Background
Hepatocellular carcinoma (HCC) is one of the most lethal cancer types worldwide and also the most common type of liver cancer [
1‐
3]. The exact mechanisms that drive hepatocarcinogenic processes are not yet completely understood. Identification of genetic and epigenetic changes involved in hepatocellular carcinoma development is of high interest for a better understanding of this aggressive malignancy.
Smad interacting protein-1 (SIP1, also known as ZEB2) is encoded by
ZFHX1B at chromosome 2q22 and is a two-handed zinc finger transcription factor that contains a central homeodomain as well as CtBP-binding and Smad-interacting domains. SIP1 has been shown to act predominantly as transcriptional repressor but can also act as transcriptional activator
in vivo [
4‐
8].
SIP1 was originally identified in a transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling pathway by its binding to the MH2 domain of receptor-activated SMADs [
9]. SIP1 has been thoroughly studied for its role in repressing E-cadherin expression, which is a central event in the epithelial-to-mesenchymal transition (EMT) [
5‐
7,
10,
11]. Accordingly, an elevated SIP1/E-cadherin ratio was shown to correlate with invasive disease and poor prognosis in gastric, pancreatic, esophageal and ovarian carcinomas [
12‐
15]. Overexpressed
SIP1 also caused resistance to DNA damage-induced apoptosis and correlated with poor survival in patients with bladder cancer [
16]. In contrast, only a few studies exist with regard to the role of SIP1 in suppressing tumorigenesis. For instance, repression of human telomerase reverse transcriptase (
hTERT) expression in breast and liver cancer cells was shown to be partly mediated by SIP1 [
17,
18]. Also, by directly inhibiting cyclin D1, SIP1 caused G1 arrest in squamous carcinoma cells [
19].
SIP1 was strongly expressed in, and with another transcriptional repressor,
SNAIL, increased invasion of HCC cells [
20]. We recently reported an immunohistochemistry study on tissue arrays and described decreased SIP1 levels in a group of tumors, including HCC [
21]. In mature hepatocytes
in vitro, TGF-β induces EMT by downregulation of Claudin-1, which is also associated with upregulation of
SIP1 and
SNAIL and downregulation of E-cadherin [
22]. Our recent observations also implicated
SIP1 as a candidate regulator of replicative senescence in HCC cells [
18]. Taken together, these findings indicate that
SIP1 may play a role in hepatocarcinogenesis.
Epigenetic regulation of
SIP1 expression by miRNAs [
23‐
26] and a natural antisense transcript (NAT) [
27] were recently described. Studies on the promoter methylation of
SIP1 were also reported. The
SIP1 gene was found to be hypermethylated and silenced in a poorly metastatic breast cancer cell line [
28]. In a more recent study,
SIP1 downregulation in pancreatic cancer was shown to be mediated through promoter hypermethylation [
29]. However, genetic and epigenetic mechanisms regulating
SIP1 expression have never been studied in HCC.
In the present study, we investigated the expression of SIP1 at genetic, epigenetic and protein levels in a series of HCC cell lines and primary tumors. Downregulation of SIP1 in HCC cell lines and tumors was found to be mediated by aberrant promoter methylation. Therefore, epigenetic inactivation of SIP1 may play a critical role in hepatocarcinogenesis.
Discussion
SIP1 is a member of the ZEB family of transcription factors and, along with other E-cadherin repressors, it was repeatedly shown to induce the EMT phenotype both
in vivo and
in vitro and correlate with a poor prognosis in cancer patients [
5,
12,
15,
19]. On the other hand, SIP1 was also shown to be a negative regulator of
hTERT transcription in breast cancer cells [
17]. Consistent with this, we have recently reported that SIP1 was partly responsible for inducing senescence in hepatocellular carcinoma-derived cells through
hTERT repression, and hypothesized that it may act as a tumor suppressor gene in HCC [
18]. In support of this hypothesis, a recent microarray study showed downregulation of
SIP1 in early and advanced HCC [
44]. Also, induced expression of SIP1 has recently been shown to directly inhibit cyclin D1 in the A431 squamous carcinoma cell line, leading to the accumulation of cells in the G1 phase [
19]. Other studies described posttranscriptional regulation mechanisms, such as those mediated by miR-200 family [
24‐
26] and
SIP1 NAT [
27], in the downregulation of SIP1 in different pathophysological contexts.
Obviously, DNA methylation and alterations of chromatin structure are predominant mechanisms that epigenetically inactivate tumor suppressor genes in tumors [
45]. For instance,
SIP1 was found to be hypermethylated in poorly metastatic breast adenocarcinoma cells, but hypomethylated in a more aggressive variant of this cell line [
28]. Yet more recently, silencing of
SIP1 expression was shown to be mediated by promoter hypermethylation in a substantial proportion of pancreatic cancer cell lines and tissues [
29].
Herein, we explored the expression of SIP1 in HCC at the transcriptional and protein levels and provided a mechanistic insight by demonstrating that promoter hypermethylation operates as one of the mechanisms in the epigenetic regulation and downregulation of SIP1 in the majority of HCC samples.
Our initial expression studies in HCC cell lines revealed two groups of cells that differentially express
SIP1. Most fibroblastoid-like cells displayed strong
SIP1 transcripts, while cell lines with an epitheloid appearance had no or low expression. This
in vitro expression pattern of
SIP1 was in accordance with its role in inducing EMT, but was neither informative about a tumor versus normal comparison of SIP1 levels nor the behaviour of SIP1 in a liver tissue context. We therefore proceeded with normal liver and HCC tissues and found a significant decrease of
SIP1 transcripts in 74% of tumors. By using a previously described anti-SIP1 monoclonal antibody, clone 6E5, an immunoblot was performed with the lysates of HCC cell lines [
21]. This assay not only proved the specificity of this antibody, but confirmed our initial observation that SIP1 is downregulated in tumors (Figure
2). Even a higher rate of
SIP1 downregulation was observed in our IHC experiments. Compared to normal, 83% of HCC cases displayed no immunoreactivity and the remaining tumor samples were stained with only a weak intensity. This small difference between transcript and protein levels might be explained by the aforementioned posttranscriptional regulatory mechanisms of
SIP1 expression. In fact, a miRNA profiling study that showed upregulation of miR-200c in HCCs but not in benign liver tumors could partly explain the downregulation of SIP1 in HCCs [
46]. It would be interesting to analyze the expression levels of these regulators in HCCs.
Genetic screening of cell lines did not reveal any mutational alterations in the SIP1 gene, suggesting the implication of epigenetic regulatory mechanisms for the silencing of SIP1 in HCC. Upon our observation that SIP1 mRNA expression was restored after treatment of cells with 5-azaC and TSA, we decided to explore promoter hypermethylation as a possible mechanism of SIP1 downregulation in HCC.
In vitro activities of three alternative
SIP1 promoter regions have been previously described in experiments with mouse tissue and the one located around the first exon (P2) exhibited the highest activity. The other alternative promoter regions (P1 and P3) were of low, but detectable, activity [
42]. Studies in human cancer cell lines revealed a similar pattern of promoter activation, but this time in an AKT-dependent manner [
43]. In striking contrast to these findings, we detected only one methylated tumor sample among 26 paired tissues when we examined the P2 promoter region. However, nearly half of the HCC cases displayed tumor-specific hypermethylation at both the P1 and P3 putative promoter sites. In fact, the number of CpG sites were more restricted in P2 than in the P1 and P3 regions. We also noticed only one
BstUI restriction site in P2 but two in the P1 region. P3 amplicons, which were devoid of
BstUI sites, were digested with the
TaqI enzyme. Hypermethylation in the P3 alternative promoter region (43%) might inhibit
SIP1 translation in a different context. The regulation of
SIP1 translation by a NAT has been previously shown [
27]. After the completion of EMT, a NAT is expressed and makes translation of
SIP1 mRNA possible. This NAT expression was shown to be controlled by elements placed at the 5' site of the second exon, which corresponds to the P3 alternative promoter region [
27]. Therefore, hypermethylation at the P3 site might inhibit the expression of the NAT, which in turn negatively affects
SIP1 translation. It would be interesting to study the expression of the NAT in HCC.
Downregulation of SIP1 in HCC is also in accordance with the dual role of TGF-β in tumorigenesis. The tumor suppressor role of TGF-β in the premalignant stage was shown to switch to an EMT-inducing role in the later stages of cancers, leading to metastasis [
47]. This former failsafe mechanism might partly explain higher levels of SIP1 expression in normal liver compared to HCCs. Despite our previous description that SIP1 is partly responsible for replicative senescence in liver cancer cells, its role in inducing apoptosis in distinct pathophysiolocal contexts should also be thoroughly investigated in HCC. Given the downregulation and possible tumor suppressor role of SIP1 in HCC, we also propose the assessment of this regulator as a prognostic factor for patients affected by this aggressive form of liver cancer.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TA processed all the experiments except the IHC study and participated in the design of the study and in drafting the manuscript and data interpretation. EO performed the IHC study. TY participated in the IHC study and data interpretation and helped draft the manuscript. MCY designed and coordinated the study, participated in the data interpretation and in drafting the manuscript. All authors have read and approved the final manuscript.