The microenvironment of tumors is characterized by structural changes in the fibronectin matrix, which include increased deposition of the EDA isoform of fibronectin and the unfolding of the fibronectin Type III domains. The impact of these structural changes on tumor progression is not well understood. The fibronectin EDA (FnEDA) domain and the partially unfolded first Type III domain of fibronectin (FnIII-1c) have been identified as endogenous damage-associated molecular pattern molecules (DAMPs), which induce innate immune responses by serving as agonists for Toll-Like Receptors (TLRs). Using two triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, we show that FnEDA and FnIII-1c induce the pro-tumorigenic cytokine, IL-8, by serving as agonists for TLR5 and TLR2, the canonical receptors for bacterial flagellin and lipoprotein, respectively. We also find that FnIII-1c is not recognized by MDA-MB-468 cells but is recognized by MDA-MB-231 cells, suggesting a cell type rather than ligand specific utilization of TLRs. As IL-8 plays a major role in the progression of TNBC, these studies suggest that tumor-induced structural changes in the fibronectin matrix promote an inflammatory microenvironment conducive to metastatic progression.

Changes in the organization and structure of the fibronectin matrix are believed to contribute to dysregulated wound healing and subsequent tissue inflammation and tissue fibrosis. These changes include an increase in the EDA isoform of fibronectin as well as the mechanical unfolding of fibronectin type III domains. In previous studies using embryonic foreskin fibroblasts, we have shown that fibronectin’s EDA domain (FnEDA) and the partially unfolded first Type III domain (FnIII-1c) function as Damage Associated Molecular Pattern (DAMP) molecules to stimulate the induction of inflammatory cytokines by serving as agonists for Toll-Like Receptor-4 (TLR4). However, the role of signaling molecules downstream of TLR-4 such as TGF-β Activated Kinase 1 (TAK1) and Mitogen activated protein kinases (MAPK) in regulating the expression of fibronectin DAMP induced inflammatory genes in specific cell types is not known. In the current study, we evaluate the molecular steps regulating the fibronectin driven induction of inflammatory genes in three human fibroblast cell lines: embryonic foreskin, adult dermal, and adult kidney. The fibronectin derived DAMPs each induce the phosphorylation and activation of TAK1 which results in the activation of two downstream signaling arms, IKK/NF-κB and MAPK. Using the specific inhibitor 5Z-(7)-Oxozeanol as well as siRNA, we show TAK1 to be a crucial signaling mediator in the release of cytokines in response to fibronectin DAMPs in all three cell types. Finally, we show that FnEDA and FnIII-1c induce several pro-inflammatory cytokines whose expression is dependent on both TAK1 and JNK MAPK and highlight cell-type specific differences in the gene-expression profiles of the fibroblast cell-lines.