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Tumor Biology

, Volume 37, Issue 7, pp 9045–9057 | Cite as

Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures

  • Elizabete Bagordakis
  • Iris Sawazaki-Calone
  • Carolina Carneiro Soares Macedo
  • Carolina M. Carnielli
  • Carine Ervolino de Oliveira
  • Priscila Campioni Rodrigues
  • Ana Lucia C. A. Rangel
  • Jean Nunes dos Santos
  • Juha Risteli
  • Edgard Graner
  • Tuula Salo
  • Adriana Franco Paes Leme
  • Ricardo D. Coletta
Original Article

Abstract

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-β1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.

Keywords

Cancer-associated fibroblasts Secretome Extracellular matrix Type I collagen FNDC1 SERPINE1 STC2 

Notes

Acknowledgments

This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo-FAPESP, São Paulo, Brazil; Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, Brasília, Brazil, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES, Brasília, Brazil (AUXPE-PVES-570/2013).

Compliance with ethical standard

Conflicts of interest

None

Supplementary material

13277_2015_4629_Fig9_ESM.gif (46 kb)
Supplementary Figure 1

TGF-β1 induces transdifferentiation of NOFs to CAFs. NOFs were cultured with 10 ng/ml of TGF-β1 in culture media containing 0.1% of calf serum for 2 days. Following treatment, cells were collected and subjected RNA purification and quantitative PCR analysis. The levels of the CAF marker α-SMA were markedly increased after incubation with TGF-β1. (GIF 45 kb)

13277_2015_4629_MOESM1_ESM.tif (15.2 mb)
(TIF 15526 kb)
13277_2015_4629_MOESM2_ESM.docx (17 kb)
Supplementary Table 1 Primers used in the quantitative PCR. (DOCX 17 kb)
13277_2015_4629_MOESM3_ESM.docx (84 kb)
Supplementary Table 2 Proteins identified in NOF-1 and CAF-1 cell lines by LC-MS/MS. (DOCX 84 kb)
13277_2015_4629_MOESM4_ESM.docx (59 kb)
Supplementary Table 3 Overrepresented GO terms for the dataset of differentially expressed proteins between CAF-1 and NOF-1 cell lines. (DOCX 58 kb)
13277_2015_4629_MOESM5_ESM.docx (71 kb)
ESM 1 (DOCX 71 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Elizabete Bagordakis
    • 1
  • Iris Sawazaki-Calone
    • 2
  • Carolina Carneiro Soares Macedo
    • 1
  • Carolina M. Carnielli
    • 3
  • Carine Ervolino de Oliveira
    • 1
  • Priscila Campioni Rodrigues
    • 1
  • Ana Lucia C. A. Rangel
    • 2
  • Jean Nunes dos Santos
    • 4
  • Juha Risteli
    • 5
    • 6
  • Edgard Graner
    • 1
  • Tuula Salo
    • 5
    • 6
    • 7
    • 8
  • Adriana Franco Paes Leme
    • 3
  • Ricardo D. Coletta
    • 1
  1. 1.Department of Oral Diagnosis, School of DentistryState University of CampinasPiracicabaBrazil
  2. 2.Oral Pathology and Oral Medicine, Dentistry SchoolWestern Paraná State UniversityCascavelBrazil
  3. 3.Brazilian Biociences National Laboratory-CNPEMCampinasBrazil
  4. 4.Laboratory of Surgical Pathology, Dental SchoolFederal University of Bahia-UFBASalvadorBrazil
  5. 5.Cancer and Translational Medicine Research UnitUniversity of OuluOuluFinland
  6. 6.Medical Research CenterOulu University HospitalOuluFinland
  7. 7.Oral and Maxillofacial Diseases UnitUniversity of HelsinkiHelsinkiFinland
  8. 8.Helsinki University HospitalHelsinkiFinland

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