ERα Methylation Research Articles

Zuogui Pills Improve the Learning and Memory Ability of Brain-Aging Mice through the Estrogen Receptor ERα Methylation Pathway.

Sex hormones are important factors in maintaining brain function and acting as brain protectors. Recent research suggests that neuronal damage in brain aging may be linked to the methylation of the estrogen receptor α (ERα). However, the mechanism of Zuogui Pills (ZGW) in brain-aging ERα DNA methylation and neuronal repair remains unknown. D-galactose-induced ovary removal mice were used as a model of aging. Changes in estrous cycle were detected in mice by vaginal cell smear. Animal behavior tests, including the Morris water maze (MWM) and new object recognition (NOR) test, were conducted. Hematoxylin-eosin (HE) and Nissl-staining were carried out to assess hippocampal neurogenesis. Enzyme-linked immunosorbent assay (ELISA) was performed for 5- methylcytosine methylation levels, and immunohistochemistry (IHC) and western blotting (WB) experiments were performed to assess ERα/DNA methyltransferase 1 (DNMT1) expression after ZGW treatment. Finally, bisulfite sequencing PCR (BSP) analysis was performed to identify methylated differentially expressed estrogen receptor 1 (ESR1) gene in D-gal-induced senescent neurons before and after ZGW treatment. We found that ERα methylation was involved in the delayed brain ageing process of ZGW. Mechanistically, ZGW can improve the learning and memory ability of brain-aging mice, reduce the expression of 5-methylcytosine (5-mc) in serum, increase the amount of ERα, inhibit the expression of DNMT1, and significantly reduce methylated expression of the ESRI gene. Our data suggested that ZGW slowed down D-gal-induced brain aging in mice, and these results showed that ZGW is beneficial for aging. It may be used for neuronal protection in aging.

Nuclear PRMT5 is a biomarker of sensitivity to tamoxifen in ERα+ breast cancer

Endocrine therapies targeting estrogen signaling, such as tamoxifen, have significantly improved management of estrogen receptor alpha (ERα)‐positive breast cancers. However, their efficacy is limited by intrinsic and acquired resistance to treatment, and there is currently no predictive marker of response to these anti‐estrogens to guide treatment decision. Here, using two independent cohorts of breast cancer patients, we identified nuclear PRMT5 expression as an independent predictive marker of sensitivity to tamoxifen. Mechanistically, we discovered that tamoxifen stimulates ERα methylation by PRMT5, a key event for its binding to corepressors such as SMRT and HDAC1, participating in the inhibition of the transcriptional activity of ERα. Although PRMT5 is mainly localized in the cytoplasm of tumor cells, our analyses show that tamoxifen triggers its nuclear translocation in tamoxifen‐sensitive tumors but not in resistant ones. Hence, we unveil a biomarker of sensitivity to tamoxifen in ERα‐positive breast tumors that could be used to enhance the response of breast cancer patients to endocrine therapy, by fostering its nuclear expression.

Methylation of ERβ 5'-untranslated region attenuates its inhibitory effect on ERα gene transcription and promotes the initiation and progression of papillary thyroid cancer.

Normal thyroid tissue displays a prevalent expression of ERβ than ERα, which drastically turns upside down in the initiation and progression of papillary thyroid cancer (PTC). The underlying molecular mechanism of this phenomenon remains unclear. Here, we demonstrated that ERα and ERβ were coexpressed in human thyroid tissues and cells. ERα mRNA (A-1) and ERβ mRNA (0N-1), transcribed from Promoter A of ERα gene and Promoter 0N of ERβ gene, respectively, were the major mRNA isoforms which mainly contributed to total ERα mRNA and total ERβ mRNA in human thyroid-derived cell lines and tissues. The expression levels of ERα mRNA (A-1) and total ERα mRNA were gradually increased, and those of ERβ mRNA (0N-1) and total ERβ mRNA were decreased by degree in the initiation and progression of PTC. No aberrant DNA methylation of ERα 5'-untranslated region was involved in its up-regulation; however, aberrant DNA methylation in Promoter 0N and Exon 0N of ERβ gene was found to be involved in its down-regulation in the initiation and progression of PTC. ERβ can repress ERα gene transcription via recruitment of NCoR and displacement of RNA polymerase II at the Sp1 site in ERα Promoter A-specific region in thyroid-derived cells. It is suggested that DNA methylation of CpG islands in Promoter 0N and Exon 0N of ERβ gene leads to a decreased ERβ gene expression, which attenuates its inhibitory effect on ERα gene transcription and results in an increased ERα gene expression, cell proliferation, initiation, and progression of PTC.

Liuwei Dihuang prevents postmenopausal atherosclerosis and endothelial cell apoptosis via inhibiting DNMT1-medicated ERα methylation

Ethnopharmacological relevanceThe classical and traditional Chinese medicine prescription, Liuwei Dihuang (LWDH), has been commonly used to treat the menopausal syndrome. It has been reported that LWDH could improve estrogen receptor α (ERα) expression to prevent atherosclerosis (AS), while the mechanism of LWDH on regulating ERα expression was still unknown. Aim of the studyTo reveal the mechanism of LWDH on regulating the ERα expression. Materials and methodsThe protective effect of LWDH on Hcy-induced apoptosis of human umbilical vein endothelial cells (HUVECs) was examined. The expression of ERα and DNA methyltransferases 1 (DNMT1) were detected by Western blot and real-time polymerase chain reaction (RT-PCR). The methylation rate of the ERα gene was assayed by the bisulfite sequencing PCR (BSP). High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was applied to determine the level of S-Adenosyl methionine (SAM) and S-Adenosyl homocysteine (SAH). In vivo, the ApoE−/- mice were ovariectomized to establish postmenopausal atherosclerosis (AS) model. ResultsIn vitro study showed that LWDH protects HUVECs from Hcy-induced apoptosis. Treatment with LWDH significantly increased the ERα expression and reduced the methylation rate of the ERα gene by inhibiting the DNMT1 expression. The level of main methyl donor SAM and the ration of SAM/SAH were reduced by LWDH. In vivo, LWDH prevented the formation of plaque and reduced the concentration of Hcy. In addition, LWDH upregulated the ERα expression, as well as inhibiting the expression of DNMT1 in atherosclerotic mice. ConclusionsLWDH exerted protective effects on postmenopausal AS mice, and HUVECs treated with Hcy. LWDH increased of ERα expression via inhibiting DNMT1-dependent ERα methylation.

The arginine methyltransferase PRMT1 regulates IGF-1 signaling in breast cancer

Aside from its well-known nuclear routes of signaling, estrogen also mediates its effects through cytoplasmic signaling. Estrogen signaling involves numerous posttranslational modifications of its receptor ERα, the best known being phosphorylation. Our research group previously showed that upon estrogen stimulation, ERα is methylated on residue R260 and forms the mERα/Src/PI3K complex, central to the rapid transduction of nongenomic estrogen signals. Regulation of ERα signaling via its phosphorylation by growth factors is well recognized, and we wondered whether they could also trigger ERα methylation (mERα). Here, we found that IGF-1 treatment of MCF-7 cells induced rapid ERα methylation by the arginine methyltransferase PRMT1 and triggered the binding of mERα to IGF-1R. Mechanistically, we showed that PRMT1 bound constitutively to IGF-1R and that PRMT1 became activated upon IGF-1 stimulation. Moreover, we found that expression or pharmacological inhibition of PRMT1 impaired mERα and IGF-1 signaling. Our findings were substantiated in a cohort of breast tumors in which IGF-1R expression was positively correlated with ERα/Src and ERα/PI3K expression, hallmarks of nongenomic estrogen signaling, reinforcing the link between IGF-1R and mERα. Altogether, these results provide a new insight into ERα and IGF-1R interference, and open novel perspectives for combining endocrine therapies with PRMT1 inhibitors in ERα-positive tumors.

A motif in HSP90 and P23 that links molecular chaperones to efficient estrogen receptor α methylation by the lysine methyltransferase SMYD2

Heat shock protein 90 (HSP90) is a molecular chaperone that supervises folding of cellular signaling proteins such as steroid receptors and many protein kinases. HSP90 relies on ATP hydrolysis for powering a conformational circuit that helps fold the client protein. To that end, HSP90 binds to co-chaperone proteins that regulate ATP hydrolysis rate or interaction with client proteins. Co-chaperones such as P23, cell division cycle 37 (CDC37), or activator of HSP90 ATPase activity 1 (AHA1) interact with the N-terminal or middle domain of HSP90, whereas others, such as HSP70/HSP90-organizing protein (HOP), use tetratricopeptide repeat (TPR) domains to bind the EEVD motif at the very C-terminal end of HSP90. Recently, the lysine methyltransferase SET and MYND domain-containing 2 (SMYD2) has been proposed as an HSP90-binding partner, and interaction analyses indicate that SMYD2 binding to HSP90 is independent of the EEVD motif. Using the amplified luminescence proximity homogeneous assay (Alpha) technique, I identified a new (M/I/L/V)PXL motif at the C termini of HSP90 and P23 that mediates an interaction with SMYD2, and synthetic peptides harboring this motif dissociated this complex. Of note, the HSP90- and P23-dependent client estrogen receptor α (ERα), was a major methylation target of SMYD2. In a reconstituted system in bacteria, I analyzed HSP90/P23-associated, SMYD2-mediated ERα methylation and found that when SMYD2 binds to the molecular chaperones, it considerably increases methylation of Lys-266 in ERα. Because methylation represses ERα activity, the observed complex formation between SMYD2 and HSP90/P23 may contribute to ERα regulation.

Estrogen receptor-α promoter methylation is a biomarker for outcome prediction of cisplatin resistance in triple-negative breast cancer.

A number of previous studies have indicated the presence of a link between estrogen receptor-α (ERα) methylation and triple-negative breast cancer (TNBC). However, the association between ERα methylation and drug resistance during the treatment of TNBC remains unclear. Methylation-specific polymerase chain reaction was used to investigate the methylation of ERα in the genomic DNA of 35 patients with TNBC who were defined as cisplatin-based chemotherapy-resistant using chemosensitivity testing. Survival probabilities by covariates were assessed using Kaplan-Meier estimator survival analysis and Cox's proportional hazards models, adjusting for age, menopausal status, tumor size, lymph node metastasis and ERα promoter DNA methylation. Of the 35 patients with TNBC analyzed, 8 exhibited ERα promoter DNA methylation. Cisplatin resistance was confirmed to be overwhelmingly associated with ERα methylation by univariate and multivariate analysis. Even in a limited analysis in patients with ERα methylation, the results generated from methylated tumor tissue and unmethylated tumor tissue revealed that expression of breast cancer type 1/2 susceptibility proteins was increased in ERα-methylated breast tumor tissue compared with in unmethylated tissue. The ERα methylation group tended to have significantly shorter progression-free (P=0.010) and overall (P=0.023) survival times compared with those in the unmethylated group. Similarly, shorter progression-free (P=0.024) and overall (P=0.018) survival times were observed in the cisplatin-resistant group compared with the cisplatin-non-resistant group. ERα methylation predicts a poor clinical outcome for patients with TNBC. The results of the present study indicated that ERα methylation may be a candidate surrogate biomarker for outcome prediction and cisplatin resistance in TNBC. Further investigation is required to identify potential biomarkers in a larger cohort in a prospective study.

Promoter methylation of MCAM, ERα and ERβ in serum of early stage prostate cancer patients.

BackgroundProstate cancer (PC) is the second most common cancer among men worldwide. Currently, the most common non-invasive approach for screening and risk assessment of PC is measuring the level of serum prostate-specific antigen (PSA). However, the sensitivity of PSA is 42.8 % and specificity is 41.1%. As a result, the serum PSA test leads to numerous unneeded biopsies. Therefore, a rigorous search for biomarkers for early detection of PC is ongoing. In this study, we aim to assess a panel of epigenetic markers in an intend to develop an early detection test for PC.ResultsThe sensitivity and specificity of hypermethylation of MCAM was 66% and 73% respectively which is an improvement from the sensitivity and specificity of PSA. Considering a combination marker panel of MCAM, ERα and ERβ increased the sensitivity to 75% and the specificity became 70% for the minimally invasive early detection test of PC.Materials and MethodsSixteen primary matched tumor and serum were analyzed by quantitative methylation specific PCR (QMSP) to determine analytical and clinical sensitivity of the genes tested (SSBP2, MCAM, ERα, ERβ, APC, CCND2, MGMT, GSTP1, p16 and RARβ2). Additionally, serum samples from eighty four cases of PC, thirty controls and seven cases diagnosed as high grade Prostatic Intraepithelial Neoplasia (HGPIN) were analyzed.ConclusionsPromoter methylation of MCAM, ERα and ERβ have a potential to be utilized as biomarker for the early detection of prostate PC as their sensitivity and specificity seem to be better than serum PSA in our cohort of samples. After robust validation in a larger prospective cohort, our findings may reduce the numbers of unwarranted prostate biopsies.

Impact of Distinct Genetic and Epigenetic Aberrations on Survival and Response in Acute Myeloid Leukemia Patients Receiving Epigenetic Therapy

Background: Treatment with hypomethylating agents such as decitabine, which results in complete and partial remission rates of up to 50%, has become standard of care in older patients with acute myeloid leukemia (AML) who are no candidates for intensive chemotherapy. While clinical parameters such as reduced performance status, high leukocyte counts, elevated lactate dehydrogenase (LDH) levels as well as poor-risk cytogenetics are associated with lower response rates and shorter overall survival, there exists only limited data on molecular biomarkers that enables for selection of AML patients who are likely to benefit from epigenetic therapy. Shen et al. (JCO 2010) could show that distinct DNA methylation changes are prognostic for overall survival in myelodysplastic syndrome patients, however, response to decitabine therapy could not be predicted. Additionally, mutations in the epigenetic-modifier gene DNMT3A were recently reported to be associated with response and survival in AML patients treated with hypomethylating agents, however, while some of these studies revealed a positive association with outcome, other studies showed no association with response and overall survival (Metzeler et al., Leukemia 2012, DiNardo et al., Leuk Lymph 2014, Coombs et al., Blood 2015).Results: In order to contribute further knowledge regarding prognosis and response to hypomethylating therapy in AML patients, we investigated distinct genetic (FLT3-ITD, NPM1, DNMT3A) and epigenetic (estrogen receptor alpha (ERα), C/EBPα and OLIG2) aberrations in 87 AML patients from the recently published phase II decitabine trial (AML00331, Lübbert et al. Haematologica 2012) to identify potential molecular biomarkers. While FLT3-ITD and NPM1 mutational status were not associated with survival or response to therapy, in our cohort patients harboring DNMT3A R882 mutations showed shorter overall survival (hazard ratio (HR): 2.15, 95%-confidence interval (CI): 0.91-5.12, p=0.08). Promoter DNA methylation analyses using pyrosequencing revealed shorter overall survival of patients with higher levels of methylation of ERα (HR: 1.50, CI: 0.97-2.32, p=0.07) and OLIG2 CpG4 (HR: 1.52, CI: 0.96-2.41, p=0.08), while DNA methylation of C/EBPα showed no association with outcome. Importantly, in multivariate analyses adjusted for clinical baseline parameters, the impact of ERα and OLIG2 CpG4 methylation was conserved (HR: 1.74, CI: 1.03-2.96, p=0.04 and HR: 1.61, CI: 0.96-2.71, p=0.07, respectively) whereas the effect of DNMT3A R882 mutations was reduced to HR: 1.94 (CI: 0.79-4.73, p=0.15). In contrast, none of the investigated molecular markers was associated with response to treatment.Conclusion: In addition to the well established adverse prognostic clinical parameters such as patients’ age, reduced performance status and elevated LDH levels, we provide further evidence that pretreatment genetic and especially epigenetic analyses including DNMT3A R882 mutation status, as well as ERα and OLIG2 CpG4 DNA methylation status may be potential molecular biomarkers in AML patients undergoing epigenetic therapy. DisclosuresLübbert:Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding.

Expression pattern and methylation of estrogen receptor α in breast intraductal proliferative lesions

Intraductal proliferative lesions of the breast including usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) are associated with increased risk, albeit of greatly different magnitudes, for the subsequent development of invasive carcinoma. Estrogen receptor α (ERα) has been widely accepted as a prognostic marker and a predictor for endocrine therapy response of breast cancer. To investigate the ERα expression and methylation in breast intraductal proliferative lesions, we analyzed ERα expression in breast intraductal proliferative lesions including pure UDH (N=98), ADH without DCIS (N=160), DCIS without invasive breast cancer (N=149) by immunohistochemistry. Furthermore, the methylation status of ERα by methylation-specific PCR (MSP) was defined in 217 cases of breast intraductal proliferative lesions. Immunohistochemistry showed that 98/98 (100%) of the UDH cases were positive for ERα expression. ERα protein expression in ADH (132/160) (92.5%) was higher than in DCIS (101/149) (67.8%). But the ERα expression pattern was different with histological diversity of breast intraductal proliferative lesions. The average percent cells staining positive for ERα was 35.33% in UDH, 87.75% in ADH and 71.45% in DCIS. ERα methylation in 32/60 (53.3%) UDH, 11/77 (10.2%) ADH and 32/80 (40.0%) DCIS. Our results demonstrated a strong negative correlation between the percent of cells staining positive for ERα and ERα methylation (r=−0.831, p<0.001). Taken together, our results underlined that ERα expression or methylation may be involved in the breast carcinogenesis and advancement, thus it is not parallel to breast cancer risk in breast intraductal proliferative lesions. No obvious watershed between ERα-positive and -negative breast carcinogenesis was established. Estrogen receptor (ER) methylation or expression is a reversible signal in breast carcinogenesis which affected biological behavior of cells.

Identification of Cyproheptadine as an Inhibitor of SET Domain Containing Lysine Methyltransferase 7/9 (Set7/9) That Regulates Estrogen-Dependent Transcription.

SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.

G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression.

The correlation between DNMT1 and ERα expression and the methylation status of ERα, and its clinical significance in breast cancer.

The correlation between the expression of DNA methyltransferase-1 (DNMT1) and estrogen receptor α (ERα), as well as the methylation status of ERα, was analyzed to investigate the clinical significance of DNMT1 and ERα in breast cancer. Substance P immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) were utilized to detect the protein and mRNA expression levels of DNMT1 and ERα in 112 breast cancer and 20 normal breast specimens. Methylation specific PCR was utilized to detect the methylation status of ERα in ERα-positive and -negative breast cancer specimens and 20 normal breast specimens. The results of the present study revealed that DNMT1 protein and mRNA levels were low in normal breast specimens (10.00 and 46.05%, respectively) and ERα-positive breast cancer specimens (15.00 and 48.68%, respectively), compared with increased levels in ERα-negative breast cancer specimens (81.11 and 88.89%, respectively; P<0.05). The methylation rate of ERα was highest in ERα-negative breast cancer specimens (86.11%) compared with normal breast specimens and ERα-positive breast cancer specimens (10.00 and 36.84%, respectively; P<0.05). Positive expression of ERα protein was observed to be associated with progesterone receptor expression (P<0.05), however, no such association was observed for age, menopause state, tumor size, number of positive nodes, Tumor-Node-Metastasis stage or tumor type (P>0.05). The protein and mRNA expression levels of DNMT1 were negatively correlated with ERα expression (P<0.05). DNMT1 expression was positively correlated with methylation of ERα (P<0.05), and was positively correlated with the methylation of CpG islands of ERα, indicating that the detection of DNMT1 expression may be significant for the diagnosis and typing of breast cancer.

Abstract B29: Maintenance of a lean phenotype is associated with increased ERβ expression and ERβ gene intron methylation in murine MMTVneu luminal mammary cancer

Abstract Background: Obesity is associated with increased breast cancer mortality in premenopausal women, who predominately develop estrogen receptor (ER)-α negative breast tumors. New mechanism-based targets and intervention strategies for offsetting the procancer effects of obesity are urgently needed given the rising rates of obesity in women throughout the world and the lack of effective targeted therapies for ER-negative breast cancer. Unfortunately, the mechanisms by which obesity impacts ER-negative breast cancer prognosis remain unclear. Methods: In this study, we utilized the MMTV-neu transgenic mouse model of Her2+ breast cancer to test the hypothesis that dietary energy balance modulation alters mammary tumor development and progression through epigenetic regulation of ER in the mammary epithelium of these mice. MMTV-neu transgenic mice form spontaneous mammary tumors that progress from an ERα-positive hyperplasia to aggressive ERα-negative ductal adenocarcinomas. Female MMTV-neu transgenic mice were randomized to 3 diet regimens (30/regimen), resulting in either an obese, overweight or lean phenotype. A subset of mice were killed at baseline, 1, 3, and 5 months following diet initiation, and tissues were collected for analysis; remaining animals were followed for up to a 22 month survival study. Results: Gene and protein expression analysis revealed that mammary ERα expression, known to be lost by 8 weeks of age in normoweight MMTV-neu transgenic mice, was maintained in lean mice but lost in the overweight and obese mice (which did not statistically differ in any parameter studied). Additionally, we found lean mice had significantly increased mammary ERβ gene expression and delayed onset of hyperplasia relative to the overweight/obese mice. ERα and ERβ gene intron methylation increased from 5 months to 12 months on diet in lean mice and conversely decreased in overweight/obese mice, which parallels the ER gene expression data. After 22 months of feeding, lean mice had significantly increased tumor-free survival in relative to the overweight/obese mice. Interestingly, human and mouse HER2+ (but not but not HER2-) breast cancer cell lines, when treated with serum from obese (BMI&amp;gt;30), women resulted in a significant down-regulation of ERβ gene expression. Conclusions: In MMTV-neu transgenic mice, dietary energy balance modulation impacts spontaneous mammary tumor development and ERα and ERβ levels in normal and tumor tissue, possibly through epigenetic mechanisms. Moreover, increased mammary ERβ expression may represent a novel target for interventions to offset the enhancing effects of obesity on breast cancer. Citation Format: Emily L. Rossi, Sarah M. Dunlap, Nikki A. Ford, Laura W. Bowers, Stephen D. Hursting. Maintenance of a lean phenotype is associated with increased ERβ expression and ERβ gene intron methylation in murine MMTVneu luminal mammary cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B29.

Correlation of expression of DNA methy transferases 1 and estrogen receptor alpha with methylation status of estrogen receptor alpha and its clinical significance in breast cancer

Objective To investigate the correlation of DNA methy transferases 1(DNMT1)and estrogen receptor alpha(ERα)expression with ERαmethylation and its clinical significance in normal breast specimens and breast cancer specimens. Methods Immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the protein and m RNA expression of DNMT1 and ERαin 20 cases of normal breast specimens and 112 cases of breast cancer specimens.Mutagenically separated polymerase chain reaction(MS-PCR) was used to detect the methylation status of ERαin different specimens. Results Immunohistochemistry and reverse RT-PCR results showed that the positive rate of DNMT1 protein and m RNA was low in normal breast specimens and ERαpositive breast cancer specimens, which was 10. 00% and 46. 05%, and 15. 00% and 48. 68% respectively, while it was high in ERα-negative breast cancer specimens, which was 81. 11% and 88. 89% respectively(P< 0. 05). MS-PCR results revealed that the methylation rate of ERαwas increased by turns in normal breast specimens, ERα-positive breast cancer specimens and ERαnegative breast cancer specimens, which was 10. 00%, 36. 84% and 86. 11% respectively(P< 0. 05).The expression of DNMT1 was positively correlated to the methylation of ERα(P< 0. 05). Conclusion In breast cancer specimens, the protein and mRNA expression of DNMT1 was negatively correlated to the expression of ERα; the expression of DNMT1 was positively correlated to the methylation of Cp G island of ERα. Key words: Breast cancer; DNA methyltransferase 1; Estrogen receptorα; Methylation

Folate- and vitamin B12-deficient diet during gestation and lactation alters cerebellar synapsin expression via impaired influence of estrogen nuclear receptor α.

Deficiency in the methyl donors vitamin B12 and folate during pregnancy and postnatal life impairs proper brain development. We studied the consequences of this combined deficiency on cerebellum plasticity in offspring from rat mothers subjected to deficient diet during gestation and lactation and in rat neuroprogenitor cells expressing cerebellum markers. The major proteomic change in cerebellum of 21-d-old deprived females was a 2.2-fold lower expression of synapsins, which was confirmed in neuroprogenitors cultivated in the deficient condition. A pathway analysis suggested that these proteomic changes were related to estrogen receptor α (ER-α)/Src tyrosine kinase. The influence of impaired ER-α pathway was confirmed by abnormal negative geotaxis test at d 19-20 and decreased phsophorylation of synapsins in deprived females treated by ER-α antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP). This effect was consistent with 2-fold decreased expression and methylation of ER-α and subsequent decreased ER-α/PPAR-γ coactivator 1 α (PGC-1α) interaction in deficiency condition. The impaired ER-α pathway led to decreased expression of synapsins through 2-fold decreased EGR-1/Zif-268 transcription factor and to 1.7-fold reduced Src-dependent phosphorylation of synapsins. The treatment of neuroprogenitors with either MPP or PP1 (4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline, 6,7-dimethoxy-N-(4-phenoxyphenyl)-4-quinazolinamine, SKI-1, Src-l1) Src inhibitor produced similar effects. In conclusion, the deficiency during pregnancy and lactation impairs the expression of synapsins through a deregulation of ER-α pathway.

Abstract P1-06-03: Maintenance of a lean phenotype is associated with increased ERβ expression and ERβ gene intron methylation in murine MMTVneu luminal mammary cancer

Abstract Background: Obesity is associated with increased breast cancer mortality in premenopausal women, who predominately develop estrogen receptor (ER)-α negative breast tumors. New mechanism-based targets and intervention strategies for offsetting the procancer effects of obesity are urgently needed given the rising rates of obesity in women throughout the world and the lack of effective targeted therapies for ER-negative breast cancer. Unfortunately, the mechanisms by which obesity impacts ER-negative breast cancer prognosis remain unclear. Methods: In this study, we utilized the MMTV-neu transgenic mouse model of Her2+ breast cancer to test the hypothesis that dietary energy balance modulation alters mammary tumor development and progression through epigenetic regulation of ER in the mammary epithelium of these mice. MMTV-neu transgenic mice form spontaneous mammary tumors that progress from an ERα-positive hyperplasia to aggressive ERα-negative ductal adenocarcinomas. Female MMTV-neu transgenic mice were randomized to 3 diet regimens (30/regimen), resulting in either an obese, overweight or lean phenotype. A subset of mice were killed at baseline, 1, 3, and 5 months following diet initiation, and tissues were collected for analysis; remaining animals were followed for up to a 22 month survival study. Results: Gene and protein expression analysis revealed that mammary ERα expression, known to be lost by 8 weeks of age in normoweight MMTV-neu transgenic mice, was maintained in lean mice but lost in the overweight and obese mice (which did not statistically differ in any parameter studied). Additionally, we found lean mice had significantly increased mammary ER gene expression and delayed onset of hyperplasia relative to the overweight/obese mice. ERα and ERβ gene intron methylation increased from 5 months to 12 months on diet in lean mice and conversely decreased in overweight/obese mice, which parallels the ER gene expression data. After 22 months of feeding, lean mice had significantly increased tumor-free survival in relative to the overweight/obese mice. Interestingly, human and mouse HER2+ (but not but not HER2-) breast cancer cell lines, when treated with serum from obese (BMI&amp;gt;30), women resulted in a significant down-regulation of ERβ gene expression. Conclusions: In MMTV-neu transgenic mice, dietary energy balance modulation impacts spontaneous mammary tumor development and ERα and ERβ levels in normal and tumor tissue, possibly through epigenetic mechanisms. Moreover, increased mammary ERβ expression may represent a novel target for interventions to offset the enhancing effects of obesity on breast cancer. Citation Format: Emily L Rossi, Sarah M Dunlap, Nikki A Ford, Laura W Bowers, Stephen D Hursting. Maintenance of a lean phenotype is associated with increased ERβ expression and ERβ gene intron methylation in murine MMTVneu luminal mammary cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-06-03.

Abstract 2105: Regulation of estrogen receptor turnover by lysine 302 methylation

Abstract The estrogen pathway promotes growth of breast cancer, the leading cancer diagnosis among US women. The antiestrogen therapy Tamoxifen has been the mainline therapy for the 70% of women whose tumors are estrogen receptor alpha (ERα) positive for over thirty years. Understanding how ERα is regulated, particularly in the context of Tamoxifen, is essential for the treatment of breast cancer. In previous work, we showed that SetD7 mediated methylation of ERα at lysine 302 (K302) is a key mediator of estrogen receptor alpha stability. Downregulation of SetD7 or mutation of K302 increased the rate of ERα turnover resulting in a compromised estrogen driven transcriptional response. However, the precise mechanism by which lysine 302 methylation regulates ERα stability and transcriptional activity is not yet known. In this study, we used a novel small molecule inhibitor of SetD7, (R)-PFI-2, as a chemical probe to further investigate the role of K302 methylation in the regulation of ERα. Treatment of ERα expressing MCF7 breast cancer cells with (R)-PFI-2 resulted in a dose-dependent shift in the 66kDa ERα species to a slower migrating form as detected by protein electrophoresis and immunoblotting. Molecular mass estimates of this slower migrating form are consistent with a sumoylation event. Interestingly, (R)-PFI-2-induced accumulation of the slower migrating form was only observed upon inhibition of the proteasome with MG132, suggesting that this alternately modified form of ERα represents an intermediate in the pathway to degradation. A similar time-dependent shift to the same slower migrating form was observed upon estrogen depletion of MCF7 cells stably knocked down for SetD7, but not in control cells. Furthermore, an inverse relationship was observed between endogenous levels of SetD7 and the levels of modified ER in two strains of MCF7 cell line that differ in their sensitivity to Tamoxifen. These data suggest that methylation of ERα at K302 by SetD7 may stabilize ERα by blocking another post-translational modification, possibly sumoylation, necessary for its turnover. The relationship between ER methylation, sumoylation and Tamoxifen sensitivity will be discussed. Citation Format: Elizabeth L. Zoeller, Dalia Barsyte-Lovejoy, Peter J. Brown, Dafydd R. Owen, Cheryl H. Arrowsmith, Paula M. Vertino. Regulation of estrogen receptor turnover by lysine 302 methylation. [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 2105. doi:10.1158/1538-7445.AM2014-2105

Abstract 2108: Estrogen non genomic signalling is activated in tamoxifen-resistant breast cancer

Abstract Introduction: Although the presence of nuclear estrogen receptor (ERα) is widely used to guide breast cancer therapy, less attention has been paid to the receptor cytoplasmic signalling. We have already shown that arginine methylation of ERα is essential for E2-non genomic pathway triggered by ERα interaction with PI3K and c-Src thereby propagating the signal to downstream transduction cascades (Le Romancer et al, Mol Cell, 2008). Recently, we have found that this pathway is operative in vivo and is activated in aggressive tumors representing a new potential target for breast cancer therapy (Poulard et al, Embo Mol Med, 2012). In addition, the cross talk between ERα and growth factor signalling has emerged as a critical factor in endocrine resistance. The aim of this study was to investigate the receptor cytoplasmic signalling in human ERα-positive breast cancer with acquired resistance to tamoxifen. Experimental procedures: The activation of cytoplasmic ER signalling pathway was investigated in tamoxifen resistant MCF-7 cells and in patient-derived xenografts (PDX) of ERα-positive breast cancer with acquired resistance in vivo to tamoxifen. In addition, tamoxifen-resistant PDX models were treated with tamoxifen, fulvestrant and everolimus, during 90 days. ERα interactions with Src and PI3K were investigated by Proximity Ligation Assay (PLA) in formalin-fixed tumors, a technology which enables to investigate protein/protein interactions in tissues. Results: Interestingly, we detected an increase of ERα/Src/PI3K expression in tamoxifen resistant versus parental MCF-7 cells. This result was confirmed in one PDX model where we found a significant increase of ERα/Src/PI3K expression in the tamoxifen-resistant tumor compared to the corresponding parental xenograft. Finally, the analysis of treated tumors revealed a significant decrease of ERα/Src/PI3K expression in the fulvestrant-treated tumors as well as in tumors treated by the everolimus and fulvestrant combination. In conclusion these results show that ERα cytoplasmic signalling is associated to acquisition of tamoxifen resistance both in cell line and patients-derived xenografts. In addition, ERα cytoplasmic signalling appears to be affected in tumors responding to alternative treatments such as fulvestrant or everolimus. Targeted therapies against ERα, Src and PI3K are in progress and could constitute a new approach to circumvent resistance to endocrine therapies. Citation Format: Coralie Poulard, Juliette Rambaud, Pascale Cohen, Antimo Migliaccio, Laura Corbo, Elisabetta Marangoni, Muriel Le Romancer. Estrogen non genomic signalling is activated in tamoxifen-resistant breast cancer. [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 2108. doi:10.1158/1538-7445.AM2014-2108

Epigenetic marks in estrogen receptor alpha CpG island correlate with some reproductive risk factors in breast cancer.

Reproductive backgrounds, such as age at menarche and menopause, age of first full-term pregnancy (FFTP), number of full-term deliveries and oral contraceptive use are main hormone-related risk factors of breast cancer. It seems that the mentioned factors may affect the risk of breast cancer by enhancing the duration of exposure to estrogen as a potent carcinogen for breast tissue, but the molecular mechanism which links each risk factor to breast cancer is unclear. Estrogen mainly works via its nuclear receptor (ERα). As epigenetic alterations such as CpG methylation are potential links between endogenous or exogenous exposures and genome, we hypothesized that hormone-related risk factors may correlate with the epigenetic marks of the ERα promoter in breast tumors. In the present study, the CpG methylation status of the ERα gene in 99 samples of breast tumors belonged to women with different reproductive histories was evaluated. The reproductive history data were collected from patients. ERα CpG methylation was investigated by methylation specific PCR in DNA samples were obtained from the breast tumors. We could show that some of the hormone-related risk factors (early FFTP and increased number of pregnancies) were inversely correlated with epigenetic marks in ERα gene in breast tumors. Other hormone-related risk factors such as age of menarche and menopause and oral contraceptive use did not show any association with ERα methylation. It seems that pregnancy-related risk factors in comparison with other hormone-related factors work via different mechanism. As ERα methylation is a poor prognosis marker in breast tumors, its association with some modifiable reproductive risk factors (FFTP age and numbers of pregnancies) reiterates the importance of programming reproductive life style not only for prevention of breast cancer but also in favoring the prognosis of the affected women. The exact molecular mechanisms of the observed correlation need more investigation in the future.