Transforming growth factor-ß enhances invasion and metastasis in Ras-transfected human malignant epidermal keratinocytes.

Transforming growth factor-ß (TGF-ß) is known to act as a tumour suppressor early in carcinogenesis, but then switches to a pro-metastatic factor in some late stage cancers. However, the actions of TGF-ß are context dependent, and it is currently unclear how TGF-ß influences the progression of human squamous cell carcinoma (SCC). This study examined the effect of overexpression of TGF-ß1 or TGF-ß2 in Ras-transfected human malignant epidermal keratinocytes that represent the early stages of human SCC. In vitro, the proliferation of cells overexpressing TGF-ß1 or TGF-ß2 was inhibited by exogenous TGF-ß1; cells overexpressing TGF-ß1 also grew more slowly than controls, but the growth rate of TGF-ß2 overexpressing cells was unaltered. However, cells that overexpressed either TGF-ß1 or TGF-ß2 were markedly more invasive than controls in an organotypic model of SCC. The proliferation of the invading TGF-ß1 overexpressing cells in the organotypic assays was higher than controls. Similarly, tumours formed by the TGF-ß1 overexpressing cells following transplantation to athymic mice were larger than tumours formed by control cells and proliferated at a higher rate. Our results demonstrate that elevated expression of either TGF-ß1 or TGF-ß2 in cells that represent the early stages in the development of human SCC results in a more aggressive phenotype.

Minor salivary gland squamous cell carcinoma of the lower lip demonstrating striking perineural invasion.

Squamous cell carcinomas (SCC) of minor salivary gland origin are extremely rare. We present an unusual case of a 29-year-old female patient who presented with a well-differentiated SCC of minor salivary gland origin arising in the lower lip. Wedge resections of the lip, including bilateral mental nerve excision, were required to clear the tumor because of striking pathological evidence of perineural invasion distant from the primary tumor site.

TGF-ß inhibits metastasis in late stage human squamous cell carcinoma of the skin by a mechanism that does not involve Id1.

It is now generally accepted that TGF-ß acts as a pro-metastatic factor in advanced human breast cancer. However, it is well documented, that TGF-ß is context dependent, and whether the TGF-ß pathway switches to promote metastasis during the progression of squamous cell carcinoma (SCC) is unknown. This study examined the role of TGF-ß signalling in SCC using a series of genetically related keratinocyte cell lines representing later stages of the disease, stably transduced with a dominant negative TßRII cDNA (dnTßRII). We demonstrated that clones expressing dnTßRII lost their growth inhibitory response to TGF-ßin vitro, while ligand expression remained unchanged. Following transplantation of transduced cells to athymic mice in vivo, we showed that attenuation of the TGF-ß signal resulted in a loss of differentiation and increased metastasis. In human tissue samples loss of TGF-ß signal transduction as measured by pSmad2 activity also correlated with a loss of differentiation. Id1, previously shown to be down regulated by TGF-ß, an inhibitor of differentiation and associated with metastasis, was weakly expressed in focal areas of a small number of human tumours but expression did not correlate with low levels of pSmad2. Our data demonstrate that TGF-ß does not switch to promote metastasis in late stage human SCC of the skin and that inhibition of TGF-ß signalling results in a loss of differentiation and increased metastasis in the later stages of this disease.

Sialosis: 35 cases of persistent parotid swelling from two countries.

Diffuse, non-inflammatory, non-neoplastic enlargement of the major salivary glands (sialosis) is uncommon and has various systemic causes. This paper examines 35 patients whose persistent swelling of the parotid was diagnosed as sialosis, and shows that diabetes mellitus and alcoholism are the most common causes.

Myokymia (fasciculation) of the tongue as a unique presentation of mucoepidermoid carcinoma.

Neural invasion is a relatively common feature of squamous cell carcinomas of the head and neck and malignant salivary gland tumours. The symptoms depend on the location and the particular nerves involved, and include pain, anaesthesia, paraesthesia and cranial nerve palsy. The present case appears to be unique. A mucoepidermoid carcinoma showed evidence of neural involvement and presented with nerve stimulation inducing myokymia, rather than nerve destruction, which is the usual consequence of tumoral nerve invasion.

Expression of Ki-67 and p53 in cutaneous free flaps used to reconstruct soft tissue defects following resection of oral squamous cell carcinoma.

Radial forearm free flaps are used routinely to reconstruct oro-facial tissues following resection of oral squamous cell carcinoma. Surprisingly, there is little information regarding their behaviour following engraftment. The present report is a clinico-pathological study of 10 patients who had incisional biopsies of cutaneous free flaps after the presence of a white patch or erythema raised clinical suspicion. Tissues were stained with haematoxylin and eosin, diastase-periodic acid-Schiff reagent and labelled immunohistochemically for Ki-67 and p53. Four of 10 specimens showed severe epithelial dysplasia within the graft, which was contiguous with dysplasia in the adjacent oral mucosa; the remaining grafts had features typical of candidosis (n=4) or hyperkeratosis (n=2). Grafts with dysplasia had a significantly higher Ki-67 labelling index than lesions in the 'non-dysplastic' group. There were no significant differences in the Ki-67 labelling index between areas of dysplasia in the graft and areas of dysplasia in the adjacent oral epithelium. p53 staining was present in all strata of the epithelium in the dysplastic grafts and adjacent dysplastic mucosa, but was absent or weakly expressed in the stratum basale of grafts showing reactive changes only. None of the dysplastic lesions progressed to carcinoma despite a mean follow-up period of 32 months; one patient developed a recurrent mucosal tumour at the resection margin. These observations indicate that cutaneous free flaps grafted to a site of field cancerisation can develop severe epithelial dysplasia with concomitant deregulation of proliferation and increased p53 expression. Such changes raise the possibility that these lesions have the potential for malignant transformation.

Lichenoid and granulomatous stomatitis: an entity or a non-specific inflammatory process?

BACKGROUND: The presence of lichenoid or granulomatous inflammation in an oral mucosal biopsy usually suggests a distinct range of diagnostic possibilities. However, the presence of both patterns of inflammation in the same biopsy is uncommon. METHODS: A clinico-pathological study of six patients. RESULTS: All the patients in this study presented with similar mucosal lesions of the upper lip. Microscopically the lesions were characterized by the presence of lichenoid inflammation with concomitant granulomatous inflammation. The lesions were persistent and refractory to treatment with steroid medications, but remained localized and did not appear to herald the onset of systemic inflammatory or neoplastic disease. CONCLUSION: We propose the designation 'lichenoid and granulomatous stomatitis' for the cases described in this study. The clinico-pathological features of a subset of these cases suggest an unusual drug eruption.

Caspase-3 expression is reduced, in the absence of cleavage, in terminally differentiated normal oral epithelium but is increased in oral squamous cell carcinomas and correlates with tumour stage.

Oral carcinomas are known to have a greater apoptotic index than normal oral epithelium, evident as shrinking cells with condensed chromatin. In this study, these morphologically apoptotic cells stained positively for cleaved (active) caspase-3. In normal oral epithelium, cleaved caspase-3 positive-cells were only rarely detected. The terminally differentiated surface epithelial layers did not express cleaved caspase-3. The caspase-3 pro-enzyme showed a gradient of expression in normal oral epithelium, decreasing with differentiation. No expression was detectable in surface epithelial layers. Lack of expression of the major 'executioner' caspase-3 may, at least in part, explain differences in morphology between terminally differentiated and apoptotic cells. In cancers of different tissue origins, caspase-3 pro-enzyme expression can be either increased or decreased compared with normal tissue counterparts. To determine how caspase-3 expression alters during oral carcinogenesis, caspase-3 expression was compared in 39 samples of normal oral epithelium and 54 oral squamous cell carcinomas. Squamous cell carcinomas had more intense caspase-3 staining than normal epithelium (p < 0.001). Moreover, within the oral squamous cell carcinoma series, there was significantly more intense nuclear and cytoplasmic staining with increasing STNMP stage (p = 0.017 and 0.03, respectively). This was a reflection of significant associations with site (S), palpable lymph nodes (N), and differentiation (P). Both caspase-3 staining intensity and the percentage of cells positive for caspase-3 were inversely associated with differentiation. Studies of the mechanisms by which high levels of caspase-3 expression are tolerated in oral carcinoma cells may identify targets that can be used to harness caspase-3 overexpression for therapeutic benefit.

Attenuated type II TGF-beta receptor signalling in human malignant oral keratinocytes induces a less differentiated and more aggressive phenotype that is associated with metastatic dissemination.

We examined the effect of stable transfection of dominant negative TbetaR-II (dn TbetaR-II) cDNA in a human oral carcinoma cell line that contained normal Ras and was growth inhibited by TGF-beta1. Two clonal cell lines containing dn TbetaR-II were isolated and compared to the vector-only control and parent cell line. The treatment of cells with exogenous TGF-beta1 resulted in a decrease in ligand-induced growth inhibition and loss of c-myc downregulation in test cells compared to controls; transcriptional activation of certain genes including fra-1 and collagenase was retained. Cells containing dn TbetaR-II grew faster in monolayer culture, expressed less keratin 10 and exhibited increased motility and invasion in vitro compared to control cell lines. Endogenous TGF-beta1 production and the regulation of MMP-2 and MMP-9 by TGF-beta1 remained unchanged. After orthotopic transplantation to the floor of the mouth in athymic mice, cells containing dn TbetaR-II formed comparable numbers of primary tumours at the site of inoculation as controls but the tumours were less differentiated as demonstrated by the absence of keratin 10 immunostaining. Further, metastatic dissemination to the lungs and lymphatics was more evident in grafts of cells containing dn TbetaR-II than controls. Taken together, the results demonstrate that attenuation of TGF-beta signalling through transfection of dn TbetaR-II cDNA leads to an enhanced growth rate, a loss of tumour cell differentiation and an increase in migration and invasion, characteristics that corresponded to the development of the metastatic phenotype.

The cyclooxygenase 2-selective inhibitor NS398 inhibits proliferation of oral carcinoma cell lines by mechanisms dependent and independent of reduced prostaglandin E2 synthesis.

PURPOSE: We investigated the potential of cyclooxygenase (COX)-2 as anappropriate chemopreventive and/or therapeutic target for oral cancer. EXPERIMENTAL DESIGN: Immunohistochemical analysis of COX-2 expression was carried out on 37 oral squamous cell carcinomas (OSCCs) and 23 normal oral epithelium samples. We investigated whether the COX-2-selective inhibitor NS398 induced growth inhibition in four human OSCC cell lines and whether this was COX-2 dependent. RESULTS: COX-2 staining was more intense in the carcinomas compared with normal epithelium (P < 0.001). Early-stage tumors (stages I and II) had significantly higher epithelial COX-2 staining than late-stage tumors (stages III and IV; P = 0.034), and overexpression of COX-2 was detected in hyperplastic and dysplastic epithelium. Treatment of OSCC cells with NS398 for 72 h at concentrations of 50 micro M and above resulted in growth inhibition accompanied by a reversible G(0)-G(1) arrest, but no apoptosis or terminal differentiation. However, a concentration of 10 micro M was sufficient to abolish secreted prostaglandin E(2) (PGE(2)) production. Over a longer treatment time, lower concentrations of NS398 were growth inhibitory. Growth inhibition of the OSCC cell line H357 was detected after treatment with 5 micro M NS398 as well as 100 micro M NS398 for 6-12 days. In cultures treated with 5 micro M NS398, but not in those treated with 100 micro M NS398, restoration of PGE(2) to control levels abrogated growth inhibition. CONCLUSIONS: NS398 inhibits the growth of OSCC cells by mechanisms that are dependent and independent of suppression of PGE(2) synthesis. Molecular targeting of COX-2, PGE(2) synthase, or PGE(2) receptors may be useful as a chemopreventive or therapeutic strategy for oral cancer.

Deregulated Bag-1 protein expression in human oral squamous cell carcinomas and lymph node metastases.

Bag-1 is an anti-apoptotic protein that promotes metastasis in some tumour cell types. To determine whether Bag-1 expression is altered in 64 oral squamous cell carcinomas, tumour samples were compared with 17 samples of normal oral epithelium. Normal oral epithelia had pronounced nuclear staining in the basal and maturation layers and weak cytoplasmic staining that was most pronounced in the basal and suprabasal layers. Oral squamous cell carcinomas demonstrated a tendency for reduced nuclear staining intensity (p=0.036). Cytoplasmic staining intensity was not significantly different between tumour and normal tissue. However, many tumours were observed to have less of a difference between nuclear staining intensity and cytoplasmic staining intensity than normal oral epithelium. Furthermore, in lymph node metastases, cytoplasmic Bag-1 staining was stronger in 8/13 cases than in corresponding primary tumours (p=0.021). Western blotting using nine oral primary carcinoma cell lines and four normal keratinocyte cultures showed that the isoforms Bag-1s, Bag-1M, and Bag-1L were expressed in normal and malignant oral epithelial cells. Bag-1L unique sequences were shown to adopt an exclusively nuclear, and predominantly nucleolar, localization by use of transiently transfected N-terminal Bag-1L-EGFP. However, levels of Bag-1L in carcinoma cells did not differ significantly from those of normal keratinocytes. Therefore the reduced nuclear staining observed in oral squamous cell carcinomas compared with normal epithelium may reflect changes in the localization of Bag-1 isoforms, rather than decreased expression of Bag-1L. Alterations in the relative proportions of Bag-1S, Bag-1M, and Bag-1L were detected in 6/9 oral carcinoma cell lines; 5/9 oral carcinoma cell lines had a significantly greater proportion of Bag-1M than normal keratinocytes and in another cell line, Bag-1L was significantly underrepresented. Overall, the results suggest that Bag-1 deregulation plays a role in oral carcinogenesis at two different stages: during primary carcinoma development and during lymph node metastasis.

TGF-beta1 acts as a tumor suppressor of human malignant keratinocytes independently of Smad 4 expression and ligand-induced G(1) arrest.

This study examined the role of TGF-beta1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGF-beta1-induced G(1) arrest, the presence of wild type TGF-beta cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-beta responsive cell line, and treatment of the cells with TGF-beta1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-beta1 resulted in a failure to induce transcription from an artificial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and fibronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-beta1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-beta1 resulted in the attenuation of cell growth in vitro. To over-express TGF-beta1, both cell lines were transfected with latent TGF-beta1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that over-expressed endogenous TGF-beta1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-beta1 to the floor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-beta type II receptor in cells that over-expressed endogenous TGF-beta1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor effect of the ligand in vivo, indicating that the endogenous TGF-beta1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-beta1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-beta1-induced G(1) arrest.