Serdar Bulun - US grants

The long range goal is to characterize the molecular and cellular mechanisms that are responsible for local biosynthesis of estrogen in endometriosis. The findings of our preliminary studies include (i) significant levels of aromatase P450 (P450arom) mRNA, protein and activity in stromal cells of endometriotic tissue but not in eutopic endometrium; (ii) P450arom gene expression directed by promoter II and aromatase activity in endometriotic stromal cells are induced strikingly by PGE2 via EP2 receptors or by cAMP analogs; (iii) differential binding of stimulatory (SF-1) and inhibitory (COUP-TFs) transcription factors upstream of promoter II account for the difference in aromatase expression in endometriotic and eutopic endometrial stromal cells; (iv) an unusually severe case of recurrent postmenopausal endometriosis resolved after treatment with an aromatase inhibitor. Thus, molecular aberrations in endometriotic tissue in contrast to eutopic endometrium give rise to increased local concentration of estrogen that promotes the growth and development of pelvic endometriosis. To determine the molecular basis for estrogen and PGE2 formation and estrogen action in endometriosis, we propose the following studies: Initially, we will characterize regulatory elements and differential binding of nuclear proteins to these sequences upstream of P450arom promoter II in endometriotic and eutopic endometrial stromal cells using deletion mutations of this regulatory region, site-directed mutagenesis and electrophoretic mobility shift assays. Transcription factors that bind to these regulatory sequences will be defined and their roles will be characterized in the regulation of aromatase expression in endometriotic stromal cells. This will be accomplished by screening expression libraries using DNA binding sites as probes and determining the effects of these factors on promoter II activity and aromatase expression. We will define mechanisms whereby PGE2 action and production are regulated in endometriotic tissue. The regulation of expression of EP2 receptors and COX-2 will be evaluated in both endometriotic tissue and eutopic endometrium. Finally, the in vivo significance of local estrogen biosynthesis and estrogen (and progesterone) action will be determined in a mouse model of endometriosis. The rate of formation and the site of surgically transplanted endometriotic lesions will be quantified in transgenic mice with disrupted genes of P450arom, estrogen receptor-alpha and progesterone receptor. The role of aromatase inhibitors in the treatment of endometriosis (in comparison with conventional treatments) will also be characterized in this model.

The long-range objective is to develop a clinically useful primate model for endometriosis-associated infertility in order to define underlying molecular biological and physiologic mechanisms and to test the efficacy of experimental medical treatments. Our preliminary results are indicative that the aberrant expression of aromatase and cyclo-oxygenase (COX)-2 and the deficiency of 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD-2) in endometriosis (in comparison with the eutopic endometrium) give rise to elevated local levels of estradiol, which is mitogenic for endometriotic tissue. These findings are clinically relevant, because treatment of a postinenopausal woman with an unusually-long- standing and severe case of recurrent endometriosis using an aromatase inhibitor nearly eradicated the disease. We hypothesize that excessive estrogen formation in endometriosis represents an important abnormality associated with infertility and a potential target in its treatment. Additionally, the deficiency of the progesterone-induced enzyme 17beta- HSD-2 in endometriotic tissue and severely altered ratio of progesterone receptor isoforms represent, in part, the molecular basis of a general progesterone resistance in endometriosis, which is also confirmed clinically by resistance of this disease to treatment with progestins. Results of our preliminary findings, however, can only be interpreted with caution, since the models used were either human endometriotic cell cultures or mice with transplanted uterine tissues. Baboon is an appropriate model, since these animals develop endometriosis spontaneously similar to the human disease and can be manipulated surgically and hormonally to answer fundamental questions. Consequently, we obtained institutional bridge funds for a year to perform a limited study on 6 baboons. The following studies, however, require much larger numbers of baboons. These can be performed extremely efficiently in the Institute for Primate Research in Nairobi, given the size of the colony and the level of enthusiasm and expertise in baboon endometriosis in this institution. The first specific aim of this application is to characterize the molecular and cellular mechanisms responsible for excessive estrogen formation and progesterone resistance in a baboon endometriosis model. Endometriosis will be induced by injection of menstrual material into the pelvic cavity. Alternatively, baboons that develop endometriosis spontaneously will be identified by laparoscopy. Activities of aberrant or deficient enzymes in endometriotic lesions will be determined using in vivo conversion assays. We will then determine the expression of aromatase, COX-2, 17beta-HSD- 2 and steroid receptor isoforms in endometriotic tissue biopsies. Finally, end results such as proliferation and apoptotic cell death in baboon endometriotic lesions will be determined in response to various hormonal treatments including aromatase inhibitors. The second specific aim is to determine the mechanism of infertility associated with endometriosis and the effectiveness of early medical treatment for prevention. Baboons with or without endometriosis will be monitored for ovulation. Possible defects in fertilization, implantation and early miscarriage will be detected. Once major mechanisms are characterized, we will determine whether infertility associated with endometriosis can be prevented or treated medically using novel strategies.

DESCRIPTION (provided by applicant): The long-range goal is to define the epithelial-stromal interactions responsible for aromatase expression and estrogen production in breast cancer tissue. The overall hypothesis is that upregulated aromatase expression in breast fibroblasts increases the tissue concentration of estradiol (E2), which enhances development and growth of malignant breast epithelial cells. This clinically pertains, since aromatase inhibitors (Als) are the most effective hormonal treatment of breast tumors. A single gene encodes aromatase, the key enzyme in estrogen biosynthesis, the inhibition of which by an Al effectively eliminates E2 production. We showed that 81% of estrogen production in breast cancer tissue was accounted for by the aberrant activation of the alternatively used promoter I.3/II region, which is coordinately regulated by PGE2 and its downstream signaling effectors/transcription factors p38/ATF-2, JNK/c-jun and BRCA1 in breast adipose fibroblasts. Selective inhibition of this promoter region may treat beast cancer while permitting aromatase expression at other body sites and thus obviate the key side effects of the current Als. The mechanisms that regulate the promoter I3/II region will be investigated under the following aims:1A. To identify p38 and JNK-dependent transcriptional regulators that stimulate aromatase expression in breast adipose fibroblasts. PGE2 secreted by malignant cells upregulates the aromatase promoters I.3/II significantly both in vivo in breast tumor tissue and in vitro in cultured breast adipose fibroblasts. We will test the hypothesis that the transcription factors ATF-2 and c-jun activated by p38 and JNK are necessary for PGE2-dependent transcriptional activation of aromatase via the promoter I.3/II region in breast adipose fibroblasts. 1B. To determine in vivo phosphorylation of p38, ATF-2, JNK and c-jun in fibroblasts in breast tumors. 2A. To define the role of BRCA1 in the regulation of aromatase expression in breast adipose fibroblasts. We will test the hypothesis that BRCA1 interacts with the transcriptional complex at promoters I.3/II to inhibit their transactivation, whereas reduced BRCA1 levels fail to repress aromatase expression. 2B. To determine in vivo differential expression of aromatase and other key PGE2/estradiol-related genes in BRCA1 haplo insufficient breast tissues from mutation carriers vs. noncarrier controls. Proposed studies may lead to new pilot trials of treatment and prevention using p38/JNK inhibitors in BRCA1 mutation carriers.

DESCRIPTION (provided by applicant): Uterine leiomyomata (fibroids) represent the most prevalent benign gynecologic disorder in the US. The cellular and molecular mechanisms regulating the development and growth of leiomyoma are not well understood. Our multidisciplinary team has designed 3 well-integrated projects focusing on Interactions between biologically critical hormonal pathways in uterine leiomyoma involving the transcription factors progesterone receptor (PR) and FOXO, the signaling pathway PI3K/AKT and the pro-fibrotic factor TGF-beta. Project I (Bulun) will be pursued to understand the mechanisms as to how antl-progestins such as RU486 reduce tumor size. We hypothesize that progesterone regulates a number of critical genes, that favors increased proliferation and decreased apoptosis of leiomyoma smooth muscle cells, whereas anti-progestins reverse this effect by enhancing apoptosis and decreasing proliferation. Project II (Kim/Chakravarti) will determine the role of the PI3K/AKT/F0X0 signaling pathway regulating leiomyoma cell growth and survival in response to progesterone. We hypothesize that progesterone Induces proliferation of leiomyoma cells through activation of the PI3K/AKT/F0X0 signaling pathway and that Inhibitors of the AKT pathway should override the proliferative effects of progesterone and promote apoptosis. Project III (Nowak) will define the mechanisms as to how antifibrotic drugs regulate leiomyoma growth. We hypothesize that the increased proliferation exhibited by leiomyoma smooth muscle cells Is due to a major shift in the extracellular matrix environment caused by increased synthesis of new, monomeric collagen type I by these cells. We will determine whether antifibrotic drugs may be an effective new treatment for leiomyomas. These projects are supported by an Administrative Core (Bulun) and Tissue Procurement and Cell Culture Core (Kurita). Overall, as part of our long range goal, all projects investigate local hormonal signaling regulating apoptosis and proliferation as biologic endpoints and test existing and upcoming pharmaceutical compounds that target these pathways in uterine leiomyomata. RELEVANCE (See instructions): Symptomatic uterine leiomyomata affect millions of US women and cause irregular uterine bleeding, anemia, recurrent pregnancy loss leading to more than 200,000 hysterectomies per year. Available treatments are limited due in large part to the fact that the mechanisms regulating the development and growth of these tumors are unclear. We propose integrated molecular, cellular and translational studies that should lead to a better understanding and future development of novel therapeutics for uterine leiomyomata.

The objective of the Administrative Core is to coordinate the scientific efforts, provide cost-effective administrative and fiscal support, coordinate the activities of the Internal and External Advisory Committees, and interact with NICHD project staff regarding initiative for the program grant. All projects will utilize this Core equally. The Core Director, Dr. Bulun, has responsibilities for the day-to-day management of activities and direction of personnel (Figure 1). Priorities with respect to use of Core services will be determined in consultation with program faculty member under the guidance of PI, and the Co-PI and Internal Advisory Committee when necessary. Policies related to Core functions are reviewed and approved at monthly meetings of the project investigators.

Uterine leiomyomas occur in approximately 77% of all women in the United States and can cause severe morbidity and infertility. To date, there is no effective treatment for leiomyomas besides hysterectomies. In this application, we hope to better understand the biology behind uterine leiomyoma growth at the cellular and molecular level. Specifically, there is evidence that the hormone progesterone causes leiomyomas to grow and once we understand how progesterone, through its receptor, PR, promotes leiomyoma growth, we can begin to study ways to inhibit this process. We have gathered evidence that progesterone can activate the AKT pathway which is a pathway that is involved in cell proliferation and survival. In addition, we have found that progesterone, through PR, can attenuate the action of a transcription factor, FOXOI, which is a member ofthe AKT pathway as well as a molecule that inhibits cell proliferation and promotes cell death. In light of this, we have gathered preliminary evidence testing the efficacy of a chemical compound, an AKT inhibitor for treating leiomyomas in an animal model. We observe that the AKT inhibitor causes significant tissue necrosis in leiomyomas that have been grown in immunocompromised mice. In this proposal, we hypothesize that progesterone can act at both non-genomic (signaling events) as well as genomic (at the gene) levels to promote leiomyoma growth. It does this by targeting the AKT signaling pathway and the downstream effector, FOXOI. Thus, inhibition of this pathway should provide a means of inhibiting leiomyoma growth and promoting tissue death. To test this hypothesis, we propose three aims. Specific aim 1 will investigate the elucidating the mechanisms that are involved in PR attenuation of FOXOI action on a gene called BM, that is involved in apoptosis (cell death). Specific aim 2 will explore how progesterone activates the AKT pathway and what the consquence of inhibiting this pathway would be on cell proliferation. Specific aim 3 investigates the efficacy of the AKT inhibitor in inducing apoptosis in leiomyoma cells as well as human leiomyoma tissues that are growing in immunocompromised mice. RELEVANCE (See instructions): Millions of women in the US are affected by symptomatic uterine leiomyomata causing significant morbidity. The studies in this project explore an important signaling pathway, PI3K/AKT/F0X01, that is activated by progesterone and associated with cell proliferation and survival. This study will provide a potential mechanism of action of PR in promoting growth of leiomyomas and translate these data into a potential treatment nf leiomyomas.; PROJEeT/

DESCRIPTION (provided by applicant): Endometriosis, the pathologic endometrium-like tissue on pelvic organs, develops and persists in part due to defective apoptosis and is associated with pelvic pain and infertility. During the previous funding period, we showed that retinoic acid (RA) induces endometrial expression of HSD17B2, an enzyme responsible for inactivating estradiol. Estradiol is a critical mitogen for endometriosis that is deficient of HSD17B2. A genome-wide gene expression profiling indicated strikingly lower expression of multiple RA-related genes in endometriotic tissue compared with matched endometrium: STRA6, a membrane receptor responsible for retinol (vitamin A) uptake, and CRABP2, which channels cytosolic RA to its nuclear receptor RAR1 to enhance apoptosis and reduce proliferation, were expressed at severely lower levels in endometriosis vs. endometrium. We find that these genes are primarily expressed in endometrial stromal cells and regulated by progesterone receptor (PR). We provide functional evidence that retinol and RA levels and expression of RA-target genes are significantly lower in endometriotic compared with endometrial stromal cells. Our overall hypothesis is that RA production and action is deficient in endometriosis compared with normal endometrium. This is responsible for deficient inactivation of estradiol and enhanced survival leading to persistence of endometriosis. The following aims will be pursued using established human and baboon experimental model systems of endometriosis. 1) To determine whether strikingly lower levels of STRA6 result in deficient RA production and action in endometriotic compared with normal endometrial stromal cells. We will test the hypothesis whether PR-dependent expression of STRA6 regulates retinol uptake leading to normal RA production in normal endometrial stromal cells, whereas reduced STRA6 expression causes RA deficiency and enhanced survival in endometriotic stromal cells. 2) To define the pathologic consequences of CRABP2 deficiency in human endometriotic stromal cells. We will test the hypothesis whether CRABP2 is necessary for RA/RAR1-dependent apoptosis and inhibition of proliferation in endometrial stromal cells, whereas in its absence, RA enhances cell survival via an alternative pathway. 3) To define the in vivo roles of the key genes for RA production and action in the baboon endometriosis model. We will test the hypothesis whether the key genes for RA production and RAR1-targeted action are expressed in a cycle-dependent manner in normal endometrium and significantly reduced in eutopic endometrium of baboons with endometriosis and severely deficient in pelvic endometriotic implants, where cell survival is predominant.

DESCRIPTION (provided by applicant): The long-term goal of the WRHRCDC at Northwestern University is to continue to foster and develop an outstanding mentored research training program for obstetrician-gynecologists, and to prepare our WRHR trainees (Scholars) to become independent investigators in women's health research. Since it has been first funded in 2005, our WRHRCDC has made major progress in reaching this goal. We have successfully trained 4 Scholars, who published 27 papers and received 18 grant awards including 6 from NIH, and met all our WRHRCDC benchmarks. The Dept of Ob/Gyn will continue to coordinate and administer the WRHRCDC Program and make full use of the scientific wealth and reproductive research infrastructure at Northwestern in its entirety. The key leadership at Northwestern made strong institutional commitments to the WRHRCDC Program. Sherman Elias, MD, Chair of Ob/Gyn and Serdar Bulun, MD, the Division Director of Reproductive Biology Research will continue to serve as the PI and WRHR Research Director, respectively. The Department of Ob/Gyn at Northwestern has traditionally recruited high quality residents, subspecialty fellows and junior faculty; currently 16% of this group are underrepresented minorities (URM). One of our current WRHR Scholars is an URM. Thus, we have a large and active pool of candidates for WRHR Scholar selection. These Scholars will have an opportunity to choose between highly competitive clinical research teams or laboratories conducting research in the areas of Reproductive Endocrinology and Infertility, Maternal-Fetal Medicine, Gynecologic Oncology and Reproductive Genetics and covering the reproductive portion of hypothalamic-pituitary axis, ovary, uterus, placenta and the fetus. The WRHR Scholars will interact with existing two P01, two U54, and one SCOR Center grants and one T32 training grant specifically in the area of obstetrics and gynecology. As the Northwestern WRHRCDC Program, we are fully equipped to continue to recruit top-quality obstetrician-gynecologists and train them to conduct research and compete for federal grants and retain them as they become independent investigators. Our excellent track record during the past funding period strongly supports our application.

PROJECT SUMMARY ? ADMIN CORE The Administrative Core will provide structure to facilitate the activities and meet the needs of the individual research Projects of the Center. The principal missions of the Core are: to reduce administrative barriers within and between Center Projects so researchers can focus on advancing reproductive science; foster team science by facilitating communication within the Center; engage the next generation of independent researchers; and evaluate Center progress in order to speed the pace and quality of research. The Core will reduce administrative barriers by providing centralized accounting, budgeting, and administrative services to the Project teams and to the overall Center. Furthermore, the Admin Core will ease the burden on the research teams by facilitating all grant renewals and providing regulatory support to ensure compliance for all projects and research personnel. The Core will also handle any human resources and immigration considerations. Utilizing established methodologies, the Core will foster team science to integrate the Center's research efforts and will support frequent and straightforward communication between the individual research Projects and Cores. The Core will organize in-person and virtual scientific meetings, which will increase collaboration and accelerate the pace and quality of translational research. The Core will send out request for proposals for pilot projects and assemble the Pilot Project Advisory and Assessment Committee to evaluate the applications. The Core will also evaluate Center progress by supporting Internal and External Advisory Committees, and by monitoring the progress of each Project. Through these means, the Core will facilitate and coordinate research efforts and support the dissemination of the Center's work to the reproductive health research community to further the understanding and knowledge base surrounding uterine fibroids in order to address the substantial health impact the disease has upon the nation.

Leiomyomas disrupt uterine function and cause recurrent pregnancy loss, excessive uterine bleeding, and anemia in 15-30% of reproductive-age women. There are few medical treatments available for leiomyomas, and many women opt to undergo hysterectomy. Understanding how leiomyomas develop is essential for identifying new non-surgical treatments. Preliminary data suggest that the clonal expansion of a defined stem/progenitor cell population is responsible for leiomyoma growth. Employing antibody-based sorting and in vivo tumor reconstruction, we revealed 3 distinct cell populations within a leiomyoma: 1-tumor progenitor CD34+/CD49b+ cells (5%) with self-renewing capacity, which express stem cell markers and the cytokine receptor RANK but are deficient in estrogen and progesterone (E+P) receptors (ER?/PR); 2-intermediately differentiated CD34+/CD49b- ?support? cells (7%) with lower tumorigenicity and high levels of ER?/PR and the cytokine RANKL; and 3-fully differentiated CD34-/CD49b- cells (88%) with negligible tumorigenic potential and high ER?/PR levels. Only the CD34+/CD49b+ cell population was indispensable for robust tumor formation in response to E+P in vivo. These cells, however, are ER?/PR deficient, suggestive of a paracrine mechanism. Our long-range objective is to define the molecular interactions between distinct uterine leiomyoma cell populations that are responsible for self-renewal, proliferation, and E+P responsiveness. The overall hypothesis is that a small stem cell population, devoid of ER? or PR, is essential for E+P-dependent growth, and that steroid-initiated alterations in ER?/PR-expressing support cells are transduced to the stem cells by RANKL/RANK signals. Microarray expression analysis suggested a hierarchical differentiation pattern: CD34+/CD49b+? CD34+/CD49b-? CD34-/CD49b- and multiple paracrine interactions between the CD34+/CD49b+ and CD34+/CD49b- populations, with the latter acting as support cells. E+P induced RANKL expression by over 100-fold in leiomyoma tissue. RANKL induced Cyclin D1 and BCL2 expression and expanded the CD34+/CD49b+ stem cell population. RANKL also activated ERK and NF?B in leiomyoma cells. Using xenografts of sorted leiomyoma cell populations under the mouse kidney capsule and cultures of tissue explants, we will test our hypothesis in vivo and in vitro in the following Aims: 1-Determine whether a small but distinct leiomyoma stem cell population serves as tumor progenitors. We will test the hypothesis that CD34+/CD49b+ cells are responsible for self-renewal, proliferation and robust tumor growth. 2-Define the biological roles of the RANKL/RANK pathway in leiomyoma progenitor/stem cell function. We will test the hypothesis that RANKL/RANK signaling acts in response to E+P and mediates self-renewal and proliferation of CD34+/CD49b+ tumor-initiating cells. In summary, our work will shift the therapeutic focus from the total leiomyoma cell mass to a small stem cell population and paracrine signaling. We expect to identify new therapeutic targets to help prevent tumorigenesis and reduce leiomyoma size and associated symptoms.

Uterine leiomyomas (UL), also known as uterine fibroids, are benign smooth muscle tumors with excessive depostition of extracellular matrix proteins. UL is a major health problem worldwide, because it affects almost 70-80% of all women and disproportionally African Americans, but still remains poorly understood. The long term goal of our team is to systematically discover novel mechanisms regulating key molecular events that contribute to leiomyoma. The immediate goal of this R01 application is to test the hypothesis that altered epigenomic signatures define normal myometrial tissues and leiomyomas, and therapy treated human tissues. Using genome-wide studies integrated with bioinformatic and other analyses, we will determine the epigenomic signatures in uterine fibrosis for the first time. This unbiased study will identify epigenomic differentiating features of normal and diseased tissues and may allow for development of Epitherapy (targeting the epigenome) for leiomyomas The proposed work is scientifically, translationally, and clinically significant and highly innovative because it represents the first systematic exploration of the epigenome in leiomyomas. Results obtained from this analysis will be used to generate new hypotheses to better understand the molecular underpinning of leiomyomas.

Uterine leiomyoma (LM, fibroids) are the most common tumor in women, disproportionately affect African- Americans, and cause irregular uterine bleeding and anemia, necessitating more than 200,000 hysterectomies annually in the US. Our long-term objective is to understand novel clinically relevant and medically targetable mechanisms responsible for the pathogenesis and growth of uterine LM in order to reduce associated morbidity. We propose and request funds to support the Northwestern Uterine Leiomyoma Research Center, comprising three highly coordinated and synergistic Research Projects, an Administrative Core and an Education and Outreach Core. Two mutually exclusive key driver somatic mutations (mut-) or rearrangements (-ra) affecting the MED12 and HMGA2 genes have been found in 85% of all LM, but the underlying mechanisms that cause tumorigenesis and tumor growth remain unknown. We propose to ascertain the effects of mut-MED12 and HMGA2-ra on epigenomic programming of LM cells, LM stem cell (LSC) function, development of heterogenic cell populations in these tumors, and genome-wide progesterone (P4) action. We will use cutting-edge in vivo models and high-throughput technologies to uncover novel mechanisms and identify genotype-specific therapeutic targets for developing precision medical treatments for LM. Project 1 (Bulun/Yin/Dai) will test the hypothesis that mut-MED12 alter genome-wide progesterone receptor (PR)- chromatin interaction signatures and associated histone modifications, thereby enhancing P4 action in the LM intermediate cell population (LICs), which provides a support niche for LSC survival and proliferation. Project 2 (Rajkovic) will test the hypothesis that distinct driver mutations affecting MED12 and HMGA2 determine cellular and molecular heterogeneity during LM tumorigenesis. Through cell fate tracing studies, we will determine whether mut-MED12 cells give rise to different cell populations in the myometrium that drive the formation of LM. Project 3 (Chakravarti/Wei) will test the hypothesis that overexpression of HMGA2 in LM alters 3D chromatin interactions and the epigenome to modify the development, progression, and therapeutic response of LM. As model systems, we will use LM tissues, antibody-sorted human LM cell populations and a human- equivalent mouse model of LM with mut-MED12 in uterine tissue. The Education and Outreach Core will support research activities performed within and across the Center by developing communication, outreach, and education strategies to promote health equity and eliminate disparities in LM, engaging the general public, students in the Chicago area, and the scientific and medical community. The Administrative Core will ensure that the Center achieves its aims and will synergize the individual Research Projects with the work of the Education-Outreach Core and other institutional cores; it will also solicit and coordinate the review of Pilot Projects. We anticipate that our synergistic approach will lead to the development of mutation- or epigenetic signature-selective therapeutic approaches to LM, moving the field into the realm of personalized medicine.

Although more than 1 in 4 men can be expected to develop symptomatic inguinal hernia, its mechanism is currently unknown. A subset of hernias may develop due to muscle fibrosis and myofiber atrophy leading to lower abdominal wall weakness. The long-term objective of this application is to determine the role of estrogen action in the etiology of lower abdominal muscle tissue (LAMT) fibrosis and atrophy associated with a subset of inguinal hernias. Aromatase, which converts testosterone to estradiol, is expressed only in the brain and testes of male mice. However, in men, aromatase is expressed in many additional tissues (muscle, fat) to provide physiologically necessary local quantities of estrogen. We generated transgenic humanized aromatase (Aromhum) mouse lines, each containing a single copy of the full-length human aromatase gene including its regulatory region, to mimic human patterns of estrogen production. Aromhum mice express the aromatase gene in peripheral tissues including the fibroblast component of the skeletal muscle tissue. LAMT has been found to be more sensitive to estradiol than the upper abdominal or quadriceps muscles, because the stroma of LAMT contains strikingly larger amounts of estrogen receptor-? (ER?)-expressing fibroblasts. Locally increased concentrations of estradiol in LAMT was associated with LAMT fibrosis characterized by progressive replacement of atrophic myocytes (muscle fibers) with ER?-rich fibroblasts and excessive extracellular matrix, resulting in formation of large inguinal hernias in >90% of Aromhum male mice by 24 weeks. However, there were no hernias observed in any of the wild-type (WT) littermates. Microarray expression analysis of LAMT at four weeks (before the appearance of hernias) showed activated profibrotic pathways in Aromhum vs. WT mice. We hypothesize that enhanced estrogen action caused by locally formed estradiol drives muscle fibrosis and myocyte atrophy, leading to the hernia phenotype affecting highly estrogen-sensitive portions of skeletal muscle tissue, which is LAMT in Aromhum mice. This resonates with the remarkable and parallel increases in inguinal hernia incidence and increased aromatase expression in skeletal muscle and fat in aging men. To ascertain the underlying mechanisms, we propose the following aims: 1. Determine whether treatment with an aromatase inhibitor, an estradiol antagonist, or a highly selective ER? antagonist prevents fibrosis, LAMT muscle atrophy, and hernia formation in Aromhum mice. The estradiol/ER?-mediated genomic mechanisms responsible for disordered proliferation of fibroblasts and extracellular matrix formation will be determined using integrative analysis of RNA-seq and ER?-ChIP-seq on LAMT and fibroblasts. 2. Determine whether the genetic disruption of ER? selectively in skeletal muscle fibroblasts affects LAMT fibrosis and hernia formation in Aromhum mice. In parallel, we will assess tissue steroid levels, aromatase and ER??expression, and estrogen responsive genes in abdominal muscle biopsies of men with or without hernia. We anticipate that this novel proposal will identify new drug targets and likely lead to the discovery of preventive approaches for hernia in high-risk populations.

Uterine leiomyoma (LM, fibroid) is the most common tumor in women. No long-term medical treatment is available. Each LM seems to originate from the clonal expansion of a single mutated LM stem cell (LSC) in the myometrium (MYO). LSC comprise 5% of tumor mass and differentiate into an intermediate cell population (LIC, 7%), which then become terminally differentiated cells (LDC) comprising 88% of the tumor bulk. Driver mutations of mediator complex subunit 12 (mut-MED12) occur in 70% of all LM. Progesterone (P4) and its receptor PR are essential for LM growth. PR-rich LIC transduce P4 signaling to PR-deficient LSC via paracrine factors to activate their proliferation. Ulipristal acetate (UPA), a PR-selective P4 antagonist, shrank LM and reduced its symptoms, but its use was halted because of risk of liver injury. Our overall goal is to define the role of genomewide P4 action in the etiology of mut-MED12?associated LM tumorigenesis and identify novel therapeutic targets. We found that mut-MED12 physically interacts with PR and genomewide PR-chromatin interaction landscapes are dramatically dysregulated in LM expressing mut-MED12 vs. normal MYO tissue carrying wild type MED12. Mut-MED12 enhances PR recruitment to cis-regulatory elements of P4 target genes encoding paracrine growth factors, cytokines and extracellular matrix proteins critical for LSC proliferation and LM tumorigenesis. Furthermore, the uteri of mice with a human-equivalent gain-of-function mutation in Med12 develop LM in response to P4, whereas Med12 knockout blocks P4/PR signaling in mouse uterus. We hypothesize that mut-MED12 alters PR-chromatin interaction signatures and enhances P4 action in LIC, providing a support niche for LSC survival and proliferation. Using ChIP-seq, RNA-seq, STARR-seq, and CRISPR/Cas9-gene editing strategies, and in vivo PDX and mut-Med12 knock-in mouse models, we propose the following Aims: (1) Determine whether PR-chromatin interaction loci specifically associated with mut- MED12 stimulate P4 action in a distinct stem-support cell population (LIC), which then send tumorigenic paracrine signals to increase LSC activity and tumor growth. We will test the hypothesis that mut-MED12 interacts with PR and alters its interaction with chromatin, thereby enhancing P4 responsiveness of LICs to activate gene transcription and paracrine signaling that support the function of adjacent LSC. (2) Define whether tumorigenic activity of mut-Med12 is mediated via altering PR-chromatin interaction landscapes in a human-equivalent mut-Med12 mouse model. We will test the hypothesis that mut-Med12 disrupts chromatin features surrounding PR-binding sites, thereby supporting gene transcription and pathways critical for LM tumorigenesis, whereas UPA shrinks LM via altering the aberrant PR-chromatin-epigenomic interactions and reversing inappropriate expression of disease-associated genes. Deciphering the genomewide mechanisms at a defined cell population level will help us identify genotype-specific novel targets associated with mut-MED12 for pharmacogenomics and precision medicine in the treatment of LM.

The overall goal of the Administrative Core is to provide a robust centralized administrative structure that will coordinate the activities of the proposed NCTRI Northwestern Uterine Leiomyoma Center researchers and provide operational support that will reduce the burden on its investigators, allowing them to focus on their research. The Core will ensure that the proposed Center achieves its aims and will synergize the efforts of the Research Project investigators with the work of the Education and Outreach Core and the Tissue Procurement and Cell Culture Core. The Administrative Core will coordinate the participation of the Research Project investigators in the community engagement and research training activities of the Education and Outreach Core. We have developed an optimally organized Tissue/Cell Core, run by an experienced gynecologic pathologist to support LM research at Northwestern; this will provide an efficient facility for procuring and banking tissue samples and for cell preparation and storage. This facility is entirely supported by intramural funds from Northwestern. The Administrative Core will ensure and coordinate the distribution of human tissues and cells to all Center investigators. The Core will also solicit and coordinate a rigorous review process for Pilot Project applications. Below are the aims of the Administrative Core, which capture the overall objectives. AIM 1. Reduce administrative barriers within and between Center Research Projects by providing funds and resource management services. AIM 2. Foster team science through established communication methodologies to integrate cross-disciplinary research and coordinate scientific efforts within the Center and externally. AIM 3. Provide an administrative framework to evaluate the progress of the Research Projects and the Cores to ensure a rapid pace of high-quality research and to solicit Pilot Project applications and rigorously select the best Pilot Project. All aims will be achieved through the efforts of the Core Director, Dr. Bulun and the Research Administrator, Erin Simpson, who will provide overall and comprehensive administrative support. Aim 1 is designed to provide support for financial and human resources management for the investigators, projects and cores; the Bulun and Simpson team will serve as the liaison to NIH and to central offices at Northwestern. Aim 2 will be accomplished through a series of communication efforts, including recurring meetings for research progress and communication updates and the coordination of the Research Projects and Core functions. Aim 3 will support the efforts of the Internal and External Advisory Committees that will review each project's progress and provide feedback on the overall Center and help review the Pilot Project applications and select the best Pilot Project. By aligning the Center through these activities, the Administrative Core will ensure the uninhibited progress of the Center's research, leverage team science and cross-disciplinary efforts to accelerate the pace of discovery, and evaluate progress and trajectory so the Center is consistently on target.

ABSTRACT GI tract microbiome is highly metabolically active, comparable to the host's liver. It has a significant role in the bioavailability and the physiological effects of chemicals within foods and medications, esp. those that undergo enterohepatic circulation (with excretion from the liver into the bile and the reabsorption back from the intestines). One group of chemicals that are extensively metabolized in the GI tract and/or undergo enterohepatic circulation are steroid hormones (such as estrogens, progestogens, androgens). The overall goal of this translational R21 proposal is to identify bacterial taxa and their candidate genes that contribute to the metabolism of steroid hormones within the GI tract. Thereby, this proposal lays the groundwork for individualized microbiome-based precision medicine therapies that can target steroid hormone metabolism in the GI tract. One specific example in which steroid hormones are related to a disease is breast cancer (BC): Exposure to high levels of estrogens is a well-known risk factor for BC. Although many hypotheses have been put forth that the GI tract microbiota play a role in BC primarily in terms of the enterohepatic circulation of estrogens, alterations in bacterial taxa in BC are not known. We undertook the first study to look at bacterial taxa in the gut mucosa of breast cancer patients and our data support our model for a role for bacterial taxa in breast cancer. We also identified two novel associations between steroid hormones and bacterial genera. This preliminary data suggests that a person's own gut microbiota may contribute to the development of BC by directly affecting the availability of steroid hormones. Importantly however, the majority of the bacterial taxa and their genes responsible for steroid hormone metabolism in the gut are still unknown. We hypothesize that the GI tract microbiome is different in BC; and that there are GI tract bacteria and their genes/proteins that are yet to be identified that directly metabolize steroid hormones. Hence, we propose the following Specific Aims: Aim 1. Characterize fecal bacterial taxa and steroid hormone levels in BC patients and controls with metagenomic sequencing and also with a second sample set. Aim 2. Identify bacterial taxa and their candidate genes that metabolize steroid hormones. We will perform metagenomics sequencing in patient and control samples. We will also determine the ability of whole bacterial communities from feces of BC patients and controls and two specific bacterial taxa in metabolizing steroid hormones. Sample will be examined with 16S rDNA sequencing, shot-gun metagenomics and metatranscriptomics to identify bacterial communities and their metabolic genes that are enhanced with steroid hormone exposure. Understanding which bacterial taxa may play a role in the metabolism of steroid hormones in the GI tract and identification of bacterial taxa and genes that are involved in steroid metabolism can potentially be used to design individualized microbiome-based therapies directed at these organisms.