Integrin-uPAR signaling leads to FRA-1 phosphorylation and enhanced breast cancer invasion.

BACKGROUND: The Fos-related antigen 1 (FRA-1) transcription factor promotes tumor cell growth, invasion and metastasis. Phosphorylation of FRA-1 increases protein stability and function. We identify a novel signaling axis that leads to increased phosphorylation of FRA-1, increased extracellular matrix (ECM)-induced breast cancer cell invasion and is prognostic of poor outcome in patients with breast cancer. METHODS: While characterizing five breast cancer cell lines derived from primary human breast tumors, we identified BRC-31 as a novel basal-like cell model that expresses elevated FRA-1 levels. We interrogated the functional contribution of FRA-1 and an upstream signaling axis in breast cancer cell invasion. We extended this analysis to determine the prognostic significance of this signaling axis in samples derived from patients with breast cancer. RESULTS: BRC-31 cells display elevated focal adhesion kinase (FAK), SRC and extracellular signal-regulated (ERK2) phosphorylation relative to luminal breast cancer models. Inhibition of this signaling axis, with pharmacological inhibitors, reduces the phosphorylation and stabilization of FRA-1. Elevated integrin αVß3 and uPAR expression in these cells suggested that integrin receptors might activate this FAK-SRC-ERK2 signaling. Transient knockdown of urokinase/plasminogen activator urokinase receptor (uPAR) in basal-like breast cancer cells grown on vitronectin reduces FRA-1 phosphorylation and stabilization; and uPAR and FRA-1 are required for vitronectin-induced cell invasion. In clinical samples, a molecular component signature consisting of vitronectin-uPAR-uPA-FRA-1 predicts poor overall survival in patients with breast cancer and correlates with an FRA-1 transcriptional signature. CONCLUSIONS: We have identified a novel signaling axis that leads to phosphorylation and enhanced activity of FRA-1, a transcription factor that is emerging as an important modulator of breast cancer progression and metastasis.

Aberrant glycosylation of αvß3 integrin is associated with melanoma progression.

N-glycosylation of integrins plays an important role in cancer progression. Increased αvß3 integrin expression during melanoma progression is well-documented but the role of its glycans in tumorigenesis is still poorly understood. In the present study we used the WM793 primary melanoma cell line and its highly metastatic variant, WM1205Lu, to examine αvß3 glycosylation. Lectin precipitation, enzyme digestion and the use of swainsonine (SW) showed that αvß3 integrin glycosylation differs significantly between primary and metastatic melanoma cells. High-mannose structures and complex glycans with bisecting N-acetylglucosamine (GlcNAc) were more abundant in both subunits of primary cells. We also observed a shift in the sialylation of αvß3 integrin related to reduction of α2-6-linked sialic acid expression and an increase of α2-3 sialylation of both subunits in melanoma progression. Metastatic melanoma migration on vitronectin (VN) was reduced in the presence of antibody against αvß3 and the lectins phytohemagglutinin-L (PHA-L), Sambucus nigra agglutinin (SNA) and Maackia amurensis (MAA) in woundhealing assays. Our results show that the acquisition of metastatic competence by melanoma cells is accompanied by alteration of αvß3 integrin glycosylation and that both αvß3 and ß1-6-branched sialylated complex-type N-glycans promote metastatic melanoma migration on VN.

A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach.

Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications.

Cationic surface charge combined with either vitronectin or laminin dictates the evolution of human embryonic stem cells/microcarrier aggregates and cell growth in agitated cultures.

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 µm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 µm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment.

Vitronectin absorbed on nanoparticles mediate cell viability/proliferation and uptake by 3T3 Swiss albino mouse fibroblasts: in vitro study.

We study the interaction of 3T3 Swiss albino mouse fibroblasts with polymeric nanoparticles (NPs) and investigate cellular behaviour in terms of viability/cytotoxicity, cell cycle, NPs uptake, MAP kinase (ERK1/2), and focal adhesion kinase (FAK) activation. After incubation of NPs with cell culture media, western blot analysis showed that Vitronectin is retained by NPs, while Fibronectin is not detected. From cytotoxicity studies (MTT and BrdU methods) an LD50 of about 1.5 mg/mL results for NPs. However, NPs in the range 0.01-0.30 mg/mL are able to trigger a statistically significant increase in proliferation and cell cycle progression in dose and time depending manner. Also, biochemical evaluation of ERK1/2 and FAK clearly shows an increasing phosphorylation in a dose and time depending manner. Finally, we found by transmission electron microscopy that NPs are internalised by cells. Competitively blocking VN-integrin receptors with echistatin (1 µg/mL) results in a decrease of viability/proliferation, cell cycle progression, cellular uptake, and FAK/ERK activation showing the involvement of Vitronectin receptors in signal transduction. In conclusion, our results show that cell surface NPs interactions are mediated by absorbed plasma proteins (i.e., Vitronectin) that represent an external stimuli, switched to the nucleus by FAK enzyme, which in turn modulate fibroblasts viability/proliferation.

Human pilot studies reveal the potential of a vitronectin: growth factor complex as a treatment for chronic wounds.

Several different advanced treatments have been used to improve healing in chronic wounds, but none have shown sustained success. The application of topical growth factors (GFs) has displayed some potential, but the varying results, high doses and high costs have limited their widespread adoption. Many treatments have ignored the evidence that wound healing is driven by interactions between extracellular matrix proteins and GFs, not just GFs alone. We report herein that a clinical Good Manufacturing Practice-grade vitronectin:growth factor (cVN:GF) complex is able to stimulate functions relevant to wound repair in vitro, such as enhanced cellular proliferation and migration. Furthermore, we assessed this complex as a topical wound healing agent in a single-arm pilot study using venous leg ulcers, as well as several 'difficult to heal' case studies. The cVN:GF complex was safe and re-epithelialisation was observed in all but 1 of the 30 patients in the pilot study. In addition, the case studies show that this complex may be applied to several ulcer aetiologies, such as venous leg ulcers, diabetic foot ulcers and pressure ulcers. These findings suggest that further evaluation is warranted to determine whether the cVN:GF complex may be an effective topical treatment for chronic wounds.

Hyaluronic acid: evaluation as a potential delivery vehicle for vitronectin:growth factor complexes in wound healing applications.

We have previously reported that novel vitronectin:growth factor (VN:GF) complexes significantly increase re-epithelialization in a porcine deep dermal partial-thickness burn model. However, the potential exists to further enhance the healing response through combination with an appropriate delivery vehicle which facilitates sustained local release and reduced doses of VN:GF complexes. Hyaluronic acid (HA), an abundant constituent of the interstitium, is known to function as a reservoir for growth factors and other bioactive species. The physicochemical properties of HA confer it with an ability to sustain elevated pericellular concentrations of these species. This has been proposed to arise via HA prolonging interactions of the bioactive species with cell surface receptors and/or protecting them from degradation. In view of this, the potential of HA to facilitate the topical delivery of VN:GF complexes was evaluated. Two-dimensional (2D) monolayer cell cultures and 3D de-epidermised dermis (DED) human skin equivalent (HSE) models were used to test skin cell responses to HA and VN:GF complexes. Our 2D studies revealed that VN:GF complexes and HA stimulate the proliferation of human fibroblasts but not keratinocytes. Experiments in our 3D DED-HSE models showed that VN:GF complexes, both alone and in conjunction with HA, led to enhanced development of both the proliferative and differentiating layers in the DED-HSE models. However, there was no significant difference between the thicknesses of the epidermis treated with VN:GF complexes alone and VN:GF complexes together with HA. While the addition of HA did not enhance all the cellular responses to VN:GF complexes examined, it was not inhibitory, and may confer other advantages related to enhanced absorption and transport that could be beneficial in delivery of the VN:GF complexes to wounds.

Endovascular histologic effects of ultrathin gold- or vitronectin-coated platinum aneurysm coils in a rodent arterial occlusion model: a preliminary investigation.

BACKGROUND AND PURPOSE: Novel stratagems to improve the efficacy of platinum coils in occluding cerebral aneurysms have primarily involved coating coils with materials thought likely to provoke more desirable histologic reactions. No investigations to date, however, have evaluated the utility of gold or vitronectin coatings, despite known endovascular histologic effects of these agents, which may be favorable for treating cerebral aneurysms. This study was conducted to evaluate the degree of endovascular histologic change associated with ultrathin gold- or vitronectin-coated platinum coils. It was hypothesized that such coatings would increase intra-aneurysmal intimal hyperplasia and the degree of luminal occlusion compared with standard platinum coils. MATERIALS AND METHODS: The ligated carotid artery rat model was used to study 4 different aneurysm coil conditions: no coil (sham-surgery controls), uncoated platinum coil, and gold- or vitronectin-coated platinum coil. Two weeks postimplantation, the aneurysms were harvested and stained with hematoxylin-eosin. Slides were evaluated for the degree of neointimal response by a pathologist blinded to treatment. Additional quantitative evaluation was performed blindly by using the ratio of intimal-to-luminal cross-sectional area. RESULTS: A gold- or vitronectin-coated platinum aneurysm coil produced a statistically significant increase in neointimal response compared with a sham (no coil). Arterial segments treated with gold-coated platinum coils also demonstrated a statistically significant 100% increase in neointimal response compared with those treated with bare platinum coils. CONCLUSIONS: In concordance with our hypothesis, ultrathin coatings of gold provoked a neointimal response and degree of luminal occlusion greater than that of plain platinum aneurysm coils in a rat arterial occlusion model.

Photolithographic patterning of C2C12 myotubes using vitronectin as growth substrate in serum-free medium.

The C2C12 cell line is frequently used as a model of skeletal muscle differentiation. In our serum-free defined culture system, differentiation of C2C12 cells into myotubes required surface-bound signals such as substrate-adsorbed vitronectin or laminin. On the basis of this substrate requirement of myotube formation, we developed a photolithography-based method to pattern C2C12 myotubes, where myotubes formed exclusively on vitronectin surface patterns. We have determined that the optimal line width to form single myotubes is approximately 30 mum. To illustrate a possible application of this method, we patterned myotubes on the top of commercial substrate-embedded microelectrodes. In contrast to previous experiments where cell patterning was achieved by selective attachment of the cells to patterned surfaces in a medium that contained all of the factors necessary for differentiation, this study illustrates that surface patterning of a signaling molecule, which is essential for skeletal muscle differentiation in a defined system, can result in the formation of aligned myotubes on the patterns. This technique is being developed for applications in cell biology, tissue engineering, and robotics.

Evaluation of silicon dioxide-based coating enriched with bioactive peptides mapped on human vitronectin and fibronectin: in vitro and in vivo assays.

A wide range of biochemical signals promoting cell functions (adhesion, migration, proliferation, and differentiation) and thereby improving the osseointegration process are currently investigated. Unfortunately, their application for the production of bioactive implantable devices is often hampered by their insolubility; instability; and limited availability of a large amount of inexpensive, high-purity samples. An attractive alternative is the use of short peptides carrying the minimum active sequence of the natural factors. Synthetic peptides mapped on fibronectin and vitronectin have been demonstrated to enhance cell adhesion both to polystyrene and acellular bone matrix; in particular, a nonapeptide sequence from human vitronectin works via an osteoblast-specific adhesion mechanism. In this study, we incorporated these peptides into a sol-gel silica dressing applied to coat sand-blasted and acid-attacked titanium samples; measured the kinetic of peptide release; and used titanium disks, coated with a peptide-enriched film, as substrates to determine the peptide concentration that maximizes cell adhesion in vitro. We also evaluated in vivo the capacity of the vitronectin-derived peptide to improve osteogenic activity: histologic analysis revealed markedly improved osteogenic activity around peptide-enriched samples. This article also discusses the role of surface characteristics and the importance of bioactive peptides.

Potential synergies between matrix proteins and soluble factors on resorption and proteinase activities of rabbit bone cells.

Human growth hormone (GH) has recently been found to stimulate osteoclastic resorption, cysteine-proteinase and metalloproteinase activities (MMP-2 and MMP-9) in vitro via insulin-like growth factor-I (IGF-I) produced by stromal cells. The present study investigated the effects of two extracellular matrix components (vitronectin and type-I collagen) on hGH- and hIGF-1-stimulated osteoclastic resorption and proteinase activities in a rabbit bone cell model. After 4 days of rabbit bone cell culture on dentin slices with vitronectin coating, hGH and hIGF-1 stimulated bone resorption and hIGF-1 upmodulated cysteine-proteinase activities. MMP-2 expression (but not resorption, cathepsin or MMP-9 activities) was upmodulated by hGH and hIGF-1 on dentin slices coated with type I collagen as compared to those without coating. Then, vitronectin was synergistic with hIGF-1 in the regulation of cysteine-proteinase production whereas collagen showed synergy with hGH and hIGF-1 in the regulation of MMP-2 production. Anti-alphavbeta3 totally abolished the effects of hGH and hIGF-1 on metalloproteinase release, but had no influence on cathepsin release. The results suggest that cysteine-proteinase modulation is not mediated by alphavbeta3 integrin (strongly expressed on osteoclastic surface) whereas the resorption process and metalloproteinase modulation are clearly mediated by this integrin. Our finding about the collagen coating also suggests that hGH- and hIGF-1-stimulated MMP-2 activity are mediated, along with alphavbeta3 integrin, by another adhesion molecule.