Inhibitory effects of estrogen receptor beta on specific hormone-responsive gene expression and association with disease outcome in primary breast cancer

Abstract

Introduction: The impact of interactions between the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, on gene expression in breast cancer biology is not clear. The goal of this study was to examine transcriptomic alterations in cancer cells co-expressing both receptors and the association of gene expression signatures with disease outcome.

Methods: Transcriptional effects of ERbeta overexpression were determined in a stably transfected cell line derived from ERalpha-positive T-47D cells. Microarray analysis was carried out to identify differential gene expression in the cell line, and expression of key genes was validated by quantitative polymerase chain reaction. Microarray and clinical data from patient samples were then assessed to determine the in vivo relevance of the expression profiles observed in the cell line.

Results: A subset of 14 DNA replication and cell cycle-related genes was found to be specifically downregulated by ERbeta. Expression profiles of four genes, CDC2, CDC6, CKS2, and DNA2L, were significantly inversely correlated with ERbeta transcript levels in patient samples, consistent with in vitro observations. Kaplan-Meier analysis revealed better disease outcome for the patient group with an expression signature linked to higher ERbeta expression as compared to the lower ERbeta-expressing group for both disease-free survival (p = 0.00165) and disease-specific survival (p = 0.0268). These findings were further validated in an independent cohort.

Conclusion: Our findings revealed a transcriptionally regulated mechanism for the previously described growth inhibitory effects of ERbeta in ERalpha-positive breast tumor cells and provide evidence for a functional and beneficial impact of ERbeta in primary breast tumors.

Figure 1

Two clusters of genes showing disruption of estrogen-responsive expression profiles by estrogen receptor beta (ERβ) overexpression. (a) The columns represent time points arranged in chronological order, and each row represents the expression profile of a particular gene. By convention, upregulated genes are indicated by red signals and downregulated genes are indicated by green. The magnitude of change is proportional to the brightness of the signal. (b) Second cluster of genes disrupted by ERβ overexpression.

Figure 2

Validation of estrogen-responsive regulation of cell cycle and DNA replication genes suppressed by estrogen receptor beta (ERβ) overexpression by real-time quantitative polymerase chain reaction. CDC2, CDC6, DNA2L, and CKS2 were selected for further validation based on their significant correlation with ERβ transcript levels, and β actin expression was assessed as a negative control. Transcript levels in T-47Dbeta cells were measured at 30 hours following estrogen treatment (+E2) or mock treatment and under induction (-TET [+ERβ]) and non-induction (+TET) conditions. Relative fold-changes were calculated using the non-induced (+TET) samples as the reference. CDC2, cell division cycle 2; CDC6, cell division cycle 6; CKS2, cell division cycle 28 protein kinase regulatory subunit 2; DNA2L, DNA replication helicase 2-like; TET, tetracycline.

Figure 3

Clustering of 69 tumor samples by ERβ/ESR2, CDC2, CDC6, DNA2L, and CKS2 expression profiles is associated with clinical parameters and disease outcome. (a) Hierarchical clustering of tumor samples into two groups of high (black dendrogram; cluster 1) and low (red dendrogram; cluster 2) expression clusters. Clinical parameters, including relapse and death from breast cancer within 10 years of surgery and lymph node-positive (LN+) status, are indicated with a solid bar beneath each tumor sample. Tumor grade is indicated by colored bars; green, blue, and red bars denote grades 1, 2, and 3, respectively. Gray bars denote missing data. Significant distribution of clinical parameters between the two clusters was determined by Fisher exact tests, and p values were derived from the calculated distributions. (b) Kaplan-Meier plot of 10-year censored disease-free survival (DFS) curves for cluster 1 (black) and cluster 2 (red) patients is shown with the associated p values from likelihood-ratio analysis. (c) Kaplan-Meier plot of 10-year censored disease-specific survival (DSS) curves for cluster 1 (black) and cluster 2 (red) patients is shown with the associated p values from likelihood-ratio analysis. CDC2, cell division cycle 2; CDC6, cell division cycle 6; CKS2, cell division cycle 28 protein kinase regulatory subunit 2; DNA2L, DNA replication helicase 2-like; ERβ/ESR2, estrogen receptor beta.

Figure 4

Clustering of 45 previously published ERα-positive tumor samples [40] confirms that ERβ-responsive gene set expression profile is associated with disease outcome. (a) Hierarchical clustering of tumor samples into two groups of high (black dendrogram) and low (red dendrogram) ERβ expression clusters. (b) Kaplan-Meier plot of disease-free survival (DFS) curves for high (black) and low (red) expression patients is shown with the associated p values. (c) Kaplan-Meier plot of disease-specific survival (DSS) curves for high (black) and low (red) expression patients is shown with the associated p values. Due to the absence of the corresponding probes on the arrays used by Sotiriou and colleagues [40], ERβ and DNA2L transcript levels were not assessed. DNA2L, DNA replication helicase 2-like; ER, estrogen receptor.