Estrogen Receptor Binding Sites Research Articles

Synthesis, characterization, cytotoxic activity, and molecular docking of 3,4-diaminobenzophenone-CuhNFs and its major components-based nanoflowers

In this study, organic–inorganic hybrid nanoflower (hNFs) synthesis was realized using 3,4-diaminobenzophenone and Cu(II) metal ions as organic and inorganic ingredients, respectively. The characterization of the synthesized hNFs was carried out using SEM, EDX, FT-IR, XRD, and elemental mapping. The cytotoxic effect of hNFs was investigated against A549 (lung) and MCF-7 (breast) cancer cell lines. Interactions of 3,4-diaminobenzophenone were also investigated via molecular docking studies at the binding site of the human estrogen receptor (PDB ID:3ERT) and epidermal growth factor receptor tyrosine kinase (PDB ID: 1M17). Physicochemical and pharmacokinetic parameters for ligands that could be potential anticancer drug candidates were determined by ADME prediction calculations from in silico approaches. In vitro results showed an increase in cell death when the synthesized hybrid nanoflowers were applied against both cell lines, depending on the concentrations tested. Additionally, it was determined that the synthesized nanoflower was more effective on the MCF-7 cell line.

8028 Targeted Therapy-Induced ER And HER2 Chromatin Binding: A Novel Driver Of Poor Response To Treatment In Triple Positive Breast Cancer

Abstract Disclosure: S. Tam: None. M.D. McCoy: None. S. Bahnassy: None. R.B. Riggins: None. Triple positive breast cancer (TPBC) is a unique variant of breast cancer characterized by co-expression of two oncogenic drivers, estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2). Both drivers promote tumor progression and serve as targets for effective small molecule and monoclonal antibody (mAb) therapies. Previous studies showed that the co-expression of ER reduces the efficacy of anti-HER2 targeted therapy in TPBC, and vice versa. ER is a well-established nuclear receptor and transcription factor. HER2 mainly functions as a transmembrane receptor, activating cytosolic pro-tumorigenic signaling pathways. However, less-studied nuclear HER2 is involved in the transcriptional regulation of several genes, leading to increased BC cell growth and survival. In this study, we investigate the subcellular localization and transcriptional regulatory functions of ER and HER2 in the unique context of TPBC, using cell fractionation, immunofluorescence, chromatin immunoprecipitation (ChIP)-qPCR, and integrated analysis of ER ChIP-seq/RNA-seq data. We hypothesize that crosstalk between ER and HER2 in the nucleus may, in part, underlie poor response to targeted therapy. At baseline, ER has strong nuclear localization in TPBC cells, BT474 and MDA-MB-361, but is predominantly cytosolic in ER+ MCF7 cells. Activated, phospho-HER2 is also localized to the nucleus of TPBC and HER2+ SkBr3 cells at baseline. Treatment with the anti-HER2 mAb combination of trastuzumab and pertuzumab (TP) induces ER binding to specific ER binding sites (ERBSs) in the enhancers or promoters of PGR, TFF1, and FOXC1 in BT474, but this is not observed in MCF7 or SkBr3 cells. Unlike TP, which increases HER2 nuclear localization but not ERBS recruitment, fulvestrant (a selective ER degrader) induces HER2 binding at the same ERBSs in BT474, but not MCF7 or SkBr3 cells. Collectively, these results suggest that the inhibition of either ER or HER2 can promote chromatin binding of the other at ERBSs. Integration of ER ChIP-seq peaks and differentially expressed genes from RNA-seq of BT474 cells treated with E2 vs control following short-term estrogen deprivation shows the ER cistrome is enriched for genes associated with RNA binding, RNA processing, and ribosome biogenesis. Interestingly, nuclear HER2 is reported to increase BC cell growth by activating transcription of ribosomal RNA genes. By contrast, the ER cistrome in MCF7 cells is enriched for genes associated with cell cycle regulation. In summary, anti-HER2 therapy induces recruitment of ER to well-established ERBSs, while anti-ER treatment causes recruitment of HER2 to these same sites in TPBC. Ongoing and future work leverages HER2 cistrome data to further define nuclear HER2/ER functional interactions, at the level of transcriptional regulation, that may contribute to poor treatment outcomes in TPBC. Presentation: 6/2/2024

8028 Targeted Therapy-Induced ER and HER2 Chromatin Binding: a Novel Driver of Poor Response to Treatment in Triple Positive Breast Cancer

Abstract Disclosure: S. Tam: None. M.D. McCoy: None. S. Bahnassy: None. R.B. Riggins: None. Triple positive breast cancer (TPBC) is a unique variant of breast cancer characterized by co-expression of two oncogenic drivers, estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2). Both drivers promote tumor progression and serve as targets for effective small molecule and monoclonal antibody (mAb) therapies. Previous studies showed that the co-expression of ER reduces the efficacy of anti-HER2 targeted therapy in TPBC, and vice versa. ER is a well-established nuclear receptor and transcription factor. HER2 mainly functions as a transmembrane receptor, activating cytosolic pro-tumorigenic signaling pathways. However, less-studied nuclear HER2 is involved in the transcriptional regulation of several genes, leading to increased BC cell growth and survival. In this study, we investigate the subcellular localization and transcriptional regulatory functions of ER and HER2 in the unique context of TPBC, using cell fractionation, immunofluorescence, chromatin immunoprecipitation (ChIP)-qPCR, and integrated analysis of ER ChIP-seq/RNA-seq data. We hypothesize that crosstalk between ER and HER2 in the nucleus may, in part, underlie poor response to targeted therapy. At baseline, ER has strong nuclear localization in TPBC cells, BT474 and MDA-MB-361, but is predominantly cytosolic in ER+ MCF7 cells. Activated, phospho-HER2 is also localized to the nucleus of TPBC and HER2+ SkBr3 cells at baseline. Treatment with the anti-HER2 mAb combination of trastuzumab and pertuzumab (TP) induces ER binding to specific ER binding sites (ERBSs) in the enhancers or promoters of PGR, TFF1, and FOXC1 in BT474, but this is not observed in MCF7 or SkBr3 cells. Unlike TP, which increases HER2 nuclear localization but not ERBS recruitment, fulvestrant (a selective ER degrader) induces HER2 binding at the same ERBSs in BT474, but not MCF7 or SkBr3 cells. Collectively, these results suggest that the inhibition of either ER or HER2 can promote chromatin binding of the other at ERBSs. Integration of ER ChIP-seq peaks and differentially expressed genes from RNA-seq of BT474 cells treated with E2 vs control following short-term estrogen deprivation shows the ER cistrome is enriched for genes associated with RNA binding, RNA processing, and ribosome biogenesis. Interestingly, nuclear HER2 is reported to increase BC cell growth by activating transcription of ribosomal RNA genes. By contrast, the ER cistrome in MCF7 cells is enriched for genes associated with cell cycle regulation. In summary, anti-HER2 therapy induces recruitment of ER to well-established ERBSs, while anti-ER treatment causes recruitment of HER2 to these same sites in TPBC. Ongoing and future work leverages HER2 cistrome data to further define nuclear HER2/ER functional interactions, at the level of transcriptional regulation, that may contribute to poor treatment outcomes in TPBC. Presentation: 6/2/2024

7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells

Abstract Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None. Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer. Presentation: 6/3/2024

Resistance of estrogen receptor function to BET bromodomain inhibition is mediated by transcriptional coactivator cooperativity.

The bromodomain and extraterminal domain (BET) family of proteins are critical chromatin readers that bind to acetylated histones through their bromodomains to activate transcription. Here, we reveal that bromodomain inhibition fails to repress oncogenic targets of estrogen receptor because of an intrinsic transcriptional mechanism. While bromodomains are necessary for the transcription of many genes, bromodomain-containing protein 4 (BRD4) binds to estrogen receptor binding sites and activates transcription of critical oncogenes such as MYC, independently of its bromodomains. BRD4 associates with the Mediator complex and disruption of Mediator reduces BRD4's enhancer occupancy. Profiling changes of the post-initiation RNA polymerase II (Pol II)-associated factors revealed that BET proteins regulate interactions between Pol II and elongation factors SPT5, SPT6 and the polymerase-associated factor 1 complex, which associate with BET proteins independently of their bromodomains and mediate their transcription elongation effect. Our findings highlight the importance of bromodomain-independent functions and interactions of BET proteins in the development of future therapeutic strategies.

Resistance of estrogen receptor function to BET bromodomain inhibition is mediated by transcriptional coactivator cooperativity.

The Bromodomain and Extra-Terminal Domain (BET) family of proteins are critical chromatin readers that bind to acetylated histones through their bromodomains to activate transcription. Here, we reveal that bromodomain inhibition fails to repress oncogenic targets of estrogen receptor due to an intrinsic transcriptional mechanism. While bromodomains are necessary for the transcription of many genes, BRD4 binds to estrogen receptor binding sites and activates transcription of critical oncogenes independently of its bromodomains. BRD4 associates with the Mediator complex and disruption of Mediator complex reduces BRD4's enhancer occupancy. Profiling changes in the post-initiation RNA polymerase II (Pol II)-associated factors revealed that BET proteins regulate interactions between Pol II and elongation factors SPT5, SPT6, and PAF1 complex, which associate with BET proteins independently of their bromodomains and mediate their transcription elongation effect. Our findings highlight the importance of bromodomain-independent functions and interactions of BET proteins in the development of future therapeutic strategies.

Abstract 3505: Classification of cancer subtypes by cfDNA fragmentomics analysis

Abstract Introduction Fragmentation patterns of cell-free DNA (cfDNA) from whole genome sequencing offer insights into nucleosome occupancy, providing a tool to infer epigenomic and transcriptomic information. The nucleosome complex, the primary protector of cfDNA, is reflected in the size distribution of cfDNA fragments, with a mode fragment size of 167 bp corresponding to the wrapping of DNA around a single nucleosome. Nucleosome occupancy patterns at transcription factor binding sites (TFBS) exhibit differences between circulating tumor DNA in cancer patients and normal plasma controls, as well as among different tumor subtypes. Therefore, cfDNA fragmentomics can be employed for cancer subtyping analysis. Methods We utilized the Griffin framework to classify tumor subtypes based on nucleosome profiling of cancer-specific TFBS and tumor subtype-specific chromatin accessibility regions from low-pass whole genome sequencing data of cfDNA. For prostate cancer subtyping, AR and ASCL1 binding sites were used to distinguish between androgen receptor-dependent prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). ER and ERBB2 were employed for breast cancer subtyping to differentiate between ER-positive and ER-negative tumor subtypes. Results Nucleosome profiling patterns at AR binding sites (ARBS) were compared among over 500 prostate cancer plasma samples and 42 normal plasma backgrounds. The ARBS nucleosome profiling abnormality score (ARBS score) was quantified by comparing its GC-corrected fragment central coverage with the normal background using a standard Z-score.Majority of prostate cancer samples with a high ARBS score could be classified as ARPC. For 12 samples with high tumor fraction but low ARBS score and no AR copy number amplification, we found that 8 had a high ASCL1 nucleosome profiling abnormality score. All these samples also exhibited RB1 copy number loss, suggesting that these 8 samples likely belong to NEPC. Nucleosome profiling of 171 breast cancer plasma samples at breast cancer-specific ER binding sites revealed that majority of samples had a high ERBS abnormality score, indicating likely ER-positive breast cancer. Conclusion Our results demonstrate that nucleosome profiling of tumor cfDNA exhibits distinct patterns across different tumor types. Integrating nucleosome profiling at cancer-specific transcription factor binding sites can facilitate cancer subtyping, highlighting its potential clinical utility for patient stratification. Citation Format: Lisha Zhu, Chao Dai, Shuang Gan, Shidong Jia, Pan Du. Classification of cancer subtypes by cfDNA fragmentomics analysis [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 3505.

Anticancerous Properties of Tetragonula travancorica (stingless bee) Honey on Reproductive Cancer Cells

Honey from stingless bee is highly regarded as a medicine in many global communities due to its nutritional and therapeutic value. There is a comparatively restricted amount of research on the biological characteristics of stingless bee honey (SBH) in contrast to Indian bee honey (IBH). Therefore, the objective of this research is to explore the anti-cancer properties of Tetragonula travancorica honey. In the present investigation, antioxidant activity of honey was evaluated using ABTS, DPPH and SOD radical scavenging assays. Anti-proliferative effects was examined on various cell lines using the MTT assay. Liquid chromatography-mass spectrometry (LCMS) analysis was employed to characterize the honey. Binding capabilities of compounds identified in SBH on estrogen receptors (ER) α and ER β were explored through Schrödinger Maestro software. Results indicated that SBH effectively scavenged ABTS, DPPH and SOD free radicals with IC50 values of 22, 36.15, and 79.85 v/v, respectively. SBH exhibited anti-proliferative activity against MCF-7 and HeLa cell lines, with IC50 values of 58.11 and 66.68 v/v, respectively. LCMS analysis, shows the presence of methyl syringate, 2-hydroxycinnamic acid, and fumaric acid in SBH which were not present in IBH. Docking experiments determined that these compounds, with the exception of fumaric acid, interacted stronger with the binding sites of ER β than ER α. The comprehensive findings from antioxidant, anti-proliferative, and docking studies underscore the potential anti-cancer properties of Tetragonula travancorica honey, particularly its heightened cytotoxicity against reproductive system cancer cell lines, such as those in the breast and cervix. Keywords : Anti-proliferative, estrogen receptor, stingless bee, cytotoxicity.

THU558 Combination Therapy Of Acetyl-CoA Synthetase Inhibitor And Fulvestrant Reverse Epigenetic Alterations Of Metastatic Breast Cancer Cells And Reduce Cancer Cell Proliferation

Abstract Disclosure: A.N. Mogol: None. Z. Madak Erdogan: None. Endocrine therapies exploit the hormone dependencies of the cancer cells in various ways, including by targeting estrogen receptor alpha (ERα) that is expressed by approximately 70% of human breast cancers. After treatment, recurrence and metastasis are still observed in 30-40% of ER+ patients and the 5-year relative overall survival rate for these patients is just 24%. Acetyl-CoA production significantly regulates histone acetylation and gene regulation, which support tumor progression. In our previous study, we showed the synergistic effect of the ERα agonist Fulvestrant (Fulv) and Acetyl-CoA Synthase Inhibitor (ACSI) in reducing cell viability in ER+ metastatic breast cancer (MBC) cells in vitro. To study the synergistic contribution of ACSS2 to endocrine resistance, we used ChIP analysis, which showed that ACSS2 was recruited to ERα binding sites with Fulv treatment in cell line xenograft tumors and PDX samples. We used the CUT&RUN method to investigate specific protein (H3k27ac, ERα and ACSS2) and DNA interactions in ER+ MBC cells globally. Enriched chromatin samples were then used for qPCR and sequencing. Combination therapy of the ACSI and Fulv significantly altered the epigenetic landscape of the breast cancer cells in a manner to decrease cell viability. High histone acetylation at lysine 27 residue was observed in Veh treatment group and combination therapy of ACSI+Fulv further decreased the enrichment of this histone modification in classical ER binding sites. Interestingly, ACSS2 binding was increased with Fulv treatment. Targeting epigenetic alterations caused by cancer cell adaptations is a novel approach that could potentially lead to new therapies to reduce metastasis mortality. Presentation: Thursday, June 15, 2023

Identification of a novel immune-related long noncoding RNA in carp primary macrophages associated with bisphenol A’ s immunoregulatory effects

Increasing evidence suggests that long non-coding RNAs (lncRNAs) play pivotal roles in various biological processes. However, current studies on lncRNAs mostly focus on mammalian species, with little research on the functional roles of lncRNAs in teleost fish. Here, we identified a novel intergenic lncRNA (linc-93.2) in the head kidney primary macrophages of common carp (Cyprinus carpio) after exposure to a typical environmental endocrine disrupting chemical, bisphenol A (BPA). As a result, linc-93.2 was more than 3,619 bp in length and predominantly localized to the nucleus of primary macrophages other than cytoplasm, with the highest expression level in spleen followed by head kidney among different organs. Bioinformatic analysis predicted a cis-target gene, dennd1b, and 20 trans-target genes including hsp70, gna13 and rasgap, were potentially regulated by linc-93.2; NFκB and estrogen receptor (ERα) binding sites were located in the promoter region upstream of its transcription start site, which together suggested the involvement of linc-93.2 in immune and neurological functions in fish. Based on that, the expression level of linc-93.2 was determined in macrophages following acute lipopolysaccharide (LPS) and BPA treatments, both of which significantly induced linc-93.2 and IL-1β expression in cells. Moreover, a NF-κB inhibitor PDTC significantly reduced linc-93.2 expression in macrophages, but co-exposure of macrophages to PDTC with BPA or LPS could significantly rescue linc-93.2 expression, consistent with the observation on that LPS or BPA alone significantly induced both linc-93.2 and its target gene expression. Interestingly, linc-93.2 and its target gene expression was significantly suppressed by an ER antagonist ICI 182,780, however, the co-exposure of macrophages to ICI 182,780 with BPA failed to attenuate their declined expression. Overall, the current study demonstrated that linc-93.2, a novel immune-related lncRNA, may participate in the immune processes of common carp macrophages via the NF-κB and ER pathway. The results presented in this study enhance our understanding of the immunotoxin mechanisms of BPA in teleost fish.

A green synthesis, DFT calculations, and molecular docking study of some new indeno[2,1-b]quinoxalines containing thiazole moiety

A series of new indenoquinoxaline-thiazole hybrids were synthesized by combining indenoquinoxaline thiosemicarbazone with appropriate hydrazonoyl chlorides or α-halo ketones using DABCO as a catalyst. The short reaction times and excellent yields make this method simple, green, and economic to synthesize new compounds with potential biological activities. FT IR, NMR, and MS spectrum data are some of the spectroscopic techniques used to confirm the structure of the synthesized hybrids. In addition, density functional theory (DFT) calculations were conducted to study the geometry optimization of some representative compounds, and their electronic transitions in DMSO were investigated by performing time-dependent DFT calculations. Additionally, a molecular docking study was carried out to investigate the possible interactions and binding mode of the newly synthesized compounds to the estrogen receptor (ER) binding site to estimate their anticancer activity against hormonally driven breast cancer.

Abstract 3079: Characterization of ER-AF2 inhibitors in breast cancer

Abstract Up to 80% of breast cancers (BCa) rely on the estrogen receptor (ER) for their growth and progression. This dependence on ER has led to many hormonal therapies that target this receptor. However, almost 40% of these cancers will acquire resistance over the course of the treatment period. One potential cause of resistance is mutations in the estrogen binding site (EBS) of ER. As such, there is an increasing need for novel inhibitors that targets ER at a site separate from the EBS. Here, we propose targeting the activation-function (AF2) pocket of ER that is important for cofactor binding and transcription activation. Billions of compounds were screened through an in-silico deep docking method, and potential AF2 inhibitors were then validated in cell-based and biophysical assays. We tested the effect of potential AF2 inhibitors on ER transcriptional activity using luciferase reporter assay in ER-positive T47D-kbluc cells. We then evaluated the effect of molecules on cell viability of ER-positive T47D and ER-negative MDA-MB-231 cells using PrestoBlue assays in order to exclude off-target effects. From these cell-based assays, we identified several inhibitors that effectively reduced transcriptional activity and viability in ER-positive T47D cells at low micromolar concentrations. We conducted PGC-1α peptide displacement assay to confirm their AF2 binding and estradiol displacement assays to exclude any binding to the EBS. Proximity ligation assay (PLA) showed disruption of the interaction between ER and coactivator SRC-3 upon treatment with ER-AF2 inhibitors in T47D cells. Current work focuses on confirming the direct binding between the compounds and recombinant ER-ligand binding domain by various biophysical assays (MST, BLI, and ITC). Future work aims to solve the structure of ER-LBD in a complex with our lead compound by X-ray crystallography. We predict that the use of potent ER-AF2 inhibitors along with current treatments, will provide a novel tactic that can act as a complementary therapeutic to target treatment resistance in ER+ BCa. Citation Format: Jane Foo, Francesco Gentile, Joseph Lee, Helene Morin, Shabnam Massah, Maria Guo, Jason Smith, Fuqiang Ban, Artem Cherkasov, Nada Lallous. Characterization of ER-AF2 inhibitors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3079.

RF03 | PMON289 KLF4 drives estrogen driven tumor heterogeneity in breast cancer

Abstract Whilst hormonal therapies have proved effective in targeting ER positive breast cancer, intrinsic and acquired resistance to therapy has meant that the disease remains deadly. To identify potential drivers of transcriptional and epigenetic heterogeneity in breast cancer, we performed detailed single-cell multi-ome experiments +/- estrogen stimulation. Our results identified that the transcription factor KLF4 is highly associated with heterogeneity in our experimental models. KLF4 is one of four Yamanaka pluripotency factors that can reprogram cells to a stem like state and is also a pioneer factor, that possesses the ability to bind to condensed chromatin. Silencing of KLF4 significantly reduced transcriptional heterogeneity in multiple models. Importantly, we also found that KLF4 acts as a pioneer factor for ER. ER binding, transcriptional activity and chromatin accessibility at ER binding sites were lost upon KLF4 silencing. Using proteomics based interactome profiling, we find multiple other signaling pathway rely on KLF4. Together, our results suggest that KLF4 maintains plasticity in ER+ breast cancer cells and also functions as a pivotal factor for ER to function. Presentation: Saturday, June 11, 2022 1:36 p.m. - 1:41 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Polymorphic estrogen receptor binding site causes Cd2-dependent sex bias in the susceptibility to autoimmune diseases

Complex autoimmune diseases are sexually dimorphic. An interplay between predisposing genetics and sex-related factors probably controls the sex discrepancy in the immune response, but the underlying mechanisms are unclear. Here we positionally identify a polymorphic estrogen receptor binding site that regulates Cd2 expression, leading to female-specific differences in T cell-dependent mouse models of autoimmunity. Female mice with reduced Cd2 expression have impaired autoreactive T cell responses. T cells lacking Cd2 costimulation upregulate inhibitory Lag-3. These findings help explain sexual dimorphism in human autoimmunity, as we find that CD2 polymorphisms are associated with rheumatoid arthritis and 17-β-estradiol-regulation of CD2 is conserved in human T cells. Hormonal regulation of CD2 might have implications for CD2-targeted therapy, as anti-Cd2 treatment more potently affects T cells in female mice. These results demonstrate the relevance of sex-genotype interactions, providing strong evidence for CD2 as a sex-sensitive predisposing factor in autoimmunity.

Abstract 2852: A gene activating in vivo screen identifies EBAG9 as novel metastasis regulator in human colorectal cancer

Abstract As colorectal cancer (CRC) mortality remains high due to metastatic spread in patients with advanced disease stages, we here aimed at identifying novel metastasis regulators in CRC and at understanding their mechanism of action. In CRC, tumor progression and metastasis formation are driven by cells that possess tumor-initiating cell (TIC) activity. We therefore employed a collection of primary patient-derived CRC tumor spheroid cultures (TSC) that are enriched for cells with TIC activity to perform a gene activating in vivo screen, intending to identify genes that enhance metastasis formation. To this end, TSC (n=4) were transduced with a lentiviral trapping vector that integrates into the genome and thereby drives the overexpression of genes located in the vicinity of the integration site (IS). Transduced TSC were then serially transplanted under the kidney capsule of immunodeficient mice (NSG) and monitored for the induction of metastasis formation. Analysis of lentiviral IS by linear amplification-mediated (LAM)-PCR revealed an enrichment of an IS within the gene locus of Estrogen Receptor Binding Site Associated Antigen 9 (EBAG9) in metastases. RNA sequencing confirmed the trapping vector mediated upregulation of a truncated version of the EBAG9 gene (exon 5-7), which strongly suggested a contribution of EBAG9 to metastatic spread. To validate these findings, both the truncated (TR) as well as the physiologically occurring full length (FL) EBAG9 gene were overexpressed in TSC (n=4) and serially transplanted into NSG mice. In addition to an augmentation in metastasis upon EBAG9 overexpression compared to controls (FL: 1.4-fold, TR: 2.4-fold), we also detected the occurrence of lymphogenous metastases when EBAG9 levels were increased, indicating that EBAG9 not only contributes to metastatic spread but also modulates tropism. To further characterize the underlying processes, global gene and protein expression analyses were performed in primary TSC as well as in the CRC cell line DLD1. Compared to control transduced cells, EBAG9 overexpression induced expression changes in cell adhesion and microtubule molecules, which led us to investigate cell migration and adhesion as potential contributing mechanisms to metastasis formation. We here observed an increase both in cell migration as well as in adhesion to the extracellular matrix protein collagen I upon EBAG9 overexpression in DLD1. Interestingly, DLD1 adhesion to lymphatic endothelial cells was significantly higher in EBAG9 FL (p=0.008) and EBAG9 TR (p=0.016) overexpressing cells, underscoring the role of EBAG9 in tropism modulation. In summary, we here identified EBAG9 as novel metastasis regulator in CRC which will not only contribute to a better mechanistic understanding of metastatic spread but also to the development of novel future strategies for preventing and targeting advanced disease stages. Citation Format: Karin Laaber, Taronish D. Dubash, Anna Maria Melzer, Tania Christiansen, Nati Ha, Benedikt Brors, Martin Schneider, Friederike Herbst, Hanno Glimm, Claudia R. Ball. A gene activating in vivo screen identifies EBAG9 as novel metastasis regulator in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2852.

Abstract 2611: Transcription factor-nucleosome dynamics inferred from plasma cfDNA delineates tumor and tumor-microenvironment phenotype

Abstract Our current understanding of solid tumors and their progression primarily relies on in vitro models, cell lines, patient-derived xenografts, and scarce data from invasive tissue biopsies from patients. The ability to monitor changes in chromatin structure and transcription factor binding in tumor cells using a minimally invasive approach in humans has the potential to revolutionize our understanding of disease progression and treatment resistance. In this study, we use the example of estrogen receptor (ER) positive breast cancer, the most common disease subtype, and define the ER axis from plasma cell-free DNA (cfDNA). While lymphoid/myeloid cell turnover represents the dominant source of cfDNA in the bloodstream, a detectable fraction of DNA from tumor tissue-of-origin can be found in patients with solid cancers. cfDNA is the product of the action of endogenous nucleases on chromatin; and retains the map of epigenomes from cells of origin. Our method therefore non-invasively captures TF-nucleosome dynamics in tumor tissue-of-origin using plasma cfDNA. First, we show that we can reliably identify the active binding of hematopoietic pioneer factor PU.1 and CTCF from cfDNA of healthy humans and cancer patients. Then to define cfDNA binding of disease specific TF ER, we used ER+ patient-derived xenograft (PDX) models allowing for a clear separation of tumor signal from hematopoietic background. This allowed us to establish the sensitivity and specificity of our approach. We also identified the subset of CUT&RUN-defined ER binding sites that feature the strongest binding in vivo from both lymphocyte background as well as cancer cells. Furthermore, we can define the active binding sites of pioneer factor FOXA1, which facilitates ER binding by opening the chromatin. Based on the TF protection levels from cfDNA we were able to define tumor as well as hematopoietic-specific TF binding sites that can serve as potential hotspots to monitor ER+ disease state at around 1% tumor fraction. These data demonstrate our ability to simultaneously monitor TF and nucleosome dynamics at disease sites just from plasma that can enable real-time monitoring of disease phenotype in a minimally invasive manner. Citation Format: Satyanarayan Rao, Amy Han, Alexis Zukowski, Etana Kopin, Peter Kabos, Srinivas Ramachandran. Transcription factor-nucleosome dynamics inferred from plasma cfDNA delineates tumor and tumor-microenvironment phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2611.

Abstract 2848: Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer

Abstract Background: Hotspot estrogen receptor-α (ER/ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer and are associated with worse outcome. How these mutations facilitate metastasis remains ambiguous. It is necessary to identify a clear mechanism for therapeutic intervention. Methods: ESR1 mutations were detected by ddPCR. Transcriptome data were derived from cell lines and clinical samples. Y537S and D538G genome-edited MCF7 and T47D cell lines were used for in vitro phenotypic characterization. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and ibidi microfluidic assays. Altered cell-cell adhesion genes were validated using qRT-PCR, immunoblot and immunostaining. Desmosome blocking peptides and carbenoxolone were used for blockade of adhesion. ER cistromes were profiled by ChIP-seq. Tail vein injection was performed on athymic nude mice to evaluate metastasis in vivo. Circulating tumor cell (CTC) clustering propensity in vivo was assessed via intracardiac injection followed by CTC microfilter capture. CTC-cluster gene signatures were generated from ER+ breast cancer CTC RNA-seq data. Results: ESR1 mutations were significantly enriched in distant metastases (12/48) vs local (0/27) recurrences, confirming their critical role in promoting metastasis. Transcriptomic analysis revealed altered cell-cell interaction pathways in ESR1 mutant tumors. ESR1 mutant cells formed more compact spheroids in suspension culture, and exhibited stronger cell-cell adhesion in static condition. Under microfluidic condition with physiological shear stress, mutant ESR1 cells derived more and larger clusters, which were prominently preserved from pre-existing clusters. This effect was correlated with increased expression of desmosome and gap junction genes in mutant cells and pharmacological blockade significantly reduced the enhanced cell-cell adhesion. ER ChIP-seq revealed no de novo mutant ER binding sites at the loci of the target genes, suggesting indirect regulation by mutant ER. This was exemplified by a secondary regulation from cFos/AP1 signaling of GJA1 expression, and an epigenetic regulation at DSC1/DSG1 loci through enhanced H3K4me2 and H3K27ac modification. In vivo studies showed ESR1 mutant cells derived more distant metastases, and MCF7 Y537S cells formed larger CTC clusters with increased compactness compared to WT cells. Finally, ER+ CTC-cluster signatures were enriched in ESR1 mutant tumors, suggesting their unique dependence on this pathway during metastasis. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer enhanced cell-cell adhesion, which facilitates breast cancer metastasis via increased CTCs clustering propensity. These findings provide insights to the development of drugs targeting gap junction in ER mutant tumors. Citation Format: Yang Wu, Zheqi Li, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan M. Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben Ho Park, Jennifer M. Atkinson, Dorraya El-Ashry, Adrian V. Lee, Steffi Oesterreich. Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2848.

Sex hormones regulate lipid metabolism in adult Sertoli cells: A genome-wide study of estrogen and androgen receptor binding sites

Optimal functioning of Sertoli cells is crucial for spermatogenesis which is under tight regulation of sex hormones, estrogen and androgen. Adult rat Sertoli cells expresses estrogen receptor beta (ERβ) and androgen receptor (AR), both of which regulate gene transcription by binding to the DNA. The present study is aimed to acquire a genome-wide map of estrogen- and androgen-regulated genes in adult Sertoli cells. ChIP-Seq was performed for ERβ and AR in Sertoli cells under physiological conditions. 30,859 peaks in ERβ and 9,594 peaks in AR were identified with a fold enrichment >2 fold. Pathway analysis for the genes revealed metabolic pathways to be significantly enriched. Since Sertoli cells have supportive functions and provide energy substrates to germ cells during spermatogenesis, significantly enriched metabolic pathways were explored further. Peaks of the genes involved in lipid metabolism, like fatty acid, glyceride, leucine, and sphingosine metabolism were validated. Motif analysis confirmed the presence of estrogen- and androgen-response elements (EREs and AREs). Moreover, transcript levels of enzymes involved in the lipid metabolic pathways were significantly altered in cultured Sertoli cells treated with estrogen and androgen receptor agonists, demonstrating functional significance of these binding sites. This study elucidates a mechanism by which sex hormones regulate lipid metabolism in Sertoli cells by transcriptionally controlling the expression of these genes, thereby shedding light on the roles of these hormones in male fertility.

Abstract PD8-09: Androgen and ESR1 mutant receptors mechanistically collaborate for overexpression of genes associated with poor outcomes in ER-positive metastatic breast cancer

Abstract Background: Recurrences are delayed for years with endocrine therapy (ET), but resistance ultimately evolves with the development of ER+ metastatic breast cancer (MBC). ESR1 mutations are acquired in MBC undergoing ET in about 20-40% of patients. We have previously demonstrated that AR overexpression confers resistance to both tamoxifen and aromatase inhibitor treatments. We discovered that AR protein is up-regulated in tumors expressing ESR1 mutations, and herein explore the functional consequences of AR-ESR1 mutant co-expression on metastatic progression of breast cancer. Objective: We hypothesize that ET resistance mechanisms driven by AR overexpression and acquisition of ESR1 mutations co-evolve under the selective pressure of therapy to drive distant metastasis. We investigated whether AR engages in novel genomic binding interactions with mutant ER to promote tumor progression. Methods: Expression profiling of CRISPR-Cas9 engineered cell lines was integrated with chromatin immunoprecipitation-sequencing (ChIP-Seq) to define the genomic distribution of ER and AR binding sites. Genomic distribution of AR-ER co-occupied binding sites from MBC patient tumors, and a gene expression signature associated with patient outcomes was generated using using Cox Regression analysis to calculate Hazard Ratios for recurrence free survival from gene expression in the KMPlotter Breast Cancer Dataset and signature validation by Kaplan Meier analysis in the ER+ METABRIC patient cohort. Results: AR protein was post-transcriptionally elevated in ESR1 mutant cells, including distant metastatic tumors from ESR1 mutant xengraft models. Transcriptome data showed significant elevation of Cancer Hallmark pathways, including epithelial-mesenchymal transition (EMT) and estrogen response, but down-regulation of androgen response genes. However, both ER and AR functions were constitutively activated, independent of their respective ligands in ESR1 mutant-expressing models. AR was disproportionally re-distributed to selected enhancer genomic regions, but also heterochromatin in ESR1 mutant compared to cells with WT ER in the presence of estrogen. In addition, AR-ER co-occupied sites were increased in enhancer regions in ESR1 mutant cells, and these sites best discriminated distal ER enhancer regions in non-responder patients and MBC. . The expression of genes co-bound by AR and ER were significantly associated with shorter recurrence-free and overall survival in ER+ breast cancer. Treatment with AR agonists blocked distant metastasis of ESR1 mutant PDX models in vivo, in part due to reduction in ER protein and ER binding to select genomic sites. Conclusions: Our results demonstrate that unliganded AR collaborates with ESR1 mutations to regulate genes associated with the metastasis of ER+ breast cancer. We hypothesize that in an ESR1 mutant genomic background, AR acquires ER co-activator functions that are blocked with AR agonists, suggesting a unique hormonal therapeutic vulnerability in tumors with ESR1 mutations. Citation Format: Suzanne Fuqua, Thomas L Gonzalez, Guowei Gu, Sarah Herzog, Yassine Rechoum, Amanda Beyer, David Edwards, Hangqing Lin, BAsil Paul, Ralf Kittler, Nancy Weigel, Sandra Grimm, Cristian Coarfa, Tasneem Bawa-Khalfe. Androgen and ESR1 mutant receptors mechanistically collaborate for overexpression of genes associated with poor outcomes in ER-positive metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-09.

Abstract PS17-32: Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature that consists of two functional biomarker panels, HOXB13/IL17BR (H/I) and Molecular Grade Index (MGI), that interrogate important proliferation and estrogen signaling pathways in breast cancer. The BCI prognostic score reports individualized risk of overall and late distant recurrence and is based on the algorithmic combination of the H/I ratio and MGI, while the predictive component, BCI (H/I), reports a categorical prediction of high versus low likelihood of benefit from extended endocrine therapy.The homeobox transcription factor HOXB13 has previously been shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis, where it colocalizes with FOXA1 at reprogrammed AR binding sites. The aim of the current study was to characterize the potential role of HOXB13 in estrogen receptor (ER) reprogramming by analyzing changes in the global ER binding pattern induced by transient overexpression of HOXB13 in breast cancer cells. In addition, gene ontology (GO) analysis was performed to delineate the functional role of HOXB13 in modulating estrogen signaling and response to endocrine therapy.Methods: HOXB13 was overexpressed in MCF-7 cells by electroporating HOXB13 mRNA, or eGFP mRNA as control. Cells were harvested at different time points and analyzed by western blot and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) using antibodies against ER, HOXB13, FOXA1 and H3K27ac. After aligning reads with Bowtie2, peak calling and data integration were performed using HOMER v4.10. Gene ontology (GO) analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT v4.0.4) (http://great.stanford.edu/) to identify annotations enriched among genes near ER genomic binding sites.Results: ChIP-seq analysis revealed substantial binding of HOXB13 to a large number of genomic binding sites compared to the eGFP control. HOXB13 overexpression in ER+ breast cancer cells significantly increased ER binding with a majority of ER binding sites being co-bound by FOXA1 and HOXB13. GO analysis of both proximal and distal genomic regions showed significant enrichment of genes associated with mammary gland development, such as “mammary gland epithelium development”, “mammary gland development”, and “mammary gland epithelial cell differentiation”.Conclusion: Findings from this analysis show that transient HOXB13 overexpression reprograms and expands the ER binding pattern in breast cancer cells. Genomic regions newly bound by ER are enriched for genes involved in mammary gland and epithelial differentiation suggesting that HOXB13 activates ER transcriptional programs that link oncogenic and developmental pathways. These results will be compared to ongoing experiments using an inducible HOXB13 expression system to assess the effects of HOXB13 expression on ER binding and function in response to estradiol and tamoxifen. Citation Format: Kai Treuner, Dominic Schenone, Christopher Benner, Catherine A Schnabel, Sven Heinz. Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-32.