FOXM1 Induces a Global Methylation Signature That Mimics the Cancer Epigenome in Head and Neck Squamous Cell Carcinoma

<div><p>The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates <em>HELLS</em>, a SNF2/helicase involved in DNA methylation, implicating <em>FOXM1</em> in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of <em>FOXM1</em> suppressed the tumour suppressor gene <em>p16^INK4A</em> (<em>CDKN2A</em>) through promoter hypermethylation. Knockdown of <em>HELLS</em> using siRNA re-activated the mRNA expression of <em>p16^INK4A</em> and concomitant downregulation of two DNA methyltransferases <em>DNMT1</em> and <em>DNMT3B</em>. The dose-dependent upregulation of endogenous <em>FOXM1</em> (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of <em>HELLS</em>, <em>BMI1</em>, <em>DNMT1</em> and <em>DNMT3B</em> and negatively with <em>p16^INK4A</em> and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of <em>FOXM1</em> significantly induced <em>p16^INK4A</em> promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant <em>FOXM1</em> expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with <em>in vivo</em> mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of <em>FOXM1</em> orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique <em>FOXM1</em>-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.</p>