Simon A. Whawell

MicroRNA-124 suppresses oral squamous cell carcinoma motility by targeting ITGB1.

Alterations in the levels of molecules which interact with the extracellular matrix, such as integrins, are associated with invasion of oral squamous cell carcinomas (OSCC). The molecular mechanisms underlying dysregulation of integrin expression in OSCC, however, remain unclear. Here, we show that microRNA‐124, a small non‐coding RNA down‐regulated in OSCC, is able to downregulate expression of integrin beta‐1 (ITGB1) by interacting with its 3′ untranslated region. Over‐expression of miR‐124 attenuates endogenous ITGB1 expression and reduces the adherence and motility of OSCC cells, suggesting disruption of miR‐124‐mediated repression of ITGB1 may be a key factor in OSCC progression.

Internalization of Staphylococcus aureus by Human Keratinocytes

ABSTRACT Staphylococcus aureus is among the most important human pathogens and causes various superficial and systemic infections. The ability of S. aureus to be internalized by, and survive within, host cells, such as keratinocytes, may contribute to the development of persistent or chronic infections and may finally lead to deeper tissue infections or dissemination. To examine the mechanisms of internalization of S. aureus by keratinocytes, isogenic mutants lacking fibronectin-binding proteins (FnBPs), a recombinant protein consisting of the fibronectin-binding domain of S. aureus FnBPs, and an anti-α5β1 antibody were used in cocultures with immortalized keratinocytes and primary keratinocytes. We found that internalization of S. aureus by immortalized keratinocytes requires bacterial FnBPs and is mediated by the major fibronectin-binding integrin α5β1. In contrast to internalization by immortalized keratinocytes, internalization of S. aureus by primary keratinocytes could occur through FnBP-dependent and -independent pathways. S. aureus clumping factor B (ClfB), which was recently determined to bind to epithelial cells, was not involved in the uptake of this bacterium by keratinocytes. The identification of an alternate uptake pathway, which is independent of S. aureus FnBPs and host cell α5β1, has important implications for the design of therapies targeted to bacterial uptake by host cells.

Endothelin-1 stimulates motility of head and neck squamous carcinoma cells by promoting stromal-epithelial interactions.

The invasion and migration of cancer cells is increasingly recognised to be influenced by factors derived from adjacent tumour‐associated stroma. The contextual signals regulating stromal–tumour interactions, however, remain poorly understood. Here, we identify a role for endothelin‐1 (ET‐1), a mitogenic peptide elevated in a number of malignancies, in promoting pro‐metastatic cross‐talk between head and neck cancer cells and adjacent fibroblasts. We demonstrate that treatment of oral fibroblasts with ET‐1 activates ADAM17‐mediated release of epidermal growth factor receptor (EGFR) ligands, triggering EGFR signalling and increased motility in neighbouring head and neck cancer cells. ET‐1–mediated paracrine transactivation of EGFR also increased cyclo‐oxygenase‐2 levels in the cancer cells, providing a molecular insight into the mechanisms by which the elevated levels of ET‐1 observed in head and neck cancers may contribute to disease progression.

Identification of bistable populations of Porphyromonas gingivalis that differ in epithelial cell invasion.

Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen Porphyromonas gingivalis exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct subtypes within a population of P. gingivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly invasive subtype invades cells at 10-30-fold higher levels than the poorly invasive subtype and remains highly invasive for approximately 12-16 generations. Analysis of the gingipain activity of these subtypes revealed that the highly invasive type had reduced cell-associated arginine-specific protease activity. The role of Arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an Arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition, a population of ΔrgpAB bacteria did not contain a hyperinvasive subtype. Screening of the protease activity of wild-type populations of both strains identified high and low protease subtypes which also showed a corresponding reduction or enhancement, respectively, of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that includes oxidative stress resistance and iron transport genes, and which might be critical to invasion of or survival within epithelial cells.

Functional expression of the chemokine receptor XCR1 on oral epithelial cells.

Chemokines are chemoattractant cytokines which act on specific receptors and play an important role in leukocyte migration as well as physiological and pathological processes. We investigated the role of the chemokine receptor XCR1 and its ligand lymphotactin (Lptn/XCL1) in the regulation of oral epithelial cell behaviour. In vitro XCR1 mRNA and cell surface protein expression was detected in normal oral keratinocytes and oral squamous cell carcinoma cell lines. Lymphotactin mediated intracellular activation of the ERK1/2 signalling pathway and stimulated migration, invasion, and proliferation of all cells through XCR1. Oral cancer cells showed a greater response to lymphotactin than normal keratinocytes and a direct relationship between receptor expression and migration, invasion, and proliferation was observed. Exposure of normal keratinocytes to lymphotactin resulted in increased adhesion to fibronectin but not collagen and stimulated MMP‐2 and MMP‐9 but not MMP‐7 release, whereas exposure of cancer cells resulted in increased adhesion to both collagen and fibronectin and stimulated production of MMP‐2, MMP‐9, and MMP‐7. We observed XCR1 but not lymphotactin to be expressed by epithelial cells in normal oral mucosa in vivo, whilst both were expressed and up‐regulated in inflammatory oral disease and oral cancer including primary and metastatic disease. Lymphotactin mRNA and constitutive intracellular protein were detected in normal keratinocytes and oral cancer cell lines in vitro. These findings show that XCR1 and its ligand, lymphotactin, are expressed by oral epithelial cells and suggest that they play a role in regulating the behaviour of these cells. Copyright

Endothelin-1 stimulates oral fibroblasts to promote oral cancer invasion

AIMS The aims of this study were to examine the role of endothelin-1 (ET-1), a pleiotropic peptide found at elevated levels in a number of malignancies and which has been shown to influence oral cancer cell behaviour via paracrine signalling pathways, on the phenotype of oral fibroblasts. MAIN METHODS The effect of ET-1 on proliferation and migration of human primary oral fibroblasts was assessed using MTS and scratch assays, respectively. The ability of ET-1 to affect fibroblast contractility was analysed using type-I collagen gels. Changes in gene expression in oral fibroblasts exposed to ET-1 were examined using quantitative PCR. The invasiveness of oral cancer cells in the presence of conditioned media collected from ET-1 treated fibroblasts was determined using 2D Matrigel assays. KEY FINDINGS Here we provide evidence that ET-1 increases the migration of oral fibroblasts and induces a more contractile phenotype which is not associated with changes in gene expression indicative of myofibroblast transdifferentiation. In addition we provide evidence that conditioned medium of ET-1-stimulated oral fibroblasts promotes invasion of OSCC cells in vitro. SIGNIFICANCE In oral squamous cell carcinoma, a frequently fatal and increasingly common epithelial malignancy of the oral cavity, ET-1 is known to contribute to pro-migratory paracrine signalling between stromal fibroblasts and cancer cells. The ability of ET-1 to modulate the phenotype of human oral stromal fibroblasts, however, has not previously been reported. The findings presented here suggest that targeting the stromal endothelin system may be a viable and novel therapeutic strategy for invasive oral cancer.

ADAM 10 is over expressed in oral squamous cell carcinoma and contributes to invasive behaviour through a functional association with αvβ6 integrin

We conclude ADAM 10 may influence OSCC invasion by functionally interacting with αvβ6 integrin which we have previously shown is over expressed in OSCC.

Differential adhesion and invasion by Staphylococcus aureus of epithelial cells derived from different anatomical sites

Staphylococcus aureus can invade epithelial cells, and the host-cell receptor α(5)β(1) integrin is thought to mediate this process. The aim of this study was to investigate S. aureus invasion of epithelial cell lines derived from oral (H357), skin (UP) and nasopharyngeal (Detroit 562) sites and to determine whether any differences were due to the levels of α(5)β(1) integrin expressed. While the adhesion and invasion of two S. aureus strains were similar in both oral and skin-derived keratinocytes, this was markedly reduced in the nasopharyngeal cell line, despite it expressing similar levels of α(5)β(1). While this might be explainable on the basis of availability of cell receptor, adhesion to and invasion of H357 and UP cells by S. aureus were enhanced when the epithelial cells were in suspension rather than on a surface, and levels of α(5) integrin subunit mRNA were also increased. Detroit 562 cells exhibited a similar α(5) gene upregulation, but this did not result in enhanced adhesion and invasion of S. aureus. The Detroit 562 cells also showed reduced adhesion to fibronectin compared with the other cell types. This, and the low S. aureus invasion, may result from reduced α(5)β(1) integrin activity or from variation in an as-yet-unidentified additional receptor or accessory molecule. These studies shed further light on the mechanisms of S. aureus invasion of human cells.

Characterisation and optimisation of organotypic oral mucosal models to study Porphyromonas gingivalis invasion.

Porphyromonas gingivalis is a Gram-negative, keystone pathogen in periodontitis that leads to tissue destruction and ultimately tooth loss. The organism is able to infect oral epithelial cells and two-dimensional (monolayer) cultures have been used to investigate this process. However, recently there has been interest in the use of three-dimensional, organotypic mucosal models to analyse infection. These models are composed of collagen-embedded fibroblasts overlain with multilayers of oral epithelial cells. In this study we report for the first time significant differences in the response of oral mucosal models to P. gingivalis infection when compared to monolayer cultures of oral epithelial cells. Intracellular survival (3-fold) and bacterial release (4-fold) of P. gingivalis was significantly increased in mucosal models compared with monolayer cultures, which may be due to the multi-layered nature and exfoliation of epithelial cells in these organotypic models. Furthermore, marked differences in the cytokine profile between infected organotypic models and monolayer cultures were observed, particularly for CXCL8 and IL6, which suggested that degradation of cytokines by P. gingivalis may be less pronounced in organotypic compared to monolayer cultures. These data suggest that use of oral mucosal models may provide a greater understanding of the host responses to P. gingivalis invasion than simple monolayer cultures.

The chemokine receptors CXCR1 and CXCR2 regulate oral cancer cell behaviour

BACKGROUND Chemokines regulate physiological and pathological leucocyte trafficking, and chemokine receptors play a role in tumorigenesis. Expression of interleukin-8 (IL-8) receptors CXCR1 and CXCR2 has been shown in oral squamous cell carcinoma (OSCC) but remains poorly characterised. This aim of this study was to investigate CXCR1 and CXCR2 expression on normal oral keratinocytes (NOKs) and oral cancer cell lines (OCCL) and their relative response when exposed to IL-8 and growth-related oncogene-α (which selectively binds CXCR2). METHODS mRNA and protein expression was studied using RT-PCR, immunocytochemistry and flow cytometry. ELISAs were used to investigate ERK1/2 phosphorylation and MMP production, whereas a MTS-based assay was employed to study proliferation. Migration assays were carried out using modified Boyden chambers with a matrigel coating used for invasion assays. RESULTS mRNA expression of CXCR1 and CXCR2 was seen in both NOKs and OCCL with significantly higher protein expression in OCCL. Exposure to IL-8 and GROα increased intracellular ERK phosphorylation, proliferation, migration and invasion with OCCL showing a greater response than NOKs. These effects were mediated through CXCR1 and CXCR2 (for IL-8) and CXCR2 (for GROα) as receptor-blocking antibodies significantly inhibited the responses. IL-8 and GROα also increased MMP-9 release from NOKs and OCCL with significantly higher amounts released by OCCL. However, an increase in MMP-7 production was only seen in OCCL. CONCLUSIONS Functional CXCR1 and CXCR2 exist on normal and cancerous oral epithelial cells, and our data suggests a role for these receptors in oral cancer biology.