Cell and Experimental Pathology
Role of DARPP-32 in Breast Cancer Cell Signalling and Migration
Academic Dissertation by due permission of the Faculy of Medicine, Lund University, Sweden
to be defended at the main lecture hall, Pathlogy building, Malmö University Hospital , Malmö
on Friday 25th of January, 2008 at 9,15am
for the degree of Doctor of Philosophy,
Faculty of Medicine
Faculty opponent: Docent Per Svenningson,
Institute for Fysiology and Pharmacology,
Breast cancer is the most common form of cancer in women. While 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 anti-metastatic therapies that will stop, reduce or delay the spread and growth of breast cancer metastasis.
Low Wnt-5a expression in a primary breast tumor is associated with shorter recurrence free survival, suggesting that Wnt-5a expression acts to restrict breast cancer metastasis. Moreover, Wnt-5a is known to inhibit migration of breast epithelial cells in culture and expression of Wnt-5a potentiates activation of the receptor tyrosine kinase DDR1, a collagen receptor implicated in cell adhesion and migration.
This thesis describes the identification of DARPP-32 as a novel interaction partner to DDR1. We demonstrate that DARPP-32 inhibits MCF-7 cell migration and that this effect requires phosphorylation of Threonine-34, an event catalyzed by protein kinase A (PKA) and strongly induced by detachment of cells from the culture substrate. DARPP-32 mediated inhibition of cell migration proved to be dependent on DDR1 expression, consolidating a functional relevance of the interaction between DARPP-32 and DDR1. In addition, we found that Wnt-5a could directly trigger a cAMP response that resulted in phosphorylation of DARPP-32 and stimulation with Wnt-5a was necessary for DARPP-32 mediated inhibition of cell migration in wound healing assay.
The anti-migratory effects of Wnt-5a and DARPP-32 were reduced by dominant negative-CREB, which suggests that CREB plays a functional role in this signalling mechanism. Finally, we found that phospho-DARPP-32 inhibited the activity of the focal adhesion kinase (FAK) in MCF-7 breast cancer cells, and that MCF-7 cells expressing phospho-DARPP-32 displayed less filopodia formation.
These results suggest that DARPP-32 restricts the migration of breast epithelial cells via both a transcription dependent mechanism that involves CREB and a transcription independent mechanism affecting FAK. Pharmacological activation of this pathway may constitute a novel way of limiting breast cancer metastasis.