The G-protein coupled receptors (GPCRs) represent the largest superfamily of membrane proteins in charge to pass the cell signaling after binding with their cognate ligands to the cell interior. In breast cancer, a GPCR named GPER1 plays a key role in the process of growth and the proliferation of cancer cells. In a previous study, theoretical methods were applied to construct a model of GPER1, which later was submitted to molecular dynamics (MD) simulations to perform a docking calculation. Based on this preceding work, it is known that GPER1 is sensitive to structural differences in its binding site. However, due to the nature of that past study, conformational changes linked to the ligand binding were not observed. Therefore, in this study, in order to explore the conformational changes coupled to the agonist/antagonist binding, MD simulations of about 0.25μs were performed for the free and bound states, summarizing 0.75μs of MD simulation in total. For the bound states, one agonist (G-1) and antagonist (G-15) were chosen since is widely known that these two molecules cause an impact on GPER1 mobility. Based on the conformational ensemble generated through MD simulations, we found that despite G-1 and G-15 being stabilized by similar map of residues, the structural differences between both ligands impact the hydrogen bond pattern not only at the GPER1 binding site but also along the seven-helix bundle, causing significant differences in the conformational mobility along the extracellular and cytoplasmic domain, and to a lesser degree in the curvatures of helix 2, helix 3 and helix 7 between the free and bound states, which is in agreement with reported literature, and might be linked to microscopic characteristics of the activated-inactivated transition. Furthermore, binding free energy calculations using the MM/GBSA method for the bound states, followed by an alanine scanning analysis allowed us to identify some important residues for the complex stabilization.

27-Hydroxycholesterol (27OH) is a strong suppressor of cholesterol synthesis and a weak activator of LXR in vitro. The regulatory importance of 27OH in vivo is controversial. Here we utilized male mice with increased levels of 27OH either due to increased production (CYP27A1 transgenic mice) or reduced metabolism (Cyp7b1-/- mice). We also used mice lacking 27OH due to a knockout of Cyp27a1. The latter mice were treated with cholic acid to compensate for reduced bile acid synthesis. The effects of the different levels of 27OH on Srebp- and other LXR-regulated genes in the liver were investigated. In the liver of CYP27tg mice we found a modest increase of the mRNA levels corresponding to the LXR target genes Cyp7b1 and Abca1. A number of other LXR-regulated genes were not affected. The effect on Abca1 mRNA was not seen in the liver of Cyp7b1-/- mice. There were little or no effects on cholesterol synthesis. In the liver of the Cyp27-/- mice treated with 0.025% cholic acid there was no significant effect of the knockout on the LXR target genes. In a previous work triple-knockout mice deficient in the biosynthesis of 24S-hydroxycholesterol, 25-hydroxycholesterol and 27OH were shown to have impaired response to dietary cholesterol, suggesting side-chain oxidized oxysterols to be mediators in cholesterol-induced effects on LXR target genes at a transcriptional level (Chen W. et al., Cell Metab. 5 (2007) 73-79). The hydroxylated oxysterol responsible for the effect was not defined. We show here that treatment of wildtype mice with dietary cholesterol under the same conditions as in the above study induced the LXR target genes Lpl, Abcg8 and Srebp1c in wild type mice but failed to activate the same genes in mice lacking 27-hydroxycholesterol due to a knockout of Cyp27. We failed to demonstrate the above effects at the protein level (Abcg8) or at the activity level (Lpl). The results suggest that 27OH is not an important regulator of Srebp- or LXR regulated genes under basal conditions in mouse liver. On the other hand 27OH appears to mediate cholesterol-induced effects on some LXR target genes at a transcriptional level under some in vivo conditions.

Previous studies have shown that breast tissues and breast cell lines can convert progesterone to 5α-pregnane-3,20-dione (5aP), and that 5αP stimulates breast cell proliferation and detachment in vitro, and tumor formation in vivo, regardless of presence or absence of receptors for progesterone (PR) or estrogen (ER). Recently it was demonstrated, both in vitro and in vivo, that pro-cancer actions attributed to administered progesterone are due to the in situ produced 5αP. Because of the significant role of 5αP in breast cancers, it is important to understand its molecular mechanisms of action. The aims of the current studies were to identify 5αP binding sites and to determine if the mechanisms of action of 5αP involve the mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinases (ERK1/2) pathway. Binding studies, using tritium-labeled 5αP ([(3)H]5αP), carried out on membrane, cytosol and nuclear fractions from human breast cells (MCF-7, PR/ER-positive; MDA-MB-231, PR/ER-negative) and on highly enriched membrane fractions, identified the plasma membrane as the site of ligand specific 5αP receptors. Localization of 5αP receptors to the cell membrane was confirmed visually with fluorescently labeled conjugate (5αP-BSA-FITC). Treatment of cells with either 5αP or membrane-impermeable 5αP-BSA resulted in significant increases in cell proliferation and detachment. 5αP and 5αP-BSA equally activated the MAPK/ERK1/2 pathway as evidenced by phosphorylation of ERK1/2. Inhibitors (PD98059, mevastatin and genistein) of specific sites along the Ras/Raf/MEK/ERK signaling pathway, blocked the phosphorylation and concomitantly inhibited 5αP-induced stimulation of cell proliferation and detachment. The study has identified high affinity, stereo-specific binding sites for 5αP that have the characteristics of a functional membrane 5αP receptor, and has shown that the cancer-promoter actions of 5αP are mediated from the liganded receptor via the MAPK/ERK1/2 signaling cascade. The findings enhance our understanding of the role of the progesterone metabolite 5αP in breast cancer and should promote new approaches to the development of breast cancer diagnostics and therapeutics.

The association between increased serum 25-hydroxyvitamin D (25D) and reduced osteoclastic bone resorption is well known. Previously, we have demonstrated that mechanism by which this occurs, may include the conversion of 25D to 1,25-dihydroxyvitamin D (1,25D) by osteoclasts, catalysed by the CYP27B1 enzyme. Local 1,25D synthesis in osteoclasts was shown to regulate osteoclastogenesis and moderating resorptive activity. Thus, we hypothesised that osteoclasts differentiated from mice with global deletion of the Cyp27b1 gene (Cyp27b1 KO) would display enhanced resorptive capacity due to the lack of an ameliorating effect of 1,25D. Splenocytes isolated from Cyp27b1 KO mice or their wild-type (WT) littermates between 6 and 8 weeks of age were cultured under osteoclast-forming conditions for up to 14 days. Osteoclast formation was measured by staining for the osteoclast marker tartrate resistant acid phosphatase (TRAP). Bone resorption activity was measured by plating the cells on a bone-like substrate. In Cyp27b1 KO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p<0.05) when compared to wild-type (WT) levels. However, Cyp27b1 KO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts (p<0.03). In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in Cyp27b1 KO cultures, implying that these smaller osteoclasts survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of CYP27B1 expression, consistent with the notion that endogenous metabolism of 25D optimises osteoclastogenesis and ameliorates the resulting activity of mature osteoclasts.

Glucocorticoids play essential roles in lung development. We investigated for expression of CYP21A2 (21-hydroxylase) as well as for the presence of the corresponding protein and identification of CYP21A2-expressing cells in several human developing lungs. Expression of some related genes was also assessed. CYP21A2 and CYP17A1 (P450c17) mRNAs were found in all the 34 lung samples from 17 to 40 weeks' gestation at variable levels. No correlation was found according to sex but a correlation with age was detected for CYP17A1 only. In contrast, CYP11B1 (11β-hydroxylase)- and CYP11B2 (aldosterone synthase)-mRNAs were not detected. Significant levels of the CYP21A2 protein were detected in all the analyzed samples, while only very low signals were detected for CYP17A1 protein. In situ hybridization revealed that CYP21A2 was almost exclusively expressed in the distal epithelium. It was reported that the lung distal epithelium of human fetuses also express 11β-hydroxysteroid dehydrogenase type 2, which catalyzes cortisol inactivation into cortisone. Based on this information, intracrine glucocorticoid actions should take place from CYP21A2 products through the glucocorticoid receptor in the absence of cortisol. In contrast, mineralocorticoid receptor activation did not seem to depend on deoxycorticosterone produced from local activity of CYP21A2 because of the reported circulating amounts of aldosterone.

The substrate for the generation of 5α-dihydrotestosterone (DHT) is either androstenedione (4-dione) which is first converted to androstanedione and then to DHT through 17-oxoreductase activity, or testosterone, which is directly converted to DHT. Three 5α-reductase isoenzymes have been characterized and designated as types 1, 2 and 3 (SRD5A1, 2 and 3).

The research was to appraise the utility of the patient-derived tumor xenografts (PDXs) as models of estrogen receptor positive (ER+HER2- and ER+HER2+) breast cancers. We compared protein expression profiles by Reverse Phase Protein Array (RPPA) in tumors that resulted in PDXs compared to those that did not. Our overall PDX intake rate for ER+ breast cancer was 9% (9/97). The intake rate for ER+HER2+ tumors (3/16, 19%) was higher than for ER+HER2- tumors (6/81, 7%). Heat map analyses of RPPA data showed that ER+HER2- tumors were divided into 2 groups by luminal A/B signature [protein expression of ER, AR, Bcl-2, Bim (BCL2L11), GATA3 and INPP4b], and this expression signature was also associated with the rate of PDX intake. Cell survival pathways such as the PI3K/AKT signaling and RAS/ERK pathways were more activated in the specimens that could be established as PDX in both classes. Expression of the ER protein itself may have a bearing on the potential success of an ER+ PDX model. In addition, HER2 and its downstream protein expressions were up-regulated in the ER+HER2+ patient tumors that were successfully established as PDX models. Moreover, the comparison of RPPA data between original and PDX tumors suggested that the selection/adaptation process required to grow the tumors in mice is unavoidable for generation of ER+ PDX models, and we identified differences between patient tumor samples and paired PDX tumors. A better understanding of the biological characteristics of ER+PDX would be the key to using PDX models in assessing treatment strategies in a preclinical setting.

Multiple epidemiological studies have shown that high vitamin D3 status is strongly associated with improved breast cancer survival. To determine the molecular pathways influenced by 1 alpha, 25-dihydroxyvitamin D3 (1,25D) in breast epithelial cells we isolated RNA from normal human breast and cancer tissues treated with 1,25D in an ex vivo explant system. RNA-Seq revealed 523 genes that were differentially expressed in breast cancer tissues in response to 1,25D treatment, and 127 genes with altered expression in normal breast tissues. GoSeq KEGG pathway analysis revealed 1,25D down-regulated cellular metabolic pathways and enriched pathways involved with intercellular adhesion. The highly 1,25D up-regulated target genes CLMN, SERPINB1, EFTUD1, and KLK6were selected for further analysis and up-regulation by 1,25D was confirmed by qRT-PCR analysis in breast cancer cell lines and in a subset of human clinical samples from normal and cancer breast tissues. Ketoconazole potentiated 1,25D-mediated induction of CLMN, SERPINB1, and KLK6 mRNA through inhibition of 24-hydroxylase (CYP24A1) activity. Elevated expression levels of CLMN, SERPINB1, and KLK6 are associated with prolonged relapse-free survival for breast cancer patients. The major finding of the present study is that exposure of both normal and malignant breast tissue to 1,25D results in changes in cellular adhesion, metabolic pathways and tumor suppressor-like pathways, which support epidemiological data suggesting that adequate vitamin D3 levels may improve breast cancer outcome.

Ruminants, possessing the rumino-hepatic circulation, are thought to cope easily with reduced dietary nitrogen (N) supply which is of economic and environmental interest to diminish N output. Nevertheless, feeding an N reduced diet to young goats resulted in a decrease in calcitriol and calcium (Ca) plasma concentrations. Although a dietary Ca reduction alone stimulated calcitriol synthesis and plasma Ca concentrations were restored, in combination with a reduced N supply this stimulating effect was abolished. Based on the important role bone tissue plays in maintaining Ca homeostasis, aim of the present study was to determine effects of an N reduced diet with or without a concomitant Ca reduction on bone metabolism in young goats. A dietary N reduction alone resulted in a significant rise in plasma concentrations of bone resorption marker C-terminal telopeptide of type I collagen (CTX) and bone formation marker osteocalcin (OC), while reduced intake of Ca as well as the combination of both dietary interventions increased bone markers only slightly. Bone mineral content and bone mineral density of metatarsi were decreased by reduced N intake, while Ca and phosphorus (P) content of dried bones remained unaffected. In contrast, a dietary Ca reduction alone led to decreased Ca and P content of dried bones. From these data it can be concluded that a dietary N reduction alone or in combination with a reduced dietary Ca supply modulated bone metabolism in young goats.

This study investigated the role and mechanism of action of G protein-coupled estrogen receptor (GPER) in melanogenesis.

Des-acyl ghrelin is the unacylated form of the well-characterized appetite-stimulating hormone ghrelin. It affects a number of physiological processes, including increasing adipose lipid accumulation and inhibiting adipose tissue inflammation. Breast adipose tissue inflammation in obesity is associated with an increase in the expression of the estrogen biosynthetic enzyme, aromatase, and is hypothesized to create a hormonal milieu conducive to tumor growth. We previously reported that des-acyl ghrelin inhibits the expression and activity of aromatase in isolated human adipose stromal cells (ASCs), the main site of aromatase expression in the adipose tissue. The current study aimed to examine the effect of des-acyl ghrelin on the capacity of mouse macrophages (RAW264.7 cells) and human adipose tissue macrophages (ATMs) to stimulate aromatase expression in primary human breast ASCs. RAW264.7 cells were treated with 0, 10 and 100pM des-acyl ghrelin following activation with phorbol 12-myristate 13-acetate, and cells and conditioned media were collected after 6 and 24h. The effect of des-acyl ghrelin on macrophage polarization was examined by assessing mRNA expression of pro-inflammatory M1-specific marker Cd11c and anti-inflammatory M2-specific marker Cd206, as well as expression of Tnf and Ptgs2, known mediators of the macrophage-dependent stimulation of aromatase. TNF protein in conditioned media was assessed by ELISA. The effect of RAW264.7 and ATM-conditioned media on aromatase expression in ASCs was assessed after 6h. Results demonstrate des-acyl ghrelin significantly increases the expression of Cd206 and suppresses the expression of Cd11c, Tnf and Ptgs2 in activated RAW264.7 cells. Treatment of RAW264.7 and ATMs with des-acyl ghrelin also significantly reduces the capacity of these cells to stimulate aromatase transcript expression in human breast ASCs. Overall, these findings suggest that in addition to direct effects on aromatase in ASCs, des-acyl ghrelin also has the capacity to inhibit the macrophage-dependent induction of aromatase, and provides a novel mechanism for potential effects of des-acyl ghrelin to break the linkage between obesity and breast cancer.

Vitamin D has so far not fulfilled its early promise as an antineoplastic agent, in spite of compelling in vitro data. With the aim of bringing vitamin D or its derivatives (VDDs) effectively to the clinic, we developed a two-pronged approach. First, by adding the plant-derived Carnosic Acid (CA) to a vitamin D2 derivative Doxercalciferol we increased its differentiation potency without increasing it hypercalcemic properties. Second, we added these two agents together to AML cells already treated with Cytarabine (AraC), the standard drug for the treatment of patients with AML. We now report that BRAF, a part of the MAPK signaling pathway, is required for the optimally increased cell death in this system and acts upstream of BIM, the regulator of the caspase cascade that leads to cell death by apoptosis. It is proposed that this therapeutic regimen should be tested in a clinical trial.

Small molecule inhibitors of lysine deacetylases (KDACs) are approved for clinical use in treatment of several diseases. Nuclear receptors, such as the glucocorticoid receptor (GR) use lysine acetyltransferases (KATs or HATs) and KDACs to regulate transcription through acetylation and deacetylation of protein targets such as histones. Previously we have shown that KDAC1 activity facilitates GR-activated transcription at about half of all cellular target genes. In the current study we examine the role of Class I KDACs in glucocorticoid-mediated repression of gene expression. Inhibition of KDACs through two structurally distinct Class I-selective inhibitors prevented dexamethasone (Dex)-mediated transcriptional repression in a gene-selective fashion. In addition, KDAC activity is also necessary to maintain repression. Steroid receptor coactivator 2 (SRC2), which is known to play a vital role in GR-mediated repression of pro-inflammatory genes, was found to be dispensable for repression of glucocorticoid target genes sensitive to KDAC inhibition. At the promoters of these genes, KDAC inhibition did not result in altered nucleosome occupancy or histone H3 acetylation. Surprisingly, KDAC inhibition rapidly induced a significant decrease in H3K4Me2 at promoter nucleosomes with no corresponding change in H3K4Me3, suggesting the activation of the lysine demethylase, LSD1/KDM1A. Depletion of LSD1 expression via siRNA restored Dex-mediated repression in the presence of KDAC inhibitors, suggesting that LSD1 activation at these gene promoters is incompatible with transcriptional repression. Treatment with KDAC inhibitors does not alter cellular levels of LSD1 or its association with Dex-repressed gene promoters. Therefore, we conclude that Class I KDACs facilitate Dex-induced transcriptional repression by suppressing LSD1 complex activity at selected target gene promoters. Rather than facilitating repression of transcription, LSD1 opposes it in these gene contexts.

Melatonin is functionally involved in the control of circadian rhythm and hormonal secretion. In the present study, we investigated the roles of melatonin in the interaction of catecholamine synthesis with adrenocortical steroids by focusing on bone morphogenetic protein (BMP)-4 expressed in the adrenal medulla using rat pheochromocytoma PC12 cells. Melatonin treatment significantly reduced the mRNA expression of catecholamine synthases, including the rate-limiting enzyme tyrosine hydroxylase (Th), 3,4-dihydroxyphenylalanine decarboxylase and dopamine-β-hydroxylase expressed in PC12 cells. In accordance with changes in the expression levels of enzymes, dopamine production and cAMP synthesis determined in the culture medium and cell lysate were also suppressed by melatonin. The MT1 receptor, but not the MT2 receptor, was expressed in PC12 cells, and luzindole treatment reversed the inhibitory effect of melatonin on Th expression, suggesting that MT1 is a functional receptor for the control of catecholamine synthesis. Interestingly, melatonin enhanced the inhibitory effect of BMP-4 on Th mRNA expression in PC12 cells. Melatonin treatment accelerated BMP-4-induced phosphorylation of SMAD1/5/8 and transcription of the BMP target gene Id1. Of note, melatonin significantly upregulated Alk2 and Bmpr2 mRNA levels but suppressed inhibitory Smad6/7 expression, leading to the enhancement of SMAD1/5/8 signaling in PC12 cells, while BMP-4 did not affect Mt1 expression. Regarding the interaction with adrenocortical steroids, melatonin preferentially enhanced glucocorticoid-induced Th mRNA through upregulation of the glucocorticoid receptor and downregulation of Bmp4 expression, whereas melatonin repressed Th mRNA expression induced by aldosterone or androgen without affecting expression levels of the receptors for mineralocorticoid and androgen. Collectively, the results indicate that melatonin plays a modulatory role in catecholamine synthesis by cooperating with BMP-4 and glucocorticoid in the adrenal medulla.

Vitamin D has been shown to display a wide variety of antitumour effects, but their therapeutic use is limited by its severe side effects. We have designed and synthesized a Gemini vitamin D analogue of calcitriol (UVB1) which has shown to display antineoplastic effects on different cancer cell lines without causing hypercalcemia. The aim of this work has been to investigate, by employing in silico, in vitro, and in vivo assays, whether UVB1 inhibits human colorectal carcinoma progression. We demonstrated that UVB1 induces apoptotic cell death and retards cellular migration and invasion of HCT116 colorectal carcinoma cells. Moreover, the analogue reduced the tumour volume in vivo, and modulated the expression of Bax, E-cadherin and nuclear β-catenin in tumour animal tissues without producing toxic effects. In silico analysis showed that UVB1 exhibits greater affinity for the ligand binding domain of vitamin D receptor than calcitriol, and that several characteristics in the three-dimensional conformation of VDR may influence the biological effects. These results demonstrate that the Gemini vitamin D analogue affects the growth of the colorectal cancer and suggest that UVB1 is a potential chemotherapeutic agent for treatment of this disease.

In colon cancer, adenomatous polyposis coli (APC) inactivating gene mutations increase nuclear β-catenin levels and stimulate proliferation. In vitro, 1,25 dihydroxyvitamin D (1,25(OH)2D), suppresses β-catenin-mediated gene transcription by inducing vitamin D receptor (VDR)-β-catenin interactions. We examined whether acute treatment with 1,25(OH)2D could suppress β-catenin-mediated gene transcription in the hyperplastic colonic lesions of mice with colon-specific deletion of both APC gene alleles (CAC; APC(Δ580/Δ580)). At four weeks of age, CAC; APC(Δ580/Δ580) and control mice were injected with vehicle or 1,25(OH)2D (1μg/kg body weight) once a day for three days and then killed six hours after the last injection. mRNA levels of β-catenin target genes were elevated in the colon of CAC; APC(Δ580/Δ580) mice. 1,25(OH)2D increased 25 hydroxyvitamin D-24 hydroxylase mRNA levels in the colon of CAC; APC(Δ580/Δ580) and control mice indicating the treatments activated the VDR. However, 1,25(OH)2D had no effect on either β-catenin target gene mRNA levels or the proliferation index in CAC; APC(Δ580/Δ580) or control mice. VDR mRNA and protein levels were lower (-65% and -90%) in the colon of CAC; APC(Δ580/Δ580) mice compared to control mice, suggesting loss of colon responsiveness to vitamin D. Consistent with this, vitamin D-induced expression of transient receptor potential cation channel, subfamily V, member 6 mRNA was reduced in the colon of CAC; APC(Δ580/Δ580) mice. Our data show that short term exposure to 1,25(OH)2D does not suppress colonic β-catenin signaling in vivo. This article is part of a special issue entitled '17th Vitamin D Workshop'.

Cancer stem cells (CSCs) are a small subset of cells that may be responsible for initiation, progression, and recurrence of tumors. Recent studies have demonstrated that CSCs are highly tumorigenic and resistant to conventional chemotherapies, making them a promising target for the development of preventive/therapeutic agents. A single or combination of various markers, such as CD44, EpCAM, CD49f, CD133, CXCR4, ALDH-1, and CD24, were utilized to isolate CSCs from various types of human cancers. Notch, Hedgehog, Wnt, and TGF-β signalingregulate self-renewal and differentiation of normal stem cells andare aberrantly activated in CSCs. In addition, many studies have demonstrated that these stem cell-associated signaling pathways are required for the maintenance of CSCs in different malignancies, including breast, colorectal, prostate, and pancreatic cancers. Accumulating evidence has shown inhibitory effects of vitamin D and its analogs on the cancer stem cell signaling pathways, suggesting vitamin D as a potential preventive/therapeutic agent against CSCs. In this review, we summarize recent findings about the roles of Notch, Hedgehog, Wnt, and TGF-β signaling in CSCs as well as the effects of vitamin D on these stem cell signaling pathways. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.

Accumulating evidence supports the theory that breast cancer arises from a subpopulation of mammary stem/progenitor cell which posses the ability to self-renew. However, the involvement of estrogen signaling in regulation of breast cancer stem/progenitor cells has not been fully established, mainly because expression and function of ER-α in breast cancer stem cells remains controversial. Previously, our laboratory cloned a variant of ER-α, ER-α36, and found that ER-α36-mediated non-genomic estrogen signaling plays an important role in malignant growth of triple-negative breast cancer cells. In this study, we found that ER-α36 was highly expressed in ER-negative breast cancer SK-BR-3 cells and mediated non-genomic estrogen signaling such as activation of the MAPK/ERK signaling in these cells. Knock-down of ER-α36 expression in these cells using the shRNA method diminished their responsiveness to estrogen and significantly down-regulated HER2 expression. HER2 signaling activated ER-α36 transcription through an AP1 site in the ER-α36 promoter and ER-α36 physically interacted with HER2. We also found that ER-α36 is highly expressed in a subset of SK-BR-3 cells that was positive for ALDH1, a breast cancer stem cell marker, and knock-down of ER-α36 expression reduced the population of ALDH1 positive cells. Our results thus demonstrated that ER-α36 positively regulates HER2 expression and the population of ALDH1 positive breast cancer cells, and suggested that non-genomic estrogen signaling mediated by ER-α36 is involved in maintenance and regulation of breast cancer stem cells.

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the formation of the potent proliferation-stimulating hormone estradiol, and it is thus involved in the development of hormone-dependent breast cancer. Due to its high substrate specificity and the known relationships between its overexpression and disease incidence, 17β-HSD1 is considered an attractive target for drug development. Here, we have used structure-based virtual high-throughput screening to successfully identify potent nonsteroidal 17β-HSD1 inhibitors. Computational screening of a drug-like database containing 13 million compounds identified hits with a 2-benzylidenebenzofuran-3(2H)-one scaffold that we show to be highly potent 17β-HSD1 inhibitors. The most potent in the series, compound 1, showed an IC(50) of 45nM in our 17β-HSD1 inhibition assay, and also showed good selectivity for 17β-HSD1 over 17β-HSD2.