© The Author 2006. Published by Oxford University Press.
ARTICLE |
Epigenetic Modulation of Tumor Suppressor CCAAT/Enhancer Binding Protein 
Activity in Lung Cancer
Affiliations of authors: Department of Molecular Virology, Immunology, and Medical Genetics, Division of Human Cancer Genetics (YT, RMB, BH, CP), Department of Pathology (CM), Department of Internal Medicine, Division of Hematology and Oncology (GAO, CP), The Comprehensive Cancer Center, The Ohio State University, Columbus, OH; Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany (BH)
Correspondence to: Christoph Plass, PhD, The Ohio State University, Division of Human Cancer Genetics, Tzagournis Medical Research Facility 464A, 420 West 12th Ave., Columbus, OH 43210 (e-mail: christoph.plass{at}osumc.edu).
 |
ABSTRACT |
|---|
 TopNotes Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Background: Loss of tumor suppressor CCAAT/enhancer-binding protein-
(C/EBP) expression is seen in several human malignancies, including acute myelogenous leukemia and lung cancer. We hypothesized that DNA methylation and histone acetylation of the C/EBP promoter may modulate C/EBP expression in lung cancer. Methods: We analyzed C/EBP expression in 15 human lung cancer cell lines and in 122 human lung primary tumors by northern blotting, immunoblotting, and immunohistochemistry. C/EBP promoter methylation was assessed using bisulfite sequencing, combined bisulfite restriction analysis, methylation-specific polymerase chain reaction, and Southern blotting. We examined the acetylation status of histones H3 and H4 at the C/EBP promoter by chromatin immunoprecipitation. Binding of methyl-CpG–binding proteins MeCP2 and MBD2 and upstream stimulatory factor (USF) to the C/EBP promoter was assayed in cell lines that were untreated or treated with histone deacetylase inhibitor trichostatin A and demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) by chromatin immunoprecipitation and by electrophoretic mobility shift assays. Results: DNA methylation and histone acetylation in the upstream region (–1422 to –896) of the C/EBP promoter were associated with low or absent C/EBP expression in 12 of 15 lung cancer cell lines and in 81 of 120 primary lung tumors. MeCP2 and MBD binding to the upstream C/EBP promoter was detected in C/EBP-nonexpressing cell lines; USF binding was detected in C/EBP-expressing cell lines; however, in C/EBP-nonexpressing cell lines USF binding was detected only after trichostatin A and 5-aza-dC treatment. Conclusions: DNA hypermethylation of the upstream C/EBP promoter region, not the core promoter region as previously reported, is critical in the regulation of C/EBP expression in human lung cancer.
|
INTRODUCTION |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Lung cancer is the leading cause of cancer-related death worldwide (1). Despite intensive research, the overall 5-year survival for lung cancer patients has not changed substantially during the last 20 years and remains at only 15% (1). Genetic approaches have identified chromosomal regions that are frequently lost or gained in human lung tumors, and fine mapping of these regions has led to the identification of numerous oncogenes and tumor suppressor genes (2,3). In addition, data from recent studies (4–6) indicate that epigenetic mechanisms, such as DNA methylation and histone tail modifications, are major contributors to the disease phenotype. Epigenetic studies of lung tumors have identified the silencing of known tumor suppressor genes, including death-associated protein kinase 1 (DAPK1) (4), mismatch repair genes hMLH1 and hMSH2 (5), Ras-effector gene NORE1A (6), and others (7). Also, novel candidate tumor suppressor genes, including RASSF1A (8), SEMA3B and SEMA3F (9), BMP3B (10), and SMARCA4/BRG1 (11), have been identified based on their frequent inactivation by promoter hypermethylation in primary lung tumors and cell lines.
CCAAT/enhancer-binding protein- (C/EBP) is a basic leucine zipper transcription factor that is highly expressed in differentiated tissues (12,13) where it controls differentiation-dependent gene expression and inhibits cell proliferation (4,14,15). In vivo and in vitro experiments in which C/EBP was inactivated or overexpressed have provided evidence for a possible tumor suppressor function for this gene (13,16,17). C/EBP–/– knockout mice show hyperproliferation of type II pneumocytes and abnormal alveolar structure, among other defects (16,18), whereas overexpression of C/EBP in lung cancer and myeloid leukemia cell lines induces their differentiation and inhibits their proliferation (19,20).
Immunohistochemical evaluation of 36 primary lung cancers revealed low C/EBP expression that could not be accounted for by genetic abnormalities (20,21). Based on these findings, we hypothesized that epigenetic mechanisms, such as DNA methylation, might be involved in the regulation of C/EBP expression. In this study, we performed a comprehensive analysis of the DNA methylation patterns and histone modification status of the C/EBP-associated CpG island in human lung cancer cell lines and in human primary lung tumors.
|
MATERIALS AND METHODS |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Primary Lung Cancer Tissue Samples and Cell Lines
Primary human lung cancer and adjacent normal tissue samples were collected through the Cooperative Human Tissue Network at The Ohio State University James Cancer Hospital. Consent from participants was waived under 45 CFR 46, Subpart A. A total of 122 non–small-cell lung cancers, including 69 squamous cell carcinomas, two large cell carcinomas, and 51 adenocarcinomas were studied. Tissue specimens and peripheral blood mononuclear cells (PBMCs) were procured in accordance with The Ohio State University Cancer Internal Review Board guidelines. Human non–small-cell lung cancer (NSCLC) (H23, H125, H522, H1155, H1299, H2009, H2086, and A549), human small-cell lung cancer (SCLC) (H69, H82, H209, H719, H792, H841, and N417), human leukemia (U937), human kidney (293T), and mouse neuroblastoma (Neuro2A) cell lines were obtained from American Tissue Culture Collection (ATCC; Manassas, VA) and were cultured in RPMI-1640. The mouse myeloid cell line (32Dc13) was kindly provided by Dr. Danilo Perrotti (The Ohio State University, Columbus, OH).
To analyze restoration or induction of C/EBP expression, H719 and H1299 cell lines were incubated for 72 hours with 5 µM 5-aza-2'-deoxycytidine (5-aza-dC) (Sigma-Aldrich, St. Louis, MO) and/or for 24 hours with 300 nM trichostatin A (TSA; WAKO, Tokyo), a histone deacetylase inhibitor. Stock solutions of 5-aza-dC (20 mM dissolved in dimethyl sulfoxide) and TSA (3 mM dissolved in 100% ethanol) were added directly to the cell culture medium. Treated cells were incubated in trypsin-EDTA (Gibco BRL, Gaithersburg, MD), removed from culture dishes, and washed twice with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM K2HPO4) (Gibco BRL) before DNA, RNA, and protein isolation.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from lung cancer tumors and cell lines and from normal lung tissues using the RNeasy protocol (QIAGEN, Hilden, Germany) as described previously (22). Total RNA (10 or 20 µg) was separated on 1% formaldehyde–agarose gels and transferred to Zeta Bind-GT nylon membranes (Bio-Rad) by capillary transfer in 10x SSC overnight. The membranes were rinsed in 2x SSC and UV cross-linked (Stratalinker 1800; Stratagene, La Jolla, CA). The membranes were incubated in hybridization buffer (0.5 M sodium phosphate, pH 7.2, 7% w/v sodium dodecyl sulfate [SDS], and 1 mM EDTA, pH 7.0) for 2 hours at 65 °C and were subsequently hybridized in hybridization buffer for 16 hours at 50 °C with random primer (Stratagene) [-32P]dCTP-labeled C/EBP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes. The 533-base pair (bp) C/EBP cDNA probe (located in the 3'-untranslated region, at position 1595 to 2128 from the transcription start site) was amplified by polymerase chain reaction (PCR) using the following primer pair: 5'-CTCCTTCCCGAGGCTACAG-3' and 5'-TCATAACTCCGGTCCCTCTG -3' (NM_004364
[GenBank]
). The 497-bp GAPDH cDNA probe was amplified by PCR using the following primer pair: 5'-TGGAAGGACTCATGACCACA-3' and 5'-TTACTCCTTGGAGGCCATGT-3' (NM_002046
[GenBank]
). Blots were exposed to a PhosphorImager screen (ECL Amersham Biosciences, Piscataway, NJ), and C/EPB expression was normalized to that of GAPDH. Northern blot analysis was performed once.
Southern Blot Analysis
Genomic DNA was isolated from normal lung tissues, PBMCs, and lung cancer cell lines, and Southern blot analysis of C/EBP was performed as previously described (24), with minor modifications. All probes (50 ng each) were labeled with [-32P]dCTP by random primer labeling (Stratagene). Probe 1 was a 1115-bp AscI/PstI fragment (located –1583 to –467 from the C/EBP transcription start site) that was amplified using PCR from genomic DNA using the primers 5'-GACCGAAAACGAAGCAGTTG-3' and 5'-AGTCTTGGTCTTGAGCTGCTG-3' (NM_004364
[GenBank]
). Probe 2 was a 205-bp NruI/PstI fragment (+4 to +178 from the C/EBP transcription start site) that was isolated from a 1123-bp KpnI/NotI clone that was derived from the bacterial artificial chromosome RP11-939022 clone. Probe 3 was a 155-bp NotI/PstI fragment (+585 to +739 of C/EBP) that was isolated from a NotI/EcoRV clone that was identified in a genomic NotI-EcoRV library designed for restriction landmark genomic scanning spot cloning (23). Southern blot hybridizations were performed as previously described (10). Southern blot analysis was performed once.
Methylation-Specific PCR (MSP), Combined Bisulfite Restriction Analysis, and Bisulfite Sequencing
Genomic DNA was extracted as described above from H2086, H1299, and H719 cells, from primary tumors and adjacent normal tissues, and from PBMCs and treated with 3 M sodium bisulfite as previously described (22). Treatment of DNA with sodium bisulfite results in selective conversion of unmethylated cytosine to uracil, whereas methylated cytosine remains virtually unchanged. The primers and PCR conditions for combined bisulfite restriction analysis assays, MSP, and bisulfite sequencing are summarized in Supplementary Fig. 1 (available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol98/issue6) The PCR products were purified using a QiaQuick gel extraction kit (QIAGEN, Valencia CA) and incubated with BstUI at 60 °C for 4 hours or with HpyCH4IV at 37 °C for 4 hours. The digested DNA was separated on an 8% polyacrylamide gel that was then stained with ethidium bromide. Sequencing was performed once.
Cell line– and primary tumor–derived DNA was treated with sodium bisulfite, amplified by PCR, and purified as described above. The purified PCR products were ligated into pCR2.1-TOPO using the topo-TA cloning system (Invitrogen, Carlsbad, CA). Bacteria TOP10 F' were transformed with plasmids and cultured overnight, and the plasmid DNA was isolated using the Miniprep kit (QIAGEN). For each sample, four to 10 separate clones were sequenced using ABI Big Dye Terminator Chemistry Kit (Applied Biosystems, Foster City, CA).
Immunohistochemical Staining
Immunohistochemical staining of the human primary tumor samples was performed on formalin-fixed, paraffin-embedded specimens. Briefly, a rabbit polyclonal anti-C/EBP antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at 1 : 750 dilution for immunohistochemical detection. Antibody binding was detected by incubating the slides with a secondary polyclonal anti-rabbit IgG antibody (Amersham Biosciences). Positive staining was visualized by incubating the slides with diaminobenzadine (Sigma-Aldrich). The slides were examined by an experienced lung pathologist (C.M.) and reviewed with the primary investigator (Y.T.). Samples were scored as C/EBP positive when signal (diaminobenzadine) was detected in more than 5% of the tumor epithelial cells (24).
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation was carried out using the ChIP assay kit (Upstate Biotechnology) following the manufacturer's recommendations with minor modifications. Chromatin was immunoprecipitated for 16 hours at 4 °C using anti-acetyl-histone H3 (polyclonal rabbit IgG) or anti-acetyl-histone H4 (polyclonal rabbit IgG) antibodies, anti-MeCP2 (polyclonal rabbit IgG), or anti-MBD2 (polyclonal sheep IgG) antibodies (Upstate Biotechnology, Lake Placid, NY) to detect methyl-binding proteins or antibodies to upstream stimulating factor (USF)-1 or USF-2 (Santa Cruz Biotechnology), after which PCR was performed by using 1/100 of the immunoprecipitated DNA. In addition, 1/100 of the solution without antibody was amplified as a negative control. Primer sequences and conditions used for PCR are summarized in Supplementary Fig. 2 (available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol98/issue6). Five cycles of 95 °C for 30 s, 67 or 62 °C for 30 s, and 72 °C for 30 s were followed by 30 cycles of 95 °C for 30 s, 62 or 57 °C for 30 s, and 72 °C for 30 s. PCR products were isolated with phenol–chloroform, precipitated with ethanol, separated on a 2.5% agarose gel, and visualized by staining with ethidium bromide. Chromatin immunoprecipitation assays were performed twice.
Immunoblot Analysis
Whole-cell lysates from 32Dc13, U937, H2086, parental A549 cells, A549 cells transfected with C/EBP, and H719 cells that were either left untreated or were treated with 5 µM 5-aza-dC for 72 hours were prepared by incubating 2 x 106 cells in 2x Laemmli buffer (125 mM Tris-HCl, pH 6.8, 4% w/v SDS, 20% w/v glycerol, 0.2% w/v bromphenol blue, and 600 mM 2-mercaptoethanol) for 10 minutes at 100 °C. Proteins were separated by electrophoresis on 4%–15% gradient polyacrylamide gels (BioRad, Hercules CA). C/EBP protein was detected using rabbit polyclonal anti-C/EBP (Santa Cruz Biotechnology) at 1 : 1000 dilution. A horseradish peroxidase–conjugated anti-rabbit secondary antibody (Amersham Biosciences) was used at 1 : 5000 dilution. Membranes were also incubated with monoclonal mouse anti--tubulin (1 : 5000 dilution) to control for protein loading and transfer (EMD Biosciences, San Diego, CA). Protein–antibody complexes were detected via chemiluminescence (ECL Amersham Biosciences). Immunoblot analysis was performed twice.
Site-Directed Mutagenesis and Luciferase Assays
To determine which regions of the C/EBP promoter are involved in transcription, we generated the following truncated promoter constructs by cloning restriction enzyme fragments into the pGL-Basic vector (Promega, Madison WI) containing a luciferase reporter gene: p-1422 (SacI-NruI), p-1256 (PvuII-NruI), p-831 (SacII-NruI), and p-467 (PstI-NruI). Additional mutant C/EBP promoter constructs were generated using the –1256 to +4 C/EBP genomic DNA sequence cloned into a pGL3-Basic vector as template and a site-directed mutagenesis kit (QuikChange; Stratagene). The C/EBP promoter sequence contains three USF binding sites (E1–E3) and one SP1 binding site. Sequence E1 5'-CACGTG-3' from position –1206 to –1200, sequence E2 5'-CACCTG-3' from position –994 to –989, sequence Sp-1 5'-CCCCGC-3' from position –937 to –931, and sequence E3 5'-CCCGTG-3' from position –877 to –871 were changed to 5'-GAATTC-3' to generate E1, E2, and Sp-1 mutants and to 5'-GAATCC-3' to generate the E3 mutant. Individual clones that contained mutant plasmids were selected, and plasmid DNA was purified and sequenced to confirm which clones had the correct mutations.
Human lung cancer H1299 and A549 cells (4 x 104 cells/35-mm well) and 293T and Neuro2A cells (2 x 104 cells/35-mm well) were plated in RPMI-1640 medium the day before the transfection. The next day, cells were transfected by incubation for 3 hours in a mixture of 4 µL of Superfect reagent (Invitrogen), 60 ng of Renilla luciferase plasmid pRL-TK (Promega) as the internal control, or 1 µg of reporter plasmid and 1 mL of fresh medium. Either 200 ng of wild-type C/EBP p-1256 control vector containing Simian virus 40 promoter-enhancer or promoterless pGL3-Basic vector was transfected into cells as a positive and negative control for promoter activity, respectively. Luciferase activity was measured using the Dual Luciferase assay system (Promega). Experiments were performed in triplicate and were repeated at least twice. Promoter activities are shown as means and 95% confidence intervals of triplicate transfections.
In Vitro Translation and Electrophoretic Mobility Shift Assay
In vitro–translated USF-1 and USF-2 proteins were prepared with the TNT coupled transcription–translation reticulocyte lysate kit (T7 polymerase version) (Promega) according to the manufacturer's instructions. The expression plasmids were constructed using the pTNT vector (Promega). USF-1 and USF-2 inserts were generated by PCR using PfuTurbo DNA polymerase (Stratagene) and the following primer pairs: USF-1 sense, 5'-TCTCGAGAGCACTCAGGCCTGTGAATCAGGAGATACAAAGACCTCC-3' and antisense, 5'-GCTCTAGACATATCACAGGGCCTCAGTTCAAGGACACACCTTCTGAACTTC-3'; USF-2 sense, 5'-TCTCGAGCATGGACATGCTGGACCCGGGTCTGGATCCCGCTG-3' and antisense 5'-GCTCTAGACTGTGCTAAGGGCTGGGGAAGGGGGCAGCAGAGG-3'. Each primer contains a restriction enzyme site (XhoI or XbaI) at its 5' end for subsequent subcloning into the pTNT vector (Promega). The in vitro–translated USF-1 and USF-2 proteins were analyzed by immunoblotting, and 3 µL of the total product of each reaction was used for electrophoretic mobility shift assay.
Electrophoretic mobility shift assay was performed according to the Promega Gel Shift Assay Systems protocol with minor modifications. The sequences of the oligonucleotides used to generate the probes were as follows: 1/E sense, 5'-ATGCGAGGGACGCACGTGGCTGGGGGTCTCG-3' antisense, 5'- CGAGACCCCCAGCCACGTGCGTCCCTCGCAT-3'; 2/E sense, 5'-CGCCGTTGGCGCCCACCTGAATGGGGAGGCG-3', and antisense,5'-CGCCGTTGGCGCCCACCTGAATGGGGAGGCG-3'; 3/E sense, 5'-CTCGGTGCGCCCCTCCCCGTGCTCGCCCCGGCG-3' and antisense, 5'-CGCCGGGGCGAGCACGGGGAGGGGCGCACCGAG-3'.
The underlined sequences in 1/E sense and antisense and in 2/E sense and antisense were changed to GAATCC in 1/E mut and 2/E mut probes, respectively. The underlined sequences in 3/E sense and antisense were changed to GAATTC in the 3/E mut probe. Probes (50 ng of each) were end-labeled with [
-32P]ATP using T4 kinase (Gibco BRL). Labeled 1/E, 2/E, or 3/E oligonucleotides were incubated with 3 µL of each in vitro translation reaction in 20-µL mixtures consisting of binding buffer (10 mM Tris-HCl, pH 7.5, 4% glycerol, 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, and 1 mM MgCl2) and 10 ng of poly(dI-dC)–poly(dI-dC) (Pharmacia) as a nonspecific competitor for 15 min at room temperature. The binding reactions were separated on an 8% polyacrylamide gel at 200 V. Subsequently, the gel was dried under vacuum at 80 °C for 1 hour and exposed to a PhosphorImager screen (Amersham Biosciences). Assays were performed three times.
Statistical Analysis
Statistical significance of differences between groups was analyzed by unpaired Student's t test, and P<.05 was considered to be statistically significant. Statistical significance was confirmed by the 
2 test for independence. All statistical analyses were performed using StatView software (SAS Institute Inc., Cary, NC). All statistical tests were two-sided.
For the methylation studies, the percent methylation at each CpG site was calculated, and a nonparametric Wilcoxon rank-sum test was used to compare percent methylation at individual CpG sites in C/EBP-negative versus -positive samples. From the percent methylation at all CpG sites, an average methylation level was calculated for each patient, and the Wilcoxon rank-sum test was applied to compare the average methylation of the two groups. The –log(P values) were plotted against the site location, and a cutoff value for statistical significance (P<.05 or –log[P value]> –log[.05]) is indicated by the dotted line.
|
RESULTS |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
C/EBP
Expression and C/EBP Promoter Methylation Status
C/EBP is encoded by a single exon (Supplementary Fig. 3, available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol98/issue6). The promoter and exonic regions of the gene fulfill the strict definition of a CpG island, being a 200-bp or greater stretch of DNA with a C/G content of >50% and an observed CpG/expected CpG in excess of 0.6, usually located in the promoter region of genes (25). This CpG island spans almost 3.4 kilobases and has a CpG frequency of 70% and an observed/expected CpG ratio of 0.9. We first assessed C/EBP expression in 15 human lung cancer cell lines by northern blot analysis (Fig. 1, A). C/EBP expression was normalized to GAPDH expression and was quantified relative to C/EBP expression levels in N417 cells, which showed the highest C/EBP expression in our experiment. All but two (N417 and H2086) of the lung cancer cell lines tested showed low or no C/EBP expression, in sharp contrast to normal lung tissues, among which C/EBP expression was high (Fig. 1, A).
|
To investigate the molecular mechanism of the low C/EBP
expression that we observed in lung cancer cell lines, we examined whether C/EBP expression was associated with the degree of DNA methylation or the number of methylated sites detected in the C/EBP promoter using either Southern blot, combined bisulfite restriction analysis, bisulfite sequencing or MSP, depending on the sequence composition of the region analyzed (Figs. 1, B and C, and Table 1). Increased DNA methylation—compared with normal tissue—was not found in the C/EBP core promoter (from –437 to +4 relative to the transcription start site) (26) or in the C/EBP exon (positions –411, –140, –117, –11, +448, and +585) of any of the lung cancer cell lines regardless of C/EBP expression (Figs. 1, B and C, and Table 1). Positions –795 and –776/–760 were completely methylated in H1155 cells only and were partially methylated in H290 and H1299 cells, which showed a complete lack of gene expression (Figs. 1, B and C, and Table 1). DNA methylation at position –1368 was detected in all cell lines except N417, which strongly expresses C/EBP. In addition, nearby site –1164 was methylated in all C/EBP-nonexpressing cell lines but was partially methylated in H2086 cells and unmethylated in N417 cells.
|
These data suggested a possible association between C/EBP
expression and the DNA methylation status of the upstream region of the promoter in the lung cancer cell lines that we examined. Thus, we evaluated the DNA methylation status of individual CpG dinucleotides from –1422 to –896 by bisulfite sequencing (Supplementary Fig. 4, available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol98/issue6). C/EBP-expressing and -nonexpressing cell lines differed in the degree of DNA methylation between positions –1250 and –896. Samples with strong C/EBP expression had less promoter methylation (9.4%, 32 methylated CpG dinucleotides of 340 CpG dinucleotides in H2086; 0%, 0 of 340 in N417; and 9.9%, 37 of 374 in normal lung tissues) than did nonexpressing or low-expressing cell lines (58%, 197 of 340 in H719; and 56%, 189 of 340 in H1299). These data further support the hypothesis that hypermethylation of the upstream promoter region could be involved in the low C/EBP expression in human lung cancer cell lines.
C/EBP Expression in Lung Cancer Cell Lines After Treatment With 5-aza-dC and/or TSA
To determine whether the low C/EBP expression in H719 and H1299 cells, which have a high degree of DNA methylation in the upstream region of the promoter, could be modulated, we treated the cells with 5 µM 5-aza-dC for 72 hours and/or with 300 nM TSA for 24 hours and measured C/EBP expression. An increase in C/EBP expression after treatment was detected at both the RNA (Fig. 2, A) and protein (Fig. 2, C) levels. Treatment with both drugs resulted in higher C/EBP expression than treatment with either drug alone (Fig. 2, A). This additive interaction between TSA and 5-aza-dC is consistent with changes in the expression of a number of other genes previously observed in cancer cell lines (15). Next, we evaluated the DNA methylation status of the upstream region of the promoter before and after 5-aza-dC treatment. In the untreated cell lines, combined bisulfite restriction analysis revealed methylation at positions –1422 to –1121; however, after 5-aza-dC treatment, there was a marked loss of DNA methylation in this region (Fig. 2, B).
|
Loss of C/EBP
Expression and Aberrant DNA Methylation of the C/EBP Upstream Promoter in Primary Lung Cancers
To assess the frequency of C/EBP gene silencing in primary human lung tumors, we performed immunohistochemical analysis on 122 non–small-cell lung cancers. In normal lung, strong staining was observed in the basal cell layer of bronchi and in type II pneumocytes (20). Overall, greater than half (81 of 120) of the specimens had no detectable C/EBP expression by immunohistochemistry. C/EBP expression was absent in 24 of 51 adenocarcinomas and in 57 of 69 squamous cell carcinomas (Table 2).
|
To determine whether C/EBP
promoter hypermethylation and gene expression have a direct relationship, the upstream region of the C/EBP promoter was bisulfite sequenced using microdissected material from patient 1, which showed high C/EBP expression in the tumor cells, and from patient 2, which showed no detectable expression. Methylation was low in the cancerous epithelium and noncancerous adjacent tissue of patient 1 and high in tumor tissue of patient 2 (Fig. 3, A and B). Microdissected material collected for other samples that either expressed or did not express C/EBP was also analyzed. A two-sided nonparametric Wilcoxon rank-sum test was used to test the percent methylation in C/EBP-negative versus -positive samples for each CpG site and for the whole region (–1211 to –896) (Fig. 3, C). Selection of this specific region was based on combined bisulfite analysis data showing no DNA methylation in other downstream areas (data not shown). Statistically significantly higher methylation was found in C/EBP-negative samples than in C/EBP-positive samples (P = .002, Fig. 3, C).
|
Hypermethylation, Histone Deacetylation, and MBD Protein Binding of the Upstream C/EBP
Promoter Region in Lung Adenocarcinoma Cells
Methylation-induced gene silencing is thought to be associated with a modified chromatin structure that is enriched in deacetylated histones (27). To examine the histone acetylation status within the CpG island of the C/EBP gene, we performed chromatin immunoprecipitation using antibodies against the acetylated forms of histones H3 and H4 in three non–small-cell lung cancer cell lines (Fig. 2, D–E). After PCR amplification of various regions of the C/EBP CpG island, an enrichment of acetylated histones was observed in the core promoter and exonic regions of all lung cancer cell lines, regardless of C/EBP expression level (Fig. 2, D and E). No acetylated histones were found associated with the upstream promoter of the H719 and H1299 cell lines, whereas histone acetylation was detected in region –1106 to –887 in the H23 and A549 cell lines, both of which express intermediate or low levels of C/EBP. Acetylated H3 and H4 histones were detected within all regions of the C/EBP promoter in H2086, a cell line with strong C/EBP expression. Overall, the acetylation status of histones H3 and H4 was associated directly with gene expression and inversely with DNA methylation in the C/EBP upstream promoter region.
MeCP2 and MBD2 are methyl-CpG binding proteins that suppress transcription from methylated promoters (28). To determine the binding status of MeCP2 and MBD2 within the C/EBP CpG island, we used chromatin immunoprecipitation assays (Fig. 2, E). In H719 and H1299 cells, MeCP2 and MBD2 binding was detected in the upstream region of the C/EBP promoter, whereas in H23 and A549 cells, binding was detected in the –1413 to –887 area. In contrast, H2086 cells showed weak or no MBD2/MeCP2 binding in the upstream promoter region (data not shown). Thus, MBD binding was directly associated with the DNA methylation status of the C/EBP upstream promoter and inversely with C/EBP expression.
The C/EBP Upstream Promoter Region and Gene Activity in Human Cells
A previous report identified the C/EBP core promoter as from –437 to +4 relative to the transcription start site (26). Thus, luciferase reporter assays using C/EBP 5'-promoter deletion constructs were performed to determine whether the upstream promoter region of the C/EBP gene had a role in transcriptional regulation. The luciferase activities of p-1422 and of wild-type p-1256 were similar (Fig. 4, A), whereas the activities of truncated p-831 (SacII-NruI) and p-467 (PstI-NruI) were approximately 50% lower than that of p-1422. These results suggested that a putative positive cis-acting regulatory element is located in the –1256 to –831 region.
|
Several consensus motifs for USF-1/-2 and Sp-1 transcription factor binding sites were identified in the –1256 to –831 promoter region of C/EBP
(Fig. 4, B). Constructs containing the C/EBP promoter sequence with specific mutations in one of the three USF or Sp-1 binding sites or in multiple consensus sequences of the three binding elements were assayed for luciferase expression in Neuro2A, A549, and H1299 cell lines (Fig. 4, B). Compared with the wild-type p-1256 construct, the USF-1/-2 mutant reporter, in which the first upstream USF site was mutated (E1), showed a slight reduction in luciferase activity. The activity of the USF-1/-2 mutant construct, in which the second USF site (E2) was mutated, was even lower. Depending on the cell line used, the Sp-1 mutant reporter constructs showed approximately 40%–60% lower promoter activity than wild-type p-1256. The luciferase activity of E1, E2 double mutants, and E1, E2, and E3 triple mutants was decreased (by 50%–80% and 90%, respectively) when compared with wild-type p-1256. Together, these data indicate that the upstream promoter region of C/EBP is important for gene expression, possibly because of the interaction of this region with the transcriptional activators USF-1/-2 and Sp-1.
USF-1 and USF-2 Binding to the Upstream Promoter Region of C/EBP Gene in Vivo and in Vitro
Two putative USF binding sites between positions –1413 and –831 of the C/EBP promoter had been reported previously (26). A third putative USF site was identified by sequence analysis in this study. To investigate the binding of USF-1 and USF-2 to the C/EBP promoter in vivo, chromatin immunoprecipitation assays were performed. USF-1/-2 binding was not detected at any of the three candidate binding sites of the upstream promoter region in the C/EBP-nonexpressing cell lines, whereas binding was observed in C/EBP-expressing H2086 cells (Fig. 5, A). In addition, USF-1/-2 binding in C/EBP-nonexpressing cell lines was observed after TSA and 5-aza-dC treatment.
|
Overall, these data suggest that epigenetic silencing of C/EBP
is associated with inhibition of USF-1 and USF-2 binding. We could not assess USF binding at each consensus site individually using chromatin immunoprecipitation because of the close proximity of these sites. Thus, we performed electrophoretic mobility shift assays using in vitro–translated USF-1 and USF-2 proteins. Three probes, each containing a USF binding motif, were designed. 1/E represents the most upstream site, E/2 is located in between, and E/3 is closest to the core promoter. USF binding was observed with the 1/E and 2/E probes, but neither USF-1 nor USF-2 binding was detected with the 3/E probe (Fig. 5, B). These results further support the importance of USF-1/-2 binding to the upstream promoter region of C/EBP and the involvement of USF-1/-2 in the regulation of C/EBP expression. |
DISCUSSION |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study, we tested the hypothesis that C/EBP
expression was decreased in human lung cancer by aberrant DNA methylation and histone modifications in the promoter region of the C/EBP gene. To do this, we examined C/EBP expression and the DNA methylation status of its promoter in lung cancer cell lines and in primary lung tumors and adjacent normal tissues. The finding of suppressed C/EBP expression is in agreement with a previous report (20). Overall, 68% (n = 120) of human lung cancer specimens had no detectable C/EBP expression by immunohistochemistry. C/EBP expression was absent in 47% of adenocarcinomas (n = 51) and 83% of squamous cell carcinomas (n = 69). We found that the core promoter region of C/EBP was unmethylated in all lung cancer cell lines analyzed, regardless of their C/EBP expression status. However, aberrant DNA methylation in the upstream promoter region of CpG islands decreased C/EBP transcription in both lung cancer cell lines and primary human lung tumors. C/EBP expression was directly associated with CpG methylation, the acetylation status of histones H3 and H4, and the binding of MBD2 and MeCP2 in the upstream promoter regulatory region. Expression could be induced or restored by treating cell lines with DNA methyltransferase and histone deacetylase inhibitors. Our data demonstrate that USF and Sp-1 transcription factor binding sites in the upstream promoter region have an important role for C/EBP promoter activity.
Methylation-induced suppression of gene transcription is thought to occur either by direct interference with the binding of transcription factors or through the action of MBD proteins, which trigger a cascade of chromatin modifications that result in a condensed chromatin structure. Our data show that C/EBP belongs to a recently identified group of genes regulated by several MBD proteins (29). Here we found that the binding of USF-1/-2 to the C/EBP upstream promoter was reduced by the binding of MBDs, which is associated with chromatin modifications. We observed an association between low C/EBP expression, the hypermethylation of the upstream promoter region, and local histone deacetylation. In addition, MeCP2 and MBD2 binding were observed in the upstream C/EBP promoter in cells with low C/EBP expression. The distal promoter region contains several putative consensus sequences for MeCP2 binding ([A/T] 
4) adjacent to methyl-CpG) as recently described (30), whereas the core promoter contains only a single putative site. Our data suggest that MeCP2 and MBD2 binding to the upstream promoter blocks USF-1/-2 binding, which leads to lowered C/EBP expression. We also showed that the mutations in the USF/Sp-1 binding elements of the C/EBP upstream promoter reduced promoter activity. This scenario is unique because epigenetic regulation of C/EBP is not occurring at the core promoter, and further suggests that epigenetic alterations are participating in the modulation of gene expression levels rather than in the complete inactivation of the gene (Fig. 5, C). Consequently, complete silencing of C/EBP would require additional regulatory events. Thus, we hypothesize that aberrant DNA methylation could spread from the 5'-flanking regions into the upstream promoter region. However, this spreading is blocked upstream of the core promoter, which is protected from aberrant methylation by unknown mechanisms, leaving the chromatin in an open and transcriptionally permissive configuration.
The role of C/EBP in terminal differentiation of myeloid cells, hepatocytes, and adipocytes is well established. However, its role in lung epithelial cells is not completely understood. C/EBP has been identified in acute myeloid leukemia as a tumor suppressor, inactivated by genetic mutations (21,31). C/EBP is strongly expressed in type II pneumocytes and bronchial epithelial cells, and its importance in lung development was demonstrated in C/EBP–/– mice, which show hyperproliferation of type II pneumocytes and abnormal alveolar structure. It has recently been shown that C/EBP expression is low in non–small-cell lung cancers and that C/EBP re-expression results in growth reduction, proliferation arrest, differentiation, and apoptosis, which suggests that C/EBP could perform a candidate tumor suppressor function in non–small-cell lung cancer (20). In contrast to the findings of Halmos et al. (20), we found a higher frequency of low C/EBP expression in squamous cell carcinomas, possibly due to the smaller sample size (n = 9) used in the Halmos et al. study. Further studies on large well-characterized sample sets are needed to clarify the importance of reduced C/EBP expression in individual subtypes of lung cancer as well as clinical outcome. In addition, studies to determine associations between C/EBP expression and epigenetic and genetic alterations would provide a better understanding of the contribution of each of these mechanisms to C/EBP regulation.
It should be noted that the binding of MBD1 to the C/EBP promoter was not tested in our study. Thus, it is possible that C/EBP regulation is also dependent on the presence of MBD1 in the cellular environment. Given the complexity of C/EBP regulation, which depends not primarily on the core promoter but on the chromatin conformation of an upstream enhancer region, it should be considered that transcription factors other than Sp-1 and USF-1 might participate in activation of C/EBP in human lung tissue. Moreover, we would like to mention that aberrant DNA methylation in an area other than the ones analyzed in our study might also contribute to the modulation of C/EBP in the disease state.
In the lung, C/EBP has been shown to regulate the activity of another transcription factor, hepatocyte nuclear factor 3
(HNF3), which is also expressed at low levels in a large number of lung cancers (32). In non–small-cell lung cancer, C/EBP expression is observed primarily in differentiated lung tumors. This association between the stage of differentiation and C/EBP expression underlines the importance of C/EBP in lung epithelial cells. Our results further suggest that C/EBP may be an appropriate therapeutic target for reactivation through demethylation treatment with compounds such as 5-azacytidine and 5-dC (33,34) or histone deacetylase inhibitors, such as depsipeptide (35,36), some of which are currently in clinical trials (37,38). In addition, C/EBP expression should be investigated for possible use as a biomarker for early detection of lung cancer.
|
NOTES |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
C. Plass is a scientific consultant for Epigenomics, Inc.
The work was supported by grants from Translational V-Foundation Award (to C. Plass), in part by P30CA16058 and by a grant from the Dr. Mildred Scheel Foundation for Cancer Research (to B. Hackanson). C. Plass is a Scholar of the Leukemia Lymphoma Society of America. Funding agencies provided salary support and support for supply cost and had no role in the study design, data collection, analysis, and interpretation of the findings.
The authors thank Laura Rush, Aparna Raval, Kristi Bennett, and Danilo Perrotti for constant support and helpful discussions and Sandya Liyanarachchi for helping with the statistical analysis. The C/EBP construct for transfection was kindly provided by Dr. Rob Smart, Cold Spring Harbor.
|
REFERENCES |
|---|
|
TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
(1) Jemal A, Clegg LX, Ward E, Ries LA, Wu X, Jamison PM, et al. Annual report to the nation on the status of cancer, 1975–2001, with a special feature regarding survival. Cancer 2004;101:3–27.[CrossRef][ISI][Medline]
(2) Wistuba II, Gazdar AF. Characteristic genetic alterations in lung cancer. Methods Mol Med 2003;74:3–28.[Medline]
(3) Fong KM, Sekido Y, Gazdar AF, Minna JD. Lung cancer. 9: Molecular biology of lung cancer: clinical implications. Thorax 2003;58:892–900.
(4) Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature 2004;429:457–63.[CrossRef][Medline]
(5) Hsu HS, Wen CK, Tang YA, Lin RK, Li WY, Hsu WH, et al. Promoter hypermethylation is the predominant mechanism in hMLH1 and hMSH2 deregulation and is a poor prognostic factor in nonsmoking lung cancer. Clin Cancer Res 2005;11:5410–6.
(6) Irimia M, Wen CK, Tang YA, Esteller M. Promoter hypermethylation of the predominant mechanism in the context of a wild-type K-ras in lung cancer. Oncogene 2004;23:8695–9.[CrossRef][ISI][Medline]
(7) Belinsky SA. Gene-promoter hypermethylation as a biomarker in lung cancer. Nat Rev Cancer 2004;4:707–17.[CrossRef][ISI][Medline]
(8) Li J, Zhang Z, Dai Z, Popkie AP, Plass C, Morrison C, et al. RASSF1A promoter methylation and Kras2 mutations in non small cell lung cancer. Neoplasia 2003;5:362–6.[ISI][Medline]
(9) Zabarovsky ER, Lerman MI, Minna JD. Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene 2002;21:6915–35.[CrossRef][ISI][Medline]
(10) Dai Z, Popkie AP, Zhu WG, Timmers CD, Raval A, Tannehill-Gregg S, et al. Bone morphogenetic protein 3B silencing in non-small-cell lung cancer. Oncogene 2004;23:3521–9.[CrossRef][ISI][Medline]
(11) Medina PP, Carretero J, Fraga MF, Esteller M, Sidransky D, Sanchez-Cespedes M. Genetic and epigenetic screening for gene alterations of the chromatin-remodeling factor, SMARCA4/BRG1, in lung tumors. Genes Chromosomes Cancer 2004;41:170–7.[CrossRef][ISI][Medline]
(12) Birkenmeier EH, Gwynn B, Howard S, Jerry J, Gordon JI, Landschulz WH, et al. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev 1989;3:1146–56.
(13) Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc Natl Acad Sci U S A 1997;94:569–74.
(14) Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 1998;72:141–96.[ISI][Medline]
(15) Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 1999;21:103–7.[CrossRef][ISI][Medline]
(16) Flodby P, Barlow C, Kylefjord H, Ahrlund-Richter L, Xanthopoulos KG. Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein alpha. J Biol Chem 1996;271:24753–60.
(17) McKnight SL. McBindall—a better name for CCAAT/enhancer binding proteins? Cell 2001;107:259–61.[CrossRef][ISI][Medline]
(18) Wang ND, Finegold MJ, Bradley A, Ou CN, Abdelsayed SV, Wilde MD, et al. Impaired energy homeostasis in C/EBP alpha knockout mice. Science 1995;269:1108–12.
(19) Watkins PJ, Condreay JP, Huber BE, Jacobs SJ, Adams DJ. Impaired proliferation and tumorigenicity induced by CCAAT/enhancer-binding protein. Cancer Res 1996;56:1063–7.
(20) Halmos B, Huettner CS, Kocher O, Ferenczi K, Karp DD, Tenen DG. Down-regulation and antiproliferative role of C/EBP in lung cancer. Cancer Res 2002;62:528–34.
(21) Gombart AF, Hofmann WK, Kawano S, Takeuchi S, Krug U, Kwok SH, et al. Mutations in the gene encoding the transcription factor CCAAT/enhancer binding protein in myelodysplastic syndromes and acute myeloid leukemias. Blood 2002;99:1332–40.
(22) Tada Y, Wada M, Taguchi K, Mochida Y, Kinugawa N, Tsuneyoshi M, et al. The association of death-associated protein kinase hypermethylation with early recurrence in superficial bladder cancers. Cancer Res 2002;62:4048–53.
(23) Smiraglia DJ, Fruhwald MC, Costello JF, McCormick SP, Dai Z, Peltomaki P, et al. A new tool for the rapid cloning of amplified and hypermethylated human DNA sequences from restriction landmark genome scanning gels. Genomics 1999;58:254–62.[CrossRef][ISI][Medline]
(24) Dai Z, Zhu WG, Morrison CD, Brena RM, Smiraglia DJ, Raval A, et al. A comprehensive search for DNA amplification in lung cancer identifies inhibitors of apoptosis cIAP1 and cIAP2 as candidate oncogenes. Hum Mol Genet 2003;12:791–801.
(25) Takai D, Jones PA. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci U S A 2002;99:3740–5.
(26) Timchenko N, Wilson DR, Taylor LR, Abdelsayed S, Wilde M, Sawadogo M, et al. Autoregulation of the human C/EBP gene by stimulation of upstream stimulatory factor binding. Mol Cell Biol 1995;15:1192–202.[Abstract]
(27) Bird AP, Wolffe AP. Methylation-induced repression—belts, braces, and chromatin. Cell 1999;99:451–4.[CrossRef][ISI][Medline]
(28) Cross SH, Meehan RR, Nan X, Bird A. A component of the transcriptional repressor MeCP1 shares a motif with DNA methyltransferase and HRX proteins. Nat Genet 1997;16:256–9.[CrossRef][ISI][Medline]
(29) Ballestar E, Paz MF, Valle L, Wei S, Fraga MF, Espada J, et al. Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer. EMBO J 2003;22:6335–45.[CrossRef][ISI][Medline]
(30) Klose RJ, Sarraf SA, Schmiedeberg L, McDermott SM, Stancheva I, Bird AP. DNA binding selectivity of MeCP2 due to a requirement for A/T sequences adjacent to methyl-CpG. Mol Cell 2005;19:667–78.[CrossRef][ISI][Medline]
(31) Nerlov C. C/EBP mutations in acute myeloid leukaemias. Nat Rev Cancer 2004;4:394–400.[CrossRef][ISI][Medline]
(32) Halmos B, Basseres DS, Monti S, D'Alo F, Dayaram T, Ferenczi K, et al. A transcriptional profiling study of CCAAT/enhancer binding protein targets identifies hepatocyte nuclear factor 3 as a novel tumor suppressor in lung cancer. Cancer Res 2004;64:4137–47.
(33) Lubbert M. DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of action. Curr Top Microbiol Immunol 2000;249:135–64.[ISI][Medline]
(34) Cheng JC, Weisenberger DJ, Gonzales FA, Liang G, Xu GL, Hu YG, et al. Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol Cell Biol 2004;24:1270–8.
(35) Byrd JC, Stilgenbauer S, Flinn IW. Chronic lymphocytic leukemia. Hematology (Am Soc Hematol Educ Program) 2004:163–83.
(36) Byrd JC, Rai K, Peterson BL, Appelbaum FR, Morrison VA, Kolitz JE, et al. Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011. Blood 2005;105:49–53.[Medline]
(37) Belinsky SA, Klinge DM, Stidley CA, Issa JP, Herman JG, March TH, et al. Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer. Cancer Res 2003;63:7089–93.
(38) Momparler RL, Ayoub J. Potential of 5-aza-2'-deoxycytidine (Decitabine) a potent inhibitor of DNA methylation for therapy of advanced non-small cell lung cancer. Lung Cancer 2001;34 Suppl 4:S111–5.[Medline]
Manuscript received June 2, 2005; revised January 3, 2006; accepted January 26, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Li, H.-i. H. Paik, C. Balch, Y. Kim, L. Li, T. H-M. Huang, K. P. Nephew, and S. Kim Enriched transcription factor binding sites in hypermethylated gene promoters in drug resistant cancer cells Bioinformatics, August 15, 2008; 24(16): 1745 - 1748. [Abstract] [PDF]
|
|
|
|
 |
|
 G. Huang, R. Eisenberg, M. Yan, S. Monti, E. Lawrence, P. Fu, J. Walbroehl, E. Lowenberg, T. Golub, J. Merchan, et al. 15-Hydroxyprostaglandin Dehydrogenase is a Target of Hepatocyte Nuclear Factor 3{beta} and a Tumor Suppressor in Lung Cancer Cancer Res., July 1, 2008; 68(13): 5040 - 5048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Hackanson, K. L. Bennett, R. M. Brena, J. Jiang, R. Claus, S.-S. Chen, N. Blagitko-Dorfs, K. Maharry, S. P. Whitman, T. D. Schmittgen, et al. Epigenetic Modification of CCAAT/Enhancer Binding Protein {alpha} Expression in Acute Myeloid Leukemia Cancer Res., May 1, 2008; 68(9): 3142 - 3151. [Abstract] |