ERβ Expression Research Articles

Abstract 6283: Tumor repressive effects of estrogen receptor β mRNA-based therapy in aggressive breast cancers

Abstract Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are the most aggressive forms of breast cancer due to their high propensity to develop distant metastases and the lack of specific effective targeted therapies. The failure of previous research to produce viable therapeutic targets combined with the absence of estrogen receptor α (ERα) in TNBC and the majority of IBC led us to explore the second ER (ERβ) as a molecule with targeting potential. We previously reported the anti-metastatic effects of ERβ in both TNBC and IBC. Considering the anti-tumor activity together with the decreased expression of ERβ in IBC and TNBC tumors, we employed an mRNA-based approach to induce tumor-specific upregulation of the receptor and restore its anti-metastatic activity. Our findings show that complexes of ERβ mRNA with lipid nanoparticles (LNPs) enable in vitro translation of a functional protein with strong anti-invasive activity. More importantly, direct administration of ERβ mRNA-LNPs in orthotopically grown IBC and TNBC tumors results in high tumor levels of the receptor and an evident inhibition of metastasis demonstrating for the first time the ability of ERβ mRNA to prevent progression of the disease. Our study may have important clinical implications by establishing ERβ mRNA as a novel and specific therapy intervention that either alone or in combination with existing treatments can substantially repress metastasis, eliminate associated mortality and benefit patients with aggressive breast cancer. Citation Format: Kim Cuong Cap, Aireana Phillips, Karem A. Court Pinto, Kristina D. Zambo, Wei Qian, Jianying Zhou, Jenny Chang, Biana Godin, Christoforos Thomas. Tumor repressive effects of estrogen receptor β mRNA-based therapy in aggressive breast cancers [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 6283.

Abstract 1110: Estrogen receptor β signaling induced radio-resistance in bladder cancer

Abstract Introduction and Objective: Although radiotherapy (RT) combined with chemotherapy shows comparable outcomes to surgery, women tend to have higher mortality from bladder cancer (BC) compared to men. Emerging evidence indicated the involvement of estrogen receptorβ (ERβ) signaling in BC progression and the association of ER signaling with DNA repair pathways which are involved in radio-resistance. Therefore, we evaluated the association between ERβ signaling and the sensitivity to BC cells. Methods: We compared the inhibitory effect of RT on BC cell viability between ERβ positive [5637 and T24 expressing control short-heparin RNA (shRNA)] and ERβ negative (5637 and T24 expressing ERβ-shRNA) cells. Moreover, we compared DNA double-strand breaks (DSB) via γ-H2AX foci formation, as well as the expression levels of DNA repair genes, in these cells after RT. Finally, in a mouse xenograft model, we evaluated it on the growth of ERβ positive tumor treated with radiation combined with mock or tamoxifen treatment. Results: In vitro experiment showed that ionizing radiation reduced the numbers of viable cells in ERβ negative cells more significantly than those in ERβ positive cells. Moreover, tamoxifen treatment reduced the numbers of viable cells more than mock treatment in ERβ positive cells, while tamoxifen didn’t show significant effects in ERβ negative cells. The down-regulation of ERβ resulted in a delay in DSB repair 4-24 hours after RT. We then established radio-resistant cells and confirmed considerable elevation of the expression of ERβ as well as DNA repair genes including RAD51 and RAD54. Estradiol treatment induced the expression of these genes and tamoxifen abolished these effects. Finally, in vivo experiment showed that tamoxifen enhanced the cytotoxic effects of RT in xenograft bearing mice. Conclusions: These results indicated that ERβ signaling inversely correlated with sensitivity to RT in BC. Thus, ERβ inhibition have a potential of enhancing the cytotoxic effects of radiation, especially in ERβ positive BC cells. Citation Format: Hiroki Ide, Tetsushi Murakami, Eiji Kashiwagi, Sotaro Kitaoka, Akari Komatsuda, Chugo Nishimura, Shinsuke Funakoshi, Mototsugu Oya, Hiroshi Miyamoto. Estrogen receptor β signaling induced radio-resistance in bladder cancer [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 1110.

Abstract 6270: Anti-metastatic effects of estrogen receptor β in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is a rare but highly aggressive form of breast cancer with a 5-year survival rate of less than 50%. Although 20-30% of women with IBC have distant metastasis at the time of diagnosis, the biology underlying the aggressive behavior of the disease is poorly understood and molecular markers that distinguish IBC from non-IBC have not yet been identified. In response to the urgent need of discovering new targeted therapies in IBC, we have studied the second estrogen receptor (ERβ) since prior work from our lab correlated higher tumor expression of ERβ in samples from IBC patients with longer metastasis free survival. We showed that ERβ exerted anti-metastatic activity in IBC by inhibiting actin-based cell migration through the suppression of RhoC GTPase signaling. However, since estrogen receptors elicit their effects by inducing global cellular alterations, we have now adopted an integrated genomic approach to fully understand the anti-metastatic effects of ERβ in IBC and delineate the underlying transcriptional regulatory circuits. We found ERβ binding sites in IBC cells and combined this information with gene expression data to identify downstream direct target genes and associated pathways. Our findings suggest the regulation of tumor micro-environment and metabolism as the primary targets of ERβ and we are currently working to pinpoint the crucial regulatory components in these functions. Our findings thus offer an opportunity to better understand the mechanism of ERβ action in aggressive breast cancer, serving as a critical step in validating its tumor repressive role. Citation Format: Harika Nagandla, Spyros Tastsoglou, Kim Cuong Cap, Aireana Phillips, Wei Qian, Jianying Zhou, Jenny Chang, Savitri Krishnamurthy, Naoto Ueno, Artemis Hatzigeorgiou, Christoforos Thomas. Anti-metastatic effects of estrogen receptor β in inflammatory breast cancer [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 6270.

Abstract 7217: Orthogonal targeting of NAD metabolism and ErbB2/ErbB3 signaling in pancreatic adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is now the third leading cause for cancer related deaths in the US with a five-year survival of ~10% that has improved little over the past 20 years. Thus, there is a critical need to fully understand mechanisms of PDAC tumorigenesis in order to develop novel and effective therapies. Nicotinamide Adenine Dinucleotide (NAD), a critical co-factor and energy source required for cellular processes such as DNA repair and epigenetic regulation is frequently upregulated in PDAC. We recently reported a novel oncogenic axis connecting the NAD-dependent epigenetic regulator and oncogene C-terminal Binding Protein (CtBP) with the EGFR family growth factor receptors ErbB2/ErbB3 (ErbB2/3) in PDAC cells and tumors. Indeed, previous reports have shown ~30% of human PDAC tumors overexpress ErbB3, and our work has shown that PDAC tumor cell growth relies on maintenance of ErbB3 expression. Moreover, we show that the majority of PDAC cells with high levels of ErbB3 expression are uniquely sensitive to the ErbB2-targeted tyrosine kinase inhibitor lapatinib. In addition, we have recently shown that co-targeting NAD and CtBP with small molecule inhibitors is an effective and synergistic therapeutic strategy in PDAC model systems. Methods and Results: In the current study, we demonstrate NAD-dependent regulation of ErbB2/3 signaling in PDAC cells in vitro. Upon depletion of NAD using the NAD salvage pathway inhibitor GMX1778 in human PDAC cells, we observed a striking coordinate decrease in the levels of both ErbB2 and ErbB3. Furthermore, stable knockdown of the limiting enzyme of the complementing Preiss-Handler NAD synthetic pathway, Nicotinamide Adenine Phosphoribosyl Transferase, also resulted in robust downregulation of ErbB2/3 protein levels, suggesting a direct linkage in PDAC cells between the status of NAD metabolism and synthesis and activity of the key growth factor receptors ErbB2/3. Conclusions: Our findings highlight the novel connection between NAD and ErbB2/3 in PDAC. Furthermore, our data suggests combining chemical or biologic inhibitors of ErbB2/3 with an inhibitor of NAD synthesis may result in highly synergistic cytotoxic effects in PDAC cells and PDAC preclinical models. Such data could support the future development of a human PDAC clinical trial repurposing agents targeting NAD synthesis and ErbB2/3 that are already FDA-approved or currently in clinical testing. Citation Format: Kranthi Kumar Chougoni, Nari Kim, Martin M. Dcona, Diana T. Dcona, Gautam K. Malhotra, Steven R. Grossman. Orthogonal targeting of NAD metabolism and ErbB2/ErbB3 signaling in pancreatic adenocarcinoma [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 7217.

Abstract 1768: Investigating oncogene amplifications in brain metastasis of esophageal adenocarcinoma samples

Abstract Esophageal Adenocarcinoma (EAC) exhibits a rising incidence and dismal survival rate in the US, primarily attributed to frequent distant metastases. Brain metastasis, though uncommon is an aggressive tumor type, remains poorly understood in EAC. To address this knowledge gap, we conducted a comprehensive analysis of 9 brain metastasis samples, profiling their whole-genome with matched normal and comparing them to the primary EAC tumors, other EAC metastasis types and other brain metastasis types, including the Hartwig Institute and PCAWG. Overall, our sample size comprised over 4,500 tumor whole-genome data and we utilized a uniform data analysis pipeline for robust comparisons between cohorts. Our analysis revealed that brain metastasis samples have significantly higher single-nucleotide variants, however, the structural variant burden in brain metastases closely resembled those observed in the primary tumor. Notably, the incidence of whole genome doubling is significantly higher in brain metastasis compared to other primary cancer types and metastases. This significant difference stems from the higher WGD incidence in primary EACs compared to other tumor types. Mutational signature analysis revealed no significant changes compared to the primary tumor, and we observed an enrichment of SBS 17a/b, previously associated with tumor progression in primary esophageal adenocarcinoma, along with a prevalent presence of the APOBEC mutagenesis signature in most samples. Notably, ERBB2 and TP53 emerged as prominent driver genes present in most brain metastasis samples. We also observed an upregulation of ERBB2 expression in brain metastasis samples. Interestingly, ERBB2 appeared to play a more significant role in brain metastasis progression compared to the primary tumor and other metastasis types, suggesting its potential as a therapeutic target. These findings advance our comprehension of brain metastasis in EAC, providing crucial insights into genomic alterations and mutational processes that could inform future therapeutic strategies and clinical management for this previously understudied metastatic type. Citation Format: Nora Lawson, Lingqun Ye, Bo Zhao, Andy Futreal, Kadir Akdemir. Investigating oncogene amplifications in brain metastasis of esophageal adenocarcinoma samples [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 1768.

Abstract 5157: Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells

Abstract Cell surface proteins are increasingly considered in the development of novel anticancer agents, as demonstrated by recent clinical successes in the field of immuno-oncology and antibody-drug conjugates (ADCs). These conjugates specifically target antigens present on the surface of tumor cells, enabling the delivery of cytotoxic agents to specific tumor sites. During the preclinical development of such compounds, for example, when they are tested on a wide panel of cell lines, it is essential to have a rapid and reliable method for monitoring target expression and distribution. Complementing our 160 Cell line panel ProLiFiler™, we report here the development of a novel method named oncoFLOW-Profiler™ which includes steps of cell fixation, micronic storage, staining, and measurement by flow cytometry. This method is applied in a 96-well format, enabling screening of all cell lines in parallel and analysis of the expression of different targets in just one day. In this proof-of-concept study, we investigated the level of ERBB2 expression across the ProLiFiler panel of 160-cell lines in order to validate this methodology. We first compared the impact of fixation methods and sample storage on knowingly positive and negative control cell lines over a period of several months. ERBB2 measurements from the oncoFLOW-Profiler™ will be analyzed and validated by comparison with various OMICs datasets from the Cancer DataMiner platform (4HF Biotec), including RNA expression, mutational status, and protein expression levels. To determine the expression cutoff of ERBB2 amplified models, results will be analyzed according to gene copy number variation. Finally, the flow cytometry data generated will be investigated for use as a predictor of response to clinically approved ADCs targeting ERBB2: Kadcyla and Enhertu. Here we present a method that we have demonstrated is feasible for investigating cell surface receptor/antigens during preclinical phase of drug development. Our method has enabled us to successfully assess ERBB2 in biological samples and to identify models sensitive to ADCs targeting ERBB2. We will also discuss the results of ongoing experiments analyzing the applicability of this screen to intracellular targets and carbohydrate-based surface antigens, which cannot be analyzed either by transcriptomics or proteomics. Citation Format: Kaja Holtorf, Anne-Lise Peille, Daniel Feger, Vincent Vuaroqueaux, Jan Erik Ehlert, Nadine Obier. Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells [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 5157.

Linking alterations in estrogen receptor expression to memory deficits and depressive behavior in an ovariectomy mouse model

The high risk of neurological disorders in postmenopausal women is an emerging medical issue. Based on the hypothesis of altered estrogen receptors (ERα and β) after the decline of estrogen production, we investigated the changes in ERs expressions across brain regions and depressive/amnesic behaviors. C57BL/6J female mice were ovariectomized (OVX) to establish a menopausal condition. Along with behavior tests (anxiety, depression, and memory), the expression of ERs, microglial activity, and neuronal activity was measured in six brain regions (hippocampus, prefrontal cortex, striatum, raphe nucleus, amygdala, and hypothalamus) from 4 to 12 weeks after OVX. Mice exhibited anxiety- and depressive-like behaviors, as well as memory impairment. These behavioral alterations have been linked to a suppression in the expression of ERβ. The decreased ERβ expression coincided with microglial-derived neuroinflammation, as indicated by notable activations of Ionized calcium-binding adapter molecule 1 and Interleukin-1beta. Additionally, the activity of brain-derived neurotrophic factor (BDNF), particularly in the hippocampus, decreased in a time-dependent manner from 4 to 12 weeks post-OVX. Our study provides evidence shedding light on the susceptibility to memory impairment and depression in women after menopause. This susceptibility is associated with the suppression of ERβ and alteration of ERα in six brain regions.

Loss of ERβ Disrupts Gene Regulation in Primordial and Primary Follicles.

Loss of ERβ increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERβ in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erβ knockout (Erβnull) mouse ovaries, and their transcriptomes were compared with those of control Erβfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erβnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERβ has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erβnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erβfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erβ-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erβnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erβnull PrFs were downregulated. Such deregulation of genes in Erβnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erβnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERβ either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.

Abstract A063: What can we learn from a retrospective evaluation of HER2, P53 and P16INK4A protein expression in rare ovarian tumors?

Abstract A063: What can we learn from a retrospective evaluation of HER2, P53 and P16INK4A protein expression in rare ovarian tumors?

BRD4-targeting PROTACs Synergize With Chemotherapeutics Against Osteosarcoma Cell Lines.

Osteosarcoma at an advanced stage has a poor outcome, and novel targeted therapies are needed, especially for metastatic disease. Bromodomain inhibitors (BETi) are epigenetic modulators that broadly impair the expression of oncogenic proteins and exert antitumor effects. BETi can be combined with chemotherapeutics to increase therapeutic responses with superior effects in the form of proteolysis targeting chimeras (PROTACs) that degrade proteins of interest (POI) in multiple cycles. This work aimed to investigate the efficacy of BETi, such as JQ1, dBET57, and MZ1 PROTACs in combination with cytotoxic drugs against osteosarcoma cell lines. Chemosensitivity of the osteosarcoma cell lines HOS, Saos-2, MG-63, and G292 were tested with BET-directed agents alone or in combination with cytotoxic drugs comprising cisplatin, doxorubicin, topotecan, and gemcitabine using cell viability assays. The BET degraders exhibited highest toxicity to HOS cells and showed synergistic activity in combination with the chemotherapeutics, except for the degrader - topotecan/gemcitabine combinations. Highest synergy between BET agents and chemotherapeutics were found for the more chemoresistant Saos-2 cells and potentiation of toxicity in MG-63 cells for the BET agents - doxorubicin combinations and the MZ1-topotecan pair. HOS and Saos-2 cell lines had reduced protein expression of AXL, BCL-X, e-cadherin, CAIX, EpCAM, ErbB2, and vimentin in response to JQ1, MZ1, and BET57. The study suggests that the application of novel BET PROTACs in combination with chemotherapeutics could represent a new therapeutic option to improve the therapy of osteosarcomas. First orally available PROTACs have reached clinical trials.

Nutraceuticals known to promote hair growth do not interfere with the inhibitory action of tamoxifen in MCF7, T47D and BT483 breast cancer cell lines.

Hair loss/thinning is a common side effect of tamoxifen in estrogen receptor (ER) positive breast cancer therapy. Some nutraceuticals known to promote hair growth are avoided during breast cancer therapy for fear of phytoestrogenic activity. However, not all botanical ingredients have similarities to estrogens, and in fact, no information exists as to the true interaction of these ingredients with tamoxifen. Therefore, this study sought to ascertain the effect of nutraceuticals (+/- estrogen/tamoxifen), on proliferation of breast cancer cells and the relative expression of ERα/β. Kelp, Astaxanthin, Saw Palmetto, Tocotrienols, Maca, Horsetail, Resveratrol, Curcumin and Ashwagandha were assessed on proliferation of MCF7, T47D and BT483 breast cancer cell lines +/- 17β-estradiol and tamoxifen. Each extract was analysed by high performance liquid chromatography (HPLC) prior to use. Cellular ERα and ERβ expression was assessed by qRT-PCR and western blot. Changes in the cellular localisation of ERα:ERβ and their ratio following incubation with the nutraceuticals was confirmed by immunocytochemistry. Estradiol stimulated DNA synthesis in three different breast cancer cell lines: MCF7, T47D and BT483, which was inhibited by tamoxifen; this was mirrored by a specific ERa agonist in T47D and BT483 cells. Overall, nutraceuticals did not interfere with tamoxifen inhibition of estrogen; some even induced further inhibition when combined with tamoxifen. The ERα:ERβ ratio was higher at mRNA and protein level in all cell lines. However, incubation with nutraceuticals induced a shift to higher ERβ expression and a localization of ERs around the nuclear periphery. As ERα is the key driver of estrogen-dependent breast cancer, if nutraceuticals have a higher affinity for ERβ they may offer a protective effect, particularly if they synergize and augment the actions of tamoxifen. Since ERβ is the predominant ER in the hair follicle, further studies confirming whether nutraceuticals can shift the ratio towards ERβ in hair follicle cells would support a role for them in hair growth. Although more research is needed to assess safety and efficacy, this promising data suggests the potential of nutraceuticals as adjuvant therapy for hair loss in breast cancer patients receiving endocrine therapy.

Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells

Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor β (Esr2/ERβ) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERβ and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERβ and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERβ and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERβ and GPER in cumulus cells.

ERBB3 Overexpression is Enriched in Diverse Patient Populations with Castration-sensitive Prostate Cancer and is Associated with a Unique AR Activity Signature.

Despite successful clinical management of castration-sensitive prostate cancer (CSPC), the 5-year survival rate for men with castration-resistant prostate cancer is only 32%. Combination treatment strategies to prevent disease recurrence are increasing, albeit in biomarker-unselected patients. Identifying a biomarker in CSPC to stratify patients who will progress on standard-of-care therapy could guide therapeutic strategies. Targeted deep sequencing was performed for the University of Illinois (UI) cohort (n = 30), and immunostaining was performed on a patient tissue microarray (n = 149). Bioinformatic analyses identified pathways associated with biomarker overexpression (OE) in the UI cohort, consolidated RNA sequencing samples accessed from Database of Genotypes and Phenotypes (n = 664), and GSE209954 (n = 68). Neutralizing antibody patritumab and ectopic HER3 OE were utilized for functional mechanistic experiments. We identified ERBB3 OE in diverse patient populations with CSPC, where it was associated with advanced disease at diagnosis. Bioinformatic analyses showed a positive correlation between ERBB3 expression and the androgen response pathway despite low dihydrotestosterone and stable expression of androgen receptor (AR) transcript in Black/African American men. At the protein level, HER3 expression was negatively correlated with intraprostatic androgen in Black/African American men. Mechanistically, HER3 promoted enzalutamide resistance in prostate cancer cell line models and HER3-targeted therapy resensitized therapy-resistant prostate cancer cell lines to enzalutamide. In diverse patient populations with CSPC, ERBB3 OE was associated with high AR signaling despite low intraprostatic androgen. Mechanistic studies demonstrated a direct link between HER3 and enzalutamide resistance. ERBB3 OE as a biomarker could thus stratify patients for intensification of therapy in castration-sensitive disease, including targeting HER3 directly to improve sensitivity to AR-targeted therapies.

Abstract B063: Investigation of tumor heterogeneity using integrated single-cell RNA sequence data based on HER2 status in patients with breast cancer

Abstract [Introduction] Human epidermal growth factor receptor 2 (HER2), which is characterized by ERBB2 amplification, is one of the important markers for treatment decision related to breast cancer. Many targeted therapies for HER2-positive (immunohistochemistry [IHC] scores of 3+ or 2+ with an in situ hybridization [ISH] gene amplification) or HER2-low (IHC score 1+ or 2+ with no ISH gene amplification) breast cancers have been extensively developed thus far. Meanwhile, tumoral heterogeneity is considered one of the mechanisms for drug resistance; however, it has not been fully elucidated on each HER2 status at single-cell level. Therefore, an integrated analysis of the breast cancer single-cell gene expression data of both public datasets and our cohort was used to investigate tumor heterogeneity based on HER2 status. [Methods] We collected 21 and 6 scRNA-seq samples of primary breast cancer from the public Gene Expression Omnibus (GEO) and our institution, respectively. A total of 27 samples included HER2-positive cases (pure HER2 cases: estrogen receptor [ER]-negative/HER2-positive and Luminal-HER2 cases: ER-positive/HER2-positive) and Luminal cases (ER-positive/HER2-negative). These datasets were imported into R software version 4.2.0. and transformed into Seurat objects with the package Seurat version 4.3.0. UMAP plots, which is a non-linear dimension reduction method, were used for clustering analyses. Seurat in R was used to generate UMAP, feature, and violin plots. Additionally, we performed pathway enrichment analysis with the significant gene list from each cluster between the ERBB2-high and ERBB2-low groups. [Results] Clustering analysis revealed heterogeneous distribution in each gene related to breast cancer (ESR1, PGR, ERBB2, and MKI67). One of the clusters revealed high MKI67 expression. ERBB2 expressions were diffusely distributed on each cluster of pure HER2 cases; however, the expressions were considerably higher in one of the clusters in cases of Luminal-HER2. The ERBB2 expression in Luminal cases, which included HER2-low status, was lower than that in HER2-positive cases, albeit slight expression was observed. Cell proliferation factors, including IGF, IGF1R, and EGF, were included in the ERBB2-high expression group compared with the ERBB2-low expression group for pathway analysis. Further, we examined typical gene expressions which are associated with markers related to breast cancer, cancer stem cell, and epithelial-to-mesenchymal transition in each case and revealed heterogeneous patterns across patients. [Conclusion] Heterogeneous ERBB2 expression distribution was observed in HER2-positive cases, and slight ERBB2 expression was identified in the Luminal cases. Moreover, Luminal-HER2 cases could be considered a more heterogeneous subtype compared with pure HER2 cases. Additionally, gene expressions of typical gene markers varied across patients. These results indicated that breast cancer displays heterogeneous patterns on each HER2 status not only intra-tumoral heterogeneity but also inter-patient heterogeneity. Citation Format: Sho Shiino, Momoko Tokura, Jun Nakayama, Masayuki Yoshida, Akihiko Suto, Yusuke Yamamoto. Investigation of tumor heterogeneity using integrated single-cell RNA sequence data based on HER2 status in patients with breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B063.

Mechanism of Action of Yikun Yitong Ping-Containing Serum on Mast Cells and its Possible Involvement in Endometriosis-Related Dysmenorrhea

Background Previously, we showed that Yikun Yitong Ping (YKYTP) can effectively and safely relieve endometriosis-related dysmenorrhea; however, the underlying mechanism remained unclear. This study aimed to assess the effects of YKYTP-containing serum on rat basophilic leukemia cell line, as mast cells (MCs) analog, and investigate the mechanisms by which YKYTP alleviates endometriosis-related dysmenorrhea. Method In this study, YKYTP drug-containing serum was used to treat rat basophilic leukemia cell line (RBL2H3). The effect of YKYTP-containing serum on the expression of ER-α, ER-β, NGF, and NGFRp75 in RBL2H3 cells was evaluated using enzyme-linked immunosorbent assay, quantitative real-time polymerase reaction, and Western blotting. Results Different concentrations (5%-40%) of YKYTP-containing serum reduced ER expression in RBL2H3 cells, and the optimum concentration was 40%. Compared to the blank group, the expression of ER and NGF significantly increased in the E2 group ( P < .01). After co-administration with YKYTP-containing serum, the expression of ER-α, ER-β, NGF, and P75 significantly decreased ( P < .01). Conclusion YKYTP-containing serum can efficiently inhibit the expression of ER-α, ER-β, NGF, and P75 in RBL2H3 cells. YKYTP may alleviate endometriosis-related dysmenorrhea by downregulating ER expression in MCs.

ERBB3 expression in racially diverse patients with castration-sensitive prostate cancer and its association with a unique AR activity signature.

23 Background: Despite successful clinical management of castration sensitive prostate cancer (CSPC), the 5-year survival rate for men with castration-resistant prostate cancer (CRPC) is only 32%. This number is even more striking in the context of racial disparities where prostate cancer mortality rates for Black/African American (AA) men are two to four times higher than those in every other racial and ethnic group in the US. Identification of molecular expression and activity patterns that vary by racial group may provide a key to addressing outcomes related to survival disparities, as current treatments have not been developed with attention to the inclusion of diverse patient populations. Developing a biomarker in CSPC to stratify patients who will progress on standard-of-care therapy could guide intelligent intensification of therapeutic strategies and improve survival, particularly in high-risk patient populations. Methods: Targeted deep sequencing was performed as routine care for treatment naïve patients in the UI cohort (n=30), and immunostaining was performed in racially diverse tissue microarray (n=149). Bioinformatics analyses were utilized to identify signaling pathways associated with biomarker overexpression in the UI cohort, consolidated publicly available RNA-seq datasets (n=664), and a racially diverse Gene Expression Omnibus dataset (n=68). Results: We identified ERBB3 overexpression (OE) in racially diverse CSPC patient populations, where it was associated with advanced disease at diagnosis. At the protein level, HER3 expression was negatively correlated with intraprostatic androgen in Black/AA men. Bioinformatics analyses showed a positive correlation between ERBB3 expression and the Androgen Response pathway despite low intraprostatic androgen and stable expression of androgen receptor (AR) transcript in Black/AA men. Clinically, ERBB3 OE is associated with high pre-treatment serum PSA in Black/AA men and is positively correlated with a clinically adaptable AR signature score. Preliminary outcomes data suggest Black/AA men with ERBB3 OE have a shorter time to disease progression. Conclusions: In diverse CSPC patient populations, ERBB3 OE was associated with high AR signaling and serum PSA despite low intraprostatic androgen, suggesting ERBB3 OE as a prospective biomarker for ligand-independent AR activation. In this context, ERBB3 OE could stratify patients for intensification of therapy in castration-sensitive disease and emphasizes the importance of biomarker-directed clinical trials with diverse populations.

Treadmill Running Regulates Adult Neurogenesis, Spatial and Non-spatial Learning, Parvalbumin Neuron Activity by ErbB4 Signaling.

Exercise can promote adult neurogenesis and improve symptoms associated with schizophrenia and other mental disorders via parvalbumin(PV)-positive GABAergic interneurons in the dentate gyrus ErbB4 is the receptor of neurotrophic factor neuregulin 1, expressed mostly in PV-positive interneurons. Whether ErbB4 in PV-positive neurons mediates the beneficial effect of exercise and adult neurogenesis on mental disorder needs to be further investigation. Here, we first conducted a four-week study on the effects of AG1478, an ErbB4 inhibitor, on memory and neurogenesis. AG1478 significantly impaired the performance in several memory tasks, including the T-maze, Morris water maze, and contextual fear conditioning, downregulated the expression of total ErbB4 (T-ErbB4) and the ratio of phosphate-ErbB4 (p-ErbB4) to T-ErbB4, and associated with neurogenesis impairment. Interestingly, AG1478 also appeared to decrease intracellular calcium levels in PV neurons, which could be reversed by exercise. These results suggest exercise may regulate adult neurogenesis and PV neuron activity through ErbB4 signaling. Overall, these findings provide further evidence of the importance of exercise for neurogenesis and suggest that targeting ErbB4 may be a promising strategy for improving memory and other cognitive functions in individuals with mental disorders.

Abstract IA20: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer

Abstract Oncogenic KRAS (KRAS*) is a signature mutation in pancreatic ductal adenocarcinoma (PDAC). While genetic extinction or pharmacological inhibition of KRAS* elicits anti-tumor responses in preclinical models and patients, we and others have identified cancer cell intrinsic (YAP, RTKs) and paracrine immune-mediated (TGFb) mechanisms enabling KRAS* bypass and tumor recurrence. Here, we explored the potential role of cancer associated fibroblasts (CAFs) as contributors to mechanisms driving KRAS* bypass and identified CAF-derived NRG1 activation of cancer cell ERBB2 and ERBB3 receptor tyrosine kinases as a mechanism by which KRAS* independent tumor regrowth is supported. We showed that genetic extinction or pharmacological inhibition of KRAS* results in upregulation of ERBB2 and ERBB3 expression in human and murine cancer cell models. We demonstrated that CAF-derived NRG1 supports KRAS*-dependent tumor growth. These insights prompted genetic depletion or pharmacological inhibitions studies targeting ERBB2/3 or NRG1, which showed abolition of KRAS* bypass and synergy with KRASG12D inhibitor in mouse and human PDAC models. Thus, CAFs via the NRG-ERBB2/3 paracrine axis provide yet another mechanism enabling KRAS* inhibitor therapy resistance, providing a highly actionable combination therapeutic strategy to improve the effectiveness of KRAS* inhibitors in PDAC patients. Citation Format: Jincheng Han, Jiaqian Xu, Yonghong Liu, Shaoheng Liang, Kyle A. LaBella, Deepavali Chakravarti, Denise J. Spring, Yan Xia, Ronald A. DePinho. Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr IA20.

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.