ERbeta Expression Research Articles

Overexpression of Estrogen Receptor Beta and Its Prognostic Significance among Malignant Pleural Mesothelioma Patients

Abstract Background The expression of estrogen receptor beta (ER beta) and its correlation with the prognosis of malignant mesothelioma remains controversial. This study aimed at studying ER beta expression in tumour cells in patients with mesothelioma and its association with clinicopathological factors and survival outcomes. Material and Methods A prospective study to evaluate prognostic significance of ER beta overexpression by immunohistochemistry (IHC) on paraffin blocks among malignant mesothelioma patients at Ain Shams university hospital and correlate their overexpression with the patients' follow up clinical data, OS, and PFS. Results Forty-two mesothelioma cases were enrolled and followed up, High ER beta expression (modified Allred score 7-9) was reported in 9.5% of the patients (4 cases), moderate expression (modified Allred score 4-6) was reported in 28.6% of the patients(12 cases), weak and negative expression ( modified Allred score 0-3) was reported in 61% of the patients(26 cases), percent of ER beta positive cases was 69% of the total patients, there was no statistical difference as regard age (p value:0.8), sex(p value:0.4), stage (p-value: 0.71), ECOG (p-value :0.84), histological subtype(p value:0.17) and response to first-line treatment (p value:0.5) across the 3 groups of patients (high, moderate, weak and negative ER beta on tumour cells).there was no statistically significant difference in OS and PFS for ER beta overexpression. Conclusion ER beta overexpression was not associated with improved OS or PFS in mesothelioma patients, larger sample size would be recommended for more solid conclusions.

Abstract C091: Liquiritigenin, an estrogen receptor beta agonist, enhances cholesterol biosynthesis inhibitor RO 48-8071-induced growth inhibition of ovarian cancer cells

Abstract Almost 25,000 new cases of epithelial ovarian cancer (EOC) are reported each year in the United States. Cancers of the ovary have poor prognosis due to late detection, drug resistance and metastasis, and have the highest mortality rate of all the known gynecological malignancies. Despite concerted efforts to develop new strategies for preventing and treating ovarian cancer, novel and more effective non-toxic therapies are urgently needed. Recent observations from our laboratory show that RO 48-8071 ([4’-[6-(Allylmethylamino)hexyloxy]-4-bromo-2’-fluorobenzophenone fumarate] [RO], a small-molecule inhibitor of the key cholesterol biosynthesis enzyme 2, 3-oxidosqualene cyclase (OSC), inhibits breast and prostate cancer cells in vitro and in vivo. Further studies showed that RO also suppresses ovarian cancer cells, both in vitro and in vivo (Cholesterol Biosynthesis Inhibitor RO 48-8071 Suppresses Growth of Epithelial Ovarian Cancer Cells in Vitro and In Vivo. Journal of Cancer Science and Clinical Therapeutics 7 (2023): 01-08.). In short term assays (24h), pharmacological doses of RO (uM) suppressed ovarian cancer cell viability. In long-term assays (7 days) the same effects were observed in response to RO in the nM range. Interestingly, RO induced the tumor-suppressor protein estrogen receptor-beta (ER-beta) in both breast- and prostate-cancer cells and inhibited growth of xenografts of both types of tumor cells. We tested whether RO treatment also enhanced tumor-suppressor ER-beta expression in ovarian cancer cells and whether ER-beta agonists might enhance the effects of RO. Using Western blot analysis, we found that RO indeed induced ERb expression in OVCAR-3 ovarian-cancer cells, and that treatment of two ovarian-cancer cell lines (OVCAR-3 and SK-OV-3) with liquiritigenin (LQ), a naturally occurring ER-beta agonist derived from plants, enhanced RO mediated reduction in vitro. Treatment with RO (5-10 uM), LQ (100-300 uM), or RO + LQ (multiple combinations of doses) reduced cell viability; RO + LQ combination treatment synergistically reduced cell viability. We predict that our observed in vitro effects will be translatable to in vivo models, given that we have already demonstrated that RO alone quite effectively inhibits the in vivo growth of EOC xenografts. Since treatment of ovarian cancer with existing anti-cancer drugs is accompanied by extreme side effects and drug resistance, we propose that low-dose combinations of RO and LQ should be further explored as a potential new therapy for combatting this disease. Our findings contribute to ongoing efforts to identify novel, effective treatments for primary and metastatic ovarian cancer that employ new non-toxic drugs. Further in vivo studies examining the effects of RO + LQ combination treatment on EOC are warranted as a means of exploring a potential new therapeutic approach to combat ovarian cancers with minimal toxicity. Citation Format: Yayun Liang, Salman Hyder. Liquiritigenin, an estrogen receptor beta agonist, enhances cholesterol biosynthesis inhibitor RO 48-8071-induced growth inhibition of ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C091.

MP04-04 PD-L1 EXPRESSION AND RENAL CELL CARCINOMA: IS EXOGENOUS ESTROGEN DRIVING AGGRESSIVE TUMOR CHARACTERISTICS THROUGH INCREASED ONCOLOGIC EXOSOME PRODUCTION?

You have accessJournal of UrologyCME1 Apr 2023MP04-04 PD-L1 EXPRESSION AND RENAL CELL CARCINOMA: IS EXOGENOUS ESTROGEN DRIVING AGGRESSIVE TUMOR CHARACTERISTICS THROUGH INCREASED ONCOLOGIC EXOSOME PRODUCTION? Jacob W. Greenberg, Hogyoung Kim, Joshua Pincus, Ahmed A. Moustafa, Asim B. Abdel-Mageed, and L. Spencer Krane Jacob W. GreenbergJacob W. Greenberg More articles by this author , Hogyoung KimHogyoung Kim More articles by this author , Joshua PincusJoshua Pincus More articles by this author , Ahmed A. MoustafaAhmed A. Moustafa More articles by this author , Asim B. Abdel-MageedAsim B. Abdel-Mageed More articles by this author , and L. Spencer KraneL. Spencer Krane More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In 2022, SEER data projects 79,000 new cases renal cell carcinoma (RCC) with 15% of these presenting with progressed disease and distant metastasis. Prior publications have indicated that estrogen plays an important role by increasing RCC risk in female obese patients (Sun et al, EBioMedicine, 2018). However, the role of estrogen is still unknown in the setting of metastatic disease and treatment resistance. The aim of this study was to investigate whether estrogen’s role in treatment resistance could be linked to exosomal transfer of this hormone. METHODS: Overall survival data was extracted and analyzed from the TCGA based on several gene expression profiles. 786-O, 786-O Suni Resistant (SR), A498, A498 SR, Caki-2, Caki-2 SR and 293-T cells were cultured, exosomes collected, and later isolated using differential ultracentrifugation. After exposure to estrogen, downstream analysis included western blot and qNano exosome quantification. Statistical analysis was performed using R computational language. RESULTS: TCGA database analysis revealed that upregulated ER-beta expression is associated with a 25% decrease in overall survival at 5 years. Increased ER-Beta was also linked to patients with increased stage and metastatic disease. After exposing our cell lines to estrogen for 24 hr ER-beta, activated NF-kB, and PD-L1 expression increased in all 3 TKI-sensitive and resistant cell lines. However, when quantifying exosome concentrations after estrogen exposure our 3 TKI-sensitive lines displayed a decrease in exosome production. On the other hand, each of the TKI-resistant cells showed a 3-4 fold increased exosome concentrations. We then linked this discordance in exosome production with a similar pattern in EGFR expression. CONCLUSIONS: In conclusion, estrogen is an important molecule present in many patients with RCC. This study’s data shows that estrogen is a causative agent of increasing PD-L1 expression potentiated through exosome production. A graphical hypothesis based on this studies data of estrogen's role in the tumor microenvironment can be found in Figure 1. Source of Funding: U54 GM104940 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e34 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jacob W. Greenberg More articles by this author Hogyoung Kim More articles by this author Joshua Pincus More articles by this author Ahmed A. Moustafa More articles by this author Asim B. Abdel-Mageed More articles by this author L. Spencer Krane More articles by this author Expand All Advertisement PDF downloadLoading ...

Differential Expression of DDX3 and microRNAs in Response to Hormone and Cisplatin Against Cervical Cancer

Objectives: To discover micro ribonucleic acids (miRNAs) involved in the regulation of DDX3 expression using sexual hormones in combination with the well-known anticancer medication cisplatin. Methods: SiHa cells were treated with estradiol, dihydrotestosterone, and cisplatin and evaluated the expression of ER beta, Ki67, and DDX3 via quantitative reverse transcription–polymerase chain reaction. We generated a chimeric fusion construct five untranslated region (UTR)–hLUC–3UTR in the pEZX-MT06 miRNA vector under the control of the SV40 promoter. Reporter activity is measured with/without hormones, and their activity is compared with 5'- and 3'-UTR respectively. Various reporter deletion constructs were generated to identify the minimal UTR region in regulating the expression of DDX3. We identified the potential miRNA binding sites on the DDX3 UTR region, and their expression is monitored in cancer patients and cisplatin-treated SiHa cells. Results: Hormones increased the proliferation of SiHa cells and expression of DDX3. The 3'-UTR region 2135–4307bp contains miRNA sites that regulate DDX3 expression. miRNAs hsa-miR-671-5p, hsa-miR-361-5p, hsa-miR-140-5p, hsamiR-564, and hsa-miR-769-5p downregulated in patient samples but upregulated in cisplatin-treated cells. miRNA hsamiR-671-5p and hsa-miR-564 were associated with patient data and cisplatin-treated cancer cells. Conclusion: We discovered that sexual hormones enhanced DDX3 expression in SiHa cells. MiR-671-5p and miR-564 are two potential therapeutic miRNAs that can be used to treat DDX3-related malignancies. Keywords: Cancer patients, Cervical cancer, Cisplatin, DDX3, miRNA

Abstract P139: An estrogen receptor beta agonist liquiritigenin potentiates inhibition of hormone-dependent breast-cancer growth by cholesterol biosynthesis inhibitor RO 48-8071

Abstract Hormone-dependent breast cancer represents approximately 70% of all diagnosed breast cancers. Estrogens and progestins stimulate breast cancer cell proliferation, angiogenesis and metastasis. Estrogen receptor (ER)-alpha positive breast cancers are treated with anti-estrogens and aromatase inhibitors, but resistance to these agents invariably develops during the course of therapy. Our goal was to identify novel treatment strategies to suppress breast cancer growth and avoid drug resistance. Cholesterol biosynthesis plays a vital role in tumor cell growth. Cholesterol is an integral part of membrane structure and is also a precursor of steroid hormones that are synthesized by tumor cells and cause cellular proliferation by interacting with specific receptors. Cancer cells have elevated levels of cholesterol, which could be responsible for anti-hormone resistance. During studies to determine the effects of the cholesterol biosynthesis inhibitor RO 48-8071 ([4’-[6-(Allylmethylamino)hexyloxy]-4-bromo-2’-fluorobenzophenone fumarate] (RO), which inhibits oxidosqualene cyclase (OSC), an enzyme that acts downstream of HMG-CoA reductase to convert 2, 3-monoepoxysqualene to lanosterol (a key step in the biosynthesis of cholesterol), it was serendipitously discovered that RO degraded ER-alpha (ERa), and elevated levels of ER-beta (ERb), a known tumor suppressor. We tested RO alone and in combination with liquiritigenin (LQ), a selective agonist for ERb, to ascertain their inhibitory effects against tumor cell progression. RO or LQ alone reduced significantly the in vitro viability of MCF-7 and BT-474 breast-cancer cells. BT-474 cells were more sensitive than MCF-7 cells to LQ (IC50 0.15 uM vs 0.25 mM, respectively). RO (5-10 uM) + LQ (0.1-0.3 mM) treatment reduced cell viability in an additive manner in MCF-7 cells and in a synergistic manner in BT-474 cells. Combination treatment was significantly more effective than treatment with a single agent. Administration of RO (10 mg/kg/iv) or LQ (20 mg/kg/ip) significantly inhibited growth of tamoxifen-resistant BT-474 tumor xenografts in vivo; combination treatment was even more effective. Both agents appeared to be non-toxic in vivo. Mechanistically, RO, LQ, or RO + LQ reduced ERa but induced ERb expression in tumor xenografts. Both compounds significantly reduced expression of markers of angiogenesis and increased apoptosis in tumor xenografts; use of RO + LQ significantly enhanced the effects observed with a single agent. The anti-tumor properties of RO may in part be due to an off-target effect that reduces ERa and increases ERb, the latter then interacting with LQ to more effectively promote its anti-proliferative effects. The potent anti-tumor properties of RO and LQ, particularly when used together, suggest that this combination has substantial potential as a means by which to clinically manage breast-cancer growth, including in disease types that are tamoxifen resistant. Citation Format: Yayun Liang, Salman Hyder. An estrogen receptor beta agonist liquiritigenin potentiates inhibition of hormone-dependent breast-cancer growth by cholesterol biosynthesis inhibitor RO 48-8071 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P139.

Maternal Exposure to Dibutyl Phthalate (DBP) or Diethylstilbestrol (DES) Leads to Long-Term Changes in Hypothalamic Gene Expression and Sexual Behavior.

Xenobiotic exposure during pregnancy and lactation has been linked to perinatal changes in male reproductive outcomes and other endocrine parameters. This pilot study wished to assess whether brief maternal exposure of rats to xenobiotics dibutyl phthalate (DBP) or diethylstilbestrol (DES) might also cause long-term changes in hypothalamic gene expression or in reproductive behavior of the resulting offspring. Time-mated female Sprague Dawley rats were given either DBP (500 mg/kg body weight, every second day from GD14.5 to PND6), DES (125 µg/kg body weight at GD14.5 and GD16.5 only), or vehicle (n = 8–12 per group) and mild endocrine disruption was confirmed by monitoring postnatal anogenital distance. Hypothalamic RNA from male and female offspring at PND10, PND24 and PND90 was analyzed by qRT-PCR for expression of aromatase, oxytocin, vasopressin, ER-alpha, ER-beta, kisspeptin, and GnRH genes. Reproductive behavior was monitored in male and female offspring from PND60 to PND90. Particularly, DES treatment led to significant changes in hypothalamic gene expression, which for the oxytocin gene was still evident at PND90, as well as in sexual behavior. In conclusion, maternal xenobiotic exposure may not only alter endocrine systems in offspring but, by impacting on brain development at a critical time, can have long-term effects on male or female sexual behavior.

Estrogen Replacement Improves Mesenteric Arterial Relaxation in UC Davis Pre‐diabetic Rats: Possible Involvement of Estrogen Receptor Beta

The risk of cardiovascular diseases (CVD) is lower in premenopausal women compared to men, and this lower risk is related to the presumptive cardioprotective effects of female sex hormones, estrogen in particular. However, premenopausal women may lose this protection with the onset of menopause and/or when they become diabetic. Little is known about the vascular function in presence or absence of estrogen at the early stage of diabetes.The objective of this study was to investigate the effects of 17β‐estradiol (E2) replacement on mesenteric arterial (MA) function in ovariectomized (ovex) UC Davis type 2 diabetes mellitus (UCD‐T2DM) rats at the pre‐diabetic stage. MA were obtained from four groups of ovex rats implanted with a subcutaneous pellet for 45 days: (1) Control (Ctrl), ovex + placebo, (2) Ctrl, ovex + E2 (1.5mg/pellet), (3) pre‐diabetic, ovex + placebo, (4) pre‐diabetic, ovex + E2. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5 M) in MA were measured before and after indomethacin (Indo, 10μM), a cyclooxygenase (COX) inhibitor; followed by addition of L‐NAME (200μM), a nitric oxide synthase (NOS) inhibitor. The MA expression of estrogen receptors (ERs) and plasma E2 concentration were determined by Western blotting and ELISA assay, respectively.E2 treatment significantly increased plasma estradiol concentration in both Ctrl and Prediabetic groups. Analysis of ERs showed a higher expression of ER‐beta only in E2‐treated prediabetic group. ACh responses were similar in Ctrl groups, regardless of E2 replacement. ACh induced relaxation was, however, markedly reduced in MA of placebo treated pre‐diabetic group compared with other groups. Treatment of pre‐diabetic with E2 enhanced relaxation to the same level as those from Ctrl rats. Blocking of both COX and NOS completely abolished the relaxation responses to ACh in pre‐diabetic groups, regardless of E2 replacement, indicating no contribution of EDHF‐type relaxation in these groups. However, the added effect of L‐NAME, in presence of Indo, in blocking ACh‐mediated vasodilation was much more prominent in E2‐treated prediabetic group when compared with placebo treated group, indicating an enhanced role of NO. On the other hand, both NO and EDHF remained important for EDV in Ctrl groups. Addition of L‐NAME, however, caused a much smaller reduction of EDV in E2 treated than in placebo treated arteries, suggesting EDHF is the predominant mediator of mesenteric vasorelaxation in this groups.Here, we show that EDV is impaired at pre‐diabetic stage, may be due to loss of EDHF. E2 treatment restores EDV in MA of pre‐diabetic group, possibly via elevated contribution of NO‐mediated relaxation by increased E2 signaling (via ER beta) in this group. The role of ER alpha remains to be investigated.Support or Funding InformationSupported by NHLBI.

Role of preovulatory concentrations of estradiol on timing of conception and regulation of the uterine environment in beef cattle

ABSTRACTThe ability to induce ovulation with an injection of GnRH facilitated the development of fixed-time artificial insemination (AI) protocols. However, animals that exhibit estrus prior to fixed-time AI have greater pregnancy success. Thus, the objectives of the present experiments were to determine the impact of estrus expression prior to fixed-time AI on timing of conception and to characterize the role of preovulatory estradiol in regulating changes in expression of uterine genes. In experiment 1, data were collected from 4,499 beef cows inseminated by fixed-time AI in 31 different herds. Animals that did not conceive to AI but exhibited estrus before timed-AI were more likely to conceive during cycle 1 after AI, and overall conceived earlier in the breeding season compared to animals that did not exhibit estrus. In experiment 2, beef cows were synchronized using a fixed-time AI protocol. Uterine horn biopsies and blood samples were collected on Day 0, 5, 10, or 16. Concentrations of estradiol on Day 0 did not influence expression of progesterone receptor, ER beta, or oxytocin receptor. Increased concentrations of estradiol on Day 0 increased expression of ER alpha from Days 0 to 5 of the estrous cycle. Furthermore, cows with increased concentrations of estradiol on Day 0 had increased expression of inhibin beta A, and uterine milk protein precursor. Thus, animals that do not exhibit estrus prior to fixed-time AI had decreased breeding season pregnancy success and conceived later in the breeding season, and preovulatory concentrations of estradiol likely play a major role in this establishment of pregnancy, not only directly by regulating uterine gene expression, but also indirectly throughout the subsequent estrous cycle.

Abstract 5032: High ERbeta expression correlates with the tumor suppressor 15-PGDH and PGD2 synthase in a female cohort of colorectal cancer patients

Abstract Background: The estrogen receptor β (ERβ) is the predominant ER in the colon mucosa. We have previously reported that high expression of ERβ is independently associated with a better prognosis in female patient with colorectal cancer (CRC). β-catenin and cyclooxygenase-2 (COX-2) are well known to be involved in CRC tumorigenesis. We have shown that a high level of CysLT2R, a low level of CysLT1R and high mast cell density are correlated with good prognosis in CRC patients. β-catenin and some inflammatory mediators, such are cyclooxygenase-2 (COX-2), cysteinyl leukotriene receptor 1 and 2 (CysLT1R, CysLT2R), prostaglandin D2 (PGD2), 15-PGDH activity and mast cells have been associated with CRC. Aim: To investigate the association between ERβ and other tumor modulators in CRC patients, as well as the underlying mechanism in colon cancer cell lines. Material and methods: A tissue microarray of primary CRCs from 320 female patients was stained for the expression of ERβ, COX-2, 15-PGDH, CysLT1R, CysLT2R, ß-catenin, PGD synthase and mast cells. The Immunohistochemistry technique was used to evaluate the staining intensity, which was assigned an Immuno-Reactive Score (IRS). The zebrafish xenograft model was used to study colon cancer cells metastasis. ERβ -041 (an ERβ agonist) was injected into the zebrafish perivitelline space to allow for xamination of its effect on colon cancer cells. After 48 h, the tail, considered the distant metastatic site, was investigated for the presence of the metastasized cells. The effect of ERβ on colon cancer cells (SW-480 and HCT-116) after treatment with ERβ-041 was studied by wound healing and trans-well migration assay, as well as by survival assay. Results: Patients with high ERβ expression had significantly higher levels of membrane ß-catenin, CysLT2R, 15-PGDH and PGD synthase, which all are known to have an anti-tumor effect. These patients had significantly lower levels of CysLT1R, COX-2 and nuclear ß-catenin expression, which are known to have pro-tumorigenic effects. In addition, patients with high ERβ expression had higher numbers of mast cells, which are a major source of PGD2 synthase and PGD2. In the zebrafish xenograft model, the ERB-041 treated cells showed no distant metastasis, unlike the untreated cells. ERβ stimulation of colon cancer cells produced a significant decrease in migration and survival compared to that of the untreated control cells after 48 h post-stimulation. Conclusion: High ERβ expression was significantly correlated with anti-tumorigenic inflammatory mediators and lower nuclear ß-catenin in patients with CRC. These results strengthen our hypothesis regarding the beneficial role of ERβ in CRC patients. Our results raise the possibility of cross-talk between ERβ, CysLT1R and CysLT2R signalling. Citation Format: Geriolda Topi, Pujarini Dash, Shakti R. Satapathy, Syrina Mehrabi, Roy Ehrnström, Marie-Louise Lydrup, Anita Sjölander. High ERbeta expression correlates with the tumor suppressor 15-PGDH and PGD2 synthase in a female cohort of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5032.

The Effect of Perinatal Blockade of Androgen Receptors on Adult Rat Behaviors and the Expression of Estrogen and Androgen Receptors in specific brain regions

The brain, by default, differentiates into a female brain unless testosterone (T) is present during a critical window of time. In rats, there is a surge of T around the time of birth, more specifically four days prior to and four days after birth, referred to as the perinatal period. The purpose of this study was to examine the effects of blockade of androgen receptors (AR) during the perinatal period using flutamide (F) on sexual motivation (SM) and an open field (OF) test as well as the expression of AR and estrogen receptors (ER)‐alpha and ER‐beta in the hypothalamus, hippocampus, and amygdala. Four timed‐pregnant rats were divided into two groups: one group (N=2) received F during perinatal period whereas the other group (N=2) served as the control group receiving vehicle (95% Olive oil‐5% ethanol). Once the pups reached adulthood, the male rats underwent SM testing where they were exposed to either an estrus or an ovariectomized rat. Both F and control groups of male rats spent more time with the estrus rat compared to the ovariectomized rat suggesting that SM was not affected by their AR blockade during perinatal period. Open field testing recorded animal movements between the two zones, the center and the periphery, within an open arena. Male rats moved between the two zones more than the female rats did. At the end of the behavioral study, animals were sacrificed and the brains were harvested to isolate the hippocampus, hypothalamus, and amygdala. Real time PCR testing was carried out to quantify the levels of AR, ER alpha, and ER beta using B‐Actin as an internal control. Perinatal blockade of AR had no effect on the expression of any of the receptors in the three regions tested in the adult rats. Thus, the increased movement by the male rats in the OF testing is not associated with changes in the expression of AR, ER‐alpha, or ER‐beta.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vivo Quantitation of Estrogen Receptor β Subtype Expression in Ovarian Surface Epithelium Using Immunofluorescence Profiling and Confocal Microscopy.

Immunohistochemistry using formalin-fixed, paraffin-embedded tissue, chromogen label, and light microscopy has traditionally been used to semiquantify estrogen receptor (ER) to guide diagnosis and management of breast cancer. Quantitation of ER for this purpose currently only assesses levels of the ER-alpha subtype. Considerable variability in results reported has been due to protocol and fixation variability, intraobserver and interobserver variability, and different scoring systems and thresholds for scoring ER positivity. Results can also vary with low expression levels of ER. ER-beta expression is reduced in breast and ovarian cancers and requires quantitation.Herein we describe a novel approach to quantifying ERβ using older mouse ovarian surface epithelium, where ERβ is expressed at lower levels than ERα and is therefore harder to detect. We use an antibody highly specific to the ERβ1 isoform, together with immunofluorescence, confocal microscopy, and imaging and statistical software to achieve clear, reproducible, and unbiased quantitation of ERβ.

Expression of estrogen receptor beta isoforms in pancreatic adenocarcinoma.

Limited studies have shown that some patients with pancreatic adenocarcinoma (PAC) may benefit from treatment with tamoxifen. PAC has been shown to be largely negative for estrogen receptor alpha (ER-alpha). The aim of this pilot study was to investigate ER-beta expression in human PAC. Sections of tissue microarray with 18 evaluable cases of human PAC were stained by immunohistochemistry (IHC) for ER-beta1, ER-beta2, ER-beta5, and Cyclin A. The levels of ER-beta isoform expression and the S-phase fraction (SPF) were determined using quantitative digital image analysis.Higher mean and median ER-beta2 levels correlated with male sex (p = 0.057 and p = 0.035, respectively), older age (p = 0.005 and p = 0.006, respectively), and lower pT stage (p = 0.008 and p = 0.009). Mean and median ER-beta5 levels correlated negatively with SPF (p = 0.021 and p = 0.047, respectively). Mean ER-beta1 expression did not correlate with any of the above mentioned clinicopathologic factors. The findings in this pilot study, although should be considered preliminary, suggest that some ER-beta isoforms may play a role in the biology of PAC. Additional larger studies are needed to confirm our findings, and to determine whether ER-beta may be considered for future targeted therapy.

Chemoprevention of inflammation-related colorectal cancer by silymarin-, acetyl-11-keto-beta-boswellic acid-, curcumin- and maltodextrin-enriched dietetic formulation in animal model.

On the basis of preliminary in vitro experience, we assessed whether an enriched nutritional formulation with estrogen receptor (ER)-beta agonist and anti-inflammatory properties may prevent inflammation-associated colorectal cancer (CRC) in an animal model. Study sample enclosed 110 C57BL/6J male mice. Forty underwent dietary supplement safety assessment (20 standard diet and 20 enriched formulation). Seventy were treated with azoxymethane (AOM)/dextran sulfate sodium and divided into two groups: 35 received standard diet and 35 enriched formulation (curcumin, boswellic acids, silymarin and maltodextrins). Miniature colonoscopy demonstrated colitis and solid lesion development in five mice/group 100 days after first AOM injection. Mice were killed after 10 days. In each group, four subgroups received intraperitoneal bromodeoxyuridine (BrdU) injection at 24th/48th/72nd/96th hour before killing. Anti-inflammatory effect and chemoprevention were evaluated by lesion number/size, histological inflammation/dysplasia/neoplasia assessment, pro-inflammatory cytokine messenger RNA (mRNA), ER-beta/ER-alpha/BrdU immunohistochemistry and TUNEL immunofluorescence. Standard formulation assumption was associated with colon shortening compared with enriched one (P = 0.04), which reduced solid lesion number and size (P < 0.001 for both), histological inflammation score (P = 0.04), pro-inflammatory cytokine mRNA expression (P < 0.001), number of low-grade dysplasia (LGD; P = 0.03) and high-grade dysplasia (P < 0.001) areas. CRC was observed in 69.6% in standard and 23.5% in enriched formulation assuming animals (P < 0.001). Enriched formulation induced lower ER-alpha expression in CRC (P < 0.001) and higher ER-beta expression in LGD (P < 0.001) being associated to higher epithelial turnover (BrdU; P<0.001) in normal mucosa and increased apoptosis in LGD and CRC (P < 0.001 for both). Our results are promising for a successful anti-inflammatory and chemopreventive effect of enriched formulation in CRC arising from inflamed tissue.

PO-521 Expression of oestrogen receptor beta as a predictive marker of platinum and taxane-based chemotherapy in ovarian cancer patients

IntroductionPredictive markers of platinum-based chemotherapy efficacy in patients with ovarian cancer have been widely discussed in recent years. Oestrogen receptors (ER) are involved in regulation of proliferative factors responsible for tumour growth, so they could predict the effectiveness of anticancer chemotherapy. The aim of the study was to determine the predictive value of ER-beta expression in a cohort of ovarian cancer patients treated with platinum and taxanes. The level of ER-beta expression was correlated with progression-free survival and also with a number of disease relapses during 40 months of monitoring.Material and methodsThe quantitative immunofluorescence flow cytometry analysis was performed to detect ER-beta in 34 serous ovarian cancer surgical specimens. All the patients were treated with the first line platinum and taxane-based regimen. The primary anti-ER-beta (ab14C8) and the secondary antibodies (DyLight650, ab98729) were used for the analysis. The level of ER-beta expression was calculated by Kolmogorov-Smirnov statistical test as the ratio (%) of specifically fluorescent cells to the number of cells incubated only with secondary antibodies. Association of ER-beta expression levels with progression-free survival was analysed using the Kaplan-Meier method and log-rank tests.Results and discussionsER-beta were revealed in all tumour specimens tested and the median value of the expression level was 41,5%. Patients were dichotomized in groups with low and high level of ER-beta expression, below- and above-median value respectively. Significant differences were shown between these groups. First, median of progression-free survival was larger in the high-level expression group as compared to the low-level expression group – 25,5 vs 8 months. Second, the disease rate during 40 months of monitoring was lover in the high-level expression group as compared the low-level expression group – 6 vs 15 respectively.ConclusionA quantitative index of the ER-beta level expression in tumour tissue predicts the efficacy of the first line platinum and taxane-based chemotherapy in ovarian cancer patients. Progression-free survival was about 3.0 times longer and the number of the disease relapses during 40 months of monitoring was 1.5 times less in patients with high level of ER-beta expression. The study was supported in part by RSF 17-75-10212.

Investigation of hormone receptor expression and its prognostic value in endometrial stromal sarcoma.

To evaluate the immunohistochemical (IHC) expression of hormone receptors and analyze the prognostic implication of these receptors in patients with endometrial stromal sarcoma (ESS). Fifty-one patients with ESS whose paraffin blocks and pathologic slides, which were obtained after hysterectomy, were available and included in this study. Clinicopathologic data were gathered from patients' medical records, and IHC staining of hormone receptors was performed using tissue microarrays. Estrogen receptor (ER)-alpha was expressed in 37 patients (72.5%), and strong immunoreactivity was observed in 27 patients (52.9%). However, ER-beta expression was observed in only two patients (3.9%). Progesterone receptor (PR) expression was identified in 36 patients (70.6%), and strong immunoreactivity was found in 26 patients (51%). Androgen receptor (AR) expression was observed in 30 patients (58.8%), and strong immunoreactivity was noted in 14 patients (27.5%). Gonadotropin-releasing hormone receptor (GnRH-R) expression was observed in 49 patients (96.1%), but no patient exhibited strong immunoreactivity. All patients expressed CYP19A1, and 43 patients (84.3%) had strong immunoreactivity. ER-alpha, PR, and AR positivity was associated with significantly better overall survival (OS). No patient with AR positivity died of ESS. When the patients were categorized according to ER-alpha, PR, and AR immunoreactivity, triple-positive ESS had the best OS, and triple-negative ESS was linked to the worst OS. Expression of hormone receptors was associated with favorable survival outcome in ESS. Hormone receptors that showed strong expression deserve further evaluation to clarify their importance as a therapeutic target and predictor of treatment response.

Role of co-expression of estrogen receptor beta and Ki67 in prostate adenocarcinoma

PurposeTo evaluate the expression of estrogen receptor (ER)-beta and Ki67 in prostate cancer and study their relationship.Materials and MethodsWe analyzed 101 cases of prostate adenocarcinoma diagnosed from January 2011 to June 2015 in 100 patients. Immunohistochemical staining of ER-beta and Ki67 was analyzed according to Gleason score categorized into prognostic groups of 1 to 5. Double-immunofluorescent staining of ER-beta and Ki67 was performed in a total of 20 cases to study the co-expression and the relationship between these markers within the same tumor.ResultsA total of 53 of 101 cases (52.5%) were positive for ER-beta expression. There was a positive correlation whereby a high percentage of ER-beta expression was seen in the higher prognostic groups (groups 4 and 5; p=0.007). High Ki67 expression was observed in the higher prognostic group, whereas low Ki67 or negative expression was found in the lower prognostic group (p<0.001). The majority of cases evaluated with double-immunofluorescent staining (14/20) showed co-expression of ER-beta and Ki67 at the individual cell level.ConclusionsER-beta and Ki67 are independent tumor markers in high prognostic groups. Hence, co-expression of ER-beta and Ki67 indicates a more aggressive tumor with a poorer prognosis.

The sharp decline of beta estrogen receptors expression in long-lasting ulcerative-associated carcinoma

Objective. Colorectal carcinoma is an important cause of death in inflammatory bowel diseases, thus requiring surveillance for dysplasia in long-standing ulcerative colitis (UC). Females show a lower incidence probably related to hormonal factors; therefore, a role of estrogen receptors (ERs) has been supposed in carcinoma-associated colitis (CAC) development. Our aim was to identify ER beta/alpha expression in long-lasting pancolitis through each grade of dysplasia to carcinoma and, furthermore, to investigate the simultaneous epithelial apoptosis/proliferation. Materials and methods. Forty-eight patients affected by long-lasting pancolitis were retrospectively investigated. Samples were divided into four groups: UC, low-grade dysplasia/high-grade dysplasia (UC-HGD), and CAC. Normal colon samples were used as controls. ER-beta, ER-alpha, Ki-67, and TUNEL expression (labeling/H index) were evaluated by immunohistochemistry. Results. ER-beta expression revealed an impressive reduction in CAC (10.4 ± 5.1; p < 0.001) compared to controls and UC (34.3 ± 3.1 and 26.8 ± 7.8, respectively), meanwhile ER-beta level in LGD (29.4 ± 3.7) was comparable to UC. As far ER-beta/ER-alpha mean value ratio revealed a progressive reduction. Ki67 demonstrated a progressive significant increase from UC until CAC (37.9 ± 6.4 < 45.7 ± 6.2 < 60.6 ± 5.2 < 71.1 ± 5.1; p < 0.001). Apoptotic index (TUNEL) revealed a strong fall in UC-HGD and CAC. Conclusions. ER-beta fall could be considered as a biomarker of UC-dysplasia progression. It occurs in HGD and overt neoplasia, while in LGD shows a normal expression. At the moment, we are unable to use this tool in the clinical practice to predict tumor progression, but it would be appropriate to encourage ER expression investigations in large samples for the interesting perspectives of application.

ER-alpha and ER-beta expression in differentiated thyroid cancer: relation with tumor phenotype across the TNM staging and peri-tumor inflammation.

Thyroid cancer may express estrogen receptors (ERs) and various grades of peri-tumor inflammation. The aim of the study was to evaluate the expression of ERs in relation to the TNM stage and peri-tumor inflammatory infiltrate in differentiated thyroid cancers. 127 patients (109 females, 18 males) with differentiated thyroid cancer (T1 = 91, T2 = 18, T3 = 11, T4 = 7) were evaluated. In tumors and in the correspondent extra-tumor parenchyma, ERs expression was evaluated by immunohistochemistry. In 114 tumors and correspondent peri-tumor tissues, the presence of inflammatory infiltration was also recorded. ER-alpha expression was higher in clinical than in incidental tumors of the T1 subgroup (p = 0.037), and was associated with capsular invasion in T2 tumors (p < 0.0001). ER-beta expression was negatively associated with vascular invasion in T1 (p = 0.005) and T2 tumors (p = 0.015). No significant relationship between ERs expression and tumor phenotype emerged in T3 and T4 subgroups. Tumors without inflammatory cell infiltrate showed a higher expression of both ER-alpha (p = 0.035) and ER-beta (p = 0.026) than the ones with inflammatory infiltrate. The relationship between tumor phenotype and ERs expression did not vary in the presence or absence of peri-tumor inflammatory infiltration. ER-alpha positivity and ER-beta negativity are associated with a more aggressive phenotype in both T1 and T2 thyroid cancers, suggesting that tumor biology may be more relevant than tumor size for cancer risk assessment. Inflammatory status is also associated with ERs expression, but not with tumor growth or phenotype.

Icariin promote maturation of osteoblasts in vitro by an estrogen-independent mechanism

To investigate the estrogenic activity of icariin and genistein with estrogen-dependent human breast cancer (MCF-7) cells. MCF-7 cells were incubated with media containing 5% charcoal dextran-treated FBS in phenol red-free media for 48 h. CCK-8 kit was used to study the impact of defferent concentration of icariin and genistein on MCF-7 proliferation in vitro. Optimal concentration icariin and genistein were added into medium and total RNA was isolated after 12, 24, 36, 48 h. The gene expression of ERalpha, ERbeta, PS2, and PR were investigated by Real-time RT-PCR Total protein was also isolated and secretion of ERalpha, ERbeta, PS2, and PR were examined by Western blot. 10 micromol x L(-1) icariin and genistein could promote the proliferation of MCF-7 evidently. However, the ability of genistein to promote the proliferation was better than icariin. With the concentration of 10 micromol x L(-1), genistein group had a stronger expression of ERa, PS2 and PR mRNA levels than icariin while ERbetaexpression had no significant difference in two group. The same effects were detected by western blotting. Both genistein and icariin have a strong estrogen-like effect, but the estrogenic activity of genistein is stronger than icariin. It showed that the activity of icariin is stron-ger than genistein to promote ROB maturation. So it must be that icariin promotes the maturation of osteoblasts in vitro by a estogen-independent mechanism.

Expression of Ki-67 and estrogen receptor beta in primary malignant melanoma as a potential indicator of regional lymph node positivity

Background Malignant melanoma (MM) is one of the tumors with fastest-growing incidence, also deadliest form of skin cancer. In the early stages of the disease it is extremely difficult to adequately predict risk from hematogenic and lymphatic metastasis. We assumed that the determination of Ki-67 and estrogen receptor beta (ER beta) in cells of MM could predict their biological behavior. Objectives To examine whether the level of expression of Ki-67 and ER beta in cells of MM are in correlation with status of regional lymph nodes (LN), and compare the relationship between melanoma thickness (pT) and status of LN. Methods Expression of Ki-67 and ER beta was examined by immunohistochemistry, and then staining positivity assessed and correlated with relevant clinical and pathological parameters. Results Logistic regression analysis showed that when ER beta levels increase by 1% the risk of positive LN decreases by 7% (OR = 0.930, 95%CI 0.87-0.99, p = 0.036), and when the Ki67 expression increases by 1% that the risk of LN positivity increases by 10% (OR = 1.108, 95%CI 1.02 to 1.19, p = 0.009). Conclusions Correlation between melanoma thickness and the status of LN has less prognostic significance than the relationship between expression of Ki-67 and ER beta and the status of LN. Our study showed a significant prognostic value of Ki67 expression in predicting the behavior of MM, and potential prognostic significance of ER beta.