Introduction
The
WWOX (WW domain containing oxidoreductase) gene is located in the chromosome 16 region 16q23.3–24.1, also known as common fragile site FRA16D [
1], an area which was found to be frequently affected by allelic losses in breast and other cancers [
2].
WWOX expression was reported to be higher in the testis, ovary and prostate, i.e. tissues where its activity is regulated hormonally [
1]. On this basis,
WWOX was speculated to be involved in regulation of the steroids signalling pathways. Studies on biological role of
WWOX in tumourigenesis showed that its function in cellular metabolism is likely to modulate gene expression by interactions with other proteins involved in cell cycle/apoptosis control and transcription factors. Up to now several partner proteins were identified, i.e. p73, AP-2γ[
3], ErbB-4 [
4], Runx2 [
5] and members of Dvl protein family [
6]. It was also shown that WWOX protein physically binds to two cytoplasmic regions of ErbB-4, which were previously verified to be responsible for interactions with Yap proteins. This competition for the ErbB-4 binding sites may prevent ErbB-4 transactivation and may lead to dysregulation of cell signalling [
4]. Regardless of its function in cell metabolism,
WWOX is considered as a tumour suppressor gene in various types of malignancies, including: breast [
7], ovarian and lung cancer [
2]. The evidence for its tumour suppressor activity was demonstrated for the first time in several cancer cell lines [
7]. Since then numerous studies showed either loss or reduction of the
WWOX expression in a variety of human tumours of breast, ovary, liver, stomach, pancreas, oesophagus, lung and haematopoietic malignancies [
8]. Latest studies showed that
WWOX gene is a bona fide tumour suppressor gene (reviewed in [
3]), however the most common mechanism of decreasing
WWOX expression in cancer cells is through hemizygous deletions (especially in breast cancer), while point mutations are very rare [
1]. Recently, a set of complex deletions was found at FRA16D in the HCT116 colon cancer cell line, which was responsible for removing fragments of
WWOX gene [
9].
Another mechanism of reducing
WWOX transcriptional level which was vastly studied is CpG islands hypermethylation of
WWOX promoter and coding region. It seems that this mechanism may play some role in downregulation of
WWOX expression in several cancer cell lines, for example tumours of pancreas and prostrate [
10], breast, lung and bladder [
11]; however first reports on methylation at the
WWOX promoter region in thirteen breast cancer cell lines revealed that despite dramatic difference in
WWOX expression, there was no methylation present at this region in any studied cell line [
7]. Płuciennik et al. have shown that breast cancer patients exhibiting higher levels of
WWOX expression exhibited significantly longer DFS in contrast to the group with relatively lower
WWOX transcript levels [
12]. Similarly, Aqeilan et al. showed prognostic relevance of WWOX and ErbB4 proteins in breast cancer [
13].
With all results cited above, the lack of studies regarding role of
WWOX gene and its protein product in tumourigenesis in colon and especially homozygous deletions in
WWOX region found in HCT116 colon cancer cell line, as reported by [
9], prompted us to undertake present work. The aim of our research was to evaluate the role of deletions in
WWOX gene, its expression and prognostic value in patients with CRC (colorectal cancer). We also evaluated methylation of
WWOX gene promoter region and the correlations of
WWOX expression level with other well-known cancer/cell cycle-related genes, as: pro-apoptotic
BAX, anti-apoptotic
BCL2, cell cycle regulators: cyclins D1 (
CCND1) and E1 (
CCNE1) both regarded as playing an important role in tumourigenesis, tumour suppressor gene
TP73 which encodes for the p73 protein, proliferation marker -
Ki-67 and one
ERBB4 isoform transcript—JM-a/CVT-1.
Discussion
In the presented study, we analysed the expression of
WWOX gene in 99 tumours from patients with colorectal cancer. In several reports it was shown that
WWOX expression is lowered in various types of tumours (mentioned above). Moreover, many authors have shown that suppressed transcription of
WWOX is associated with more aggressive phenotype of breast cancer [
12], non-small cell lung cancer [
20] and ovarian cancer [
21]. Here, we show that relatively high
WWOX expression corresponds with better disease-free survival of CRC patients hazard ratio (HR = 0.39;
p = 0.0452; Mantel–Cox log-rank Test, Fig.
1) in comparison with those with lowered
WWOX transcription. This supports the view that loss of
WWOX expression is associated with tumourigenesis in different types of cancers. Such an idea was additionally proven by in vitro and in vivo studies which showed that elevated
WWOX expression suppresses tumourigenicity of different cancer cell lines: breast [
7], lung [
22] and prostate [
23]. However,
WWOX expression cannot be used as an independent prognostic marker in CRC, since results of multivariate analysis excluded this marker from analysis on early stages (results not shown). Despite the frequent suppression of
WWOX expression in many cancers, complete gene inactivation by deletion of one allele and second mutation or homozygous deletion is very rare [
9]. Based on the observations, it was postulated that
WWOX inactivation is driven by hemizygous deletions, which was recently proven with mouse model using targeted deletion of
WWOX gene [
24]. In our analysis of 16q23.3–24.1 region we did not find any evidence for LOH in the two studied
WWOX-associated loci in CRC. We used two STS (sequence-tagged site) markers (D16S3096 and D16S518) which are most often afflicted by hemizygous deletions in all kinds of cancers, for instance: breast ductal carcinoma in situ lesions [
16], breast cancer metastases [
25], hepatocellular carcinoma [
26], non-small cell lung cancer [
27], oesophageal squamous cell carcinoma [
28], gastric carcinoma [
29], but none of the STS markers displayed LOH in our set of colorectal cancer samples.
We also tested the status of methylation in the promoter region of
WWOX gene, presumably resulting in lowered
WWOX expression, which was shown in several studies [
11,
22]. Nevertheless, there are data showing that the methylation status of
WWOX promoter region does not contribute to the decrease of
WWOX expression in breast cancer cell lines and prostate tumours [
7,
30] which is also in the case of CRC patients studied herein. To our knowledge, this is the first report on methylation status of
WWOX gene in CRC patients or CRC cell lines. Nevertheless, results of our MethylScreen analysis were very similar to the previously cited work by Bastian et al. [
30], who analysed CpG island hypermethylation in a set of 13 gene loci (including
WWOX) in 78 prostate carcinomas, 32 benign prostate hyperplasias and four prostate cell lines (LNCaP, DU145, PC3, BPH-1) using MethyLight PCR. They found only one case showing
WWOX promoter region methylation; none of the benign samples were methylated in
WWOX locus [
30]. Moreover, none of the cell lines surveyed (LNCaP, DU145, PC3, BPH-1) exhibited methylation of
WWOX [
30]. Interestingly, previous studies showed loss of
WWOX expression in as much as 84% (37 of 44 tumour samples) [
23] and involvement of promoter methylation in decreasing of
WWOX expression in prostate cancer cell lines LNCaP, DU145 and PC-3 [
23]. We hypothesise that this striking discrepancy between the two abovementioned papers could arise because of the two different strategies of study: Bastian et al showed the exact methylation status of
WWOX by using MethyLight PCR, whereas Qin et al. used methylation-specific PCR (MSP). One should remember that MSP is gel-based technique and provides rather qualitative results, whereas PCR-based techniques are able to discriminate between different levels of methylation. Qin et al. also assumed that increased
WWOX mRNA and protein expression in prostate cancer-derived cells after treatment with 5-aza-2′-deoxycytidine (AZA; a DNA methyltransferase inhibitor) and trichostatin A (a histone deacetylase inhibitor), is a result of demethylation of only
WWOX promoter region. However, one should be aware of the fact that these agents are not specific and they change the global methylation/acetylation status of the cell, including all hypothetical and/or unknown regulators of
WWOX expression.
Recently, a paper by Kosla et al. showed that both methylation of
WWOX promoter region and LOH at D16S518, D16S3096 and D16S504 have influence on
WWOX expression in glioblastoma multiforme tumours [
31]. In this work, we did not find any evidence for such a relationship, which may suggest that these mechanisms are tissue specific.
We found that in population of Polish patients studied herein
WWOX expression correlated with several genes involved in cell cycle/apoptosis or interacting with
WWOX. The strongest correlation found was negative association of
WWOX expression level with that of
CCNE1 (−0.3579;
p = 0.0005). Cyclin E1 is thought to be a potential predictor of systemic therapy, because of the cell cycle alterations induced by its overexpression: decreased length of the G1 phase, faster transition from G1 to S phase and increased genomic instability [
32]. Moreover, overexpression of
CCNE1 and amplification in breast cancer human breast epithelial cells results in chromosomal instability and worse prognosis [
32]. In colorectal cancer cells it was found that combined treatment of these cells with various cytotoxic drugs (e.g. c-myc antisense phosphorothioate oligonucleotides, taxol, 5-fluorouracil (5-FU), doxorubicin and vinblastine) resulted in growth arrest of these cells in the G2/M and S phases, noticeable apoptotic effect and the reduction of mRNA levels of
BCL2, BCLxL, CDK2, cyclin E1, CDK1 and
cyclin B1, while increasing the mRNA levels of p21, p27
, BAX and caspase-3 [
33].
We also found correlation of
WWOX transcription level with the
BCL2/BAX expression ratio (0.3480
p = 0.0006). This relationship would mean that in CRC patients with higher
WWOX expression, the tumours/its cells are less prone to apoptosis. This seemingly paradoxical finding has been also recently reported by Reeve's group in CRC patients. The impact of tumour proliferation on the grade of malignancy in CRC is not clear, especially when markers well established for breast cancer are used (e.g. Ki-67, PCNA) that is why the group used a self-devised colon-specific gene-proliferation signature (GPS) [
34], including 36 genes commonly expressed (upregulated) in an exponentially growing in vitro CRC model and in human colon proliferative crypt compartments. Among the GPS genes, there are 15 cell cycle related, for instance
CCNA2 (cyclin A2),
CDC2 (cell division cycle 2, G1 to S and G2 to M, transcript variant 1). After stratification of colorectal tumours into high and low GPS groups by K-means clustering method, authors found that reduced GPS expression was associated with shorter DFS in CRC patients [
34]. Authors also validated the GPS on public microarray data from two independent breast cancer experiments and found that in breast cancer group with increased GPS had significantly shorter DFS [
34]. It is worth mentioning that among the 36 GPS genes there are only two involved in apoptosis—
MADL2, which is anti-apoptotic and
ITGB3BP (integrin beta 3 binding protein,
NRIF3) shown to induce rapid and profound apoptosis in various breast cancer cell lines [
35]. In a previous report, it was found that in breast cancer patients, the median expression of
WWOX was almost 13-fold lower in tumours exhibiting
BCL2/BAX ratio lower than 2 [
12]. Similarly, in the presented work: colorectal tumours in which
BCL2/BAX ratio was lower than 2, showed
WWOX median expression 0.791, whereas in samples with higher
BCL2/BAX ratio it was 4.590 (5.81-fold difference;
p = 0.0025). We also hypothesise that
WWOX expression regulation in CRC, or in colon tissue/cell lines in general, could be similar to the E-cadherin (
CDH1) gene/E-Cad protein. This well-known tumour suppressor, which is located in the vicinity of
WWOX locus (16q22.1) was reported to have decreased expression in various cancers, including CRC. However, the exact mode of
CDH1 expression regulation was largely unknown when studies were performed to identify the ‘classical’ ways of downregulating gene expression. Early works on downregulation of E-Cad expression due to the mutations in
CDH1 gene showed that the mutation rate in this gene was low [
36]. Also, polymorphisms found in the
CDH1 and its promoter region seems to have at least ambiguous significance in regulation of E-Cad expression, because studies on greater number of patients showed no such associations [
37]. Epigenetic changes (methylation status) in
CDH1 gene region in tumours were also studied, but results of these analyses are also unclear and seem to depend mostly on the technique used in the survey (this situation is very much alike to the one of
WWOX methylation). Once again, when methylation was studied using MSP-based methods, it seemed that this kind of regulation has great influence on E-cad protein level [
38], whereas study done using the qPCR-based method (
MethyLight) showed extremely modest level of
CDH1 promoter methylation and there was no correlation between DNA methylation and E-cad protein level (neither in tumour tissues nor the normal mucosae; total 142 pairs of matching tissues) [
39]. Also in a paper mentioned earlier, Bastian et al. described differences between the
CDH1 promoter methylation status they found in prostate carcinomas and previously published results of such analyses in this kind of tumour [
30]. The next step in resolving this complexity was the showing of different repressor proteins that contribute to E-cad transcription regulation. Up to this date many of these transacting factors were discovered, including: Snail, Slug, Twist, SIP1/ZEB2, deltaEF1/ZEB2, as reviewed in [
38]. Recently, a paper by Guler et al. described a relationship between the “triple negative” breast tumours phenotype and reduced expression of WWOX with elevated expression of AP-2γ (as shown by using tissue microarrays), although the authors did not find direct correlation between WWOX neither AP-2α nor Ap-2γ expression levels [
40]. In summary, in this study we found that
WWOX expression varies among patients and correlates with DFS, however we were unable to identify the molecular reason of lowered
WWOX transcription. Our data suggest that unlike other tumours,
WWOX expression in colorectal cancer is affected by different mechanisms than small deletions or methylation of promoter region. These findings, the ambiguous nature of role of the
WWOX promoter methylation in expression regulation and the previous studies showing a wide array of proteins interacting with WWOX (e.g. YAP, ErbB-4, Dvl family) seem to suggest that there is a place to hypothesise that phenomena similar to
CDH1 expression regulation may occur in
WWOX expression regulation in colon.