Cancer And Proprotein Convertases

PCs have been associated with cancer since the early 1990's. The first members identified as overexpressed in cancer were the neuroendocrine PCs such as PC1 and PC2. For instance PC2 has been identified by PCR amplification of a human insulinoma cDNA. The putative gene product was reported to have similarity to furin and proposed to function as a pro-hormone convertase [14]. Soon, PC3 was cloned and the analysis of its sequence revealed high homology with PC2 and furin. The authors anticipated that these proteins may play a different role than furin since they were expressed almost exclusively in neuroendocrine tissue and locate in different subcellular compartments [15]. PC2 and PC3 were expressed in pheochromocytomas but not in normal adrenal tissue [16] and their expression correlated with the expression of their substrate, proenkephalin [17], pointing to a direct relationship between these PCs and tumors of the endocrine glands [16].

Before long, these differences in PC expression between normal and tumors or between tumors of different aggressive potentials were observed also in the ubiquitous PCs, furin and PACE4. Furin and PACE4 were highly expressed in lung squamous cell carcinoma and adenocarcinoma. On the other hand, PC1 [3] and PC2 where expressed in small cell lung carcinoma [18]. Overexpression of PCs has also been observed in breast, head and neck and colon tumors. A clear gradation was observed between furin expression, aggressive behavior and VEGF-C expression.

Figure 1. Immunohistochemical detection of furin in normal oral epithelium, dysplastic Epithelium and Squamous Cell Carcinoma. Note increased furin expression from normal to dysplatic to carcinoma

Furin expression increased from normal epithelium to invasive SCC in oral epithelia (Figure 1). Moreover furin localized preferentially near or at the invasion front [19]. These correlative studies provided evidence for the association between PCs and cancer. PCs are proteases that cleave proteins at specific sites, modifying the protein substrates leading to alteration of their properties. For instance many proteins have a PC recognition and cleavage site between the pro-domain and the mature protein. Hence, these proteins maturate, usually becoming active, upon PC cleavage. The next step in the development of the ideas that PCs were related to invasion was the discovery of a series of MMP, enzymes responsible for ECM degradation that become activated after PC (specifically furin) cleavage.

The pioneering paper by Pei and Weiss [20] describes the furin-dependent cleavage and activation of Stromelysin-3. This metalloprotease, expressed mainly in stromal cells, had been implicated in tumor progression and degradation of the ECM [21-23]. In contrast to many other MMPs, Stromelysin-3 (MMP-11) contained an insertion of 10 aminoacids GLSARNRQKR which adds a PC cleaving site. This finding was of capital importance since it underscored a direct relationship between furin and PCs and tumor invasion or metastasis. Also, PCs were identified for the first time as possible targets for future therapy.

One year before the previous findings were published, a new MMP gene had been cloned from a cDNA library from human placenta [24]. Analysis of its sequence revealed a unique transmembrane domain; hence this new gene was referred as MT-MMP (for Membrane Type metallopreoteinase). This plasma membrane localization was confirmed by immunohistochemistry. Interestingly, MT-MMP (now renamed as MT1-MMP after the subsequent discovery of five additional members sharing its structural and physiological characteristics) was able to cleave and activate MMP-2, or collagenase A, one of the enzymes responsible for the degradation of collagen IV, a key component of the basement membrane. Surprisingly MT1-MMP had an insertion cassette similar to Stromelysin-3 generating a PC cleaving site between the pro-domain and the mature form.

These results suggested that a furin-like protease would be responsible for the activation of this new MMP [24]. The crucial role that furin pays in the cleavage and activation of MT1-MMP, was demonstrated shortly after in two independent studies. Sato et al. demonstrated that purified furin was able to process recombinant MT1-MMP in vitro and that this process resulted in stimulation of pro-gelatinase A processing [25]. Pei et al., provided further evidences that MT1-MMP was cleaved at the RRKRY112 and point mutations within the furin-like cleavage sequence resulted in inhibition of MT1-MMP processing [26]. In agreement with these experiments, treatment of the fibrosarcoma cell line HT-1080 with a synthetic furin inhibitor decreased pro-MT1-MMP processing and pro-MMP-2 processing and activation leading to a reduction in cell invasiveness [27].

More recently Hubbard et al., provided evidence that PACE 4 was overex-pressed in murine chemically-induced squamous cell carcinoma of high grade [28]. Moreover, SCC lines transfected with the full-length PACE4 cDNA resulted in the activation of stromelysin-3 and in increased cell invasiveness. These studies broadened the horizons in the field, linking this new family of proteases' expression with the specific processing of matrix-degrading enzymes. Two important substrates, Stromelysin-3 and the family of MT-MMPs were defined. New interactions between metalloproteases and other proteins, some of them PCs' substrates, would come into play increasing the complexity and stressing the role of this novel family in cancer invasion.

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