Maria Diakonova - US grants

DESCRIPTION (provided by applicant): Listeria monocytogenes is a food-borne pathogen that can cause meningitis, meningoencephalitis, septicemias, abortions and, in some cases, gastroenteritis. The overall mortality rate is >20% and fetal or neonatal infection with Listeria has an even higher mortality. Listeria invades a broad range of cell types. Intracellular Listeria replicates in the cytoplasm of host cells and induces the polymerization of host actin filaments ("actin tails") at the bacteria surface using bacterial protein ActA. Actin-based motility allows Listeria to spread from cell to cell without leaving the protective intracellular niche, and is essential for pathogenesis. However, the mechanism underlying Listeria motility and spreading remains elusive. The adapter protein SH2- Bbeta regulates cell motility. I have implicated SH2- Bbeta in the motility of Listeria. Preliminary data revealed that Listeria in cells overexpressing wild type SH2- Bbeta demonstrates increased velocity (225% of control) while expression of SH2 domain-deficient mutants of SH2- Bbeta in host cells inhibits Listeria movement (by approximately 60%). In a cell-free system using Xenopus ooeyte extracts and purified GST-SH2-Bbeta, SH2-Ba increased the velocity of Listeria by 140% of control. I have shown that SH2- Bbeta binds to VASP/profilin two proteins that have been shown to participate in actin-dependent Listeria motility. This application tests the hypothesis that SH2- Bbeta promotes Listeria infection by stimulating actin-based motility. The first aim will determine whether VASP/ profilin directly bind(s) to SH2- Bbeta. The second aim will determine whether SH2- Bbeta interaction with VASP/profilin is required for Listeria motility. The third aim will test whether SH2- Ba is required for spreading of Listeria infection. The fourth aim will examine whether SH2- Bbeta is required for the virulence of Listeria. In addition to providing insight into the molecular mechanism by which SH2- Bbeta contributes to Listeria motility, the results of the application studies will increase our understanding of the fundamental mechanism by which Listeria spreads. These studies designed to identify new proteins and signaling pathways involved in Listeria motility may identify new therapeutic targets for preventing the rapid distribution of Listeria infection and thereby protect people from listeriosis.

[unreadable] DESCRIPTION (provided by applicant): 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 2002, 203,500 new cases of breast cancer were expected to be diagnosed, and 39,600 women were expected to die of the disease. 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 to growth promoting, anti-apoptotic pathways. 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 protect cells from apoptosis. This grant proposes to examine the hypothesis that PAK1 is a substrate for JAK2 and that in response to PRL, PAK1 is activated by JAK2-dependent Tyr phosphorylation and enhances PRL-dependent cell survival. Aim1 will verify that PRL promotes Tyr phosphorylation of PAK1 in vivo. Aim2 will determine whether JAK2 phosphorylation of PAK1 alters PAK1 kinase activity and/or ability of PAK1 to bind PAK1 targets. In Aim3 the effect of JAK2 Tyr phosphorylation of PAK1 on PRL-dependent cell survival will be determined. 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. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Breast cancer is the leading type of cancer in women and is the second leading cause of cancer death among women. 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. Tumor metastasis still remains the main cause of breast cancer death. Although with chemotherapy and radiation therapy, the prognosis has improved in some cases, these approaches often result in severe side effects. Two proteins - JAK2 and PAK1-have been implicated in breast cancer. Tyrosine (Tyr) kinase JAK2 was identified as a prolactin receptor-bound signaling molecule. The prolactin receptor is detected in 80% of human breast cancers and is overexpressed in breast cancer cells. Normal and tumor mammary epithelial cells synthesize prolactin and prolactin receptor, thus the prolactin could behave as an autocrine growth factor for human breast cancer cells. Serine-threonine kinase PAK1 has been linked to breast cancer by several lines of evidence: (a) PAK1 gene amplification and/or PAK1 protein up-regulation have been reported in breast cancers; (b) activated PAK1 increased cell invasion of breast cancer cells; (c) PAK1 is involved in the activation or regulation of several distinct mitogen-activated protein kinase cascades that lead to increased proliferation; [unreadable] (d) PAK1 contributes towards cancerous phenotypes by enhancement of cell survival. Preliminary data [unreadable] demonstrate that PAK1 associates with and is Tyr phosphorylated by JAK2. Two-dimensional peptide [unreadable] mapping identified three Tyr(s) of PAK1 which are phosphorylated by JAK2. Tyr phosphorylation of PAK1 by JAK2 was also shown to protect cells from apoptosis and increase cell motility. This grant proposes to examine the hypothesis that JAK2-dependent tyrosyl phosphorylation of PAK1 activates PAK1, increases cell proliferation, and causes anchorage-independent growth and invasiveness of human breast cell. Aim1 will determine whether JAK2 phosphorylation of PAK1 alters in vitro growth properties of human breast cells by promoting cells proliferation and/or anchorage-independent growth. Aim2 will determine whether JAK2 phosphorylation of PAK1 causes human breast cell invasiveness by increasing cell motility and/or decreasing cell adhesion. Although the significance of both JAK2 and PAK1 in breast cancer is widely acknowledged, the mechanism involved remains poorly understood. There is a gap between upstream JAK2-dependent events and downstream PAK1 and PAK1-dependent functions in our understanding of the mechanism of breast cancer progression. Exciting preliminary data suggest that PAK1 is an important signaling molecule phosphorylated by JAK2 and participating in cell survival. The results of the proposed studies will provide important insight into the fundamental mechanism by which tyrosyl phosphorylation of PAK1 by JAK2 regulates cell proliferation and invasiveness. It will also provide needed insight into the possible mechanism by which JAK2 and PAK1 participate in breast cancer. We believe that tyrosyl phosphorylation of PAK1 by JAK2 is likely to represent a novel molecular target in the search for theetiology and treatment of human breast cancer. [unreadable] PUBLIC HEALTH RELEVANCE: 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. Normal cell behavior is tightly controlled by multiple signaling pathways that ensure that cells proliferate only when they are required by the body. Cancer occurs when normal growth regulation breaks down, usually because of a defect in these signaling mechanisms. Metastasis, the spread of cancer to distant sites in the body, is in fact what makes cancer so lethal. A surgeon can remove a primary tumor relatively easily, but a cancer that has metastasized usually reaches so many places that cure by surgery alone becomes impossible. The ability of cells to metastasize is also regulated by multiple signaling pathways. Two proteins - JAK2 and PAK1 -have been implicated in the regulation of cell pathways that can lead to breast cancer but how they work together and the precise mechanism of their action is unknown. Our long term goal is to understand the molecular mechanisms that control cell division and cell motility and disregulation of which leads to human breast cancer. Toward this aim, we have started to analyze the relationship between JAK2 [unreadable] and PAK1. We showed that JAK2 binds to PAK1 and makes PAK1 more active. Activated PAK1 contributes to better cell survival that may promote cancer development. In the current proposal we will study how JAK2 and PAK1 increase cell proliferation and cell migration that leads to metastasis. In understanding of how these proteins work together will help to design new therapeutic approaches and possibly drugs for treatment of human breast cancer. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): 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.