Irma Parra

Low Levels of Estrogen Receptor β Protein Predict Resistance to Tamoxifen Therapy in Breast Cancer

Purpose: Breast cancer is a hormone-dependent cancer, and the presence of estrogen receptor α (ER-α) 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-β, 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-β 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-β expression. Results: Expression of ER-β 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-α expression. However, there was no association between ER-β 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-β predicted an improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy. Conclusions: These findings provide evidence that ER-β may be an independent predictor of response to tamoxifen in breast cancer. Furthermore, these results suggest that ER-β may influence tumor progression in ways different from those mediated by the ER-α isoform.

Androgen Receptor Overexpression Induces Tamoxifen Resistance in Human Breast Cancer Cells

Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR and reduced estrogen receptor (ER) α mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERα-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex™ (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERα that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen’s agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

Elevated Expression of Mitogen-Activated Protein Kinase Phosphatase 3 in Breast Tumors: A Mechanism of Tamoxifen Resistance

Antiestrogen resistance is a major clinical problem in the treatment of breast cancer. Altered growth factor signaling with estrogen receptor (ER)-alpha is associated with the development of resistance. Gene expression profiling was used to identify mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP3) whose expression was correlated with response to the antiestrogen tamoxifen in both patients and in vitro-derived cell line models. Overexpression of MKP3 rendered ER-alpha-positive breast cancer cells resistant to the growth-inhibitory effects of tamoxifen and enhanced tamoxifen agonist activity in endometrial cells. MKP3 overexpression was associated with lower levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in the presence of estrogen but that estrogen deprivation and tamoxifen treatment decreased MKP3 phosphatase activity, leading to an up-regulation of pERK1/2 MAPK, phosphorylated Ser(118)-ER-alpha, and cyclin D1. The MAPK/ERK kinase inhibitor PD98059 blocked tamoxifen-resistant growth. Accumulation of reactive oxygen species was observed with tamoxifen treatment of MKP3-overexpressing cells, and antioxidant treatment increased MKP3 phosphatase activity, thereby blocking resistance. Furthermore, PD98059 increased the levels of phosphorylated c-Jun NH(2)-terminal kinase (JNK) in tamoxifen-treated MKP3-overexpressing cells, suggesting an interaction between MKP3 levels, activation of ERK1/2 MAPK, and JNK signaling in human breast cancer cells. MKP3 represents a novel mechanism of resistance, which may be a potential biomarker for the use of ERK1/2 and/or JNK inhibitors in combination with tamoxifen treatment.

Association between the Estrogen Receptor α A908G Mutation and Outcomes in Invasive Breast Cancer

Purpose: Estrogen receptor α (ERα) predicts the natural history of breast cancer without intervening therapy. Here, we have optimized the detection of a somatic mutation, an A908G transition of ERα, and examined its association with clinical and biological features of invasive breast cancer. Experimental Design: We compared two methods of sequencing to detect the A908G ERα mutation. We then used primer extension sequencing with genomic DNA isolated from invasive breast tumors to determine whether the mutation was associated with clinical outcome in 267 axillary node–negative and axillary node–positive breast tumors. The presence of the mutation and clinical variables were analyzed for association with recurrence-free survival and overall survival by Cox proportional hazards regression models. Results: We determined that dye-labeled terminator sequencing was not adequate for detection of the A908G ERα mutation. The mutation was detected at a high frequency (50%) in invasive breast tumors using primer extension sequencing, and was found to be associated with clinical measures of poor outcome, including larger tumor size and axillary lymph node positivity. Although the mutation was associated with recurrence-free survival in univariate analysis, it was not an independent predictor of outcomes in multivariate analysis. Conclusions: Consistent with our previous finding of this somatic ERα mutation in breast ductal hyperplasias, we now present evidence that the A908G mutation is present in invasive breast tumors using an optimized sequencing method. We find that the mutation is significantly associated with aggressive biological tumor features, and with an unfavorable prognosis, but was not an independent prognostic marker in untreated patients.