Wnt‐3a stimulates neurite outgrowth in Ewing sarcoma cells

We previously reported that various Wnt/Fz family members are expressed in Ewing sarcoma family tumor (ESFT) cell lines, and that Wnt‐3a induced the formation of elongated cytoplasmic extensions (Üren et al., Pediatr Blood Cancer, 2004). Subsequent analysis suggests that these extensions are neurites. Approximately 50% of cells exhibit a process exceeding one cell diameter in length within 3h of Wnt‐3a treatment. This extension stains positively both for polymerized actin and markers of microtubules, including acetylated and tyrosinated tubulin, as well as tau. Among the Wnt receptors expressed by ESFT cells, Fz3 has been implicated in axonal guidance. Inhibition of Fz3, either by an antiserum raised against its amino‐terminal extracellular domain or RNAi knockdown of its expression, markedly reduced neurite outgrowth. As observed in other model systems, inhibition of GSK‐3 with lithium or BIO stimulated neurite formation in ESFT cells. This implied that Wnt‐3a‐dependent, GSK‐3 inhibition might contribute to neuritogenesis. While Wnt‐3a activated the beta‐catenin/canonical Wnt pathway in ESFT cells, which is indicative of GSK‐3 inhibition, suppression of the beta‐catenin pathway with the Wnt antagonist, Dickkopf‐1, did not block neurite outgrowth. Alternatively, preliminary evidence suggests that JNK activation might have a significant role in Wnt‐dependent neurite formation.