5-Hydroxymethylation highlights the heterogeneity in keratinization and cell junctions in head and neck cancers

Siyu Liu, Marcell Costa de Medeiros, Evan M. Fernandez, Katie R. Zarins, Raymond G. Cavalcante, Tingting Qin, Gregory T. Wolf, Maria E. Figueroa, Nisha J. D’Silva, Laura S. Rozek, Maureen A. Sartor

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide, with human papillomavirus (HPV)-related HNSCC rising to concerning levels. Extensive clinical, genetic and epigenetic differences exist between HPV-associated HNSCC and HPV-negative HNSCC, which is often linked to tobacco use. However, 5-hydroxymethylation (5hmC), an oxidative derivative of DNA methylation and its heterogeneity among HNSCC subtypes, has not been studied. Results: We characterized genome-wide 5hmC profiles in HNSCC by HPV status and subtype in 18 HPV(+) and 18 HPV(−) well-characterized tumors. Results showed significant genome-wide hyper-5hmC in HPV(−) tumors, with both promoter and enhancer 5hmC able to distinguish meaningful tumor subgroups. We identified specific genes whose differential expression by HPV status is driven by differential hydroxymethylation. CDKN2A (p16), used as a key biomarker for HPV status, exhibited the most extensive hyper-5hmC in HPV(+) tumors, while HPV(−) tumors showed hyper-5hmC in CDH13, TIMP2, MMP2 and other cancer-related genes. Among the previously reported two HPV(+) subtypes, IMU (stronger immune response) and KRT (more keratinization), the IMU subtype revealed hyper-5hmC and up-regulation of genes in cell migration, and hypo-5hmC with down-regulation in keratinization and cell junctions. We experimentally validated our key prediction of higher secreted and intracellular protein levels of the invasion gene MMP2 in HPV(−) oral cavity cell lines. Conclusion: Our results implicate 5hmC in driving differences in keratinization, cell junctions and other cancer-related processes among tumor subtypes. We conclude that 5hmC levels are critical for defining tumor characteristics and potentially used to define clinically meaningful cancer patient subgroups.

Original languageEnglish (US)
Article number175
JournalClinical Epigenetics
Issue number1
StatePublished - Dec 2020


  • Head and neck cancer
  • Human papillomavirus
  • Hydroxymethylation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Developmental Biology
  • Genetics(clinical)


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