Increased Expression Of Estrogen Receptor Research Articles

Adolescent exposure to tris(1,3-dichloro-2-propyl) phosphate (TCPP) induces reproductive toxicity in zebrafish through hypothalamic-pituitary-gonadal axis disruption

Tris(1,3-dichloro-2-propyl) phosphate (TCPP), a prevalent organophosphorus flame retardant in aquatic environments, has raised significant concerns regarding its ecological risks. This study aims to explore the impacts of TCPP on the reproductive functions of zebrafish and delineate its gender-related toxic mechanisms. By assessing the effects on zebrafish of 10 mg/L TCPP exposure from 30 to 120 days post-fertilization (dpf), we thoroughly evaluated the reproductive capability and endocrine system alterations. Our findings indicated that TCPP exposure disrupted gender differentiation in zebrafish and markedly impaired their reproductive capacity, resulting in decreased egg laying and offspring development quality. Histological analyses of gonadal tissues showed an abnormal increase in immature oocytes in females and a reduction in mature sperm count and spermatogonial structure integrity in males, collectively leading to compromised embryo quality. Additionally, molecular docking results indicated that TCPP showed a strong affinity for estrogen receptors, and TCPP-treated zebrafish exhibited imbalanced sex hormones and increased estrogen receptor expression. Alterations in genes associated with the hypothalamic-pituitary-gonadal (HPG) axis and activation of the steroidogenesis pathway suggested that TCPP targets the HPG axis to regulate sex hormone homeostasis. Tamoxifen (TAM), as a competitive inhibitor of estrogen, exhibited a biphasic effect, as evidenced by the counteraction of TCPP-induced effects in both male and female zebrafish after TAM addition. Overall, our study underscored the gender-dependent reproductive toxicity of TCPP exposure in zebrafish, characterized by diminished reproductive capacity and hormonal disturbances, likely due to interference in the HPG axis and steroidogenesis pathways. These findings emphasize the critical need to consider gender differences in chemical risk assessments for ecosystems and highlight the importance of understanding the mechanisms underlying the effects of chemical pollutants on the reproductive health of aquatic species.

Immunohistochemical Detection of Indoleamine 2,3-Dioxygenase in Spontaneous Mammary Carcinomas of 96 Pet Rabbits.

For mammary carcinomas in pet rabbits, prognostic biomarkers are poorly defined, and treatment is limited to surgical excision. Additional treatment options are needed for rabbit patients for which surgery is not a suitable option. In human breast cancer, the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) represents a prognostic biomarker and possible therapeutic target. This retrospective immunohistochemical study examined IDO1 in 96 pet rabbit mammary carcinomas with known mitotic count, hormone receptor status, and percentage of stromal tumor infiltrating lymphocytes (TILs). Tumors were obtained from 96 pet rabbits with an average of 5.5 years. All rabbits with reported sex (n = 88) were female or female-spayed. Of the carcinomas, 94% expressed IDO1, and 86% had sparse TILs consistent with cold tumors. Statistically significant correlations existed between a higher percentage of IDO1-positive tumor cells, lower mitotic counts, and increased estrogen receptor expression. The threshold for significance was IDO1 staining in >10% of tumor cells. These results lead to the assumption that IDO1 expression contributes to tumorigenesis and may represent a prognostic biomarker and possible therapeutic target also in pet rabbit mammary carcinomas. They also support the value of rabbits for breast cancer research.

Oxytocin receptor expression is associated with estrogen receptor status in breast tumors

The oxytocin receptor (OXTR) plays an important role in childbirth, breastfeeding, and social interactions. There is emerging evidence that OXTR is associated with the breast cancer (BC) initiation and progression. However, the mechanisms leading to a change in its expression, the diagnostic or prognostic value of the receptor in BC are currently poorly understood. Here, we have evaluated the relative level of OXTR expression in BC samples (n=107), and also investigated the effect of estradiol on its expression in MCF-7 and MDA-MB-231 cells. The level of OXTR expression was significantly lower in breast tumor tissue than in normal tissue obtained from the same patient. The expression of OXTR was dependent on the status and expression of the estrogen receptor (ER): the level of OXTR mRNA was significantly lower in ER-negative BC samples compared to ER-positive BC samples. Moreover, OXTR expression was also lower in samples from patients with luminal subtype with a low value of ER expression (0-5 score according to the IHC assay, Allred scoring) compared with samples with high ER expression (6-8 score). In luminal BC, OXTR expression was associated with the HER2 expression level: the OXTR mRNA level was higher in tumors with a HER2 IHC score of 1+ as compared to cases with the HER2 expression score of 2+, 3+. We also showed that estradiol increased the level of OXTR mRNA in MCF-7 cells, but not in ER-negative MDA-MB-231 cells. These data indicate that changes in OXTR expression in BC tissues can be caused by increased ER expression. We found no association between OXTR and T or N stages and progesterone receptor expression.

<i>Rehmannia</i> Root Improves Extracellular Matrix Production and Epidermal Differentiation by Upregulation of the Estrogen Receptor

Estrogen is essential for the skin to maintain its physiological function. The binding of estrogen to the estrogen receptor (ER) activates gene transcription, which has biological effects on the target tissue. Estrogen levels and ER expression are known to decrease with aging and exposure to ultraviolet light (UV); therefore, increased estrogen levels and ER expression may improve age-related changes in the skin. Rehmannia root has been reported to have blood circulation-promoting and anti-inflammatory effects; however, few studies have reported the effects of Rehmannia root on skin. In this study, we examined the effects of Rehmannia glutinosa Libosch. var. purpurea Makino root extract (RE) on ER expression, and estrogen, RE, or their related ingredients increased ER expression in human epidermal keratinocytes, human dermal fibroblasts, and skin models. Moreover, RE increased the production of basic fibroblast growth factor, transforming growth factor β1, and epidermal growth factor. The mixture of estrogen and RE improved extracellular matrix (ECM) production to a greater degree than estrogen and RE independently. Although high population doubling levels (PDL) and UV irradiation downregulated ER expression, RE upregulated ER expression in high PDL cells and UV irradiated cells. In addition, RE increased the expression of epidermal differentiation marker proteins compared to their expression levels in the absence of RE. The collective findings suggest that RE aids in the prevention of skin aging by upregulating the ER expression that has been decreased by aging and UV and promoting estrogen activity, ECM production, and epidermal differentiation.

Activation of Nrf2 attenuates delayed gastric emptying in obesity induced diabetic (T2DM) female mice

Diabetic gastroparesis (GP) is a clinical syndrome characterized by delayed gastric emptying (DGE). Loss of Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) led to reduced nNOSα mediated gastric motility and DGE. The molecular signaling of cinnamaldehyde (CNM) mediated Nrf2 activation and its mechanistic role on DGE were further investigated in obese/T2D female mice. Adult female homozygous Nfe2l2−/− (C57BL/6J) and their wild-type (WT) littermates (Nfe2l2+/+) mice were fed with high fat diet (HFD; Obese/T2D model), or normal diet (ND) with or without CNM (50 mg/kg b.w; i.p). Supplementation of CNM attenuated (p < 0.05) DGE in WT female but not in Nrf2 KO Obese/T2D mice. CNM (1) normalized serum estradiol-17β levels, (2) induced gastric Nrf2 and phase II antioxidant enzymes through extracellular signal-regulated kinase, (ERK)/c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK), (3) reduced glucose synthase kinase 3 beta (GSK3β) and aryl hydrocarbon receptor (AhR) and this was associated with (4) increased estrogen receptor expression, BH4 (Cofactor of nNOS) biosynthesis enzyme GCH-1 and nNOSα dimerization in WT Obese/T2 diabetic female mice. In addition, CNM restored impaired nitrergic relaxation in hyperglycemic conditions. These findings emphasize the importance of Nrf2 in maintaining nNOSα mediated GE and may have a translational relevance to treat obese/diabetic gastroparesis in women.

Can MTHFR C677T and A1298C Polymorphisms Alter the Risk and Severity of Sporadic Breast Cancer in Brazilian Women?

IntroductionPolymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR) modify the risk and severity of sporadic breast cancer (BC). In this context, the MTHFR C677T and A1298C polymorphisms have been associated with risk and severity of sporadic BC. Patients and MethodsIn total, 253 women with BC and 257 controls were enrolled in this study. Polymorphisms were analyzed using restriction fragment length polymorphism - polymerase chain reaction. Epidemiology, tumor characteristics, and reproductive factors were considered in the analysis. Statistical tests included the χ2 test, the Fisher exact test, and the Mann-Whitney and Kruskal-Wallis tests, or parametric equivalents. ResultsMTHFR polymorphisms were not a risk factor for BC. The 677CC genotype was associated with distant metastasis (odds ratio [OR] = 5.311; 95% confidence interval [CI] = 1.124-25.09) and lower estrogen receptor expression, whereas the 1298AA genotype was associated with stage 0 (OR = 0.244; 95% CI = 0.077-0.771) and increased estrogen receptor expression. In haplotype analysis, 677CC/1298AA was associated with hypertension (OR = 1.979; 95% CI = 1.036-3.782), and 677CT/1298AC was associated with invasive carcinoma of no special type (OR = 0.472; 95% CI = 0.243-0.918) and stage 0 (OR = 3.476; 95% CI = 1.341-10.47). ConclusionThe MTHFR C677T and A1298C polymorphisms do not alter the risk of BC, but are associated with the clinical severity of BC.

Oncogenic AKT1(E17K) mutation induces mammary hyperplasia but prevents HER2-driven tumorigenesis.

One of the most frequently deregulated signaling pathways in breast cancer is the PI 3-K/Akt cascade. Genetic lesions are commonly found in PIK3CA, PTEN, and AKT, which lead to excessive and constitutive activation of Akt and downstream signaling that results in uncontrolled proliferation and increased cellular survival. One such genetic lesion is the somatic AKT1(E17K) mutation, which has been identified in 4-8% of breast cancer patients. To determine how this mutation contributes to mammary tumorigenesis, we constructed a genetically engineered mouse model that conditionally expresses human AKT1(E17K) in the mammary epithelium. Although AKT1(E17K) is only weakly constitutively active and does not promote proliferation in vitro, it is capable of escaping negative feedback inhibition to exhibit sustained signaling dynamics in vitro. Consistently, both virgin and multiparous AKT1(E17K) mice develop mammary gland hyperplasia that do not progress to carcinoma. This hyperplasia is accompanied by increased estrogen receptor expression, although exposure of the mice to estrogen does not promote tumor development. Moreover, AKT1(E17K) prevents HER2-driven mammary tumor formation, in part through negative feedback inhibition of RTK signaling. Analysis of TCGA breast cancer data revealed that the mRNA expression, total protein levels, and phosphorylation of various RTKs are decreased in human tumors harboring AKT1(E17K).

Abstract P1-12-14: Retinoic acid sensitizes triple-negative breast cancer cells to tamoxifen treatment

Abstract Tamoxifen, an estrogen receptor (ER) antagonist, is often used as an adjuvant endocrine therapy in the successful treatment of ER+ breast tumors. Tumors that lack ER, progesterone receptor (PR) and HER2 expression (i.e. triple-negative breast cancers) cannot be treated with adjuvant endocrine therapies, like tamoxifen, and are often more aggressive. Inducing ER expression is a potential strategy for sensitization of triple-negative breast cancers to adjuvant endocrine therapies. Given recent evidence suggesting cross-talk between the retinoic acid (RA) and estrogen signaling pathways, we investigated if RA induces expression of ER in triple-negative breast cancer cells. We hypothesize that this would lead to sensitization of the cells to tamoxifen treatment. Quantitative PCR of mRNA isolated from triple-negative MDA-MB-231 cells treated with RA and estradiol had increased ER transcript levels. Furthermore, treatment with estradiol and RA synergistically induced increased expression of RA-inducible genes. In cell proliferation studies, neither RA nor estradiol treatment alone significantly altered the growth of MDA-MB-231 cells; however, when treated with both estradiol and RA together, the growth of the cells increased significantly. This suggests that the RA-mediated increase in ER expression sensitizes MDA-MB-231 cells to estradiol-induced cell growth. Next, we investigated whether the increased ER expression sensitized MDA-MB-231 cells to tamoxifen treatment. Tamoxifen did not decrease the growth of MDA-MB-231 cells; however, when applied in combination with both estradiol and RA, tamoxifen significantly reduced MDA-MB-231 proliferation. Furthermore, tamoxifen treatment reduced the synergistic effects of estradiol/RA on RA-inducible gene expression. Together, these results suggest that the use of RA in combination with tamoxifen warrants further investigation as a potential treatment for triple-negative breast cancers. The success of the combination treatment of tamoxifen and RA in the reduction of triple-negative breast cancer cell tumor xenografts will provide further justification for this strategy in the treatment of triple-negative breast cancers. Citation Format: Krysta M Coyle, Cheryl A Dean, Diana B Jo, Margaret Thomas, Mohammad Sultan, Paola Marcato. Retinoic acid sensitizes triple-negative breast cancer cells to tamoxifen treatment [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 P1-12-14.

Abstract P3-06-15: Breast cancer molecular profile according to BMI and menopausal status

Abstract Introduction: High body mass index (BMI) is considered a risk factor for breast cancer onset and is correlated with bad prognosis in breast cancer patients. However, very few studies have associated clinical and molecular tumor characteristics with the patients’ BMI at the time of breast cancer diagnosis. The present study analyses how BMI influences tumor biology and correlates with different molecular subtypes according to menopausal status in a large cohort of patients with new diagnoses of early breast cancer (EBC). Methods: Overall 967 patients with a new diagnosis of EBC from 2007 till 2012 were recruited for this study. Clinical and tumor characteristics such as age, menopausal status, weight, height, tumor size (T), grading, estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2) status, were prospectively collected. BMI was categorized into two groups (low: ≤25; high: &amp;gt;25). Associations between BMI and clinicopathological variables were performed by χ2 test. Results: A total of 594 (61%) post-menopause and 373 (39%) pre-menopause women were enrolled. Regardless of menopausal status, patients with high BMI were older (p&amp;lt;0.0001) and had larger tumors (p&amp;lt;0.0001). However, among postmenopausal patients, high BMI correlates with increased ER expression (p = 0.015), lower HER2 expression (p = 0.05) and less aggressive tumor subtypes as luminal A (p = 0.043). In the same patient subset, women with low BMI were more likely to develop a Luminal B, HER2 like or TN breast cancer. No correlation was observed between tumor molecular phenotypes and BMI among premenopausal women. Conclusion: Among postmenopausal patient, high BMI is associated with less aggressive and more endocrine sensitive EBC. This is consistent with the hypotheses that higher estrogen exposure of breast tissue in women with higher BMI may drive growth of these cancers. If further confirmed, our data suggests that weight control in this subset of women may help to prevent cancer development. Table 1Clinical and Molecular CharacteristicsBMI ≤25BMI&amp;gt;25 N%N%totalp valueER .015Positive15376.132884.3481 Negative4823.96115.7109 PgR .2Positive14471.629676.1440 Negative5728.49323.9150 HER2 .05Positive3819.55013.388 Negative15780.532786.7484 MOLECULAR SUBTYPES .043Luminal A11560.225771.2372 Luminal B3015.74813.378 HER2147.3143.928 TN3216.84211.674 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-06-15.

The cross‐talk between estrogen receptor and peroxisome proliferator‐activated receptor gamma in thyroid cancer

Estrogen receptor (ER) and peroxisome proliferator-activated receptor gamma (PPARγ) are associated with thyroid tumorigenesis and treatment. However, the interaction between them has not been studied. The impact of ER over-expression or down-expression by DNA/small interfering RNA (siRNA) transfection, ERα agonists, and the ERβ agonist diarylpropiolnitrile (DPN) on PPARγ expression/activity was examined in papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC) cells. The effects of PPARγ modulation by rosiglitazone (RTZ), a PPARγ ligand, and of PPARγ siRNA on ER expression were determined. Cellular functions reflected by cell proliferation and migration were assayed. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick-end labeling, and apoptotic-related proteins were evaluated by Western blot analysis. PPARγ protein and activity were reduced by the over-expression of either ERα or ERβ, whereas repression of ERα or ERβ increased PPARγ expression. The administration of RTZ counteracted the effects of ER and also reduced their expression, particularly in PTC cells. Moreover, knockdown of PPARγ increased ER expression and activity. Functionally, ERα activation offset the inhibitory effect of PPARγ on cellular functions, but ERβ activation aggregated it and induced apoptosis, particularly in PTC cells. Finally, the interaction between ERβ and PPARγ enhanced the expression of proapoptotic molecules, such as caspase-3 and apoptosis-inducing factor. This study provides evidence supporting a cross-talk between ER and PPARγ. The reciprocal interaction between PPARγ and ERβ significantly inhibits the proliferation and migration of thyroid cancer cells, providing a new therapeutic strategy against thyroid cancer.

Calycosin protects HUVECs from advanced glycation end products-induced macrophage infiltration

Astragali radix is a traditional Chinese medicine that has long been used for treatment of diabetes and diabetes-associated disease, but its active component and mechanism on the disease is not well defined. Infiltration of leukocytes within the glomeruli and vasculature is one of the early and characteristic features of diabetic nephropathy. Advanced glycation end products (AGEs) play pivotal role in the progression of diabetic-associated diseases. The present study was designed to explore the therapeutic effect of calycosin, an active component from A. radix, on AGEs-induced macrophages infiltration in HUVECs. Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion. Immunocytochemistry was applied to examine TGF-beta1, ICAM-1 and RAGE protein expressions; real-time PCR was carried out to determine mRNA expression of TGF-beta1, ICAM-1 and RAGE. Immunofluorescence was carried out to observe estrogen receptor-alpha, ICAM-1, RAGE expression and the phosphorylation status of ERK1/2 and NF-κB. Calycosin significantly reduced AGEs-induced macrophage migration and adhesion to HUVEC. Pre-treatment with calycosin strikingly down-regulated HUVEC TGF-beta1, ICAM-1 and RAGE expressions in both protein and mRNA levels. Furthermore, calycosin incubation significantly increased estrogen receptor expression and reversed AGEs-induced ERK1/2 and NF-κB phosphorylation and nuclear translocation in HUVEC, and this effect of calycosin could be inhibited by estrogen receptor inhibitor, ICI182780. These findings suggest that calycosin can reduce AGEs-induced macrophage migration and adhesion to endothelial cells and relieve the local inflammation; furthermore, this effect was via estrogen receptor-ERK1/2-NF-κB pathway.

Gender-Specific Reduction in Contraction is Associated with Increased Estrogen Receptor Expression in Single Vascular Smooth Muscle Cells of Female Rat

Gender differences in the incidence of cardiovascular disease have been related to plasma estrogen levels; however, the role of vascular estrogen receptor (ER) subtypes in these sex differences is less clear. We tested whether the gender differences in vascular smooth muscle (VSM) function reflect differential expression/activity of ERα, ERβ and the newly-identified GPR30. Single aortic VSM cells (VSMCs) were freshly isolated from male and female Sprague-Dawley rats, and their contraction to phenylephrine (PHE, 10^-5 M), AngII (10^-7 M) and membrane-depolarization by KCl (51 mM) was measured in the absence or presence of 10^-6 M 17β-estradiol (E2, stimulant of most ERs), PPT (ERα agonist), DPN (ERβ agonist), and ICI 182,780 (an ERα/ERβ antagonist with GPR30 agonistic properties). The cells were fixed and fluorescently labeled with ERα, ERβ or GPR30 antibody, and the subcellular distribution of ERs was examined using digital imaging microscopy. The mRNA expression and protein amount of aortic ER subtypes was examined using RT-PCR and Western blots. PHE, AngII, and KCl caused less contraction in VSMCs of females than males. Pretreatment of VSMCs with E2 reduced PHE-, AngII- and KCl-induced contraction in both males and females. PPT caused similar inhibition of PHE-, AngII- and KCl-induced contraction as E2, suggesting a role of ERα. DPN mainly inhibited PHE and KCl contraction, suggesting an interaction between ERβ and Ca²⁺ channels. ICI 182,780 did not reduce aortic VSMC contraction, suggesting little role for GPR30. RT-PCR and Western blots revealed greater expression of ERα and ERβ in VSMCs of females than males, but similar amounts of GPR30. The total immunofluorescence signal for ERα and ERβ was greater in VSMCs of females than males, and was largely localized in the nucleus. GPR30 fluorescence was similar in VSMCs of males and females, and was mainly in the cytosol. In PPT treated cells, nuclear ERα signal was enhanced. DPN did not affect the distribution of ERβ, and ICI 182,780 did not significantly increase GPR30 in the cell surface. Thus, ER subtypes demonstrate similar responsiveness to specific agonists in VSMCs of male and female rats. The reduced contraction in VSMCs of females could be due to gender-related increase in the expression of ERα and ERβ.

Lapatinib can activate or supress estrogen receptor (ER) signaling in cell models of endocrine resistant breast cancer.

Abstract Abstract #3031 Background&amp;#x2028; Growth factor (GF) signaling is frequently upregulated in hormone resistant breast cancer (BC) and provides the rationale for using the dual EGFR/HER2 inhibitor, lapatinib (LAP) in an effort to overcome endocrine resistance. Hormone resistant BC can also vary in its degree of continued ER dependence. We previously demonstrated potent synergy between LAP and TAM in cell models of acquired endocrine resistance to tamoxifen (TAMR cells), and to estrogen deprivation (LTED cells)1. This study aimed to characterize the phenotype of these two models with a focus on the ER pathway, and to determine the biological basis for the observed synergy between LAP and TAM.&amp;#x2028; Methods&amp;#x2028; LTED cells and TAMR cells were both derived from hormone-sensitive MCF7 parental cells as previously described1. The differential effects of LAP, TAM, estrogen deprivation (ED) or the combination were assessed by proliferation assays, ERE reporter assays, quantitative PCR and western blot analyses.&amp;#x2028; Results&amp;#x2028; In LTEDs: Increased ER expression and ERE transactivation were seen in LTEDs compared to MCF7 cells, as well as continued sensitivity to the ER downregulator, fulvestrant (FUL), indicating that in LTEDs, ER activity is not only enhanced but is also a significant driver of growth. In LTEDs, LAP alone suppressed ER activity as measured by both ERE transactivation and ER phosphorylation at Ser118; the most effective suppression of ER activity was achieved with a combination of LAP+TAM/ED where the effects were additive.&amp;#x2028; In TAMRs: In contrast, TAMRs demonstrated decreased ER activity (low basal ERE transactivation and loss of PgR) and ER independent proliferation (FUL had minimal growth inhibitory effects despite ER downregulation). In TAMRs, neither LAP nor TAM had significant antiproliferative effects on their own: LAP alone decreased pMAPK/pAkt but increased basal ERE transactivation and transcription of the ER-responsive gene pS2. TAM alone inhibited ERE activation but increased HER2 gene and protein expression. Inhibiting both HER2 and ER pathways with LAP+TAM resulted in synergistic growth inhibition.&amp;#x2028; Conclusions&amp;#x2028; In LTED cells with enhanced ER function attributable in part to GF receptor activation, the synergistic antiproliferative effects from LAP and TAM or ED may be largely due to enhanced inhibition of ER signaling. In contrast in TAMR cells with suppressed ER activity, LAP alone appears to enhance ER signaling, while TAM alone inhibits ER activity but increases HER2 expression. In TAMRs, the combination of TAM+LAP is required to interrupt both ER and GF signaling. These results suggest that the basis for synergy between LAP and endocrine therapy in hormone resistant BC may vary depending on the nature of the cross-talk between the ER and GF pathways.&amp;#x2028; 1SABCS, 2006 Abs 303. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3031.

1183 PUBLICATION Methylenetetrahydrofolate reductase (MTHFR) polymorphisms affect chemotherapy response in lung cancer patients

Polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR) modify the risk and severity of sporadic breast cancer (BC). In this context, the MTHFR C677T and A1298C polymorphisms have been associated with risk and severity of sporadic BC.In total, 253 women with BC and 257 controls were enrolled in this study. Polymorphisms were analyzed using restriction fragment length polymorphism - polymerase chain reaction. Epidemiology, tumor characteristics, and reproductive factors were considered in the analysis. Statistical tests included the χ2 test, the Fisher exact test, and the Mann-Whitney and Kruskal-Wallis tests, or parametric equivalents.MTHFR polymorphisms were not a risk factor for BC. The 677CC genotype was associated with distant metastasis (odds ratio [OR] = 5.311; 95% confidence interval [CI] = 1.124-25.09) and lower estrogen receptor expression, whereas the 1298AA genotype was associated with stage 0 (OR = 0.244; 95% CI = 0.077-0.771) and increased estrogen receptor expression. In haplotype analysis, 677CC/1298AA was associated with hypertension (OR = 1.979; 95% CI = 1.036-3.782), and 677CT/1298AC was associated with invasive carcinoma of no special type (OR = 0.472; 95% CI = 0.243-0.918) and stage 0 (OR = 3.476; 95% CI = 1.341-10.47).The MTHFR C677T and A1298C polymorphisms do not alter the risk of BC, but are associated with the clinical severity of BC.

Autocrine Activation of the Local Insulin-Like Growth Factor I System Is Up-Regulated by Estrogen Receptor (ER)-Independent Estrogen Actions and Accounts for Decreased ER Expression in Type 2 Diabetic Mesangial Cells

Autocrine activation of the IGF-I system in mesangial cells (MC) promotes glomerular scarring in a model of type 1 diabetes. Although estrogens protect against progressive nondiabetic glomerulosclerosis (GS), women with diabetes seem to loose the estrogen-mediated protection against cardiovascular disease. However, little is known about the local IGF-I system and its interactions with estrogens in the pathogenesis of type 2 diabetic GS. Therefore, we examined db/db B6 (db/db) mice, a model of type 2 diabetes and diabetic GS. The IGF-I system was activated in the glomeruli and MC of female diabetic db/db mice, but not in nondiabetic db/+ littermates. We found increased IGF-I receptor (IGFR) expression and activation, including activation of MAPK. Surprisingly, estrogens, via an estrogen receptor (ER)-independent mechanism(s), increased IGFR expression, IGFR and insulin receptor substrate phosphorylation, and extracellular signal-regulated kinase activation in db/db MC. In contrast, ER expression was decreased in MC and glomeruli of db/db mice. Treatment with a neutralizing antibody to IGF-I or the MAPK inhibitor PD98059 increased ER expression and transcriptional activity. This suggests that the local prosclerotic IGF-I system is activated in type 2 diabetes and diminishes ER-mediated protection against GS. Although estrogens may stimulate protective ER signaling, they also activate the IGF-I system via ER-independent mechanisms in db/db MC. The later estrogen effects appear to outweigh the antisclerotic effects of ER activation. This may in part account for loss of estrogen protection against the progression of diabetic GS in women with type 2 diabetes.

Expression of estrogen receptor α and β in the epiphyseal plate of the rat

In this study we examine the spatial and temporal expression of estrogen receptor (ER) α and β mRNA and protein in the tibial growth plate of the rat after birth, as well as the hormonal regulation of their expression. Using in situ hybridization and immunohistochemistry, we demonstrated ERα and ERβ mRNA and protein in tibial growth plates from 1 to 40 weeks after birth. ERα and β mRNA and protein were localized in late proliferating and early hypertrophic chondrocytes during early life (1 and 4 weeks of age), whereas the immunohistochemistry also showed staining for ERα and β in the resting cells. A similar expression pattern was observed during sexual maturation (7 weeks of age) except that ERβ mRNA was also detected in early proliferating chondrocytes. After sexual maturation (from 12 up to 40 weeks of age) ERα and β mRNA and protein expression was confined to late proliferating and early hypertrophic chondrocytes. Apart from a relatively higher ERα mRNA expression in males after sexual maturation, we did not detect differences in expression of ERs between genders. Expression of ERβ mRNA in epiphyseal plates was increased in growth-retarded hypophysectomized rats compared with controls. Administration of growth hormone (GH) did not reverse the increased ER expression to normal. These data suggest that ERα and β are coexpressed in growth plates of the rat after birth and that the level of expression of ERs in these tissues is hormonally regulated. Furthermore, our data indicate that the absence of growth-plate closure in the rat cannot be explained by disappearance of ERα expression during sexual maturation per se.

Estradiol-induced IP 3 mediates the estrogen receptor activity expressed in human cells

The recent findings that estradiol-induced IP 3/PKC-α signalling pathway triggers DNA synthesis in HepG2 cells, containing estrogen receptor unable to stimulate gene transactivation, raises the hypothesis that this pathway represents an alternative signalling present when the amount of estrogen receptor (ER) is insufficient to mediate genomic effects. β-estradiol-stimulated DNA synthesis and target gene expression have been studied in HepG2 and, ER-α or ER-β negative, HeLa cells. We also examined whether either receptor is required for rapid effects of estrogen on DNA synthesis. Finally, the consequences of increased ER expression on estrogen-induced DNA synthesis and synthetic target gene expression have been evaluated. Our data indicate that the E2-induced IP 3 production is dependent on expression of either ER-α or ER-β in both HepG2 and HeLa cells. Moreover, inhibition of the IP 3 second messenger pathway blocks E2-induced cellular actions suggesting that this second messenger is responsible for estrogen's rapid, non-genomic effects on both DNA synthesis and gene expression.

Investigation of oncogene amplification or deletion, and oncoprotein expression in papillary thyroid cancer

AIM: Assessment of occurrence and possible prognostic significance of c-myc and Ha-ras amplification, p53 deletion and overexpression of cyclin D1, p53 and p21 in papillary thyroid cancer. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tumor tissue from 24 patients were investigated. Dot-blot DNA hybridization was used to detect oncogene amplification or deletion. The expression of oncoproteins was determined by immunohistochemical method. RESULTS: In our samples neither Ha-ras amplification nor p53 deletion were found. Low c-myc amplification (mean: 2.55) occured in 4 cases (17%). p53 protein was detected in 16 samples (66.6%), with p21 expression (chi(2)=7.02, p<0.01) in 6 cases (25%). The p53 expression did not influence the tumor fenotype. Cyclin D1 overexpression was found in 12 cases (50%), it was often associated with p21 expression (chi2=10.1, p<0.001) and in inverse relation to the tumor lymphocytic infiltration (chi(2)=5.35, p<0.05). Increased expression of estrogen receptor was shown in 4 cyclin D1 positive samples (17%). CONCLUSIONS: The p53 detected in our study is likely not to be mutant protein in all cases because its presence was associated with p21 expression that the mutant protein cannot induce and also it did not mean more aggressive tumor phenotype. The connection of cyclin D1 overexpression with the lymphocytic infiltration of the tumor suggests that the increased expression of cyclin D1 means poor prognosis. The coexpression of cyclin D1 and p21 raises the modulative character of the p21 protein, thought to be a tumor suppressor originally, but we find a CDK-independent, estrogen receptor mediated effect of cyclin D1 more likely, which has been described in breast cancer and is also proved by the coexpression of cyclin D1 and estrogen receptor detected here.

Estrogen receptors: new perspectives in breast cancer management

The imbalance between proliferative and differentiative estrogenic effect, caused by quantitative and qualitative alteration of the estrogen receptor (ER) expression, may play a determinant role in mammary neoplastic transformation. Our studies demonstrate that ER levels are significantly higher in human mammary neoplastic tissues when compared to perineoplastic tissues and that increased ER expression is associated with ER gene hypomethylation. During progressive multifactorial carcinogene, ER overexpression may represent an early step in neoplastic transformation. In fact, high levels of ER represent good markers of differentiation and can predict the likelihood of benefiting from anti-estrogen therapy. Nevertheless, about 35% of ER-positive breast cancers are resistant to endocrine therapy and 10% of ER-negative tumors behave as hormone-sensitive tumors. Recent studies on ER mRNA variants, which naturally occur in human breast tumors, demonstrated mutations, deletions and alternative splicings, yielding deletions of exons 3, 4, 5 and 7. ER variants exhibited altered functions or changed the responsiveness to hormonal therapy. Analysis of these variants could be a useful parameter to better predict tumor responsiveness to anti-estrogen therapy. Recently, a regain of hormonal responsiveness by ER-negative breast cancer cells has been reported following ER gene transfection. However, estradiol treatment inhibits rather than stimulates cell growth as well as the metastatic and invasive potential of the ER gene transduced cells. Transfer of the ER gene may be considered as a new therapeutic approach in the management of hormone-independent breast cancer.

Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors

The genomic organization of the estrogen receptor (ER) gene has been analyzed in 21 primary human breast cancers and 1 axillary metastasis. No evidence of rearrangements of the ER gene was found in the analyzed tumors. In 6 14 ER-positive tumors a certain degree of amplification of the ER gene, ranging from 1.6 to 3-fold, was detected. No correlation was observed between the level of gene amplification and the amount of ER in the tumors. In the 8 ER-negative tumors analyzed no amplification could be detected. It is concluded that ER gene amplification may be one of the mechanisms underlying the increased ER expression in some breast tumors.