Prostaglandin E 2 Induces Breast Cancer–Related Aromatase Promoters via Activation of p38 and c-Jun NH 2 -Terminal Kinase in Adipose Fibroblasts Dong Chen, Scott Reierstad, Zhihong Lin, Meiling Lu, Chris Brooks, Newton Li, Joy Innes, and Serdar E. Bulun Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois Abstract Aromatase is the key enzyme for estrogen biosynthesis. A distal promoter, PI.4, maintains baseline levels of aromatase in normal breast adipose tissue. In contrast, malignant breast epithelial cells secrete prostaglandin E 2 (PGE 2 ), which stimulates aromatase expression via proximal promoters PI.3/PII in a cyclic AMP (cAMP)– and protein kinase C (PKC)–dependent manner in adjacent breast adipose fibro- blasts (BAF), leading to increased local concentrations of estrogen. Although an effective treatment for breast cancer, aromatase inhibitors indiscriminately abolish estrogen syn- thesis in all tissues, causing major side effects. To identify drug targets to selectively block aromatase and estrogen production in breast cancer, we investigated PGE 2 -stimulated signaling pathways essential for aromatase induction down- stream of cAMP and PKC in human BAFs. Here, we show that PGE 2 or its surrogate hormonal mixture dibutyryl cAMP (Bt 2 cAMP) + phorbol diacetate (PDA) stimulated the p38, c-jun NH 2 -terminal kinase (JNK)-1, and extracellular signal– regulated kinase (ERK) mitogen-activated protein kinase pathways. Inhibition or small interfering RNA–mediated knockdown of p38 or JNK1, but not ERK, inhibited PGE 2 - or Bt 2 cAMP + PDA–induced aromatase activity and expression via PI.3/PII. Conversely, overexpression of wild- type p38A or JNK1 enhanced PGE 2 -stimulated aromatase expression via PII. PGE 2 or Bt 2 cAMP + PDA stimulated c-Jun and activating transcription factor-2 (ATF2) phosphor- ylation and binding to the PI.3/PII region. Specific activa- tion of protein kinase A (PKA) or EPAC with cAMP analogues stimulated p38 and JNK1; however, only PKA- activating cAMP analogues induced aromatase expression. The PKC activator PDA effectively stimulated p38 and JNK1 phosphorylation but not aromatase expression. Taken together, PGE 2 activation of p38 and JNK1 via PKA and PKC is necessary for aromatase induction in BAFs, and p38 and JNK1 are potential new drug targets for tissue-specific ablation of aromatase expression in breast cancer. [Cancer Res 2007;67(18):8914–22] Introduction Aromatase catalyzes the conversion of C 19 steroids to estrogens in a number of human cells and tissues, including ovarian granulosa cells and skin and adipose fibroblasts, hypothalamic neurons, bone, and the placental syncytiotrophoblast (1). A single gene, CYP19 , encodes aromatase. Aromatase expression in adipose tissue is restricted to undifferentiated fibroblasts and not detected in significant quantities in fully differentiated, lipid-filled adipo- cytes (1). Disproportionately high aromatase expression and activity in undifferentiated breast adipose fibroblasts (BAF) adjacent to malignant epithelial cells likely contributes to breast cancer development and progression (2, 3). Moreover, malignant epithelial cells secrete tumor necrosis factor (TNF) and interleukin (IL)-11, which maintain BAFs in an undifferentiated state (4). These fibroblasts are compacted around malignant cells and provide structural support for the tumor (4). This relationship, in which BAFs provide functional support for cancer growth, is supported by the observation that the breast quadrant bearing a malignant tumor consistently displays the highest levels of aromatase activity (2). Expression of aromatase is controlled by several distinct and partially tissue-specific promoters (5). The coding region of aromatase transcripts and the translated protein, however, are identical in all tissues where aromatase is expressed (6, 7). In adipose tissue, three promoters are used. In disease-free breast adipose tissue, aromatase is usually expressed at low levels via distal promoter I.4, whereas in breast adipose tissue bearing a tumor, aromatase expression is activated via two proximally located promoters, I.3 and II (1). Currently, competitive or suicidal aromatase inhibitors are the most effective endocrine treatment of breast cancer (1, 8). However, these agents lead to indiscriminate reduction of aromatase expression throughout the body, resulting in severe estrogen deprivation and major side effects, including hot flashes, bone loss, increased fracture rates, and abnormal lipid metabolism (9). That activation of promoters I.3 and II leads to up-regulation of aromatase expression in breast cancer provides an opportunity to develop new breast cancer treatments that specifically target pathways leading to PI.3/PII activation. To this end, it is important to identify the mechanisms by which aromatase PI.3/PII are activated in breast cancer adipose fibroblasts. PI.3 and PII are located within 215 bp from each other and are coordinately regulated by distinct hormonal stimuli (1). Zhao et al. (10) found that prostaglandin E 2 (PGE 2 ) was a potent stimulator of aromatase expression via PI.3/PII, and several lines of evidence suggest that PGE 2 is involved in breast cancer develop- ment and progression (11, 12). Breast tumor epithelial cells secrete large amounts of PGE 2 as a result of up-regulated cyclooxygenase-2 expression (13), and high levels of PGE 2 production are also Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Dong Chen, Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Northwestern University, 303 East Superior Street, Chicago, IL 60611. Phone: 312-503-3761; Fax: 312-503-0095; E-mail: dong- chen@northwestern.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4751 Cancer Res 2007; 67: (18). September 15, 2007 8914 www.aacrjournals.org Research Article Research. on November 18, 2015. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from