Cell type- and estrogen receptor-subtype specific regulation of selective estrogen receptor modulator regulatory elements

Abstract

Selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene can act as estrogen receptor (ER) antagonists or agonists depending on the cell type. The antagonistic action of tamoxifen has been invaluable for treating breast cancer, whereas the agonist activity of SERMs also has important clinical applications as demonstrated by the use of raloxifene for osteoporosis. Whereas the mechanism whereby SERMs function as antagonists has been studied extensively very little is known about how SERMs produce agonist effects in different tissues with the two ER types; ERalpha and ERbeta. We examined the regulation of 32 SERM-responsive regions with ERalpha and ERbeta in transiently transfected MCF-7 breast, Ishikawa endometrial, HeLa cervical and WAR-5 prostate cancer cells. The regions were regulated by tamoxifen and raloxifene in some cell types, but not in all cell lines. Tamoxifen activated similar number of regions with ERalpha and ERbeta in the cell lines, whereas raloxifene activated over twice as many regions with ERbeta compared to ERalpha. In Ishikawa endometrial cancer cells, tamoxifen activated 17 regions with ERalpha, whereas raloxifene activated only 2 regions, which might explain their different effects on the endometrium. Microarray studies also found that raloxifene regulated fewer genes than tamoxifen in U2OS bone cancer cells expressing ERalpha, whereas tamoxifen was equally effective at regulating genes with ERalpha and ERbeta. Our studies indicate that tamoxifen is a non-selective agonist, whereas raloxifene is a relative ERbeta-selective agonist, and suggest that ERbeta-selective SERMs might be safer for treating clinical conditions that are dependent on the agonist property of SERMs.

Fig. 1

Cell and ER subtype regulation by tamoxifen and raloxifene. (A) The regions regulated by the SERMs in Table 1 are shown as the % of the 32 regions that were regulated in MCF-7, HeLa, Ishikawa, U2OS or WAR-5 cell lines. (B) The sum of the total number of regions regulated by tamoxifen or raloxifene in the presence of ERα or ERβ in all five cell lines.

Fig. 2

Heatmaps of log-intensities of the regulated genes by tamoxifen and raloxifene in U2OS-ERα or U2OS-ERβ cells. Genes regulated by at least one of the compounds in U2OS-ERα (A) or U2OS-ERβ cells (B) cells are shown in rows. U2OS-ERα or U2OS-ERβ cells were treated for 6 h with 1 μM tamoxifen (Tam) or raloxifene (Ral). For each gene (row), the average log intensities are colored yellow, relatively higher expression are colored with reds of increasing intensity, and relatively lower expression are colored with blues of increasing intensity. The controls and treated samples for microarrays were done in triplicate. Each activated gene was 2-fold or greater and each repressed gene was 0.5-fold or greater with p-values < 0.05.

Fig. 3

Venn diagrams of genes regulated by tamoxifen or raloxifene. The number of genes regulated specifically by 1 μM tamoxifen (Tam) or raloxifene (Ral) in U2OS-ERα (left) or U2OS-ERβ (right) cells. The numbers in the middle represent the genes commonly regulated by tamoxifen and raloxifene.

Fig. 4

Pie charts comparing significantly enriched GO terms for genes regulated by tamoxifen or raloxifene in U2OS-ERα cells. Gene ontology (GO) terms significantly enriched in various biological processes for genes regulated by tamoxifen (A) and raloxifene (B) in U2OS-ERα cells. Threshold 0.05 was used for selecting GO terms using BH-adjusted p-values. Significant GO slim terms are presented in the pie chart. The number of genes regulated in the each biological class is shown in parentheses.

Fig. 5

Pie charts comparing significantly enriched GO terms for genes regulated by tamoxifen or raloxifene in U2OS-ERβ cells. Gene ontology (GO) terms significantly enriched in various biological processes for genes regulated by tamoxifen (A) and raloxifene (B) in U2OS-ERβ cells. Threshold 0.05 was used for selecting GO terms using BH-adjusted p-values. Significant GO slim terms are presented in the pie chart. The number of genes regulated in the each biological class is shown in parentheses.

Fig. 6

Venn diagrams of genes regulated by E₂, tamoxifen or raloxifene. The number of genes regulated by 10 nM estradiol (E₂ ), 1 μM tamoxifen (Tam) or raloxifene (Ral) in U2OS-ERβ cells. The numbers in the middle represent the genes commonly regulated by E₂ and tamoxifen (left) or E₂ and raloxifene (right). The numbers in parentheses represent the genes that were regulated in an opposite direction.