Background
Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the world, in spite of the availability of colonoscopy screening and effective prevention through a colorectal adenoma resection. Recent evidence indicates that epigenetic alterations, apart from genetic alteration, play an important mechanism in CRC carcinogenesis [
1]. Epigenetic alterations are potentially reversible in neoplasia, and they present new opportunities for the clinical management of cancer, which are different from mutation [
2]. The most common epigenetic alteration in CRC is aberrant DNA methylation, in which a methyl group is added to the cytosine base in the dinucleotide sequence CpG islands, which are often associated with the promoter [
1,
2]. Methylation in a gene promoter region generally correlates with a silenced gene [
2]. Several genes involved in CRC carcinogenesis found to be silenced by DNA methylation include:
MCC, MLH1, MGMT, APC, hMLH1, GATA-4, GATA-5, TFPI2, and
SOX17[
2‐
8].
The iron-activated alcohol dehydrogenase family had seemed to only exist in microbial organisms until Mao
et al. cloned alcohol dehydrogenase, iron containing, 1 (
ADHFE1) from a human fetal brain cDNA in 2002 [
9]. About 10 years later, just as there are only a few more studies [
10,
11]. Kim
et al. showed that
ADHFE1 transcript exhibits differentiation-dependent expression during
in vivo brown and white adipogenesis [
10]. Another study indicated that
ADHFE1 is related to bacterial γ-hydroxybutyrate dehydrogenase and has a conserved NAD-binding site [
11]. Recently, we had documented gene profiles with promoter hypermethylation using Human Methylation27 DNA Analysis BeadChip in CRC [
12]. Among these genes, the
ADHFE1 gene is the most highly methylated in CRC compared to normal colorectal mucosa and the expression is down-regulated more than twofold [
12]. It was also reported that the
ADHFE1 promoter is hypermethylated in CRC and adenoma by another research group [
13].
Here, we investigated ADHFE1 promoter methylation in CRC cell lines and CRC tissues. Additionally, we researched the functional implications of ADHFE1 methylation for CRC.
Methods
Subjects
One hundred twenty four CRC and 59 advanced adenoma tissues greater than 1 cm in diameter and/or with a villous component and/or with severe dysplasia were collected from patients treated at Samsung Medical Center, Seoul, Korea (Table
1). We also retrieved 124 adjacent normal colorectal mucosa tissues from CRC patients. None of the patients had clinically apparent polyposis syndrome or hereditary nonpolyposis colon cancer syndrome. Written informed consent was obtained from all patients. This study was reviewed and approved by the Samsung Medical Center Institutional Review Board in accordance with the Declaration of Helsinki.
Table 1
Clinical characteristics of the study population
Age, years | |
Mean ± SD | 58.9 ± 9.97 |
Median (range) | 60.0 (32–77) |
Gender (%) | |
Male/Female | 77 (62.1)/47 (37.9) |
Location (%) | |
Right/Left side | 64 (51.6)/60 (48.4) |
Differentiation (%) | |
Well/Moderate/Poorly | 13 (10.5)/92 (74.2)/19 (15.3) |
Duke stage (%) | |
I/II/III/IV | 4 (3.2)/64 (51.6)/45 (36.3)/11 (8.9) |
Microsatellite instability status (%) | |
MSS/Low MSI/High MSI | 107 (86.2)/8 (6.5)/9 (7.3) |
Advanced adenoma (n = 59)
| |
Age, years | |
Mean ± SD | 46.5 ± 1.74 |
Median (range) | 40.0 (17–72) |
Gender (%) | |
Male/Female | 47 (79.7)/12 (20.3) |
Size, mm | |
Mean ± SD | 17.4 ± 0.92 |
Median (range) | 15.0 (0.7-35) |
Location (%) | |
Right/Left side | 20 (33.9)/39 (66.1) |
Histology (%) | |
Tubular/Tubulovillous/Villous | 39 (66.1)/19 (32.2)/1 (1.7) |
Grade of dysplasia (%) | |
Low/High | 49 (83.1)/10 (16.9) |
CRC cell lines and 5-aza-2′-deoxycytidine (5-aza-2-dC) treatment
We used CoLo205, DLD-1, HT15, HT116, HT29, KM12C, KM12SM, KM20, RKO, SNU81, SW48 and WiDr as human CRC cell lines. Each of the CRC cell lines were treated for 48 hours with 0 or 2 μM 5-aza-2-dC (Sigma-Aldrich, St. Louis, Mo, USA).
DNA extraction and bisulfite modification
Genomic DNA was extracted using the QIAamp tissue kit (Qiagen, Valencia, CA, USA). A bisulfite conversion was performed using the Zymo EZ DNA methylation kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. DNA was extracted from tumor cells or the epithelial layer of formalin-fixed, paraffin embedded tissue sections.
Quantitative real time PCR (RT-PCR) to measure DNA methylation (MethyLight)
The ADHFE1 gene presents on human chromosome 8q 13.1. The promoter region of the ADHFE1 gene contains a dense CpG island located from nucleotides −257 to +265 relative to translation start site (TSS). We examined 12 CRC cell lines and CRC tissues using methylation-specific PCR with primers located from −4 to +102 relative to TSS.
After bisulfite conversion of the same amount of DNA solution, ALU-based MethyLight control reaction was done to quantify the number of input target DNA, and the threshold cycle values were confirmed to be comparable among DNA samples. The primer and probe sequences used were as follows: ADHFE1 forward primer: 5-CGTTATGGTCGTTGTCGTTC-3′, ADHFE1 probe: 6FAM-CGCCGACCCCGCACTCACGC-MGBNFQ, ADHFE1 reverse primer: 5-GTAAACACCCTACGATCCCCTACCCG-3. ALU forward primer: 5-GGTTAGGTATAGTGGTTTATATTTGTAATTTTAGTA-3, ALU probe: 6FAM-CCTACCTTAACCTCCC-MGBNFQ, ALU reverse primer: 5-ATTAACTAAACTAATCTTAAACTCCTAACCTCA-3.
MethyLight was performed by using a 7900HT Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR program was as follows: 5 minutes at 95°C, followed by 45 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C. CpGenome™ universal methylated and unmethylated DNA (Chemicon, Temecula, CA, USA) were used as a positive control for the methylated and unmethylated genes, respectively. The percentage of methylated reference (PMR) at a specific gene was calculated by dividing the GENE/ALU ratio of a sample by the GENE/ALU ratio of the in vitro fully methylated placental DNA and multiplying by 100. We used the PMR cut-off value more than 10% to define methylation-positive vs. negative and determined DNA methylation frequencies for each CpG island locus.
Bisulfite genomic sequencing
We performed bisulfite genomic sequencing for CRC tissue, and adjacent normal colorectal mucosa according to the manufacturer’s instructions. We performed PCR to amplify the sequence for CpG island located in the promoter lesion and TSS using the following primer sets: BS1F (5′-TTTGAAAAATAAGATATAGTGTTTAATTAT-3′) and BS1R (5′-AAATAACTCTAAACTCAACAAAAAC-3′); BS2F (5′-GTTTTTGTTGAGTTTAGAGTTATTT-3′) and BS2R (5′-ACCACTACCCCAAAATTACAC-3′). The PCR assays were performed at 95°C for 15 minutes, 45 cycles of 92°C for 30 seconds, 50 (BS1) or 57 (BS2)°C for 45 seconds, 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. PCR products were cloned using One Shot® TOP10 kit (Invitrogen, Carlsbad, CA, USA) and sequenced using 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA).
RT-PCR
Two μg of total RNA was reverse transcribed by M-MuLV reverse transcriptase with Oligo(dT)16 as a primer. RT-PCR was performed on the ABI PRISM 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA) by monitoring the increase of fluorescence by the binding of SYBR Green (TaKaRa, Otsu, Japan) to double-stranded DNA. A dissociation analysis was performed at the end of each PCR reaction to ensure there was only a specific product. The settings for the PCR thermal profile were as follows; initial denaturation at 95°C for 5 minutes, followed by 45 amplification cycles of 95°C for 10 seconds, annealing at 55°C for 10 seconds, and elongation at 72°C for 30 seconds. The primer and probe sequences used were as follows: ADHFE1 forward primer: 5- CACTGCCAGGATCCAAGATG −3′, ADHFE1 reverse primer: 5- GAGCTTTGGGGAATTTCCTG −3. GAPDH forward primer: 5′ - CCACCCATGGCAAATTCCATGGCA - 3′, GAPDH reverse primer: 5′ - TCTAGACGGCAGGTCAGGTCCACC -3′. Each PCR was run in triplicate. For quantification of gene expression changes, the ΔΔCt method was used to calculate relative fold changes normalized against the GAPDH gene.
Western blot
Cells lysates were prepared in ice-cold RIPA buffer (0.5% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris (pH 7.5), 0.1% SDS, and 1 mM PMSF) and cleared by microcentrifugation (14 000 rpm for 30 min at 4°C). The protein concentration in each sample was estimated by BCA Assay. Twenty to thirty μg of each protein sample was resolved by 12% SDS–polyacrylamide gel electrophoresis, and electroblotted into nitrocellulose membranes. After 1 hour incubation in blocking solution (5% non-fat milk in TBST), it was incubated with primary antibodies overnight at 4°C. The primary antibodies included: those against ADHFE1 (1:1000) (Sigma-Aldrich, St. Louis, MO, USA), carcinoembryonic antigen (CEA) (1:1000), caudal type homeobox 2 (Cdx2) (1:1000) (Cell Signaling, Danvers, MA, USA) and β-actin (1:5000) (Santa Cruz Biotchology, Santa. Cruz, CA, USA). The blots were then washed in TBST and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. The results were visualized using an ECL system (Amersham Pharmacia Biotech, Arlington Heights, IL, USA).
Immunohistochemistry
To confirm the ADHFE1 protein expression in CRC and adjacent normal colorectal mucosa, we performed immunohistochemical analysis of ADHFE1 protein expression in 243 CRC cohort groups. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections (4 μm thick). Sections were deparaffinized in xylene, rehydrated, and incubated with 0.3% hydrogen peroxide in methanol for 30 minutes. The sections were placed 10 mM Tris + 1 mM EDTA with a subsequent microwave antigen retrieval procedure. The sections were incubated with 4% BSA + Dextran/PBST to block nonspecific antibody binding, followed by incubation with the primary anti-ADHFE1 polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:2500 in PBS with 0.1% Tween and 0.5% BSA. Sections were incubated with secondary antibody against HRP-conjugated-rabbit Ig, and bound antibody was visualized using 3, 3-diaminobenzidine substrate as a chromogen (Dako, Copenhagen, Denmark) followed by hematoxylin counter staining.
To assess ADHFE1 expression during mouse gut differentiation and development, six pregnant female mice (Orient Bio Inc., Seongnam, Korea). On 15, 17 and 18 days after implantation, mice were killed by CO2 inhalation, and each embryo was resected and frozen in liquid nitrogen for ADHFE1 immunohistochemistry. The immunohistochemistry method was the same as above. Mice were handled at the institute’s (Samsung Medical Center, Seoul, Korea) animal facility, and all treatments were in accordance with the legal and institutional guidelines.
Immunoreactivity score (IS) in CRC tissues
In this study, we used the scoring method of Sinicrope
et al. for evaluation of both the intensity of immunohistochemical staining and the proportion of stained epithelial cells [
13]. The staining intensity was further classified as follows: weak, moderate, or strong. Positive cells were quantified as a percentage of the total number of epithelial cells and were assigned to one of the following five categories: 0, <5%; 1, 5%–25%; 2, 26%–50%; 3, 51%–75%; and 4, >75%. The percentage of positivity of epithelial cells and staining intensities were then multiplied to generate the IS for each case. For example, if the staining intensity was strong and the percentage of positive cells was greater than 75%, then the IS would be 3 × 4 = 12. As a result, the IS range was from 0 to 12. Each lesion was examined and scored by one pathologist (SHK). In the evaluation, membranous staining was considered positive.
Cell culture and transfection
DLD-1 was cultured in IMDM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA). The full-length ADHFE1 cDNA (Origene Technologies, Inc., Rockville, MD, USA) was subcloned into a pcDNA3.1(−) vector (Invitrogen, Carlsbad, CA, USA) to create pcDNA3.1(−)-ADHFE1. DLD-1 was transfected with pcDNA3.1(−)-ADHFE1 or empty vector (pcDNA3.1(−)) by using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.
Alkaline phosphatase (ALP) activity assay
ALP activity was determined according to the manufacturer’s protocol (Cell biolabs Inc., San Diego, CA, USA). DLD-1cells were seeded into 12 well plates at 3×105cells/ml. After 72 hours, we gently aspirated the medium from the cells and washed the cells twice with cold PBS. Cells were lyzed in Cell Lysis Buffer (each 0.2 mL for 12 well dishes), incubated for 10 minutes at 4°C and removed from the solution and the cell debris spun down at 12,000 × g for 10 minutes. We performed a BCA assay to determine the protein concentration of the cell lysate. We added 50 μL of cell lysate to a 96-well plate. And then we prepared blank wells containing 50 μL Cell Lysis Buffer. We added 50 μL of StemTAG™ AP Activity Assay Substrate and incubated for 10–30 minutes at 37°C. After stopping the reaction by adding 50 μL of 1X Stop solution, we read the absorbance of each well at 405 nm.
Statistics analysis
Statistics were calculated using SPSS software (SPSS v.20.0; IBM Corp., Armonk, NY, USA). Continuous data are presented as a mean ± standard deviation. Categorical data are presented as percentages. The comparison of IS among each group was evaluated using the Kruskal-Wallis H test because of the skewed distribution. For all tests, a p value < 0.05 was considered statistically significant. The difference in the continuous data of ALP activity assay among categorized groups was evaluated using Mann–Whitney U test because of a skewed distribution.
Discussion
We report that the
ADHFE1 gene is frequently hypermethylated in CRC cell lines and tissues, using methylation-specific PCR and bisulfite genomic sequencing.
ADHFE1 mRNA levels are frequently down-regulated in CRC cell lines and tissues. When dense methylation of the
ADHFE1 promoter was restored after 5-aza-2-dC treatment, expression of
ADHFE1 mRNA and ADHFE1 protein had increased. Therefore we confirmed that
ADHFE1 methylation is under epigenetic regulation. In our study, some CRC cell lines such as KM12SM, KM20 and WiDr didn’t have the 5-aza-2-dC effect. Mossman D
et al. found that 5-aza-2-dC induces gene expression, but is not necessarily dependent on DNA demethylation [
14]. Therefore, all CRC lines didn’t observe the definite demethylating effect of 5-aza-2-dC in our study. In addition, the SW48 cell line had periods of rapid re-methylation and restoration with exposure of 5-aza-2-dC [
14]. It is possible that the increased
ADHFE1 mRNA by 5-aza-2-dC in the SW 48 cell line was not detected.
According to the results of immunohistochemical staining, ADHFE1 expression is significantly reflected by the state of differentiation in CRC tissue. ADHFE1 expression in well differentiated CRC tissues was more intense than that in poorly differentiated CRC tissues, with statistical significance. We could confirm this finding from a representative case that simultaneously showed well and moderately differentiated CRC cells. In addition, ADHFE1 is expressed at the top of the colon with a decreasing gradient toward the crypt along the colon crypt axis in normal colon epithelium. This explains why ADHFE1 is also associated with the differentiation of normal colon epithelium. To obtain more information about the expression of ADHFE1 protein in embryo, we performed an immunohistochemical analysis according to respective mouse embryo steps. Our expression study showed that the ADHFE1 protein is highly related to mouse embryogenesis. Even though the data is not shown, ADHFE1 is expressed in the organs with active morphogenetic activities, such as the gut, lung, kidney and several glandular structures. In a mature embryo gut, ADHFE1 exhibits different expression patterns along the anterior-posterior axis of the gut. At E18.5, ADHFE1 expression is confined to the ventral part of gut. It is similar to ADHFE1 expression in the mature gut. Although immunohistochemical analysis is not a quantitative strategy, it was informative enough and a discernible method to show ADHFE1 expression in the epithelial cell layer of mouse embryo. Therefore, it seems that ADHFE1 may play a role in the differentiation and development of the developing gut in mouse embryo.
To confirm the association between ADHFE1 and differentiation, we choose DLD-1 CRC cell line without endogenous
ADHFE1 expression. The DLD-1, a typical poorly differentiated CRC cell line, has been known to have no endogenous CEA and ALP [
15,
16]. We confirmed the role of ADHFE1 for the increase of a well-known colon epithelium differentiation marker, such CEA and ALP [
17]. Cdx2 is a caudal-related homeobox transcription factor. Unlike CEA and ALP, DLD-1 is well known to express Cdx2 [
18]. Therefore, we might be able to find the definite difference of Cdx2 expression between the vector and
ADHFE1-transfected cells [
18]. Although it was not same focus, Kim
et al. have shown a differentiation-dependent expression of ADHFE1 in adipogenesis [
10].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CHT analysed the data and wrote the paper. KJY carried out experiments and analysed the data. SHK reviewed the hisopathological diagnosis of patients and provided the interpretation of the immunohistochemical staining results. SHK and HCK were involved in the collection of the study population. BHM, DKC, PLR and JCR critically read and evaluated the manuscript. JJK advised the protocol and analysed the data. YHK designed the study protocol, analysed the data and supervised the writing of the paper. All authors had final approval of the submitted and published versions.