Regulation Of Aromatase Research Articles

Insights into epigenetic regulation of cyp19a via comparative analysis using the scombrid chub mackerel as model

Sexual development and reproduction are largely linked to epigenetic changes in many fish species. However, understanding of epigenetic regulation in scombrid species, such as tunas and mackerels, is limited. This study investigates DNA methylation’s impact on cyp19a expression, crucial for estrogen synthesis, focusing on chub mackerel. Given the commercial significance of scombrids and susceptibility of marine fish to environmental changes, elucidating epigenetic mechanisms, particularly in the context of global warming, becomes imperative. We aimed to generalize observations from chub mackerel to other scombrids. Additionally, we studied DNA methylation patterns across fish with different sexual systems to understand aromatase regulation’s phenotypic plasticity. Our in silico analysis revealed highly conserved promoter sequences within scombrids, sharing TFBS like Foxl2, FOS::JUN, ESRR, and Sox3, while CpG content varies. This indicates a conserved regulatory network controlling gene expression. We found sexual dimorphism in DNA methylation, with males hypermethylated and aromatase expression downregulated. Despite similar dnmt1 expression, tet1, tet2, and tet3 were higher expressed in females, suggesting that the observed DNA methylation patterns are maintained through active demethylation rather than differential methylation. Gonochoristic Japanese anchovy and protogynous bamboo leaf wrasse displayed similar trends, but species-specific methylation patterns highlight DNA methylation’s complex role in gonadal changes. In vitro assays confirmed methylation’s regulatory role and identified an SF-1 binding site relevant for promoter activation in chub mackerel. Another studied SF-1 site, present in both chub mackerel and bamboo leaf wrasse, showed regulatory effects, indicating potential similar regulatory mechanisms for cyp19a expression. Overall, our findings suggest that while global methylation affects cyp19a transcription, the variation in CpG density and location could be introducing nuances in its epigenetic regulation. This study contributes to our understanding of the cyp19a regulation in fish gonad maturation.

Impact of Aromatase Enzyme and its Altered Regulation on Polycystic Ovary Syndrome (PCOS): A Key Factor in Pathogenesis of PCOS

Abstract Polycystic ovary syndrome (PCOS) is found to be a common endocrinopathy in a woman’s reproductive life. It is importantly characterized by hyperandrogenism. The enzyme aromatase prevents the accumulation of androgen by converting it into estrogens during the process of steroidogenesis. Down-regulation of this enzyme causes elevated levels of testosterone which leads to PCOS. This review is aims to explore the various factors and pathogenic pathways that influence the regulation of aromatase and steroidogenesis. The articles were selected from PUBMED, Google Scholar, Cross ref, and Research Gate. The results of the selected articles were analyzed and presented systematically. Polymorphism in the CYP19A1 gene specifically rs2414096 was found to be strongly associated with PCOS while other polymorphisms showed variable results. Micro RNAs such as miRNA1294 and lncRNAs exhibited an inhibitory role on CYP19A1 along with down-regulation of aromatase. In addition to this, the altered ratio between Inositol epimers and pathogenic activation of the JAK2/STAT3 pathway caused the down-regulation of aromatase and accumulation of testosterone. It was also observed that there was a direct effect of environmental toxins on endocrine balance which affected steroidogenesis that led to PCOS. In conclusion, Aromatase played a key role in steroidogenesis and it is the hotspot of research to investigate the pathophysiology of PCOS. Genetic polymorphism, microRNAs, pathogenic metabolism, and pollutants were significantly influenced the enzyme.

Protective role of vitamin E against acrylamide-induced testicular toxicity from pregnancy to adulthood: insights into oxidative stress and aromatase regulation.

Acrylamide (ACR) is a toxic chemical frequently encountered in daily life, posing health risks. This study aimed to elucidate the molecular-level mechanism of ACR's toxic effects on testicles and investigate whether Vitamin E can mitigate these effects. A total of 40 adult pregnant rats were utilized, divided into four groups: Control, ACR, Vitamin E, and ACR + Vitamin E. ACR and Vitamin E were administered to the mother rats during pregnancy and lactation, and to the male offspring until the 8th week post-birth. Serum hormone levels, oxidant-antioxidant parameters, histopathological examination of testicular tissue, and mRNA and protein levels of the testicular and liver aromatase gene were analyzed. Spermiogram analysis was conducted on the collected sperm samples from the male offspring. The results revealed that ACR exposure adversely affected hormone levels, oxidant-antioxidant parameters, histological findings, as well as aromatase gene and protein expressions. However, Vitamin E administration effectively prevented the toxic effects of ACR. These findings demonstrate that ACR application significantly impairs the reproductive performance of male offspring rats by increasing liver aromatase activity.

Effects of Ventromedial Hypothalamic Nucleus (VMN) Aromatase Gene Knockdown on VMN Glycogen Metabolism and Glucoregulatory Neurotransmission

The enzyme aromatase is expressed at high levels in the ventromedial hypothalamic nucleus (VMN), a principal component of the brain gluco-regulatory network. Current research utilized selective gene knockdown tools to investigate the premise that VMN neuroestradiol controls glucostasis. Intra-VMN aromatase siRNA administration decreased baseline aromatase protein expression and tissue estradiol concentrations and either reversed or attenuated the hypoglycemic regulation of these profiles in a VMN segment-specific manner. Aromatase gene repression down-regulated protein biomarkers for gluco-stimulatory (nitric oxide; NO) and -inhibitory (gamma-aminobutyric acid; GABA) neurochemical transmitters. Insulin-induced hypoglycemia (IIH) up- or down-regulated neuronal nitric oxide synthase (nNOS) and glutamate decarboxylase65/67 (GAD), respectively, throughout the VMN. Interestingly, IIH caused divergent changes in tissue aromatase and estradiol levels in rostral (diminished) versus middle and caudal (elevated) VMN. Aromatase knockdown prevented hypoglycemic nNOS augmentation in VMN middle and caudal segments, but abolished the GAD inhibitory response to IIH throughout this nucleus. VMN nitrergic and GABAergic neurons monitor stimulus-specific glycogen breakdown. Here, glycogen synthase (GS) and phosphorylase brain- (GPbb; AMP-sensitive) and muscle- (GPmm; noradrenergic -responsive) type isoform responses to aromatase siRNA were evaluated. Aromatase repression reduced GPbb and GPmm content in euglycemic controls and prevented hypoglycemic regulation of GPmm but not GPbb expression while reversing glycogen accumulation. Aromatase siRNA elevated baseline glucagon and corticosterone secretion and abolished hypoglycemic hyperglucagonemia and hypercorticosteronemia. Outcomes document the involvement of VMN neuroestradiol signaling in brain control of glucose homeostasis. Aromatase regulation of VMN gluco-regulatory signaling of hypoglycemia-associated energy imbalance may entail, in part, control of GP variant-mediated glycogen disassembly.

Sex-specific regulation of inhibition and network activity by local aromatase in the mouse hippocampus

Cognitive function relies on a balanced interplay between excitatory and inhibitory neurons (INs), but the impact of estradiol on IN function is not fully understood. Here, we characterize the regulation of hippocampal INs by aromatase, the enzyme responsible for estradiol synthesis, using a combination of molecular, genetic, functional and behavioral tools. The results show that CA1 parvalbumin-expressing INs (PV-INs) contribute to brain estradiol synthesis. Brain aromatase regulates synaptic inhibition through a mechanism that involves modification of perineuronal nets enwrapping PV-INs. In the female brain, aromatase modulates PV-INs activity, the dynamics of network oscillations and hippocampal-dependent memory. Aromatase regulation of PV-INs and inhibitory synapses is determined by the gonads and independent of sex chromosomes. These results suggest PV-INs are mediators of estrogenic regulation of behaviorally-relevant activity.

Abstract P4-10-15: Whole-body vibration combined with physical exercise to treat aromatase inhibitor-induced musculoskeletal symptoms in breast cancer women: Results of a pilot randomized controlled study

Abstract BACKGROUND. Aromatase inhibitors (AIs) represent a cornerstone of hormone receptor (HR)-positive breast cancers management. However, AIs-induced musculoskeletal symptoms (AIMSS) might be frequently reported during estrogen depletion, with a crucial impact on cancer treatment adherence [1-2]. To date, physical exercise has been proposed as a promising therapeutic intervention to improve symptoms in AIMSS breast cancer patients [3] and recently, also whole-body vibration (WBV) showed promising results in these women. WBV is an effective neuromuscular training proposed to enhance muscle function and proprioception through various frequencies of mechanical vibration inducing repetitive muscle contractions. However, at present, data about tailored and effective multimodal rehabilitative exercise protocols to treat AIMSS are still controversial [3]. Therefore, this pilot randomized controlled study aimed at assessing the safety and efficacy of WBV in breast cancer women suffering from AIMSS. METHODS: In this study we recruited adults women undergoing AIs treatment for breast cancer and suffering from AIMSS. Twentytwo patients were enrolled and subsequently randomly assigned 1:1 to two groups. Group A received physical exercise and WBV, while Group B received physical exercise combined with sham WBV. The patients were assessed at the baseline (T0) and after 4 weeks of treatment (T1). The primary endpoint was the numerical pain rating scale (NPRS) for pain assessment; the secondary endpoints were the handgrip strength (HGS) test to assess appendicular muscle strength, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) to assess pain, joint stiffness, and function; 10-meter walking test (10MWT) and 6MWT to evaluate physical performance; the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) to assess function, symptoms, and global health. RESULTS: Group A (n=11; mean age: 51.73±10.73 years; body mass index (BMI): 25.56±5.17 kg/m2) showed a statistically significant improvement of pain (NPRS: 6.82±1.17 vs 5.73±1.01; p=0.031). In contrast, patients in Group B, control group, (n=11; mean age: 58.55±9.71 years; BMI: 27.31±3.84 kg/m2), did not significantly improved in terms of pain (NPRS: 6.91±2.02 vs 5.91±2.51; p=0.07). Moreover, significant differences between groups at T1 were found in terms of WOMAC physical functioning (Group A: 77.56±9.853; Group B: 65.63±14.27; p=0.044). Furthermore, a significant improvement in terms of muscle strength, physical performance and quality of life (EORTC QLQ-C30) were reported in intragroup analysis in both groups, without significant differences between groups. We did not register dropouts or side effects in both groups. Lastly, both patients and the physical therapist reported a high level of satisfaction about the rehabilitative intervention performed. CONCLUSION: These findings showed that physical exercise and WBV combination therapy might be safe, well-tolerated and effective in patients with AIMSS. However, further studies are warranted to assess long terms effects of this combined rehabilitative treatment. REFERENCES: 1. Molehin D, Rasha F, Rahman RL, Pruitt K. Regulation of aromatase in cancer. Mol Cell Biochem. 2021. 2. Murphy CC, Bartholomew LK, Carpentier MY, Bluethmann SM, Vernon SW. Adherence to adjuvant hormonal therapy among breast cancer survivors in clinical practice: a systematic review. Breast Cancer Res Treat. 2012;134(2):459-78. 3. Roberts KE, Rickett K, Feng S, Vagenas D, Woodward NE. Exercise therapies for preventing or treating aromatase inhibitor-induced musculoskeletal symptoms in early breast cancer. Cochrane Database Syst Rev. 2020;1:CD012988. Citation Format: Lorenzo Lippi, Alessandro de Sire, Antonio Ammendolia, Carlo Cisari, Konstantinos Venetis, Elham Sajjadi, Nicola Fusco, Marco Invernizzi. Whole-body vibration combined with physical exercise to treat aromatase inhibitor-induced musculoskeletal symptoms in breast cancer women: Results of a pilot randomized controlled study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-10-15.

Transcriptional regulation of CYP19A1 expression in chickens: ESR1, ESR2 and NR5A2 form a functional network

Aromatase, encoded by CYP19A1, is responsible for the conversion of androgen to estrogen, which plays a vital role in the development and function of the ovary and functions in many other physiological processes in both sexes. Instead of being expressed in ovarian granulosa cells, as in mammals, CYP19A1 is expressed in chickens in the theca cells of ovarian follicles, and the mechanism of CYP19A1 expression regulation remains unknown. Here, using immunofluorescence and western blotting assay, we first confirmed that CYP19A1 and FOXL2 (Forkheadbox L2) were coexpressed in pre-granulosa cells of female chicken embryonic gonads, while FOXL2 did not affect aromatase expression at embryonic stages. Second, our research showed that CYP19A1, ESR1 (estrogen receptor alpha), ESR2 (estrogen receptor beta) and NR5A2 (liver receptor homologue-1) were coexpressed in the theca cell layers of chicken small yellow follicles. There was cross-talk between CYP19A1 and candidate transcription factors (ESR1, ESR2 and NR5A2), which was identified by generating a reliable theca cell culture model. Using luciferase assays in theca cells and chicken embryonic fibroblast (DF-1) cells, the results suggested that ESR1 and NR5A2 had potential effects on CYP19A1 promoter activity in chickens. Overexpression of ESR1, ESR2 and NR5A2 in chicken embryonic fibroblast (DF-1) cells upregulated the protein expression of CYP19A1, mutually restricted each other and formed a potential regulatory network to coordinate the expression of CYP19A1. To conclude, our results indicated that FOXL2 cannot regulate the expression of CYP19A1 at chicken embryonic stages and after sexual maturity, ESR1, ESR2 and NR5A2 form a functional network to affect the expression of CYP19A1. These results laid a foundation for further research on the transcriptional regulation of chicken aromatase.

MeCP2 duplication causes hyperandrogenism by upregulating LHCGR and downregulating ROR\u03b1

Duplication of MECP2 (methyl-CpG-binding protein 2) gene causes a serious neurological and developmental disorder called MECP2 duplication syndrome (MDS), which is usually found in males. A previous clinical study reported that MDS patient has precocious puberty with hyperandrogenism, suggesting increased MeCP2 may cause male hyperandrogenism. Here we use an MDS mouse model and confirm that MECP2 duplication significantly upregulates androgen levels. We show for the first time that MeCP2 is highly expressed in the Leydig cells of testis, where androgen is synthesized. Mechanistically, MECP2 duplication increases androgen synthesis and decreases androgen to estrogen conversion through either the upregulation of luteinizing hormone receptor (LHCGR) in testis, as a result of MeCP2 binds to G-quadruplex structure of Lhcgr promoter and recruits the transcription activator CREB1 or the downregulation of the expression of aromatase in testis by binding the CpG island of Rorα, an upstream regulator of aromatase. Taken together, we demonstrate that MeCP2 plays an important role in androgen synthesis, supporting a novel non-CNS function of MeCP2 in the process of sex hormone synthesis.

FSH stimulated bovine granulosa cell steroidogenesis involves both canonical and noncanonical WNT signaling

Gonadotrophins play key roles in follicular development; however, the underlying molecular mechanisms are not fully understood. Follicle stimulating hormone (FSH) regulation of aromatase and subsequent estradiol (E2) production relies on β-catenin, a key effector of WNT signaling. We previously demonstrated that treatment with the canonical WNT inhibitor, IWR-1, reduced FSH induced bovine granulosa cell E2 production in vitro. Here we demonstrated that intrafollicular injection in vivo with IWR-1 alters steroidogenesis and triggers a significant decrease in estrogen to progesterone ratio in the IWR-1 treated follicles compared to diluent injected control follicles. We next examined markers of canonical and noncanonical WNT signaling in dominant and subordinate follicles collected at different stages of follicular development and showed that protein for both CTNNB1 (canonical pathway) and phosphorylated (p)-LEF1 (noncanonical pathway) was significantly elevated in dominant compared to subordinate follicles at the early dominance stage of development. Therefore, we hypothesized that canonical and/or noncanonical WNT ligands modulate FSH stimulated E2 production. Hence, we examined the effects of specific WNT ligands on FSH stimulated E2 production in the absence of endogenous WNT production in vitro. Universal WNT signaling inhibitor, LGK-974 was able to inhibit FSH stimulation of E2 and reduce the abundance of proteins linked to canonical and noncanonical WNT pathway activation. Supplementation with the canonical ligand WNT2b did not affect the inhibitory effects of LGK-974 on FSH stimulated E2 production but rescued the LGK-974 mediated inhibition of CTNNB1 (canonical pathway) but not p-LEF1, p-JNK or p-P38 abundance (noncanonical pathway) abundance. In contrast, WNT5a treatment rescued FSH stimulated estradiol production and indices of activation of both the canonical (CTNNB1) and noncanonical (p-LEF1, p-JNK and p-P38) WNT signaling pathways in LGK-974 treated granulosa cells. Taken together, these results suggest that both canonical and noncanonical WNT pathways activation is linked to FSH stimulation of E2 production by bovine granulosa cells.

Regulation of aromatase expression: Potential therapeutic insight into breast cancer treatment

Estrogen signaling has been implicated in hormone-dependent breast cancer which constitutes >75% of breast cancer diagnosis and other malignancies. Aromatase, the key enzyme involved in the synthesis of estrogen, is often dysregulated in breast cancers. This has led to the administration of aromatase-inhibitors (AIs), commonly used for hormone-dependent breast cancers. Unfortunately, the increasing development of acquired resistance to the current AIs and modulators of estrogen receptors, following initial disease steadiness, has posed a serious clinical challenge in breast cancer treatment. In this review we highlight historical and recent advances on the transcriptional and post-translational regulation of aromatase in both physiological and pathological contexts. We also discuss the different drug combinations targeting various tumor promoting cell signaling pathways currently being developed and tested both in laboratory settings and in the clinic.

CC chemokine ligand 2 (CCL2) stimulates aromatase gene expression in mammary adipose tissue.

Obesity is a risk factor for postmenopausal breast cancer. Obesity-related inflammation upregulates aromatase expression, the rate-limiting enzyme for estrogen synthesis, in breast adipose tissue (BAT), increasing estrogen production and cancer risk. The regulation of aromatase gene (CYP19A1) in BAT is complex, and the mechanisms linking obesity and aromatase dysregulation are not fully understood. An obesity-associated factor that could regulate aromatase is the CC chemokine ligand (CCL) 2, a pro-inflammatory factor that also activates signaling pathways implicated in CYP19A1 transcription. By using human primary breast adipose stromal cells (ASCs) and aromatase reporter (hARO-Luc) mouse mammary adipose explants, we demonstrated that CCL2 enhances the glucocorticoid-mediated CYP19A1 transcription. The potential mechanism involves the activation of PI.4 via ERK1/2 pathway. We also showed that CCL2 contributes to the pro-inflammatory milieu and aromatase expression in obesity, evidenced by increased expression of CCL2 and CYP19A1 in mammary tissues from obese hARO-Luc mice, and subcutaneous adipose tissue from obese women. In summary, our results indicate that postmenopausal obesity may promote CCL2 production in BAT, leading to exacerbation of the menopause-related inflammatory state and further stimulation of local aromatase and estrogens. These results provide new insights into the regulation of aromatase and may aid in finding approaches to prevent breast cancer.

Estrogen disorders: Interpreting the abnormal regulation of aromatase in granulosa cells (Review).

Ovarian granulosa cells (GCs) are the most important source of estrogen. Therefore, aromatase (estrogen synthase), which is the key enzyme in estrogen synthesis, is not only an important factor of ovarian development, but also the key to estrogen secretion by GCs. Disorders of the ovarian estrogen secretion are more likely to induce female estrogen-dependent diseases and fertility issues, such as ovarian cancer and polycystic ovary syndrome. Hence, aromatase is an important drug target; treatment with its inhibitors in estrogen-dependent diseases has attracted increasing attention. The present review article focuses on the regulation and mechanism of the aromatase activity in the GCs, as well as the specific regulation of aromatase promoters. In GCs, follicle-stimulating hormone (FSH) is dependent on the cyclic adenosine monophosphate (cAMP) pathway to regulate the aromatase activity, and the regulation of this enzyme is related to the activation of signaling pathways, such as phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK). In addition, endocrine-disrupting substance and other related factors affect the expression of aromatase, which eventually create an imbalance in the estrogen secretion by the target tissues. The present review highlights these useful factors as potential inhibitors for target therapy.

Regulation of aromatase in cancer.

The regulation of aromatase, an enzyme involved in the biosynthesis of estrogen in normal and cancer cells, has been associated with growth factor signaling and immune response modulation. The tissue-specific regulatory roles of these factors are of particular importance as local aromatase expression is strongly linked to cancer development/progression and disease outcomes in patients. Therefore, aromatase has become a chemotherapeutic target and aromatase inhibitors (AIs) are used in the clinic for treating hormone-dependent cancers. Although AIs have shown promising results in the treatment of cancers, the emerging increase in AI-resistance necessitates the development of new and improved targeted therapies. This review discusses the role of tumor and stromal-derived growth factors and immune cell modulators in regulating aromatase. Current single-agent and combination therapies with or without AIs targeting growth factors and immune checkpoints are also discussed. This review highlights recent studies that show new connections between growth factors, mediators of immune response, and aromatase regulation.

Sex-dimorphic aromatase regulation of ventromedial hypothalamic nucleus glycogen content in euglycemic and insulin-induced hypoglycemic rats

Estrogen receptors control hypothalamic astrocyte glycogen accumulation in vitro. Glycogen metabolism impacts metabolic transmitter signaling in the ventromedial hypothalamic nucleus (VMN), a key glucoregulatory structure. Aromatase, the enzyme that converts testosterone to estradiol, is expressed at high levels in the VMN. Here, the aromatase inhibitor letrozole (Lz) was used alongside high-resolution microdissection/UPHLC-electrospray ionization-mass spectrometric methods to determine if neuroestradiol imposes sex-specific control of VMN glycogen content during glucostasis and/or glucoprivation. Testes-intact male and estradiol-replaced ovariectomized female rats were pretreated by lateral ventricular letrozole (Lz) infusion prior to subcutaneous insulin (INS) injection. Vehicle-treated female controls exhibited higher VMN glycogen content compared to males. Lz increased VMN glycogen levels in males, not females. INS-induced hypoglycemia (IIH) elevated (males) or diminished (females) rostral VMN glycogen accumulation. Induction of IIH in Lz-pretreated animals reduced male VMN glycogen mass, but augmented content in females. Data provide novel evidence for regional variation, in both sexes, in glycogen reactivity to IIH. Results highlight sex-dimorphic neuroestradiol regulation of VMN glycogen amassment during glucostasis, e.g. inhibitory in males versus insignificant in females. Locally-generated estradiol is evidently involved in hypoglycemic enhancement of male VMN glycogen, but conversely limits glycogen content in hypoglycemic females. Further research is needed to characterize mechanisms that underlie the directional shift in aromatase regulation of VMN glycogen in eu- versus hypoglycemic male rats and gain in negative impact in hypoglycemic females.

CC chemokine ligand 2 (CCL2) stimulates local aromatase expression in mammary adipose tissue via MAPK/ERK signaling pathway

Obesity is an important risk factor for the most common type of breast cancer, i.e., estrogen‐dependent postmenopausal breast cancer. Obesity‐related inflammation, defined by enhanced tissue macrophage infiltration and increased levels of proinflammatory factors, stimulates estrogen production and, in turn, promotes tumor formation and progression. The augmented inflammatory milieu activating aromatase expression, the rate‐limiting enzyme for estrogen synthesis, is suggested to be the major mechanism behind the increased risk for breast cancer. The regulation of aromatase (CYP19A1) gene expression in breast tissue is complex and not fully understood. This study focuses on the role of CCL2, a macrophage recruiter often overexpressed in the obese and cancerous breast tissue, on the aromatase expression in mammary adipose tissue. We utilized mammary adipose tissue explants obtained from female hARO‐Luc aromatase reporter mice and cultured primary human breast adipose stromal cells (hASCs). In tissue explants, aromatase expression was measured by quantification of luciferase reporter activity, while in cultured hASCs, qPCR was used to examine expression of CYP19A1 and its specific promoter I.4 and II transcripts. Both, in mammary adipose tissue and in hASCs, CCL2 induced aromatase expression via the distal promoter I.4. Moreover, inhibitors of CCL2 receptor and MAPKs pathway, ERK1/2, blocked CCL2–induced CYP19A1 and PI.4 mRNA expression. In conclusion, these findings indicate that CCL2, an intrinsic pro‐inflammatory chemokine in adipose tissue, may stimulate aromatase expression and play an important role in controlling estrogen biosynthesis in the breast adipose stroma.Support or Funding InformationThis research is supported by the University of Turku Graduate School, the Finnish Cultural Foundation, the Turun yliopistosäätiö and the Institute of Biomedicine, University of TurkuThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rational design of allosteric modulators of the aromatase enzyme: An unprecedented therapeutic strategy to fight breast cancer

Estrogens play a key role in cellular proliferation of estrogen-receptor-positive (ER+) breast cancers (BCs). Suppression of estrogen production by competitive inhibitors of the enzyme aromatase (AIs) is currently one of the most effective therapies against ER + BC. Yet, the development of acquired resistance, after prolonged treatments with AIs, represents a clinical major concern. Serendipitous findings indicate that aromatase may be non-competitively inhibited by clinically employed drugs and/or industrial chemicals. Here, by performing in silico screening on two putative allosteric sites, molecular dynamics and free energy simulations, supported by enzymatic and cell-based assays, we identified five leads inhibiting the enzyme via a non-active site-directed mechanism. This study provides new compelling evidences for the existence of an allosteric regulation of aromatase and for the possibility of exploiting it to modulate estrogens biosynthesis. Such modulation can aptly reduce side effects caused by the complete estrogen deprivation therapy, and, possibly, delay/avoid the onset of resistance.

BRCA1 and Breast Cancer: a Review of the Underlying Mechanisms Resulting in the Tissue-Specific Tumorigenesis in Mutation Carriers.

Since the first cloning of BRCA1 in 1994, many of its cellular interactions have been elucidated. However, its highly specific role in tumorigenesis in the breast tissue—carriers of BRCA1 mutations are predisposed to life-time risks of up to 80%—relative to many other tissues that remain unaffected, has not yet been fully enlightened. In this article, we have applied a universal model of tissue-specificity of cancer genes to BRCA1 and present a systematic review of proposed concepts classified into 4 categories. Firstly, tissue-specific differences in levels of BRCA1 expression and secondly differences in expression of proteins with redundant functions are outlined. Thirdly, cell-type specific interactions of BRCA1 are presented: its regulation of aromatase, its interaction with Progesterone- and receptor activator of nuclear factor-κB ligand-signaling that controls proliferation of luminal progenitor cells, and its influence on cell differentiation via modulation of the key regulators jagged 1-NOTCH and snail family transcriptional repressor 2. Fourthly, factors specific to the cell-type as well as the environment of the breast tissue are elucidated: distinct frequency of losses of heterozygosity, interaction with X inactivation specific transcript RNA, estrogen-dependent induction of genotoxic metabolites and nuclear factor (erythroid-derived 2)-like 2, and regulation of sirtuin 1. In conclusion, the impact of these concepts on the formation of hormone-sensitive and -insensitive breast tumors is outlined.

Oocyte-Secreted Factors Synergize With FSH to Promote Aromatase Expression in Primary Human Cumulus Cells.

The role of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) on aromatase regulation is poorly understood in humans. Determine GDF9 and BMP15 effects on FSH stimulation of estradiol production in primary human cumulus granulosa cells (GCs). We hypothesized that the combination of GDF9 and BMP15 potentiates FSH-induced aromatase expression. Primary human cumulus GCs in culture. University infertility center. GCs of 60 women undergoing in vitro fertilization were collected. Cells were treated with GDF9 and/or BMP15 (GB) in the presence or absence of FSH, dibutyryl cAMP, or SMAD inhibitors. Promoter activity, mRNA, protein, and estradiol levels were quantified. FSH and GB treatment increased CYP19A1 promoter activity, mRNA, and protein levels as well as estradiol when compared with cells treated with FSH only. GB treatment potentiated cAMP stimulation of aromatase and IGF2 stimulation by FSH. GB effects were inhibited by SMAD3 inhibitors and IGF1 receptor inhibitors. GB, but not FSH, stimulates SMAD3 phosphorylation. The combination of GDF9 and BMP15 potently stimulates the effect of FSH and cAMP on CYP19a1 promoter activity and mRNA/protein levels. These effects translate into an increase in estradiol production. This potentiation seems to occur through activation of the SMAD2/3 and SMAD3 signaling pathway and involves, at least in part, the effect of the IGF system.

Sexually Dimorphic Effects of Aromatase on Neurobehavioral Responses.

Aromatase is the enzyme responsible for converting testosterone to estradiol. In mammals, aromatase is expressed in the testes, ovaries, brain, and other tissues. While estrogen is traditionally associated with reproduction and sexual behavior in females, our current understanding broadens this perspective to include such biological functions as metabolism and cognition. It is now well-recognized that aromatase plays a vital lifetime role in brain development and neurobehavioral function in both sexes. Thus, ongoing investigations seek to highlight potentially vital sex differences in the role of aromatase, particularly regarding its centrally mediated effects. To characterize the role of aromatase in mediating such functions, effects of aromatase inhibitor (AI) treatments on humans and animal models have been determined. Aromatase knockout (ArKO) mice that systemically lack the enzyme have also been employed. Humans possessing mutations in the gene encoding aromatase, CYP19, have also provided critical insight into how aromatase affects brain function in a possible sex-dependent manner. A better understanding of how AIs, used to treat breast cancer and other clinical conditions, may detrimentally affect neurobehavioral responses will likely promote development of future therapies to combat these effects. Herein, we will provide a critical review of the current knowledge of sex differences in aromatase regulation of various neurobehavioral functions. Although many species have been used to better understand the functions of aromatase, this review focuses on rodent models and humans. Critical gaps in our present understanding of this area will be considered, and important future research directions will be discussed.

Aromatase Acetylation Patterns and Altered Activity in Response to Sirtuin Inhibition.

Aromatase, a cytochrome P450 member, is a key enzyme involved in estrogen biosynthesis and is dysregulated in the majority of breast cancers. Studies have shown that lysine deacetylase inhibitors (KDI) decrease aromatase expression in cancer cells, yet many unknowns remain regarding the mechanism by which this occurs. However, advances have been made to clarify factors involved in the transcriptional regulation of the aromatase gene (CYP19A1). Yet, despite aromatase being a primary target for breast cancer therapy, its posttranslational regulation has been virtually unexplored. Acetylation is a posttranslational modification (PTM) known to alter the activity and stability of many oncoproteins, and given the role of KDIs in regulating aromatase expression, we postulate that aromatase acetylation acts as a novel posttranslational regulatory mechanism that impacts aromatase expression and/or activity in breast cancer. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that aromatase is basally acetylated on several lysine residues (108, 169, 242, 262, 334, 352, and 354) in MCF-7 cells, and treatment with a SIRT-1 inhibitor induced additional acetylation (376, 390, 440, and 448). These acetylated lysine residues are in regions critical for aromatase activity. Site-directed mutagenesis and overexpression studies demonstrated that K108R/Q or K440R/Q mutations significantly altered aromatase activity in breast cancer cells without altering its subcellular localization.Implications: These findings demonstrate a novel posttranslational regulation of aromatase and uncover novel anticancer effects of deacetylase inhibitors, thus providing new insight for ongoing development of deacetylase inhibitors as cancer therapeutics. Mol Cancer Res; 16(10); 1530-42. ©2018 AACR.