Bert W. O'Malley, MD - US grants

SUBPROJECT ABSTRACT NOT AVAILABLE

Breast development and tumorigenesis are influenced by hormonal and growth factor signals whose responses are mediated by intracellular and cell membrane receptors. The involvement of intracellular receptors for estrogen and progesterone in the regulation of these processes is well documented. The function of estrogen and progesterone receptors in mammary tissue is influenced by the composition of the receptors themselves and by the factors (hormones, etc.) that regulate their transcriptional regulatory activity. For example, post-transcriptional variants of estrogen and progesterone receptors have been identified that display altered functional activity relative to wild-type receptors. The expression of some of these variants is increased in breast cancer cells. The activity of these variants clearly influences the overall activity of estrogen and progesterone receptors. Secondly, we and others have shown that the activity of steroid receptors for estrogen and progesterone, can be activated in the absence of a s specific steroidal ligand by extracellular signals including growth factors that stimulate intracellular phosphorylation pathways. Our results suggest that these receptors may provide common mediators through which hormone and non-hormone signaling pathways converge to regulate the expression of tar get genes that influence mammary cellular phenotype. The overall objective of this proposal is to examine the mechanistic contribution of estrogen and progesterone receptor composition and pathways for activation of receptors to the oncogenic refractory state in mammary tissue which is induced by high dose estrogen and progesterone treatment. The specific aims to accomplish the overall objectives are as follows. 1) To establish the quantitative complement of wild-type and variant estrogen and progesterone receptors in rat mammary epithelial and stromal tissue during mammary development, during MNU-induced tumorigenesis and after induction of a mammary state that is refractory to carcinogenesis by early treatment with estrogen and progesterone; 2) To establish the functional capacity of estrogen and progesterone receptor variants in vitro and in estrogen and progesterone receptor negative cell cultures.; 3) To determine whether the temporal and spatial expression of estrogen and progesterone receptor regulated target genes is age, hormone and growth factor regulated and to establish whether the refractory mammary phenotype that is induced by early administration of estrogen and progesterone is accompanied by alterations in the subsequent responses of these target genes to estrogen, or progesterone receptor can directly contribute to the oncogenic potential of rat mammary tissue. Together, these studies will provide valuable new insight into the biochemical mechanisms that underlie mammary carcinogenesis as well as th prevention of tumorigenesis by estrogen and progesterone.

The steroid hormones (estrogen and progesterone) stimulate growth, maturation and the development of new biochemical capacities in their endocrine target organs. Although it is widely accepted that steroid hormones exert major influences on the transcriptional process, the detailed mechanisms and the associated co-regulator proteins involved in gene transactivation are not yet defined in precise detail. The general objectives of our studies are to define the mechanism of steroid hormones and their receptors in regulating morphologic differentiation and biochemical specialization in target tissues. This will be accomplished by coordinating a network of investigations designed to uncover the mechanisms by which steroid receptors interact with nuclear regulatory proteins (co-activators, co-repressors, general transcription factors, chromatin modifiers, etc.) to effect target gene expression. We will emphasize experimental dissection of the protein-protein interactions which occur inside a living cell and during transcriptional process; we will define the co-regulator's role in modulating transcription and chromatin structure. In addition, we must understand the mechanisms of positive and negative regulation of receptor functional domains effected by various co-regulators. Our studies will utilize the human progesterone receptor, but to establish regulatory concepts, we will carry out selected experiments using human estrogen, thyroid hormone, and retinoic acid receptors. Cell-free binding and transcription approaches, technologies developed over the past five-year period of this grant, will be a corner stone of our methodology; all new concepts will tested finally in the milieu of the intact cell. These studies will involve aspects of nucleic acid and protein biochemistry, protein purification, cell biology and molecular endocrinology. It is expected that the understanding derived from this project will be relevant to the actions of natural sex steroid hormones relative to human physiology. The following proposed studies should also be pertinent to development of more precise theories for the biochemical mechanism of action of intracellular hormones and receptors in general.

Breast development and tumorigenesis are influenced by hormonal and growth factor signals whose responses are mediated by both intracellular and cell membrane receptors. Estrogen receptor (ER) and progesterone receptor (PR) function in mammary tissue is influenced by the overall receptor composition and by factors that regulate their transactivation potential. For example, some post-transcriptional ER and PR variants, whose expression is increased in certain breast cancer cells, display altered functional activity relative to wild-type receptors and clearly modulate overall receptor function. In addition to steroidal ligands, recent evidence reveals that ERs and PRs can be activated also by certain growth factors that initiate responses at membrane receptors that result in a ligand-independent stimulation of intracellular phosphorylation pathways. We wish to determine whether steroid receptors act as common mediators through which intracellular and membrane signaling pathways converge to regulate target gene expression and thereby influence mammary cellular phenotype, growth pattern, and neoplasia. Our specific objective is to examine the mechanistic contribution of ER and PR composition and receptor activation pathways to the oncogenic refractory state in mammary tissue that is induced by high dose estrogen and progesterone treatment. We propose to: 1) establish the quantitative complement of wild-type and variant ERs and PRs in rat mammary tissue during development, MNU-induced tumorigenesis and after induction of an oncogenic refractory state by early estrogen and progesterone treatment; 2) quantify the functional capacity of ER and PR variants in cells in tissue culture; and 3) determine the ability of growth factor signaling pathways to activate endogenous ER- and PR-dependent gene expression in rat mammary tissue (in vivo) during tumorigenesis and in the refractory phenotype induced by early estrogen plus progesterone treatment.

DESCRIPTION (provided by applicant): Much of our general understanding of coactivator function stems from characterizations of the p160/Steroid Receptor Coactivator (SRC) family of coregulators, which comprises SRC-1, -2, and -3 full length (SRC-3FL). Our prior work revealed that coactivators are controlled by a complex posttranslational modification (PTM) code that triggers a multitude of changes to regulate their molecular functions. Such PTM-induced changes explain how a single SRC coregulates diverse transcriptional outputs that enable the execution of wide-ranging physiological and pathophysiological responses, including even nongenomic activities in the cytoplasm and cell membrane. These non-nuclear roles for coactivators have been significantly expanded through our discovery of a new SRC-3 isoform (SRC-3?4) which functions as an essential molecular adaptor for growth factor induced signaling at the plasma membrane. Because our recent data suggesting that crosstalk between SRC- 3FL (from the nucleus) and SRC-3 ?4 (at the plasma membrane) is critical for coordinate control of cell proliferation and motility, expansion of this concept will be a major focus of this renewal application. Finally, our preliminary data show that Gene Regulated by Estrogen in Breast -1 (GREB1 (previously known as an estrogen target)) plays a critical role in the determination of the ER- positive luminal epithelial cell type and in the response of the differentiated target cell to estrogen. Therefore, a major element of this proposal will be to determine how ER, SRC-3FL and GREB1 interact to maintain identity of the luminal epithelial cell during its normal proliferative response to estrogen and promote loss of differentiation during mammary tumorigenesis.

The Baylor College of Medicine Center for Reproductive Biology Research is a goal-oriented Population Research Center, dedicated to innovative basic research into mechanisms involved in regulation of mammalian reproductive processes at the cell, physiological and molecular levels. The Center has created a rich environment fostering basic research of the highest quality, and fostering interdisciplinary exchanges and contacts suitable for further studies in this important area of biomedical science. The basic science interests of participating faculty involve all aspects of modem reproductive biology research, including steroid and peptide hormones, the ovary, testis, central nervous system, and approaches ranging from endocrine physiology to gene structure, function and regulation. Although concentrated in the Cell Biology Department, the Center has wide visibility in the local research environment, through its seminar, visitor and materials distribution functions. The Center operates ten research excellence Core Laboratories, making available to scientists conducting reproductive research the most rigorous and up-- to-date methodology and resources. These laboratories are directed by skilled and experienced research faculty members, all of whom are themselves innovative investigators in reproductive biology. The Center attempts through its unique setting in the Texas Medical Center to be a focus for interaction between basic scientists and the clinical investigators. A further goal is to apply new information and insights into new approaches to contraceptive development, to problems of infertility, and to problems arising from genetic disorders common to reproduction.

Squamous cell cancer of the oral cavity is a devastating disease in which the present therapies result in severe functional and cosmetic deformities. The primary objective of this project is to develop a new treatment modality for oral cancer which results in improved survival and less morbidity than standard surgery, radiation, or chemotherapy. The new treatment is based on a replication defective adenoviral vector that will transfer select therapeutic genes into established oral tumors. Adenoviral-mediated transfer of the "suicide gene", herpes thymidine kinase (tk), will result in direct cancer cell death, but in previous studies, has not resulted in long-term tumor eradication in more than 50% of experimental animals. Two cytokine genes which stimulate anti-tumor immune responses have therefore been chosen to investigate the synergistic effects of their actions in combination with the tk gene transfer. Local secretion of interleukin-2 (IL-2) has been effective in stimulating cytotoxic T-cell responses and causing tumor regression . Local secretion of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) results in helper T-cell stimulation and induces systemic anti-tumor immunity. A synergistic effect between the direct cancer cell killing of tk and the anti-tumor immune responses of IL-2 and GM-CSF should provide a powerful new treatment regimen for oral cancer. We will test our hypothesis that gene transfer can be effectively used to treat oral squamous cell carcinoma by addressing the following specific aims: 1) Maximize the efficacy of adenoviral-mediated delivery of the herpes thymidine kinase gene; 2) Determine the effects of cytokine gene transfer alone and in combination with tk therapy; and 3) Define the mechanism of active immunization against oral cavity carcinoma after adenoviral-mediated gene transfer. We anticipate that our investigations in this novel therapy will provide the foundation for innovative clinical trials in our oral cancer patient population.

Cationic lipid formulations complexed with the plasmid carrying hIL-2 have been shown to produce a significant anti-tumor response in animal squamous cell carcinomas of the head and neck. Based on these preclinical investigations, a Phase I clinical trial has been initiated. The primary purpose of this trial is to define the safety and tolerability of formulated hIL-2 plasmid when administered as a single intratumoral injection at three dose levels in patients with unresectable or recurrent/refractory squamous cell carcinoma of the head and neck. A secondary goal is to document expression of the IL-2 gene in squamous cell carcinoma by molecular biologic, histologic and immuno- histochemical methods.

The Center for Reproductive Biology Research has been serving the scientific community for approximately 25 years as a P30 Center. Since the center for Population Research of NICHD has changed their application format so that P-30 Center Grants will be eliminated, we have been advised to move to the U54 Center Grant mechanism. The theme chosen and approved for the renewal application is "Mechanisms Involved in Female Reproduction". The purpose of this Center will be to establish a comprehensive research and training program to investigate the molecular mechanisms governing female reproduction. This research will be accomplished by four research projects. Project I is titled "Steroid receptor activation pathways in female reproductive behavior", and will investigate the interactions of steroid hormone receptors and neurotransmitters in the regulation of female hypothalamic function. Project II, titled "Transgenic mouse models to study ovarian function," will investigate the role of Novel. 1, a member of the TGFb gene family, and Germ Cell Nuclear Factor, a member of the steroid hormone receptor superfamily, in oogenesis and folliculogenesis. Project III, titled "Cellular signals in ovulation and luteinization", will investigate the regulation and interactions of members of the steroid hormone receptor superfamily in folliculogenesis and luteinization. Finally, Project IV titled "Molecular approaches for the evaluation of human fertility", will identify the molecular interactions of human zona pellucida proteins with human sperm in the evaluation of human infertility. These research projects will be supported by four Cores. The Cores chosen are the Administrative Core (Core A), the Animal Core (Core B), the Cell Culture Core (Core C) and an Integrated Microscopy Core (Core D). The Administrative Core will provide the Center with centralized management. The Animal Core will serve as a resource for the maintenance and identification of transgenic and knockout mice, as well as, a centralization of the technology required to generate these animals and perform technical surgical manipulations on these animals. The Cell Culture Core will provide investigators with specific reagents for the culture of primary cells and cell lines, as well as, the expertise required to manipulate these cells. The Integrated Microscopy Core will aid investigators in the histological immunohistochemical and ultrastructural analysis required in the execution of their projects. The research in this Center for Reproductive Biology Research application will investigate the molecular regulation of female reproduction and fertility and foster the training of investigators in the field of reproductive biology.

Circulating ovarian steroid hormones, estrogen (E) and progesterone (P), have a profound modulatory influence upon an extensive network of neuronal and astroglial cells in the brain that ensure both the release of pituitary gonadotropins which triggers ovulation and the synchronous expression of behavioral receptivity. We have recently reported that the neurotransmitter dopamine (DA) exerts influences on hypothalamic steroid receptors in the absence of E and/or P. At present, the molecular, cellular and interneuronal mechanisms underlying ligand-independent regulation of steroid receptor-dependent function in the central nervous system (CNS) are undefined. Receptors for DA are expressed in those regions of the hypothalamus linked to reproduction. Since receptors for E and P have a profound influence on hypothalamic control of gonadotropin release required for ovulation and synchronous display of reproductive behavior, it is critical to understand the molecular mechanisms by which the membrane-bound DA receptor activate hypothalamic steroid receptors and their function. The goal of this project is to characterize critical signaling molecules for ligand- dependent (P) and ligand-independent (DA) progesterone receptor (PR)- dependent reproductive behavior. Since activation of PR-dependent gene transcription and reproductive behavior by DA appears to include the powerful effects of signal transduction and phosphorylation cascades initiated from the membrane, the specific aims of this project are: 1. To determine predominant second messengers and protein kinases in rat VMN associated with the induction of reproductive behavior by DA agonist and/or P treatment. 2. To ascertain whether changes in phosphorylated state of dopamine and cAMP-regulated phosphoprotein-32 (DARPP-32) and protein phosphatase inhibitor-1 (Inh-1) are associated with the induction of reproductive behavior by DA agonist and P. 3. To ascertain whether nuclear receptor co-activator molecules mediate PR-dependent reproductive behavior. 4. To characterize PR-containing cells in the ventrolateral (VL) region of the VMN and determine the specific cellular localization of PR, D5 DA receptor, DARPP-32, Inh-1, and SRC-1. 5. To characterize downstream, molecular targets in the VMN in PR-dependent transcription. The accomplishments of these aims will allow a determination of the molecular interactions of steroid hormone receptors and neurotransmitters in the regulation of female sexual behavior.

Steroid receptors regulate the expression of target genes involved in metabolism, development and reproduction. Steroid ligand hormones bind their cognate nuclear receptors and relocate to hormone response elements within target gene units. The activated receptors than accumulate coactivators which link them to the promoter complex and remodel chromatic locally. The p160/SRC family of coactivators appears to play a fundamental role in this regard. We hypothesize that individual coactivators for nuclear receptors have evolved as regulators of cellular metabolic pathways. Toward the goal of elucidating such pathways, we plan to carry out investigations of the genetic, regulatory and metabolic and metabolic functions of the SRC family of coactivators in vivo. We intend to (1) develop a genetic fingerprint of cellular expression (mRNA) and protein for the SRC family coactivators and determine the relative affinities of test receptors for these specific coactivators; (2) develop in vivo mouse models to understand the tissue preference of SRC family coactivators for estrogen (ER-alpha, ER-beta), progesterone and androgen receptors; (3) develop genetic fingerprints of downstream genes altered in specific and combinatorial Kos; and (4) do a thorough metabolic assessment of the effects of these co-activator alterations on carbohydrate and lipid metabolism in the mouse KO models. This information should lead to a greater understanding of steroid hormone tissue kinetics, individual phenotypic responses to hormones during development and following maturation, syndromes of partial hormone resistance, tissue-specific actions of Selective Receptor Modulators (SRMs), hormone-dependent cancers of breast, uterus and prostate, and the cross-communication of coactivators of cAMP/cytokine/Jak-State pathways which regulate carbohydrate metabolism, blood sugar levels, and lipid metabolism.

DESCRIPTION (provided by applicant): One of the most important issues facing the O&NR field is the curation, annotation, management and dissemination of the data that the prodigious pace of research in this field has generated. The quality of the tools and resources that have been placed at the disposal of researchers in the field, and the efficiency with which they have been used, have not yet been matched by the design and and execution of a coherent resource within which the data can be efficiently managed and evaluated. In order to sustain the pace of research in the field, we propose the implementation of a Functional Atlas for ONRs within which bioinformatic and research efforts can be pursued in a synergistic, multidisciplinary approach, on a common intellectual and technological platform. The primary directive of this Atlas is to gather and organize information, quantitative and qualitative, existing and new, relating to a number of key aspects of ONR biology. Commensurate with this directive, the goals of the Atlas can be distilled into two broad aims: (i) to execute research strategies designed to rapidly and efficiently elucidate those facets of ONR biology we deem most critical to its understanding; and (ii) to facilitate the generation of hypotheses, design of experiments and communication of results by scientists active in this field. Our initiative will provide a valuable service to the ONR community and the wider NR field by developing a web-accessible bioinformatics resource, in which current and emerging information on ONRs will be organized into more accessible and "user-mineable" forms. Should the formula we propose meet with the success we envisage, it could conceivably be applied to promote research in the field of NRs in its entirety.

[unreadable] DESCRIPTION (provided by applicant): Squamous cell carcinoma of the oral cavity and head and neck (HNSCC) is a devastating disease in which surgery, radiation and/or chemotherapy have not improved the 50 percent overall 5 year survival over the past 20 years. In an attempt to improve survival and reduce morbidity, gone therapy strategies are being developed for oral cancer. Despite encouraging preclinical data in many tumor types, initial clinical studies with adenovirus gene therapy have been disappointing. We posit that cellular differences exist even among head and neck cancers of the same histology that limit gone therapy responses. We further posit that variations in shared Coxsackie and adenovirus receptor (CAR) and integrin receptors play a major role in the transduction efficiency and translates to a significant variation in multi-tumor responses to adenovirus gene therapy strategies. We will test five hypotheses by addressing the following Specific Aims: 1) Determine the concentration of CAR, integrins, and FGF2 receptor on fresh human HNSCC samples and derived cell lines; 2) Establish the correlation between expression of CAR or integrin and Ad-tk anti-tumor effects and develop a FGF2 retargeting strategy in vitro that circumvents these limitations; 3) Quantify gene expression and therapeutic response to Ad-tk using both standard adenovirus and FGF2-R retargeted vectors in tumors established from 11NSCC lines. 4) Optimize direct linter-tumor injection therapy using circumventing treatment strategies and introduce systemic FGF2 retargeting therapy. We focus on a newly created fibroblast growth factor (FGF) conjugated adenovirus vector to develop a! Circumventing strategy that will improve gone transfer efficiency and corresponding therapeutic response. This novel FGF-2 receptor-based retargeting strategy may also allow safe and effective systemic delivery of tumor targeted adenovirus vectors. Five investigations regarding the role of adenovirus receptor and integrin expression on tumor cells will provide a platform of important gone therapy information that will lead to more effective and applicable preclinical animal studies and human clinical investigation. Adenovirus receptor or integrin testing prior to enrollment into a clinical trial may provide a means of selecting, stratifying, or assessing outcomes in head and neck cancer patients. This platform of information will also prove valuable to investigators who wish to circumvent limitations by developing and using alternative strategies such as FGF adenovirus retargeting.

DESCRIPTION (provided by applicant): The steroid hormones (estrogen and progesterone) stimulate growth, maturation and the development of new biochemical capacities in their endocrine target organs. Although it is widely accepted that steroid hormones exert major influences on the transcriptional process and induce gene activation, the detailed mechanisms of action of the receptors and their associated coregulator proteins are not yet defined in precise detail. The general objectives of our studies are to define the mechanism of action of steroid hormones and their receptors in regulating morphologic differentiation and biochemical specialization in target tissues. This will be accomplished by coordinating a network of investigations designed to uncover the mechanisms by which steroid receptors interact with nuclear coactivator proteins to mediate their effects on expression of protein products from target genes. We will emphasize experimental dissection of the coactivator's role in modulating initiation of transcription, alternative RNA processing, and importantly, in communicating signals to the genome from membrane receptors via intracellular signaling cascades and pathways. In addition, we must understand the role of sequential actions of distinct multiple high molecular weight coactivator complexes in the various substeps of nuclear receptor regulation of gene transcription. Finally, we will investigate the mechanisms by which specific steroid receptors accumulate coactivators at target promoters in a gene and cell specific manner to carry out these functions. These studies will involve aspects of nucleic acid and protein biochemistry, molecular biology, cell biology, endocrinology, and molecular endocrinology. It is expected that the understanding derived from this project will be relevant to the actions of the natural steroid hormones as well as selective receptor modulator drugs in human physiology. The following proposed studies should also be pertinent to development of more precise theories for the biochemical mechanism of action of intracellular hormones and receptors in general.

The objective of the Administrative Core is to provide centralized scientific and administrative management, financial controls and clerical and materials management support to all Projects and the Animal Core. The Administrative Core will provide necessary support for the organization and conduct of the Steering Committee Meetings, the annual meeting of the External Advisory Board, liaison with institutional officials and staff, and ongoing administration of the overall grant. The Core will also provide web and data-sharing support between BCM and Consortium members. RELEVANCE (See instructions): Efficient co-ordination of the activities of a multi-laboratory, multi-institutional research consortium requires a central administrative unit encompassing issues related to financing, purchasing, staffing and reporting, but extending also to issues related to the day-to-day scientific effort, data distribution and resource sharing. The Administrative Core of this PPG has competently handled these responsibilities over the last ten years and will remain an important component ofthe initiative going forward.

Description (provided by applicant): The Integrated Microscopy Core is organized to provide state-of-the-art imaging facilities in support of the projects comprising the Center for Reproductive Biological Research. This Core will provide access and training for light and electron microscopy, from routine brightfield to highly sophisticated, multispectral imaging and live cell studies, to digital ultrastructural studies, as necessary for Center investigators to achieve their project goals. The concentrated collection of expertise and diligently maintained high-end imaging equipment provides a resource readily available to Center investigators not possible for individual laboratories due to issues of cost, space and expertise.

Description (provided by applicant): The ovarian steroid hormones, estrogen and progesterone (P), have profound modulatory influences upon an extensive network of hypothalamic cells that ensures the release of pituitary gonadotropins which trigger ovulation and synchronous expression of reproductive behavioral receptivity in female rodents. Although we have found that the neurotransmitter dopamine (DA) exerts an influence on hypothalamic P receptors (PRs) in the absence of ligand, the underlying molecular and cellular mechanisms are defined only in part. The goal of this project is to characterize critical signaling molecules in the ventromedial nucleus (VMN) that regulate PR-dependent reproductive behavior. Relative to the female VMN and reproductive behavior, the specific aims are to: 1) identify the signaling mechanisms by which membrane GTP-coupled pathways modify the PR-mediated genomic pathway; 2) ascertain the membrane pathway for membrane anchored proteins by which cAMP/PKA signaling is augmented or "reinforced"; 3) determine the patterns of receptor-coactivator phosphorylation and histone posttranslational modifications that are inherent to the genomic and reinforcing pathways; and 4) identify potential target genes for PR- and DA-induced transcription in specific cells within the VMN that regulate reproductive behavior. In our studies, we will employ a combination of animal models (rats, PR knockout mice expressing a PRE-driven reporter protein), pharmacologic agents (antagonists, antisense oligonucleotides and small interfering RNAs), well-established procedures (membrane binding assays, blot analysis, in situ hybridization, immunohistochemistry) fluorescence, confocal/deconvolution microscopy, PR-dependent behavior and new technologies (real-time RT-PCR, ChIP assays for patterns of targeted histone modifications, phospho-specific antibodies, single cell expression profiling, microarrays, and laser capture of individual cells). By monitoring coordinate changes in PR-dependent behavior, intracellular signaling, molecular changes within the nucleus, and gene expression induced by P and DAergic stimulation of individual VMN cells that mediate this behavior, we should gain unique insight into the underlying cellular and molecular mechanisms associated with steroid receptor regulation in the female hypothalamus that results in PR-dependent behavior and ovulation. In addition, such analyses should contribute to the development of new therapeutic strategies for the regulation of reproduction.

[unreadable] DESCRIPTION (provided by applicant): Interactions between developing gametes and their neighboring somatic cells are essential for normal fertility. Studies over the last decade established that bidirectional communication between the oocyte and companion granulosa cells is essential for folliculogenesis and development of an egg competent to undergo fertilization and embryogenesis. The Matzuk laboratory and others delineated roles for oocyte-secreted factors such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in the regulation of folliculogenesis in mammals. In the process, the Matzuk laboratory produced key reagents (i.e., recombinant mouse GDF9, monoclonal antibodies that detect mouse GDF9 by immunohistochemistry and Gdf9 and Gdf9/Bmp15 knockout mice that are infertile) that were useful for the above studies and helped to identify cumulus granulosa cell genes that are directly regulated by GDF9. Recent evidence supports the presence of GDF9 and BMP 15 mRNA and protein in the ovary of primates, and the downregulation of GDF9 mRNA in women with polycystic ovarian syndrome. However, little is known about the significance of GDF9 and BMP 15 in oocyte-somatic cell communication during follicular development in primates or in human diseases. An important role for GDF9 and/or BMP15 can be implied from the recent data of Dr. Matzuk and colleagues that levels of expression of GDF9-regulated cumulus cell genes correlate with subsequent embryo development during assisted reproductive protocols in infertile women. The following translational study was designed to: 1) develop important GDF9 and BMP 15 related reagents; and 2) begin investigations to unravel the functions of GDF9 and BMP15 in oocyte-granulosa cell interactions during preovulatory follicular development in primates. The Specific Aims of the proposed U54 collaborative research initiative are as follows: 1) Produce large amounts of purified recombinant human GDF9 and BMP15 and anti-GDF9 and anti-BMP 15 neutralizing antibodies for in vitro and in vivo studies; and 2) Use bioactive GDF9 and BMP15 and anti-human GDF9/BMP15 antibodies for comparative analysis of gene expression and functional assays in mouse, nonhuman primate, and human pre-ovulatory granulosa cells/cumulus cell-oocyte complex systems. The current collaboration between Drs. Martin M. Matzuk and Richard L. Stouffer should yield important and clinically relevant findings for understanding human ovarian function, fertility, and reproductive diseases. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): BRCA1 is for the cellular to DNA and for reco`mbination, although required response damage homologous it is not yet clear exactly what role it plays in these critical processes. BRCA1 also acts as a co-activator of p53-responsive promoter elements, suggesting that modulating expression of genes that mediate apoptosis and cell cycling is part of this process. Yet, given the universal nature of these cellular processes, it is difficult to explain the striking differences in cancer risk between breast and other tissues in women with BRCA1 germline mutations. The answer will lie in a meticulous dissection of events that initiate and drive BRCAl-associated tumorigenesis. One important clue is that of hormone responsiveness in the tissues at highest risk. During the last funding period, we developed a murine model that recapitulates a known disease-associated human germline BRCA1 mutation with a conditional deletion of the C-terminus of Brca1. We will use that model to ask: What genetic events are necessary for BRCA1-related tumors to develop? The specific aims of this proposal are: Aim 1: To generate murine mammary hyperplasia and carcinomas in mice with a homozygous deletion of the Brcal BRCT domain. Aim 2: To define the early genetic changes in BRCA1 related tumorigenesis using genomic analyses of murine and human tissue Aim3: To define the role of estrogen in the initiation and progression of Brcal-associated mammary tumors. Aim 4: To evaluate the role of haploinsufficiency in BRCA1 mut/wt cells. The completion of this work will define pathways that are altered in BRCAl-associated cancers and will determine whether ER-negative breast cancers predominate in women with BRCA1 mutations because they arise from cells that are intrinsically ER negative or because other proliferative advantages replace ER signaling. We will determine whether heterozygous BRCA1 wt/mut cells have higher mutation rates than BRCA1 wt/wt cells, and whether this effect, if present, is enhanced by estrogen. A complete understanding of these events will lead to testable strategies for prevention, early diagnosis and treatment for women with BRCA1 mutations as well as an enhanced understanding of the molecular events that drive cancer development in general.

Steroid receptors regulate the expression of a myriad of target genes involved in metabolism, development,[unreadable] and reproduction. Nuclear receptors (NRs) are activated, usually by ligands, and relocate to specific DMA[unreadable] binding sites at target gene promoters where they accumulate an array of coactivators (or corepressors) that[unreadable] carry out the series of transcriptional substeps required for modulating gene expression. The SRC/p160[unreadable] family of coactivators appears to play a fundamental role in this latter regard. We hypothesize that NRs and[unreadable] their attendant coactivators have evolved as the primary regulators of adipocyte devevelopment and of[unreadable] metabolic pathways in fat cells and other metabolic tissues.[unreadable] Toward the goal of elucidating these pathways that control lipid and carbohydrate metabolism, we plan to[unreadable] carry out investigations of the genetic, structural, regulatory and metabolic functions of SRC family[unreadable] coactivators in cell extracts, in cells and in animals. Specific Aim 1: Definition of SRC-3 target genes[unreadable] regulated during adipocyte differentiation and study of the epigenetic regulators of SRC-3 function in fat[unreadable] cells. Specific Aim 2: Study of the role of protein stabilization of SRC-3 during adipocyte differentiation.[unreadable] Specific Aim 3: Characterization of adipocyte SRC-coactivator complexes. Specific Aim 4: Study of the[unreadable] specific contribution of each of the three p160 SRC family members and COUPTF-II in the cascade of[unreadable] events responsible for the adipogenic process.[unreadable] To accomplish these tasks, we will employ an integrative methodological approach to prove our hypothesis[unreadable] which uses the technoloigies of cellular biology, biochemistry, physical chemistry, physical chemistry,[unreadable] microscopy, nucleic acid and transgenic biology, and genetics and animal physiology. This information will[unreadable] lead to a much greater understanding of the contributions of coactivator biology to adipocyte function and[unreadable] should uncover new intervention points that would aid in the design of novel therapies for metabolic[unreadable] disorders in humans.

The objective of the Administrative Resource is to provide centralized scientific &administrative management, financial control services and clerical and materials management support to all Projects and Resources. The Administrative Resourcewill provide necessary support for the organization and conduct of the Steering Committee Meetings, the annual meetings of the External Advisory Board and ongoing administration of the overall grant. The Resource will also provide web support for communication and data sharing between consortium members.

[unreadable] DESCRIPTION (provided by applicant): The estrogen proliferative signal, which plays important roles in reproductive tissue function and tumorigenesis, is tightly controlled by the counterbalancing influences of coactivators and corepressors. Our objectives are to determine the importance of 'phosphorylation codes' in maintaining the balance in protein levels between steroid receptor coactivator-3 (SRC-3) and the corepressors, Represser of Estrogen Action (REA) and prohibitin (PHB). Together, they provide a balance which is necessary for normal cellular responses to estrogen. We will determine the physiological consequences of perturbing this coregulator homeostasis on steroid-induced mammary ontogenesis and oncogenesis. For SRC-3, we have identified two distinct phosphorylation codes which exert opposing effects on its protein stability. While atypical protein kinase C zeta promotes SRC-3 stabilization, glucose synthase kinase 3 (GSK3) promotes SRC-3 degradation. Like SRC-3, we have evidence that REA and PHB also operate as phosphoproteins. Based on the foregoing, we hypothesize that permutations of phosphorylation sites on SRC-3, REA and PHB constitute a 'phosphorylation code', which not only codes for coactivator/corepressor activation states but defines the stability of these coregulators. We predict that the phosphorylation code is a pivotal post- translational mechanism that maintains a critical balance between the countervailing influences of SRC-3 (growth promotion) and the prohibitin family (growth suppression) which together ensures a normal proliferative response to estrogen. To advance this hypothesis, we will (1) demonstrate that phosphorylation status determines SRC-3 protein stability with consequences for steroid-dependent cell proliferation in vitro; (2) disclose the in vivo effects of perturbing SRC-3 phosphorylation status on steroid-induced epithelial proliferation; (3) define the regulatory events which control REGgamma mediated degradation of SRC-3; (4) determine the mechanism by which phosphorylation status coordinately controls the protein levels and repressive functions of REA and PHB in vitro; and (5) define the in vivo corepressor functions of REA and PHB in steroid-induced epithelial cell proliferation. Mass spectrometric analysis, transcript profiling and state-of-the-art mouse genetics will be employed to successfully execute our stated aims. Apart from addressing a new control paradigm for estrogen signaling in the epithelial cell, these studies will furnish a broader conceptual framework for understanding abnormal proliferative responses to steroid-exposure. Beyond providing important new information on coregulator control of estrogen action during breast development and cancer, we believe the results from these studies will aid in the design of novel diagnostic approaches and/or therapies for the future treatment and prevention of breast cancer, regulation of fertility and other reproductive diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION, OVERALL (provided by applicant): The initial period of funding for the Functional Atlas of Nuclear Receptors has demonstrated the ability of the Consortium members to pursue the collective goal of generation and validation of high-content datasets and the distribution of these data to the wider community through a central web portal. In doing so, we greatly exceeded all of the specific aims articulated in the original application and fulfilled our mission of providing discovery-driven research datasets to the wider community with a rich data framework that provides a basis for new ideas and directions for their research. Moreover, we also demonstrated our ability to engage the wider nuclear receptor community to participate in the annotation of the resource by initiating the only active, peer reviewed, free access journal dedicated entirely to nuclear receptors (NRs). Over the four years of funding to date however, there has been an increasing emphasis placed by the sponsoring agencies involved on the need for the Consortium to establish strategies that will result in the generation and dissemination of datasets and methodologies of a more translational character. Accordingly, the scientific scope of the Consortium has shifted to reflect this emphasis. Our goals for the second phase of funding are: (1) to define the temporal and spatial physiology of NRs and to relate normal mechanisms to that of pathologic states in obesity, diabetes, cardiovascular, metabolic, toxic/environmental, senescent, oncologic, and reproductive diseases;(2) to define normal hormonal/environmental signaling to coregulator (coactivators and corepressors) complexes and to relate such signaling to the aforementioned diseases by construction of new animal models for human diseases. As in the previous funding period, we will continue to: acquire large data sets of information on NRs and coregulators that are not easily possible in R01 funded laboratories and to quickly disseminate this data world-wide to investigators in the NR/coregulator field;refine and develop new approaches to manipulate data and distribute it via the NURSA website;collect protocols, new techniques, investigative materials, animal models, and expert scientific opinions/perspectives, and provide them to the non-NURSA community for their use in laboratory/clinical investigations;and finally, we will accomplish all of the above in the context of modern genomic and proteomic technologies, and in the most cost effective and efficient manner possible.

Administrative Management; Grant; Institution; Investigators; NIDDK; National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Digestive Diseases and Kidney Disorders; Office of Administrative Management; Phone; Research Personnel; Research Resources; Researchers; Resources; Services; Telephone; conference; symposium

Steroid receptors regulate the expression of a myriad of target genes involved in metabolism, development,[unreadable] and reproduction. Nuclear receptors (NRs) are activated, usually by ligands, and relocate to specific DMA[unreadable] binding sites at target gene promoters where they accumulate an array of coactivators (or corepressors) that[unreadable] carry out the series of transcriptional substeps required for modulating gene expression. The SRC/p160[unreadable] family of coactivators appears to play a fundamental role in this latter regard. We hypothesize that NRs and[unreadable] their attendant coactivators have evolved as the primary regulators of adipocyte devevelopment and of[unreadable] metabolic pathways in fat cells and other metabolic tissues.[unreadable] Toward the goal of elucidating these pathways that control lipid and carbohydrate metabolism, we plan to[unreadable] carry out investigations of the genetic, structural, regulatory and metabolic functions of SRC family[unreadable] coactivators in cell extracts, in cells and in animals. Specific Aim 1: Definition of SRC-3 target genes[unreadable] regulated during adipocyte differentiation and study of the epigenetic regulators of SRC-3 function in fat[unreadable] cells. Specific Aim 2: Study of the role of protein stabilization of SRC-3 during adipocyte differentiation.[unreadable] Specific Aim 3: Characterization of adipocyte SRC-coactivator complexes. Specific Aim 4: Study of the[unreadable] specific contribution of each of the three p160 SRC family members and COUPTF-II in the cascade of[unreadable] events responsible for the adipogenic process.[unreadable] To accomplish these tasks, we will employ an integrative methodological approach to prove our hypothesis[unreadable] which uses the technoloigies of cellular biology, biochemistry, physical chemistry, physical chemistry,[unreadable] microscopy, nucleic acid and transgenic biology, and genetics and animal physiology. This information will[unreadable] lead to a much greater understanding of the contributions of coactivator biology to adipocyte function and[unreadable] should uncover new intervention points that would aid in the design of novel therapies for metabolic[unreadable] disorders in humans.

[unreadable] Steroid receptors regulate the expression of myriad target genes involved in metabolism,[unreadable] development, and reproduction. Nuclear receptors (NRs) are activated, usually by ligands, and[unreadable] relocate to specific DMA binding sites at target gene promoters where they accumulate an array of[unreadable] coactivators (or corepressors) that carry out the series of transcriptional substeps required for[unreadable] modulating gene expression. The SRC/p160 family of coactivators appears to play a fundamental[unreadable] role in this latter regard. We hypothesize that NRs and their attendant coactivators have evolved as[unreadable] the primary regulators of adipocyte development and of metabolic pathways in fat cells and other[unreadable] metabolic tissues. Toward the goal of elucidating these pathways that control lipid and[unreadable] carbohydrate metabolism, we plan to carry out investigations of the genetic, structural, regulatory[unreadable] and metabolic fingerprints of COUP-TFs and SRC family coactivators in cell extracts, in cells and in[unreadable] animals. We intend to: (1) elucidate the mechanisms by which COUP-TFII regulates adipocyte[unreadable] differentiation; (2) define the role by which SRC-3 controls the onset of the chain of transcriptional[unreadable] events leading to adipocyte developmental function; (3) define the roles of SRC coactivators in[unreadable] coordinately activating (or repressing) their target genes to effect spatiotemporal regulation of[unreadable] metabolic pathways in liver, muscle and fat cells; and (4) define the functional phenotypic effects of[unreadable] the SRC coactivators in controlling lipid and carbohydrate functions in the whole animal and[unreadable] examine the mechanism by which animal genotype modifies such coactivator phenotypic functional[unreadable] diversity. We will employ an integrative methodological approach to prove our hypothesis which[unreadable] uses cellular biology, biochemistry, physical chemistry, microscopy, nucleic acid and transgenic[unreadable] biology, bioinformatics, genetics and animal physiology in 4 Projects and 2 Core Labs. This[unreadable] information will lead to a much greater understanding of nuclear receptor and coactivator biology[unreadable] that would uncover new intervention points to aid in the design of novel therapies for metabolic[unreadable] disorders.

[unreadable] DESCRIPTION: Tumor cell invasion is the primary reason for morbidity and morality in patients with oral squamous cell carcinomas (OSCC). While it is clear that tumor cell invasion involves extracellular matrix (ECM) components, ECM receptors and proteases, the mechanism remains elusive. The goal of this application is to define the function of laminin-5 (Ln-5) in OSCC tumor invasion. Ln-5 is an ECM component that is proteolytically processed after secretion. Ln-5 has been found in invasive carcinomas and is important in both adhesion and migration of cells. The different proteolytic forms of Ln-5 appear to be responsible for this dual function. We have discovered that OSCC cells synthesize and secrete Ln-5 and that OSCC cells that fail to express processed Ln-5 are invasive while OSCC cells that process Ln-5 are non-invasive. Furthermore, our studies indicate that forced expression of processed Ln-5 in invasive OSCC cells suppresses migration while forced expression of unprocessed Ln-5 in non-invasive OSCC cells increases motility. Finally, we have found that processing of Ln-5 requires procollagen C-proteinase (BMP-1) expression, which is lost in invasive cells. The experiments in this application test the hypothesis that failure to process Ln-5 by BMP-1 leads to weak OSCC adhesion and an increase in OSCC motility and invasion while processing of Ln-5 by BMP-1 leads to stable OSCC adhesion and a reduction in OSCC cell motility and invasion. We will evaluate the expression and processing of Ln-5 and its receptors and assess whether Ln-5 processing influences OSCC migration and invasion. In addition, we will modulate OSCC adhesion, migration and invasion by altering Ln-5 expression and processing and the expression of the Ln-5 receptors. Finally, we will examine the contribution of BMP-1 in processing Ln-5 and determine whether manipulating expression and activity of this protease alters OSCC migration and invasion. This information will help expand our understanding of how the ECM and proteases influence tumor cell invasion in oral cancer. [unreadable] [unreadable]

Description (provided by applicant): The ovarian steroid hormones, estrogen and progesterone (P), have profound modulatory influences upon an extensive network of hypothalamic cells that ensures the release of pituitary gonadotropins which trigger ovulation and synchronous expression of reproductive behavioral receptivity in female rodents. Although we have found that the neurotransmitter dopamine (DA) exerts an influence on hypothalamic P receptors (PRs) in the absence of ligand, the underlying molecular and cellular mechanisms are defined only in part. The goal of this project is to characterize critical signaling molecules in the ventromedial nucleus (VMN) that regulate PR-dependent reproductive behavior. Relative to the female VMN and reproductive behavior, the specific aims are to: 1) identify the signaling mechanisms by which membrane GTP-coupled pathways modify the PR-mediated genomic pathway; 2) ascertain the membrane pathway for membrane anchored proteins by which cAMP/PKA signaling is augmented or "reinforced"; 3) determine the patterns of receptor-coactivator phosphorylation and histone posttranslational modifications that are inherent to the genomic and reinforcing pathways; and 4) identify potential target genes for PR- and DA-induced transcription in specific cells within the VMN that regulate reproductive behavior. In our studies, we will employ a combination of animal models (rats, PR knockout mice expressing a PRE-driven reporter protein), pharmacologic agents (antagonists, antisense oligonucleotides and small interfering RNAs), well-established procedures (membrane binding assays, blot analysis, in situ hybridization, immunohistochemistry) fluorescence, confocal/deconvolution microscopy, PR-dependent behavior and new technologies (real-time RT-PCR, ChIP assays for patterns of targeted histone modifications, phospho-specific antibodies, single cell expression profiling, microarrays, and laser capture of individual cells). By monitoring coordinate changes in PR-dependent behavior, intracellular signaling, molecular changes within the nucleus, and gene expression induced by P and DAergic stimulation of individual VMN cells that mediate this behavior, we should gain unique insight into the underlying cellular and molecular mechanisms associated with steroid receptor regulation in the female hypothalamus that results in PR-dependent behavior and ovulation. In addition, such analyses should contribute to the development of new therapeutic strategies for the regulation of reproduction.

Patients with node positive oral squamous cell carcinomas (OSee) have a 5-year survival of only 20-30% even with multi-modality treatment. Unfortunately, the current staging system does not predict osee tumor nodal disease or patient outcome, and no single gene has been shown to have sufficient prognostic utility. Moreover, state-of-the-art clinical imaging techniques can miss sub-clinical metastases. Discovering a more accurate and powerful way of predicting lymph node positive primary tumors and patient outcomes will require identification and characterization of the genes expressed within the tumor microenvironment. Using Affymetrix microarrays and tumor specimens comprised of both osee neoplastic and stromal components, we have discovered a gene signature that predicts which osee primary tumors will metastasize to the lymph nodes. In this proposal, using this osee preliminary node positive signature for comparison, we will test the hypothesis that a more accurate and reliable signature for prediction of osee lymph node disease can be identified by separately acquiring gene signatures from the osee neoplastic and the stromal components of lymph node negative and positive osee primary tumors. In Aim-1 we will use microarrays, laser capture microdissection (LeM), and statistics to identify the osee Neoplastic cell component node positive signature using osee lymph node positive and negative primary tumors. In Aim-2 we will use microarrays, LeM and statistics to define a node positive signature for the osee Stromal compartment using osee lymph node positive and negative primary tumors. Both molecular signatures will be validated at the RNA and protein level. Additionally, each signature will be tested for its ability to predict nodal disease using a blinded. cohort of osee tumors. Finally, all signatures will be compared using statistical platforms, and the best set of genes will be selected that can predict node positivity using the same cohort of osee tumors. This study will involve a multi-disciplinary team of collaborators with expertise in DNA and tissue microarrays, laser capture microdissection, tumor and molecular biology, statistics, medicine, head and neck surgery, and pathology as well as multi-institutional collaborations. Besides identifying the best signature for prediction of osee nodal disease, the results from this study will identify genes, biomarkers and signaling pathways in the tumor microenvironment that can be targeted for diagnostic, prognostic and therapeutic studies. The accurate and reliable lymph node metastasis gene signature identified in this proposal will have a profound clinical utility for reducing osee patient mortality and morbidity.

Project Summary The estrogen receptor-? (ER) and androgen receptor (AR) are the primary sex steroid hormone receptors in females and males, respectively. They drive reproduction-related gene expression programs by recruiting a series of coactivators (CoA) that form large, dynamic protein complexes at gene promoters and enhancers. While we know a great deal about these sex steroid hormone receptors at a functional and structural level, much less is known about the CoAs that interact with them at gene promoters and enhancers. Using advanced cryo-electron microscopy (cryo-EM) approaches, we were able to provide the first 3D understanding of ER engaged with CoA complexes at a gene enhancer. In this proposal, we plan to expand upon these findings by resolving the structure of receptor-CoA complexes on chromatin and with a more complete repertoire of proteins that comprise transcriptional supercomplexes. Also, in preliminary studies, we have made new breakthrough progress toward resolving an androgen receptor (AR)/SRC-2/DNA holocomplex. Importantly, this will generate a better understanding of NR/CoA holocomplexes to reveal new insights into the distinct roles for the N-terminal and C- terminal activation functions (AF-1 and AF-2) of both ER and AR. We will functionally interrogate the structures identified in our cryo-EM studies using innovative cell-free in vitro assay systems to understand how ER, AR, SRCs and co-CoAs distinctly regulate chromatin post-translational modifications, chromatin conformation and gene expression. Steroid receptor coactivators (SRCs) are the central scaffolding components of multi-protein NR/CoA/co-CoA complexes. New mechanistic insights into NR/CoA function must take into consideration the 3D structural conformation of the modular domains of NRs (DNA binding, AF-1 and AF-2 domains), CoAs and chromatin topology to generate conceptually novel insights into CoA biology and gene regulation by NRs. The studies proposed here should form a roadmap to therapy that can be used to develop new therapeutic drugs for reproductive disease states.

Coordinated control of the synergistic and antagonistic actions of estrogen (E2) and progesterone (P4) is required to achieve endometrial receptivity and successful embryo implantation. Steroid receptor coregulator-2 (SRC-2), a member of the SRC family of coregulators, is essential for murine endometrial receptivity and decidualization. SRC-2 can act as a coactivator or as a corepressor of P4 receptor (PR) mediated transcriptional responses in the murine uterus. Importantly, recent studies show that (a) SRC-2 is expressed in the same endometrial cell-types in the human as observed in the mouse;(b) knockdown of SRC-2 blocks the decidualization of human endometrial stromal cells in culture;and (c) dysregulation of normal SRC-2 expression is observed in the eutopic endometrium of patients diagnosed with endometriosis. Together, these findings suggest that SRC-2 represents a potent coregulator of steroid hormone-dependent endometrial function in the human, and that its dysregulation may be linked to endometrial pathologies, such as endometriosis. Based on the aforementioned, the following hypothesis is proposed: Steroid receptor coregulator-2 is an essential coregulator of P4 signaling which controls proliferative and differentiatlve programs required for endometrial receptivity and decidualization. To advance this hypothesis, we will (1) delineate the cellular and molecular mechanisms which underpin murine SRC-2 action in normal endometrial responses to steroid hormone exposure;(2) define the importance of human SRC-2 in normal endometrial responses to steroid hormone exposure;(3) establish the importance of unscheduled upregulation of SRC-2 expression in the breakdown of normal P4 induced endometrial responses that are required for the establishment of pregnancy;and (4) determine the role of SRC-2 in P4 control of endometriotic lesion growth. State-of-the-art mouse genetics, lentiviral technology to deregulate SRC-2 expression in an innovative experimental model for human endometrial function and endometriosis, and judiciously designed transcript profiling and microRNA array approaches will constitute a number of the inventive methodologies that will be used to successfully execute our stated aims. By addressing the limitations of conventional methods, this research program will significantly expand the field of endometrial coregulator study far beyond the current descriptive mode of analysis to a more interrogative field of investigation. Results from these studies promise not only to further our understanding of endometrial coregulator function but also aid in the design of novel diagnostic approaches and/or therapies for infertility.

The Administrative Core will support the Research Advisory Committee in its review of the scientific performance of the individual Projects and Cores and their Directors, and the Program's operation and direction as a whole. The Administrative Core will carry out all planning and arrangements for the annual review, and will assist the Committee members in the preparation of a report of their recommendations to the Program Director. Follow-up information on the implementation of recommendations will be provided to the members of the Advisory Committee, through the Administrative Core. As detailed below, all projects will require substantial statistical and bioinformatics support. In particular, analysis of data from molecular profiling experiments (be it at the DMA, mRNA, protein, or microRNA levels) is especially demanding and complex and requires expertise in bioinformatics.

Over the last decade, a number of transcription factors and coregulators have been characterized as important regulators of energy homeostasis. Despite these recent advances, relatively few molecular targets have been described that control energy balance in a holistic manner. Interestingly, our recently published data and preliminary studies now show that Steroid Receptor Coactivator-2 (SRC-2) is involved not only in energy dissipation, but also in the dietary absorption of fat. SRC-2 achieves this function through the coordinated regulation of a hepatic gene cassette, which includes the Bile Salt Export Pump (BSEP) that is necessary for bile acid secretion and reabsorption. Interfering with this process by hepatic-specific ablation of SRC-2 results in fat malabsorption in the gut, which can be rescued by dietary administration of bile acids, or by hepatic re-expression of BSEP. Furthermore, SRC-2 not only modulates the energy balance at both ends of the energy equation (accretion and dissipation), but itself is also sensitive to changes in energy status. In response to low cellular energy, we show that AMPK phosphorylates SRC-2, which increases its intrinsic transcriptional activity and drives these two metabolic regulators to target genes necessary for energy intake. Moreover, we found that SRC-2 enhances AMPK phosphorylation of histones, an event that has been reported to enhance target gene expression. Collectively, these findings form the basis of our PPG renewal for Project 1, where we hypothesize that SRC-2 functions as a master regulator of energy homeostasis in coordination with AMPK. RELEVANCE (See instructions): The precise mechanistic underpinnings ofthe AMPK/SRC-2 'energy sensor' axis are completely unexplored, as Project 1 is based upon entirely new findings. Regulation of whole body energetics has profound implications for the exploding co-morbidities of the metabolic syndrome that plague our country. As such, detailing such molecular pathways would open new intervention avenues for developing small molecule inhibitors to treat these metabolic diseases.

DESCRIPTION (provided by applicant): Over the past decade the field of NR signaling has generated a large volume of global datasets that collectively describe sequences of NR and coregulator genes (genomics); the regulation by NRs and coregulators of gene networks in specific target tissues (transcriptomics); protein-protein interactions required for the efficient function of NRs and coregulators (proteomics); specific sites of action of NRs in target gene promoters (cistromics); covalent modification of chromatin (epigenomics); and, more recently, specific functional endpoints in the form of regulation of cellular metabolic pathways (metabolomics). In order to effectively leverage these resources, we propose the continued development of the Nuclear Receptor Signaling Atlas (NURSA) web resource that will assemble and integrate these datasets, build user-friendly data analysis tools and present these to the community for hypothesis generation and validation. To fully engage the research community, we propose to administer a program of NURSA Data Source Projects (NDSPs) that will generate new global scale datasets that will be submitted to the website and integrated with existing datasets. We will also engage in outreach efforts that will offer members of the research community to participate in NURSA as Affiliate members, as well as in testing software tools during their development. With the involvement of the community, we anticipate that NURSA will be an important research resource for this field.

DESCRIPTION (provided by applicant): Nuclear receptors (NRs) and their coregulators act in concert to regulate the expression of myriad target genes involved in maintenance of metabolic homeostasis. Over the past ten years of this PPG, we have shown that NRs (specifically COUP-TFII) and their attendant coactivators, in particular the Steroid Receptor Coactivators (SRCs), have evolved as primary regulators of metabolic pathways in fat, liver and skeletal muscle tissues. This phase of the PPG will extend these observations by focusing our investigations on the molecular, cellular and physiological metabolic contributions of SRC-2 in tissues that contribute to the development of Metabolic Syndrome (MS). Our overarching hypothesis is that SRC-2 is a 'Master Genetic Regulator' for organs affected by MS. Specifically, Project 1 will focus on the hepatic functions of the AMPK/SRC-2 signaling axis as it pertains to dietary fat absorption and whole body energy accretion. This project will be dovetailed with Project 2, which is focused on defining the physiological role of SRC-2 as a master circadian regulator that controls liver and adipose metabolism. Continuing with this theme, Project 3 is aimed at defining the role of SRC-2 as an essential mediator of the beneficial effects of LRH-1 activation that combat NASH induced by MS. Finally, Project 4 will dissect the functional interactions of SRC-2 and COUP-TFII in skeletal muscle energy metabolism. The realignment of these individual projects within this central hypothesis has created a PPG application that is both highly innovative and extensively integrated. These research efforts will be tightly coordinated with a centralized animal core (Core A) and administrative core (Core B) that will serve to expedite availability of animal resources and promote the free exchange of information generated from these research initiatives. Overall, these proposed studies will utilize state of the art technologies and methodologies to test our hypotheses, which utilize cell biology, biochemistry, bioinformatics, transgenic and traditional genetic animal models. When complete, this PPG will afford a much greater understanding of NR and coregulator biology and will help define novel therapeutic leverage points for intervention of diseases associated with MS.

DESCRIPTION (provided by applicant): Nuclear receptors (NRs) and their coregulators act in concert to regulate the expression of myriad target genes involved in maintenance of metabolic homeostasis. Over the past ten years of this PPG, we have shown that NRs (specifically COUP-TFII) and their attendant coactivators, in particular the Steroid Receptor Coactivators (SRCs), have evolved as primary regulators of metabolic pathways in fat, liver and skeletal muscle tissues. This phase of the PPG will extend these observations by focusing our investigations on the molecular, cellular and physiological metabolic contributions of SRC-2 in tissues that contribute to the development of Metabolic Syndrome (MS). Our overarching hypothesis is that SRC-2 is a 'Master Genetic Regulator' for organs affected by MS. Specifically, Project 1 will focus on the hepatic functions of the AMPK/SRC-2 signaling axis as it pertains to dietary fat absorption and whole body energy accretion. This project will be dovetailed with Project 2, which is focused on defining the physiological role of SRC-2 as a master circadian regulator that controls liver and adipose metabolism. Continuing with this theme, Project 3 is aimed at defining the role of SRC-2 as an essential mediator of the beneficial effects of LRH-1 activation that combat NASH induced by MS. Finally, Project 4 will dissect the functional interactions of SRC-2 and COUP-TFII in skeletal muscle energy metabolism. The realignment of these individual projects within this central hypothesis has created a PPG application that is both highly innovative and extensively integrated. These research efforts will be tightly coordinated with a centralized animal core (Core A) and administrative core (Core B) that will serve to expedite availability of animal resources and promote the free exchange of information generated from these research initiatives. Overall, these proposed studies will utilize state of the art technologies and methodologies to test our hypotheses, which utilize cell biology, biochemistry, bioinformatics, transgenic and traditional genetic animal models. When complete, this PPG will afford a much greater understanding of NR and coregulator biology and will help define novel therapeutic leverage points for intervention of diseases associated with MS.

DESCRIPTION (provided by applicant): Exposure of cells or tissues to environmental stressors during critical periods of development can permanently reprogram normal physiological responses to increase susceptibility to disease later in life, a process termed developmental reprogramming. Developmental reprogramming is thought to occur via disruption of the epigenome, and is now appreciated to increase risk in adulthood for metabolic diseases, including obesity, diabetes, cardiovascular disease, and cancer. We were the first to identify non-genomic (or more accurately pre-genomic) signaling as a direct mechanism for endocrine disrupting chemicals (EDCs) to disrupt the epigenetic machinery and induce developmental reprogramming. Activation of kinases in pre-genomic signaling pathways that phosphorylate readers, writers and erasers is extremely attractive as a central mechanism for environmental stressors to engage the cell's epigenetic machinery. However, tremendous knowledge gaps remain in our understanding of how the epigenomic machinery is disrupted by pre-genomic signaling during developmental reprogramming. We do not know: 1) Which kinases/pre-genomic signaling pathways beyond PI3K/AKT are activated by obesogens in the liver (or other tissues); 2) Which epigenomic programmers are targeted by these kinases or how phosphorylation (or other PTMs) modifies their activity (up or down); nor 3) Which specific epigenetic marks placed on reprogrammed genes are altered to change gene expression and increase susceptibility to obesity (or other diseases) in adulthood. The goal of this application i to fill these knowledge gaps with a detailed mechanistic understanding of how EDCs utilize pre-genomic signaling to disrupt the epigenome, and to better understand how this developmental reprogramming alters metabolic set-points in the liver to promote obesity.

Steroid receptor coacfivator-3/amplified in breast cancer-1 (SRC-3/AIB1) is frequently overexpressed in breast carcinomas and can promote cell growth and resistance to endocrine therapies. In breast carcinomas overexpressing SRC-3, especially when in combination with activated HER2 signaling, the selective ER modulator (SERM) tamoxifen functions as an ER agonist. ER+/HER2+ tumors have a very poor response to tamoxifen treatment if SRC-3 is also overexpressed. Experimental targeting of SRC-3 can both a) augment the anti-estrogenic and anti-proliferative activity of tamoxifen in hormone-naive breast cancer cell lines, and b) restore the anti-estrogenic and anti-proliferative activity of tamoxifen in refractory breast cancer cell lines. Therefore, targeting SRC-3 expression and/or function is expected to enhance the activity of first-line conventional therapy and restore sensitivity in treatment-refractory breast cancer. Unfortunately, up to this point, SRC-3 has been considered undruggable because it lacks a natural ligand-binding site that can be inhibited by small molecule compounds and protein:protein interacfions are difficult to disrupt. Importanfiy, the stability and activity of the SRC-3 protein are strongly regulated via its post-translational modificafion (PTM) by upstream kinase signaling networks, including protein kinase C (PKC). PKC family members can phosphorylate and protect SRC-3 from proteasome-mediated degradation. Given the critical role of SRC-3 in breast cancer and the lack of FDA-approved SRC-3 targeting agents, this proposal represents an innovafive hypothesis-driven approach, based on key basic research and robust preclinical evidence, to establish PKC SMls as first-in-class inhibitors of SRC-3 expression and function and as targeted therapies for use in combination with conventional agents for ER+ breast cancer. Preliminary studies demonstrate that PKC inhibitors (including agents that have been well-tolerated in clinical trials in other diseases) can, at clinically achievable and tolerated concentrafions, decrease SRC-3 protein expression, exert anticancer activity in SRC-3-overexpressing cell lines, enhance the anticancer activity of endocrine therapy in sensifive cells lines, and restore sensitivity to endocrine therapy in resistant breast cancer cell lines and xenografts. RELEVANCE

No abstract provided

? DESCRIPTION (provided by applicant): The long-term objective of the Penn Multidisciplinary Consortium (PMC), specifically the Dental, Oral, Craniofacial Tissue Resource Center (DOCTRC) at Penn, is to enable rapid translation of tissue engineered and regenerative medicine (TE/RM) products via preclinical and clinical pipeline to address a broad spectrum of fundamental clinical needs in the reconstruction of the injured or dysfunctional orofacial complex sustained by ablative surgery, trauma and warfare. We capitalize on the University of Pennsylvania's great institutional strengths, including established translational research institutes, their geographic proximity linking the interdisciplinary schools within one city block, and a collaborative, integrated environment that allows faculty, researchers and practicing clinicians to transcend disciplines with research, technology, and patient care, specifically to develop safe, predictive and effective clinical strategies for regeneration of functional tissues o the human DOC complex. The following objectives are proposed: 1) To establish a robust DOCTRC infrastructure and effectively streamline translational research pipeline supported by several key translational and clinical research institutes, centers, and inter-disciplinary schools 2) To organize integrated Working Groups to specifically define and address the clinical needs of patients sustaining injuries to the craniofacial tissues, ablative surgery, and trauma; 3) To strategize development of innovative Interdisciplinary Translational Projects synergizing with the expertise of the PMC; and 4) To facilitate communication of research results, data and methods within the consortium and outreach to the research and healthcare community, and to the industry, to safely, rapidly and effectively translate TE/RM products to clinics Significance: The Planning grant will allow the development of an effective supporting infrastructure and organizational team to facilitate and catalyze the translation of TE/RM candidate products from pre-clinical studies to effective clinical strategies for regeneration of functional tissues of the human DOC complex.

The goal of this supplement request is to continue support for NURSA biocuration and analysis took development as they relate to NIDDK's mission. NURSA biocurators will carry out detailed, systematic curation of datasets using consistent structured vocabularies and constrained unique identifiers to provide for more accurate data retrieval. NURSA-curated datasets will be citable in full concordance with the FAIR principles. Datasets will be exposed for discovery through two major dataset search engines, NIH bioCADDIE DataMed and Thomson Reuters Web Of Science. Fourthly, journal staff in two major publishers in the field of mammalian cellular signal transduction and metabolic disease, Elsevier and Public Library of Science, will facilitate interactions between NURSA staff and authors of accepted articles to gain access to datasets. The web development team will integrate transcriptomics, ChIP-Seq, protein-protein interactions, and protein translational modifications in a single searchable resource. To ensure that we deliver to the community the curated content of most immediate use and impact, we will prioritize our curational efforts in descending order of dataset date of publication (newer > older), dataset throughput (discovery-scale vs focused hypothesis-drven) and signaling pathway level of interest (greater > lesser).

Over the past decade the field of NR signaling has generated a large volume of global datasets that collectively describe sequences of NR and coregulator genes (genomics); the regulation by NRs and coregulators of gene networks in specific target tissues (transcriptomics); protein-protein interactions required for the efficient function of NRs and coregtjiators (proteomics); specific sites of action of NRs in target gene promoters (cistromics); covalent modification of chromatin (epigenomics); and, more recently, specific functional endpoints in the form of regulation of cellular metabolic pathways (metabolomics). In order to effectively leverage these resources, we propose the continued development ofthe Nuclear Receptor Signaling Atlas (NURSA) web resource that will assemble and integrate these datasets, build user-friendly data analysis tools and present these to the community for hypothesis generation and validation. To fully engage the research community, we propose to administer a program of NURSA Data Source Projects (NDSPs) that will generate new global scale datasets that will be submitted to the website and integrated with existing datasets. We will also engage in outreach efforts that will offer members of the research community to participate in NURSA as Affiliate members, as well as in testing software tools during their development. With the involvement of the community, we anticipate that NURSA will be an important research resource for this field. RELEVANCE (See instructions): Nuclear receptors (NRs) and coregulators are important therapeutic targets in many different disease states including cancer, obesity, diabetes, inflammation, neurological disorders and senescent diseases. This application proposes a web resource for information and data analysis tools for the NR research community, as well as a community research grant program that will generate global-scale 'omics datasets for distribution via the web resource. These initiatives will have tangible benefits for the progress of research in the field towards developing novel NR- and coregulator-based therapeutics.

Project 3 -­ Project Summary    In addition to the well-­studied circadian rhythm, a cluster of genes that cycle at the second (12h period) harmonic  of circadian rhythmicity was discovered in several peripheral mouse tissues in vivo. Genes exhibiting apparent  12h  rhythmicity  were  enriched  in  endoplasmic  reticulum  (ER)  stress  and  unfolded  protein  response  (UPR)  pathways, which are universally conserved adaptive responses to cope with accumulation of unfolded protein in  the  ER.  Despite  these  initial  findings,  the  exact  prevalence  of  the  12h  rhythm  in  vivo,  its  relationship  with  the  circadian clock, how the 12h rhythm is established at the molecular level and its precise roles in regulating both  physiology  and  pathology  still  remain  elusive.  We  recently  developed  a  novel  mathematical  approach  to  decompose time-­series gene expression data and reveal hidden oscillations separate from the 24h rhythmicity.  We unexpectedly discovered that, in addition to the UPR genes, the 12h rhythmicity is much more prevalent than  was initially thought and is widely found in metabolic genes in mouse liver. We further uncovered prevalent 12h  oscillations in liver metabolism in vivo. The fact that the 12h rhythmicity remains intact in the absence of functional  24h  circadian  clock  suggests  that  the  12h  rhythm  is  established  and  maintained  by  an  independent  clock  component  distinct  from  the  24h  circadian  clock.  The  objective  of  this  proposal  is  to  use  the  liver  as  a  model  organ  to  test  the  hypothesis  that:  1)  there  is  an  equally  important  molecular  clock  establishing  the  12h  period  rhythmicity, which coordinates oscillations of ER stress and dynamic bioenergetic metabolism to ensure systemic  homeostasis, and 2) that the hepatic 12h clock is transcriptionally regulated by SRC-­3 and XBP1s. In Aim 1 the  transcriptional regulation of the 12h rhythm of gene expression and metabolism will be investigated with a focus  on the interplay between UPR TF XBP1 and coactivator SRC-­3 using ChIP-­Seq, RNA-­Seq, metabolomics and  mathematical  modeling  approaches.  In  Aim  2,  whether  XBP1s/SRC-­3  dependent  12-­hour  clock  dysregulation  contributes to chronic ER stress-­induced NAFLD will be determined. In Aim 3, whether XBP1s/SRC-­3 dependent  12-­hour clock dysregulation contributes to nutritional challenge-­induced NAFLD will be determined.   

Overall -­ Project Summary    Obesity is approaching epidemic proportions as a nutritional and pathological disorder in the U.S. and is closely  associated  with  important  syndromes  including  type  2  diabetes,  insulin  resistance,  hepatic  steatosis,  dyslipidemias,  hypertension,  coronary  heart  disease,  osteoarthritis  and  cancer.  Obesity  rates  in  the  U.S.  have  continued  to  worsen  whereby  nearly  70%  of  the  adult  population  is  overweight  and  the  yearly  medical  cost  of  treatment  for  obesity-­related  disease  is  estimated  at  $190  billion,  currently  6X  the  annual  NIH  budget.  Consequently,  obesity  is  anticipated  to  overtake  smoking  as  the  most  preventable  cause  of  adult  mortality.  Therefore, public health initiatives focused on identifying therapeutic options to combat the detrimental effects of  obesity-­related disease rely on an improved understanding of how alterations in metabolic tissue crosstalk alters  metabolism  to  favor  energy  accretion  and  deposition.  Our  highly  synergistic  and  integrated  Program  Project  team has identified previously unappreciated molecular mechanisms that highlight the coordinated actions of the  hypothalamus (brain), liver and white adipose tissues as gatekeepers of metabolic energy balance that become  dysregulated by overnutrition. By focusing on the metabolic actions of the Steroid Receptor Coactivator (SRC)  family  as  amplifiers  of  nuclear  hormone  receptor  (NR)/transcription  factor  (TF)  function  in  this  triad  of  energy-­ responsive  tissues,  we  have  exposed  transcriptional  reprogramming  as  a  key  molecular  determinant  in  the  disruption  of  normal  energy  homeostasis  arising  from  chronic  exposure  to  caloric  excess.  Such  a  complex  metabolic regulatory axis, which involves the interplay of multiple tissue systems (i.e. brain, liver, adipose), and  underlying transcriptional machinery (NR/SRCs) that maintain their homeostatic balance, can only be adequately  studied by a multidisciplinary research team with unique, yet synergistic, expertise. Leveraging the collaborative  framework of the NIDDK P01 Program Project, we have assembled such a team of scientists who are dedicated  to the overall objective of understanding the ?mechanisms? for the downstream tissue-­specific metabolic functions  of NR/SRC action that govern whole body energy balance.   

Core A (Administrative Core) -­ Project Summary    All  research  projects  of  this  Program  Project  (PP)  will  rely  on  the  use  of  administrative,  bioinformatics  and  statistical  support,  which  will  be  directed  by  Core  A.  The  Administrative  Component  of  Core  A  will  provide  centralized  scientific,  administrative  and  financial  management  as  well  as  computer  hardware,  software  and  network  support  services  (which  includes  data  sharing,  storage,  and  maintenance).  The  Administrative  Component of Core A will be responsible for monitoring the research progress attained by Projects 1-­4 as well  as the efficient use of Core B by each of the four projects. Core A also will be responsible for general support  of cost areas common to Projects 1-­4 and Cores A-­B. The Bioinformatics and Statistics Component of Core A  will  integrate  both  data  generated  from  Projects  1-­4  as  well  as  publicly  available  data  across  a  continuum  of  assays  ranging  from  cistromics  (via  ChIP-­Seq),  transcriptomics  (via  RNA-­Seq/microarray),  proteomics  (RPPA  and  DNA-­IP),  and  metabolomics  (via  mass  spec  platforms).  Data  acquisition  will  occur  in  established  core  facilities currently in place within the Department of Molecular and Cellular Biology at Baylor College of Medicine.  The Bioinformatics and Statistics Component of Core A will perform primary data analysis, extracting significant  features  from  each  series  of  OMICs  assays  proposed  in  Projects  1-­4.  The  major  contribution  of  the  Bioinformatics and Statistics Component of Core A consists of integrative analysis, wherein data from multiple  OMICs platforms are combined, leading to comprehensive biological insight and testable hypotheses. Core A  will use data from Encode, the NIH Epigenomic Roadmap, International Human Epigenome Consortium (IHEC),  and  scientific  data  repositories  such  as  the  NIH  Gene  Expression  Omnibus  (GEO)  and  Nuclear  Receptor  Signaling Atlas (NURSA). For scientific publications, Core A will generate visualizations of these data and results  by employing both off-­line and online software tools. To meet data dissemination guidelines, Core A will perform  data  deposition  to  the  appropriate  repositories,  including  but  not  limited  to  the  NIDDK  sponsored  NURSA.  Collectively,  these  efforts  will  provide  the  necessary  infrastructure  to  support  the  various  research  and  core  components  of  this  Program  Project  application.  The  successful  operation  of  this  administrative  team  is  unparalleled  and  is  essential  to  completion  of  the  overarching  scientific  objectives  of  our  integrative  research  teams.