The role of FOXM1 in oral squamous cell carcinoma

PhDFOXM1 transcription factor regulates the expression of a multitude of genes, which are important for cell proliferation, mitosis, and differentiation. Although it is abundantly expressed in majority of human solid tumours, its role in early stages of human neoplasia remains unclear. Oral squamous cell carcinoma (OSCC) is characterized by sequential genomic alterations, which lead to invasive malignancy. In this study, it is shown that FOXM1 is significantly upregulated in early oral pre-malignant and OSCC tissues and cultured keratinocytes. Furthermore, the current study suggests that FOXM1B is the main isoform driving the cell cycle dependent expression of FOXM1, and that it is expressed mainly at the G2 phase of human epithelial keratinocytes. In an attempt to understand why FOXM1 precedes epithelial malignancy, the present study investigated 1) the genomic profile of FOXM1B overexpressing human epithelial keratinocytes, and 2) whether FOXM1B overexpression interferes with the innate program of keratinocyte differentiation, which is frequently reported as being the earliest oncogenic event in epithelial neoplasia. First, by using a high-resolution Affymetrix single nucleotide polymorphism (SNP) mapping technique, this study provides the first evidence that FOXM1B overexpression alone in primay human keratinocytes was sufficient to induce genomic instability, mainly in the form of copy number alterations. FOXM1B overexpression also cooperated with damaging agents relevant to human epidermal (UVB) and oral epithelial cancer (Nicotine), to promote genomic instability in human keratinocytes. Second, by using a 3D-organotypic culture model of oral mucosa, sustained overexpression of FOXM1 was found to induce a hyper-proliferative phenotype with suprabasal proliferation, exhibiting perturbed markers of epithelial differentiation such as cytokeratin 13 and filaggrin, resembling early oral dysplastic epithelium. Based on these observations it is hypothesised that aberrant upregulation of FOXM1B serves as a ‘first hit’ whereby cells acquire genomic instability, and an abnormal differentiation program. The latter event promotes epithelial proliferation at the expense of terminal differentiation, allowing sufficient time for the accumulation of additional genetic aberrations/mutations required for tumour promotion and expansion. The Role of FOXM1 in Oral Squamous Cell Carcinom

Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes

<p>Abstract</p> <p>Background</p> <p>The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation.</p> <p>Results</p> <p>The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP) mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH) and copy number variations (CNV). FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3) and segmental LOH (6q25.1-6q25.3).</p> <p>Conclusion</p> <p>We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant upregulation of FOXM1 serves as a 'first hit' where cells acquire genomic instability which in turn predisposes cells to a 'second hit' whereby DNA-damage checkpoint response (eg. p53 or p16) is abolished to allow damaged cells to proliferate and accumulate genetic aberrations/mutations required for cancer initiation.</p

Anti-stromal treatment together with chemotherapy targets multiple signalling pathways in pancreatic adenocarcinoma.

Stromal targeting for pancreatic ductal adenocarcinoma (PDAC) is rapidly becoming an attractive option, due to the lack of efficacy of standard chemotherapy and increased knowledge about PDAC stroma. We postulated that the addition of stromal therapy may enhance the anti-tumour efficacy of chemotherapy. Gemcitabine and all-trans retinoic acid (ATRA) were combined in a clinically applicable regimen, to target cancer cells and pancreatic stellate cells (PSCs) respectively, in 3D organotypic culture models and genetically engineered mice (LSL-Kras(G12D) (/+) ;LSL-Trp53(R172H) (/+) ;Pdx-1-Cre: KPC mice) representing the spectrum of PDAC. In two distinct sets of organotypic models as well as KPC mice, we demonstrate a reduction in cancer cell proliferation and invasion together with enhanced cancer cell apoptosis when ATRA is combined with gemcitabine, compared to vehicle or either agent alone. Simultaneously, PSC activity (as measured by deposition of extracellular matrix proteins such as collagen and fibronectin) and PSC invasive ability were both diminished in response to combination therapy. These effects were mediated through a range of signalling cascades (Wnt, hedgehog, retinoid, and FGF) in cancer as well as stellate cells, affecting epithelial cellular functions such as epithelial-mesenchymal transition, cellular polarity, and lumen formation. At the tissue level, this resulted in enhanced tumour necrosis, increased vascularity, and diminished hypoxia. Consequently, there was an overall reduction in tumour size. The enhanced effect of stromal co-targeting (ATRA) alongside chemotherapy (gemcitabine) appears to be mediated by dampening multiple signalling cascades in the tumour-stroma cross-talk, rather than ablating stroma or targeting a single pathway. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.This work was supported by project grants from the Knowledge Transfer Network (Engineering and Physical Sciences Research Committee) and Pancreatic Cancer Research Fund (UK) to HMK. CF was supported by an EMBO long term fellowship and by a Marie Curie Intra8European Fellowship within the 7th European Community Framework Programme. TB and FR were supported by Cancer Research UK (grant C14303/A17197). Other grant funding includes project grants from Pancreatic Cancer Research Fund, Cancer Research UK and Barts Charity.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/path.472

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

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 HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (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 HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16INK4A 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 FOXM1 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 in vivo 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 FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions

Topical 4-thiothymidine is a viable photosensitiser for the photodynamic therapy of skin malignancies

The nucleoside analogue 4-thiothymidine has shown great potential in vitro as a photosensitiser for the photodynamic therapy of numerous cancer cell lines. However, the limited penetrating power of UV-A radiation, to which it responds, raises doubts as to its practical usefulness in clinical applications. We addressed this issue by studying the penetration extent of topical thiothymidine and the antiproliferative effect of its combination with UV-A radiation on ex vivo basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) skin cancer biopsies, and normal skin. Our results show that both the intralesional concentration of the drug and the intensity of UV-A radiation are sufficient to activate the molecule and cause extensive death by apoptosis of the malignant cells. Normal skin biopsies were not significantly affected by the treatment

<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