A t-butyloxycarbonyl-modified Wnt5a-derived hexapeptide functions as a potent antagonist of Wnt5a-dependent melanoma cell invasion

Abstract

The influential role of Wnt5a in tumor progression underscores the requirement for developing molecules that can target Wnt5a-mediated cellular responses. In the aggressive skin cancer, melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches can be used to counteract these effects: inhibition of Wnt5a expression or direct blockade of Wnt5a signaling. We have investigated both options in the melanoma cell lines, A2058 and HTB63. Both express Frizzled-5, which has been implicated as the receptor for Wnt5a in melanoma cells. However, only the HTB63 cell line expresses and secretes Wnt5a. In these cells, the cytokine, TGFbeta1, controlled the expression of Wnt5a, but due to the unpredictable effects of TGFbeta1 signaling on melanoma cell motility, targeting Wnt5a signaling via TGFbeta1 was an unsuitable strategy to pursue. We therefore attempted to target Wnt5a signaling directly. Exogenous Wnt5a stimulation of A2058 cells increased adhesion, migration and invasion, all crucial components of tumor metastasis, and the Wnt5a-derived N-butyloxycarbonyl hexapeptide (Met-Asp-Gly-Cys-Glu-Leu; 0.766 kDa) termed Box5, abolished these responses. Box5 also inhibited the basal migration and invasion of Wnt5a-expressing HTB63 melanoma cells. Box5 antagonized the effects of Wnt5a on melanoma cell migration and invasion by directly inhibiting Wnt5a-induced protein kinase C and Ca(2+) signaling, the latter of which we directly demonstrate to be essential for cell invasion. The Box5 peptide directly inhibits Wnt5a signaling, representing an approach to anti-metastatic therapy for otherwise rapidly progressive melanoma, and for other Wnt5a-stimulated invasive cancers.

Fig. 1.

Wnt5a and Foxy5 promote melanoma cell migration. (A) A2058 cells (low Wnt5a expressing melanoma cell line, Fig. S1) were stimulated with the indicated concentrations of Wnt5a for 16 h, detached with versene and allowed to adhere for 1 h. Non-adherent cells were washed away, while the adherent cells were stained and their relative number determined. This number is represented as a percentage of non-Wnt5a-stimulated cells. (B) Wnt5a (0.2 μg/mL, open symbols and solid line) increased the migration of A2058 cells in a wound-healing assay compared to untreated cells (control, closed symbols and broken line) over a 2-day time course. The wound-healing data are expressed as a percentage of the wound area closed after 0, 16, 24, 40, and 48 h. (C) Foxy5 structure (formyl group in blue). (D) Foxy5 (50 μM; solid squares and solid line) increased the migration of A2058 cells in a wound-healing assay compared to untreated cells (control, closed circles and broken line) over a 2-day time course. The wound-healing data are expressed as a percentage of the wound area closed after 0, 16, 24, 40, and 48 h. Error bars, SEM. Paired t-tests; *, P < 0.05.

Fig. 2.

Box5 is a modified analogue of Foxy5 and functions as an antagonist of Wnt5a in melanoma cells. (A) Box5 structure (t-boc group in yellow and blue, yellow represents the additional modification as compared to the formyl group). (B) Box5 (open symbols and solid line) inhibits Wnt5a-mediated A2058 cell migration (wound-healing analysis) in a dose-dependent manner. A t-boc-conjugated random peptide, Boc-Ran (closed symbols and broken line), could not antagonize Wnt5a-mediated migration. Cells were incubated with either of the peptides at the indicated concentrations, and 0.2 μg/mL recombinant Wnt5a (rWnt5a) for 40 h. (C) Wound-healing analysis of A2058 cells during a 48-h time course. The cells were incubated with rWnt5a (0.2 μg/mL) alone (open squares and solid line) or rWnt5a (0.2 μg/mL) and 100 μM Box5 (open triangles and solid line), or without any additive (control, closed symbols and broken line). (D) Wound-healing analysis of HTB63 cells (high Wnt5a expressing melanoma cell line) (Fig. S1) during a 40-h time course. The HTB63 cells were incubated in conditioned medium (open squares and solid line), or conditioned medium containing and 100 μM Box5 (open triangles and solid line), or fresh serum-free medium without any additive (control, closed symbol and broken line). * t-tests indicate conditioned medium values compared to control, while analysis of conditioned medium compared to Box5 are indicated by ⋀, P < 0.05, ⋀⋀, P < 0.01. In A–D, error bars, SEM. Paired t-tests: *, P < 0.05; **, P < 0.01.

Fig. 3.

(A) Box5 inhibits Wnt5a-mediated melanoma cell invasion through matrigel. A2058 cells were preincubated with/without Box5 (100 μM) for 40 min, followed by the addition of rWnt3a (0.05 μg/mL), rWnt5a (0.2 μg/mL), or no stimuli at all. (B) Invasion of HTB63 cells is inhibited by addition of 100 μM Box5. All error bars, SEM. Paired t-tests for all experiments; *, P < 0.05, **, P < 0.01.

Fig. 4.

The Wnt/Ca²⁺ signaling pathway is essential for Wnt5a-mediated melanoma cell invasion. (A) rWnt5a (0.1 μg/mL; addition indicated by arrow) triggers a rapid cytosolic Ca²⁺ signal in A2058 cells. (B) Preincubation of A2058 cells with 10 μM MAPT/AM for 30 min abolishes rWnt5a (0.1 μg/mL) stimulation (indicated by arrow) of cytosolic Ca²⁺. (C) MAPT/AM abolishes Wnt5a-induced A2058 cell invasion. Cells were preincubated with 10 μM MAPT/AM or vehicle alone for 30 min, then stimulated with/without rWnt5a (0.2 μg/mL), and then with 1 μM MAPT/AM or vehicle alone throughout the duration of the invasion experiment (24 h), where the latter treatment condition had the same chelating effect on Ca²⁺ as 10 μM of MAPT/AM for 30 min, shown in A. Error bars, SEM. Paired t-tests; *, P < 0.05, ***, P < 0.001.

Fig. 5.

Box5 functions as a Wnt5a antagonist by inhibiting Wnt5a-mediated Ca²⁺ signaling. (A) Preincubation with Box5 (100 μM) overnight blocks rWnt5a-induced (0.1 μg/mL) Ca²⁺ release. Representative Ca²⁺ traces of A2058 cells stimulated with rWnt5a (first arrow), and then with (second arrow) either endothelin-1 (ET-1; 10 nM) or carbacol (5 μM). (B) Accumulated ΔCa²⁺ changes in ratio values recorded from A2058 cells stimulated with the various ligands described for A, either in the presence or absence of 100 μM Box5. Error bars, SEM. Paired t-test; ***, P < 0.001. (C) Preincubation with Box5 (100 μM) overnight inhibits myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation after 45 min of rWnt5a stimulation (0.2 μg/mL). 1 nM of the PKC activator, phorbol 12-myristate 13-acetate (PMA) was used as a positive indicator for MARCKS phosphorylation.