J Oral Pathol Med (2014) 43: 652–657 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
doi: 10.1111/jop.12178
wileyonlinelibrary.com/journal/jop
Frequent hypermethylation of WNT pathway genes in laryngeal squamous cell carcinomas Jarosław Paluszczak1, Dawid Hemmerling1, Magdalena Kostrzewska-Poczekaj2, Małgorzata Jarmu_z-Szymczak2, Reidar Grenman3, Małgorzata Wierzbicka4, Wanda Baer-Dubowska1 1
Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Pozna n, Poland; 2Department of Mutagenesis, 3 Institute of Human Genetics, Polish Academy of Sciences, Pozna n, Poland; Department of Otorhinolaryngology, Head and Neck Surgery and Department of Medical Biochemistry, Turku University Central Hospital and Turku University, Turku, Finland; 4Department of Otolaryngology and Laryngeal Oncology, Poznan University of Medical Sciences, Pozna n, Poland
BACKGROUND: Aberrations in the function of the WNT signaling pathway have been recently implicated in the pathogenesis of head and neck cancer, and the hypermethylation of several WNT cascade inhibitors were shown to be useful in disease prognosis. However, the extent of deregulation of WNT pathway by DNA hypermethylation has not been studied in detail in laryngeal cancer so far. The aim of this study was to establish the frequency of methylation of WNT pathway negative regulators in laryngeal squamous cell carcinomas and evaluate its prognostic significance. METHODS: Twenty-six laryngeal squamous cell carcinoma cell lines and samples obtained from twenty-eight primary laryngeal carcinoma patients were analyzed. The methylation status of DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1 was assessed using the methylation-specific polymerase chain reaction. RESULTS: Frequent hypermethylation of DKK1, PPP2R2B, SFRP1, SFRP2, and WIF-1 was detected, and a high methylation index was usually observed. Half of the cell lines analyzed and seventy percent of primary laryngeal carcinoma cases were characterized by the methylation of at least four genes. The hypermethylation of PPP2R2B or WIF-1 was associated with longer survival in laryngeal carcinoma cell lines. Moreover, the concurrent methylation of PPP2R2B and SFRP1 differentiated primary from recurrent laryngeal carcinoma cell lines. CONCLUSIONS: Frequent hypermethylation of WNT pathway negative regulators is observed in laryngeal squamous cell carcinomas. The possible prognostic significance of the methylation of DKK1, PPP2R2B, and SFRP1 needs to be evaluated in further prospective studies. J Oral Pathol Med (2014) 43: 652–657 Correspondence: Jarosław Paluszczak, PhD, Department of Pharmaceutical z cickiego 4, Biochemistry, Pozna n University of Medical Sciences, ul. Swie 60-781 Pozna n, Poland. Tel: +48 61 8546624, Fax: +48 61 8546620, E-mail: [email protected] Accepted for publication February 3, 2014
Keywords: laryngeal cancer; DNA methylation; WNT pathway; epimarker
Abbreviations HNSCC head and neck squamous cell carcinoma LSCC
laryngeal squamous cell carcinoma
Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide with approximately 600 000 new cases yearly (1), and laryngeal squamous cell carcinomas (LSCC) are the most frequent tumors of the head and neck region. Despite the growing understanding of the risk factors and molecular mechanisms which drive HNSCC, the treatment outcomes remain unchanged with 5-year survival rates below 50%. This is attributed to frequent tumor recurrence and the development of second primary tumors or metastases (2). The establishment of new molecular prognostic biomarkers may potentially improve therapy outcome. In the last decade, epimarkers based on DNA methylation analysis have emerged as one of the most promising diagnostic tools which may be applied in both cancer detection and prognosis and in predicting therapeutic response (3). The canonical WNT pathway which is responsible for the regulation of cell proliferation, migration, and apoptosis is frequently deregulated in cancer (4). This pathway is activated upon the binding of WNT ligands to transmembrane receptors of the Frizzled family and/or low-densitylipoprotein receptor-related family receptors (LRP5 or LRP6). This leads to the activation of Dishevelled protein that inhibits the GSK-3b-mediated phosphorylation and subsequent ubiquitin-dependent proteasomal degradation of b-catenin. The resulting stabilization of b-catenin enables its translocation to the nucleus where it forms complexes with LEF and TCF transcription factors and stimulates expression of Myc, CCND1, or MMP-7. A number of proteins regulate the activity of this pathway. WNT signaling may be
WNT pathway methylation in larynx tumors Paluszczak et al.
inhibited by proteins which disrupt the binding or activation of Frizzled/LRP by WNT ligands such as DKK1, WIF-1, or SFRP family proteins. The downstream activation of b-catenin may be, on the other hand, inhibited by such proteins as APC or the components of the protein phosphatase PP2A complex (e.g., PPP2R2B/B55b). Finally, the activity of the transcriptional complex incorporating nuclear b-catenin may be inhibited by RUNX3 protein. The aberrations in the canonical WNT signaling pathway have been recently implicated in head and neck carcinogenesis and especially well documented in the case of oral cancers (5). There is however little information on the occurrence and role of WNT pathway changes specifically in laryngeal cancers. It seems that the activating mutations of b-catenin are rare in HNSCC and that the aberrant overstimulation of WNT pathway relies on other mechanisms. Both WNT ligands and Frizzled and Dishevelled proteins are frequently upregulated in HNSCC (6, 7). Moreover, the activity of WNT negative feedback regulators that are active antagonists of this pathway is often lost due to genetic or epigenetic changes. The inactivation of APC gene by loss of heterozygosity was observed in over a quarter of HNSCC cases (8, 9). Importantly, the expression of many WNT antagonists may undergo silencing due to the methylation of their promoter regions. In this regard, recent investigations show that oral cancers are characterized by frequent hypermethylation of SFRP1, SFRP2, SFRP4, SFRP5, WIF-1, and DKK3 genes (10). Interestingly, the assessment of the methylation of SFRP1, WIF-1, DKK3, and RUNX3 was proven useful in the prediction of recurrence of esophageal squamous cell carcinoma (11). The aim of the present study was to assess the frequency of hypermethylation of WNT pathway antagonists (DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1) in laryngeal squamous cell carcinomas and find correlations of gene promoter methylation with such clinicopathological features as tumor recurrence or disease-free survival. We report that the analysis of the methylation of promoter regions of PPP2R2B, SFRP1, and WIF-1 genes may constitute a new promising biomarker strategy in LSCC patients.
Materials and methods Laryngeal cancer cell lines Twenty-six cell lines established from laryngeal squamous cell carcinoma patients at the University of Turku, Finland, were analyzed in the study. The characteristics of the original material taken to establish the cell line are presented in Table 1. Fourteen cell lines were derived from primary laryngeal tumors, while eight and four cell lines were derived from recurrent tumors and metastases, respectively. The cytogenetic characteristics of these cell lines were described elsewhere (12–15). Patients The study group consisted of twenty-eight patients with primary T3 or T4 laryngeal squamous cell carcinoma who were primarily treated surgically (total laryngectomy) at the Department of Otolaryngology and Laryngeal Oncology, Pozna n University of Medical Sciences between 2005 and
Table 1 The characteristics of the patients and tumors that were taken to establish the analyzed laryngeal squamous cell carcinoma cell lines
UT-SCC
Age
Sex
TNM classification
4 6A 6B 8 9 11 13 17 19A 19B 22 23 29 34 38 49 50 57 75 96 103 106A 106B 107 108 116
43 51 51 42 81 58 53 65 44 44 79 66 82 63 66 76 70 76 56 65 51 59 59 46 68 60
F F F M M M M M M M M M M M M M M M M M M M M M M M
T3N0M0 T2N1M0 T2N1M0 T2N0M0 T2N1M0 T1N0M0 T3N0M0 T2N0M0 T4N0M0 T4N0M0 T1N0M0 T3N0M0 T2N0M0 T4N0M0 T2N0M0 T2N0M0 rT2N0 T2N0M0 T2N2BM0 T2N0M0 T3N0M0 T1AN0M0 rT3N0M0 T4N2CM0 T2N0M0 T4N1M0
Type of lesion
Survival (months)
Metastasis Recurrent tumor Metastasis Primary tumor Metastasis Recurrent tumor Recurrent tumor Metastasis Primary tumor Primary tumor Recurrent tumor Primary tumor Primary tumor Primary tumor Primary tumor Primary tumor Recurrent tumor Recurrent tumor Primary tumor Recurrent tumor Primary tumor Primary tumor Recurrent tumor Primary tumor Primary tumor Primary tumor
4 31 31 35 7 >5 years 11 No data >5 years >5 years 28 >5 years >5 years 10 16 No data >5 years >4 years No data No data No data >4 years >4 years 5 days 19 9
653
2007. For comparison, tumor sections were analyzed along with the fragments of macroscopically normal mucosa from a distant region of the resection field. Almost all of the patients were further treated with radiotherapy. All the patients were cigarette smokers and nearly half of them (43%) admitted frequent consumption of strong alcohols. Clinical data are collected in Table 2. Methylation-specific PCR The methylation status of DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1 was assessed using the methylation-specific polymerase chain reaction (MSP) (16). DNA was extracted from tissue samples with GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, St. Louis, MO, USA) according to manufacturer’s instructions and, subsequently, bisulfite converted using the EZ DNA Methylation Kit from ZymoResearch (Orange, CA, USA). Hot FIREPol DNA Polymerase from Solis BioDyne (Tartu, Estonia) was used in all the reactions. The primers and reaction conditions for MSP were chosen based on previously published data (17–21). All the primers were obtained from Oligo.pl (Warsaw, Poland). DNA extracted from the lymphocytes of healthy blood donors and a completely methylated human DNA (Fermentas, Burlington, Canada) were used as the negative and positive controls, respectively. Amplification products were resolved on 2% agarose gels and visualized in UV light after ethidium bromide staining. Statistical analysis The correlation between clinicopathological features and gene methylation was assessed with chi-square or Fisher’s exact test and F-Cox test (P ≤ 0.1) using STATISTICA 10. J Oral Pathol Med
WNT pathway methylation in larynx tumors Paluszczak et al.
654
Table 2 Patient and tumor characteristics
Table 4 The frequency of simultaneous methylation of genes in primary laryngeal carcinomas and laryngeal carcinoma cell lines
Age (years) Range Mean Sex Male Female T classification T2 T3 T4 No data N classification N0 N1 N2 N3 No data Adjuvant therapy Radiotherapy Chemoradiotherapy No data
43–75 57.4
Number of methylated genes
23 5
Primary laryngeal carcinomas Laryngeal carcinoma cell lines
1 10 16 1 19 3 2 3 1 21 3 4
Results The frequency of gene promoter hypermethylation in laryngeal squamous cell carcinoma cell lines and primary LSCC samples is shown in Table 3. Out of the seven genes analyzed, two (LKB1, RUNX3) were practically unmethylated (0% and 4.5% in the group of patients and LSCC cell lines, respectively). Intermediate frequency of methylation (8% and 42.3% in the group of patients and LSCC cell lines, respectively) was observed in the case of DKK1. The other four genes (SFRP1, SFRP2, PPP2R2B, and WIF-1) showed high frequency of methylation. DNA methylation profiles were compared with gene expression level of SFRP1, SFRP2, PPP2R2B, and WIF-1 in ten LSCC cell lines using our previously described chip data (15). In the case of SFRP2, PPP2R2B, and WIF-1, cell lines characterized by lack of gene methylation generally showed higher expression than cell lines where gene methylation was observed (data not shown). Such a correlation was however not described in the case of SFRP1. There was an overall agreement in the rates of gene methylation between cell lines and primary tumor samples with the exception of WIF-1 and DKK1. The cell lines under analysis were characterized by greater heterogeneity with representatives of all the stages of tumor progression (T1– T4), while the clinical group consisted of only patients with T3/T4 LSCC; however, the above-mentioned variation could not be attributed merely to the observed differences in tumor stage. All the patients but one showed the methylation of at least one gene (Table 4). A high methylation index was usually observed. Half of the cell
0
1
2
3
4
5
4.16%
0
8.3%
16.6%
25%
45.8%
0
5%
25%
20%
35%
15%
lines analyzed and seventy percent of primary LSCC cases were characterized by the methylation of at least four genes. No correlation was found between the hypermethylation of any of the analyzed genes and age, sex, tumor stage, or lymph node involvement. A difference in the frequency of methylation of DKK1 was observed between T3 (62.5%) and T4 (26.7%) primary LSCC; however, this was not statistically significant. On the other hand, there was a significant difference (P = 0.0515) in the frequency of joint methylation of PPP2R2B and SFRP1 between cell lines derived from either primary or recurrent LSCC, with either half or all of the cases showing the methylation of both genes, respectively. Moreover, the hypermethylation of PPP2R2B was associated with longer survival (P = 0.0769) in the case of LSCC cell lines (Fig. 1). Also the methylation of WIF-1 predicted longer survival (P = 0.0126) in the case of LSCC cell lines derived from primary tumors (Fig. 2); however, this result should be interpreted with caution because of a low number of samples analyzed. The methylation of the genes was also analyzed in normal mucosa derived from LSCC patients undergoing laryngectomy. With the exception of LKB1 and RUNX3, all the
Figure 1 Disease-free survival estimated by PPP2R2B methylation in laryngeal squamous cell lines. M—cell lines showing PPP2R2B methylation and U—cell lines showing lack of PPP2R2B methylation.
Table 3 The frequency of methylation of genes in primary laryngeal carcinomas and laryngeal carcinoma cell lines Frequency of methylation Primary laryngeal carcinoma Laryngeal carcinoma cell lines
J Oral Pathol Med
RUNX3
SFRP1
SFRP2
LKB1
PPP2R2B
WIF-1
DKK1
0% (0/28) 4.5% (1/22)
89.3% (25/28) 88.5% (23/26)
75.0% (21/28) 72.0% (18/25)
0% (0/26) 4.5% (1/22)
76.0% (20/25) 72.0% (18/25)
92% (23/25) 62.5% (15/24)
42.3% (11/26) 8.0% (2/25)
WNT pathway methylation in larynx tumors Paluszczak et al.
Figure 2 Disease-free survival estimated by WIF-1 methylation in primary laryngeal squamous cell lines. M—cell lines showing WIF-1 methylation and U—cell lines showing lack of WIF1 methylation.
genes showed frequent methylation in epiglottic mucosa and there was a high level of concordance of methylation profiles between tumor samples and corresponding normal epithelium (Table 5).
Discussion Aberrations in WNT signaling have been implicated in the pathogenesis of numerous types of cancer (4). Recent findings indicate a role of WNT/b-catenin deregulation also in head and neck carcinogenesis (5). Genetic mutations of APC or b-catenin are rarely observed in HNSCC (19). Therefore, the upregulation of this pathway must be related to other mechanisms including epigenetic changes. In this regard, the aberrant hypermethylation of promoters of genes encoding WNT pathway negative regulators including DKK3, RUNX3, SFRP1, SFRP2, and WIF-1 was observed in oral (10, 19, 22, 23) and nasopharyngeal (24–26) carcinomas. The methylation of these genes has not been studied specifically in laryngeal squamous cell carcinomas so far, with the exception of RUNX3 (27). Thus, in this study, we evaluated the methylation status of seven negative feedback regulators of the WNT pathway: DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1. We compared gene methylation rates assessed in clinical tissue samples and in cell lines because the latter can be easily used in further functional studies. Frequent methylation of all the studied genes except LKB1 and RUNX3 was found both in laryngeal carcinoma cell lines and clinical tissue samples.
The lack of LKB1 methylation observed in our study is in agreement with the findings of other authors. The loss of LKB1 function by either genetic mutations (28, 29) or epimutations (30) is very rare in HNSCC/LSCC. Moreover, significant downregulation of nuclear LKB1 is associated with metastatic and not primary HNSCC (31). On the other hand, the lack of methylation of RUNX3 is in contradiction to the results of the study by Tang et al. (27) where 95% of LSCC cases showed the hypermethylation of this gene. Also, a quarter of tongue cancer cases were characterized by RUNX3 methylation (23). However, a tumor suppressor role for RUNX3 in HNSCC has been recently questioned because its overexpression was shown to correlate with enhanced cell growth, inhibition of apoptosis, and malignant tumor behavior (32). Thus, more research is required to elucidate the function of this gene in head and neck carcinogenesis. The frequency of hypermethylation of WIF1 or SFRP2 observed in our study was similar to that reported in other studies related to HNSCC (19, 24, 26, 33). On the other hand, we observed a higher frequency of methylation of SFRP1 than reported by other authors (19, 33) and this may possibly result from patients’ heterogeneity. DKK1 was less frequently methylated in patients with T4 than T3 primary LSCC, which suggests a possible role of this gene in disease progression. Similarly, the overexpression of DKK1 was associated with increased proliferation rate and progression of esophageal carcinoma (11). Interestingly, the allelic loss of DKK1 correlated with longer survival and reduction in the risk of distant metastasis in HNSCC (34). These results put into question the tumor suppressor function of DKK1 in HNSCC and indicate the requirement for further investigation of its role in head and neck carcinogenesis, similarly as in the case of RUNX3. Although the concurrent methylation of PPP2R2B and SFRP1 differentiated primary from recurrent LSCC cell lines, our results cannot indicate whether this finding could suggest its application as a prospective marker for the estimation of recurrence risk. However, the methylation of SFRP1 was recently found to predict recurrence in esophageal squamous cell carcinoma (11). Interestingly, high immunoreactivity of CIP2A, which can normally inhibit the PP2A complex containing B55b encoded by PPP2R2B, characterized aggressive disease and was a prognostic factor in tongue cancer patients (35). Thus, the results of our study implicate the potential suitability of the analysis of the methylation of PPP2R2B and SFRP1 in the assessment of the risk of recurrence; however, subsequent prospective analyses are required to validate its laboratory utility.
655
Table 5 The analysis of concordance of methylation between tumor tissue (T) and the normal mucosa from epiglottis (E)
T+ T+ T T
E+ E E+ E
SFRP1
SFRP2
PPP2R2B
WIF-1
24/28 (85.7%) 1/28 (3.6%) 3/28 (10.7%) 0/28
20/28 (71.4%) 1/28 (3.6%) 7/28 (25%) 0/28
18/25 (72%) 1/25 (4%) 4/25 (16%) 2/25 (8%)
23/25 (92%) 0/25 2/25 (8%) 0/25
DKK1 8/26 3/26 8/26 7/26
(30.7%) (11.5%) (30.7%) (26.9%)
‘ ’ lack of methylation, ‘+’ methylation of gene in the sample. The number of cases showing gene hypermethylation in relation to all cases tested and the respective percentage values are given. J Oral Pathol Med
WNT pathway methylation in larynx tumors Paluszczak et al.
656
Moreover, both the frequent hypermethylation of PPP2R2B in laryngeal carcinomas and frequent hyperactivation of CIP2A in oral cancers (36) indicate a possible role of the PP2A/CIP2A system in the pathogenesis of HNSCC (37), which may open the way to developing drugs targeting PP2A/CIP2A for treating LSCC/HNSCC. PP2A has been already shown to be a druggable anticancer target with forskolin and its derivatives acting as its inducers (38). The methylation of the analyzed genes was frequently found in the normal epiglottic mucosa that was derived from LSCC patients along with tumor sections. This fact indicates that the methylation of WNT pathway antagonists may not be cancer-specific but rather associated with the molecular changes induced by the exposure to tobacco and alcohol, which predispose epithelial cells for tumorigenesis. This is in agreement with our previous findings that laryngeal cancer patients are characterized by a broad field of epigenetically changed mucosa which is in line with the field cancerization model for HNSCC (39). The frequent hypermethylation of the molecular inhibitors of the WNT pathway in laryngeal carcinomas reported in this study seems to further underscore the importance of the deregulation of this signaling cascade in the pathogenesis of LSCC/HNSCC and makes its components candidate therapeutic targets. It has been recently shown that the knockdown of b-catenin results in the induction of apoptotic and autophagic death in head and neck cancer cell lines (40). Moreover, all-trans retinoic acid which is the only efficient chemopreventive compound in HNSCC was shown to interfere with the WNT pathway (41). It would be interesting to assess whether the use of DNA demethylating compounds could alleviate WNT signaling in laryngeal/ head and neck carcinomas. Overall, we report frequent hypermethylation of DKK1, PPP2R2B, SFRP1, SFRP2, and WIF-1 in laryngeal squamous cell carcinomas. The possible prognostic significance of the methylation of DKK1, PPP2R2B, and SFRP1 needs to be evaluated in further prospective studies. A high rate of methylation of the studied genes indicates an important role of the silencing of WNT pathway negative regulators for the pathogenesis of laryngeal carcinoma at all the stages of disease development.
7.
8. 9. 10. 11. 12. 13.
14. 15.
16.
17. 18. 19. 20.
References 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90. 2. Leemans CR, Braakhuis BJM, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer 2011; 11: 9– 22. 3. Paluszczak J, Baer-Dubowska W. Epigenetic diagnostics of cancer – the application of DNA methylation markers. J Appl Genet 2006; 47: 365–75. 4. Reya T, Clevers H. WNT signaling in stem cells and cancer. Nature 2005; 434: 843–50. 5. Molinolo AA, Amornphimoltham P, Squarize CH, Castilho RM, Patel V, Gutkind JS. Dysregulated molecular networks in head and neck carcinogenesis. Oral Oncol 2009; 45: 324– 34. 6. Leethanakul C, Patel V, Gillespie J, et al. Distinct pattern of expression of differentiation and growth-related genes in squamous cell carcinomas of the head and neck revealed by J Oral Pathol Med
21.
22.
23.
24.
the use of laser capture microdissection and cDNA arrays. Oncogene 2000; 19: 3220–4. Baker H, Patel V, Molinolo AA, et al. Proteome-wide analysis of head and neck squamous cell carcinomas using laser-capture microdissection and tandem mass spectrometry. Oral Oncol 2005; 41: 183–99. Worsham MJ, Chen KM, Meduri V, et al. Epigenetic events of disease progression in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2006; 132: 668–77. Chang KW, Lin SC, Mangold KA, et al. Alterations of adenomatous polyposis Coli (APC) gene in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2000; 29: 223–6. Pannone G, Bufo P, Santoro A, et al. WNT pathway in oral cancer: epigenetic inactivation of WNT-inhibitors. Oncol Rep 2010; 24: 1035–41. Liu JB, Qiang FL, Dong J, et al. Plasma DNA methylation of WNT antagonists predicts recurrence of esophageal squamous cell carcinoma. World J Gastroenterol 2011; 17: 4917–21. Giefing M, Martin-Subero JI, Kiwerska K, et al. Characterization of homozygous deletions in laryngeal squamous cell carcinoma cell lines. Cancer Genet Cytogenet 2008; 184: 38–43. Jarmu_z M, Golusi nski W, Grenman R, Szyfter K. Analysis of chromosome aberrations in cell lines derived from laryngeal cancer in relation to tumor progression. Eur Arch Otorhinolaryngol 2002; 259: 269–73. Jarmu_z M, Grenman R, Golusi nski W, Szyfter K. Aberrations of 11q13 in laryngeal squamous cell lines and their prognostic significance. Cancer Genet Cytogenet 2005; 160: 82–8. Jarmu_z-Szymczak M, Pelinska K, Kostrzewska-Poczekaj M, et al. Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization. Mol Biol Rep 2013; 40: 4161–71. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93: 9821–6. Song HJ, Shim KN, Joo YH, Kim SE, Jung SA, Yoo K. Methylation of the tumor suppressor gene RUNX3 in human gastric carcinoma. Gut Liver 2008; 2: 119–25. Esteller M, Avizienyte E, Corn PG, et al. Epigenetic inactivation of LKB1 in primary tumors associated with the PeutzJeghers syndrome. Oncogene 2000; 19: 164–8. Sogabe Y, Suzuki H, Toyota M, et al. Epigenetic inactivation of SFRP genes in oral squamous cell carcinoma. Int J Oncol 2008; 32: 1253–61. Tan J, Lee PL, Li Z, et al. B55b-associated PP2A complex controls PDK1-directed myc signaling and modulates rapamycin sensitivity in colorectal cancer. Cancer Cell 2010; 18: 459–71. Dhir M, Montgomery EA, Gl€ ockner SC, et al. Epigenetic regulation of WNT signaling pathway genes in inflammatory bowel disease (IBD) associated neoplasia. J Gastrointest Surg 2008; 12: 1745–53. Gao F, Huang C, Lin M, et al. Frequent inactivation of RUNX3 by promoter hypermethylation and protein mislocalization in oral squamous cell carcinomas. J Cancer Res Clin Oncol 2009; 135: 739–47. Supic G, Kozomara R, Jovic N, Zeljic K, Magic Z. Hypermethylation of RUNX3 but not WIF1 gene and its association with stage and nodal status of tongue cancers. Oral Dis 2011; 17: 794–800. Chan SL, Cui Y, Van Hasselt A, et al. The tumor suppressor WNT inhibitory factor 1 is frequently methylated in nasopharyngeal and esophageal carcinomas. Lab Invest 2007; 87: 644– 50.
WNT pathway methylation in larynx tumors Paluszczak et al.
25. Lin YC, You L, Xu Z, et al. WNT signaling activation and WIF-1 silencing in nasopharyngeal cancer cell lines. Biochem Biophys Res Commun 2006; 341: 635–40. 26. Fendri A, Khabir A, Hadri-guiga B, et al. Epigenetic alteration of the WNT inhibitory factor-1 promoter is common and occurs in advanced stage of Tunisian nasopharyngeal carcinoma. Cancer Invest 2010; 28: 896–903. 27. Tang J, Bai W, Ji W, Gao H, Liu J. Relationship between hypermethylation of Runx3 gene and the development and metastasis of laryngeal carcinoma. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2010; 24: 834–7. 28. Chen RW, Avizienyte E, Roth S, et al. PTEN and LKB1 genes in laryngeal tumours. J Med Genet 1999; 36: 943–4. 29. Qiu W, Schnleben F, Thaker HM, Goggins M, Ugh S. A novel mutation of STK11/LKB1 gene leads to the loss of cell growth inhibition in head and neck squamous cell carcinoma. Oncogene 2006; 25: 2937–42. 30. Ekizoglu S, Dalay N, Karaman E, Akdeniz D, Ozaydin A, Buyru N. LKB1 downregulation may be independent of promoter methylation or FOXO3 expression in head and neck cancer. Transl Res 2013; 162: 122–9. 31. Kline ER, Muller S, Pan L, Tighiouart M, Chen ZG, Marcus AI. Localization-specific LKB1 loss in head and neck squamous cell carcinoma metastasis. Head Neck 2011; 33: 1501–12. 32. Kudo Y, Tsunematsu T, Takata T. ONCOGENIC role of RUNX3 in head and neck cancer. J Cell Biochem 2011; 112: 387–93. 33. Marsit CJ, Mcclean MD, Furniss CS, Kelsey KT. Epigenetic inactivation of the SFRP genes is associated with drinking, smoking and HPV in head and neck squamous cell carcinoma. Int J Cancer 2006; 119: 1761–6.
34. Katase N, Gunduz M, Beder LB, et al. Frequent allelic loss of Dkk-1 locus (10q11.2) is related with low distant metastasis and better prognosis in head and neck squamous cell carcinomas. Cancer Invest 2010; 28: 103–10. 35. Bockelman C, Hagstrom J, Makinen LK, et al. High CIP2A immunoreactivity is an independent prognostic indicator in early-stage tongue cancer. Br J Cancer 2011; 104: 1890–5. 36. Katz J, Jakymiw A, Ducksworth MK, et al. CIP2A expression and localization in oral carcinoma and dysplasia. Cancer Biol Ther 2010; 10: 694–9. 37. Basile JR, Czerninski R. The role of CIP2A in oral squamous cell carcinoma. Cancer Biol Ther 2010; 10: 700–2. 38. Perrotti D, Neviani P. Protein phosphatase 2A: a target for anticancer therapy. Lancet Oncol 2013; 14: e229–38. 39. Paluszczak J, Misiak P, Wierzbicka M, Wozniak A, BaerDubowska W. Frequent hypermethylation of DAPK, RARbeta, MGMT, RASSF1A and FHIT in laryngeal squamous cell carcinomas and adjacent normal mucosa. Oral Oncol 2011; 47: 104–7. 40. Chang HW, Lee YS, Nam HY, et al. Knockdown of b-catenin controls both apoptotic and autophagic cell death through LKB1/AMPK signaling in head and neck squamous cell carcinoma cell lines. Cell Signal 2013; 25: 839–47. 41. Lim YC, Kang HJ, Kim YS, Choi EC. All-trans-retinoic acid inhibits growth of head and neck cancer stem cells by suppression of WNT/b-catenin pathway. Eur J Cancer 2012; 48: 3310–8.
657
Conflict of interest None declared.
J Oral Pathol Med
doi: 10.1111/jop.12178
wileyonlinelibrary.com/journal/jop
Frequent hypermethylation of WNT pathway genes in laryngeal squamous cell carcinomas Jarosław Paluszczak1, Dawid Hemmerling1, Magdalena Kostrzewska-Poczekaj2, Małgorzata Jarmu_z-Szymczak2, Reidar Grenman3, Małgorzata Wierzbicka4, Wanda Baer-Dubowska1 1
Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Pozna n, Poland; 2Department of Mutagenesis, 3 Institute of Human Genetics, Polish Academy of Sciences, Pozna n, Poland; Department of Otorhinolaryngology, Head and Neck Surgery and Department of Medical Biochemistry, Turku University Central Hospital and Turku University, Turku, Finland; 4Department of Otolaryngology and Laryngeal Oncology, Poznan University of Medical Sciences, Pozna n, Poland
BACKGROUND: Aberrations in the function of the WNT signaling pathway have been recently implicated in the pathogenesis of head and neck cancer, and the hypermethylation of several WNT cascade inhibitors were shown to be useful in disease prognosis. However, the extent of deregulation of WNT pathway by DNA hypermethylation has not been studied in detail in laryngeal cancer so far. The aim of this study was to establish the frequency of methylation of WNT pathway negative regulators in laryngeal squamous cell carcinomas and evaluate its prognostic significance. METHODS: Twenty-six laryngeal squamous cell carcinoma cell lines and samples obtained from twenty-eight primary laryngeal carcinoma patients were analyzed. The methylation status of DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1 was assessed using the methylation-specific polymerase chain reaction. RESULTS: Frequent hypermethylation of DKK1, PPP2R2B, SFRP1, SFRP2, and WIF-1 was detected, and a high methylation index was usually observed. Half of the cell lines analyzed and seventy percent of primary laryngeal carcinoma cases were characterized by the methylation of at least four genes. The hypermethylation of PPP2R2B or WIF-1 was associated with longer survival in laryngeal carcinoma cell lines. Moreover, the concurrent methylation of PPP2R2B and SFRP1 differentiated primary from recurrent laryngeal carcinoma cell lines. CONCLUSIONS: Frequent hypermethylation of WNT pathway negative regulators is observed in laryngeal squamous cell carcinomas. The possible prognostic significance of the methylation of DKK1, PPP2R2B, and SFRP1 needs to be evaluated in further prospective studies. J Oral Pathol Med (2014) 43: 652–657 Correspondence: Jarosław Paluszczak, PhD, Department of Pharmaceutical z cickiego 4, Biochemistry, Pozna n University of Medical Sciences, ul. Swie 60-781 Pozna n, Poland. Tel: +48 61 8546624, Fax: +48 61 8546620, E-mail: [email protected] Accepted for publication February 3, 2014
Keywords: laryngeal cancer; DNA methylation; WNT pathway; epimarker
Abbreviations HNSCC head and neck squamous cell carcinoma LSCC
laryngeal squamous cell carcinoma
Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide with approximately 600 000 new cases yearly (1), and laryngeal squamous cell carcinomas (LSCC) are the most frequent tumors of the head and neck region. Despite the growing understanding of the risk factors and molecular mechanisms which drive HNSCC, the treatment outcomes remain unchanged with 5-year survival rates below 50%. This is attributed to frequent tumor recurrence and the development of second primary tumors or metastases (2). The establishment of new molecular prognostic biomarkers may potentially improve therapy outcome. In the last decade, epimarkers based on DNA methylation analysis have emerged as one of the most promising diagnostic tools which may be applied in both cancer detection and prognosis and in predicting therapeutic response (3). The canonical WNT pathway which is responsible for the regulation of cell proliferation, migration, and apoptosis is frequently deregulated in cancer (4). This pathway is activated upon the binding of WNT ligands to transmembrane receptors of the Frizzled family and/or low-densitylipoprotein receptor-related family receptors (LRP5 or LRP6). This leads to the activation of Dishevelled protein that inhibits the GSK-3b-mediated phosphorylation and subsequent ubiquitin-dependent proteasomal degradation of b-catenin. The resulting stabilization of b-catenin enables its translocation to the nucleus where it forms complexes with LEF and TCF transcription factors and stimulates expression of Myc, CCND1, or MMP-7. A number of proteins regulate the activity of this pathway. WNT signaling may be
WNT pathway methylation in larynx tumors Paluszczak et al.
inhibited by proteins which disrupt the binding or activation of Frizzled/LRP by WNT ligands such as DKK1, WIF-1, or SFRP family proteins. The downstream activation of b-catenin may be, on the other hand, inhibited by such proteins as APC or the components of the protein phosphatase PP2A complex (e.g., PPP2R2B/B55b). Finally, the activity of the transcriptional complex incorporating nuclear b-catenin may be inhibited by RUNX3 protein. The aberrations in the canonical WNT signaling pathway have been recently implicated in head and neck carcinogenesis and especially well documented in the case of oral cancers (5). There is however little information on the occurrence and role of WNT pathway changes specifically in laryngeal cancers. It seems that the activating mutations of b-catenin are rare in HNSCC and that the aberrant overstimulation of WNT pathway relies on other mechanisms. Both WNT ligands and Frizzled and Dishevelled proteins are frequently upregulated in HNSCC (6, 7). Moreover, the activity of WNT negative feedback regulators that are active antagonists of this pathway is often lost due to genetic or epigenetic changes. The inactivation of APC gene by loss of heterozygosity was observed in over a quarter of HNSCC cases (8, 9). Importantly, the expression of many WNT antagonists may undergo silencing due to the methylation of their promoter regions. In this regard, recent investigations show that oral cancers are characterized by frequent hypermethylation of SFRP1, SFRP2, SFRP4, SFRP5, WIF-1, and DKK3 genes (10). Interestingly, the assessment of the methylation of SFRP1, WIF-1, DKK3, and RUNX3 was proven useful in the prediction of recurrence of esophageal squamous cell carcinoma (11). The aim of the present study was to assess the frequency of hypermethylation of WNT pathway antagonists (DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1) in laryngeal squamous cell carcinomas and find correlations of gene promoter methylation with such clinicopathological features as tumor recurrence or disease-free survival. We report that the analysis of the methylation of promoter regions of PPP2R2B, SFRP1, and WIF-1 genes may constitute a new promising biomarker strategy in LSCC patients.
Materials and methods Laryngeal cancer cell lines Twenty-six cell lines established from laryngeal squamous cell carcinoma patients at the University of Turku, Finland, were analyzed in the study. The characteristics of the original material taken to establish the cell line are presented in Table 1. Fourteen cell lines were derived from primary laryngeal tumors, while eight and four cell lines were derived from recurrent tumors and metastases, respectively. The cytogenetic characteristics of these cell lines were described elsewhere (12–15). Patients The study group consisted of twenty-eight patients with primary T3 or T4 laryngeal squamous cell carcinoma who were primarily treated surgically (total laryngectomy) at the Department of Otolaryngology and Laryngeal Oncology, Pozna n University of Medical Sciences between 2005 and
Table 1 The characteristics of the patients and tumors that were taken to establish the analyzed laryngeal squamous cell carcinoma cell lines
UT-SCC
Age
Sex
TNM classification
4 6A 6B 8 9 11 13 17 19A 19B 22 23 29 34 38 49 50 57 75 96 103 106A 106B 107 108 116
43 51 51 42 81 58 53 65 44 44 79 66 82 63 66 76 70 76 56 65 51 59 59 46 68 60
F F F M M M M M M M M M M M M M M M M M M M M M M M
T3N0M0 T2N1M0 T2N1M0 T2N0M0 T2N1M0 T1N0M0 T3N0M0 T2N0M0 T4N0M0 T4N0M0 T1N0M0 T3N0M0 T2N0M0 T4N0M0 T2N0M0 T2N0M0 rT2N0 T2N0M0 T2N2BM0 T2N0M0 T3N0M0 T1AN0M0 rT3N0M0 T4N2CM0 T2N0M0 T4N1M0
Type of lesion
Survival (months)
Metastasis Recurrent tumor Metastasis Primary tumor Metastasis Recurrent tumor Recurrent tumor Metastasis Primary tumor Primary tumor Recurrent tumor Primary tumor Primary tumor Primary tumor Primary tumor Primary tumor Recurrent tumor Recurrent tumor Primary tumor Recurrent tumor Primary tumor Primary tumor Recurrent tumor Primary tumor Primary tumor Primary tumor
4 31 31 35 7 >5 years 11 No data >5 years >5 years 28 >5 years >5 years 10 16 No data >5 years >4 years No data No data No data >4 years >4 years 5 days 19 9
653
2007. For comparison, tumor sections were analyzed along with the fragments of macroscopically normal mucosa from a distant region of the resection field. Almost all of the patients were further treated with radiotherapy. All the patients were cigarette smokers and nearly half of them (43%) admitted frequent consumption of strong alcohols. Clinical data are collected in Table 2. Methylation-specific PCR The methylation status of DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1 was assessed using the methylation-specific polymerase chain reaction (MSP) (16). DNA was extracted from tissue samples with GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, St. Louis, MO, USA) according to manufacturer’s instructions and, subsequently, bisulfite converted using the EZ DNA Methylation Kit from ZymoResearch (Orange, CA, USA). Hot FIREPol DNA Polymerase from Solis BioDyne (Tartu, Estonia) was used in all the reactions. The primers and reaction conditions for MSP were chosen based on previously published data (17–21). All the primers were obtained from Oligo.pl (Warsaw, Poland). DNA extracted from the lymphocytes of healthy blood donors and a completely methylated human DNA (Fermentas, Burlington, Canada) were used as the negative and positive controls, respectively. Amplification products were resolved on 2% agarose gels and visualized in UV light after ethidium bromide staining. Statistical analysis The correlation between clinicopathological features and gene methylation was assessed with chi-square or Fisher’s exact test and F-Cox test (P ≤ 0.1) using STATISTICA 10. J Oral Pathol Med
WNT pathway methylation in larynx tumors Paluszczak et al.
654
Table 2 Patient and tumor characteristics
Table 4 The frequency of simultaneous methylation of genes in primary laryngeal carcinomas and laryngeal carcinoma cell lines
Age (years) Range Mean Sex Male Female T classification T2 T3 T4 No data N classification N0 N1 N2 N3 No data Adjuvant therapy Radiotherapy Chemoradiotherapy No data
43–75 57.4
Number of methylated genes
23 5
Primary laryngeal carcinomas Laryngeal carcinoma cell lines
1 10 16 1 19 3 2 3 1 21 3 4
Results The frequency of gene promoter hypermethylation in laryngeal squamous cell carcinoma cell lines and primary LSCC samples is shown in Table 3. Out of the seven genes analyzed, two (LKB1, RUNX3) were practically unmethylated (0% and 4.5% in the group of patients and LSCC cell lines, respectively). Intermediate frequency of methylation (8% and 42.3% in the group of patients and LSCC cell lines, respectively) was observed in the case of DKK1. The other four genes (SFRP1, SFRP2, PPP2R2B, and WIF-1) showed high frequency of methylation. DNA methylation profiles were compared with gene expression level of SFRP1, SFRP2, PPP2R2B, and WIF-1 in ten LSCC cell lines using our previously described chip data (15). In the case of SFRP2, PPP2R2B, and WIF-1, cell lines characterized by lack of gene methylation generally showed higher expression than cell lines where gene methylation was observed (data not shown). Such a correlation was however not described in the case of SFRP1. There was an overall agreement in the rates of gene methylation between cell lines and primary tumor samples with the exception of WIF-1 and DKK1. The cell lines under analysis were characterized by greater heterogeneity with representatives of all the stages of tumor progression (T1– T4), while the clinical group consisted of only patients with T3/T4 LSCC; however, the above-mentioned variation could not be attributed merely to the observed differences in tumor stage. All the patients but one showed the methylation of at least one gene (Table 4). A high methylation index was usually observed. Half of the cell
0
1
2
3
4
5
4.16%
0
8.3%
16.6%
25%
45.8%
0
5%
25%
20%
35%
15%
lines analyzed and seventy percent of primary LSCC cases were characterized by the methylation of at least four genes. No correlation was found between the hypermethylation of any of the analyzed genes and age, sex, tumor stage, or lymph node involvement. A difference in the frequency of methylation of DKK1 was observed between T3 (62.5%) and T4 (26.7%) primary LSCC; however, this was not statistically significant. On the other hand, there was a significant difference (P = 0.0515) in the frequency of joint methylation of PPP2R2B and SFRP1 between cell lines derived from either primary or recurrent LSCC, with either half or all of the cases showing the methylation of both genes, respectively. Moreover, the hypermethylation of PPP2R2B was associated with longer survival (P = 0.0769) in the case of LSCC cell lines (Fig. 1). Also the methylation of WIF-1 predicted longer survival (P = 0.0126) in the case of LSCC cell lines derived from primary tumors (Fig. 2); however, this result should be interpreted with caution because of a low number of samples analyzed. The methylation of the genes was also analyzed in normal mucosa derived from LSCC patients undergoing laryngectomy. With the exception of LKB1 and RUNX3, all the
Figure 1 Disease-free survival estimated by PPP2R2B methylation in laryngeal squamous cell lines. M—cell lines showing PPP2R2B methylation and U—cell lines showing lack of PPP2R2B methylation.
Table 3 The frequency of methylation of genes in primary laryngeal carcinomas and laryngeal carcinoma cell lines Frequency of methylation Primary laryngeal carcinoma Laryngeal carcinoma cell lines
J Oral Pathol Med
RUNX3
SFRP1
SFRP2
LKB1
PPP2R2B
WIF-1
DKK1
0% (0/28) 4.5% (1/22)
89.3% (25/28) 88.5% (23/26)
75.0% (21/28) 72.0% (18/25)
0% (0/26) 4.5% (1/22)
76.0% (20/25) 72.0% (18/25)
92% (23/25) 62.5% (15/24)
42.3% (11/26) 8.0% (2/25)
WNT pathway methylation in larynx tumors Paluszczak et al.
Figure 2 Disease-free survival estimated by WIF-1 methylation in primary laryngeal squamous cell lines. M—cell lines showing WIF-1 methylation and U—cell lines showing lack of WIF1 methylation.
genes showed frequent methylation in epiglottic mucosa and there was a high level of concordance of methylation profiles between tumor samples and corresponding normal epithelium (Table 5).
Discussion Aberrations in WNT signaling have been implicated in the pathogenesis of numerous types of cancer (4). Recent findings indicate a role of WNT/b-catenin deregulation also in head and neck carcinogenesis (5). Genetic mutations of APC or b-catenin are rarely observed in HNSCC (19). Therefore, the upregulation of this pathway must be related to other mechanisms including epigenetic changes. In this regard, the aberrant hypermethylation of promoters of genes encoding WNT pathway negative regulators including DKK3, RUNX3, SFRP1, SFRP2, and WIF-1 was observed in oral (10, 19, 22, 23) and nasopharyngeal (24–26) carcinomas. The methylation of these genes has not been studied specifically in laryngeal squamous cell carcinomas so far, with the exception of RUNX3 (27). Thus, in this study, we evaluated the methylation status of seven negative feedback regulators of the WNT pathway: DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1. We compared gene methylation rates assessed in clinical tissue samples and in cell lines because the latter can be easily used in further functional studies. Frequent methylation of all the studied genes except LKB1 and RUNX3 was found both in laryngeal carcinoma cell lines and clinical tissue samples.
The lack of LKB1 methylation observed in our study is in agreement with the findings of other authors. The loss of LKB1 function by either genetic mutations (28, 29) or epimutations (30) is very rare in HNSCC/LSCC. Moreover, significant downregulation of nuclear LKB1 is associated with metastatic and not primary HNSCC (31). On the other hand, the lack of methylation of RUNX3 is in contradiction to the results of the study by Tang et al. (27) where 95% of LSCC cases showed the hypermethylation of this gene. Also, a quarter of tongue cancer cases were characterized by RUNX3 methylation (23). However, a tumor suppressor role for RUNX3 in HNSCC has been recently questioned because its overexpression was shown to correlate with enhanced cell growth, inhibition of apoptosis, and malignant tumor behavior (32). Thus, more research is required to elucidate the function of this gene in head and neck carcinogenesis. The frequency of hypermethylation of WIF1 or SFRP2 observed in our study was similar to that reported in other studies related to HNSCC (19, 24, 26, 33). On the other hand, we observed a higher frequency of methylation of SFRP1 than reported by other authors (19, 33) and this may possibly result from patients’ heterogeneity. DKK1 was less frequently methylated in patients with T4 than T3 primary LSCC, which suggests a possible role of this gene in disease progression. Similarly, the overexpression of DKK1 was associated with increased proliferation rate and progression of esophageal carcinoma (11). Interestingly, the allelic loss of DKK1 correlated with longer survival and reduction in the risk of distant metastasis in HNSCC (34). These results put into question the tumor suppressor function of DKK1 in HNSCC and indicate the requirement for further investigation of its role in head and neck carcinogenesis, similarly as in the case of RUNX3. Although the concurrent methylation of PPP2R2B and SFRP1 differentiated primary from recurrent LSCC cell lines, our results cannot indicate whether this finding could suggest its application as a prospective marker for the estimation of recurrence risk. However, the methylation of SFRP1 was recently found to predict recurrence in esophageal squamous cell carcinoma (11). Interestingly, high immunoreactivity of CIP2A, which can normally inhibit the PP2A complex containing B55b encoded by PPP2R2B, characterized aggressive disease and was a prognostic factor in tongue cancer patients (35). Thus, the results of our study implicate the potential suitability of the analysis of the methylation of PPP2R2B and SFRP1 in the assessment of the risk of recurrence; however, subsequent prospective analyses are required to validate its laboratory utility.
655
Table 5 The analysis of concordance of methylation between tumor tissue (T) and the normal mucosa from epiglottis (E)
T+ T+ T T
E+ E E+ E
SFRP1
SFRP2
PPP2R2B
WIF-1
24/28 (85.7%) 1/28 (3.6%) 3/28 (10.7%) 0/28
20/28 (71.4%) 1/28 (3.6%) 7/28 (25%) 0/28
18/25 (72%) 1/25 (4%) 4/25 (16%) 2/25 (8%)
23/25 (92%) 0/25 2/25 (8%) 0/25
DKK1 8/26 3/26 8/26 7/26
(30.7%) (11.5%) (30.7%) (26.9%)
‘ ’ lack of methylation, ‘+’ methylation of gene in the sample. The number of cases showing gene hypermethylation in relation to all cases tested and the respective percentage values are given. J Oral Pathol Med
WNT pathway methylation in larynx tumors Paluszczak et al.
656
Moreover, both the frequent hypermethylation of PPP2R2B in laryngeal carcinomas and frequent hyperactivation of CIP2A in oral cancers (36) indicate a possible role of the PP2A/CIP2A system in the pathogenesis of HNSCC (37), which may open the way to developing drugs targeting PP2A/CIP2A for treating LSCC/HNSCC. PP2A has been already shown to be a druggable anticancer target with forskolin and its derivatives acting as its inducers (38). The methylation of the analyzed genes was frequently found in the normal epiglottic mucosa that was derived from LSCC patients along with tumor sections. This fact indicates that the methylation of WNT pathway antagonists may not be cancer-specific but rather associated with the molecular changes induced by the exposure to tobacco and alcohol, which predispose epithelial cells for tumorigenesis. This is in agreement with our previous findings that laryngeal cancer patients are characterized by a broad field of epigenetically changed mucosa which is in line with the field cancerization model for HNSCC (39). The frequent hypermethylation of the molecular inhibitors of the WNT pathway in laryngeal carcinomas reported in this study seems to further underscore the importance of the deregulation of this signaling cascade in the pathogenesis of LSCC/HNSCC and makes its components candidate therapeutic targets. It has been recently shown that the knockdown of b-catenin results in the induction of apoptotic and autophagic death in head and neck cancer cell lines (40). Moreover, all-trans retinoic acid which is the only efficient chemopreventive compound in HNSCC was shown to interfere with the WNT pathway (41). It would be interesting to assess whether the use of DNA demethylating compounds could alleviate WNT signaling in laryngeal/ head and neck carcinomas. Overall, we report frequent hypermethylation of DKK1, PPP2R2B, SFRP1, SFRP2, and WIF-1 in laryngeal squamous cell carcinomas. The possible prognostic significance of the methylation of DKK1, PPP2R2B, and SFRP1 needs to be evaluated in further prospective studies. A high rate of methylation of the studied genes indicates an important role of the silencing of WNT pathway negative regulators for the pathogenesis of laryngeal carcinoma at all the stages of disease development.
7.
8. 9. 10. 11. 12. 13.
14. 15.
16.
17. 18. 19. 20.
References 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90. 2. Leemans CR, Braakhuis BJM, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer 2011; 11: 9– 22. 3. Paluszczak J, Baer-Dubowska W. Epigenetic diagnostics of cancer – the application of DNA methylation markers. J Appl Genet 2006; 47: 365–75. 4. Reya T, Clevers H. WNT signaling in stem cells and cancer. Nature 2005; 434: 843–50. 5. Molinolo AA, Amornphimoltham P, Squarize CH, Castilho RM, Patel V, Gutkind JS. Dysregulated molecular networks in head and neck carcinogenesis. Oral Oncol 2009; 45: 324– 34. 6. Leethanakul C, Patel V, Gillespie J, et al. Distinct pattern of expression of differentiation and growth-related genes in squamous cell carcinomas of the head and neck revealed by J Oral Pathol Med
21.
22.
23.
24.
the use of laser capture microdissection and cDNA arrays. Oncogene 2000; 19: 3220–4. Baker H, Patel V, Molinolo AA, et al. Proteome-wide analysis of head and neck squamous cell carcinomas using laser-capture microdissection and tandem mass spectrometry. Oral Oncol 2005; 41: 183–99. Worsham MJ, Chen KM, Meduri V, et al. Epigenetic events of disease progression in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2006; 132: 668–77. Chang KW, Lin SC, Mangold KA, et al. Alterations of adenomatous polyposis Coli (APC) gene in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2000; 29: 223–6. Pannone G, Bufo P, Santoro A, et al. WNT pathway in oral cancer: epigenetic inactivation of WNT-inhibitors. Oncol Rep 2010; 24: 1035–41. Liu JB, Qiang FL, Dong J, et al. Plasma DNA methylation of WNT antagonists predicts recurrence of esophageal squamous cell carcinoma. World J Gastroenterol 2011; 17: 4917–21. Giefing M, Martin-Subero JI, Kiwerska K, et al. Characterization of homozygous deletions in laryngeal squamous cell carcinoma cell lines. Cancer Genet Cytogenet 2008; 184: 38–43. Jarmu_z M, Golusi nski W, Grenman R, Szyfter K. Analysis of chromosome aberrations in cell lines derived from laryngeal cancer in relation to tumor progression. Eur Arch Otorhinolaryngol 2002; 259: 269–73. Jarmu_z M, Grenman R, Golusi nski W, Szyfter K. Aberrations of 11q13 in laryngeal squamous cell lines and their prognostic significance. Cancer Genet Cytogenet 2005; 160: 82–8. Jarmu_z-Szymczak M, Pelinska K, Kostrzewska-Poczekaj M, et al. Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization. Mol Biol Rep 2013; 40: 4161–71. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93: 9821–6. Song HJ, Shim KN, Joo YH, Kim SE, Jung SA, Yoo K. Methylation of the tumor suppressor gene RUNX3 in human gastric carcinoma. Gut Liver 2008; 2: 119–25. Esteller M, Avizienyte E, Corn PG, et al. Epigenetic inactivation of LKB1 in primary tumors associated with the PeutzJeghers syndrome. Oncogene 2000; 19: 164–8. Sogabe Y, Suzuki H, Toyota M, et al. Epigenetic inactivation of SFRP genes in oral squamous cell carcinoma. Int J Oncol 2008; 32: 1253–61. Tan J, Lee PL, Li Z, et al. B55b-associated PP2A complex controls PDK1-directed myc signaling and modulates rapamycin sensitivity in colorectal cancer. Cancer Cell 2010; 18: 459–71. Dhir M, Montgomery EA, Gl€ ockner SC, et al. Epigenetic regulation of WNT signaling pathway genes in inflammatory bowel disease (IBD) associated neoplasia. J Gastrointest Surg 2008; 12: 1745–53. Gao F, Huang C, Lin M, et al. Frequent inactivation of RUNX3 by promoter hypermethylation and protein mislocalization in oral squamous cell carcinomas. J Cancer Res Clin Oncol 2009; 135: 739–47. Supic G, Kozomara R, Jovic N, Zeljic K, Magic Z. Hypermethylation of RUNX3 but not WIF1 gene and its association with stage and nodal status of tongue cancers. Oral Dis 2011; 17: 794–800. Chan SL, Cui Y, Van Hasselt A, et al. The tumor suppressor WNT inhibitory factor 1 is frequently methylated in nasopharyngeal and esophageal carcinomas. Lab Invest 2007; 87: 644– 50.
WNT pathway methylation in larynx tumors Paluszczak et al.
25. Lin YC, You L, Xu Z, et al. WNT signaling activation and WIF-1 silencing in nasopharyngeal cancer cell lines. Biochem Biophys Res Commun 2006; 341: 635–40. 26. Fendri A, Khabir A, Hadri-guiga B, et al. Epigenetic alteration of the WNT inhibitory factor-1 promoter is common and occurs in advanced stage of Tunisian nasopharyngeal carcinoma. Cancer Invest 2010; 28: 896–903. 27. Tang J, Bai W, Ji W, Gao H, Liu J. Relationship between hypermethylation of Runx3 gene and the development and metastasis of laryngeal carcinoma. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2010; 24: 834–7. 28. Chen RW, Avizienyte E, Roth S, et al. PTEN and LKB1 genes in laryngeal tumours. J Med Genet 1999; 36: 943–4. 29. Qiu W, Schnleben F, Thaker HM, Goggins M, Ugh S. A novel mutation of STK11/LKB1 gene leads to the loss of cell growth inhibition in head and neck squamous cell carcinoma. Oncogene 2006; 25: 2937–42. 30. Ekizoglu S, Dalay N, Karaman E, Akdeniz D, Ozaydin A, Buyru N. LKB1 downregulation may be independent of promoter methylation or FOXO3 expression in head and neck cancer. Transl Res 2013; 162: 122–9. 31. Kline ER, Muller S, Pan L, Tighiouart M, Chen ZG, Marcus AI. Localization-specific LKB1 loss in head and neck squamous cell carcinoma metastasis. Head Neck 2011; 33: 1501–12. 32. Kudo Y, Tsunematsu T, Takata T. ONCOGENIC role of RUNX3 in head and neck cancer. J Cell Biochem 2011; 112: 387–93. 33. Marsit CJ, Mcclean MD, Furniss CS, Kelsey KT. Epigenetic inactivation of the SFRP genes is associated with drinking, smoking and HPV in head and neck squamous cell carcinoma. Int J Cancer 2006; 119: 1761–6.
34. Katase N, Gunduz M, Beder LB, et al. Frequent allelic loss of Dkk-1 locus (10q11.2) is related with low distant metastasis and better prognosis in head and neck squamous cell carcinomas. Cancer Invest 2010; 28: 103–10. 35. Bockelman C, Hagstrom J, Makinen LK, et al. High CIP2A immunoreactivity is an independent prognostic indicator in early-stage tongue cancer. Br J Cancer 2011; 104: 1890–5. 36. Katz J, Jakymiw A, Ducksworth MK, et al. CIP2A expression and localization in oral carcinoma and dysplasia. Cancer Biol Ther 2010; 10: 694–9. 37. Basile JR, Czerninski R. The role of CIP2A in oral squamous cell carcinoma. Cancer Biol Ther 2010; 10: 700–2. 38. Perrotti D, Neviani P. Protein phosphatase 2A: a target for anticancer therapy. Lancet Oncol 2013; 14: e229–38. 39. Paluszczak J, Misiak P, Wierzbicka M, Wozniak A, BaerDubowska W. Frequent hypermethylation of DAPK, RARbeta, MGMT, RASSF1A and FHIT in laryngeal squamous cell carcinomas and adjacent normal mucosa. Oral Oncol 2011; 47: 104–7. 40. Chang HW, Lee YS, Nam HY, et al. Knockdown of b-catenin controls both apoptotic and autophagic cell death through LKB1/AMPK signaling in head and neck squamous cell carcinoma cell lines. Cell Signal 2013; 25: 839–47. 41. Lim YC, Kang HJ, Kim YS, Choi EC. All-trans-retinoic acid inhibits growth of head and neck cancer stem cells by suppression of WNT/b-catenin pathway. Eur J Cancer 2012; 48: 3310–8.
657
Conflict of interest None declared.
J Oral Pathol Med
