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.
|Harrington, Katherine M; Clevenger, Charles V (2016) Identification of NEK3 Kinase Threonine 165 as a Novel Regulatory Phosphorylation Site That Modulates Focal Adhesion Remodeling Necessary for Breast Cancer Cell Migration. J Biol Chem 291:21388-21406|
|Fang, Feng; Zheng, Jiamao; Galbaugh, Traci L et al. (2010) Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells. J Mol Endocrinol 44:319-29|
|Galbaugh, Traci; Feeney, Yvonne B; Clevenger, Charles V (2010) Prolactin receptor-integrin cross-talk mediated by SIRPÎ± in breast cancer cells. Mol Cancer Res 8:1413-24|
|Kotsopoulos, Joanne; Tworoger, Shelley S; Campos, Hannia et al. (2010) Reproducibility of plasma and urine biomarkers among premenopausal and postmenopausal women from the Nurses' Health Studies. Cancer Epidemiol Biomarkers Prev 19:938-46|
|McHale, Kevin; Tomaszewski, John E; Puthiyaveettil, Ragunath et al. (2008) Altered expression of prolactin receptor-associated signaling proteins in human breast carcinoma. Mod Pathol 21:565-71|
|Swaminathan, Gayathri; Varghese, Bentley; Thangavel, Chellappagounder et al. (2008) Prolactin stimulates ubiquitination, initial internalization, and degradation of its receptor via catalytic activation of Janus kinase 2. J Endocrinol 196:R1-7|
|Clevenger, Charles V; Zheng, Jiamao; Jablonski, Elizabeth M et al. (2008) From bench to bedside: future potential for the translation of prolactin inhibitors as breast cancer therapeutics. J Mammary Gland Biol Neoplasia 13:147-56|
|Miller, S L; Antico, G; Raghunath, P N et al. (2007) Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization and motility of breast cancer cells. Oncogene 26:4668-78|
|Miller, Sommer L; DeMaria, Jamie E; Freier, David O et al. (2005) Novel association of Vav2 and Nek3 modulates signaling through the human prolactin receptor. Mol Endocrinol 19:939-49|
|Clevenger, Charles V (2004) Roles and regulation of stat family transcription factors in human breast cancer. Am J Pathol 165:1449-60|