Suzanne D. Conzen - US grants

Suzanne Conzen, M.D., recieved a balaurate degree, magna cum laude from Brown University, Her first exposure to bench science was in Dr. Charles Janeway, Jr's labortory at Yale Medical School . Following her second year of medical school she obtained an M.Sc. At the University of Tropical Medicine and Science before graduating from Yale in 1987. Following an internal medince residency at Cornell Medical Center she did her hematology and oncology fellowships at Dartmounth. Dr. Conzen wa awarded a Howard Hughes Physician Fellowship to study mechanism OS Sv40 mediated transformation in the Dartmouth Biochemistry under the mentorship of Dr. Chuck Cole. Dr. Conzen then moved to the University of Chicago's Ben May Institute to do a postdoctoral fellowship in the labortory of Dr. Nauseam Hay where she studied tumor-associated mutant c-Myc-medication apoptosis andd cell cycle progression. In 1998 Dr. Conzen was appointed Assistant Prefacer at the University of Chicago and established a research program studying mammary epithelial cell survival under the mentioship of Dr. Geoffrey Greene, director of the Breast Cancer Program at the Inversed of Chicago Cancer Cent. Dr. Conzen's work has shown that the glucocorticoid receptor (GR) has potent signaling effects in mammary epithelium and can transcriptionaly activate cell cycle-related kinases hypothesized to be important for cell survival and division. Because Dr. Conzen has little previous experience in steriod receptor biology or the analysis of intracelluar kinnase-mediated signaling pathways, the proposed research career development plan requests support to provide a comprehensive training period to work closely with Drs. Greene andd Marsha Rosner to determine the mechanism of GR- medicated survival signaling in breast cancer. Dr. Conzen proposes to characterize the gluocorticoid repersiveness and GR alpha and beta expression in breast tumor cell lines and to conurrently investigate the signaling pathways of GR-induced or repressed cellular kinase including SGK( serum and glucocorticoid induible kinases). Under the mentorship of Dr. Greene and with the assistance of Dr. Shutsung Liao and Dr. Mark Ratain, Dr. Conzen also proposes to eastablish anddan in vivo system to evaluate the GR in human breast cancer using an ER/PR negative human mammary carsinoma xenograft nude mouse model. The proposed career development plan will formulize Dr. Conzen's mentoring program with a defined research plan and ensure the protected research time needed for training in the methods and anyalytical skills required to develop an independent research program in survival signaling in malignant and normal breast epithelium.

DESCRIPTION (provided by applicant): The identification of anti-apoptotic signaling mechanisms has provided an experimental framework for understanding how pre-malignant and malignant cells can escape environmental signals that would normally initiate programmed cell death in human mammary epithelial cells (MECs). we have recently defined a novel glucocorticoid receptor (GR)-mediated anti-apoptotic signaling pathway that is induced by physiological concentrations of glucocorticoid and inhibited by GR-specific antagonists (anti-glucocorticoids). Although it is well established that glucocorticoids activate the glucocorticoid receptor (GR) and in turn regulate transcription, the precise mechanism of glucocorticoid-mediated survival signaling in epithelial cells is not known. Recently, our laboratory has found that ectopic expression of a GR-transactivated gene, serum and glucocorticoid-inducible kinase (SGK), inhibits apoptosis in MECs. This finding suggests that SGK, a putative serinethreonine kinase, is a bona fide transcriptional target of GR activation that orchestrates UR-initiated survival signaling. In addition to transcriptional activation by the GR and post-translational phosphorylation by upstream kinases, we have discovered that SGK protein expression is regulated via proteasome-mediated degradation, revealing yet another level of regulation of SGK activity. The overall goal of this grant is to understand the extent and manner through which OR-induced SGK expression contributes to MEC survival. To this end, we will first determine the specific steps in the apoptotic cascade that are inhibited by glucocorticoids and then determine whether or not SGK activity can substitute for GR activation. Second, we will examine the role of OR activation and PI3-K/PDK-l.2 signaling in activating SOK's kinase activity and in subsequently inhibiting apoptosis. Finally, we will characterize the proteasome-mediated mechanism underlying wild type SGK's rapid proteolysis. Together these studies are likely to lead to a detailed understanding of the mechanisms regulating the GR-SGK anti-apoptotic pathway.

DESCRIPTION (provided by applicant: Identifying anti-apoptotic signaling pathways driving therapy resistance and relapse in breast cancer (BC) is essential for developing effective strategies for improving outcome in patients with aggressive breast cancer (BC). To this end, we have identified an important role for glucocorticoid receptor (GR)-mediated anti- apoptotic signaling in both in vitro and in vivo pre-clinical models of GR-expressing human breast cancer (BC). Specifically, in the previous funding period we identified mechanisms underlying direct cross-talk between GR signaling and the PI3-K and MAPK pathways through identifying GR-regulated genes encoding kinases (e.g. SGK1) and phosphatases (e.g. MKP1) required for GR-mediated cell survival in estrogen receptor, progesterone receptor and HER2-negative BC (triple-negative BC or TNBC) models. This renewal proposes to expand our studies to achieve a comprehensive understanding of pathways mediated by glucocorticoid receptor (GR)-mediated cell survival in TNBC. We have collected, curated, and analyzed data from several early BC gene expression studies with long-term clinical follow-up and examined the association between high GR expression in primary BCs and risk of relapse in over 1300 patients. We were excited to find that high GR expression in estrogen receptor-alpha (ER)-negative BCs indeed associates with a significantly increased risk of early relapse, supporting our previous discoveries. More interestingly, we unexpectedly found that risk of relapse was reduced in patients with ER-positive, GR over expressing tumors. This exciting finding opens up a new area of study: Investigating how GR-signaling differs in BC depending upon ER context. We hypothesize that GR-mediated gene expression in ER-negative BC (including TNBC) activates genes and pathways that are specifically antagonized by ER activity in ER+ tumors. We now propose to 1) use ER antagonism of GR transactivation as a tool to identify additional critical GR target genes and pathways underlying therapy- resistant TNBC, 2) to identify mechanisms underlying ER antagonism of GR transactivation, and 3) to test the function of ER-antagonized GR target genes and pathways in preclinical TNBC models. The results of these experiments are expected to expand options for high-risk ER-negative and TNBC patients by identifying previously unknown GR-mediated cell survival pathways contributing to therapy resistance and early relapse.

DESCRIPTION (provided by applicant): Numerous studies have concluded that pre-menopausal African-American (AA) women have a significantly higher proportion of estrogen receptor (ER)-negative breast cancers compared to non-AA women. More recently it has become clear that many ER-negative tumors also lack progesterone receptor (PR) and HER2 expression- these tumors are termed "triple negative cancers" (TNBC). Because the proportion of TNBC breast cancer in AA women (55%) is more than double than that found in tumors of white women (23%), the refractory nature of TNBC is now recognized as one important component to the health disparity between young AA and non-AA breast cancer patient outcome. Thus, even after adjustments for socioeconomic factors- including access to screening and appropriate care- improving treatment of TNBC will likely improve the health disparity observed in women of color worldwide. Among the factors that might contribute to chemoresistance of TNBC is overexpression of SGK1, a very potent anti-apoptotic kinase downstream of PI3-K activation that our laboratory discovered is overexpressed in about 30% of TNBC and which we hypothesize contributes to chemo-resistance. In this proposal, we propose that increased SGK1 expression levels contribute to therapeutic resistance of TNBC to conventional chemotherapy (doxorubicin and paclitaxel) as well as novel therapies (e.g. Hsp90 inhibitors). To test this hypothesis we propose three specific aims: 1) To determine whether SGK1 overexpression inhibits paclitaxel or doxorubicin-induced apoptosis in TNBC cell lines;2) to examine the mechanisms of predicted SGK1-mediated resistance to Hsp90 inhibitor-induced apoptosis in TNBC, and 3) to determine whether SGK1 expression contributes to in vivo resistance of TNBC to conventional chemotherapy and/or Hsp90 inhibitors. Completion of this project will increase our knowledge of the role of SGK1 biology in therapeutic resistance to both traditional and new therapies for TNBC, paving the way for rationale use of PI3K/SGK1 inhibitors in these cancers that disproportionately affect young AA women. PUBLIC HEALTH RELEVANCE: Triple negative breast cancer (TNBC) is a subtype of breast cancer that disproportionately affects young AA women. Due to TNBC's relatively rapid rate of growth in relapse and limited treatment options beyond conventional chemotherapy, mechanisms of TNBC chemoresistance must be identified. We propose to study the role of SGK1, an anti-apoptotic kinase that is phosphorylated and activated by the PI3-K pathway, for its likely role in TNBC resistance to chemotherapy. Targeting SGK1 in tumors that overexpress this protein is likely to overcome resistance to apoptosis from a variety of treatments.

DESCRIPTION (provided by applicant): Understanding human cancer in the context of the social environment is essential for optimizing cancer prevention and care. By identifying stress mechanisms that impact on a patient's neuroendocrine physiology and subsequent tumor biology, we will increase our understanding of tumor biology. The neuroendocrine system links behavior and experience with hormone secretion (e.g. estrogen and cortisol) resulting in hormone-induced gene expression changes within both tumor cells and their microenvironment. However, the cellular and molecular mechanisms underlying the role of chronic stress in breast tumor biology remain poorly understood. Because of the complex genetic and environmental variation found in human populations, identifying the cellular and molecular mechanisms through which stress responses affect cancer biology will require transdisciplinary approaches to traditional models already used for studying cancer. The Conzen and McClintock laboratories have developed such an approach to studying the role of social stress in two complementary rodent models of human breast cancer. We discovered that chronic social isolation leads to a heightened glucocorticoid response to a superimposed stressor; in turn, mammary gland gene expression and morphology suggest an alteration in adipose tissue architecture during gland development. Moreover, social isolation and the ensuing increased stress (glucocorticoid-mediated) reactivity are associated with a significant increase in mammary gland fat metabolism, even prior to invasive cancer development. Based on these data, we propose to study mammary gland fat tissue and its paracrine effects on tumor growth by identifying the gene expression changes as well as the secreted proteins and factors that can contribute to increased tumor growth rates. We predict that completion of these studies will uncover novel stress-induced microenvironment mechanisms affecting mammary tumor growth.

ABSTRACT The overarching goal of the University of Chicago Medicine Comprehensive Cancer Center (UCCCC) Molecular Mechanisms of Cancer (MMC) Program is to identify and characterize molecular mechanisms underlying cancer cell growth and metastasis leading to development of improved treatment options through discovery-based science. To meet these overall objectives, the MMC Program has been organized by its leadership around three key scientific themes: 1) mechanisms of altered gene expression in cancer that encompasses understanding the significance for cancer etiology of genomic rearrangements, context-specific gene expression patterns and altered gene signatures, chromatin modifications and epigenetic marks and RNA biology; 2) mechanisms of transformation and altered cell growth in cancer, that includes analysis of how proliferation, differentiation, metabolism, cell death and autophagy are deregulated in cancers, and the role these processes play in cancer stem cells and therapy responses; and 3) mechanistic analysis of the tumor microenvironment and cancer metastasis, with an emphasis on defining novel mechanisms of altered cell motility, loss of adhesion, extra-cellular matrix control, acquisition of invasiveness, cell-cell signaling in the tumor microenvironment, tumor hypoxia, cancer-associated fibroblasts, and tumor-associated macrophages. The MMC Program consists of 37 faculty members from 14 Departments, including key faculty from the Department of Chemistry. In the current funding period (2013-2016), Program members published 564 cancer-related articles (18% intraprogrammatic, 34% interprogrammatic, and 60% interinstitutional). W2Program members are supported by $8.52 M (direct costs) in peer-reviewed funding, and $2.80 M (direct costs) from the National Cancer Institute (NCI), as well as $4.2 M (direct costs) in non-peer-reviewed funding. The focused development of MMC during the past 5 years provides the requisite infrastructure and knowledge base to forge translational research interactions within our own Cancer Center and with other Cancer Centers. A major strength of the Program over the past 5 years has been the expansion of research into mechanisms of metastasis and epigenetic signaling, as well as the new development of chemical approaches in cancer research. In summary, the MMC Program has a major impact on all components of the UCCCC as the primary driver of basic scientific discovery in molecular mechanisms of cancer using systems approaches, model organisms and primary human tumor samples. The interactions of MMC Program members with other UCCCC faculty through intra- and interprogrammatic collaborations further enable the key scientific steps needed for the discovery and development of promising therapies. Moving forward, MMC Program leadership recognizes new opportunities by leveraging our growing strengths especially in chemical biology, tumor metabolism, tumor microenvironment and immunology, and cancer metastasis, with the overarching goal of increasing successful basic science-clinical partnerships in these areas.

PROJECT ABSTRACT Approximately 70% of invasive breast cancer expresses estrogen receptor-alpha (ER). Our lab and others have found that 50-60% of early-stage ER+ breast cancers also express glucocorticoid receptor (GR), and that high GR expression provides favorable prognostic information in early-stage breast cancer independently of PR expression. Specifically, in an analysis of primary tumor GR expression in ER+ invasive breast tumors from over 1000 ER+ early-stage patients with >20 year clinical follow-up, we discovered that high tumor GR mRNA expression (and by implication, high GR activity) was associated with a significantly lower risk of relapse. More recently, we found that in ER+ breast cancer cell line models, GR activation remodels chromatin so that ER- chromatin association and ER-target gene expression are significantly altered. Based on these data, we hypothesize that ER and GR can provide coordinated regulation of good prognosis, anti-proliferative and pro- differentiation genes. We propose to determine the molecular mechanisms underlying GR-mediated modulation of both wild-type and mutant ER transcriptional activity, and to define the specific patterns of ER/GR-mediated gene expression in breast cancer. In Aim 1, we will characterize how GR ligand binding domain activation [by either dexamethasone (dex) or novel selective GR modulators (SGRMs)] affects ER activity, consequent ER-mediated gene expression, and ultimately ER-associated tumor cell proliferation. In Aim 2, we will investigate GR and ER chromatin and co-regulator association in the presence of dex in comparison to the novel SGRMs. This will allow us to better understand the requirements of chromatin conformation and GR/ER co-regulator assembly in modifying ER+ breast cancer's proliferative gene expression. Finally, in Aim 3, we will use in vivo ER+ breast cancer patient-derived xenograft models, mutant ER-expressing cell lines, and tamoxifen-resistant ER+ models to define SGRM anti-tumor activity with the aim of defining clinical contexts in which GR modulators are most likely to be effective. Together, these experiments will both increase our understanding of GR/ER crosstalk in breast cancer (and possibly other hormone-sensitive cancers e.g. endometrial) and are expected to lead to new GR-targeted therapies that harness our discovery of anti-proliferative activity following GR modulation in ER+/GR+ breast cancer.

PROJECT ABSTRACT The glucocorticoid receptor (GR) is a cortisol-activated transcription factor and chromatin remodeling protein that is variably expressed in ovarian cancer (OvCa). GR-mediated gene expression encoding anti-apoptotic pathway proteins is associated with chemotherapy-resistance in OvCa, while pre-treatment with a selective GR antagonist enhances chemotherapy sensitivity. Our group also recently discovered that amongst a range of OvCa histologic subtypes, both higher GR (NR3C1) mRNA and higher GR protein levels are associated with overall worse clinical outcome. We now propose to develop a ?GR transcriptional activity signature? (GRSig) for OvCa to better assess GR activity in OvCa with the goal of identifying patients most likely to benefit from addition of GR antagonism to chemotherapy. Using cell line models of GR+ OvCa representing a variety of histologies (Aim 1), we will identify genes whose expression is either significantly upregulated or repressed following GR activation by physiological glucocorticoid concentrations as well as significantly reversed in expression by GR antagonism. GR ChIP-seq will be employed to determine which of these genes are putative direct (rather than indirect) GR target genes with promoter or enhancer region GR chromatin association, further refining the GRSig to represent canonical OvCa GR activity. The resulting GRSig will then be used to assess relative GR activity in existing OvCa samples from a pooled public GEO dataset (Discovery set) containing normalized RNA expression and outcome (PFS and OS). A GRSig score cutoff will be calculated that identifies patients with worse prognosis in the GEO dataset. A second, well-annotated Mayo Clinic OvCa cohort (Validation set) will then be used to validate the cutoff. We will also test the hypothesis that higher GRSig scores (representing higher GR transcriptional activity) will associate with PFS and OS more strongly than GR (NR3C1) mRNA expression alone. In Aim 2, we will examine the GRSig in N=131 OvCa PDX models with chemotherapy response data to test the hypothesis that higher GRSig score associates with relative chemotherapy resistance. Furthermore, we will explore the relationship of GRSig score to GR protein expression by performing GR IHC in the PDX tumors, and then evaluating the strength of association of the GRSig score relative to GR IHC score with respect to chemotherapy response. In an exploratory analysis, GRSig-scored PDX models will be treated with chemotherapy +/- a GR antagonist (GRA) to test the hypothesis that pre-treatment with a GRA improves tumor shrinkage and/or lengthens time to tumor regrowth in GRSig high PDX models. Completion of this project will identify a GR transcriptional signature designed to identify women with OvCa who have a worse prognosis and to for whom addition of a selective GR antagonist to chemotherapy is expected to improve outcome compared to standard treatment with chemotherapy alone.

PROJECT/SUMMARY ABSTRACT Early-stage triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype in which, despite standard of care, approximately 1 in 3 patients relapse within the first three years of diagnosis. Through our group?s previous identification of the glucocorticoid receptor (GR) signaling pathway as a risk factor for recurrence in TNBC, we discovered that the gene encoding nicotinamide N-methyltransferase (NNMT) is a direct GR transcriptional target. We also found that high NNMT expression, independently of GR expression, is associated with a significantly worse outcome in early-stage chemotherapy-treated TNBC, suggesting a role for high NNMT activity in tumor cell chemotherapy-resistance and metastasis. Recently, accumulating evidence suggests that NNMT activity may play a role in the biology of various human cancers; however, NNMT?s mechanism of action in TNBC is completely unknown. Based on our preliminary data suggesting that NNMT activity induces hypomethylation of an important subset of TNBC mRNAs, we hypothesize that NNMT alters an oncogenic epitranscriptome of aggressive TNBC. Our central hypothesis is that high NNMT activity promotes TNBC cell survival and metastasis by reducing m6A mRNA modification of target transcripts, increasing oncogenic mRNA stability, and upregulating resultant cell survival and metastatic protein expression. In Aim 1, we will determine whether NNMT depletion or inhibiting NNMT activity biochemically alters m6A mRNA modification. We will also determine whether high versus depleted NNMT-expressing TNBC cells exhibit differential motility/invasion, adherence-independent viability and protection from chemotherapy- induced apoptosis in two dimensional (in vitro) assays. In Aim 2, using control, NNMT-knockdown, and NNMT- overexpression-rescue TNBC xenografted cells and a syngeneic model, we will determine whether NNMT expression/activity contributes to altered expression of m6A mRNA modified tumor-promoting genes and increases tumor regrowth. The same experiments will also be performed with and without NNMT inhibitor treatment. In summary, NNMT is a putative driver of epitranscriptomic mRNA modification that we will test as a driver of chemoresistance and metastatic gene expression and will test as a novel therapeutic target in TNBC.

PROJECT ABSTRACT The developing mammary gland (MG) is vulnerable to environmental and lifestyle risk factors that increase breast cancer (BC) burden in later adulthood. Therefore, optimizing BC prevention and care requires a lifespan approach to identify specific early life risk factors, to understand these risk factors? underlying molecular mechanisms in promoting cancer risk, and to design appropriate interventions that reduce BC in adulthood. Using a Sprague-Dawley rat model of human BC, we have established a dynamic and successful transdisciplinary collaboration among a breast cancer biologist, an endocrinologist, and a biopsychologist to understand how adverse early life exposures lead to increased mammary cancer risk in adulthood. We find that glucocorticoid (GC) reactivity to everyday stressors is heighted by social isolation in puberty and young adulthood and is associated with increased adult mammary cancer burden. Moreover, heightened GC reactivity during puberty impairs ductal development and increases mammary stem cell populations, two characteristics that have been linked to increased mammary cancer. We now propose to determine how heightened GC reactivity disrupts MG development and increases cancer burden by examining the underlying molecular mechanisms connecting glucocorticoid receptor (GR) activation with MG developmental defects (Aim 1). In Aim 2 we will introduce both pharmacological- and social environmental-interventions in early adulthood to reverse heightened stress reactivity. We predict these interventions will restore normal MG ductal differentiation and thereby decrease later cancer risk. In Aim 3, we will examine how heightened GC reactivity during puberty inappropriately preserves mammary stem cell (MaSC) populations that are known to increase later cancer risk. We will also investigate the association between circulating steroid hormone levels, in conjunction with their localized production within the MG microenvironment, and ductal maturation and MaSC biology. Completion of these studies will uncover novel stress-mediated molecular and cellular mechanisms of disrupted MG development linked to subsequent mammary cancer and determine whether these stress- mediated events are reversible with early adulthood interventions.