ERβ Expression Research Articles

Benzo[a]pyrene exposure affects colorectal cancer susceptibility by regulating ERβ-mediated LINC02977 transcription

Environmental pollutants known as polycyclic aromatic hydrocarbons (PAHs) are produced through the incomplete combustion of organic material. While PAHs have been investigated as genotoxicants, they can also operate through nongenotoxic pathways in estrogen-dependent malignancies, such as breast, cervical and ovarian cancer. However, whether PAHs induce colorectal cancer (CRC) risk through estrogenic effects is still illusive. Here, we systematically investigated the abnormal expression and activation of estrogen receptor beta (ERβ) regulated by PAHs in CRC as well as the underlying mechanisms of ERβ-mediated CRC risk. Based on the 300 plasma samples from CRC patients and healthy controls detected by GC–MS/MS, we found that the plasma concentrations of benzo[a]pyrene (BaP) were significantly higher in CRC cases than in healthy controls, with significant estrogenic effects. Moreover, histone deacetylase 2 (HDAC2)-induced deacetylation of the promoter decreases ERβ expression, which is associated with poor overall survival and advanced tumor stage. The study also revealed that BaP and estradiol (E2) had different carcinogenic effects, with BaP promoting cell proliferation and inhibiting apoptosis, while E2 had the opposite effects. Additionally, this study mapped ERβ genomic binding regions by performing ChIP-seq and ATAC-seq and identified genetic variants of rs1411680 and its high linkage disequilibrium SNP rs6477937, which were significantly associated with CRC risk through meta-analysis of two independent Chinese population genome-wide association studies comprising 2,248 cases and 3,173 controls and then validation in a large-scale European population. By integrating data from functional genomics, we validated the regulatory effect of rs6477937 as an ERβ binding-disrupting SNP that mediated allele-specific expression of LINC02977 in a long-range chromosomal interaction manner, which was found to be highly expressed in CRC tissues. Overall, this study suggests that the different active effects on ERβ by PAHs and endogenous E2 may play a crucial role in the development and progression of CRC and highlights the potential of targeting ERβ and its downstream targets for CRC prevention and treatment.

Dynamic effects of ventral hippocampal NRG3/ERBB4 signaling on nicotine withdrawal-induced responses

Tobacco smoking remains a leading cause of preventable death in the United States, with approximately a 5% success rate for smokers attempting to quit. High relapse rates have been linked to several genetic factors, indicating that the mechanistic relationship between genes and drugs of abuse is a valuable avenue for the development of novel smoking cessation therapies. For example, various single nucleotide polymorphisms (SNPs) in the gene for neuregulin 3 (NRG3) and its cognate receptor, the receptor tyrosine-protein kinase erbB-4 (ERBB4), have been linked to nicotine addiction. Our lab has previously shown that ERBB4 plays a role in anxiety-like behavior during nicotine withdrawal (WD); however, the neuronal mechanisms and circuit-specific effects of NRG3-ERBB4 signaling during nicotine and WD are unknown. The present study utilizes genetic, biochemical, and functional approaches to examine the anxiety-related behavioral and functional role of NRG3-ERBB4 signaling, specifically in the ventral hippocampus (VH) of male and female mice. We report that 24hWD from nicotine is associated with altered synaptic expression of VH NRG3 and ERBB4, and genetic disruption of VH ErbB4 leads to an elimination of anxiety-like behaviors induced during 24hWD. Moreover, we observed attenuation of GABAergic transmission as well as alterations in Ca2+-dependent network activity in the ventral CA1 area of VH ErbB4 knock-down mice during 24hWD. Our findings further highlight contributions of the NRG3-ERBB4 signaling pathway to anxiety-related behaviors seen during nicotine WD.

In vitro effects of heparin-binding epidermal growth factor on adhesion stage of implantation.

A member of the epidermal growth factor (EGF) family, the heparin-binding EGF (HB-EGF) is expressed in the uteri of both humans and mice during the implantation process. To study the effects of HB-EGF on adhesion stage, we developed an in vitro implantation model employing Ishikawa cell line and JAR cell line, which may attach to Ishikawa cells. For 1, 6, 12, and 24 hours, co-cultures of JAR spheroids grown on Ishikawa monolayers were treated with 1, 10, and 100 ng∕mL doses of HB-EGF. Using immunocytochemistry and Western blot analysis, the effects of HB-EGF on the protein expressions of E-cadherin, Erb-B2 receptor tyrosine kinase 4 (ErbB4), and integrin ανβ3 in Ishikawa and JAR cells were examined semi-quantitatively and quantitatively. Ultrastructural changes of in vitro implantation model were investigated by transmission electron microscopy. We revealed that HB-EGF influenced trophoblast cell adhesion to endometrial cells by upregulating the expression of the proteins ErbB4 and trophoblastic integrin ανβ3. Decrease in trophoblastic E-cadherin expression and increase in endometrial E-cadherin expression were demonstrated accompanying morphological variations in cells required for the invasion. We discovered ultrastructurally that Ishikawa cells acquired uterodome-like appearance, including the organelles, when 10 and 100 ng∕mL dosages of HB-EGF were administered for 12 and 24 hours. However, following additional hours of adhesion and invasion, their intercellular spaces enlarged. The trafficking of vesicular transport was enhanced by JAR spheroids. We therefore discovered that in this implantation paradigm, HB-EGF may enhance the receptivity of Ishikawa cells and the adherence of JAR cells.

Codonopsis lanceolata polysaccharide ameliorates high-fat diet induced-postpartum hypogalactia via stimulating prolactin receptor-mediated Jak2/Stat5 signaling

This study aims to investigate the ameliorative effect of Codonopsis lanceolata polysaccharide (PCL) on mice with hypogalatia induced by a high-fat diet (HFD) and the potential underlying mechanism. We found that oral administration of PCL demonstrated significant benefits in countering the negative effects of HFD, including weight gain, hepatic steatosis, mesenteric adipocyte hypertrophy, and abnormal glucose/lipid metabolism. In addition, PCL improved mammary gland development and enhanced lactogenesis performance. Histologically, PCL ameliorated the retardation of ductal growth, reduced mammary fat pad thickness, improved the incomplete linear encapsulation of luminal epithelium and myoepithelium, and increased the proliferation of mammary epithelial cells. Flow cytometry analysis showed that PCL mitigated the detrimental effects of HFD on mammary gland development by promoting the proliferation and differentiation of mammary epithelial cells. Mechanistic studies revealed that PCL upregulated the levels of prolactin (PRL) and its receptor (PRLR) in the mammary gland, activated JAK2/STAT5 signaling pathway, and increased the expression of p63, ERBB4, and NRG1. Overall, PCL can ameliorate HFD-induced hypogalactia by activating PRLR-mediated JAK2/STAT5 signaling. Our findings offer a methodological and theoretical foundation for investigating the functional constituents of traditional Chinese medicine in the treatment of hypogalactia.

Tonic ErbB signaling underlies TGFβ-induced activation of ERK and is required for lens cell epithelial to myofibroblast transition.

Fibrosis is a major, but incompletely understood, component of many diseases. The most common vision-disrupting complication of cataract surgery involves differentiation of residual lens cells into myofibroblasts. In serum-free primary cultures of lens epithelial cells (DCDMLs), inhibitors of either ERK or of ErbB signaling prevent TGFβ from upregulating both early (fibronectin) and late (αSMA) markers of myofibroblast differentiation. TGFβ stimulates ERK in DCDMLs within 1.5 h. Kinase inhibitors of ErbBs, but not of several other growth factor receptors in lens cells, reduce phospho ERK to below basal levels in the absence or presence of TGFβ. This effect is attributable to constitutive ErbB activity playing a major role in regulating the basal levels pERK. Additional studies support a model in which TGFβ-generated reactive oxygen species serve to indirectly amplify ERK signaling downstream of tonically active ErbBs to mediate myofibroblast differentiation. ERK activity is in turn essential for expression of ErbB1 and ErbB2, major inducers of ERK signaling. By mechanistically linking TGFβ, ErbB, and ERK signaling to myofibroblast differentiation, our data elucidate a new role for ErbBs in fibrosis and reveal a novel mode by which TGFβ directs lens cell fate.

Evodiamine suppresses endometriosis development induced by early EBV exposure through inhibition of ERβ.

Introduction: Endometriosis (EMS) is characterized as a prevalent gynecological inflammatory disorder marked by the existence of endometrial tissues situated beyond the uterus. This condition leads to persistent pelvic pain and may contribute to infertility. In this investigation, we explored the potential mechanism underlying the development of endometriosis (EMS) triggered by transient exposure to either latent membrane protein 1 (LMP1) or Epstein-Barr virus (EBV) in a mouse model. Additionally, we examined the potential inhibitory effect of evodiamine (EDM) on EMS. Methods: Immortalized human endometrial stromal cells (HESC) or epithelial cells (HEEC) were transiently exposed to either EBV or LMP1. The presence of evodiamine (EDM) was assessed for its impact on estrogen receptor β (ERβ) expression, as well as on cell metabolism parameters such as redox balance, mitochondrial function, inflammation, and proliferation. Additionally, a mixture of LMP1-treated HESC and HEEC was administered intraperitoneally to generate an EMS mouse model. Different dosages of EDM were employed for treatment to evaluate its potential suppressive effect on EMS development. Results: Transient exposure to either EBV or LMP1 triggers persistent ERβ expression through epigenetic modifications, subsequently modulating related cell metabolism for EMS development. Furthermore, 4.0µM of EDM can efficiently reverse this effect in in vitro cell culture studies. Additionally, 20mg/kg body weight of EDM treatment can partly suppress EMS development in the in vivo EMS mouse model. Conclusion: Transient EBV/LMP1 exposure triggers permanent ERβ expression, favoring later EMS development, EDM inhibits EMS development through ERβ suppression. This presents a novel mechanism for the development of endometriosis (EMS) in adulthood stemming from early Epstein-Barr virus (EBV) exposure during childhood. Moreover, evodiamine (EDM) stands out as a prospective candidate for treating EMS.

MiR-497-5p Expression and Biological Activity in Gastric Cancer.

Background: This research aims to investigate the expression and biological roles of miR-497-5p in gastric cancer (GC), and its possible mechanisms. Methods: Real Time Quantitative PCR (RT-qPCR) was performed to detect miR-497-5p in GC and normal tissues, as well as GC cell lines versus normal gastric mucosal cells (GES-1). The effects of miR-497-5p overexpression on proliferation were measured by the cell counting kit-8 (CCK8) assay and ethidium bromide (EdU) assay. Flow cytometry was used to assess the cell cycle. The migration and invasion were evaluated by scratch assay and Transwell assay, respectively. Gene targets of miR-497-5p were predicted using "multiMiR" R package combined with mirTarPathway database. And then luciferase reporter experiment was used to evaluate the activity of ERBB2 by miR-497-5p mimics in GC cell line. Besides, functional experiments were performed to verify the impact of miR-497-5p /ERBB2 on phenotypes of GC cells. Results: Compared with the normal tissues and mucosal cells, miR-497-5p was reduced in GC tissues and GC cell lines. miR-497-5p significantly decreased proliferation, migration, and invasion capacity, with an elevated apoptosis ratio of gastric cancer cells. Bioinformatics indicated that ERBB2 might be the potential target of miR-497-5p Dual-luciferase reporter experiments showed it adversely regulated ERBB2 3'UTR luciferase activity. The expression of ERBB2 in GC tissues and cells is significantly higher compared to normal tissues and cells. Over-expression of ERBB2 in gastric cancer cells significantly reduced miR-497-5p's inhibitory effect on the malignant behavior of GC cells. Conclusion: miR-497-5p was significantly down-regulated in GC tissues and cells, which inhibited the malignant features of GC cells by targeting ERBB2.

Overexpression of estrogen receptor beta and its prognostic significance among malignant pleural mesothelioma patients

Abstract Background The overexpression of estrogen receptor beta (ERβ) and its correlation with prognosis of malignant mesothelioma remains controversial and unexplored. This study aimed at studying ERβ expression in tumour tissue in patients with pleural mesothelioma and its association with clinical and pathological factors and survival analysis. Material and methods A prospective cohort study was conducted on patients who were diagnosed with malignant pleural mesothelioma (MPM) in Ain Shams university hospitals, the primary end point was to correlate ERβ overexpression that was analysed by immunohistochemistry (IHC) on paraffin tissue blocks with 1-year overall survival (OS) among MPM patients, the secondary endpoint was to correlate ERβ overexpression with 1-year progression free survival (PFS) and response to platinum-based chemotherapy among MPM patients. Results A total 42 MPM patients were included, High ERβ expression (modified Allred score 7-9) was reported in 9.5% of the patients (4 cases), moderate expression (modified Allred score 4-6) was reported in 28.6% of the patients(12 cases), weak and negative expression ( modified Allred score 0-3) was reported in 61% of the patients(26 cases), percent of ERβ positive cases was 69% of the total patients, there was no statistical difference as regard age (p value:0.8), sex(p value:0.4), stage (p value: 0.71), ECOG (p value :0.84) , histological subtype(p value:0.17) and response to platinum based treatment (p value:0.5) across the 3 groups of patients (high, moderate , weak and negative ER beta on tumour tissues).there was no statistically significant difference in OS and PFS for ERβ overexpression. Conclusion The overexpression of ERβ was not associated with improved OS or PFS in mesothelioma patients, despite high rate of ERβ positive cases in our analysis, larger sample size would be recommended for more solid conclusions

CircPVT1 Inhibits the Proliferation and Aids in Prediction of the Prognosis of Bladder Cancer.

Circular RNA PVT1 (circPVT1) is aberrantly expressed in several cancers, but its functional role and clinical relevance in bladder urothelial carcinoma (BLCA) remain unknown. This study aimed to identify the expression level of circPVT1 in BLCA and investigated its functional relevance with BLCA progression both in vitro and in vivo. GEPIA, UALCAN, and OncoLnc were referred to presented data. Quantitative real-time PCR (qPCR) was used for the measurement of transnational expression of genes in BLCA specimens and cell lines. Immunohistochemistry (IHC) and fluorescence in situ hybridization analysis (FISH) assays were performedto detect HER2 amplification, Pearson's correlation analysis to analyze the correlation between circPVT1 expression and clinical characteristics, Cox regression and K-M survival analyses to analyze prognostic factors. A nomogram was constructed for predicting prognosis. The proliferation of cells was measured by CCK-8 and colony formation assay, and the proliferation in vivo was evaluated using nude mouse models. qPCR was used to detect the expression of proliferation-related genes. circPVT1 was but mRNA PVT1 was not significantly overexpressed in BLCA. A high circPVT1 expression was associated with a better survival and negative HER2, but not with age, gender, and T stage. circPVT1 was an independent prognostic factor for the overall survival of BLCA patients. Knocking down circPVT1 promoted BLCA proliferation in vitro and in vivo. Knocking down circPVT1 upregulated ERBB2, MKI67, and PCNA expression and downregulated TP53 expression, but exerted no influence on CCND1 and CCNB1 expression. circPVT1 is a tumor suppressor and novel prognostic biomarker for BLCA.

Involvement of microRNA-4680-3p against phenytoin-induced cell proliferation inhibition in human palate cells.

Cleft palate (CP) is one of the most common birth defects and is caused by a combination of genetic and/or environmental factors. Environmental factors such as pharmaceutical exposure in pregnant women are known to induce CP. Recently, microRNA (miRNA) was found to be affected by environmental factors. The aim of the present study was to investigate the involvement of miRNA against phenytoin (PHE)-induced inhibition of proliferation in human embryonic palatal mesenchymal (HEPM) cells. We demonstrated that PHE inhibited HEPM cell proliferation in a dose-dependent manner. We found that treatment with PHE downregulated cyclin-D1 and cyclin-E expressions in HEPM cells. Furthermore, PHE increased miR-4680-3p expression and decreased two downstream genes (ERBB2 and JADE1). Importantly, an miR-4680-3p-specific inhibitor restored HEPM cell proliferation and altered expression of ERBB2 and JADE1 in cells treated with PHE. These results suggest that PHE suppresses cell proliferation via modulation of miR-4680-3p expression.

Parvalbumin interneurons dysfunction is potentially associated with FαMNs decrease and NRG1-ErbB4 signaling inhibition in spinal cord in amyotrophic lateral sclerosis.

To investigate the alteration of PV interneurons in ALS mainly focusing its dynamic changes and its relationship with motor neurons and ErbB4 signaling. SOD1G93A mice were used as ALS model. ALS animals were divided into different groups according to birth age: symptomatic prophase (50~60 days), symptomatic phase (90~100 days), and symptomatic progression (130~140 days). Immunofluorescence was performed for measurement of PV-positive interneurons, MMP-9, ChAT, NeuN and ErbB4. RT-qPCR and western blot were used to determine the expression of PV and MMP-9. PV expression was remarkably higher in the anterior horn of gray matter compared with posterior horn and area in the middle of gray matter in control mice. In ALS mice, PV, MMP-9 and ErbB4 levels were gradually decreased along with onset. PV, MMP-9 and ErbB4 levels in ALS mice were significantly down-regulated than control mice after onset, indicating the alteration of PV interneurons, FαMNs and ErbB4. SαMNs levels only decreased remarkably at symptomatic progression in ALS mice compared with control mice, while γMNs levels showed no significant change during whole period in all mice. MMP-9 and ErbB4 were positively correlated with PV. NRG1 treatment significantly enhanced the expression of ErBb4, PV and MMP-9 in ALS mice. PV interneurons decrease is along with FαMNs and ErbB4 decrease in ALS mice.

RETRACTED: The novel antibody fusion protein rhNRG1-HER3i promotes heart regeneration by enhancing NRG1-ERBB4 signaling pathway

Stimulating cardiomyocyte proliferation in the adult heart has emerged as a promising strategy for cardiac regeneration following myocardial infarction (MI). The NRG1-ERBB4 signaling pathway has been implicated in the regulation of cardiomyocyte proliferation. However, the therapeutic potential of recombinant human NRG1 (rhNRG1) has been limited due to the low expression of ERBB4 in adult cardiomyocytes. Here, we investigated whether a fusion protein of rhNRG1 and an ERBB3 inhibitor (rhNRG1-HER3i) could enhance the affinity of NRG1 for ERBB4 and promote adult cardiomyocyte proliferation. In vitro and in vivo experiments were conducted using postnatal day 1 (P1), P7, and adult cardiomyocytes. Western blot analysis was performed to assess the expression and activity of ERBB4. Cardiomyocyte proliferation was evaluated using Ki67 and pH 3 immunostaining, while fibrosis was assessed using Masson staining. Our results indicate that rhNRG1-HER3i, but not rhNRG1, promoted P7 and adult cardiomyocyte proliferation. Furthermore, rhNRG1-HER3i improved cardiac function and reduced cardiac fibrosis in post-MI hearts. Administration of rhNRG1-HER3i inhibited ERBB3 phosphorylation while increasing ERBB4 phosphorylation in adult mouse hearts. Additionally, rhNRG1-HER3i enhanced angiogenesis following MI compared to rhNRG1. In conclusion, our findings suggest that rhNRG1-HER3i is a viable therapeutic approach for promoting adult cardiomyocyte proliferation and treating MI by enhancing NRG1-ERBB4 signaling pathway.

Ameliorative effects of androstenediol against acetic acid-induced colitis in male wistar rats via inhibiting TLR4-mediated PI3K/Akt and NF-κB pathways through estrogen receptor β activation

5-androstenediol (ADIOL) functions as a selective estrogen receptor β (ERβ) ligand with a protective effect against many diseases. So, we conducted a novel insight into its role in acetic acid (AA)-induced colitis and investigated its effect on TLR4-Mediated PI3K/Akt and NF-κB Pathways and the potential role of ERβ as contributing mechanisms. Methods: Rats were randomized into 5 Groups; Control, Colitis, Colitis + mesalazine (MLZ), Colitis + ADIOL, and Colitis + ADIOL + PHTPP (ER-β antagonist). The colitis was induced through a rectal enema of acetic acid (AA) on the 8th day. At the end of treatment, colons were collected for macroscopic assessment. Tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), nuclear factor kappa b (NF-κB), toll-like receptor (TLR4), and phosphorylated Protein kinase B (pAKT) were measured. Besides, Gene expression of interleukin-1beta (IL-1β), metalloproteases 9 (Mmp9), inositol 3 phosphate kinase (PI3K), Neutrophil gelatinase-associated lipocalin (NGAL), ERβ and NLRP6 were assessed. Histopathological and immunohistochemical studies were also investigated. Results: Compared to the untreated AA group, the disease activity index (DAI) and macroscopic assessment indicators significantly decreased with ADIOL injections. Indeed, ADIOL significantly decreased colonic tissue levels of MDA, TLR4, pAKT, and NF-κB immunostainig while increased SOD activity and β catenin immunostainig. ADIOL mitigated the high genetic expressions of IL1β, NGAL, MMP9, and PI3K while increased ERβ and NLRP6 gene expression. Also, the pathological changes detected in AA groups were markedly ameliorated with ADIOL. The specific ERβ antagonist, PHTPP, largely diminished these protective effects of ADIOL. Conclusion: ADIOL could be beneficial against AA-induced colitis mostly through activating ERβ.

Anti-inflammatory effects of neuregulin-1 in HaCaT keratinocytes and atopic dermatitis-like mice stimulated with Der p 38

Neuregulin (NRG)-1 plays fundamental roles in several organ systems after binding to its receptors, ErbB2 and ErbB4. This study examines the role of NRG-1 in atopic dermatitis (AD), a chronic skin disease that causes dryness, pruritus, and inflammation. In mice administered Der p 38, the skin presents AD-like symptoms including filaggrin downregulation and infiltration of neutrophils and eosinophils. Noticeably, there is an increased expression of NRG-1, ErbB2, and ErbB4 in the skin. Upregulation of these proteins is significantly correlated to the clinical skin severity score. In human keratinocyte HaCaT cells, exposure to Der p 38 decreased filaggrin expression, and NRG-1 alone had no effect on the expression. However, co-treatment of Der p 38 with NRG-1 enhanced the filaggrin expression decreased by Der p 38. Pre-treatment with AG879 (an ErbB2 inhibitor) or ErbB4 siRNA blocked the recovery of filaggrin expression in the cells after co-treatment with Der p 38 and NRG-1. Der p 38 treatment enhanced the secretion of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1). Co-treatment of Der p 38 with NRG-1 lowered the cytokine secretion increased by Der p 38, although NRG-1 alone was not effective on cytokine alteration. Neutrophil apoptosis was not altered by NRG-1 or supernatants of cells treated with NRG-1, but the cell supernatants co-treated with Der p 38 and NRG-1 blocked the anti-apoptotic effects of Der p 38-treated supernatants on neutrophils, which was involved in the activation of caspase 9 and caspase 3. Taken together, we determined that NRG-1 has anti-inflammatory effects in AD triggered by Der p 38. These results will pave the way to understanding the functions of NRG-1 and in the future development of AD treatment.

The PES1/FOXM1 heterodimer suppresses TCF21 and ERβ expression in ovarian endometriosis

AbstractTranscription factor 21 (TCF21) and estrogen receptor beta (ERβ, encoded by ESR2) are highly expressed in endometriotic stromal cells (ESCs) and contribute to the pathogenesis of endometriosis. However, the exploration of TCF21 and ERβ expression regulation at the molecular level remains limited. Here, by using bioinformatics analysis and experimental verification, we identified PES1, also known as Pescadillo, as a negative regulator in the development of endometriosis that downregulates TCF21 and ERβ expression in ESCs. PES1 overexpression regulated critical biological processes involved in endometriosis development, such as invasion and apoptosis. A coimmunoprecipitation assay showed that PES1 could form a complex with Forkhead box M1 (FOXM1). Further analyses elucidated that siPES1 in ectopic lesions decreased the stability of FOXM1 protein and reduced the binding activities of FOXM1 to TCF21 and ESR2 promoters, thus weakening the transcriptional inhibition of TCF21 and ERβ by FOXM1. Moreover, in an endometriosis mouse model, overexpressing PES1 effectively reduced the growth of ectopic lesions and suppressed TCF21 and ERβ expression, which suggests a promising therapeutic strategy for endometriosis. Collectively, our results indicate that the loss of PES1 in ectopic lesions contributes to endometriosis progression by upregulating ERβ and TCF21 expression through heterodimer formation with FOXM1. Moreover, targeting PES1 could serve as a treatment method for endometriosis.

Exploration and validation of key genes associated with early lymph node metastasis in thyroid carcinoma using weighted gene co-expression network analysis and machine learning.

Thyroid carcinoma (THCA), the most common endocrine neoplasm, typically exhibits an indolent behavior. However, in some instances, lymph node metastasis (LNM) may occur in the early stages, with the underlying mechanisms not yet fully understood. LNM potential was defined as the tumor's capability to metastasize to lymph nodes at an early stage, even when the tumor volume is small. We performed differential expression analysis using the 'Limma' R package and conducted enrichment analyses using the Metascape tool. Co-expression networks were established using the 'WGCNA' R package, with the soft threshold power determined by the 'pickSoftThreshold' algorithm. For unsupervised clustering, we utilized the 'ConsensusCluster Plus' R package. To determine the topological features and degree centralities of each node (protein) within the Protein-Protein Interaction (PPI) network, we used the CytoNCA plugin integrated with the Cytoscape tool. Immune cell infiltration was assessed using the Immune Cell Abundance Identifier (ImmuCellAI) database. We applied the Least Absolute Shrinkage and Selection Operator (LASSO), Support Vector Machine (SVM), and Random Forest (RF) algorithms individually, with the 'glmnet,' 'e1071,' and 'randomForest' R packages, respectively. Ridge regression was performed using the 'oncoPredict' algorithm, and all the predictions were based on data from the Genomics of Drug Sensitivity in Cancer (GDSC) database. To ascertain the protein expression levels and subcellular localization of genes, we consulted the Human Protein Atlas (HPA) database. Molecular docking was carried out using the mcule 1-click Docking server online. Experimental validation of gene and protein expression levels was conducted through Real-Time Quantitative PCR (RT-qPCR) and immunohistochemistry (IHC) assays. Through WGCNA and PPI network analysis, we identified twelve hub genes as the most relevant to LNM potential from these two modules. These 12 hub genes displayed differential expression in THCA and exhibited significant correlations with the downregulation of neutrophil infiltration, as well as the upregulation of dendritic cell and macrophage infiltration, along with activation of the EMT pathway in THCA. We propose a novel molecular classification approach and provide an online web-based nomogram for evaluating the LNM potential of THCA (http://www.empowerstats.net/pmodel/?m=17617_LNM). Machine learning algorithms have identified ERBB3 as the most critical gene associated with LNM potential in THCA. ERBB3 exhibits high expression in patients with THCA who have experienced LNM or have advanced-stage disease. The differential methylation levels partially explain this differential expression of ERBB3. ROC analysis has identified ERBB3 as a diagnostic marker for THCA (AUC=0.89), THCA with high LNM potential (AUC=0.75), and lymph nodes with tumor metastasis (AUC=0.86). We have presented a comprehensive review of endocrine disruptor chemical (EDC) exposures, environmental toxins, and pharmacological agents that may potentially impact LNM potential. Molecular docking revealed a docking score of -10.1 kcal/mol for Lapatinib and ERBB3, indicating a strong binding affinity. In conclusion, our study, utilizing bioinformatics analysis techniques, identified gene modules and hub genes influencing LNM potential in THCA patients. ERBB3 was identified as a key gene with therapeutic implications. We have also developed a novel molecular classification approach and a user-friendly web-based nomogram tool for assessing LNM potential. These findings pave the way for investigations into the mechanisms underlying differences in LNM potential and provide guidance for personalized clinical treatment plans.

The Genomic Landscape of Urothelial Carcinoma with High and Low ERBB2 Expression.

Recent data suggests that HER2-targeted treatment is efficacious in urothelial carcinoma (UC). We investigated the genomic, transcriptomic, and immune landscapes and clinical outcomes in UC segmented by ERBB2 expression. NextGen DNA/RNA sequencing was performed for 4743 UC tumors. A total of 3% (124/4125) of tumors had HER2 IHC and whole transcriptome sequencing (WTS) data. ERRB2-high and -low tumors were defined by ≥75th and <25th percentiles of ERBB2 expression, respectively. PD-L1 (SP142) positive staining was defined as ≥2+ and ≥5%. HER2 (4B5) positive staining was defined as ≥3+ and >10% or 2+ and >10% with positive HER2 in situ hybridization (ISH). Of the patients who were ERBB2-high, 79% (61/77) were HER2 positive via IHC. Tumors from lower tract UC had higher ERBB2 expression compared to upper tract UC (50 v 40 median TPM (mTPM), p < 0.001). ERBB2 expression was similar between primary and metastatic tumors (47 v 47 mTPM, p = 0.95). ERBB2-high tumors had a higher prevalence of pathogenic mutations in pTERT, ERBB2, and ELF3 versus ERBB2-low tumors, p < 0.001. ERBB2-high tumors had higher expressions of ADC target genes NECTIN4 (12 v 8 mTPM) and TACSTD2 (366 v 74 mTPM) versus ERBB2-low (p < 0.001), as well as better overall survival from time of tissue sampling than ERBB2-low (HR 1.71, p < 0.001). Our study demonstrated a high concordance between HER2 expression by IHC and ERBB2 gene expression by WTS in UC. Differences in ADC target expression between ERBB2-high vs. ERBB2-low UC may provide a rationale for combination treatment strategies with HER2-ADC. The association between high ERBB2 expression and survival advantage warrants further investigation.

Estrogen Receptor β in the Lateral Septum Mediates Estrogen Regulation of Social Anxiety-like Behavior in Male Mice

17β-estradiol (E2) regulates various forms of social behavior through the activation of two types of estrogen receptors, ERα and ERβ. The lateral septum (LS) is thought to be one of the potential target sites of E2, but the role played by ERα and ERβ in this brain area remains largely unknown. In the present study, we first analyzed the distribution of ERα and ERβ with double fluorescent immunohistochemistry in a transgenic mouse line in which red fluorescent protein (RFP) signal has been a reliable marker of ERβ expression. The overall number of ERβ-RFP-expressing cells was significantly higher (about 2.5 times) compared to ERα-expressing cells. The distribution of the two types of ERs was different, with co-expression only seen in about 1.2% of total ER-positive cells. Given these distinctive distribution patterns, we examined the behavioral effects of site-specific knockdown of each ER using viral vector-mediated small interference RNA (siRNA) techniques in male mice. We found ERβ-specific behavioral alterations during a social interaction test, suggesting involvement of ERβ-expressing LS neurons in the regulation of social anxiety and social interest. Further, we investigated the neuronal projections of ERα- and ERβ-expressing LS cells by injecting an anterograde viral tracer in ERα-Cre and ERβ-iCre mice. Dense expression of green fluorescence protein (GFP) in synaptic terminals was observed in ERβ-iCre mice in areas known to be related to the modulation of anxiety. These findings collectively suggest that ERβ expressed in the LS plays a major role in the estrogenic control of social anxiety-like behavior.

Abstract LB_A22: Advancing personalized therapies for small cell lung cancer: Exploring HER2-targeting antibody-drug conjugates (ADCs) and SLFN11 biomarker insights

Abstract Background: Small-cell lung cancer (SCLC) is a highly aggressive malignancy constituting 15% of all lung cancers, with a 5-year survival rate of less than 7%. Our group has established four transcriptionally defined subtypes of SCLC, each with distinct cell surface targets (Gay et al, Cancer Cell 2021). Further, we identified SLFN11 (a putative DNA/RNA helicase) as a candidate biomarker of sensitivity to DNA-damaging agents, including topoisomerase I inhibitors. Despite these advances, SCLC is still treated clinically with a “one-size-fits-all” approach. Human epidermal growth factor receptor 2 (HER2/ERBB2) has been previously described in a subset of SCLCs and has been linked to poor prognosis. Notably, HER2-targeting antibody-drug conjugates (ADCs) like Trastuzumab Deruxtecan (T-DXd) (where the payload is a topoisomerase I inhibitor) have received FDA approval for various solid tumors, highlighting their therapeutic potential. Methods: We analyzed ERBB2 mRNA expression in publicly available patient datasets and profiled HER2 protein expression in cell lines and patient-derived xenograft models by flow cytometry and IHC. We then assessed the cytotoxicity of Trastuzumab Deruxtecan in a panel of cell lines representative of the heterogeneity of SCLC to test whether levels of HER2 and/or SLFN11 predicted response. Results: Analyses of HER2/ERBB2 in SCLC cell lines and patient-derived xenograft (PDX) models revealed an enrichment in the SCLC-POU2F3 (SCLC-P) subtype. HER2 expression was validated by IHC and flow cytometry in a subset of SCLC cell lines and PDX models. ERBB2 mRNA expression data was analyzed using publicly available SCLC patient datasets, where we uncovered high expression of ERBB2 in a subset of patient tumors. Consistent with this, single-cell RNA-Seq data revealed ERBB2 expression in POU2F3+ cells in tumors from patients with relapsed SCLC. As predicted, we found a strong correlation between the in vitro cytotoxicity of Trastuzumab Deruxtecan and both the level of surface HER2 expression and SLFN11 level. Importantly, the SCLC cell lines with the highest HER2 surface expression exhibited the lowest IC50 of Trastuzumab Deruxtecan, and the response was predicted by the presence of SLFN11. Conclusion: We demonstrate HER2/ERBB2 mRNA and protein expression in a subset of SCLC, with enrichment in the SCLC-P subtype. Further, the FDA-approved HER2-targeting ADC Trastuzumab Deruxtecan had significant cytotoxicity, correlating with expression of the surface target (HER2) as well as with SLFN11 (a candidate predictive biomarker for the topoisomerase inhibitor payload). These findings suggest the potential of tailoring HER2-targeting strategies based on HER2 and SLFN11 expression in SCLC as an effective and personalized approach for tackling SCLC. Citation Format: Ali H. Ibrahim, Bingnan Zhang, C. Allison Stewart, Alejandra Serrano, Luisa M. Solis, Wei-Lien Wang, Kavya Ramkumar, Robert J. Cardnell, Runsheng Wang, Alberto Duarte Jr, Lixia Diao, Yuanxin Xi, Jing Wang, Lauren A. Byers, Carl M. Gay. Advancing personalized therapies for small cell lung cancer: Exploring HER2-targeting antibody-drug conjugates (ADCs) and SLFN11 biomarker insights [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A22.

Abstract PR004: PRISM high-throughput screening of antibody-drug conjugates uncovers clinically relevant targets

Abstract Antibody-drug conjugates (ADCs) are a rapidly emerging class of cancer therapeutics composed of an antibody scaffold, a cytotoxic payload, and a joining linker. They exhibit properties of both biologics and small molecule inhibitors, can exert activity through multiple mechanisms, and some are capable of inducing target-independent cell killing via a phenomenon known as the bystander effect. Due to this inherent structural and biological complexity, cell-based functional screening is an essential component of ADC development. However, the majority of preclinical in vitro studies for ADCs are limited to a small number of cell lines and thus provide minimal insights into their activity. High-throughput drug-screening platforms, such as PRISM (Profiling Relative Inhibition Simultaneously in Mixtures), that have been highly effective in characterizing small molecule inhibitors could facilitate characterization of ADCs at an unprecedented scale and provide more comprehensive insights into ADC mechanisms, target specificity, and potency. To test the suitability of the PRISM platform for ADC screening, we investigated several ADCs conjugated to the HER2-targeting antibody trastuzumab that differed in terms of linker type (cleavable and non-cleavable), payload class, and drug-to-antibody ratio. ADCs were screened in parallel with controls including unconjugated antibodies and free payloads across a panel of nearly 500 cell lines consisting of diverse cell lineages. We explored whether ADC selectivity was associated with target antigen expression and found a clear association between cell line sensitivity and ERBB2 (HER2) gene expression, with the strongest effects on viability observed in a subset of cell lines overexpressing ERBB2. Univariate biomarker analysis identified ERBB2 gene expression as a significantly correlated feature of sensitivity to all HER2-targeting ADCs. Notably, this correlation was retained across ADCs with payloads capable of exerting bystander killing of cells with low ERBB2 expression. Upon further comparison of biomarker profiles across different ADCs, established relationships between payload resistance and ABCB1 efflux pumps emerged for a subset of ADC payloads. These results indicate that PRISM pooled cell line screening is able to distinguish ADCs with different payload characteristics while maintaining selectivity of the primary target. Overall, our study highlights how PRISM can be leveraged to analyze ADC activity and uncover clinically relevant targets. This approach can aid in evaluating potential ADC candidates by providing a better understanding of ADC mechanisms of action and how their unique structural components drive cytotoxic activity. Citation Format: Jillian N Eskra, Ellen Nguyen, Aydin Golabi, Shiker Nair, Antonella Masciotti, Anthony Fazio, Mustafa Kocak, Melissa Ronan, Matthew G Rees, Jennifer A Roth. PRISM high-throughput screening of antibody-drug conjugates uncovers clinically relevant targets [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR004.