Wnt-5a-induced Phosphorylation of DARPP-32 Inhibits Breast Cancer Cell Migration in a CREB-dependent Manner

Tumor cell migration plays a central role in the process of cancer metastasis. We recently identified dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) as an antimigratory phosphoprotein in breast cancer cells. Here we link this effect of DARPP-32 to Wnt-5a signaling 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 knockdown of Frizzled-3 receptor or Gα_s expression abolished Wnt-5a-induced phosphorylation of DARPP-32. Furthermore, Wnt-5a induced DARPP-32-dependent inhibition of MCF-7 cell migration. Phospho-Thr-34-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 antimigratory effect of Wnt-5a-induced phospho-Thr-34-DARPP-32. Phalloidin-staining revealed that that the presence of phospho-Thr-34-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-Thr-34-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/Gα_s/cAMP/PKA signaling pathway that triggers a DARPP-32- and CREB-dependent antimigratory response in breast cancer cells, representing a novel mechanism whereby Wnt-5a can inhibit breast cancer cell migration.Breast cancer is the most common form of cancer in women. Whereas the prognosis for breast cancer patients without local or distal dissemination is relatively favorable, the prognosis is considerably worse once distal metastasis has been established. It is therefore imperative to identify molecular targets and develop novel antimetastatic therapies that will stop, reduce, or delay the dissemination and growth of breast cancer metastasis.We recently isolated the dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32),² from a human breast expression library, as a DDR1-binding partner (1). Introduction of DARPP-32 in breast cancer cells lacking endogenous expression of this protein inhibited cell migration in a phospho-Thr-34-DARPP-32-dependent manner (1). DARPP-32 was originally identified 25 years ago as a dopamine and cAMP target enriched in dopaminoceptive neurones (2). Since then, a large body of work has shown that DARPP-32 is crucial for signal transmission by a wide array of neurotransmitters and drugs of abuse. DARPP-32 can act as either a phosphatase inhibitor or a kinase inhibitor depending on its phosphorylation state. Phosphorylation of Thr-34 by protein kinase A (PKA) converts DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP1) (3), whereas phosphorylation at Thr-75 by Cdk5 turns DARPP-32 into an inhibitor of PKA (4). Downstream of DARPP-32 it has been shown that the activity of CREB and c-fos are strongly attenuated in DARPP-32 knockout mice (5). Despite the substantial amount of work done on DARPP-32 over the past 25 years, the role of this phosphoprotein outside the neuronal system has only recently started to be explored.Regarding the role of DARPP-32 in cancer, overexpression of DARPP-32 has been reported in gastric, colon, and prostate cancer (6, 7). In contrast, patients with esophageal squamous cell carcinoma that lacks DARPP-32 expression have reduced survival when compared with patients with DARPP-32-expressing esophageal squamous cell carcinomas (8, 9). Furthermore, DARPP-32 is needed to get a fully differentiated thyroid cell phenotype, and transformation of thyroid cells by constitutively activated Ras resulted in a loss of DARPP-32 expression (10). Thus, the role of DARPP-32 in cancer is somewhat uncertain, and little is known about the cell signaling mechanisms behind a possible suppressor or promotor role of DARPP-32 in cancer.Wnt-5a is a non-canonical member of the Wnt family of secreted glycoproteins that acts through the family of Frizzled G-protein-coupled receptor, Ror2, and co-receptors such as, LRP5/6, to mediate important events during development and cancer (11–15). In the breast, the non-transforming Wnt-5a has been shown to inhibit epithelial cell migration (16), and in breast cancer, expression of Wnt-5a has been shown to be a predictor of longer disease-free survival (17). Wnt-5a expression has been shown to increase activation of the receptor tyrosine kinase DDR1 in breast epithelial cells upon plating on collagen (18). As DDR1 is a collagen-binding adhesion receptor important for cell migration (19), and its binding partner DARPP-32 is a phospho-dependent antimigratory molecule (1), we wanted to test whether the functional overlaps between DARPP-32 and Wnt-5a, could be a result of Wnt-5a acting upstream in the signaling process leading to DARPP-32 phosphorylation.Here we demonstrate that Wnt-5a can trigger a Frizzled-3/Gαs/cAMP signal that results in PKA-dependent phosphorylation of DARPP-32. Furthermore, we show that phospho-DARPP-32 potentiates Wnt-5a-mediated CREB activity and suppresses filopodia formation.