Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors.

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.

Matrix metalloproteinase 14 overexpression reduces corneal scarring.

Once a corneal scar develops, surgical management remains the only option for visual rehabilitation. Corneal transplantation is the definitive treatment for a corneal scar. In addition to the challenges posed by graft rejections and other postoperative complications, the lack of high-quality donor corneas can limit the benefits possible with keratoplasty. The purpose of our study was to evaluate a new therapeutic strategy for treating corneal scarring by targeting collagen deposition. We overexpressed a fibril collagenase (matrix metalloproteinase 14 (MMP14)) to prevent collagen deposition in the scar tissue. We demonstrated that a single and simple direct injection of recombinant adeno-associated virus-based vector expressing murine MMP14 can modulate gene expression of murine stromal keratocytes. This tool opens new possibilities with regard to treatment. In a mouse model of corneal full-thickness incision, we observed that MMP14 overexpression reduced corneal opacity and expression of the major genes involved in corneal scarring, especially type III collagen and α-smooth muscle actin. These results represent proof of concept that gene transfer of MMP14 can reduce scar formation, which could have therapeutic applications after corneal trauma.

[Corneal collagen cross-linking with ultraviolet-A light and riboflavin for the treatment of progressive keratoconus]. / Cross-linking du collagène cornéen induit par ultraviolet-A et riboflavine dans le traitement du kératocône évolutif.

INTRODUCTION: The aim of the present study was to evaluate the clinical usefulness of riboflavin- and ultraviolet-A (UV-A)-induced collagen cross-linking (CXL) on progressive keratoconus. PATIENTS AND METHODS: Twenty eyes of 20 patients with progressive keratoconus were included in a prospective, nonrandomized pilot study. The preoperative progression of keratoconus was documented by an increase in maximum keratometry (Kmax) on serial corneal topographies. Preoperative corneal thickness was at least 400 microm. The postoperative progression of keratoconus was evaluated from corneal topography, best-corrected visual acuity (BCVA) testing, and corneal biomechanical properties assessed with the Ocular Response Analyzer (ORA). The follow-up time was between 3 and 18 months. RESULTS: The steepening decreased significantly by 1.68+/-2.18 diopters (D) in the first year (p<0.05). The Kmax value decreased in 66% of eyes and remained stable in 17% (within+/-0.50 D). The mean preoperative BCVA was 0.49+/-0.22. Six months after the CXL treatment, the mean BCVA was 0.63+/-0.23 (p<0.05) and improved at least one line in 56.25% of eyes. The ORA values remained stable over the entire follow-up period. DISCUSSION: Extensive experimental study showed a significant increase in corneal rigidity after riboflavin/UV-A-induced CXL. CONCLUSION: Preliminary results on the clinical effects of riboflavin/UV-A-induced CXL are encouraging in reducing the progression of keratoconus. Long-term results are necessary to evaluate the duration of the stiffening effect.

Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair?

In vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.

Role of collagenase in mediating in vitro alveolar epithelial wound repair.

Type II pneumocytes are essential for repair of the injured alveolar epithelium. The effect of two MMP collagenases, MMP-1 and MMP-13 on alveolar epithelial repair was studied in vitro. The A549 alveolar epithelial cell line and primary rat alveolar epithelial cell cultures were used. Cell adhesion and cell migration were measured with and without exogenous MMP-1. Wound healing of a cell monolayer of rat alveolar epithelial cell after a mechanical injury was evaluated by time lapse video analysis. Cell adhesion on type I collagen, as well as cytoskeleton stiffness, was decreased in the presence of exogenous collagenases. A similar decrease was observed when cell adhesion was tested on collagen that was first incubated with MMP-1 (versus control on intact collagen). Cell migration on type I collagen was promoted by collagenases. Wound healing of an alveolar epithelial cell monolayer was enhanced in the presence of exogenous collagenases. Our results suggest that collagenases could modulate the repair process by decreasing cell adhesion and cell stiffness, and by increasing cell migration on type I collagen. Collagen degradation could modify cell adhesion sites and collagen degradation peptides could induce alveolar type II pneumocyte migration. New insights regarding alveolar epithelial cell migration are particularly relevant to investigate early events during alveolar epithelial repair following lung injury.