FOXM1 Upregulation Is an Early Event in Human Squamous Cell Carcinoma and it Is Enhanced by Nicotine during Malignant Transformation

Abstract

BackgroundCancer associated with smoking and drinking remains a serious health problem worldwide. The survival of patients is very poor due to the lack of effective early biomarkers. FOXM1 overexpression is linked to the majority of human cancers but its mechanism remains unclear in head and neck squamous cell carcinoma (HNSCC).Methodology/Principal FindingsFOXM1 mRNA and protein expressions were investigated in four independent cohorts (total 75 patients) consisting of normal, premalignant and HNSCC tissues and cells using quantitative PCR (qPCR), expression microarray, immunohistochemistry and immunocytochemistry. Effect of putative oral carcinogens on FOXM1 transcriptional activity was dose-dependently assayed and confirmed using a FOXM1-specific luciferase reporter system, qPCR, immunoblotting and short-hairpin RNA interference. Genome-wide single nucleotide polymorphism (SNP) array was used to ‘trace’ the genomic instability signature pattern in 8 clonal lines of FOXM1-induced malignant human oral keratinocytes. Furthermore, acute FOXM1 upregulation in primary oral keratinocytes directly induced genomic instability. We have shown for the first time that overexpression of FOXM1 precedes HNSCC malignancy. Screening putative carcinogens in human oral keratinocytes surprisingly showed that nicotine, which is not perceived to be a human carcinogen, directly induced FOXM1 mRNA, protein stabilisation and transcriptional activity at concentrations relevant to tobacco chewers. Importantly, nicotine also augmented FOXM1-induced transformation of human oral keratinocytes. A centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, both located within a consensus loci (10q23), were found to be novel targets of FOXM1 and their expression correlated tightly with HNSCC progression.Conclusions/SignificanceThis study cautions the potential co-carcinogenic effect of nicotine in tobacco replacement therapies. We hypothesise that aberrant upregulation of FOXM1 may be inducing genomic instability through a program of malignant transformation involving the activation of CEP55 and HELLS which may facilitate aberrant mitosis and epigenetic modifications. Our finding that FOXM1 is upregulated early during oral cancer progression renders FOXM1 an attractive diagnostic biomarker for early cancer detection and its candidate mechanistic targets, CEP55 and HELLS, as indicators of malignant conversion and progression.

Downstream targets of FOXM1: CEP55 and HELLS are cancer progression markers of head and neck squamous cell carcinoma

“…29 The gene expression data of CEP55 and HELLS were extracted from the microarray dataset (GDS1584) using the NCBI's Gene Expression Omnibus online research tool as described previously. 7 The expression patterns of FOXM1, CEP55 and HELLS all showed similar bell-shaped profile across the different TNM tumour stages (Fig. 1C) with peak expression at early-mid tumour stages (T1N2bM0 to T4N2bM0) and decrease expression found in more advance tumour stages (T4N2M0 and T4N3M0).…”

“…Primer sequences for FOXM1B, CEP55, HELLS and two stable reference genes (POLR2A and YAP1) were described previously. 7 Statistical analysis was performed using the GraphPad InStat software (V2.04a, GraphPad Software, San Diego, CA) for Student's t-test analysis.…”

“…5,6 We have recently reported that transcriptional upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. 7 It has been shown that FOXM1 interacts with the human papilloma virus (HPV) 16 E7 protein 8 and that HPV16 E6/E7 has been implicated in HNSCC. 9 It is likely that HPV may play a role in HPV-positive HNSCC through FOXM1 activation.…”

“…10,11 We have recently shown that aberrant upregulation of FOXM1 serves as a first oncogenic hit by destabilising the genome and subsequent activation of downstream targets which regulates cytokinesis (CEP55, centrosomal protein 55kDa) [12][13][14] and epigenetic/stem-cell factor re-programming (HELLS, helicase lymphoid specific) [15][16][17][18] leading to malignant transformation (anchorage-independent growth) of a premalignant oral keratinocyte line (SVpgC2a). 7 However, the protein expression profiles of CEP55 and HELLS during HNSCC progression have not been described. In this study, we have characterised the expression patterns of CEP55 and HELLS using immunohistochemistry and digital densitometry to quantify their protein levels in an HNSCC progression tissue panel consisting of normal oral mucosa, dysplasias, HNSCC and lymph node metastasis (LnMet).…”

Phenotype-assisted transcriptome analysis identifies FOXM1 downstream from Ras–MKK3–p38 to regulate in vitro cellular invasion

“…Moreover, this transcription factor appears to play a major role in early tumorigenesis of head and neck squamous cell cancers (Gemenetzidis et al, 2009) and seems not to induce apoptosis (Kalinina et al, 2003;Gusarova et al, 2007;Park et al, 2009). It displays a proliferation-specific expression pattern.…”