Estrogen-estrogen Receptor Research Articles

Estrogen/estrogen receptor activation protects against DEN-induced liver fibrosis in female rats via modulating TLR-4/NF-kβ signaling

AimMen are more susceptible to liver fibrosis (LF) than women. However, the underlying molecular mechanism, especially the role of estrogen/estrogen receptor (ER) activation in this sexual dimorphism is unclear. Therefore, the aim of the current study was to investigate the impact and the underlying molecular mechanisms of estrogen/ER activation on diethyl nitrosamine (DEN)-induced LF. Main methodsThirty ovariectomized (OVX) female rats were randomly allocated into five groups (n = 6), and received no treatment, diethyl nitrosamine (DEN), DEN/fulvestrant, DEN/silymarin or DEN/estradiol benzoate (EB). In addition, three sham groups received no treatment, DEN or DEN/fulvestrant, and one control group that neither ovariectomized nor treated. Directly after treatment, liver injury biomarkers were measured. In addition, hepatic tissue hydroxyproline, TNF- α, TGF- β, and IL-10 were evaluated. Expression of NF-kβ, CD68 (a marker for macrophage infiltration), ER-β and TLR-4 were measured. Finally, liver tissue histopathology was assessed. Key findingsOvariectomy aggravates DEN-induced LF, as it significantly elevated all liver tissue injury biomarkers. This effect has become even worse after blocking ER by fulvestrant, indicating a protective role of estrogen/ER activation against DEN-induced LF. Inhibition of TLR-4/NF-kβ signaling pathway contributed to this protective effect, as estrogen deprivation or blocking of ER significantly activates this pathway during the onset of LF. While administration of EB or silymarin (selective ER-β activator) improved LF indices and deactivated this pathway. SignificanceThese results provide new insight into the pivotal role of estrogen/ER activation via modulation of TLR-4/NF-kβ, in the alleviation of LF pathogenesis.

Abstract 3439: Evaluating nuclear receptor heterodimerization using live cell proximity assays: Implications for novel therapeutic approaches for hormone driven cancers

Abstract Breast and prostate cancer are the leading cancer diagnoses for women and men, respectively, second only to cutaneous cancers. Breast and prostate cancer arise from aberrant estrogen/estrogen receptor (ER) and testosterone/androgen receptor signaling (AR), respectively. These sex hormone receptors are members of the steroid hormone receptor family, which also includes the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and progesterone receptor (PR). Although these receptors canonically function through homodimerization, recent evidence has suggested that receptor crosstalk can amplify or dampen the actions of heterologous receptors. However, these interactions have not been extensively studied in all possible combinations of steroid hormone receptor crosstalk. In this project, we hypothesize that heterodimerization may amplify or repress the transcriptional response(s) elicited by the various receptor complexes depending upon conditions such as concentration of ligands, type of ligand, physiological states, and the types/functions of promoter regions. To test this hypothesis, we investigated the significance of ligand-induced interactions between different homo- and heterodimer combinations of AR, ARV7, GR, MR, PR, and ERα in HEK293 cells using IncuCyte live-cell imaging and NanoBRET technology. Ultimately, the combination of transfection experiments, live-cell imaging, and NanoBRET assays provided insight into which combination of steroid receptors successfully interact to amplify (or dampen) the HaloTag expression, luminescence intensity at 450nM (i.e., NanoLuc expression), and NanoBRET ratio (i.e., acceptor emission luminescence at 610nM divided by donor emission luminescence at 450nM) in HEK293 cells. Our studies revealed that heterodimerization of ERα-PR, ERα-MR, AR-GR, AR-PR, ARV7-MR, ARV7-PR, and GR-MR produced significant ligand-induced changes in signaling intensity compared to no ligand conditions for each fusion protein construct. Additionally, to demonstrate the value of the NanoBRET method for modelling clinically relevant disease conditions, we assessed the physical interaction of ERα and PR in the context of the ERα Y537S mutation - a mutation associated with endocrine therapy resistance in breast cancer that results in a constitutively active ERα. The results showed that ERα-Y537S mutation increases ERα/PR physical interaction upon PR-B stimulation in a NanoBRET proximity-based assay. Future directions include testing these interactions in breast and prostate cancer cell lines and identifying the receptors’ localization within the cell, the results of which may be of clinical importance. Citation Format: Amira Ishag-Osman, David Hosfield, Rosemary Huggins, Geoffrey Greene. Evaluating nuclear receptor heterodimerization using live cell proximity assays: Implications for novel therapeutic approaches for hormone driven cancers [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 3439.

SCM-198 Prevents Endometriosis by Reversing Low Autophagy of Endometrial Stromal Cell via Balancing ERα and PR Signals.

BackgroundEndometriosis (EMS), an endocrine-related inflammatory disease, is characterized by estrogen and progesterone imbalance in ectopic lesions. However, its pathogenic mechanism has not been fully elucidated. While SCM-198 is the synthetic form of leonurine and has multiple pharmacological activities such as antioxidation and anti-inflammation, it remains unknown whether it could inhibit the progress of EMS by regulating estrogen signaling and inflammation.MethodsThe therapeutic effects of SCM-198 on EMS and its potential mechanism were analyzed by establishing EMS mouse models and performing an RNA sequencing (RNA-seq) assay. ELISA was performed to detect estrogen and tumor necrosis factor (TNF) -α concentrations in normal endometrial stromal cells (nESCs) and ectopic endometrial stromal cells (eESCs) with or without SCM-198 treatment. Western blotting, RNA silencing, and plasmid overexpression were used to analyze the relationship between inflammation, endocrine factors, and autophagy and the regulatory activity of SCM-198 on the inflammation-endocrine-autophagy axis.ResultsIncreased estrogen-estrogen receptor (ER) α signaling and decreased progesterone receptor isoform B (PRB) expression synergistically led to a hypo-autophagy state in eESCs, which further inhibited the apoptosis of eESCs. The high expression of TNF-α in eESCs enhanced the antiapoptotic effect mediated by low autophagy through the activation of the aromatase-estrogen-ERα signaling pathway. SCM-198 inhibited the growth of ectopic lesions in EMS mice and promoted the apoptosis of eESCs both in vivo and in vitro. The apoptotic effect of SCM-198 on eESCs was attained by upregulating the autophagy level via the inhibition of the TNF-α-activated aromatase-estrogen-ERα signal and the increase in PRB expression.ConclusionInflammation facilitated the progress of EMS by disrupting the estrogen regulatory axis. SCM-198 inhibited EMS progression by regulating the inflammation-endocrine-autophagy axis.

MP07-03 THE PROTECTIVE ROLE OF ESTROGEN RECEPTOR Β IN RENAL CALCIUM OXALATE FORMATION VIA REDUCING THE LIVER OXALATE BIOSYNTHESIS AND RENAL CELL INJURY

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology (MP07)1 Sep 2021MP07-03 THE PROTECTIVE ROLE OF ESTROGEN RECEPTOR Β IN RENAL CALCIUM OXALATE FORMATION VIA REDUCING THE LIVER OXALATE BIOSYNTHESIS AND RENAL CELL INJURY Wei Zhu, Wenbiao Ren, Chawnshang Chang, Edward M. Messing, Jean V. Joseph, Rajat Jain, David Levy, David Bushinsky, Guohua Zeng, and Shuyuan Yeh Wei ZhuWei Zhu More articles by this author , Wenbiao RenWenbiao Ren More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Jean V. JosephJean V. Joseph More articles by this author , Rajat JainRajat Jain More articles by this author , David LevyDavid Levy More articles by this author , David BushinskyDavid Bushinsky More articles by this author , Guohua ZengGuohua Zeng More articles by this author , and Shuyuan YehShuyuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001980.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Kidney stone is a common disorder that poses a significant health care burden. The incidence of kidney stone disease is lower in premenopausal women than age-matched men, as well as in female animal models with renal calcium oxalate (CaOx) crystal deposition. However, it is unclear if this gender difference is related to altered estrogen/estrogen receptor (ER) signaling. METHODS: Kidney and liver cell lines, HK-2, HKC-8, HepG2, and HEK-293, were used. The shRNA and cDNA of NOX2 and ERβ were applied to study their functions. Hydrogen Peroxide Assay and DHE Staining were used to detect Oxalate treatment effects and ROS in renal cells. The q-PCR and western blot assays were applied to assess the RNA and protein expressions, respectively. ERβ knockout (ERβKO,) and WT female mice were used to compare CaOx formation. Effects of ERβ antagonist was tested in hydroxyl-L-proline (HLP) diet induced CaOx crystal deposition rat model. RESULTS: CaOx is one major composition accounts for majority of kidney stones. We found that ERβ signals could suppress hepatic oxalate biosynthesis via transcriptional up-regulation of the glyoxylate aminotransferase (AGT1) expression in liver cells. Results from renal cell lines also showed that ERβ could function via suppressing the oxalate-induced injury to reduce the reactive oxygen species (ROS) production and decrease the renal calcium oxalate (CaOx) deposition. Mechanism studies showed that ERβ could suppress the oxalate-induced oxidative stress via transcriptional suppression of the NADPH oxidase subunit 2 (NOX2) via binding to the NOX2 5′ promoter. We further applied two in vivo animal models rat fed with 5% HLP diet-induced renal CaOx deposition. Our data demonstrated that mice lacking ERβ (ERβKO) as well as mice or rats treated with ERβ antagonist PHTPP had increased urinary oxalate excretion, renal ROS production as well as renal CaOx crystal deposition. Importantly, targeting ERβ-regulated NOX2 with the NADPH oxidase inhibitor, apocynin, can reduce the renal CaOx crystal deposition in the in vivo animal model. CONCLUSIONS: Results from multiple in vitro cell lines and in vivo mouse/rat models all demonstrate that ERβ may protect against renal CaOx deposition via reducing the hepatic oxalate biosynthesis and oxidative stress-induced renal injury. Source of Funding: This work was partially supported by URMC Urology Research fund, National Natural Science Foundation of China, and George Whipple Professorship Endowment at U of Rochester © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e140-e140 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wei Zhu More articles by this author Wenbiao Ren More articles by this author Chawnshang Chang More articles by this author Edward M. Messing More articles by this author Jean V. Joseph More articles by this author Rajat Jain More articles by this author David Levy More articles by this author David Bushinsky More articles by this author Guohua Zeng More articles by this author Shuyuan Yeh More articles by this author Expand All Advertisement Loading ...

Estrogen Receptors in Epithelial-Mesenchymal Transition of Prostate Cancer.

Prostate cancer (PC) remains a widespread malignancy in men. Since the androgen/androgen receptor (AR) axis is associated with the pathogenesis of prostate cancer, suppression of AR-dependent signaling by androgen deprivation therapy (ADT) still represents the primary intervention for this disease. Despite the initial response, prostate cancer frequently develops resistance to ADT and progresses. As such, the disease becomes metastatic and few therapeutic options are available at this stage. Although the majority of studies are focused on the role of AR signaling, compelling evidence has shown that estrogens and their receptors control prostate cancer initiation and progression through a still debated mechanism. Epithelial versus mesenchymal transition (EMT) is involved in metastatic spread as well as drug-resistance of human cancers, and many studies on the role of this process in prostate cancer progression have been reported. We discuss here the findings on the role of estrogen/estrogen receptor (ER) axis in epithelial versus mesenchymal transition of prostate cancer cells. The pending questions concerning this issue are presented, together with the impact of the available data in clinical management of prostate cancer patients.

Expression of estrogen receptor alpha is associated with pathogenesis and prognosis of human papillomavirus‐positive oropharyngeal cancer

Human papillomavirus (HPV) has been identified as a causative agent of cervical cancer and oropharyngeal cancer (OPC). Intriguingly, estrogen and HPV were shown to play synergistic roles in cervical carcinogenesis. We recently demonstrated that the apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3, A3) family, which is inducible by estrogen, could lead to HPV DNA hypermutation and cause viral DNA integration. In the present study, we examined the relationships between estrogen-estrogen receptor α (ERα) and A3s in HPV-positive OPC. ERα expression was associated with HPV positivity in OPC biopsy samples using immunohistochemical analysis and reverse-transcription quantitative polymerase chain reaction. In addition, ERα was significantly associated with improved overall survival in HPV-positive OPC (hazard ratio, 0.26; p = 0.029). APOBEC3A (A3A) mRNA was induced by estrogen in HPV and ERα-positive OPC cells. Furthermore, A3A mRNA and protein expression were significantly higher in ERα-positive cases than in ERα-negative ones, among HPV-positive biopsy samples (p = 0.037 and 0.047). These findings suggest that A3A is associated with a good prognosis in ERα-positive OPC, and indicate the prognostic significance of ERα in HPV-positive OPC. This is the first study to demonstrate the prognostic role of ERα in HPV-positive OPC.

The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury.

Females develop kidney stones less frequently than males do. However, it is unclear if this gender difference is related to altered estrogen/estrogen receptor (ER) signaling. Here, we found that ER beta (ERβ) signals could suppress hepatic oxalate biosynthesis via transcriptional upregulation of the glyoxylate aminotransferase (AGT1) expression. Results from multiple in vitro renal cell lines also found that ERβ could function via suppressing the oxalate-induced injury through increasing the reactive oxygen species (ROS) production that led to a decrease of the renal calcium oxalate (CaOx) crystal deposition. Mechanism study results showed that ERβ suppressed oxalate-induced oxidative stress via transcriptional suppression of the NADPH oxidase subunit 2 (NOX2) through direct binding to the estrogen response elements (EREs) on the NOX2 5′ promoter. We further applied two in vivo mouse models with glyoxylate-induced renal CaOx crystal deposition and one rat model with 5% hydroxyl-L-proline-induced renal CaOx crystal deposition. Our data demonstrated that mice lacking ERβ (ERβKO) as well as mice or rats treated with ERβ antagonist PHTPP had increased renal CaOx crystal deposition with increased urinary oxalate excretion and renal ROS production. Importantly, targeting ERβ-regulated NOX2 with the NADPH oxidase inhibitor, apocynin, can suppress the renal CaOx crystal deposition in the in vivo mouse model. Together, results from multiple in vitro cell lines and in vivo mouse/rat models all demonstrate that ERβ may protect against renal CaOx crystal deposition via inhibiting the hepatic oxalate biosynthesis and oxidative stress-induced renal injury.

Estrogen affects cell growth and IGF-1 receptor expression in renal cell carcinoma.

PurposeBoth obesity and gender are important etiological factors in renal cell carcinoma (RCC) development, suggesting a pivotal role of sex hormone signaling pathway and insulin-like growth factor (IGF) family in RCC carcinogenesis. Here, we aimed to investigate the effect of estrogen on RCC growth and the possible interaction between estrogen/estrogen receptor (ER) signaling pathway and the IGF axis.MethodsER-α and ER-β were detected in four human RCC cell lines. Cells were treated with 17β-estradiol (E2), and cell proliferation was determined using the cell counting kit-8 assay. Using siRNA, ER-β was downregulated in RCC cells and the effect of E2 on cell growth and IGF-1 receptor (IGF-1R) expression was examined.ResultsE2 inhibited 786-O cell but not A498 cell growth significantly. After the downregulation of ER-β, E2 showed no obvious inhibitory role in 786-O cells. E2 stimulation increased the expression of IGF-1R in 786-O cells. Downregulation of ER-β, as well as fulvestrant, attenuated the stimulatory effect of E2 on IGF-1R expression.ConclusionOur results revealed that estrogen induced RCC growth inhibition via an ER-β-dependent pathway. Estrogen also upregulated the expression of IGF-1R, suggesting a link between estrogen/ER and IGF axis.

Short-term early exposure to lapatinib confers lifelong protection from mammary tumor development in MMTV-erbB-2 transgenic mice

BackgroundAlthough chemopreventative agents targeting the estrogen/estrogen receptor (ER) pathway have been effective for ER+ breast cancers, prevention of hormone receptor-negative breast cancers, such as Her2/erbB-2+ breast cancers, remains a significant issue. Previous studies have demonstrated that administration of EGFR/erbB-2-targeting lapatinib to MMTV-erbB-2 transgenic mice inhibited mammary tumor development. The prevention, however, was achieved by prolonged high dose exposure. The tolerance to high dose/long-term drug administration may hinder its potential in clinical settings. Therefore, we aimed to test a novel, short-term chemopreventative strategy using lapatinib during the premalignant risk window in MMTV-erbB-2 mice.MethodsWe initially treated cultured cells with lapatinib to explore the anti-proliferative effects of lapatinib in vitro. We used a syngeneic tumor graft model to begin exploring the in vivo anti-tumorigenic effects of lapatinib in MMTV-erbB-2 mice. Then, we tested the efficacy of brief exposure to lapatinib (100 mg/kg/day for 8 weeks), beginning at 16 weeks of age, in the prevention of mammary tumor development in MMTV-erbB-2 mice.ResultsIn the syngeneic tumor transplant model, we determined that lapatinib significantly inhibited tumor cell proliferation. Furthermore, we demonstrated that short-term lapatinib exposure resulted in life-long protective effects, as supported by increased tumor latency in lapatinib-treated mice compared to the control mice. We further established that delayed tumor development in the treated mice was preceded by decreased BrdU nuclear incorporation and inhibited mammary morphogenesis. Molecular analysis indicated that lapatinib inhibited phosphorylation and expression of EGFR, erbB-3, erbB-2, Akt1, and Erk1/2 in premalignant mammary tissues. Also, lapatinib drastically inhibited the phosphorylation and expression of ERα and the transcription of ER target genes in premalignant mammary tissues. We also determined that lapatinib suppressed the stemness of breast cancer cell lines, as evidenced by decreased tumorsphere formation and ALDH+ cell populations.ConclusionsTaken together, these data demonstrate that brief treatment with EGFR/erbB-2-targeting agents before the onset of tumors may provide lifelong protection from mammary tumors, through the concurrent inhibition of erbB-2 and ER signaling pathways and consequential reprogramming. Our findings support further clinical testing to explore the benefit of shorter lapatinib exposure in the prevention of erbB-2-mediated carcinogenesis.

HPV関連中咽頭癌における内因性免疫APOBEC3とエストロゲン–エストロゲン受容体の予後との関係

HPV関連中咽頭癌における内因性免疫APOBEC3とエストロゲン–エストロゲン受容体の予後との関係

ERRF is essential for Estrogen-Estrogen Receptor alpha signaling pathway in ER positive breast cancer cells

Estrogen-Estrogen Receptor alpha (ERα) belongs to one of the most important signaling pathways controlling breast tissue development and progression of breast cancer. ERRF was recently identified as a candidate breast cancer associated protein and showed positive association with ERα status in clinical samples and cell lines. To further explore the relationship between ERRF and ERα, we studied whether ERRF plays any roles in estrogen-ERα pathway. Knockdown of ERRF in ER positive breast cancer cells T-47D and BT-474 reduced the level of p-AKT, p-MAPK, and phosphorylation of ERα at Ser 118 and Ser 167, and the transcriptional activity of ERα was inhibited as well. Further mechanism study proved ERRF to be an interacting partner of ERα. In total, these data revealed that ERRF is essential for the activity of E2-ERα pathway.

Expression levels of estrogen receptor α mRNA in peripheral blood cells are an independent biomarker for postmenopausal osteoporosis

BackgroundThe up- and down-regulation of the osteoclastogenesis response depends on the estrogen/estrogen receptor (ER) signaling pathway. Previous reports have shown that the promoter hypermethylation and gene polymorphism of ERα are risks for menopausal osteoporosis. No previous study has evaluated the expression levels of ERα mRNA in menopausal osteoporosis using human subjects. We hypothesized that ERα mRNA expression may show less resistance to postmenopausal osteoporosis. MethodsIn this study, we enrolled 107 women older than 45years without menstruation and classified them into control, osteopenia, and osteoporosis groups depending on their T-scores. The ERα mRNA levels in peripheral blood cells (PBCs) were analyzed via quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), and estrogen in the serum was detected via ELISA. ResultsERα mRNA levels in PBCs had a negative correlation with age and a positive correlation with estrogen and BAP in the osteopenia and osteoporosis groups, but not in the control group. Additionally, multivariate analysis showed that older age (>55years), and low ERα mRNA levels in PBLs (≦250.39 copies/μg DNA) were associated with an approximately 9.188-, and 31.25-fold risk of osteoporosis. ConclusionWe conclude that ERα mRNA levels in PBLs could be used as an independent risk factor for postmenopausal osteoporosis. General significanceOur findings suggested that ERα mRNA levels in PBLs may be more important than age and serum estrogen levels.

Estrogen receptor-α is localized to neurofibrillary tangles in Alzheimer's disease.

The female predominance for developing Alzheimer disease (AD) suggests the involvement of gender specific factor(s) such as a reduced estrogen-estrogen receptor signaling in the pathogenesis of AD. The potential role of ERα in AD pathogenesis has been explored by several groups with mixed results. We revisited this issue of expression and distribution of ERα in AD brain using a specific ERα antibody. Interestingly, we found that ERα co-localized with neurofibrillary pathology in AD brain and further demonstrated that ERα interacts with tau protein in vivo. Immunoprecipitaion experiments found increased ERα-tau interaction in the AD cases, which may account for ERα being sequestered in neuronal tau pathology. Indeed, tau overexpression in M17 cells leads to interruption of estrogen signaling. Our data support the idea that sequestration of ERα by tau pathology underlies the loss of estrogen neuroprotection during the course of AD.

Abstract 5342: Hardwiring mechanism into predicting cancer phenotypes by computational learning

Abstract Rationale. Despite promising beginnings, molecular classifiers derived from statistical learning do not yet appear to be sufficiently mature for clinical use. Besides known limitations, the nearly universal absence of mechanistic underpinnings for such signatures represents as major barrier toward successful implementation of clinically useful biomarkers. To overcome this limitation we constrained the search for predictive models to those with mechanistic justification, by incorporating microRNA (miR) and transcription factor (TF) gene regulatory networks directly into the learning process of cancer phenotypes. Methods. To illustrate the impact of embedding such regulatory motifs into computational learning, we analyzed the ability to predict estrogen receptor (ER) status from transcriptional data. We applied this approach to two independent breast cancer studies used as training and validation sets respectively. This analysis provided a test case with well-characterized clinical attributes, in which the ER itself is a TF engaged in regulatory miR/TF motifs. We built our predictors using Top Scoring Pair (TSP), a two-gene parameter-free classifier returning one class (ER positive) or the other (ER negative) based on the relative ordering of the two genes. We compared classification performance between TSPs chosen from all possible gene pairs and TSPs constructed under network-based constraints - “random” and “mechanistic” TSPs respectively hereafter. Each “mechanistic” TSP consists of a gene pair: the first gene regulates a miR or a TF “hub”, which in turn regulates the second gene. We started from a network of 200 TFs, 373 miRs, and 2772 target genes based on regulatory information from the miRgen v2.0 and TarBase v5.0 databases. Results. We assessed the classification accuracy of the TSP classifiers derived from the training dataset in the validation set and nearly all top-performing predictors were based on regulatory motifs. A Wilcoxon rank-sum test comparing the “random” classifiers with either TF or miR based TSPs had P-values of 10−14 and 10−26, respectively. Most of such top “mechanistic” predictors involved the ER gene (ERS1), consistent with the underlying biology. The mechanistic predictor also paired ERS1 expression with genes relevant to the biology. For instance, TSP selected POU2F1 _ a TF member of the POU family also known as OCT1 _ which physically interacts with the ER itself and BRCA1, recruiting BRCA1 to the ESR1 promoter modulating ER expression. Consistent with the classifier, BRCA1-mutant breast tumors are typically estrogen ER negative. Conclusions. We have implemented a novel class of mechanistic predictors by ”hardwiring” gene regulatory network information into statistical learning of cancer phenotypes. This approach has intrinsic added value for knowledge discovery and disease treatment design, and will ultimately move the field towards a successful transition to personalized health care. Citation Format: Bahman Afsari, Elana Judith Fertig, Laurent Younes, Donald Geman, Luigi Marchionni. Hardwiring mechanism into predicting cancer phenotypes by computational learning. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5342. doi:10.1158/1538-7445.AM2014-5342

Is there a role for base excision repair in estrogen/estrogen receptor-driven breast cancers?

Estrogen and estrogen metabolite-induced reactive oxygen species generation can promote oxidative DNA base damage. If unrepaired, base damaging lesions could accelerate mutagenesis, leading to a “mutator phenotype” characterized by aggressive behavior in estrogen-estrogen receptor (ER)-driven breast cancer. To test this hypothesis, we investigated 1406 ER+ early-stage breast cancers with 20 years' long-term clinical follow-up data for DNA polymerase β (pol β), flap endonuclease 1 (FEN1), AP endonuclease 1 (APE1), X-ray cross-complementation group 1 protein (XRCC1), single-strand monofunctional uracil glycosylase-1 (SMUG1), poly (ADP-ribose) polymerase 1 (PARP1), ataxia telangiectasia mutated and Rad3 related (ATR), ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Chk1, Chk2, p53, breast cancer susceptibility gene 1 (BRCA1), and topoisomerase 2 (TOPO2) expression. Multivariate Cox proportional hazards model was used to calculate a DNA repair prognostic index and correlated to clinicopathological variables and survival outcomes. Key base excision repair (BER) proteins, including XRCC1, APE1, SMUG1, and FEN1, were independently associated with poor breast cancer-specific survival (BCSS) (ps≤0.01). Multivariate Cox model stratified patients into four distinct prognostic sub-groups with worsening BCSS (ps<0.01). In addition, compared with prognostic sub-group 1, sub-groups 2, 3, and 4 manifest increasing tumor size, grade, mitosis, pleomorphism, differentiation, lymphovascular invasion, high Ki67, loss of Bcl-2, luminal B phenotype (ps≤0.01), and poor survival, including in patients who received tamoxifen adjuvant therapy (p<0.00001). Our observation supports the hypothesis that BER-directed stratification could inform appropriate therapies in estrogen-ER-driven breast cancers. Antioxid. Redox Signal. 21, 2262–2268.

Estrogen Receptor-Alpha Promotes Alternative Macrophage Activation during Cutaneous Repair

Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERβ, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.

Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

Regulation of bcl-2 transcription by estrogen receptor-α and c-Jun in human endometrium

The estrogen-estrogen receptor (ER) signaling pathway plays crucial physiologic roles in not only the control of reproduction, but also in the generation of cancer in the breast and uterus. While some ER target genes have been identified containing the estrogen-responsive element (ERE), others are activated eventually by ER via protein-protein interaction without binding to ERE. In a previous study, we identified that the proliferative phase-specific expression of the bcl-2 gene in glandular cells could be regulated by the binding of c-Jun to its motifs in the promoter. Results from our present study indicate that the menstrual cyclic expression of bcl-2 could be controlled by either direct binding of ERα to ERE in the c-Jun promoter or the interaction of ERα with c-Jun that binds to its motifs in the bcl-2 gene. Intriguingly, the transcriptionally active form of c-Jun phosphorylated at Ser63 was identified binding to its motifs in the bcl-2 gene in a menstrual cyclic non-specific manner. Our study revealed a novel mechanism that transcriptionally regulates the expression of bcl-2 in the normal human endometrium.

Differential effects of estrogen/androgen on the prevention of nonalcoholic fatty liver disease in the male rat

It is important to clarify the distinct contributions of estrogen/estrogen receptor (ER) and androgen/androgen receptor (AR) signaling and their reciprocal effects on the regulation of hepatic lipid homeostasis. We studied the molecular mechanisms underlying the preventive effects of estradiol (E2), dihydrotestosterone (DHT), or E2+DHT on high-fat diet-induced nonalcoholic fatty liver disease (NAFLD) in an orchidectomized Sprague-Dawley (SD) rat model. E2 is shown to be associated with decreased fatty acid synthesis in hepatic zone 3-specific manner by increasing the phosphorylation of acetyl coenzyme-A carboxylase via an ERα-mediated pathway. DHT is shown to be associated with decreased lipid accumulation and cholesterol synthesis in a hepatic zone 1-specific manner by increasing expression of carnitine palmitotyltransferase1 and phosphorylation of 3-hydroxy-3-methyl-glutaryl-CoA reductase via an AR-mediated pathway. E2+DHT showed an additive positive effect and normalized all three impaired zones of the liver. Gene expression changes in human severe liver steatosis were similar to those of experimental rat NAFLD. Steroids reversed the histopathological NAFLD changes, likely by decreasing fatty acid and cholesterol synthesis and increasing β-oxidation. The diverse steroid effects (ER/AR) on NAFLD prevention in male rats indicate the potential applicability of ER/AR modulators for NAFLD treatment.

Estrogen receptor potentiates mTORC2 signaling in breast cancer cells by upregulating superoxide anions

The estrogen receptor (ER) plays a cardinal role in estrogen-responsive breast carcinogenesis. It is, however, unclear as to how estrogen–ER interaction potentiates breast cancer progression. Compelling evidence supports estrogen-induced redox alterations, such as augmented reactive oxygen species (ROS) levels, as having a crucial role in breast carcinogenesis. Despite ER being a biological mediator of the majority of estrogen-induced cellular responses; its role in estrogen-induced tissue-specific ROS generation remains largely debatable. We examined a panel of human breast cancer specimens and found that ER-positive breast cancer specimens exhibited a higher incidence of augmented O2•− levels compared to matched normal tissue. ROS are known to function as signal transducers and ROS-mediated signaling remains a key complementary mechanism that drives carcinogenesis by activating redox-sensitive oncogenic pathways. Additional studies revealed that augmented O2•− levels in breast cancer specimens coincided with mammalian target of rapamycin complex 2 (mTORC2) hyperactivation. Detailed investigations using in vitro experiments established that 17β-estradiol (E2)-stimulated breast cancer cells exhibited transiently upregulated O2•− levels, with the presence of ER being a crucial determinant for the phenomenon to take place. Gene expression, ER transactivation, and confocal studies revealed that the E2-induced transient O2•− upregulation was effected by ER through a nongenomic pathway possibly involving mitochondria. Furthermore, E2 treatment activated mTORC2 in breast cancer cells in a characteristically ER-dependent manner. Interestingly, altering O2•− anion levels through chemical/genetic methods caused significant modulation of the mTORC2 signaling cascade. Taken together, our findings unravel a novel nongenomic pathway unique to estrogen-responsive breast cancer cells wherein, upon stimulation by E2, ER may regulate mTORC2 activity in a redox-dependent manner by transiently modulating O2•− levels particularly within mitochondria. The findings suggest that therapies aimed at counteracting these redox alterations and/or resultant signaling cascades may complement conventional treatments for estrogen-responsive breast cancer.