Background
Human colon cancer is the second leading cause of cancer-related death in developed countries. About 50% of the Western population develops adenomatous polyps (a benign colon tumor) by the age of 70, and the lifetime risk for colon cancer is estimated to be 5% [
1]. The formation of colon cancer involves a multiple-step process, starting from a small adenomatous polyp and followed by the development of a large adenoma with dysplasia that ultimately leads to the formation of invasive carcinoma (see the recent review by Fearon ER [
2]). It is widely accepted that most human colon cancers are initiated by the inactivation of the Adenomatous Polyposis Coli (APC)/Wnt signaling pathway and then progress as the result of a series of mutational activation of oncogenes coupled with the inactivation of tumor-suppressor genes [
2,
3]. Apart from genetic mutations, epigenetic alterations, particularly aberrant CpG island methylation, have been demonstrated as a major alternative mechanism for suppressing gene function during the development of colon cancer [
4‐
6]. To date, many genes that are epigenetically silenced in human colon cancers as well as in colonic adenomas have been identified. However, the function of many of these genes in colon carcinogenesis is still largely unknown. In this study, we have characterized the role of one of these methylated genes, termed Helicase-like Transcription Factor (HLTF), in intestinal carcinogenesis.
HLTF (SMARCA3 in OMIM) is homologous to the SWI/SNF family of chromatin remodelers [
7‐
10]. Although HLTF was originally identified as a DNA-binding protein that could interact with several gene promoters and enhancers [
7‐
11], recent studies indicate that this DNA helicase is more involved in the DNA-damage repair pathway. First, HLTF has been shown to exhibit an E3 ubiquitin ligase activity for the polyubiquitination of proliferating cell nuclear antigen (PCNA), which is required for the initiation of an error-free replication through DNA damage lesions [
12,
13]. Second, HLTF has also been found to display a double-stranded DNA translocase activity, which promotes the resolution of stalled replication forks at DNA damage lesions [
14,
15]. Third, a recent study indicates that HLTF also possesses a chromatin remodeling activity, which leads to the displacement of DNA-bound proteins on stalled replication forks and facilitates DNA-damage repair [
16]. These findings demonstrate that HLTF may be a functional homologue of yeast rad5 and that it plays an important role in an error-free post-replicative repair pathway. The requirement of HLTF for repair of damaged DNA may also implicate a tumor suppression role in human colon cancers, where HLTF has been identified as a common target for methylation and epigenetic gene silencing.
Epigenetic inactivation of HLTF gene expression by promoter hypermethylation has been reported in more than 40% of human colon cancers [
17‐
20]. The frequency of
HLTF promoter methylation was found to increase drastically between early stage of adenomas and advanced adenomas, suggesting that this epigenetic alteration could be a later event in colon carcinogenesis [
17,
19]. In addition,
HLTF methylation was demonstrated to be significantly correlated with poor prognosis in human colon cancer patients [
21,
22]. Besides colon tumors,
HLTF methylation was also commonly detected in human gastric tumors or cancers, but not in other human cancers, such as lung and breast cancers [
17,
23‐
25], suggesting that this epigenetic alteration is unique to the tumors of the gastrointestinal tract. However, although
HLTF methylation has been demonstrated as a common event in human colon cancer and it has been suggested as a prognostic biomarker [
22,
26], it is largely unclear whether this epigenetic event is important for the development of this cancer. In the present study, we generated an
Hltf deficient mouse allele, and applied this mouse model to characterize the role of loss of HLTF function in the development of intestinal cancer. Our data demonstrates that loss of Hltf has a promoting effect on the transition of intestinal adenomas to invasive cancers, which highly suggests that aberrant HLTF methylation, as found in most human colon cancers, may have an important pathogenetic role in the development of this cancer.
Discussion
There is now strong evidence that a series of genetic alterations are required for the pathogenesis of human colon cancer. Specific gene mutations, such as in the
APC gene, initiate the formation of colonic adenomas and others (
e.g. TP53 mutations as well as other alterations, including KRAS activation) drive the malignant transformation of the adenomas in a multistep progression model [
2,
3]. More recently, epigenetic alterations, specifically aberrant DNA methylation, have been found to occur commonly in colon cancers [
4,
5]. Although there is controversy regarding the significance of these alterations in the pathogenesis of colon cancer, more and more data indicate that the aberrant methylation of at least some of these genes, such as
MLH1 (MutL homolog 1),
MGMT (O-6-methylguanine-DNA methytransferase) and
HIC1 (hypermethylated in cancer 1) can be pathogenetic in colorectal carcinogenesis [
2,
6]. Here, we have added another example to demonstrate the importance of epigenetic alterations in this carcinogenesis.
In this study, we focused on determining the role of loss of HLTF function in the development of colon cancer. The
HLTF promoter has been found to be hypermethylated in more than 40% of human colon cancers [
17‐
20], suggesting that
HLTF is a common target for methylation in this cancer. All colon cancer cells that lacked HLTF expression had the methylation of CpG islands within the
HLTF promoter, while methylation was not detected in the HLTF-expressing cells [
17] (Figure
6A), further indicating that this epigenetic event leads to a complete inactivation of HLTF expression in colon cancer cells. To determine the role of loss of HLTF function, in this study, we generated
Hltf deficient mice (Figure
1). Although these mice did not develop intestinal or colonic tumors, the majority of them were found to develop invasive intestinal and colonic cancers upon Apc mutation-mediated tumor initiation (Figures
4 and
5). Our results strongly suggest that aberrant methylation of HLTF, which leads to the loss of this gene's function as found in most human colon cancers, could have a pathogenetic role rather than being a consequence of colorectal carcinogenesis. This epigenetic event could be important for driving the transition of benign adenomas to the malignant adenocarcinomas, which is consistent with human tumor data showing that
HLTF promoter methylation is mainly detected in the advanced colonic adenomas and cancers, but not in the early stage of adenomatous polyp [
17,
19]. The high frequency of
HLTF promoter hypermethylation observed in these tumors indicates that the silencing of this gene could confer a selective advantage for the progression of these tumors to the malignant cancers.
Although the mechanism by which loss of HLTF function results in this malignant transformation still needs to be defined, our data demonstrating gross chromosomal abnormalities in
Hltf deficient colon tumor cells may suggest that genomic instability induced in these tumors is a driving force for this carcinogenesis. Genomic instability is a characteristic of human colon cancers, and has been implicated in the initiation and progression of these cancers by increasing the rate of genetic alterations [
2]. Two types of genetic instability have been found in human colon cancer: microsatellite instability (MIN) and chromosomal instability (CIN). Tumors with MIN display normal diploid with no obvious chromosomal defects but have high incidence of instability in microsatellite repeats linked to impaired mismatch repair (MMR) [
40]. The predominant mechanism identified to inactivate MMR in these tumors is epigenetic silencing through promoter methylation [
41], although somatic mutations in MMR genes also occur [
42]. CIN, which accounts for 85% of colorectal cancers, exhibits aneuploid, allelic losses and other chromosomal abnormalities, such as translocation, fusion and breaks [
32]. Colon cancers with CIN phenotype have been found to behave more aggressively in terms of invasiveness and metastasis [
32]. Although its molecular basis remains largely elusive, CIN is thought to arise from structural defects involving centromere or centrosome, microtubule dysfunction, telomere defects, chromosomal fragility and cell cycle checkpoint failure [
2,
6]. In this work, we have provided solid evidence that
Hltf deficient mouse colon tumors display severe chromosomal abnormalities, similar to CIN as described in human colon cancers (Figure
5). We have also demonstrated that down-regulation of HLTF increased chromosomal abnormalities in human colon cancer cells (Figure
7). Furthermore, similar to human colon cancers with a CIN phenotype, we have also found that Hltf deficient mouse colon tumors or
HLTF knockdown human colon cancer cells developed more malignant features, such as invasiveness, as compared to the tumor cells that contain functional HLTF (Figures
4,
5 and
6). All these findings indicate that the genomic instability induced in HLTF deficient colon tumors likely plays an important role in the malignant transformation of these tumors. Future studies using our established
Hltf knockout mouse colon tumor model to identify the genetic mutations involved in this pathogenesis have the potential to provide a deeper understanding of colon cancer development.
The presence of a CIN phenotype in
Hltf knockout or knockdown tumor cells also implicates loss of HLTF function as an additional mechanism to induce CIN phenotypes in human colon cancer. The involvement of HLTF in the post-replicative DNA damage-repair pathway has been demonstrated by several recent studies [
12‐
16]. In these work, HLTF was shown to display a similar function as yeast rad5 in the polyubiquitination of PCNA, which is required for initiating an error-free post-replicative repair activity [
12,
13]. HLTF was also found to have both DNA translocase and chromatin remodeling activities to facilitate the repair of damaged DNA on stalled replication forks [
14‐
16]. Therefore, HLTF could be an important factor in the post-replicative repair pathway. Without HLTF, it is likely that the progression of the replication fork would be blocked at DNA damage lesions, resulting in an accumulation of DNA intermediates that trigger recombination and genomic instability [
43]. Our cytogenetic data demonstrating the gross chromosomal abnormalities present in
Hltf deficient mouse tumor or in HLTF knockdown HCT116 colon cancer cells strongly support this notion. Our data also agree with many other studies indicating that aberrant post-replicative repair activity is a major source of the mutations and chromosome rearrangements that could be important for tumorigenesis. Interestingly, mice deficient in
Hltf alone were found to develop normally and did not display any pathological disorders associated with genetic instability. This may due to the robust replication checkpoints present in normal mouse cells, which, upon DNA damage, could boost a DNA damage response that arrests cells that harbor unrepaired DNA damage [
44]. These unrepaired cells could undergo apoptosis and then be replenished by cells differentiated from a pool of progenitor and stem cells during development. However, when these checkpoint pathways are not functional, the cells that contain defective or unrepaired DNA lesions could still progress through the cell cycle, resulting in broken chromosomes, genome aberrations and an accumulation of mutations [
44,
45]. A recent study indicates that APC could play an important role in the DNA replication checkpoints by stabilizing the association of DNA replication complexes at stalled DNA replication forks [
46]. Consistent with this finding, tumor cells that have lost APC function, such as human SW480 colon cancer cells, have been found to lose the capacity to arrest cells with damaged DNA [
46]. Therefore,
Hltf -/-/
Apc
min/+
colon tumor cells may have impaired replication checkpoints, which could facilitate cell cycle progression to enhance the genomic instability in these tumor cells.
HLTF has also been demonstrated to interact with several different gene promoters and enhancers [
7‐
11], suggesting its potential role in the regulation of gene expression. We recently performed micoarray-based gene expression analyses and did not find significant changes of gene expression between
Hltf +/+ and
Hltf -/- mouse ES cells or in HCT116 cells with and without HLTF knockdown. In both cases, a very few genes showed 2-fold change with the statistical significance (Additional file
4). None of these genes have been implicated in tumorigenesis or in the maintenance of genomic stability. Therefore, the loss of HLTF function is unlikely to mis-regulate the expression of specific genes, leading to the development of colon cancers.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SS, XW, SM and HD conceived and designed the experiments. SS, XW and HD generated the gene-targeting vector and produced the knockout mice and characterized the tumor phenotypes. SS and SM performed cytogenetic analysis. ZN, MR and SK provided reagents and technical supports for this study. SS and HD wrote the manuscript with subsequent contributions from all authors. All authors read and approved the final manuscript.