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. 2013;8(1):e54613.
doi: 10.1371/journal.pone.0054613. Epub 2013 Jan 28.

Endoxifen's molecular mechanisms of action are concentration dependent and different than that of other anti-estrogens

John R Hawse  1 , Malayannan Subramaniam, Muzaffer Cicek, Xianglin Wu, Anne Gingery, Sarah B Grygo, Zhifu Sun, Kevin S Pitel, Wilma L Lingle, Matthew P Goetz, James N Ingle, Thomas C Spelsberg
Affiliations

Endoxifen's molecular mechanisms of action are concentration dependent and different than that of other anti-estrogens

John R Hawse et al. PLoS One. 2013.

Abstract

Endoxifen, a cytochrome P450 mediated tamoxifen metabolite, is being developed as a drug for the treatment of estrogen receptor (ER) positive breast cancer. Endoxifen is known to be a potent anti-estrogen and its mechanisms of action are still being elucidated. Here, we demonstrate that endoxifen-mediated recruitment of ERα to known target genes differs from that of 4-hydroxy-tamoxifen (4HT) and ICI-182,780 (ICI). Global gene expression profiling of MCF7 cells revealed substantial differences in the transcriptome following treatment with 4HT, endoxifen and ICI, both in the presence and absence of estrogen. Alterations in endoxifen concentrations also dramatically altered the gene expression profiles of MCF7 cells, even in the presence of clinically relevant concentrations of tamoxifen and its metabolites, 4HT and N-desmethyl-tamoxifen (NDT). Pathway analysis of differentially regulated genes revealed substantial differences related to endoxifen concentrations including significant induction of cell cycle arrest and markers of apoptosis following treatment with high, but not low, concentrations of endoxifen. Taken together, these data demonstrate that endoxifen's mechanism of action is different from that of 4HT and ICI and provide mechanistic insight into the potential importance of endoxifen in the suppression of breast cancer growth and progression.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ChIP analysis of ERα binding to a consensus ERE and endogenous target genes.
ChIP assays were performed in MCF7 cells transiently transfected with a consensus ERE and treated as indicated for either 1 hour (A) or 24 hours (B). Data are expressed as the relative abundance of the target following indicated treatments relative to vehicle treated controls as detected by real-time PCR. All data were normalized using input values. Experiments were conducted in triplicate and a representative data set is shown. Asterisks denote significance at the P<0.05 level (ANOVA) compared to vehicle controls. # denotes significant differences (P<0.05) between estrogen and anti-estrogen treatments.
Figure 2
Figure 2. Microarray analysis of 4HT, endoxifen and ICI action in the presence of estrogen.
(A) Venn diagram of genes whose expression levels were significantly altered by 1.5 fold or greater in MCF7 cells treated with 10 nM estrogen, or estrogen plus 100 nM concentrations of indicated anti-estrogens, relative to vehicle control treated cells following 24 hours of exposure. (B) Venn diagram of genes whose expression was altered by estrogen plus anti-estrogen treatments relative to vehicle treatment alone. (C) Graph depicting the number of genes up-regulated (red) and down-regulated (green) by indicated treatments relative to vehicle control treated cells. (D) Heat map depicting the hierarchical clustering of genes that were differentially expressed in at least one of the indicated treatment groups relative to vehicle control and which had average fold-changes >3 standard deviations from all other genes in the comparison. Red indicates increased gene expression while green indicates decreased gene expression relative to vehicle treated controls.
Figure 3
Figure 3. Real-time PCR confirmation of selected genes whose expression levels were either increased (red) or decreased (green) by a specific anti-estrogen.
Genes whose expression levels were determined to be specifically increased (red) or decreased (green) by only one of the three anti-estrogen treatments were randomly selected for confirmation of the microarray data. Darkly shaded bars depict relative fold changes from vehicle treated cells (dashed line) as detected by microarray analysis while lightly shaded bars depict fold change as detected by RT-PCR analysis. Solid lines represent the 1.5 fold cut-off used in the microarray analysis. Data represent the mean ± the standard error of three independent treatments. Asterisks denote values with significant differences at the P<0.05 level (ANOVA) relative to vehicle treated controls which also met the 1.5 fold cut-off parameter used in the microarray analysis.
Figure 4
Figure 4. Venn diagrams depicting the anti-estrogen specific effects on estrogen-dependent and -independent genes.
(A) Venn of genes whose expression levels were significantly altered by 1.5 fold or greater in MCF7 cells treated with 10 nM estrogen plus 100 nM concentrations of indicated anti-estrogens, relative to cells treated with estrogen alone following 24 hours of exposure. (B) Venn diagram of genes whose expression levels were significantly altered by 1.5 fold or greater in MCF7 cells treated with 10 nM estrogen plus 100 nM concentrations of indicated anti-estrogens, but not by estrogen treatment alone, relative to vehicle treated controls following 24 hours of exposure.
Figure 5
Figure 5. Microarray analysis of 4HT, endoxifen and ICI action in the absence of estrogen.
(A) Venn diagram of genes whose expression levels were determined to be significantly altered by 100 nM concentrations of 4HT, endoxifen or ICI in the absence of estrogen following 24 hours of regardless of fold-change levels. (B) A second Venn diagram was developed using only those genes which exhibited fold-changes of 1.4 fold or greater relative to vehicle treated control cells. (C) Heat map depicting the hierarchical clustering of genes that were differentially expressed in at least one of the indicated treatment groups relative to vehicle control and which had average fold-changes >3 standard deviations from all other genes in the comparison. Red indicates increased gene expression while green indicates decreased gene expression relative to vehicle treated controls.
Figure 6
Figure 6. Microarray analysis of endoxifen concentration-dependent changes in gene expression in the presence of estrogen and physiologically relevant levels tamoxifen and its metabolites.
(A) Venn diagram of genes whose expression levels were significantly altered by 1.5 fold or greater in MCF7 cells treated with 10 nM estrogen (E2), 300 nM tamoxifen (TAM), 7 nM 4-hydroxy-tamoxifen (4HT) and 700 nM N-desmethyl-tamoxifen (NDT), plus indicated concentrations of endoxifen, relative to cells treated with all compounds minus endoxifen for 24 hours. (B) Venn diagram of genes detected to be regulated by the 100 nM and 20 nM endoxifen treatments. (C) Graph depicting the number of genes up-regulated (red) and down-regulated (green) by indicated treatments relative to E2, TAM, 4HT and NDT treated cells. (D) Heat map depicting the hierarchical clustering of genes that were differentially expressed in at least one of the indicated treatment groups relative to E2, TAM, 4HT and NDT treated cells and which had average fold-changes >3 standard deviations from all other genes in the comparison. Red indicates increased gene expression while green indicates decreased gene expression relative to vehicle treated controls.
Figure 7
Figure 7. Real-time PCR confirmation of randomly selected genes whose expression levels were either increased (red) or decreased (green) by a specific endoxifen concentration.
Genes whose expression levels were determined to be specifically increased (red) or decreased (green) by only one of the three endoxifen concentrations were randomly selected for confirmation of the microarray data. Darkly shaded bars depict relative fold changes from E2 (10 nM), TAM (300 nM), 4HT (7 nM) and NDT (700 nM) treated cells (combo/dashed line) as detected by microarray analysis while lightly shaded bars depict fold change as detected by RT-PCR analysis. Solid lines represent the 1.5 fold cut-off used in the microarray analysis. Data represent the mean ± the standard error of three independent treatments. Asterisks denote values with significant differences at the P<0.05 level (ANOVA) relative to combo treated controls which also met the 1.5 fold cut-off parameter used in the microarray analysis.
Figure 8
Figure 8. Pathway analysis of genes regulated by 100 nM or 20 nM endoxifen treatments in the presence of estrogen and physiologically relevant levels tamoxifen and its metabolites.
The sub-categories of biological pathways determined to be significantly altered and which were unique to the 100 nM endoxifen treatment (A) or the 20 nM endoxifen treatment (B), or which were commonly regulated by both endoxifen concentrations (C), are shown. The specific biological pathways which are comprised within these sub-categories are listed in Table 1.
Figure 9
Figure 9. Profile of cell cycle changes induced by endoxifen.
MCF7 cells were treated as indicated for 24 hours and cell cycle profiles were determined by propidium iodide staining and flow cytometry. (A) The percentage of cells from each treatment in G2/M phase (blue), S phase (green) and G1 phase (red) are shown. Asterisks within each cell cycle phase denote significance at the P<0.05 level (ANOVA) compared to vehicle controls. # within each cell cycle phase denotes significant differences (P<0.05) compared to estrogen treated cells. (B) Representative flow cytometry plots for each treatment condition.
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