Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein–coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry. Exposure to 17β-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase–protein kinase B–mechanistic target of rapamycin pathway: -knockout and -knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. The accession number in the Gene Expression Omnibus is GSE92544.
In response to cell stress, cancer cells often activate the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR). Little was known about the potential role in cancer of a different mode of UPR activation, anticipatory activation of the UPR prior to accumulation of unfolded protein or cell stress. We show that estrogen, acting via estrogen receptor alpha (ER alpha), induces rapid anticipatory activation of the UPR, resulting in increased production of the antiapoptotic chaperone BiP/GRP78, preparing cancer cells for the increased protein production required for subsequent estrogen-ER alpha-induced cell proliferation. In ER alpha-containing cancer cells, the estrogen, 17 beta-estradiol (E-2) activates the UPR through a phospholipase C gamma (PLC gamma)-mediated opening of EnR IP3R calcium channels, enabling passage of calcium from the lumen of the EnR into the cytosol. siRNA knockdown of ER alpha blocked the estrogen-mediated increase in cytosol calcium and UPR activation. Knockdown or inhibition of PLC gamma, or of IP3R, strongly inhibited the estrogen-mediated increases in cytosol calcium, UPR activation and cell proliferation. E-2-ER alpha activates all three arms of the UPR in breast and ovarian cancer cells in culture and in a mouse xenograft. Knockdown of ATF6 alpha, which regulates UPR chaperones, blocked estrogen induction of BiP and strongly inhibited E-2-ER alpha-stimulated cell proliferation. Mild and transient UPR activation by estrogen promotes an adaptive UPR response that protects cells against subsequent UPR-mediated apoptosis. Analysis of data from ER alpha(+) breast cancers demonstrates elevated expression of a UPR gene signature that is a powerful new prognostic marker tightly correlated with subsequent resistance to tamoxifen therapy, reduced time to recurrence and poor survival. Thus, as an early component of the E-2-ER alpha proliferation program, the mitogen estrogen, drives rapid anticipatory activation of the UPR. Anticipatory activation of the UPR is a new role for estrogens in cancer cell proliferation and resistance to therapy.
BACKGROUND: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. OBJECTIVES: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) and demonstrate the efficacy of using predictive computational models trained on high-throughput screening data to evaluate thousands of chemicals for ER-related activity and prioritize them for further testing. METHODS: CERAPP combined multiple models developed in collaboration with 17 groups in the United States and Europe to predict ER activity of a common set of 32,464 chemical structures. Quantitative structure-activity relationship models and docking approaches were employed, mostly using a common training set of 1,677 chemical structures provided by the U.S. EPA, to build a total of 40 categorical and 8 continuous models for binding, agonist, and antagonist ER activity. All predictions were evaluated on a set of 7,522 chemicals curated from the literature. To overcome the limitations of single models, a consensus was built by weighting models on scores based on their evaluated accuracies. RESULTS: Individual model scores ranged from 0.69 to 0.85, showing high prediction reliabilities. Out of the 32,464 chemicals, the consensus model predicted 4,001 chemicals (12.3%) as high priority actives and 6,742 potential actives (20.8%) to be considered for further testing. CONCLUSION: This project demonstrated the possibility to screen large libraries of chemicals using a consensus of different in silico approaches. This concept will be applied in future projects related to other end points.
Non‐technical summary Women during their child‐bearing years have longer QT intervals in their electrocardiograms than men and are more susceptible to lethal arrhythmias elicited by drugs that delay repolarization. Current theories posit that women have a reduced ‘repolarization reserve’ due to reduced potassium currents resulting in longer QT and greater repolarization delays. We proposed an alternative mechanism of higher calcium currents in women which would likewise prolong QT intervals, delay repolarization while increasing the force of contractions and intracellular calcium load. Here, we show that physiological concentrations of oestrogen increase the calcium current only in cells from the base of the heart, by increasing messenger RNA and proteins levels that encode for the calcium current. Moreover, oestrogen acts by interacting with oestrogen receptors (ER)α but not ERβ which may explain why hormone replacement therapy increases the risk of arrhythmia and offers a possible protective solution of using an oestrogen mimetic that selectively binds to ERβ. Abstract In type‐2 long QT (LQT2), adult women and adolescent boys have a higher risk of lethal arrhythmias, called Torsades de pointes (TdP), compared to the opposite sex. In rabbit hearts, similar sex‐ and age‐dependent TdP risks were attributed to higher expression levels of L‐type Ca 2+ channels and Na + –Ca 2+ exchanger, at the base of the female epicardium. Here, the effects of oestrogen and progesterone are investigated to elucidate the mechanisms whereby I Ca,L density is upregulated in adult female rabbit hearts. I Ca,L density was measured by the whole‐cell patch‐clamp technique on days 0–3 in cardiomyocytes isolated from the base and apex of adult female epicardium. Peak I Ca,L was 28% higher at the base than apex ( P < 0.01) and decreased gradually (days 0–3), becoming similar to apex myocytes, which had stable currents for 3 days. Incubation with oestrogen (E2, 0.1–1.0 n m ) increased I Ca,L (∼2‐fold) in female base but not endo‐, apex or male myocytes. Progesterone (0.1–10 μ m ) had no effect at base myocytes. An agonist of the α‐ (PPT, 5 n m ) but not the β‐ (DPN, 5 n m ) subtype oestrogen receptor (ERα/ERβ) upregulated I Ca,L like E2. Western blots detected similar levels of ERα and ERβ in male and female hearts at the base and apex. E2 increased Cav1.2α (immunocytochemistry) and mRNA (RT‐PCR) levels but did not change I Ca,L kinetics. I Ca,L upregulation by E2 was suppressed by the ER antagonist ICI 182,780 (10 μ m ) or by inhibition of transcription (actinomycin D, 4 μ m ) or protein biosynthesis (cycloheximide, 70 μ m ). Therefore, E2 upregulates I Ca,L by a regional genomic mechanism involving ERα which is a known determinant of sex differences in TdP risk in LQT2.
BACKGROUND: Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a "liquid biopsy." METHODS: We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-alpha-positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). RESULTS: Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. CONCLUSIONS: Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease. 2015 American Association for Clinical Chemistry
The findings of the current study indicate that concurrent neoadjuvant chemotherapy and estrogen deprivation could be the preferred neoadjuvant treatment option for patients with estrogen receptor–positive, human epidermal growth factor receptor 2–negative breast cancer, especially for those with higher Ki‐67 levels. These results support concurrent treatment as part of a promising therapeutic strategy for this patient population.
The protective effects of estrogen in the cardiovascular system result from both systemic effects and direct actions of the hormone on the vasculature. Two estrogen receptors have been identified, ERα and ERβ . We demonstrated previously that estrogen inhibits the response to vascular injury in both wild-type and ERα -deficient mice, and that ERβ is expressed in the blood vessels of each, suggesting a role for ERβ in the vascular protective effects of estrogen. In the present study, we examined the effect of estrogen administration on mouse carotid arterial injury in ERβ -deficient mice. Surprisingly, in ovariectomized female wild-type and ERβ knockout mice, 17β -estradiol markedly and equally inhibited the increase in vascular medial area and the proliferation of vascular smooth muscle cells after vascular injury. These data demonstrate that ERβ is not required for estrogen-mediated inhibition of the response to vascular injury, and suggest that either of the two known estrogen receptors is sufficient to protect against vascular injury, or that another unidentified estrogen receptor mediates the vascular protective effects of estrogen.