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

Abstract

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 siRNAmediated knockdown of Frizzled-3 receptor or G␣ 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 phosphoThr-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/ G␣ 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 prote...

Wnt5a Induces a Tolerogenic Phenotype of Macrophages in Sepsis and Breast Cancer Patients

“…One explanation is that a nonclassical NF-kB activity (p50-or p52-homodimer formation) induces IL-10 transcription together with certain transcription factors (e.g., cAMP-induced CREB activity) (39). Wnt5a induces cAMP and CREB activa- tion (40). We suggest that the mechanism behind Wnt5a-induced macrophage reprogramming is that Wnt5a acts as a feedback antagonist of TLR signaling with subsequent induction of antiinflammatory cytokines such as IL-10.…”

International Union of Basic and Clinical Pharmacology. LXXX. The Class Frizzled Receptors

“…The mechanistic contribution of heterotrimeric G proteins and downstream second messengers is still unclear, as discussed in section IX.A. However, second messengers, such as inositol phosphates, inositol pentakisphosphates (Gao and Wang, 2007), inositol 3-phosphates (Slusarski et al, 1997), phosphatidylinositol 4-trisphosphate (Qin et al, 2009), phosphatidylinositol 3,4,5-trisphosphate (Pan et al, 2008), calcium, or the cyclic nucleotides cGMP (Ahumada et al, 2002) and cAMP (Hansen et al, 2009), were reported to mediate WNT effects (see also Fig. 5).…”

“…RAP is closely related to RHO-like proteins and, among other stimuli, is regulated by cAMP through a cAMP-binding GEF called exchange protein directly activated by cAMP (de Rooij et al, 1998). With the above-mentioned signaling axis through cAMP (Hansen et al, 2009) in mind, it seems likely that exchange protein directly activated by cAMP could mediate RAP activation. This connection however, is purely hypothetical and awaits experimental confirmation.…”

“…Furthermore, WNT-5A stimulation of breast cancer cells induces cAMP production. Subsequent phosphorylation of a classic cAMP target, the Thr34 of dopamine and cAMP-regulated phosphoprotein of 32 kDa, through FZD 3 inhibits the formation of filopodia, necessary for cancer cell migration (Hansen et al, 2009). This suggests the existence of a FZD-G s -cAMP-PKA-CREB signaling axis.…”

Response to Wnt Signaling Pathways

“…In 2005, Chen and colleagues (6) first revealed PKA involvement in Wnt signaling. In 2008, Torii and colleagues (7) documented noncanonical Wnt/PKA signaling in dermal fibroblasts, and in 2009, Hansen and colleagues (8) demonstrated Wnt/cAMP/PKA signaling in breast cancer cells. Thus activation of this pathway by Wnts is not unique to osteoclast precursors, nor is it completely unexpected.…”