Open Access Research article

Investigation of HOXA9 promoter methylation as a biomarker to distinguish oral cancer patients at low risk of neck metastasis

Kenichiro Uchida13, Ratna Veeramachaneni1, Bing Huey1, Aditi Bhattacharya14, Brian L Schmidt12 and Donna G Albertson12*

Author Affiliations

1 Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, 1450 Third Street, San Francisco CA 94158-9001, USA

2 Bluestone Center for Clinical Research, New York University College of Dentistry, 421 First Avenue, New York NY 10010-4086, USA

3 Current address, Department of Oral and Maxillofacial Surgery, Yamaghuchi University Hospital, 1-1-1,Minamikogushi, Ube City, Yamaguchi Prefecture 755-8505, Japan

4 Current address, Department of Diagnostic Sciences Texas A&M HSC Baylor College of Dentistry, 3302 Gaston Ave, Room 214, Dallas, TX 75246, USA

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BMC Cancer 2014, 14:353  doi:10.1186/1471-2407-14-353

Published: 21 May 2014

Abstract

Background

Metastasis to the cervical (neck) lymph nodes is one of the most significant clinical factors responsible for death from oral squamous cell carcinoma (SCC). Therefore, the lymph nodes are frequently removed when the tumor is excised (neck dissection), even though the majority of patients will not benefit from the extra surgery. Two subtypes of oral SCC distinguished by the presence of tumor genomic aberrations +3q, -8p, +8q and/or +20 differ in risk for metastasis – high for the 3q8pq20 subtype, harboring one or more of the aberrations and low for the non-3q8pq20 subtype, lacking these alterations. A prior analysis of the literature suggested genes differentially methylated in the two subtypes. Therefore, the goal of this study was to further investigate the methylation status of candidate biomarkers of the non-3q8pq20 subtype, and evaluate their utility for identifying patients at low risk for metastasis.

Methods

Methylation status of genes in a cohort of 52 oral SCC patients with at least five year follow up was determined by pyrosequencing. Gene expression levels were determined by quantitative RT-PCR. Growth following re-expression of HOXA9 in cultured oral SCC cells was assessed by proliferation and colony formation assays.

Results

A pilot study evaluating methylation levels of HOXA9, MT1A and HOXA11 promoters in DNA from 12 tumors (six each of the 3q8pq20 and non-3q8pq20 subtypes) revealed that only HOXA9 was differentially methylated. Significant differences in methylation levels of HOXA9 were observed amongst the 52 oral SCCs with respect to genomic subtype and nodal status (p = 0.014, and p = 0.024, respectively, Wilcoxon rank sum test). High levels of HOXA9 methylation and low levels of expression in oral SCC cell lines were observed compared to HaCaT, a non-tumorigenic keratinocyte cell line. Re-expression of HOXA9 in the SCC4 oral cancer cell line resulted in diminished proliferation and colony formation.

Conclusions

HOXA9 methylation is frequent in oral cancers and levels are higher in tumors with greater risk of metastasis. Expression of HOXA9 is low in cells with high levels of methylation and reduced expression appears to confer a growth advantage.

Keywords:
Oral cancer; Metastasis; HOXA9; Methylation; Pyrosequencing