Characterization of dipeptidyl peptidase IV (DPPIV) as an inhibitor of melanoma invasion

Metastasis of tumor cells to distant organs remains the primary cause of cancer fatalities. Yet, the number of genes that have been identified as playing a major role in the dissemination of tumor cells to distant organs lags far behind the vast array of genes that have been implicated in the tumorgenic process. We have identified Dipeptidyl Peptidase IV (DPPIV) as a melanocyte gene that is able to inhibit the invasion and metastasis of malignant melanoma cells. DPPIV was isolated by an expression cloning strategy due to its ability to suppress the anchorage independent colony forming potential of a melanoma cell line. DPPIV is a 110kD, trans-membrane, mainly extra-cellular glycoprotein, with unknown function. Constitutively expressed on numerous epithelial cell types, DPPIV is often disregulated in a variety of human malignancies. The most striking evidence of DPPIV downregulation is found in transformed melanocytes, where nearly 100% of melanomas lack its expression. By transfecting the full-length cDNA of DPPIV, we have established stable melanoma cell lines that express comparable levels of the protein as normal epidermal melanocytes. Matrigel invasion assays were utilized to study the effects of DPPIV expression on the invasive potential of these cells. The parental and mock vector transfectants were invasive, and readily migrated across the Matrigel, while the invasiveness of DPPIV transfected cells was reduced by greater than 75% in at least two independent melanoma cell lines. Pilot in-vivo metastasis assays have indicated that cells transfected with the DPPIV gene form fewer pulmonary metastases than vector controls when injected into immuno-suppressed mice. The effects on cellular invasion are not attributed to overall growth characteristics, as both DPPIV transfected and untransfected cells behave comparably in culture and form subcutaneous tumors when implanted in-vivo. We have also constructed mutants of DPPIV that lack either the extra-cellular serine protease activity or the six amino acid cytoplasmic domain. Both mutants were stably expressed in melanoma cells. Matrigel invasion assays performed with cells expressing the two mutant forms of the protein revealed phenotypic effects similar to wild type DPPIV transfected cells. Preliminary evidence also suggests that the anti-invasive activity of DPPIV requires the presence of Seprase, a pro-invasive member of the DPPIV family of extra-cellular membrane bound proteases. Ectopic expression of DPPIV has no effect on the invasive potential of melanoma cells that lack Seprase. Furthermore, the inhibition of Seprase activity in melanoma cells that express endogenous Seprase results in a decrease of cellular invasion similar to DPPIV transfection.;In this study, we have demonstrated that expression of the DPPIV protein inhibits the invasiveness of malignant melanoma cell lines lacking its endogenous expression. Furthermore, we have shown that neither the protease activity nor the cytoplasmic domain of DPPIV is required for its activity. The anti-invasive activity of DPPIV, however, is restricted to cells that also express Seprase.