Aromatase Expression In Adipose Tissue Research Articles

PPARγ antagonists induce aromatase transcription in adipose tissue cultures

Aromatase is the rate-limiting enzyme in the biosynthesis of estrogens and a key risk factor for hormone receptor-positive breast cancer. In postmenopausal women, estrogens synthesized in adipose tissue promotes the growth of estrogen receptor positive breast cancers. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) in adipose stromal cells (ASCs) leads to decreased expression of aromatase and differentiation of ASCs into adipocytes. Environmental chemicals can act as antagonists of PPARγ and disrupt its function. This study aimed to test the hypothesis that PPARγ antagonists can promote breast cancer by stimulating aromatase expression in human adipose tissue.Primary cells and explants from human adipose tissue as well as A41hWAT, C3H10T1/2, and H295R cell lines were used to investigate PPARγ antagonist-stimulated effects on adipogenesis, aromatase expression, and estrogen biosynthesis. Selected antagonists inhibited adipocyte differentiation, preventing the adipogenesis-associated downregulation of aromatase. NMR spectroscopy confirmed direct interaction between the potent antagonist DEHPA and PPARγ, inhibiting agonist binding. Short-term exposure of ASCs to PPARγ antagonists upregulated aromatase only in differentiated cells, and a similar effect could be observed in human breast adipose tissue explants. Overexpression of PPARG with or without agonist treatment reduced aromatase expression in ASCs.The data suggest that environmental PPARγ antagonists regulate aromatase expression in adipose tissue through two mechanisms. The first is indirect and involves inhibition of adipogenesis, while the second occurs more acutely.

Aromatase Blockade Is Associated With Increased Mortality in Acute Illness in Male Mice.

Context:The increase in circulating estrogen levels with acute illness in humans is accompanied by increased aromatase expression in adipose tissue and increased peripheral aromatization of estrogens to androgens. Animal studies indicate that estrogen may be beneficial in acute illness.Objective:We hypothesized that blockade of aromatase in acute illness would decrease survival.Design:Prospective sham controlled.Setting:Maine Medical Center Research Institute animal facility.Animals:Six- to 8-week-old male black 6 mice.Intervention:Mice underwent cecal ligation and puncture (CLP) to induce acute illness and were administered letrozole to block aromatase or saline. Mice undergoing sham surgery with or without letrozole served as controls. Adipose and cardiovascular tissue was harvested for preliminary evaluation of aromatase expression.Main outcome measurements:Survival was the main outcome measurement. Evidence for aromatase expression in tissue samples was assessed using western blot and/or immunohistochemistry.Results:With aromatase blockade, survival in CLP mice was decreased (P = 0.04). The presence of aromatase in adipose tissue was observed by western blot in CLP but not control mice. Similarly, the presence of aromatase was observed in cardiac tissue of CLP but not in control mice.Conclusions:The decreased survival during sepsis with aromatase blockade suggests that this response to acute illness may be important both physiologically and clinically. The preliminary observation of aromatase expression in adipose and cardiovascular tissue during acute illness in this mouse model indicates that this model has parallels to human physiology and may be useful for further studying the aromatase response to acute illness.

Abstract A12: Urinary levels of PGE-M and estrogens are independently associated with postmenopausal breast cancer risk

Abstract Prostaglandin E2 (PGE2) induces aromatase expression in adipose tissue leading to increased estrogen production that may promote the development and progression of breast cancer. However, few studies have simultaneously investigated systemic levels of PGE2 and estrogen in relation to postmenopausal breast cancer risk. In a case-cohort study of postmenopausal women (295 cases and 294 subcohort) we previously reported that high levels of PGE-M, a major metabolite of PGE2, were associated with an increased risk of breast cancer among postmenopausal women who did not regularly use nonsteroidal anti-inflammatory drugs (NSAIDs). Here we determined urinary estrogen metabolites (EMs) using mass spectrometry in the same case-cohort set and using linear regression estimated the effect of PGE-M on EMs. Hazard ratios (HRs) for the risk of developing breast cancer in relation to PGE-M and EMs were evaluated in Cox regression models with and without mutual adjustment. PGE-M was a significant predictor of estrone (E1), but not estradiol (E2) levels in multivariable analysis. Elevated E2 levels were associated with an increased risk of developing breast cancer (HRQ5vs.Q1 =1.54, 95% CI: 1.01-2.35), and this association remained unchanged after adjustment for PGE-M (HRQ5vs.Q1 =1.52, 95% CI: 0.99-2.33). Similarly, elevated levels of PGE-M were associated with increased risk of developing breast cancer (HRQ4vs.Q1 =2.01, 95% CI: 1.01-4.29), and this association was only nominally changed after consideration of E1 or E2 levels. Urinary levels of PGE-M and parent estrogens were independently associated with future risk of developing breast cancer among these postmenopausal women. Increased breast cancer risk associated with PGE-M might be attributable both to PGE2-mediated increases in estrogens, and to additional effects related to inflammation. Note: This abstract was not presented at the conference. Citation Format: Sangmi Kim, Jeff Campbell, Wonsuk Yoo, Jack A. Taylor, Dale P. Sandler. Urinary levels of PGE-M and estrogens are independently associated with postmenopausal breast cancer risk. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A12.

Ghrelin and Breast Cancer: Emerging Roles in Obesity, Estrogen Regulation, and Cancer.

Local and systemic factors have been shown to drive the growth of breast cancer cells in postmenopausal obese women, who have increased risk of estrogen receptor-positive breast cancer. Estrogens, produced locally in the breast fat by the enzyme aromatase, have an important role in promoting cancer cell proliferation. Ghrelin, a 28-amino acid peptide hormone, may also influence cancer growth. This peptide is produced in the stomach and acts centrally to regulate appetite and growth hormone release. Circulating levels of ghrelin, and its unacylated form, des-acyl ghrelin, are almost always inversely correlated with obesity, and these peptide hormones have recently been shown to inhibit adipose tissue aromatase expression. Ghrelin and des-acyl ghrelin have also been shown to be produced by some tumor cells and influence tumor growth. The ghrelin/des-acyl ghrelin–cancer axis is complex, one reason being that tumor cells have been shown to express splice variants of ghrelin, and ghrelin and des-acyl ghrelin might act at receptors other than the cognate ghrelin receptor, growth hormone secretagogue receptor 1a, in tumors. Effects of ghrelin and des-acyl ghrelin on energy homeostasis may also affect tumor development and growth. This review will summarize our current understanding of the role of ghrelin and des-acyl ghrelin in hormone-dependent cancers, breast cancer in particular.

Systemic Levels of Estrogens and PGE2 Synthesis in Relation to Postmenopausal Breast Cancer Risk

Background: Prostaglandin E2 (PGE2) induces aromatase expression in adipose tissue, leading to increased estrogen production that may promote the development and progression of breast cancer. However, few studies have simultaneously investigated systemic levels of PGE2 and estrogen in relation to postmenopausal breast cancer risk.Methods: Here, we determined urinary estrogen metabolites (EM) using mass spectrometry in a case-cohort study (295 incident breast cancer cases and 294 subcohort members), and using linear regression estimated the effect of urinary levels of a major PGE2 metabolite (PGE-M) on EMs. HRs for the risk of developing breast cancer in relation to PGE-M and EMs were compared between Cox regression models with and without mutual adjustment.Results: PGE-M was a significant predictor of estrone (E1), but not estradiol (E2) levels in multivariable analysis. Elevated E2 levels were associated with an increased risk of developing breast cancer [HRQ5vs.Q1, 1.54; 95% confidence interval (CI), 1.01-2.35], and this association remained unchanged after adjustment for PGE-M (HRQ5vs.Q1, 1.52; 95% CI, 0.99-2.33). Similarly, elevated levels of PGE-M were associated with increased risk of developing breast cancer (HRQ4vs.Q1, 2.01; 95% CI, 1.01-4.29), and this association was only nominally changed after consideration of E1 or E2 levels.Conclusions: Urinary levels of PGE-M and estrogens were independently associated with future risk of developing breast cancer among these postmenopausal women.Impact: Increased breast cancer risk associated with PGE-M might not be fully explained by the estrogens-breast cancer association alone but also by additional effects related to inflammation. Cancer Epidemiol Biomarkers Prev; 26(3); 383-8. ©2016 AACR.

Osteopontin promotes aromatase expression and estradiol production in human adipocytes.

Breast and endometrial cancer are often estrogen dependent, and their incidence and mortality are increased by obesity in postmenopausal women. Osteopontin (OPN) is a cytokine strongly upregulated in adipose tissue (AT) in obesity. OPN function is potentiated by cleavage by matrix metalloproteinases (MMP). OPN and MMPs play a role in cancer development and are prognostic markers in breast cancer progression. While induction of the estrogen-synthesizing enzyme aromatase by TNFa and IL1 has been shown in preadipocytes, an impact of OPN on aromatase expression in AT has not been investigated yet. Gene expression was determined in AT samples of 21 morbidly obese and matched non-obese subjects. Primary human adipocytes were treated with full-length OPN or MMP-cleaved OPN (cOPN). Protein and mRNA expressions were analyzed from cell lysates, or cells were subsequently supplied with testosterone to determine estradiol production and for indirect co-culture with the estrogen-dependent MCF-7 cell line. Aromatase expression strongly correlated with gene expression of OPN and various MMPs in visceral and MMPs in subcutaneous AT, but not with TNFα expression in both tissues. In vitro, cOPN more effectively than full-length OPN upregulated aromatase mRNA in adipocytes and significantly increased aromatase protein level and estradiol production, leading to increased MCF-7 growth in indirect co-culture. OPN and MMPs are upregulated in AT in obesity, and MMP-cleaved OPN is particularly effective in inducing aromatase activity in human adipocytes. Thereby, obesity-induced OPN expression in AT may contribute to estradiol production and thus to the association of obesity with estrogen-dependent cancers.

IL6 genotype, tumour ER-status, and treatment predicted disease-free survival in a prospective breast cancer cohort.

BackgroundIn breast cancer, high levels of the inflammatory cytokine interleukin-6 (IL-6) have been associated with disease-free survival and treatment resistance. Increased serum levels of IL-6 have been correlated with increased levels of NF-κβ and aromatase expression in adipose tissue. Several IL6 single nucleotide polymorphisms have been associated with breast cancer prognosis, but the impact may differ depending on tumour oestrogen receptor (ER) status. This translational study investigated the association between IL6 genotypes, ER-status, and treatment on the risk of early events among breast cancer patients.MethodsThe study included 634 25- to 99-year-old primary breast cancer patients in Sweden from 2002–2008. Genotyped IL6 single nucleotide polymorphisms rs1800797, rs1800796, rs1800795, and rs2069849 were analysed separately and as diplotypes. Disease-free survival was assessed for 567 patients. Clinical data, patient-, and tumour-characteristics were obtained from questionnaires, patient charts, population registries, and pathology reports.ResultsThe median follow-up time was 5.1 years. IL6 diplotype was not associated with early events for all 567 patients, but AGCC/AGCC diplotype-carriers with ER-negative tumours had an increased risk, (adjusted Hazard Ratio (HR) = 5.91, 95% CI: 1.28–27.42). Any C-carriers (rs1800795) with ER-negative tumours had a higher risk of early events than GG-carriers with ER-negative tumours, (adjusted HR = 3.76, 95% CI: 1.05–13.43), particularly after radiotherapy (adjusted HR = 7.17, 95% CI: 1.16–32.28). Irrespective of ER-status, chemotherapy-treated Any C-carriers had a higher risk of early events than GG-carriers (adjusted HR = 3.42, 95% CI: 1.01–11.54).ConclusionsThe main finding of the present study was that IL6 genotype was strongly associated with early events among patients with ER-negative tumours, particularly among radiotherapy-treated patients, and among chemotherapy-treated patients irrespective of ER-status. The high risk for early events observed in these subgroups of patients suggests that combined information on IL6 genotype, tumour ER-status, and breast cancer treatment may represent a tool for identifying patients who require more personalised treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-759) contains supplementary material, which is available to authorized users.

Abstract 2102: Phenformin down-regulates mammary aromatase expression via the AMPK pathway in humanized aromatase expressing ERBB2 mice

Abstract Local mammary estrogen production may be vital for breast cancer development because about 70% of breast cancers occur in postmenopausal women. Aromatase is a key enzyme essential for estrogen biosynthesis, and aromatase inhibitors (AIs) are the most effective endocrine treatment for estrogen-responsive breast cancer in postmenopausal women. However, they are also accompanied a number of severe side-effects on bone and joints. Many researchers are currently focusing on developing the novel mammary-specific AIs to prevent or treat breast cancer to minimize the side effects of treatment. Phenformin, an antidiabetic drug, lowers serum glucose levels via activating the AMP-activated protein kinase (AMPK) pathway and reduces tumor burden and prolongs survival in non-small cell lung cancers. In vitro studies show that activated AMPK inhibits human mammary adipose tissue aromatase expression via suppression of binding of cAMP-responsive element binding protein (CREB) to aromatase promoters I.3/II, leading to decreased estrogen formation. We hypothesized that phenformin will be an effective adipose-specific AI for prevention and treatment of breast cancer. However, one of the major obstacles to define in vivo effects of phenformin on mammary aromatase expression is the lack of suitable mouse models because aromatase expression, present in the human breast, is absent in the mouse mammary gland. We developed a unique humanized aromatase mouse model (Aromhum) to mimic the human aromatase expression pattern and physiology in the mouse. We observed that tumorigenesis was accelerated in the mice overexpressing ERBB2 that were crossed with Aromhum mice (AE mice). Phenformin treatment (i.p., 70 mg/kg /day for 14 days) significantly decreased blood glucose levels by 37% in AE mice as compared to vehicle treatment. Phosphorylation of CREB, a downstream effector of the AMPK pathway, which mediates aromatase expression, was significantly decreased. Furthermore, after phenformin administration, mammary aromatase mRNA expression in AE mice was significantly decreased by 58% and estrogen response genes (SUSD3 and cyclin D1) were also reduced by about 65%. Real-time PCR showed that mammary AMPKα1 mRNA expression is 10-fold higher compared with AMPKα2 mRNA. Moreover, phenformin treatment significantly decreased AMPKα1 mRNA levels by 53% but not AMPKα2 mRNA expression in AE mice. Interestingly, daily oral phenformin treatment for 7 days significantly reduced mammary tumor volume of AE mice by 76%. In conclusion, phenformin decreased mammary aromatase expression and possible estrogen production, and reduced tumorigenesis via upregulation of the AMPK pathway in breast tissue of AE mice. This study provides justification for future testing of the adipose-specific preventive and therapeutic potential of the metabolic drug phenformin in postmenopausal breast cancer with minimal side-effects. Citation Format: Hong Zhao, Robert T. Chatterton, Timothy Prajka, Lin Li, Serdar E. Bulun. Phenformin down-regulates mammary aromatase expression via the AMPK pathway in humanized aromatase expressing ERBB2 mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2102. doi:10.1158/1538-7445.AM2014-2102

NSAID use reduces breast cancer recurrence in overweight and obese women: role of prostaglandin-aromatase interactions.

Obesity is associated with a worse breast cancer prognosis and elevated levels of inflammation, including greater cyclooxygenase-2 (COX-2) expression and activity in adipose-infiltrating macrophages. The product of this enzyme, the proinflammatory eicosanoid prostaglandin E2 (PGE2), stimulates adipose tissue aromatase expression and subsequent estrogen production, which could promote breast cancer progression. This study demonstrates that daily use of a nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 activity, is associated with reduced estrogen receptor α (ERα)-positive breast cancer recurrence in obese and overweight women. Retrospective review of data from ERα-positive patients with an average body mass index of >30 revealed that NSAID users had a 52% lower recurrence rate and a 28-month delay in time to recurrence. To examine the mechanisms that may be mediating this effect, we conducted in vitro studies that utilized sera from obese and normal-weight patients with breast cancer. Exposure to sera from obese patients stimulated greater macrophage COX-2 expression and PGE2 production. This was correlated with enhanced preadipocyte aromatase expression following incubation in conditioned media (CM) collected from the obese-patient, sera-exposed macrophages, an effect neutralized by COX-2 inhibition with celecoxib. In addition, CM from macrophage/preadipocyte cocultures exposed to sera from obese patients stimulated greater breast cancer cell ERα activity, proliferation, and migration compared with sera from normal-weight patients, and these differences were eliminated or reduced by the addition of an aromatase inhibitor during CM generation. Prospective studies designed to examine the clinical benefit of NSAID use in obese patients with breast cancer are warranted.

Abstract P1-06-01: NSAID use attenuates breast cancer recurrence in obese women: Role of prostaglandin-aromatase interactions

Abstract Introduction: Obesity is associated with a worse breast cancer prognosis, with the most prominent effects seen in hormone responsive postmenopausal patients. It has also been linked to elevated levels of inflammation, including greater cyclooxygenase-2 (COX-2) expression and activity in adipose-infiltrating macrophages. The product of this enzyme, the pro-inflammatory eicosanoid prostaglandin E2 (PGE2), stimulates adipose tissue aromatase expression and subsequent estrogen production, which could promote breast cancer progression. Consequently, we hypothesized that non-steroidal anti-inflammatory drug (NSAID) use decreases estrogen receptor (ER) positive breast cancer recurrence in the obese population via inhibition of PGE2-mediated local aromatase expression. Methods: Four-hundred and forty women treated for invasive, ER positive breast cancer at San Antonio area clinics were retrospectively classified according to NSAID use, body mass index category (Normal Weight (NW): 18.5-24.9 kg/m2; Overweight (OW): 25.0-29.9 kg/m2; Obese (OB): ≥30.0 kg/m2), and disease recurrence. To examine the role of obesity-induced local aromatase expression in the link between NSAID use and disease recurrence, we utilized an in vitro model of obesity in which we exposed macrophages to pooled sera samples from NW or OB postmenopausal breast cancer patients. Adipose stromal cells’ (ASC) aromatase expression was measured following exposure to conditioned media (CM) collected from these sera-exposed macrophages. ER activity and in vitro measures of cancer aggression were assessed in MCF-7 and T47D breast cancer cells cultured in CM from sera-exposed macrophage/ASC co-cultures. Results: Within our patient population, which had an average BMI in the obese range, NSAID users had significantly lower recurrence rates (p = 0.05) and their time to disease progression was delayed by almost 28 months in comparison to nonusers. Our in vitro studies demonstrated that growth in OB macrophage CM significantly enhances ASC aromatase expression in comparison to NW (p<0.05), while macrophage treatment with celecoxib during the generation of CM neutralizes the difference between OB and NW. This was correlated with significantly greater macrophage PGE2 production following OB versus NW sera exposure (p<0.05). In addition, CM from macrophage/ASC co-cultures exposed to OB patient sera stimulates more breast cancer cell ER activity, proliferation, and S phase activity, and these differences are eliminated by the addition of an aromatase inhibitor during the generation of CM. We also plan to examine how the co-culture CM impacts breast cancer cell expression of a panel of genes related to cancer aggression. Conclusions: Our results indicate that NSAID use can improve the recurrence rate for hormone-responsive breast cancer patients, particularly those with an elevated BMI. The in vitro model suggests that obesity-related enhancement of PGE2-induced local aromatase expression and estrogen production may be a key mechanism mediating this effect. Further studies designed to examine the clinical benefit of NSAID use in the obese breast cancer patient population are warranted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-01.

Abstract P6-02-02: Altered matrix homeostasis regulates estrogen biosynthesis in adipose tissue

Abstract Several estrogen-dependent pathological conditions including breast cancer, uterine fibroids, and endometriosis are associated with local overexpression of aromatase, a key enzyme in estrogen biosynthesis and important therapeutic target for postmenopausal ER+ breast cancer. In addition, excessive aromatase expression in adipose tissue at least partially accounts for obesity-associated breast cancer risk among postmenopausal women. While altered matrix homeostasis is associated with these aromatase-overexpressing tissues, little is known as to whether it can directly impact local steroidogenic gene expression and estrogen biosynthesis. A paucity of knowledge in this area is partly due to the lack of proper model systems that can recapitulate the mechanical properties of a cell's microenvironment. Our data indicate that matrix alone can significantly stimulate aromatase transcription in breast stromal cells (BSCs) via distinct signaling pathways. With the help of a repertoire of molecular and biophysical tools, such as micropost, micropattern and polyacrylamide gel and 3-dimensional matrix culture, we determined that matrix rigidity and cellular shape play important role in the regulation of aromatase transcription in the BSCs. We further discovered that cytoskeleton dynamics can significantly alter the ECM-stimulated aromatase transcription in BSCs. Following careful and elaborate studies with the help of siRNA-mediated knockdown and over-expression of the individual factors, we found that a non-canonical collagen receptor member of discoidin domain receptor (DDR) family, DDR1, transduce the external signal inside the cells to facilitate the aromatase transcription. Further studies with siRNA-mediated knockdown and specific inhibitors revealed that DDR1 activates a signaling cascade, which promotes activation of the cJun kinase (JNK-1) by phosphorylation and subsequently the activation of Activation Protein-1 family member JunB. Activated JunB physically binds at the cancer-specific promoters of aromatase and induce transcription in stromal cells. To determine the consequence of the altered matrix generated aromatase on the growth of estrogen receptor (ER)-positive breast cancer cell lines we set up in vitro co-culture system. Proliferation of ZR75-1 and its ability to produce estrogen-stimulated genes were enhanced in multiple fold, when the cells were co-cultured physically with BSCs inside collagen or fed with BSC conditioned media in the presence of testosterone. The link between matrix homeostasis and hormone metabolism is a vastly under-explored topic. Results from our work has bridged a major gap of knowledge in this field. Findings from the study also identified new factors that are key for the tissue-specific activation of estrogen biosynthesis and provide novel prognostic tools and markers, as well as new therapeutic targets for reducing local estrogen production, thus overcoming the side effects often associated with systemic inhibition of aromatase. The proposed work promises to establish a novel paradigm for regulation of steroidogenic gene expression and estrogen production and may have a far-reaching impact on the etiology and treatment of endocrine diseases that are associated with altered matrix homeostasis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-02.

Red wine increases adipose tissue aromatase expression and regulates body weight and adipocyte size

Objective Obesity is an important component of the metabolic syndrome in constituting a risk factor for cardiovascular disease, diabetes, and cancer. Estrogens influence lipid accumulation in adipocytes, acting indirectly or directly on adipose tissue. In this study we aimed to investigate the influence of red wine ingestion on the expression of aromatase (estrogen synthase) in adipose tissue. Methods Red wine or ethanol solution, in the concentration found in red wine, was provided to Wistar rats as the sole drinking fluid for 8 wk. Food and drink intakes and body weight were monitored throughout treatment and adipocyte size and aromatase expression in the adipose tissue were determined at the end of the experimental period. Results Red wine and ethanol increased aromatase expression in the adipose tissue and red wine decreased adipocyte size ( P < 0.05). In addition, animals treated with red wine or ethanol had significantly lower weight gain than controls, despite a similar energy intake. Conclusion Thus, the ingestion of red wine may alter the production of estrogens by adipose tissue, body weight gain, and adipocyte size. Some of these red wine effects are attributable to ethanol. This relation among estrogen availability, adipocyte biology, and weight gain is most interesting and deserves further study because it may lead to new strategies to reduce metabolic syndrome incidence.

Cell shape is a key determinant of estrogen biosynhesis in adipose stromal cells.

Abstract Abstract #5061 BACKGROUND: Accumulating evidence suggests that obesity is a high risk factor for breast cancer among post-menopausal women, which can at least partially be explained by the contribution of adipose tissue to local and circulating estrogen. In response to tumor cell-secreted cytokines, adipose stromal cells (ASCs) express a large amount of aromatase, a key enzyme in estrogen biosynthesis and a clinically proven target for breast cancer therapy. Increased estrogen production from ASCs in turn stimulates hormone-dependent tumor growth, creating a “vicious cycle” between estrogen-dependent tumor cells and estrogen-producing ASCs that is conducive to mammary tumorigenesis. In contrast to our knowledge of aromatase expression in tumor-associated ASCs, relatively little is known about its regulation in ASCs under disease-free conditions. Could aromatase expression in ASCs be induced without the presence of tumor cells? Is there any genetic or epigenetic difference in the inducibility of aromatase expression in cancer-free individuals? If so, might the difference be related to breast cancer risk?&amp;#x2028; Material and Methods: ASCs were isolated from cancer-free individuals by using blendzyme. ACSs were plated in confluent and subconfluent states for several days and then were harvested for RNA isolation, enzymatic activity and protein study. Specific genes were expressed in ASCs using lentiviral expression system and were depleted by siRNA method. Co-culture system was established using ASC and ZR-75 cell line. In a xenograft model, ASCs were injected with the ZR-75 cells in mammary fat pad.&amp;#x2028; Results: Aromatase expression in ASCs can be stimulated up to several hundred folds by cell shape changes induced by various physical or chemical stimuli. Our work uncovered several key signal-transducing molecules including Inhibitory kB Kinases (IKKs) in mediating the cell shape-triggered aromatase expression. Unexpectedly, activation of aromatase gene expression following cell shape change does not involve activation of NFkB, a downstream effector of IKKs. Rather, aromatase induction following cell shape change positively correlated with the expression level of CEBPβ; and CEBPβ knockdown severely impaired aromatase expression. Significant increase in aromatase expression was due to activation of the same breast cancer-associated promoters that are robustly stimulated in intratumoral adipose tissue. In a coculture and invivo xenograft model, ACSs were able to trigger E2 responsive genes as well as stimulate early tumor occurence in ZR-75 cells, respectively.&amp;#x2028; Discussion: Our study suggests that genetic and epigenetic alterations in aromatase-inducing signaling pathway may result in excessive estrogen production and thus breast cancer development. Excess aromatase produced by the stromal cells are capable of initiating tumor in surrounding breast epithelial cells. Study further uncovers a previously unappreciated paradigm for regulation of aromatase expression in adipose tissue, which should have a far-reaching impact on risk assessment and prevention of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5061.

Chronic Green Tea Consumption Decreases Body Mass, Induces Aromatase Expression, and Changes Proliferation and Apoptosis in Adult Male Rat Adipose Tissue

Green tea (GT) and its components have been shown to possess antiobesity properties and the corresponding mechanisms of action are being investigated, given the epidemic proportions of obesity incidence. In the current work, we used 12-mo-old male Wistar rats to test the effect of 6 mo of treatment with GT as the sole drinking beverage (52.8 ± 6.4 mL/d) on adipose tissue (AT). AT aromatase expression was determined by Western blotting, plasma concentrations of 17 β-estradiol and testosterone were determined by RIA, and adipocyte size determined by measuring diameter in tissue sections. Proliferation and apoptosis were also assessed by Ki67 immunostaining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, respectively. Evaluations were made in subcutaneous (sc) AT and visceral (v) AT. Body weight increased over time in both groups (P < 0.001), but the increase was more pronounced in controls (P < 0.001) and food and fluid intake did not influence that effect. At the end of the experiment, aromatase expression increased in the AT (318.5 ± 60.6% of control in scAT, P < 0.05, and 285.5 ± 82.9% of control in vAT, P < 0.01). AT of GT-treated rats had a higher percentage of proliferating cells (204.1 ± 19.5% of control in scAT, P < 0.01, and 246.6 ± 50.2% of control in vAT, P < 0.01) and smaller adipocytes (78.3 ± 1.7% of control in scAT, P < 0.001, and 87.9 ± 3.2% of control in vAT, P < 0.05). GT also increased the number of apoptotic cells in vAT (320.4 ± 21.9% of control; P < 0.001). These results suggest new mechanisms for GT on body weight and highlight its potential benefit to prevent or treat obesity and the metabolic syndrome.

Expression of aromatase increases in both the myocardium and the aortic endothelium during acute illness

OBJECTIVE: Serum estrogen levels rise in humans during acute illness due to increased aromatase expression in adipose tissue. We have also demonstrated increased expression of aromatase in adipose and myocardial tissue in a mouse model of sepsis. Because estrogen appears beneficial to the cardiovascular system, we determined if vascular endothelium as well as myocardial tissue can locally express aromatase and if that expression increases during acute illness.

Characterisation of aromatase expression in the human adipocyte cell line SGBS

Aromatase is a member of the cytochrome P450 superfamily of enzymes which catalyses the rate-limiting step in the biosynthesis of estrogens. A number of clinical studies have highlighted the importance of local estrogen production in adipose tissue. In particular, in the postmenopausal woman, the degree of her estrogenization is mainly determined by the extent of her adiposity and it is this extragonadal source of estrogen that likely contributes to breast cancer development and progression. The mechanisms regulating aromatase expression in adipose tissue however, have not been fully elucidated. In this study, we have characterised the expression of aromatase and its activity in a human preadipocyte cell strain, SGBS. Aromatase is expressed in SGBS cells and its expression and activity are strongly stimulated by forskolin (FSK) and phorbol 12-myristate-13-acetate (PMA) treatment. Consistent with this, FSK and PMA treatment also increased activation of the proximal aromatase promoter, promoter II. These findings mimic those that have previously been shown in isolated primary human preadipocytes. These data suggest that SGBS cells are a valuable model with which to further elucidate the mechanisms regulating aromatase expression, and therefore local estrogen synthesis in human adipose tissue.

No evidence of a role for PPAR?? Pro12Ala polymorphism in endometrial cancer susceptibility

Endogenous oestrogens play a crucial role in endometrial cancer pathogenesis, with most endometrial cancer risk factors causing an increase in oestrogens. Adipose tissue, where androgens are converted to oestrogens by the enzyme aromatase, is an important source of endogenous oestrogen production in the postmenopausal woman. The peroxisome proliferator-activated receptor-gamma (PPARgamma), a key transcriptional regulator of adipogenesis, may also play a role in the regulation of aromatase expression in adipose tissue. We hypothesized that the functional PPARgamma ProAla polymorphism may alter aromatase expression, ultimately affecting endometrial cancer susceptibility. We genotyped the PPARgamma ProAla polymorphism in a study of invasive endometrial cancer cases (n = 222) and matched controls (n = 666) nested within the Nurses' Health Study Cohort. We found little or no evidence of an association between the Ala allele of the PPARgamma codon 12 polymorphism and endometrial cancer risk (adjusted odds ratio = 1.18, 95% confidence interval = 0.80-1.76). Furthermore, we found no association with the PPARgamma ProAla polymorphism and the ratio of oestrone to androstenedione or oestradiol to testosterone plasma hormone levels, measures of aromatase activity. Consistent with previous findings for breast cancer, these results suggest that the PPARgamma ProAla polymorphism does not play a major role in mediating circulating oestrogen levels or endometrial cancer susceptibility.

Estrogen production and action

Estradiol production is most commonly thought of as an endocrine product of the ovary; however, there are many tissues that have the capacity to synthesize estrogens from androgen and to use estrogen in a paracrine or intracrine fashion. In addition, other organs such as the adipose tissue can contribute significantly to the circulating pool of estrogens. There is increasing evidence that in both men and women extraglandular production of C18 steroids from C19 precursors is important in normal physiology as well as in pathophysiologic states. The enzyme aromatase is found in a number of human tissues and cells, including ovarian granulosa cells, the placental syncytiotrophoblast, adipose and skin fibroblasts, bone, and the brain, and it locally catalyzes the conversion of C19 steroids to estrogens. Aromatase expression in adipose tissue and possibly the skin primarily accounts for the extraglandular (peripheral) formation of estrogen and increases as a function of body weight and advancing age. Sufficient circulating levels of the biologically active estrogen estradiol can be produced as a result of extraglandular aromatization of androstenedione to estrone that is subsequently reduced to estradiol in peripheral tissues to cause uterine bleeding and endometrial hyperplasia and cancer in obese anovulatory or postmenopausal women. Extraglandular aromatase expression in adipose tissue and skin (via increasing circulating levels of estradiol) and bone (via increasing local estrogen concentrations) is of paramount importance in slowing the rate of postmenopausal bone loss. Moreover, excessive or inappropriate aromatase expression was demonstrated in adipose fibroblasts surrounding a breast carcinoma, endometriosis-derived stromal cells, and stromal cells in endometrial cancer, giving rise to increased local estrogen concentrations in these tissues. Whether systemically delivered or locally produced, elevated estrogen levels will promote the growth of these steroid-responsive tissues. Finally, local estrogen biosynthesis by aromatase activity in the brain may be important in the regulation of various cognitive and hypothalamic functions. The regulation of aromatase expression in human cells via alternatively used promoters, which can be activated or inhibited by various hormones, increases the complexity of estrogen biosynthesis in the human body. Aromatase expression is under the control of the classically located proximal promoter II in the ovary and a far distal promoter I.1 (40 kilobases upstream of the translation initiation site) in the placenta. In skin, the promoter is I.4. In adipose tissue, 2 other promoters (I.4 and I.3) located between I.1 and II are used in addition to the ovarian-type promoter II. In addition, promoter use in adipose fibroblasts switches between promoters II/I.3 and I.4 upon treatments of these cells with PGE2 versus glucocorticoids plus cytokines. Moreover, the presence of a carcinoma in breast adipose tissue also causes a switch of promoter use from I.4 to II/I.3. Thus there can be complex mechanisms that regulate the extraglandular production of estrogen in a tissue-specific and state-specific fashion. (J Am Acad Dermatol 2001;45:S116-24.)

Aromatase in aging women.

Cessation of ovarian estrogen secretion is the key event during the climacteric. An enzyme termed aromatase in a number of human tissues and cells, including ovarian granulosa cells, the placental syncytiotrophoblast, adipose and skin fibroblasts, bone, and the brain, catalyzes the conversion of C19 steroids to estrogens. Aromatase expression in adipose tissue and possibly the skin primarily accounts for the extraglandular (peripheral) formation of estrogen and increases as a function of body weight and advancing age. Sufficient circulating levels of the biologically active estrogen, estradiol, can be produced as a result of extraglandular aromatization of androstenedione to estrone, which is subsequently reduced to estradiol in peripheral tissues, to cause uterine bleeding and endometrial hyperplasia and cancer in obese anovulatory or postmenopausal women. Extraglandular aromatase expression in adipose tissue and skin (via increasing circulating levels of estradiol) and bone (via increasing local estrogen concentrations) is of paramount importance in slowing the rate of postmenopausal bone loss. Moreover, excessive or inappropriate aromatase expression was demonstrated in adipose fibroblasts surrounding a breast carcinoma, endometriosis-derived stromal cells, and stromal cells in endometrial cancer and gave rise to increased local estrogen concentrations in these tissues. Whether systemically delivered or locally produced, elevated estrogen levels promote the growth of these steroid-responsive tissues. Finally, local estrogen biosynthesis by aromatase activity in the brain may be important in the regulation of various cognitive and hypothalamic functions. The regulation of aromatase expression in human cells via alternatively used promoters, which can be activated or inhibited by various hormones, increases the complexity of estrogen biosynthesis in the human body. Aromatase expression is under the control of the classically located proximal promoter II in the ovary and a far distal promoter I.1 (40 kb upstream of the translation initiation site) in the placenta. In adipose tissue, two other promoters (I.4 and I.3) located between I.1 and II are used in addition to the ovarian-type promoter II. To add a further twist, promoter use in adipose fibroblasts switches between promoters II/I.3 and I.4 upon treatment of these cells with prostaglandin E2 (PGE2) versus glucocorticoids plus cytokines. Moreover, the presence of a carcinoma in breast adipose tissue causes a switch of promoter use from I.4 to II/I.3. Molecular and cellular mechanisms responsible for estrogen formation and their physiologic and clinical relevance will be reviewed in this article.

Cellular characterization of adipose tissue from various body sites of women

Adipose tissue is the primary site of estrogen biosynthesis in postmenopausal women. The two main histologic components of adipose tissue are mature adipocytes and fibroblasts. Aromatase P450 expressed in the fibroblast component of adipose tissue is responsible for catalyzing conversion of C19 steroids to estrogens. We previously have demonstrated that in women, aromatase expression in adipose tissue of various body sites increases with age and that aromatase expression in the hip is markedly higher than in the abdomen. To determine whether this age- and regional-dependent variation in aromatase expression is caused by an alteration in the ratio of fibroblasts to mature adipocytes, we collected sc adipose tissue samples from 19 women (age range: 21-93 yr) at the time of autopsy. Using a computerized image analysis system, we determined by morphometry the proportions of adipocytes, fibroblasts, and vascular endothelial cells within histologic sections of adipose tissue from midabdomen, both breasts, and both hips. The percentage of each cell component at each body site was expressed as the mean of triplicate replicates. Statistical analysis of our results did not indicate any correlation between advancing age and fibroblast to adipocyte ratios in the breast, abdomen, or hip. Fibroblast to adipocyte ratios were found to be significantly higher in the breast and abdomen compared with the hip ( P < 0.05). No statistical differences were found between the breast and abdomen. These findings suggest that the increase in aromatase expression with advancing age and the higher aromatase expression in the hip compared with the abdomen in women may be caused by alterations in specific signal transduction mechanisms rather than a simple increase in local adipose fibroblast numbers.