An extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks.

Extracellular domains of cell surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein "interactome" data sets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on a set of 202 proteins composed of the Drosophila melanogaster immunoglobulin superfamily (IgSF), fibronectin type III (FnIII), and leucine-rich repeat (LRR) families, which are known to be important in neuronal and developmental functions. Out of 20,503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We "deorphanized" the 20 member subfamily of defective-in-proboscis-response IgSF proteins, showing that they selectively interact with an 11 member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common "orphan" LRR protein. We also observed interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs.

Integrin mechanosensing relies on a pivot-clip mechanism to reinforce cell adhesion.

Cells intricately sense mechanical forces from their surroundings, driving biophysical and biochemical activities. This mechanosensing phenomenon occurs at the cell-matrix interface, where mechanical forces resulting from cellular motion, such as migration or matrix stretching, are exchanged through surface receptors, primarily integrins, and their corresponding matrix ligands. A pivotal player in this interaction is the α5ß1 integrin and fibronectin (FN) bond, known for its role in establishing cell adhesion sites for migration. However, upregulation of the α5ß1-FN bond is associated with uncontrolled cell metastasis. This bond operates through catch bond dynamics, wherein the bond lifetime paradoxically increases with greater force. The mechanism sustaining the characteristic catch bond dynamics of α5ß1-FN remains unclear. Leveraging molecular dynamics simulations, our approach unveils a pivot-clip mechanism. Two key binding sites on FN, namely the synergy site and the RGD (Arg-Gly-Asp) motif, act as active points for structural changes in α5ß1 integrin. Conformational adaptations at these sites are induced by a series of hydrogen bond formations and breaks at the synergy site. We disrupt these adaptations through a double mutation on FN, known to reduce cell adhesion. A whole-cell finite-element model is employed to elucidate how the synergy site may promote dynamic α5ß1-FN binding, resisting cell contraction. In summary, our study integrates molecular- and cellular-level modeling to propose that FN's synergy site reinforces cell adhesion through enhanced binding dynamics and a mechanosensitive pivot-clip mechanism. This work sheds light on the interplay between mechanical forces and cell-matrix interactions, contributing to our understanding of cellular behaviors in physiological and pathological contexts.

Periostin Is a Disulfide-Bonded Homodimer and Forms a Complex with Fibronectin in the Human Skin.

The protein periostin is a matricellular protein that is expressed in connective tissue. It is composed of five globular domains arranged in an elongated structure with an extensive disordered C-terminal tail. Periostin contains 11 cysteine residues, of which one is unpaired and the rest form five intramolecular disulfide bonds. Periostin plays an important role during wound healing and is also involved in driving the inflammatory state in atopic diseases. This study provides a comprehensive biochemical characterization of periostin in human skin and in dermal and pulmonary fibroblasts in vitro. Through the application of Western blotting, co-immunoprecipitation, and LC-MS/MS, we show for the first time that periostin is a disulfide-bonded homodimer and engages in a novel disulfide-bonded complex with fibronectin both in vivo and in vitro. This inherent characteristic of periostin holds the potential to redefine our approach to exploring and understanding its functional role in future research endeavors.

Nucleation of fibronectin fibril assembly requires binding between heparin and the 13th type III module of fibronectin.

Fibronectin (FN), a critical component of the extracellular matrix, is assembled into fibrils through a cell-mediated process. Heparan sulfate (HS) binds to the III13 module of FN, and fibroblasts lacking this glycosaminoglycan exhibit reduced FN fibril assembly. To determine if HS depends on III13 to control FN assembly, we deleted both III13 alleles in NIH 3T3 cells using the CRISPR-Cas9 system. ΔIII13 cells assembled fewer FN matrix fibrils and less DOC-insoluble FN matrix than wildtype cells. Little if any mutant FN matrix was assembled when purified ΔIII13 FN was provided to Chinese hamster ovary (CHO) cells, showing that lack of III13 caused the deficiency in assembly by ΔIII13 cells. Addition of heparin promoted the assembly of wildtype FN by CHO cells, but it had no effect on the assembly of ΔIII13 FN. Furthermore, heparin binding stabilized the folded conformation of III13 and prevented it from self-associating with increasing temperature suggesting that stabilization by HS/heparin binding might regulate interactions between III13 and other FN modules. This effect would be particularly important at matrix assembly sites where our data show that ΔIII13 cells require both exogenous wildtype FN and heparin in the culture medium to maximize assembly site formation. Our results show that heparin-promoted growth of fibril nucleation sites is dependent on III13. We conclude that HS/heparin binds to III13 to promote and control the nucleation and development of FN fibrils.

Cell-Free Nonequilibrium Assembly for Hierarchical Protein/Peptide Nanopillars.

Cells contain intricate protein nanostructures, but replicating them outside of cells presents challenges. One such example is the vertical fibronectin pillars observed in embryos. Here, we demonstrate the creation of cell-free vertical fibronectin pillar mimics using nonequilibrium self-assembly. Our approach utilizes enzyme-responsive phosphopeptides that assemble into nanotubes. Enzyme action triggers shape changes in peptide assemblies, driving the vertical growth of protein nanopillars into bundles. These bundles, with peptide nanotubes serving as a template to remodel fibronectin, can then recruit collagen, which forms aggregates or bundles depending on their types. Nanopillar formation relies on enzyme-catalyzed nonequilibrium self-assembly and is governed by the concentrations of enzyme, protein, peptide, the structure of the peptide, and peptide assembly morphologies. Cryo-EM reveals unexpected nanotube thinning and packing after dephosphorylation, indicating a complex sculpting process during assembly. Our study demonstrates a cell-free method for constructing intricate, multiprotein nanostructures with directionality and composition.

Engineering a 3D Biomimetic Peptides Functionalized-Polyethylene Glycol Hydrogel Model Cocultured with Endothelial Cells and Astrocytes: Enhancing In Vitro Blood-Brain Barrier Biomimicry.

The blood-brain barrier (BBB) poses a significant challenge for drug delivery and is linked to various neurovascular disorders. In vitro BBB models provide a tool to investigate drug permeation across the BBB and the barrier's response to external injury events. Yet, existing models lack fidelity in replicating the BBB's complexity, hindering a comprehensive understanding of its functions. This study introduces a three-dimensional (3D) model using polyethylene glycol (PEG) hydrogels modified with biomimetic peptides that represent recognition sequences of key proteins in the brain. Hydrogels were functionalized with recognition sequences for laminin (IKVAV) and fibronectin peptides (RGD) and chemically cross-linked with matrix metalloprotease-sensitive peptides (MMPs) to mimic the extracellular matrix of the BBB. Astrocytes and endothelial cells were seeded within and on the surface of the hydrogels, respectively. The barrier integrity was assessed through different tests including transendothelial electrical resistance (TEER), the permeability of sodium fluorescence (Na-F), the permeability of Evan's blue bound to albumin (EBA), and the expression of zonula occluden-1 (ZO-1) in seeded endothelial cells. Hydrogels with a combination of RGD and IKVAV peptides displayed superior performance, exhibiting significantly higher TEER values (55.33 ± 1.47 Ω·cm2) at day 5 compared to other 2D controls including HAECs-monoculture and HAECs-cocultured with NHAs seeded on well inserts and 3D controls including RGD hydrogel and RGD-IKVAV monoculture with HAECs and RGD hydrogel cocultured with HAECs and NHAs. The designed 3D system resulted in the lowest Evan's blue permeability at 120 min (0.215 ± 0.055 µg/mL) compared to controls. ZO-1 expression was significantly higher and formed a relatively larger network in the functionalized hydrogel cocultured with astrocytes and endothelial cells compared to the controls. Thus, the designed 3D model effectively recapitulates the main BBB structure and function in vitro and is expected to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

Assembly of fibronectin extracellular matrix.

In the process of matrix assembly, multivalent extracellular matrix (ECM) proteins are induced to self-associate and to interact with other ECM proteins to form fibrillar networks. Matrix assembly is usually initiated by ECM glycoproteins binding to cell surface receptors, such as fibronectin (FN) dimers binding to α5ß1 integrin. Receptor binding stimulates FN self-association mediated by the N-terminal assembly domain and organizes the actin cytoskeleton to promote cell contractility. FN conformational changes expose additional binding sites that participate in fibril formation and in conversion of fibrils into a stabilized, insoluble form. Once assembled, the FN matrix impacts tissue organization by contributing to the assembly of other ECM proteins. Here, we describe the major steps, molecular interactions, and cellular mechanisms involved in assembling FN dimers into fibrillar matrix while highlighting important issues and major questions that require further investigation.

Modification of 316L Stainless Steel, Nickel Titanium, and Cobalt Chromium Surfaces by Irreversible Immobilization of Fibronectin: Towards Improving the Coronary Stent Biocompatibility.

An extracellular matrix protein, fibronectin (Fn), was covalently immobilized on 316L stainless steel, L605 cobalt chromium (CoCr), and nickel titanium (NiTi) surfaces through an 11-mercaptoundecanoic acid (MUA) self-assembled monolayer (SAM) pre-formed on these surfaces. Polarization modulation infrared reflection adsorption spectroscopy (PM-IRRAS) confirmed the presence of Fn on the surfaces. The Fn monolayer attached to the SAM was found to be stable under fluid shear stress. Deconvolution of the Fn amide I band indicated that the secondary structure of Fn changes significantly upon immobilization to the SAM-functionalized metal substrate. Scanning electron microscopy and energy dispersive X-ray analysis revealed that the spacing between Fn molecules on a modified commercial stent surface is approximately 66 nm, which has been reported to be the most appropriate spacing for cell/surface interactions.

Chirality and Curvature on Protein Adsorption and Osteogenesis.

Here we designed enantiomeric lipid-mimetic glutamic acid derivatives (L/D-UG) and investigated their self-assembled chiral nanostructures' interaction with the protein adsorption as well as the osteogenesis. It was found that L or D-UG molecules can self-assemble into vesicle bilayers and two-dimensional (2D) nanocrystals via a kinetic and thermodynamic control, respectively. These chiral vesicles and 2D crystals showed differentiated adsorption of proteins, determined by their curvature and chirality. Specifically, fibronectin constituted by L-amino acids adsorbed preferentially on L-UG 2D crystal in a semi-random pattern and L-2D nanocrystal show as the most effective structures to promote bone regeneration. The controlled vesicle and 2D crystal assemblies with different chirality and curvature helps to clarify their determine roles in protein adsorption and osteogenesis.

Dynamics of integrin α5ß1, fibronectin, and their complex reveal sites of interaction and conformational change.

Integrin α5ß1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by α5ß1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5ß1-binding affinities and stabilities. We also interrogated binding of the α5ß1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two ß-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit ß-propeller domain for the Fn9 synergy site and in the ß1-subunit ßI domain for Fn10 based on decreases in α5ß1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5ß1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5ß1 binding to Fn and dynamics associated with this interaction.

Myeloid immune checkpoint ILT3/LILRB4/gp49B can co-tether fibronectin with integrin on macrophages.

LILRB4 (B4, also known as ILT3/CD85k) is an immune checkpoint of myeloid lineage cells, albeit its mode of function remains obscure. Our recent identification of a common ligand for both human B4 and its murine ortholog gp49B as the fibronectin (FN) N-terminal 30 kDa domain poses the question of how B4/gp49B regulate cellular activity upon recognition of FN in the plasma and/or the extracellular matrix. Since FN in the extracellular matrix is tethered by FN-binding integrins, we hypothesized that B4/gp49B would tether FN in cooperation with integrins on the cell surface, thus they should be in close vicinity to integrins spatially. This scenario suggests a mode of function of B4/gp49B by which the FN-induced signal is regulated. The FN pull-down complex was found to contain gp49B and integrin ß 1 in bone marrow-derived macrophages. The confocal fluorescent signals of the three molecules on the intrinsically FN-tethering macrophages were correlated to each other. When FN-poor macrophages adhered to culture plates, the gp49-integrin ß 1 signal correlation increased at the focal adhesion, supporting the notion that gp49B and integrin ß 1 become spatially closer to each other there. Adherence of RAW264.7 and THP-1 cells to immobilized FN induced phosphorylation of spleen tyrosine kinase, whose level was augmented under B4/gp49B deficiency. Thus, we concluded that B4/gp49B can co-tether FN in cooperation with integrin in the cis configuration on the same cell, forming a B4/gp49B-FN-integrin triplet as a regulatory unit of a focal adhesion-dependent pro-inflammatory signal in macrophages.

CAR-T Therapy Targets Extra Domain B of Fibronectin Positive Solid Tumor Cells.

BACKGROUND: CAR-T cell immunotherapy has achieved remarkable success in malignant B-cell malignancies, but progress in solid tumors is slow, and one of the key reasons is the lack of ideal targets. Cancer-specific extra domain B of fibronectin (EDB-FN) is widely upregulated in solid tumors and expressed at low levels in normal tissues. Many imaging and targeted cancer therapies based on EDB-FN targets have been developed and tested in clinical trials, making EDB-FN an ideal target for immunotherapy. METHODS: We constructed two EDB-FN-targeted CAR-Ts based on the peptide APT0 and the single-chain antibody CGS2 in a lentiviral infection manner for the first time. Luciferase cytotoxicity assay to assess CAR-T killing of tumor cells. An enzyme-linked immunosorbent assay was used to detect the release of the cytokine IFN-γ. Fluorescence imaging to evaluate the dynamics of CAR-T cell and tumor cell coculture. Knockdown assays were used to validate the target specificity of CAR-T cells. RESULTS: In this research, two CAR-Ts targeting EDB-FN, APT0 CAR-T, and CGS2 CAR-T, were constructed. In vitro, both CAR-T cells produced broad-spectrum killing of multiple EDB-FN-positive solid tumor cell lines and were accompanied by cytokine IFN-γ release. Regarding safety, the two CAR-T cells did not affect T cells' normal growth and proliferation and were not toxic to HEK-293T human embryonic kidney epithelial cells. CONCLUSION: APT0 CAR-T and CGS2 CAR-T cells are two new CAR-Ts targeting EDB-FN. Both CAR-T cells can successfully identify and specifically kill various EDB-FN-positive solid tumor cells with potential clinical applications.

Molecular basis of conformational changes and mechanics of integrins.

Integrin, as a mechanotransducer, establishes the mechanical reciprocity between the extracellular matrix (ECM) and cells at integrin-mediated adhesion sites. This study used steered molecular dynamics (SMD) simulations to investigate the mechanical responses of integrin αvß3 with and without 10th type III fibronectin (FnIII10) binding for tensile, bending and torsional loading conditions. The ligand-binding integrin confirmed the integrin activation during equilibration and altered the integrin dynamics by changing the interface interaction between ß-tail, hybrid and epidermal growth factor domains during initial tensile loading. The tensile deformation in integrin molecules indicated that fibronectin ligand binding modulates its mechanical responses in the folded and unfolded conformation states. The bending deformation responses of extended integrin models reveal the change in behaviour of integrin molecules in the presence of Mn2+ ion and ligand based on the application of force in the folding and unfolding directions of integrin. Furthermore, these SMD simulation results were used to predict the mechanical properties of integrin underlying the mechanism of integrin-based adhesion. The evaluation of integrin mechanics provides new insights into understanding the mechanotransmission (force transmission) between cells and ECM and contributes to developing an accurate model for integrin-mediated adhesion. This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.

VEGF and VEGFR2 bind to similar pH-sensitive sites on fibronectin, exposed by heparin-mediated conformational changes.

Physical interactions between vascular endothelial growth factor (VEGF), a central player in blood endothelial cell biology, and fibronectin, a major fibrillar protein of the extracellular matrix, are important determinants of angiogenic activity in health and disease. Conditions signaling the need for new blood vessel growth, such as hypoxia and low extracellular pH, increase VEGF-fibronectin interactions. These interactions can be further fine-tuned through changes in the availability of the VEGF-binding sites on fibronectin, regulated by conformational changes induced by heparin and heparan sulfate chains within the extracellular matrix. These interactions may alter VEGF bioavailability, generate gradients, or alter the way VEGF is recognized by and activates its cell-surface receptors. Here, using equilibrium and kinetic studies, we discovered that fibronectin can also interact with the extracellular domain of the VEGF receptor 2 (VEGFR2). The VEGFR2-binding sites on fibronectin show great similarity to the VEGF-binding sites, as they were also exposed upon heparin-induced conformational changes in fibronectin, and the interaction was enhanced at acidic pH. Kinetic parameters and affinities for VEGF and VEGFR2 binding to fibronectin were determined by surface plasmon resonance measurements, revealing two populations of fibronectin-binding sites for each molecule. Our data also suggest that a VEGF/VEGFR2/fibronectin triple complex may be formed by VEGF or VEGFR2 first binding to fibronectin and subsequently recruiting the third binding partner. The formation of such a complex may lead to the activation of distinct angiogenic signaling pathways, offering new possibilities for clinical applications that target angiogenesis.

Cross-linking and modification of fibronectin by peroxynitrous acid: Mapping and quantification of damage provides a new model for domain interactions.

Fibronectin (FN) is an abundant glycoprotein found in plasma and the extracellular matrix (ECM). It is present at high concentrations at sites of tissue damage, where it is exposed to oxidants generated by activated leukocytes, including peroxynitrous acid (ONOOH) formed from nitric oxide (from inducible nitric oxide synthase) and superoxide radicals (from NADPH oxidases and other sources). ONOOH reacts rapidly with the abundant tyrosine and tryptophan residues in ECM proteins, resulting in the formation of 3-nitrotyrosine, di-tyrosine, and 6-nitrotryptophan. We have shown previously that human plasma FN is readily modified by ONOOH, but the extent and location of modifications, and the role of FN structure (compact versus extended) in determining these factors is poorly understood. Here, we provide a detailed LC-MS analysis of ONOOH-induced FN modifications, including the extent of their formation and the sites of intramolecular and intermolecular cross-links, including Tyr-Tyr, Trp-Trp, and Tyr-Trp linkages. The localization of these cross-links to specific domains provides novel data on the interactions between different modules in the compact conformation of plasma FN and allows us to propose a model of its unknown quaternary structure. Interestingly, the pattern of modifications is significantly different to that generated by another inflammatory oxidant, HOCl, in both extent and sites. The characterization and quantification of these modifications offers the possibility of the use of these materials as specific biomarkers of ECM modification and turnover in the many pathologies associated with inflammation-associated fibrosis.

A computational study on strontium ion modified hydroxyapatite-fibronectin interactions.

Protein adsorption is the first key step in cell-material interactions. The initial phase of such an adsorption process can only be probed using modelling approaches like molecular dynamics (MD) simulations. Despite a large number of studies on the adsorption behaviour of proteins on different biomaterials including calcium phosphates (CaP), little attention has been paid towards the quantitative assessment of the effects of various physicochemical influencers like surface modification, pH, and ionic strength. In the case of doped CaPs, surface modification through isomorphic substitution of foreign ions inside the apatite structure is of particular interest in the context of protein-HA interactions, as it is widely used to tailor the biological response of HA. Given this background, we present here the molecular-level understanding of the fibronectin (FN) adsorption mechanism and kinetics on a Sr2+-doped hydroxyapatite, HA, (001) surface at 300 K by means of all-atom molecular dynamics simulations. Electrostatic interactions involved in the adsorption of FN on HA were found to be significantly modified due to Sr2+ doping into the apatite lattice. In harmony with the published experimental observations, the Sr-doped surfaces were found to better support FN adhesion compared to pure HA, with 10 mol% Sr-doped HA exhibiting the best FN adsorption. The observed altered adsorption behaviour of FN on Sr-doped HA was correlated with the Hofmeister effect. Moreover, the non-monotonous trend of the FN-material interaction energy can be attributed to the spatial rearrangement of the functional groups (PO43-, OH-) in the apatite crystal. Sr2+ ions also influence the stability of the secondary structure of FN, as observed from the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analysis. The presence of Sr2+ enhances the flexibility of specific residues (residue nos. 20-44, 74-88) of the FN module. Rupture forces to disentangle FN from the biomaterial surface, obtained from steered molecular dynamics (SMD) simulations, were found to corroborate well with the results of equilibrium MD simulations. One particular observation is that the availability of an RGD motif (Arginine-Glycine-aspartate sequence, which interacts with cell surface receptor integrin to form a focal adhesion complex) for the interaction with cell surface receptor integrin is not significantly influenced by Sr2+ substitution.

Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition.

During the cell cycle, DNA duplication in S phase must occur before a cell divides in mitosis. In the intervening G2 phase, mitotic inducers accumulate, which eventually leads to a switch-like rise in mitotic kinase activity that triggers mitotic entry. However, when and how activation of the signaling network that promotes the transition to mitosis occurs remains unclear. We have developed a system to reduce cell-cell variation and increase accuracy of fluorescence quantification in single cells. This allows us to use immunofluorescence of endogenous marker proteins to assess kinetics from fixed cells. We find that mitotic phosphorylations initially occur at the completion of S phase, showing that activation of the mitotic entry network does not depend on protein accumulation through G2. Our data show insights into how mitotic entry is linked to the completion of S phase and forms a quantitative resource for mathematical models of the human cell cycle.

Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands.

Staphylococcus aureus adhesion to the host's skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.

Anoikis resistance conferred by tenascin-C-derived peptide TNIIIA2 and its disruption by integrin inactivation.

Glioblastoma multiforme (GBM), the most common brain tumor in adults, has an extremely poor prognosis, which is attributed to the aggressive properties of GBM cells, such as dysregulated proliferation and disseminative migration. We recently found that peptide TNIIIA2, derived from tenascin-C (TNC), which is highly expressed in GBM, contributes to the acquisition of these aggressive properties through ß1-integrin activation. In general, cancer cells often acquire an additional malignant property that confers resistance to apoptosis due to loss of adhesion to the extracellular matrix, termed anoikis resistance. Our present results show that regulation of ß1-integrin activation also plays a key role in both the development and loss of anoikis resistance in GBM cells. Despite being derived from a GBM with an extremely poor prognosis, the human GBM cell line T98G was susceptible to anoikis but became anoikis resistant via treatment with peptide TNIIIA2, which is able to activate ß1-integrin. The TNIIIA2-conferred anoikis resistance of T98G cells was disrupted by further addition of peptide FNIII14, which has the ability to inactivate ß1-integrin. Moreover, anchorage-independent survival of GBM cells in suspension culture was abrogated by peptide FNIII14, but not by RGD and CS-1 peptides, which are antagonistic for integrins α5ß1, αvß3, and α4ß1. These results suggest that GBM cells develop anoikis resistance through activation of ß1-integrin by TNC-derived peptide TNIIIA2, which is abundantly released into the tumor microenvironment of GBM. Inactivation of ß1-integrin may provide a promising strategy to overcome the apoptosis resistance of cancer cells, including GBM.

Fibronectin and colorectal cancer: signaling pathways and clinical implications.

Colorectal cancer (CRC) is the fourth leading cause of cancer deaths worldwide, with poor prognosis mainly related to metastasis. Fibronectin (FN), a vital component of the extracellular matrix (ECM), has been found involved in tumorigenesis and malignant progression in different types of malignancy. Numerous studies have indicated the distinct expression of FN in various cancers and demonstrated the different functions of FN in the proliferation, migration, and invasion of cancers. Meanwhile, FN isoforms have been extensively used for targeted drug delivery and imaging for tumors. Although a growing number of studies on FN in CRC have been reported, integrated reviews on the relationship between FN and CRC are rare. In this review, we will summarize the association between FN and CRC, including the signaling pathways and molecules involved in, as well as potential diagnostic and therapeutic values of FN for patients with CRC.