Estrogen-induced Gene Research Articles

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

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

Abstract 1886: BRY812, an anti-LIV-1 antibody drug conjugate with novel conjugation method for cancer therapeutics

Abstract LIV-1, also known as SLC39A6 or ZIP6, belongs to the zinc transporter family and was initially identified as an estrogen-inducible gene in breast cancer. Immunohistochemical (IHC) analysis have revealed escalated LIV-1 expression in estrogen receptor-positive (ER+), hormone-treated tumors (both primary and metastatic sites) as well as ER-/PR-/Her2- (triple-negative) breast cancers. Notably, healthy human tissues exhibit limited LIV-1 expression, primarily in hormone regulated organs (prostate, uterus, and breast). The escalated expression of LIV-1 in breast and prostate cancer, as well as melanoma and other tumors, positions LIV-1 as an excellent target for ADC therapeutics. BRY812 is a monomethyl auristatin E (MMAE)-conjugated humanized IgG1 ADC against LIV-1. With Bioray’s irreversible conjugation method CysLinkTM, BRY812 enables superior in vivo stability and eliminates off-target payload migration. The PK studies in rat and monkey demonstrated that the free MMAE released from ADC is 1 to 2 logs lower compared with approved ADCs prepared by the standard conjugation method. BRY812 is well tolerated in cynomolgus monkeys with repeated doses. With best-in-class anti-tumor efficacy in various models, BRY812 demonstrated a wider therapeutic window for cancer treatment. BRY812 is currently in Phase Ia dose escalation stage clinical trial to explore the maximum tolerate dosage (MTD) from 0.25 to 3.6 mg/kg Q3W in patients with advanced malignancies. Citation Format: Xiaobei Zhao, Jie Zhu, Yaqiong Zhou, Jing Li, Lei Nie, Haibin Wang, Gang Chen. BRY812, an anti-LIV-1 antibody drug conjugate with novel conjugation method for cancer therapeutics [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 1886.

The role of mesenchymal estrogen receptor 1 in mouse uterus in response to estrogen

Estrogens play important roles in uterine growth and homeostasis through estrogen receptors (ESR1 and ESR2). To address the role of ESR1-mediated tissue events in the murine uterus, we analyzed mice with a mesenchymal tissue-specific knockout of Esr1. Isl1-driven Cre expression generated Esr1 deletion in the uterine stroma and endometrium (Isl-Esr1KO). We showed that overall structure of the Isl1-Esr1KO mouse uterus developed normally, but estrogen responsiveness and subsequent growth were defective, suggesting that mesenchymal ESR1 is necessary for both epithelial and mesenchymal cell proliferation. Furthermore, RNA-seq analysis revealed that the majority of estrogen-induced genes were regulated by stromal ESR1. In control mice, E2 administration induced 9476 up-regulated differentially expressed genes (DEGs), whereas only 1801 up-regulated DEGs were induced by E2 in Isl1-Esr1KO mice. We further showed that stromal ESR1-regulated genes in the mouse uterus included several growth factors and cytokines, which are potential factors that regulate epithelial and stromal tissue interaction, and also genes involved in lipid homeostasis. Therefore, we infer that stromal ESR1 expression is indispensable for most estrogen actions in the mouse uterus and the current results provide new insights into estrogen-mediated homeostasis in female reproductive organs.

Abstract B075: Anti-human LIV-1 antibody drug conjugate for treatment of metastatic prostate cancer

Abstract In the worldwide male population, prostate cancer has the most frequent malignancy; out of all the cancer-related causes of deaths, it is also one of the most common. With the advance of new therapies of androgen receptor pathway inhibitors, patient survival rates have improved significantly. There are still tremendous unmet needs, however, for the patients with treat-refractory metastatic castration-resistant prostate cancer (mCRPC). LIV-1, also known as SLC39A6 or ZIP6, is a member of the zinc transporter family and was first identified as an estrogen-inducible gene in breast cancer. Previous studies with immunohistochemical (IHC) analysis showed that LIV-1 is expressed by estrogen receptor-positive (ER+), hormone-treated tumors (both primary and metastatic sites) and ER-/PR-/Her2- (triple-negative) breast cancers. These studies also showed that in healthy human tissues, LIV-1 expression is limited to hormonally-regulated organs (prostate, uterus, and breast). The broad expression of LIV-1 in prostate and breast cancer tumors in combination with the limited expression in vital organs makes LIV-1 an excellent target for an antibody-drug conjugate (ADC). We generated an ADC, BRY812, consisting of a humanized anti-LIV-1 mAb conjugated to monomethyl auristatin E (MMAE), via a novel conjugation method that prevents MMAE from coming off of the antibody during the circulation. The PK studies in rat and monkey showed that the free MMAE released from ADC is 1 to 2 log lower compared with approved ADCs prepared by the standard conjugation method. Low free MMAE concentration significantly lowers the risk of off target toxicity by the ADC. In vitro and in vivo studies demonstrated the antitumor activity of BRY812 for the treatment of prostate and ER+ and triple-negative breast cancers with a better safety profile and a larger therapeutic window. The humanized LIV-1 ADC, BRY812 is currently in preclinical toxicity studies and will advance to First-in-Human trials in April, 2023. Citation Format: Xiaobei Zhao, Gang Chen. Anti-human LIV-1 antibody drug conjugate for treatment of metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B075.

Coagulation Factor V (F5) is an Estrogen-Responsive Gene in Breast Cancer Cells.

Most breast cancers express estrogen receptor (ER) where estrogen signaling plays an important role. Cancer contributes to activation of the coagulation system leading to an imbalance in the hemostatic system, and coagulation factor (F) V, which is a key regulator of blood coagulation, has been shown to be increased in breast tumors. Thus, the molecular association between estrogens and FV was explored. Stimulation with 17-β-estradiol (E2) or 17-β-ethinylestradiol (EE2) resulted in a time- and dose-dependent increase in F5 messenger RNA and FV protein in ERα-positive MCF-7 cells. Pretreatment with the ER antagonist fulvestrant or knockdown of ERα prior to stimulation with E2 counteracted this effect. Three ERα-binding half-sites were identified in the promoter region of the F5 gene in silico. Reporter gene analysis showed that all three half-sites were involved in the estrogen-induced gene regulation in vitro, as the effect was abolished only when all half-sites were mutated. High F5 levels in ER-positive breast tumors were associated with increased relapse-free survival of breast cancer patients.

Long non-coding RNA H19 mediates N-acetyltransferase 1 gene methylation in the development of tamoxifen resistance in breast cancer.

Long non-coding RNA (lncRNA) H19 is associated with proliferation, invasion and metastasis in numerous types of cancer. H19 lncRNA has been demonstrated to be an estrogen-inducible gene, the expression of which is significantly increased in tamoxifen (TAM)-resistant MCF-7 breast cancer cells. The aim of the present study was to investigate the role and molecular mechanism of lncRNA H19 in the development of TAM resistance. TAM-resistant MCF-7 (MCF-7R) cells were developed by the treatment of wild-type MCF-7 cells with 4-hydroxytamoxifen. Analysis of H19 expression in the cells indicated that upregulation of H19 contributed to the resistance of the MCF-7R cell line. Furthermore, when H19 was knocked down in the MCF-7R cells, the sensitivity to 4-hydroxytamoxifen was markedly restored. The results further demonstrated that N-acetyltransferase 1 (NAT1) may serve an important role in TAM-resistant cells, as NAT1 expression was notably downregulated in the MCF-7R cells but significantly elevated following the knockdown of H19. In addition, lower expression of NAT1 and higher expression of H19 were indicated to be associated with poor prognosis in patients with breast cancer treated with TAM. The results of bisulfite genomic sequencing PCR analysis indicated that the methylation rate of NAT1 in MCF-7R cells was significantly higher compared with that in MCF-7 cells, while the methylation rate of NAT1 in TAM-resistant cells transfected with small interfering RNA against H19 was significantly lower than that in the corresponding untransfected cells. Therefore, the present study suggests that the H19 gene regulates NAT1 expression in TAM-resistant cells via the mediation of NAT1 promoter methylation.

Combined Use of Immunoreactivities of RIG-I with Efp/TRIM25 for Predicting Prognosis of Patients With Estrogen Receptor-positive Breast Cancer

BackgroundWe previously identified estrogen-responsive finger protein (Efp) as an estrogen-induced gene, and showed that the positive immunoreactivity of Efp is a poor prognostic factor for patients with breast cancer. We also demonstrated that Efp has distinctive roles in innate immunity by activating pattern recognition receptor retinoic acid-inducible gene I (RIG-I). The clinical value of RIG-I protein expression in breast cancer had not been evaluated in relationship with patients’ prognosis. Patients and MethodsTissue samples of estrogen receptor-positive invasive breast cancer were obtained from 145 female patients with breast cancer who underwent surgical treatment. Immunoreactivities of RIG-I and Efp were analyzed with the antibodies generated for the present study. ResultsPositive immunoreactivity of RIG-I was correlated with lower disease-free survival (P = .032) and was an independent poor prognostic factor (P = .043). RIG-I immunoreactivity was positively correlated with that of Efp (P = .0004). Patients with positive immunoreactivities of both RIG-I and Efp proteins were associated with a lower disease-free survival rate (P = .005). ConclusionsPositive immunoreactivity of RIG-I has clinical significance as a poor prognostic factor in patients with estrogen receptor-positive breast cancer. A positive correlation of RIG-I and Efp immunoreactivities was observed, and the combination of their immunoreactivities can be used to predict patients’ prognosis.

Estrogen receptor-dependent and independent roles of benzo[a]pyrene in Ishikawa cells.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants generated from the incomplete combustion of organic material. PAHs have been studied as genotoxicants, but some also act via non-genotoxic mechanisms in estrogen-dependent malignancies, such as breast cancer. PAHs require metabolic activation to electrophilic metabolites to exert their genotoxicity but non-genotoxic properties may also contribute to their carcinogenicity. The role of PAHs in endometrial cancer, a cancer associated with unopposed estrogen action is unknown. We assessed the metabolism of the representative PAH, benzo[a]pyrene (B[a]P), to estrogenic compounds in Ishikawa human endometrial cells in the presence and absence of cytochrome P450 induction. Using stable-isotope dilution high-performance liquid chromatography and APCI tandem mass spectrometry in the selected reaction monitoring mode, we analyzed B[a]P metabolism in Ishikawa cells. Estrogenic activity of B[a]P metabolites was determined by the endogenous estrogen inducible alkaline phosphatase reporter gene and an exogenous estrogen response element (ERE) luciferase reporter gene construct. We also assessed whether PAHs can induce a proliferative phenotype via estrogen receptor (ER)- and non-ER-regulated pathways. We demonstrate that B[a]P can be metabolized in human endometrial cells into 3-OH-B[a]P and B[a]P-7,8-dione in sufficient amounts to activate ERs. We also show that only B[a]P-7,8-dione induces endometrial cell proliferation at concentrations lower than required to activate the ER; instead non-genomic signaling by the EGF receptor (EGFR) and activation of the mitogen-activated protein kinase (MAPK) pathway was responsible. This work indicates that human endometrial cells can metabolize PAHs into estrogenic metabolites, which may induce cell proliferation through non-ER-regulated pathways.

An H3K4me3 reader, BAP18 as an adaptor of COMPASS-like core subunits co-activates ERα action and associates with the sensitivity of antiestrogen in breast cancer.

Estrogen receptor alpha (ERα) signaling pathway is essential for ERα-positive breast cancer progression and endocrine therapy resistance. Bromodomain PHD Finger Transcription Factor (BPTF) associated protein of 18kDa (BAP18) has been recognized as a crucial H3K4me3 reader. However, the whole genomic occupation of BAP18 and its biological function in breast cancer is still elusive. Here, we found that higher expression of BAP18 in ERα-positive breast cancer is positively correlated with poor prognosis. ChIP-seq analysis further demonstrated that the half estrogen response elements (EREs) and the CCCTC binding factor (CTCF) binding sites are the significant enrichment sites found in estrogen-induced BAP18 binding sites. Also, we provide the evidence to demonstrate that BAP18 as a novel co-activator of ERα is required for the recruitment of COMPASS-like core subunits to the cis-regulatory element of ERα target genes in breast cancer cells. BAP18 is recruited to the promoter regions of estrogen-induced genes, accompanied with the enrichment of the lysine 4-trimethylated histone H3 tail (H3K4me3) in the presence of E2. Furthermore, BAP18 promotes cell growth and associates the sensitivity of antiestrogen in ERα-positive breast cancer. Our data suggest that BAP18 facilitates the association between ERα and COMPASS-like core subunits, which might be an essential epigenetic therapeutic target for breast cancer.

The proapoptotic gene interferon regulatory factor-1 mediates the antiproliferative outcome of paired box 2 gene and tamoxifen

Tamoxifen is the most prescribed selective estrogen receptor (ER) modulator in patients with ER-positive breast cancers. Tamoxifen requires the transcription factor paired box 2 protein (PAX2) to repress the transcription of ERBB2/HER2. Now, we identified that PAX2 inhibits cell growth of ER+/HER2− tumor cells in a dose-dependent manner. Moreover, we have identified that cell growth inhibition can be achieved by expressing moderate levels of PAX2 in combination with tamoxifen treatment. Global run-on sequencing of cells overexpressing PAX2, when coupled with PAX2 ChIP-seq, identified common targets regulated by both PAX2 and tamoxifen. The data revealed that PAX2 can inhibit estrogen-induced gene transcription and this effect is enhanced by tamoxifen, suggesting that they converge on repression of the same targets. Moreover, PAX2 and tamoxifen have an additive effect and both induce coding genes and enhancer RNAs (eRNAs). PAX2–tamoxifen upregulated genes are also enriched with PAX2 eRNAs. The enrichment of eRNAs is associated with the highest expression of genes that positivity regulate apoptotic processes. In luminal tumors, the expression of a subset of these proapoptotic genes predicts good outcome and their expression are significantly reduced in tumors of patients with relapse to tamoxifen treatment. Mechanistically, PAX2 and tamoxifen coexert an antitumoral effect by maintaining high levels of transcription of tumor suppressors that promote cell death. The apoptotic effect is mediated in large part by the gene interferon regulatory factor 1. Altogether, we conclude that PAX2 contributes to better clinical outcome in tamoxifen treated ER-positive breast cancer patients by repressing estrogen signaling and inducing cell death related pathways.

Endometrial biomarkers in premenopausal women with obesity: an at-risk cohort

Obesity is a well-known risk factor for endometrial cancer, but the mechanisms of obesity-related carcinogenesis are not well defined, particularly for premenopausal women. With the continuing obesity epidemic, increases in the incidence of endometrial cancer and a younger age of diagnosis are often attributed to a hyperestrogenic state created by hormone production in adipose tissue, but significant knowledge gaps remain. The balance of estrogen-responsive signals has not been defined in the endometrium of premenopausal women with obesity, where obesity may not create hyperestrogenism in the context of ovaries being the primary source of estrogen production. Obesity is associated with a state of low-grade, chronic inflammation that can promote tumorigenesis, and it is also known that hormonal changes alter the immune microenvironment of the endometrium. However, limited research has been conducted on endometrial immune-response changes in women who have an increased risk for cancer due to obesity. Endometrial estrogen-regulated biomarkers, previously shown to be dysregulated in endometrial cancer, were evaluated in a cohort of premenopausal women to determine if obesity is associated with differences in the biomarker expression levels, which might reflect an altered risk of developing cancer. The expression of a multiplexed panel of immune-related genes was also evaluated for expression differences related to obesity. Premenopausal women with a body mass index of ≥30 kg/m2 (n=97) or a body mass index of ≤25 kg/m2 (n=33) were prospectively enrolled in this cross-sectional study, which included the assessment of serum metabolic markers and a timed endometrial biopsy for pathologic evaluation, hormone-regulated biomarker analysis, and immune response gene expression analysis. Medical and gynecologic histories were obtained. Endometrial gene expression markers were also compared across the body mass index groups in a previous cohort of premenopausal women with an inherited cancer risk (Lynch syndrome). In addition to known systemic metabolic differences, histologically normal endometria from women with obesity showed a decrease in gene expression of progesterone receptor (P=.0027) and the estrogen-induced genes retinaldehyde dehydrogenase 2 (P=.008), insulin-like growth factor 1 (P=.016), and survivin (P=.042) when compared with women without obesity. The endometrial biomarkers insulin-like growth factor 1, survivin, and progesterone receptor remained statistically significant in multivariate linear regression models. In contrast, women with obesity and Lynch syndrome had an increased expression of insulin-like growth factor 1 (P=.017). There were no differences in endometrial proliferation, and limited endometrial immune differences were observed. When comparing premenopausal women with and without obesity in the absence of endometrial pathology or an inherited cancer risk, the expression of the endometrial biomarkers does not reflect a local hyperestrogenic environment, but it instead reflects a decreased cancer risk profile that may be indicative of a compensated state. In describing premenopausal endometrial cancer risk, it may be insufficient to attribute a high-risk state to obesity alone; further studies are warranted to evaluate individualized biomarker profiles for differences in the hormone-responsive signals or immune response. In patients with Lynch syndrome, the endometrial biomarker profile suggests that obesity further increases the risk of developing cancer.

Abstract 4669: Pharmacologic inhibition of CBP/p300 suppresses estrogen-induced gene expression and proliferation in ER+ luminal breast cancer cells

Abstract Luminal breast cancer, defined by the expression of the estrogen receptor alpha (ERα), comprises nearly two thirds of all breast cancer cases and most patients with breast cancer die of this subtype. ERα is a steroid nuclear receptor that functions as a transcription factor to upregulate genes such as MYC that are involved in cell cycle progression. Antagonists of ERα are the standard of care treatment for ER+ luminal breast cancer, but resistance to these antagonists in advanced patients is a major clinical issue. Therefore, there is an unmet medical need to develop more effective therapies for treating ER+ breast cancer. CBP/p300 are two paralogous acetyltransferases that catalyze histone acetylation at enhancers and promoters to activate gene expression. They serve as transcriptional co-activators for several oncogenic signaling pathways, including the pro-growth androgen and estrogen signaling pathways in cancer. Due to their emerging role in promoting tumor growth, the oncogenic functions of CBP/p300 and means of their pharmacologic inhibition are under intense investigation for developing potential therapeutics for cancer treatment. CBP/p300's acetyltransferase (HAT) domain and their bromodomain represent tractable drug targets and potent and specific small molecule inhibitors have been developed for both. However, the pharmacologic effects and anticancer efficacy of these compounds in breast cancer remain largely unexplored. Previous research shows that CBP/p300 serve as co-activators for ERα and are responsible for catalyzing Histone 3, lysine 18 and 27 acetylation (H3K18ac and H3K27ac) at ERα target genes. We hypothesize that CBP/p300 are critical for ERα function in driving oncogene expression and their pharmacologic inhibition will block ERα-mediated gene expression underpinning the tumor growth of luminal breast cancer. In support of our hypothesis, we report that small molecule inhibitors of the CBP/p300 HAT domain and bromodomain strongly inhibit estrogen- induced MYC expression and prevent colony formation in vitro of a panel of ER+ cell lines. Furthermore, inhibition of CBP/p300 also downregulates ERα activity in a luciferase assay using a construct driven by the conical ERα-binding DNA sequence. H3K27ac ChIP-seq data in the ER+ luminal breast cancer MCF-7 cells indicate that pharmacologic inhibition of CBP/p300 catalytic function blocks specific enhancer function. Our study suggests pharmacologically targeting CBP/p300 may be an effective strategy for treating luminal breast cancer. (Supported by James and Esther King Biomedical Research Program, and Bankhead-Coley Cancer Research Program, Florida Department of Health) Citation Format: Aaron Richard Waddell, Iqbal Mahmud, Zhiguang Huo, Daiqing Liao. Pharmacologic inhibition of CBP/p300 suppresses estrogen-induced gene expression and proliferation in ER+ luminal breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4669.

Early estrogen-induced gene 1 facilitates osteoclast formation through the inhibition of interferon regulatory factor 8 expression.

Osteoclast-mediated inflammatory bone resorption is a major cause of many inflammatory bone disorders, including rheumatoid arthritis and periodontitis. However, the mechanisms regulating osteoclast differentiation in inflammatory settings are not well understood. We demonstrate here that early estrogen-induced gene 1 (EEIG1)-deficient mice are protected from inflammatory bone loss as determined with the use of models of lipopolysaccharide (LPS)-induced bone destruction. EEIG1-deficient macrophages markedly decreased RANKL- and TNFα-mediated osteoclastogenesis due to the downregulation of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which is an essential transcription factor for osteoclast formation. In contrast, expression of interferon regulatory factor 8 (IRF8), a transcriptional repressor that blocks osteoclast differentiation, is elevated in EEIG1-deficient macrophages relative to wild-type cells. We found that reduced expression of B lymphocyte-induced maturation protein-1 (Blimp1) by siRNA downregulated RANKL-induced EEIG1 levels, whereas overexpression of Blimp1 potentiated EEIG1 levels. Mechanistic studies revealed that EEIG1 forms a complex with Blimp1 to negatively regulate the expression of the anti-osteoclastogenic gene, Irf8. We elucidated a novel mechanism by which EEIG1 restricts IRF8 expression and function, thereby enhancing the osteoclast formation by contributing to Blimp1-mediated IRF8 regulation. Together, these findings identify EEIG1 as a key regulator of osteoclastogenesis and a possible therapeutic target for pathological bone destruction.

OR12-04 Regulatory Sharing between Estrogen Receptor Alpha Bound Enhancers

Mammalian genomes encode an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by combinations of enhancers. We previously found that neighboring estrogen-responsive enhancers exhibit cooperative/synergistic contributions to an estrogenic transcriptional response1. However, when the same combinations of enhancers are targeted with synthetic activators in the absence of estrogens, then the regulatory regions exhibit independent effects on gene expression2. Taken together, these findings indicate that estrogen receptor alpha (ER) bound enhancers cooperate with each other in cis but influence target gene promoters independently. To determine the molecular underpinnings of enhancer cooperativity, we generated genetic deletions of individual ER bound enhancers. We discovered “regulatory sharing” between enhancers in which loci containing full estrogen response elements (EREs) contribute ER binding to neighboring sites, while enhancers with pre-existing histone acetylation/accessibility contribute this permissible chromatin environment to the neighboring enhancers upon estrogen induction. Genome engineering revealed that a cluster of two half ERE enhancers could not compensate for a full ERE site loss within the cluster. However, two full ERE enhancers produced a transcriptional response greater than the wild-type locus, suggesting that combinations of enhancers are not necessarily configured for a maximal response. By swapping genomic sequences, we found that the genomic location in which a full ERE resides strongly influences enhancer activity. Our results lead to a model in which a full ERE is critical for ER recruitment, but the presence of pre-existing histone acetylation and accessibility within an enhancer cluster is also needed in order for estrogen-induced gene regulation to occur.

A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis.

While protein arginine methyltransferases (PRMTs) and PRMT-catalyzed protein methylation have been well-known to be involved in a myriad of biological processes, their functions and the underlying molecular mechanisms in cancers, particularly in estrogen receptor alpha (ERα)-positive breast cancers, remain incompletely understood. Here we focused on investigating PRMT4 (also called coactivator associated arginine methyltransferase 1, CARM1) in ERα-positive breast cancers due to its high expression and the associated poor prognosis.Methods: ChIP-seq and RNA-seq were employed to identify the chromatin-binding landscape and transcriptional targets of CARM1, respectively, in the presence of estrogen in ERα-positive MCF7 breast cancer cells. High-resolution mass spectrometry analysis of enriched peptides from anti-monomethyl- and anti-asymmetric dimethyl-arginine antibodies in SILAC labeled wild-type and CARM1 knockout cells were performed to globally map CARM1 methylation substrates. Cell viability was measured by MTS and colony formation assay, and cell cycle was measured by FACS analysis. Cell migration and invasion capacities were examined by wound-healing and trans-well assay, respectively. Xenograft assay was used to analyze tumor growth in vivo.Results: CARM1 was found to be predominantly and specifically recruited to ERα-bound active enhancers and essential for the transcriptional activation of cognate estrogen-induced genes in response to estrogen treatment. Global mapping of CARM1 substrates revealed that CARM1 methylated a large cohort of proteins with diverse biological functions, including regulation of intracellular estrogen receptor-mediated signaling, chromatin organization and chromatin remodeling. A large number of CARM1 substrates were found to be exclusively hypermethylated by CARM1 on a cluster of arginine residues. Exemplified by MED12, hypermethylation of these proteins by CARM1 served as a molecular beacon for recruiting coactivator protein, tudor-domain-containing protein 3 (TDRD3), to CARM1-bound active enhancers to activate estrogen/ERα-target genes. In consistent with its critical role in estrogen/ERα-induced gene transcriptional activation, CARM1 was found to promote cell proliferation of ERα-positive breast cancer cells in vitro and tumor growth in mice.Conclusions: our study uncovered a “hypermethylation” strategy utilized by enhancer-bound CARM1 in gene transcriptional regulation, and suggested that CARM1 can server as a therapeutic target for breast cancer treatment.

Interplay of Guanine Oxidation and G-Quadruplex Folding in Gene Promoters.

Living in an oxygen atmosphere demands an ability to thrive in the presence of reactive oxygen species (ROS). Aerobic organisms have successfully found solutions to the oxidative threats imposed by ROS by evolving an elaborate detoxification system, upregulating ROS during inflammation, and utilizing ROS as messenger molecules. In this Perspective, recent studies are discussed that demonstrate ROS as signaling molecules for gene regulation by combining two emergent properties of the guanine (G) heterocycle in DNA, namely, oxidation sensitivity and a propensity for G-quadruplex (G4) folding, both of which depend upon sequence context. In human gene promoters, this results from an elevated 5'-GG-3' dinucleotide frequency and GC enrichment near transcription start sites. Oxidation of DNA by ROS drives conversion of G to 8-oxo-7,8-dihydroguanine (OG) to mark target promoters for base excision repair initiated by OG-glycosylase I (OGG1). Sequence-dependent mechanisms for gene activation are available to OGG1 to induce transcription. Either OGG1 releases OG to yield an abasic site driving formation of a non-canonical fold, such as a G4, to be displayed to apurinic/apyrimidinic 1 (APE1) and stalling on the fold to recruit activating factors, or OGG1 binds OG and facilitates activator protein recruitment. The mechanisms described drive induction of stress response, DNA repair, or estrogen-induced genes, and these pathways are novel potential anticancer targets for therapeutic intervention. Chemical concepts provide a framework to discuss the regulatory or possible epigenetic potential of the OG modification in DNA, in which DNA "damage" and non-canonical folds collaborate to turn on or off gene expression. The next steps for scientific discovery in this growing field are discussed.

AIB1 sequestration by androgen receptor inhibits estrogen-dependent cyclin D1 expression in breast cancer cells

BackgroundAndrogens, through their own receptor, play a protective role on breast tumor development and progression and counterbalance estrogen-dependent growth stimuli which are intimately linked to breast carcinogenesis.MethodsCell counting by trypan blu exclusion was used to study androgen effect on estrogen-dependent breast tumor growth. Quantitative Real Time RT–PCR, western blotting, transient transfection, protein immunoprecipitation and chromatin immunoprecipitation assays were carried out to investigate how androgen treatment and/or androgen receptor overexpression influences the functional interaction between the steroid receptor coactivator AIB1 and the estrogen- or androgen receptor which, in turn affects the estrogen-induced cyclin D1 gene expression in MCF-7 breast cancer cells. Data were analyzed by ANOVA.ResultsHere we demonstrated, in estrogen receptor α (ERα)-positive breast cancer cells, an androgen-dependent mechanism through which ligand-activated androgen receptor (AR) decreases estradiol-induced cyclin D1 protein, mRNA and gene promoter activity. These effects involve the competition between AR and ERα for the interaction with the steroid receptor coactivator AIB1, a limiting factor in the functional coupling of the ERα with the cyclin D1 promoter. Indeed, AIB1 overexpression is able to reverse the down-regulatory effects exerted by AR on ERα-mediated induction of cyclin D1 promoter activity. Co-immunoprecipitation studies indicated that the preferential interaction of AIB1 with ERα or AR depends on the intracellular expression levels of the two steroid receptors. In addition, ChIP analysis evidenced that androgen administration decreased E2-induced recruitment of AIB1 on the AP-1 site containing region of the cyclin D1 gene promoter.ConclusionsTaken together all these data support the hypothesis that AIB1 sequestration by AR may be an effective mechanism to explain the reduction of estrogen-induced cyclin D1 gene activity. In estrogen-dependent breast cancer cell proliferation, these findings reinforce the possibility that targeting AR signalling may potentiate the effectiveness of anti-estrogen adjuvant therapies.

Propagation of functional estrogen receptor positive normal human breast cells in 3D cultures.

Understanding how differentiation, microenvironment, and hormonal milieu influence human breast cell susceptibility to malignant transformation will require the use of physiologically relevant in vitro systems. We sought to develop a 3D culture model that enables the propagation of normal estrogen receptor alpha (ER) + cells. We tested soluble factors and protocols for the ability to maintain progenitor and ER + cells in cultures established from primary cells. Optimized conditions were then used to profile estrogen-induced gene expression changes in cultures from three pathology-free individuals. Long-term representation of ER + cells was optimal in medium that included three different TGFβ/activin receptor-like kinase inhibitors. We found that omitting the BMP signaling antagonist, Noggin, enhanced the responsiveness of the PGR gene to estradiol exposure without altering the proportions of ER + cells in the cultures. Profiling of estradiol-exposed cultures showed that while all the cultures showed immediate and robust induction of PGR, LRP2, and IGFB4, other responses varied qualitatively and quantitatively across specimens. We successfully identified conditions for the maintenance and propagation of functional ER + cells from normal human breast tissues. We propose that these 3D cultures will overcome limitations of conventional 2D cultures of partially or fully transformed cell lines by sustaining normal endocrine function and growth regulation of the cell populations that comprise intact breasts.

Abstract P3-10-14: LIV-1 expression in primary breast cancers in the I-SPY 2 TRIAL

Abstract Background: LIV-1 is an estrogen-inducible gene that has been implicated in epidermal-to-mesenchymal transition (EMT) in preclinical models of progression and metastasis. Its expression is associated with node-positivity in breast cancer; and has been detected in a variety of cancer types, including estrogen receptor positive breast cancers. SGN-LIV1A is a novel antibody drug conjugate targeting LIV-1 that is currently being evaluated in the I-SPY 2 TRIAL. In this pilot study, we evaluated LIV-1 levels by IHC within HR/HER2/MammaPrint (MP) defined subtypes among patients screening for the I-SPY 2 TRIAL and its correlation to microarray assessed LIV-1 expression levels. Method: In a pilot study, LIV-1 IHC staining was performed by Quest Diagnostics on the pre-treatment samples of 38 patients screening for the I-SPY 2 TRIAL. Pre-treatment expression data generated on a custom Agilent 44K platform was also available. We summarized the LIV-1 H-Scores and percent (%)-positivity across the population and within HR/HER2/MP subtypes; and we assessed the Pearson correlation between LIV-1 H-Score and LIV-1 gene expression levels. In addition, we compared the pre-treatment LIV-1 expression levels within HR/HER2/MP subtypes across I-SPY 2 TRIAL patients from completed arms and their relevant controls (n=989) using ANOVA and post-hoc Tukey tests. Our statistics are descriptive rather than inferential; and does not take into account multiplicities of other biomarkers outside of this study. Results: Of the 38 patients evaluated, 37 have LIV-1 %-positivity > 0; and 18 (47%) have 100% LIV1 positivity. The median LIV-1 H-Score is 200; and 89% of patients (34/38) have moderate/high LIV-1 staining (with H-Score≥100). Of the 34 patients who proceeded onto the trial (and have known HR/HER2/MP status), 9 are triple negative, 19 are HR+HER2-, and 6 are HER2+. Due to our small sample size, we did not further subset the triple negative and HER2+ cases; but within the HR+HER2- patients, 10 are MP1 compared to 9 who are MP2 class. LIV1 H-Score appears highest within the HR+HER2-MP1 cases (median: 290), followed by the HER2+ (median: 216), then the HR+HER2-/MP2 (median: 155), and the TN (median: 120) subtype. LIV1 H-score is significantly correlated with LIV-1 mRNA expression levels (Rp=0.79, p<0.0001). Consistent with these observations, LIV-1 pre-treatment expression levels are significantly higher in the HR+HER2-MP1 group relative to all other HR/HER2/MP defined subtypes (Tukey HSD p < 0.0001) across the I-SPY 2 TRIAL population. The HR+HER2+MP1 group also have high LIV-1 expression levels. Conclusion: Our result suggest that although LIV-1 expression differs by subtype, it is expressed at a moderate/high level in the majority of patients. The good correlation between IHC and array-based LIV-1 expression levels enables us to leverage the entire existing I-SPY 2 dataset and confirm the high rates of LIV-1 expression across the I-SPY 2 population. Further studies to evaluate LIV-1 expression as a biomarker of response to LIV-1 targeting therapies for the neoadjuvant treatment of breast cancer are warranted and ongoing in I-SPY 2. Citation Format: Yau C, Brown-Swigart L, Asare S, I-SPY 2 TRIAL Consortium, Esserman L, van' t Veer L, Beckwith H, Forero A, Rugo H. LIV-1 expression in primary breast cancers in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-14.

SOX9/FXYD3/Src Axis Is Critical for ER+ Breast Cancer Stem Cell Function.

The presence of cancer stem cells (CSC), which possess the ability of self-renewal and cancer initiation, is correlated with poor prognosis and drug resistance of breast cancer patients. But the molecular regulatory networks for maintenance of CSC function still remain unclear. Here, we identified that an estrogen-inducible gene FXYD3, whose expression is significantly upregulated in ER+ breast CSCs, is a critical player for regulating ER+ breast CSC function. FXYD3 amplification is crucial in mediating tamoxifen resistance in ER+ breast cancer cells. Interestingly, we also find that stem cell-related transcription factor SOX9 directly promotes FXYD3 expression, and FXYD3 is indispensable for SOX9 nucleus localization, thus forming a positive regulatory feedback loop for FXYD3 amplification and function. In terms of mechanism, FXYD3 interacts with Src and ERα to form an activated complex and triggers Src to transduce nongenomic estrogen signaling for facilitating ER+ breast CSCs. Collectively, these results establish a critical role for SOX9/FXYD3/Src axis in boosting nongenomic estrogen signaling and SOX9 nucleus entry, which is required for maintenance of ER+ breast CSCs and endocrine resistance. Targeting FXYD3-mediated pathway might be a promising therapeutic strategy for hormone therapy-refractory ER+ breast cancer. IMPLICATIONS: SOX9/FXYD3/Src axis is critical for promoting CSC function and tamoxifen resistance in ER+ breast cancer.