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Fibroblast activation protein-α promotes tumor growth and invasion of breast cancer cells through non-enzymatic functions

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Abstract

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 (FAPS624A) also produced tumors that grew rapidly. In vitro studies revealed that cells expressing wild type FAP or FAPS624A 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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Abbreviations

ANOVA:

Analysis of variance

GIP:

Glucose dependent and insulinotropic polypeptide

GLP-1:

Glucagon-like peptide-1

ECM:

Extracellular matrix

FACS:

Fluorescence activated cell sorting

FAP:

Fibroblast activation protein-α

FAPS624A :

MDA MB-231 cells expressing a catalytically inactive mutant of FAP

FITC:

Fluorescien isothiocyanate

F19:

Mouse monoclonal antibody directed against human FAP

LAF-237:

(vildagliptin), 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine, an inhibitor of DPPIV and related proteases

MDA MB-231:

A human breast adenocarcinoma cell line

MMPs:

Matrix metalloproteinases

MMP-9:

Matrix metalloproteinase-9

pcDNA3.1:

A plasmid mammalian expression vector

PT-100:

Val-boroPro, an inhibitor of FAP and related proteases

PT-630:

Glu-boroPro, an inhibitor of FAP and related proteases

RT-PCR:

Reverse transcription-polymerase chain reaction

SCID:

Severe combined immune deficient

WTY-1:

Stable transfectant of MDA MB-231 engineered to express high levels of FAP

WTY-6:

Stable transfectant of MDA MB-231 engineered to express high levels of FAP

4G10:

Monoclonal antibody to phosphotyrosine

6-6B:

Monoclonal antibody to MMP-9

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Acknowledgments

The authors thank Amir Khan for help producing the cDNA encoding the S624A mutant FAP. The authors thank Steven Post for critical review of the manuscript. Supported by grants from the DoD CDMRP-BCRP-BC074331, Arkansas Breast Cancer Research Program and funds from Point Therapeutics, Inc.

Conflict of interest

The study was funded in part by Point Therapeutics who produced the talabostat and PT-630 compounds used in this research.

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Authors and Affiliations

  1. Department of Pathology, Winthrop P. Rockefeller Cancer Institute, Arkansas Cancer Research Center, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 753, Little Rock, AR, 72205-7199, USA

    Yan Huang, Avis E. Simms, Anna Mazur, Sophie Wang, Noel R. León & Thomas Kelly

  2. Point Therapeutics, Inc., Boston, MA, USA

    Barry Jones & Nazneen Aziz

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  1. Yan Huang
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  2. Avis E. Simms
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Correspondence to Thomas Kelly.

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Huang, Y., Simms, A.E., Mazur, A. et al. Fibroblast activation protein-α promotes tumor growth and invasion of breast cancer cells through non-enzymatic functions. Clin Exp Metastasis 28, 567–579 (2011). https://doi.org/10.1007/s10585-011-9392-x

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