Fibroblast activation protein-α (FAP) is a cell surface, serine protease of the post-prolyl peptidase family that is expressed in human breast cancer but not in normal tissues. Previously, we showed that FAP expression increased tumor growth rates in a mouse model of human breast cancer. Here the role of the proteolytic activities of FAP in promoting tumor growth, matrix degradation and invasion was investigated. Mammary fat pads of female SCID mice were inoculated with breast cancer cells that express FAP and the mice treated with normal saline or Val-boroPro (talabostat); Glu-boroPro (PT-630); or 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine (LAF-237) that inhibit prolyl peptidases. Other mice were injected with breast cancer cells expressing a catalytically inactive mutant of FAP and did not receive inhibitor treatment. PT-630 and LAF-237 did not slow growth of tumors produced by any of the three cell lines expressing FAP. Talabostat slightly decreased the growth rates of the FAP-expressing tumors but because PT-630 and LAF-237 did not, the growth retardation was likely not related to the inhibition of FAP or the related post-prolyl peptidase dipeptidyl peptidase IV. Breast cancer cells expressing a catalytically inactive mutant of FAP (FAP(S624A)) also produced tumors that grew rapidly. In vitro studies revealed that cells expressing wild type FAP or FAP(S624A) degrade extracellular matrix (ECM) more extensively, accumulate higher levels of matrix metalloproteinase-9 (MMP-9) in conditioned medium, are more invasive in type I collagen gels, and have altered signaling compared to control transfectants that do not express FAP and form slow growing tumors. We conclude that the proteolytic activity of FAP participates in matrix degradation, but other functions of the protein stimulate increased tumor growth.

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Our overarching hypothesis is that FAP functions with other proteases in an extracellular communication network to digest certain proteins, thereby exposing signals stored in peptide regions that enable breast cancer cells to thrive in diverse microenvironments. FAP likely has important functions in two parts of the metastatic cascade: 1) FAP and proteases such as MMP-1 and MMP-9 cooperate to produce fragments of ECM proteins during adjacent tissue remodeling and these derivative peptides promote fibroblast growth, ECM deposition and angiogenesis; 2) cancer cell membrane FAP cleaves precursive A2AP to generate the more effective derivative for protecting and stabilizing fibrin within ECM margins of the expanding neoplastic cell mass as well as fibrin within cancer cell/fibrin/platelet emboli that lead to hematogenous metastasis. We believe that peptides that target and inhibit FAP on FAP-expressing cells can be produced by taking advantage of the substrate/active-site binding specificity of FAP.