Fibroblast Activation Protein Peptide Substrates Identified from Human Collagen I Derived Gelatin Cleavage Sites

Abstract

A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-α (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest K m of 21 μM. Overall the K m values were relatively similar for both high and low ranked substrates, whereas the k cat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAPselective peptide carriers for incorporation into FAP-activated prodrugs.The growth of epithelial neoplasms requires the formation of a supporting tumor stroma to supply nutrients and growth factors for tumor cell survival and continued growth. This invasive growth is associated with characteristic changes in the supporting stroma that include induction of tumor blood vessel formation, the recruitment of reactive stromal † This work supported by funding from the Susan G. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript myofibroblasts, lymphocytes, and macrophages, the release of peptide signaling molecules and proteases, and the production of an altered extracellular matrix (1-5). The tumor stroma compartment represents a major component of the mass of most carcinomas, with 20-50% commonly seen in breast, lung, and colorectal cancers and reaching >90% in carcinomas that have desmoplastic reactions such as breast and pancreatic cancers (5, 6).Unlike malignant epithelial cells, activated tumor stromal fibroblasts are not transformed genetically and do not demonstrate the genetic and phenotypic heterogeneity seen in malignant cells. Reactive tumor stromal fibroblasts differ from fibroblasts of normal adult tissues in regard to morphology, gene expression profiles, and production of important biological ...

Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice

“…Despite a lack of direct evidence, FAP is assumed to degrade ECM components, including type I collagen in vivo (9,22,23). In support of this idea, we have observed that FAP deficiency leads to increased tumor collagen content in a syngeneic transplant model of colon cancer and an endogenous K-ras-driven murine lung tumor model (24).…”

“…FAP has also been detected on pericytes, bone marrow-derived mesenchymal stem cells (18,19), and a small population of macrophages (20,21 FAP's in vivo substrates remain unclear. Despite a lack of direct evidence, FAP is assumed to degrade ECM components, including type I collagen in vivo (9,22,23). In support of this idea, we have observed that FAP deficiency leads to increased tumor collagen content in a syngeneic transplant model of colon cancer and an endogenous K-ras-driven murine lung tumor model (24).…”

“…The DPP family of serine proteases cleaves amino-terminal dipeptides from polypeptides with L-proline or L-alanine at the penultimate position. FAP is unique in that it displays additional in vitro endopeptidase (7), gelatinase, and potentially collagenase activity (8,9). FAP expression is restricted, occurring at high levels on mesenchymal cells during embryogenesis (10) and then is repressed shortly after birth.…”

Colorectal Cancer Cells Activate Adjacent Fibroblasts Resulting in FGF1/FGFR3 Signaling and Increased Invasion

“…8 FAP is a membrane-bound glycoprotein belonging to the serine protease family that has been demonstrated to cleave gelatin and human collagen I, and therefore has been implicated in the remodeling of the extracellular matrix. 9 It is normally expressed transiently in healing wounds, and abundantly by activated stromal fibroblasts in CRC and other human epithelial malignancies (breast, lung, and ovary carcinomas). 10 -12 Abundant FAP expression has been implicated as a negative prognostic factor in patients with metastatic CRC, 13 but its biological role is still not clear.…”

Theranostic Probes for Targeting Tumor Microenvironment: An Overview

“…CAFs are widely seen in breast and pancreatic cancer [81,82]. The overexpression of fibroblast activation protein-α (FAP) (membrane bound serine protease), on the CAFs, makes them different from the normal stromal cells [83]. FAP has a unique post-prolyl endopeptidase activity to cleave the dipeptide at NH2-terminal [84,85].…”