ERα Levels Research Articles

A high throughput method to study the physiology of E2:ERα signaling in breast cancer cells.

17β-estradiol (E2) regulates diverse physiological effects including cell proliferation through the estrogen receptor α (ERα), which as a transcription factor drives gene transcription and as an extra-nuclear localized receptor triggers the membrane-dependent activation of diverse kinase cascades. E2 also modifies ERα intracellular levels via diverse intracellular mechanisms. In this way, the E2-acivated ERα integrates signaling cascades with the modulation of receptor intracellular concentration and with the induction of DNA synthesis and ultimately drives cell proliferation. In turn, E2 signaling deregulation can cause many diseases including breast cancer (BC). Recently, we performed a Western blotting (WB)-based screen to identify novel pathways affecting ERα intracellular levels and BC cell proliferation. However, because WB lacks high throughput potential, a high-content method to detect all aspects of E2:ERα signaling (nuclear and extra-nuclear receptor activity, ERα levels, E2-induced DNA synthesis) is desirable. Here, we set up a rapid way to measure E2:ERα signaling in 96-well plate format. To demonstrate its robustness, we also challenged 4OH-tamoxifen resistant (Tam-Res) BC cells with a library of anti-cancer drugs and identified methotrexate (MTX) as a molecule inducing ERα degradation and preventing BC cell proliferation. Overall, our research provides a high-content technique to study the physiology of E2:ERα signaling in cells and further suggests a possible anti-ERα and anti-proliferative use for MTX in Tam-Res BCs.

MiR-1271 inhibits ERα expression and confers letrozole resistance in breast cancer.

Attenuation of estrogen receptor α (ERα) expression via unknown mechanism(s) is a hallmark of endocrine-resistant breast cancer (BCa) progression. Here, we report that miR-1271 was significantly down-regulated in letrozole-resistant BCa tissues and in letrozole-resistant BCa cells. miR-1271 directly targeted the chromatin of DNA damage-inducible transcript 3 (DDIT3) gene. miR-1271 expression level was inversely correlated to DDIT3 mRNA level in BCa biopsies. Form a mechanistic standpoint, reintroduction of exogenous miR-1271 could effectively restore ERα level via inhibiting DDIT3 expression, thereby potentiating letrozole sensitivity in BCa cells. Moreover, DDIT3 deregulation promoted letrozole-resistance by acting as a potent corepressor of ESR1 transcription. Taken together, we have identified that disruption of the miR-1271/DDIT3/ERα cascade plays a causative role in the pathogenesis of letrozole resistance in BCa.

Bazedoxifene, a selective estrogen receptor modulator, reduces cerebral aneurysm rupture in Ovariectomized rats

BackgroundEstrogen deficiency is thought to be responsible for the higher frequency of aneurysmal subarachnoid hemorrhage in post- than premenopausal women. Estrogen replacement therapy appears to reduce this risk but is associated with significant side effects. We tested our hypothesis that bazedoxifene, a clinically used selective estrogen receptor (ER) modulator with fewer estrogenic side effects, reduces cerebral aneurysm rupture in a new model of ovariectomized rats.MethodsTen-week-old female Sprague-Dawley rats were subjected to ovariectomy, hemodynamic changes, and hypertension to induce aneurysms (ovariectomized aneurysm rats) and treated with vehicle or with 0.3 or 1.0 mg/kg/day bazedoxifene. They were compared with sham-ovariectomized rats subjected to hypertension and hemodynamic changes (HT rats). The vasoprotective effects of bazedoxifene and the mechanisms underlying its efficacy were analyzed.ResultsDuring 12 weeks of observation, the incidence of aneurysm rupture was 52% in ovariectomized rats. With no effect on the blood pressure, treatment with 0.3 or 1.0 mg/kg/day bazedoxifene lowered this rate to 11 and 17%, almost the same as in HT rats (17%). In ovariectomized rats, the mRNA level of ERα, ERβ, and the tissue inhibitor of metalloproteinase-2 was downregulated in the cerebral artery prone to rupture at 5 weeks after aneurysm induction; the mRNA level of interleukin-1β and the matrix metalloproteinase-9 was upregulated. In HT rats, bazedoxifene restored the mRNA level of ERα and ERβ and decreased the level of interleukin-1β and matrix metalloproteinase-9. These findings suggest that bazedoxifene was protective against aneurysmal rupture by alleviating the vascular inflammation and degradation exacerbated by the decrease in ERα and ERβ.ConclusionsOur observation that bazedoxifene decreased the incidence of aneurysmal rupture in ovariectomized rats warrants further studies to validate this response in humans.

Effects of Atrial Natriuretic Peptide on p53 and Estrogen Receptor in Breast Cancer Cells.

The atrial natriuretic peptide (ANP) hormone is secreted by cardiac atrial myocytes and acts to regulate blood pressure homeostasis in humans. Previous research indicates ANP treatment significantly decreases the proliferation of human prostate cancer cells, pancreatic adenocarcinoma, and breast cancer cells. Minimal studies have been conducted with regard to ANP regulating tumor suppressor genes and steroid hormone receptors in breast cancer cells. Our study analyzed the effects of ANP in combination with 17β-estradiol (E2) and antiestrogen treatments on p53 and ERα levels in T-47D breast cancer cells. Preliminary studies through Western blot analysis showed that ANP treatment decreases p53 and ERα expression levels in a concentration-dependent (10–100 nM) manner. Treatment with ANP alone, at a 100 nM concentration, causes a decrease of p53 and ERα expression compared with Cs (control stripped), but with E2 and antiestrogen combinations, expression of both protein levels decreased compared with treatments without ANP. Combined treatment with E2, an estrogen antagonist, and ANP decreased cellular proliferation compared with treatments without ANP, except in the case of raloxifene (RAL). Our studies indicate that ANP has potential as a therapeutic breast cancer treatment and should inspire further studies on the molecular mechanism of ANP in T-47D breast cancer cells.

Evaluation of long term low dose exposure to mixtures on the lymphocytes of the peripheral blood of rats

The environmental risks of environmental estrogens (EEs) are often assessed via the same mode of action in the concentration addition (CA) model, neglecting the complex combined mechanisms at the genetic level. In this study, the cell proliferation effects of estrone, 17α-ethinylestradiol, 17β-estradiol, estriol, diethylstilbestrol, estradiol valerate, bisphenol A, 4-tert-octylphenol and 4-nonylphenol were determined individually using the CCK-8 method, and the proliferation effects of a multicomponent mixture of estrogenic chemicals mixed at equipotent concentrations using a fixed-ratio design were studied using estrogen-sensitive MCF-7 cells. Furthermore, transcription factors related to cell proliferation were analyzed using RT-PCR assays to explore the potential molecular mechanisms related to the estrogenic proliferative effects. The results showed that the estrogenic chemicals act together in an additive mode, and the combined proliferative effects could be predicted more accurately by the response addition model than the CA model with regard to their adverse outcomes. Furthermore, different signaling pathways were involved depending on the different mixtures. The RT-PCR analyses showed that different estrogens have distinct avidities and preferences for different estrogen receptors at the gene level. Furthermore, the results indicated that estrogenic mixtures increased ERα, PIK3CA, GPER, and PTEN levels and reduced Akt1 level to display combined estrogenicity. These findings indicated that the potential combined environmental risks were greater than those found in some specific assessment procedures based on a similar mode of action due to the diversity of environmental pollutions and their multiple unknown modes of action. Thus, more efforts are needed for mode-of-action-driven analyses at the molecular level. Furthermore, to more accurately predict and assess the individual responses in vivo from the cellular effects in vitro, more parameters and correction factors should be taken into consideration in the addition model.

Transcriptional activation of Nlp by estrogen-ERα in breast cancer

Estrogen Receptor-α (ERα) is the key transcription factor that regulates cell proliferation and homeostasis. In this pathway, estrogen plays an important role in genomic instability and cell cycle regulation processes and the mechanisms of its action are multifaceted. In this study, we showed that estrogen regulates genomic instability through promoting the expression of Nlp, a BRCA1-associated centrosomal protein which is involved in microtubule nucleation, spindle formation, chromosomal missegregation and abnormal cytokinesis. We demonstrated that the expression of Nlp is strongly associated with ERα and FOXA1 level in clinical breast cancer samples with poor clinical outcomes to breast cancer patients. Addition of estrogen in the ER-positive breast cancer cells resulted in elevation of NLP mRNA. Significantly, we identified that estrogen-ERα is capable of regulating Nlp expression through specifically binding ERα to the proximal region and the Estrogen Responsive Elements (ERE) enhancer in the distal region of NLP gene. Reporter assays demonstrated that estrogen directly activated Nlp promoter. ChIP assay results showed that E2-ERα directly bound to the EREs of Nlp. Therefore, overexpression of Nlp in breast cancer exhibits a hormone-dependent pattern, and estrogen participates in the regulation of genome instability and cell cycle in breast cancer cells partially through transcriptional activation of NLP gene. Overexpression of Nlp enhances the malignant progression of ERα-positive breast cancer cells in vitro, whereas knockdown of Nlp suppresses this biological effects in ERα-positive breast cancer cells. ERα/Nlp axis may serve as a promising target against breast cancer.

Inhibitory effects of 17β-estradiol or a resveratrol dimer on hypoxia-inducible factor-1α in genioglossus myoblasts: Involvement of ERα and its downstream p38MAPK pathways.

Deficiency in the functioning of the genioglossus, which is one of the upper airway dilator muscles, is an important cause of obstructive sleep apnea/hypopnea syndrome (OSAHS). Estrogens have been reported to inhibit hypoxia-inducible factor-1α (HIF-1α) expression in hypoxia, regulating its target genes and exerting protective effects on the genioglossus in chronic intermittent hypoxia (CIH). This study aimed to investigate the role of 17β-estradiol (E2) and a resveratrol dimer (RD) on HIF-1α and the underlying mechanism. Mouse genioglossus myoblasts were isolated and cultured, and the estrogen receptor α (ERα) shRNA lentivirus was used for gene knockdown. Then MTT assay was used to determine the effects of E2 and RD on the viability of the cells. Cells in different groups were treated with different agents (E2, or RD, or E2 and SB203580), incubated under normoxia or hypoxia for 24 h, and then expression levels of HIF-1α, ERα, ERβ, total-p38 MAPK and phospho-p38 MAPK were detected. We observed that both E2 and RD inhibited the overexpression of HIF-1α induced by hypoxia at the mRNA and protein levels, and these effects were eliminated by genetic silencing of ERα by RNAi. In addition, we found that E2 activated p38 MAPK pathways to inhibit HIF-1α expression. On the whole, ERα may be responsible for downregulation of HIF-1α by E2 or RD via activation of downstream p38 MAPK pathways.

Role of metformin in inhibiting estrogen-induced proliferation and regulating ERα and ERβ expression in human endometrial cancer cells.

Diabetes mellitus (DM) is an important factor that contributes to the development of type I endometrial cancer (EC). Previous studies have demonstrated that metformin decreases mortality and risk of neoplasms in patients with DM. Since estrogen and estrogen receptor (ER) expression has been associated with the development of EC, the present study aimed to investigate the effects of metformin on cell proliferation and ER expression in EC cell lines that are sensitive to estrogen. The viability and proliferation of Ishikawa and HEC-1-A cells were measured following treatment with metformin and/or a 5' AMP-activated protein kinase (AMPK) inhibitor (compound C) with or without treatment with estradiol (E2). In addition, the levels of ERα, ERβ, AMPK, ribosomal protein S6 kinase β-1 (p70S6K), myc proto-oncogene protein (c-myc) and proto-oncogene c-fos (c-fos) were measured following treatment. Metformin significantly decreased E2-stimulated cell proliferation; an effect that was rescued in the presence of compound C. Metformin treatment markedly increased the phosphorylation of AMPK while decreasing p70S6K phosphorylation, indicating that metformin exerts its effects through stimulation of AMPK and subsequent inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. In addition, metformin significantly inhibited ERα expression while increasing ERβ expression, whereas treatment with compound C reversed these effects. Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that c-fos and c-myc expression were attenuated by metformin, an effect that was rescued in the presence of compound C. Therefore, metformin regulates the expression of ERs, and inhibits estrogen-mediated proliferation of human EC cells through the activation of AMPK and subsequent inhibition of the mTOR signaling pathway.

Bazedoxifene and raloxifene protect neocortical neurons undergoing hypoxia via targeting ERα and PPAR-γ

Selective estrogen receptor modulators (SERMs) such as bazedoxifene and raloxifene are recognized to mainly act via estrogen receptors (ERs), but there is no study examining the involvement of PPAR-γ in their actions, especially in neurons undergoing hypoxia. Little is also known about age-dependent actions of the SERMs on neuronal tissue challenged with hypoxia. In this study, bazedoxifene and raloxifene protected neocortical cells against hypoxia at early and later developmental stages. Both SERMs evoked caspase-3-independent neuroprotection and increased protein levels of ERα (66 and 46 kDa isoforms) and PPAR-γ. In addition, bazedoxifene enhanced expression of ERα-regulated Cyp19a1 mRNA. Using double siRNA silencing, for the first time we demonstrated a key role of ERα and PPAR-γ in the neuroprotective action of the SERMs in neocortical neurons undergoing hypoxia. This study provides prospects for the development of a new therapeutic strategies against hypoxic brain injury that selectively target ERα and/or PPAR-γ.

Repression of ESR1 transcription by MYOD potentiates letrozole-resistance in ERα-positive breast cancer cells

Transcriptional silencing of estrogen receptor α (ERα) expression is an important etiology contributing to the letrozole-resistance in ERα-positive breast cancer (BCa) cells, but the transcription factors responsible for this transcriptional repression remain largely unidentified. Here we report that the expression of the basic helix-loop-helix myogenic regulatory factor MYOD was abnormally up-regulated in letrozole-resistant BCa tissues and in experimentally-induced letrozole-resistant BCa cells. Overexpression of the exogenous MYOD impaired ERα expression and potentiated letrozole-resistance in letrozole-sensitive MCF7 cells, whereas MYOD knockdown could effectively restore ERα expression and thereby promote letrozole-sensitivity in letrozole-resistant MCF-7/LR cells. Mechanistically, MYOD was shown to be a potent corepressor of ESR1 transcription, and this transcriptional repression was significantly enhanced in the presence of letrozole treatment. Thus, targeted inhibition of MYOD may restore ERα level and lead to resensitization to letrozole-based hormone therapy, providing a novel therapeutic strategy for relapsed ERα-positive BCa patients. Our data also underscore an unexpected chemotherapeutic facet of MYOD, which may operate as a novel regulator of BCa biology.

The study of endometrium at gestational days 5 and 15 in dairy goats (Capra hircus)

Endometrium receptivity, an absolutely necessary part of successful embryo implantation, and several morphological and biochemical endometrial receptivity biomarkers have already been studied and proposed in some animals. However, to our knowledge, no such a study has as yet been undertaken in dairy goat. In the present study, the serum and uterus of dairy goat were studied at gestational days 5 and 15, the estrogen (E2) and progesterone (P4) concentrations in serum were determined by electrochemiluminescence immunoassay, the surface topography of endometrium was observed by scanning electron microscopy. Furthermore, the estrogen receptors (ER), prolactin (PRL), some marker genes of receptive endometrium, and cell proliferation and apoptosis in the uterus were also detected. Well-formed pinopodes were found on the surface of endometrium at day 15 with higher E2 and P4 concentrations in the serum and higher estrogen receptors ERα and ERβ expression levels and a lower PRL level in the endometrium. Moreover, some expression levels of marker genes of receptive endometrium (OPN, VEGF, LIF, PRLR) were increased at day 15 compared to day 5, but no significant differences were observed in the cell proliferation and apoptosis in the uterus. The results showed that the endometrium reached the receptive state at gestational day 15 in dairy goats.

Regulation of hepatic Na+/K+-ATPase in obese female and male rats: involvement of ERK1/2, AMPK, and Rho/ROCK.

In this study, we assessed whether the disturbed regulation of sodium/potassium-adenosine-triphosphatase (Na+/K+-ATPase) occurs as a consequence of obesity-induced IR in sex-specific manner. We also assessed whether alterations of IRS/PI3K/Akt, ERK1/2, AMPKα, and RhoA/ROCK signaling cascades have an important role in this pathology. Female and male Wistar rats (150-200g, 8weeks old) were fed a standard laboratory diet or a high-fat (HF) diet (42% fat) for 10weeks. The activity of hepatic Na+/K+-ATPase and Rho, and the association of IRS-1/p85 were assessed in liver. Furthermore, the protein level of α1 Na+/K+-ATPase in plasma membrane fractions, and protein levels of IRS-1, PI3K-p85, -p110, RhoA, ROCK1, ROCK2, ERK1/2, AMPKα, ERα, and ERβ in liver lysates were assessed. The expression of hepatic α1 Na+/K+-ATPase mRNA was also analyzed by qRT-PCR. The results show that HF-fed female rats exhibited an increase in hepatic ERK1/2 (p<0.05) and AMPKα (p<0.05) phosphorylation levels, unchanged level of Na+/K+-ATPase α1 mRNA, decreased level of Na+/K+-ATPase activity (p<0.05), and decreased α1 Na+/K+-ATPase protein expression (p<0.01). In liver of HF-fed male rats, results show decreased levels of Na+/K+-ATPase activity (p<0.01), both protein and mRNA of α1 subunit (p<0.05), but significant increase in Rho activity (p<0.05). Our results indicate significant sex differences in α1 Na+/K+-ATPase mRNA expression and activation of ERK1/2, AMPKα, and Rho in the liver. Exploring the sex-specific factors and pathways that promote obesity-related diseases may lead to a better understanding of pathogenesis and discovering new therapeutic targets.

Gene expression profile of estrogen receptors alpha and beta in rat brain during aging and following high fat diet

The “sex-hormone” estrogen-17β promotes several cognitive functions and is a master regulator of brain bioenergetics via the estrogen receptors α and β (ERα and ERβ). In this work, by using Real-Time PCR analysis, we evaluated the effect of aging and high fat diet (HFD) on ERα and ERβ expression in rat hippocampus and cortex. In young rats, ERβ is abundant in cortex and ERα in hippocampus. During the aging, in the cortex, we observe a general decrease in ERα and ERβ expression; in hippocampus ERα increases and ERβ decreases. ER expression patterns in rat brain are also affected by the administration of an HFD. In cortex, after 4weeks of HFD, ERβ transcripts are down-regulated, whereas ERα levels remain unchanged; after 12weeks, both ERα and ERβ expression is up-regulated. In the hippocampus, the level of ERβ transcripts does not change following HDF, whereas ERα expression is affected by HDF, in a time-dependent manner: it increases after the 4-week treatment and decreases after 12weeks. Possible involvements of these receptors in the control of cortex and hippocampus functions during aging and in the modulation of energetic metabolism and feeding behaviour are discussed.

Vitamin E-rich nanoemulsion enhances the antitumor efficacy of low-dose paclitaxel by driving Th1 immune response and promoting dendritic cell activation

Transcriptional silencing of estrogen receptor α (ERα) expression is an important etiology contributing to the letrozole-resistance in ERα-positive breast cancer (BCa) cells, but the transcription factors responsible for this transcriptional repression remain largely unidentified. Here we report that the expression of the basic helix-loop-helix myogenic regulatory factor MYOD was abnormally up-regulated in letrozole-resistant BCa tissues and in experimentally-induced letrozole-resistant BCa cells. Overexpression of the exogenous MYOD impaired ERα expression and potentiated letrozole-resistance in letrozole-sensitive MCF7 cells, whereas MYOD knockdown could effectively restore ERα expression and thereby promote letrozole-sensitivity in letrozole-resistant MCF-7/LR cells. Mechanistically, MYOD was shown to be a potent corepressor of ESR1 transcription, and this transcriptional repression was significantly enhanced in the presence of letrozole treatment. Thus, targeted inhibition of MYOD may restore ERα level and lead to resensitization to letrozole-based hormone therapy, providing a novel therapeutic strategy for relapsed ERα-positive BCa patients. Our data also underscore an unexpected chemotherapeutic facet of MYOD, which may operate as a novel regulator of BCa biology.

A functional drug re-purposing screening identifies carfilzomib as a drug preventing 17β-estradiol: ERα signaling and cell proliferation in breast cancer cells

Most cases of breast cancer (BC) are estrogen receptor α-positive (ERα+) at diagnosis. The presence of ERα drives the therapeutic approach for this disease, which often consists of endocrine therapy (ET). 4OH-Tamoxifen and faslodex (i.e., fulvestrant - ICI182,780) are two ETs that render tumor cells insensitive to 17β-estradiol (E2)-dependent proliferative stimuli and prevent BC progression. However, ET has limitations and serious failures in different tissues and organs. Thus, there is an urgent need to identify novel drugs to fight BC in the clinic. Re-positioning of old drugs for new clinical purposes is an attractive alternative for drug discovery. For this analysis, we focused on the modulation of intracellular ERα levels in BC cells as target for the screening of about 900 Food and Drug Administration (FDA) approved compounds that would hinder E2:ERα signaling and inhibit BC cell proliferation. We found that carfilzomib induces ERα degradation and prevents E2 signaling and cell proliferation in two ERα+ BC cell lines. Remarkably, the analysis of carfilzomib effects on a cell model system with an acquired resistance to 4OH-tamoxifen revealed that this drug has an antiproliferative effect superior to faslodex in BC cells. Therefore, our results identify carfilzomib as a drug preventing E2:ERα signaling and cell proliferation in BC cells and suggest its possible re-position for the treatment of ERα+ BC as well as for those diseases that have acquired resistance to 4OH-tamoxifen.

MiR-203 inhibits estrogen-induced viability, migration and invasion of estrogen receptor α-positive breast cancer cells.

Breast cancer is common in females, and accounts for a large proportion of cancer-related cases of mortality. MicroRNAs (miRs) have been found to be involved in the progression of breast cancer via mediation of tumor suppressor genes or oncogenes. Previously, miR-203 has been reported to play a suppressive role in breast cancer. In the present study, the effects of miR-203 on the malignant phenotypes of estrogen receptor α (ERα)-positive breast cancer cells were investigated. It was found that treatment with estradiol (E2) significantly enhanced the viability, migration and invasion of ERα-positive breast cancer MCF-7 cells, accompanied by the significant downregulation of miR-203 in a dose-dependent manner. Furthermore, MCF-7 cells were transfected with miR-203 mimics, resulting in a significant increase in miR-203 levels. Upregulation of miR-203 was found to significantly inhibit E2-induced upregulation of MCF-7 cell viability, migration and invasion. Upregulation of miR-203 also led to a significant decrease in the protein expression of ERα in MCF-7 cells. Using a luciferase reporter assay, ERα was identified as a direct target of miR-203 in MCF-7 cells. Finally, it was demonstrated that miR-203 was significantly downregulated in ERα-positive breast cancer tissues compared to their matched normal adjacent tissues. The expression levels of miR-203 were inversely correlated to the ERα levels in ERα-positive breast cancer tissues. Based on these results, it is proposed that miR-203 inhibits E2-induced viability, migration and invasion of ERα-positive breast cancer cells, and that this may be via direct targeting of ERα. Therefore, the present study highlights the importance of miR-203 and ERα in breast cancer progression.

Involvement of estrogen receptor β in androgen receptor-induced growth inhibition in prostate cancer PC-3 cells.

Previous studies have suggested that changes in sex hormone receptor expression may be associated with the initiation and progression of prostate cancer (PCa). Therefore, the present study aimed to investigate the association and possible pathways between two sex hormone receptors and PCa by measuring the expression levels of the androgen receptor (AR) and the estrogen receptor subtypes alpha (ERα) and beta (ERβ) in prostatic cancer PC-3 cell lines. The pcDNA3.1-hERβ plasmid was transfected into PC-3 cell lines. The expression levels of AR, ERα and ERβ were detected at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR). The results demonstrated that the expression levels of AR, ERβ and ERα were downregulated to different degrees: ERβ test group vs. PC-3 cell group (P=0.000; 95% confidence interval: 0.9803–1.6331). ERβ and AR expression was detected continuously in the PC-3 cells, but the expression of ERα was not. AR expression levels exhibited an upward trend whilst the expression of ERβ demonstrated a marked downward trend. There is a correlation between the expression levels of ERβ and the incidence of PCa, and ERβ may inhibit the growth of PC-3 cell lines by regulating the expression levels of AR. ERβ may provide a novel target for PCa therapies.

Expression of Estrogen and Progesterone Receptor in Tumor Stroma Predicts Favorable Prognosis of Cervical Squamous Cell Carcinoma.

The aim of this study was to investigate the expression of estrogen receptor α (ERα) and progesterone receptor B (PRB) in the stroma and carcinoma tissues of cervical cancer and their relationship to clinical characteristics and the status of human papillomavirus (HPV) infection. Expressional levels of ERα and PRB in tissue blocks of 95 cervical carcinomas were independently scored by 2 pathologists. Human papillomavirus DNA, viral load, and genotypes were determined by polymerase chain reaction. Clinical characteristics were reviewed from chart and cancer registry. Estrogen receptor α and PRB were mainly expressed in the stroma but not in the carcinoma tissues of the cervical cancer, and their expressions were highly correlated. More stromal ERαs were found in early-stage tumors than in advanced-stage tumors. Greater stromal expressions of ERα and PRB were associated with a more favorable prognosis (P = 0.018 and P = 0.004, respectively). The expressions were not related to the differentiation of cancer, the status of HPV infection, the HPV load, or the genotype. In multivariate analysis, stromal ERα and PRB expressions were independently associated with a lower risk of mortality. The adjusted hazard ratios of mortality for low and high expressions of ERα were 0.19 (95% confidential interval [95% CI], 0.04-0.87) and 0.15 (95% CI, 0.03-0.81), respectively, whereas for low and high expressions of PRB hazard ratios were 0.46 (95% CI, 0.19-1.16) and 0.24 (95% CI, 0.06-0.96), respectively. This study showed that stromal ERα and PRB expressions are independent prognostic indicators of cervical squamous cell carcinoma.

The effects of estradiol valerate and remifemin on liver lipid metabolism

To investigate the lipid metabolism dysregulation in the liver of ovariectomized (OVX) rats and effects of estradiol valerate (E) and remifemin (ICR) thereon, forty female Sprague-Dawley rats were randomly divided into sham-operated (SHAM), OVX, OVX+E, and OVX+ICR group. After 4 weeks’ E or ICR treatment, serum estrogen, cholesterol, and triglyceride levels; lipid droplets in hepatocytes; hepatocyte morphology; and the expression of estrogen receptor α (ERα), liver X receptor (LXR), and sterol regulatory element binding proteins (SREBPs) in the liver of the rats were assessed. OVX rats had significantly decreased serum estrogen levels, which significantly increased after treatment with E but not with ICR. Serum triglyceride levels and the amount of lipid droplets in hepatocytes increased after ovariectomy, and significantly decreased after E treatment. In addition, ICR treatment markedly increased serum triglyceride levels and lipid droplet size. No significant differences in the serum cholesterol levels were observed among the four groups. After ovariectomy, hepatocyte mitochondria became hypertrophic and misformed, which were reversed with E or ICR treatment. ICR-treated rats also showed endoplasmic reticulum disorganization. After ovariectomy, ERα and LXR levels significantly decreased while SREBP expression increased. E treatment increased ERα and LXR levels while ICR treatment only increased LXR expression. E treatment decreased SREBP-1c levels, whereas SREBP-1c levels increased with ICR treatment. Treatment with E significantly reversed the ovariectomy-induced dysregulation of hepatocyte lipid metabolism, which was, however, exacerbated with ICR treatment. The effects of E and ICR on hepatocyte lipid metabolism may involve the regulation of LXR and SREBP-1c.

Abstract 5741: Evaluating the role of recurrent ESR1-CCDC170 fusion in breast cancer endocrine resistance

Abstract Background: Recurrent gene fusions resulting from chromosome rearrangements are central to the formation of cancer. Previously, our lab identified recurrent rearrangements between the estrogen receptor α (ERα) gene ESR1 and its neighbor gene CCDC170, in ~6-8% of luminal B tumors, a more aggressive form of ER+ breast cancer that has worse clinical outcome after endocrine therapy. ESR1-CCDC170 rearrangements enable the expression of different-sized N-terminally truncated CCDC170 (ΔCCDC170) under ESR1 promoter, such as the E2-E7 or E2-E10 variants in which exon 2 of ESR1 fused with exon 7 or exon 10 of CCDC170. Consistent with the behavior of luminal B tumors, ectopic ΔCCDC170 expression in ER+ breast cancer cells transformed cells to a more aggressive form and led to reduced endocrine sensitivity in vitro, as well as enhanced xenograft growth in vivo. In the present study, we examined the role of ESR1-CCDC170 in breast cancer endocrine resistance in vivo and further elucidated the potential engaged mechanisms of its actions. Results: Our in vivo endocrine sensitivity study showed that, while tamoxifen (Tam) treatment rendered tumor regression in the vector-expressing T47D xenograft tumors, it made the E2-E7 overexpressing T47D xenografts static and less likely to regress, whereas the E2-E10 overexpressing xenografts, though grew initially upon Tam treatment, became cytostatic and showed significant reduction in tumor regression. Kaplan-Meier analysis revealed a significantly worse progression-free survival for E2-E7 (p&amp;lt;0.01) and E2-E10 (p&amp;lt;0.001) overexpressing tumors treated with Tam compared to the control tumors. These data suggest that ESR1-CCDC170 variants differ in level of reduced responsivity to Tam, and may render the T47D xenografts less sensitive or resistant to Tam in vivo. Further studies suggest that ΔCCDC170 protein preferentially localizes to cytoplasm (different from wild-type CCDC170 protein enriched in nucleus), physically interacts with ERα and HER2, and forms homodimers. RPPA analysis showed that silencing of ESR1-CCDC170 repressed ERα and BCL2 protein levels, as well as total/phospho-HER2 levels, consistent with the findings of ΔCCDC170 interactions with ERα and HER2. Importantly, analysis of ΔCCDC170 protein sequence revealed a potential high-affinity ATP-binding pocket at its C-terminus, suggesting that ΔCCDC170 may be directly druggable. Conclusion: These data suggest a potential role of ESR1-CCDC170 in mediating breast cancer endocrine responsivity, which may possibly through cytoplasmic mislocalization of ΔCCDC170 and interactions with ERα and HER2. Further studies will affirm and elucidate the role of overexpressed or endogenous ESR1-CCDC170 in breast cancer endocrine resistance, and pinpoint the precise mechanisms of its oncogenic function, in order to develop a novel target therapy against it for the fusion-carrying patient community around the world. Citation Format: Yiheng Hu, Jamunarani Veeraraghavan, Xian Wang, Ying Tan, Jin-Ah Kim, Rachel Schiff, Xiao-Song Wang. Evaluating the role of recurrent ESR1-CCDC170 fusion in breast cancer endocrine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5741. doi:10.1158/1538-7445.AM2017-5741