Expression Of ERβ5 Research Articles

The ERβ5 splice variant increases oestrogen responsiveness of ERαpos Ishikawa cells.

Endometrial cancer is a common gynaeological malignancy: life time exposure to oestrogen is a key risk factor. Oestrogen action is mediated by receptors encoded by ESR1 (ERα) and ESR2 (ERβ): ERα plays a key role in regulating endometrial cell proliferation. A truncated splice variant isoform (ERβ5) encoded by ESR2 is highly expressed in cancers. This study explored whether ERβ5 alters oestrogen responsiveness of endometrial epithelial cells. Immunhistochemistry profiling of human endometrial cancer tissue biopsies identified epithelial cells co-expressing ERβ5 and ERα in stage I endometrial adenocarcinomas and post menopausal endometrium. Induced co-expression of ERβ5 in ERαpos endometrial cancer cells (Ishikawa) significantly increased ligand-dependent activation of an ERE-luciferase reporter stimulated by either E2 or the ERα-selective agonist 1,3,5-(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) compared to untransfected cells. Fluorescence recovery after photobleaching (FRAP) analysis of tagged yellow fluorescent protein (YFP)-ERβ5 transfected into Ishikawa cells revealed that incubation with E2 induced a transient reduction in intra-nuclear mobility characterised by punctate protein redistribution which phenocopied the behaviour of ERα following ligand activation with E2. In ERαneg MDA-MD-231 breast cancer cells, there was no E2-dependent change in mobility of YFP-ERβ5 and no activation of the ERE reporter in cells expressing ERβ5. In conclusion, we demonstrate that ERβ5 can act as heterodimeric partner to ERα in Ishikawa cells and increases their sensitivity to E2. We speculate that expression of ERβ5 in endometrial epithelial cells may increase the risk of malignant transformation and suggest that immunostaining for ERβ5 should be included in diagnostic assessment of women with early grade cancers.

Interaction of estrogen receptor β5 and interleukin 6 receptor in the progression of non-small cell lung cancer.

Numerous studies have shown that the estrogen receptor beta (ERβ) and interleukin 6 receptor (IL-6R) had interaction in many tumors, including lung cancer. Previous studies found that ERβ5 exhibits a different biological function compared with the other subtypes of ERβ. Therefore, this study mainly explores the interaction between ERβ5 and IL-6R in the progression of lung cancer. We found that the expression of ERβ5, IL-6 and glycoprotein 130 (GP130) were significantly increased (P < 0.001) and the 5-year survival rate with the co-expression of ERβ5 and GP130 is significantly lower (P = 0.0315) in non-small cell lung cancer (NSCLC) patients. The cell proliferation, invasion, and cell cycle were markedly increased, and the cell apoptotic was markedly inhibited with the concurrent action of ERβ5 and IL-6 in A549 cells (P < 0.05). In addition, the expression of ERβ5, GP130, p-AKT, and p-44/42 MAPK was also significantly increased in A549 cells (P < 0.05). These results indicate that ERβ5 and GP130 can synergistically promote the progression of NSCLC and maybe combined as an independent prognostic factor in patients. In addition, these results also provide a theoretical basis for the combined targeting therapy of ERβ5 and GP130 in NSCLC.

Differential Effects of Estrogen Receptor β Isoforms on Glioblastoma Progression

The estrogen receptor β (ERβ) functions as a tumor suppressor in glioblastoma (GBM) cells. However, the in vivo significance of endogenous ERβ and the roles of its isoforms in GBM are incompletely understood. Using ERβ isoform-specific PCR screening, we found that GBM cells predominantly express ERβ1 and ERβ5, along with low levels of ERβ2 and ERβ4. We observed greater ERβ5 expression in higher grades of glioma than in lower grades. In CRISPR-based ERβ knockout (KO) cells and ERβ KO cells uniquely expressing ERβ1 or ERβ5 only, ERβ1 significantly reduced proliferation. Compared with parental GBM cells, ERβ KO cells exhibited high migratory and invasive potentials, and reexpression of ERβ1 resulted in the reduction of this phenotype. Interestingly, ERβ5 expression increased foci formation and anchorage-independent growth of NIH3T3 cells and increased motile structure formation, including filopodia and ruffles in GBM cells. Only ERβ1-expressing tumors resulted in longer mouse survival. RNA-Seq analysis revealed unique pathways modulated by ERβ1 and ERβ5. Compared with ERβ KO cells, ERβ1 cells exhibited lower activation of mTOR signaling molecules, including p-mTOR, p-S6K, and p-S6, and ERβ5-expressing cells had enhanced mTOR downstream signaling. Unique proteins including several that function as regulators of mTOR, immunomodulatory, and apoptosis pathways bound to ERβ1 and ERβ5 isoforms. Our work confirms the tumor-suppressive potential of ERβ1 and reveals the acquired oncogenic ability of ERβ5 in GBM cells. ERβ isoform status and their unique interactions with oncogenic pathways may have important implications in GBM progression.Significance: These findings suggest that only ERβ isoform 1 has tumor suppressor function in GBM and that ERβ isoform switching contributes to GBM progression. Cancer Res; 78(12); 3176-89. ©2018 AACR.

Oestrogen receptor β5 and epidermal growth factor receptor synergistically promote lung cancer progression

Oestrogen receptor beta (ERβ) and epidermal growth factor receptor (EGFR) pathway can synergistically promote the proliferation, invasion, and metastasis of non-small-cell lung cancer (NSCLC) cells. ERβ has five subtypes, and the selective splicing of exon 8 in ERβ5 transcription translational phase makes its biological function different from other subtypes. The following study investigates whether ERβ5 interacts with EGFR pathway in lung cancer. Briefly, we found that the overexpression of ERβ5 and EGFR is associated with poor prognosis and decreased overall survival in NSCLC patients. Furthermore, the effects of ERβ5 and EGFR on cell biological behaviour were investigated in vitro. These results indicated that the combination of ERβ5 and EGF induces cell proliferation and invasion, while the combination of ERβ5 and Gefitinib (EGFR inhibitors, Gef) induces cell apoptosis and promotes cell mitosis in A549 cell line. In addition, the combination of ERβ5 and EGF increases the expression of ERβ5, EGFR, and p-ERK1/2 in lung cancer cells. To sum up, the obtained results suggest that ERβ5 and EGFR synergistically promote the progression of lung cancer by activating MEK/ERK signalling pathway, which provides a theoretical basis for more accurate combined targeted therapy.

Abstract P3-04-04: Estrogen receptor β5 increases aggressiveness of the triple negative breast cancer cell line SUM159

Abstract Recent clinical studies are indicating that the estrogen receptor β variant β5 (ERβ5) expression correlates to worse prognosis. We wanted to know if expression of ERβ5 is changing the growth behavior of the triple negative cell line SUM159. Estrogen receptor β5 is highly similar to estrogen receptor β1 except for a truncated C-terminus making the remaining ligand binding domain incapable of binding to estrogen. In addition a 4 amino acid unique peptide is added to the C-terminal end. Stably expressing ERβ5 using a transposon integrated tetracycline regulated expression system we find that expression of ERβ5 increases proliferation of the triple negative SUM159 cells especially in reduced serum condition compared to control cells. Since SUM159 have been shown to depend on autocrine stimulation for growth we are suggesting that expression of ERβ5 is affecting the production of autocrine growth factors. Citation Format: Faria M, Tin-U C, Dey P, Gustafsson J-A, Strom AM. Estrogen receptor β5 increases aggressiveness of the triple negative breast cancer cell line SUM159. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-04.

Cytoplasm estrogen receptor β5 as an improved prognostic factor in thymoma and thymic carcinoma progression

A number of previous studies have reported that sex steroid hormones, including estrogens, are involved in the regulation of the thymic function. The aim of the present study was to investigate the expression of estrogen receptor β5 (ERβ5) in thymic tumors and the correlation between ERβ5 expression and thymoma biological characteristics. The expression levels of ERβ5 in thymic epithelial tumors was evaluated in 103 patents using immunohistochemical staining and reverse transcription-quantitative polymerase chain reaction. In addition, an indirect immunofluorescence assay was performed to evaluate the ERβ5 expression levels in the TC1889 and T1682 cell lines. The survival outcome was estimated using Kaplan-Meier plots. The results indicated that ERβ5 expression was mainly located in the thymic tumor cell cytoplasm (87.37%; 90/103 cases) and overexpression was observed in thymic tumors compared with normal thymic tissues (P=0.001). Using the Kruskal-Wallis test, a statistically significant association was observed between cytoplasmic ERβ5 (cERβ5) expression and thymic tumor subtypes (P=0.024) and stages (P=0.003 and R=-0.376). The Kaplan-Meier plots revealed that cERβ5 expression was significantly associated with improved overall and progression-free survival (P=0.008 and P=0.004, respectively). The present study suggested that overexpression of cERβ5 may indicate an improved prognosis and may be involved in the underlying mechanism through which estrogen inhibits thymoma and thymic carcinoma development.

Estrogen Receptor β Isoform 5 Confers Sensitivity of Breast Cancer Cell Lines to Chemotherapeutic Agent-Induced Apoptosis through Interaction with Bcl2L12

Alternative splicing of estrogen receptor β (ERβ) yields five isoforms, but their functions remain elusive. ERβ isoform5 (ERβ5) has been positively correlated with better prognosis and longer survival of patients with breast cancer (BCa) in various clinical studies. In this study, we investigated the inhibitory role of ERβ5 in BCa cells. Although ERβ5 does not reduce proliferation of BCa cell lines MCF-7 and MDA-MB-231, its ectopic expression significantly decreases their survival by sensitizing them to doxorubicin- or cisplatin-induced apoptosis through the intrinsic apoptotic pathway. Moreover, we discovered Bcl2L12, which belongs to the Bcl-2 family regulating apoptosis, to be a specific interacting partner of ERβ5, but not ERβ1 or ERα, in an estradiol-independent manner. Knockdown of Bcl2L12 enhanced doxorubicin- or cisplatin-induced apoptosis, and this process was further promoted by ectopic expression of ERβ5. Whereas Bcl2L12 was previously shown to inhibit apoptosis through binding to caspase 7, such interaction is reduced in the presence of ERβ5, suggesting a mechanism by which ERβ5 sensitizes cells to apoptosis. In conclusion, ERβ5 interacts with Bcl2L12 and functions in a novel estrogen-independent molecular pathway that promotes chemotherapeutic agent-induced in vitro apoptosis of BCa cell lines.

Abstract 501: Effect of estrogen receptor beta isoforms on the function of estrogen receptor beta in estrogen recpetor alpha negative breast cancer cell lines.

Abstract African American (AA) women with breast cancer are more likely to be diagnosed at a more advanced stage of the disease, have a higher recurrence rate and a poorer prognosis than Caucasian (CA) women. African American women are more often diagnosed with high-grade breast tumors at an earlier age (pre-menopausal), most of which are negative for the estrogen and progesterone receptors (ER-, PR-). These factors result in a higher mortality rate among AA breast cancer patients. This survival disparity can be attributed to socioeconomic status and other related factors, however, there are biological factors that may also be involved. The insulin-like growth factor II (IGF-II) plays a crucial role in fetal and cancer development by signaling through the IGF-I receptor (IGF-IR), the insulin receptor (Ins-R) and also crosstalk with the estrogen receptor alpha (ER-α). Compared to CA women, AA women have an increased level of proIGF-II expression in breast tissues and higher signaling activation through the IGF-1 and Ins-R. Studies have shown that the estrogen receptor beta (ER-β) is also expressed in a majority of breast cancers. The estrogen receptor (ER) as well as the progesterone receptor and Her2Neu have been the most common markers used for determining breast cancer status. Most research done in breast cancer as it pertains to the ER status has mainly been done in cells and tissues expressing the Estrogen Receptor Alpha (ER-α). However, with its discovery in 1996, the Estrogen Receptor Beta (ER-ß) has lead to a better understanding the breast cancer disease. Studies have shown that the estrogen receptor beta (β) is expressed in a majority of breast cancers, including cancers designated as ER negative (ER-). The tumor suppressing function of ER-β has been described based on its interaction with ER-α, however it's function in the absence of ER-α has not been clearly assessed. ER-β has 5 isoforms of which ER-β isoform 5 (ER-β5) is the most abundantly expressed among AA women with triple negative breast cancer. ER-β5 expression is associated with a poor prognosis and decreased survival of breast cancer patients. Therefore, we hypothesize that the heterodimerization and subsequent activation of ER-β with its isoforms, mainly ER-β5, leads to a suppression of its function in breast cancers not expressing ER-α, thus preventing cell death. To test this hypothesis we analyzed the basal expression of ER-β and all of its isoforms (ER- β1-5) in AA and CA normal and breast cancer cell lines. The expression of ER-β5 increased in cells lines in response to insulin like growth factor-II (IGF-II). IGF-II promotes the phosphorylation of ER-β and ER-β5 and may regulate the ER-β signaling pathway in ER-α negative cell lines. Citation Format: Chane’ N. O'Bannon. Effect of estrogen receptor beta isoforms on the function of estrogen receptor beta in estrogen recpetor alpha negative breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 501. doi:10.1158/1538-7445.AM2013-501

Involvement of estrogen receptor β5 in the progression of glioma

Emerging evidence suggests a decline of ERβ expression in various peripheral cancers. ERβ has been proposed as a cancer brake that inhibits tumor proliferation. In the current study, we have identified ERβ5 as the predominant isoform of ERβ in human glioma and its expression was significantly increased in human glioma as compared with non-neoplastic brain tissue. Hypoxia and activation of hypoxia inducible factor (HIF) increased ERβ transcription in U87 cells, suggesting elevated ERβ expression in glioma might be induced by the hypoxic stress in the tumor. Over-expression of either ERβ1 or ERβ5 increased PTEN expression and inhibited activation of the PI3K/AKT/mTOR pathway. In addition, ERβ5 inhibited the MAPK/ERK pathway. In U87 cells, ERβ1 and ERβ5 inhibit cell proliferation and reduced cells in the S+G2/M phase. Our findings suggest hypoxia induced ERβ5 expression in glioma as a self-protective mechanism against tumor proliferation and that ERβ5 might serve as a therapeutic target for the treatment of glioma.

Abstract 1114: IGF-II regulates the expression of estrogen receptor beta 5: Role in the survival disparity between African-American and Caucasian women with breast cancer

Abstract African American (AA) women with breast cancer are more likely to be diagnosed at a more advanced stage of the disease, have a higher recurrence rate and a poorer prognosis than Caucasian (CA) women. African American women are more often diagnosed with high-grade breast tumors at an earlier age most of which are negative for the estrogen and progesterone receptors. These factors result in a higher mortality rate among AA patients. This survival disparity can be attributed to socieconomic status and other related factors, however there are biological factors that may also be involved. The insulin-like growth factor II (IGF-II) plays a crucial role in fetal and cancer development by signaling through the IGF-I receptor (IGF-IR), the insulin receptor (InsR) and crosstalk with the estrogen receptor alpha (ER-α). Compared to CA women, AA women have an increased level of proIGF-II expression in breast tissues and higher signaling activation through the IGF-1 and InsRs. Since the estrogen receptor beta (ER-β) is also expressed in a majority of breast cancers we propose that IGF-II may also activate the ER-β signaling pathway. ER-β has 5 isoforms of which ER-β5 is the most abundantly expressed among AA women with breast cancer and it is associated with a poor prognosis and decreased survival. Thus, our hypothesis is that IGF-II activation of the ER-β5 contributes to the survival disparity between AA women and CA women. To test this hypothesis we analyzed the expression of ER-β5 in AA and CA breast cancer cell lines by western blot, siRNA analysis and IGF-II stimulation. IGF-II not only modulated the phosphorylation of ER-β5, but it also promoted the translocation of ER-β5 to different organelles. Thus, we conclude that ER-β5 may contribute to the survival disparity among AA breast cancer patients since ER-β5 impairs the inhibitory effect of other ER-β isoforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1114. doi:10.1158/1538-7445.AM2011-1114

The changes of estrogen receptor-β variants expression in breast carcinogenesis: Decrease of estrogen receptor-β2 expression is the key event in breast cancer development

Although more than five variant forms of estrogen receptor-beta (ERbeta) have been identified, their role has not been identified. This study was carried out to investigate the changes of ERbeta variants in breast cancer development. Using reverse transcription polymerase chain reaction (RT-PCR) and triple primer PCR (TP-PCR), the expression levels of ERbeta variants mRNA were measured in 66 paired normal and cancer tissues. The relative expression level of ERbeta variants were compared between normal and cancer tissues, and also compared according to various clinicopathological parameters. Among ERbeta variants, ERbeta2 and ERbeta5 consist of the major proportion of ERbeta expression both in normal and cancer tissues. The ERbeta and ERbeta2 expression levels decreased significantly in the cancers compared with corresponding normal tissues, particularly in ERalpha-expressing cancers. However, ERbeta5 expression level increased significantly in the cancers, especially in those of postmenopausal patients. The relative increase of ERbeta5 expression in cancer tissues was associated with favorable differentiation. Decrease of ERbeta2 is thought to be the key reason for the decrease in ERbeta expression in cancer tissues, and it is particularly associated with the development of ERalpha-expressing breast cancer.

Correlation of mRNA for oestrogen receptor beta splice variants ERβ1, ERβ2/ERβcx and ERβ5 with outcome in endocrine-treated breast cancer

This study has been performed to test the hypothesis that different oestrogen receptor beta (ERbeta) splice variants may be important determinants of clinical parameters, including outcome, in post-menopausal women with breast cancer receiving adjuvant endocrine treatment but no chemotherapy. Splice variants ERbeta1, ERbeta2 and ERbeta5 have been analysed by semi-quantitative RT-PCR in a cohort of 105 patients with primary breast cancer. Clinical correlates included age, grade, size, nodal status, ERalpha, progesterone receptor, Ki67, relapse-free survival (RFS) and overall survival (OS). Seventy per cent of cases were ERbeta1 positive, 69% ERbeta2 positive and 70% ERbeta5 positive. Within the cohort, 47% were positive for all three variants while 10% were negative for all three. ERbeta1 exhibited no discernible relationship with disease outcome. ERbeta2 and ERbeta5 expression was significantly associated with better RFS (P<0.005), and ERbeta2 with better OS (P=0.0002). In multivariate analysis, ERbeta2 (P=0.006), nodal status and the level of Ki67 expression were independent predictors for RFS while ERbeta2 (P=0.0008) and Ki67 status were independent predictors for OS. In the ERalpha-positive cases, or in the subset of those receiving adjuvant tamoxifen, ERbeta2 was significantly associated with good RFS (P<0.0005) and was the only independent marker of OS. We conclude that precise identification of splice variants of ERbeta are more important assessors than is ERbeta1 alone of the biological status of individual breast cancers, and hence in predicting their response to endocrine therapy.

Altered expression pattern of alternatively spliced estrogen receptor β transcripts in breast carcinoma

Dysregulation of total estrogen receptor β ( ERβ) expression has been implicated in breast tumorigenesis. The ERβ gene yields five exon 8 alternatively spliced transcripts ( ERβ1–5), which encode proteins with different C-terminal amino acids. Individual expression analysis of these transcripts may provide new insights into estrogen signaling in breast cancer. We measured mRNA levels of total ERβ and its five isoforms in normal tissues, breast carcinomas from post-menopausal patients, and breast cancer cell lines by means of real-time reverse transcription-polymerase chain reaction and fluorescent fragment analysis. In various normal human tissues, ERβ1–5 isoforms displayed different qualitative and quantitative expression patterns that were consistent with previous reports. Total ERβ mRNA levels were significantly lower in breast tumors than in normal breast tissues (38-fold lower, P<0.001), mainly due to lower expression of ERβ1 and ERβ2 ( ERβ5 expression was similar in the two tissue types). This altered expression pattern of ERβ isoforms in breast cancer should be taken into account in future ERβ-based clinical applications.