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, TGF␤1, controlled the expression of Wnt5a, but due to the unpredictable effects of TGF␤1 signaling on melanoma cell motility, targeting Wnt5a signaling via TGF␤1 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.inhibitory peptide ͉ malignant melanoma ͉ tumor cell invasion W nt ligands comprise a family of 19 human secreted signaling proteins, which coordinate essential processes required for development and maintenance of tissue homeostasis. Misregulation of Wnt signaling can lead to cancer progression (1). The Wnt ligands are secreted glycoproteins that can be divided based on their ability to activate different intracellular signals, in a tissue-dependent manner. One group primarily activates canonical signaling that controls ␤-catenin stability, while the other is loosely described as ␤-catenin-transcriptionally independent (non-canonical Wnt signaling). However, cross-talk between the two signaling networks does exist (2).Wnt5a is in most situations characterized as a non-canonical Wnt ligand that elicits intracellular signals through association with distinct receptors and co-receptors in a cell specific manner. Wnt5a has been shown to stimulate increases in intracellular Ca 2ϩ levels in developmental models (3) and mammalian cell lines, including breast and thyroid cancer cells (4-6), giving rise to the model of a non-canonical Wnt/Ca 2ϩ signaling pathway. Wnt5a-mediated intracellular increases in Ca 2ϩ levels enables the activation of Ca 2ϩ -regulated proteins, such as protein kinase C (PKC) in a context dependent manner, as reviewed recen...

LRP5/6 directly bind to Frizzled and prevent Frizzled-regulated tumour metastasis

“…Wnt5a has been reported to activate the non-canonical pathway and induce migration of human melanoma cell line A2058 (ref. 16). We further used Wnt5a to examine the inhibitory role of LRP5/6 in endogenous Frz in A2058 cells.…”

“…The non-canonical Wnt5a can promote normal cell motility as well as tumour metastasis through the activation PCP/JNK or Ca 2 þ /PKC signalling cascade [16][17][18] . Thus, noncanonical pathways are involved in cancer development through promoting tumour cell motility.…”

Controlled levels of canonical Wnt signaling are required for neural crest migration

“…Another chemical modulator used in our studies is Box5, a Wnt5a-derived hexapeptide which has been shown to antagonize activities mediated by Wnt5a and Fz5 such as calcium response, cell invasion and migration in tumor cell lines (Jenei et al, 2009). Wnt5a can also bind Ror2 and this interaction is well established and classified as a separated branch of the Wnt pathway, namely Wnt5a/Ror2 pathway (Grenn et al, 2008, Minami et al, 2010.…”

WNT Signaling in Cutaneous Squamous Cell Carcinoma: A Future Treatment Strategy?

“…DVL antagonises destruction complex activity, causing β-catenin accumulation to allow nuclear translocation where in consort with the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors, β-catenin elicits activation of WNT target gene expression (Cadigan 2012). WNT/β-catenin-independent signaling commonly occurs through Ca 2+ signaling in tumours, resulting in activation of Ca 2+ -dependent enzymes to elicit transcriptional changes and increase small GTPase activity, causing cytoskeletal rearrangements and alteration of cell polarity/migration (Jenei, et al 2009). …”

“…However a WNT5A-derived hexapeptide, termed Box5, 12 developed to inhibit WNT/Ca 2+ signaling in melanoma (Jenei, et al 2009), has recently been shown to antagonise WNT5A/Ca 2+ signaling in HNSCC (Prgomet, et al 2015). Future efforts should be focused on identifying which WNT network inhibitors could provide potent therapeutic activity in cSCC.…”