Association of promoter methylation of ERα and ERβ with sporadic breast cancer—a study from North India

Methylations in estrogen receptor (ER) α and ERβ are known to be involved in the pathogenesis of breast cancer. Here, we explore the role of promoter methylation of estrogen receptors, ERα and ERβ, in sporadic breast cancer cases from a North Indian population. To this end, association between ERα and ERβ methylation status along with different clinicopathological parameters and its correlation with protein expression was examined. Four hundred eighty paired breast cancer tissue samples and adjacent normal controls from 240 sporadic breast cancer patients were included, and their clinical and demographic profiles were recorded. ERα and ERβ methylation was determined by methylation-specific polymerase (MSP) chain reaction. Our findings demonstrate that methylation of ERα and ERβ occurs in high frequency and appears to be a mechanism of gene silencing in our population. Furthermore, on performing stratified analysis, we observed strong associations between ERα/ERβ methylation and ER, PR, and HER2 status, tumor size, clinical stage, and triple negative tumors. Thus, our study not only highlights the role of ERα/ERβ methylation in breast cancer but also suggests the ERα/ERβ methylation pattern as a biomarker for assessing breast cancer risk.     

Attenuation of Estrogen Receptor α (ERα) Signaling by Selenium in Breast Cancer Cells via Downregulation of ERα Gene Expression

Summary Numerous studies have shown that selenium provides beneficial effects as a cancer chemoprevention agent. Although long-term intervention trials failed to confirm selenium protection against breast cancer in humans because of insufficient cases, the evidence of effective selenium chemoprevention in animal mammary tumor models or human breast cancer cells is substantial and convincing. The present study demonstrates that the selenium compound methylseleninic acid (MSA) inhibits estrogen receptor α (ERα) signaling in ER-positive MCF-7 breast cancer cells as evidenced by decreased estradiol-dependent cell growth and gene expression. MSA diminishes estradiol induction of endogenous ER-regulated pS2 and c-myc genes as well as the expression of an ER-regulated reporter gene. A major mode of MSA action on ER signaling is through a downregulation of ERα gene expression that precedes a decrease in ERα protein level. This study provides a mechanism driven rationale for using selenium as a chemopreventive agent for women at high risk for developing breast cancer or as a therapeutic strategy for ER-positive breast cancer.     

Significance of ER–Src axis in hormonal therapy resistance

The estrogen receptor (ER) is implicated in the progression of breast cancer. Despite positive effects of hormonal therapy, initial or acquired resistance to endocrine therapies frequently occurs. Recent studies suggested ERα-coregulator PELP1 and growth factor receptor ErbB2/HER2 play an essential role in hormonal therapy responsiveness. Src axis couples ERα with HER2 and PELP1, thus representing a new pathway for targeted therapy resistance. To establish the significance of ER–Src axis in PELP1 and HER2 mediated therapy resistance, we have generated model cells that stably express Src-shRNA under conditions of PELP1, HER2 deregulation. Depletion of Src using shRNA substantially reduced E2 mediated activation of Src and MAPK activation in resistant model cells. Pharmacological inhibition of Src using dasatinib, an orally available inhibitor substantially inhibited the growth of therapy resistant MCF7–PELP1, MCF7–HER2, and MCF7–Tam model cells in proliferation assays. In post-menopausal xenograft based studies, treatment with dasatinib significantly inhibited the growth of therapy resistant cells. IHC analysis revealed that the tumors were ERα positive, and dasatinib treated tumors exhibited alterations in Src and MAPK signaling pathways. Combinatorial therapy of tamoxifen with dasatinib showed better therapeutic effect compared to single agent therapy on the growth of therapy resistant PELP1 driven tumors. The results from our study showed that ER–Src axis play an important role in promoting hormonal resistance by proto-oncogenes such as HER2, PELP1, and blocking this axis prevents the development of hormonal independence in vivo. Since PELP1, HER2, and Src kinase are commonly deregulated in breast cancers, combination therapies using both endocrine agents and dasatinib may have better therapeutic effect by delaying the development of hormonal resistance.     

乳腺癌中ERα和ERβ研究进展

雌激素主要是通过和靶细胞雌激素受体(ER)结合而起作用,在乳腺癌发生发展中起重要作用.近年来,ERβ的发现促使人们对乳腺癌中雌激素作用的分子基础重新评价.研究结果提示配体通过ERα和ERβ可能存在不同的作用机制,两者表达关系可能在肿瘤发生、临床生物学特性、内分泌治疗以及预后中有重要价值.     

乳腺癌中ERα和ERβ研究进展

雌激素主要是通过和靶细胞雌激素受体（ER）结合而起作用,在乳腺癌发生发展中起重要作用。近年来,ERβ的发现促使人们对乳腺癌中雌激素作用的分子基础重新评价。研究结果提示配体通过ERα和ERβ可能存在不同的作用机制,两者表达关系可能在肿瘤发生、临床生物学特性、内分泌治疗及预后中有重要价值。     

Nuclear and cytoplasmic expressions of ERβ1 and ERβ2 are predictive of response to therapy and alters prognosis in familial breast cancers

Estrogen receptor (ER) α has been studied extensively in familial breast cancers but there are limited data on ERβ and its isoforms. This is an important issue since many BRCA1-associated tumours are “triple negative” and are resistant to conventional and targeted therapies. We performed an immunohistochemical study of pan-ERβ, ERβ1 and ERβ2 in a cohort of 123 familial breast carcinomas (35 BRCA1, 33 BRCA2 and 55 BRCAX) using a cut-off for positivity at 20% (Shaaban et al. in Clin Cancer Res 14:5228–5235, 2008). BRCA1 cancers were more likely to be nuclear ERα negative and nuclear pan-ERβ positive (21/32, 66%) when compared with BRCA2 (2/29, 7%) and BRCAX cancers (11/49, 22%) (both P < 0.001). For survival analysis, expression was also stratified using cut-offs defined by Bates et al. (Breast Cancer Res Treat 111:453–459, 2008) (score out of 7). Cytoplasmic ERβ2 expression correlated with shorter overall survival at 15 years regardless of cut-off used (both P < 0.046) At a cut-off score of 6 out of 7, cytoplasmic ERβ2 expression correlated with a poorer response to chemotherapy in both univariate (P = 0.011) and multivariate analyses including grade, lymph node status and chemotherapy as an interaction variable (P = 0.045, Hazard ratio 1.22, 95% CI 1.004–9.87). A similar trend was seen in a univariate analysis with a cut-off of 20% although this did not reach statistical significance (P = 0.057). Expression of nuclear ERβ1 was associated with a favourable response to endocrine therapy at 15 years regardless of cut-offs employed (both P < 0.025). However, this did not reach statistical significance in a multivariate analysis (P > 0.05). Since a significant proportion of ERα negative familial breast carcinomas are positive for nuclear ERβ1 and cytoplasmic ERβ2, the different ERβ isoforms and their intracellular location may need to be assessed, to identify patients that may benefit from hormonal and chemotherapy.     

Comparison of increased aromatase versus ERα in the generation of mammary hyperplasia and cancer

Factors associated with increased estrogen synthesis increase breast cancer risk. Increased aromatase and estrogen receptor α (ERα) in both normal epithelium and ductal carcinoma in situ lesions are found in conjunction with breast cancer, leading to the idea that altered estrogen signaling pathways predispose the mammary gland to cancer development. Here, we developed a transgenic mouse that conditionally expresses aromatase in the mammary gland, and used it along with a deregulated ERα expression model to investigate the molecular pathways involved in the development of mammary gland preneoplasia and carcinoma. Both increased ERα and aromatase expression led to the development of preneoplasia, but increased preneoplasia, in addition to carcinoma, was found in aromatase overexpressing mice. Increased prevalence of mammary pathologic changes in mice expressing aromatase correlated with increased cyclin E and cyclin-dependent kinase 2 expression. Gain of both ERα and aromatase increased expression of ERα and progesterone receptor, but aromatase produced a higher increase than ERα, accompanied by higher levels of downstream target genes Ccnd1, Myc, and Tnfsf11. In summary, whereas gain of both ERα and aromatase activate abnormal growth pathways in the mammary gland, aromatase induced a wider range of abnormalities that was associated with a higher prevalence of mammary preneoplasia and cancer progression.     

乳腺癌ER蛋白及ER mRNA含量的预后价值

雌激素受体(ER)及孕激素受体(PgR)状况对乳腺癌复发及内分泌治疗效果的预测有肯定意义,但仍有大约40％ER阳性和25％～30％ER,PgR均阳性乳腺癌患者对内分泌治疗无反应。研究发现,单纯ER蛋白浓度测定的临床意义有其局限性。目前人们已对异常ER蛋白与抗激素基因表现型的关系     

Transactivation of ERα by Rosiglitazone induces proliferation in breast cancer cells

In the present study, we demonstrate that Rosiglitazone (Rosi), a thiazolidinedione and PPARγ agonist, induces ERE (Estrogen Receptor Response Element) reporter activity, pS2 (an endogenous ER gene target) expression, and proliferation of ER positive breast cancer (MCF-7) cells. By performing a dose–response assay, we determined that high concentrations of Rosi inhibit proliferation, while low concentrations of Rosi induce proliferation. Using the anti-estrogen ICI, ER negative breast cancer (MDA-MB-231) cells, and a prostate cancer cell line (22Rv1) deficient in both ERα and PPARγ, we determined that Rosiglitazone-induced ERE reporter activation and proliferation is through an ERα dependent mechanism. Rosiglitazone-induced ERE activation is also dependent on activation of the Extracellular Signal-Regulated Kinase–Mitogen Activated Protein Kinase (ERK–MAPK) pathway, since it is inhibited by co-treatment with U0126, a specific inhibitor of this pathway. We also demonstrate that when ERα and PPARγ are both present, they compete for Rosi, inhibiting each others transactivation. To begin to unravel the pharmacological mechanism of Rosi-induced ER activation, sub-maximally effective concentrations of E₂ were used in combination with increasing concentrations of Rosi in luciferase reporter assays. From these assays it appears that E₂ and Rosi both activate ERα via similar pharmacological mechanisms. Furthermore sub-maximally effective concentrations of E₂ and Rosi additively increase both ERE reporter activity and MCF-7 cell proliferation. The results of this study may have clinical relevancy for Rosi’s use both as an anti-diabetic in post-menopausal women and as an anti-cancer drug in women with ER positive breast cancer     

乳腺癌组织中ERβ及其异构体ERβ1、ERβcx的表达及其临床意义

目的 探讨雌激素受体β（ERβ）及其异构体（ERβ1、ERβcs）在人类表皮生长因子受体2（HER-2）阳性与阴性乳腺癌组织中的表达差异,分析其与雌激素受体α（ERα）、孕激素受体（PR）表达及临床分期和腋窝淋巴结状况的关系。方法采用简单随机抽样的方法选择中山大学附属第一医院2004年12月至2007年12月收治的HER-2高表达（卅）51例与无表达（一）53例乳腺浸润性导管癌病例,分别检测乳腺癌组织的ERα、ERβ、ERβ1、ERβcx及PR表达水平,分析其相互关系及与肿瘤临床分期、腋窝淋巴结转移等临床指标的相关性。采用x^2检验对HER-2（卅）组与（一）组淋巴结表达情况进行对比分析,对HER-2（卅）及HER-2（一）组内各指标采用Spearman相关性分析。结果 在HER-2（卅）与（一）两组中临床分期及淋巴结转移率差异均无统计学意义（P〉0．050）;HER-2（卅）组ERα、ERβ1、ERβcx、PR的阳性表达率均低于HER-2（一）组,差异均有统计学意义（P〈0．050）。HER-2（卅）组中,ERβ1与ERα的表达、ERβcx与PR的表达均呈正相关（P〈0．050）,但ERG、ERβ1及ERβx均未显示与淋巴结转移状况及临床分期相关（P〉0．050）。HER-2（一）组中,HEβ1及ERβcx的表达均与ERα、ERβ1、ERβ、PR呈正相关（P〈0．050）,但ERα、ERβ、ERβ1及ERβcx均与淋巴结转移情况及临床分期无显著相关性（P〉0．050）。结论 ERβ1和ERβcx可能作为乳腺癌预后良好的指标,有必要进一步研究其与乳腺癌治疗后的生存关系。     

乳腺癌ER与FES-PET

雌激素受体fER）是乳腺癌发生、发展最为重要的分子生物学标志物．其状态在乳腺癌的预后、临床治疗策略、内分泌治疗疗效预测等方面具有非常重要的临床价值。当前ER状态测定方法以肿瘤标本免疫组化法为代表，但均为有创、离体测定，存在取材等诸多因素影响和限制，临床内分泌治疗疗效的预测也远不够完美。FES是一种雌激素类似物，能够特异地与雌激素受体结合，FES-PET能够有效表达肿瘤ER的生物活性，采用标准摄取值客观定性、定量分析。FES-PET与病理测定结果密切相关，能有效评估乳腺癌原发灶及转移灶ER状态，并预测内分泌治疗疗效，有望成为临床无创测定肿瘤ER状态的有效方法。     

The greater impact of menopause on ER– than ER+ breast cancer incidence: a possible explanation (United States)

Objective: Analysis of 3359 Danish breast cancer cases indicated that menopause exerted a greater protective effect on estrogen- receptor negative (ER–) breast cancer than on estrogen- receptor positive (ER+) breast cancer. We examined US age-specific breast cancer rates by hormone receptor status in white and black women and men to investigate this unexpected result. Methods: Age-specific breast cancer incidence rates from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute were analyzed by joint estrogen receptor and progesterone receptor (ER/PR) status of 101,140 white female and 8870 black female cases and by ER status in 706 white male and black male cases diagnosed from 1992 to 1998. Changes in the rate of increase in rates with age were identified using Poisson regression analyses. Results: For both white women and black women the age-specific rates of ER– breast cancer cease increasing after 50 years of age, but age-specific rates of ER+ breast cancer continue to increase after 50 years of age. For men the incidence of ER– cancers may increase at a slower rate than incidence of ER+ cancers in older ages. In women the black rates of ER+ cancers are greater than white rates only until age 35, but black rates of ER– cancers are greater than white rates for all ages. Conclusions: Differences in age-specific breast cancer incidence patterns by hormone receptor status are similar for black women and white women. The incidence pattern for ER– cancers is consistent with a paracrine model for hormone-stimulated growth in normal breast tissue. The continued increase in ER+ cancers after menopause may be explained by both the paracrine growth model and an increase in the proliferation rate of ER+ cells with age.     

Clinical implications of ERβ methylation on sporadic breast cancers in Chinese women

Estrogens play a key role in the genesis and progression of breast cancer, activating two estrogen receptors, alpha and beta (ERα and ERβ). We have previously observed that ERα methylation occurs in high frequency and may be one of the mechanisms of ERα expression silence in a subset of Chinese sporadic breast cancers. However, the ERβ promoter methylation status and the relationship between clinicopathological characteristics and ERβ methylation in sporadic breast cancer are still unknown, especially in Chinese women. This study acted to determine the methylation status of the ERβ promoter and its correlation with clinicopathological features of sporadic breast cancers in Chinese women, and lay a foundation for the management of breast cancer. In total, 178 cases with sporadic breast cancers were enrolled in the study. ERβ methylation was determined using a methylation-specific polymerase chain reaction (MSP). In general, ERβ promoter methylation was found in 44.9% (80/178) of breast tumor samples, significantly higher than the benign breast hyperplasia (44.9% vs. 14.3%, X(2) = 4.986, P = 0.026). A total of 58% (40/69) of ERβ-negative tumors got methylation compared with 36.7% (40/109) of ERβ-positive cases being methylated (X(2) = 7.728, P = 0.005). The levels of ERβ protein expression diminished with the frequency of ERβ methylation (r = -0.249, P < 0.0001). In addition, we observed a strong correlation between ERα promoter and ERβ promoter methylation (odds ratio 2.054, 95% confidence interval 1.086-3.886, P = 0.026), and the triple-negative tumors showed a significantly higher methylation rate of ERβ. This study presents, for what we believe to be the first time, that ERβ methylation is also a frequent event in breast cancer and maybe also one of the mechanisms of ERβ expression silence in a subset of Chinese sporadic breast cancers. Epigenetic alteration of the ERβ gene may play an important role in the pathogenesis of breast cancer.     

ERα和ERβ在乳腺癌组织中的表达及临床意义

目的:观察ERα和ERβ蛋白表达与乳腺癌临床病理参数的相关性及临床意义。方法:收集散发性乳腺癌标本214例,乳腺纤维腺瘤组织25例,应用SP免疫组化法检测ERα和ERβ蛋白的表达情况,并分析与乳腺癌患者临床指标及病理参数的相关性。结果:乳腺癌中ERα、ERβ蛋白表达阳性率分别为56.5%（121/214）、62.1%（133/214）,乳腺纤维腺瘤中ERα、ERβ蛋白表达阳性率分别76.0%（19/25）、84.0%（21/25）,与乳腺纤维腺瘤组织比较,乳腺癌组织ERα蛋白阳性表达率无差异,ERβ蛋白阳性表达率显著低于乳腺纤维腺瘤组织（P=0.031）。在乳腺癌组织中,ERα蛋白表达水平与患者年龄、病理类型、临床分级及淋巴结转移、HER-2、p53蛋白表达均未见相关性,与PR蛋白表达正相关（P<0.000 1）。 ERβ蛋白表达与患者年龄和绝经状态相关,在不同的病理类型中,浸润性小叶癌的ERβ蛋白表达阳性率明显高于其它类型肿瘤（P=0.029）;ERβ蛋白表达与临床分级、淋巴结转移情况、HER-2、p53蛋白表达比较均未见相关性。生存分析发现,ERα和ERβ蛋白表达对乳腺癌患者总体生存期未见显著影响,ERα和ERβ均阳性表达的乳腺癌患者,OS时间明显延长（P<0.05）。结论:ERα、ERβ蛋白在乳腺癌组织的异常表达与患者年龄、绝经状态、病理类型相关,二者协同表达对于乳腺癌的发生和发展更具指导意义。     

乳腺癌组织中ERα、ERβ蛋白表达及其意义

[目的]探讨雌激素受体亚型在乳腺癌发生和发展中的作用。[方法]应用免疫组化技术及医学图像分析仪检测正常乳腺组织(30例)、单纯性增生组织(26例)、不典型增生组织(22例)及乳腺癌组织(26例)中ERα、ERβ及Ki67的阳性细胞百分率及灰度值,分析相应抗体的表达量。[结果]①与正常乳腺组织比较,乳腺癌患者组织中ERα阳性细胞百分率(58.34±64.43)及灰度值明显升高(102.17±11.89)(P=0.05,P=0.07);②ERβ的阳性细胞百分率在正常乳腺组织(58.40±63.36)中高于不典型增生组织(37.79±33.35)(P=0.047),而后者又高于乳腺癌组织(18.42±49.24)(P=0.001),ERβ的灰度值在正常乳腺组织(91.63±104.68)中也高于不典型增生(62.09±111.05)(P=0.035);③不典型增生周围组织及癌旁组织中ERβ的表达水平高于病变组织中;④Ki67的表达水平与ERβ的表达呈负相关(rs=-0.61,P=0.001),与ERα表达无显著性相关(rs=-0.013,P=0.989)。[结论]在乳腺癌发生的不同病理阶段ERβ表达量是逐渐降低的,推测ERβ的表达降低与乳腺癌的发生有关。     

Trichostatin A enhances acetylation as well as protein stability of ERα through induction of p300 protein

Introduction  

Trichostatin A (TSA) is a well-characterized histone deacetylase (HDAC) inhibitor. TSA modifies the balance between HDAC and histone acetyltransferase activities that is important in chromatin remodeling and gene expression. Although several previous studies have demonstrated the role of TSA in regulation of estrogen receptor alpha (ERα), the precise mechanism by which TSA affects ERα activity remains unclear.