Conformation-dependent binding of a Tetrastatin peptide to αvβ3 integrin decreases melanoma progression through FAK/PI3K/Akt pathway inhibition

Abstract

Tetrastatin, a 230 amino acid sequence from collagen IV, was previously demonstrated to inhibit melanoma progression. In the present paper, we identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin in vivo and in vitro on melanoma cell proliferation, migration and invasion. We demonstrated that QS-13 binds to SK-MEL-28 melanoma cells through the α_vβ₃ integrin using blocking antibody and β3 integrin subunit siRNAs strategies. Relevant QS-13 conformations were extracted from molecular dynamics simulations and their interactions with α_Vβ₃ integrin were analyzed by docking experiments to determine the binding areas and the QS-13 amino acids crucial for the binding. The in silico results were confirmed by in vitro experiments. Indeed, QS-13 binding to SK-MEL-28 was dependent on the presence of a disulfide-bound as shown by mass spectroscopy and the binding site on α_Vβ₃ was located in close vicinity to the RGD binding site. QS-13 binding inhibits the FAK/PI₃K/Akt pathway, a transduction pathway that is largely involved in tumor cell proliferation and migration. Taken together, our results demonstrate that the QS-13 peptide binds α_vβ₃ integrin in a conformation-dependent manner and is a potent antitumor agent that could target cancer cells through α_Vβ₃.

Figure 1

QS-13 peptide inhibits in vivo tumor growth, in vitro SK-MEL-28 melanoma cell proliferation, colony formation, migration and invasion. Tumor growth was measured at day 20 in a mouse melanoma model (a). Cell proliferation was measured after 72 h of incubation (b). Colony formation in soft agar was measured after 10 days of incubation (c). Cell migration in scratch wound assay was measured after 48 h of incubation. (d) Cell invasion through Matrigel-coated membranes was measured after 48 h of incubation (e) **p < 0.01, ***p < 0.001.

Figure 2

QS-13 peptide binds to SK-MEL-28 through α_vβ₃ integrin. Cells were pre-incubated for 30 min with an anti-α_vβ₃ blocking antibody or an irrelevant IgG (10 µg/mL) before seeding on QS-13 peptide and adhesion was assessed (a). Cell extracts were submitted to affinity chromatography on a QS-13 peptide-bounded column and analyzed by western blot. Total cell extract (line 1), fraction eluted with 0.15 M (line 2), 0.6 M (line 3), 1 M NaCl (line 4) (b). Cells were transfected with β₃ subunit siRNA or control siRNA. Gene expression was measured by RT-qPCR 24 h after transfection (c). Protein expression was assessed by western-blot 48 h after transfection. Lane 1: control; lane 2: control siRNA; lane 3: β₃ subunit siRNA (d). Cells were seeded on QS-13 peptide 48 h after transfection and adhesion was measured (e) ***p < 0.001.

Figure 3

QS-13 peptide forms intra chain disulfide bond in solution. MALDI-ToF MS analysis of the QS-13 peptide at t 0 h (no disulfide bond) (a), t 24 h (presence of disulfide bond) (b), t 24 h + dithiothreitol (DTT) (no disulfide bond) (c).

Figure 4

Molecular Dynamics simulation and docking of the QS-13 peptide. (a) The snapshots extracted from MD simulations of the QS-13-db (left panel) and QS-13 (central panel) peptides were superimposed using the α carbons of the CQVC central sequence. A first type of display (upper panel) is obtained with the licorice representation picturing the CQVC central residues (atom color coded) and the Arginine residue (in light green). The second type of display (lower panel) is obtained using the licorice representation for the CQVC central residues (atom color coded) and the new cartoon representation for the backbone (in silver). Finally, superimposition of the first cluster of the disulfide bonded peptides was performed (right panel). The structures of the AS-13-db (in red), QS-13-R5A-db (in silver) and QS-13-db (in blue) were superimposed using the α carbons of the CQVC central sequence. The backbone is displayed using the new cartoon representation, the CQVC sequence using the licorice representation and the Arginine residues using the Van der Waals representation. (b) Specification of the QS-13 peptides characteritics related to the origin of the cluster (family of conformation) and the nature of the termini. (c) Representative conformation of QS-13 peptides. N-termini are displayed in green licorice and C-termini in red licorice. The backbone of the peptides is colored according to its local secondary structure (cyan for turn and white for coil) and the sulfur atoms are shown as yellow spheres. (d) The results of the QS-13-3 docking experiment highlight the existence of 5 Preferred Area of Interaction (PAI) (magenta surfaces). These PAI are also evidenced through the docking experiments performed with the other peptides. The protein is represented using the New Cartoon scheme and colored according to the nature of the chain (grey for α_V and brown for β₃) (left panel). Identification of contacts between QS-13-6 and the PAI shared with RGD peptides: contacts made with the α_V subunit (central panel) and contacts made with the β3 subunit (right panel).

Figure 5

Characterization of the QS-13 residues crucial for α_vβ₃ integrin interaction. SK-MEL-28 melanoma cells were pre-incubated 30 min in the presence or absence of the RGDS peptide (1 to 20 µg/mL) or anti-α_vβ₃ blocking antibody (20 µg/mL) and adhesion was assessed. *p < 0.05, ***p < 0.01, ***p < 0.001 (a). SK-MEL-28 melanoma cells were incubated with different substituted peptides (b) tagged with biotin and with AF-488 streptavidin complex and analyzed by flow cytometry (c) and results are reported on a histogram (d).

Figure 6

Kinetic analysis of FAK, PI₃K p85 and Akt phosphorylation in SK-MEL-28 cells after incubation with QS-13. Western blot analysis of phosphorylated-FAK^Y397 compared to total FAK (a), phosphorylated-PI₃K p85^Y458 compared to total PI₃K p85 subunit (b), phosphorylated-Akt^T308 compared to total Akt (c). Bands were quantified by densitometric analysis and phosphorylated proteins were reported to corresponding total proteins.

Figure 7

Summary diagram of QS-13 binding and mechanism of action. The QS-13 peptide is located at the C-terminal extremity of the Tetrastatin or Non Collagenous (NC)-1 domain of the α4 chain from type IV collagen. QS-13 conformation was deducted from Molecular Dynamics simulation. Molecular docking experiments suggest the presence of 5 preferred areas of interaction (PAI-1 to PAI-5); 4 of them appear in red on α_Vβ₃ integrin that is represented in blue and violet. Magnifications of PAI-1 allow to visualize QS-13 binding (up) or RGD binding (down) [20]. This explains that RGD pre-incubation abolishes QS-13 interaction with α_Vβ₃ at the cell surface. QS-13 binding inhibits cell proliferation, migration and invasion through the FAK/PI₃K/Akt pathway.