Expression of protease-activated receptors 1 and 2 in melanocytic nevi and malignant melanoma

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 “dysplastic” 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.

Introduction

Recently, there has been considerable interest in understanding the biologic significance of protease-activated receptors (PARs) in physiological and pathological conditions, including neoplastic diseases [1], [2]. PARs constitute a family of G protein–coupled receptors that show a unique mechanism of activation being enzymatically activated by serine proteases through cleavage of their amino terminal domain. So far, 4 PARs have been cloned, PAR-1 and PAR-3, which are activated by thrombin; PAR-4, which is activated by thrombin and trypsin; and PAR-2, which is activated by trypsin, mast cell tryptase, and coagulation factors [3], [4], [5], [6], [7]. PARs are expressed in several tissues by a variety of cells, and their activation is implicated in numerous biologic effects, including coagulation, inflammation, mitogenesis, and cell proliferation [1], [2], [8].

There is emerging evidence that PAR-1 modulates cell proliferation and motility in physiopathologic cell invasion processes, suggesting that it plays a role in the setting of cancer growth and metastasis [9], [10], [11], [12], [13]. Indeed, overexpression of PAR-1 has been detected in numerous human cancers, including colon [14], [15], laryngeal [16], breast [10], pancreatic [17], [18], and oral cavity carcinomas [19]. Of interest, an up-regulation of PAR-1 and PAR-2 has been demonstrated in stromal fibroblasts surrounding neoplastic aggregates in human malignant tissues [20]. In cutaneous melanomas, it has been recently shown that loss of expression of the transcription factor activator protein 2α correlates with overexpression of PAR-1, which in turn contributes to the acquisition of a malignant phenotype [21]. Less is known concerning the role of PAR-2 in cell growth, although it may also contribute to tumor development and metastasis by stimulating proliferation of neoplastic cells [22]. More recently, it has been reported that both PAR-1 and PAR-2 contribute to tumor cell motility and metastasis [23].

In the development of malignant melanoma, the critical steps of tumor progression were originally postulated as follows: from benign common melanocytic nevi, to atypical, “dysplastic” nevi, to early radial growth phase melanomas (incapable of metastasis), to vertical growth phase invasive melanomas with competence for metastasis to metastatic melanomas [24]. Although it has been suggested that such stepwise melanocytic tumor progression may reflect distinct gene expression patterns and messenger RNA (mRNA) profiles, the exact molecular pathogenetic events underlying the transition from normal melanocytes into various forms of melanocytic nevi and melanomas are not fully understood [25], [26], [27]. Great attention has been recently focused on the identification of markers of tumor progression, including growth factors, cell surface antigens, extracellular matrix proteins, and angiogenetic factors, which are differently expressed in common benign nevi, “dysplastic nevi,” and in situ, radial, vertical, and metastatic phases of melanomas. However, data on PAR-1 and PAR-2 activity in situ in benign melanocytic nevi, atypical nevi, and human melanoma tissue samples are not available.

To define the possible role of PAR-1 and PAR-2 in melanocytic tumor progression, we evaluated PAR-1 and PAR-2 immunohistochemical expression in benign and malignant melanocytic lesions. Because we found a differential expression in PAR-1 protein, but not in PAR-2 protein, in melanocytic nevi and melanomas, we next sought to confirm these results at mRNA level through analysis of PAR-1 expression by ribonuclease protection assay (RPA) applied to selected archival formalin-fixed paraffin-embedded tissue samples of the same series.