Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

FAP

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101596

Synonyms

Historical Background

Fibroblast activation protein (FAP; FAP-α; seprase) is a type II 170 kDa post-proline cleaving atypical serine protease, closely related to the dipeptidyl peptidases (DPP) DPP-4, DPP-8, and DPP-9 in the prolyl oligopeptidase gene and enzyme family. FAP is conserved throughout chordate evolution, with homologs in human (hFAP), mouse (mFAP), and Xenopus laevis (89% and 50% protein sequence identity to hFAP, respectively). FAP expression correlates with tissue remodeling events (Hamson et al. 2014; Liu et al. 2015; Jiang et al. 2016). FAP is also phylogenetically linked to the prototype prolyl oligopeptidase and prolyl endopeptidase (PEP; PREP). FAP has both an endopeptidase activity that is similar to but distinct from that of PEP and a DPP activity that is similar to those of DPP4, DPP8, and DPP9 as well as the unrelated peptidase DPP7 (DPP-II).

FAP was originally discovered in the late 1980s as a cell surface protein expressed on cultured human fibroblasts, astrocytomas, and sarcomas (Garin-Chesa et al. 1990; Pineiro-Sanchez et al. 1997). A soluble form of circulating FAP was later identified as antiplasmin-cleaving enzyme (APCE), which increases the activity of α2-antiplasmin (Lee et al. 2006). The monoclonal antibody mAb F19 was used in isolating the FAP-encoding cDNA and in further characterization of 95 kDa plasma membrane–associated FAP glycoprotein in human tissues. The human FAP gene spans 72.8 kb and comprises 26 exons at chromosome 2q24.3.

The FAP polypeptide is 52% identical to DPP4 and has five residues that bear N-linked glycosylation. Immunochemical analyses of FAP multimers have identified high molecular mass protein complexes comprising of FAP and a FAP associated 105 kDa protein, which was DPP4 but named FAP-β in that study (Rettig et al. 1994). DPP4 was found to form cell surface heterometric complexes with FAP only on cultured fibroblast cells and melanocytes. Immunoblot experiments revealed that the epitope defined by mAb F19 is only carried by FAP-α and not by FAP-β/DPP4.

Activity, Specificity, and Structural Chemistry

FAP has the unusual ability to hydrolyze substrates at a post-proline bond. FAP is an atypical serine protease in that the catalytic triad, Ser-Asp-His, is in the reverse order to trypsin. The DPP activity is specific for N-terminal Xaa-(Pro/Ala) sequences (Aertgeerts et al. 2005; Lee et al. 2006; Keane et al. 2011; Dunshee et al. 2016). Degradation of FGF-21, α2-antiplasmin, and denatured type I collagen is attained by the FAP endopeptidase activity (Lee et al. 2006; Christiansen et al. 2007; Dunshee et al. 2016). The DPP and PEP activities of FAP are both ablated by alanine substitution of the catalytic serine (Aertgeerts et al. 2005).

FAP activity is assayed at 37 °C in pH 7.6 Tris/EDTA, usually with 1 mM DTT. Its enzymatic activity is heightened in tumor stroma (Hamson et al. 2014; Liu et al. 2015; Jiang et al. 2016) and cirrhotic livers (Wang et al. 2005). Within human collagen I, the FAP cleavage sites take place following Pro-Pro-Gly-Pro and (Asp/Glu)-(Arg/Lys)-Gly/(Glu/Asp)/(Thr/Ser)-Gly-Pro (Christiansen et al. 2007). Human α2-antiplasmin, a physiological FAP substrate, is cleaved by FAP/APCE following Glycine11-Proline12 (Lee et al. 2006). FAP-DPP activity more rapidly hydrolyzes Ala-Pro-AFC over Gly-Pro-AFC (Rettig et al. 1994), whereas its endopeptidase activity requires Gly-Pro following a benzoyloxycarbonyl (Z) moiety. FAP has or prefers a positive charge at P1’ (Keane et al. 2011; Zi et al. 2015).

FAP monomer (1–760) appears as a single protein band at about 95 kDa on SDS-PAGE. Each FAP monomer has five residues capable of N-linked glycosylation. Digestions of 94 kDa FAP with neuroaminidase or endoglycosidase H result in reduced molecular size on SDS-PAGE and in removal of all sugars to generate a 75 kDa polypeptide. FAP exists in a membrane bound and a soluble form, and like DPP-4, its enzymatic activity requires the dimer complex. The crystal structure of FAP is composed of a C-terminal α/β-hydrolase domain and an N-terminal 8-blade β-propeller domain (Protein Data Bank Code 1Z68) (Aertgeerts et al. 2005). The catalytic pocket is enclosed by these two domains and contains essential catalytic residues Ser624, Asp702, and His734, and, from the β-propeller domain, Glu203 and Glu204 (Wang et al. 2005). Along with these residues, Arg123, Tyr656, and Asn657 contribute to FAP catalysis and confer transition-state stabilization. The small conserved active site residue Ala657 contributes to lower affinity for N-terminal amines and allows FAP to act as both a dipeptidyl peptidase and endopeptidase. The transmembrane-bound domain of FAP is formed by residues 7–26 (Hamson et al. 2014).

FAP activity is inhibited by PT-100 and PT-630, which are also known as DPP4 inhibitors. Linagliptin, an incretin-based type-2 diabetes therapeutic, is the only gliptin that has ability to inhibit FAP activity in addition to DPP4 (Liu et al. 2015). FAP is not inhibited by the PEP – selective inhibitor S17092 – but is inhibited by zinc. Thus, FAP is distinct from matrix metalloproteinases in not requiring a metal ion for activity.

Expression and Distribution

FAP is selectively expressed in fetal cells, reactive stromal fibroblasts, wounded tissues, and the stromal tumor fibroblasts of more than 90% of common human epithelial carcinomas examined (breast, colorectal, lung, and ovarian) (Garin-Chesa et al. 1990). Its expression is low or absent in benign tumors and normal healthy fibroblasts. FAP is involved in extracellular matrix (ECM) pericellular proteolysis, endothelial cell migration and invasion into the ECM, and in granulation tissue of healing wound (Wang et al. 2005; Hamson et al. 2014; Jiang et al. 2016).

In liver cirrhosis, FAP colocalizes with fibronectin and collagen and with collagen fibrils alongside activated hepatic stellate cells (Wang et al. 2005). Its expression is found to be increased and to correlate with severity of liver fibrosis. In livers, FAP is expressed by myofibroblasts (in the fibrotic septum) and activated hepatic stellate cells at the portal-parenchymal interface, where the tissue remodeling takes place (Wang et al. 2005). Upregulation of FAP is also present during remodeling interface in pulmonary fibrosis (Fan et al. 2016).

FAP has both an extra-enzymatic and enzymatic functions. FAP can bind to urokinase plasminogen activator receptor (uPAR). This and α2-antiplasmin and collagen cleavage results in altered degradation of fibrin and ECM complexes and might contribute to the invasive and metastatic ability of tumors. Cellular invasiveness can be induced also by interacting with other proteinases and receptors, such as α3β1 and α5β1 integrin, DPP4, MMP-2, and membrane-type 1 MMP (MMP14).

Little is known about the FAP enzymatic role in the tumor microenvironment. There is limited evidence that invasion of tumor cells is promoted by the collagenolytic role of FAP. FAP-expressing cells can have an immunosuppressive role in murine cancer. Also, FAP has been shown to promote angiogenesis in the tumor microenvironment, as inhibition of both DPP4 and FAP can cause decreased tumor vascularization (partly mediated by FAP cleavage of NPY) in FAP-dependent manner (Hamson et al. 2014; Liu et al. 2015; Zi et al. 2015; Jiang et al. 2016). FAP-enzyme activity has also been implicated with promoting plaque instability, by cleavage of CN-1, and in progression of atherosclerotic plaques. The collagenolytic activity of FAP may be important in promoting fibrous cap rupture (Hamson et al. 2014; Jiang et al. 2016).

Basal, endogenous expression of FAP has recently been examined in normal tissue (Keane et al. 2014) utilizing 3144-AMC, a FAP-specific substrate. This substrate is able to quantify both soluble and cell-bound forms of FAP from different range of tissues, with potential as a useful diagnostic tool in clinical settings.

FAP Depletion in Mouse Models

FAP expression is observed in primitive mesenchymal cells during mouse embryogenesis and in tissues that are undertaking tissue remodeling. FAP gene knockout (gko) mice are viable and have a normal phenotype for body weight, organ weight, histological examination of major organs, and hematological analysis. Similar to human, mFAP expression is not detected in normal murine tissues, outside of areas undergoing remodeling. In a mouse model of the syngeneic CT26 colon tumor and endogenous K-rasG12D-driven lung cancer, FAP depletion inhibits tumor cell proliferation indirectly, decreases myofibroblast content, and decreases blood vessel density (Jiang et al. 2016).

Overexpression of FAP has antitumorigenic effects in vitro, similar to DPP4. These resulted in cell cycle arrest at G0-G1 phase, increased susceptibility to stress induced apoptosis, and restoration of contact inhibition. FAP has a role in in vitro cell adhesion, invasion, migration, and Staurosporine-induced apoptosis (Wang et al. 2005). In nude mice, overexpression of FAP in melanoma cells leads to suppression of malignancy.

The role FAP in cartilage degradation was shown in FAP(−/−) human TNF transgenic (hTNFtg) mice, where less cartilage degradation but similar inflammation and bone erosion was observed in these animals, compared to wild-type hTNFtg mice (Brennen et al. 2012). FAP role in wound healing was confirmed in its knockout mice, however with no increased susceptibility towards cancer.

FAP in Cellular Functions and Implications in Cancer and Immunology

FAP is expressed in stroma associated with carcinomas of the breast, lung, stomach, pancreatic ductal adenocarcinoma, liver, colorectum, uterine cervix, and oral squamous cell carcinoma. Cell types known to express FAP in vivo include fetal mesenchymal cells, placenta, and pancreatic A cells, which are glucagon producing endocrine cells. The exact biological role of FAP in these cells is unknown.

FAP has important roles in fibrinolysis and fibrosis via cleaving α2-antiplasmin, gelatin, and collagen I but not collagen IV, fibronectin, or laminin (Christiansen et al. 2007; Hamson et al. 2014). In the complex extracellular microenvironment of tumors or other remodeling tissues, FAP may work alongside other proteases in ECM degradation, and thereby contribute to increased migratory abilities of tumors (Liu et al. 2015; Jiang et al. 2016).

The primary indicator of reactive stroma formation is the presence of activated fibroblasts that have acquired a myofibroblast – like phenotype within the tumor microenvironment. These activated fibroblasts, also known as carcinoma-associated fibroblasts (CAFs), are central to regulating the dynamic and reciprocal interactions that occur among malignant epithelial cells, the ECM, and noncancerous cells found within the tumor milieu. Exogenous FAP protein expression by FAP-overexpressing CAFs probably promotes tumorogenesis via multiple mechanisms, including proliferation, invasion, angiogenesis, and cell death inhibition. These effects can be mediated directly or indirectly. Direct effects are mediated through expression and secretion of proteins such as VEGF, TGF-β, SDF-1, HGF, LOX, MMPs, and FGF2. Paracrine signals of adipocytes, inflammatory cells, and immune cells, influenced by CAFs indirectly, promote tumor growth (Lo et al. 2015; Zi et al. 2015; Jiang et al. 2016).

A number of cytokines (e.g., TNFα; TGFβ1), chemical substances (e.g., 12-o-tetradecanoyl phorbol-13-acetate (TPA), retinol, retinoic acid), and physical stimulants (e.g., ultraviolet radiation) are able to elevate FAP expression in fibroblasts, melanocytes, and primary melanoma cells to promote cell invasion and migration. The highly regulated expression and restricted distribution of FAP suggests that FAP inhibition may be useful as a therapeutic target in tumors. To date, FAP has been targeted in cancer models using chemical inhibitors, prodrugs, T-cells, antibodies, and RNA interference (Brennen et al. 2012; Hamson et al. 2014; Lo et al. 2015).

Chimeric antigen receptors (CARs) reactive against FAP (FAP-CAR) and genetically engineered and expressed by T-cells have been examined in mouse models of cancer, and in human pancreatic cancer xenografts (Brennen et al. 2012; Hamson et al. 2014; Lo et al. 2015). Significant decreases in FAP-positive stromal cells and decreases in tumor growth have been observed in both lung cancer xenograft and other models (Hamson et al. 2014). FAP-CAR T cell treatment combined with administration of cancer cell-targeted T cells can prolong inhibition of tumor growth. Off-target effects in bone marrow and skeletal muscles are possible and are being explored (Lo et al. 2015).

FAP induced increased STAT3-CCL2 signaling in CAF is sufficient to program an inflammatory component of the tumor microenvironment, which may have particular significance in desmoplasia-associated cancers. Increased stromal expression of FAP, p-STAT3, and CCL2 in human intrahepatic cholangiocarcinoma (ICC) predicts a poor survival outcome.

FAP plays role in inflammatory diseases, and it is upregulated in fibroblast-like synoviocytes of rheumatoid arthritis patients compared to osteoarthritis controls. FAP is expressed by myofibroblasts in the submucosa strictures of patients with Crohn’s disease and is upregulated after TNFα or TGFβ stimulation. Expression of FAP is downregulated in patients with systemic lupus erythematosus.

FAP-targeted cancer therapy continues to show promise in mice (Meng et al. 2016) but not in humans (Liu et al. 2015). Talabostat is an inhibitor of FAP that has been subjected to clinical trials, but trials with non–small cell lung cancer and malignant melanoma were suspended. Sibrotuzumab is a human monoclonal antibody against FAP intended for cancer treatment but failed a clinical trial for metastatic colorectal cancer (Brennen et al. 2012).

FAP has potential as a therapeutic target for the treatment of atherosclerosis. Its expression is enhanced in some types of human atheromata (Hamson et al. 2014). On smooth muscle cells, FAP expression correlates with macrophage burden. FAP is mainly associated with collagen-deficient regions of the plaque and can digest type I collagen and gelatin. This may influence plaque instability. FAP is in tumor-associated macrophages in human breast cancer.

Summary

FAP, fibroblast activation protein alpha, is a 170 kDa homodimer membrane-bound gelatinase belonging to the S9B prolyl oligopeptidase family. FAP is closely related to DPP4 but has both dipeptidyl peptidase activity and endopeptidase activity. High levels of FAP expression occur in cancer-associated fibroblasts of over 90% of human epithelial malignancies but not in benign tumor or healthy adult tissue. FAP is generally expressed by CAFs and pericytes, rarely by tumor cells. The level of expression of FAP is associated with histological grade, invasion, and metastatic progression. FAP is highly induced during inflammation and is strongly expressed by mesenchymal cells of remodeling tissue. All these processes require degradation of the ECM, which appears to involve FAP activity. Few endogenous substrates of FAP have been identified (Hamson et al. 2014; Jiang et al. 2016; Wilson et al. 2016).

Notes

Acknowledgment

MDG is supported by project grants 1105238 and 1113842 from the Australian National Health and Medical Research Council.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Centenary Institute and Sydney Medical School, The University of SydneyNewtownAustralia