A Functional Proteomics Screen of Proteases In Colorectal Carcinoma

James H. McKerrow, Vivek Bhargava, Elizabeth Hansell, Sandra Huling, Thomas Kuwahara, Mary Matley, Lisa Coussens, Robert Warren

Research output: Contribution to journalArticlepeer-review

85 Scopus citations

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), ca-thepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metallopro-teases 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 metastases. The major proteases detected by activity assays were then localized in biopsy sections by im-munohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. Conclusions: This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, 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 molecular weight protease inhibitors as potential chemotherapy.

Original languageEnglish (US)
Pages (from-to)450-460
Number of pages11
JournalMolecular Medicine
Volume6
Issue number5
DOIs
StatePublished - May 2000
Externally publishedYes

Keywords

  • Adjacent Normal Colon
  • Chymase
  • Major Protease Activity
  • Mast Cell Serine Proteases
  • Matrix Metalloproteases (MMPs)

ASJC Scopus subject areas

  • Genetics(clinical)
  • Genetics
  • Molecular Medicine
  • Molecular Biology

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