A functional proteomics screen of proteases in colorectal carcinoma

Background: Proteases facilitate several steps in cancer progression. To id entify proteases most suitable for drug targeting, actual enzyme activity a nd not messenger RNA levels or immunoassay of protein is the ideal assay re adout. Materials and Methods: An automated microtiter plate assay format was modif ied to allow detection of all four major classes of proteases in tissue sam ples. Fifteen sets of colorectal carcinoma biopsies representing primary tu mor, adjacent normal colon, and liver metastases were screened for protease activity. Results: The major proteases detected were matrix metalloproteases (MMP9, M MP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine protease s, tryptase and chymase. Matrix metalloproteases were expressed at higher l evels in the primary tumor than in adjacent normal tissue. The mast cell pr oteases, in contrast, were at very high levels in adjacent normal tissue, a nd not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major prot eases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matri x metalloproteases were localized to the tumor cells themselves; whereas, c athepsin B was predominantly expressed by macrophages at the leading edge o f invading tumors. Although only low levels of urinary plasminogen activato r were detected by direct enzyme assay, immunohistochemistry showed abundan t protein within the tumor. Conclusions: This analysis, surveying all major classes of proteases by ass ays of activity rather than immunolocalization or in situ hybridization alo ne, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low mole cular weight protease inhibitors as potential chemotherapy.