The actions of prolactin (PRL), a hormone utilized in normal and malignant mammary tissues, are mediated through its receptor, the PRLr. PRL acts to stimulate the growth, motility, and progression of human breast cancer. Following binding of ligand, a complex of the Nek3 serine/threonine kinase and the Vav2 guanine nucleotide exchange factor (GEF) associates with the PRLr. Our lab has demonstrated that Nek3 is required for Vav2 phosphorylation and GEF activity and is highly overexpressed in malignant (vs. normal) breast tissues. As a consequence of SiRNA-mediated Nek3 knockdown, the PRL-induced formation of filamentous actin, motility, and invasion of breast cancer cells was inhibited. To further correlate these in vitro findings with in vivo biology, a congenic Nek3-/- mouse was recently generated for our lab. The Nek3-/- mouse demonstrates a distinct mammary phenotype of attenuated terminal end buds and altered branching, and demonstrates a significant loss of epithelial cell progenitors. To better understand the function of the Nek3/Vav2 complex during the pathogenesis of breast cancer our lab has sought to identify downstream substrates of this complex through targeted proteomic analysis. These studies have demonstrated a PRL-induced association of the Nek3/Vav2 with the focal adhesion protein paxillin and the transcription factors Stat5 and NFAT. Indeed, our analysis demonstrates that Nek3 regulates paxillin phosphorylation and that the Nek3/Vav2 complex associates with and modifies the the chromatin of PRL-regulated genes, regulating the activity of NFAT and Stat5. It is the central hypothesis, therefore, of this proposal that the Nek3/Vav2 complex coordinately regulates the function of paxillin, Stat5, and NFAT, proteins all implicated in the pathogenesis of breast cancer. This hypothesis will be tested in three specific aims using representative breast cancer lines and models. First, the sites of PRL-induced, Nek3-mediated serine phosphorylation on paxillin will be identified and characterized as to functional significance through the use of mutagenesis and phospho-specific antibodies. Second, the intranuclear dynamics and regulation of the Stat5- and NFAT-mediated gene expression by the Nek3/Vav2 complex will be assessed. Third, the in vivo biologic relevance of the Nek3/Vav2 complex will be addressed through the study of Nek3-/- mice and breast cancer cell xenografts with altered levels of Nek3 activity.
The proposed studies on the Nek3/Vav2 complex in human breast cancer are highly relevant as they will detail how this complex, that is overexpressed in a breast cancer cell, regulates cancer cell metastasis and gene expression. In addition, these studies will provide a basis for the development and testing of targeted inhibitors of the Nek3/Vav2 complex in the treatment of this disease.
