A Functional Proteomics Screen of Proteases In Colorectal Carcinoma

Abstract

Background: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. Materials and Methods: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. Results: The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the

Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer

“…Thus there is not only more MMP-2 present in tumour than in normal tissue, but the ratio active to proenzyme also increases substantially. A similar finding was reported by Parsons et al (1998) and McKerrow et al (2000) and supports the view that the activation of MMP-2 is a crucial step in tumour invasion. Expression of MMP-2 mRNA in tumour has been described in stromal fibroblastic cells (Poulsom et al, 1992;Newell et al, 1994), but MMP-2 protein appears to be localised predominantly in the cytoplasm of tumour cells (Ring et al, 1997).…”

“…By the use of a specific MMP inhibitor as a control in this activity assay and the use of a general protease inhibitor, only actual active MMPs are measured and not other proteinases or protein-inhibitor complexes. This is an important difference with assays used by others (Garbett et al, 1999b;Baker et al, 2000;McKerrow et al, 2000). The results obtained here indicate that in the tumours studied MMP (production and) activation is elevated compared to the production of TIMPs.…”

Reduced tumour cell proliferation and delayed development of high-grade mammary carcinomas in cathepsin B-deficient mice

“…These findings indicate that Ctsb expressed by cells of tumour-associated stroma and/or inflammatory cells participates in the processes leading to induction of tumour cell proliferation, despite its function of proteolysis of extracellular matrix proteins. Increased Ctsb expression by stromal cells at the invasive front of the tumour node has been reported (McKerrow et al, 2000;Fernandez et al, 2001). In addition, there is growing evidence supporting the regulation of tumour cell proliferation by different stromal cells (for example stromal fibroblasts, myeloid cells) through secretion of mitogenic and growth factors (van Roozendaal et al, 1996;Loffek et al, 2006).…”

“…These cell types are reckoned to contribute to the tumour-promoting effects of the stroma. In particular, macrophages strongly upregulate Ctsb expression on recruitment to a tumour (McKerrow et al, 2000;Fernandez et al, 2001;Vasiljeva et al, 2006).…”

The Extracellular Matrix in Digestive Cancer

“…Eighteen MMPs are soluble enzymes secreted into the ECM, six are membrane-type MMPs (MT-MMPs) that are attached to a cell surface and one, MMP-23, is initially a membranebound MMP but can also be cleaved to form a soluble factor [23]. Expression of important MMPs and MT-MMPs occurs within, but is not restricted to, the neoplastic cells [52]. Stromal fibroblasts and endothelial cells also express these molecules to promote their own migration into the activated stroma and thus contribute to the microenvironment in terms of remodeling and angiogenesis [53][54][55].…”