Supplementary Figure 1 from Subcellular Localization of Cyclic AMP-Responsive Element Binding Protein-Regulated Transcription Coactivator 2 Provides a Link between Obesity and Breast Cancer in Postmenopausal Women

ABSTRACT As adipose tissue depots are active endocrine organs, they secrete a variety of hormones (including estrogens from white adipose) and inflammatory mediators, which have important implications in numerous obesity‐associated diseases. Adipose tissues are broadly characterized as consisting of white, beige, and brown depot types. The endocrine, metabolic, and inflammatory profiles of adipose are depot dependent and influenced by the estrogenic and androgenic status of the adipose tissue. Estrogen receptors mediate both the genomic and nongenomic actions of estrogens and are expressed in the brain, heart, and other peripheral tissues. All three known estrogen receptor α (ERα) and estrogen receptor β (ERβ), and the G‐protein coupled estrogen receptor (GPER/GPR30) are expressed in white adipose and can modulate adipose mass. Expression of each receptor is dependent on depot location, adipose cell type, and estrogen levels. Estrogen receptor expression profiles in beige and brown adipocytes are less well established. This review will discuss the effects of estrogens on the differential deposition of the major adipose tissues and the impact of estrogens within white adipose depots. © 2019 American Physiological Society. Compr Physiol 9:457‐475, 2019.

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

We investigated the effects of estrogens on glucose homeostasis using the Aromatase Knockout (ArKO) mouse, which is unable to convert androgens into estrogens. The ArKO mouse is a model of total estrogen ablation which develops symptoms of metabolic syndrome.

A dynamic partnership between follicle-stimulating hormone (FSH) and activin is required for normal Sertoli cell development and fertility. Disruptions to this partnership trigger Sertoli cells to deviate from their normal developmental pathway, as observed in inhibin α-knockout (Inha-KO) mice, which feature Sertoli cell tumours in adulthood. Here, we identified the developmental windows by which adult Sertoli cell tumourigenesis is most FSH sensitive. FSH was suppressed for 7 days in Inha-KO mice and wild-type littermates during the 1st, 2nd or 4th week after birth and culled in the 5th week to assess the effect on adult Sertoli cell development. Tumour growth was profoundly reduced in adult Inha-KO mice in response to FSH suppression during Weeks 1 and 2, but not Week 4. Proliferative Sertoli cells were markedly reduced in adult Inha-KO mice following FSH suppression during Weeks 1, 2 or 4, resulting in levels similar to those in wild-type mice, with greatest effect observed at the 2 week time point. Apoptotic Sertoli cells increased in adult Inha-KO mice after FSH suppression during Week 4. In conclusion, acute FSH suppression during the 1st or 2nd week after birth in Inha-KO mice profoundly suppresses Sertoli cell tumour progression, probably by inhibiting proliferation in the adult, with early postnatal Sertoli cells being most sensitive to FSH action.

Obesity is associated with an increased risk of estrogen-dependent breast cancer after menopause. Adipose tissue undergoes important changes in obesity due to excess storage of lipids, leading to adipocyte cell death and the recruitment of macrophages. The resultant state of chronic low-grade inflammation is associated with the activation of NFkB signaling and elevated levels of aromatase, the rate-limiting enzyme in estrogen biosynthesis. This occurs not only in the visceral and subcutaneous fat, but also in the breast fat. The regulation of aromatase in the breast adipose stromal cell in response to inflammatory mediators is under the control of complex signaling pathways, including metabolic pathways involving LKB1/AMPK, p53, HIF1α and PKM2. Interventions aimed at modifying weight, including diet and exercise, are associated with changes in adipose tissue inflammation and estrogen production that are likely to impact breast cancer risk. This review will present an overview of these topics.

Although oestrogens are essential for spermatogenesis and their biosynthesis is dependent on aromatase expression, the molecular mechanism of aromatase regulation is poorly understood. Our laboratory has demonstrated that liver kinase B1 (LKB1) is a negative regulator of aromatase in the breast by phosphorylating AMP-activated protein kinase (AMPK) and inhibiting the nuclear translocation of the cAMP response element-binding protein-regulated transcription co-activator (CRTC) 2. The aim of this study was to determine the location of testis-associated proteins in the LKB1-CRTC pathway. Aromatase, LKB1, phosphorylated AMPK (pAMPK) and CRTC1-3 were examined by selected immunofluorescent antibodies in testis samples from a prepubertal boy and three fertile men. Aromatase, pAMPK and LKB1 proteins were present in the seminiferous epithelium and interstitium of the testis and were expressed in a differential and developmental manner in particular cell types. The expression pattern of LKB1 was similar to that of pAMPK and inversely related to aromatase expression. CRTC1 and CRTC3 were localised in the seminiferous epithelium, whereas CRTC2 was barely detectable in testis. These results lead to the conclusion that LKB1 is involved in the molecular pathway that underpins aromatase regulation in the testis via CRTC1 and CRTC3 and may be important for the oestrogen-mediated development of germ cells.

AIMS:Controversy surrounds the gender basis of progression in chronic kidney disease. Unfortunately, most experimental studies addressing this question do not distinguish between direct effects of estrogen and indirect activation of estrogen receptors through conversion of testosterone to 17β-estradiol by aromatase. We examined the pathogenesis of renal fibrosis in female aromatase knockout (ArKO) mice, which lack circulating and stored estrogens, while having normal levels of testosterone.MAIN METHODS:ArKO mice and their wild-type (ArWT) counterparts were subjected to unilateral ureteric obstruction (UUO), with kidney tissue collected at day(D) 0, 3 and 9 post-UUO. Effects of 5α-dihydrotestosterone (DHT) administration on each genotype were also studied. Tissue was assessed biochemically and histochemically for fibrosis. Western blot analysis was used to measure α-smooth muscle actin (α-SMA) expression and TGF-β1 signalling. Matrix metalloproteinase-2 (MMP-2) activity was measured by zymography.KEY FINDINGS:UUO increased collagen content over time (p<0.05 (D3) and p<0.01 (D9) vs day 0), with no difference between genotypes in qualitative (collagen IV staining) and quantitative (hydroxyproline concentration) analyses. Systemic administration of non-aromatizable DHT increased collagen content after 3days of UUO in both genotypes. This was not paralleled by any change in α-SMA (myofibroblast burden) or TGF-β1 signalling but was commensurate with DHT reducing MMP2 activity in both genotypes (p<0.05 vs genotype controls).SIGNIFICANCE:Physiological concentrations of estrogens do not protect the injured kidney from fibrosis progression. Androgens rather than estrogens are the relevant factor involved in regulating disease-related renal scarring in this model.

I. IntroductionII. The Concept of Local Estrogen BiosynthesisIII. Aromatase and Its GeneIV. The ArKO MouseV. The ArKO Mouse and the Metabolic SyndromeVI. The Metabolic Syndrome in Humans with Natural Mu-tations in AromataseVII. Summary of the Metabolic Effects of EstrogensVIII. Local Aromatase Expression in the Breast and BreastCancerIX. Role of LRH-1 in Aromatase Expression in the Breast

The role of sex steroids in cardioprotection is contentious, with large clinical trials investigating hormone supplementation failing to deliver outcomes expected from observational studies. Mechanistic understanding of androgen/estrogen myocardial actions is lacking. Using a genetic model of aromatase tissue deficiency (ArKO) in female mice, the goal of this investigation was to evaluate the capacity of a shift in cardiac endogenous steroid conversion to influence ischemia-reperfusion resilience by optimizing cardiomyocyte Ca2+ handling responses. In isolated normoxic cardiomyocytes, basal Ca2+ transient amplitude and extent of shortening were greater in ArKO myocytes, with preservation of diastolic Ca2+ levels. Isolated ArKO cardiomyocytes exposed to a high Ca2+ load exhibited greater Ca2+ transient and contractile amplitudes, associated with a greater postrest spontaneous sarcoplasmic reticulum Ca2+ load-release. Microarray differential gene expression analysis of normoxic ventricular tissues from ArKO vs wild-type identified a significant influence of aromatase on genes involved in cardiac Ca2+ handling and signaling [including calmodulin dependent kinase II (CaMKII)-δ], myofilament structure and function, glucose uptake and signaling, and enzymes controlling phosphorylation-specific posttranslational modification status. CaMKII expression was not changed in ventricular tissues, although CaMKIIδ activation and phosphorylation of downstream targets was enhanced in ArKO hearts subjected to ischemia-reperfusion. Overall, this investigation shows that relative withdrawal of estrogen in favor of testosterone through genetically induced tissue aromatase deficiency in females modifies the gene expression profile to effect inotropic support via optimized Ca2+ handling in response to stress, with a modest impact on basal function. Consideration of aromatase inhibition, acutely or chronically, may have a role in cardioprotection, of particular relevance to women.

The maintenance of glucose homeostasis within the body is crucial for constant and precise performance of energy balance and is sustained by a number of peripheral organs. Estrogens are known to play a role in the maintenance of glucose homeostasis. Aromatase knockout (ArKO) mice are estrogen-deficient and display symptoms of dysregulated glucose metabolism. We aim to investigate the effects of estrogen ablation and exogenous estrogen administration on glucose homeostasis regulation. Six month-old female wildtype, ArKO, and 17β-estradiol (E2) treated ArKO mice were subjected to whole body tolerance tests, serum examination of estrogen, glucose and insulin, ex-vivo muscle glucose uptake, and insulin signaling pathway analyses. Female ArKO mice display increased body weight, gonadal (omental) adiposity, hyperinsulinemia, and liver triglycerides, which were ameliorated upon estrogen treatment. Tolerance tests revealed that estrogen-deficient ArKO mice were pyruvate intolerant hence reflecting dysregulated hepatic gluconeogenesis. Analyses of skeletal muscle, liver, and adipose tissues supported a hepatic-based glucose dysregulation, with a down-regulation of Akt phosphorylation (a key insulin signaling pathway molecule) in the ArKO liver, which was improved with E2 treatment. Concurrently, estrogen treatment lowered ArKO serum leptin and adiponectin levels and increased inflammatory adipokines such as tumour necrosis factor alpha (TNFα) and interleukin 6 (IL6). Furthermore, estrogen deficiency resulted in the infiltration of CD45 macrophages into gonadal adipose tissues, which cannot be reversed by E2 treatment. This study describes the effects of estrogens on glucose homeostasis in female ArKO mice and highlights a primary phenotype of hepatic glucose dysregulation and a parallel estrogen modified adipokine profile.

The tumor suppressor p53 has established functions in cancer. Specifically, it has been shown to cause cell-cycle arrest and apoptosis in response to DNA damage. It is also one of the most commonly mutated or silenced genes in cancer and for this reason has been extensively studied. Recently, the role of p53 has been shown to go beyond its effects on cell cycle and apoptosis, with effects on metabolism emerging as a key contributor to cancer growth in situations where p53 is lost. Beyond this, the role of p53 in the tumor microenvironment is poorly understood. The publication by Wang and colleagues demonstrates for the first time that p53 is a key negative regulator of aromatase and, hence, estrogen production in the breast tumor microenvironment. It goes further by demonstrating that an important regulator of aromatase, the obesity-associated and tumor-derived factor prostaglandin E2, inhibits p53 in the breast adipose stroma. This review presents these findings in the context of established and emerging roles of p53 and discusses possible implications for the treatment of breast cancer.

Aromatase is the critical enzyme that converts androgens to estrogens. It is frequently highly expressed in the tumour bearing breast of women diagnosed with estrogen receptor positive tumours, resulting in dramatically increased local estrogen production to drive tumour progression. Expression of aromatase is regulated primarily at the transcriptional level of its encoding gene CYP19A1, located on chromosome 15 of the human genome. A characteristic feature of CYP19A1 expression is its use of alternative promoters to regulate transcription in a tissue-specific manner. In breast cancer, the increase in aromatase expression is mediated via higher expression of the distal adipose-specific promoter 1.4 and a switch to the preferential use of proximal promoters 1.3 and II. This results in a net increase of CYP19A1 transcripts in tumour-bearing breast up to 3-4-fold higher than normal breast. Current aromatase inhibitors - whilst efficacious - exhibit significant side effects that reduce patient compliance. Understanding the transcription factors and signalling pathways that control aromatase expression will lead to opportunities to develop breast-specific inhibitors with an improved side-effects profile.This article is part of a Special Issue entitled 'Essential role of DHEA'. (c) 2014 Elsevier Ltd. All rights reserved.

It gives me great pleasure to contribute to this special issue of Seminars which honors the career of Bruce Carr. As it happens, Bruce was my first Fellow upon my arrival at the Green Center for Reproductive Biology Sciences at UT Southwestern Medical Center in 1977. At that time, the Center was filled with luminaries of Reproductive Endocrinology, such as John Porter, Jack Johnston, Norman Gant, and of course the Director, Paul MacDonald, so to be given the responsibility of mentoring a new Fellow was a daunting responsibility. However, Bruce quickly rolled up his sleeves and plunged straight in, and we forged a relationship which led to some 36 manuscripts in 4 years. The first of these was entitled "The Role of Serum Lipoproteins in Steroidogenesis by the Human Fetal Adrenal Cortex," published in the Journal of Clinical Endocrinology and Metabolism, volume 49, pages 146-148, in 1979, and the authors were Simpson ER, Carr BR, Parker CR Jr, Milewich L, Porter JC, and MacDonald PC. Bruce quickly moved up the ranks of the Obstetrics/Gynecology Department to become full Professor and we went our separate ways professionally, but we remain close friends to this day. This special issue is indeed a worthy tribute to an outstanding career and especially to Bruce's role as editor-in-chief of Seminars which he has guided through the rapid evolution of the specialty, always maintaining a strong research focus and thus carrying on the rich tradition of the Green Center and the Obstetrics/Gynecology Department.

The number of breast cancer cases has increased in the last a few decades and this is believed to be associated with the increased prevalence of obesity worldwide. The risk of breast cancer increases with age beyond menopause and the relationship between obesity and the risk of breast cancer in postmenopausal women is well established. The majority of postmenopausal breast cancers are estrogen receptor (ER) positive and estrogens produced in the adipose tissue promotes tumor formation. Obesity results in the secretion of inflammatory factors that stimulate the expression of the aromatase enzyme, which converts androgens into estrogens in the adipose tissue. Evidence demonstrating a link between obesity and breast cancer has led to the investigation of metabolic pathways as novel regulators of estrogen production, including pathways that can be targeted to inhibit aromatase specifically within the breast. This review aims to present some of the key findings in this regard.

Obesity is a risk factor for postmenopausal breast cancer and the majority of these cancers are estrogen dependent. Aromatase converts androgens into estrogens and its increased expression in breast adipose stromal cells (ASC) is a major driver of estrogen receptor-positive breast cancer. In particular, obesity-associated and tumor-derived factors, such as prostaglandin E2 (PGE2), have been shown to drive the expression of aromatase by stimulating the activity of the proximal promoter II (PII). The tumor-suppressor p53 is a key regulator of cell-cycle arrest and apoptosis and is frequently mutated in breast cancer. Mutations in p53 are rare in tumor-associated ASCs. Therefore, it was hypothesized that p53 is regulated by PGE2 and involved in the PGE2-mediated regulation of aromatase. Results demonstrate that PGE2 causes a significant decrease in p53 transcript and nuclear protein expression, as well as phosphorylation at Ser15 in primary human breast ASCs. Stabilization of p53 with RITA leads to a significant decrease in the PGE2-stimulated aromatase mRNA expression and activity, and PII activity. Interaction of p53 with PII was demonstrated and this interaction is decreased in the presence of PGE2. Moreover, mutation of the identified p53 response element leads to an increase in the basal activity of the promoter. Immunofluorescence on clinical samples demonstrates that p53 is decreased in tumor-associated ASCs compared with ASCs from normal breast tissue, and that there is a positive association between perinuclear (inactive) p53 and aromatase expression in these cells. Furthermore, aromatase expression is increased in breast ASCs from Li-Fraumeni patients (germline TP53 mutations) compared with non-Li-Fraumeni breast tissue. Overall, our results demonstrate that p53 is a negative regulator of aromatase in the breast and its inhibition by PGE2 provides a novel mechanism for aromatase regulation in obesity and breast cancer.