Kristy A. Brown

Obesity and Breast Cancer: Progress to Understanding the Relationship

Epidemiological evidence supports a correlation between obesity and breast cancer in women. AMP-activated protein kinase (AMPK) is recognized to be a master regulator of energy homeostasis. One of its actions is to phosphorylate and inhibit the actions of cAMP-responsive element binding protein (CREB)-regulated transcription coactivator 2 (CRTC2). In postmenopausal women, the CREB-dependent regulation of aromatase is a crucial determinant of breast tumor formation through local production of estrogens. We report here that the regulation of aromatase expression in the breast by AMPK and CRTC2, in response to the altered adipokine milieu associated with obesity, provides an important link between obesity and breast cancer risk.

Anti-Müllerian hormone reduces follicle sensitivity to follicle-stimulating hormone in human granulosa cells

OBJECTIVE To determine that anti-Müllerian hormone (AMH) has been shown to inhibits E(2) production in rodents and in luteinized granulosa cells (GC). We determined whether this occurs in human cells most highly expressing AMH (i.e., from small antral follicles) and whether this is an effect on aromatase promoter activity. We also investigated the effects of AMH on other factors determining FSH sensitivity. DESIGN Granulosa cells were exposed to AMH with and without gonadotropins for 48 hours. SETTING University laboratory. PATIENT(S) Not applicable. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Aromatase and FSH receptor messenger RNA expression measured using real time quantitative polymerase chain reaction (PCR). Aromatase promoter II activity measured using a luciferase assay. Estradiol, inhibin A and B, and vascular endothelial growth factor production were measured in the conditioned medium. RESULT(S) The AMH decreased gonadotropin-stimulated aromatase expression and decreased forskolin-stimulated aromatase in KGN cells and this effect was through a dose-dependent inhibition of promoter II. Surprisingly, AMH also reduced FSH receptor mRNA expression. High AMH doses had no effect on inhibin B, whereas a low dose stimulated production. There was no effect on inhibin A or vascular endothelial growth factor. CONCLUSION(S) The AMH inhibits factors affecting FSH sensitivity. As AMH levels decrease with follicle growth, this inhibition would be removed. The AMH overproduction in anovulatory polycystic ovaries (PCO) may therefore restrict folliculogenesis by an inhibitory effect on FSH sensitivity, thereby contributing to anovulation.

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

Epidemiologic evidence supports a correlation between obesity and breast cancer in women. AMP-activated protein kinase plays an important role in energy homeostasis and inhibits the actions of cyclic AMP-responsive element binding protein-regulated transcription coactivator 2 (CRTC2). In postmenopausal women, the cyclic AMP-responsive element binding protein-dependent regulation of aromatase is a determinant of breast tumor formation through local production of estrogens. The present work aimed to examine the effect of adipokines on aromatase expression and identify additional mechanisms by which prostaglandin E(2) causes increased aromatase expression in human breast adipose stromal cells. Treatment of human adipose stromal cells with forskolin and phorbol 12-myristate 13-acetate (PMA), to mimic prostaglandin E(2), resulted in nuclear translocation of CRTC2. Aromatase promoter II (PII) activity assays showed that CRTC2 in addition to forskolin/PMA treatment significantly increased PII-induced activity. CRTC2 binding to PII was examined by chromatin immunoprecipitation, and forskolin/PMA treatment was associated with increased binding to PII. Treatment of human adipose stromal cells with leptin significantly up-regulated aromatase expression associated with nuclear translocation of CRTC2 and increased binding of CRTC2 to PII. Adiponectin treatment significantly decreased forskolin/PMA-stimulated aromatase expression, consistent with the decreased nuclear translocation of CRTC2 and the decreased binding of CRTC2 to PII. The expression and activity of the AMP-activated protein kinase LKB1 was examined and found to be significantly decreased following either forskolin/PMA or leptin treatment. In contrast, adiponectin significantly increased LKB1 expression and activity. In conclusion, the regulation of aromatase by CRTC2, in response to the altered hormonal milieu associated with menopause and obesity, provides a critical link between obesity and breast cancer.

Minireview: Obesity and Breast Cancer: A Tale of Inflammation and Dysregulated Metabolism

In addition to the spectrum of conditions known collectively as the Metabolic Syndrome, obesity is now recognized to be associated with increased risk of several cancers including colon, endometrial, and breast cancer. Obesity and carcinogenesis share 2 characteristics in common. On the one hand, they involve inflammatory pathways, and on the other hand, they involve dysregulated metabolism. In this review we focus on postmenopausal breast cancer and discuss the metabolic and cellular mechanisms whereby obesity and breast cancer are related. Because a majority of postmenopausal breast tumors are estrogen responsive, we include a discussion of the action of obesity-related factors on estrogen formation within the breast.

p53: Protection against Tumor Growth beyond Effects on Cell Cycle and Apoptosis

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.

Endocrine-related cancers and the role of AMPK.

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis involved in the regulation of a number of physiological processes including β-oxidation of fatty acids, lipogenesis, protein and cholesterol synthesis, as well as cell cycle inhibition and apoptosis. Important changes to these processes are known to occur in cancer due to changes in AMPK activity within cancer cells and in the periphery. This review aims to present findings relating to the role and regulation of AMPK in endocrine-related cancers. Obesity is a known risk factor for many types of cancers and a number of endocrine factors, including adipokines and steroid hormones, are regulated by and regulate AMPK. A clear role for AMPK in breast cancer is evident from the already impressive body of work published to date. However, information pertaining to its role in prostate cancer is still contentious, and future work should unravel the intricacies behind its role to inhibit, in some cases, and stimulate cancer growth in others. This review also presents data relating to the role of AMPK in cancers of the endometrium, ovary and colon, and discusses the possible use of AMPK-activating drugs including metformin for the treatment of all endocrine-related cancers.

Obesity and breast cancer: role of inflammation and aromatase

Obesity is now recognised to be an inflammatory condition in which dysregulated metabolism plays an integral role. Inflammatory mediators regulate aromatase expression in the human breast as one mechanism whereby they increase the risk of breast cancer, especially in women who are obese.

Regulation of LKB1 expression by sex hormones in adipocytes

Objective:In the adipose tissue, activation of AMP-activated protein kinase (AMPK) by phosphorylation favours local fatty acid oxidation and inhibition of lipogenesis. We have previously shown that the potent androgen dihydrotestosterone (DHT) can inhibit phosphorylation of AMPK in adipose tissue and 3T3-L1 adipocytes in a dose-dependent manner. This negative effect of DHT was reversed by oestrogen treatment. The purpose of this current study was to determine the underlying mechanisms whereby androgens and oestrogens can regulate AMPK phosphorylation in adipocytes, and whether this mechanism is receptor dependent.Results:Phosphorylation of AMPK was assessed by western blot in cells treated for 24 h with testosterone or DHT (1–1000 nM). Testosterone and DHT significantly inhibited basal phosphorylation of AMPK. Addition of the androgen receptor antagonist Flutamide (1 μM) to the media reversed the negative effect of testosterone and DHT by returning AMPK phosphorylation levels to those of basal. To further dissect the mechanism underlying AMPK inhibition by testosterone or DHT, we examined the mRNA expression of the upstream activator of AMPK, namely LKB1. Testosterone and DHT treatment of murine 3T3-L1 or human SGBS adipocytes for 24 h significantly decreased the mRNA expression of LKB1. In contrast, 17β-estradiol treatment increased LKB1 mRNA, an effect mediated by oestrogen receptor alpha.Conclusion:We conclude that regulation of AMPK phosphorylation by androgens and oestrogens is receptor-dependent, and demonstrate for the first time that LKB1 is regulated by sex hormones in adipocytes.

Prostaglandin E2 inhibits p53 in human breast adipose stromal cells: a novel mechanism for the regulation of aromatase in obesity and breast cancer.

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.

Promoter-specific effects of metformin on aromatase transcript expression.

Phase III aromatase inhibitors (AIs) are proving successful in the treatment of hormone-dependent postmenopausal breast cancer. Side-effects associated with total body aromatase inhibition have prompted new research into the development of breast-specific AIs. The identification of tissue- and disease-specific usage of aromatase promoters has made the inhibition of aromatase at the transcriptional level an interesting approach. We have previously demonstrated that AMPK-activating drugs, including metformin, were potent inhibitors of aromatase expression in primary human breast adipose stromal cells (hASCs). This study examines the promoter-specific effects of metformin on inhibiting aromatase expression in hASCs. Tumour-associated promoters PII/PI.3 were activated using forskolin (FSK)/phorbol ester (PMA), whereas normal adipose associated promoter PI.4 was activated using dexamethasone (DEX)/tumour necrosis factor-α (TNFα). Results demonstrate that metformin significantly decreased the FSK/PMA-, but not the DEX/TNFα-mediated expression of total aromatase at concentrations of 10, 20, and 50 μM (P ≤ 0.05). Using PCR to amplify promoter-specific transcripts of aromatase, it appears that the inhibition of the FSK/PMA-mediated expression of aromatase is due to decreases in PII/PI.3-specific transcripts, whereas no effect of metformin is observed on any promoter-specific transcript, including PI.4, in DEX/TNFα-treated hASCs. This report therefore supports the hypothesis that metformin would act as a breast-specific inhibitor of aromatase expression in the context of postmenopausal breast cancer.