Clinicopathological characteristics of estrogen receptor-beta-positive human breast cancers.

Recent studies have identified the presence of estrogen receptor (ER)-beta in addition to ER-alpha in human breast cancers, but the clinicopathological characteristics of ER-beta-positive tumors remain to be established. In this study, we have conducted an immunohistochemical analysis of ER-alpha and ER-beta expression in human breast cancers. In addition, we investigated the correlation of ER-alpha and ER-beta expression with progesterone receptor (PR) status, determined by enzyme immunoassay, and with various clinicopathological factors. Of 79 tumors, 49 (62%) were positive for ER-alpha and 24 (30%) were positive for ER-beta, and there was no significant association between ER-alpha and ER-beta expression. ER-alpha-positive tumors were significantly more likely to be PR-positive than were ER-alpha-negative tumors (P < 0.0001), but there was no significant association between ER-beta expression and PR status. However, the PR values of ER-alpha-positive and ER-beta-positive tumors (65 +/- 17 fmol / mg protein, mean +/- SE) were marginally significantly lower than those of ER-alpha-positive and ER-beta-negative tumors (340 +/- 109) (P = 0.08). ER-beta positivity was significantly associated with small tumor size ( < or = 2 cm) and high histological grade (P < 0.05), and this association was also observed when only ER-alpha-positive tumors were considered. These results suggest that determination of ER-beta status might be clinically useful for further defining the characteristics of ER-alpha-positive tumors.     

Estrogen receptor alpha and beta are prognostic factors in non-small cell lung cancer.

PURPOSE: Estrogen receptor-alpha (ER-alpha) and -beta (ER-beta) play important roles in the carcinogenesis of breast tumors. Similarly, there have been several reports of ER expression in lung cancers, but the results have not been consistent, and the receptors' prognostic value remains unclear. Our goal was to investigate ER expression in non-small cell lung cancer (NSCLC) and to assess whether their expression correlates with prognosis. EXPERIMENTAL DESIGN: ER expression was examined using immunohistochemical methods with sections from 132 resected NSCLC specimens. Kaplan-Meier survival curves were analyzed to determine the significance of ER expression in the prognosis of NSCLC patients. RESULTS: ER-alpha was detected in the cytoplasm of 73% of the specimens analyzed, whereas ER-beta was detected in the nucleus of 51%. ER-alpha expression correlated with poorer overall survival (P < 0.001), as did the absence of ER-beta expression (P = 0.048). Likewise, at histopathologic stage I, ER-alpha expression (P = 0.028) or the absence of ER-beta (P = 0.037) correlated with a poorer prognosis, and ER-alpha(+)ER-beta(-) patients had a significantly worse prognosis than ER-alpha(-)ER-beta(+) patients (P = 0.00007). Multivariate Cox regression analysis revealed the absence of ER-beta to be an independent factor predictive of poor disease outcome (hazard ratio, 1.9; 95% confidence interval, 1.1-3.4; P = 0.0264). CONCLUSIONS: ER-alpha expression and the absence of ER-beta expression are associated with a poorer prognosis among NSCLC patients. In particular, the absence of ER-beta could serve as a marker identifying patients at high risk even at an early clinical stage.     

Low levels of estrogen receptor beta protein predict resistance to tamoxifen therapy in breast cancer.

PURPOSE: Breast cancer is a hormone-dependent cancer, and the presence of estrogen receptor alpha (ER-alpha) in tumors is used clinically to predict the likelihood of response to hormonal therapies. The clinical value of the second recently identified ER isoform, called ER-beta, is less clear, and there is currently conflicting data concerning its potential role as a prognostic or predictive factor. EXPERIMENTAL DESIGN: To assess whether ER-beta expression is associated with clinical outcome, protein levels were measured by immunoblot analysis of a retrospective bank of tumor cell lysates from 305 axillary node-positive patients. A total of 119 received no adjuvant therapy, and 186 were treated with tamoxifen only. The median follow-up time was 65 months. Univariate and multivariate Cox regression modeling was done to assess the prognostic and predictive significance of ER-beta expression. RESULTS: Expression of ER-beta protein did not correlate significantly with any other clinical variables, including ER and progesterone levels (as measured ligand binding assay), tumor size, age, or axillary nodal status. In the untreated population, those patients whose tumors who expressed both receptor isoforms exhibited the most favorable outcome as compared with those patients who had lost ER-alpha expression. However, there was no association between ER-beta levels alone and either disease-free or overall survival in the untreated patient population. In contrast, in both univariate and multivariate analyses, high levels of ER-beta predicted an improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy. CONCLUSIONS: These findings provide evidence that ER-beta may be an independent predictor of response to tamoxifen in breast cancer. Furthermore, these results suggest that ER-beta may influence tumor progression in ways different from those mediated by the ER-alpha isoform.     

Quantitative analysis of estrogen receptor-alpha and -beta messenger RNA expression in breast carcinoma by real-time polymerase chain reaction

BACKGROUND: Estrogen action is mediated not only through a classic estrogen receptor (ER) (ER-alpha) but also through a second ER (ER-beta) that has a structure and function similar to ER-alpha. A correlation between ER-beta mRNA expression with ER and progesterone receptor (PR) protein levels as well as prognostic factors remains to be established in breast carcinoma. METHODS: The authors conducted a quantitative analysis of ER-alpha and ER-beta mRNA expression in 116 breast tumors using real-time polymerase chain reaction (PCR), and investigated a possible correlation between ER-alpha and ER-beta mRNA expression and ER and PR status as determined by enzyme immunoassay as well as with various prognostic factors. RESULTS: ER-alpha mRNA levels were significantly (P < 0.01) higher in ER positive compared with ER negative tumors. Conversely, ER-beta mRNA levels were significantly (P < 0.01) lower in ER positive compared with ER negative tumors. Accordingly, the ratio of ER-beta to ER-alpha was significantly (P < 0.01) higher in ER negative compared with ER positive tumors. A subset analysis based on ER and PR status showed that ER-beta mRNA levels as well as the ratios of ER-beta to ER-alpha mRNA level were highest in ER negative and PR negative tumors (P < 0.05). ER-alpha mRNA levels were significantly (P < 0.05) higher in postmenopausal compared with premenopausal tumors. Histologic Grade 3 tumors showed a significant decrease in ER-alpha mRNA levels compared with Grade 1 and 2 tumors (P < 0.01 and P < 0.05, respectively). No significant correlation between ER-alpha and ER-beta mRNA levels and histologic type, tumor size, or lymph node status was observed. CONCLUSIONS: An absolute and relative increase in ER-beta mRNA levels in ER negative and PR negative breast tumors, which rarely respond to endocrine therapy, suggests the possible involvement of up-regulation of ER-beta mRNA in the development of estrogen-independent tumors.     

Expression of Estrogen Receptor Alpha and Beta in Breast Cancers of Pre- and Post-menopausal Women

Expression of estrogen receptors (ER) is clinically relevant in designing therapeutic strategies. The relative importance of the two types of estrogen receptors (ER-alpha and ER-beta) in human breast cancers in pre- and post-menopausal women has not been properly defined. To determine the possible association between the expression of estrogen receptor and serum estradiol levels in pre- and post-menopausal women with breast cancer. 44 patients with invasive ductal carcinoma of the breast were studied and a breast tissue biopsy was taken. ER-alpha and ER-beta were detected by immunocytochemistry. Serum levels of estradiol and estrone were measured by radioimmunoassay and FSH was measured using IRMA. We studied 21 pre- and 23 post-menopausal women with breast carcinoma. Examining the number of cases with tumors positive for ER, we found no differences in the frequency of ER-alpha between pre- and post-menopausal women, but ER-beta decreased marginally after menopause (p < 0.051). In cases with tumors positive for ER, the proportion of cells positive for ER-alpha was similar post-menopausally (53.95%) and pre-menopausally (57.21%), but for ER-beta the number of positive cells decreased significantly after menopause (p < 0.051). In pre-menopausal women there was a correlation between serum estradiol levels and ER-beta; in post-menopausal women there was a correlation between serum FSH levels and ER-alpha. These results indicate that estradiol levels in women with mammary carcinoma are related to ER-beta expression in the breast tumor tissue.     

17beta-hydroxysteroid dehydrogenase type 1 is an independent prognostic marker in breast cancer

Estrogens have an important role in the development and progression of breast cancer. 17beta-Hydroxysteroid dehydrogenase type 1 (17HSD1), type 2 (17HSD2), and type 5 (17HSD5) are associated with sex steroid metabolism in normal and cancerous breast tissue. The mRNA expressions of the 17HSD1, 17HSD2, and 17HSD5 enzymes were analyzed in 794 breast carcinoma specimens by using tissue microarrays and normal histologic sections. The results were correlated with the estrogen receptor alpha (ER-alpha) and beta (ER-beta), progesterone receptor, Ki67, and c-erbB-2 expressions analyzed by immunohistochemical techniques and with the Tumor-Node-Metastasis classification, tumor grade, disease-free interval, and survival of the patients. Signals for 17HSD1 mRNA were detected in 16%, 17HSD2 in 25%, and 17HSD5 in 65% of the breast cancer specimens. No association between the 17HSD1, 17HSD2, and 17HSD5 expressions was detected. A significant association was observed between ER-alpha and ER-beta (P = 0.02; odds ratio, 1.96) expressions. There was also a significant inverse association between ER-alpha and 17HSD1 (P = 0.04; odds ratio, 0.53), as well as ER-alpha and 17HSD5 (P = 0.001; odds ratio, 0.35). Patients with tumors expressing 17HSD1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients (P = 0.0010 and 0.0134, log rank). The expression of 17HSD5 was significantly higher in breast tumor specimens than in normal tissue (P = 0.033; odds ratio, 5.56). The group with 17HSD5 overexpression had a worse prognosis than the other patients (P = 0.0146). ER-alpha also associated with survival (P = 0.045). Cox multivariate analyses showed that 17HSD1 mRNA, tumor size, and ER-alpha had independent prognostic significance.     

Estrogen receptor alpha and beta profiling in human breast cancer.

BACKGROUND: The identification of a second estrogen receptor (ER-beta) has significant implications for therapeutic strategy in breast cancer management arising from the potential agonist effect of Tamoxifen at estrogen receptor sites and as such, antiestrogen therapy may be inappropriate in patients with a dominance of ER-beta. METHODS: To determine the proportion of breast cancer patients who may be so at risk, we developed a novel multiplexed RT-PCR technique to establish the relative ER-alpha and ER-beta levels in 53 primary breast cancers, 11 normal breast tissues and six cell lines. We further assessed the prognostic significance of receptor status relative to the Nottingham prognostic index (NPI). The ER-alpha and ER-beta status was also determined by immunohistochemistry using previously published and 'in-house' scoring systems. RESULTS: Using RT-PCR analysis, 46 tumours were hormone receptor positive (ER+) with 42 displaying ER-alpha predominance. Comparison with immunohistochemistry demonstrated 44/53 (ER-alpha) and 27/50 (ER-beta) concordance rates. There was no significant difference in the NPI between ER-alpha and ER-beta predominant cohorts or between ER+ and ER- cohorts. CONCLUSION: This study identifies the existence of a subgroup of ER+ patients in whom Tamoxifen therapy may be inappropriate and has significant implications for adjuvant therapy of primary breast cancer.     

Clinical significance of expression of estrogen receptor alpha and beta mRNAs in ovarian cancers

Novel human estrogen receptor (ER)-beta was identified in cDNA libraries from human testis. ER-beta specifically expresses in testis, ovary, thymus, spleen, osteoblasts and fetus. ER-beta might not conserve the same physiological functions as does ER-alpha. Therefore, the clinical significance of the expression of ER-alpha and ER-beta mRNAs in ovarian cancers was investigated. The percentage of ER-beta mRNA to ER-alpha mRNA ranged from 1.5 to 10% in normal ovaries. On the other hand, the ratios of ER-beta mRNA to ER-alpha mRNA were in a wide range in ovarian cancers. There was no significant difference in the ratios among ovarian cancers classified according to histological types or clinical stages. In a 48-month survival rate, the patient prognosis in ovarian cancers with a low or high ratio of ER-beta mRNA to ER-alpha mRNA (<1.5 or >10% of ER-beta mRNA to ER-alpha mRNA) was significantly worse than that in ovarian cancers with a medium ratio (>==1.5 to <==10% of ER-beta mRNA to ER-alpha mRNA). In conclusion, the intact synchronized expression of ER-beta mRNA interacting with ER-alpha mRNA might be damaged in some ovarian cancers, which might lead to poor patient prognosis.     

Inverse relationship between ER-beta and SRC-1 predicts outcome in endocrine-resistant breast cancer

The oestrogen receptor (ER) interacts with coactivator proteins to modulate genes central to breast tumour progression. Oestrogen receptor is encoded for by two genes, ER-alpha and ER-beta. Although ER-alpha has been well characterized, the role of ER-beta as a prognostic indicator remains unresolved. To determine isoform-specific expression of ER and coexpression with activator proteins, we examined the expression and localisation of ER-alpha, ER-beta and the coactivator protein steroid receptor coactivator 1 (SRC-1) by immunohistochemistry and immunofluorescence in a cohort of human breast cancer patients (n=150). Relative levels of SRC-1 in primary breast cultures derived from patient tumours in the presence of beta-oestradiol and tamoxifen was assessed using Western blotting (n=14). Oestrogen receptor-beta protein expression was associated with disease-free survival (DFS) and inversely associated with the expression of HER2 (P=0.0008 and P<0.0001, respectively), whereas SRC-1 was negatively associated with DFS and positively correlated with HER2 (P<0.0001 and P<0.0001, respectively). Steroid receptor coactivator 1 protein expression was regulated in response to beta-oestradiol or tamoxifen in 57% of the primary tumour cell cultures. Protein expression of ER-beta and SRC-1 was inversely associated (P=0.0001). The association of ER-beta protein expression with increased DFS and its inverse relationship with SRC-1 suggests a role for these proteins in predicting outcome in breast cancer.     

Expression and regulation of estrogen receptor beta in human breast tumors and cell lines

Expression of estrogen receptor beta (ER-beta) and its regulation by estradiol and anti-estrogens was analyzed in breast cancer cells. We determined that ER-beta is expressed in normal and tumor human breast tissue as well as in breast cancer cell lines. We observed moderate levels of ER-beta expression in both T47D and T47D-V22 (a T47D variant cell line) cells, in contrast with T47DCo (a T47D variant cell line) cells when compared to ER-alpha expression. While T47DCo (a T47D variant cell line), BT474, MDA-MB-231, MDA-MB-453, MDA-MB-468 and MCF-7 express low levels of ER-beta, other cell lines including the T47D-Y (a T47D variant cell line), MDA-MB-435, BT-549, and SKBr-3 cells express undetectable levels of ER-beta. Interestingly, ER-beta and ER-alpha are apparently not co-expressed in the breast tissue analyzed. Estradiol induced 30-40-fold increased ER-beta mRNA expression in T47D cells over control untreated cells. Moreover, the anti-estrogen, 4-hydroxy-tamoxifen (4OH-Tam) strongly inhibited estradiol induction of ER-beta expression, but had little or no effect on estradiol induction of ER-alpha. A pure anti-estrogen, ICI-182,780, completely abolished the ability of estradiol to up-regulate the expression of ER. In addition, both actinomycin D and cyclohexymide inhibited estradiol induction of ER-beta mRNA, indicating that de novo mRNA and protein synthesis are probably required for this induction. In summary, this study demonstrates that ER-beta is expressed in breast cancer, and it is regulated by estradiol. Moreover, the studies demonstrate that estradiol up-regulation of ER-beta mRNA in T47D cells can be abolished by anti-estrogens. Thus, ER-beta expression may serve as a prognostic, diagnostic and/or therapeutic marker for breast cancer. To the best of our knowledge, this is the first report regarding hormonal regulation of ER-beta in human mammary cells.     

Estrogen in obesity-associated colon cancer: friend or foe? Protecting postmenopausal women but promoting late-stage colon cancer

Obesity is associated with the increased incidence of colon cancer. Many cancer risk factors have been identified including increased blood levels of insulin, leptin, interleukin-6, interleukin-17, tumor necrosis factor-alpha, and decreased blood levels of adiponectin. However, the role of blood levels of estrogen in obesity-associated colon cancer is controversial. Evidence showed that obesity affected men more strongly than women in the carcinogenesis of colon cancer, indicating protective effect of estrogen which is increased in obesity. However, an epidemiological study has also shown that endogenous estradiol level is an independent risk factor for colon cancer, positively associated with colon cancer after normalizing insulin, IGF-1. The controversial opinions may be caused by different effects of ER-alpha and ER-beta. ER-alpha can increase colon cancer cell proliferation and increase cancer incidence. ER-beta has the opposite effect to ER-alpha, and it causes apoptosis of colon cancer cells. The normal colonocytes mainly express ER-beta. Therefore, increased estrogen in obesity may have protective effect via ER-beta in obesity-associated colon cancer. However, with the development of colon cancer, ER-alpha is increased and ER-beta is decreased. In the late stage of colon cancer, estrogen may promote cancer development via ER-alpha. The different effects and expression of ER-alpha and ER-beta may explain the different results observed in several epidemiological studies as well as several animal experiments. Therefore, manipulation of estrogen-caused signal pathways to inhibit ER-alpha and stimulate ER-beta may have preventive and therapeutic effect for obesity-associated colon cancer.     

Human kallikrein gene 11 (KLK11) mRNA overexpression is associated with poor prognosis in patients with epithelial ovarian cancer.

PURPOSE: The purpose of this study was to examine expression levels of the human tissue kallikrein 11 gene (KLK11) in epithelial ovarian tumors and to identify the relationship between KLK11 expression and patient survival. EXPERIMENTAL DESIGN: KLK11 mRNA expression was examined by semiquantitative PCR in 64 epithelial ovarian tumors (7 adenomas, 6 low malignant potential tumors, and 51 adenocarcinomas) and in 10 normal ovaries. Semiquantitative PCR results were correlated with clinicopathologic variables and overall survival. cDNA from human normal tissues and tumor tissues was also analyzed. RESULTS: KLK11 mRNA expression was detected in various human cancer tissues including breast, lung, colon, prostate, pancreas, and ovarian carcinoma. The mean value of relative KLK11 expression ratio was significantly higher in ovarian tumor samples than in normal ovary samples (compared with normal samples: adenoma, P = 0.0006; low malignant potential tumor, P = 0.0049; and carcinoma, P < 0.0001). No statistically significant associations between KLK11 mRNA expression level and clinical stage, histological type, or histological grade were observed. The log-rank test showed that high KLK11 mRNA expression and advanced clinical stage significantly correlated with poor patient survival (P = 0.0185 and P = 0.0043, respectively). High KLK11 mRNA expression and clinical stage remained significantly associated with overall survival (P = 0.0225 and P = 0.0202, respectively) after multivariate analysis. CONCLUSIONS: KLK11 expression may play an important role in ovarian cancer development and act as an independent prognostic marker in ovarian cancer patients.     

Expression of estrogen receptor beta1, beta2, and beta5 messenger RNAs in human breast tissue

A triple-primer PCR assay was developed, based on the coamplification of estrogen receptor (ER)-beta1, -beta2, and -beta5 cDNAs, to investigate the relative expressions of the corresponding mRNAs in breast cancer lines and in 53 independent breast tumors. The expression of ER-beta2 and ER-beta5 mRNAs was higher than that of ER-beta1 mRNA in both cancer cell lines and breast tumors. In breast tumors, increases in the ER-beta2:ER-beta1 and ER-beta5:ER-beta1 mRNA expression ratios were observed, which positively correlated with the level of tumor inflammation and tumor grade, respectively. A trend toward an increase of these ratios was also found in tumors, as compared to the normal adjacent breast tissue available for 13 cases. Our data suggest that changes in the relative expression of ER-beta1, -beta2, and -beta5 mRNAs occur during breast tumorigenesis and tumor progression.