Summary Protease-activated receptors (PARs) are members of the G protein-coupled receptor superfamily that are activated by the proteolytic cleavage of their amino terminal domain. PAR-1 activation by thrombin results in several biologic effects, including platelet adhesion to other cells or extracellular matrix, fibroblast, and endothelial cell growth, whereas PAR-2, activated by trypsin, has mainly a proinflammmatory and angiogenetic role. PAR-1 and PAR-2 modulate cell proliferation in physiopathologic cell invasion processes, suggesting that they may play a role in the setting of cancer growth and metastasis. Here, we have investigated the expression of PAR-1 and PAR-2 proteins by immunohistochemistry in a series of benign and malignant melanocytic lesions: 20 melanocytic lesions (10 common melanocytic nevi and 10 atypical or bdysplasticQ melanocytic nevi) and 50 melanomas (10 in situ melanomas, 10 melanomas T1, 10 melanomas T2, 10 melanomas T3 to T4, and 10 metastatic melanomas). PAR-1 was significantly overexpressed in atypical nevi and melanomas in comparison with common melanocytic nevi. PAR-2 was strongly and diffusely expressed by immunohistochemistry in all melanocytic lesions, with no statistically significant differences between nevi and melanomas. Because we found a differential expression in PAR-1 protein, but not in PAR-2, we next investigated the expression of PAR-1 messenger RNA (mRNA) by ribonuclease protection assay in paraffin-embedded tissues using a paraffin block RNA isolation procedure. Similarly to immunohistochemical results, PAR-1 mRNA expression was significantly higher in atypical nevi and melanomas in comparison with common nevi and controls. Overexpression of PAR-1 in atypical nevi and melanomas supports a role for PAR-1 in the initial phases of melanoma development as well as in tumor progression and metastasis. Conversely, the significance of PAR-2 up-regulation in both benign and malignant melanocytic lesions requires further research. D

Nitric oxide (NO) is a diatomic free radical molecule that has been implicated in tumour angiogenesis and progression of head and neck squamous cell carcinoma (HNSCC). However, the mechanism underlying the effect of NO on tumour spread remains largely unknown. Tumour lymphangiogenesis has recently received considerable attention and there is increasing evidence that it is relevant for metastasis to lymph nodes in HNSCC. Here, we study the correlation between inducible NOS synthase (iNOS) activity and lymphangiogenesis in a series of 60 HNSCCs and the possible involvement of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C. HNSCC presenting with lymph node metastasis had a significantly higher lymphatic vessel density in both the tumour mass and the peritumour area (p = 0.006 and p = 0.001, respectively). Similarly, tumours with lymph node metastasis showed greater lymphatic vessel area than tumours with no lymph node involvement (p = 0.001 for intratumour lymphatics and p < 0.001 for peritumour lymphatics). iNOS activity measured in specimens from the tumour periphery correlated strongly with both lymphatic vessel density and lymphatic vessel area (p = 0.01, rs = 0.45 and p < 0.001, rs = 0.725, respectively). Conversely, these correlations were not observed in specimens from the tumour core. In addition, VEGF-C mRNA expression was significantly elevated in tumours with high iNOS activity (p = 0.008, rs = 0.563), and VEGF-C expression correlated positively with the presence of lymph node metastases (p = 0.03). In vitro, in the A431 human squamous carcinoma cell line, exogenous and endogenous stimulation of the iNOS pathway led to up-regulation of VEGF-C, which was blocked by the NOS inhibitor L-NNA. Taken together, our results indicate that iNOS activity may promote lymphangiogenesis and spread to lymph nodes in HNSCC, with the possible involvement of VEGF-C.

Protease-activated receptor (PAR)-1 and PAR-2 are reported to contribute to the fibrotic process in a number of organs, including lung, liver, pancreas, and kidney. The aim of this study was to localize expression and biological activity of PAR-1 and PAR-2 in normal and pathological cutaneous scars. First, we investigated the immunohistochemical expression of PAR-1 and PAR-2 proteins in a series of human normal scars (NS, n = 10), hypertrophic scars (HS, n = 10), and keloids (K, n = 10). Expression of PAR-1 and PAR-2 was observed in all types of scar. Specifically, in HS and K, diffuse PAR-1 and PAR-2 positivity was found in dermal cellular areas composed of myofibroblasts, while no or minor staining was observed in the scattered fibroblasts embedded in abundant extracellular matrix in the context of the more collagenous nodules, irrespective of the type of scar. The hyperplastic epidermis overlying K was also found to be strongly PAR-1 and PAR-2 positive, whilst in most NS and HS the epidermis was faintly to moderately stained. Second, ribonuclease protection assay on paraffin-embedded specimens showed overexpression of PAR-1 and PAR-2 mRNA in K compared to NS and HS. Third, cultured human fibroblasts exposed to TGF-beta1 expressed a myofibroblast phenotype associated with overexpression of PAR-2, while PAR-1 expression was unaffected. Intracellular Ca(2+) mobilization by PAR-2 agonists in myofibroblasts was increased as compared to fibroblasts, whereas the effect of PAR-1 agonists was unchanged. Our in vivo study indicates that PAR-1 and PAR-2 are expressed in cells involved in physiological and pathological scar formation and suggests that in vitro overexpression and exaggerated functional response of PAR-2 may play a role in the function of myofibroblasts in scar evolution from a physiological repair process to a pathological tissue response.

Protease-activated receptor-1 (PAR-1) is a unique G-protein-coupled receptor belonging to the protease-activated receptor family. Its activation leads to downstream signaling events that launch a variety of cellular responses related to tumor progression. PAR-1 expression has been associated to a variety of human cancers, and our previous studies reveal a high PAR-1 expression in melanoma specimens as compared to common nevi. In the present study, we investigated the contribution of PAR-1 to the malignant phenotype of human melanoma cell lines obtained from cutaneous primary lesions, capable of different metastatic behaviors in the patients from which they have been derived. We found that melanoma cells isolated from lesions giving rise to metastases in patients (WM115, WM278A, WM1361A, WM983A), had higher PAR-1 mRNA and protein expression, as compared to those obtained from lesions that did not develop metastatic disease (WM793, WM35). The cells isolated from metastatic primary lesions were able to colonize the lungs of immunodeficient SCID mice while those isolated from non-metastatic lesions were not. Additionally, cells expressing elevated PAR-1 had higher migratory and invasive abilities than those holding minimal PAR-1 expression. The migration and invasion capabilities of the melanoma cells expressing high PAR-1 were hampered by genetic and pharmacological interventions. The reduction of PAR-1 expression by siRNA and the inhibition of PAR-1 function by the SCH79797 specific antagonist significantly decreased the melanoma cell motility and invasiveness, down to an extent similar to that of the non-metastatic and low PAR-1 expressing cells. Our results provide strong evidence supporting the implication of PAR-1 in the malignant progression of human melanoma.

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