Wnt-5a-induced phosphorylation of DARPP-32 inhibits breast cancer cell migration in a CREB-dependent manner.

Tumour cell migration plays a central role in the process of cancer metastasis. We recently identified dopamine and cAMP regulated phospho-protein of 32 kDa (DARPP-32) as an anti-migratory phospho-protein in breast cancer cells. Here we link this effect of DARPP-32 to Wnt-5a signalling by demonstrating that recombinant Wnt-5a triggers cAMP elevation at the plasma membrane and Thr34-DARPP-32 phosphorylation in MCF-7 cells. In agreement, both protein kinase A (PKA) inhibitors and siRNA-mediated knock down of Frizzled-3 receptor or Galphas expression abolished Wnt-5a-induced phosphorylation of DARPP-32. Furthermore, Wnt-5a induced DARPP-32 dependent inhibition of MCF-7 cell migration. Phospho-Thr34-DARPP-32 interacted with protein phosphatase-1 (PP1) and potentiated the Wnt-5a-mediated phosphorylation of CREB, a well-known PP1 substrate, but had no effect on CREB phosphorylation by itself. Moreover, inhibition of the Wnt-5a/DARPP-32/CREB pathway, by expression of dominant negative CREB (DN-CREB), diminished the anti-migratory effect of Wnt-5a-induced phospho-Thr34-DARPP-32. Phalloidin-staining revealed that that the presence of phospho-Thr34-DARPP-32 in MCF-7 cells results in reduced filopodia formation. In accordance, the activity of the Rho GTPase Cdc42, known to be crucial for filopodia formation, was reduced in MCF-7 cells expressing phospho-Thr34-DARPP-32. The effects of DARPP-32 on cell migration and filopodia formation could be reversed in T47D breast cancer cells that were depleted of their endogenous DARPP-32 by siRNA targeting. Consequently, Wnt-5a activates a Frizzled-3/Galphas /cAMP/PKA signalling pathway that triggers a DARPP-32- and CREB-dependent anti-migratory response in breast cancer cells, representing a novel mechanism whereby Wnt-5a can inhibit breast cancer cell migration.