Increased Estrogen Receptor Research Articles

Brain Derived Neurotrophic Factor Stimulates Hypothalamic and Gonadal Reproductive Hormones and Oocyte Maturation in Zebrafish.

Brain-derived neurotrophic factor (BDNF) is a peptide widely known for its role in neurogenesis and synaptic plasticity. Its expression in non-neuronal tissues has been reported. In mammals, it is involved in ovarian development, follicle growth, oocyte maturation, and early embryonic development. In zebrafish, it was demonstrated that BDNF increases food intake and regulates metabolism. Reproduction and metabolism are tightly linked. We hypothesized that BDNF modulates reproductive hormones and reproductive functions in zebrafish. This study aimed to determine BDNF expression in the zebrafish reproductive axis and whether it modulates the reproductive endocrine milieu and oocyte biology in zebrafish. Our results show that bdnf and its receptor trkb, and BDNF-like immunoreactivity are detected in zebrafish gonads and liver cells. This suggests BDNF local production and possible actions within the gonads and liver. Intraperitoneal administration of 1, 10, or 100 ng/g bodyweight BDNF significantly (ANOVA, p<0.05) increased sgnrh/cgnrh-II, kiss1, and cyp19a1b mRNAs in the zebrafish brain; steroidogenic enzymes (star and cyp19a1a) and key receptors in the zebrafish gonads. In vitro incubation of zebrafish liver cells with BDNF significantly (ANOVA, p<0.05) increased estrogen receptor mRNAs and vitellogenin concentrations (ELISA) in the cells. BDNF (100 ng/mL) induced (ANOVA, p<0.05) oocyte maturation in vitro at 24 hours post-incubation and significantly upregulated cumulus-expansion related genes (ANOVA, p<0.05). Overall, our findings indicate a stimulatory role for BDNF in the reproductive axis of zebrafish. This provides impetus for future research on its mechanism of action and potential practical applications to enhance reproduction in aquaculture.

Effects of royal jelly and its extracts on endometrial receptivity and MCF-7 cell growth in rats with thin endometrium

Royal jelly (RJ) is rich in various nutrients, including proteins and amino acids. RJ consumption improves sexual and urinary function in postmenopausal women, possibly owing to its hormone-like effects. Currently, hormone regulation and assisted reproductive technologies are widely used to treat thin endometria; however, high doses of these drugs can add to liver burden. In this study, RJ proteins and fatty acids were isolated, and their therapeutic effects on thin endometria were investigated in vivo and in vitro to verify their hormone-like regulatory effects. The results showed that RJ and its extracts could regulate endometrial receptivity-related factor expression levels in rats with a thin endometrium, accelerate MCF-7 cell apoptosis, and increase estrogen receptor expression in MCF-7 cells wherein. The estrogen-like effects of RJ and the fatty acids may be the main contributors. RJ may be a potential natural hormone replacement therapy.

Adolescent exposure to tris(1,3-dichloro-2-propyl) phosphate (TCPP) induces reproductive toxicity in zebrafish through hypothalamic-pituitary-gonadal axis disruption

Tris(1,3-dichloro-2-propyl) phosphate (TCPP), a prevalent organophosphorus flame retardant in aquatic environments, has raised significant concerns regarding its ecological risks. This study aims to explore the impacts of TCPP on the reproductive functions of zebrafish and delineate its gender-related toxic mechanisms. By assessing the effects on zebrafish of 10 mg/L TCPP exposure from 30 to 120 days post-fertilization (dpf), we thoroughly evaluated the reproductive capability and endocrine system alterations. Our findings indicated that TCPP exposure disrupted gender differentiation in zebrafish and markedly impaired their reproductive capacity, resulting in decreased egg laying and offspring development quality. Histological analyses of gonadal tissues showed an abnormal increase in immature oocytes in females and a reduction in mature sperm count and spermatogonial structure integrity in males, collectively leading to compromised embryo quality. Additionally, molecular docking results indicated that TCPP showed a strong affinity for estrogen receptors, and TCPP-treated zebrafish exhibited imbalanced sex hormones and increased estrogen receptor expression. Alterations in genes associated with the hypothalamic-pituitary-gonadal (HPG) axis and activation of the steroidogenesis pathway suggested that TCPP targets the HPG axis to regulate sex hormone homeostasis. Tamoxifen (TAM), as a competitive inhibitor of estrogen, exhibited a biphasic effect, as evidenced by the counteraction of TCPP-induced effects in both male and female zebrafish after TAM addition. Overall, our study underscored the gender-dependent reproductive toxicity of TCPP exposure in zebrafish, characterized by diminished reproductive capacity and hormonal disturbances, likely due to interference in the HPG axis and steroidogenesis pathways. These findings emphasize the critical need to consider gender differences in chemical risk assessments for ecosystems and highlight the importance of understanding the mechanisms underlying the effects of chemical pollutants on the reproductive health of aquatic species.

Immunohistochemical Detection of Indoleamine 2,3-Dioxygenase in Spontaneous Mammary Carcinomas of 96 Pet Rabbits.

For mammary carcinomas in pet rabbits, prognostic biomarkers are poorly defined, and treatment is limited to surgical excision. Additional treatment options are needed for rabbit patients for which surgery is not a suitable option. In human breast cancer, the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) represents a prognostic biomarker and possible therapeutic target. This retrospective immunohistochemical study examined IDO1 in 96 pet rabbit mammary carcinomas with known mitotic count, hormone receptor status, and percentage of stromal tumor infiltrating lymphocytes (TILs). Tumors were obtained from 96 pet rabbits with an average of 5.5 years. All rabbits with reported sex (n = 88) were female or female-spayed. Of the carcinomas, 94% expressed IDO1, and 86% had sparse TILs consistent with cold tumors. Statistically significant correlations existed between a higher percentage of IDO1-positive tumor cells, lower mitotic counts, and increased estrogen receptor expression. The threshold for significance was IDO1 staining in >10% of tumor cells. These results lead to the assumption that IDO1 expression contributes to tumorigenesis and may represent a prognostic biomarker and possible therapeutic target also in pet rabbit mammary carcinomas. They also support the value of rabbits for breast cancer research.

Estrogens and Olfactomedins May Induce a Feedback Loop that Increases Nicotine Consumption in Females via the Brain Reward Circuitry

Estrogen is associated with enhanced nicotine use in women, with enhanced vulnerability to nicotine use disorders being associated with higher levels of circulating estrogen. In ovariectomized (OVX) female rats, estradiol (E2) treatments increased estrogen receptor β (ERβ) and not ERα in a key brain reward region, the nucleus accumbens core (NAcore). Therefore, we hypothesized that ERβ might be important in the reward circuitry for regulating nicotine use in females. To determine ERβ-specific functions that might regulate nicotine use, we analyzed large sequencing datasets of estrogen-induced genes and narrowed the list to those expressed in the brain and that have a hormone function. We found one class of genes known as the olfactomedins (OLFMs), which meet these criteria. Therefore, we wanted to determine whether ERα or ERβ regulates the expression of the OLFM isoforms. We used human uterine cells (Ishikawa) that express both ER isoforms and the OLFMs and found that two-hour E2 treatments induced the expression of OLFM1 and OLFM3. However, nicotine only suppressed the E2-induction of OLFM1 and not the others. We next performed chromatin immunoprecipitation (ChIP) assays with these cells and treatments using antibodies for ERα, ERβ, or GFP control. We found that the OLFM1 promoter was only enriched by ERβ, and no binding of ERα was observed. We next treated OVX female rats with E2, nicotine, E2+nicotine, or vehicle and found that Olfm1 and Olfm2 significantly increased in the NAcore, and all three OLFM isoforms were significantly higher in the ventral tegmental area (VTA), another key region within the mesolimbic reward pathway. Nicotine suppressed the E2-induced OLFM expression in the NAcore and VTA. These new findings suggest that ERβ-induction of the olfactomedins might serve as a feedback loop for driving nicotine addiction processes, which is suppressed when nicotine is on board. These results indicate a potential new target for therapeutic cessation of nicotine use in women. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

5-O-Acetylpinostrobin derivatives inhibit estrogen alpha and progesterone receptors through a molecular docking approach

Background: Among all cancers, breast cancer accounts for 11.7% of new cases and 6.9% of deaths worldwide. This is driven by increased estrogen receptor alpha (ERα) and progesterone receptor (PgR) expression. Many breast cancer drugs cause various side effects. Modifying the structure of pinostrobin by adding acyl groups to obtain 5-O-acetylpinostrobin derivatives can increase its activity and selectivity. Objective: This study aimed to predict the interaction of 5-O-acetylpinostrobin derivatives with ERα and PgR. Method: A molecular docking approach using AutodockTool. The Protein Data Bank (PDB) was used to obtain ID 3ERT (ERα) and 2W8Y (PgR). Result: The analysis showed the value of free energy binding (ΔG) to ERα with a range of -8.58 to -5.76 kcal/mol and an inhibition concentration (Ki) of 0.51 to 59.91 μM. PgR had ΔG values of -12.37 to -8.30 kcal/mol and Ki of 0.86 to 830.64 nM. Conclusion: The study showed that 5-O-4-(dimethylamino)benzoylpinostrobin, 5-O-cyclohexancarbonylpinostrobin, 5-O-2-phenylacetylpinostrobin, 5-O-3-phenylpropanoylpinostrobin, and 5-O-cyclobutanecarbonylpinostrobin have the potential to be synthesised and serve as the basis for the development of new anticancer compounds that inhibit ERα and PgR in breast cancer.

Estrogenic endocrine disruptor exposure directly impacts erectile function

Erectile dysfunction (ED) is an extremely prevalent condition which significantly impacts quality of life. The rapid increase of ED in recent decades suggests the existence of unidentified environmental risk factors contributing to this condition. Endocrine Disrupting Chemicals (EDCs) are one likely candidate, given that development and function of the erectile tissues are hormonally dependent. We use the estrogenic-EDC diethylstilbestrol (DES) to model how widespread estrogenic-EDC exposure may impact erectile function in humans. Here we show that male mice chronically exposed to DES exhibit abnormal contractility of the erectile tissue, indicative of ED. The treatment did not affect systemic testosterone production yet significantly increased estrogen receptor α (Esr1) expression in the primary erectile tissue, suggesting EDCs directly impact erectile function. In response, we isolated the erectile tissue from mice and briefly incubated them with the estrogenic-EDCs DES or genistein (a phytoestrogen). These acute-direct exposures similarly caused a significant reduction in erectile tissue contractility, again indicative of ED. Overall, these findings demonstrate a direct link between estrogenic EDCs and erectile dysfunction and show that both chronic and acute estrogenic exposures are likely risk factors for this condition.

Abstract 756: Differences in composition, diversity, and function of the fecal microbiota of post-menopausal breast cancer patients compared to healthy, age-matched controls

Abstract Gut microbiota are a community of organisms that colonize the intestinal tract with essential roles in immune function, gut epithelial integrity, and metabolite production. Dysbiosis occurs when the gut microbiota environment is negatively altered causing detrimental changes to these functions, which may predispose an individual to breast cancer development. To understand the relationship between dysbiosis and breast cancer development, fecal microbiota samples were collected from 27 post-menopausal breast cancer patients of varying hormone receptor statuses (ER+HER2-, ER+Her2+, ER-HER2+, or ER-Her2-) prior to starting chemotherapeutic treatment. Samples were compared to 25 healthy controls, and all samples underwent 16srRNA sequencing. Samples from breast cancer patients and control groups were compared for significant differences (P&amp;lt;.05) in alpha (within sample) and beta (between sample) diversity. Using the Shannon Index and Generalized Least Squares Model, significant differences in alpha diversity were identified between all breast cancer types vs. controls (P=.0104), and between ER+HER2- breast cancer vs. controls (P=.00153). Healthy controls exhibited a higher mean Shannon Index than breast cancer patients, thus higher within sample diversity. Using principal coordinate analyses, significant differences in beta diversity were identified between all breast cancer types vs. controls (P = .01199), and ER+HER2- breast cancer vs. control (P = .008991). Further analyses identified significant taxa of microbiota with functions related to breast cancer development and progression. Of note, Lachnoclostridium, a genus of bacteria that can produce β-glucuronidase, which can deconjugate estrogen, was increased in all breast cancer types and ER+HER2- breast cancer when compared to controls. Deconjugation of estrogen can cause elevated blood estrogen levels, potentially increasing Estrogen Receptor binding in hormone-receptor-positive breast cancer. Additionally, Akkermansia, a genus implicated in gut barrier dysfunction, was decreased in ER+HER2- patients, while Parvimonas, a tumorigenic cell-cycle regulator, was increased. Elucidating if these effects are causative or reactionary may have important implications for understanding breast cancer etiology. Samples are currently undergoing additional metagenomic analyses using long-read sequencing to identify specific bacteria, viruses, fungi, parasites, and phages that may be contributing to dysbiosis. Overall, this study is the first, to our knowledge, to employ short and long-read sequencing to the fecal samples of untreated postmenopausal breast cancer patients. We provide mechanistic insight into the relationship between microbiota and breast cancer development, with the intent of informing future patient screening and prevention. Citation Format: Nadia Kabbej, Frederick Ashby, Erika Atencio, Erinn Rosenkrantz, Dominique Day, Khloe Dang, Josee Gauthier, Rachel Newsome, John Sommerville, Taqwa Naas, Raad Gharaibeh, Christian Jobin, Amira Quevedo, Coy D. Heldermon. Differences in composition, diversity, and function of the fecal microbiota of post-menopausal breast cancer patients compared to healthy, age-matched controls [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 756.

Sex differences in inflammation correlated with estrogen and estrogen receptor-β levels in azoxymethane/dextran sodium sulfate-induced colitis-associated colorectal cancer mice

Colorectal cancer (CRC) is a type of cancer that develops in the colon or rectum and is the second leading cause of cancer-related death worldwide. Several epidemiology studies have identified a significant sexual dimorphism in CRC, with women exhibiting a lower incidence rate and delayed onset compared to men. This study aims to investigate the sexual dimorphism in the inflammatory response in colitis-associated CRC and its relationship with estrogen and estrogen receptors. An azoxymethane (AOM)/dextran sodium sulfate (DSS) mouse model was used to induce colitis-associated CRC. Five-week-old male and female mice were randomly assigned into either the control group or the AOM/DSS CRC group, with 10 mice in each group. Colitis-associated CRC was induced by injecting AOM (10 mg/kg) and administering two-cycles of DSS treatment in the drinking water. The results revealed a significant decrease in colon length exclusively in the female group, indicating more severe colonic inflammation (P < 0.01). A significant interaction was identified between sex and AOM/DSS treatment in the female AOM/DSS group, with higher visceral fat weight compared to their male counterparts (P < 0.05). The female AOM/DSS group also exhibited elevated production of M1 macrophage-related pro-inflammatory cytokines, suggesting increased tumor-associated macrophage activity. Surprisingly, the male AOM/DSS group showed a marked increase in serum estradiol levels, while the female AOM/DSS group exhibited a decrease compared to the normal control group. Additionally, a notable upregulation of both estrogen receptor α and estrogen receptor β expression was observed in the colon tissues of the AOM/DSS groups compared to the normal control groups, with estrogen receptor β expression being particularly pronounced in females. Taken together, our findings suggest that a decline in endogenous estrogen and increased estrogen receptors potentially contribute to the pro-inflammatory response in early CRC by augmenting cytokine expressions associated with M1 macrophage polarization in females.

Ictal activity is sustained by the estrogen receptor β during the estrous cycle

Catamenial epilepsy, defined as a periodicity of seizure exacerbation during the menstrual cycle, affects up to 70 % of epileptic women. Seizures in these patients are often non-responsive to medication; however, our understanding of the relation between menstrual cycle and seizure generation (i.e. ictogenesis) remains limited. We employed here field potential recordings in the in vitro 4-aminopyridine model of epileptiform synchronization in female mice (P60–P130) and found that: (i) the estrous phase favors ictal activity in the entorhinal cortex; (ii) these ictal discharges display an onset pattern characterised by the presence of chirps that are thought to mirror synchronous interneuron firing; and (iii) blocking estrogen receptor β-mediated signaling reduces ictal discharge duration. Our findings indicate that the duration of 4AP-induced ictal discharges, in vitro, increases during the estrous phase, which corresponds to the human peri-ovulatory period. We propose that these effects are caused by the presumptive enhancement of interneuron excitability due to increased estrogen receptor β-mediated signaling.

Noncanonical HPV carcinogenesis drives radiosensitization of head and neck tumors

We analyzed transcriptional data from 104 HPV+ (Human papillomavirus) HNSCC (head and neck squamous cell carcinoma) tumors together with two publicly available sources to identify highly robust transcriptional programs (modules) which could be detected consistently despite heterogeneous sequencing and quantification methodologies. Among 22 modules identified, we found a single module that naturally subclassifies HPV+ HNSCC tumors based on a bimodal pattern of gene expression, clusters all atypical features of HPV+ HNSCC biology into a single subclass, and predicts patient outcome in four independent cohorts. The subclass-defining gene set was strongly correlated with Nuclear factor kappa B (NF-κB) target expression. Tumors with high expression of this NF-κB module were rarely associated with activating PIK3CA alterations or viral integration, and also expressed higher levels of HPHPV E2 and had decreased APOBEC mutagenesis. Alternatively, they harbored inactivating alterations of key regulators of NF-κB, TNF receptor associated factor 3 (TRAF3), and cylindromatosis (CYLD), as well as retinoblastoma protein (RB1). HPV+ HNSCC cells in culture with experimental depletion of TRAF3 or CYLD displayed increased expression of the subclass-defining genes, as well as robust radio-sensitization, thus recapitulating both the tumor transcriptional state and improved treatment response observed in patient data. Across all gene sets investigated, methylation to expression correlations were the strongest for the subclass-defining, NF-κB-related genes. Increased tumor-infiltrating CD4+ T cells and increased Estrogen receptors alpha (ERα) expression were identified in NF-κB active tumors. Based on the relatively high rates of cure in HPV+ HNSCC, deintensification of therapy to reduce treatment-related morbidity is being studied at many institutions. Tumor subclassification based on oncogenic subtypes may help guide the selection of therapeutic intensity or modality for patients with HPV+ HNSCC.

Estetrol Increases Progesterone Genetic Response without Triggering Common Estrogenic Effects in Endometriotic Cell Lines and Primary Cultures

Estetrol (E4), a natural estrogen produced by the human fetal liver, is actively studied for menopause and breast cancer treatment. It has low side effects and preferential estrogen receptor alpha (ERα) affinity. There are no data about its effects on endometriosis, a common gynecological disease affecting 6-10% of cycling women, generating painful pelvic lesions and infertility. Current combined hormone treatment (progestins and estrogens) is safe and efficient; nevertheless, one-third of patients develop progesterone (P4) resistance and recurrence by reducing P4 receptors (PRs) levels. We aimed to compare E4 and 17β-estradiol (E2) effects using two human endometriotic cell lines (epithelial 11Z and stromal Hs832 cells) and primary cultures from endometriotic patients. We evaluated cell growth (MTS), migration (wound assay), hormone receptors levels (Western blot), and P4 response by PCR array. Compared to E2, E4 did not affect cell growth or migration but increased estrogen receptor alpha (ERα) and PRs, and reduced ERβ. Finally, the incubation with E4 improved the P4 gene response. In conclusion, E4 increased PRs levels and genetic response without inducing cell growth or migration. These results suggest that E4 might be useful for endometriosis treatment avoiding P4 resistance; however, evaluating its response in more complex models is required.

Reproductive factors and the risk of breast cancer among Nigerian women by age and oestrogen receptor status

The aim of the study was to investigate the association between reproductive factors and breast cancer risk in Nigeria. This has not been widely investigated in sub-Saharan Africa. We conducted a hospital-based case-control study involving participants from five hospitals in Lagos and Abuja. Women were interviewed in-person between October 2016 and May 2017 using a semi-structured questionnaire. We collected data on parity, breastfeeding, age at first and last birth, age at menarche, oral contraceptive use and history of abortion. The data were analysed using multivariable logistic regression adjusting for relevant confounders. Every additional 6months of breastfeeding over a lifetime reduced breast cancer odds by: 7% (95% CI: 1%, 12%) in all women, 15% (95% CI: 5%, 24%) in women < 50years, and 8% (95% CI: 0%, 12%, p for trend = 0.043) in oestrogen receptor negative (ER-) cases. Each additional 1-year delay before the first full-term pregnancy increased oestrogen receptor positive breast cancer odds by 9% (95% CI: 2%, 17%). Each additional 1-year delay before the last full-term pregnancy increased breast cancer odds by: 7% (95% CI: 2%, 12%) in all women, 12% (95% CI: 4%, 21%) in ER- breast cancer patients, and 14% (95% CI: 4%, 25%) in triple negative breast cancer patients. Other reproductive factors did not significantly increased breast cancer odds. While advanced age at first and last full-term pregnancies increased breast cancer odds, breastfeeding reduced it. These associations varied by age and oestrogen receptor status. Improved breastfeeding practices and timely births should be promoted in Nigeria.

BPA disrupted the testis testosterone levels by interfering ER enrichments within StAR 5′ flanking region in rare minnow Gobiocypris rarus

Bisphenol A (BPA) is a widely used endocrine disruptor, which has attracted much attention due to its harmful effects on male reproduction. To investigate the interference of BPA on steroid synthesis in males, male rare minnows (Gobiocypris rarus) were exposed to 15 μg L−1 BPA for 7, 14 and 21 d. Meanwhile, a positive control group was performed with 25 ng L−1 17α-ethynyl estradiol (EE2). Results showed that BPA exposure induced lower testosterone (T) levels, while affecting the transcripts of steroidogenic gene StAR. Moreover, BPA induced abnormal germ cells proliferation in the testis in rare minnow. Transcriptome analysis showed that 354 transcripts significantly differentially expressed after BPA exposure for 14 d, several of them were enriched in the signaling pathways of cell cycle process, PPAR signaling pathway, the steroid synthesis pathway and estrogen signaling pathway. BPA significantly increased estrogen receptor (ER) levels and induced abnormal protein levels of PPARγ. BPA disrupted the StAR expression by interfering ER enrichments within StAR 5′ flanking region. Additionally, our study also revealed that BPA and EE2 might have different mechanisms for interfering with steroid hormone levels and germ cells proliferation in the testis.

RNA Binding Motif 5 Gene Deletion Modulates Cell Signaling in a Sex-Dependent Manner but Not Hippocampal Cell Death.

RNA-binding motif 5 (RBM5) is a pro-death tumor suppressor gene in cancer cells. It remains to be determined if it is neurotoxic in the brain or rather if it plays a fundamentally different role in the central nervous system (CNS). Brain-specific RBM5 knockout (KO) mice were given a controlled cortical impact (CCI) traumatic brain injury (TBI). Markers of acute cellular damage and repair were measured in hippocampal homogenates 48 h post-CCI. Hippocampal CA1/CA3 cell counts were assessed 7 days post-CCI to determine if early changes in injury markers were associated with histological outcome. No genotype-dependent differences were found in the levels of apoptotic markers (caspase 3, caspase 6, and caspase 9). However, KO females had a paradoxical increase in markers of pro-death calpain activation (145/150-spectrin and breakdown products [SBDP]) and in DNA repair/survival markers. (pH2A.x and pCREB). CCI-injured male KOs had a significant increase in phosphorylated calcium/calmodulin-dependent protein kinase II (pCaMKII). Despite sex/genotype-dependent differences in KOs in the levels of acute cell signaling targets involved in cell death pathways, 7 day hippocampal neuronal survival did not differ from that of wild types (WTs). Similarly, no differences in astrogliosis were observed. Finally, gene analysis revealed increased estrogen receptor α (ERα) levels in the KO hippocampus in females and may suggest a novel mechanism to explain sex-dimorphic effects on cell signaling. In summary, RBM5 inhibition did not affect hippocampal survival after a TBI in vivo but did modify targets involved in neural signal transduction/Ca2+ signaling pathways. Findings here support the view that RBM5 may serve a purpose in the CNS that is dissimilar from its traditional pro-death role in cancer.

Abstract P5-06-11: Secreted frizzled related protein 1: From lobular involution to breast osteoimmunological disorder

Abstract Breast cancer is the leading cause of cancer-related death in women worldwide. Our group previously performed a human transcriptome array on normal tissue, atypical ductal hyperplasia, ductal carcinoma in situ and estrogen receptor-α positive invasive ductal carcinoma. Among the transcripts down-regulated during carcinogenesis: Secreted Frizzled-Related Protein 1 (SFRP1), a Wnt signaling pathway antagonist inversely associated with age-related lobular involution has been identified.Interestingly, SFRP1 expression is higher in presence of hydroxyapatite microcalcifications in non-tumoral breast tissue. SFRP1 is also known to be involved in the increase of bone resorption, notably by downregulating osteoblastic differentiation. This suggests that the lack of SFRP1 could be involved in both osteoblast-like phenotype acquisition and early breast carcinogenesis. Unfortunately, data on the mechanism of microcalcifications formation and their impacts on breast carcinogenesis are still lacking.We hypothesize that loss of SFRP1 is responsible for microcalcifications accumulation and early breast carcinogenesis. Our objectives are:1. To define the impact of microcalcifications on SFRP1 expression in non-tumoral mammary gland and 2. to decipher the causal role of SFRP1 in microcalcifications formation. Organoid culture using freshly purified epithelial cells from nulliparous and multiparous mice mammary glands, in presence or in absence of hydroxyapatite crystals has been performed. Organoids number and phenotype have been studied (aim 1). We also have modulated SFRP1 expression in mammary cell lines by lentiviral transduction and performed proliferation, migration and alizarin assays to assess the tumoral aggressivity and cells abilities to produce calcium deposits in vitro (aim 2). SFRP1 and markers of osteoblastic differentiation (Runt-related transcription factor 2, RUNX2) and bone formation (Alkaline Phosphatase, ALPL; Osteopontin, SPP1; Periostin, POSTN) will also be quantified by quantitative PCR in both mice organoids and human mammary cell lines (aims 1 and 2). 1. The ratio between the mammary gland and body weight obtained from nulliparous mice (n = 12) is significantly lower compared with multiparous mice (n = 12; fold-change = 1.84; p-value &amp;lt; 0.001). Hydroxyapatite crystals induced an increase in the proportion of organoids with lumen and branching compared with the control without hydroxyapatite specifically in organoids obtained from nulliparous mice. Organoid’s size and gene expression profiles are still to be assessed. 2. SFRP1 downregulation in a non-tumoral atypical ductal hyperplasia cell line (MCF10AT1) induced an increase in proliferative and migratory cells abilities. It was also responsible for an increase in estrogen receptor 1 (ESR1) expression, suggesting that SFRP1 is related to luminal carcinogenesis. Results also demonstrate that decreasing SFRP1 in MCF10AT1 induced a profile similar to growth stage in bone tissue, which notably means an increase of expression of the osteoblastic transcription factor RUNX2 and the marker of mineralization SPP1, also involved in lactation-related pink adipose tissue formation. We also observed an increase of POSTN expression, known to be involved in decreased bone mineral density during lactation.SFRP1 upregulation in the invasive luminal A cell line MCF7 reduced cell migration and ESR1 expression. Likewise, it increased MCF7 proliferation compared to control. In addition, it induced an increase of RUNX2 and SPP1 expression, similar to what is reported in matrix mineralization profile in bone. These results support our hypothesis that SFRP1 has a causal role in the acquisition of a malignant phenotype but also in osteoblast-like phenotype in mammary epithelial cells. The cause of SFRP1 downregulation in women remains to be explored. Citation Format: Alisson Clemenceau, Aurélie Lacouture, Juliette Bherer, Audet-Walsh Étienne, Caroline Diorio, Francine Durocher. Secreted frizzled related protein 1: From lobular involution to breast osteoimmunological disorder [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 P5-06-11.

White Adipose Tissue Depots Respond to Chronic Beta-3 Adrenergic Receptor Activation in a Sexually Dimorphic and Depot Divergent Manner.

Beta-3 adrenergic receptor activation via exercise or CL316,243 (CL) induces white adipose tissue (WAT) browning, improves glucose tolerance, and reduces visceral adiposity. Our aim was to determine if sex or adipose tissue depot differences exist in response to CL. Daily CL injections were administered to diet-induced obese male and female mice for two weeks, creating four groups: male control, male CL, female control, and female CL. These groups were compared to determine the main and interaction effects of sex (S), CL treatment (T), and WAT depot (D). Glucose tolerance, body composition, and energy intake and expenditure were assessed, along with perigonadal (PGAT) and subcutaneous (SQAT) WAT gene and protein expression. CL consistently improved glucose tolerance and body composition. Female PGAT had greater protein expression of the mitochondrial uncoupling protein 1 (UCP1), while SQAT (S, p < 0.001) was more responsive to CL in increasing UCP1 (S×T, p = 0.011) and the mitochondrial biogenesis induction protein, PPARγ coactivator 1α (PGC1α) (S×T, p = 0.026). Females also displayed greater mitochondrial OXPHOS (S, p < 0.05) and adiponectin protein content (S, p < 0.05). On the other hand, male SQAT was more responsive to CL in increasing protein levels of PGC1α (S×T, p = 0.046) and adiponectin (S, p < 0.05). In both depots and in both sexes, CL significantly increased estrogen receptor beta (ERβ) and glucose-related protein 75 (GRP75) protein content (T, p < 0.05). Thus, CL improves systemic and adipose tissue-specific metabolism in both sexes; however, sex differences exist in the WAT-specific effects of CL. Furthermore, across sexes and depots, CL affects estrogen signaling by upregulating ERβ.

GPR30 Activation by 17β-Estradiol Promotes p62 Phosphorylation and Increases Estrogen Receptor α Protein Expression by Inducing Its Release from a Complex Formed with KEAP1.

Estrogens can elicit rapid cellular responses via the G-protein-coupled receptor 30 (GPR30), followed by estrogen receptor α (ERα/ESR1)-mediated genomic effects. Here, we investigated whether rapid estrogen signaling via GRP30 may affect ESR1 expression, and we examined the underlying molecular mechanisms. The exposure of human endometrial cancer cells to 17β-estradiol promoted p62 phosphorylation and increased ESR1 protein expression. However, both a GPR30 antagonist and GPR30 silencing abrogated this phenomenon. GPR30 activation by 17β-estradiol elicited the SRC/EGFR/PI3K/Akt/mTOR signaling pathway. Intriguingly, unphosphorylated p62 and ESR1 were found to form an intracellular complex with the substrate adaptor protein KEAP1. Upon phosphorylation, p62 promoted ESR1 release from the complex, to increase its protein expression. Given the critical role played by p62 in autophagy, we also examined how this process affected ESR1 expression. The activation of autophagy by everolimus decreased ESR1 by promoting p62 degradation, whereas autophagy inhibition with chloroquine increased ESR1 expression. The treatment of female C57BL/6 mice with the autophagy inhibitor hydroxychloroquine—which promotes p62 expression—increased both phosphorylated p62 and ESR1 expression in uterine epithelial cells. Collectively, our results indicate that 17β-estradiol-mediated GPR30 activation elicits the SRC/EGFR/PI3K/Akt/mTOR signaling pathway and promotes p62 phosphorylation. In turn, phosphorylated p62 increased ESR1 expression by inducing its release from complexes that included KEAP1. Our findings may lead to novel pharmacological strategies aimed at decreasing ESR1 expression in estrogen-sensitive cells.

Mechanisms of OCT4 on 3,5,3'-Tri-iodothyronine and FSH-induced Granulosa Cell Development in Female Mice.

Octamer-binding transcription factor 4 (OCT4) regulates the pluripotency of stem cells and also plays important roles in granulosa cells growth, which is regulated by follicle-stimulating hormone (FSH). Thyroid hormone (TH) is important for the development and maturation of follicles and the maintenance of various endocrine functions. Although 3,5,3'-triiodothyronine (T3) enhances the effects of FSH on the regulation of the growth of granulosa cells and development of follicles, it is unclear whether and, if so, how TH combines with FSH to regulate OCT4 expression in granulosa cells during the preantral to early antral transition stage. Our results showed that T3 enhanced FSH-induced OCT4 expression. However, T3/FSH-induced cellular growth was reduced by OCT4 small interfering RNA. OCT4 knockdown significantly increased the number of apoptotic cell. Moreover, T3 combined with FSH to increase estrogen receptor β (ERβ) expression but did not significantly affect estrogen receptor α expression. ERβ knockdown dramatically decreased T3/FSH-induced OCT4 expression and cell development and increased cell apoptosis. The phosphoinositide 3-kinases/protein kinase B pathway was involved in hormones inducing OCT4 and ERβ expressions. Furthermore, the hormones regulating OCT4 and ERβ expressions were regulated by cytochrome P450 lanosterol 14a-demethylase (CYP51), a key enzyme in sterol and steroid biosynthesis. T3 and FSH cotreatment potentiated cellular development by upregulating OCT4 expression, which is mediated by CYP51 and ERβ. These regulatory processes are mediated by the phosphoinositide 3-kinase/protein kinase B signaling pathway. These findings suggest that OCT4 mediates the T3 and FSH-induced development of follicles.

O12: IHC ANALYSIS OF RESIDUAL DISEASE POST NEOADJUVANT TRASTUZUMAB FOR EARLY STAGE BREAST CANCER REVEALS ER/HER2 CROSSTALK THROUGH INCREASED ER SIGNALLING

Abstract Introduction Therapeutic pressure functionally affects oncogenes and related signalling pathways through dynamic alterations in transcriptional and epigenetic alterations. Altered receptor status occurs throughout tumour progression and may be influenced by adjuvant and neoadjuvant therapies. Recurrent transcriptional remodelling events have been described in the progression of primary breast cancer to metastasis, including increased tyrosine kinase signalling, specifically Her2, and loss of ESR1 gene expression. We hypothesise that in the setting of tyrosine kinase inhibition, an increase in estrogen receptor (ER) signalling is observed. Method A database of patients recruited to ICORG trial 07/09 was queried to identify patients with histologically confirmed, Her2-overexpressing or Her2 amplified, nonmetastatic, invasive breast cancer who received neoadjuvant trastuzumab, alone or in combination with neoadjuvant systemic chemotherapy. Clinicopathological characteristics recorded include age at diagnosis, clinical stage, receptor status and percentage positivity, and pathological complete response. Result A total of 55 patients identified on ICORG trial 09/07 received neoadjuvant trastuzumab. Of these, 27 achieved a complete pathological response (49%; n=27/55). In those with residual disease, a gain in mean ER staining percentage positivity was observed in the residual disease compared to diagnostic biopsy staining (59.22 vs 45.11; p=0.03). A corresponding loss in Her2 percentage staining positivity was also observed (p=0.006). Conclusion An inverse correlation was observed between loss of Her2 positivity and percentage gain in ER staining in patients with residual disease following treatment with neoadjuvant trastuzumab. Further study is needed to elucidate the regulatory mechanism of ER/Her2 crosstalk, which may be epigenetically regulated through DNA methylation. Take-home message ER/Her2 crosstalk can be demonstrated clinically in IHC analysis of patients with residual disease post neoadjuvant trastuzumab. Tyrosine kinase inhibition in the form of neoadjuvant trastuzumab results in loss of Her2 signalling and corresponding gain in ER signalling.