Ahmad Waseem

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

Background Cancer 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 Findings FOXM1 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/Significance This 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.

Keloids and hypertrophic scars: are they two different sides of the same coin?

Keloids and hypertrophic scars are different forms of excessive dermal fibrosis thought to be caused by regulation of cellularity increase and decrease during the wound-healing process in predisposed individuals. Differences between keloids and hypertrophic scars include distinct clinical features, histologic evidence, and cellular function in response to molecular events. Keloids and hypertrophic scars are the results of increased fibroblast density and extracellular matrix substances. Interactions between epidermal keratinocytes and dermal fibroblasts play an important role in regulating tissue homeostasis and processing scar formation. Keloids and hypertrophic scars are the two different stages of the same process that is based on separate clinical and histochemical entities. The aim of this review is to provide updated information regarding similarities and differences between keloids and hypertrophic scars as two different sides of the same coin. This article will also enable the dermatologist to better understand fundamental biology of the scarring.

Induction of Human Epithelial Stem/Progenitor Expansion by FOXM1

Stem cells are permanent residents of tissues and thought to be targets of cancer initiation. The frequent, and often early, upregulation of the FOXM1 transcription factor in the majority of human cancers suggests that it may participate in the initiation of human tumorigenesis. However, this hypothesis has not been tested. Herein, we show that targeting the ectopic expression of FOXM1 to the highly clonogenic cells of primary human keratinocytes with stem/progenitor cell properties, but not to differentiating cells, caused clonal expansion in vitro. We show, using a functional three-dimensional organotypic epithelial tissue regeneration system, that ectopic FOXM1 expression perturbed epithelial differentiation generating a hyperproliferative phenotype reminiscent of that seen in human epithelial hyperplasia. Furthermore, transcriptional expression analysis of a panel of 28 epithelial differentiation-specific genes reveals a role for FOXM1 in the suppression of epithelial differentiation. This study provides the first evidence that FOXM1 participates in an early oncogenic pathway that predisposes cells to tumorigenesis by expanding the stem/progenitor compartment and deregulating subsequent keratinocyte terminal differentiation. This finding reveals an important window of susceptibility to oncogenic signals in epithelial stem/progenitor cells prior to differentiation, and may provide a significant benefit to the design of cancer therapeutic interventions that target oncogenesis at its earliest incipient stage.

Keratin down-regulation in vimentin-positive cancer cells is reversible by vimentin RNA interference, which inhibits growth and motility

At later stages of tumor progression, epithelial carcinogenesis is associated with transition to a mesenchymal phenotype, which may contribute to the more aggressive properties of cancer cells and may be stimulated by growth factors such as epidermal growth factor and transforming growth factor-β. Previously, we found that cells derived from a nodal metastatic squamous cell carcinoma are highly proliferative and motile in vitro and tumorigenic in vivo. In the current study, we have investigated the role of vimentin in proliferation and motility. Cells derived from nodal metastasis express high levels of vimentin, which is undetectable in tumor cells derived from a synchronous primary lesion of tongue. Vimentin expression was enhanced by epidermal growth factor and transforming growth factor-β both independently and in combination. Use of RNA interference resulted in the generation of stable cell lines that express constitutively low levels of vimentin. RNA interference-mediated vimentin knockdown reduced cellular proliferation, migration, and invasion through a basement membrane substitute by 3-fold compared with nontargeting controls. In addition, cells with reduced vimentin reexpressed differentiation-specific keratins K13, K14, and K15 as a result of increased gene transcription as judged by quantitative PCR and promoter-reporter assays. Furthermore, cells in which vimentin expression was reduced showed a greatly decreased tumorigenic potential, as tumors developing from these cells were 70% smaller than those from control cells. The data suggest that reversal of the mesenchymal phenotype by inhibiting vimentin expression results in reexpression of epithelial characteristics and reduced tumor aggressiveness. [Mol Cancer Ther 2008;7(9):2894–903]

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

We recently showed that upregulation of a key oncogene FOXM1 precedes head and neck squamous cell carcinoma (HNSCC) malignancy. Furthermore, we also identified a centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, which are both downstream targets of FOXM1. In this study, we have investigated the expression profiles of CEP55 and HELLS using immunohistochemistry and quantified by digital densitometry in a tissue panel (20 samples) consisting of normal oral mucosa, dysplasias, HNSCC and lymph node metastasis (LnMet) samples. Furthermore, we corroborated our findings using absolute real-time PCR (qPCR) on a panel of 12 primary normal human oral keratinocytes, five dysplasia and 10 HNSCC cell lines. Finally, we validated our study using bioinformatics microarray analysis on an independent HNSCC patient cohort (four normal and 16 tumours). In normal oral mucosa, CEP55 protein was detected at very low level within the upper differentiated layers. In contrast, CEP55 was highly expressed in oral dysplasia whereas only moderate expression was detected in HNSCC and LnMet. Low level of HELLS expression was detected in the basal cell layer of the normal oral mucosa, moderate level was seen in dysplasia and high levels in both HNSCC and LnMet. These expression patterns were consistent with both qPCR data from the cell line panel and microarray data analysis of TNM-stage defined HNSCC samples confirming the progressive expression pattern of CEP55 and HELLS. To our knowledge, this is the first pilot study demonstrating that both CEP55 and HELLS mRNA and protein expression positively correlate with pre-malignancy and HNSCC progression. This study provides strong evidence that CEP55 and HELLS may be used in conjunction with FOXM1 as a biomarker set for early cancer detection and indicators of malignant conversion and progression.

Expression of keratin K2e in cutaneous and oral lesions: association with keratinocyte activation, proliferation, and keratinization.

The cytoskeleton in keratinocytes is a complex of highly homologous structural proteins derived from two families of type I and type II polypeptides. Keratin K2e is a type II polypeptide that is expressed in epidermis late in differentiation. Here we report the influence of keratinocyte activation, proliferation, and keratinization on K2e expression in samples of cutaneous and oral lesions. The normal expression of K2e in the upper spinous and granular layers of interfollicular epidermis is increased in keloid scars but showed distinct down-regulation in psoriasis and hypertrophic scars where keratinocytes are known to undergo activation. Unlike normal and psoriatic skin, K2e expression in hypertrophic and keloid scars began in the deepest suprabasal layer. In cutaneous basal and squamous cell carcinomas, K2e was absent in most tumor islands but the overlying epidermis showed strong expression. No significant K2e expression in nonkeratinized or keratinized oral epithelia, including buccal mucosa, lateral border of tongue and gingiva was detected. In oral lichen planus K2e expression was undetectable, but in benign keratoses of lingual mucosa induction of K2e along with K1 and K10 was observed. In mild-to-moderate oral dysplasia with orthokeratinization, K2e was highly expressed compared with parakeratinized areas but in severe dysplasia as well as in oral squamous cell carcinoma, K2e expression was undetectable. Taken together, the data suggest that K2e expression in skin is sensitive to keratinocyte activation but its up-regulation in oral lesions is a reflection of the degree of orthokeratinization.

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.

Thyroid Hormones and Gamma Interferon Specifically Increase K15 Keratin Gene Transcription

ABSTRACT Basal layers of stratified epithelia express keratins K5, K14, and K15, which assemble into intermediate filament networks. Mutations in K5 or K14 genes cause epidermolysis bullosa simplex (EBS), a disorder with blistering in the basal layer due to cell fragility. Nonkeratinizing stratified epithelia, e.g., in the esophagus, produce more keratin K15 than epidermis, which alleviates the esophageal symptoms in patients with K14 mutations. Hypothesizing that increasing the cellular content of K15 could compensate for the mutant K14 and thus ease skin blistering in K14 EBS patients, we cloned the promoter of the K15 gene and examined its transcriptional regulation. Using cotransfection, gel mobility shifts, and DNase I footprinting, we have identified the regulators of K15 promoter activity and their binding sites. We focused on those that can be manipulated with extracellular agents, transcription factors C/EBP, AP-1, and NF-κB, nuclear receptors for thyroid hormone, retinoic acid, and glucocorticoids, and the cytokine gamma interferon (IFN-γ). We found that C/EBP-β and AP-1 induced, while retinoic acid, glucocorticoid receptors, and NF-κB suppressed, the K15 promoter, along with other keratin gene promoters. However, the thyroid hormone and IFN-γ uniquely and potently activated the K15 promoter. Using these agents, we could boost the amounts of K15 in human epidermis. Our findings suggest that treatments based on thyroid hormone and IFN-γ could become effective agents in therapy for patients with EBS.

Keratin K15 as a Biomarker of Epidermal Stem Cells

Keratin 15 (K15) is type I keratin protein co-expressed with the K5/K14 pair present in the basal keratinocytes of all stratified epithelia. Although it is a minor component of the cytoskeleton with a variable expression pattern, nonetheless its expression has been reported as a stem cell marker in the bulge of hair follicles. Conversely, suprabasal expression of K15 has also been reported in both normal and diseased tissues, which is inconsistent with its role as a stem cell marker. Our recently published work has given evidence of the molecular pathways that seem to control the expression of K15 in undifferentiated and differentiated cells. In this article, we have critically reviewed the published work to establish the reliability of K15 as an epidermal stem cell marker.

Isolation, sequence and expression of the gene encoding human keratin 13.

Keratins are a family of highly homologous proteins expressed as pairs of acidic and basic forms which make intermediate filaments in epithelial cells. Keratin 13 (K13) is the major acidic keratin, which together with K4, its basic partner, is expressed in the suprabasal layers of non-cornified stratified epithelia. The mechanism which allows mucosal-specific expression of this keratin remains unknown. To provide insight into the tissue-specific expression, we have isolated the human K13 gene by screening a chromosome 17 library with a specific K13 cRNA probe. Sequence analysis of unidirectional deletions produced by transposon Tn3 has revealed that the gene is 4601 nucleotides long and contains seven introns and eight exons. When driven by the CMV promoter, the gene produced K13 protein in MCF-7 cells, which normally do not express this protein. Two transcription-start sites were identified, the major being at 61 and the minor at 63 nucleotides upstream of ATG. The upstream sequence contained a TATA box and several other putative transcription factor binding sites. A single copy of the K13 gene was detected in the human genome by Southern hybridisation and polymerase chain reaction. K13 mRNA shows differential expression in cultured keratinocytes, and in A431 cells the RNA levels remained independent of calcium concentrations in the culture medium. Characterisation of the human K13 gene will facilitate elucidation of the molecular mechanism regulating K13 expression in mucosal tissues.