Estrogen receptors ß1 and ß2 are associated with distinct responses of estrogen receptor α-positive breast carcinoma to adjuvant endocrine therapy.

Our purpose was to assess whether and how ERß1 and/or ERß2 expression status could predict response of early stage ERα-positive breast carcinoma to adjuvant endocrine therapy (AET). ERß1 and ERß2 expression were determined using immunohistochemistry. ERß1- and ERß2-positivity were derived from receiver operating characteristic analysis and the median percentage of immunostained tumor cells, respectively. Patients with recurrent disease were grouped according to whether they relapsed within 4 years or after 4 years from surgery. The predictive significance of ERß1 and ERß2 was determined using Kaplan-Meier survival analysis and Cox proportional hazards regression analysis. ERß1-positivity in the first-4-year relapse patient group was lower and ERß2-positivity in the post-4-year relapse group was higher compared with no-relapse group. ERß1-positivity was associated with lower tumor size and longer first-4-year disease-free survival, while ERß2-positivity was associated with shorter post-4-year disease-free survival. Cox multivariate analysis including ERß1, ERß2 and established clinico-pathological variables showed that ERß1-positivity was an independent predictor of lower first-4-year risk of relapse. Thus, low ERß1 expression and high ERß2 expression are markers for identification of AET-treated ERα-positive breast carcinoma patients at risk of early and late relapse, respectively.

Estrogen receptor beta 2 is associated with poor prognosis in estrogen receptor alpha-negative breast carcinoma.

PURPOSE: Our aim was to examine the prognostic significance of ERbeta1 and ERbeta2 expression in ERalpha-negative breast carcinomas. MATERIALS AND METHODS: We evaluated nuclear and cytoplasmic expression of ERbeta1 and ERbeta2 by immunohistochemistry in a group of 95 patients with long follow-up. ERbeta1 and ERbeta2 status was correlated with clinicopathological parameters and disease outcome. Univariate and multivariate analyses of ERbeta1 and ERbeta2 as independent markers of disease-free survival (DFS) were carried out using the Cox proportional hazards model. RESULTS: Nuclear ERbeta1 (nERbeta1) and nERbeta2 status was positively correlated (p = 0.01). nERbeta1 positivity was associated with low histological grade (p = 0.01) in all patients and in the nERbeta2-positive subgroup (p = 0.03) but not in the nERbeta2-negative (p = 0.27). nERbeta2 positivity was associated with lymph node involvement and tumor relapse in all cases (p < 0.00 and p < 0.00, respectively) and in the nERbeta1-negative subgroup (p < 0.00 and p < 0.00, respectively) but not in the nERbeta1-positive (p = 0.09 and p = 0.20, respectively). nERbeta2 positivity was associated with poor DFS in all patients (log-rank p <0.00), in the post-menopausal patient subgroup (log-rank p = 0.02) and in the HER2-negative (triple-negative) subgroup (log-rank p = 0.04). Cox multivariate analysis including ERbeta1, ERbeta2 and established clinicopathological variables highlighted ERbeta2 as an independent marker of early disease recurrence (hazard ratio 4.87; 95 % confidence interval 1.07-22.3; p = 0.04). CONCLUSION: High nERbeta2 is an independent marker of early relapse in ERalpha-negative breast carcinoma, and in particular, in the nERbeta1-negative, the post-menopausal patient and the triple-negative subgroups. These findings suggest that inhibition of expression and/or function of ERbeta2 could improve disease outcome.

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer.

BACKGROUND: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting. RESULTS: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells. CONCLUSION: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

Nuclear localization of cytokeratin 8 and the O-linked N-acetylglucosamine-containing epitope H in epithelial cells of infiltrating ductal breast carcinomas: a combination of immunogold and EDTA regressive staining methods.

In a previous study, the authors have shown cytokeratin 8 (CK8) and epitope H ultrastructural localization in breast cancer cell nuclei. Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H. In this study, double immunogold labeling of CK8 and epitope H combined with the EDTA regressive staining method was applied in biopsy material from infiltrating ductal breast carcinomas and fibroadenomas, to localize both antigens in correlation to RNPs distribution in the nuclear subcompartments of cancer cells. CK8 and epitope H were localized mostly over condensed chromatin, whereas staining was weaker over interchromatin granule clusters and perichromatin fibers. These results revealed, the distribution of CK8 in the nucleus as MAR-binding protein, contributing in the organization of the nuclear DNA in the neoplastic cell, as well as the distribution of O-GlcNAc glycosylated polypeptides bearing the epitope H. The latter finding indicates that these polypeptides might play a significant role in the neoplastic behavior of breast cancer cells because they colocalize in the same nuclear subcompartments with proteins modified by O-GlcNAc, such as hnRNPs G and A1, RNA polymerase II, its transcription factors, and the oncogene product of c-myc. These proteins are known to participate in coordinated transcription/RNA processing events, contributing in the neoplastic behavior of breast cancer cells.

Ultrastructural immunostaining of infiltrating ductal breast carcinomas with the monoclonal antibody H: a comparative study with cytokeratin 8.

The monoclonal antibody H (mAbH) detects an epitope consisting of an O-linked N-acetyl glucosamine (O-GlcNAc) and neighboring amino acids. This epitope has been found by using extracts from the MCF-7 human breast carcinoma cell line in immunoblotting experiments, on cytokeratin 8 (CK8) and 5 other polypeptides. In the present study, a double immunogold method was applied for the colocalization of CK8 and mAbH epitope on epoxy thin sections in 18 cases of infiltrating ductal breast carcinomas (IDBC) and in 6 cases of fibroadenomas, to study the accurate subcellular distribution of CK8 in breast cancer cells, as compared to the 5 polypeptides, recognized by mAbH. Furthermore, a detailed quantitative evaluation of the double immunolocalization over the cellular compartments of cancer cells was undertaken with the aid of a computerized image analysis system and the results were assessed statistically. The distribution pattern of CK8 and the mAbH epitope in the neoplastic mammary epithelial cells was similar in IDBC as compared to fibroadenomas, while the gold labeling intensity of these epitopes differed over the cellular compartments between malignant and benign biopsies. The results reveal the significance of the role of CK8 and O-GlcNAc glycosylation in the biology of the neoplastic mammary cells in vivo, determining their malignant potential.