Ian C. Paterson

Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF‐β1 involves MAPK, Smad and AP‐1 signalling pathways

Recent data indicate that transforming growth factor‐β1 (TGF‐β1) can act to promote tumour progression in the late stages of carcinogenesis. The mechanism by which this occurs is unknown although a ligand‐induced epithelial–mesenchymal transition (EMT) is thought to be important. In this study, we demonstrate that active Ras is required for TGF‐β1‐induced EMT in human keratinocytes and that epidermal growth factor (EGF) can substitute for mutant Ras. EMT was reversed by the removal of TGF‐β1. Under conditions of TGF‐β1‐induced EMT, cells were growth inhibited by the ligand resulting in G1 arrest. In cells containing normal Ras, TGF‐β1‐activated ERK and p38 mitogen‐activated protein kinases (MAPKs), and levels of activation were further increased by co‐treatment with EGF. Inhibition of MAPK pathways and Smad2/3 signalling blocked the induction of EMT by TGF‐β1. Further, inhibition of the AP‐1 transcriptional complex by [6]‐Gingerol, or by the ectopic expression of JDP2, blocked TGF‐β1‐induced EMT and conversely, stimulation of AP‐1 by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) substituted for EGF in the induction of EMT by TGF‐β1 in cells containing normal Ras. The presence of oncogenic Ras, the treatment of cells with EGF, or the treatment of cells with TPA to activate AP‐1, potentiated TGF‐β1‐induced Smad‐dependent transcription, an effect that was attenuated by the inhibition of MAPKs and AP‐1. The results demonstrate that active Ras and TGF‐β1 co‐operate to reversibly induce EMT in human keratinocytes by mechanisms that involve MAPKs, Smad2/3 and AP‐1. Further we demonstrate that MAPK/AP‐1 signalling enhances Smad transcriptional activity under conditions associated with TGF‐β1‐induced EMT.

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi.

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae.

Decreased expression of TGF‐β cell surface receptors during progression of human oral squamous cell carcinoma

This study examined the immunocytochemical expression of the transforming growth factor‐β (TGF‐β) isoforms TGF‐β1, TGF‐β2, and TGF‐β3, together with the TGF‐β cell surface receptors TβR‐I and TβR‐II, in patient‐matched tissue pairs of normal human oral epithelium, primary squamous cell carcinomas, and metastatic lymph node tumour deposits. There were no significant differences in the intensity of TGF‐β isoform specific staining between the normal oral epithelium, the primary tumours, and the lymph node metastases. By contrast, there was significantly less TβR‐II in the metastases than in the primary tumour and between the primary tumour and the normal oral epithelium. Similar trends were evident with TβR‐I, but not at a statistically significant level. This study also examined the structure of TβR‐I and TβR‐II in normal human oral keratinocytes in vitro and in 14 human oral carcinoma cell lines with known responses to TGF‐β1. No structural abnormalities of TβR‐II were present in the normal keratinocytes or in 13 of 14 malignant cell lines; in one line, there were both normal and mutant forms of TβR‐II, the latter being in the form of a frameshift mutation with the insertion of a single adenine base (bases 709–718, codons 125–128), predicting a truncated receptor having no kinase domain. No defects were present in TβR‐I. The structures of TβR‐I and TβR‐II did not correlate with growth inhibition by TGF‐β1. The data suggest that decreased expression of TGF‐β receptors, rather than structural defects of these genes, may be important in oral epithelial tumour progression. In order to examine the functional significance of a specific decrease in TβR‐II expression, a dominant‐negative TβR‐II construct (dnTβR‐II) was transfected into a human oral carcinoma cell line with a normal TGF‐β receptor profile and known to be markedly inhibited by TGF‐β1. In those clones that overexpressed the dnTβR‐II, growth inhibition and Smad binding activity were decreased, whilst the regulation of Fra‐1 and collagenase‐1 remained unchanged following treatment with TGF‐β1. The results demonstrate that a decrease in TβR‐II relative to TβR‐I leads to selective gene regulation with loss of growth inhibition but continued transcription of AP‐1‐dependent genes that are involved in the regulation of the extracellular matrix. Copyright

Partial characterization of specific inducers of a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae.

The insect pathogenic fungus Metarhizium anisopliae produces several extracellular cuticle-degrading proteases and evidence is consistent with one of these, PR1, which is a chymoelastase, being a determinant of pathogenicity. We have shown previously that PR1 production is regulated by both carbon catabolite and nitrogen metabolite repression and also by specific induction under derepressed conditions by insect cuticle. In the present work we have established that an enzymically released proteinaceous component(s) of insect cuticle is capable of inducing PR1 (based on appearance of extracellular activity). Cuticle of the desert locust Schistocerca gregaria treated with KOH to remove protein failed to induce PR1 production, whereas cuticle treated with either chloroform or ether to remove lipids still induced PR1. Cuticle digested with either PR1 or the trypsin-like PR2 of M. anisopliae released peptides mainly in the range 150-2000 Da; addition of these peptides generated by PR1 or PR2 at 3 micrograms alanine equivalents ml-1 induced PR1 production to a level similar (75%) to that obtained with untreated insect cuticle. Several amino acids and peptides which are abundant in insect cuticular protein (Ala, Gly, Ala-Ala, Ala-Ala-Ala, Ala-Pro and Pro-Ala) were tested at a range of concentrations and in restricted cultures for their ability to induce PR1. None induced the protease to the levels seen with cuticle or peptides enzymically released from cuticle, although some dimers and notably the monomers Ala and Gly gave 2-2.7-fold enhanced PR1 activity above depressed basal levels (up to 48-57% of that achieved with induced synthesis on cuticle).(ABSTRACT TRUNCATED AT 250 WORDS)

Specific induction of a cuticle-degrading protease of the insect pathogenic fungus Metarhizium anisopliae.

Summary: The insect pathogenic fungus Metarhizium anisopliae produces several extracellular cuticle-degrading proteases and evidence is consistent that one of these, a chymoelastase PR1, is a determinant of pathogenicity. We have shown previously that the wide-domain regulatory circuits of carbon and nitrogen derepression regulate PR1 production. In the present work we have established in addition that PR1 is specifically induced by insect cuticle, but not by other soluble or insoluble proteinaceous substrates. The feeding of elastin or collagen to derepressed established mycelium (starved for carbon and nitrogen) did not enhance PR1 production significantly and the soluble proteins BSA and gelatin rapidly and completely repressed PR1. The carbohydrate polymers cellulose and xylan gave derepressed basal levels of PR1. However, addition of locust cuticle enhanced PR1 production to a level approximately 10-fold that of derepressed mycelium. In order to establish if the enhancing effect of insect cuticle on PR1 production was due to specific induction or merely a reflection of enhanced growth on this insoluble dual carbon and nitrogen source, ergosterol was used as a measure of fungal growth. Expressing enzyme activity per mg dry weight showed that PR1 production in cuticle cultures increased approximately five- and ninefold after 12 and 24 h growth compared with elastin-grown cultures. Thus, the substantial increase in PR1 production on cuticle was shown not to be a function of fungal growth and this confirms that PR1 is induced by a component of insect cuticle; we believe this is the first report of induction by a specific substrate for any microbial protease.

The Role of TGF-β in Epithelial Malignancy and its Relevance to the Pathogenesis of Oral Cancer (Part II)

The role of transforming growth factor-beta (TGF-beta) in epithelial malignancy is complex, but it is becoming clear that, in the early stages of carcinogenesis, the protein acts as a potent tumor suppressor, while later, TGF-beta can function to advance tumor progression. We review the evidence to show that the pro-oncogenic functions of TGF-beta are associated with (1) a partial loss of response to the ligand, (2) defects of components of the TGF-beta signal transduction pathway, (3) over-expression and/or activation of the latent complex, (4) epithelial-mesenchymal transition, and (5) recruitment of signaling pathways which act in concert with TGF-beta to facilitate the metastatic phenotype. These changes are viewed in the context of what is known about the pathogenesis of oral cancer and whether this knowledge can be translated into the development of new therapeutic modalities.

Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation

Oral squamous cell carcinoma (OSCC) is a lethal disease and early death usually occurs as a result of local invasion and regional lymph node metastases. Current treatment regimens are, to a certain degree, inadequate, with a 5-year mortality rate of around 50% and novel therapeutic targets are urgently required. Using expression microarrays, we identified the eps8 gene as being overexpressed in OSCC cell lines relative to normal oral keratinocytes, and confirmed these findings using RT–PCR and western blotting. In human tissues, we found that Eps8 was upregulated in OSCC (32% of primary tumors) compared with normal oral mucosa, and that expression correlated significantly with lymph node metastasis (P=0.032), suggesting a disease-promoting effect. Using OSCC cell lines, we assessed the functional role of Eps8 in tumor cells. Although suppression of Eps8 produced no effect on cell proliferation, both cell spreading and migration were markedly inhibited. The latter cell functions may be modulated through the small GTP-ase, Rac1 and we used pull-down assays to investigate the role of Eps8 in Rac1 signaling. We found that αvβ6- and α5β1-integrin-dependent activation of Rac1 was mediated through Eps8. Knockdown of either Eps8 or Rac1, inhibited integrin-dependent cell migration similarly and transient expression of constitutively active Rac1 restored migration of cells in which Eps8 expression had been suppressed. We also showed that knockdown of Eps8 inhibited tumor cell invasion in an organotypic model of OSCC. These data suggest that Eps8 and Rac1 are part of an integrated signaling pathway modulating integrin-dependent tumour cell motility and identify Eps8 as a possible therapeutic target.

A review of inherited cancer syndromes and their relevance to oral squamous cell carcinoma.

This paper examines the genetic defects associated with inherited cancer syndromes and their relevance to oral cancer. Tumour suppressor genes are now thought of as either gatekeepers or caretakers according to whether they control cell growth directly by inhibiting cell proliferation and/or promoting cell death (gatekeepers) or whether they maintain the integrity of the genome by DNA repair mechanisms (caretakers). In disorders such as xeroderma pigmentosum, ataxia telangiectasia, Bloom syndrome and Fanconis anaemia, where there are defective caretaker genes, there is an increased incidence of second primary malignancies, including oral cancer. By contrast, with the exception of Li Fraumeni syndrome, abnormalities of gatekeeper genes do not predispose to oral cancer. Not only do Li Fraumeni patients develop second primary malignancies, but defects of the p53 pathway (p53 mutation, MDM2 over-expression, CDKN2A deletion) appear to be a ubiquitous feature of sporadic oral cancer as it occurs in the West. The findings suggest that genetic instability is of fundamental importance in the pathogenesis of oral cancer.

TGF-β Signal Transduction in Oro-facial Health and Non-malignant Disease (Part I)

The transforming growth factor-beta (TGF-) family of cytokines consists of multi-functional polypeptides that reg- ulate a variety of cell processes, including proliferation, differentiation, apoptosis, extracellular matrix elaboration, angiogenesis, and immune suppression, among others. In so doing, TGF- plays a key role in the control of cell behavior in both health and disease. In this report, we review what is known about the mechanisms of activation of the peptide, together with details of TGF- signal transduction pathways. This review summarizes the evidence implicating TGF- in normal physiological processes of the craniofacial complex—such as palatogenesis, tooth formation, wound healing, and scarring—and then evaluates its role in non-malignant disease processes such as scleroderma, submucous fibrosis, periodontal disease, and lichen planus.

DNA studies underestimate the major role of CDKN2A inactivation in oral and oropharyngeal squamous cell carcinomas

Loss of CDKN2A expression was demonstrated by immunohistochemistry in 87% of oral and oropharyngeal squamous cell carcinoma (OSCC) primary tumor samples. By contrast, DNA studies showed a much lower frequency of loss of the CDKN2A gene. Point mutations and promoter methylation of CDKN2A were seen in 7% and 23%, respectively, of primary tumors. Loss of heterozygosity analysis using a dense set of 9p markers showed allelic imbalance that included CDKN2A in only 31% of samples, but a further 47% showed loss at loci near CDKN2A with apparent retention of CDKN2A. No tumor with any allelic imbalance expressed CDKN2A, whether or not the imbalance appeared to involve the CDKN2A locus. We interpret these data as showing partially overlapping deletions on the two 9p homologues, with homozygous deletion of CDKN2A masked by amplification of contaminating stromal material. Our data show that inactivation of the CDKN2A gene products is a near‐universal step in the development of oral and oropharyngeal squamous cell carcinomas, and we suggest that homozygous deletion is the most common mechanism of inactivation. The CDKN2A locus may be particularly prone to deletion because it encodes two unrelated tumor suppressor proteins, CDKN2A (p16INK4a) and p19ARF, and deletion, but not point mutation or methylation, would inactivate both gene products. However, our results also suggest that complex patterns of allelic imbalance in primary squamous carcinomas in general may not provide reliable evidence for the existence of multiple tumor suppressor genes within a single chromosomal region. Genes Chromosomes Cancer 25:16–25, 1999.