Fibronectin Knockdown Research Articles

Fibronectin mediates activin A-promoted human trophoblast migration and acquisition of endothelial-like phenotype

BackgroundDuring human early placentation, a proportion of extravillous trophoblasts (EVTs) migrate to the maternal decidua, differentiating into endovascular EVTs to remodel spiral arteries and ensure the establishment of blood circulation at the maternal-fetal interface. Inadequate EVT migration and endovascular differentiation are closely associated with adverse pregnancy outcomes such as miscarriage. Activin A and fibronectin are both secretory molecules abundantly expressed at the maternal-fetal interface. Activin A has been reported to regulate EVT biological functions. However, whether fibronectin mediates activin A-promoted EVT migration and acquisition of endothelial-like phenotype as well as the underlying molecular mechanisms remain unknown. Additionally, the role of fibronectin in pregnancy establishment and maintenance warrants further investigation.MethodsPrimary and immortalized (HTR8/SVneo) human EVTs were used as in vitro study models. Cultured human first-trimester chorionic villous explants were utilized for ex vivo validation. A local fibronectin knockdown model in ICR mouse uteri, achieved by nonviral in vivo transfection with small interfering RNA (siRNA) targeting fibronectin 1 (si-Fn1), was employed to explore the roles of fibronectin in the establishment and maintenance of early pregnancy.ResultsOur results showed that activin A treatment significantly induced fibronectin 1 (FN1) mRNA expression and fibronectin protein production, which is essential for human trophoblast migration and endothelial-like tube formation. Both basal and activin A-upregulated fibronectin expression were abolished by the TGF-β type I receptor inhibitor SB431542 or siRNA-mediated knockdown of activin receptor-like kinase (ALK4) or SMAD4. Moreover, activin A-increased trophoblast migration and endothelial-like tube formation were attenuated following the depletion of fibronectin. Fibronectin knockdown via intrauterine siRNA administration reduced CD31 and cytokeratin 8 (CK8) expression at the maternal-fetal interface, resulting in a decrease in the number of implantation sites and embryos.ConclusionsOur study demonstrates that activin A promotes trophoblast cell migration and acquisition of endothelial-like phenotype via ALK4-SMAD2/3-SMAD4-mediated fibronectin upregulation. Furthermore, through a local fibronectin knockdown model in mouse uteri, we found that the absence of fibronectin at the maternal-fetal interface impedes endovascular migration of trophoblasts and decidual vascularization, thereby interfering with early embryo implantation and the maintenance of pregnancy. These findings provide novel insights into placental development during early pregnancy establishment and contribute to the advancement of therapeutic approaches for managing pregnancy complications related to trophoblast dysfunction.

The histone deacetylase inhibitor MS-275 enhances the matrix mineralization of dental pulp stem cells by inducing fibronectin expression

Background/purposeThe acetylation of histone H3 proteins keeps local chromatin regions open and accessible, thereby facilitating transcriptional events. We recently reported integrative epigenomic and transcriptome analyses of differentiating dental pulp stem cells (DPSCs). A significant increase in the number of super-enhancers, which are local genomic locations marked by condensed open chromatin peaks that facilitate transcriptional events, in differentiating DPSCs were observed. However, it is still unclear whether histone deacetylase (HDACs) inhibitors (HDACis) have beneficial effects on the odontogenic differentiation of DPSCs and on the matrix mineralization-inducing ability of DPSCs. Materials and methodsDPSCs were cultured in an odontogenic induction medium for a prolonged period in the presence of HDACis, MS-275 and Trichostatin A (TSA). ATAC-seq and RNA-seq samples were collected from differentiating DPSCs to explore the epigenomic and transcriptomic alterations induced by HDACis and identify key target proteins that mediate HDACis-induced phenotypic changes. ResultsMS-275 and TSA did not change whole-genome open chromatin accessibility or increase odontogenic differentiation, as assessed by alkaline phosphate activity. However, the matrix mineralization-inducing ability assessed by calcified nodule formation was significantly increased by MS-275 but not by TSA. FN1, which encodes fibronectin, was identified as upregulated by MS-275. The knockdown of fibronectin evidently suppressed MS-275-induced calcified nodule formation. ConclusionMS-275 induced calcified nodule formation by the mechanistic upregulation of FN1, independent of epigenomic alterations. Hence, the application of MS-275 as direct capping materials has therapeutic potential for promoting reparative dentin formation by constructing a fibronectin-organizing physiological extracellular matrix environment that is adequate for matrix mineralization.

Hypoxia-Induced Biosynthesis of the Extracellular Matrix Molecules, Perlecan and Fibronectin, Promotes the Growth of Pleomorphic Adenoma Cells In Vitro Models.

Salivary pleomorphic adenoma is histopathologically characterized by its colorful stroma with myxoid, chondroid, and hyaline appearances, due to enhanced biosynthesis of extracellular matrix (ECM) molecules and poor vascularity. Thus, pleomorphic adenoma cells embedded in the stroma typically survive under hypoxic conditions. We determined the expression kinetics of ECM molecules, such as perlecan and fibronectin (FN), under hypoxia in SM-AP1 cells which are duct epithelial differentiated cells, and in SM-AP4 cells, which are myoepithelial differentiated cells, cloned from pleomorphic adenoma of the parotid gland. We investigated hypoxia-inducible factor-1α (HIF-1α)-inducing pathways through a variety of ECM molecules in association with their cellular proliferation and migration. We observed that hypoxic conditions with elevated HIF-1α protein levels induced increased expression of perlecan and FN in SM-AP cells than in controls. Moreover, perlecan and FN knockdown reduced the proliferation of SM-AP1 and SM-AP4 cells under hypoxia. Further, SM-AP1 cell migration was enhanced by both perlecan and FN knockdown, whereas SM-AP4 cell migration was increased by perlecan knockdown and inhibited by fibronectin knockdown. The results indicated that pleomorphic adenoma cells can survive under hypoxic conditions by promoting cell proliferation via enhanced synthesis of ECM molecules. Overall, ECM molecules may be a new anti-tumor target under hypoxic conditions.

Aspirin Inhibits Fibronectin Expression and Reverses Fibronectin-Mediated Cell Invasiveness by Activating Akt Signaling in Preeclampsia.

Preeclampsia is a severe gestational hypertensive disorder that may lead to maternal multiple organ dysfunction and adverse fetal outcomes. Aspirin provides a protective effect by reducing the risk of preeclampsia; however, its mechanism of action is unclear. Fibronectin (FN) is a key factor in cell motility and is associated with preeclampsia. Here, we demonstrated that cellular FN expression was elevated in the placenta of preeclamptic patients. The functional roles of plasma and cellular FN in trophoblasts were investigated by treating HTR-8/SVneo cells with exogenous recombinant human FN protein (rhFN) and siRNA, respectively. Trophoblast migration and invasion were inhibited by rhFN and facilitated by FN knockdown. Moreover, rhFN activated ERK and Akt signaling in trophoblasts, and FN-suppressed cell motility was rescued by ERK and/or Akt inhibitors. In this study, aspirin suppressed trophoblast cellular FN expression and reversed FN-mediated cell functions, including cell migration, invasion, and ERK/Akt signal changes. Taken together, the results of this study revealed the effects of FN on trophoblast motility and signaling; aspirin inhibits FN expression and reverses FN-mediated trophoblast biology. These results provide a drug mechanism for disease prevention and a target for preeclampsia intervention.

Uptake of Staphylococcus aureus by keratinocytes is reduced by interferon–fibronectin pathway and filaggrin expression

Staphylococcus aureus (S.aureus) is frequently detected in the skin of patients with atopic dermatitis (AD). AD skin-derived strains of S.aureus (AD strain) are selectively internalized into keratinocytes (HaCaT cells) compared to standard strains. However, the mechanism of AD strain internalization by keratinocytes and effect of the skin environment on internalization remain unclear. HaCaT cells were exposed to heat-killed AD or standard strains of fluorescently labeled S.aureus, with or without interferon (IFN)-γ, interleukin (IL)-4, and IL-13 cytokines, for 24 h. Filaggrin and fibronectin expression in HaCaT cells was knocked down using small interfering RNA. The amount of internalized S.aureus was evaluated using a cell imaging system. The effects of INF-γ, IL-4, and S.aureus exposure on mRNA expression in HaCaT cells were analyzed using single-cell RNA sequencing. AD strains adhered to HaCaT cells in approximately 15 min and were increasingly internalized for up to 3h (2361 ± 467 spots/100 cells, mean ± SD), whereas the standard strain was not (991 ± 71 spots/100 cells). In the presence of IFN-γ, both the number of internalized strains and fibronectin expression significantly decreased compared to in the control, whereas Th2 cytokines had no significant effects. The number of internalized AD strains was significantly higher in filaggrin knockdown and lower in fibronectin knockdown HaCaT cells compared to in the control. RNA sequencing revealed that IFN-γ decreased both fibronectin and filaggrin expression. Keratinocyte internalization of the AD strain may be predominantly mediated by the INF-γ-fibronectin pathway and partially regulated by filaggrin expression.

Discoidin Domain Receptor 2 Regulates AT1R Expression in Angiotensin II-Stimulated Cardiac Fibroblasts via Fibronectin-Dependent Integrin-β1 Signaling.

This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-β1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-β1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-β1.

Increased Fibronectin Impairs the Function of Excitatory/Inhibitory Synapses in Hirschsprung Disease.

Although approximately 50% of cases have a known genetic defect, the precise pathogenesis of Hirschsprung disease (HSCR) is still unclear. We recently reported that expression of fibronectin (FN), which is involved in the migration, colonization, and differentiation of enteric neural crest cells (ENCCs), is increased in aganglionic colonic segments obtained from patients. We hypothesized that abnormally high levels of FN might play a role in the etiology of HSCR. Here, to test this hypothesis, we investigated aganglionic, transitional, and ganglionic colon segments from 63 children with HSCR and distal colon from thirty healthy Wistar rats at embryonic day 20, in addition to in vitro studies with PC12 Adh neural crest cells. We measured the protein and mRNA expression levels of FN, together with a panel of excitatory (VGLUT1, GluA1, GluN1, PSD-95, and NL-1) and inhibitory (GAD67, GABA AR-α1, NL-2, and SLC32) synaptic markers. Expression of all these synaptic markers was significantly decreased in aganglionic colon, compared to ganglionic colon, whereas expression of FN was significantly increased. In a neural crest cell line, PC12 Adh, knockdown of FN with small-interfering RNA increased the expression of synaptic markers. Co-culture of colons from embryonic day 20 rats with RGD recombinant protein, which contains the RGD motif of FN, reduced the expression of excitatory and inhibitory synaptic markers. These results are consistent with the idea that the etiology of HSCR involves aberrant overexpression of FN, which may impair synaptic function and enteric nervous system development, leading to motor dysfunction of intestinal muscles.

Fibrin As a Target for Glioblastoma Detection and Treatment

Glioblastoma is a highly aggressive brain tumor characterized by diffuse growth and resistance to therapy. Angiogenesis in glioblastoma is poorly organized and therefore, tends to be associated with tumor cell necrosis, hemorrhage and thrombosis. This leads to the formation of a fibrin-rich extracellular matrix, which could provide important adhesive cues for glioblastoma growth and proliferation. To establish that blood clotting takes place in the extracellular matrix of malignant brain tumors, we assessed fibrin formation in tumor samples from patients with astrocytoma and glioblastoma using immunohistochemistry. Compared to normal brain tissue, which is essentially fibrin-free, this analysis revealed a marked upregulation of clot formation in the interstitial spaces of patients with high-grade tumors (i.e. astrocytoma 3 and GBM). The low-grade astrocytoma 2, however, expressed 3-3.5 fold less fibrin than was found in tissues from patients with astrocytoma 3 and GBM indicating that the degree of clot formation positively correlates with tumor grade. Paralleling these data, we found that primary GBM cells, that were freshly isolated from patients after tumor surgery, infiltrated and proliferated strongly after embedding in a three dimensional (3D) matrix of clotted plasma ex vivo. Primary tumor cells from patients with astrocytoma 2 and 3, on the other hand, infiltrated clot but were unable to proliferate in 3D. GBM proliferation in 3D depended on fibrin, which mediated upregulation of the stem cell marker nestin, whereas culturing glioblastoma cells in a 3D matrix of matrigel™ failed to promote nestin expression as well as glioblastoma proliferation. Therefore, these data suggest that the presence of clotted plasma in the tumor extracellular matrix represents a niche for glioblastoma stem cells and, as such, contributes to GBM progression. To determine the interaction of GBM cells with fibrin on a molecular basis, we transfected GBM cells with siRNA against integrin β3, which completely abolished invadopodia formation and, at the same time, caused a sustained growth inhibition. GBM cell proliferation in 3D fibrin also depended on the formation of a fibronectin matrix as knockdown of fibronectin led to complete growth arrest. These findings appear to be clinically relevant since freshly isolated tumor cells from patients with glioblastoma colonized 3D fibrin most efficiently when they express fibronectin in combination with integrin β3. This suggests that fibrin stimulates adhesive interactions between integrin β3 and fibronectin and that these interactions in turn support glioblastoma stemness. To assess fibrin formation in glioblastoma in vivo, mice with orthotopic U87MG xenografts were injected intravenously with the fluorescein-coupled dekapeptide CGLKIQKNEC, which is a derivative of the clot-binding peptide CLT1. Using a fluorescence endoscope in situ, we detected strong green fluorescence over the parietal lobe of the right cerebral hemisphere, where tumor growth had been established by MRI beforehand. Subsequently, we confirmed tumor binding of the peptide in isolated brain tissue by fluorescence microscopy ex vivo, which demonstrated specific green fluorescence in the tumor xenograft while adjacent normal brain tissue as well as tissues from distant organs only exhibited background fluorescence. Together, our data demonstrate a specific upregulation of fibrin in high-grade astrocytoma, which promotes infiltration and proliferation of glioblastoma stem cells via integrin β3 and fibronectin. Moreover, we present a strategy to identify fibrin in the tumor extracellular matrix as a possible means to identify astrocytoma progression in vivo. Disclosures Eichler: Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Pilch:CSL Behring: Other: Grants (investigator initiated), Speakers Bureau; ASPIRE Award/Pfizer: Other: Grants (investigator initiated); Bayer: Consultancy, Speakers Bureau; Roche: Consultancy.

Abstract 178: MYO10 aberrant methylation and overexpression in leader cells regulates lung cancer collective cell invasion and fibronectin patterning

Abstract Myosin-X (MYO10) is a noncanonical myosin that drives filopodia formation and extension, and is necessary for invasion and metastasis of breast cancer, prostate cancer and melanoma. It is not yet known by what mechanism MYO10 increases cancer cell invasion and metastasis. Two independently published MYO10 knockout mouse models showed severe developmental defects dependent upon collective migration of several cell types, suggesting that MYO10 may also regulate collective invasion of cancer cells. In order to study collective cancer cell invasion, our lab previously developed a technique to isolate and culture purified highly-invasive leader cells and highly-proliferative follower cells from collectively invading lung cancer cell lines. Using this method, we demonstrated distinct gene expression patterns, DNA methylation patterns, and phenotypic properties of leader versus follower cells. MYO10 is highly overexpressed in leader cells but is not expressed in follower cells, and MYO10 overexpression in leaders correlates with highly significant CpG island promoter DNA hypomethylation and gene body DNA hypermethylation. We hypothesize that MYO10 regulates cancer cell collective invasion by driving the formation of long filopodia necessary for leading-edge fibronectin patterning and subsequent leader cell invasion. MYO10 localizes at leader cell filopodia tips but not in follower cell filopodia within both 2D culture and 3D invading spheroids of four non-small cell lung cancer cell lines. MYO10 knockdown in purified leader cells and in these four lung cancer cell lines decreases the number and length of filopodia, 2D cell motility and 3D spheroid invasion; in contrast, MYO10 overexpression in follower cells increases filopodia length, cell motility and spheroid invasion. In addition, proteomic analysis shows that leader cells produce and secrete fibronectin (FN1), unlike follower cells. Immunofluorescence within invading spheroids shows the formation of long FN1 fibrils, i.e. elongated parallel bundles of FN1 that extend far past the leader cell body in multiple cell lines. FN1 fibrils preferentially localized with MYO10+ filopodia. Knockdown of MYO10 disrupts the formation of these fibronectin fibrils. We previously reported that knockdown of fibronectin completely abrogates lung cancer spheroid collective invasion. Therefore, our data suggest that MYO10 regulates cancer cell collective invasion by driving the formation of long filopodia that then regulate fibronectin architecture and subsequent invasion by leader cells. Note: This abstract was not presented at the meeting. Citation Format: Emily R. Summerbell, Jessica Konen, Jeanne Kowalski, Paula Vertino, Adam Marcus. MYO10 aberrant methylation and overexpression in leader cells regulates lung cancer collective cell invasion and fibronectin patterning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 178.

Fibronectin promotes nasopharyngeal cancer cell motility and proliferation

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)-associated carcinoma. Fibronectin is regarded as a prognosticator in NPC and its involvement in cell motility has been reported in EBV infection and viral latent membrane protein 1 (LMP1) overexpression NPC cell lines. However, its malignant potential in NPC cell lines without harbouring the EBV genome has not been investigated. We investigatd and compared among four NPC cell lines, and the results revealed a positive association between fibronectin levels and NPC cell motility as well as proliferation. Studies of antibody neutralization, exogenous addition, overexpression, and RNA interference confirmed a migration role of fibronectin in NPC cells involving integrin α5, Src, Rac1, and Cdc42, implying a mesenchymal-like cell movement. Furthermore, hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1) were identified as alternative activators of fibronectin expression and NPC cell migration. Besides cell migration, studies of RNA interference also showed a stimulatory effect of fibronectin in NPC cell proliferation. Mechanistic studies further revealed a subsequent reduction of HIF-1α, TGF-β1, cyclin D1, β-catenin, vimentin, and Slug together with decreased Src and Akt phosphorylation after fibronectin knockdown. Parallel studies in a xenograft tumor mice model further showed that tumor growth correlated well with elevation of circulating fibronectin and activation of the identified intracellular signaling molecules. The results of our study highlight a role of fibronectin in NPC cell motility and proliferation in concerted action with HIF-1α and TGF-β1 possibly through linking molecules Src and Akt. Fibronectin overexpression and autoantibody are known to have potential prognostic value in patients with NPC. Our findings shed light on the biochemical and molecular mechanisms underlying the pathogenic role of fibronectin in this disease.

High expression of fibronectin is associated with poor prognosis, cell proliferation and malignancy via the NF-κB/p53-apoptosis signaling pathway in colorectal cancer.

Fibronectin is a glycoprotein of the extracellular matrix, and regulates the processes of self-renewal and cell cycle progression. This study aimed to investigate fibronectin expression in colorectal cancer (CRC) and elucidate the effects of fibronectin on CRC by using a knockdown approach. Immunohistochemistry was used to evaluate the expression of fibronectin in 107 CRC patient tissues and gene expression was detected by real-time quantitative PCR (qPCR) and western blot analysis. Based on the above findings, the association among fibronectin expression, clinicopathological features and prognosis was analyzed. Next, fibronectin expression was silenced by small-interfering RNAs (siRNAs) and the effects of fibronectin siRNA transfection on CRC cells and tumor growth in nude mice were assessed. Expression of genes in the NF-κB/p53-apoptosis signaling pathway were analyzed after fibronectin siRNA transfection both in vitro and in vivo. Based on the results, high expression of fibronectin was observed both in the CRC tissues and CRC cell lines. The expression level was positively correlated with TNM stage (P=0.0025) and distant metastasis (P=0.0013). By Kaplan-Meier analysis, the patients with low fibronectin expression had a longer survival time comparing to those with relatively high expression. Knockdown of fibronectin suppressed SW480 cell proliferation, migration and invasion. In addition, knockdown of fibronectin led to S phase cell cycle arrest. The following study showed that the NF-κB/p53-apoptosis signaling pathway in CRC was affected by fibronectin knockdown. Tumor formation was also depressed by fibronectin siRNA transfection of CRC cells. These results showed the significant role of fibronectin in CRC tissues and cell lines. Therefore, fibronectin may be regarded as a potential target for CRC treatment.

The complexity of microenvironment-mediated drug resistance.

The complexity of microenvironment-mediated drug resistance.

The P2Y₂ receptor promotes Wnt3a- and EGF-induced epithelial tubular formation by IEC6 cells by binding to integrins.

Epithelial tubular structures are essential units in various organs. Here, we used rat intestinal epithelial IEC6 cells to investigate tubulogenesis and we found that tubular formation was induced by a combination of Wnt3a and EGF signaling during three-dimensional culture. Wnt3a and EGF induced the expression of the P2Y2 receptor (P2Y2R, also known as P2RY2), and knockdown of P2Y2R suppressed tubular formation. A P2Y2R mutant that lacks nucleotide responsiveness rescued the phenotypes resulting from P2Y2R knockdown, suggesting that nucleotide-dependent responses are not required for P2Y2R functions in tubular formation. The Arg-Gly-Asp (RGD) sequence of P2Y2R has been shown to interact with integrins, and a P2Y2R mutant lacking integrin-binding activity was unable to induce tubular formation. P2Y2R expression inhibited the interaction between integrins and fibronectin, and induced cell morphological changes and proliferation. Inhibition of integrin and fibronectin binding by treatment with the cyclic RGD peptide and fibronectin knockdown induced tubular formation in the presence of EGF alone, but a fibronectin coat suppressed Wnt3a- and EGF-induced tubular formation. These results suggest that Wnt3a- and EGF-induced P2Y2R expression causes tubular formation by preventing the binding of integrins and fibronectin rather than by mediating nucleotide responses.

A fibrin antibody binding to fibronectin induces potent inhibition of angiogenesis

Antiserum from rabbits immunised with pure human fibrinogen was affinity purified on immobilised fibrin fragment E (FFE). This FFE antibody (Ab) induced significant growth inhibition of a human cancer xenograft in mice and suppression of tumour angiogenesis, leaving no formed vessels and only CD31-staining endothelial fragments in place. Tubule formation of HUVEC on MatrigelTM was also significantly inhibited by FFE Ab. Since MatrigelTM is fibrin-free, this effect implicated a different FFE Ab binding site than FFE. Flow cytometry of HUVEC showed that FFE Ab bound to HUVEC, but with a broad range of 55-98 %. Immunofluorescent staining of HUVEC explained this range, since FFE Ab was seen not to bind to human umbilical vein endothelial cells (HUVEC) directly but instead to a matrix protein variably adherent to HUVEC. This protein was identified as fibronectin (FN) by appearance, staining with FN Ab, and by a FN knockdown study. Neither HUVEC nor matrix reacted with fibrin D-dimer (DD) Ab. Immunofluorescent stains of HUVEC matrix with FFE and FN Ab's showed that these Ab's bound to the same epitopes on FN, as also seen on Western blots of purified FN. These findings indicate the presence of an antigenic determinant in fibrinogen/FFE that is homologous with an epitope(s) in FN recognised by FFE Ab, and critical for angiogenesis in this xenograft. The FN epitope(s) remains to be identified, but the present findings can be used for the selection of the appropriate clones from mice immunised with fibrinogen which can facilitate this identification, and which may also be of clinical use.

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis.

Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation.

Abstract PR11: Ovarian cancer cells activate the mesothelium in the peritoneal microenvironment promoting fibronectin production and the early steps of metastasis

Abstract The main site of ovarian cancer (OvCa) metastasis is the omentum, which is covered by a single layer of mesothelial cells overlaying an extracellular matrix interspersed with fibroblasts (mesothelium). A 3D model of the human peritoneal surface (in vitro), fragments of human omentum (ex vivo), and a mouse xenograft model (in vivo) of OvCa were used to investigate the early effects of cancer cells on the host stromal microenvironment. Our results reveal that fibronectin is overexpressed in the stroma of more than 93% of OvCa omental metastases. OvCa cells induce fibronectin matrix assembly in omental stromal cells, and stimulate fibronectin expression in mesothelial cells. The fibronectin produced by human mesothelial stromal cells promotes OvCa cell adhesion, invasion and proliferation. Inhibition of fibronectin production in mesothelial cells or omental surface cells using a fibronectin specific siRNA decreases OvCa cell adhesion, invasion and proliferation. Genetic knock-down of fibronectin in vitro and in vivo, using fibronectinfl/fl mice, also impaired OvCa cell adhesion/invasion and metastasis. Re-analysis of an Australian OvCa study gene array data set suggested that OvCa, which highly express fibronectin, show co-activation of TGFβ1/Rac1 dependent signaling pathways. Indeed, OvCa cells increase phosphorylation of Smad2/3 in mesothelial cells upon co-culture. Inhibition of TGFβ1 in OvCa cells or inhibition of Smad 3, as well as TGFβRI or TGFβRII, in mesothelial cells resulted in decreased fibronectin production. Moreover, Rac 1 activity was increased upon the co-culture of OvCa and mesothelial cells. Subsequent inhibition of Rac 1 in mesothelial cells led to a decrease in fibronectin secretion. Our data suggest that OvCa cells regulate fibronectin expression in mesothelial cells through a TGFβ1/Rac1 dependent pathway. In vitro and in vivo therapy with a fibronectin inhibitor, ATN161, a peptide derived from the synergy region of fibronectin, reduced OvCa invasion, proliferation and metastasis. In summary, we show that abdominally metastasizing cancer cells induce fibronectin production in the peritoneal and omental microenvironment and that inhibition of this fibronectin-response prevents the first steps of peritoneal metastasis. By understanding the mechanism of OvCa metastasis we will be better able to target cells in the tumor microenvironment (i.e. mesothelial cells, fibroblasts and macrophages) during early ovarian cancer metastasis. This abstract is also presented as Poster A60. Citation Format: Hilary A. Kenny, Chun-Yi Chiang, Andras Ladanyi, Joshy George, David D. Bowtell, Katja Gwin, Andrew Mazar, Ernst Lengyel. Ovarian cancer cells activate the mesothelium in the peritoneal microenvironment promoting fibronectin production and the early steps of metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR11.

Fibronectin Regulates Wnt7a Signaling and Satellite Cell Expansion

The influence of the extracellular matrix (ECM) within the stem cell niche remains poorly understood. We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a coreceptor complex in satellite cells and that binding of the ECM glycoprotein Fibronectin (FN) to Sdc4 stimulates the ability of Wnt7a to induce the symmetric expansion of satellite stem cells. Newly activated satellite cells dynamically remodel their niche via transient high-level expression of FN. Knockdown of FN in prospectively isolated satellite cells severely impaired their ability to repopulate the satellite cell niche. Conversely, invivo overexpression of FN with Wnt7a dramatically stimulated the expansion of satellite stem cells in regenerating muscle. Therefore, activating satellite cells remodel their niche through autologous expression of FN that provides feedback to stimulate Wnt7a signaling through the Fzd7/Sdc4 coreceptor complex. Thus, FN and Wnt7a together regulate the homeostatic levels of satellite stem cells and satellite myogenic cells during regenerative myogenesis.

A Crucial Sequence for Transglutaminase Type 2 Extracellular Trafficking in Renal Tubular Epithelial Cells Lies in Its N-terminal β-Sandwich Domain

Transglutaminase type 2 (TG2) catalyzes the formation of an ε-(γ-glutamyl)-lysine isopeptide bond between adjacent peptides or proteins including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlie tissue scarring and fibrosis. The extracellular trafficking of TG2 is crucial to its role in ECM homeostasis; however, the mechanism by which TG2 escapes the cell is unknown as it has no signal leader peptide and therefore cannot be transported classically. Understanding TG2 transport may highlight novel mechanisms to interfere with the extracellular function of TG2 as isoform-specific TG2 inhibitors remain elusive. Mammalian expression vectors were constructed containing domain deletions of TG2. These were transfected into three kidney tubular epithelial cell lines, and TG2 export was assessed to identify critical domains. Point mutation was then used to highlight specific sequences within the domain required for TG2 export. The removal of β-sandwich domain prevented all TG2 export. Mutations of Asp(94) and Asp(97) within the N-terminal β-sandwich domain were identified as crucial for TG2 externalization. These form part of a previously identified fibronectin binding domain ((88)WTATVVDQQDCTLSLQLTT(106)). However, siRNA knockdown of fibronectin failed to affect TG2 export. The sequence (88)WTATVVDQQDCTLSLQLTT(106) within the β-sandwich domain of TG2 is critical to its export in tubular epithelial cell lines. The extracellular trafficking of TG2 is independent of fibronectin.

Polypeptide N-acetylgalactosaminyltransferase 6 Disrupts Mammary Acinar Morphogenesis through O-glycosylation of Fibronectin

A high expression of short and immature O-glycans is one of the prominent features of breast cancer cells, which would be attributed to the upregulated expression of glycosyltransferases. Therefore, a detailed elucidation of glycosyltransferases and their substrate(s) may improve our understandings for their roles in mammary carcinogenesis. Here we report that overexpression of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), a glycosyltransferase involved in the initial step of O-glycosylation, has transformational potentials through disruptive acinar morphogenesis and cellular changes similar to epithelial-to-mesenchymal transition in normal mammary epithelial cell, MCF10A. As one of the critical O-glycan substrates, we identified fibronectin that was O-glycosylated in vivo and thereby stabilized by GALNT6. Because knockdown of fibronectin abrogated the disruptive proliferation caused by introduction of GALNT6 into epithelial cells, our findings suggest that GALNT6-fibronectin pathway should be a critical component for breast cancer development and progression.

Short Hairpin RNA-Mediated Fibronectin Knockdown Delays Tumor Growth in a Mouse Glioma Model

Glioblastoma multiforme is the most common and lethal primary brain tumor. Glioma progression depends on the rapid proliferation of tumor cells accompanied by an acute immunosuppressive environment, facilitated mainly by tumor infiltration of regulatory T cells (Tregs). In this study, we characterize the role of fibronectin, a high-molecular weight extracellular matrix glycoprotein secreted by tumor cells, in controlling glioma progression and in mediating immunosuppression. Fibronectin binds to membrane-spanning integrin receptors and plays an important role in cell signaling, in defining cellular shape, in mobility, and in regulating the cell cycle. We found that inhibition of fibronectin expression in glioma cells, using short hairpin RNA-mediated silencing of gene expression, delayed cell proliferation in vitro. This delayed growth is explained, in part, by the observed reduced expression of integrin β1 fibronectin receptor, which was restored by the inhibition of proteosomal activity. In our analysis of the downstream signaling targets of integrin β1, we demonstrated reduced phosphorylation of Src kinase and STAT-3. We also observed reduced survivin expression that induced a three-fold increased accumulation of fibronectin-knockdown cells in the G2/M phase. In an experimental animal model, the fibronectin knockdown tumors had a mean survival advantage of 23 days over wild-type tumors. Moreover, brain samples of animals bearing fibronectin-knockdown tumors showed delayed Treg recruitment. Collectively, we propose that fibronectin is a key mediator of glioma progression because its inhibition delays both tumor progression and immunosuppression.