Polycystic ovary syndrome (PCOS) affects between 5 and 8% of women, making it one of the most common endocrinopathies in women. The disorder typically has its onset at puberty with evidence of excessive androgen production, obesity, and insulin resistance. Women with PCOS are more insulin resistant than weight-matched controls and have an exceptionally high prevalence of early-onset impaired glucose tolerance (30 - 40 percent), and type 2 diabetes (up to 10 percent). Over the past several years, chronic decreases in sleep duration and/or quality have been identified as a risk for the development of a number of metabolic derangements that are strikingly similar to those seen in PCOS. Specifically, decreased sleep quality due to obstructive sleep apnea (OSA) has been causally linked to insulin resistance, glucose intolerance, dyslipidemia and hypertension independent of body mass index (BMI). Until recently, however, it had not been recognized that OSA is present in a disproportionate number of women with PCOS: the risk for OSA is at least 5-10 fold higher compared to the risk in similarly obese women without PCOS. The causes and consequences of OSA in women with PCOS are addressed in this manuscript.
Polycystic ovary syndrome; obstructive sleep apnea; metabolic syndrome; type 2 diabetes mellitus; impaired glucose tolerance; insulin resistance; obesity; cardiovascular; dyslipidemia
Chronic low-grade inflammation has emerged as a key contributor to the pathogenesis of Polycystic Ovary Syndrome (PCOS). A dietary trigger such as glucose is capable of inciting oxidative stress and an inflammatory response from mononuclear cells (MNC) of women with PCOS, and this phenomenon is independent of obesity. This is important because MNC-derived macrophages are the primary source of cytokine production in excess adipose tissue, and also promote adipocyte cytokine production in a paracrine fashion.
The proinflammatory cytokine tumor necrosis factor-α (TNFα) is a known mediator of insulin resistance. Glucose-stimulated TNFα release from MNC along with molecular markers of inflammation are associated with insulin resistance in PCOS. Hyperandrogenism is capable of activating MNC in the fasting state, thereby increasing MNC sensitivity to glucose; and this may be a potential mechanism for promoting diet-induced inflammation in PCOS.
Increased abdominal adiposity is prevalent across all weight classes in PCOS, and this inflamed adipose tissue contributes to the inflammatory load in the disorder. Nevertheless, glucose ingestion incites oxidative stress in normal weight women with PCOS even in the absence of increased abdominal adiposity.
In PCOS, markers of oxidative stress and inflammation are highly correlated with circulating androgens. Chronic suppression of ovarian androgen production does not ameliorate inflammation in normal weight women with the disorder. Furthermore, in vitro studies have demonstrated the ability of pro-inflammatory stimuli to upregulate the ovarian theca cell steroidogenic enzyme responsible for androgen production. These findings support the contention that inflammation directly stimulates the polycystic ovary to produce androgens.
Hyperandrogenism; inflammation; oxidative stress; insulin resistance; ovarian dysfunction; abdominal adiposity
Polycystic ovary syndrome (PCOS) is associated with metabolic derangements including insulin resistance, dyslipidemia, systemic inflammation and endothelial dysfunction. There is a growing need to develop pharmacologic interventions to improve metabolic function in women with PCOS. Medications that have been tested in patients with PCOS include metformin, thiazolidinediones, acarbose, naltrexone, orlistat, vitamin D and statins.
Metformin decreases hepatic gluconeogenesis and free fatty acid oxidation while increasing peripheral glucose uptake. Early studies in PCOS suggested that metformin indirectly reduces insulin level, dyslipidemia and systemic inflammation; however, recent placebo-controlled trials failed to demonstrate significant metabolic benefit. Thiazolidinediones act primarily by increasing peripheral glucose uptake. Most studies in PCOS have demonstrated that thiazolidinediones reduce insulin resistance; however, effects on dyslipidemia were disappointing. Use of thiazolidinediones is associated with weight gain and major complications. Acarbose reduces digestion of polysaccharides. Studies in PCOS yielded inconsistent effects of acarbose on insulin sensitivity and no significant improvement of dyslipidemia. Naltrexone reduces appetite and modulates insulin release; its use in PCOS may reduce hyperinsulinemia. Orlistat decreases absorption of dietary fats; studies in PCOS suggest beneficial effects on insulin sensitivity. Vitamin D may improve insulin sensitivity but mixed results on lipid profile in PCOS have been reported. Statins are competitive inhibitors of the key enzyme regulating the mevalonate pathway; their effects are related to reduced cholesterol production as well as anti-inflammatory and anti-oxidant properties. In women with PCOS, statins reduce hyperandrogenism, improve lipid profile and reduce systemic inflammation while the effects on insulin sensitivity are variable. Use of statins is contraindicated in pregnancy.
Polycystic ovary syndrome; Metabolic dysfunction; Insulin sensitivity; Dyslipidemia; Inflammation
Polycystic ovarian syndrome (PCOS) is a common disorder characterized by ovulatory dysfunction and hyperandrogenemia (HA). Neuroendocrine abnormalities including increased gonadotropin-releasing hormone (GnRH) pulse frequency, increased luteinizing hormone (LH) pulsatility, and relatively decreased follicle stimulating hormone contribute to its pathogenesis. HA reduces inhibition of GnRH pulse frequency by progesterone, causing rapid LH pulse secretion and increasing ovarian androgen production. The origins of persistently rapid GnRH secretion are unknown but appear to evolve during puberty. Obese girls are at risk for HA and develop increased LH pulse frequency with elevated mean LH by late puberty. However, even early pubertal girls with HA have increased LH pulsatility and enhanced daytime LH pulse secretion, indicating the abnormalities may begin early in puberty. Decreasing sensitivity to progesterone may regulate normal maturation of LH secretion, potentially related to normally increasing levels of testosterone during puberty. This change in sensitivity may become exaggerated in girls with HA. Many girls with HA—especially those with hyperinsulinemia—do not exhibit normal LH pulse sensitivity to progesterone inhibition. Thus, HA may adversely affect LH pulse regulation during pubertal maturation leading to persistent HA and the development of PCOS.
Polycystic ovarian syndrome; Gonadotropin releasing hormone; Obesity; Puberty; Hyperandrogenemia; Neuroendocrine
Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH)2D3 analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25-N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner–Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80–1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH)2D3, further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH)2D3 and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1.
1; 25-Dihydroxyvitamin D3; Cytochrome P450 inhibitors; CYP24A1; Cyclopropylamines; Cancer therapy; Molecular docking
Although exogenous glucocorticoids (GC) play a role in the regulation of bone marrow mesenchymal stem/stromal cells (MSCs) proliferation and differentiation, the function of endogenous GC is not well understood. The purpose of this study was to investigate the effect of the blockage of endogenous GC using RU486, an antagonist of the glucocorticoid receptor, on the in vitro proliferation and differentiation capabilities of human MSCs. We quantitatively measured cell proliferation of human MSCs after treatment with increasing concentrations of RU486. We also evaluated multiple MSC differentiation capabilities, as well as the expression of stemness and senescence genes after proliferation of these human cells in vitro in the presence of RU486 at 10−8M. It was observed that RU486 treatment significantly increases the proliferation of human MSCs, although the optimal dose of RU486 for this increase in proliferation differs depending on the gender of the MSC donor. This improvement in MSC proliferation with RU486 treatment was higher in MSCs from male donors than that from females. No effect of RU486 on MSC proliferation was observed in a steroid-free medium. RU486 pretreatment significantly increased the expression of mRNA for alkaline phosphatase in human MSCs and the mRNA expression of osteocalcin of these cells up-regulated earlier after their exposure to osteogenic differentiation medium. Although no statistical significance in terms of chondrogenic differentiation markers was detected, mRNA expression for aggrecan and collagen type 2 were higher in a majority of the RU486-pretreated donor MSCs than their untreated controls. No significant difference in terms of MSC adipogenic differentiation capabilities were observed after RU486 treatment. RU486 treatment up-regulated the expressions of FGF-2 and Sox-11 in human MSCs. These results indicate that blockage of endogenous GCs may be developed as a novel approach to effectively improve the proliferation and osteochondral differentiation capabilities of human MSCs for potential clinical applications. Additional studies will be required to determine the potential long-term effects of RU486 treatment on these bone marrow cells.
Glucocorticoids; Antagonist; Bone marrow mesenchymal stem/stromal cells; Proliferation; Differentiation; Senescence
Aromatase is expressed in multiple tissues, indicating a crucial role for locally produced oestrogens in the differentiation, regulation and normal function of several organs and processes. This review is an overview of the role of aromatase in different tissues under normal physiological conditions and its contribution to the development of some oestrogen-related pathologies.
Aromatase; Oestrogen function
The mineralocorticoid receptor (MR) is a widely distributed ligand activated nuclear transcription factor that is bound by various chaperone proteins that alter its conformation depending upon its location in the cell and whether it is ligand-bound. We describe the development and characterization of new monoclonal antibodies produced against a rat recombinant protein corresponding to aminoacids 5-550 of the MR to produce antibodies that recognize the receptor in specific conformations. Most of the resulting monoclonal antibodies studied were similar to those we produced by immunization with peptide isotopes, however two detected a single band at the appropriate molecular mass as the MR and had distinct immunostaining characteristics in neurons. One labeled cytosolic MR, the other labeled membranes and cytosol, including axons. These antibodies will permit study of the subcellular localization of the MR under various physiological and pathological conditions. We have also confirmed that the MR is highly unstable and requires special handling.
Mineralocorticoid receptor; Aldosterone; Monoclonal antibodies
Little is currently known about the substrate binding site of the human UDP-glucuronosyltransferases (UGTs) and the structural elements that affect their complex substrate selectivity. In order to further understand and extend our earlier findings with phenylalanines 90 and 93 of UGT1A10, we have replaced each of them with Gly, Ala, Val, Leu, Ile or Tyr, and tested the activity of the resulting 12 mutants toward 8 different substrates. Apart from scopoletin glucuronidation, the F90 mutants other than F90L were nearly inactive, while the F93 mutants’ activity was strongly substrate dependent. Hence, F93L displayed high entacapone and 1-naphthol glucuronidation rates, whereas F93G, which was nearly inactive in entacapone glucuronidation, was highly active toward estradiol, estriol and even ethinylestradiol, a synthetic estrogen that is a poor substrate for the wild-type UGT1A10. Kinetic analyses of 4-nitrophenol, estradiol and ethinylestradiol glucuronidation by the mutants that catalyzed the respective reactions at considerable rates, revealed increased Km values for 4-nitrophenol and estradiol in all the mutants, whilst the Km values of F93G and F93A for ethinylestradiol were lower than in control UGT1A10. Based on the activity results and a new molecular model of UGT1A10, it is suggested that both F90 and F93 are located in a surface helix at the far end of the substrate binding site. Nevertheless, only F93 directly affects the selectivity of UGT1A10 toward large and rigid estrogens, particularly those with substitutions at the D ring. The effects of F93 mutations on the glucuronidation of smaller or less rigid substrates are indirect, however.
In a continuing study of our clinical candidate 5 (VN/124-1 or TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure-activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3β-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ~2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.
Androgen receptor; androgen receptor ablative agents CYP17; CYP17 inhibitors; prostate cancer therapy; VN/124-1 (TOK-001); abiraterone; abiraterone acetate; potent anti-prostate cancer agents
To evaluate the role of physiologic levels of androgens and their precursors in the regulation of body composition, energy and substrate metabolism and aerobic capacity in healthy, cycling, premenopausal women.
We evaluated 30 young (27 ± 1 yr) premenopausal, non-obese (23 ± 0.5 kg/m2), normally-cycling women, without clinical or chemical evidence of hyperandrogenism or hyperinsulinemia, for parameters of total and regional body composition, glucose tolerance, aerobic capacity and resting energy expenditure and substrate oxidation. Serum was assayed for androgens and androgen precursors by techniques optimized to assess the low androgen levels in this population.
Higher serum testosterone levels correlated with greater fat mass (r= .377; p=0.04), but not abdominal adiposity or other metabolic/physiologic variables. Additionally, dehydroepiandrosterone (DHEA) was negatively related to visceral fat content (r=−.569; p=0.02). Other serum androgens did not correlate with total or regional adiposity, skeletal muscle mass, aerobic capacity, glucose tolerance, or resting energy and substrate metabolism.
In this group of non-obese, premenopausal women with no clinical or chemical evidence of hyperandrogenemia, serum testosterone levels were positively related with fat mass, but not with abdominal adiposity; whereas, DHEA was negatively related to visceral adiposity. Our data suggest that within the normal physiologic range, testosterone is a predictor of overall adiposity, but that this effect does not appear to be associated with concomitant alterations in resting energy or substrate metabolism that could predispose to weight gain.
androgen; testosterone; premenopausal women; adiposity; body composition; metabolism
Cellular cholesterol homeostasis is regulated by oxygenated cholesterol metabolites called oxysterols. While the importance of oxysterols in the acute regulation of cholesterol homeostasis is known, the precise molecular mechanisms through which oxysterols exert their effects remain to be elucidated. LY295427 (1) is a known antagonist of the cholesterol-homeostatic effects of 25-hydroxycholesterol (25-HC), a biologically active oxysterol. In order to examine the mechanism of action of this antagonism, and to further explore recent evidence suggesting that the membrane effects of 25-HC contribute to acute cholesterol regulation, we synthesized the enantiomer of LY295427 (ent-LY295427). ent-LY295427 (2) will serve as a unique probe to provide insight into the role of transcription-independent mechanisms in regulation of cholesterol homeostasis.
enantiomer; oxysterol; LY295427
Few studies have examined the potential effects of childbirth on the responses of the female vasculature – especially the resistance microvasculature of non-reproductive tissues. In the present study we have investigated the response of mesenteric microvascular resistance vessels to estrogen (E2), an important vasoactive hormone. Vessels were obtained from either nulliparous or postpartum female Sprague-Dawley rats, and isometric tension studies were performed. We found that E2 induced a concentration-dependent, endothelium-independent relaxation of microvessels precontracted with 10−5M phenylephrine; however, E2-induced relaxation was reduced by nearly half in vessels from postpartum animals compared to nulliparous controls. Inhibiting nitric oxide synthase activity with 10−4M L-NMMA or L-NPA (which exhibits selectivity for type 1 or nNOS) attenuated the relaxation effect of E2 on arteries from nulliparous animals. In contrast, L-NPA had little effect on arteries from postpartum animals, suggesting a reduced influence of nNOS after parturition. Moreover, expression of nNOS protein in microvessels was decreased 39% in the postpartum state compared to arteries from nulliparous animals. We propose that the impaired E2-induced relaxation response of microvessels from postpartum animals reflects a downregulation of NO production due to lower nNOS expressed in vascular smooth muscle cells. We measured a 73% decrease in serum E2 levels in the postpartum state compared to nulliparous animals. Because E2 has been shown to increase nNOS protein expression, we propose that lower E2 levels after parturition decrease expression of nNOS, leading to a reduced vasodilatory capacity of resistance microvessels.
estrogen; parturition; postpartum; NOS; vascular smooth muscle cells
Thyroid hormone receptors (TRs) mediate the critical activities of the thyroid hormone (T3) in growth, development, and differentiation. Decreased expression and/or somatic mutations of TRs have been shown to be associated with several types of human cancers including liver, breast, lung, and thyroid. A direct demonstration that TRβ mutants could function as oncogenes is evidenced by the spontaneous development of follicular thyroid carcinoma similar to human cancer in a knockin mouse model harboring a mutated TRβ (denoted as PV; ThrbPV/PV mice). PV is a dominant negative mutation identified in a patient with resistance to thyroid hormone. Analysis of altered gene expression and molecular studies of thyroid carcinogenesis in ThrbPV/PV mice show that the oncogenic activity of PV is mediated by both nucleus-initiated transcription and extranuclear actions to alter gene expression and signaling transduction activity. This article focuses on recent findings of novel extranuclear actions of PV that affect signaling cascades and thereby the invasiveness, migration, and motility of thyroid tumor cells. These findings have led to identification of potential molecular targets for treatment of metastatic thyroid cancer.
thyroid hormone receptors; thyroid hormone receptor mutants; mouse model; thyroid cancer; carcinogenesis; extranuclear signaling
The therapeutic use of progesterone following traumatic brain injury has recently entered phase III clinical trials as a means of neuroprotection. Although it has been hypothesized that progesterone protects against calcium overload following excitotoxic shock, the exact mechanisms underlying the beneficial effects of progesterone have yet to be determined. We found that therapeutic concentrations of progesterone to be neuroprotective against depolarization-induced excitotoxicity in cultured striatal neurons. Through use of calcium imaging, electrophysiology and the measurement of changes in activity-dependent gene expression, progesterone was found to block calcium entry through voltage-gated calcium channels, leading to alterations in the signaling of the activity-dependent transcription factors NFAT and CREB. The effects of progesterone were highly specific to this steroid hormone, although they did not appear to be receptor mediated. In addition, progesterone did not inhibit AMPA or NMDA receptor signaling. This analysis regarding the effect of progesterone on calcium signaling provides both a putative mechanism by which progesterone acts as a neuroprotectant, as well as affords a greater appreciation for its potential far-reaching effects on cellular function.
Brain Injury; Calcium; Excitotoxicity; Ion Channels; Neuroprotection; Progesterone; Glutamate
Lung cancer is the most common cause of cancer mortality in male and female patients in the US. Although it is clear that tobacco smoking is a major cause of lung cancer, about half of all women with lung cancer worldwide are never-smokers. Despite a declining smoking population, the incidence of non-small cell lung cancer (NSCLC), the predominant form of lung cancer, has reached epidemic proportions particularly in women. Emerging data suggest that factors other than tobacco, namely endogenous and exogenous female sex hormones, have a role in stimulating NSCLC progression. Aromatase, a key enzyme for estrogen biosynthesis, is expressed in NSCLC. Clinical data show that women with high levels of tumor aromatase (and high intratumoral estrogen) have worse survival than those with low aromatase. The present and previous studies also reveal significant expression and activity of estrogen receptors (ERα, ERβ) in both extranuclear and nuclear sites in most NSCLC. We now report further on the expression of progesterone receptor (PR) transcripts and protein in NSCLC. PR transcripts were significantly lower in cancerous as compared to non-malignant tissue. Using immunohistochemistry, expression of PR was observed in the nucleus and/or extranuclear compartments in the majority of human tumor specimens examined. Combinations of estrogen and progestins administered in vitro cooperate in promoting tumor secretion of vascular endothelial growth factor and, consequently, support tumor-associated angiogenesis. Further, dual treatment with estradiol and progestin increased the numbers of putative tumor stem/progenitor cells. Thus, ER- and/or PR-targeted therapies may offer new approaches to manage NSCLC.
Progesterone; Estrogen; Steroid hormone receptor; Non-small cell lung cancer; VEGF; Progenitor cells; Cancer stem cells; Angiogenesis
The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.
membrane progesterone receptor; mPR; nuclear progesterone receptor; PR; truncated progesterone receptors; breast cancer cells
Various ovarian cell types including granulosa cells and ovarian surface epithelial cells express the progesterone (P4) binding protein, Progesterone Receptor Membrane Component-1 (PGRMC1). PGRMC1 is also expressed in ovarian tumors. PGRMC1 plays an essential role in promoting the survival of both normal and cancerous ovarian cell in vitro. Given the clinical significance of factors that regulate the viability of ovarian cancer, this review will focus on the role of PGRMC1 in ovarian cancer, while drawing insights into the mechanism of PGRMC1’s action from cell lines derived from healthy ovaries as well as ovarian tumors.
Studies using PGRMC1 siRNA demonstrated that P4’s ability to inhibit ovarian cells from undergoing apoptosis in vitro is dependent on PGRMC1. To confirm the importance of PGRMC1, the ability of PGRMC1-deplete ovarian cancer cell lines to form tumors in intact nude mice was assessed. Compared to PGRMC1-expressing ovarian cancer cells, PGRMC1-deplete ovarian cancer cells formed tumors in fewer mice (80% compared to 100% for controls). Moreover, the number of tumors derived from PGRMC1-deplete ovarian cancer cells was 50% of that observed in controls. Finally, the tumors that formed from PGRMC1-deplete ovarian cancer cells were about a fourth the size of tumors derived from ovarian cancer cells with normal levels of PGRMC1. One reason for PGRMC1-deplete tumors being smaller is that they had a poorly developed microvasculature system. How PGRMC1 regulates cell viability and in turn tumor growth is not known but part of the mechanism likely involves the regulation of genes that promote cell survival and inhibit apoptosis.
Progesterone; PGRMC1; Ovary; Ovarian Cancer
Aromatase inhibitors (AIs) have become the front-line choice for treatment of ER+ breast cancer. Nevertheless, although patients are responsive initially, they may acquire resistance and become unresponsive to further treatment. In addition, approximately 25% of breast cancers do not express the estrogen receptor (ERα) and consequently, are innately resistant to endocrine therapy. We have investigated the mechanisms associated with this lack of treatment response using xenograft models. We found that in cells and tumors that acquired resistance to the AI letrozole therapy, expression of the ER was reduced whereas growth factor signally was enhanced, including a marked increase in HER2 expression. Treatment with trastuzumab (HER2 antibody) resulted in a significant down-regulation of HER2 and p-MAPK as well as restoration of ERα expression. Thus, when trastuzumab was added to letrozole treatment at the time of tumor progression, there was significantly prolonged tumor suppression compared to trastuzumab or letrozole alone. This suggests that inhibition of both HER2 and ERα signaling pathways are required for overcoming resistance and restoring treatment sensitivity. ER negative tumors are innately resistant to endocrine therapy. Repression of the ERα has been found to be due to epigenetic modifications such as increased methylation and histone deacetylation. We found that entinostat (ENT), a histone deacetylase inhibitor (HDACi), activated not only expression of ERα but also aromatase in MDA-MB-231 ER-negative breast cancer cells, resulting in their ability to respond to estrogen and letrozole. Treatment with ENT in combination with letrozole significantly reduced tumor growth rate in xenografts compared to control tumors (p <0.001). ENT plus letrozole treatment also prevented the colonization and growth of MDA-MB-231 cells in the lung with a significant reduction (p<0.03) in both visible and microscopic foci. These results provide a strong indication for possible use of AIs in combination with HDAC inhibitors for the treatment of ER-negative breast cancer.
Evidence that aromatase expression in tumor-associated breast stroma is elevated, provides a rationale for use of aromatase inhibitors (AIs) in breast cancer treatment. However, regulation of local aromatase expression in cancer-free breast stroma is poorly understood. Recent clinical work indicates that stromal cells in dense breast tissue tend to express higher levels of aromatase than their counterpart from non-dense tissue. Consistent with the clinical observation, our cell culture-based study indicated that cell density, cell shape, and extracellular matrix (ECM) significantly induced stromal aromatase expression via a distinct signal transduction pathway. In addition, we identified a number of cell surface markers that are commonly associated with aromatase-expressing stromal cells. As mammographic density is one of the strongest and most prevalent risk factors for breast cancer, these findings provide a potential mechanistic link between alterations in tissue composition of dense breast tissue and increased stromal aromatase expression. Further exploration of the in vitro model system may advance understanding of an important problem in breast cancer biology.
aromatase; mechanical phenotype; cell shape; cell density; ECM; mammographic density
Aromatase (CYP19A1) is an integral membrane enzyme that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens. All human estrogens are synthesized from their androgenic precursors by this unique cytochrome P450. The crystal structure of active aromatase purified from human placenta has recently been determined in complex with its natural substrate androstenedione in the high-spin ferric state of heme. Hydrogen bond forming interactions and tight packing hydrophobic side chains closely complement puckering of the steroid backbone, thereby providing the molecular basis for the androgenic specificity of aromatase. In the crystal, aromatase molecules are linked by a head-to-tail intermolecular interaction via a surface loop between helix D and helix E of one aromatase molecule that penetrates the heme-proximal cavity of the neighboring, crystallographically-related molecule, thus forming in tandem a polymeric aromatase chain. This intermolecular interaction is similar to the aromatase-Cytochrome P450 reductase coupling and is driven by electrostatics between the negative potential surface of the D-E loop region and the positively charged heme-proximal cavity. This loop-to-proximal site link in aromatase is rather unique - there are only a few of examples of somewhat similar intermolecular interactions in the entire P450 structure database. Furthermore, the amino acids involved in the intermolecular contact appear to be specific for aromatase. Higher order organization of aromatase monomers may have implications in lipid integration and catalysis.
Aromatase is the rate-limiting enzyme in estrogen biosynthesis. As a cytochrome P450, it utilizes electrons from NADPH–cytochrome P450 reductase (CPR) to produce estrogen from androgen. Estrogen is a key factor in the promotion of hormone-dependent breast cancer growth. Aromatase inhibitors (AIs) are drugs that block estrogen synthesis, and are widely used to treat estrogen-dependent breast cancer. Structure–function experiments have been performed to study how CPR and AIs interact with aromatase to further the understanding of how these drugs elicit their effects. Our studies have revealed a strong interaction between aromatase and CPR, and that the residue K108 is situated in a region important to the interaction of aromatase with CPR. The published X-ray structure of aromatase indicates that the F221, W224 and M374 residues are located in the active site. Our site-directed mutagenesis experiments confirm their importance in the binding of the androgen substrate as well as AIs, but these residues interact differently with steroidal inhibitors (exemestane) and non-steroidal inhibitors (letrozole and anastrozole). Furthermore, our results predict that the residue W224 also participates in the mechanism-based inhibition of exemestane, as time-dependent inhibition is eliminated with mutation on this residue. Together with previous research from our laboratory, this study confirms that W224, E302, D309 and S478 are important active site residues involved in the suicide mechanism of exemestane against aromatase.
Aromatase; Androstenedione; Letrozole; Exemestane; Site-directed mutagenesis
Human steroid 5β-reductase (Aldo-keto Reductase 1D1) catalyzes the stereospecific NADPH-dependent reduction of the C4-C5 double bond of Δ4-ketosteroids to yield an A/B cis-ring junction. This cis-configuration is crucial for bile acid biosynthesis and plays important roles in steroid metabolism. The biochemical properties of the enzyme have not been thoroughly studied and conflicting data have been reported, partially due to the lack of highly homogeneous protein. In the present study, we systematically determined the substrate specificity of homogeneous human recombinant AKR1D1 using C18, C19, C21, and C27 Δ4-ketosteroids and assessed the pH-rate dependence of the enzyme. Our results show that AKR1D1 proficiently reduced all the steroids tested at physiological pH, indicating AKR1D1 is the only enzyme necessary for all the 5β-steroid metabolite present in humans. Substrate inhibition was observed with C18 to C21 steroids provided that the side-chain at C17 was unsubstituted. This structure activity relationship can be explained by the existence of a small alternative substrate binding pocket revealed by the AKR1D1 crystal structure. Non-steroidal anti-inflammatory drugs which are potent inhibitors of the related AKR1C enzymes do not inhibit AKR1D1 by contrast chenodeoxycholate and ursodeoxycholate were found to be potent non-competitive inhibitors suggesting that bile-acids may regulate their own synthesis at the level of AKR1D1 inhibition.
Bile acids; steroid hormones; substrate inhibition; non-steroidal anti-inflammatory drugs
Insights from vitamin D-resistant New World primates and their human homologues as models of natural and pathological insensitivity to sterol/steroid action have uncovered a family of novel intracellular vitamin D and estrogen regulatory proteins involved in hormone action. The proteins, known as “vitamin D or estrogen response element-binding proteins”, behave as potent cis-acting, transdominant regulators to inhibit steroid receptor binding to DNA response elements and is responsible for vitamin D and estrogen resistances. This set of interactors belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family of previously known pre-mRNA-interacting proteins. This review provides new insights into the mechanism by which these novel regulators of signaling and metabolism can act to regulate responses to vitamin D and estrogen. In addition the review also describes other molecules that are known to influence nuclear receptor signaling through interaction with hormone response elements.