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

Christian Hansen1, Jillian Howlin, Anders Tengholm

  • 1Experimental Pathology, Department of Laboratory Medicine, Lund University, CRC, 205 02 Malmö, Sweden.

The Journal of Biological Chemistry
|August 5, 2009
PubMed

Insights

Wnt-5a signaling inhibits breast cancer cell migration by activating dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). This novel pathway involves DARPP-32 phosphorylation, leading to reduced cell motility and filopodia formation.

Area of Science:

  • Cell Biology
  • Molecular Oncology
  • Cancer Metastasis Research

Background:

  • Tumor cell migration is critical for cancer metastasis.
  • Dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) is identified as an antimigratory phosphoprotein in breast cancer.
  • The precise molecular mechanisms linking DARPP-32 to cell migration require further elucidation.

Purpose of the Study:

  • To investigate the role of DARPP-32 in Wnt-5a signaling pathways.
  • To elucidate the downstream molecular events mediating DARPP-32's antimigratory effects.
  • To characterize the Wnt-5a/DARPP-32 signaling cascade in breast cancer cell migration.

Main Methods:

  • Recombinant Wnt-5a stimulation of MCF-7 cells.
  • Assessment of cAMP levels and DARPP-32 phosphorylation at Thr34.
  • Inhibition studies using protein kinase A (PKA) inhibitors and siRNA-mediated knockdown of Frizzled-3 or Galpha(s).
  • Analysis of cell migration, filopodia formation (Phalloidin staining), and Rho GTPase Cdc42 activity.
  • Investigation of DARPP-32 interaction with protein phosphatase-1 (PP1) and CREB phosphorylation.
  • Functional studies using dominant-negative CREB (DN-CREB) and DARPP-32 depleted T47D cells.

Main Results:

  • Wnt-5a triggers cAMP elevation and Thr34-DARPP-32 phosphorylation in MCF-7 cells via Frizzled-3/Galpha(s)/PKA.
  • Wnt-5a induces DARPP-32-dependent inhibition of MCF-7 cell migration and reduces filopodia formation by decreasing Cdc42 activity.
  • Phospho-Thr-34-DARPP-32 interacts with PP1, potentiates Wnt-5a-mediated CREB phosphorylation, and this pathway is crucial for the antimigratory effect.
  • The antimigratory effects of DARPP-32 were reversible in T47D cells depleted of endogenous DARPP-32.

Conclusions:

  • Wnt-5a activates a Frizzled-3/Galpha(s)/cAMP/PKA signaling pathway.
  • This pathway triggers a novel DARPP-32- and CREB-dependent antimigratory response in breast cancer cells.
  • The findings reveal a new mechanism by which Wnt-5a can inhibit breast cancer cell migration and metastasis.

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