<i>FOXM1</i> induces promoter hypermethylation of <i>p16^INK4A</i> gene in primary human oral keratinocytes.

<p>(<b>A</b>) Bisulfite modification and methylation specific absolute qPCR for the quantification of <i>p16^INK4A</i> promoter methylation status. Genomic DNA was first treated with sodium bisulfite prior to PCR pre-amplification of the promoter region of <i>p16^INK4A</i> (PCR^BS, 273 bp). Methylation specific (p16M-R/F) and methylation-independent (p16U-F/R) primers were then used to quantify the relative levels of methylated and unmethylated products within the PCR^BS sample using standard-curve based absolute qPCR method for each product, respectively. Melting analysis was performed to validate the qPCR specificity in detecting the two M and U products. (<b>B</b>) Bisulfite conversion and methylation specific qPCR were performed to measure the relative levels of unmethylated (U, melting temperature at 85.8°C) and methylated (M, 91.2°C) in either EGFP- or FOXM1-transduced primary NOK treated with either vehicle (DMSO) or 5Aza (1 µM, 3-day incubation with fresh drug replenishment daily). A total of n = 11 replicates from at least 4 independent experiments were performed. Statistical t-test significance notations *P<0.05 and ***P<0.001.</p

Upregulation of <i>FOXM1</i> (isoform B) induces a global shift in methylation pattern that mimics the cancer epigenome.

<p>(<b>A</b>) Genome-wide promoter microarray analysis of primary normal oral human keratinocytes expressing either <i>EGFP</i> (NOKG, black dots) or <i>FOXM1</i> (NOKF, yellow dots) and an established squamous cell carcinoma cell line (SCC15, red dots). Each dot represents a single gene. (<b>B</b>) A non-linear 2^nd order polynomial regression analyses were performed on the relative methylation patterns between NOKG vs NOKF (inverse correlation), NOKG vs SCC15 (inverse correlation) and NOKF vs SCC15 (positive correlation). (<b>C</b>) Gene selection criteria for differentially methylated genes between control (NOKG) and tests groups (NOKF and SCC15). 100-most hypermethylated and 100-most hypomethylated genes were inversely matched with differentially methylated genes from NOKF and SCC15. The adjacent gene lists show the shortlisted FOXM1-induced (also found in SCC15) differentially hypermethylated (red) and hypomethylated (green) genes compared to control NOKG cells. The CDKN2A (encodes <i>p16^INK4A</i>) gene, its promoter known to be hypermethylated in HNSCC, was included as a positive control for promoter hypermethylation. (<b>D</b>) Clinical tumour tissue sample correlation between the relative levels of methylation and gene expression of each shortlisted gene in a cohort of 10 patients with paired normal margin and HNSCC tumour tissue samples. Each dot represents mean ± SEM of each gene. Vertical error bars were derived from relative gene expression of 10 margin-tumour tissue pairs and horizontal error bars were derived from relative promoter methylation of 3 independent primary NOK (NOKG/NOKF) experiments. Correlation coefficient (R²) of a non-linear 2^nd order polynomial regression analyses were performed on all 30 candidate genes (left panel), 16 hypermethylated genes (middle panel) or 14 hypomethylated genes (right panel), respectively.</p

Upregulation of FOXM1 suppressed <i>p16^INK4A</i> expression in primary human oral keratinocytes.

<p>(<b>A</b>) FOXM1 significantly supresses <i>p16^INK4A</i> mRNA and protein expression (inset figure) in primary normal human keratinocytes. GAPDH was used as a control for protein loading. Control cells (mock-transduced with empty retroviral particles or EGFP-transduced) did not show significant suppression of p16^INK4A expression. (<b>B</b>) Knockdown of a FOXM1-target gene <i>HELLS</i>, which regulates genome-wide methylation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034329#pone.0034329-Dennis1" target="_blank">[14]</a>, induced <i>p16^INK4A</i> and simultaneously suppressed <i>DNMT1</i> and <i>DNMT3B</i>, but not <i>DNMT3A</i> mRNA expression in a FOXM1-transformed malignant cell line (SVFN5) expressing constitutive levels of endogenous <i>HELLS </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034329#pone.0034329-Gemenetzidis1" target="_blank">[8]</a>. Each bar represents a mean ± SEM of triplicate transfection (48 h) with either siCTRL or siHELLS. *P<0.05, **P<0.01 and ***P<0.001 indicate the level of statistical significance compared to controls. (<b>C</b>) Endogenous <i>FOXM1</i> (isoform B) mRNA expression levels in 8 strains of primary human normal oral keratinocytes, 5 dysplastic and 11 HNSCC cell lines. Total <i>FOXM1</i> mRNA expression levels were measured in the EGFP and FOXM1-transduced NOK (NOKG and NOKF), respectively. (<b>D</b>–<b>J</b>) Third-order polynomial regression analyses were performed to obtain the R² coefficient of determination values which indicate the significance of co-expression between each gene with <i>FOXM1</i> across the 24 cell strains/lines indicated in panel C.</p