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Background: Alternative splicing of EDA fibronectin (FN) region is a cell type- and development-regulated mechanism controlled by pathological processes, growth factors and extracellular matrix (ECM). Classic and vascular Ehlers-Danlos syndrome (cEDS and vEDS) are connective tissue disorders caused by COL5A1/COL5A2 and COL3A1 gene mutations, leading to an in vivo abnormal collagen fibrillogenesis and to an in vitro defective organisation in the ECM of type V (COLLV) and type III collagen (COLLIII). These defects induce the FN-ECM disarray and the decrease of COLLs and FN receptors, the α2β1 and α5β1 integrins. Purified COLLV and COLLIII restore the COLL-FN-ECMs in both EDS cell strains., Methods: Real-time PCR, immunofluorescence microscopy, and Western blotting were used to investigate the effects of COLLs on FN1 gene expression, EDA region alternative splicing, EDA(+)-FN-ECM assembly, α5β1 integrin and EDA(+)-FN-specific α9 integrin subunit organisation, α5β1 integrin and FAK co-regulation in EDS fibroblasts., Results: COLLV-treated cEDS and COLLIII-treated vEDS fibroblasts up-regulate the FN1 gene expression, modulate the EDA(+) mRNA maturation and increase the EDA(+)-FN levels, thus restoring a control-like FN-ECM, which elicits the EDA(+)-FN-specific α9β1 integrin organisation, recruits the α5β1 integrin and switches on the FAK binding and phosphorylation., Conclusion: COLLs regulate the EDA(+)-FN-ECM organisation at transcriptional and post-transcriptional level and activate the α5β1-FAK complexes. COLLs also recruit the α9β1 integrin involved in the assembly of the EDA(+)-FN-ECM in EDS cells., General Significance: The knowledge of the COLLs-ECM role in FN isotype expression and in EDA(+)-FN-ECM-mediated signal transduction adds insights in the ECM remodelling mechanisms in EDS cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer., Methods: We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses., Results: We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated., Conclusion: Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions., (© 2011 Lee et al; licensee BioMed Central Ltd.)

Mammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma, yet the associated molecular mechanisms remain largely unknown. Importantly, regions of high breast density are associated with increased stromal collagen and epithelial cell content. We set out to determine whether increased collagen-matrix density, in the absence of stromal cells, was sufficient to promote proliferation and invasion characteristic of a malignant phenotype in non-transformed mammary epithelial cells. We demonstrate that increased collagen-matrix density increases matrix stiffness to promote an invasive phenotype. High matrix stiffness resulted in increased formation of activated three-dimensional (3D)-matrix adhesions and a chronically elevated outside-in/inside-out focal adhesion (FA) kinase (FAK)-Rho signaling loop, which was necessary to generate and maintain the invasive phenotype. Moreover, this signaling network resulted in hyperactivation of the Ras-mitogen-activated protein kinase (MAPK) pathway, which promoted growth of mammary epithelial cells in vitro and in vivo and activated a clinically relevant proliferation signature that predicts patient outcome. Hence, the current data provide compelling evidence for the importance of the mechanical features of the microenvironment, and suggest that mechanotransduction in these cells occurs through a FAK-Rho-ERK signaling network with extracellular signal-regulated kinase (ERK) as a bottleneck through which much of the response to mechanical stimuli is regulated. As such, we propose that increased matrix stiffness explains part of the mechanism behind increased epithelial proliferation and cancer risk in human patients with high breast tissue density.

Background: A number of adhesion-mediated signaling pathways and cell-cycle events have been identified that regulate cell proliferation, yet studies to date have been unable to determine which of these pathways control mitogenesis in response to physiologically relevant changes in tissue elasticity. In this report, we use hydrogel-based substrata matched to biological tissue stiffness to investigate the effects of matrix elasticity on the cell cycle., Results: We find that physiological tissue stiffness acts as a cell-cycle inhibitor in mammary epithelial cells and vascular smooth muscle cells; subcellular analysis in these cells, mouse embryonic fibroblasts, and osteoblasts shows that cell-cycle control by matrix stiffness is widely conserved. Remarkably, most mitogenic events previously documented as extracellular matrix (ECM)/integrin-dependent proceed normally when matrix stiffness is altered in the range that controls mitogenesis. These include ERK activity, immediate-early gene expression, and cdk inhibitor expression. In contrast, FAK-dependent Rac activation, Rac-dependent cyclin D1 gene induction, and cyclin D1-dependent Rb phosphorylation are strongly inhibited at physiological tissue stiffness and rescued when the matrix is stiffened in vitro. Importantly, the combined use of atomic force microscopy and fluorescence imaging in mice shows that comparable increases in tissue stiffness occur at sites of cell proliferation in vivo., Conclusions: Matrix remodeling associated with pathogenesis is in itself a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1.

Invadopodia are actin-rich subcellular protrusions with associated proteases used by cancer cells to degrade extracellular matrix (ECM) [1]. Molecular components of invadopodia include branched actin-assembly proteins, membrane trafficking proteins, signaling proteins, and transmembrane proteinases [1]. Similar structures exist in nontransformed cells, such as osteoclasts and dendritic cells, but are generally called podosomes and are thought to be more involved in cell-matrix adhesion than invadopodia [2-4]. Despite intimate contact with their ECM substrates, it is unknown whether physical or chemical ECM signals regulate invadopodia function. Here, we report that ECM rigidity directly increases both the number and activity of invadopodia. Transduction of ECM-rigidity signals depends on the cellular contractile apparatus [5-7], given that inhibition of nonmuscle myosin II, myosin light chain kinase, and Rho kinase all abrogate invadopodia-associated ECM degradation. Whereas myosin IIA, IIB, and phosphorylated myosin light chain do not localize to invadopodia puncta, active phosphorylated forms of the mechanosensing proteins p130Cas (Cas) and focal adhesion kinase (FAK) are present in actively degrading invadopodia, and the levels of phospho-Cas and phospho-FAK in invadopodia are sensitive to myosin inhibitors. Overexpression of Cas or FAK further enhances invadopodia activity in cells plated on rigid polyacrylamide substrates. Thus, in invasive cells, ECM-rigidity signals lead to increased matrix-degrading activity at invadopodia, via a myosin II-FAK/Cas pathway. These data suggest a potential mechanism, via invadopodia, for the reported correlation of tissue density with cancer aggressiveness.