The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvβ3 integrin interaction

Abstract

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.

Keywords: FAK/PI3K/Akt/mTOR; NC1 domain; collagen XIX; integrin; tumor invasion.

Figure 1. NC1(XIX) inhibits migration of SK-MEL-28 melanoma cells

(A) SK-MEL-28 cells were seeded in 12-well plates. Cell layer was scratched with a pipet tip and then incubated with or without 20 μM of NC1(XIX) up to 72 h at 37°C. Wound closure was measured by microscopy at 0, 24, 48, 72 h respectively. (B) Wound closure was significantly delayed after NC1(XIX) treatment compared to control. Histogram represents the mean ± SEM of 4 replicates. *Significantly different from control (*p < 0.05).

Figure 2. αvβ3 integrin specifically binds NC1(XIX)

(A) Effect of divalent cations and EDTA on SK-MEL-28 cell adhesion. Cells were seeded in DMEM containing 5 mM CaCl₂ + 2.5 mM MgCl₂ and/or 5 mM EDTA, in wells coated with 0.1 μg BSA used as negative control, 0.1 μg fibronectin used as positive control or 1 μg of NC1(XIX) for 2 h at 37°C. (B) Effect of Mn²⁺ on SK-MEL-28 cell adhesion. Cells were seeded in HEPES buffer containing 0.1 mM Mn²⁺ in well coated with 1 μg of NC1(XIX) for 2 h at 37°C. (C) Effect of αvβ3 antibody on SK-MEL-28 cell adhesion. Cells were preincubated in DMEM containing 20 μg/mL of αvβ3 antibody for 30 min at 37°C and then incubated in wells coated with 1 μg of NC1(XIX) for 2 h at 37°C. After washing, cells were fixed with glutaraldehyde and stained with crystal violet. Absorbance was read at 560 nm. The values are the means ± SD of 4 replicates. *Significantly different from control (***p < 0.001). (D) SK-MEL-28 extracts were submitted by affinity chromatography on a NC1(XIX) peptide-bounded column. Bound proteins were eluted with increasing concentrations of NaCl (0.15, 0.6 and 1 M) and eluted samples were then analyzed by SDS-PAGE and western blot. The 0.6 M eluted sample revealed a band which matched the 105 kDa band of the denaturated recombinant αvβ3 integrin used as positive control. (E) In the liquid phase binding assay, recombinant αvβ3 integrin was incubated with NC1(XIX) biotinylated peptide with or without an excess of NC1(XIX) (lane 1 and 2 respectively). Integrin-peptide complexes were precipitated using sepharose streptavidin beads and revealed by western blot analysis. Recombinant αvβ3 integrin was used a positive control (lane 3).

Figure 3. NC1(XIX) colocalizes with the αvβ3 integrin in SK-MEL-28 melanoma cells

(A) Fluorescent microscopy visualization of biotinylated NC1(XIX) on the cell surface of SK-MEL-28 melanoma cells (top panel). Competition between NC1(XIX) and biotinylated NC1(XIX) (low panel). (B) Fluorescent microscopy visualization of biotinylated NC1(XIX) (green) and αvβ3 integrin (red). Yellow staining corresponds to areas where biotinylated NC1(XIX) and anti-αvβ3 integrin colocalize. Cells were cultured on glass slides, fixed with paraformaldehyde and labeled with biotinylated NC1(XIX) peptide and anti-αvβ3 integrin antibody. Scale bar: 20 μm. (C) SK-MEL-28 melanoma cells were cultured on glass slides, fixed with paraformaldehyde and incubated with biotinylated NC1(XIX) peptide and anti-αvβ3 integrin antibody (23C6).

Figure 4. cRGDfV blocking peptide inhibits the NC1(XIX) binding on αvβ3 integrin

(A) Effect of cRGDfV blocking peptide on SK-MEL-28 cell adhesion. Cells were preincubated in DMEM containing 0.1, 1 or 10 μg/mL of cRGDfV blocking peptide for 30 min at 37°C and then incubated in wells coated with 1 μg of NC1(XIX) for 2 h at 37°C. After washing, cells were fixed with glutaraldehyde and stained with crystal violet. Absorbance was read at 560 nm. The values are the means ± SD of 4 replicates. *Significantly different from control (***p < 0.001). (B) Flow cytometry studies showed NC1(XIX) peptide (100 μM) binding on SK-MEL-28 cell surface. (C) NC1(XIX) peptide (100 μM) binding was inhibited by the addition of αvβ3 integrin antibody (LM609) or cRGDfV blocking peptide.

Figure 5. Kinetic analysis of FAK, PI3K p85 subunit of PI3 kinase and PDK1 phosphorylation in SK-MEL-28 melanoma cells after incubation with NC1(XIX)

Western blot analysis of: (A) phosphorylated-FAK^Y397, (B) phosphorylated-FAK^Y861, (C) phosphorylated-PI3K p85^Y458 and (D) phosphorylated-PDK1^S241 compared to total FAK, total PI3K p85 and total PDK1 expression respectively after incubation of SK-MEL-28 melanoma cells with NC1(XIX) for 0, 1, 5, 15, 30 and 60 min. Bands were quantified by densitometric analysis. Phosphorylated protein was reported to corresponding total protein.

Figure 6. Kinetic analysis of Akt, mTOR and GSK3β phosphorylation in SK-MEL-28 melanoma cells after incubation with NC1(XIX)

Western blot analysis showing expression of: (A) phosphorylated-Akt^T308, (B) phosphorylated-Akt^S473, (C) phosphorylated-mTOR^S2448, (D) phosphorylated-mTOR^S2481 and (E) phosphorylated-GSK3β^S9 compared to total Akt, total mTOR and total GSK3β expression, respectively, after incubation of SK-MEL-28 melanoma cells with NC1(XIX) for 0, 1, 5, 15, 30, 60 and 360 min. Bands were quantified by densitometric analysis. Phosphorylated protein was reported to corresponding total protein.

Figure 7. Schematic representation of the FAK / PI3K / Akt / mTOR pathway and modifications of phosphorylated proteins after incubation with NC1(XIX)