Tumor cell and carcinoma-associated fibroblast interaction regulates matrix metalloproteinases and their inhibitors in oral squamous cell carcinoma

Abstract

Co-culture of periodontal ligament (PDL) fibroblasts and SCC-25 oral squamous carcinoma cells (OSCC), results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs). Paracrin circuits between CAFs and OSCC cells were hypothesized to regulate the gene expression of matrix remodeling enzymes in their co-culture, which was performed for 7days, followed by analysis of the mRNA/protein expression and activity of metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and other relevant genes. Interleukin1-β, transforming growth factor-β1, fibronectin and αvβ6 integrin have shown to be involved in the regulation of the MMP and TIMP gene expression in co-culture of CAFs and tumor cells. In addition, these cells also cooperated in activation of MMP pro-enzymes. It is particularly interesting that the fibroblast-produced inactive MMP-2 has been activated by the tumor-cell-produced membrane-type 1 matrix metalloproteinase (MT1-MMP). The crosstalk between cancer- and the surrounding fibroblast stromal-cells is essential for the fine tuning of cancer cells invasivity.

Graphical abstract

Fig. 1

Summary of the suggested mechanism for the regulation of MMPs and TIMPs in the paracrine interplay between SCC-25 cells and fibroblasts. MMP-9 showed a tumor specific expression, regulated presumably by the fibronectin ITGA5B6 pathway. The ITGA5 was inducible in both SCC-25 and PDL fibroblasts in co-culture, but ITGB6 expression was tumor (SCC-25) specific. Based on a previous report , MMP-9 might be activated in interaction with CD-44, and according to our gelatinase assay results, it remains bound with the tumor cells (A). The results of this study suggest that MMP-2 is secreted in its pro- (inactive-) form by CAFs surrounding the tumor cells, and at a lower extent also by the tumor cells themselves. Activation of MMP-2 either requires MT1-MMP localized on the SCC-25 cancer cells , or integrins, where the involvement of αv integrins (ITGA5) is expected (A). MMPs-1, -3 and TIMPs-1, -3 are produced in the PDL fibroblasts, and their expression might be regulated by inflammatory cytokines, including IL1-β produced by SCC-25 cells. The gene expression of MMP-1, MMP-2, TIMP-1 and TIMP-3 was reduced by dexamethasone (DEX) (B).

Fig. 2

mRNA expression of MMP-1 (A), MMP-2 (B), MMP-3 (C), TIMP-1 (D), TIMP-3 (E) and MMP-9 (F) in PDL fibroblasts and SCC-25 cells in control and co-cultured conditions. *: p < 0.05; **: p < 0.01, ***: p < 0.001.

Fig. 3

Western blot analysis of MMP-2 (A, B), TIMP-1 (A, C) and TIMP-3 (A, D) related to loading control (β-actin) in PDL fibroblasts in control (1–2) and co-cultured (3–4) conditions. Densitometry of the detected bands normalized to the β-actin band densities. MMP-2 (B), TIMP-1 (C), TIMP-3 (D), *: p < 0.05.

Fig. 4

mRNA expression of TGF-β1 (A), TGFBR2 (B), LTBP-1 (C) and TIEG (D) in fibroblasts and SCC-25 cells in control and co-cultured conditions. *: p < 0.05; **: p < 0.01, ***: p < 0.001.

Fig. 5

mRNA expression of ITGA5 (A), ITGB6 (B) and fibronectin (C), in PDL fibroblasts and SCC-25 cells in control and co-cultured conditions. *: p < 0.05; **: p < 0.01, ***: p < 0.001. Western blot of ITGA5 and loading control β-actin (D) in nuclear-free extracts of control (1) and co-cultured (2) fibroblasts, control (3) and co-cultured (4) SCC-25 cells. Confocal microscopy of EDA-fibronectin (red detected by Alexa fluor 647–conjugated anti-mouse IgG) in PDLs, bar: 100 μm.

Fig. 6

Detection of MMP-2 and MMP-9 gelatinase activity in cell lysate (A) and supernatant (B) of fibroblasts and SCC-25 cells by combined gelatinase zymography. 1–2: fibroblast control, 3–4: SCC-25 control, 5–6: fibroblast co-culture, 7–8: SCC-25 co-culture. Densitometry of MMP-2 and MMP-9 gelatinase zymography (C–F). C and D represent the densitometry of pro-MMP-9 (C) and active MMP-9 (D) bands in cell lysates. E and F show the densitometry of pro-MMP-2 (E) and active MMP-2 (F) form from supernatants.