ERα Gene Expression Research Articles

SUN-011 Identification Of Regulators Of Estrogenic Signalling In ER-positive Breast Cancer Cells By Whole Genome shRNA Screening

Over 70% of breast tumours express estrogen receptor alpha (ERα), a ligand-inducible transcription factor. Stimulation with estradiol leads to receptor binding to estrogen response elements (EREs) in target genes regulatory sequences in association with transcriptional cofactors. This results in altered gene expression, increased cell proliferation and accelerated tumour growth. To systematically identify genes contributing to ERα expression and signalling in breast cancer, we performed a genome-wide screen in human ERα+ luminal breast cancer cell lines stably expressing estrogen-induced luciferase reporters using an arrayed format. A primary screen (Sigma Mission lentiviral library, 16083 genes, three shRNA/gene) and secondary screens (615 genes identified as hits by at least two shRNAs with concordant impact on reporter gene expression without major effects on cell survival) were carried out using estrogen-sensitive cells (T47D-ERE3-Luc, MCF-7-ERE-Luc) and insensitive controls (antioxidant response reporter T47D-ARE-Luc). We ultimately characterized by RNASeq the impact of suppressing expression of each of 30 prioritized hits (2-3 shRNAs with >50% reduction of RNA levels per hit) on gene expression in ERα+ breast cancer cells. Knockdown of several of these hits had extensive impacts on expression of direct estrogen target genes and on ERα+ breast cancer cell proliferation. Some hits regulated ERα gene expression and several interacted with ERα in co-IP experiments, indicating roles as upstream regulators and/or transcriptional cofactors of ERα.

Vitamin D Receptor in Breast Cancer Tissues and Its Relation to Estrogen Receptor Alpha (ER-α) Gene Expression and Serum 25-hydroxyvitamin D Levels in Egyptian Breast Cancer Patients: A Case-control Study

IntroductionThis study aimed to explore the role of vitamin D receptor (VDR) in breast cancer tissues and its relation to serum 25-hydroxyvitamin D [25(OH)D] levels and estrogen receptor alpha (ER-α) gene expression in patients with breast cancer. Patients and MethodsCancerous and normal breast tissues from 40 women with breast cancer were analyzed for quantification of VDR levels and ER-α gene expression. The serum levels of 25(OH)D were measured in patients with breast cancer and controls by radioimmunoassay. ResultsPatients with breast cancer had serum levels of 25(OH)D significantly lower than normal control subjects. The levels of VDR and ER-α were significantly higher in breast cancer tissues than in normal breast tissues. The serum levels of 25(OH)D were indirectly and significantly correlated with the tissue levels of both VDR and ER-α gene expression. There was a significant direct correlation between the tissue levels of VDR and ER-α gene expression. The serum 25(OH) D levels, tissue VDR levels, and ER-α gene expression levels were inversely and significantly correlated with breast cancer histopathologic grade. Women with serum 25(OH)D levels ≤ 30 nmol/L, tissue levels of VDR > 5 ng/mL, and tissue levels of ER-α gene expression > 17.7 copies had significantly increased risk for breast cancer incidence. ConclusionWomen with low serum 25(OH)D levels, high tissue levels of VDR, and ER-α gene expression had increased risk for breast cancer. VDR are upregulated in breast cancer tissues thus it may be used for target therapy especially in hormone-negative breast cancer.

Epigenetic disruption of estrogen receptor alpha is induced by a glyphosate-based herbicide in the preimplantation uterus of rats

Previously, we have shown that perinatal exposure to a glyphosate-based herbicide (GBH) induces implantation failures in rats. Estrogen receptor alpha (ERα) is critical for successful implantation. ERα transcription is under the control of five promoters (E1, OT, O, ON, and OS), which yield different transcripts. Here, we studied whether perinatal exposure to a GBH alters uterine ERα gene expression and prompts epigenetic modifications in its regulatory regions during the preimplantation period. Pregnant rats (F0) were orally treated with 350 mg glyphosate/kg bw/day through food from gestational day (GD) 9 until weaning. F1 females were bred, and uterine samples were collected on GD5 (preimplantation period). ERα mRNA levels and its transcript variants were evaluated by RT-qPCR. Enzyme-specific restriction sites and predicted transcription factors were searched in silico in the ERα promoter regions to assess the methylation status using the methylation-sensitive restriction enzymes-PCR technique. Post-translational modifications of histones were studied by the chromatin immunoprecipitation assay. GBH upregulated the expression of total ERα mRNA by increasing the abundance of the ERα-O transcript variant. In addition, different epigenetic changes were detected in the O promoter. A decrease in DNA methylation was observed in one of the three sites evaluated in the O promoter. Moreover, histone H4 acetylation and histone H3 lysine 9 trimethylation (H3K9me3) were enriched in the O promoter in GBH-exposed rats, whereas H3K27me3 was decreased. All these alterations could account for the increase in ERα gene expression. Our findings show that perinatal exposure to a GBH causes long-term epigenetic disruption of the uterine ERα gene, which could be associated with the GBH-induced implantation failures.

Beneficial Effects of Curcumin on Rats with Polycystic Ovary Syndrome: Evaluation of the Gene Expression of GLUT4, Erα and Insulin Resistance

Beneficial Effects of Curcumin on Rats with Polycystic Ovary Syndrome: Evaluation of the Gene Expression of GLUT4, Erα and Insulin Resistance

Amelioration by quercetin of insulin resistance and uterine GLUT4 and ERα gene expression in rats with polycystic ovary syndrome (PCOS).

Insulin resistance (IR) and infertility are two major complications of polycystic ovary syndrome (PCOS), which are the results of changes in certain parts of the reproductive and metabolic systems. We aimed to observe the effect of quercetin on dehydroepiandrosterone (DHEA)-induced PCOS and insulin resistance in rats. All animals were divided into five groups and DHEA was used to induce PCOS. Bodyweight and ovarian morphology of all groups were observed. Fasting blood glucose and insulin levels were analysed. The homeostasis model assessment of insulin resistance (HOMA-IR) method was used for IR level determination. The expression of oestrogen receptor α (ERα) and glucose transporter 4 (GLUT4) genes in the uterus was examined by real-time polymerase chain reaction. Liver hexokinase (HK) and glucokinase (GK) activity was determined using spectrophotometry. Quercetin significantly improved the IR state in PCOS rats. PCOS resulted in a decrease in liver GK and an increase in liver HK specific activity, whereas quercetin increased both liver HK and GK activity. Our data also showed a significant reduction in uterine ERα and GLUT4 expression in the PCOS group, which was increased by quercetin. A remarkable effect of quercetin was the intensive reduction of PCOS-IR and significant induction of uterine GLUT4 and ERα gene expression; it could thus be a possible effective treatment for PCOS and its complications, IR and infertility.

Stress urinary incontinent women, the influence of age and hormonal status on estrogen receptor alpha and beta gene expression and protein immunoexpression in paraurethral tissues.

The underlying cause of stress urinary incontinence (SUI) is an anatomical abnormality associated with paraurethral connective tissue dysfunction. The question as to whether estrogens affect the quality of that tissue remains unexplained. Samples of paraurethral connective tissue from 81 women were examined (the SUI's n = 49; the control's n = 32). In both groups, the patients were subdivided into pre- and postmenopausals. Primary study outcome was comparison of the estrogen receptor alpha (ERα) and the estrogen receptor beta (ERβ) gene and protein in paraurethral tissue between SUI and control group. Secondary study outcome was comparison of these receptors according to hormonal status of the patients and their age. In both examined groups, we found both ER proteins. The ERα gene expression was detected in-19/32 (SUI) samples and in 24/31 (control), and ERβ gene expression 31/32 and 30/31 samples, respectively. The SUI's had significantly lower ERa gene expression premenopausally than the control's. The analysis found considerably lower ERβ and reduced ERα gene expression in postmenopausals, approaches the significance level. There was also significant decrease in both receptors' genes expression in post-53 women, compared to younger patients. Spearman's correlation test revealed a statistically significant decrease in ERβ gene with age. Both estrogen receptors are found in women's paraurethral tissue, so this tissue is an estrogen target. No correlation between ERβ gene expression and immunoexpression and SUI was found. The ERα gene seems to play a key role in SUI in the premenopausal period, but ERβ gene expression in the paraurethral connective tissue decreases with age.

Neuronal Dnmt1 Deficiency Attenuates Diet-Induced Obesity in Mice.

Aberrant neuronal DNA methylation patterns have been implicated in the promotion of obesity development; however, the role of neuronal DNA methyltransferases (Dnmts), enzymes that catalyze DNA methylation, in energy balance remains poorly understood. We investigated whether neuronal Dnmt1 regulates normal energy homeostasis and obesity development using a neuronal Dnmt1 knockout (ND1KO) mouse model, Dnmt1fl/fl Synapsin1Cre, which specifically deletes Dnmt1 in neurons. Neuronal Dnmt1 deficiency reduced adiposity in chow-fed mice and attenuated obesity in high-fat diet (HFD)-fed male mice. ND1KO male mice had reduced food intake and increased energy expenditure with the HFD. Furthermore, these mice had improved insulin sensitivity, as measured using an insulin tolerance test. The HFD-fed ND1KO mice had smaller fat pads and upregulation of thermogenic genes in brown adipose tissue. These data suggest that neuronal Dnmt1 plays an important role in regulating energy homeostasis. Notably, ND1KO male mice had elevated estrogen receptor-α (ERα) gene expression in the medial hypothalamus, which previously has been shown to control body weight. Immunohistochemistry experiments revealed that ERα protein expression was upregulated specifically in the dorsomedial region of the ventromedial hypothalamus, a region that might mediate the central effect of leptin. We conclude that neuronal Dnmt1 regulates energy homeostasis through pathways controlling food intake and energy expenditure. In addition, ERα expression in the dorsomedial region of the ventromedial hypothalamus might mediate these effects.

Characterizing combined effects of antiestrogenic chemicals on vitellogenin production in rainbow trout (Oncorhynchus mykiss) hepatocytes

ABSTRACTFish are exposed to a complex mixture of endocrine disrupting compounds (EDC), some of which display antiestrogenic activity leading to suppression of estrogen receptor (ER)- mediated reproductive processes. Although the main mode of action (MoA) of these antiestrogens is to directly interfere with natural ligand binding of the ER, several other MoA have been proposed. The aim of the present study was to characterize single and combined antiestrogenic effects of the aryl hydrocarbon receptor (AhR)-agonist β-naphthoflavone (BNF) and ER-antagonist 4-hydroxytamoxifen (OHT) on vitellogenin (Vtg) protein using primary rainbow trout (Oncorhynchus mykiss) hepatocytes. Supporting transcriptional analysis of ER-responsive genes (estrogen receptor-α (er-α), vitellogenin-1 (vtg-1), eggshell zona radiata protein (zrp)) and AhR-mediated genes (aryl hydrocarbon receptor-2β, cytochrome p450-1a (cyp1a)) was performed by qPCR to characterize the antiestrogenic influence on ER- and AhR-mediated responses. Data demonstrated that both BNF and OHT significantly reduced 17β-estradiol (E2)-induced Vtg protein expression in a concentration responsive manner, whereas exposure to a mixture of these produced an additive antiestrogenic effect. The results observed at the protein level were further supported by transcriptional analysis of ER-responsive genes (er-α, vtg-1, zrp), where only E2-induced vtg-1 gene expression was significantly decreased by OHT and the mixture of OHT and BNF. E2-induced er-α and zrp gene expression was not markedly altered. The significant reduction of E2-induced vtg-1 gene expression by OHT suggested that the antiestrogenic effect of this compound may be associated with ER signaling pathway. Specific genes involved in putative AhR-ER cross-talk were also investigated, however none were directly associated with the compound anti-estrogenic MoA. Although the MoA of the single compounds and mixture were not completely characterized, the present study enhanced our knowledge of the combined toxicity mediated by antiestrogens acting through different MoA.

Synergistic effect of 1α,25-dihydroxyvitamin D3 and 17β-estradiol on osteoblast differentiation of pediatric MSCs

Vitamin D is essential for mineral homeostasis and contributes to bone metabolism by stimulating osteoblast differentiation of marrow stromal cells (MSCs). In this study, we used MSCs from pre-pubertal girls and boys to test the hypothesis that 1α,25(OH)2D and 17β-estradiol have synergistic effects on these MSCs, and what mechanism is involved. With IRB approval, we isolated MSCs from discarded excess iliac marrow graft from children undergoing alveolar cleft repair. Plasma was available from 8 female (9.3 ± 0.2 years) and 8 male (9.6 ± 0.1 years) subjects for hormone assays [25(OH)D, total testosterone, 17β-estradiol, estrone, DHEA-S, Growth Hormone, IGF-I]. RT-PCR was used for gene expression. Alkaline phosphatase (ALP) activity was used to measure osteoblast differentiation at day 7; alizarin red was used to measure matrix mineralization at day 21. All subjects were pre-pubertal based on their hormone levels. Serum 25(OH)D levels ranged from 13.1 to 26.4 ng/mL, with 75% below 20 ng/mL. Constitutive gene expression of VDR and ERα, β varied from subject to subject with no association with sex or serum chemistries. In osteoblastogenic medium, 1α,25(OH)2D3 (10 nM) increased ALP activity by 36% (p < 0.05) in MSCs; 10 nM of E2 was not stimulatory but the combination of 1α,25(OH)2D3 and E2 increased ALP 151% (p < 0.05 vs. control) and by 84.5% (p < 0.05 vs. 1α,25(OH)2D3 alone). The combination of 1α,25(OH)2D3 and E2 significantly increased mineralization 11-fold, compared with either agent alone. Twenty-four hour treatment with 1α,25(OH)2D3 (10 nM) or E2 (10 nM) upregulated each other’s receptor by as much as 5.8-fold for ERα and 2.9-fold for the VDR. In summary, 1α,25(OH)2D3 stimulated osteoblast differentiation and matrix mineralization with MSCs from pre-pubertal subjects, with a synergistic effect of E2, mediated by upregulated receptor levels, at least in part. These studies add new information about the regulation of human osteoblast differentiation, effects of 1α,25(OH)2D3 and E2 on MSCs, and the importance of vitamin D for skeletal health.

Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents

The safety, including the endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (≥10,000 μg/L or 59 μM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-KBluc cells, which was blocked by the estrogen antagonist ICI 182,780. Commercial GBH formulations or their adjuvants alone did not exhibit estrogenic effects in either assay. Transcriptomics analysis of MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced cell proliferation but did not overlap with an ERα gene expression biomarker. Calculation of glyphosate binding energy to ERα predicts a weak and unstable interaction (−4.10 kcal mol−1) compared to estradiol (−25.79 kcal mol−1), which suggests that activation of this receptor by glyphosate is via a ligand-independent mechanism. Induction of ERE-luc expression by the PKA signalling activator IBMX shows that ERE-luc is responsive to ligand-independent activation, suggesting a possible mechanism of glyphosate-mediated activation. Our study reveals that glyphosate, but not other components present in GBHs, can activate ERα in vitro, albeit at relatively high concentrations.

Williams syndrome transcription factor (WSTF) acts as an activator of estrogen receptor signaling in breast cancer cells and the effect can be abrogated by 1α,25-dihydroxyvitamin D3

A majority of estrogen receptor positive (ER+) breast cancers are growth stimulated by estrogens. The ability to inhibit the ER signaling pathway is therefore of critical importance in the current treatment of ER+ breast cancers. It has been reported that 1α,25-dihydroxyvitamin D3 down-regulates the expression of the CYP19A1 gene, encoding the aromatase enzyme that catalyzes the synthesis of estradiol. Furthermore, 1α,25-dihydroxyvitamin D3 has also been reported to down-regulate the expression of estrogen receptor α (ERα), the main mediator of ER signaling.This study reports a novel transcription factor critical to 1α,25-dihydroxyvitamin D3-mediated regulation of estrogenic signaling in MCF-7 breast cancer cells. We have investigated the molecular mechanisms for the 1α,25-dihydroxyvitamin D3-mediated down-regulation of CYP19A1 and ERα gene expression in human MCF-7 breast cancer cells and found that Williams syndrome transcription factor (WSTF) plays a key role by binding to the promoters of CYP19A1 and ERα. Although sometimes reported as an inhibitor of gene expression, we found that WSTF acts as an activator of the promoter activity of both CYP19A1 and ERα. Silencing of WSTF by siRNA transfection resulted in decreased aromatase-dependent cell growth as well as decreased ER signaling in the cells. When cells were treated with 1α,25-dihydroxyvitamin D3, WSTF was dissociated from the promoters and the promoter activities of CYP19A1 and ERα were decreased. We have measured the expression of WSTF in ER-positive tumor-samples from breast cancer patients and found that WSTF is expressed in the majority of the investigated samples and that the expression is higher in cancer tissue than in normal tissue. However, we were not able to show any significant association between the WSTF expression in the tumor and the disease free and overall survival in this patient group who have received adjuvant tamoxifen treatment, nor between the WSTF expression and the expression of ERα, progesterone receptor or HER2. The major conclusions of this study are that WSTF acts as an activator of ER signaling in MCF-7 breast cancer cells, that this action can be inhibited by 1α,25-dihydroxyvitamin D3, and that the expression of WSTF is higher in breast cancer tissue than in normal tissue. WSTF may by a new target for treatment of estrogen-dependent breast cancer cell growth.

Editor's Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives Activate Estrogen Receptor Alpha in Human Breast Cancer Cells.

Plasticizers with estrogenic activity, such as bisphenol A (BPA), have potential adverse health effects in humans. Due to mounting evidence of these health effects, BPA is being phased out and replaced by other bisphenol variants in “BPA-free” products. We have compared estrogenic activity of BPA with 6 bisphenol analogues [bisphenol S (BPS); bisphenol F (BPF); bisphenol AP (BPAP); bisphenol AF (BPAF); bisphenol Z (BPZ); bisphenol B (BPB)] in 3 human breast cancer cell lines. Estrogenicity was assessed (10−11–10−4 M) by cell growth in an estrogen receptor (ER)-mediated cell proliferation assay, and by the induction of estrogen response element-mediated transcription in a luciferase assay. BPAF was the most potent bisphenol, followed by BPB > BPZ ∼ BPA > BPF ∼ BPAP > BPS. The addition of ICI 182,780 antagonized the activation of ERs. Data mining of ToxCast high-throughput screening assays confirm our results but also show divergence in the sensitivities of the assays. Gene expression profiles were determined in MCF-7 cells by microarray analysis. The comparison of transcriptome profile alterations resulting from BPA alternatives with an ERα gene expression biomarker further indicates that all BPA alternatives act as ERα agonists in MCF-7 cells. These results were confirmed by Illumina-based RNA sequencing. In conclusion, BPA alternatives are not necessarily less estrogenic than BPA in human breast cancer cells. BPAF, BPB, and BPZ were more estrogenic than BPA. These findings point to the importance of better understanding the risk of adverse effects from exposure to BPA alternatives, including hormone-dependent breast cancer.

Effect of Silibinin on Maspin and ERα Gene Expression in MCF-7 Human Breast Cancer Cell Line

According to reports, a serine protease inhibitor (Maspin) suppresses metastasis, invasion and angiogenesis in breast and prostate cancers. Silibinin is a natural polyphenolic flavonoid with anti-cancer activity. We assessed the effects of silibinin on cell viability, maspin and ERα gene expression in MCF-7 cell line. The human MCF-7 breast cancer cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with different concentrations of silibinin (100-600 μg/mL) for 24, 48 and 72 hours. The cytotoxic effect of silibinin on MCF-7 viability was determined using Methyl-Thiazolyl-Tetrazolium (MTT) assay by IC50 determination. The fold changes of Maspin and ERα expression were determined by reverse-transcription real-time Polymerase Chain Reaction (PCR). All experiments on the cells were performed in triplicates. The maximum inhibitory effect of silibinin on cell viability was observed at 600 μg/mL after 72-hour incubation (p = 0.001). Incubation of the cells with silibinin for 48 and 72 hours significantly decreased IC50 values to 250 and 207 μg/mL (p = 0.005 and p= 0.006), respectively. The expression of maspin and ERα in the treated cells compared to controls was significantly decreased following treatment with different concentrations of silibinin during a 24-hour period. Silibinin reduces both maspin and ERα gene expression in MCF-7 cell line. The therapeutic effect of silibinin on the treatment of breast cancer may be mediated by the reduction of ERα expression. For verifying this hypothesis and the possible therapeutic implication of silibinin on breast cancer, further studies in this direction are necessary.

Behavioral response and gene expression changes in fipronil-administered male Japanese quail (Coturnix japonica)

Fipronil is an important member of the phenylpyrazole group of insecticides and is widely used for various crops and vegetables to control insects, thereby exposing birds, animals, and humans to fipronil. Currently, there is limited information on the effects of fipronil exposure in Japanese quail. Therefore, our aim was to assess the reproductive toxicological effects of fipronil in the Japanese quail in a 15-day gavage study and then its recovery over a period of 60 days. Fipronil-administration led to significant losses in both feed intake and body weight. Whereas, the gonadosomatic index was not affected, and histological changes observed in the testes were reversible, particularly by day 45 and day 60 of recovery. Cloacal gland atrophy, reduced foam quantity and a reduction in fertility, sexual and aggressive behaviors, and serum testosterone with elevated estradiol (E2) hormone levels were also observed. All these changes gradually reversed during various recovery periods. Further, alterations in hepatic vitellogenin (Vtg) and estrogen receptor α (ERα) gene expression, assessed by quantitative polymerase chain reaction, were also observed. Specifically, ERα1 was induced after fipronil administration, while the Vtg transcript was elevated during both exposure and recovery periods. Our results showed that fipronil exposure has a profound negative influence on reproductive traits in the male Japanese quail and exhibits an estrogenic activity that can raise the incidence of infertility in males. Nevertheless, most of the changes could be reversed after a recovery period of 30–45 days.

Pitaya Extracts Induce Growth Inhibition and Proapoptotic Effects on Human Cell Lines of Breast Cancer via Downregulation of Estrogen Receptor Gene Expression.

Breast cancer is one of the most prevalent cancers in the world and is also the leading cause of cancer death in women. The use of bioactive compounds of functional foods contributes to reduce the risk of chronic diseases, such as cancer and vascular disorders. In this study, we evaluated the antioxidant potential and the influence of pitaya extract (PE) on cell viability, colony formation, cell cycle, apoptosis, and expression of BRCA1, BRCA2, PRAB, and Erα in breast cancer cell lines (MCF-7 and MDA-MB-435). PE showed high antioxidant activity and high values of anthocyanins (74.65 ± 2.18). We observed a selective decrease in cell proliferation caused by PE in MCF-7 (ER+) cell line. Cell cycle analysis revealed that PE induced an increase in G0/G1 phase followed by a decrease in G2/M phase. Also, PE induced apoptosis in MCF-7 (ER+) cell line and suppressed BRCA1, BRCA2, PRAB, and Erα gene expression. Finally, we also demonstrate that no effect was observed with MDA-MB-435 cells (ER−) after PE treatment. Taken together, the present study suggests that pitaya may have a protective effect against breast cancer.

Genistein and Trichostatin A Induction of Estrogen Receptor Alpha Gene Expression, Apoptosis and Cell Growth Inhibition in Hepatocellular Carcinoma HepG 2 Cells

Epigenetic changes such as DNA methylation and histone acetylation play important roles in determining gene expression. Hypermethylation of CpG islands of the promoter region of tumor suppressor genes can greatly influence carcinogenesis through transcriptional silencing. Acetylation of lysine in histone tails causes relaxation of chromatin, which facilitates gene transcription, while deacetylation is associated with condensed chromatin resulting in gene silencing. DNA demethylating agents such as genistein (GE) and histone deacetylase inhibitors (HDACIs) such as trichostatin A (TSA) may strongly reactivate silenced genes and exposure to these two agents in combination is reported to enhance estrogen receptor alpha (ERα) reactivation and induction of apoptosis. The present study was designed to evaluate the effect of these compounds on ERα gene expression, cell viability and apoptosis in hepatocellular carcinoma (HCC) Hep G2 cells. GE exerted biphasic effects; it stimulated cell growth at a low concentration (1 μM) but inhibitory influence was noted with high concentrations (10, 20 and 40 μM). In contrast, TSA demonstrated inhibitory effects on growth at all of concentrations tested. Furthermore, GE and GE/TSA significantly induced apoptosis at all concentrations, but TSA only after 72 h. GE induced ERα re-expression and this was maximal in combined treatment groups treated with GE/TSA for 72 h.Discussion:Our finding clearly indicates that GE and TSA have an inhibitory cell growth, induce apoptosis and reactivate the ERα gene expression.Conclusion:GE and TSA can significantly inhibit the growth of HCC cells and play a significant role in apoptosis and reactivation of ERα gene.

Pathological and immunohistochemical studies in mammary gland tumours affecting male dogs

The study was conducted on 40 clinical cases suspected of canine mammary tumours collected over a period of 2 years. Out of total 38 cases diagnosed as CMT, two (5.26%) cases were noticed in male Labrador dogs of 2.5 and 7 yrs age which were observed in the caudal abdominal and cranial thoracic pair of glands, respectively. The male dog exhibited generalized clinical signs with variability in size. Serum estrogen and progesterone levels in both dogs were higher than the basal normal values. High levels of heavy metals concentration viz. iron, zinc, mercury and cadmium were noticed in the mammary tissues and serum samples of tumour affected dogs. Grossly, growth was hard, firm lobulated pattern with ulceration in one case and soft diffuse round appearance in another case. Tumours were classified as carcinosarcoma and lipoma, respectively. Carcinosarcoma was classified as complex adenocarcinoma forming papillary growths with chondrosarcoma and myxomsarcoma components. Lipoma revealed adipocytes arranged in variable sized lobules separated by connective tissue septa. Immunohistochemical expression of p53 and estrogen receptor alpha (ERα) gene was found to be negative in both cases. Epithelial component (complex carcinoma) in carcinosarcoma showed intense positive immunoreactivity for pancytokeratin in luminal epithelial cells and moderate cytokeratin-14 expression in proliferated myoepithelial cells forming papillary growth.

Identification of MYST3 as a novel epigenetic activator of ER\u03b1 frequently amplified in breast cancer

Estrogen receptor α (ERα) is a master driver of a vast majority of breast cancers. Breast cancer cells often develop resistance to endocrine therapy via restoration of the ERα activity through survival pathways. Thus identifying the epigenetic activator of ERα that can be targeted to block ERα gene expression is a critical topic of endocrine therapy. Here, integrative genomic analysis identified MYST3 as a potential oncogene target that is frequently amplified in breast cancer. MYST3 is involved in histone acetylation via its histone acetyltransferase domain (HAT) and, as a result, activates gene expression by altering chromatin structure. We found that MYST3 was amplified in 11% and/or overexpressed in 15% of breast tumors, and overexpression of MYST3 correlated with worse clinical outcome in estrogen receptor+ (ER+) breast cancers. Interestingly, MYST3 depletion drastically inhibited proliferation in MYST3-high, ER+ breast cancer cells, but not in benign breast epithelial cells or in MYST3-low breast cancer cells. Importantly, we discovered that knocking down MYST3 resulted in profound reduction of ERα expression, while ectopic expression of MYST3 had the reversed effect. Chromatin immunoprecipitation revealed that MYST3 binds to the proximal promoter region of ERα gene, and inactivating mutations in its HAT domain abolished its ability to regulate ERα, suggesting MYST3 functioning as a histone acetyltransferase that activates ERα promoter. Furthermore, MYST3 inhibition with inducible MYST3 shRNAs potently attenuated breast tumor growth in mice. Together, this study identifies the first histone acetyltransferase that activates ERα expression which may be potentially targeted to block ERα at transcriptional level.

Different epigenetic mechanisms of ERα implicated in the fate of fulvestrant-resistant breast cancer

Approximately 70% of breast cancers express estrogen receptor α (ERα), which plays critical roles in breast cancer development. Fulvestrant has been effectively used to treat ERα-positive breast cancer, although resistance remains a critical problem. To elucidate the mechanism of resistance to fulvestrant, we established fulvestrant-resistant cell-lines named MFR (MCF-7 derived fulvestrant resistance) and TFR (T-47D derived fulvestrant resistance) from the ERα-positive luminal breast cancer cell lines MCF-7 and T-47D, respectively. Both fulvestrant-resistant cell lines lost sensitivity to estrogen and anti-estrogens. We observed diminished ERα expression at both the protein and mRNA levels. To address the mechanism of gene expression regulation, we examined epigenetic alteration, especially the DNA methylation level of ERα gene promoters. MFR cells displayed high methylation levels upstream of the ERα gene, whereas no change in DNA methylation was observed in TFR cells. Hence, we examined the gene expression plasticity of ERα, as there are differences in its reversibility following fulvestrant withdrawal. ERα gene expression was not restored in MFR cells, and alternative intracellular phosphorylation signals were activated. By contrast, TFR cells exhibited plasticity of ERα gene expression and ERα-dependent growth; moreover, these cells were resensitized to estrogen and anti-estrogens. The difference in epigenetic regulation among individual cells might explain the difference in the plasticity of ERα expression. We also identified an MFR cell-activating HER/Src-Akt/MAPK pathway; thus, the specific inhibitors effectively blocked MFR cell growth. This finding implies the presence of multiple fulvestrant resistance mechanisms and suggests that the optimal therapies differ among individual tumors as a result of differing epigenetic mechanisms regulating ERα gene expression.

Calcitriol stimulates gene expression of cathelicidin antimicrobial peptide in breast cancer cells with different phenotype.

BackgroundIn normal and neoplastic cells, growth-promoting, proangiogenic, cytotoxic and pro-apoptotic effects have all been attributed to cathelicidin antimicrobial peptide (CAMP). Nevertheless, little is known about the factors regulating this peptide expression in breast cancer. Herein we asked if the well-known antineoplastic hormone calcitriol could differentially modulate CAMP gene expression in human breast cancer cells depending on the cell phenotype in terms of efficacy and potency.MethodsThe established breast cancer cell lines MCF7, BT-474, HCC1806, HCC1937, SUM-229PE and a primary cell culture generated from invasive ductal breast carcinoma were used in this study. Calcitriol regulation of cathelicidin gene expression in vitro and in human breast cancer xenografts was studied by real time PCR. Tumorigenicity was evaluated for each cell line in athymic mice.ResultsEstrogen receptor (ER)α + breast cancer cells showed the highest basal CAMP gene expression. When incubated with calcitriol, CAMP gene expression was stimulated in a dose-dependent and cell phenotype-independent manner. Efficacy of calcitriol was lower in ERα + cells when compared to ERα- cells (<10 vs. >70 folds over control, respectively). Conversely, calcitriol lowest potency upon CAMP gene expression was observed in the ERα-/EGFR+ SUM-229PE cell line (EC50 = 70.8 nM), while the highest was in the basal-type/triple-negative cells HCC1806 (EC50 = 2.13 nM) followed by ERα + cells MCF7 and BT-474 (EC50 = 4.42 nM and 14.6 nM, respectively). In vivo, lower basal CAMP gene expression was related to increased tumorigenicity and lack of ERα expression. Xenografted triple-negative breast tumors of calcitriol-treated mice showed increased CAMP gene expression compared to vehicle-treated animals.ConclusionsIndependently of the cell phenotype, calcitriol provoked a concentration-dependent stimulation on CAMP gene expression, showing greater potency in the triple negative HCC1806 cell line. Efficacy of calcitriol was lower in ERα + cells when compared to ERα- cells in terms of stimulating CAMP gene expression. Lower basal CAMP and lack of ERα gene expression was related to increased tumorigenicity. Our results suggest that calcitriol anti-cancer therapy is more likely to induce higher levels of CAMP in ERα- breast cancer cells, when compared to ERα + breast cancer cells.