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 siRNA-mediated knockdown of Frizzled-3 receptor or Galpha(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/Galpha(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.

FIGURE 1.

Wnt-5a induces Thr-34 phosphorylation of DARPP-32 in MCF-7 and HB2 breast epithelial cells. A, left panel, expression of DARPP-32 in five different breast epithelial cell lines, detected by anti-DARPP-32 Western blotting. Right panel, comparison of endogenous levels of DARPP-32 in HB2 cells to exogenous levels of Myc-tagged DARPP-32 in transfected MCF-7 cells, detected by anti-DARPP-32 Western blotting. B, expression of Wnt-5a in five different breast epithelial cell lines, detected by an anti-Wnt-5a antibody. C, Western blot showing Thr-34 phosphorylation of DARPP-32 in MCF-7 cells expressing Myc-DARPP-32, after 5 min of stimulation with different concentrations of rWnt-5a, as detected by an anti-phospho-Thr-34-DARPP-32 antibody. D, upper panel, Western blot showing the kinetics of Thr-34-DARPP-32 phosphorylation upon stimulation with 0.4 μg/ml rWnt-5a in DARPP-32-expressing MCF-7 cells. Lower panel, densitometric analysis of three different Western blots. The data are given as means ± S.D. E, Western blot showing Thr-34 phosphorylation of endogenous DARPP-32 induced by Wnt-5a in HB2 cells as detected by anti-phospho Thr-34-DARPP-32 antibody. F, Western blot showing the time kinetics of Thr-75 DARPP-32 phosphorylation upon stimulation with 0.4 μg/ml rWnt-5a in Myc-DARPP-32-expressing MCF-7 cells, anti-phospho Thr-75 DARPP-32 antibody. G, left panel, Thr-34-DARPP-32 phosphorylation after 5 min of stimulation with either 0.4 μg/ml rWnt-5a or 0.1 μg/ml rWnt-3a. Right panel, densitometric analysis of five different Western blots. Wnt-3a or Wnt-5a stimulation was measured relative to no stimulation, which was set to 1. All of the Western blots shown are representative of at least three independent experiments.

FIGURE 2.

Wnt-5a-induced Thr-34 phosphorylation of DARPP-32 involves cAMP elevation and activation of PKA. A, Western blot showing that the PKA inhibitor RPcAMPS inhibits rWnt-5a-induced Thr-34-DARPP-32 phosphorylation in DARPP-32-expressing MCF-7 cells, as detected by anti-pThr34 antibody. RPcAMPS was added in serum-free medium 1 h prior to stimulation with 0.4 μg/ml rWnt-5a for 5 min. Stimulation with 1 μm forskolin for 20 min was used as a positive control. B, Western blot showing H89 inhibition of rWnt-5a-induced pThr34-DARPP-32 phosphorylation in Myc-DARPP-32-expressing MCF-7 cells, as detected by anti-pThr34 antibody. H89 was added in serum-free medium 1 h prior to stimulation with 0.4 μg/ml rWnt-5a for 5 min. Stimulation with 1 μm forskolin for 20 min was used as a positive control. C and D, evanescent wave microscopy recording of CFP and YFP fluorescence and the cAMP-dependent CFP/YFP ratio from individual MCF-7 cells expressing a fluorescent cAMP biosensor. The cells are stimulated with 10 μm forskolin (C, n = 8) or 0.4 μg/ml Wnt-5a (D, n = 12) and the prestimulatory CFP/YFP ratio was normalized to unity. E, Thr-34-DARPP-32 phosphorylation after 5 min of stimulation with 0.4 μg/ml rWnt-5a in cells treated with siRNA targeting the Gα_s protein or a scrambled sequence as a control. All of the Western blots shown are representative of at least three independent experiments.

FIGURE 3.

Wnt-5a induced phosphorylation of DARPP-32 leads to inhibition of cell migration. A, upper panel, migration of MCF-7 cells transfected with Myc-DARPP-32 or empty vector through collagen-coated wells (n = 8). All statistical significances were calculated in comparison with the empty vector control; Lower panel, Western blot showing comparison of rWnt-5a and detachment induced pThr34-phosphorylation, as detected by anti-pThr34 antibody. B, upper panel, wound-healing assay on MCF-7 cells in collagen-coated plates, transfected with either empty vector or Myc-DARPP-32 and left untreated or stimulated with 0.4 μg/ml rWnt-5a for 24 h (n = 12); lower panel, representative photographs from the wound-healing experiments. C, left panel, migration of T47D cells expressing endogenous DARPP-32 or depleted for DARPP-32 by siRNA targeting (n = 6), right panel, DARPP-32 expression level in T47D cells treated with siRNA targeting DARPP-32 or a scrambled control sequence. Both of the Western blots shown are representative of at least three independent experiments.

FIGURE 4.

DARPP-32 interacts with PP1 in MCF-7 cells. Upper panel, co-immunoprecipitation of PP1 and DARPP-32. MCF-7 cells transfected with either empty vector, Myc-T34ADARPP-32, or Myc-DARPP-32 expression plasmid. Binding of PP1 to DARPP-32 was verified by anti-PP1 immunoprecipitation followed by anti-DARPP-32 Western blotting. Lower panel, aliquots of the lysates to be used for immunoprecipitation experiments were tested for Myc-DARPP-32 expression to verify equal DARPP-32 levels in all Myc-DARPP-32-expressing MCF-7 lysates. The Western blot shown is a representative of three independent co-immunoprecipitation experiments.

FIGURE 5.

Involvement of CREB in Wnt-5a- and DARPP-32-mediated inhibition of cell migration. A, Western blot showing rWnt-5a-induced CREB phosphorylation in MCF-7 cells, as detected by anti-pCREB antibody. B, Western blot showing CREB phosphorylation in MCF-7 cells transfected with empty vector or Myc-DARPP-32 expression plasmid, non-stimulated or stimulated with 0.4 μg/ml rWnt-5a for or 1 μm forskolin for 10 min. Each Western blot is representative of six independent experiments. C, wound-healing assay with MCF-7 cells in collagen-coated plates, transfected with either empty vector, Myc-DARPP-32 vector, or both Myc-DARPP-32 and DN-CREB vectors. All cells were stimulated with 0.4 μg/ml rWnt-5a for 24 h (n = 12). D, migration of MCF-7 through collagen-coated wells transfected with empty vector, Myc-DARPP-32 vector, or both Myc-DARPP-32 and DN-CREB vectors (n = 6).

FIGURE 6.

Thr-34 phosphorylation of DARPP-32 leads to inhibition of filopodia formation. MCF-7 cells expressing Myc-DARPP-32 were plated onto collagen-coated glass slides overnight. A, non-stimulated cells or B, cells stimulated with 1 μm forskolin for 20 min were fixed and stained for Myc and pThr34-DARPP-32. C, upper panel, cells expressing Myc-DARPP-32 were stimulated with 1 μm forskolin for 20 min were fixed and stained for F-actin (with FITC-phalloidin) and pThr34-DARPP-32. Lower panel, quantitative analysis of filopodia formation in pThr34-DARPP-32-positive and -negative MCF-7 cells. The left panel gives the percentage of cells in each category (black bars: + pThr34-DARPP-32, gray bars: - pThr34-DARPP-32). The right panel gives the ratio of pThr34-DARPP-32-positive to pThr34-DARPP-32-negative for each category. D, Myc-T34A-DARPP-32 was expressed in MCF-7 cells, and cells were plated onto collagen-coated glass slides overnight, stimulated with 1 μm forskolin for 20 min, fixed, and stained F-actin (with FITC-phalloidin) and DARPP-32 (n = 5 for all experiments in this figure).

FIGURE 7.

Effect of Wnt-5a and involvement of DARPP-32 and CDC42 in filipodia formation. A, upper panel, Myc-DARPP-32 was expressed in MCF-7 cells, that were then plated onto collagen-coated glass slides overnight and subsequently stimulated with 0.4 μg/ml Wnt-5a for 24 h before being fixed and stained for F-actin (with FITC-phalloidin) and DARPP-32. Lower panel, quantitative analysis of filopodia formation in DARPP-32-positive and -negative MCF-7 cells. The left panel gives the percentage of cells in each category (black bars: + DARPP-32, gray bars: - DARPP-32) (n = 5). The right panel gives the ratio of DARPP-32-positive to DARPP-32-negative for each category. B, upper panel, T47D cells were treated with siRNA against DARPP-32 or scrambled control siRNA and stained for F-actin by FITC-phalloidin. Lower panel, quantitative analysis of filopodia formation in DARPP-32 siRNA- or scrambled siRNA-treated T47D cells. The left panel gives the percentage of cells in each category (black bars: DARPP-32 siRNA, gray bars: scrambled siRNA) (n = 5). The right panel gives the ratio of DARPP-32 siRNA to scrambled siRNA for each category. C, MCF-7 cells transfected with either Myc-DARPP-32 expression vector or empty vector were detached and re-plated onto collagen-coated plates for 0–60 min. Cells were lysed, and active Cdc42 was precipitated in a GST-PAK1 pull-down assay. A representative anti-Cdc42 Western blot of these pulldowns (n = 5) is shown.

FIGURE 8.

Frizzled-3 mediates Wnt-5a-induced phosphorylation of DARPP-32. A, Western blots showing Ror2 and Ryk expression in MCF-7 and T47D cells. B, RT-PCR expression analysis of Frizzled -2,-3,-4,-5, Ryk, and actin. For each expression analysis a negative control lacking reverse transcriptase (−RT) has been included. C, upper panel, RT-PCR of Frizzled-3 in MCF-7 cells transfected with either Frizzled-3 or scrambled siRNA. Actin served as a positive control and for each expression analysis a negative control lacking reverse transcriptase (−RT) has been included. Lower panel, knockdown of Frizzled-3 expression in MCF-7 cells verified by Western blot analysis using an anti-Frizzled-3 antibody. D, Western blot analysis showing Wnt-5a (0.4 μg/ml, 5 min)-mediated Thr-34 phosphorylation in DARPP-32-expressing MCF-7 cells co-transfected with either Frizzled-3 siRNA or scrambled siRNA. The Western blots and RT-PCRs shown are representative of at least three independent experiments.

FIGURE 9.

Model of Wnt-5a/DARPP-32-mediated inhibition of breast cancer cell migration. Autocrine or paracrine acting Wnt-5a ligand activates Frizzled-3 receptors that via Gα_s stimulates cAMP formation by adenylyl cyclases (AC). cAMP activates PKA, which phosphorylates DARPP-32 at Thr-34 and CREB at Ser-133. pThr34-DARPP-32 suppresses filopodia formation in part by reducing CDC42 activity. In addition, pThr34-DARPP-32 inhibits PP1, which in turn leads to enhanced CREB phosphorylation. CREB mediates transcription of genes that also regulate cell migration by an as yet unidentified mechanism.