Breast cancer aromatase expression evaluated by the novel antibody 677: Correlations to intra-tumor estrogen levels and hormone receptor status

Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E₂; estrone, E₁; estrone sulfate, E₁S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E₂ levels comparing 677+ versus 677? tumors, although a non-significant trend towards higher tumor E₁ and E₁S levels was observed in 677+ breast cancers. In contrast, tumor levels of E₂ were significantly higher in ER+ tumors compared to ER? tumors (P < 0.001) and to benign breast tissue from the same breast (P < 0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E₂ (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E₁ (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E₁S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677? tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E₂ were also found to be significantly higher among 677+ compared to 677? tumors: 873.2 fmol/g (95% CI 395.9–1925.6) versus 217.9 fmol/g (95% CI 88.8–534.9) (P = 0.015).In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis.     

The anti-inflammatory activities of Tanshinone IIA, an active component of TCM, are mediated by estrogen receptor activation and inhibition of iNOS

Tanshinone IIA (Tan IIA) is a major compound extracted from a traditional herbal medicine Salvia miltiorrhiza BUNGE, which is used to treat cardiovascular diseases, cerebrovascular diseases and postmenopausal syndrome. It has also been shown to possess anti-inflammatory activity. Since Tan IIA has a similar structure to that of 17β-estradiol (E₂), the present study was undertaken to characterize the estrogenic activity of Tan IIA and to demonstrate a functional role of this activity in RAW 264.7 cells. In transient transfection assay, Tan IIA (10 μM) increases ERE-luciferase activity in an estrogen receptor (ER) subtype-dependent manner when either ERα or ERβ were co-expressed in Hela cells. In LPS-induced RAW 264.7 cells, Tan IIA exerts anti-inflammatory effects by inhibition of iNOS gene expression and NO production, as well as inhibition of inflammatory cytokine (IL-1β, IL-6, and TNF-α) expression via ER-dependent pathway. Therefore, it could serve as a potential selective estrogen receptor modulator (SERM) to treat inflammation-associated neurodegenerative and cardiovascular diseases without increasing the risk of breast cancer.     

Activation of ERα Signaling Differentially Modulates IFN-γ Induced HLA-Class II Expression in Breast Cancer Cells

The coordinate regulation of HLA class II (HLA-II) is controlled by the class II transactivator, CIITA, and is crucial for the development of anti-tumor immunity. HLA-II in breast carcinoma is associated with increased IFN-γ levels, reduced expression of the estrogen receptor (ER) and reduced age at diagnosis. Here, we tested the hypothesis that estradiol (E₂) and ERα signaling contribute to the regulation of IFN-γ inducible HLA-II in breast cancer cells. Using a panel of established ER⁻ and ER⁺ breast cancer cell lines, we showed that E₂ attenuated HLA-DR in two ER⁺ lines (MCF-7 and BT-474), but not in T47D, while it augmented expression in ER⁻ lines, SK-BR-3 and MDA-MB-231. To further study the mechanism(s), we used paired transfectants: ERα⁺ MC2 (MDA-MB-231 c10A transfected with the wild type ERα gene) and ERα⁻ VC5 (MDA-MB-231 c10A transfected with the empty vector), treated or not with E₂ and IFN-γ. HLA-II and CIITA were severely reduced in MC2 compared to VC5 and were further exacerbated by E₂ treatment. Reduced expression occurred at the level of the IFN-γ inducible CIITA promoter IV. The anti-estrogen ICI 182,780 and gene silencing with ESR1 siRNA reversed the E₂ inhibitory effects, signifying an antagonistic role for activated ERα on CIITA pIV activity. Moreover, STAT1 signaling, necessary for CIITA pIV activation, and selected STAT1 regulated genes were variably downregulated by E₂ in transfected and endogenous ERα positive breast cancer cells, whereas STAT1 signaling was noticeably augmented in ERα⁻ breast cancer cells. Collectively, these results imply immune escape mechanisms in ERα⁺ breast cancer may be facilitated through an ERα suppressive mechanism on IFN-γ signaling.     

Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells

Select changes in microRNA (miRNA) expression correlate with estrogen receptor α (ERα) expression in breast tumors. miR-21 is higher in ERα positive than negative tumors, but no one has examined how estradiol (E₂) regulates miR-21 in breast cancer cells. Here we report that E₂ inhibits miR-21 expression in MCF-7 human breast cancer cells. The E₂-induced reduction in miR-21 was inhibited by 4-hydroxytamoxifen (4-OHT), ICI 182 780 (Faslodex), and siRNA ERα indicating that the suppression is ERα-mediated. ERα and ERβ agonists PPT and DPN inhibited and 4-OHT increased miR-21 expression. E₂ increased luciferase activity from reporters containing the miR-21 recognition elements from the 3′-UTRs of miR-21 target genes, corroborating that E₂ represses miR-21 expression resulting in a loss of target gene suppression. The E₂-mediated decrease in miR-21 correlated with increased protein expression of endogenous miR-21-targets Pdcd4, PTEN and Bcl-2. siRNA knockdown of ERα blocked the E₂-induced increase in Pdcd4, PTEN and Bcl-2. Transfection of MCF-7 cells with antisense (AS) to miR-21 mimicked the E₂-induced increase in Pdcd4, PTEN and Bcl-2. These results are the first to demonstrate that E₂ represses the expression of an oncogenic miRNA, miR-21, by activating estrogen receptor in MCF-7 cells.     

Mammographic density and hormone receptor expression in breast cancer: the Multiethnic Cohort Study

Background: It is unclear whether mammographic breast density, a strong risk factor for breast cancer, predicts subtypes of breast cancer defined by estrogen receptor (ER) and/or progesterone receptor (PR) expression. Methods: In a nested case–control study, we compared the breast density of 667 controls and 607 breast cancer cases among women of Caucasian, Japanese, and Native Hawaiian ancestry in the Hawaii component of the Multiethnic Cohort Study. A reader blinded to disease status performed computer assisted density assessment on prediagnostic mammograms. Receptor status was obtained from the statewide Hawaii Tumor Registry. Tumors were classified into ER+PR+ (n = 341), ER−PR− (n = 50), ER+PR−/ER−PR+ (n = 64), and unstaged/unknown (n = 152). Mean percent density values were computed for women with more than one mammogram. Polytomous logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) while adjusting for confounders. Results: Mean percent density was significantly greater for ER+PR+ but not for ER−PR− tumors compared to controls after adjusting for age: 37.3%, 28.9% versus 29.4%, respectively. The overall OR per 10% increase in percent density were similar for ER+PR+ and ER+PR−/ER−PR+ tumors: 1.26 (95% CI 1.17–1.36) and 1.23 (95% CI 1.07–1.42), respectively. However, percent density was not found to be a predictor for ER−PR− tumors (OR 1.00, 95% CI 0.84–1.18). The results did not differ by ethnicity, nor by menopausal status, parity, or HRT use. Conclusions: Our findings indicate that within a multiethnic population, women with higher breast density have an increased risk for ER+PR+ but not ER−PR− tumors.     

Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer

An increase in the expression of estrogen receptors (ER) and the expanded population of ER-positive cells are two common phenotypes of breast cancer. Detection of the aberrantly expressed ERα in breast cancer is carried out using ERα-antibodies and radiolabelled ligands to make decisions about cancer treatment and targeted therapy. Capitalizing on the beneficial advantages of aptamer over the conventional antibody or radiolabelled ligand, we have identified a DNA aptamer that selectively binds and facilitates the detection of ERα in human breast cancer tissue sections. The aptamer is identified using the high throughput sequencing assisted SELEX screening. Biophysical characterization confirms the binding and formation of a thermodynamically stable complex between the identified DNA aptamer (ERaptD4) and ERα (Ka = 1.55±0.298×10⁸ M^-1; ΔH = 4.32×10⁴±801.1 cal/mol; ΔS = -108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERα-positive breast cancer cells in a target-selective manner and localizes specifically in the nuclear region. To harness these characteristics of ERaptD4 for detection of ERα expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERα-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, p-value <0.05 for strong ERα positive and the ERα negative samples; kappa value = 0.823, p-value <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERα-positive cells in breast tissues without cross-reacting to ERα-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERα in strong ERα positive, moderate ERα positive and ERα negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERα may potentially be used for an efficient grading of ERα expression in cancer tissues.     

Estrogen-dependent regulation of sodium/hydrogen exchanger-3 (NHE3) expression via estrogen receptor β in proximal colon of pregnant mice

Although constipation is very common during pregnancy, the exact mechanism is unknown. We hypothesized that the involvement of estrogen receptor (ER) in the regulation of electrolyte transporter in the colon leads to constipation. In this study, the intestines of normal female ICR mouse and pregnant mice were examined for the expression of ERα and ERβ by immunohistochemistry and in situ hybridization. ERβ, but not ERα, was expressed in surface epithelial cells of the proximal, but not distal, colon on pregnancy days 10, 15, and 18, but not day 5, and the number of ERβ-positive cells increased significantly during pregnancy. Expression of NHE3, the gene that harbors estrogen response element, examined by immunohistochemistry and western blotting, was localized in the surface epithelial cells of the proximal colon and increased in parallel with ERβ expression. In ovariectomized mice, NHE3 expression was only marginal and was up-regulated after treatment with 17β-estradiol (E₂), but not E₂ + ICI 182,780 (estrogen receptor antagonist). Moreover, knock-down of ERβ expression by electroporetically transfected siRNA resulted in a significant reduction of NHE3 expression. These results indicate that ERβ regulates the expression of NHE3 in the proximal colon of pregnant mice through estrogen action, suggesting the involvement of increased sodium absorption by up-regulated NHE3 in constipation during pregnancy.     

Estrogens Correlate with PELP1 Expression in ER Positive Breast Cancer

The Proline-, glutamic acid- and leucine-rich protein 1 (PELP1) is an estrogen receptor (ER) coactivator and a proto-oncogene known to be deregulated in endocrine cancers. In breast cancer, PELP1 overexpression has been associated with endocrine therapy resistance. Although PELP1 is known to be regulated by estrogens in vitro, its association with estrogen levels within the tissue of breast cancer patients has not previously been assessed. Here, we determined PELP1 mRNA expression levels in paired samples of normal and malignant breast tissue obtained from 32 postmenopausal and 11 premenopausal women. In the total sample set, PELP1 levels were higher in tumors compared to normal breast tissue (P = 0.041). Among postmenopausal women, PELP1 tumor levels correlated positively with estrone (E₁) and estradiol (E₂) levels in both normal tissue (r = 0.543, P = 0.003 and r = 0.601, P = 0.001, respectively) and plasma (r = 0.392, P = 0.053 and r = 0.403, P = 0.046, respectively). Analyzing all ER+ tumors (n = 26), PELP1 correlated positively with E₁ and E₂ in tumor tissue (r = 0.562, P = 0.003 and r = 0.411, P = 0.037, respectively) and normal tissue (r = 0.461, P = 0.018 and r = 0.427, P = 0.030, respectively) in addition to plasma E₁, E₂ and estrone sulphate (E₁S) concentrations (r = 0.576, P = 0.003, r = 0.456, P = 0.025 and r = 0.406, P = 0.049, respectively). Finally, PELP1 correlated positively with ER mRNA (ESR1) (r = 0.553, P = 0.026) in ER+ tumors, whereas a negative association between PELP1 and ESR1 (r = -0.733, P = 0.010) was observed in ER- breast tumors. Taken together, tumor PELP1 mRNA expression is associated with estrogen levels in breast cancer, suggesting a potentially important role of PELP1 in ER+ breast cancer growth in vivo.     

Reciprocal regulation of 17β-estradiol, interleukin-6 and interleukin-8 during growth and progression of epithelial ovarian cancer

Estrogens have been associated with risk for epithelial ovarian cancer (OVCA). Both IL-6 and IL-8 are also likely involved in the progression of OVCA. In order to discover the underline molecular mechanism, we investigated the modulation of estrogen and two cytokines in the growth and progression of epithelial OVCA. In these studies, the effect of 17β-estradiol (E₂) on the expression levels of IL-6, IL-8 and their receptors was investigated. The effect of IL-6 and IL-8 on activation of estrogen-responsive promoter as well as estrogen receptor (ER)α and ERβ expression was also analyzed. Gene expression profile analysis revealed that CAOV-3 and OVCAR-3 cells, which express ER, IL-6 and IL-8 receptors, are suitable model for this study. We found that E₂ not only enhanced IL-6 and IL-8 production via NF-κB signaling pathway, but also modulated their respective receptor expression. Tamoxifen (Txf), an ER antagonist, completely abolished E₂-stimulated cell growth and the expression of IL-6 and IL-8. IL-6/IL-8-induced cell proliferation was completely blocked by their specific neutralizing antibodies, which partially inhibited E₂-induced cell growth. In the absence of estrogen, both cytokines activated estrogen-responsive promoter, which was completely blocked by Txf, and caused a dose-dependent ERα increase and ERβ decrease. Pretreatment of OVCAR-3 with p38 MAPK, MEK1/2 or ErbB2 MAPK inhibitors, respectively, blocked IL-6-mediated induction of estrogen-responsive promoter while Src inhibitor blocked IL-8-induced activation of estrogen-responsive promoter. These results provide a novel mechanism that estrogens, IL-6 and IL-8 may form a common amplifying signaling cascade to modulate OVCA growth and progression. Estrogen-induced OVCA proliferation is partially occurring via enhanced IL-6 and IL-8 production and modulated their receptors, and IL-6/IL-8 could also promote OVCA growth through an ERα pathway.     

Recent data on intratumor estrogens in breast cancer

The contribution of local synthesis versus circulatory delivery of normal breast as well as breast cancer tissue estrogens has remained a controversial area for decades. Novel data on tissue estrogen levels confirm a positive normal breast tissue to plasma concentration gradient for estrone, and to a smaller extent estradiol. Remarkably, this gradient is similar for pre- and post-menopausal women. Together with pharmacokinetic data on estrogen disposition, these findings suggest plasma and breast tissue estrogens to rapidly equilibrate, with circulating estrogens being a major contributor to breast tissue estrone levels. A likely explanation to the concentration gradient could be the fact that non-polar estrogens easily dissolve into tissue fat compartments as compared to plasma. While intratumor estrone levels are low as compared to benign tissue concentrations, intratumor estradiol is elevated in ER+ tumors. The correlation between intratumor estradiol levels and expression levels of dehydrogenases reducing estrone into estradiol but also intratumor ER concentrations are consistent with intratumor estrogen activation but also a scavenger effect of the ER.     

Inverse Regulation of EGFR/HER1 and HER2-4 in Normal and Malignant Human Breast Tissue

Cross-talk between the estrogen and the EGFR/HER signalling pathways has been suggested as a potential cause of resistance to endocrine therapy in breast cancer. Here, we determined HER1-4 receptor and neuregulin-1 (NRG1) ligand mRNA expression levels in breast cancers and corresponding normal breast tissue from patients previously characterized for plasma and tissue estrogen levels. In tumours from postmenopausal women harbouring normal HER2 gene copy numbers, we found HER2 and HER4, but HER3 levels in particular, to be elevated (2.48, 1.30 and 22.27 –fold respectively; P<0.01 for each) compared to normal tissue. Interestingly, HER3 as well as HER4 were higher among ER+ as compared to ER- tumours (P=0.004 and P=0.024, respectively). HER2 and HER3 expression levels correlated positively with ER mRNA (ESR1) expression levels (r=0.525, P=0.044; r=0.707, P=0.003, respectively). In contrast, EGFR/HER1 was downregulated in tumour compared to normal tissue (0.13-fold, P<0.001). In addition, EGFR/HER1 correlated negatively to intra-tumour (r=-0.633, P=0.001) as well as normal tissue (r=-0.556, P=0.006) and plasma estradiol levels (r=-0.625, P=0.002), suggesting an inverse regulation between estradiol and EGFR/HER1 levels. In ER+ tumours from postmenopausal women, NRG1 levels correlated positively with EGFR/HER1 (r=0.606, P=0.002) and negatively to ESR1 (r=-0.769, P=0.003) and E2 levels (r=-0.542, P=0.020). Our results indicate influence of estradiol on the expression of multiple components of the HER system in tumours not amplified for HER2, adding further support to the hypothesis that cross-talk between these systems may be of importance to breast cancer growth in vivo.