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Vanida A Serna, Xin Wu, Wenan Qiang, Justin Thomas, Michael L Blumenfeld, and Takeshi Kurita

Cellular mechanisms of uterine leiomyoma (LM) formation have been studied primarily utilizing in vitro models. However, recent studies established that the cells growing in the primary cultures of MED12-mutant LM (MED12-LM) do not carry causal mutations. To improve the accuracy of LM research, we addressed the cellular mechanisms of LM growth and regression utilizing a patient-derived xenograft (PDX) model, which faithfully replicates the patient tumors in situ. The growth and maintenance of MED12-LMs depend on 17β-estradiol (E2) and progesterone (P4). We determined E2 and P4-activated MAPK and PI3K pathways in PDXs with upregulation of IGF1 and IGF2, suggesting that the hormone actions on MED12-LM are mediated by the IGF pathway. When hormones were removed, MED12-LM PDXs lost approximately 60% of volume within 3 days through reduction in cell size. However, in contrast to general belief, the survival of LM cells was independent of E2 and/or P4, and apoptosis was not involved in the tumor regression. Furthermore, it was postulated that abnormal collagen fibers promote the growth of LMs. However, collagen fibers of actively growing PDXs were well aligned. The disruption of collagen fibers, as found in human LM specimens, occurred only when the volume of PDXs had grown to over 20 times the volume of unstimulated PDXs, indicating disruption is the result of growth not the cause. Hence, this study revises generally accepted theories on the growth and regression of LMs.

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A Rody, U Holtrich, C Solbach, K Kourtis, G von Minckwitz, K Engels, S Kissler, R Gätje, T Karn, and M Kaufmann

The function of estrogen receptor beta (ER-β) in mammary tissue is not completely understood. While early observations were often conflicting, more recent data suggest an important role as a tumor-suppressor gene. A decrease of ER-β expression has been observed in ductal carcinoma in situ and invasive carcinoma as compared with benign mammary epithelial cells. The loss of ER-β resulted in abnormal growth of mammary epithelial cells. We have previously shown that the mRNA expression of the ER-β gene is almost totally suppressed in breast carcinomas from patients with a poor prognosis. Here we analyzed whether methylation changes in the different promoters of ER-β are responsible for the loss of expression of the gene. A methylation assay with high specificity and sensitivity was developed, and a panel of breast tissue samples (n = 175) was characterized for methylation status. In contrast to benign breast, more than two-thirds of invasive breast cancers showed a high degree of methylation. Importantly, increased methylation was also detectable in numerous premalignant lesions. By analysis of breast tumors, previously characterized by gene-expression profiling, methylation was predominantly detected in a subgroup of patients with an unfavorable prognosis, suggesting a possible prognostic value of the ER-β methylation status. We also investigated the structural characteristics of the two ER-β promoters, which were both found to be closely associated with a second, downstream, localized and opposite-oriented promoter. However, we could not detect endogenous antisense RNA transcribed from these promoters, which may be involved in epigenetic gene silencing. We also failed to induce ER-β promoter methylation by expressing siRNAs in cell lines. Interestingly, by comparing the promoter sequences of ER-β with other genes known to be epigenetically inactivated in breast cancers, we identified a sequence motif possibly involved in promoter methylation.

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Joanna E Burdette and Teresa K Woodruff

Activin is a member of the transforming growth factor β superfamily that regulates mammary cell function during development, lactation, and in cancer. Activin slows the growth of breast cancer cells by inducing G0/G1 cell cycle arrest. Estrogen is a steroid hormone that stimulates the proliferation of mammary epithelial cells in development and oncogenesis. The crosstalk between estrogen and activin that regulates activin ligand expression, activin and estrogen signal transduction, and cell cycle arrest was investigated in this study. Estrogen antagonized activin-dependent production of plasminogen activator inhibitor 1 (PAI-1) mRNA, while activin repressed estrogen-dependent transcription of trefoil factor 1. The repression of estrogen signaling by activin was recapitulated using a simple estrogen response element-luciferase construct and was enhanced in the presence of overexpressed estrogen receptor α (ERα). In contrast, estrogen-mediated repression of activin signaling could not be recapitulated on a simple CAGA Smad-binding element but did inhibit the short PAI-1 promoter, p3TP-luciferase, especially when ERα was overexpressed. Repression of both estrogen- and activin-regulated transcription was found to be ligand induced and Smad3 dependent. In addition to transcriptional repression, estrogen also reduced the amount of activin B mRNA and protein produced by MCF7 breast cancer cells. These studies demonstrate the importance of activin and estrogen crosstalk during mammary cell growth and cancer initiation.

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Takashi Suzuki, Yasuhiro Miki, Yasuhiro Nakamura, Takuya Moriya, Kiyoshi Ito, Noriaki Ohuchi, and Hironobu Sasano

It is well known that sex steroids are involved in the growth of breast cancers, and the great majority of breast carcinomas express estrogen (ER), progesterone (PR), and androgen (AR) receptors. In particular, recent studies have demonstrated that estrogens and androgens are locally produced in breast carcinoma tissues, and total blockade of in situ estrogen production potentially leads to an improvement in prognosis of breast cancer patients. Therefore, it is important to obtain a better understanding of sex steroid-producing enzymes in breast carcinoma tissues. In this review, we summarize recent studies on the expression and regulation of enzymes related to intratumoral production of estrogens (aromatase, 17β-hydroxysteroid dehydrogenase type 1 (17βHSD1), and steroid sulfatase (STS) etc) and androgens (17βHSD5 and 5α-reductase) in human breast carcinoma tissues, and discuss the biological and/or clinical significance of these enzymes. The cellular localization of aromatase in breast carcinoma tissues still remains controversial. Therefore, we examined localization of aromatase mRNA in breast carcinoma tissues by laser capture microdissection/real time-polymerase chain reaction. Aromatase mRNA expression was detected in both carcinoma and intratumoral stromal cells, and the expression level of aromatase mRNA was higher in intratumoral stromal cells than in carcinoma cells in the cases examined. We also examined an association among the immunoreactivity of enzymes related to intratumoral estrogen production and ERs in breast carcinoma tissues, but no significant association was detected. Therefore, the enzymes responsible for the intratumoral production of estrogen may not always be the same among breast cancer patients, and not only aromatase but also other enzymes such as STS and 17βHSD1 may have important therapeutic potential as targets for endocrine therapy in breast cancer patients.

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Shu Zhang, Ping Lei, Xinyi Liu, Xiangrong Li, Kelcey Walker, Leela Kotha, Craig Rowlands, and Stephen Safe

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the relatively non-toxic selective aryl hydrocarbon receptor (AhR) modulator 6-methyl-1,3,8-trichlorodibenzo-furan (MCDF) induced CYP1A1-dependent ethoxyresorufin O-deethylase activity and inhibited proliferation of seven estrogen receptor (ER) negative breast cancer cell lines. MCDF, TCDD and structurally related 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 3,3′,4,4′,5-pentachlorobiphenyl induced CYP1A1 and inhibited proliferation of BT-474 and MDA-MB-468 cells. In BT474 and MDA-MB-468 cells transfected with a small inhibitory RNA for the AhR, the antiproliferative activity of the chlorinated aromatic compounds was reversed, whereas for MCDF, only partial reversal was observed, suggesting that this compound acts through both AhR-dependent and AhR-independent pathways in these two cell lines. MCDF also inhibited tumor growth in athymic nude mice in which MDA-MB-468 cells were injected directly into the mammary fat pad. These results suggest that the AhR is a potential drug target for treatment of ER-negative breast cancer.

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Frauke Döll, Josef Pfeilschifter, and Andrea Huwiler

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17β-estradiol (E2). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E2 is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E2 cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

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R J Santen, W Yue, F Naftolin, G Mor, and L Berstein

Substantial evidence supports the concept that estrogens cause breast cancer in animals and in women but the precise mechanism is unknown. The most commonly held theory is that estrogens stimulate proliferation of breast cells and thus statistically increase the chances for genetic mutations which could result in cancer. Another theory is that estrogen metabolism generates oxygen-free radicals and quinones which produce both stable and unstable DNA adducts. Both result in genetic mutations which accumulate and could ultimately cause cancer. A major criticism of the latter hypothesis is that breast tissue contains insufficient concentrations of estrogen for accumulation of genotoxic metabolites. Our hypothesis is that breast tissue estrogen levels, as a result of in situ synthesis, are much higher than previously thought. We and others have shown that estrogen can be made in the breast itself through conversion of androgens to estrogens, a process catalyzed by the enzyme aromatase. The levels of estrogen in the breast increase when aromatase is overexpressed. With sufficient amounts of aromatase in breast tissue, enough estradiol as substrate should be available to allow formation of substantial amounts of genotoxic metabolites. We postulate that aromatase overexpression may in this way cause breast cancer. As evidence supporting this concept, four animal models of aromatase overexpression and either breast cancer or premalignant lesions have been described. We have provided evidence that normal breast tissue can make estrogen and that certain stimulatory compounds can increase aromatase activity in the breast by nearly 10,000-fold. If our concepts are correct, it might be possible to prevent breast cancer by blocking the aromatase enzyme. Drugs are currently available to inhibit aromatase nearly completely without causing significant side-effects. Aromatase inhibitors might be more effective than antiestrogens in preventing breast cancer because of their dual role to block both initiation and promotion of breast cancer. To inhibit the initiation process, these inhibitors would reduce levels of the genotoxic metabolites of estradiol by lowering estradiol concentrations in tissue. At the same time, aromatase inhibitors would inhibit the process of tumor promotion by lowering tissue levels of estradiol and thus blocking cell proliferation. These concepts provide a strong rationale for studies of aromatase inhibitors to prevent breast cancer.

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S E Bulun, K Zeitoun, K Takayama, L Noble, D Michael, E Simpson, A Johns, M Putman, and H Sasano

Estrogen is the most important known factor that stimulates the growth of endometriosis. Estrogen delivery to endometriotic implants was classically viewed to be only via the circulating blood in an endocrine fashion. We recently uncovered an autocrine positive feedback mechanism, which favored the continuous production of estrogen and prostaglandin (PG)E2 in the endometriotic stromal cells. The enzyme, aromatase, is aberrantly expressed in endometriotic stromal cells and catalyzes the conversion of C19 steroids to estrogens, which then stimulate cyclooxygenase-2 to increase the levels of PGE2. PGE2, in turn, is a potent inducer of aromatase activity in endometriotic stromal cells. Aromatase is not expressed in the eutopic endometrium. Aromatase expression in endometriosis and its inhibition in eutopic endometrium are controlled by the competitive binding of a stimulatory transcription factor, steroidogenic factor-1, and an inhibitory factor, chicken ovalbumin upstream promoter-transcription factor to a regulatory element in the aromatase P450 gene promoter. In addition, we find that endometriotic tissue is deficient in 17beta-hydroxysteroid dehydrogenase type 2, which is normally expressed in eutopic endometrial glandular cells and inactivates estradiol-17beta to estrone. This deficiency is another aberration that favors higher levels of estradiol-17beta in endometriotic tissues in comparison with the eutopic endometrium. The clinical relevance of local aromatase expression in endometriosis was exemplified by the successful treatment of an unusually aggressive form of recurrent endometriosis in a postmenopausal woman using an aromatase inhibitor.

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C J Fabian, B F Kimler, M S Mayo, and S A Khan

Breast tissue and duct fluid provide a rich source of biomarkers to both aid in the assessment of short-term risk of developing breast cancer and predict and assess responses to prevention interventions. There are three methods currently being utilized to sample breast tissue in asymptomatic women for risk assessment: nipple-aspirate fluid (NAF), random periareolar fine-needle aspiration (RPFNA) and ductal lavage. Prospective single-institution trials have shown that the presence of atypical cells in NAF fluid or RPFNA specimens is associated with an increased risk of breast cancer. Furthermore, RPFNA-detected atypia has been observed to further stratify risk based on the commonly used Gail risk-assessment model. A prospective trial evaluating risk prediction on the basis of atypical cells in ductal-lavage fluid is ongoing. The ability of other established non-genetic biomarkers (mammographic breast density; serum levels of bioavailable estradiol, testosterone, insulin-like growth factor-1 and its insulin like growth factor binding protein-3) to stratify risk based on the Gail model is as yet incompletely defined. Modulation of breast intra-epithelial neoplasia (i.e. hyperplasia with or without atypia) with or without associated breast-tissue molecular markers, such as proliferation, is currently being used to evaluate response in Phase II chemoprevention trials. RPFNA has been the method most frequently used for Phase II studies of 6–12 months duration. However, ductal lavage, RPFNA and random and directed core needle biopsies are all being utilized in ongoing multi-institutional Phase II studies. The strengths and weaknesses of each method are reviewed.

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Patricia de Cremoux, Dan Rosenberg, Jacques Goussard, Catherine Brémont-Weil, Frédérique Tissier, Carine Tran-Perennou, Lionnel Groussin, Xavier Bertagna, Jérôme Bertherat, and Marie-Laure Raffin-Sanson

Adrenal tumors occur more frequently in women and are the leading cause of Cushing's syndrome during pregnancy. We aimed to evaluate the potential role of sex steroids in the susceptibility of women to adrenocortical tumors. We evaluated the presence of the progesterone receptor (PR), estradiol receptors (ERs), and aromatase in 5 patients with primary pigmented nodular adrenal disease (PPNAD), 15 adrenocortical adenomas (ACAs) and adjacent normal tissues, 12 adrenocortical carcinomas (ACCs), and 3 normal adrenal glands (NA). The expression of PR and ERα was evaluated by enzyme immunoassays, real-time RT-PCR, immunohistochemistry, and cytosol-based ligand-binding assays. ERβ and aromatase levels were evaluated by real-time RT-PCR. ERα concentrations were low in NA, in adrenal tissues adjacent to ACA (51±33), in ACC (53±78), and lower in ACA (11±11 fmol/mg DNA). Conversely, PR concentrations were high in NA and adrenal tissues adjacent to ACA, at 307±216 fmol/mg DNA, and were even higher in tumors – 726±706 fmol/mg DNA in ACA and 1154±1586 fmol/mg DNA in ACC – and in isolated PPNAD nodules. Binding study results in four tumors were compatible with binding to a steroid receptor. In patients with PPNAD, a strong positive immunohistochemical signal was associated with the sole isolated nodular regions. ERβ transcript levels were very high in all samples except those for two ACCs, whereas aromatase levels were low. PR and ERβ are clearly present in normal adrenal glands and adrenal tumors. Further studies may shed light on the possible pathogenic role of these receptors in adrenal proliferation.