Charles V. Clevenger - US grants

DESCRIPTION (adapted from applicant's abstract): The proposed research will identify at the molecular level the pathways utilized by the PRL-R in regulating the growth of normal and malignant T lymphocytes. First, the structural-functional determinants of the transmembrane and intracytoplasmic domains of the PRL-R will be mapped through the transfection of chimeric receptors into T cell lines. These chimeric receptors will be composed of the extracellular domain of the granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R) and deletional mutants of the intracellular domain of the PRL-R. The T-cell clones containing the chimeric receptors will then be used to study the contributions of the intracellular and transmembrane domains of the PRL-R to T lymphocyte proliferation and the nuclear translocation of PRL. Second, PRL-R binding proteins will be identified by coimmunop- recipitation studies, and their ability to bind to the mutant chimeric receptors will be examined.

The growth and differentiation of normal and malignant breast tissues is regulated by several growth factors. Prolactin (PRL), a known neuroendocrine hormone, is an important growth and differentiation factor for the human breast. We have recently shown widespread expression of PRL and its receptor (PRLr) in malignant and normal breast epithelium. These studies have also demonstrated that treatment of a breast cell line with anti-PRL antibody inhibited its proliferation and survival. these data indicate that breast epithelium utilizes PRL at the autocrine and endocrine level. The autocrine expression of PRL, therefore, may regulate the growth, differentiation, and pathology of the human breast. This hypothesis will be tested in this proposal by three specific aims using normal and malignant human tissues and cells. First, the function of autocrine PRL will be examined in breast tissues and cells in vitro under defined conditions. Second, the functional differences between dimers of PRLr isoforms involved in signal transduction will be investigated through the use of transfected homo- and hetero-dimeric receptor chimeras. Third, PRLr structure will be correlated with function through the in vitro and in vivo use of recombinant PRLr mutants. These aims will test whether the function and mechanism of action of autocrine PRL and PRLr contribute to the normal and pathologic development of the human breast. The studies proposed here will delineate this autocrine regulatory loop in human breast tissues and characterize the molecular basis for PRL-driven signal at interrupting the autocrine PRL loop in human breast cancer.

DESCRIPTION (Adapted from the Investigator's Abstract): The mandate of this proposal is to test the hypothesis that discrete structural motifs of the proto-oncogene, Vav, may significantly contribute to PRLr-Vav interaction, Vav function, and Vav nuclear import, using the PRL-dependent Nb2 T-cell model system. Specifically, three aims are proposed, including: (1) to identify the Vav domains necessary for PRLr-Vav interaction using mutant Vav and PRLr recombinants in in vitro binding assays and confirmed in vivo by transient co-transfection; (2) to examine the association of Vav structure with function in two independent model systems by the ability of mutant Vav expression constructs to mediate PRLr signaling; and, (3) to determine the functional role of intranuclear Vav in lymphocyte proliferation and to examine its mechanism of nuclear importation.

The growth and differentiation of normal and malignant breast tissues is regulated by several growth factors. Prolactin (PRL), a known neuroendocrine hormone, is an important growth and differentiation factor for the human breast. We have recently shown widespread expression of PRL and its receptor (PRLr) in malignant and normal breast epithelium. These studies have also demonstrated that treatment of a breast cell line with anti-PRL antibody inhibited its proliferation and survival. these data indicate that breast epithelium utilizes PRL at the autocrine and endocrine level. The autocrine expression of PRL, therefore, may regulate the growth, differentiation, and pathology of the human breast. This hypothesis will be tested in this proposal by three specific aims using normal and malignant human tissues and cells. First, the function of autocrine PRL will be examined in breast tissues and cells in vitro under defined conditions. Second, the functional differences between dimers of PRLr isoforms involved in signal transduction will be investigated through the use of transfected homo- and hetero-dimeric receptor chimeras. Third, PRLr structure will be correlated with function through the in vitro and in vivo use of recombinant PRLr mutants. These aims will test whether the function and mechanism of action of autocrine PRL and PRLr contribute to the normal and pathologic development of the human breast. The studies proposed here will delineate this autocrine regulatory loop in human breast tissues and characterize the molecular basis for PRL-driven signal at interrupting the autocrine PRL loop in human breast cancer.

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 neuroendocrine hormone prolactin (PRL) is an important growth and differentiation factor for the human breast. The mediation of these effects of PRL on breast issues occurs through the prolactin receptor (PRLr), a Type I transmembrane receptor and member of the cytokine receptor superfamily. Within the breast, four structurally and functionally distinct PRLr isoforms (the long, intermediate, deltaS1, and PRLBP) are expressed. The extracellular binding of ligand by the PRLr isoforms initially induces receptor dimerization and phosphorylation that activates intracellular PRLr-associated signaling proteins The triggering of these PRLr associated signaling networks results in the enhanced growth and motility of human breast cancer cells. Our laboratory has demonstrated that tyrosine phosphorylation of the PRLr is necessary for these actions. PRLr action is also modulated by stimulation with extracellular matrix, indicating the presence of signaling intermediaries between the integrins and the PRLr. We have recently demonstrated that the complex of the transmembrane protein SHPS1 and the protein tyrosine phosphatases (PTP) SHP1 and SHP associate with the deltaS1 PRLr and undergo tyrosine phosphorylation following PRL stimulation. Additional data have also revealed that the SHPS1/SHP1/SHP2 complex, in turn, can modulate the signaling and function of associated receptors. Given these findings, it is the central hypotheses of this proposal that phosphorylation of the PRLr isoforms and their interaction with the SHPS1/SHP1/SHP2 complex contributes to the in vitro motility and in vivo progression of human breast cancer. This hypothesis will be tested by three specific aims using tissue culture and xenograft models of human breast cancer. First, the mechanism and functional significance of PRLr isoform phosphorylation will be examined through molecular approaches. Second, the phosphorylation, association, and role of the SHPS1/SHP1/SHP2 complex during the PRLr signaling will be assessed by over-expression of wild type and mutant forms of these proteins. Third, the role of the phosphorylated PRLr isoforms and the SHPS/SHP1/SHP2 complex to breast cancer motility and metastasis will be evaluated. These studies will provide insight into the function of the newly discovered PRLr isoform and the associated SHPS1 complex, further mapping the function of PRL within the breast. Such structure/function analysis of the PRLr isoforms may ultimately provide the basis for novel therapeutic strategies aimed at interrupting the function of the PRL/PRLr complex in human breast cancer.

[unreadable] DESCRIPTION (provided by applicant): The Stat family of transcription factors is necessary for the growth and differentiation of mammary tissues. In mammary tissues, a significant means of Stat activation is the binding of the neuroendocrine hormone prolactin (PRL) to its receptor, the PRLr. PRL/PRLr binding results in the activation of receptor-associated signaling networks, such as the Jak/Stat pathway and the endocytosis and nuclear retrotransport of a complex between PRL and the prolyl isomerase cyclophilin B (CypB). The intranuclear PRL/CypB complex regulates Stat5-mediated gene expression and DNA binding. Using multiple approaches including yeast two-hybrid and transcription factor array analysis, we have demonstrated that the PRL/CypB complex regulates the interaction of Stat5 with multiple intranuclear proteins including the peptide inhibitor of activated Stat, PIAS3, the small ubiquitin-related modifier, SUMO2, and the transcription factor and proto-oncogene, c-Myb. It is the central hypothesis of this proposal that the temporal and spatial interaction of Stat5 with these proteins, as modulated by the PRL/CypB complex, regulates Stat5 function. This hypothesis will be tested by three specific aims using breast cancer and PRL-responsive cell lines both in vitro and in vivo. First, the structural basis for Stat5/PIAS3 interaction and the functional significance of this interaction will be mapped using complementary mutagenesis/overexpression and knockdown strategies. Second, the functional consequences of Stat5 sumolyation will be evaluated by cellular and biochemical approaches. Third, the association between Stat5 and co-activators/repressors, such as c-Myb will be assessed by targeted mutagenesis, small-interfering RNA (SiRNA), and chromatin immunoprecipitation (ChlP)-based methodologies. Given that our preliminary evidence indicates that the manipulation of these interactions results in the alteration of Stat5-mediated gene expression and the survival of breast cancer cells, our proposed studies are directly relevant to a better understanding of the pathophysiology of: human breast cancer, and as such, may provide the basis for novel therapeutic strategies aimed at modifying Stat function in this disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Receptor-associated tyrosine kinases, such as Jak2 and Src, serve as proximal mediators of ligand binding. Abundant data indicate that both of these kinases significantly contribute to the pathogenesis of breast cancer, and yet the mechanisms leading to their activation has remained uncertain. Our research, which has focused on the breast cancer-relevant receptor for prolactin (PRLr), has revealed that the peptidly prolyl isomerase (PPI), cyclophilin A (CypA) is required for the activation of these kinases. These findings would indicate that PPI activity of CypA is involved in a conformational restructuring of this receptor-kinase complex that contributes to its activation. Additional studie have revealed that the formation of a multimeric complex between the PRLr, Jak2, Src, and CypA is necessary of ligand-induced activation of the PRLr-associated signaling complex. At a translational level, these results have been further corroborated by our evaluation of a CypA knockout model and the successful use in breast cancer models of the PPI inhibitors cyclosporine A (CsA) and NIM811 both in vitro and in vivo. Taken together, our findings lead us to hypothesize that the intermolecular interactions between these proteins and the PPI activity of CypA result in the triggering of Jak2 and Src following ligand engagement, and that such events are highly relevant to the pathogenesis of breast cancer. This hypothesis will be tested in three specific aims, as follows: First, the functional role of protein- protein interactions withinthe PRLr/Jak2/Src/CypA will be evaluated using mutagenic, overexpression, and knockdown approaches within breast cancer cells. Second, in vitro kinase and soluble and solid state NMR spectroscopy will be used to assess conformer status with the PRLr/Jak2/Src complex as regulated by the PPI activity of CypA. Third, both gain- and loss-of-function approaches will be used in genetic and xenograft-based murine models to assess the role of protein interactions and PPI function within the PRLr complex during the pathogenesis of mammary cancer. The studies proposes are highly significant in that the will provide a molecular foundation for our understanding of the structure/function relationships during receptor-Jak2/Src activation as modulated by the PPI activity CypA and translate these discoveries through cellular to mouse models of breast cancer.