Cystatin E/M suppresses legumain activity and invasion of human melanoma.

BACKGROUND: High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied. METHODS: A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay. RESULTS: Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay. CONCLUSIONS: These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.

Immunostimulatory effects of CpG-ODN upon dendritic cell-based immunotherapy in a murine melanoma model.

In this study, we examined the protective and therapeutic efficacy of the immunoadjuvant CpG in combination with dendritic cell (DC) immunotherapy in a murine melanoma model. We found that murine bone-marrow derived DC stimulated in vitro with CpG displayed both enhanced expression of maturation markers and secretion of IL-12p70 and IL-10. In addition, these matured DC demonstrated enhanced ability to stimulate antigen specific CD4+ and CD8+ T cell responses in vitro. In a protection model, C57BL/6 mice vaccinated with either antigen-pulsed immature or CpG matured DC were unable to reject a lethal B16 melanoma challenge. In contrast, long-term protection was achieved in mice vaccinated with both CpG and antigen-pulsed DC, which correlated with an enhanced antigen specific T cell immune response. In a therapeutic model of established subcutaneous B16 melanoma, C57BL/6 mice treated intratumorally with CpG and B16 lysate-pulsed DC demonstrated a reduced tumor burden and prolonged survival. In a similar model of established subcutaneous tumor, mice treated with CpG-matured DC pulsed with a melanoma peptide, TRP-2, alone were unable to achieve tumor regression. Conversely, mice that received the combined vaccine of CpG and peptide-pulsed DC displayed a reduced tumor burden. These experiments provide evidence that combined immunization with both antigen-pulsed DC and the immunoadjuvant, CpG, can lead to tumor regression and long-term survival in a murine B16 melanoma model.