Scientific Report 2005

Immunology

The Microenvironment of Tumors and Protease-Activated Prodrugs

W. Wu, P. Kuo, L. Wong, C. Liu

Invasion and metastasis are critical features that define a malignant tumor. The ability of cancer cells to break through tissue boundaries and penetrate into surrounding normal tissues involves the actions of diverse extracellular proteases from multiple enzymatic classes. This enrichment of proteolytic activity is one of the distinctive features of the tumor microenvironment, in contrast to the microenvironment of normal tissues. These cellular proteases also participate in a wide range of biological and pathologic processes, such as the formation of new blood vessels, signal transduction, and cell survival.According to recent reports, humans have 553 genes that encode proteases. We use an integrated approach to characterize the repertoire of extracellular proteases operating in the tumor microenvironment (i.e., the “cancer degradome”) and the functional modulators, target substrates, and specificity of the enzymes. We call this approach cancer degradomics. Currently, we are focusing on a subset of cell-surface anchored or associated proteases, such as legumain, type II membrane serine protease 4, prostate-specific membrane antigen, and a number of novel protease-encoding genes, that are drastically overexpressed in a high percentage of human cancers. Certain protease inhibitors (survivin) and cofactor molecules (tissue factor) are also upregulated in tumors. We showed that legumain activates both cathepsin cysteine proteases and matrix metalloprotease 2 and that overexpression of legumain in tumors increases tumor invasion and metastasis. Similarly, type II membrane serine protease 4 activates matrix metalloprotease 9 and promotes cancer cell migration and metastasis. In contrast, expression of prostate-specific membrane antigen in prostate cancers reportedly reduces invasive potentials. These findings indicate a high level of complexity involving the interactive protease network in cancers.

The cellular proteases and the inhibitors that constitute the cancer degradome are valuable prognostic and diagnostic markers as well as attractive targets for cancer imaging and therapy. The proteolytic specificities of peptide substrates provide modular chemical tools for the rational design of protease-activated prodrugs. For example, a novel legumain-activated, cell-impermeable doxorubicin prodrug, LEG-3 (Fig. 1), is activated exclusively in the tumor microenvironment.

Fig. 1. Chemical structure (A) and optimized structural model (B) of prodrug LEG-3.

After administration of LEG-3, a profound increase occurs in the end product doxorubicin in the nuclei of cells in tumors, but little increase in other tissues. This protease-activated prodrug completely arrested growth of a variety of neoplasms, including multidrug-resistant tumors, in vivo and significantly extended survival without evidence of myelosuppression or cardiac toxic effects. The design of prodrugs activated by proteases in the tumor microenvironment can be extended to other proteases and chemotherapeutic compounds and therefore provides new potential for the rational development of more effectively targeted cancer therapeutic agents.

Publications

Luo, Y., Zhou, H., Mizutani, M., Mizutani, N., Liu, C., Xiang, R., Reisfeld, R. A DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 induces long-lived T-cell memory against tumor recurrence. Cancer Res. 65:3419, 2005.