ERβ Levels Research Articles

The Co-Expression of Estrogen Receptors ERα, ERβ, and GPER in Endometrial Cancer.

Estrogens have important roles in endometrial cancer (EC) and exert biological effects through the classical estrogen receptors (ERs) ERα and ERβ, and the G-protein-coupled ER, GPER. So far, the co-expression of these three types of ERs has not been studied in EC. We investigated ERα, ERβ, GPER mRNA and protein levels, and their intracellular protein distributions in EC tissue and in adjacent control endometrial tissue. Compared to control endometrial tissue, immunoreactivity for ERα in EC tissue was weaker for nuclei with minor, but unchanged, cytoplasmic staining; mRNA and protein levels showed decreased patterns for ERα in EC tissue. For ERβ, across both tissue types, the immunoreactivity was unchanged for nuclei and cytoplasm, although EC tissues again showed lower mRNA and protein levels compared to adjacent control endometrial tissue. The immunoreactivity of GPER as well as mRNA levels of GPER were unchanged across cancer and control endometrial tissues, while protein levels were lower in EC tissue. Statistically significant correlations of estrogen receptor α (ESR1) versus estrogen receptor β (ESR2) and GPER variant 3,4 versus ESR1 and ESR2 was seen at the mRNA level. At the protein level studied with Western blotting, there was significant correlation of ERα versus GPER, and ERβ versus GPER. While in clinical practice the expression of ERα is routinely tested in EC tissue, ERβ and GPER need to be further studied to examine their potential as prognostic markers, provided that specific and validated antibodies are available.

Current Evidence for the Involvement of Hypogonadism and Modulation of Steroid Receptors in Prostate Carcinoma

Prostate cancer (PCa) is the most prevalent cancer in the Nigerian male population, similar to other black populations. It is postulated that exposure to endogenous or environmental steroids prompts prostatic mediated changes via steroid receptors as well as a decrease in the androgen/estrogen ratio and aging. Thereby contributing to prostatic carcinogenesis and disease progression. This study is aimed to determine the plasma levels of testosterone, 17β-estradiol as well as the pattern of expression of steroid receptors in subjects with prostate cancer, benign prostatic hyperplasia, and controls. Study participants are made up of a total of 195 consented volunteers consisting of 65 Prostate cancer (PCa) and 65 benign prostatic hyperplasia (BPH) treatment naïve participants and 65 apparently healthy subjects as controls. Anthropometric data were measured using standard methods and biochemical parameters determined by enzyme-linked immunosorbent assay (ELISA). The gene expression is quantified by Real-Time PCR with PerfeCTa SYBR Green SuperMix on CFX96 Bio-Rad, USA. The results of this study showed increased levels of 17β-estradiol, total androgen receptor (AR) and estrogen receptor-beta (Erβ) in prostate cancer participants compared with controls (P<0.05). A Significant reduction in plasma levels of testosterone in prostate cancer subjects compared with BPH and controls was observed (P<0.001). The plasma levels of androgen receptors were significantly increased in PCa and BPH participants (p<0.05). We observed a positive correlation between ERβ levels and PSA levels in the PCa group (r=0.32, p=0.02). There was a differential expression of AR, ESR1 and ESR2 in the studied group. This study shows hypogonadism and an increased 17β-estradiol, ERβ, and AR levels in subjects with prostate cancer. This data suggests that modulation of these hormones and their receptors may be associated with initiation and progression of prostate cancer and could be valuable in the interpretation of PSA kinetics and stratification of cases after screening.

Expression of transcription, growth factors, steroid hormone receptors, LC3B in papillary thyroid cancer tissue, association with prognosis and risk of recurrence

Introduction. Biological characteristics of the tumor play a major role in it’s development and progression. Currently, using the molecular markers aimed at resolving the problems in clinical oncology is becoming more important, including thyroid carcinomas. Heterogeneous contradictory data had been accumulated to date showing the ability of tumors genetic and biological parameters to predict the diseases outcome.Aim. To investigate prognostic value of transcription, growth factors, components of AKT / mTOR signaling pathway and autophagy protein LC3B in patients with papillary thyroid cancer in relation to recurrences and overall survival.Materials and methods. The study included 65 patients with T1–4N0–1M0 papillary thyroid cancer. According to the criteria of the American Thyroid Association (ATA) (2015), patients were divided into groups of patients with high, low and intermediate risk. 30 patients were classified as low risk, 23 as intermediate risk, and 12 as high risk. The BRAFV600 mutation was identified in 18 samples. The expression of transcription factors (p65 and p50 subunits of nuclear factor kappa B (NF-κB p65, NF-κB p50), hypoxia-inducible factor 1 (HIF-1), hypoxia-inducible factor 2 (HIF-2), growth factors (vascular endothelial growth factor (VEGF), receptor VEGF (VEGF-2), carbonic anhydrases of type 9 (CAIX)), AKT, c-RAF, GSK- 3β, p70S6, mammalian target of rapamycin (m-TOR), PDK, PTEN, 4E-BP1 in the tumor was assessed by real-time polymerase chain reaction (PCR). The BRAFV600 mutation was investigated using real-time allele-specific PCR. The content of the LC3B protein was examined using the Western Blot method.Results. As a result of the study, there is an increase in c-RAF expression with an increase in risk from low to high, which was accompanied by a decrease in 4E-BP1 expression. c-RAF mRNA levels were increased 3.0- and 2.8‑fold in the intermediate and high-risk groups, respectively, compared to low risk patients. There is a change in the expression of Brn-3α depending on the relapse risk. The maximum mRNA levels were found in patients with intermediate risk, where the figure was 4.3 and 6.2 times higher than in patients with low and high risk, respectively. An increase in LC3B expression by 56.0 and 28.0 times was shown in the tumor tissue of patients with intermediate risk compared with patients with low and high risk. This fact corresponds with an increasing content of the protein itself, which was higher in patients with intermediate risk. Patients with a negative BRAF gene status had an intermediate and high risk of tumor recurrence. The prognostic significance of the estrogen receptor β (ER-β) and NF-κB p50 expression level had been revealed in relation with relapse-free and overall survival of patients with papillary thyroid cancer.Conclusion. As a result of the study, additional molecular markers were found in order to for predict the tumors recurrence risk. The study showed the significance of ERβ and NF-κB p50 expression levels for predicting disease outcomes.

Melatonin protects against visible light-induced oxidative stress and promotes the implantation potential of mouse blastocyst in vitro

Improvement of embryo culture media using antioxidant agents could help to improve embryo quality against environmental factors such as visible light and could overcome implantation failures. The usefulness of the melatonin against the effect of light on the expression of the primary implantation receptors, ErbB1 and ErbB4 on pre-implantation mouse embryo was investigated. Two-cell mouse embryos were exposed to the 1600 LUX light for 30 min then randomly divided into 3 groups including: Melatonin-Treated; Luzindole Treated and Simple media as a Control group. After 72–96 The expanded blastocysts were examined for morphological quality of the embryos by Hoechst and propidium iodide staining and for the expression of ErbB1 and ErbB4 by Real-time PCR and immunocytochemistry. The expression of the Sirt3 gene was also assayed. Furthermore, intracellular reactive oxygen species (ROS) levels and the total antioxidant capacity (TAC) were examined by DCFH-DA fluorescence intensity and radical cation respectively.The number of cells in the inner cell mass (ICM) and outer cell mass (OCM) were elevated significantly in the Melatonin-treated group suggesting increased viability and proliferation. Furthermore, we found that melatonin significantly increased the expression levels of ErbB1, ErbB4, and Sirt3 genes, and the protein expression of ErbB1, ErbB4 correlated with intracellular ROS levels and TAC significantly increased after melatonin treatment. Together, these results demonstrate that melatonin could be helpful to improve preimplantation embryos through its effects in decreasing ROS levels and increasing expression of implantation-related genes.

Compound of Cynanchum wilfordii and Humulus lupulus L. Ameliorates Menopausal Symptoms in Ovariectomized Mice.

Cynanchum wilfordii and Humulus lupulus L. have been used for their various pharmacological properties in South Korea as a traditional medicine or health functional food, respectively, and their intake may relieve menopausal symptoms. The purpose of current study was to determine the effect of compound of Cynanchum wilfordii and Humulus lupulus L. (CWHL) in menopausal symptoms of ovariectomized (OVX) mice. OVX mice received CWHL or caudatin (an active ingredient of CWHL) once daily for 7weeks. Values for hypothalamic serotonin (5-HT), dopamine, norepinephrine, estrogen receptor (ER)-β, 5-HT1A, and 5-HT2A were significantly enhanced, while value for hypothalamic monoamine oxidase A was reduced in CWHL and caudatin groups compared with the OVX group. CWHL and caudatin significantly reduced tail skin temperature and rectal temperature of OVX mice through partial recovering of the levels of serum estrogen, nitric oxide, follicle-stimulating hormone, luteinizing hormone, and receptor-activator of the NF-κB ligand (RANKL). Moreover, CWHL and caudatin improved bone mineral density via decreasing levels of serum RANKL, tartrate-resistant acid phosphatase, and collagen type 1 cross-linked N-telopeptide and improving levels of serum alkaline phosphatase, osteoprotegerin, and osteocalcin compared with the OVX group without adverse effects such as dyslipidemia. CWHL increased uterine ER-β levels but did not change uterus and vaginal weights. Taken together, the results indicate that CWHL may relieve menopausal symptoms by controlling depression-, hot flashes-, and osteoporosis-associated biomarkers. Therefore, we propose that CWHL might be a safe and potential candidate for management of menopause as a health functional food.

Safety and Outcomes of a Plasmid DNA Vaccine Encoding the ERBB2 Intracellular Domain in Patients With Advanced-Stage ERBB2-Positive Breast Cancer

High levels of ERBB2 (formerly HER2)-specific type 1 T cells in the peripheral blood are associated with favorable clinical outcomes after trastuzumab therapy; however, only a minority of patients develop measurable ERBB2 immunity after treatment. Vaccines designed to increase ERBB2-specific T-helper cells could induce ERBB2 immunity in a majority of patients. To determine the safety and immunogenicity of 3 doses (10, 100, and 500 μg) of a plasmid-based vaccine encoding the ERBB2 intracellular domain (ICD). Single-arm phase 1 trial including 66 patients with advanced-stage ERBB2-positive breast cancer treated in an academic medical center between 2001 and 2010 with 10-year postvaccine toxicity assessments. Data analysis was performed over 2 periods: January 2012 to March 2013 and July 2021 to August 2022. Patients were sequentially enrolled to the 3 dose arms. The vaccine was administered intradermally once a month with soluble granulocyte-macrophage colony-stimulating factor as an adjuvant for 3 immunizations. Toxicity evaluations occurred at set intervals and yearly. Peripheral blood mononuclear cells were collected for evaluation of immunity. Biopsy of vaccine sites at weeks 16 and 36 measured DNA persistence. Safety was graded by Common Terminology Criteria for Adverse Events, version 3.0, and ERBB2 ICD immune responses were measured by interferon-γ enzyme-linked immunosorbent spot. Secondary objectives determined if vaccine dose was associated with immunity and evaluated persistence of plasmid DNA at the vaccine site. A total of 66 patients (median [range] age, 51 [34-77] years) were enrolled. The majority of vaccine-related toxic effects were grade 1 and 2 and not significantly different between dose arms. Patients in arm 2 (100 μg) and arm 3 (500 μg) had higher magnitude ERBB2 ICD type 1 immune responses at most time points than arm 1 (10 μg) (arm 2 compared with arm 1, coefficient, 181 [95% CI, 60-303]; P = .003; arm 3 compared with arm 1, coefficient, 233 [95% CI, 102-363]; P < .001) after adjusting for baseline factors. ERBB2 ICD immunity at time points after the end of immunizations was significantly lower on average in patients with DNA persistence at week 16 compared with those without persistence. The highest vaccine dose was associated with the greatest incidence of persistent DNA at the injection site. In this phase 1 nonrandomized clinical trial, immunization with the 100-μg dose of the ERBB2 ICD plasmid-based vaccine was associated with generation of ERBB2-specific type 1 T cells in most patients with ERBB2-expressing breast cancer, and it is currently being evaluated in randomized phase 2 trials. ClinicalTrials.gov Identifier: NCT00436254.

Alteration in Neuregulin 1/ERbB4 in Absence Epilepsy: Regulatory Effect on TRPV1 Expression.

The footprint of Neuregulin 1 (NRG1) / ERbB4 in the pathophysiology of some neurological disorders and TRPV1 regulation has been indicated. The alterations in NRG1 and ErbB4 as well as the TRPV1 signaling pathway were investigated during the development of absence epilepsy in the genetic animal model of absence epilepsy. Male WAG/Rij and Wistar rats were divided into four experimental groups of two and six months of age. The protein levels of NRG1, ERbB4, and TRPV1 were measured in the somatosensory cortex and hippocampus. The cortical protein levels of NRG1 and ErbB4 in the 6-month-old WAG/Rij rats were lower than in Wistar rats. Protein levels of TRPV1 were lower in two- and six-month-old WAG/Rij rats compared to age-matched Wistar rats.Hippocampal protein levels of NRG1 in 6-month-old WAG/Rij rats were lower than two-month-old WAG/Rij rats. Low levels of ErbB4 protein in two-month-old and high levels in six-month-old WAG/Rij rats were found compared to Wistar rats. Protein levels of TRPV1 were lower in the two-month-old and higher in the six-month-old WAG/Rij rats compared to age-matched Wistar rats.Furthermore, a high correlation between NRG1/ERbB4 and TRPV1 expressions in the cortex and hippocampus was indicated. The expression of NRG1/ERbB4 and TRPV1 followed a similar pattern during the life span of Wistar and WAG/Rij rats. Our findings indicated the potential role of the NRG1/ErbB4 pathway as well as TRPV1 in the pathogenesis of absence epilepsy. The regulatory effect of the ERbB4 receptor on the TRPV1 expression has been suggested following the similar pattern of expression.

Satellite Noncoding RNAs (ncRNA) as Cancer Biomarkers? New Insights from FA-SAT ncRNA Molecular and Clinical Profiles in Feline Mammary Tumors.

Satellite noncoding RNAs (ncRNAs) are a new frontier of cancer biology research and biomarkers. While the knowledge on ncRNAs in human cancers is still limited, studies in other species can be informative to guide future translational research and development for cancer molecular targets and diagnostics. In this context, FA-SAT is the major satellite DNA of the cat genome, which is also present in humans, being transcribed in both species. In this study, we report new insights on FA-SAT (DNA and RNA) profile in feline mammary tumors, using disease-free tissues from the same animals as reference. We quantified the FA-SAT DNA and RNA levels (long and small transcripts) by real-time quantitative polymerase chain reaction (qPCR) and RT-qPCR. The comparison of the FA-SAT DNA and RNA levels with clinicopathological parameters revealed several associations, such as (1) the FA-SAT DNA levels' positive relation with lymphovascular invasion, (2) the FA-SAT long RNA negative correlation with Ki-67 index, and its positive association with Estrogen Receptor status, and (3) the FA-SAT small RNA level positive correlation with tumor size and skin ulceration. Also, FA-SAT long RNA is correlated with ERBB2 and c-MYC RNA levels. These data collectively suggest that FA-SAT ncRNA offers prospects as a potential cancer biomarker in cats. Further studies in humans are also needed to decipher the emerging role of ncRNAs in cancer biology and precision medicine fields. This work brings new information on the relation of FA-SAT ncRNAs with the oncogenic process, uncovering a new potential cancer biomarker.

Intervention effects of estradiol on myocardial ischemia- reperfusion injury of rat and its mechanisms

Objective: To study the effects of estradiol (E2) on alleviating myocardial ischemia/reperfusion(I/R) injury through estrogen receptorβ(ERβ) mediated extracellular regulated protein kinases(ERK) pathway activation. Methods: Eighty-four adult female SD rats were ovariectomized and randomly divided into control group, NC siRNA adeno-associated virus (AAV) group received sham operation, the myocardial I/R injury model was prepared by ligation of the left anterior descending coronary artery in I/R group, E2+I/R group, NC siRNA AAV+I/R group, NC siRNA AAV+E2+I/R group and ERβ-siRNA AAV+E2+I/R group. E2+I/R group, NC siRNA AAV+E2+I/R group and ERβ-siRNA AAV+E2+I/R group were treated with E2 0.8 mg/kg by gavage for 60 days before modeling. NC siRNA AAV+I/R group, NC siRNA AAV+E2+I/R group, and ERβ-siRNA AAV+E2+I/R group were treated with AAV by caudal vein injection 24 h before modeling. After 120 min of reperfusion, the contents of serum lactate dehydrogenase (LDH), phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), myocardial infarction area and the expressions of ERβ, p-ERK, the contents of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1 β), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in myocardium were measured. Results: The contents of serum LDH, CK, CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β, MDA in myocardium of I/R group were higher than those of the control group, the expression levels of ERβ and p-ERK and the content of T-AOC were lower than those in the control group (P＜0.05). The contents of serum LDH, CK and CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β and MDA in myocardium of E2+I/R group were lower than those of the I/R group, the expression levels of ERβ and p-ERK and the content of T-AOC were higher than those of the I/R group(P＜0.05). After knockdown ERβ by caudal vein injection of ERβ-siRNA AAV, the contents of serum LDH, CK and CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β and MDA in myocardium of ERβ-siRNA AAV+E2+I/R group were higher than those of NC-siRNA AAV+E2+I/R, the expression levels of ERβ and p-ERK and the content of T-AOC were lower than those of NC-siRNA AAV+E2+I/R(P＜0.05). Conclusion: E2 has protective effects on myocardial I / R injury in ovariectomized rats, which are related to the promotion of ERβ mediating the activation of ERK pathway, reducing inflammatory and oxidative stress responses.

Cerulenin suppresses ErbB2-overexpressing breast cancer by targeting ErbB2/PKM2 pathway.

Cerulenin is a fungal metabolite and a specific inhibitor of fatty acid synthase (FASN), which has shown a potential anticancer activity. 20-25% of breast cancer patients with ErbB2-overexpressing develop resistance to treatment. Therefore, it is urgent to find an effective new target for the treatment of ErbB2-overexpressing breast cancer. Our previous study found that cerulenin inhibits the glycolysis and migration of SK-BR-3 cells, but the effect of cerulenin on other malignant phenotypes of breast cancer is still unknown. Furthermore, the mechanism by which cerulenin displays its inhibitory effects is not fully understood. In this study, we systematically investigate the inhibitory effects of cerulenin on proliferation, migration, invasion and glycolysis of ErbB2-overexpressing breast cancer cells and its molecular mechanism. We found that cerulenin obviously suppresses the proliferation, migration, invasion as well as glycolysis. Through bioinformatic analyses, we found that PKM2 might be a target of cerulenin. In addition, ErbB2 and its signaling pathway upregulated PKM2 protein levels. Furthermore, we demonstrated that cerulenin downregulated the protein levels of ErbB2, PKM2 and EMT markers (MMP9, MMP2 and Snail2) in a dose- and time-dependent manner. Finally, the inhibitory of cerulenin on colony formation, migration, invasion and glycolysis, as well as protein levels of EMT markers were rescued by replenishing with PKM2. These findings illustrated that cerulenin inhibits proliferation, migration, invasion and glycolysis by targeting ErbB2/PKM2 pathway in ErbB2-overexpressing breast cancer cells.

Chaigui granule exerts anti-depressant effects by regulating the synthesis of Estradiol and the downstream of CYP19A1-E2-ERKs signaling pathway in CUMS-induced depressed rats

Background: There is a significant gender difference in the prevalence of depression. Recent studies have shown that estrogen plays a crucial role in depression. Therefore, studying the specific mechanism of estrogen's role in depression can provide new ideas to address the treatment of depression. Chaigui granule has been shown to have exact antidepressant efficacy, and the contents of saikosaponin (a, b1, b2, d) and paeoniflorin in Chaigui granule are about 0.737% and 0.641%, respectively. Some studies have found that they can improve depression-induced decrease in testosterone (T) levels (∼36.99% decrease compared to control). However, whether Chaigui granule can exert antidepressant efficacy by regulating estrogen is still unclear. This study aimed to elucidate the regulation of estrogen levels by Chaigui granule and the underlying mechanism of its anti-depressant effect. Methods: Eighty-four male Sprague-Dawley (SD) rats were modeled using a chronic unpredictable mild stress (CUMS) procedure. The administration method was traditional oral gavage administration, and behavioral indicators were used to evaluate the anti-depressant effect of Chaigui granule. Enzyme-linked immunosorbent assay (ELISA) was adopted to assess the modulating impact of Chaigui granule on sex hormones. Then, reverse transcription-quantitative PCR (RT-qPCR), and Western blot (WB) techniques were employed to detect extracellular regulated protein kinases (ERK) signaling-related molecules downstream of estradiol in the hippocampus tissue. Results: The administration of Chaigui granule significantly alleviated the desperate behavior of CUMS-induced depressed rats. According to the results, we found that Chaigui granule could upregulate the level of estradiol (E2) in the serum (∼46.56% increase compared to model) and hippocampus (∼26.03% increase compared to model) of CUMS rats and increase the levels of CYP19A1 gene and protein, which was the key enzyme regulating the synthesis of T into E2 in the hippocampus. Chaigui granule was also found to have a significant back-regulatory effect on the gene and protein levels of ERβ, ERK1, and ERK2. Conclusion: Chaigui granule can increase the synthesis of E2 in the hippocampus of CUMS-induced depressed rats and further exert antidepressant effects by activating the CYP19A1-E2-ERKs signaling pathway.

Loss of HOXA10 causes endometrial hyperplasia progressing to endometrial cancer.

Endometrial cancer is the fourth most common malignancy in women and the precursor lesion is endometrial hyperplasia. HOXA10 is a transcription factor that plays key roles in endometrial functions such as the endowment of receptivity, embryo implantation, and trophoblast invasion. Herein, using testicular transgenesis, we developed transgenic mice that expressed a shRNA against HOXA10 and there was a nearly 70% reduction in the expression of HOXA10 in these animals. We observed that downregulation of HOXA10 led to the development of endometrial hyperplasia in the young animals (3 months), and as they aged (>1 year), most animals developed well-differentiated endometrial adenocarcinoma. In the endometrium of animals with reduced HOXA10, there was increased proliferation and elevated levels of ERα and ERβ. In parallel, there was increased expression of Wnt4 and β-Catenin, SOX9, and YAP1. We propose that chronic reduction in HOXA10 expression disrupts multiple pathways in the uterus that aids in the development of endometrial hyperplasia which progresses to endometrial cancer with age.

Paeoniflorin exhibits antidepressant activity in rats with postpartum depression via the TSPO and BDNF‑mTOR pathways.

Postpartum depression (PPD) is the most common type of puerperal mental syndrome and affects maternal physical and mental health and even the growth and development of infants. Paeoniflorin exerts a potential antidepressive effect; however, the functional roles and potential mechanisms of paeoniflorin in PPD are still largely unknown. PPD rat models were prepared by withdrawing hormone‑simulated pregnancy (HSP), and subjects were treated with paeoniflorin and fluoxetine or plasmids. The sucrose preference test (SPT), forced swimming test (FST) and tail suspension test (TST) were used to monitor depression‑like behavior in rats. A radioimmunoassay was utilized for estradiol (E2) and progesterone (P) measurements. ELISA was performed to detect serum corticosterone (Cor), hippocampal allopregnanolone (Allo), IL‑1β and TNF‑α levels. Expression of the E2 receptors ERα and ERβ was detected by qPCR. Western blotting was used to detect TSPO, BDNF and mTOR phosphorylation. Paeoniflorin drastically increased the sucrose preference of rats while decreasing the immobility time in the FST and TST in PPD models. Moreover, paeoniflorin intervention upregulated serum E2, hippocampal Allo, ERα, and ERβ levels but degraded P, serum Cor, IL‑1β, TNF‑α and ERα/ERβ levels. Mechanistically, paeoniflorin promoted TSPO and BDNF‑mTOR pathway activation in PPD rats. Furthermore, suppression of TSPO or the BDNF‑mTOR pathway partially reversed the effects of paeoniflorin on depression‑like behaviors, hormone levels, and inflammatory cytokine release. Paeoniflorin may improve symptoms of PPD by regulating the TSPO and BDNF‑mTOR pathways, indicating that paeoniflorin may be an effective anti‑PPD and antidepressant drug, providing evidence for the future treatment of PPD.

High concentration of estradiol has a negative correlation with free thyroxine during the second trimester of pregnancy.

ObjectiveTo explore the relationship between estradiol (E2) and thyroid function during the second trimester of pregnancy and the effect of E2 on sodium iodide transporter (NIS) expression in cultured thyroid cells.Materials and methodsWe analyzed relationships between E2 and thyroid function in 196 pregnant women during the second trimester. Multiple linear regression analysis was performed between E2 and thyroid function. The human thyroid Nthy-ori3-1 cells were cultured in different E2 concentrations, and the mRNA levels of NIS, estrogen receptor (ER)-α, and ER-β were measured by quantitative real-time PCR. Their protein levels were assessed by western blot.ResultsE2 was positively correlated with thyroid-stimulating hormone (TSH) and negatively correlated with free thyroxine (FT4) (P < 0.05). When we corrected for age, BMI, alanine aminotransferase, and serum creatinine, E2 was still negatively correlated with FT4 (P < 0.5) during the second trimester. In Nthy-ori3-1 cells treated with 10 nM E2, NIS and ER-β mRNA levels were significantly reduced, while ER-α mRNA level was not altered (P > 0.5). Moreover, 10 nM E2 significantly decreased protein levels of ER-β, phosphorylated versions of protein kinase A (p-PKA), phosphorylated versions of cAMP response element-binding protein (p-CREB), and NIS, while treatment with the ER-β inhibitor restored the expression of p-PKA, p-CREB, and NIS (P < 0.05).ConclusionHigh concentration of E2 has a negative correlation with FT4. High concentration of E2 can inhibit the NIS expression through the ER-β-mediated pathway, which may cause thyroid hormone fluctuations during pregnancy.

Expression of Sex Hormone Receptors in Canine Osteosarcoma.

Simple SummaryOsteosarcoma is a bone tumor that arises from normal bone cells that become malignant. This is a disease that is relatively common in dogs and also affects humans. Dogs are a useful model for human osteosarcoma as they share many similarities including genetic changes, manner of spread and how they respond to treatment. In dogs, the tumors are frequently seen in large breeds and may be caused by environmental and genetic factors. Sex steroids have been shown to regulate bone metabolism directly and indirectly through receptors on bone. The aim of this study was to investigate the expression levels of sex hormone receptors in canine osteosarcoma tissue and cell lines. Our results demonstrate varying levels of receptors in tissue and a high expression of progesterone receptors in the canine osteosarcoma cell lines. Low oxygen levels further increased progesterone receptor expression. Lastly, estradiol decreased the expression of progesterone receptor in one of the cell lines, and progesterone decreased cell proliferation in the same cell line. Further investigation is recommended to determine whether or not sex steroid receptor levels and sensitivity to hormones are related.Sex steroids regulate bone metabolism directly and indirectly through receptors on bone. Estrogen receptors (ER-∝, ER-β), progesterone receptor (PR), and androgen receptor (AR), have been previously identified on human osteosarcoma (OSA) cells, and are considered to influence tumor growth, but their expression and role in canine OSA is unknown. The aim of this study was to characterize sex hormone receptor expression levels in naturally occurring OSA tissue and in three canine OSA cell lines. The expression of ER-α, ER-β, PR, and AR was investigated using RT-PCR. PR expression levels were also quantified in OSA cells cultured under hypoxic conditions or in the presence of estradiol. The effects of progesterone on cell proliferation were quantified. Results demonstrated varying expression levels of these receptors in five OSA subtypes. OSA cell lines demonstrated high gene expression levels of PR and low gene expression levels of ER-α and ER-β and no gene expression of AR. PR expression was increased in OSA cells cultured under hypoxic conditions in a HIF-∝ independent manner. Interestingly, one cell line expressed very high levels of PR, expression of which decreased in response to estradiol. In addition, progesterone decreased OSA cell proliferation in this particular cell line. Further investigation of the role of sex steroids, particularly PR and its ligands, in regulation of canine OSA is recommended.

Effects of maternal deprivation and environmental enrichment on anxiety-like and depression-like behaviors correlate with oxytocin system and CRH level in the medial-lateral habenula

The medial-lateral habenula (LHbM)’s role in anxiety and depression behaviors in female mice remains unclear. Here, we used neonatal maternal deprivation (MD) and post-weaning environmental enrichment (EE) to treat female BALB/c offspring and checked anxiety-like and depression-like behaviors as well as the corticotropin-releasing hormone (CRH), oxytocin receptor (OTR), estrogen receptor-beta (ERβ) levels in their LHbM at adulthood. We found that MD enhanced state anxiety-like behaviors in the elevated plus-maze test, and EE caused trait anxiety-like behaviors in the open field test and depression-like behaviors in the tail suspension test. The immunochemistry showed that MD reduced OT immunoreactive neuron numbers in the hypothalamic paraventricular nucleus but increased OTR levels in the LHbM; EE increased CRH levels in the LHbM but decreased OTR levels in the LHbM. The additive effects of EE and MD maintained the behavioral parameters, OT-ir neuronal numbers, CRH levels, and OTR levels similar to the additive of non-MD and non-EE. The correlation analysis showed that CRH levels correlated with synaptic connection levels, OTR levels correlated with nucleus densities, and ERβ levels correlated with Nissl body levels and body weights in female mice. Neither MD nor EE affected ERβ levels in the LHbM. Together, the study revealed the relationships between behaviors and neuroendocrine and neuronal alterations in female LHbM and the effects of experiences including MD and EE on them.

140MO HER2DX genomic test in HER2-positive/hormone receptor-positive (HER2+/HR+) breast cancer (BC) treated with neoadjuvant trastuzumab (T) and pertuzumab (P): A correlative analysis from the PerELISA trial

HER2DX is a prognostic and predictive assay in early-stage HER2+ BC based on clinical data and the expression of 4 gene signatures (immune, proliferation, luminal differentiation and HER2 amplicon), including ERBB2 levels. Here, we evaluated the ability of HER2DX to predict efficacy of a de-escalated, chemotherapy (CT)-free neoadjuvant regimen in HER2+/HR+ BC.

Mechanism of Key Ingredient of Astragalus membranaceus on Lung Adenocarcinoma via PI3K/AKT Signaling Clarified by Utilizing Network Pharmacology Approach and Experimental Validation.

To investigate the mechanism of the effect of Astragalus membranaceus (A. membranaceus) on lung adenocarcinoma at the molecular level to elucidate the specific targets according to the network pharmacology approach. The active components of A. membranaceus and their potential targets were collected from the Traditional Chinese Medicine Systems Pharmacology Database. Lung adenocarcinoma-associated genes were acquired based on GeneCards, Online Mendelian Inheritance in Man (OMIM), PharmGKB, and Therapeutic Targets databases. The PI3K/AKT signaling pathway-related genes were obtained using Reactome portal. Networks of "ingredient-target" and "ingredient-target-pathway-disease" were constructed using the Cytoscape3.6.0 software. The relationships among targets were analyzed according protein-protein interaction (PPI) network. Finally, molecular docking was applied to construct the binding conformation between active ingredients and core targets. Cell counting kit 8 (CCK8) and Western blot assays were performed to determine the mechanism of the key ingredient of A. membranaceus. A total of 20 active components and their 329 targets, and 7,501 lung adenocarcinoma-related genes and 130 PI3K/AKT signaling pathway-related genes were obtained. According to Venn diagram and PPI network analysis, 2 mainly active ingredients, including kaempferol and quercetin, and 6 core targets, including TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR, were identified. The two important active ingredients of A. membranaceus, kaempferol and quercetin, exert the therapeutic effect in lung adenocarcinoma partly by acting on the 6 core targets (TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR) of PI3K/AKT signaling pathway. Expressions of potential targets in lung adenocarcinoma and normal samples were analyzed by using UALCAN portal and found that ERBB2 was overexpressed in lung adenocarcinoma tissues and upregulation of it correlated with clinicopathological characteristics. Finally, quercetin repressed viabilities of lung adenocarcinoma cells by targeting ERBB2 on PI3K/AKT signaling confirmed by CCK8 and Western blot. Our finding unraveled that an active ingredient of A. membranaceus, quercetin, significantly inhibited the lung adenocarcinoma cells proliferation by repressing ERBB2 level and inactivating the PI3K/AKT signaling pathway.

Prenatal di-(2-ethylhexyl) phthalate exposure induced myocardial cytotoxicity via the regulation of the NRG1-dependent ErbB2/ErbB4-PI3K/AKT signaling pathway in fetal mice

Environmental sanitation of maternal contact during pregnancy is extremely important for the development of different fetal tissues and organs. In particular, during early pregnancy, any adverse exposure may cause abnormal fetal growth or inhibit the development of embryogenic organs. The potential risks of phthalate exposure, which affects the development of humans and animals, are becoming a serious concern worldwide. However, the specific molecular mechanism of di-(2-ethylhexyl) phthalate (DEHP)-induced cardiotoxicity in fetal mice remains unclear. In this study, animal models of DEHP gavage at concentrations of 250, 500, and 1000 mg/kg/day within 8.5–18.5 days of pregnancy were established. The cell proliferation, survival, and apoptosis rates were evaluated using CCK8, EdU, TUNEL and flow cytometry. The molecular mechanism was assessed via transcriptome sequencing, immunohistochemistry, immunofluorescence, reverse transcription-quantitative polymerase chain reaction, and Western blot analysis. In vivo, DEHP increased apoptosis, decreased Ki67 and CD31 expression, reduced heart weight and area, slowed down myocardial sarcomere development, and caused cardiac septal defect in fetal mice heart. Transcriptome sequencing showed that DEHP decreased NRG1 expression and downregulated the ErbB2/ErbB4-PI3K/AKT signaling pathway-related target genes. In vitro, primary cardiomyocytes were cultured with DEHP at a concentration of 150 μg/mL combined with ErbB inhibitor (AG1478, 10 μmol/L) and/or NRG1 protein (100 ng/mL) for 72 h. After DEHP intervention, the expression of NRG1 and the phosphorylation level of ErbB2, ErbB4, PI3K, and AKT decreased, and the apoptosis-related protein levels increased. Moreover, the apoptosis rate increased. After adding exogenous NRG1, the phosphorylation level of the NRG1/ERbB2/ERbB4-PI3K/AKT pathway increased, and the apoptosis-related protein levels decreased. Further, the apoptosis rate reduced. Interestingly, after exposure to DEHP and AG1478 + NRG1, the anti-apoptotic effect of NRG1 and cardiomyocyte proliferation decreased by inhibiting the NRG1/ERbB2/ERbB4-PI3K/AKT pathway. Hence, the NRG1-dependent regulation of the ERbB2/ERbB4-PI3K/AKT signaling pathway may be a key mechanism of DEHP-induced myocardial cytotoxicity.

Abstract 819: ERBB4 mediates IL10-induced growth of EGFR-independent colon tumors

Abstract ERBB4 is commonly over-expressed in human CRC, however the utility of ERBB4 as a target for colorectal cancer (CRC) therapeutics is largely unexplored. Our group recently identified a molecular subtype of CRC that arises independent of EGFR and displays a more aggressive growth phenotype than those dependent on EGFR. The robust growth phenotype of EGFR-independent tumors can be in part attributed to upregulation of IL10 signaling. In addition, EGFR-independent tumors display a significant upregulation of Erbb2 and Erbb4 transcripts in both spontaneous and sporadic CRC mouse models, perhaps compensating for the loss of EGFR. To investigate the importance of other ERBB receptors in EGFR-independent CRC tumor progression, we ablated Erbb4 to interrogate its influence on tumor development in vivo using a conditional allele of Erbb4tm1Fej (Erbb4f). ERBB4-deficient ApcMin/+ mice (ApcMin/+, ErbB4f/f, Tg(Vil1-Cre)) were established and used to show that ERBB4 ablation in the intestinal epithelia results in a significant decrease in the number of intestinal and colon tumors. Polyps lacking ERBB4 were also significantly reduced size, contrary to what is observed with loss of EGFR. We also recently developed ERBB2-deficient ApcMin/+ mice, and preliminary results suggest that Erbb2 ablation also results in a decrease in intestinal and colon tumor number and size. Consistent with data from EGFR-independent tumors, transcriptomic analysis of ERBB4-deficient intestinal tumors predicted down-regulation of IL10 signaling, which was validated through Il10 and Socs3 qPCR. To complement our findings, the observed down-regulation of Il10 and Socs3 at the transcript level corresponded with a significant decrease in IL10 levels in the serum of mice harboring ERBB4 deficient tumors when compared to mice without tumors. Furthermore, we found that transcript levels of Erbb4 significantly decreased after anti-IL10 treatment of EGFR-independent tumors in vivo. This illustrates a possible negative feedback loop whereby neutralizing IL10 also leads to a reduction of Erbb4 expression, further reducing IL10 signaling. Taken together, the data suggest that the absence of EGFR triggers Erbb4 upregulation, implicating an important role for ERBB4 in EGFR-independent colon tumor growth. These results suggest that therapeutic targeting of ERBB4 may lead to a reduction in IL10 signaling and reduced intestinal polyp multiplicity and size in EGFR-independent tumors and inform the development of novel approaches to treat CRC that exhibit aberrant expression of EGFR and ERBB4. Citation Format: Michael P. McGill, Carolina Mantilla Rojas, David W. Threadgill. ERBB4 mediates IL10-induced growth of EGFR-independent colon tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 819.