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Clinical Cancer Research Vol. 10, 3825-3830, June 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Differential Methylation Status of Tumor-Associated Genes in Head and Neck Squamous Carcinoma

Incidence and Potential Implications

Shin-ichiro Maruya1, Jean-Pierre J. Issa2, Randal S. Weber3, David I. Rosenthal4, Joie C. Haviland1, Reuben Lotan5 and Adel K. El-Naggar1

Departments of 1 Pathology, 2 Leukemia, 3 Head and Neck Surgery, 4 Radiation Oncology, and 5 Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Promoter hypermethylation is one of the major mechanisms in the transcriptional inactivation of certain carcinoma-associated genes. Concurrent methylation analysis of multiple, functionally distinct genes may provide important information on their differential alterations and potential association in head and neck squamous carcinogenesis.

Experimental Design: Methylation-specific PCR analysis of the CpG islands of 8 cancer-related genes was performed on 19 cell lines and 32 primary head and neck squamous cell carcinoma (HNSC) specimens with matched histologically normal mucosa and 6 dysplastic lesions. The methylation status and histological features of the specimens were investigated.

Results: In histologically normal squamous mucosa, no to low-level methylation (0–22%) was noted in some specimens at all genes except RARß2 (50%). Considerable variation in the incidence of methylation of these genes within and between cell lines and tumor specimens was noted. The highest incidences of methylation in the cell lines and primary tumors were noted in RARß2 (53%), MGMT (37%), p16 (33%), and DAP-K (25%); low incidence of methylations were noted in E-cadherin (2%), p73 (2%) RASSF1A (10%), and p14 (20%) genes. The incidences of methylation of each gene were almost similar between the HNSC cell lines and primary cancer specimens, although methylation of RASSF1A was observed in cell line (26%), but not in dysplasia and primary tumor. RARß, p16, and MGMT genes showed the highest incidences of methylation in premalignant and invasive carcinomas.

Conclusions: Methylation of p16, RARß, and MGMT may constitute early events in HNSC tumorigenesis. The infrequent methylation at certain genes suggests a minimal role for this feature in their functional assessment in HNSC. The variability within and between cell lines and tumor specimens supports a heterogeneous and dynamic state of methylation in genes associated with HNSC tumorigenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Head and neck squamous cell carcinoma (HNSC) is the fifth most common cancer worldwide and accounts for approximately 400,000 new cases annually (1) . Despite considerable improvements in diagnosis, treatment, and understanding of the molecular mechanisms of this disease, the overall survival rate has remained constant at approximately 50% over the past 30 years (2) . Such poor outcome is related primarily to the high incidence of locoregional failure and lymph node metastasis (3) . Efforts to identify novel molecular predictors of behavior and therapeutic targets for HNSC have reported the association of several genetic and epigenetic alterations associated with their early and progressive development (4 , 5) . Among the epigenetic modifications is the cytosine methylation of the CpG islands of certain genes in human cancers including HNSC (6, 7, 8) .

Previous studies of HNSC have been limited to a single or small number of target genes and yielded inconsistent results on the methylation rates of the p16, p14, DAP-K, and RASSF1A genes in these tumors (9, 10, 11, 12, 13, 14, 15) . Recently, methylation of RARß2 was observed in several HNSC cell lines (16) , and evidence for reduced RARß2 gene expression in premalignant and malignant lesions has also been reported (17) . Other genes that may play a role in HNSC are MGMT, a DNA repair gene that functions to remove mutagenic (O6-guanine) adducts from DNA (18) , and E-cadherin, a Ca2+-dependent cell adhesion molecule that functions in cell-cell adhesion, cell polarity, and morphogenesis (19) . However, little is known about their differential involvement of methylation in HNSC tumorigenesis. Concurrent methylation analysis of genes that play key roles in various genetic pathways in HNSC carcinogenesis may provide important information on their involvement in the development and progression of this disease.

We investigated the promoter methylation patterns of 8 different genes (DAP-K, E-cadherin, MGMT, p14, p16, p73, RARß2, and RASSF1A) in 19 HNSC cell lines, including paired primary tumor and metastatic cell lines, and in 32 matched specimens of normal epithelium, dysplastic epithelium, and carcinoma to determine their relative frequency in early and invasive HNSC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
Nineteen HNSC cell lines (UMSCC-10A, UMSCC-10B, UMSCC-11B, UMSCC-14B, UMSCC-17A, UMSCC-17B, UMSCC-22A, UMSCC-22B, UMSCC-35, UMSCC-38, MDA-183, MDA-886, MDA-1186, SCC-19, SCC-30, TR146, SqCC/Y1, Tu159, and 1483), including metastatic cell lines, were used for this study (Figs. 1Citation and 2Citation ). Three pairs of cell lines (UMSCC-10A and UMSCC-10B, UMSCC-17A and UMSCC-17B, and UMSCC-22A and UMSCC-22B) were derived from primary (UMSCC-10A, UMSCC-17A, and UMSCC-22A) and metastatic tumor specimens (UMSCC-10B, UMSCC-17B, and UMSCC-22B B), respectively.



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  		Fig. 1. Representative examples of methylation-specific PCR of eight tested genes in head and neck squamous cell carcinoma cell lines (A) and surgical specimens (B). U and M denote PCR product with primers specific for unmethylated and methylated sequences, respectively. N, normal tissue; T, primary tumor; D, dysplasia.

 


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  		Fig. 2. Methylation profile by PCR analysis in head and neck squamous cell carcinoma cell lines. {rectangle}, unmethylated; , methylated; A, primary; B, metastatic tumor.

 
Tissue Samples.
Matched specimens of fresh histologically normal squamous mucosa, 6 dysplastic lesions, and carcinoma specimens from 32 patients with primary HNSC treated at The University of Texas M. D. Anderson Cancer Center (Houston, TX) formed the materials of this study. Both dysplastic and carcinoma specimens were harvested by one pathologist (A. K. E-N.) and subjected to frozen section evaluation and stored at –80°C until use. Histologically, normal mucosa was obtained from the farthest margin of resection after frozen section verification had been carried out.

Methylation-Specific PCR.
Genomic DNA was extracted from the cell lines and freshly frozen tissues using DNAzol (Molecular Research Center, Inc., Cincinnati, OH) in accordance with the manufacturer’s protocol. Extracted DNA samples were bisulfite modified using the CpGenome DNA modification kit (Intergen, Purchase, NY) in accordance with the manufacturer’s instructions. Bisulfite-modified DNAs were then by PCR amplified using primer sets specific for unmethylated and methylated sequences in each gene. The primer sequences of each gene, for unmethylated and methylated alleles, are listed in Table 1Citation . The primer sequences for DAP-K, E-cadherin, p14, p16, and p73 have been described previously (20, 21, 22, 23, 24) . PCR was performed in a final volume of 25 µl containing template DNA, 1x PCR buffer, 2.5 mM MgCl2, 0.25 mM deoxynucleotide triphosphate, 50 pM each primer, and 1 unit of AmpliTaq Gold (Applied Biosystems, Branchburg, NJ). PCR conditions included amplification at 94°C for 10 min; 35 cycles of 94°C for 45 s, the specific annealing temperature for each gene for 45 s, and 72°C for 1 min; and a final extension at 72°C for 5 min. PCR products were electrophoresed on 2% agarose gels. DNA from normal salivary gland tissue modified by SssI methylase was used as a positive control.


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  		Table 1 Primer sequences and PCR conditions for methylation-specific PCR

 
Statistical Analysis.
Statistical analysis was performed using StatView statistical software (version 5.0; SAS, Cary, NC). For each specimen, Fisher’s exact test was used to compare the methylation status of the genes with the clinicopathological findings. Ps of <0.05 were considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinicopathological Findings.
Table 2Citation presents the clinicopathological information of the patients. The 32 patients comprised 26 men and 6 women, who ranged in age from 31 to 81 years (mean age, 58.3 years). Tumor sites included the tongue (n = 16), oral cavity (n = 10), maxillary sinus (n = 4), and larynx (n = 2). Pathological stages of the patients included 4 cases of stage II disease, 10 cases of stage III disease, and 18 cases of stage IV disease. Histopathologically, tumors were classified as 6 well-differentiated carcinomas, 14 moderately differentiated carcinomas, and 12 poorly differentiated carcinomas.


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  		Table 2 Clinicopathological features of patients with head and neck squamous cell carcinoma in the study

 
Methylation Findings.
The incidences of methylation at individual loci in cell lines and surgical specimens are shown in Table 3Citation , and Figs. 2Citation and 3Citation show the profile for each gene. The frequency of methylation at an individual gene in cell lines was 37% (7 of 19) for DAP-K, 5% (1 of 19) for E-cadherin, 47% (9 of 19) for MGMT, 26% (5 of 19) for p14, 37% (7 of 19) for p16, 0% (0 of 19) for p73, 63% (12 of 19) for RARß2, and 26% (5 of 19) for RASSF1A. Fig. 1Citation shows representative examples of the methylation-specific PCR analysis in the HNSC cell lines and surgical specimens. Except for one pair of primary and metastatic cell lines, the pattern of methylation in cell lines was similar. Paired primary and metastatic cell lines UMSCC-10A and UMSCC-10B showed DAP-K and RARß2 methylation only in the primary cell line (UMSCC-10A). The rates of methylation in primary tumors were 19% (6 of 32) for DAP-K, 0% (0 of 32) for E-cadherin, 31% (10 of 32) for MGMT, 16% (5 of 32) for p14, 31% (10 of 32) for p16, 3% (1 of 32) for p73, 47% (15 of 32) for RARß2, and 0% (0 of 32) for RASSF1A.


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  		Table 3 Methylation status of multiple genes in head and neck squamous cell carcinoma cell lines and surgical tissues

 


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  		Fig. 3. Methylation profile by PCR analysis in surgical specimens.

 
To examine the occurrence of gene methylation in the cancer field, methylation-specific PCR was performed on matching histologically normal mucosa and six dysplastic lesions. Histologically normal mucosa showed a low level of methylation ranging from 0% to 22% (Table 3)Citation in all genes except for RARß2, in which 50% methylation was noted. Interestingly, methylation for E-cadherin was detected only in histologically normal mucosa (13%). Methylation for p16 was detected mostly in cell lines and carcinoma (P < 0.01). In dysplastic lesions, the incidence of methylation was 33% at the p14 and p16 genes and 67% at the RARß2 gene.

Correlation with Clinicopathological Data.
We found no significant correlation between the methylation status of an individual gene and clinicopathological parameters including age, stage, and histological differentiation. However, methylation of p16 may be associated with tumor progression (T2/T3 versus T4, P = 0.068).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results show that HNSC cell lines and carcinoma specimens manifest variable levels of methylation, with high incidence at the p16, MGMT, and RARß2 genes and low and infrequent incidence at the E-cadherin, DAP-K, RASSF1A, and p14 genes. We also noted that, in general, comparable incidences of methylation were noted between cell lines and tumor specimens in most genes. Our results are consistent with those of previous methylation studies including the DAP-K, MGMT, p14, p16, and RASSF1A genes in their analyses (10, 11, 12, 13, 14, 15, 16) . These findings suggest that a limited number of genes are methylated in HNSC tumorigenesis and that cell lines may reflect the methylation pattern of primary tumors for certain genes. We, however, noted a marked difference in the incidence of RASSF1A methylation between cell line (26%) and primary tumor (0%). A recent study by Dong et al. (25) also reported a similar discrepancy in methylation status of this gene. Such discrepancies could be attributed to the differential distribution of methylated cytosines at CpG island between cell line and primary tumor (26) .

In our study, p14 showed lower and infrequent methylation than the p16 gene, supporting previous studies of these genes in HNSC (10 , 14 , 27 , 28) . However, we noted more p14 methylation in the dysplastic lesions (33%) than in matched invasive tumor (16%). Although the numbers of such cases are too small to draw a firm conclusion, the results suggest that this may represent either regional heterogeneity or reduced methylation level during progression. In addition, the infrequent methylation of the p73 gene in both cell lines and tumor specimens suggests a minimal role of methylation at this gene in HNSC carcinogenesis.

Kulkarni et al. (29) have recently reported frequent methylation of the p16, DAP-K, and MGMT genes in primary tumor and adjacent mucosa in Indian patients with smoking history (29) . These findings, together with our results, indicate that methylation at these genes reflects field epigenetic instability. Unlike other genes, E-cadherin manifested higher incidence of methylation in histologically normal mucosa than in HNSC cell lines and carcinoma specimens. Similarly, E-cadherin methylation was reported in noncancerous tissues and infrequently in corresponding oral squamous cell carcinoma in other studies (13 , 30) . However, Waki et al. (31) reported frequent E-cadherin methylation in nonneoplastic gastric tissue and attributed these findings to aging. These results indicate a constitutional methylation of this gene in normal mucosa and that hypomethylation is associated with progression. Additional studies of this gene in the mucosa of noncancer patients are needed to support this contention. Alternatively, age-related methylation changes, as reported previously in colon mucosa, might underlie these findings. The latter explanation is unlikely in our study due to the marked variation in methylations between genes and the similar incidence of methylation in mucosa from older and younger patients.

We also noted that RARß2 was frequently methylated in the dysplastic and malignant tumors as well as in the normal tissues, suggesting early association with HNSC progression. Similar evidence for reduced expression of RARß2 in early and differentiated head and neck and esophageal squamous cell carcinomas has been reported (17 , 32) . Other studies using the same HNSC cell lines have also shown correlation between methylation of RARß, gene expression, and retinoid resistance (16) . In addition, previous studies have also reported RARß2 gene methylation in premalignant lesions of tobacco-exposed animals (33) and sputum from heavy smokers who have no evidence of cancer (34) . Similarly, immortalized cell lines from oral dysplasia manifest reduced expression of RARß due to promoter hypermethylation with restored expression by demethylating agent (35) . These findings support the critical involvement of RARß gene methylation in an early event during HNSC tumorigenesis.

In conclusion, our study demonstrates that differential epigenetic alterations in HNSC occur at several specific genes including p16, MGMT, and RARß2. These findings imply that concurrent methylation analysis provides important information on the relative functional status of these genes in HNSC tumorigenesis.


   ACKNOWLEDGMENTS
 
We are grateful to Sue M. Martinez for secretarial assistance and Kate O’Suilleabhain for critical editing and constructive comments.


   FOOTNOTES
 
Grant support: The Kenneth D. Muller Professorship and Specialized Programs of Research Excellence for the Head and Neck.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Adel K. El-Naggar, Department of Pathology, Unit 85, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3109; Fax: (713) 792-5532; E-mail: anaggar{at}mdanderson.org

Received 10/14/03; revised 3/ 1/04; accepted 3/ 8/04.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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