The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer

  • Authors:
    • Elżbieta Płuciennik
    • Magdalena Nowakowska
    • Karolina Pospiech
    • Anna Stępień
    • Mateusz Wołkowicz
    • Małgorzata Gałdyszyńska
    • Marta Popęda
    • Katarzyna Wójcik-Krowiranda
    • Andrzej Bieńkiewicz
    • Andrzej K. Bednarek
  • View Affiliations

  • Published online on: April 16, 2015     https://doi.org/10.3892/ijo.2015.2964
  • Pages: 2639-2648
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Abstract

This study defines the role of WWOX in the regulation of epithelial to mesenchymal transition. A group of 164 endometrial adenocarcinoma patients was studied as well as an ECC1 well-differentiated steroid-responsive endometrial cell line, which was transducted with WWOX cDNA by a retroviral system. The relationship between WWOX gene and EMT marker (CDH1, VIM, ZEB1, SNAI1) expression on mRNA (RT-qPCR) and protein levels (western blotting) was evaluated. The EMT processes were also analysed in vitro by adhesion of cells to extracellular matrix proteins, migration through a basement membrane, anchorage-independent growth and MMP activity assay. DNA microarrays (HumanOneArray™) were used to determine WWOX-dependent pathways in an ECC1 cell line. A positive correlation was observed between WWOX and ZEB1, and a negative correlation between CDH1 and VIM. WWOX expression was found to inversely correlate with the risk of recurrence of tumors in patients. However, in the WWOX-expressing ECC1 cell line, WWOX expression was found to be inversely related with VIM and positively with CDH1. The ECC1/WWOX cell line variant demonstrated increased migratory capacity, with increased expression of metalloproteinases MMP2/MMP9. However, these cells were not able to form colonies in suspension and revealed decreased adhesion to fibronectin and fibrinogen. Microarray analysis demonstrated that WWOX has an impact on the variety of cellular pathways including the cadherin and integrin signalling pathways. Our results suggest that the WWOX gene plays a role in the regulation of EMT processes in endometrial cancer by controlling the expression of proteins associated with cell motility, thus influencing tissue remodeling, with the suppression of mesenchymal markers.

Introduction

Endometrial cancer is one of the most common malignant tumors of the reproductive tract among women in developed counties. In Poland in 2010, over five thousand women were diagnosed with endometrial cancer of whom over a thousand died (http://epid.coi.waw.pl/krn/). The average age at diagnosis is 65, and mortality rate increases with the age of patient (1,2). Many potential risk factors have been characterized, such as obesity, menopausal status, unopposed estrogens, low parity and nullparity and history of cancer in family (13) however, the mechanisms by which they work are not fully understood.

Moreover, conditions such as diabetes mellitus and polycystic ovarian syndrome increase the risk of endometrial cancer development (4). Most endometrial cancers are sporadic, and can be divided into two general groups based on histopathological features and molecular profiles: endometroid endometrial cancers (EECs) and non-endometroid endometrial cancers (NEECs) (13). Type I, or endometrioid endometrial cancers (EEC), are more common and characterized by estrogen-dependence, microsatellite instability, loss of heterozygosity of the PTEN tumor suppressor gene and genetic alterations in the RAS and WNT pathways. Type II, non-endometrioid endometrial cancers (NEECs), are characterized by estrogen-independence with an abnormal accumulation of p53 protein, inactivation of the p16 gene, overexpression of the HER2/neu gene and decreased expression of the CDH1 gene (5). However, it is important to note that the molecular pathways of the two cancer types are not fully understood.

The epithelial-mesenchymal transition (EMT) is a complex process which results in transformation of epithelial to mesenchymal cells. Cells of the epithelium are characterized by polarity and non-motility, while mesenchymal cells are loosely organized and able to migrate (68). These features are a consequence of changes in gene expression profile i.e., increases in expression of mesenchymal markers such as vimentin and N-cadherin, and donwregulation of epithelial markers such as E-cadherin and cytokeratins, which act as thigh and adherence junction proteins (9,10). Although EMT is associated with embryogenesis and organ development under physiological conditions, it has also been associated with cancer progression and metastasis (1012). A number of transcription factors, i.e., SNAI1, SNAI2, TWIST and ZEB1/2, which are known as the E-cadherin repressors, are the key regulators triggering EMT progression (9,11,13). Changes in EMT marker expression are important in endometrial cancer, as cancer cells invade the myometrium, and this invasion is considered one of the most important prognostic factors for types I and II EC (14).

The WW domain-containing oxidoreductase gene (WWOX), also known as FOR or WOX1, is a tumor suppressor gene which spans more than one million base pairs mapped at locus 16q23.3-q24 in the common chromosomal fragile site FRA16D. The WWOX gene encodes a chain of 414 amino acids constituting a 46-kDa protein containing two WW domains in the NH2-terminal region with a short-chain dehydrogenase/reductase domain, which suggests that it may interact with the receptors regulating steroid hormones. The first WW domain interacts with many partners such as ERBB 4 (15,16), transcription factor AP2γ (17,18), YAP (15), Jun (19), HIF1α (20) and others (21). A high level of WWOX expression has been observed in hormone-dependent tissues such as those of the ovary, mammary gland or testis, which together with the enzymatic specificity of the SDR domain, indicates its role in the metabolism of steroid hormones (22,23). Loss of WWOX expression has been observed during the development of various types of cancer. Moreover, numerous studies have demonstrated correlations between the decrease of WWOX expression and the presence of adverse prognostic factors, including shorter survival and a higher degree of malignancy in various types of carcinoma, especially such hormone-dependent ones as those of the breast (5,16,24), prostate (25), ovary (26) and others (2731). The suppressive character of the WWOX gene has been confirmed in many in vitro studies, where WWOX overexpression resulted in inhibition of cell proliferation, increased apoptosis and inhibition of anchorage-independent growth (3234). Moreover, mice with a knocked out WWOX gene resulted in a greater incidence of numerous disorders such as bone metabolic defects, improper glandular development and disruption of steroidogenesis (23). However, the experiments performed in our laboratory on variety of tumor types, indicated that WWOX functioning in the cell appears to be more complex and is probably not only limited to well-known tumor suppressor properties.

We redirected our interest into WWOX regulation of cell adhesion and motility properties, as Gourley et al reported that overexpression of WWOX in an ovarian cancer cell line resulted in decreased adhesion to fibronectin (35), and WWOX ectopic overexpression in breast cancer and colon cancer cell lines has also been observed to result in increased potential of invasion with the modulation of cell adhesive properties (36,37). Moreover, in our previous study on endometrial cancer, we observed that WWOX negatively correlated with CDH1 expression, a well known epithelial marker downregulated during EMT (38). The aim of the study, as a continuation of previous observations, was to determine its role in the EMT process using both an in vitro model and endometrial tumor samples.

The overexpression of WWOX in an ECC1 well-differentiated steroid-responsive endometrial cell line was induced via stable retroviral transduction. The changes in global gene expression profile after WWOX induction were assessed by microarray and the biological effects connected with EMT process were assessed by migration through a basement membrane, anchorage-independent growth, adhesion to ECM proteins and MMPs activity. RT-qPCR analysis of chosen EMT markers was also performed on 164 tumor samples.

Materials and methods

Tumor samples

The study included 164 endometrioid endometrial carcinomas (EECs, type I) obtained from the Department of Gynecological Oncology, Medical University of Lodz. The samples were classified according to FIGO classification system and also classified into risk of recurrence category according to Pecorelli (39). The mean age was 62 years (median 61; range 31–83 years). Tumor samples were stored at −80°C in RNA Later buffer (Ambion®; Thermo Fisher Scientific, Waltham, MA, USA).

The study was performed after receiving the consent of the patients, and was approved by the the Ethics Committee of the Medical University of Lodz (RNN/208/11/KE).

Cell lines and reagents

The ECC-1 cell line was obtained from the American Type Culture Collection (ATCC). This endometrial cancer cell line is hormone-responsive, as well as estrogen (α and β), androgen and progesterone receptor-positive (40). Cells were cultured in advanced RPMI-1640 medium supplemented with heat-inactivated fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, USA), L-glutamine (Invitrogen Life Technologies), HEPES buffer (Invitrogen Life Technologies) and antibiotics (penicillin 50 U/ml; streptomycin 50 μg/ml; neomycin 100 μg/ml) (Invitrogen Life Technologies). The cells were incubated at 37°C in a humidified incubator with 5% CO2.

Transduction

The process was performed with retroviral vector pLNCX2, either with or without WWOX cDNA. To increase the effectiveness of transduction, the infectious medium was supplemented with 8 μg/ml polybrene (Sigma-Aldrich, MO, USA). After 24 h, the medium was removed and replaced with advanced RPMI-1640 (Invitrogen Life Technologies) with standard supplements. For selection, the cells were cultured in the presence of 400 mg/ml G418 (Sigma-Aldrich) for 14 days. The stable transductants were analyzed by RT-qPCR and western blot assay.

RT-qPCR

Total RNA from cells and tumor tissues was isolated using TRIzol reagent (Invitrogen Life Technologies), and 10 μg of RNA was reverse-transcribed with ImProm RT-II reverse transcriptase (Promega Corp., Madison, WI, USA).

RT-qPCR was performed with LightCycler 480 II (Roche Diagnostics GmbH, Mannheim, Germany). PCR products were detected with SYB R Green I and qPCR Core kit for SYBR Green I (Eurogentec, Southampton, UK). Reactions were performed in duplicate. The relative expression of WWOX, CDH1, and VIM were analysed, as were two CDH1 repressors: ZEB1 and SNAI1. The relative expression of all investigated genes was normalized to three reference genes: RPLPO, RPS17 and H3F3A. All information regarding primer sequences, PCR reaction conditions and the length of received products is available upon request. The relative expression level of all investigated genes was calculated using the Roche algorithm (41) with the Universal Human Reference RNA (Stratagene, La Jolla, CA, USA) as a calibrator.

Microarray assay

The microarrays were performed using Human OneArray Whole Genome Microarray v 5.1 (Phalanx Biotech, USA) in four replicates (dual labelling protocol). A ULS™ Labeling kit (Kreatech Diagnostics, The Netherlands) was used to alternately/cross label single-stranded cDNA of two variants (ECC1/vector, ECC1/WWOX) with Cy3 and Cy5 as well as Universal Human Reference RNA (Stratagene).

The slide was prepared for hybridization and pre-hybridization according to the manufacturer’s protocol. The hybridization process was performed in a 2X SSPE humidity chamber at 42°C for 16–18 h. The following buffers were used for post-hybridization washes: 1X SSPE/0.03% SDS (2 min, 42°C), 1X SSPE (2 min, RT), 0.1X SSPE (rinsed several times, RT) (Phalanx Biotech).

ProScanArray (Perkin-Elmer, USA) was used for scanning and ScanArray Express for preliminary normalization. Lowess and statistical analysis were performed with TM4 software suite provided by The Institute for Genomic Research (http://www.tm4.org/). After modified t-test analysis, the fold change was calculated and the Pantherdb online ontology application (www.pantherdb.org) was used to classify the received genes according to ontological terms. The results were regarded as significant at p<0.05.

Protein extraction and western blotting

The endometrial cancer cells were lysed in RIPA protein extraction buffer supplemented with proteases, phosphatase inhibitor cocktail and PMSF (Sigma-Aldrich). The protein concentration was measured using the Bradford method (Bio-Rad Laboratories, CA, USA), run on 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Sigma-Aldrich). The membranes were blocked in a buffer of 5% non-fat milk in TBST for 1 h at room temperature and then incubated for 19 h at 4°C with primary antibodies to goat anti-human WWOX, 1:100; mouse anti-E-cadherin, 1:100; mouse anti-vimentin, 1:100 (sc:20529, sc:8426, sc:51721, Santa Cruz Biotechnology Inc., Dallas, TX, USA). Following incubation, the membranes were washed three times with TBST and incubated with secondary antibodies conjugated with alkaline phosphatase (Sigma-Aldrich) for 1 h. The membranes were washed three times in TBST and developed using Novex® AP Chromogenic Substrate (Invitrogen Life Technologies). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at a dilution of 1:1,000 was used as the reference protein (sc-59540, Santa Cruz Biotechnology Inc.). All biological tests were performed in a minimum of three replicates.

Adhesion assay

Cell adhesion was evaluated using Cell Biolabs CytoSelect™ Cell Adhesion assay kit (Cell BioLabs, San Diego, CA, USA) according to the manufacturer’s instructions. This test provides a rapid and quantitative method for accessing the cell adhesion potential. The ECC1 cells were seeded at a density of 1.5×105 cells in a total volume of 150 μl serum-free media to each well. The cells were incubated for 1 h at 37°C. The absorbance was measured at 560 nm with a BioTek plate reader (BioTek US, USA).

Cell invasion assay

The cell invasiveness potential was determined using a Colorimetric CytoSelect™ Cell Invasion Assay Kit Cell (Cell BioLabs) according to the manufacturer’s protocol. The cell suspension was incubated at a density of 3×105/well in serum-free media for 48 h. The absorbance was measured at 560 nm on a BioTek reading plate (BioTek US). Each variant was tested in quadruplicate.

Zymography

ECC1 cells were seeded in 6-well plates at a density of 1.5×106/well. Twenty-four hours before collection, the medium was replaced with serum-free RPMI medium. Qubit® Protein assay kits (Invitrogen Life Technologies) were used to quantify protein content. Samples containing 15 μg of protein were separated in 10% SDS-page gel containing 2 mg/ml gelatin. After electrophoresis, the gel was washed twice for 30 min in 2.5% Triton X-100 wash buffer and then incubated at 37°C for 18 h in a solution containing 50 mM Tris (pH 7.5) (Sigma-Aldrich), 10 mM CaCl2 (Sigma-Aldrich) and 200 mM NaCl (Sigma-Aldrich). Following this, the gel was stained with 0.125% Coomassie brilliant blue R-250 (Sigma-Aldrich) and de-stained with solution (30% methanol, 10% acetic acid, 60% distilled water) (Sigma-Aldrich). A protein marker was used to measure the molecular weights of the analysed enzymes. Densitometric analysis with ImageJ Analysis 1.34s software (Wayane Rasband, National Institutes of Health, USA, http://rsb.info.nih.gov/ij/) was used to quantify MMP activities.

Soft agar

A soft agar assay was performed to analyze the differences in anchorage-independent growth between ECC1/WWOX and ECC1/vec. Six-well plates were coated with 2 ml 0.9% low melting agarose (AppliChem, MO, USA) in RPMI-1640 with 10% FBS. Both variants of ECC1 cells were suspended in 0.3% low melting agarose at a concentration of 104 cells per well. The cells were incubated at 37°C in a humidified incubator for 14 days. The plates were stained with 1 ml of 0.005% crystal violet (Sigma-Aldrich) in water and colonies were counted using ImageJ Analysis 1.34 s software (Wayane Rasband, National Institutes of Health, USA, http://rsb.info.nih.gov/ij/).

Statistical analysis

The correlation of expression was performed using the non-parametric Spearman’s rank correlation test. The Mann-Whitney t-test was used to determine the differences between the levels of expression of the investigated genes and clinical factors in patients with endometrial cancer. The Aspin-Welsch t-test was used to determine the statistical significance of the difference observed between cell variants in all biological tests.

Results

Confirmation of WWOX gene transduction in ECC1

The transduction resulted in the increase of WWOX expression both on the mRNA and protein levels. A >180-fold change of WWOX gene expression was observed on the mRNA level: the mean relative expression values being 0.019 and 3.34 for ECC1/vector and ECC1/WWOX respectively. The change on the protein level is shown (Fig. 1).

Microarray analysis

Our microarray analysis revealed more than 800 genes with changed expression as the result of WWOX ectopic overexpression (p<0.05). The identified genes belong to several signalling pathways associated with regulation of cell differentiation, apoptosis and cellular communication/tissue architecture (Table I). The predominant ones being the Wnt signalling pathway (13 genes), integrin signalling pathway (10 genes), inflammation mediated by chemokine and cytokine signalling pathway (6 genes), gonadotropin-releasing hormone receptor pathway (11 genes), angiogenesis (6 genes), TGF-β signalling pathways (8 genes), apoptosis (5 genes), and the cadherin signaling pathway (6 genes). The ontology classification (by cellular pathways) by WWOX overexpression is included in Table II. The two main EMT markers found in the cadherin signalling pathway were vimentin and E-cadherin.

Table I

Ontological analysis of genes in biological process regulated by WWOX gene in ECC1 cell line.

Table I

Ontological analysis of genes in biological process regulated by WWOX gene in ECC1 cell line.

Biological processNo. of genes
Metabolic process368
Primary metabolic process299
Unclassified266
Cellular process262
Cell communication147
Biological regulation138
Nucleobase-containing compound metabolic process138
Protein metabolic process136
Localization124
Transport120
Developmental process117
RNA metabolic process102
Regulation of biological process87
Transcription, DNA-dependent82
Transcription from RNA polymerase II promoter80
Multicellular organismal process70
Single-multicellular organism process70
Regulation of nucleobase-containing compound metabolic process69
System development69
Cell cycle68
Response to stimulus65
Immune system process62
Regulation of transcription from RNA polymerase II promoter62
Cellular protein modification process61
Protein transport60
Intracellular protein transport60
System process60
Regulation of catalytic activity51
Regulation of molecular function51
Neurological system process48
Cellular component organization or biogenesis46
Cellular component organization43
Mesoderm development43
Nervous system development42
Ectoderm development42
Cell adhesion41
Biological adhesion41
Cell-cell signaling41
Anatomical structure morphogenesis28
Cell-cell adhesion27
Cellular component morphogenesis26
Death26
Cell death26
Apoptotic process26
Mitosis26

Table II

The number of genes in pathways regulated by WWOX gene in ECC1 cell line.

Table II

The number of genes in pathways regulated by WWOX gene in ECC1 cell line.

PathwayNo. of genes
Unclassified667
Wnt signalling pathway13
Gonadotropin-releasing hormone receptor pathway11
Integrin signalling pathway10
TGF-β signalling pathway8
Alzheimer disease-presenilin pathway7
Angiogenesis6
Inflammation mediated by chemokine and cytokine signalling pathway6
Huntington disease6
Cadherin signalling pathway6
Apoptosis signalling pathway5
Heterotrimeric G-protein signalling pathway-Gq α and Go α mediated pathway5

A 1.6-fold increase in expression of the CDH1 gene and a 1.9-fold decrease in expression of VIM mRNA were observed in the ECC1 cell line overexpressing WWOX. Microarray data were submitted to the GEO Database (reference series: GSE61041).

Microarray validation

The observed change in expression of CDH1 and VIM was confirmed with RT-qPCR: a 3.5-fold increase of CDH1 (mean relative expression 0.48 vs 1.67 for ECC1/vector and ECC1/WWOX respectively); 2-fold decrease for VIM (0.00035 vs 0.00019 for ECC1/vector and ECC1/WWOX respectively). Similar changes were also noted on protein level (Figs. 2 and 3).

The influence of WWOX on invasiveness, adhesion potential and cell growth in suspension

Overexpression of the WWOX gene resulted in a 1.3-fold increase in migration in comparison to the ECC1/vector (p<0.05) (Fig. 4) and greater induction of metalloproteinase activity i.e., MMP-2 and -9 (Fig. 5). These results suggest that increased expression of WWOX influences the motility and invasive potential of the cells. However, the soft agar assay, which measures the ability of cells to grow in suspension, in a similar way to cancer cells being able to survive after detachment, revealed a reduced colony number for the ECC1/WWOX cell line variant (Fig. 6, p<0.05).

The invasion and growth in soft agar assay results indicate that even though ECC1/WWOX cells demonstrated a higher basic membrane transition ability, they were unable to survive after detachment. The cells overexpressing WWOX demonstrated a significantly lower ability to adhere to fibrinogen (1.8-fold change; p<0.05) (Fig. 7).

Expression analysis in tumor samples

In the tumor samples, the EMT markers were also analyzed in relation to WWOX. ZEB1 and SNAI1 were included in the EMT marker gene panel. A positive correlation was observed between WWOX and ZEB1 (Rs=0.26; p=0.0004), and a negative correlation between WWOX and VIM (Rs=-0.268, p=0.0005). A negative correlation is known to exist between WWOX and CDH1 (38) and this was confirmed by the increased number of samples. The correlation with SNAI1 was statistically insignificant (Rs=0.029; p>0.05). The remaining correlations are presented in Table III. Similar to other tumor types, the expression of WWOX in endometrial cancer inversely correlated with the risk of cancer recurrence, i.e., tumors with low risk demonstrated higher WWOX expression than those with medium and high risk. A statistically significant difference was found between the low (n=38, median expression 2.33) and medium (n=83, median expression 1.12) risks of disease recurrence (p=0.023). More detailed information of all correlations with clinical factors is presented in Table IV.

Table III

The correlation analysis between the levels of expression of WWOX and other EMT tumor-related genes in endometrial carcinoma patients.

Table III

The correlation analysis between the levels of expression of WWOX and other EMT tumor-related genes in endometrial carcinoma patients.

GeneSpearman’s rank correlationP-value
WWOX/VIM−0.2680.0005
WWOX/ZEB10.2600.0004
WWOX/CDH1−0.5120.0001
WWOX/SNAI10.0290.7114 (NS)

Table IV

The correlation analysis between WWOX, ZEB1, VIM, CDH1 genes and clinical factors in endometrial carcinoma patients.

Table IV

The correlation analysis between WWOX, ZEB1, VIM, CDH1 genes and clinical factors in endometrial carcinoma patients.

Clinical factornMedian of WWOX mRNA level (range)P-valueMedian of ZEB1 mRNA level (range)P-valueMedian of VIM mRNA level (range)P-valueMedian of CDH1 mRNA level (range)P-value
Risk of reccurence
 Low382.33 (1.24–3.40)0.012 (low/medium)0.35 (0.18–0.76)0.06 (low/medium)0.06 (0.02–0.17)0.44 (low/medium)0.34 (0.05–0.69)0.023 (low/medium)
 Medium831.12 (0.75–1.43)0.19 (medium/high)0.15 (0.10–0.23)0.13 (high-medium)0.05 (0.01–0.10)0.68 (medium/high)0.69 (0.51–0.94)0.15 (medium/high)
 High251.24 (0.86–2.77)0.44 (low/high)0.25 (0.11–0.51)0.58 (low/high)0.04 (0.01–0.12)0.23 (low/high)0.34 (0.10–0.95)0.69 (low/high)
 NS18
Grade
 1481.86 (1.05–2.57)0.05 (1 vs 2)0.15 (0.08–0.45)0.42 (1 vs 2)0.03 (0.01–0.08)0.06 (1 vs 2)0.26 (0.07–0.70)0.048 (1 vs 2)
 2791.03 (0.75–1.37)0.14 (2 vs 3)0.30 (0.18–0.47)0.06 (2 vs 3)0.09 (0.04–0.14)0.14 (2 vs 3)0.79 (0.53–1.03)0.1 (2 vs 3)
 3301.59 (0.83–4.21)0.86 (1 vs 3)0.13 (0.09–0.21)0.44 (1 vs 3)0.05 (0.01–0.09)0.83 (1 vs 3)0.39 (0.16–0.77)0.86 (1vs 3)
 NS7
FIGO stage
 I981.30 (0.90–1.72)0.87 (I vs II)0.19 (0.12–0.34)0.70 (I vs II)0.06 (0.03–0.11)0.48 (I vs II)0.64 (0.47–0.79)0.35 (I vs II)
 II251.36 (0.64–2.86)0.44 (II vs III)0.19 (0.09–0.43)0.38 (II vs III)0.03 (0.03–0.19)0.96 (II vs III)0.94 (0.4–1.28)0.11 (II vs III)
 III261.34 (0.86–4.27)0.43 (I vs III)0.23 (0.11–0.51)0.60 (I vs III)0.05 (0.01–0.12)0.34 (I vs III)0.29 (0.10–0.95)0.33 (I vs III)
 NS15
Myometrium invasion
 <1/2831.61 (1.24–2.12)0.120.20 (0.12–0.34)0.960.07 (0.04–0.11)0.180.53 (0.32–0.70)0.30
 >1/2690.97 (0.81–1.36)0.19 (0.12–0.31)0.04 (0.01–0.08)0.65 (0.24–1.15)
 Without40.720.180.130.33
 NS8
Lymph node metastasis
 Negative1251.34 (0.97–1.66)0.420.17 (0.11–0.27)0.130.05 (0.02–0.08)0.65 (0.49–0.83)0.17
 Positive241.34 (0.86–2.65)0.27 (0.11–0.63)0.05 (0.01–0.13)0.29 (0.10–0.64)
 NS15

[i] NS, not specified. Bold indicates statistically significant correlation.

Discussion

The process of epithelial to mesenchymal transition and its regulation seems to be important in endometrial carcinogenesis and disease progression. A review of the literature indicates that this is the first report to investigate the influence of the WWOX gene on EMT in endometrial tumor samples and in an endometrial cancer cell line model.

The present study is a continuation of previous observations that WWOX inversely correlates with CDH1 expression, which suggests that alterations in the expression of this tumor suppressor gene probably influence the process of cell adhesion (38).

WWOX overexpression was induced in a well-differentiated endometrial cancer cell line and its effect on EMT progression was analysed on microarrays and by multiple biological assays. The microarray analysis revealed significant changes (p<0.05) in the expression of two main EMT markers, oppositely regulated in relation to WWOX expression level i.e., CDH1 was upregulated and VIM was downregulated. This result was confirmed on both the protein and mRNA levels.

This observed upregulation of CDH1 in the cancer cell line stands in contrast to our previous report, which revealed a negative correlation between CDH1 and WWOX. However, in the present study, the larger, tumor sample population also demonstrated a negative correlation. This discrepancy between the cell line model and the tumor samples in the present study may be explained by comparing the complexity of tumor architecture between the ECM matrix and the cell line in a 2D in vitro culture. The well-differentiated nature of the cell line within the experiment may also have an impact on WWOX functioning, as shown in previous studies on colon cancer cell lines (36). However, the relationship between CDH1 and WWOX implies that the WWOX gene has an influence on the regulation of the adhesion process. The loss of E-cadherin expression is a hallmark of EMT, and its reduced transcription in endometrial cancer was found to correlate with the depth of myometrial invasion (14,42).

In the increased number of tumor samples used in the present study, loss of CDH1 expression tended to be observed in grade 3 tumors and those with the highest risk of recurrence (p>0.05). However, interestingly, grade 2 tumors and those with medium risk of recurrence demonstrated significantly higher CDH1 expression on the mRNA level in comparison to grade 1 tumors and those with a low risk of recurrence.

WWOX mRNA expression was found to have an inverse association with risk of recurrence and tumor grade. Tumors with low risk of recurrence and grade 1 demonstrated significantly higher WWOX expression, which confirms that loss of WWOX is connected with tumor progression, as seen in other tumor types (30,43,44). The inverse correlation with WWOX and CDH1 seen in tumor samples may also suggest WWOX regulatory role in the process of differentiation, since reduced CDH1 protein level is associated with diminished differentiation (42).

Even though the overexpression of WWOX resulted in the increase of CDH1 expression in the in vitro model, the result of the invasion assay revealed that the ECC1/WWOX cell line variant had increased invasion capacity and migratory potential, as overexpression of WWOX resulted in increased expression of metalloproteinases MMP2/MMP9. This observation is consistent with previous findings in breast and colon cancer cell lines ectopically overexpressing WWOX protein (36,37), but also implies that the interaction between WWOX and CDH1 may be modulated by other EMT-related genes. Hence, WWOX expression was compared with ZEB1 and SNAIL1 expression on the mRNA level. Of those two genes, a positive correlation was only noticed between WWOX and ZEB1.

The observed positive correlation between WWOX and ZEB1 may further confirm the idea that WWOX may play a important role in EMT induction in terms of cell remodelling, with suppressed acquisition of the mesenchymal phenotype; this negative correlation was previously noted with vimentin, a mesenchymal marker, both in an in vitro model and in tumor samples.

In a normal human uterus, ZEB1 expression is regulated by steroid hormones. It is higher in both stroma and the myometrium in the secretory phase than in the proliferative phase. While the ZEB1 gene is silenced in the normal endometrial epithelium (45), it is expressed in the tumor-associated stroma of endometrial cancers, as well as in epithelial-derived cancer cells, with its level correlating with disease staging (45,46). Thus, activation of ZEB1 expression in epithelial cancer cells causes EMT, through repression of CDH1 and induction of vimentin (46). In the present study, especially in tumor samples, it seems that the negative correlation between WWOX and CDH1 may be a result of ZEB1 interplay in the functional axis of WWOX-CDH1, and is not present in the stable microenvironment of cell culture.

However, ZEB1-regulated acquisition of mesenchymal phenotype was found to be incompleted in both the in vitro model and tumor samples. High WWOX expression resulted in decreased VIM expression. This negative correlation was confirmed on the protein level in the in vitro ECC1 cultures and has recently also been observed for ovarian cancer (47). The possibility that mesenchymal progression was incomplete as a result of VIM downregulation is confirmed by the observation that the WWOX-transducted cells were not able to form colonies in suspension, suggesting that they have lost some of their aggressiveness. This observation is consistent with previous findings on ovarian and cervical cancer cell lines (32,33) and confirms the tumor suppression properties of WWOX. Moreover, our findings also support those of Gourley et al that WWOX may be involved in tissue architecture organization through modulation of the adhesion potential of cells to different ECM proteins (35). Significantly reduced adhesion to fibrinogen and tendency to fibrinonectin was demonstrated by ECC1 cells with high ectopic expression of WWOX.

The received tendency for decreased adhesion to fibronectin is consistent with previous findings in ovarian cancer (35), but this is the first study to reveal reduced adhesion to fibrinogen after WWOX overexpression. Importantly, it has been hypothesized that fibrinogen is able to facilitate metastasis by enhancing adhesion, thus promoting the survival of tumor cells circulating in blood vessels. Elevated fibrinogen expression was associated with metastasis in lung, colon, ovarian and breast cancers (48,49). Moreover, in breast cancer, fibrinogen-mediated migration was observed only in such malignant tumor cell lines as MDA-MB-231 and MCF-7, whereas this ability was decreased in the non-malignant MCF-10A cell line (49). Hence, our observations on the endometrial cancer cell line seems to confirm that WWOX overexpression mediates the loss of aggressiveness and that this gene is involved in the regulation of adhesion potential.

In conclusion, this study investigated the role of the WWOX gene in the process of EMT in endometrial cancer. On both tumor and cell line models, statistically significant correlations were noted with CDH1 and vimentin, which are the main markers of EMT. Our observations indicate that WWOX may be involved in the initiation of EMT, leading to changes in cell adhesion and motility. They also suggest that WWOX plays a suppressive role in the process of mesenchymal phenotype acquisition, resulting in reduction of cell features associated with aggressiveness. However, further research in this direction is required.

Acknowledgements

We acknowledge Dr hab Agnieszka Piastowska-Ciesielska for access to BioTek reading plate and methodological support to western blot assay. This study was supported by the National Center of Sciences N N407 168940.

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June 2015
Volume 46 Issue 6

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APA
Płuciennik, E., Nowakowska, M., Pospiech, K., Stępień, A., Wołkowicz, M., Gałdyszyńska, M. ... Bednarek, A.K. (2015). The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer. International Journal of Oncology, 46, 2639-2648. https://doi.org/10.3892/ijo.2015.2964
MLA
Płuciennik, E., Nowakowska, M., Pospiech, K., Stępień, A., Wołkowicz, M., Gałdyszyńska, M., Popęda, M., Wójcik-Krowiranda, K., Bieńkiewicz, A., Bednarek, A. K."The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer". International Journal of Oncology 46.6 (2015): 2639-2648.
Chicago
Płuciennik, E., Nowakowska, M., Pospiech, K., Stępień, A., Wołkowicz, M., Gałdyszyńska, M., Popęda, M., Wójcik-Krowiranda, K., Bieńkiewicz, A., Bednarek, A. K."The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer". International Journal of Oncology 46, no. 6 (2015): 2639-2648. https://doi.org/10.3892/ijo.2015.2964