermo Fischer, Darmstadt, Germany). The expression was normalized using 18SrRNA and GAPDH as reference genes. Due to lack of differences, 18SrRNA data are presented only. Primer sequences are listed in Additional file
3: Table S2.
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM), which was calculated using Microsoft Excel 2010 (Microsoft Corporation, Redmond, Washington, USA). Differences between the subgroups were analyzed using the Mann-Whitney U-test (SPSS, IBM Inc., Nuremberg, Germany). Significances were adjusted using post hoc Bonferroni testing. P-values of less than 0.05 were considered as statistically significant.
Discussion
Taking the role of
RARRES1 as a tumor suppressor with a high methylation pattern in a variety of cancer types into consideration, we predicted that the
RARRES1 expression matched promoter methylation in placental tissues and cell lines [
9,
21‐
23]. DNA from 3rd trimester placental tissues showed a low methylation pattern of CpG region 1 compared to a significantly higher overall methylation of CpG region 2. Consequently, we assumed CpG region 2 of the
RARRES1 promotor might be less relevant for RA-induced transcriptional activation compared to CpG region 1. In all observed choriocarcinoma cell lines both analyzed CpG regions were hypermethylated, which was accompanied by a significant decrease of
RARRES1 mRNA expression.
One limitation of the provided results is that AZA treatment of primary trophoblasts could not be performed due to the lack of proliferative capacity of third trimester trophoblasts. Trophoblasts of the third trimester undergo a terminal differentiation leading to an irreversible exit from the cell cycle and thus, AZA treatment of primary trophoblasts would not originate in the incorporation of 5′-AZA-2-dC into the DNA [
24]. However, treatment of choriocarcinoma cell lines with AZA abolished the effect of high methylation resulting in an increased
RARRES1 gene expression. Nevertheless, we cannot rule out that increase of
RARRES1 expression was due to secondary side-effects, e.g. AZA-induced increase of transcription factors. It is well known that genes involved in RA-signaling are commonly methylated in cancer and that their expression can be induced by AZA treatment [
19,
25]. For example, methylation of
RARRES1 correlates with
RARβ promoter methylation in prostate cancer and treatment of colon and breast carcinoma cells with AZA induced a demethylation of the
RARβ promoter region and a restoration of
RARβ expression [
19,
26,
27].
Regardless of secondary side-effects, we hypothesize that
RARRES1 expression and functional activity might be lost in choriocarcinomas, supporting the concept that placental RARRES1 might act in a suppressive manner and in cases of choriocarcinoma as a tumor suppressor. This is in line with our observation that RARRES1 protein expression in the intramolar choriocarcinoma tissue is limited to cells of the molar trophoblast. In comparison to the choriocarcinoma cell lines BeWo, JAR and Jeg-3, the first-trimester trophoblast-like cell line Swan71 showed a hypomethylation pattern at CpG region 1, while CpG region 2 was hypermethylated as well. Unlike Jeg-3, JAR and BeWo, this cell line does not originate from choriocarcinoma tissue. Swan71 is a telomerase immortalized cell line that is used as a model for first trimester trophoblasts [
15]. In Swan71 cells the methylation of the entire
RARRES1 promoter was similar to the methylation pattern observed in placental tissues and isolated trophoblasts of the third trimester.
Peng et al. [
9] previously analyzed
RARRES1 promoter methylation (up to 600 bp upstream of the ATG codon) in primary breast tumors along with matched adjacent benign tissues. In these patients, the methylation pattern of CpG region 1 (~200 bp upstream of the ATG codon) of the benign tissues was comparable to the pattern we observed in healthy placental tissue. Their data showed a low-to-high methylation pattern with increasing distance to the start codon of
RARRES1. Even though Peng et al. solely investigated methylation up to 600 bp upstream from the ATG, this increase of methylation resembles the higher methylation pattern at CpG region 2 (>1000 bp upstream) observed by us. As CpG region 1 was hypomethylated in our placental tissues and subsequently open for transcriptional regulation, we hypothesized that the promoter area around CpG region 1 could be responsible for maintenance of
RARRES1 gene expression and that hypomethylation of this region might be essential for transcriptional regulation of
RARRES1 in trophoblasts
.
With regard to functional aspects of RARRES1 expression, earlier publications showed that
RARRES1 is associated with a regulatory role in cancer invasiveness and tumorigenicity [
5,
10]. For example, it is capable of suppressing the invasion and colony-forming ability of prostate cancer cells [
10]. Here, we detected a high RARRES1 expression in VTs and EVTs and a loss of RARRES1 in malignant choriocarcinoma cells. Thus, we hypothesized that RARRES1 might influence trophoblast function during gestational development, possibly negatively regulating trophoblast invasion and/or fusion ability. Furthermore, our preliminary results in RARRES1 transfected Jeg-3 cells might indicate a potential role in cell connectivity.
Overall, RARRES1 is known to influence many cellular processes. It is able to interact with the transmembrane protein 192 (TMEM192) and thus induces the expression of autophagy related proteins [
28]. Additionally, it was shown that RARRES1 affects the expression of valosine-containing protein (VCP), which induces the degradation of free polypeptides and thus is essential for autophagy, too [
23,
29]. During human placental development autophagy is associated with the formation of syncytial knots and a reduced trophoblast invasion [
30,
31]. This is in line with RARRES1 expression in syncytial knots and apoptotic cells seen by us. Consequently, we hypothesized that loss of RARRES1 in choriocarcinoma cells might induce an increased invasiveness and hinder autophagy as seen during malignant transformation [
32]. On the other hand, RARRES1 is capable of inducing EB1 protein expression, which is essentially involved in the regulation of spindle dynamics and the spindle assembly checkpoint machinery [
23,
33]. Furthermore, loss of both RARRES1 and EB1 protein expression is thought to be associated with the presence of cancer [
33]. In Swan71 cells RARRES1 is strongly expressed during cell division (ana-, meta- and telophase). Thus, it might be of importance for cell cycle regulation in human trophoblasts and loss of protein expression might be associated with cell cycle deregulation in term of malignant transformation.
Another hallmark of cancer progression and especially metastasis is the epithelial to mesenchymal transition (EMT). In breast cancer cells RARRES1 was reported to interfere with beta-Catenin and AGBL2 function [
34,
35]. Both proteins are involved in EMT of cancer cells or EVTs [
35‐
38]. Expression of RARRES1 during the differentiation of EVTs to giant cells might thus be important to limit myometrial trophoblast invasiveness. On the other hand, loss of RARRES1 expression in choriocarcinoma cells might be essential for cancer cell invasion into the surrounding tissue. Additionally, we observed an induction of
RARRES1 and
RARβ expression at high cell densities of Swan71 cells.
RARβ is important for the transcriptional regulation of
RARRES1 expression and thus might be an essential inductor of high cell-density related
RARRES1 expression. In the light of high
RARRES1 expression in the SCT-neighboring VTs, we predict that
RARRES1 might also be involved in the regulation of cell-cell or cell-matrix contacts. As a tumor suppressor it might induce contact inhibition either through transmembrane connections or cell cycle regulation, and thus influence trophoblast behavior. Through its interaction with AGBL2, RARRES1 can inhibit the detyrosination of α-Tubulin [
39]. α-Tubulin detyrosination is an important hallmark of EMT [
40]. An increase of detyrosinated α-Tubulin induces the formation of microtentacles, which promote the penetration of endothelial cell layers and thus are directly linked to cancer invasiveness and metastasis [
41]. Additionally, beta-Catenin is essential for cell adhesion and increases during cancer cell EMT [
42]. Loss of RARRES1 expression stimulates the nuclear localization of beta-Catenin, which leads to the dissociation of E-Cadherin/beta-Catenin/alpha-Catenin complexes [
34]. This process is important for the induction of cancer cell EMT and might explain the loss of RARRES1 expression in choriocarcinoma cases. In addition to increased
RARRES1 expression with high cell densities, we also observed rising
E-Cadherin expression. This underscores the possible connection between RARRES1 and the E-Cadherin/beta-Catenin/alpha-Catenin complex. Thus, the ability of RARRES1 to reduce invasiveness and tumorigenicity might be due to its properties as a molecule regulating cell-adhesion [
19]. RARRES1-triggered enhanced cell-cell contact of cancer cells might mediate contact inhibition of cell proliferation as well as decreased invasiveness and a reduced migratory capability [
43]. Future studies should consider the in-depth investigation of RARRES1/E-Cadherin signaling and its potential regulatory role for cell-cell contact.