Fibroblast activation protein: Purification, epitope mapping and induction by growth factors

Abstract

The human fibroblast activation protein (FAP) defined by monoclonal antibody (MAb) F19 is a cell surface antigen expressed in reactive stromal fibroblasts of breast, colorectal, lung and other epithelial cancers. In contrast to its stroma-specific localization in epithelial neoplasms, FAP is expressed in the malignant mesenchymal cells of bone and soft tissue sarcomas. FAP is transiently expressed in some fetal mesenchymal tissues but is absent or expressed at low levels in most adult tissues. FAP is induced in cultured fibroblasts and, in these cells, consists of a M(r) 95,000 subunit (FAP alpha) carrying the F19 epitope and a non-covalently bound M(r) 105,000 subunit (FAP beta) lacking the F19 epitope. Using MAb F19 and 5 newly derived MAbs, we identify 3 distinct epitopes on FAP alpha and tentatively assign one epitope to FAP beta. Analysis of detergent extracts of a FAP alpha high beta- sarcoma cell line by size exclusion-high performance liquid chromatography (HPLC) revealed that FAP alpha does not elute as a M(r) 95,000 species but as part of a high-molecular weight complex (M(r) > 400,000) that dissociates into M(r) 95,000 subunits in SDS gels. Immunoaffinity purification of FAP alpha followed by tryptic digestion, reversed-phase HPLC and microsequencing identified 3 unique FAP alpha peptides, with 2 showing sequence similarity (23/38 identical amino acids) to segments of CD26, a T-cell activation antigen. CD26 is a membrane-bound enzyme (dipeptidyl aminopeptidase IV), but immunopurified FAP alpha has little if any dipeptidase activity with typical CD26 substrates. Finally, studies with FAPlow leptomeningeal fibroblasts revealed that transforming growth factor-beta, 12-O-tetradecanoyl phorbol-13-acetate and retinoids can upregulate FAP expression, whereas serum and several other factors had no or little effect on FAP levels. FAP and CD26 may belong to a family of structurally related but functionally distinct activation proteins that are expressed on different cell types and show unique modes of regulation in normal and malignant cells.

FAPα, a surface peptidase expressed during wound healing, is a tumor suppressor

“…Our experiments demonstrate that FAP expression slows growth and decreases the threshold for cell death under stress conditions. FAP is not expressed in resting epidermal melanocytes but is upregulated when these cells are activated to proliferate (Rettig et al, 1993). Consistent with these observations, common melanocytic nevi, which are hyperplastic lesions of melanocytes, express FAP (Huber et al, 2003).…”

“…Immunohistochemical analysis of human skin lesions showed that FAP is present in a proportion of melanocytic nevi and absent from both primary and metastatic melanoma lesions (Huber et al, 2003). FAP expression in human melanocytes is abrogated when they undergo malignant transformation and acquire tumorigenic potential and growth factor independence (Rettig et al, 1993). Overall, these observations reveal that FAP is lost during malignant transformation of proliferating melanocytes, suggesting that its expression may abrogate tumorigenicity.…”

“…Previous studies have suggested that FAP encodes tumor-suppressor activity, although without direct evidence (Rettig et al, 1993;Tsujimoto et al, 1999;Wesley et al, 1999). In the melanocytic lineage, primary and metastatic human melanomas do not express FAP, while proliferating normal human melanocytes from fetal, newborn and adult skin and benign nevi express FAP (Rettig et al, 1993;Huber et al, 2003).…”

“…FAP and DPPIV each form homodimers, but also can form heterodimers. Fap expression is tightly regulated and is only detected in early development in mesenchyme during tissue remodeling (Niedermeyer et al, 2001), wound healing and in tumor stroma of epithelial cancers (Garin-Chesa et al, 1990;Rettig et al, 1993;Scanlan et al, 1994). Whether the tumor-suppressor activity is dependent on DPPIV or FAP expression, or both, is unanswered.…”

“…In the melanocytic lineage, primary and metastatic human melanomas do not express FAP, while proliferating normal human melanocytes from fetal, newborn and adult skin and benign nevi express FAP (Rettig et al, 1993;Huber et al, 2003). Immunohistochemical analysis of human skin lesions showed that FAP is present in a proportion of melanocytic nevi and absent from both primary and metastatic melanoma lesions (Huber et al, 2003).…”