Much interest is currently focused on the emerging role of tumorstroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-β and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-β and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.CXCR4 | Smad | tumor microenvironment | alpha-smooth muscle actin M yofibroblasts are often observed in the stroma of various human carcinomas that include those of the breast (1). The presence of these cells in large numbers is also associated with higher-grade malignancy and poor prognosis in patients (2-4). Myofibroblasts express α-smooth muscle actin (α-SMA) that distinguishes these cells from fibroblasts and represents a hallmark of activated fibroblasts (5-10). The activated myofibroblast state of stromal fibroblasts also correlates with their ability to promote tumor growth (11)(12)(13)(14). Although different types of mesenchymal cells and epithelial cells are proposed to be precursors of the myofibroblasts present in tumors (15-20), their precise cellular origins and functional contributions to tumor growth still remain unclear.In recent years, the tumor-promoting roles of stromal fibroblasts and α-SMA-positive myofibroblasts, collectively termed carcinoma-associated fibroblasts (CAFs), have been studied (21). CAFs, when inoculated with carcinoma cells, have potently promoted the in vivo proliferation of carcinoma cells and tumor growth in mouse xenograft models (14,(21)(22)(23)(24)(25). We previously demonstrated that CAFs, prepared directly from invasive human mammary carcinomas, contain substantial numbers of myofibroblasts that secrete elevated levels of the proangiogenic chemokine, stromal cell-derived factor-1 (SDF-1, also called CXCL12) (14). SDF-1 signaling via its cognate receptor CXCR4, expressed on the surface of carcinoma cells, directly boosts the proliferation of these cells and can stimulate neoangiogenesis by recruiting circulating endot...
Tumours are highly complex tissues composed of carcinoma cells and surrounding stroma, which is constructed by various different types of mesenchymal cells and an extracellular matrix (ECM). Carcinoma-associated fibroblasts (CAFs), which consist of both fibroblasts and myofibroblasts, are frequently observed in the stroma of human carcinomas, and their presence in large numbers is often associated with the development of high-grade malignancies and poor prognoses. Moreover, in human tumour xenograft models, CAFs extracted from the tumour are more capable of promoting tumour growth through their interactions with carcinoma cells when compared to those isolated from non-cancerous stroma. Taken together, these observations strongly suggest that CAFs actively contribute to tumour progression. In this review we highlight the emerging roles of these cells in promoting tumourigenesis, and we discuss the molecular mechanisms underlying their tumour-promoting capabilities and their cellular origin.
Newly deposited microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As integrin receptors are key determinants of fibronectin assembly, we investigated whether they also influenced microfibril deposition. Analysis of β1-integrin-receptor-null fibroblasts, blockage of cell surface integrin receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of β1 integrins in fibronectin-depleted cultures is insufficient to rescue microfibril assembly. In fibronectinRGE/RGE mutant mouse fibroblast cultures, which do not engage α5β1 integrin, extracellular assembly of both fibronectin and microfibrils is markedly reduced. Thus, pericellular microfibril assembly is regulated by fibronectin fibrillogenesis.
Dentine dysplasia type II is an autosomal dominant disorder in which mineralization of the dentine of the primary teeth is abnormal. On the basis of the phenotypic overlap between, and shared chromosomal location with, dentinogenesis imperfecta type II, a second disorder of dentine mineralization, it has been proposed that the two conditions are allelic. As recent studies have shown that dentinogenesis imperfecta type II results from mutation of the bicistronic dentine sialophosphoprotein gene (DSPP ), we have tested this hypothesis by sequencing DSPP in a family with a history of dentine dysplasia type II. Our results have shown that a missense change, which causes the substitution of a tyrosine for an aspartic acid in the hydrophobic signal peptide domain of the protein, underlies the phenotype in this family. Biochemical analysis has further demonstrated that this mutation causes a failure of translocation of the encoded proteins into the endoplasmic reticulum, and is therefore likely to lead to a loss of function of both dentine sialoprotein and dentine phosphoprotein.
We have defined the homotypic interactions of fibrillin-1 to obtain new insights into microfibril assembly. Dosedependent saturable high affinity binding was demonstrated between N-terminal fragments, between furin processed C-terminal fragments, and between these Nand C-terminal fragments. The N terminus also interacted with a downstream fragment. A post-furin cleavage site C-terminal sequence also interacted with the N terminus, with itself and with the furin-processed fragment. No other homotypic fibrillin-1 interactions were detected. Some terminal homotypic interactions were inhibited by other terminal sequences, and were strongly calcium-dependent. Treatment of an N-terminal fragment with Nethylmaleimide reduced homotypic binding. Microfibrilassociated glycoprotein-1 inhibited N-to C-terminal interactions but not homotypic N-terminal interactions. These fibrillin-1 interactions are likely to regulate pericellular fibrillin-1 microfibril assembly.
We have defined the molecular basis of cell adhesion to fibrillin-1, the major structural component of extracellular microfibrils that are associated with elastic fibres. Using human dermal fibroblasts, and recombinant domain swap fragments containing the Arg-Gly-Asp motif, we have demonstrated a requirement for upstream domains for integrin-α5β1-mediated cell adhesion and migration. An adjacent heparin-binding site, which supports focal adhesion formation, was mapped to the fibrillin-1 TB5 motif. Site-directed mutagenesis revealed two arginine residues that are crucial for heparin binding, and confirmed their role in focal adhesion formation. These integrin and syndecan adhesion motifs juxtaposed on fibrillin-1 are evolutionarily conserved and reminiscent of similar functional elements on fibronectin, highlighting their crucial functional importance.
Fibulin-5 plays an important role in elastic fibre formation in vivo. We have investigated the molecular interactions between fibulin-5 and components of fibrillin-rich microfibrils which form a template for elastin. Fibulin-5 interacted in a dose-dependent manner with a fibrillin-1 N-terminal sequence and with tropoelastin, but not with MAGP-1 (microfibril-associated glycoprotein-1) or decorin. Fibulin-5 did not inhibit interactions between fibrillin-1 N- and C-terminal fragments, or fibrillin-1 interactions with tropoelastin. Fibulin-5 may provide a link between tropoelastin and microfibrils in the pericellular space during elastic fibre assembly.
We have investigated the molecular basis of elastic fiber formation on fibrillin microfibrils. Binding assays revealed high affinity calcium-independent binding of two overlapping fibrillin-1 fragments (encoded by central exons 18 -25 and 24 -30) to tropoelastin, which, in microfibrils, map to an exposed "arms" feature adjacent to the beads. A further binding site within an adjacent fragment (encoded by exons 9 -17) was within an eightcysteine motif designated TB2 (encoded by exons 16 and 17). Binding to TB2 was ablated by the presence of Nterminal domains (encoded by exons 1-8) and reduced after deleting the proline-rich region. A novel transglutaminase cross-link between tropoelastin and fibrillin-1 fragment (encoded by exons 9 -17) was localized by mass spectrometry to a sequence encoded by exon 17. The high affinity binding and cross-linking of tropoelastin to a central fibrillin-1 sequence confirm that this association is fundamental to elastic fiber formation. Microfibril-associated glycoprotein-1 showed calcium-dependent binding of moderate affinity to fibrillin-1 N-terminal fragment (encoded by exons 1-8), which localize to the beads. Microfibril-associated glycoprotein-1 thus contributes to microfibril organization but may also form secondary interactions with adjacent microfibril-bound tropoelastin.
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