Promoter hypermethylation and silencing of tissue factor pathway inhibitor-2 in oral squamous cell carcinoma
© Lai et al.; licensee BioMed Central Ltd. 2014
Received: 16 May 2014
Accepted: 21 August 2014
Published: 2 September 2014
The treatment of oral squamous cell carcinoma (OSCC) following early detection is associated with good outcomes. Therefore, the survival and prognosis of OSCC patients could be hugely improved by identifying reliable biomarkers for the early diagnosis of the disease. Our previous methylation microarray analysis results have suggested that the gene encoding tissue factor pathway inhibitor-2 (TFPI-2) is a potential clinical predictor as well as a key regulator involved in OSCC malignancy.
Methylation of the TFPI-2 promoter in oral tissue specimens was evaluated by bisulfite sequencing assay, quantitative methylation-specific PCR, and pyrosequencing assay. The differences in methylation levels among the groups were compared using the Mann-Whitney U test. The area under the receiver operating characteristic curve (AUROC) was used to evaluate the discrimination ability for detecting OSCC. Cellular TFPI-2 expression was analyzed by quantitative reverse-transcription PCR before and after treatment with 5′-aza-2′-deoxycytidine and trichostatin A, to confirm whether TFPI-2 was epigenetically silenced in OSCC cells. We investigated whether TFPI-2 plays a role as a tumor suppressor by establishing TFPI-2-overexpressing OSCC cells and subjecting them to in vitro cellular proliferation, migration, and invasion assays, as well as an in vivo metastasis assay.
TFPI-2 was hypermethylated in OSCC tissues versus normal oral tissues (P < 0.0001), with AUROC = 0.91, when using a pyrosequencing assay to quantify the methylation level. TFPI-2 silencing in OSCC was regulated by both DNA methylation and chromatin histone modification. Restoration of TFPI-2 counteracted the invasiveness of OSCC by inhibiting the enzymatic activity of matrix metalloproteinase-2, and consequently interfered with OSCC metastasis in vivo.
Our data suggest strongly that TFPI-2 is a down-regulated tumor suppressor gene in OSCC, probably involving epigenetic silencing mechanisms. The loss of TFPI-2 expression is a key event for oral tumorigenesis, especially in the process of tumor metastasis.
KeywordsOral squamous cell carcinoma DNA methylation Biomarker TFPI-2 Tumor suppressor gene Metastasis Matrix-associated serine protease inhibitor Matrix metalloproteinase-2
Oral squamous cell carcinoma (OSCC), which constitutes more than 90% of oral cancers arising from the oral cavity , is not only one of the most frequently occurring cancers worldwide, but is also particularly prevalent in Taiwan. Oral cancer has been the fourth leading cause of death from cancer among males in Taiwan since 2003 . As a molecularly heterogeneous disease, OSCC is strongly associated with risk factors including alcohol, tobacco, and betel nut consumption [3-6]. OSCC is a particularly troublesome cancer due to its rapid progression and frequent metastasis , causing deaths at an increasing rate in Taiwan . The treatment of oral cancer following early detection is associated with good outcomes, but the 5-year survival rate is <30% among patients with stage IV disease . Therefore, identifying reliable biomarkers for the early diagnosis of OSCC may help to improve the patients’ survival and prognosis.
In addition to genetic alterations such as gene mutation , loss of heterozygosity , and microsatellite instability , epigenetic alterations (particularly hypermethylation in the promoter region of regulatory genes) have been increasingly recognized as key events in oral tumorigenesis . By taking advantage of the genome-wide screening approach, we have recently employed the Illumina GoldenGate Methylation Array to identify methylated genes in OSCC tissues (unpublished data). One of the genes identified with this approach was that encoding tissue factor pathway inhibitor-2 (TFPI-2). With a molecular weight of 27, 31, or 33 kDa, depending upon the level of glycosylation , TFPI-2 is synthesized and secreted extracellularly mainly by keratinocytes, fibroblasts, smooth-muscle cells, synoviocytes, and endothelial cells [14-17]. As a known Kunitz-type serine protease inhibitor and placental protein 5 ,,, TFPI-2 counteracts the activity of several extracellular matrix (ECM)-associated serine proteases, including trypsin, plasmin, chymotrypsin, cathepsin G, plasma kallikrein, and the factor VIIa-tissue factor complex ,. Previous studies have demonstrated that TFPI-2 suppresses tumor invasion and metastasis via its inhibitory activity on ECM degradation and remodeling ,. Roles of TFPI-2 in induction of the apoptosis pathway and in angiogenesis have also been demonstrated [24-27]. It has been shown that TFPI-2 is down-regulated via epigenetic silencing mechanisms including promoter hypermethylation and histone deacetylation in several types of tumor, such as pancreatic ductal adenocarcinoma , melanoma , hepatocellular carcinoma , gastric carcinoma , and glioma . The level of TFPI-2 methylation was also found to differ between preoperative and postoperative saliva DNA in oral cancer patients, highlighting its potential diagnostic value as a biomarker for oral cancer .
In the present study we first examined the methylation level of TFPI-2 in clinical OSCC specimens. Current techniques used to measure DNA methylation, including bisulfite sequencing assay, quantitative methylation-specific PCR (qMSP), and pyrosequencing assay, were applied to uncover the DNA methylation status of the TFPI-2 promoter region. The methylation level of TFPI-2 was further statistically analyzed to determine whether there was any correlation with the pathological stages of OSCC patients. We then restored the gene expression of TFPI-2 in OSCC cell lines by using epigenetic drugs and employing lentivirus vector-mediated gene transfer of TFPI-2. Restoration of TFPI-2 significantly suppressed the invasion and metastasis of OSCC cells. Our data strongly suggest that epigenetic silencing of TFPI-2 plays an important role in oral tumorigenesis.
Collection of oral tissue specimens and bisulfite conversion of genomic DNA
Normal oral tissues, OSCC tissues and their corresponding non-tumor tissues were obtained from the tissue banks of China Medical University Hospital and Buddhist Tzu Chi General Hospital in Taiwan. Genomic DNA of the tissues was isolated using Gentra Puregene Tissue Kit (Qiagen, Valencia, CA) and 500 ng of genomic DNA was subjected to bisulfite conversion using EZ DNA methylation kit (Zymo Research, Orange, CA). Bisulfite conversed Universal Methylated Genomic DNA (Millipore, Billerica, MA,) was used as in-vitro methylated DNA (IVD) control for the methylation level determined by qMSP and pyrosequencing assays.
Bisulfite sequencing assay and real-time quantitative methylation-specific PCR
For bisulfite sequencing, the primers targeting the promoter region near the TFPI-2 transcription start site were used for PCR as previously described . The PCR products were separated by gel electrophoresis, purified with a QIAquick gel extraction kit (Qiagen, Valencia, CA, USA), cloned into the yT&A cloning vector (Yeastern Biotech, Taipei, Taiwan), and sequenced. For real-time qMSP, primers targeting the promoter region of TFPI-2 were as follows: forward, 5′-ATAAAGCGGGTATTCGGGTC-3′; reverse, 5′-CTCCGCCGATTAAAAAAA-3′. Real-time qMSP was performed using ABI StepOne real-time PCR system according to the manufacturer’s instructions (Applied Biosystems, Forster City, CA). As an input control for real-time qMSP, a DNA fragment devoid of any CpG dinucleotide in ACTB was amplified using the following primers: forward, 5′-TGGTGATGGAGGTTTAGTAAGT-3′; reverse, 5′-AACCAATAAAACCTACTCCCTTAA-3′. The extents of methylated TFPI-2 and ACTB were determined by the threshold cycle number for each sample. The percentage of TFPI-2 methylation was calculated as the ratio of TFPI-2 to ACTB of a sample divided by the same ratio of IVD.
Pyrosequencing methylation assay
To further verify the results of bisulfite sequencing and qMSP, pyrosequencing primers were designed for the region of interest using Pyromark Assay Design v2.0 (Qiagen): forward, 5′-Bio+GGGTGATAGTTTTAGTGTATGAATTAGTT-3′; reverse, 5′-CTAAACAACATCCCCCAATACAACCTC-3′; reverse sequencing primer, 5′-ACTTTCTACTCCAAAC-3′. Pyrosequencing assay was carried out using the PyroMark Q24 System (Qiagen) according to the manufacturer’s instructions.
Epigenetic drug treatment
1 × 106 OSCC cells were seeded onto 10-cm culture dishes and treated with 0.5 μM or 5 μM 5′-aza-2′-deoxycytidine (5-azaDC) (Sigma, St Louis, MO) for 72 h followed by 0.25 μM trichostatin A (TSA) (Sigma) or DMSO for 12 h. For TSA treatment alone, cells were incubated with DMSO for 72 h followed by 0.25 μM TSA for 12 h. Drugs and culture medium were refreshed every 24 h during the treatments.
Quantitative reverse-transcription PCR
Total RNA was extracted from cell lines using REzol (Protech, Taipei, Taiwan) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using Superscript III reverse transcriptase (Invitrogen). Quantitative reverse-transcription PCR (qRT-PCR) was performed using ABI StepOne real-time PCR system as the following steps: 95°C for 10 min, followed by 50°Cycles of successive incubation at 95°C for 15’sec and at 68°C for 45’sec. TFPI-2 and GAPDH cDNA were amplified with the following primers: TFPI-2 forward, 5′-GCGATGCTGCTCAGGAG-3′ and reverse, 5′-TCTGCGTGTACCTGTCGTAGTAG-3′; GAPDH forward, 5′-TTGACGGTGCCATGGAATTT-3′ and reverse, 5′-GCCATCAATGACCCCTTCATT-3′. TFPI-2 expression was normalized against that of GAPDH.
Quantitative chromatin immunoprecipitation-PCR
Chromatin immunoprecipitation (ChIP)-PCR was performed as previously described . In brief, 2 × 106 OSCC cells were cross-linked with 1% formaldehyde and washed with PBS in the presence of protease inhibitors. Cells were homogenized and their chromatin was subjected to ChIP using magnetic Dynal beads (Invitrogen) and antibody against acetylated or trimethylated lysine 9 of histone H3 (H3K9) (Millipore, Temecula, CA). Fold-enrichment of amplified DNAs by ChIP was assessed using protocols as previously described . Specific primers targeting the promoter region of TFPI-2 were as follows: R1-forward, 5′-GCAGGTCATTTCCGTCTAGCTT-3′ and R1-reverse, 5′-ACCTGCCTCCCAAACTTTCTC-3′; R2-forward, 5′-ACCACTTTCCCTCTCTTTTGCT-3′ and R2-reverse, 5′-TCGTAGTAGTAACGGAGAAGTAGGGC-3′.
The OSCC cell lines OC2 and OCSL , derived from Taiwanese male patients who had habits of alcohol drinking, cigarette smoking, and betel nut chewing, were maintained in RPMI medium supplemented with 10% FBS (Invitrogen, Federick, MD). HEK293T cells were grown in DMEM medium supplemented with 10% FBS.
Plasmid construction and cell infection
The full-length human TFPI-2 cDNA (~0.7 kb) was cloned from the immortalized ovarian surface epithelia cell line IOSE  by RT-PCR with primers 5′-TTTCTCGGACGCCTTGCC-3′ and 5′-GAATGTTTAAAATTGCTTC-3′. The cDNA was introduced into a lentiviral vector plasmid pSin-IRES-GFP (pIG)  at the EcoRI and XbaI sites to generate pSin-TFPI2-IRES-GFP (pTIG). The plasmids pIG, pTIG and pSin-FLuc-IRES-GFP (pFIG, a firefly luciferase-expressing lentiviral vector plasmid) were transiently transfected into HEK293T cells and at 48 h posttransfection the lentivirus-containing supernatants were collected for infection. Successful infection was monitored by GFP expression and the infected cells were sorted by FACSAria III Cell Sorter (BD Bioscience, Franklin Lakes, NJ).
Extracellular matrix protein extraction and immunoblot assay
Cells were grown to ~90% confluence in culture dishes and extracellular matrix (ECM) proteins was harvested from the cells as described by Ehrlich et al.. Briefly, cells were washed three times with PBS and lysed through the incubation with PBS containing 0.5% Triton X-100 for 20 min at room temperature. The cells were washed three times with PBS and another three times with 20 nM Tris hCl [pH 7.4] containing 100 mM NaCl and 0.1% Tween 20. Finally, 200 μl of 1 × SDS-PAGE sample buffer was added to the culture dishes and agitated for 20 min at room temperature. The collected ECM proteins were boiled and 50 μl of aliquots of the extracts was assayed using 12% polyacrylamide gels. The expression of TFPI-2 protein was detected by a polyclonal antibody against TFPI-2 (Santa Cruz Biotechnology, Santa Cruz, CA).
Cell proliferation assay
The lentiviral vector-infected cells were seeded onto replicate 96-well plates (500 cells per well). Cell proliferation was determined daily with MTS assay using the CellTiter Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA). Relative cell number was measured on an ELISA plate reader with an absorbance set at a wavelength of 490 nm.
Cell cycle analysis
The lentiviral vector-infected cells were seeded onto replicate 6-well plates (1 × 105 cells per well). After 48 h, the cells were collected, washed, and fixed in 75% ethanol at -20°C for 48 h. The cells were then treated with 0.1 mg/ml RNase A (Macherey-Nagel, Düren, Germany), stained with 10 μg/ml propidium iodide (Sigma), and analyzed by FACSCalibur (BD Biosciences). The percentage of apoptotic cells in sub-G1 area was quantified using CellQuest Pro software (BD Biosciences).
Matrigel invasion assay
Invasion assay was done on 6-well Transwells (Millipore) with Matrigel-coated polycarbonate filters (8 μM pore size). Two × 104 cells were suspended in 200 μl of RPMI medium supplemented with 0.5% FBS and seeded on the upper chamber well. The lower chamber well was filled with RPMI medium containing 10% FBS. After 48 h of incubation at 37°C, nonpenetrating cells were removed from the upper surface of the filter and penetrating cells on the lower surface of the filter were fixed with methanol and stained with Giemsa solution (Sigma). The numbers of penetrating cells were counted under a light microscope.
The inhibitory effect of TFPI-2 on enzymatic activity of matrix metalloproteinases (MMPs) was assayed by collagen zymography. Two × 105 OC2 cells were suspended in complete medium and plated onto culture dishes. After 48 h of incubation, the medium was changed to serum-free RPMI and the cells were incubated for another 24 h. The conditioned medium was collected and the protein concentrations were determined by BCA Protein Assays (Bio-rad, Hercules, CA). Fifty μg of total proteins was mixed with non-reducing sample buffer containing 315 mM Tris [pH 6.8], 50% glycerol, 5% SDS and 0.025% bromophenol for electrophoresis. Without boiling, the mixed solution was loaded on a 10% polyacrylamide gel containing 0.5 mg/ml collagen (Sigma). After electrophoresis, gel was incubated for 1 h at 25°C in a 2.5% Triton X-100 solution followed by 20-min wash with deionized water for 2 times, and then incubated overnight at 37°C in a 50 mM Tris hCl [pH 8.0], containing 5 mM CaCl2. The gel was stained with 0.25% Coomassie blue and destained with 10% methanol and 7% acetic acid. Enzymatic activity attributed to MMPs can be visualized as clear bands against a blue background. The relative intensity of the bands was quantified by ImageJ.
In vivo tumor metastasis assay
Athymic BALB/c nude mice were obtained from National Laboratory Animal Center, Taiwan. Cells at a density of 1 × 106 in 100 μl of PBS were intravenously injected into the tail veins of athymic BALB/c nude mice. Forty days after tumor cell inoculation, the mice were sacrificed and their lungs were excised. The lung tissues were fixed with 10% formaldehyde and the number of pulmonary tumor nodules on each lung was counted.
The Mann-Whitney U test was used to compare the methylation levels between or among groups of tissue specimen. Receiver operating characteristic (ROC) curve and the area under the ROC curve (AUROC) was calculated to summarize the accuracy of using methylation level for detecting OSCC. The odds ratios between methylation level and OSCC were measured by logistic regression models. Differences with P < 0.05 were deemed significant. Analyses were performed in SAS 9.3 (SAS Institute, Cary, NC). Box plots were generated by SigmaPlot version 10 (Systat Software, San Jose, CA).
TFPI-is frequently hypermethylated in OSCC tissues
Selected patients’ characteristics
(a) TFPI-2 methylation level by qMSP
(b) TFPI-2 methylation level by pyrosequencing
TFPI-methylation is a good biomarker for OSCC detection
TFPI-2 hypermethylation could be used to discriminate tumor tissues from the normal ones. The AUROC values for using methylation level to detect OSCC were 0.79 (95% confidence interval (CI) = 0.69-0.89; Figure 2B) and 0.91 (95% CI = 0.83-0.98; Figure 3C) for the qMSP and pyrosequencing assays, respectively. A 1% increase in methylation level, as quantified by pyrosequencing assay, was associated with a 1.49-fold higher of risk of OSCC (95% CI = 1.06-2.10, P = 0.023), while the odds ratio was 1.13 (95% CI = 1.05-1.22, P = 0.001) per 1% increase of methylation level when quantified by qMSP. Moreover, TFPI-2 hypermethylation could be used to discriminate the early-stage tumor tissues from the normal ones. The AUROC values for using methylation level to detect early-stage OSCC (P1 and P2) were 0.82 (95% CI = 0.70-0.94; Figure 2C) and 0.94 (95% CI = 0.87-1.00; Figure 3D) for the qMSP and pyrosequencing assays, respectively.
Promoter hypermethylation and histone modification contribute to TFPI-inactivation in OCSL cells
Effects of TFPI-on the growth of OSCC cells
TFPI-2 suppresses OSCC cell invasion and blocks MMP-2 activity to reduce tumor metastasis in vivo
In this study we applied a high-throughput methylation array to investigate the DNA methylation status in oral tissue specimens. Comparison of gene methylation profiling between normal oral and OSCC tissues has suggested that TFPI-2 is hypermethylated in OSCC tissues, an observation that has been confirmed by qMSP and pyrosequencing assays. In agreement with the concept that TFPI-2 methylation is an early event in esophageal carcinogenesis , a significant difference in methylation level of TFPI-2 was found between normal oral tissues and tumor tissues at early stages, further validating the clinical value of TFPI-2 methylation profiling for the early detection of OSCC. In addition, the TFPI-2 methylation level in tumor tissues was higher at the P4 stage than at earlier stages (P1 and P2). Most of the 11 CpG sites analyzed by pyrosequencing assay displayed significant differences in methylation level between early and late stages, indicating the trend of increased methylation with progressive oral tumorigenesis.
While it is clear that TFPI-2 methylation profiling allows clinical samples to be discriminated, the difference in TFPI-2 mRNA expression between tumor and normal tissues was not as convincing as was expected (data not shown), mostly due to the heterogeneity of OSCC tissue specimens, in which both TFPI- 2-expressing and nonexpressing cells were present . Therefore, we measured TFPI-2 mRNA expression in OSCC cell lines and determined whether TFPI-2 down-regulation was caused by DNA methylation and histone deacetylation. We successfully reactivated TFPI-2 expression in OCSL cells but not in OC2 cells after combined treatment with 5-azaDC and TSA. In addition, the extent of TFPI-2 restoration in OCSL was comparable to that in TIG-transduced OCSL (3.5-fold vs 4.4-fold changes in TFPI-2 expression relative to IOSE). It is thus possible that for cells that are nonresponsive to 5-azaDC and TSA, such as OC2, the alternatives of viral or non-viral vector-mediated gene transfer for achieving sufficient TFPI-2 restoration are candidates for OSCC therapy.
The mechanism underlying the effect of DNA methylation on the repression of TFPI-2 in breast cancer cell lines has been reported previously . Sequence analysis of the TFPI-2 promoter region identified a potential Kruppel-like factor 6 (KLF6) binding site, suggesting that the aberrant DNA methylation in the KLF6 binding site diminishes the binding of transcription factor KLF6 to the TFPI-2 promoter to decrease TFPI-2 expression. Furthermore, Konduri et al. reported that methyl-CpG-binding protein 2 (MeCP2) was associated with the methylated TFPI-2 promoter using a ChIP assay in human glioma cells . The loss of MeCP2 from the activated TFPI-2 after combined treatment with 5-azaDC and TSA delineated the interplay between histone deacetylation and DNA methylation in the gene-silencing machinery. Together our findings corroborate the earlier conclusion that both promoter methylation and chromatin histone modification play important roles in TFPI-2 down-regulation.
Consistent with our in vitro data showing that TFPI-2 restoration neither suppressed cell proliferation nor promoted cell apoptosis, the tumor size did not differ significantly between IG- and TIG- transduced OSCC cells in a subcutaneous OSCC xenograft model in animals (data not shown). Although TFPI-2 has been found to reduce the cell proliferation rate in various human tumors ,[43-46] by triggering apoptosis ,, it has been suggested by some that TFPI-2 has no antiproliferation effect [47-49]. Therefore, the effect of TFPI-2 on cell proliferation and apoptosis may be complex and cell-type specific.
In the present study we demonstrated that TFPI-2 restoration could significantly abolish the invasiveness of OCSL and OC2 cells, but had no inhibitory effect on cell migration. Although TFPI-2 was previously shown to reduce tumor cell invasiveness, the findings regarding its influences on cell migration have been controversial ,,,,. As an inhibitor of ECM degradation, TFPI-2 has been described as an indirect inhibitor of MMPs via plasmin inhibition ,,. Our data also showed that TFPI-2 counteracts tumor invasion by negatively regulating MMP-2 activation, consistent with the finding that TFPI-2 is associated mainly with the inhibition of ECM degradation . As also reported by Izumi et al., TFPI-2 suppressed cell invasion via the down-regulation of the level of active MMP-2 to interfere with the process of tumor metastasis. In addition, we successfully established a metastatic tumor model in animals by the intravenous injection of TFPI-2-expressing OC2 cells, and observed a marked inhibitory effect of TFPI-2 in the formation of lung tumor nodules, demonstrating that TFPI-2 is a silenced tumor suppressor gene in OSCC.
The significance of the reported differential pattern of TFPI-2 hypermethylation between normal oral and OSCC tissues makes it a promising biomarker for the early detection of OSCC. TFPI-2 expression in OSCC can be reactivated after combined treatment with 5-azaDC and TSA, revealing a synergistic regulation of DNA methylation and histone deacetylation in TFPI-2 silencing. Our data suggest that TFPI-2 is a down-regulated tumor suppressor gene in OSCC, and that loss of TFPI-2 expression is a key event in oral tumorigenesis.
This study was supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004) and the National Science Council of Taiwan (NSC99-2320-B-194-004-MY3).
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