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.
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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).
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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)
Supplementary Table 1
Primers used in the quantitative PCR. (DOCX 17 kb)
Supplementary Table 2
Proteins identified in NOF-1 and CAF-1 cell lines by LC-MS/MS. (DOCX 84 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)
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(DOCX 71 kb)
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Bagordakis, E., Sawazaki-Calone, I., Macedo, C.C.S. et al. Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures. Tumor Biol. 37, 9045–9057 (2016). https://doi.org/10.1007/s13277-015-4629-y
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DOI: https://doi.org/10.1007/s13277-015-4629-y