In normal mammary development, the hormone prolactin (PRL) is critical for alveolar proliferation and differentiation. Increasing evidence supports the involvement of PRL in breast cancer, the leading type of cancer in women and the second leading cause (after lung cancer) of cancer death among women. In 2008, 40,480 women are expected to die from breast cancer in the U.S. The prolactin receptor (PRLR) is detected in 80% of human breast cancers and is overexpressed in breast cancer cells. Normal and tumor mammary epithelial cells synthesize PRL and PRLR, thus the PRL could behave as an autocrine growth factor for human breast cancer cells. These results suggest the need for a more complete understanding of PRLR signaling in breast cancer. Tyrosine (Tyr) kinase JAK2 was identified as a PRLR-bound signaling molecule. Identification of the proteins recruited to the PRLR-JAK2 and dissection of the signaling pathways that are subsequently activated will ultimately provide a basis for understanding PRL action. Preliminary data demonstrate that the serine-threonine kinase PAK1 associates with and is Tyr phosphorylated by JAK2. Two-dimensional peptide mapping identified three Tyr(s) of PAK1 which are phosphorylated by JAK2. Tyr phosphorylation of PAK1 by JAK2 was also shown to increase cell motility. In this grant we propose to examine the hypothesis prolactin-dependent JAK2 phosphorylation of PAK1 regulates PAK1 activity. Activated PAK1 regulation of target proteins may depend on phosphorylation events or/and protein-protein interactions, leading to the formation of a multiprotein complex that modulates the actin cytoskeleton, increases cell motility and invasiveness, mediates cyclin D1 gene transcription and affects tumorigenicity of human breast cancer cells.
Aim1 will determine the role of JAK2-phosphorylated PAK1 in regulating PRL-dependent actin cytoskeleton rearrangement, cell motility and invasiveness.
Aim2 will determine the role of PAK1 in regulating PRL-activated cyclin D1 gene transcription. Finally, Aim3 will determine whether JAK2 phosphorylation of PAK1 affects the tumorigenicity of human breast cancer cells in vivo. Because both PAK1 and PRL have been implicated in breast cancer, the proposed studies may ultimately fill out the existing gap between upstream PRL-PRLR-JAK2 events and downstream PAK1-dependent functions in our understanding of the mechanism of human breast cancer. Tyr phosphorylation of PAK1 by JAK2 is likely to represent a novel molecular target in the search for the etiology and treatment of human breast cancer.

Public Health Relevance

Prolactin (PRL) was discovered in 1928 as a pituitary factor able to stimulate mammary gland development and lactation in rabbits, as well as the production of crop milk in pigeons. A few years later, the name prolactin was given, based on its ability to stimulate milk production. More than 300 separate biological activities have been attributed to PRL. These biological functions are mediated by specific membrane receptors. These receptors are non tyrosine kinases, they transduce the signal via associated kinases that are recruited by the receptor and activated upon ligand binding. One of these kinases is JAK2 tyrosine kinase. We have recently shown that another protein - PAK1 - is a novel substrate of JAK2. We showed that JAK2 binds to PAK1 and makes PAK1 more active. Activated PAK1 contributes to better cell survival and cell migration. However, how these two proteins work together and the precise mechanism of their action is unknown. PRL is also involved in breast cancer. Breast cancer is the most commonly diagnosed cancer in women - nearly 1 in 3 (30%) of all cancers in women occur in the breast. Based on the current life expectancy for women in the United States, 1 out of 9 women will develop breast cancer in her lifetime - a risk that was 1 out of 14 in 1960. Much is still unknown regarding the molecular biological mechanism by which a normal cell becomes a cancer cell. Our long term goal is to understand the molecular mechanisms of PRL action and disregulation of which leads to human diseases including breast cancer. Toward this aim, we have started to analyze the relationship between JAK2 and PAK1. In the current proposal we will study how JAK2 and PAK1 increase cell migration and cell survival, and which genes are regulated by these two proteins. Understanding how these proteins work together will help to design new therapeutic approaches and possibly drugs for treatment of different human diseases and breast cancer.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Molecular and Cellular Endocrinology Study Section (MCE)
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Silva, Corinne M
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University of Toledo
Schools of Arts and Sciences
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Oladimeji, Peter; Skerl, Rebekah; Rusch, Courtney et al. (2016) Synergistic Activation of ER? by Estrogen and Prolactin in Breast Cancer Cells Requires Tyrosyl Phosphorylation of PAK1. Cancer Res 76:2600-11
Oladimeji, Peter; Diakonova, Maria (2016) PAK1 translocates into nucleus in response to prolactin but not to estrogen. Biochem Biophys Res Commun 473:206-211
Hammer, Alan; Diakonova, Maria (2016) Prolactin-induced PAK1 tyrosyl phosphorylation promotes FAK dephosphorylation, breast cancer cell motility, invasion and metastasis. BMC Cell Biol 17:31
Oladimeji, Peter; Kubohara, Yuzuru; Kikuchi, Haruhisa et al. (2015) A Derivative of Differentiation-Inducing Factor-3 Inhibits PAK1 Activity and Breast Cancer Cell Proliferation. Int J Cancer Clin Res 2:1-6
Hammer, Alan; Oladimeji, Peter; De Las Casas, Luis E et al. (2015) Phosphorylation of tyrosine 285 of PAK1 facilitates ?PIX/GIT1 binding and adhesion turnover. FASEB J 29:943-59
Hammer, Alan; Diakonova, Maria (2015) Tyrosyl phosphorylated serine-threonine kinase PAK1 is a novel regulator of prolactin-dependent breast cancer cell motility and invasion. Adv Exp Med Biol 846:97-137
Hammer, Alan; Laghate, Sneha; Diakonova, Maria (2015) Src tyrosyl phosphorylates cortactin in response to prolactin. Biochem Biophys Res Commun 463:644-9
Jay, Jennifer; Hammer, Alan; Nestor-Kalinoski, Andrea et al. (2015) JAK2 tyrosine kinase phosphorylates and is negatively regulated by centrosomal protein Ninein. Mol Cell Biol 35:111-31
Rider, Leah; Oladimeji, Peter; Diakonova, Maria (2013) PAK1 regulates breast cancer cell invasion through secretion of matrix metalloproteinases in response to prolactin and three-dimensional collagen IV. Mol Endocrinol 27:1048-64
Hammer, Alan; Rider, Leah; Oladimeji, Peter et al. (2013) Tyrosyl phosphorylated PAK1 regulates breast cancer cell motility in response to prolactin through filamin A. Mol Endocrinol 27:455-65

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