doi: 10.1111/jop.12227
J Oral Pathol Med (2015) 44: 185–192 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
The role of p300 in the tumor progression of oral squamous cell carcinoma Young-Ah Cho1, Ji-Soo Hong2, Eun-Jin Choe2, Hye-Jung Yoon2, Seong-Doo Hong2, Jae-Il Lee2, Sam-Pyo Hong2 1
Department of Oral and Maxillofacial Pathology, School of Dentistry and Research Center for Tooth and Periodontal Regeneration (MRC), Kyung Hee University, Seoul, Korea; 2Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea
BACKGROUND: EP300 gene encoding p300 is a candidate tumor suppressor gene. This study investigated p300 expression and gene alteration in oral squamous cell carcinoma (OSCC) specimens to assess its role in OSCC development. METHODS: Genomic DNA extracted from 13 human OSCC cell lines and 40 OSCC patient specimens was subjected to methylation-specific PCR and exon sequencing. Immunohistochemical staining with primary antibodies against p300 and p53 was performed in 48 patients with OSCC. We analyzed the association between the data and clinicopathological factors of OSCC patients. RESULTS: Methylation-specific PCR revealed that the EP300 promoter region was not hypermethylated in OSCC. Only one cell line demonstrated a point mutation at exon 31. On immunohistochemical examination, patients with metastatic lymph nodes (P = 0.009) and advanced clinical stage (P = 0.046) tended to show increased expression of p300. There was no statistically significant relationship between p300 expression and p53 accumulation in OSCC tissue samples. Patient survival was not correlated with p300 expression. CONCLUSIONS: EP300 is not a tumor suppressor gene because there was neither epigenetic inactivation of the gene nor a mutation resulting in functional impairment. Based on p300 overexpression and its association with clinical factors in patients with OSCC, it is likely that p300 itself or one of its target genes plays a key role in the aggressive phenotypes of OSCC. J Oral Pathol Med (2015) 44: 185–192 Keywords: oncogene; oral squamous cell carcinoma; p300; p53; tumor suppressor gene
Correspondence: Seong-Doo Hong, DDS, PhD, Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, 28 Yeongeon-dong, Jongno-gu, Seoul 110-749, Korea. Tel: +82 2 2072 2625, Fax: +82 2 740 8682, E-mail: [email protected] Accepted for publication May 26, 2014
Introduction Tumor suppressor genes (TSGs) maintain the integrity of the genome by regulating the cell cycle, transcription of a certain gene, and intracellular signaling (1). Because TSGs are inactivated by mutation or loss, their location is usually suspected by the detection of chromosomal deletions or by analysis of loss of heterozygosity (LOH) in neoplastic cells (1, 2). A high frequency of LOH at chromosome arm 22q has been reported in various types of malignant tumors, including colon, breast, and ovarian carcinomas (3–5). In patients with oral cancer, Miyakawa et al. (6) reported that 41% of a total of 33 cases showed LOH on chromosome 22q13. It has been also been reported that oral squamous cell carcinoma (OSCC) exhibits LOH on chromosome 22q13, whereas LOH was frequently observed on 22q11-12 in laryngeal squamous cell carcinoma (7), suggesting that a novel TSG on chromosome 22q13 might be relevant to the progression of OSCC. The EP300 gene that encodes the p300 protein is located on chromosome 22q13.2 and is considered a candidate TSG. It is known that EP300 knockout induces early embryonic lethality and that EP300 null chimeras develop hematological malignancies (2, 3, 8). p300 is a member of the histone acetyltransferase family of transcriptional coactivators. It catalyzes the acetylation of lysine residues on histones, promoting transcription through chromatin remodeling (4, 8, 9). Transcription factors such as Egr-1 and E2F1 are known targets of p300’s function as a transcriptional coactivator (10). p300 also acetylates non-histone proteins and thereby modifies the ability of these proteins to bind to a specific promoter or to other proteins (4, 8, 11). The relationship between p300 and the p53 tumor suppressor is especially well documented. p300 increases the stability of p53 by acetylating the lysine residue of p53 and promotes p53-dependent transcription of target genes such as p21Waf1/ Cip1 and p16INK4A (11), resulting in growth suppression, cell cycle arrest, and/or further epithelial differentiation (12–14). It has been also reported that binding of HPV E6 to p300 inhibits p300-mediated p53 acetylation (15). However, although wild-type p53 is a critical substrate of p300 in
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mediating tumor suppression (16), the relationship between p300 expression and mutation of p53 in human cancer has rarely been evaluated. Because EP300 is a putative TSG, mutation analyses have been performed in various carcinomas. Although the frequency of mutation was low among several cancers (8), point mutations have been reported in one human OSCC cell line (12). There have also been studies of epigenetic inactivation of EP300 caused by hypermethylation within the promoter sequence (17, 18). One of those studies, which used specimens of patients with esophageal squamous cell carcinoma, revealed that DNA methylation of the p300 promoter was significantly correlated with lymph node metastasis and depth of tumor invasion (17). In contrast to studies showing repressed EP300 expression in carcinomas, it has also been reported that increased expression of p300 mRNA and protein is associated with poor prognosis of patients with several malignancies including colon, breast, prostate, and esophageal carcinomas (19–21). Some authors speculated that the function of p300 as a histone acetyltransferase could enhance the expression of other oncoproteins, leading to increased tumor aggressiveness (20). The role of p300 in the carcinogenesis of OSCC has not been studied. Therefore, we examined mutations in the exons of EP300 and the methylation status of EP300 promoter sequence in OSCC cell lines and tissue. In addition, we investigated p300 protein expression in OSCC patient samples and analyzed the relationship between p300 and clinicopathological behavior of OSCC including their p53 expression status.
Materials and methods Cell lines A total of 13 human OSCC cell lines (SCC-4, SCC-9, SCC15, SCC-25, HSC-2, HSC-3, HSC-4, Ca9-22, HO-1-N1, HO-1-U1, KOSCC-11, KOSCC-25B, and KOSCC-33A) were included in this study. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), a mixture of F12 medium and DMEM, or Roswell Park Memorial Institute 1640 medium, supplemented with 10% fetal bovine serum and 1% antibiotics. The culture plates were incubated at 37°C in a humidified atmosphere of 5% CO2. Patients and tissue samples This study was approved by the Institutional Review Board (IRB) at Seoul National University Dental Hospital (IRB No. CRI12009G) and carried out according to institutional guidelines. OSCC cases that were documented between 1999 and 2004 were retrieved from the electronic pathology records of the Seoul National University Dental Hospital. After exclusion of patients who had not undergone radical resection of the lesions and those for whom paraffin blocks were unavailable, a total of 48 patients were included in this study. The patients’ clinical factors including age, sex, tumor location, recurrence, and survival were examined, and the histopathologic grade of each case was assessed by two experienced pathologists based on the WHO classification. Pathological staging was established according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (22). J Oral Pathol Med
Genomic DNA extraction Genomic DNA was prepared from OSCC cell lines using a G-spinTM Genomic DNA Extraction Kit for Cell/Tissue (iNtRON Biotechnology, Seongnam, Korea). For genomic DNA extraction from OSCC tissue specimens, 5–10-lmthick paraffin-embedded tissue sections were prepared. The tumor areas were separated from stroma under a microscope using sterilized scalpels. DNA extraction buffer solution consisting of 50 mM Tris buffer (pH 8.3), 1 mM EDTA (pH 8.0), 5% Tween-20, 200 lg/ml proteinase K, and 10% resin was added as previously described (23). After incubation at 56°C for 90 min, tubes containing tissue and DNA extraction buffer solution were heated to 100°C for 10 min, followed by centrifugation at 14 000 g for 10 min at 4°C. The supernatant was used for subsequent experiments. Methylation-specific PCR Sodium bisulfite modification of the genomic DNA extracted from cell lines and of the patient samples was performed using the EpiTectâ Plus Bisulfite Kit (QIAGEN, Hilden, Germany). Primers that distinguish unmethylated (U) and methylated (M) alleles were designed as described previously (17): EP300-U-forward: TGTTGTTTGGTTT GGTTTTTTT and EP300-U-reverse: CACAAAAAAC TCACCCAAACCA; EP300-M-forward: CGTTGTTCG GTTCGGTTTTTTC and EP300-M-reverse: CGCAAAA AACTCGCCCGAACCG. Each methylation-specific PCR contained 1 U of TaKaRa Ex TaqTM Hot Start Version (TAKARA BIO Inc., Otsu, Japan), 19 Ex Taq buffer, 2.5 mM deoxynucleoside triphosphates, 10 pmol forward and reverse primers, and 50–100 ng of bisulfite-modified DNA in a final reaction volume of 20 ll. Reactions were hot-started at 95°C for 5 min, followed by 38 cycles at 95°C for 30 s, 57°C (U) or 57°C (M) for 30 s, and 72°C for 30 s in a thermocycler, with water blank as a negative control. Bisulfite-modified DNA and unmodified DNA were also used as templates in PCRs with wild-type primers (EP300-W-forward: CGCTGCTCGGCCCGGCC CCCTC; EP300-W-reverse: CGCAGAGAACTCGCCC GAGCCG) as controls to ensure completeness of DNA conversion. PCR products were electrophoresed on 1.5% agarose gels. Mutational analysis Genomic DNA extracted from OSCC cell lines was subjected to PCR. Each of the 31 coding exons of EP300 was amplified with a pair of primers complementary to surrounding intronic sequences, as previously described (3). After DNA purification with GenoAidTM PCR/Gel Combo Kit (Genotech, Daejeon, Korea), the PCR products were sequenced on an ABI PRISM 3700 Genetic Analyzer (Applied Biosystems Inc., Foster City, CA, USA). Sequence data were analyzed using RefSeqGene Nucleotide Basic Local Alignment Search Tool BLAST software (Bethesda, MD, USA) at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) Web site. Based on the results from OSCC cell lines, genomic DNA extracted from the patient samples was analyzed only for exons 17 and 18.
p300 in oral squamous cell carcinoma Cho et al.
Immunohistochemical staining Paraffin-embedded specimens were sectioned at a thickness of 4 lm, deparaffinized in xylene, and rehydrated through graded alcohol solutions. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 10 min. The slides were boiled four times for 5 min in 0.01 M citrate buffer (pH 6.0) for p300 or Tris/EDTA buffer (pH 9.0) for p53. The primary antibodies used were rabbit polyclonal antihuman p300 antibody (clone sc-584, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a concentration of 2 lg/ml and mouse monoclonal antihuman p53 antibody (clone DO-7, Dako, Glostrup, Denmark) at a concentration of 3.35 lg/ml. After incubation with each primary antibody at room temperature for 1 h, detection was performed using the REALTM EnVisionTM/HRP kit (Dako). Immunohistochemical reactions were visualized with 3,30 -diaminobenzidine (Dako) and counterstained with Mayer’s hematoxylin. Sections from human breast carcinoma tissue served as the positive control for p300 staining. For the negative control, the primary antibody was replaced with mouse IgG isotype (Sigma, St. Louis, MO, USA). Evaluation of immunohistochemical results All immunostained sections were blindly scored by two investigators. Expression of p300 was defined as nuclear immunoreactivity. In normal oral squamous epithelium, the parabasal cell layer exhibited weak p300 expression (Fig. 1A). The intensity of the staining of tumor cells was categorized into four grades based on the following criteria: 0, negative staining; 1, equal to the staining intensity of the spinous cells in normal epithelium (weak, Fig. 1B); 2, slightly increased staining (moderate, Fig. 1C); and 3, strongly increased staining (strong, Fig. 1D). If there was a
difference in intensity among the tumor cells in a single high-power field, the dominant grade was chosen. Scores for the distribution of staining were assigned as described previously (20): zero (0% of cells), 1 (1–10%), 2 (11–50%), 3 (51–80%), and 4 (81–100%). After multiplying the score of the distribution by the score of the intensity (total range, 0 to 12) for five different high-power fields, the final p300 scores of each patient were calculated by averaging the five figures. These scores were dichotomized by median split. Nuclear expression of p53 in tumor cells was categorized as low (A transversion in exon 18. Because these mutations were not predicted to change the encoded amino acid, these two types of single-
nucleotide substitution were classified as synonymous substitution. EP300 exon sequencing in OSCC patient samples and their association with clinicopathological features Among 40 patient samples with available DNA, the exon 17 synonymous substitution observed in the OSCC cell lines (Fig. 3) was detected in 21 patients, and the exon 18 synonymous substitution was detected in nine patients. Chisquare tests revealed that the mutation status of exon 17 or exon 18 was not significantly associated with the patients’ clinicopathological parameters. However, there was a weak association between the exon 18 synonymous substitution and histological grade of tumor, with most patients with the altered exon 18 showing well-differentiated OSCC (P = 0.091).
Figure 3 Direct sequencing results of genomic DNA from OSCC cell lines. EP300 carries point mutations in exons 17, 18, and 31. J Oral Pathol Med
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Table 1 Mutations of EP300 identified in oral squamous cell carcinoma (OSCC) cell lines
Table 2 Relationships between clinicopathological factors and p300 expression of 48 OSCC patients
Cell line name
Exon (nucleotide change)
Codon change
Variable
SCC-9
17 (3183T>A) 17 (3183T>A) 31 (6668A>C)
T1061T T1061T Q2223P
17 17 17 18 17 17 18 17 18 17 17 17
T1061T T1061T T1061T Q1116Q T1061T T1061T Q1116Q T1061T Q1116Q T1061T T1061T T1061T
SCC-25 HSC-2 HSC-3 HSC-4 Ca9-22 HO-1-N1 HO-1-U1 KOSCC-11 KOSCC-25B KOSCC-33A
(3183T>A) (3183T>A) (3183T>A) (3348G>A) (3183T>A) (3183T>A) (3348G>A) (3183T>A) (3348G>A) (3183T>A) (3183T>A) (3183T>A)
n
Low p300
High p300
189
P-value
Motif (38) Bromo_cbp_like domain Bromo_cbp_like domain Interaction with NCOA2 region and Q-rich region Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain
Association between p300 protein expression and clinicopathological features of OSCC Although normal oral epithelium consistently showed weak nuclear expression of p300, the OSCC cases exhibited varying staining intensities for p300, with 94% (n = 45) of all 48 cases showing positive staining. The p300 scores ranged from 0 to 12, with an average of 5.6. Patients with low p300 expression showed no metastasis into the regional lymph node (P = 0.009) and low clinical stage (P = 0.046). Other clinical factors were not significantly related to p300 expression (Table 2). We also found that p300 protein expression was not associated with the synonymous substitutions in EP300 exon 17 (P = 0.516) and exon 18 (P = 0.280; Table 2). Twenty-four (50%) of 48 patients with OSCC exhibited clear positivity for p53 in ≥10% of tumor cell nuclei. However, neither p300 overexpression (P = 0.564) nor the examined clinical parameters were associated with p53 accumulation.
Age 50%) Ki-67 index (P = 0.002), suggesting an influence of p300 on cell proliferation (9). It has also been reported that transfection of p300-specific siRNA into prostate cancer cell lines inhibited cell proliferation (28) and increased apoptosis of tumor cells (29). Correlations between p300 expression and clinical factors such as tumor size, lymph node metastasis, and/or tumor stage have been reported in carcinomas of the esophagus, nasopharynx, liver, and prostate (9, 20, 28, 30). We also demonstrated that lymph node metastasis and tumor stage correlated with p300 expression in patients with OSCC. However, the prognosis of OSCC was not affected by p300 expression. Even though it has recently been reported that the overexpression of p300 may be a favorable prognostic factor in patients with colorectal adenocarcinoma (31), p300 was revealed as an independent prognostic factor for poor survival in nasopharyngeal carcinoma, hepatocellular carcinoma, esophageal carcinoma, and non-small cell lung cancers, indicating an important role of p300 protein in oncogenic processes (9, 20, 30, 32). Although the mechanism underlying the increased p300 expression in various carcinomas is not known, Vleugel et al. (21) have demonstrated that p53 accumulation correlates with high/moderate levels of p300 in breast carcinoma (P = 0.001) and hypothesized that the accumulation of mutant p53, which is thought to be acetylated, might lead to p300 overexpres-
sion. It is well established that p300 binding enhances the stability of p53 by protecting the p53 N terminus against inhibitors such as MDM2 (33) and that p300/CBP (CREBbinding protein)-mediated p53 acetylation increases its transcriptional activity by recruiting p300 onto histones at the promoter regions of p53 target genes (11, 34). Because mutation of the DNA-binding domain (residues 102–292) accounts for 80–90% of all p53 mutations (16, 35), the two p300-binding domains of p53, the transactivation domain (residues 1–57), and the C-terminal regulatory domain (residues 364–393) (16, 33) can be retained even in mutant p53, thereby allowing acetylation by p300. Accordingly, Vleugel’s hypothesis is thought to be appropriate. However, the present study failed to reveal a significant relationship between p53 nuclear accumulation and p300 expression by immunohistochemical examination. Therefore, apart from the critical role of p53 mutation in OSCC development (36), accumulation of mutant p53 is not likely to result in p300 overexpression in patients with OSCC. Our mutation analyses clearly showed that the frequent LOH on chromosome 22q13 observed in OSCC is not caused by EP300 deletion or mutation. Instead, we found synonymous substitutions, or silent mutations, in EP300 exons 17 and 18. This is consistent with the previous literature reporting several silent alterations in EP300, although the precise loci of the substitutions were not mentioned (3). Even though synonymous substitutions are expected to be translated to the same amino acid, several systematic studies have reported that they are evidently capable of affecting protein activity (37). There are two proposed mechanisms by which synonymous substitutions might change protein function. The first one, presented by Kimchi-Sarfaty et al., proposes that ‘rare codons’ that are infrequently used within the entire human genome would be translated more slowly than frequently used codons and could be clustered upstream and downstream of a synonymous substitution. Therefore, translation would pause around the substitution, changing the timing of cotranslational protein folding. The resulting proteins with changed conformation are likely to interact with their substrates or inhibitors in a different manner (38). The other mechanism is linked to mRNA secondary structure and stability. Because the translation rate is inversely proportional to the stability of mRNA structure, mRNA with reduced stability caused by synonymous substitution would be
Figure 5 Codon usage frequencies of the codons around the synonymous substitutions (red) in EP300 exons 17 and 18. The numbers indicate the frequency of codon appearance per thousand codons in the human genome. By reference to the Codon Usage Table (http://www.kazusa.or.jp/codon/), we have designated the codons in gray squares as codons with rare usage based on the report by Kimchi-Sarfaty (37). (*maximum frequency for the corresponding amino acid; † frequency for the actual codon). J Oral Pathol Med
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translated faster, leading to an increased level of protein expression (39). It appears that these two mechanisms do not always simultaneously affect the fate of a certain gene (37). It is currently unclear whether the synonymous substitutions present in the OSCC cell lines and tissues lower mRNA stability and enhance p300 translation or are associated with translational pausing at surrounding rare codon clusters. Using a codon usage database based on the study of Kimchi-Sarfaty (38), we identified clusters of rare codons around two synonymous substitutions (Fig. 5). These rare codons might regulate the translation rate sufficiently to change the interaction between p300 and substrates. Histones are the main substrates of p300, and exons 17 and 18 of p300 are within the bromodomain (exons 15–18) that is conserved in most histone acetyltransferases (including p300) and exclusively recognizes acetyl-lysine residues on histones (4). The bromodomain is essential for maintaining the basal level of histone acetylation and for coactivation of target gene transcription (4, 10). Therefore, altered interaction between bromodomain and histone might affect transcription of p300-dependent genes such as p21, Egr1, and E2F1 (10, 16). To investigate this possibility, the first priority will be to determine whether transient expression of synonymous substitutions affect the interaction between p300 and histones. On the other hand, because there was no correlation between the exon sequencing results and p300 level by immunohistochemistry, we think that the increased p300 level does not result from changes in mRNA secondary structure caused by synonymous substitutions in exons 17 and 18. Mutation analysis also revealed that a single OSCC cell line, SCC-25, carried a missense mutation in exon 31 that changes glutamine to proline. This mutation is in the region that interacts with nuclear receptor coactivator 2 (NCoA-2) (40) and is also within the glutamine-rich region (25). A missense mutation in the NCoA-2-binding region of EP300 has previously been reported in a colorectal cancer cell line, similar to the present study (25). NCoA-2SRC2/TIF2/GRIP1 is a 160-kDa protein that coactivates hormone receptors such as estrogen receptor, progesterone receptor, and retinoic acid receptor alpha (41). NCoA-2 is also capable of recruiting acetyltransferases including p300, CBP, and p300/CBPassociated factor (PCAF) (42). Although fusion of NCOA2 with another gene has been reported in acute myeloid leukemia, rhabdomyosarcoma, mesenchymal chondrosarcoma, and soft tissue angiofibroma (43), there are no studies of NCoA-2 in OSCC. In conclusion, our study provides evidence that EP300 does not function as a TSG in OSCC because it showed neither epigenetic repression nor mutation leading to defective protein structure and function. In OSCC patient samples, a high level of p300 expression correlated with positive lymph node metastasis and advanced tumor stage, suggesting that p300 may play an important role in the acquisition of an aggressive OSCC phenotype or that specific p300 target genes may promote tumor progression. Despite the functional link with p53, p300 expression was not related to p53 accumulation. In addition, synonymous substitutions in the bromodomain of EP300 might affect its interaction with histones. Further investigation into the
relationship between synonymous substitutions in EP300 and the aggressiveness of OSCC cells might elucidate the role of p300 in the pathogenesis of OSCC.
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Acknowledgements This research was supported by a grant from Clinical Dental Research Institute of the Seoul National University Dental Hospital (No.03-2012-0030)
Conflict of Interest None declared.
J Oral Pathol Med (2015) 44: 185–192 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
The role of p300 in the tumor progression of oral squamous cell carcinoma Young-Ah Cho1, Ji-Soo Hong2, Eun-Jin Choe2, Hye-Jung Yoon2, Seong-Doo Hong2, Jae-Il Lee2, Sam-Pyo Hong2 1
Department of Oral and Maxillofacial Pathology, School of Dentistry and Research Center for Tooth and Periodontal Regeneration (MRC), Kyung Hee University, Seoul, Korea; 2Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea
BACKGROUND: EP300 gene encoding p300 is a candidate tumor suppressor gene. This study investigated p300 expression and gene alteration in oral squamous cell carcinoma (OSCC) specimens to assess its role in OSCC development. METHODS: Genomic DNA extracted from 13 human OSCC cell lines and 40 OSCC patient specimens was subjected to methylation-specific PCR and exon sequencing. Immunohistochemical staining with primary antibodies against p300 and p53 was performed in 48 patients with OSCC. We analyzed the association between the data and clinicopathological factors of OSCC patients. RESULTS: Methylation-specific PCR revealed that the EP300 promoter region was not hypermethylated in OSCC. Only one cell line demonstrated a point mutation at exon 31. On immunohistochemical examination, patients with metastatic lymph nodes (P = 0.009) and advanced clinical stage (P = 0.046) tended to show increased expression of p300. There was no statistically significant relationship between p300 expression and p53 accumulation in OSCC tissue samples. Patient survival was not correlated with p300 expression. CONCLUSIONS: EP300 is not a tumor suppressor gene because there was neither epigenetic inactivation of the gene nor a mutation resulting in functional impairment. Based on p300 overexpression and its association with clinical factors in patients with OSCC, it is likely that p300 itself or one of its target genes plays a key role in the aggressive phenotypes of OSCC. J Oral Pathol Med (2015) 44: 185–192 Keywords: oncogene; oral squamous cell carcinoma; p300; p53; tumor suppressor gene
Correspondence: Seong-Doo Hong, DDS, PhD, Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, 28 Yeongeon-dong, Jongno-gu, Seoul 110-749, Korea. Tel: +82 2 2072 2625, Fax: +82 2 740 8682, E-mail: [email protected] Accepted for publication May 26, 2014
Introduction Tumor suppressor genes (TSGs) maintain the integrity of the genome by regulating the cell cycle, transcription of a certain gene, and intracellular signaling (1). Because TSGs are inactivated by mutation or loss, their location is usually suspected by the detection of chromosomal deletions or by analysis of loss of heterozygosity (LOH) in neoplastic cells (1, 2). A high frequency of LOH at chromosome arm 22q has been reported in various types of malignant tumors, including colon, breast, and ovarian carcinomas (3–5). In patients with oral cancer, Miyakawa et al. (6) reported that 41% of a total of 33 cases showed LOH on chromosome 22q13. It has been also been reported that oral squamous cell carcinoma (OSCC) exhibits LOH on chromosome 22q13, whereas LOH was frequently observed on 22q11-12 in laryngeal squamous cell carcinoma (7), suggesting that a novel TSG on chromosome 22q13 might be relevant to the progression of OSCC. The EP300 gene that encodes the p300 protein is located on chromosome 22q13.2 and is considered a candidate TSG. It is known that EP300 knockout induces early embryonic lethality and that EP300 null chimeras develop hematological malignancies (2, 3, 8). p300 is a member of the histone acetyltransferase family of transcriptional coactivators. It catalyzes the acetylation of lysine residues on histones, promoting transcription through chromatin remodeling (4, 8, 9). Transcription factors such as Egr-1 and E2F1 are known targets of p300’s function as a transcriptional coactivator (10). p300 also acetylates non-histone proteins and thereby modifies the ability of these proteins to bind to a specific promoter or to other proteins (4, 8, 11). The relationship between p300 and the p53 tumor suppressor is especially well documented. p300 increases the stability of p53 by acetylating the lysine residue of p53 and promotes p53-dependent transcription of target genes such as p21Waf1/ Cip1 and p16INK4A (11), resulting in growth suppression, cell cycle arrest, and/or further epithelial differentiation (12–14). It has been also reported that binding of HPV E6 to p300 inhibits p300-mediated p53 acetylation (15). However, although wild-type p53 is a critical substrate of p300 in
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mediating tumor suppression (16), the relationship between p300 expression and mutation of p53 in human cancer has rarely been evaluated. Because EP300 is a putative TSG, mutation analyses have been performed in various carcinomas. Although the frequency of mutation was low among several cancers (8), point mutations have been reported in one human OSCC cell line (12). There have also been studies of epigenetic inactivation of EP300 caused by hypermethylation within the promoter sequence (17, 18). One of those studies, which used specimens of patients with esophageal squamous cell carcinoma, revealed that DNA methylation of the p300 promoter was significantly correlated with lymph node metastasis and depth of tumor invasion (17). In contrast to studies showing repressed EP300 expression in carcinomas, it has also been reported that increased expression of p300 mRNA and protein is associated with poor prognosis of patients with several malignancies including colon, breast, prostate, and esophageal carcinomas (19–21). Some authors speculated that the function of p300 as a histone acetyltransferase could enhance the expression of other oncoproteins, leading to increased tumor aggressiveness (20). The role of p300 in the carcinogenesis of OSCC has not been studied. Therefore, we examined mutations in the exons of EP300 and the methylation status of EP300 promoter sequence in OSCC cell lines and tissue. In addition, we investigated p300 protein expression in OSCC patient samples and analyzed the relationship between p300 and clinicopathological behavior of OSCC including their p53 expression status.
Materials and methods Cell lines A total of 13 human OSCC cell lines (SCC-4, SCC-9, SCC15, SCC-25, HSC-2, HSC-3, HSC-4, Ca9-22, HO-1-N1, HO-1-U1, KOSCC-11, KOSCC-25B, and KOSCC-33A) were included in this study. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), a mixture of F12 medium and DMEM, or Roswell Park Memorial Institute 1640 medium, supplemented with 10% fetal bovine serum and 1% antibiotics. The culture plates were incubated at 37°C in a humidified atmosphere of 5% CO2. Patients and tissue samples This study was approved by the Institutional Review Board (IRB) at Seoul National University Dental Hospital (IRB No. CRI12009G) and carried out according to institutional guidelines. OSCC cases that were documented between 1999 and 2004 were retrieved from the electronic pathology records of the Seoul National University Dental Hospital. After exclusion of patients who had not undergone radical resection of the lesions and those for whom paraffin blocks were unavailable, a total of 48 patients were included in this study. The patients’ clinical factors including age, sex, tumor location, recurrence, and survival were examined, and the histopathologic grade of each case was assessed by two experienced pathologists based on the WHO classification. Pathological staging was established according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (22). J Oral Pathol Med
Genomic DNA extraction Genomic DNA was prepared from OSCC cell lines using a G-spinTM Genomic DNA Extraction Kit for Cell/Tissue (iNtRON Biotechnology, Seongnam, Korea). For genomic DNA extraction from OSCC tissue specimens, 5–10-lmthick paraffin-embedded tissue sections were prepared. The tumor areas were separated from stroma under a microscope using sterilized scalpels. DNA extraction buffer solution consisting of 50 mM Tris buffer (pH 8.3), 1 mM EDTA (pH 8.0), 5% Tween-20, 200 lg/ml proteinase K, and 10% resin was added as previously described (23). After incubation at 56°C for 90 min, tubes containing tissue and DNA extraction buffer solution were heated to 100°C for 10 min, followed by centrifugation at 14 000 g for 10 min at 4°C. The supernatant was used for subsequent experiments. Methylation-specific PCR Sodium bisulfite modification of the genomic DNA extracted from cell lines and of the patient samples was performed using the EpiTectâ Plus Bisulfite Kit (QIAGEN, Hilden, Germany). Primers that distinguish unmethylated (U) and methylated (M) alleles were designed as described previously (17): EP300-U-forward: TGTTGTTTGGTTT GGTTTTTTT and EP300-U-reverse: CACAAAAAAC TCACCCAAACCA; EP300-M-forward: CGTTGTTCG GTTCGGTTTTTTC and EP300-M-reverse: CGCAAAA AACTCGCCCGAACCG. Each methylation-specific PCR contained 1 U of TaKaRa Ex TaqTM Hot Start Version (TAKARA BIO Inc., Otsu, Japan), 19 Ex Taq buffer, 2.5 mM deoxynucleoside triphosphates, 10 pmol forward and reverse primers, and 50–100 ng of bisulfite-modified DNA in a final reaction volume of 20 ll. Reactions were hot-started at 95°C for 5 min, followed by 38 cycles at 95°C for 30 s, 57°C (U) or 57°C (M) for 30 s, and 72°C for 30 s in a thermocycler, with water blank as a negative control. Bisulfite-modified DNA and unmodified DNA were also used as templates in PCRs with wild-type primers (EP300-W-forward: CGCTGCTCGGCCCGGCC CCCTC; EP300-W-reverse: CGCAGAGAACTCGCCC GAGCCG) as controls to ensure completeness of DNA conversion. PCR products were electrophoresed on 1.5% agarose gels. Mutational analysis Genomic DNA extracted from OSCC cell lines was subjected to PCR. Each of the 31 coding exons of EP300 was amplified with a pair of primers complementary to surrounding intronic sequences, as previously described (3). After DNA purification with GenoAidTM PCR/Gel Combo Kit (Genotech, Daejeon, Korea), the PCR products were sequenced on an ABI PRISM 3700 Genetic Analyzer (Applied Biosystems Inc., Foster City, CA, USA). Sequence data were analyzed using RefSeqGene Nucleotide Basic Local Alignment Search Tool BLAST software (Bethesda, MD, USA) at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) Web site. Based on the results from OSCC cell lines, genomic DNA extracted from the patient samples was analyzed only for exons 17 and 18.
p300 in oral squamous cell carcinoma Cho et al.
Immunohistochemical staining Paraffin-embedded specimens were sectioned at a thickness of 4 lm, deparaffinized in xylene, and rehydrated through graded alcohol solutions. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 10 min. The slides were boiled four times for 5 min in 0.01 M citrate buffer (pH 6.0) for p300 or Tris/EDTA buffer (pH 9.0) for p53. The primary antibodies used were rabbit polyclonal antihuman p300 antibody (clone sc-584, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a concentration of 2 lg/ml and mouse monoclonal antihuman p53 antibody (clone DO-7, Dako, Glostrup, Denmark) at a concentration of 3.35 lg/ml. After incubation with each primary antibody at room temperature for 1 h, detection was performed using the REALTM EnVisionTM/HRP kit (Dako). Immunohistochemical reactions were visualized with 3,30 -diaminobenzidine (Dako) and counterstained with Mayer’s hematoxylin. Sections from human breast carcinoma tissue served as the positive control for p300 staining. For the negative control, the primary antibody was replaced with mouse IgG isotype (Sigma, St. Louis, MO, USA). Evaluation of immunohistochemical results All immunostained sections were blindly scored by two investigators. Expression of p300 was defined as nuclear immunoreactivity. In normal oral squamous epithelium, the parabasal cell layer exhibited weak p300 expression (Fig. 1A). The intensity of the staining of tumor cells was categorized into four grades based on the following criteria: 0, negative staining; 1, equal to the staining intensity of the spinous cells in normal epithelium (weak, Fig. 1B); 2, slightly increased staining (moderate, Fig. 1C); and 3, strongly increased staining (strong, Fig. 1D). If there was a
difference in intensity among the tumor cells in a single high-power field, the dominant grade was chosen. Scores for the distribution of staining were assigned as described previously (20): zero (0% of cells), 1 (1–10%), 2 (11–50%), 3 (51–80%), and 4 (81–100%). After multiplying the score of the distribution by the score of the intensity (total range, 0 to 12) for five different high-power fields, the final p300 scores of each patient were calculated by averaging the five figures. These scores were dichotomized by median split. Nuclear expression of p53 in tumor cells was categorized as low (A transversion in exon 18. Because these mutations were not predicted to change the encoded amino acid, these two types of single-
nucleotide substitution were classified as synonymous substitution. EP300 exon sequencing in OSCC patient samples and their association with clinicopathological features Among 40 patient samples with available DNA, the exon 17 synonymous substitution observed in the OSCC cell lines (Fig. 3) was detected in 21 patients, and the exon 18 synonymous substitution was detected in nine patients. Chisquare tests revealed that the mutation status of exon 17 or exon 18 was not significantly associated with the patients’ clinicopathological parameters. However, there was a weak association between the exon 18 synonymous substitution and histological grade of tumor, with most patients with the altered exon 18 showing well-differentiated OSCC (P = 0.091).
Figure 3 Direct sequencing results of genomic DNA from OSCC cell lines. EP300 carries point mutations in exons 17, 18, and 31. J Oral Pathol Med
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Table 1 Mutations of EP300 identified in oral squamous cell carcinoma (OSCC) cell lines
Table 2 Relationships between clinicopathological factors and p300 expression of 48 OSCC patients
Cell line name
Exon (nucleotide change)
Codon change
Variable
SCC-9
17 (3183T>A) 17 (3183T>A) 31 (6668A>C)
T1061T T1061T Q2223P
17 17 17 18 17 17 18 17 18 17 17 17
T1061T T1061T T1061T Q1116Q T1061T T1061T Q1116Q T1061T Q1116Q T1061T T1061T T1061T
SCC-25 HSC-2 HSC-3 HSC-4 Ca9-22 HO-1-N1 HO-1-U1 KOSCC-11 KOSCC-25B KOSCC-33A
(3183T>A) (3183T>A) (3183T>A) (3348G>A) (3183T>A) (3183T>A) (3348G>A) (3183T>A) (3348G>A) (3183T>A) (3183T>A) (3183T>A)
n
Low p300
High p300
189
P-value
Motif (38) Bromo_cbp_like domain Bromo_cbp_like domain Interaction with NCOA2 region and Q-rich region Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain Bromo_cbp_like domain
Association between p300 protein expression and clinicopathological features of OSCC Although normal oral epithelium consistently showed weak nuclear expression of p300, the OSCC cases exhibited varying staining intensities for p300, with 94% (n = 45) of all 48 cases showing positive staining. The p300 scores ranged from 0 to 12, with an average of 5.6. Patients with low p300 expression showed no metastasis into the regional lymph node (P = 0.009) and low clinical stage (P = 0.046). Other clinical factors were not significantly related to p300 expression (Table 2). We also found that p300 protein expression was not associated with the synonymous substitutions in EP300 exon 17 (P = 0.516) and exon 18 (P = 0.280; Table 2). Twenty-four (50%) of 48 patients with OSCC exhibited clear positivity for p53 in ≥10% of tumor cell nuclei. However, neither p300 overexpression (P = 0.564) nor the examined clinical parameters were associated with p53 accumulation.
Age 50%) Ki-67 index (P = 0.002), suggesting an influence of p300 on cell proliferation (9). It has also been reported that transfection of p300-specific siRNA into prostate cancer cell lines inhibited cell proliferation (28) and increased apoptosis of tumor cells (29). Correlations between p300 expression and clinical factors such as tumor size, lymph node metastasis, and/or tumor stage have been reported in carcinomas of the esophagus, nasopharynx, liver, and prostate (9, 20, 28, 30). We also demonstrated that lymph node metastasis and tumor stage correlated with p300 expression in patients with OSCC. However, the prognosis of OSCC was not affected by p300 expression. Even though it has recently been reported that the overexpression of p300 may be a favorable prognostic factor in patients with colorectal adenocarcinoma (31), p300 was revealed as an independent prognostic factor for poor survival in nasopharyngeal carcinoma, hepatocellular carcinoma, esophageal carcinoma, and non-small cell lung cancers, indicating an important role of p300 protein in oncogenic processes (9, 20, 30, 32). Although the mechanism underlying the increased p300 expression in various carcinomas is not known, Vleugel et al. (21) have demonstrated that p53 accumulation correlates with high/moderate levels of p300 in breast carcinoma (P = 0.001) and hypothesized that the accumulation of mutant p53, which is thought to be acetylated, might lead to p300 overexpres-
sion. It is well established that p300 binding enhances the stability of p53 by protecting the p53 N terminus against inhibitors such as MDM2 (33) and that p300/CBP (CREBbinding protein)-mediated p53 acetylation increases its transcriptional activity by recruiting p300 onto histones at the promoter regions of p53 target genes (11, 34). Because mutation of the DNA-binding domain (residues 102–292) accounts for 80–90% of all p53 mutations (16, 35), the two p300-binding domains of p53, the transactivation domain (residues 1–57), and the C-terminal regulatory domain (residues 364–393) (16, 33) can be retained even in mutant p53, thereby allowing acetylation by p300. Accordingly, Vleugel’s hypothesis is thought to be appropriate. However, the present study failed to reveal a significant relationship between p53 nuclear accumulation and p300 expression by immunohistochemical examination. Therefore, apart from the critical role of p53 mutation in OSCC development (36), accumulation of mutant p53 is not likely to result in p300 overexpression in patients with OSCC. Our mutation analyses clearly showed that the frequent LOH on chromosome 22q13 observed in OSCC is not caused by EP300 deletion or mutation. Instead, we found synonymous substitutions, or silent mutations, in EP300 exons 17 and 18. This is consistent with the previous literature reporting several silent alterations in EP300, although the precise loci of the substitutions were not mentioned (3). Even though synonymous substitutions are expected to be translated to the same amino acid, several systematic studies have reported that they are evidently capable of affecting protein activity (37). There are two proposed mechanisms by which synonymous substitutions might change protein function. The first one, presented by Kimchi-Sarfaty et al., proposes that ‘rare codons’ that are infrequently used within the entire human genome would be translated more slowly than frequently used codons and could be clustered upstream and downstream of a synonymous substitution. Therefore, translation would pause around the substitution, changing the timing of cotranslational protein folding. The resulting proteins with changed conformation are likely to interact with their substrates or inhibitors in a different manner (38). The other mechanism is linked to mRNA secondary structure and stability. Because the translation rate is inversely proportional to the stability of mRNA structure, mRNA with reduced stability caused by synonymous substitution would be
Figure 5 Codon usage frequencies of the codons around the synonymous substitutions (red) in EP300 exons 17 and 18. The numbers indicate the frequency of codon appearance per thousand codons in the human genome. By reference to the Codon Usage Table (http://www.kazusa.or.jp/codon/), we have designated the codons in gray squares as codons with rare usage based on the report by Kimchi-Sarfaty (37). (*maximum frequency for the corresponding amino acid; † frequency for the actual codon). J Oral Pathol Med
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translated faster, leading to an increased level of protein expression (39). It appears that these two mechanisms do not always simultaneously affect the fate of a certain gene (37). It is currently unclear whether the synonymous substitutions present in the OSCC cell lines and tissues lower mRNA stability and enhance p300 translation or are associated with translational pausing at surrounding rare codon clusters. Using a codon usage database based on the study of Kimchi-Sarfaty (38), we identified clusters of rare codons around two synonymous substitutions (Fig. 5). These rare codons might regulate the translation rate sufficiently to change the interaction between p300 and substrates. Histones are the main substrates of p300, and exons 17 and 18 of p300 are within the bromodomain (exons 15–18) that is conserved in most histone acetyltransferases (including p300) and exclusively recognizes acetyl-lysine residues on histones (4). The bromodomain is essential for maintaining the basal level of histone acetylation and for coactivation of target gene transcription (4, 10). Therefore, altered interaction between bromodomain and histone might affect transcription of p300-dependent genes such as p21, Egr1, and E2F1 (10, 16). To investigate this possibility, the first priority will be to determine whether transient expression of synonymous substitutions affect the interaction between p300 and histones. On the other hand, because there was no correlation between the exon sequencing results and p300 level by immunohistochemistry, we think that the increased p300 level does not result from changes in mRNA secondary structure caused by synonymous substitutions in exons 17 and 18. Mutation analysis also revealed that a single OSCC cell line, SCC-25, carried a missense mutation in exon 31 that changes glutamine to proline. This mutation is in the region that interacts with nuclear receptor coactivator 2 (NCoA-2) (40) and is also within the glutamine-rich region (25). A missense mutation in the NCoA-2-binding region of EP300 has previously been reported in a colorectal cancer cell line, similar to the present study (25). NCoA-2SRC2/TIF2/GRIP1 is a 160-kDa protein that coactivates hormone receptors such as estrogen receptor, progesterone receptor, and retinoic acid receptor alpha (41). NCoA-2 is also capable of recruiting acetyltransferases including p300, CBP, and p300/CBPassociated factor (PCAF) (42). Although fusion of NCOA2 with another gene has been reported in acute myeloid leukemia, rhabdomyosarcoma, mesenchymal chondrosarcoma, and soft tissue angiofibroma (43), there are no studies of NCoA-2 in OSCC. In conclusion, our study provides evidence that EP300 does not function as a TSG in OSCC because it showed neither epigenetic repression nor mutation leading to defective protein structure and function. In OSCC patient samples, a high level of p300 expression correlated with positive lymph node metastasis and advanced tumor stage, suggesting that p300 may play an important role in the acquisition of an aggressive OSCC phenotype or that specific p300 target genes may promote tumor progression. Despite the functional link with p53, p300 expression was not related to p53 accumulation. In addition, synonymous substitutions in the bromodomain of EP300 might affect its interaction with histones. Further investigation into the
relationship between synonymous substitutions in EP300 and the aggressiveness of OSCC cells might elucidate the role of p300 in the pathogenesis of OSCC.
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Acknowledgements This research was supported by a grant from Clinical Dental Research Institute of the Seoul National University Dental Hospital (No.03-2012-0030)
Conflict of Interest None declared.
