Therefore, the pro-angiogenetic effect of meprin-�� released by cancer cells may be promoted by its proteolytic activity outside and distant from pro-angiogenic/vasculogenic cells. Secreted meprin-�� may thus condition the tumor environment to promote enhanced angiogenesis together with other pro-angiogenic factors, whereas meprin-�� tethered at the cell this site surface may promote the migration of cancer cells themselves. Both meprin-dependent processes are expected to jointly promote tumor progression. In conclusion, this study reveals a complex and dynamic regulation pattern for meprin-�� in tumor progression. The transition to malignant stages in colorectal tumors correlates with increased meprin�� activity at primary tumor sites, consistent with a role in invasion and metastatic dissemination of cancer cells.
A role for meprin-�� in this process is further supported by its pro-angiogenic and pro-migratory activity. Our data also show that the promotion of migration depends on the simultanous expression of meprin-�� tethering meprin-�� onto the cell surface. The lack of detectable meprin-�� mRNA in the whole tumor does not exclude its induction in a subpopulation of cancer cells. Our data are in agreement with the view that the meprin-��/�� co-expressing cancer cells are likely to migrate away from the tumor mass. We previously described that meprin-�� weakens intercellular junctions [41]. Future studies should focus on the analysis of the role of meprins in emigration of these cancer cells.
The inhibitory activity towards meprin-�� is lower in sera from patients with colorectal cancer, which might facilitate dissemination of meprin-expressing cancer cells. Therapeutic approaches with protease inhibitors targeting meprin-�� in primary tumors and in the bloodstream might counteract tumor progression. On the other hand, meprin-�� activity is low in liver metastases, and therefore inhibition of this protease at this site is not expected to be an effective treatment. Materials and Methods Recombinant human meprin Recombinant active meprin-�� and meprin-�� was purified from insect cells as described previously [7], [42]. No residual trypsin activity was detectable in the purified recombinant meprins. Cell migration assay Madin-Darby canine kidney (MDCK) cells were grown in minimum essential medium (MEM) supplemented with 5% FCS, 100 U/ml of penicillin and 100 ��g/ml of streptomycin (cell culture media and supplements from Invitrogen, Basel, Switzerland).
Meprin-transfected MDCK cells [31] and wild-type parental MDCK cells were seeded in laminin-1 coated 12-well plates at a density of 10’000 cells/well (12-well plates from Costar, Cambridge, MA, U. S. A., purified laminin-1 from Engelbreth-Holm-Swarm mouse sarcoma, Sigma, St. Louis, MO, U. S. A) and incubated overnight in MEM with Carfilzomib 5% FCS.