Expression of estrogen-related gene markers in breast cancer tissue predicts aromatase inhibitor responsiveness.

Aromatase inhibitors (AIs) are the most effective class of drugs in the endocrine treatment of breast cancer, with an approximate 50% treatment response rate. Our objective was to determine whether intratumoral expression levels of estrogen-related genes are predictive of AI responsiveness in postmenopausal women with breast cancer. Primary breast carcinomas were obtained from 112 women who received AI therapy after failing adjuvant tamoxifen therapy and developing recurrent breast cancer. Tumor ERα and PR protein expression were analyzed by immunohistochemistry (IHC). Messenger RNA (mRNA) levels of 5 estrogen-related genes-AKR1C3, aromatase, ERα, and 2 estradiol/ERα target genes, BRCA1 and PR-were measured by real-time PCR. Tumor protein and mRNA levels were compared with breast cancer progression rates to determine predictive accuracy. Responsiveness to AI therapy-defined as the combined complete response, partial response, and stable disease rates for at least 6 months-was 51%; rates were 56% in ERα-IHC-positive and 14% in ERα-IHC-negative tumors. Levels of ERα, PR, or BRCA1 mRNA were independently predictive for responsiveness to AI. In cross-validated analyses, a combined measurement of tumor ERα and PR mRNA levels yielded a more superior specificity (36%) and identical sensitivity (96%) to the current clinical practice (ERα/PR-IHC). In patients with ERα/PR-IHC-negative tumors, analysis of mRNA expression revealed either non-significant trends or statistically significant positive predictive values for AI responsiveness. In conclusion, expression levels of estrogen-related mRNAs are predictive for AI responsiveness in postmenopausal women with breast cancer, and mRNA expression analysis may improve patient selection.

JunD and JunB integrate prostaglandin E2 activation of breast cancer-associated proximal aromatase promoters.

Aromatase is the key enzyme in estrogen biosynthesis. Normal breast adipose tissue expresses low levels of aromatase via the distal promoter I.4. Breast adipose tissue surrounding a tumor exhibits excessive aromatase expression controlled by proximal aromatase promoters I.3/II, leading to high local levels of estrogen and breast cancer progression. Prostaglandin E(2) (PGE(2)) secreted by malignant breast epithelial cells activates breast cancer-associated aromatase promoters I.3/II, but silences promoter I.4, in cultured human breast adipose fibroblasts (BAF). The c-Jun N-terminal kinase 1 and p38α mitogen activated protein kinases are necessary for PGE(2) activation of aromatase promoters I.3/II; thus, we examined the roles of downstream targets, c-Jun, JunB, JunD, and activating transcription factor 2, in PGE(2)-mediated regulation of aromatase expression in BAF. PGE(2) induced JunB and JunD protein expression through protein kinase A and protein kinase C, respectively. JunB or JunD knockdown by small interfering RNA markedly reduced PGE(2)-induced total aromatase mRNA level and enzyme activity via promoters I.3/II. JunB knockdown also abrogated JunD expression. JunB stimulated, whereas JunD inhibited, aromatase promoter I.4 activity. Activating transcription factor 2 knockdown did not affect promoter-specific or total aromatase mRNA levels. c-Jun knockdown increased promoter I.4-specific and PGE(2)-induced promoters I.3/II-specific aromatase mRNA levels, leading to enhanced PGE(2)-induced total aromatase mRNA level and enzyme activity. JunD, c-Jun, and JunB bound to a CRE(-211/-199) essential for PGE(2) induction of aromatase promoters I.3/II. Taken together, JunD and c-Jun repress aromatase promoter I.4. JunD mediates, whereas c-Jun modulates, PGE(2) activation of aromatase promoters I.3/II via CRE(-211/-199). JunB also activates aromatase promoters I.3/II by maintaining JunD expression. Targeting JunD may abolish aromatase expression selectively in breast cancer tissue.

Altered retinoid uptake and action contributes to cell survival in endometriosis.

CONTEXT: Retinoic acid (RA) controls multiple biological processes via exerting opposing effects on cell survival. Retinol uptake into cells is controlled by stimulated by RA 6 (STRA6). RA is then produced from retinol in the cytosol. Partitioning of RA between the nuclear receptors RA receptor α and peroxisome-proliferator-activated receptor ß/δ is regulated by cytosol-to-nuclear shuttling proteins cellular RA binding protein 2 (CRABP2) and fatty acid binding protein 5 (FABP5), which induce apoptosis or enhance survival, respectively. The roles of these mechanisms in endometrium or endometriosis remain unknown. OBJECTIVE: The aim was to determine the regulation of retinoid uptake and RA action in primary stromal cells from endometrium (n = 10) or endometriosis (n = 10). RESULTS: Progesterone receptor was necessary for high STRA6 and CRABP2 expression in endometrial stromal cells. STRA6, which was responsible for labeled retinoid uptake, was strikingly lower in endometriotic cells compared to endometrial cells. CRABP2 knockdown in endometrial cells increased survival, and FABP5 knockdown in endometriotic cells decreased survival without altering the expression of downstream nuclear retinoic acid receptor α and peroxisome-proliferator-activated receptor ß/δ. CONCLUSIONS: In endometrial stromal cells, progesterone receptor up-regulates expression of STRA6 and CRABP2, which control retinol uptake and growth-suppressor actions of RA. In endometriotic stromal cells, decreased expression of these genes leads to decreased retinol uptake and dominant FABP5-mediated prosurvival activity.

Transcription factor KLF11 integrates progesterone receptor signaling and proliferation in uterine leiomyoma cells.

Uterine leiomyoma is the most common tumor of the female genital tract and the leading cause of hysterectomy. Although progesterone stimulates the proliferation of uterine leiomyoma cells, the mechanism of progesterone action is not well understood. We used chromatin immunoprecipitation (ChIP)-cloning approach to identify progesterone receptor (PR) target genes in primary uterine leiomyoma smooth muscle cells. We identified 18 novel PR-binding sites, one of which was located 20.5 kb upstream of the transcriptional start site of the Krüppel-like transcription factor 11 (KLF11) gene. KLF11 mRNA levels were minimally downregulated by progesterone but robustly upregulated by the progesterone antagonist RU486. Luciferase reporter assays showed significant baseline and RU486-inducible promoter activity in the KLF11 basal promoter or distal PR-binding region, both of which contained multiple Sp1-binding sequences but lacked classic progesterone response elements. RU486 stimulated recruitment of Sp1, RNA polymerase II, PR, and the coactivators SRC-1 and SRC-2 to the distal region and basal promoter. siRNA knockdown of PR increased KLF11 expression, whereas knockdown of KLF11 increased leiomyoma cell proliferation and abolished the antiproliferative effect of RU486. In vivo, KLF11 expression was significantly lower in leiomyoma tissues compared with adjacent myometrial tissues. Taken together, using a ChIP-cloning approach, we uncovered KLF11 as an integrator of PR signaling and proliferation in uterine leiomyoma cells.

Progesterone and mifepristone regulate L-type amino acid transporter 2 and 4F2 heavy chain expression in uterine leiomyoma cells.

CONTEXT: Progesterone and its receptor (PR) play key roles in uterine leiomyoma growth. Previously, using chromatin immunoprecipitation-based cloning, we uncovered L-type amino acid transporter 2 (LAT2) as a novel PR target gene. LAT2 forms heterodimeric complexes with 4F2 heavy chain (4F2hc), a single transmembrane domain protein essential for LAT2 to exert its function in the plasma membrane. Until now, little is known about the roles of LAT2/4F2hc in the regulation of the growth of human uterine leiomyoma. OBJECTIVE: The aim of the study is to investigate the regulation of LAT2 and 4F2hc by progesterone and the antiprogestin mifepristone and their functions in primary human uterine leiomyoma smooth muscle (LSM) cells and tissues from 39 premenopausal women. RESULTS: In primary LSM cells, progesterone significantly induced LAT2 mRNA levels, and this was blocked by cotreatment with mifepristone. Progesterone did not alter 4F2hc mRNA levels, whereas mifepristone significantly induced 4F2hc mRNA expression. Small interfering RNA knockdown of LAT2 or 4F2hc markedly increased LSM cell proliferation. LAT2, PR-B, and PR-A levels were significantly higher in freshly isolated LSM cells vs. adjacent myometrial cells. In vivo, mRNA levels of LAT2 and PR but not 4F2hc were significantly higher in leiomyoma tissues compared with matched myometrial tissues. CONCLUSION: We present evidence that progesterone and its antagonist mifepristone regulate the amino acid transporter system LAT2/4F2hc in leiomyoma tissues and cells. Our findings suggest that products of the LAT2/4F2hc genes may play important roles in leiomyoma cell proliferation. We speculate that critical ratios of LAT2 to 4F2hc regulate leiomyoma growth.

A novel promoter controls Cyp19a1 gene expression in mouse adipose tissue.

BACKGROUND: Aromatase, the key enzyme in estrogen biosynthesis, is encoded by the Cyp19a1 gene. Thus far, 3 unique untranslated first exons associated with distinct promoters in the mouse Cyp19a1 gene have been described (brain, ovary, and testis-specific). It remains unknown whether aromatase is expressed in other mouse tissues via novel and tissue-specific promoters. METHODS: Real-time PCR was used to examine the aromatase expression levels in various C57BL/6 mouse tissues. 5'-rapid amplification of cDNA ends (5'-RACE) was used to determine the transcriptional start sites of Cyp19a1 transcripts. Promoter activity was measured using serial deletion mutants of DNA fused to the luciferase reporter gene. Primary mouse adipose fibroblasts were isolated and cultured from 16-week-old mouse gonadal fat pads. RESULTS: We systematically analyzed Cyp19a1 expression in a large number of mouse tissues, and demonstrated for the first time that aromatase was expressed in the male but not female gonadal fat pad. Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA. We used 5'-RACE to clone a novel gonadal fat-specific untranslated first exon, which is spliced onto a common junction 15 bp upstream of the translation start site. This adipose-specific first exon was mapped to approximately 75 kb upstream of the translation start site. Transfection of luciferase reporter gene plasmids containing the promoter region upstream of the adipose-specific first exon into murine 3T3-L1 adipose fibroblasts demonstrated significant basal promoter activity conferred primarily by the sequence located at -343/-1 bp. Dexamethasone significantly induced activity of this adipose-specific promoter region. Adipose-specific Cyp19a1 mRNA was expressed in primary mouse adipose fibroblasts and significantly induced by dexamethasone alone or serum plus dexamethasone. CONCLUSION: Taken together, this research identified a novel, adipose-specific first exon of Cyp19a1 and its hormonally regulated promoter region in male murine gonadal fat. These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site. Cyp19a1 expression in mouse adipose tissue may play an important role in reproductive biology and lipid metabolism.

Estrogen receptor-beta mediates cyclooxygenase-2 expression and vascular prostanoid levels in human placental villous endothelial cells.

OBJECTIVE: Regulation of fetoplacental blood flow is likely mediated by factors such as prostanoids. Estrogen and its receptors affect prostanoid biosynthesis. Previously, we demonstrated that villous endothelial cells express estrogen receptor-beta (ESR2), and we sought to determine its role in the mediation of fetoplacental vascular function. STUDY DESIGN: Villous endothelial cells from uncomplicated pregnancies were isolated, cultured, and treated with estrogen. RNA interference, real-time polymerase chain reaction, Western blotting, and enzyme immunoassays were performed. RESULTS: Cyclooxygenase-2 (COX-2) expression levels were not altered consistently by estrogen. RNA interference of ESR2 led to a concomitant decrease in COX-2 messenger RNA (P < .0001) and protein (P < .05) in the presence and absence of estradiol. ESR2 knock-down also led to diminished prostacyclin and thromboxane concentrations in the absence of estradiol (P < .005). CONCLUSION: ESR2 mediates COX-2 expression levels and both prostacyclin and thromboxane concentrations in the basal state, which suggests the possibility of ligand-independent regulation of COX-2 activity and prostaglandin H2 substrate availability. Further investigation regarding ESR2 regulation of prostanoid biosynthesis and its effects on the fetoplacental vasculature is warranted.

High aromatase expression in uterine leiomyoma tissues of African-American women.

CONTEXT: Symptomatic uterine leiomyoma is associated with irregular uterine bleeding, anemia, and recurrent pregnancy loss. African-American women develop uterine leiomyomas at an earlier age and with higher frequency compared with Caucasian-American women or other races; however, the underlying mechanism for this discrepancy is unknown. OBJECTIVE: Our objective was to determine whether gene targets of emerging leiomyoma therapeutics such as aromatase inhibitors and antiprogestins, which reduce tumor size and symptoms, are differentially expressed in tissues of African-American (n = 31), Caucasian-American (n = 34), and Japanese women (n = 36). RESULTS: We found strikingly higher aromatase mRNA levels in leiomyoma compared with adjacent myometrium in African-American (83 fold), Caucasian-American (38 fold), and Japanese women (33 fold). Among the four major promoters that regulate aromatase expression in leiomyoma, the proximal promoter II accounted for higher aromatase mRNA levels in tissues from African-American women. Estrogen receptor subtype alpha mRNA levels were significantly, and 1.8- to 2.6-fold, higher in leiomyoma compared with adjacent myometrium in all groups, whereas leiomyoma estrogen receptor subtype beta mRNA levels were significantly elevated only in Japanese women. Leiomyoma progesterone receptor mRNA levels were significantly higher in Japanese women compared with African-American or Caucasian-American women. CONCLUSIONS: Leiomyoma tissues from African-American women contained the highest level of aromatase expression, which may result in elevated tissue concentrations of estrogen, and account for the higher prevalence and earlier incidence. Analysis of leiomyoma tissue for biomarkers may predict the response to hormonal treatments such as aromatase inhibitors.

Steroidogenic factor-1 and endometriosis.

Endometriosis is a common and chronic disease characterized by persistent pelvic pain and infertility. Estradiol is essential for growth and inflammation in endometriotic tissue. The complete cascade of steroidogenic proteins/enzymes including aromatase is present in endometriosis leading to de novo estradiol synthesis. PGE(2) induces the expression of the genes that encode these enzymes. Upon PGE(2) treatment, coordinate recruitment of the nuclear receptor SF-1 to the promoters of these steroidogenic genes is the key event for estradiol synthesis. SF-1 is the key factor determining that an endometriotic cell will respond to PGE(2) by increased estradiol formation. The presence of SF-1 in endometriosis and its absence in endometrium is determined primarily by the methylation of its promoter. The key steroidogenic enzyme in endometriosis is aromatase encoded by a single gene because its inhibition blocks all estradiol biosynthesis. Aromatase inhibitors diminish endometriotic implants and associated pain refractory to existing treatments in affected women.

Regulation of breast cancer-associated aromatase promoters.

By converting androstenedione to estrone, or testosterone to estradiol, aromatase is a key enzyme in estrogen biosynthesis. Encoded by a single gene CYP19, aromatase is expressed in various tissues, including ovary, placenta, bone, brain, skin, and adipose tissue, via partially tissue-specific promoters, and is essential for normal estrogen-dependent physiological functions. In disease-free breast tissue, aromatase mRNA is primarily transcribed from the weak promoter I.4 and maintained at low levels in breast adipose stromal fibroblasts. In breast cancer a distinct set of aromatase promoters, i.e. I.3, II, and I.7, is activated, leading to a marked increase in aromatase expression in breast tumors and breast adipose tissue adjacent to a breast tumor, and a consequent local overproduction of estrogen that promotes growth and progression of breast cancer. In addition, the total amount of promoter I.4-specific aromatase transcript in breast adipose fibroblasts may also be increased due to both cytokine-induced desmoplastic reaction and cytokine-stimulated promoter I.4 activity in breast cancer. Targeting aromatase has proven beneficial in treating breast cancer, since aromatase inhibitors are the most effective endocrine treatment of breast cancer to date. However, aromatase inhibitors cause major side effects such as bone loss and abnormal lipid metabolism, due to indiscriminate reduction of aromatase activity in all expression sites of the body. Therefore, inhibition of aromatase expression via breast cancer-associated aromatase promoters is a useful strategy to selectively block local aromatase production, and hence estrogen synthesis, in breast cancer. This review will summarize the significant findings on regulation of the breast cancer-associated aromatase promoters, and highlight the discovery of chemical compounds and nuclear receptor ligands that specifically inhibit activation of these aromatase promoters. Clinical side effects of these agents require development of new drugs with better specificity and efficacy, and epigenetic therapies with breast cancer tissue-selective aromatase siRNA-conjugated nanoparticles.

Estrogen receptor (ER) beta regulates ERalpha expression in stromal cells derived from ovarian endometriosis.

CONTEXT: Estradiol and its nuclear receptors, estrogen receptor (ER) alpha and ERbeta, play critical roles in endometrium and endometriosis. Levels of ERbeta, due to pathological hypomethylation of its promoter, are significantly higher in endometriotic vs. endometrial tissue and stromal cells, whereas ERalpha levels are lower in endometriosis. Estradiol regulates ERalpha gene expression via its alternatively used promoters A, B, and C. OBJECTIVE: The aim of the study was to determine whether high levels of ERbeta in endometriotic stromal cells from ovarian endometriomas regulate ERalpha gene expression. RESULTS: ERbeta knockdown significantly increased ERalpha mRNA and protein levels in endometriotic stromal cells. Conversely, ERbeta overexpression in endometrial stromal cells decreased ERalpha mRNA and protein levels. ERbeta knockdown significantly decreased proliferation of endometriotic stromal cells. Chromatin immunoprecipitation assays demonstrated that estradiol enhanced ERbeta binding to nonclassical activator protein 1 and specificity protein 1 motifs in the ERalpha gene promoters A and C and a classic estrogen response element in promoter B in endometriotic stromal cells. CONCLUSIONS: High levels of ERbeta suppress ERalpha expression and response to estradiol in endometrial and endometriotic stromal cells via binding to classic and nonclassic DNA motifs in alternatively used ERalpha promoters. ERbeta also regulates cell cycle progression and might contribute to proliferation of endometriotic stromal cells. We speculate that a significantly increased ratio of ERbeta:ERalpha in endometriotic tissues may also suppress progesterone receptor expression and contribute to progesterone resistance. Thus, ERbeta may serve as a significant therapeutic target for endometriosis.

Novel promoter I.8 and promoter usage in the CYP19 (aromatase) gene.

To date, 10 promoters were reported to regulate the expression of the human aromatase (CYP19) gene, giving rise to transcripts with an identical coding region but tissue-specific first exons comprising unique 5'-untranslated regions. We describe the identification and characterization of a new CYP19 exon I, designated exon I.8, in a 5'-rapid amplification of complementary DNA ends-generated library of human THP-1 monocytic cells. A construct containing exon I.8 and its 5'-flanking sequence was sufficient to drive transcription in THP-1 cells. This novel promoter was located approximately 2-kb upstream of promoter I.4 and approximately 75-kb upstream of the common splice junction. We detected several I.8-containing splice variants, 2 of which also contained a sequence from exon I.4. Analysis of human tissues revealed a unique pattern of promoter I.8 usage. The placenta contained the highest level of I.8-specific transcripts. This work underscores the complexity of the mechanisms that regulate normal and pathologic aromatase expression.

Upstream stimulatory factor-2 regulates steroidogenic factor-1 expression in endometriosis.

Local estrogen biosynthesis is a major factor in the pathogenesis of endometriosis. Aberrant expression of steroidogenic acute regulatory protein (StAR) and aromatase in endometriotic tissue leads to an up-regulation of estrogen production. The transcription factor steroidogenic factor-1 (SF-1) activates the promoters of both StAR and aromatase in endometriotic tissue. We investigated differences in SF-1 expression in endometriotic tissue and normally located endometrium to elucidate the mechanism underlying increased StAR and aromatase activities in endometriosis. Serial deletion and site-directed mutants of the SF-1 promoter showed that an E-box sequence was critical for its activity in endometriotic stromal cells. EMSAs showed that the upstream stimulatory factor (USF) 1 and 2 in nuclear extracts from endometrial and endometriotic stromal cells bound to the E-box. Chromatin-immunoprecipitation-PCR assay, however, demonstrated in intact cells that binding activity of USF2 to the SF-1 promoter was strikingly higher than that of USF1 in endometriotic stromal cells and that USF1 or USF2 binding activity was hardly detectable in endometrial stromal cells. Moreover, knockdown of USF2 but not USF1 resulted in robust and consistent down-regulation of SF-1 and its target genes StAR and aromatase in endometriotic stromal cells. USF2 but not USF1 mRNA and protein levels were significantly higher in endometriotic vs. endometrial stromal cells. In vivo, USF2 mRNA and immunoreactive USF2 levels in endometriotic tissues were strikingly higher than those in endometrium. Taken together, the elevated levels of USF2 in endometriosis account for, in part, the aberrant expression of SF-1 and its target gene StAR and aromatase.

Prostaglandin E(2) induces breast cancer related aromatase promoters via activation of p38 and c-Jun NH(2)-terminal kinase in adipose fibroblasts.

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 fibroblasts (BAF), leading to increased local concentrations of estrogen. Although an effective treatment for breast cancer, aromatase inhibitors indiscriminately abolish estrogen synthesis 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 downstream 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 p38alpha 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) phosphorylation and binding to the PI.3/PII region. Specific activation 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.

Progesterone receptor regulates Bcl-2 gene expression through direct binding to its promoter region in uterine leiomyoma cells.

CONTEXT: Uterine leiomyomas are smooth muscle cell tumors that cause irregular uterine bleeding and pregnancy loss in many reproductive-age women. Progesterone stimulates their growth, whereas treatment with progesterone receptor (PR) antagonists or selective progesterone receptor modulators shrinks these tumors. Molecular mechanisms underlying these observations are unknown. OBJECTIVE: Bcl-2 is a key protein that inhibits apoptosis. It was proposed that growth enhancement of leiomyoma cells by progesterone was mediated via bcl-2 induction. Here we test the hypothesis that PR regulates the bcl-2 gene by directly binding to its promoter. RESULTS: The pure progesterone agonist R5020 increased the total number of viable primary human leiomyoma smooth muscle (LSM) cells in culture. Progesterone or R5020 (10(-6) m) significantly increased bcl-2 mRNA levels after 2 and 4 h by 9.2- and 3.4-fold, respectively, in LSM cells. Transient transfection with deletion mutants of bcl-2 promoter showed that the -1281/-258-bp region conferred responsiveness to progesterone induction in the presence of PR-A. We identified a palindromic progesterone response element (PRE) at -553/-539 bp. EMSA showed that PR in nuclear extracts from LSM cells bound specifically to this PRE. Chromatin immunoprecipitation-PCR confirmed in situ recruitment of PR to the -629/-388-bp region bearing the PRE. In vivo, bcl-2 mRNA levels correlated significantly with total PR mRNA levels in leiomyoma tissues. CONCLUSION: Taken together, progesterone via PR interacts with the bcl-2 promoter to induce its expression in leiomyoma tissue. This may explain, in part, the progesterone-dependent enhancement of growth in uterine leiomyoma.

Promoter methylation regulates estrogen receptor 2 in human endometrium and endometriosis.

Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (-197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2'-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (-197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.

Regulation of 17-beta hydroxysteroid dehydrogenase type 2 in human placental endothelial cells.

17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone. HSD17B2 is highly expressed in human placental tissue where it is localized to placental endothelial cells lining the fetal compartment. The aim of this study was to investigate the effects of potential regulatory factors including progesterone, estradiol, and retinoic acid (RA) onHSD17B2 expression in primary human placental endothelial cells in culture.HSD17B2 mRNA expression was not regulated by progesterone, the progesterone agonist R5020, or estradiol treatment. RA significantly induced HSD17B2 mRNA levels and enzyme activity in a dose- and time-dependent manner. Maximal stimulation occurred at Hour 48 at an RA concentration of 10(-6) M. Both retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA) were readily detected by immunoblotting in isolated placental endothelial cells. RNA interference directed against RARA or RXRA led to reduced basal levels of HSD17B2 mRNA levels and significantly abolished RA-stimulated HSD17B2 expression. Together, these data indicate that regulation of HSD17B2 mRNA levels and enzymatic activity by RA in the placenta is mediated by RARA and RXRA.

Aromatase excess in cancers of breast, endometrium and ovary.

Pathogenesis and growth of three common women's cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215 bp from each other and are coordinately stimulated by PGE(2) via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE(2) secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE(2) may play important roles in inducing local production of estrogen that promotes tumor growth.

Novel estrogen receptor-alpha binding sites and estradiol target genes identified by chromatin immunoprecipitation cloning in breast cancer.

Estrogen receptor-alpha (ERalpha) and its ligand estradiol play critical roles in breast cancer growth and are important therapeutic targets for this disease. Using chromatin immunoprecipitation (ChIP)-on-chip, ligand-bound ERalpha was recently found to function as a master transcriptional regulator via binding to many cis-acting sites genome-wide. Here, we used an alternative technology (ChIP cloning) and identified 94 ERalpha target loci in breast cancer cells. The ERalpha-binding sites contained both classic estrogen response elements and nonclassic binding sequences, showed specific transcriptional activity in reporter gene assay, and interacted with the key transcriptional regulators, including RNA polymerase II and nuclear receptor coactivator-3. The great majority of the binding sites were located in either introns or far distant to coding regions of genes. Forty-three percent of the genes that lie within 50 kb to an ERalpha-binding site were regulated by estradiol. Most of these genes are novel estradiol targets encoding receptors, signaling messengers, and ion binders/transporters. mRNA profiling in estradiol-treated breast cancer cell lines and tissues revealed that these genes are highly ERalpha responsive both in vitro and in vivo. Among estradiol-induced genes, Wnt11 was found to increase cell survival by significantly reducing apoptosis in breast cancer cells. Taken together, we showed novel genomic binding sites of ERalpha that regulate a novel set of genes in response to estradiol in breast cancer. Our findings suggest that at least a subset of these genes, including Wnt11, may play important in vivo and in vitro biological roles in breast cancer.

Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5' CpG island in endometriosis.

CONTEXT: Endometriosis is an estrogen-dependent disease. Steroidogenic factor-1 (SF-1), a transcriptional factor essential for activation of multiple steroidogenic genes for estrogen biosynthesis, is undetectable in normal endometrial stromal cells and aberrantly expressed in endometriotic stromal cells. OBJECTIVE: The objective of the study was to unravel the mechanism for differential SF-1 expression in endometrial and endometriotic stromal cells. DESIGN: We identified a CpG island flanking the SF-1 promoter and exon I region and determined its methylation patterns in endometrial and endometriotic cells. SETTING: The study was conducted at Northwestern University. PATIENTS OR OTHER PARTICIPANTS: Eutopic endometrium from disease-free subjects (n = 8) and the walls of cystic endometriosis lesions of the ovaries (n = 8) were investigated. INTERVENTION(S): Stromal cells were isolated from these two types of tissues. MAIN OUTCOME MEASURE(S): Measures are mentioned in Results. RESULTS: SF-1 mRNA and protein levels in endometriotic stromal cells were significantly higher than those in endometrial stromal cells (P < 0.001). Bisulfite sequencing showed strikingly increased methylation in endometrial cells, compared with endometriotic cells (P < 0.001). Demethylation by 5-aza-2'-deoxycytidine increased SF-1 mRNA levels by up to 55.48-fold in endometrial cell (P < 0.05). Luciferase assays showed that the -85/+239 region bearing the CpG island regulated its activity (P < 0.01). Natural or in vitro methylation of this region strikingly reduced SF-1 promoter activity in both cell types (P < 0.01). Chromatin immunoprecipitation assay showed that methyl-CpG-binding domain protein 2 binds to the SF-1 promoter in endometrial but not endometriotic cells. CONCLUSIONS: This is the first demonstration of methylation-dependent regulation of SF-1 in any mammalian tissue. These findings point to a new mechanism for targeting local estrogen biosynthesis in endometriosis.