Estrogen Stimulation Research Articles

The genitourinary syndrome of menopause.

Genitourinary syndrome of menopause (GSM) refers to a collection of symptoms resulting from diminished hormonal, primarily estrogenic stimulation to the vulvovaginal or lower urinary tract and may affect up to 50% of postmenopausal women. Symptoms, which are typically progressive and unlikely to resolve spontaneously, may include, but are not limited to, vulvovaginal dryness, burning or irritation, dyspareunia, or urinary symptoms of urgency, dysuria or recurrent urinary tract infection. These symptoms are typically progressive and unlikely to resolve spontaneously. Diagnosis is clinical. Telemedicine may play a role in diagnosis, initiation of treatment, and follow-up of women with GSM. Effective treatments include moisturizers and lubricants, local hormonal therapy with estrogen or dehydroepiandrosterone, and oral selective estrogen receptor agonists. Laser or radiofrequency procedures, although currently utilized, are being studied to comprehensively understand their overall effectiveness and safety. Additionally, the influence and effect of the vaginal microbiome, as well as potential of treatment via its manipulation, is being studied. We performed a literature search of PubMed, Google Scholar, and Ovid with search terms of vulvovaginal atrophy and GSM and reviewed major US Society Guidelines to create this narrative review of this topic. The literature suggests that healthcare providers can make a significant impact of the health and quality of life of women by being proactive about discussing and providing interventions for GSM. A systematic approach with consideration of current guidelines and attention to developing protocols for interventions should be employed.

Hormone Receptor Loss in Breast Cancer: Molecular Mechanisms, Clinical Settings, and Therapeutic Implications.

Hormone receptor-positive breast cancer (HR+ BC) accounts for approximately 75% of new BC diagnoses. Despite the undisputable progresses obtained in the treatment of HR+ BC in recent years, primary or acquired resistance to endocrine therapies still represents a clinically relevant issue, and is largely responsible for disease recurrence after curative surgery, as well as for disease progression in the metastatic setting. Among the mechanisms causing primary or acquired resistance to endocrine therapies is the loss of estrogen/progesterone receptor expression, which could make BC cells independent of estrogen stimulation and, consequently, resistant to estrogen deprivation or the pharmacological inhibition of estrogen receptors. This review aims at discussing the molecular mechanisms and the clinical implications of HR loss as a result of the therapies used in the neoadjuvant setting or for the treatment of advanced disease in HR+ BC patients.

The Role of Testosterone in Aggression

This review article explores the evidence that testosterone is significantly correlated with certain forms of aggression in a number of animals, although firm evidence is lacking for humans. Studies have revealed that structures within the limbic system are particularly involved in the elicitation of aggression and are sexually dimorphic. Testosterone can exert its effects in one of two ways: either on androgen receptors after conversion to 5-alpha dihydrotestosterone or on estrogen receptors after aromatization to estradiol. It can act via genomic mechanisms to induce production of proteins or via non genomic mechanisms to modulate neural activity. Androgen and estrogen receptors are also found along neurotransmitter pathways. As such, testosterone is able to modulate levels of various neurotransmitters that show evidence of mediating effects on aggressive behaviour. In addition, recent evidence suggests that these neurotransmitters are involved in processes such as olfaction and arousal and suggestions have been put forward explaining how testosterone may modulate these processes. However there is a critical time period early in life, usually within the first few days after birth, during which testosterone exposure is essential to elicit aggression in adulthood. It is thought that testosterone and its metabolites sensitize an androgen-responsive system, while estrogenic metabolites establish the capacity to fight in response to estrogenic stimulation later in life. Despite this, testosterone is only one of a myriad of factors that influence aggression and the effects of previous experience and environmental stimuli have at times been found to correlate more strongly.

Estrogen-regulated expression of SK3 channel in rat colonic smooth muscle contraction

AimsEstrogen can induce inhibition of colonic smooth muscle contraction in male and female mice, which may lead to constipation; however, the mechanisms of inhibition are poorly understood. Hence, this study investigated the effect of estrogen on rat colonic smooth muscle contraction and role of small-conductance Ca2+-activated K+ 3 (SK3) and transcription factors (Sp1 and Sp3) in the underlying mechanisms. Main methodsThe experiment included 24 female Sprague-Dawley (SD) rats divided into 4 groups. The rats were oophorectomized surgically, and a silicone tube containing blank solvent, 0.3 mg/mL estrogen (E2), equal-concentration of estrogen and estrogen receptor antagonist (EI), and bovine serum albumin-E2 (BSA-E2) was implanted. The rats were sacrificed on day 14. The molecular insights were confirmed using real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analyses to determine the effect of estrogenic stimulation on gene and protein expression analyses, respectively. Key findingsThe E2 group showed significantly greater SK3 expression (P < .005) compared with other groups and significantly lowers smooth muscle cell (SMC) contractility (P < .005). Estrogen stimulation and SK3 overexpression resulted in a significant decrease (P < .05) in Ca2+ mobilization in the E2 group versus the control group. Further, the E2 group showed significantly higher Sp1 mRNA (P < .05) but lower Sp3 mRNA expression (P < .05) and protein expression (P < .001) compared with other groups. SignificanceE2 may promote SK3 expression by its genomic effect and inhibit colonic contraction by affecting SK3 expression via an interaction between Sp1 and Sp3.

Endometrial thickness in the evaluation of clinical response to medical treatment for deep infiltrating endometriosis: a retrospective study.

Deep infiltrating endometriosis (DIE) is associated with severe pelvic pain and functional impairment of bowel, urinary, and sexual functions. Though hormone therapy with progestins, either as single agents or combined with estrogens, is effective in managing symptoms, some patients may experience a suboptimal response. Endometrial thickness assessed by transvaginal ultrasound examination, reflecting the overall estrogen stimulation, may correlate with the clinical response to hormonal treatments. A retrospective study was carried out on 61 women with DIE affecting the bowel or the recto-vaginal septum, undergoing hormone treatment. The symptoms of patients were evaluated at the baseline and after 12months of therapy, calculating a global Visual Analogue Scale score (gVAS) encompassing dysmenorrhea, dyspareunia, chronic pelvic pain, dyschezia, abdominal pain and dysuria. Patients were divided into two subgroups using, as a calculated cut-off value, the mean endometrial thickness in our population at 12months. The change in gVAS score during the 12months of treatment was then compared between the two groups. Women with a thinner endometrium (< 3.3mm) showed a better response to treatment in terms of symptoms control as compared to patients with a thicker endometrium (mean gVAS score reduction 9.2 ± 1.3 vs. 5.2 ± 1.3, p = 0.036). The correlation between endometrial thickness and symptomatic response was also confirmed (p = 0.041) on multivariate linear regression analysis including as covariates age, size of lesions of DIE, presence of uterine adenomyosis, ovarian endometriosis and type of medical treatment. Endometrial thickness on ultrasound transvaginal examination is correlated with better response rates to hormone therapy in terms of symptoms control. A thinner endometrium, probably resulting from a more efficient suppression of estrogen stimulation, is associated with improved symptoms. These results may aid clinicians in monitoring and tailoring hormonal treatments during follow-up of women with symptomatic DIE.

Genistein Improves Bone Healing via Triggering Estrogen Receptor Alpha-Mediated Expressions of Osteogenesis-Associated Genes and Consequent Maturation of Osteoblasts.

Osteoporosis-associated fractures may cause higher morbidity and mortality. Our previous study showed the effects of genistein, a phytoestrogen, on the induction of estrogen receptor alpha (ERα) gene expression and stimulation of osteoblast mineralization. In this study, rat calvarial osteoblasts and an animal bone defect model were used to investigate the effects of genistein on bone healing. Treatment with genistein caused a time-dependent increase in alkaline phosphatase (ALP) activity in rat osteoblasts. Levels of cytosolic and nuclear ERα significantly augmented following exposure to genistein. Subsequently, genistein elevated levels of ALP mRNA and protein in rat osteoblasts. Moreover, genistein induced other osteogenesis-associated osteocalcin and Runx2 mRNA and protein expressions. Knocking-down ERα using RNA interference concurrently inhibited genistein-induced Runx2, osteocalcin, and ALP mRNA expression. Attractively, administration of ICR mice suffering bone defects with genistein caused significant increases in the callus width, chondrocyte proliferation, and ALP synthesis. Results of microcomputed tomography revealed that administration of genistein increased trabecular bone numbers and improved the bone thickness and volume. This study showed that genistein can improve bone healing via triggering ERα-mediated osteogenesis-associated gene expressions and subsequent osteoblast maturation.

FOXA1 Mutations Reveal Distinct Chromatin Profiles and Influence Therapeutic Response in Breast Cancer

Mutations in the pioneer transcription factor FOXA1 are a hallmark of estrogen receptor-positive (ER+) breast cancers. Examining FOXA1 in ∼5,000 breast cancer patients identifies several hotspot mutations in the Wing2 region and a breast cancer-specific mutation SY242CS, located in the third β strand. Using a clinico-genomically curated cohort, together with breast cancer models, we find that FOXA1 mutations associate with a lower response to aromatase inhibitors. Mechanistically, Wing2 mutations display increased chromatin binding at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility. In contrast, SY242CS shows neomorphic properties that include the ability to open distinct chromatin regions and activate an alternative cistrome and transcriptome. Structural modeling predicts that SY242CS confers a conformational change that mediates stable binding to a non-canonical DNA motif. Taken together, our results provide insights into how FOXA1 mutations perturb its function to dictate cancer progression and therapeutic response.

Abstract 3439: FOXA1 mutations reveal distinct chromatin profiles and influence therapeutic response in breast cancer

Abstract FOXA1 is an evolutionary conserved pioneer factor that binds to condensed chromatin allowing the recruitment of other transcription factors to the DNA. Although mutations in FOXA1 are a hallmark of estrogen receptor-positive (ER+) breast cancers, it is currently unknown whether and how these mutations affect breast cancer development and response to endocrine therapies. In this work, we studied how the genome-wide chromatin recruitment, accessibility and transcriptional outcomes of recurrent FOXA1 mutations can affect therapeutic response in ER+ breast cancer. By examining the landscape of FOXA1 mutations in a cohort of 4,952 breast cancer patients, we identified several hotspot mutations, some of them present also in other malignancies and some others specific to breast cancer. In particular, we characterized three mutations in the Wing2 region and a breast-cancer specific third β strand mutation, namely SY242CS. We also showed that FOXA1 mutations are enriched in metastatic tumors and mutually exclusive with ESR1 mutations, well-known drivers of resistance to endocrine therapy. Using a clinico-genomicaly curated cohort of patients, together with in vitro and in vivo breast models, we associated FOXA1 missense mutations with a lower response to endocrine therapy. Mechanistically, by means of ChIP-seq, ATAC-seq and RNA-seq analyses, we found that FOXA1 mutations in the Wing2 loop display increased chromatin binding affinity at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility, decoupling FOXA1 Wing2 mutant binding from their pioneering function. These data correlated with the highly organized 3D conformation conferred by FOXA1 Wing2 mutations we predicted by structural modeling. Thus. the enhanced chromatin binding affinity gained by FOXA1 Wing2 mutations might be a mechanism to sustain active estrogen response even in the presence of therapeutic pressure. In contrast, breast specific SY242CS mutant shows neomorphic properties including the ability to open novel chromatin regions, and activate an alternative cistrome and transcriptome. Using an engineered luciferase reporter system, we validated that a non-canonical motif, shared by both gained accessibility and binding sites, is SY242CS-specific. Structural modeling of the binding of WT or SY242CS FOXA1 to this new motif revealed that SY242CS undergoes conformational change that results in a tight interaction with the new DNA motif, not observed in WT FOXA1. Taken together, our findings provide mechanistic insights into how FOXA1 mutations modulate its function in breast cancer to dictate malignant progression and response to endocrine therapy. More broadly, these results position FOXA1 mutations as potential biomarkers of response and potential targets for the treatment of metastatic ER+ breast cancer. Citation Format: Amaia Arruabarrena-Aristorena, Jesper LV Maag, Srushti Kittane, Yanyan Cai, Jane Park, Pedram Razavi, Lorenzo Ferrando, Pier Selenica, Wouter R. Karthaus, Srinivasaraghavan Kannan, Emiliano Cocco, Sik Y. Ho, Daisylyn Senna Tan, Mirna Sallaku, Charles L. Sawyers, Jorge S. Reis-Fihlo, Chandra S. Verma, Ralf Jauch, Richard Koche, José Baselga, Eneda Toska, Maurizio Scaltriti. FOXA1 mutations reveal distinct chromatin profiles and influence therapeutic response in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3439.

Abstract 3490: Investigation of 27-hydroxycholesterol as a biomarker for aggressive endometrial cancer

Abstract Background: Strong risk factors for the most common (endometrioid) type of endometrial cancer (EC) include obesity and exposure to high estrogen levels. While EC has a favorable prognosis overall, one third of women present with advanced stage disease at diagnosis which has high recurrence and mortality rates. Given the estrogen-mediated etiology of EC, the current study focuses on the selective estrogen receptor modulator (SERM) 27-hydroxycholesterol (27HC) pathway. SERMs like 27HC can mimic estrogenic actions, and experimental work indicates 27HC exposure leads to continuous estrogenic stimulation and increases proliferation in the endometrium. Objective: to identify biomarkers for more aggressive disease, particularly markers present in early stages of carcinogenesis, to help guide decisions regarding treatment options. Methods: We conducted immunohistochemical staining on two tissue microarrays (TMAs) representing EC and its precursor endometrial intraepithelial neoplasia (EIN) tissues to measure protein levels of the enzymes that metabolize cholesterol into 27HC (CYP27A1) and 27HC into bile acids (CYP7B1), the receptors that bind 27HC (Estrogen Receptor [ER])-alpha, ER-beta as well as Liver Receptor X [LXR]-alpha and LXR-beta) and progesterone receptor (PR). We will use Fisher's exact test to evaluate 27HC associated biomarkers (categories split at the median) in relation to the presence of myometrial invasion (MI) and final diagnosis (EC or not) in the EIN TMA, and to assess clinico-pathologic factors including disease stage and grade in the EC TMA. Results: We created a new EIN TMA using formalin-fixed paraffin-embedded endometrial curettage or Pipelle endometrial biopsies from 144 EIN cases of diverse racial/ethnic backgrounds (Japanese, Hawaiian, White, Filipino and Asian/Pacific Islander) who were treated between 2007-16 in the Hawaii Pacific Health System. The EIN TMA contains both abnormal EIN tissues and neighboring benign/non-atypical tissues. In total, 50% of the EIN cases had no EC, 30% had cancer with no myometrial invasion (MI) and 20% had cancer with myometrial invasion. We are concurrently evaluating 27HC pathway markers in a TMA representing EC tumors from 96 cases who were treated in the same hospitals between 2001-07. The EC TMA represents diverse racial/ethnic groups including 28% Japanese American, 25% Native Hawaiian and 23% white cases (24% other). The EC TMA contains representative tissues from 91% endometrioid histotype cases and 72% localized and 28% regional/distant stage cases. We will examine associations between 27HC pathway biomarkers and clinico-pathologic characteristics of EC and EIN and will present our findings at the meeting. Conclusions: This study provides new data to determine whether 27HC pathway biomarkers may relate to characteristics of aggressive EC, such as the presence of myometrial invasion and higher stage and grade at diagnosis. Citation Format: Danja Sarink, Lenora W. Loo, Jeffrey Killeen, Brenda Y. Hernandez, Owen T. Chan, Melissa A. Merrit. Investigation of 27-hydroxycholesterol as a biomarker for aggressive endometrial cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3490.

Rapid Effects of Oestrogen on Intracellular Ca2+ in the Uterine Junctional Myometrium of Patients With and Without Adenomyosis in Different Phases of the Menstrual Cycle.

We investigated the role of oestrogen receptor 1 (ESR1) in regulating the [Ca2+]i concentration in the junctional zone (JZ) and its effect on adenomyosis. JZ smooth muscle cells (JZSMCs) were isolated from 17 control and 24 adenomyotic uteri, and membrane proteins were extracted from the cells. In the control group, the levels of membrane ESR1 and [Ca2+]i in the proliferative phase were significantly greater than they were in the secretory phase. While no difference was detected between the two phases, ESR1 and [Ca2+]i levels in the adenomyosis group were significantly higher in the proliferative and secretory phases than they were in the control groups. Oestradiol induced a rapid increase in [Ca2+]i in the JZSMCs of both groups. When pretreated with the ESR1 antagonist ICI 182,780, the increase in [Ca2+]i was clearly reduced in both groups compared with the control, but the differences were not significant. Filtered E-6-BSA also induced [Ca2+]i, and its actions were similar to those of oestrogen. Removal of extracellular Ca2+ did not alter the effect of oestradiol, but the phospholipase C inhibitor U73122 (10μM) and 2-aminoethoxydiphenyl borate (5μM) significantly reduced the oestradiol-induced [Ca2+]i flux. Oestradiol was unable to induce a [Ca2+]i flux in thapsigargin-depleted cells; this result indicated that oestradiol mediates the [Ca2+]i flux in JZSMCs through ESR1, which activates the phospholipase C pathway. ESR1 levels were assessed by Western blotting. Changes in the [Ca2+]i concentration induced by oestrogen stimulation were analysed by immunofluorescence. The ΔFCa2+ was calculated as the difference between baseline and peak fluorescence response to stimulation. We found that the abnormal intracellular [Ca2+]i response to oestrogen could account for aberrant JZ peristalsis.

DEC1 directly interacts with estrogen receptor (ER) α to suppress proliferation of ER-positive breast cancer cells

Aberrant ERα signaling and altered circadian rhythms are both features of ER-positive breast cancer, however, the molecular interaction between them is still not fully understood. Herein, we analyzed the interplay between the circadian rhythm molecule DEC1 and ERα and its effect on the proliferation of ER-positive breast cancer cells, providing a new clue for clarifying the pathogenesis of breast cancer. In this study, we revealed that DEC1 negatively regulates the proliferation of ER-positive breast cancer MCF-7 cells through interaction with ERα protein. DEC1 co-localized with ERα in the nucleus of MCF7 cells, stabilized ERα protein independently of its transcriptional activity and without affecting by estrogen stimulation and inhibited the degradation of ERα mediated by CHX in a time-dependent manner. Moreover, results from luciferase reporter assay showed that overexpression of DEC1 significantly inhibits ERα-mediated transcriptional activity in a dose-dependent manner. These results together suggested that DEC1 may serve as a co-repressor of ERα in ER-positive breast cancer. Although DEC1 improved the stability of ERα and alleviated protein degradation, DEC1 inhibited the proliferation of MCF7 cells by decreasing ERα-mediated signal transduction.

Gene UCHL1 expresses specifically in mouse uterine decidual cells in response to estrogen.

UCHL1 is expressed specifically in the brain and gonads of almost all studied model organisms including Drosophila, zebrafish, amphibians, and mammals, suggesting a high degree of evolutionary conservation in its structure and function. Although UCHL1 has been involved in spermatogenesis in mice, its specific expression in mammal placenta remains elusive. Our previous work has revealed that UCHL1 is highly expressed in oocytes, and has been involved in mouse ovarian follicular development. Here, we further examined UCHL1 expression change in endometria during early natural pregnancy, with different stages of the estrous cycle and pseudopregnancy as control. The UCHL1 gene deletion model showed that UCHL1 protein is associated with endometrial development, and its deletion leads to infertility. Notably, we demonstrate evidence showing the distinct expression pattern of UCHL1: weak expression over the uterine endometria, strong expression in decidualized stromal cells at the implantation site with a peak at pregnancy D6, and a shift with primary decidualization to secondary decidualized zones. Using the delayed implantation, the delayed implantation activation, and the artificial decidualization models, we have demonstrated that strong expression of UCHL1 occurred in response to decidualization and estrogen stimulation. These observations suggest that during the early proliferation and differentiation of mouse uterine decidua, UCHL1 expression is up-regulated, and formed an unique intracellular distribution mode. Therefore, we proposed that UCHL1 is involved in decidualization, and possibly in response to estrogen regulation.

GPER1 Signaling Initiates Migration of Female V-SVZ-Derived Cells.

SummaryIn the rodent ventricular-subventricular zone (V-SVZ) neurons are generated throughout life. They migrate along the rostral migratory stream (RMS) into the olfactory bulb before their final differentiation into interneurons and integration into local circuits. Estrogen receptors (ERs) are steroid hormone receptors with important functions in neurogenesis and synaptic plasticity. In this study, we show that the ER GPER1 is expressed in subsets of cells within the V-SVZ of female animals and provide evidence for a potential local estrogen source from aromatase-positive astrocytes surrounding the RMS. Blocking of GPER1 in Matrigel cultures of female animals significantly impairs migration of V-SVZ-derived cells. This outgrowth is accompanied by regulation of phosphorylation of the actin-binding protein cofilin by GPER1 signaling including an involvement of the p21-Ras pathway. Our results point to a prominent role of GPER1 in the initiation of neuronal migration from the V-SVZ to the olfactory bulb.

Mechanism of Reproductive Effect of Acetyl acetate‐methanol Fraction of Adansonia digitata (Linn) Root Bark Extract in Female Wistar Rats

The aim of this work was to identify the exclusive components of Acetyl acetate‐methanol fraction (AAMF) of Adansonia digitata (Linn) root bark extract and to elucidate its mechanism of action in modulating hormone signaling. Gas chromatography and Mass spectrometry was done to identify the compounds present in fractions of A. digitata (Linn) root bark extract. Thirty female Wistar rats had their estrous cycles synchronized and were randomly distributed into three groups of ten rats each. Each group received 0, 150, and 300mg/kg body weight of AAMF per os from the day observed as proestrus by vaginal cytology to day‐seven. On day‐one, day‐four, day‐five, day‐six and day‐seven, blood was collected from rats in each group. Blood collected was analyzed for estrogen, progesterone and follicle stimulating hormone levels. Uterus and ovary were processed for histopathology and immunohistochemistry to study the regulation of estrogen and progesterone receptor proteins. Where a receptor protein was regulated, further investigation was done to determine its effect on the gene expression of that receptor in endometrial stromal cells through RNA extraction, OneStep reverse transcription‐pCR amplification, Agarose gel electrophoresis and quantification of pCR product. Results showed that AAMF uniquely contained 15.25% of oleic acid and caused mass atresia of antral follicles at proestrus. AAMF treated rats had significantly (p&lt;0.05) lowered serum estradiol levels at proestrus when compared to control rats and had up‐regulated estrogen receptor beta proteins in endometrium and downregulated estrogen receptor alpha (ERα) gene expression in endometrial cells. It was concluded that the oleic acid component of AAMF may have lowered estrogen level by inhibiting aromatase and hydroxysteroid 17 beta dehydrogenase enzyme activities in estrogen steroidogenesis. Also, oleic acid may have induced overexpression of Tumour Suppressor Factor (P53) which promotes follicular apoptosis and indirectly downregulates ERα when p53 activities become exacerbated. It is recommended that AAMF be investigated as a possible therapy for some ERα sensitive clinical conditions like cancer.

Profile of patients with breast diseases subjected for histopathological diagnosis

Breast tissue reacts to estrogen and progesterone stimulation, not only during puberty, pregnancy and lactation, but during each menstrual cycle. As the menopausal period is approached and postmenopausal period evolves, progressive atrophy of the epithelial and connective tissue components of the breast occurs. The loose connective tissue becomes dense and hyalinized and finally, the lobule is converted into ordinary stroma which in the process of involution is replaced by fat. All patients undergoing core needle biopsy of breast were selected for the study. An informed consent will be taken for the procedure. Under local anesthesia using 14 G needle, ultrasound guided core needle biopsy will be performed by radiologist. The biopsy specimen will be sent to histopathology department in 10% formalin. The tissue will be processed, stained with haematoxylin and eosin and examined microscopically. In present study, the most common presenting complaint is breast lump comprising of 59 cases (98.3%). Out of 59 cases 31 cases (51.7%) had lump on left side, 28 cases (46.7%) had lump on right side.1 case had no lump (1%). In the present study 41 cases underwent mammogram, 24 cases (58.5%) showed BIRADS 5 scoring followed by BIRADS 4 in 12 cases (29.3%) and BIRADS 2 in 5 cases (12.2%).

Alterations of endometrial epithelial-mesenchymal transition and MAPK signalling components in women with PCOS are partially modulated by metformin in vitro.

Growing evidence suggests that epithelial-mesenchymal transition (EMT) and its regulator mitogen-activated protein kinase (MAPK) contribute to endometria-related reproductive disorders. However, the regulation of EMT and MAPK signalling components in the endometrium from polycystic ovary syndrome (PCOS) patients has not been systematically investigated and remains elusive. In humans, how metformin induces molecular alterations in the endometrial tissues under PCOS conditions is not completely clear. Here, we recruited 7 non-PCOS patients during the proliferative phase (nPCOS), 7 non-PCOS patients with endometrial hyperplasia (nPCOSEH), 14 PCOS patients during the proliferative phase (PCOS) and 3 PCOS patients with endometrial hyperplasia (PCOSEH). Our studies demonstrated that compared with nPCOS, PCOS patients showed decreased Claudin 1 and increased Vimentin and Slug proteins. Similar to increased Slug protein, nPCOSEH and PCOSEH patients showed increased N-cadherin protein. Western blot and immunostaining revealed increased epithelial phosphorylated Cytokeratin 8 (p-CK 8) expression and an increased p-CK 8:CK 8 ratio in PCOS, nPCOSEH and PCOSEH patients compared to nPCOS patients. Although nPCOSEH and PCOSEH patients showed increased p-ERK1/2 and/or p38 protein levels, the significant increase in p-ERK1/2 expression and p-ERK1/2:ERK1/2 ratio was only found in PCOS patients compared to nPCOS patients. A significant induction of the membrane ERβ immunostaining was observed in the epithelial cells of PCOS and PCOSEH patients compared to nPCOS and nPCOSEH patients. While in vitro treatment with metformin alone increased Snail and decreased Claudin 1, N-cadherin and α-SMA proteins, concomitant treatment with metformin and E2 increased the expression of CK 8 and Snail proteins and decreased the expression of Claudin 1, ZO-1, Slug and α-SMA proteins. Our findings suggest that the EMT contributes to the switch from a healthy state to a PCOS state in the endometrium, which might subsequently drive endometrial injury and dysfunction. We also provide evidence that metformin differentially modulates EMT protein expression in PCOS patients depending on oestrogenic stimulation.

Estrogen activates pyruvate kinase M2 and increases the growth of TSC2-deficient cells.

Lymphangioleiomyomatosis (LAM) is a devastating lung disease caused by inactivating gene mutations in either TSC1 or TSC2 that result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). As LAM occurs predominantly in women during their reproductive age and is exacerbated by pregnancy, the female hormonal environment, and in particular estrogen, is implicated in LAM pathogenesis and progression. However, detailed underlying molecular mechanisms are not well understood. In this study, utilizing human pulmonary LAM specimens and cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we tested the hypothesis that estrogen promotes the growth of mTORC1-hyperactive cells through pyruvate kinase M2 (PKM2). Estrogen increased the phosphorylation of PKM2 at Ser37 and induced the nuclear translocation of phospho-PKM2. The estrogen receptor antagonist Faslodex reversed these effects. Restoration of TSC2 inhibited the phosphorylation of PKM2 in an mTORC1 inhibitor-insensitive manner. Finally, accumulation of phosphorylated PKM2 was evident in pulmonary nodule from LAM patients. Together, our data suggest that female predominance of LAM might be at least in part attributed to estrogen stimulation of PKM2-mediated cellular metabolic alterations. Targeting metabolic regulators of PKM2 might have therapeutic benefits for women with LAM and other female-specific neoplasms.

Characterization of basal and estrogen-regulated antisense transcription in breast cancer cells: Role in regulating sense transcription

Estrogen-responsive breast cancer cells exhibit both basal and estrogen-regulated transcriptional programs, which lead to the transcription of many different transcription units (i.e., genes), including those that produce coding and non-coding sense (e.g., mRNA, lncRNA) and antisense (i.e., asRNA) transcripts. We have previously characterized the global basal and estrogen-regulated transcriptomes in estrogen receptor alpha (ERα)-positive MCF-7 breast cancer cells. Herein, we have mined genomic data to define three classes of antisense transcription in MCF-7 cells based on where their antisense transcription termination sites reside relative to their cognate sense mRNA and lncRNA genes. These three classes differ in their response to estrogen treatment, the enrichment of a number of genomic features associated with active promoters (H3K4me3, RNA polymerase II, open chromatin architecture), and the biological functions of their cognate sense genes as analyzed by DAVID gene ontology. We further characterized two estrogen-regulated antisense transcripts arising from the MYC gene in MCF-7 cells, showing that these antisense transcripts are 5′-capped, 3′-polyadenylated, and localized to different compartments of the cell. Together, our analyses have revealed distinct classes of antisense transcription correlated to different biological processes and response to estrogen stimulation, uncovering another layer of hormone-regulated gene regulation.

Estrogen/estrogen receptor promotes the proliferation of endometrial carcinoma cells by enhancing hMOF expression.

BackgroundThis study aims to analyse the expression of human MOF in endometrial carcinoma cells and its relationship with estrogen and estrogen receptor and to investigate the effect of estrogen–human MOF on the malignant biological behaviours of endometrial carcinoma cells.MethodsThe expression of human MOF was detected in different endometrial tissues by immunohistochemistry. The effects of human MOF, human MOF combined with estrogen stimulation and estrogen plus anti-human MOF antibody blocking on the proliferation of endometrial carcinoma cells were evaluated by western blotting, real-time polymerase chain reaction, cell proliferation assay and cell cycle distribution. Bioinformatics was used to identify the correlations of human MOF and estrogen and involved pathways.ResultsThe expression levels of human MOF in endometrial carcinoma tissues were significantly higher than that in atypical hyperplasia and normal endometrial tissues. High expression of human MOF was associated with late-stage cancer, lymph node metastasis and short survival time, and it was also an independent prognostic risk factor for endometrial carcinoma. After human MOF knockdown, the proliferation, migration and invasive capacity of Ishikawa cells decreased and cell apoptosis increased. After stimulation with estrogen, the PI3K/Akt and Ras–Raf–MEK–ERK signalling pathways were activated, and the expression of the human MOF protein was increased. human MOF (KAT8) expression showed a positive correlation with ESR1 expression, and KAT8-associated genes were enriched in the cell cycle pathways and splicing pathways.ConclusionHuman MOF was highly expressed in endometrial carcinoma and associated with proliferation. Estrogen/estrogen receptor enhanced human MOF expression; promoted the proliferation, migration and invasion of Ishikawa cells; and inhibited cell apoptosis by activating the PI3K/Akt and Ras–Raf–MEK–ERK signalling pathways.

Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity.

Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.