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© The Author 2006. Published by Oxford University Press.
ARTICLE |
Decreased STAT1 Expression by Promoter Methylation in Squamous Cell Carcinogenesis
Affiliations of authors: Departments of Otolaryngology (SX, KFD, RLC, JRG), Pharmacology (QZ, JRG), Molecular Genetics and Biochemistry (JRC), Pathology (JH), University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Department of Biostatistics (WEG, BZ), Department of Human Genetics (MK, REF), University of Pittsburgh, Pittsburgh, PA
Correspondence to: Jennifer Rubin Grandis, MD, The Eye and Ear Institute, 200 Lothrop St., Ste. 500, Pittsburgh, PA 15213 (e-mail: jgrandis{at}pitt.edu).
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ABSTRACT |
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 TopNotes Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Background: Dysregulation of signal transducers and activators of transcription (STATs) is associated with many cancers, but no role of STAT1 in human tumor progression has been demonstrated. Methods: We compared STAT1 protein expression in squamous cell carcinoma of the head and neck (SCCHN) tumors (n = 28) and normal oropharyngeal mucosa samples (n = 10) from patients without cancer as assessed by immunoblotting. Stable clones were established from SCCHN 1483 cells that were transfected with a STAT1 expression construct; cell growth and cisplatin-induced apoptosis of the clones and vector control–transfected 1483 cells were compared using trypan blue exclusion and Annexin V staining and expression of the cyclin-dependent kinase inhibitor p21 was assayed by immunoblotting. The growth of STAT1-overexpressing SCCHN 1483 xenograft tumors was compared with that of xenograft tumors derived from cells transfected with vector control DNA. DNA from SCCHN tumors (n = 16) and paired peripheral blood lymphocytes were analyzed for STAT1 mutations and promoter methylation using methylation-specific polymerase chain reaction and bisulfite sequencing. SCCHN cell lines (PCI-15b, 1483, and UM-22B) were treated with the demethylating agent azacytidine alone or in combination with the cytotoxic drug cisplatin, and expression of STAT1 and p21 were monitored by immunoblotting. All statistical tests were two-sided. Results: STAT1 levels were statistically significantly lower in the SCCHN tumors than normal mucosa (median = 0.8 relative units versus 2.4, difference = 1.6, 95% confidence interval [CI] = 1.3 to 2.0, P<.001). Overexpression of STAT1 abrogated the growth of SCCHN cells and xenograft tumors and increased p21 expression. STAT1 expression levels of the tumors with STAT1 promoter methylation (n = 12) were lower than those of tumors (n = 4) without promoter methylation of STAT1 (P = .008). Azacytidine treatment increased expression of STAT1 and p21 in SCCHN cell lines and increased apoptosis in cisplatin-treated 1483 cells compared with cisplatin treatment alone (mean = 61.3% versus 25.8%, difference = 35.5%, 95% CI = 24.5% to 43.4%; P = .028). Conclusion: STAT1 can function as a tumor suppressor in SCCHN cells. Silencing of the STAT1 gene via promoter methylation may contribute to SCCHN tumor cell growth.
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INTRODUCTION |
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The signal transducer and activator of transcription (STAT) proteins are a family of transcription factors with seven members: STAT1, 2, 3, 4, 5A, 5B, and 6 (1). Dysregulation of STAT signaling has been implicated in tumor formation and progression (2). STATs 3 and 5 are constitutively active in many hematopoietic and epithelial malignancies, in which strategies to target these STATs inhibit growth and induce apoptosis (1,3). In contrast, cumulative and largely indirect evidence supports a tumor suppressor function for STAT1. STAT1–/– mice are highly susceptible to cancers that are induced by chemical carcinogens (4). Also, restoration of STAT1 in RAD-105 tumor cells derived from a fibrosarcoma of a STAT1-knockout mouse suppresses tumorigenicity and metastasis (5). The biologic significance of the tumor suppressive function of STAT1 is suggested by the finding that increased expression of phosphotyrosine STAT1 in human breast cancers was associated with a statistically significant improvement in survival (6). The human STAT1 gene is on the long arm of chromosome 2, and loss of heterozygosity (LOH) at 2q has been shown to be predictive of early recurrence and death in squamous cell carcinoma of the head and neck (SCCHN) (7). These cumulative, albeit indirect, results suggest that the loss of STAT1 signaling enhances oncogenesis.
Studies implicating STATs in proapoptotic signaling have focused largely on STAT1 in the context of interferon (IFN)-induced growth inhibition and apoptosis, in which IFN-
–mediated STAT1 activation leads to growth suppression via induction of the cyclin-dependent kinase inhibitor p21/waf1 (8). The promoter region of p21 has been shown to contain binding sites for STAT1 (8). However, studies to date have not linked the regulation of p21 by STAT1 with STAT1's tumor suppressor function. Demonstration of p21 regulation by STAT1 would provide insight into the molecular mechanisms underlying the tumor suppressive role of STAT1 in human cancers.
Although STAT1 activation in tumors appears to contribute to growth arrest rather than to cell survival, it is not clear how altered STAT1 leads to tumor formation and progression. In this study, we analyzed the role of STAT1 in SCCHN by determining expression levels in human tissues and the consequences of STAT1 overexpression on p21 transcription and chemotherapy-induced apoptosis in SCCHN cell lines. We examined the effect of STAT1 overexpression on the growth of SCCHN xenograft tumors. To determine the tumor suppressive mechanism of STAT1 in SCCHN, we collected fresh tumor and paired peripheral blood lymphocytes from SCCHN patients and for gene mutations of STAT1 and methylation of CpG islands in the STAT1 promoter. SCCHN cell lines with methylated STAT1 promoters were treated with the demethylating agent, 5'-aza-2'-deoxycytidine (5-Aza-dC, azacytidine) to verify that promoter methylation is the primary mechanism decreasing STAT1 expression in SCCHN.
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MATERIALS AND METHODS |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Tissues
Tissues were collected according to a University of Pittsburgh internal review board–approved protocol, and written informed consent was obtained from all subjects. Tumor and adjacent normal mucosa were collected from 28 SCCHN patients at the time of curative surgical resection. Normal mucosa was collected from 10 subjects without cancer who underwent soft palate resection (uvulopalatopharyngoplasty). All tissues were snap-frozen after histologic confirmation by a head and neck pathologist (J. Hunt) and processed for DNA and protein extraction. Blood was obtained from 16 of the SCCHN subjects and processed for DNA extraction For DNA extraction, genomic DNA from fresh-frozen tissue samples and blood samples was extracted using the QiaAmp DNA mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. DNA quantitation was determined by a spectrophotometry apparatus (NanoDrop Technologies, Inc., Wilmington, DE, USA) (9).
Cell Culture, Transfection, and Treatment
SCCHN (1483, PCI-15b, UM-22B) cells were derived from SCCHN tumors as described previously (10–12) and were grown in Dulbecco's modified Eagle medium (DMEM; Mediatech, Herndon, VA) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA). SCCHN 1483 cells were transfected with STAT1 cDNA containing an internal neomycin resistance marker (13) or a control vector (13) using Lipofectamine (Invitrogen) followed by isolation and expansion of individual clones as described previously (3). Stable clones (1–5) were selected in DMEM with 10% fetal bovine serum containing 500 µg/mL of G418 sulfate (Geneticin; Invitrogen). Cell survival was determined by trypan blue dye exclusion (14) in triplicate and repeated three times. For demethylation studies, azacytidine (Sigma, St. Louis, MO) was added to cells at a final concentration of 1 µM in acetic acid–water (1 : 1) for 48 hours (15). SCCHN cells were stably transfected with a dominant-negative STAT1 construct, in which tyrosine 701 has been replaced by a phenylalanine, HA-STAT1F, and characterized in our laboratory as described previously (3). Cisplatin (20 µM) was added to the stable cell lines, which were incubated for 24 hours before being harvested.
Luciferase Reporter Assays
SCCHN cells (1483) that were stably transfected with STAT1 cDNA (DA STAT1) or HA-STAT1F (DN STAT1) respectively, were transiently transfected with p21-luciferase construct (a generous gift from Dr. Xian-Fan Wang, Duke University, Durham, NC) (16) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The 1483 cells were plated at a density of 5 x 105 cells/mL in six-well plates (Falcon Plastics, Cockeysville, MD) the day before transfection in DMEM. Two days after transient transfection, when cells reached 60%–70% confluence, they were washed three times with cold phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), lysed with lysis buffer (10 mM Tris HCl, pH 7.6, 50 mM Na4P2O7, 50 mM NaF, 1 mM NaV3O4, 10 mM phenanthroline, 1% Triton X-100, and 1x protease inhibitor cocktail), and scraped off the plate. The lysate was then centrifuged at 4 °C, 1000g for 20 minutes. Supernatant was collected for protein quantification using the Protein Assay Solution (BioRad Laboratories, Hercules, CA) and bovine serum albumin of known concentration as the standard. Cell lysates were assayed for luciferase activity as described previously (17). Relative luciferase activity units (RLUs) were normalized to micrograms of total protein (17). All samples in experiments were performed in duplicate.
Western Blot Analysis
Whole-cell extracts from homogenized frozen SCCHN tumors, control mucosa samples, and SCCHN cell pellets were analyzed by western blotting, as described previously (18) with rabbit anti–human STAT1 polyclonal antibody (1 : 2000; Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti-p21 (1 : 2000), or polyclonal STAT3 antibody (1 : 2000, Santa Cruz Biotechnology), and mouse anti–human 
-actin monoclonal antibody as a control for protein loading and transfer (1 : 5000; Oncogene Research Products, Boston, MA). The blot was then incubated with the secondary antibody (1 : 3000, goat anti–rabbit/Mouse immunoglobulin G–horseradish peroxidase conjugate; BioRad Laboratories) for 1 hour. Band intensities were quantified by densitometry with Molecular Analyst software (Alpha Digidoc, Alpho-Innotech, San Leandro, CA). Three independent experiments were performed for each tissue/cell type.
In Vitro Apoptosis
Following treatment with cisplatin, 1483 cells that were transfected with STAT1 cDNA (clone 4) or with control vector were detached by trypsinization, counted, and pelleted (1000g) at 4 °C for 5 minutes. Cell pellets were washed once with phosphate-buffered saline, pH 7.4, and resuspended in 100 µL of Annexin V binding buffer (Biovision, Mountain View, CA), as previously described (19). The ratio (percentage) of apoptotic to total cells (apoptotic plus nonapoptotic cells) was calculated for each high-power (x40) field. For each treatment, cells in five to 10 fields were quantified per sample.
Tumor Xenograft Studies
Female athymic nude mice (n = 10) 4–6 weeks of age (nu/nu; Harlan Sprague Dawley, Indianapolis, IN) were injected subcutaneously with 106 STAT1-overexpressing 1483 cells on the right flank (clone 4) and 106 1483 vector control cells on the left flank. Tumors were measured with calipers at 3, 5, 7, 9, 11, 13, 15, and 17 days after inoculation, and tumor volumes were calculated with the following formula: volume = length x width2/2. Animal care was in strict compliance with institutional guidelines established by the University of Pittsburgh, the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, 1996) (20), and the Association for Assessment and Accreditation of Laboratory Animal Care International. The animal studies were approved by the Institutional Animal Care and Use Committee.
Sequencing
Tumor genomic DNA was isolated from peripheral blood lymphocytes of 16 of the 28 tumor specimens from patients for whom peripheral blood lymphocytes were available. Control genomic DNA was isolated from EDTA anticoagulated whole blood from the same patients by the method of Miller et al. (21). Methods for analysis of the markers used in the genome scan are reported at the National Heart, Lung, and Blood Institute Mammalian Genotyping Service Web site (22,23). Amplification and sequencing primers were synthesized by the DNA Synthesis Facility at the University of Pittsburgh. Amplimers were subjected to cycle sequencing using the dRhodamine terminator ready reaction kit or the Dye Primer ready reaction kit for –M13 and M13 Rev primers (Perkin Elmer, Wellesley, MA) and analyzed on the Prism ABI 377 fluorescent sequencer. Sequences were aligned for further analysis using SEQUENCHER 3.0 (Gene Codes, Milpitas, CA).
Bisulfite Modification and Methylation-Specific Polymerase Chain Reaction Assay
Genomic DNA (2 mg) from tumor and matched peripheral blood mononuclear cells from 16 SCCHN patients was denatured with 1 M NaOH. Bisulfite treatment—during which methylated DNA is protected and unmethylated cytosine is converted to uracil—was carried out for 16 hours at 50 °C on denatured genomic DNA, as described previously (24). DNA samples were then purified using the Wizard DNA Cleanup System (Promega, Madison WI), treated with 1 M NaOH, ethanol precipitated, and resuspended in 100 µL of water. The modified DNA was used as a template for methylation-specific polymerase chain reaction (PCR) using primers specific for either the methylated or modified unmethylated sequences. Appropriate negative and positive controls were included in each PCR. The primers for methylation-specific PCR were designed using the Web site http://www.urogene.org/methprimer, as previously described (25). Primer sequences used to amplify a 269-bp unmethylated product were 5'-AAATTTGTTTTTTGTTTGGATTTTT-3' (sense) and 5'-ACCAATTAAACACAACTATTCCATA-3' (antisense), and primer sequences for the methylated reaction were 5'-AAATTTGTTTTTTGTTTGGATTTTC-3' (sense) and 5'-AATTAAACGCGACTATTCCGTA-3' (antisense), generating a 266-bp product. Stepdown PCRs were performed in a 25-µL reaction volume containing 1x PCR buffer (Invitrogen Life Technologies), 2.5 mM MgCl2, 200 mM deoxynucleoside triphosphates, 0.5 µM of each PCR primer, 0.75 U of AmpliTaq polymerase, and approximately 25 ng of bisulfite-modified DNA, as described previously (24). Reactions were hot-started at 95 °C for 5 minutes. This step was followed by 33 cycles at 95 °C for 45 seconds, 57 °C for 30 seconds, and 72 °C for 30 seconds, and a 10-min extension at 72 °C in a PTC 200 DNA Engine Thermocyler (MJ Research, Waltham, MA). Human lymphocytic DNA that was artificially methylated by treatment with SssI methylase was used as a positive control for the methylated primer set in each PCR, whereas untreated genomic DNA was used as a positive control for the unmethylated reaction. A water blank was used as a negative control with every PCR amplification. The amplification products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining and UV transillumination. The results obtained by methylation-specific PCR of the STAT1 promoter were subsequently confirmed by bisulfite sequencing (26).
Statistical Analysis
Differences between matched samples from the same SCCHN patients, such as tumor and normal tissue or tumor and peripheral blood were tested with the signed-rank test. Groups were compared with the Wilcoxon–Mann-Whitney test. Confidence intervals for the differences in medians between two groups were computed by the bias-corrected and accelerated bootstrapping methods. Differences in tumor volumes between opposing flanks of tumor-bearing mice were tested with the Wilcoxon-signed rank test at 2-day intervals. P values were adjusted for multiple testing by complete resampling (27). Confidence intervals for the median of flank differences were computed as exact non-parametric two-tailed confidence intervals based on the Wilcoxon signed-rank test. Comparisons of frequencies of promoter hypermethylation or mutation found in the tumors and peripheral blood of SCCHN patients were tested with McNemar's test. All tests were exact and two-sided.
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RESULTS |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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STAT1 Expression in SCCHN Tumors and Normal Oral Mucosa
In the few studies of STAT1 using human cancer specimens, expression and/or activation have generally been analyzed by immunoblotting or immunostaining using monoclonal anti-STAT1 or phosphotyrosine-specific antibodies, respectively (6,28,29). There are no reports comparing expression levels of STAT1 in tumors and corresponding normal tissue. To determine whether STAT1 expression was lower in SCCHN tumors than in normal oropharyngeal mucosa, STAT1 expression levels were examined by immunoblotting in 28 SCCHN tumors (Table 1) and in 10 normal tissue samples from patients without cancer. STAT1 expression levels were decreased 82.4% in SCCHN tumors compared with the levels in normal tissue harvested from individuals without cancer (median normal tissue level = 2.4 relative units, median tumor level = 0.8, difference = 1.6, 95% confidence interval = 1.3 to 2.0; P<.001, Fig. 1). Histologically normal oral mucosa from the SCCHN patients was also analyzed and the levels of STAT1 were similar to those observed in the tumors (data not shown).
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Overexpression of STAT1 and Tumor Growth In Vitro and In Vivo
Studies using STAT1-deficient cell lines have demonstrated that STAT1 is required for the antiproliferative effects of both IFN-
and - (30). Also, restoration of STAT1 in a fibrosarcoma cell line that was derived from a tumor in a STAT1-knockout mouse resulted in tumor growth inhibition in vivo (5). To test the consequences of restoring STAT1 in SCCHN, a representative SCCHN cell line (1483) was stably transfected with STAT1 cDNA. Several clones (1–5) were isolated, all of which had higher STAT1 levels than the vector-transfected control (Fig. 2, A). Growth assays showed that SCCHN cells overexpressing STAT1 grew more slowly in vitro than vector-transfected control cells (Fig. 2, B). To determine if the growth inhibitory effects of STAT1 overexpression observed in vitro could also be detected in vivo, SCCHN cells transfected with STAT1 were grown as xenograft tumors in nude mice. In each mouse, the tumor expressing increased STAT1 levels grew more slowly from day 3 onward than the tumor grown on the contralateral flank that were derived from vector-transfected control SCCHN cells (all P = .006 after day 3, Fig. 2, C).
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p21 Expression and Cisplatin-induced Apoptosis in SCCHN Cells Overexpressing STAT1
Decreased tumor volumes can result from reduced proliferation or increased apoptosis. Several potential mechanisms have been proposed to explain how STAT1 modulates apoptosis, including the increased expression of cell cycle regulatory genes. To examine the effect of overexpression of STAT1 on p21 expression, p21 protein levels were examined in vector-transfected control and STAT1-overexpressing cells by immunoblotting. Levels of p21 were increased in SCCHN cells overexpressing STAT1 compared with vector-transfected controls (Fig. 3, A). To verify that STAT1 directly regulates p21 in SCCHN cells, STAT1 overexpressing clones were transiently transfected with a p21 promoter–luciferase reporter construct, and luciferase activity was assayed as a measure of p21 promoter activity. p21 promoter activity was higher in STAT1 transfected SCCHN cells than in vector-transfected controls (Fig. 3, B).
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SCCHN cells undergo minimal spontaneous apoptosis in tissue culture. To determine whether STAT1-overexpressing cells were more susceptible to chemotherapy-induced apoptosis than cells that express low levels of STAT1, vector-transfected control and STAT1 overexpressing cells (clone 4) were treated with cisplatin (10 µM for 24 hours) and then assayed with Annexin 5–Cy3 for apoptosis using fluorescence microscopy. More of the cisplatin-treated STAT1-overexpressing SCCHN cells underwent apoptosis than did cisplatin-treated vector-transfected controls (median apoptosis for STAT1 clone + cisplatin = 57.2%, median apoptosis for control + cisplatin = 21.6%, difference = 35.6%, 95% confidence interval = 26.6% to 43.7%; P = .001, Fig. 3, C). In contrast, similar numbers of cisplatin-treated SCCHN cells expressing dominant-negative STAT1 and vector-transfected controls underwent apoptosis (Fig. 3, D).
Sequencing of STAT1 Gene in Human SCCHN Tumors and Paired Peripheral Blood Lymphocytes
The mechanisms of STAT alterations in human cancer are not completely understood. For oncogenic STAT3 and STAT5, a combination of upstream receptor and nonreceptor kinases has been implicated in their activation by phosphorylation (2). To determine whether an inactivating mutation of the STAT1 gene was responsible for the lower expression observed in tumors as compared with normal oral mucosa, we collected fresh tumor and paired peripheral blood lymphocytes from 16 of the 28 SCCHN patients, for whom STAT1 expression in the primary tumor had been analyzed. DNA was isolated and primers designed to sequence all exons of human STAT1. In these samples, there was a low frequency of variation in the proximal promoter (data not shown), but this variation appeared to constitute normal population polymorphisms. No de novo activating promoter mutations or missense mutations were observed in STAT1 in the SCCHN tumors or in the corresponding peripheral blood lymphocytes. In addition to finding no specific mutations of STAT1 in the 16 SCCHN tumors sequenced, we also detected no activating STAT3 mutations in these specimens (data not shown).
Methylation of STAT1 Promoter in SCCHN Tumors
Because the results of the STAT1 gene mutation studies did not explain the statistically significant difference in STAT1 expression levels observed in SCCHN tumors and control normal mucosa of cancer-free control subjects, methylation of CpG islands was examined as a possible mechanism to decrease STAT1 expression and to dysregulate tumor suppression gene function. The methylation of STAT1 and its association with antitumor functions of STAT1 have not been reported to date. To determine if the STAT1 promoter was methylated in SCCHN tumors, bisulfite-treated DNA (i.e., which protects methylated DNA and coverts unmethylated cytosine to uracil) from the tumors of 16 SCCHN patients was amplified with methylated- and unmethylated-specific STAT1 primers. The 269-bp product is indicative of an unmethylated STAT1 allele, whereas the 266-bp product indicates a methylated STAT1 allele (Fig. 4, A). Methylation-specific PCR results indicating tumor-specific methylation of the STAT1 promoter was confirmed by determining the methylation of CpG dinucleotides within the promoter using bisulfite genomic sequencing. Methylation-specific PCR of peripheral blood DNA demonstrated a lack of methylation, whereas methylation-specific PCR of tumor-derived DNA showed a relatively high incidence of methylation (Fig. 4, B and C). Representative methylation patterns of the STAT1 promoter from different tumor and control samples are shown in Fig. 4, D. CpG islands in the STAT1 promoter (–895 to –626 from the start site of exon 1 of STAT1) were hypermethylated in 12 of 16 tumors compared with zero of 16 of the paired peripheral blood lymphocytes from the same 16 SCCHN patients (P<.001). Further analysis demonstrated that STAT1 expression levels in the 12 tumors with STAT1 promoter methylation were lower than those in the remaining four tumors without promoter methylation of STAT1 (mean = 0.67 versus 1.49 relative units, difference = 0.82, 95% CI = 1.03 to 0.59; P = .008, Wilcoxon text, Fig. 4, E), suggesting a role for promoter methylation in the lower STAT1 expression.
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Three SCCHN cell lines (PCI-15b, 1483, and UM22B) were also used to directly test the consequences of altered DNA methylation on STAT1 function. All three cell lines showed evidence of STAT1 promoter methylation, whereas no methylation of STAT1 was observed in either of two normal immortalized epithelial cell lines (HET1-A, CCD-1106 KERTr) (ATCC, Manassas, VA) that were analyzed (Fig. 5, A and data not shown). To investigate whether demethylation could increase STAT1 expression in SCCHN, SCCHN cell lines were treated with the demethylating agent azacytidine. The STAT1 promoter was demethylated by azacytidine, as determined by methylation-specific PCR (Fig. 5, B). Demethylation of the STAT1 promoter by azacytidine increased STAT1 and p21 expression (Fig. 5, C) in all three SCCHN cell lines studied. In contrast to what was found in a previous study in a colon cancer cell line, inhibition of DNA methyltransferase did not stimulate expression of STAT3 in SCCHN cells (31) (Fig. 5, C). Also, cisplatin treatment induced increased apoptosis of 1483 cells treated with azacytidine compared with cisplatin-treated 1483 cells that were not treated with azacytidine (mean = 61.3% versus 25.8%, difference = 35.5%, 95% CI = 24.5% to 43.4%; P = .028, Wilcoxon test, Fig. 5, D), suggesting that reduced STAT1 expression makes tumors more sensitive to cytotoxic chemotherapeutic agents.
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DISCUSSION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, we observed that STAT1 expression was lower in samples from human head and neck cancers than in samples from normal oropharyngeal mucosa, primarily as a result of promoter methylation. Forced overexpression of STAT1 in SCCHN cell lines inhibited growth and increased chemotherapy-induced apoptosis. These cumulative results are consistent with STAT1 functioning as a tumor suppressor in SCCHN.
Tumor suppressor genes can regulate a diverse array of cellular functions including cell cycle progression, mitogenic signaling, and apoptotic responses (32). Studies implicating STATs in proapoptotic signaling have focused largely on STAT1 in the context of interferon-mediated growth inhibition and apoptosis. We previously demonstrated that, in contrast to the growth inhibitory effects of blocking STAT3, inhibiting STAT1 (using dominant-negative mutants or antisense oligonucleotides) actually stimulated the proliferation of SCCHN cells (3). Furthermore, activation of STAT1 is associated with increased survival in breast cancer patients (6). In this study, overexpression of STAT1 slowed growth of SCCHN cells in vitro and SCCHN tumors in vivo (Fig. 2, C). STAT1 expression levels were also decreased in all tumors compared with levels in control normal mucosa from subjects without cancer (Fig. 1, B). These results are consistent with a tumor suppressor function of STAT1 in SCCHN.
The role of STAT1 in growth arrest and apoptosis of many cell types may be explained by its capacity to induce expression of the cyclin-dependent kinase inhibitor p21/waf1 (33,34). The promoter region of p21 contains binding sites for STAT1 (8). Normally, high p21 expression is associated with cell growth arrest, but increased p21 expression has also been observed in many human cancers (35). This apparent contradiction has been explained by the requirement of p21 for the correct assembly of the D1/CDK cyclin complex; thus, increased p21 expression may be necessary for cell cycle progression (36). STAT1 has been reported to inhibit tumor cell growth and to induce apoptosis of many cell types by increasing p21/waf1 expression (33,34). In this study, overexpression of STAT1 induced expression of p21 (Fig. 3, A and B), which suggests that loss of STAT1 may contribute to increased susceptibility to apoptotic agents, at least in part via decreased p21 activation. Also, restoration of STAT1 following demethylation treatment increased p21 expression, implicating a role for p21 in STAT1 inactivation.
To test the role of STAT1 in apoptosis signal pathways in SCCHN, cells stably transfected with STAT1 were examined for apoptosis induced by chemotherapy (Fig. 3, C). We found that cisplatin-induced apoptosis was elevated in the SCCHN cells that overexpressed STAT1 compared with control subjects. Therefore, STAT1 appears to inhibit tumor growth at least in part by inducing apoptosis of SCCHN cells, an effect that may be augmented by chemotherapy. Also, the increased cell death observed when the demethylating agent azacytidine was combined with cisplatin (Fig. 5, D) suggests that therapeutic strategies that restore STAT1 (via demethylation) may be most effective when they are combined with cytotoxic chemotherapy.
Genetic alterations are often found in both tumors and normal mucosa from SCCHN patients due to field carcinogenesis (37). The relative loss of STAT1 in the tumor was also observed in the adjacent normal mucosa (data not shown), suggesting that decreased STAT1 is a result of a field effect in SCCHN carcinogenesis. This observation is consistent with our previous findings of altered (albeit increased) STAT3 and STAT5 levels in both tumor and corresponding normal mucosa from SCCHN patients, suggesting a coordinate STAT dysregulation (18,38).
Because gene mutation did not explain the statistically significant difference in STAT1 expression levels between SCCHN tumors and control normal mucosa, we hypothesized that the reduced STAT1 expression may be due to methylation of CpG islands in the STAT1 promoter. Aberrant promoter methylation of tumor suppressor genes is a common feature of primary cancer cells, including SCCHN (39), and this fact has stimulated the development of DNA methylation inhibitors as potential cancer therapies (40). The methylation of the STAT1 promoter and its association with antitumor functions of STAT1 has not been reported to date, to our knowledge. We found methylation of CpG islands (–895 to –626 from the start site of exon 1 of STAT1) in the promoter of STAT1 in 75% of SCCHN tumors compared with none of the matched peripheral blood lymphocyte samples from SCCHN patients (Fig. 4). Furthermore, expression levels of STAT1 in the tumors with promoter methylation were decreased compared with those without methylation (Fig. 4). Because lower STAT1 expression in the corresponding normal mucosa from the SCCHN cancer patients was also observed, it will be important to determine if the STAT1 promoter is also methylated in these tissues.
In addition to finding indirect evidence of an association between STAT1 promoter methylation and STAT1 downregulation in SCCHN, we used cell lines to specifically test the consequences of modulation of DNA methylation on STAT1 function. All three SCCHN cell lines examined showed evidence of STAT1 promoter methylation, whereas no promoter methylation was observed in either of two normal immortalized epithelial cells analyzed (Fig. 5, A and data not shown). Furthermore, demethylation of the STAT1 promoter by azacytidine increased STAT1 expression levels in all three SCCHN cell lines studied. The DNA methylation inhibitor azacytidine has recently been shown to restore STAT1 expression in human tumor cells (41). These cumulative results suggest that promoter methylation is the primary mechanism of STAT1 downregulation in human tumors.
This study has several potential limitations. They include the examination of STAT1 expression and promoter methylation status in a relatively small cohort of SCCHN patients, the lack of DNA from the corresponding normal mucosa for analysis, and the uncertain physiologic significance of forced overexpression of STAT1 in SCCHN cell lines. Nevertheless, the results implicate STAT1 as a tumor suppressor in head and neck cancer as a result of promoter methylation. Strategies to restore STAT1 function are worth exploring for possible therapeutic benefit in cancers in which decreased STAT1 contributes to umorigenesis.
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NOTES |
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We are grateful to Ms. Denise Boozer for her excellent secretarial assistance.
Supported by National Institutes of Health grant R01 CA77308 (to J. Rubin Grandis). The funding agency had no role in the study design, data collection, or analysis; in the interpretation of the results; or in the preparation of the manuscript.
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Manuscript received July 22, 2005; revised November 17, 2005; accepted December 19, 2005.
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J Natl Cancer Inst 2007 99: 1343-1344.
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