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Fibroblast activation protein increases metastatic potential of fibrosarcoma line HT1080 through upregulation of integrin-mediated signaling pathways

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Abstract

The serine protease fibroblast activation protein (FAP) is selectively expressed on tumour-associated fibroblasts in most human epithelial tumours, as well as on some mesenchymal tumours such as sarcoma. High FAP expression is most often associated with poor outcome and increased metastasis. Here, we compare the in vitro metastatic potential of HT1080 fibrosarcoma cells with and without FAP expression in order to elucidate the mechanism by which FAP may influence metastasis. In the presence of FAP, cells were more adhesive to extracellular matrix proteins and migrated and invaded through Matrigel to a greater degree. The anti-FAP antibody ESC11, which caused internalization of FAP, decreased adhesion and migration, but only when cells expressed FAP. It was also found that blocking activity of integrins β1 and αvβ3 reduced both cell adhesion and migration and this effect was much more marked in FAP-expressing HT1080 cells than mock-transfected HT1080 cells. The expression or activation of intracellular proteins that form part of the downstream signaling of integrins, including integrin-linked kinase, Rac1 and focal adhesion kinase, was also upregulated when FAP was expressed, suggesting that FAP not only upregulates metastatic-like cell behaviours through interaction with integrins, but also influences the intracellular signaling of integrins. This was confirmed using both PI3 kinase and Src kinase inhibitors, which decreased adhesion and migration in FAP-expressing cells, but did not affect mock-transfected HT1080 cells. FAP is therefore a useful target for anti-cancer therapy, as not only is its expression tumour-selective, but its downregulation has the potential to reduce incidence of metastasis.

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Abbreviations

FAP:

Fibroblast activation protein

ECM:

Extracellular matrix

CDR:

Complementarity determining region

HAHA:

Human anti-human antibody response

mAb:

Monoclonal antibody

G418:

Geneticin

DMEM:

Dulbecco’s Modified Eagle Medium

FBS:

Fetal bovine serum

EDTA:

Ethylenediaminetetraacetic acid

FAK:

Focal adhesion kinase

pFAK:

Phosphorylated focal adhesion kinase

PBS:

Phosphate buffered saline

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

DMSO:

Dimethyl sulphoxide

BSA:

Bovine serum albumin

R-PE:

R-phycoerythrin

FITC:

Fluorescein isothiocyanate

DAPI:

4′,6-diamidino-2-phenylindole

SEM:

Standard error of the mean

ANOVA:

Analysis of variance

ILK:

Integrin-linked kinase

ATP:

Adenosine triphosphate

PI3K:

Phosphoinositide 3-kinase

uPA:

Urokinase-type plasminogen activator

MMP-2, -9:

Matrix metalloproteinase-2,-9

References

  1. Cheng JD, Valianou M, Canutescu AA, Jaffe EK, Lee HO, Wang H et al (2005) Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth. Mol Cancer Ther 4:351–360

    Article  CAS  PubMed  Google Scholar 

  2. Garin-Chesa P, Old LJ, Rettig WJ (1990) Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. PNAS USA 87:7235–7239

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Rettig WJ, Garin-Chesa P, Healey JH, Su SL, Ozer HL, Schwab M et al (1993) Regulation and heteromeric structure of the fibroblast activation protein in normal and transformed cells of mesenchymal and neuroectodermal origin. Cancer Res 53:3327–3335

    CAS  Google Scholar 

  4. Dohi O, Ohtani H, Hatori M, Sato E, Hosaka M, Nagura H, Itoi E, Kokubun S (2009) Histogenesis-specific expression of fibroblast activation protein and dipeptidylpeptidase IV in human bone and soft tissue tumours. Histopathology 55(4):432–440

    Article  PubMed Central  PubMed  Google Scholar 

  5. Rettig WJ, Garin-Chesa P, Beresford HR, Oettgen HF, Melamed MR, Old LJ (1988) Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells. PNAS USA 85:3110–3114

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Scott AM, Wiseman G, Welt S, Adjei A, Lee F-T, Hopkins W, Divgi CR, Hanson LH, Mitchell P, Gansen DN, Larson SM, Ingle JN, Hoffman EW, Tanswell P, Ritter G, Cohen LS, Bette P, Arvay L, Amelberg A, Vlock D, Rettig WJ, Old LJ (2003) A phase I dose-escalation study of Sibrotuzumab in patients with advanced or metastatic fibroblast activation protein-positive cancer. Clin Cancer Res 9:1639–1647

    CAS  PubMed  Google Scholar 

  7. Hofheinz RD, al-Batran SE, Hartmann F, Hartung G, Jager D, Renner C et al (2003) Stromal targeting by a humanized monoclonal antibody: an early phase II trial of sibrotuzumab in patients with metastatic colorectal cancer. Onkologie 26(1):22–28

    Article  Google Scholar 

  8. Fischer E, Chaitanya K, Wuest T, Wadle A, Scott AM, van der Broek M, Schibli R, Bauer S, Renner C (2012) Radioimmunotherapy of fibroblast activation protein positive tumours by rapidly internalizing antibodies. Clin Can Res 18:6208–6218

    Article  CAS  Google Scholar 

  9. Ariga N, Sato E, Ohuchi N, Nagura H, Ohtani H (2001) Stromal expression of fibroblast activation protein/seprase, a cell membrane serine proteinase and gelatinase, is associated with longer survival in patients with invasive ductal carcinoma of breast. Int J Cancer 95:67–72

    Article  CAS  PubMed  Google Scholar 

  10. Jia J, Martin TA, Ye L, Jiang WG (2014) FAP-alpha is involved in the control of human breast cancer cell line growth and motility via the FAK pathway. BMC Cell Biology 15:1630

    Article  Google Scholar 

  11. Henry LR, Lee HO, Lee JS, Klein-Szanto A, Watts P, Ross EA et al (2007) Clinical implications of fibroblast activation protein in patients with colon cancer. Clin Cancer Res 13:1736–1741

    Article  CAS  PubMed  Google Scholar 

  12. Wikberg ML, Edin S, Lundberg IV, Van Guelpen B, Dahlin AM, Rutegård J, Stenling R, Öberg A, Palmqvist R (2013) High intratumoral expression of fibroblast activation protein (FAP) in colon cancer is associated with poorer patient prognosis. Tumor Biol 34:1013–1020

    Article  CAS  Google Scholar 

  13. Zhang MZ, Qiao YH, Nesland JM, Trope C, Kennedy A, Chen WT et al (2007) Expression of seprase in effusions from patients with epithelial ovarian carcinoma. Chin Med J (Engl) 120:663–668

    CAS  Google Scholar 

  14. Saadi A, Shannon NB, Lao-Sirieix P, O’Donovan M, Walker E, Clemons NJ, Hardwick JS, Zhang C, Das M, Save V, Novelli M, Balkwill F, Fitzgerald RC (2010) Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers. PNAS USA 107(5):2177–2182

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Saigusa S, Toiyama Y, Tanaka K, Yokoe T, Okugawa Y, Fujikawa H, Matsusita K, Kawamura M, Inoue Y, Miki C, Kusunoki M (2011) Cancer-associated fibroblasts correlate with poor prognosis in rectal cancer after chemoradiotherapy. Int J Oncol 38(3):655–663

    Article  CAS  PubMed  Google Scholar 

  16. Cohen SJ, Alpaugh RK, Palazzo I, Meropol NJ, Rogatko A, Xu Z et al (2008) Fibroblast activation protein and its relationship to clinical outcome in pancreatic adeno-carcinoma. Pancreas 37:154–158

    Article  CAS  PubMed  Google Scholar 

  17. Liao Y, Ni Y, He R, Liu W, Du J (2013) Clinical implications of fibroblast activation protein-alpha in non-small cell lung cancer after curative resection: a new predictor for prognosis. J Cancer Res Clin Oncol 139(9):1523–1528

    Article  CAS  PubMed  Google Scholar 

  18. Ju MJ, Qiu SJ, Fan J, Xiao YS, Gao Q, Zhou J, Li YW, Tang ZY (2009) Peritumoral activated hepatic stellate cells predict poor clinical outcome in hepatocellular carcinoma after curative resection. Am J Clin Pathol 131(4):498–510

    Article  CAS  PubMed  Google Scholar 

  19. Wang XM, Yu DM, McCaughan GW, Gorrell MD (2005) Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line. Hepatology 42(4):935–945

    Article  CAS  PubMed  Google Scholar 

  20. Waester P, Rosdahl I, Gilmore BF, Seifert O (2011) Ultraviolet exposure of melanoma cells induces fibroblast activation protein-alpha in fibroblasts: implications for melanoma invasion. Int J Oncol 39:193–202

    Google Scholar 

  21. Ding L, Ye L, Xu J, Jiang WG (2014) Impact of fibroblast activation protein on osteosarcoma cell lines in vitro. Oncol Lett 7:699–704

    CAS  PubMed Central  PubMed  Google Scholar 

  22. Wang H, Wu Q, Liu Z, Fan Y, Liu Y, Zhang Y, Hua S, Fu Q, Zhao M, Chen Y, Fang W, Lu X (2014) Downregulation of FAP suppressed cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-ERK signaling in oral squamous cell carcinoma. Cell Death Dis. doi:10.1038/cddis.2014.122

    Google Scholar 

  23. Chung KM, Hsu SC, Chu YR, Lin MY, Jiaang WT, Chen RH, Chen X (2014) Fibroblast activation protein (FAP) is essential for the migration of bone marrow mesenchymal stem cells through RhoA activation. PLoS One 9(2):e88772

    Article  PubMed Central  PubMed  Google Scholar 

  24. Monsky WL, Lin CY, Aoyama A, Yeh Y, Stetler-Stevenson WG, Mueller SC et al (1994) A potential marker protease of invasiveness, seprase, is localized on invadopodia of human malignant melanoma cells. Cancer Res 54:5702–5710

    CAS  PubMed  Google Scholar 

  25. Mueller SC, Ghersi G, Akiyama SK et al (1999) A novel protease-docking function of integrin at invadopodia. J Biol Chem 274(35):24947–24952

    Article  CAS  PubMed  Google Scholar 

  26. Yilmaz M, Christofori G (2009) EMT, the cytoskeleton and cancer cell invasion. Cancer Metastasis Rev 28:15–33

    Article  PubMed  Google Scholar 

  27. Stroeken PJ, van Rijthoven EA, van der Valk MA, Roos E (1998) Targeted disruption of the β1integrin gene in a lymphoma cell line greatly reduces metastatic capacity. Cancer Res 58:1569–1577

    CAS  PubMed  Google Scholar 

  28. White DE, Kurpios NA, Zuo D, Hassell JA, BlaessS Mueller U, Muller WJ (2004) Targeted disruption of β1-integrin in a transgenic mouse model of human breast cancer reveals an essential role in mammary tumor induction. Cancer Cell 6:159–170

    Article  CAS  PubMed  Google Scholar 

  29. Wang W, Goswami S, Lapidus K, Wells AL, Wyckoff JB, Sahai E et al (2004) Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors. Cancer Res 64:8585–8594

    Article  CAS  PubMed  Google Scholar 

  30. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  CAS  PubMed  Google Scholar 

  31. Hehlgans S, Haase M, Cordes N (2007) Signalling via integrins: implications for cell survival and anticancer strategies. Biochim Biophys Acta 1775:163–180

    CAS  PubMed  Google Scholar 

  32. Forget MA, Desrosiers RR, Beliveau R (1999) Physiological roles of matrix metalloproteinases: implications for tumor growth and metastasis. Can J Physiol Pharmacol 77:465–480

    Article  CAS  PubMed  Google Scholar 

  33. Artym VV, Kindzelskii AL, Chen WT et al (2002) Molecular proximity of seprase and the urokinase-type plasminogen activator receptor on malignant melanoma cell membranes: dependence on beta1 integrins and the cytoskeleton. Carcinogenesis 23:1593–1601

    Article  CAS  PubMed  Google Scholar 

  34. Zhao J, Guan JL (2009) Signal transduction by focal adhesion kinases in cancer. Cancer Metastasis Rev 28:35–49

    Article  PubMed  Google Scholar 

  35. Hall A (2009) The cytoskeleton and cancer. Cancer Metastasis Rev 28:5–14

    Article  PubMed  Google Scholar 

  36. Yang WW, Han W, Ye SQ, Liu DY, Wu J, Liu HY, Li CH, Chen H (2013) Fibroblast activation protein-alpha promotes ovarian cancer cell proliferation and invasion via extracellular and intracellular signaling mechanisms. Exp Mol Pathol 95:105–110

    Article  CAS  PubMed  Google Scholar 

  37. Santos AM, Jung J, Aziz N et al (2009) Targeting fibroblast activation protein inhibits tumour stromagenesis and growth in mice. J Clin Invest 119:3613–3625

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Austin Health Medical Research Foundation, NHMRC Program Grant No. 487922 and Operational Infrastructure Support Program funding from the Victorian Government.

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, 18 Frederick St, Dunedin, 9016, New Zealand

    Sarah K. Baird

  2. Tumour Targeting Laboratory, Olivia Newton-John Cancer Research Institute, La Trobe University, Melbourne, VIC, Australia

    Laura Allan, Fiona E. Scott & Andrew M. Scott

  3. Department of Oncology, University Hospital Basel, Basel, Switzerland

    Christoph Renner

Authors
  1. Sarah K. Baird
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  2. Laura Allan
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  4. Fiona E. Scott
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Correspondence to Sarah K. Baird.

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Baird, S.K., Allan, L., Renner, C. et al. Fibroblast activation protein increases metastatic potential of fibrosarcoma line HT1080 through upregulation of integrin-mediated signaling pathways. Clin Exp Metastasis 32, 507–516 (2015). https://doi.org/10.1007/s10585-015-9723-4

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  • DOI: https://doi.org/10.1007/s10585-015-9723-4

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