Erica E. Marsh

Aromatase excess in cancers of breast, endometrium and ovary

Pathogenesis and growth of three common womens 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.

Promoter Methylation Regulates Estrogen Receptor 2 in Human Endometrium and Endometriosis

Abstract 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.

Decrease in gonadotropin-releasing hormone (GnRH) pulse frequency with aging in postmenopausal women

UNLABELLED Increasing evidence suggests that aging is associated with dynamic changes in the hypothalamic and pituitary components of the reproductive axis that are independent of changes in gonadal hormone secretion. This study was designed to determine the effect of age on GnRH pulse frequency in women in the absence of gonadal feedback using gonadotropin free alpha-subunit (FAS) and LH as neuroendocrine markers of endogenous GnRH secretion. All studies were performed in healthy, euthyroid postmenopausal women (PMW) during daytime hours. The impact of sampling interval and duration on assessment of pulse frequency in PMW was first examined in 10 women with a mean age of 61.6 +/- 8 yr (mean +/- SD), in whom blood was sampled every 5 min for 12 h. Each 5-min series was then reduced to simulate a 10-min series and then a 15-min series for pulse analysis, and the effect of 8 h compared with 12 h of sampling was determined. To define the changes in the frequency and amplitude of pulsatile hormone secretion with aging, 11 younger (45-55 yr) and 11 older (70-80 yr) PMW were then studied over 8 h at a 5-min sampling interval. In the initial series, the mean interpulse intervals (IPIs) for FAS were 53.8 +/- 3.6, 69.2 +/- 3.9, and 87.6 +/- 7.3 min at sampling intervals of 5, 10, and 15 min, respectively (P < 0.0005). The LH IPI also increased progressively with sampling intervals of 5, 10, and 15 min (54.4 +/- 2.5, 70.4 +/- 2.3, and 91.1 +/- 4.4 min; P < 0.0001). At the 5-min sampling interval, the calculated number of pulses/24 h was not different between a 12-h series compared with an 8-h series for either FAS or LH. In the second series of studies, the older PMW had lower gonadotropin levels (LH, 86.5 +/- 8.8 vs. 51.3 +/- 7.7 IU/L, P < 0.01; FSH, 171.6 +/- 16.9 vs. 108.2 +/- 10.5 IU/L, P < 0.005; FAS, 1021.5 +/- 147.4 vs. 425.6 +/- 89.6 ng/L, P < 0.005, in younger and older PMW, respectively) despite no differences in estrone or estradiol levels. The older PMW also demonstrated a slower FAS pulse frequency compared with their younger counterparts, as reflected in an increased FAS IPI (52.6 +/- 3.1 and 70.6 +/- 5.9 min; P < 0.002). The difference in IPIs between younger and older PMW was not statistically significant for LH (65.4 +/- 5.6 and 71.8 +/- 6.6 min for younger and older PMW, respectively). FAS pulse amplitude was decreased in older PMW compared with younger PMW (431.7 +/- 66.2 vs. 224.6 +/- 81.9 ng/L; P < 0.01), whereas the decrease in LH pulse amplitude with age was of borderline statistical significance (23.2 +/- 3.1 vs. 15.9 +/- 2.1 IU/L; P = 0.09). IN CONCLUSION 1) the use of a 5-min sampling interval and measurement of FAS as the primary marker of GnRH pulse generator activity indicate that GnRH pulse frequency in younger PMW is faster than previously reported, but not increased over that seen in the late follicular phase and midcycle surge in women with intact ovarian function; and 2) the marked decrease in FAS pulse frequency with age provides evidence of age-related changes in the hypothalamic component of the reproductive axis that are independent of changes in gonadal function.

Reactive Oxygen Species Mediate Mitogenic Growth Factor Signaling Pathways in Human Leiomyoma Smooth Muscle Cells

Abstract Uterine leiomyomas are benign uterine tumors characterized by extracellular matrix remodeling, increased collagen deposition, and increased smooth muscle cell (SMC) proliferation. The reactive oxygen species (ROS) producing NADPH oxidase complex has been shown to be involved in the signaling pathways of several growth factors, cytokines, and vasoactive agents that stimulate proliferation of a variety of cell types. Our objective was to test the hypothesis that ROS derived from NADPH oxidase is a necessary component of the MAP kinase mitogenic pathway activated by platelet derived growth factor (PDGF) and epidermal growth factor (EGF) in leiomyoma SMCs (LSMCs). Primary cell cultures of LSMCs were used as our experimental model. Our results showed that stimulation of these cells with PDGF or EGF caused a marked increase in intracellular ROS production and that the NADPH oxidase inhibitor, DPI, blocks ROS production. In addition, inhibition of ROS production by NADPH oxidase inhibitors blocked, in a dose-dependent manner, the EGF- and PDGF-induced increase in [3H]thymidine incorporation by LSMCs. Furthermore, an exogenous source of ROS, hydrogen peroxide, was sufficient to stimulate [3H]thymidine incorporation in LSMCs but did not affect COL1A2 and COL3A1 mRNA levels. Inhibition of the NADPH oxidase complex decreased PDGF-induced MAPK1/MAPK3 activation, whereas exogenous hydrogen peroxide induced MAPK1/MAPK3 activation. This article is the first report suggesting the presence of the NADPH oxidase system and its importance in mitogenic signaling pathways in LSMCs. The necessity of NADPH oxidase-derived ROS for EGF and PDGF signaling pathways leading to cell proliferation points to another potential therapeutic target for treatment and/or prevention of uterine leiomyomas.

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.

Ovarian steroids, stem cells and uterine leiomyoma: therapeutic implications

BACKGROUND Uterine leiomyoma is the most common benign tumor in women and is thought to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a cellular insult. Leiomyomas cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are the most common indication for hysterectomy in the USA. A slow rate of cell proliferation, combined with the production of copious amounts of extracellular matrix, accounts for tumor expansion. A common salient feature of leiomyomas is their responsiveness to steroid hormones, thus providing an opportunity for intervention. METHODS A comprehensive search of PUBMED was conducted to identify peer-reviewed literature published since 1980 pertinent to the roles of steroid hormones and somatic stem cells in leiomyoma, including literature on therapeutics that target steroid hormone action in leiomyoma. Reviewed articles were restricted to English language only. Studies in both animals and humans were reviewed for the manuscript. RESULTS Estrogen stimulates the growth of leiomyomas, which are exposed to this hormone not only through ovarian steroidogenesis, but also through local conversion of androgens by aromatase within the tumors themselves. The primary action of estrogen, together with its receptor estrogen receptor α (ERα), is likely mediated via induction of progesterone receptor (PR) expression, thereby allowing leiomyoma responsiveness to progesterone. Progesterone has been shown to stimulate the growth of leiomyoma through a set of key genes that regulate both apoptosis and proliferation. Given these findings, aromatase inhibitors and antiprogestins have been developed for the treatment of leiomyoma, but neither treatment results in complete regression of leiomyoma, and tumors recur after treatment is stopped. Recently, distinct cell populations were discovered in leiomyomas; a small population showed stem-progenitor cell properties, and was found to be essential for ovarian steroid-dependent growth of leiomyomas. Interestingly, these stem-progenitor cells were deficient in ERα and PR and instead relied on the strikingly higher levels of these receptors in surrounding differentiated cells to mediate estrogen and progesterone action via paracrine signaling. CONCLUSIONS It has been well established that estrogen and progesterone are involved in the proliferation and maintenance of uterine leiomyoma, and the majority of medical treatments currently available for leiomyoma work by inhibiting steroid hormone production or action. A pitfall of these therapeutics is that they decrease leiomyoma size, but do not completely eradicate them, and tumors tend to regrow once treatment is stopped. The recent discovery of stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma has the potential to provide the missing link between developing therapeutics that temper leiomyoma growth and those that eradicate them.

17β-Hydroxysteroid Dehydrogenase-2 Deficiency and Progesterone Resistance in Endometriosis

Estradiol (E2) stimulates the growth and inflammation in the ectopic endometriotic tissue that commonly resides on the pelvic organs. Several clinical and laboratory-based observations are indicative of resistance to progesterone action in endometriosis. The molecular basis of progesterone resistance in endometriosis may be related to an overall reduction in the levels of progesterone receptor (PR). In normal endometrium, progesterone acts via PR on stromal cells to induce secretion of paracrine factor(s) that in turn stimulate neighboring epithelial cells to express the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (HSD17B2). HSD17B2 is an extremely efficient enzyme and rapidly metabolizes the biologically potent estrogen E2 to weakly estrogenic estrone. In endometriotic tissue, progesterone is incapable of inducing epithelial HSD17B2 expression due to a defect in stromal cells. The inability of endometriotic stromal cells to produce progesterone-induced paracrine factors that stimulate HSD17B2 may be due to the very low levels of PR observed in vivo in endometriotic tissue. The end result is deficient metabolism of E2 in endometriosis giving rise to high local concentrations of this mitogen. The molecular details of this physiological paracrine interaction between the stroma and epithelium in normal endometrium and its lack thereof in endometriosis are discussed.

Absence of Circadian Rhythms of Gonadotropin Secretion in Women

CONTEXT Diurnal rhythms of LH and FSH have been reported in normal women, but it is unclear whether these reflect underlying circadian control from the suprachiasmatic nucleus and/or external influences. OBJECTIVE The aim of this study was to determine whether endogenous circadian rhythms of LH, FSH, and the glycoprotein free α-subunit (FAS) are present in reproductive-aged women. DESIGN AND SETTING Subjects were studied in the early follicular phase using a constant routine protocol in a Clinical Research Center at an academic medical center. SUBJECTS Subjects were healthy, normal-cycling women aged 23-29 yr (n = 11). MAIN OUTCOME MEASURES Temperature data were collected, and blood samples were assayed for LH, FSH, FAS, and TSH. RESULTS Core body temperature and TSH were best fit by a sinusoid model, indicating that known circadian rhythms were present in this population. However, the patterns of FSH, LH, and FAS over 24 h were best fit by a linear model. Furthermore, there were no differences in LH and FAS interpulse intervals or pulse amplitudes between evening, night, and morning. CONCLUSIONS Under conditions that control for sleep/wake, light/dark, activity, position, and nutritional cues, there is no circadian rhythm of LH, FSH, or FAS in women during the early follicular phase despite the presence of endogenous rhythms of TSH and core body temperature. These studies indicate that endogenous circadian control does not contribute to previously reported diurnal rhythms in reproductive-aged women and emphasizes the importance of environmental cues in controlling normal reproductive function.

CCAAT/Enhancer Binding Protein β Regulates Aromatase Expression via Multiple and Novel Cis-Regulatory Sequences in Uterine Leiomyoma

CONTEXT Control of aromatase expression in uterine leiomyoma has significant clinical implications because aromatase inhibitors reduce tumor growth and associated irregular uterine bleeding. The mechanisms that regulate aromatase expression in leiomyoma are unknown. OBJECTIVES We previously demonstrated that the cAMP-responsive proximal promoters I.3 and II regulate aromatase expression in vivo in uterine leiomyoma tissue. Here, we investigated the cellular and molecular mechanisms responsible for promoter I.3/II usage. RESULTS In smooth muscle cells isolated from leiomyoma (LSMCs), dibutyryl cAMP significantly induced aromatase mRNA and enzyme activity. Reporter constructs of promoter I.3/II deletion and site-directed mutants with selective disruption of cis-regulatory elements in the -517/-16 bp region revealed that five out of seven elements, including three CCAAT/enhancer binding protein (C/EBP) binding sites and two cAMP response elements, were essential for cAMP-induced promoter activity. EMSAs demonstrated that nuclear extracts from LSMCs contain complexes assembled on four of the five cis-elements, with C/EBP binding sites, including a novel -245/-231 bp sequence, clearly associating with C/EBPbeta. Chromatin immunoprecipitation assays revealed that C/EBPbeta binds specifically to the promoter I.3/II region in intact cells. Dibutyryl cAMP significantly induced nuclear C/EBPbeta protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBPbeta dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity. CONCLUSIONS C/EBPbeta, which binds to multiple cis-regulatory elements in promoter I.3/II, is a key factor in the transcriptional complex controlling aromatase expression in uterine leiomyoma cells. Definition of this mechanism further may assist in designing inhibitors of aromatase specific for leiomyoma tissue.

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.