5-Aza/E2 inhibits TAM-Wd cell proliferation.

<p>A. TAM-Wd and MCF-7 cell concentration response to E2 challenge (1×10⁻¹² M to 1×10⁻⁷ M) ±5-Aza (1×10⁻⁶ M). Cell counts were taken on day 7 of culture. Data shown represent E2-treated cell counts as a percentage of non-E2 treated control cells. Cell counts are significantly lower in 5-Aza treated TAM-Wd cells, co-treated with E2 at a concentration of 1×10⁻¹⁰ M and greater (*p<0.017). B. TAM-Wd cell concentration response to E2 challenge (1×10⁻¹² M to 1×10⁻⁷ M) +5-Aza (1×10⁻⁶ M) ±TAM (1×10⁻⁷ M). Cell counts were taken on day 7. Data shown represent E2-treated cell counts as a percentage of non-E2 treated control cells. The co-addition of TAM to 5-Aza treated TAM-Wd cells significantly changes the effect of E2 from a concentration of 1×10⁻⁹ M and greater (*p<0.026). C. Anchorage-dependent proliferation assay of TAM-Wd cells treated with E2 (1×10⁻⁹ M) ± TAM (1×10⁻⁷ M) in the presence of 5-Aza (1×10⁻⁶ M) for 14 days. The data shown represent actual cell number/well recorded over 3 independent experiments. By day 14, there are significantly more cells in the 5-Aza and 5-Aza/E2/TAM treated cells compared to TAM-Wd +5-Aza/E2 treated cells (*p<0.001). D. Cell cycle analysis using flow cytometric analysis of propidium iodide-stained TAM-Wd cells. Data represent percentage of cell in each phase relative to the total population.</p

Tamoxifen resistant cells retain ER expression and TAM-resistance following withdrawal from the drug.

<p>A. RT-qPCR evaluation of the basal total ER mRNA expression in MCF-7, TAM-R in the presence of TAM) and TAM-Wd cells. Data are shown as arbitrary units after normalisation to actin (n = 3) (*p<0.001). Lysates were western blotted for total ER protein and β-actin (loading control). Data are representative of 3 independent experiments. B. MCF-7, TAM-R and TAM-Wd cell concentration response to TAM (1×10⁻¹⁰M to 1×10⁻⁶ M). Cell number was assessed after 7 days. The data shown represent the percentage cell number relative to non-treated control cells (*p<0.001). The bar graph depicts flow cytometry data was used to determine the percentage of cells in S-phase following TAM treatment (1×10⁻⁷ M). A significant reduction in S-phase was observed only in the MCF-7 cells (*p<0.001). C. Cell proliferation inhibition in response to fulvestrant (1×10⁻⁷ M). Cell counts were taken on days 4, 6, 8 and 11. The data shown represent the cell number relative to time-matched non-treated control cells. Cell counts for all cell-lines treated with fulvestrant were significantly reduced compared to vehicle treated controls by day 11 of culture (*p<0.001).</p

pS2 and PgR are silenced by DNA-methylation in tamoxifen-resistant cells.

<p>RT-qPCR evaluation of pS2 (A) and PgR (C) mRNA expression in MCF-7, TAM-R and TAM-Wd cells ± E2 (1×10⁻⁹ M) for 48 hrs. Data was normalised to actin (n = 3). Gene expression is significantly induced following E2 challenge in the MCF-7 cells (*p<0.001). The right panel of the figure shows parallel ICC for pS2 (A) and PgR (B) protein (10× magnification) (representative of 3 independent experiments). Clonal bisulphite sequencing analysis was performed across pS2 (B) and PgR (D) gene promoter regions in MCF7, TAM-R and TAM-Wd. PS2 (B) and PgR (D) promoter region maps are shown on the left panel. The PCR amplicons (441 bp for pS2 and 566 bp for PgR) interrogated a total number of 12 CpG sites pS2 gene (B) and 53 CpG sites for PgR (D) located downstream the TSS. Bisulphite maps determined by direct sequencing of individual clones show the density of methylated CpG site (black circle) and unmethylated CpG site (white circle) at individual CpG sites. The line plots on the right panel show the percentage of methylation of each CpG site interrogated in each cell line for pS2 (B) and PgR (D) genes. The lines on the gene promoter maps represent CpG sites and the arrow the Transcriptional Start Site (TSS). The dashed red box denotes the genomic region with the major changes in DNA methylation that was further analyzed in the line plot.</p

5-Aza/E2 Co-treatment restored pS2 and PgR sensitivity to E2 challenge in the TAM-Wd cells.

<p>A. RT-qPCR evaluation of pS2 expression in MCF-7 and TAM-Wd cells ±5-Aza (1×10⁻⁶ M) ±E2 (1×10⁻⁹ M) for 48 hrs. Data shown represent percentage increase in pS2 detected in cells ±5-Aza following E2 challenge. Expression of pS2 is significantly increased in TAM-Wd cells treated with 5-Aza/E2 compared to 5-Aza treated cells (*p<0.001). Figure also shows ICC parallel analysis of pS2 protein expression in TAM-Wd cells (10× magnification) (representative of 3 independent experiments). B. RT-qPCR evaluation of PgR expression in MCF-7 and TAM-Wd cells ±5-Aza (1×10⁻⁶ M) ±E2 (1×10⁻⁹ M) for 48 hrs. Data shown represent percentage increase in PgR detected in cells ±5-Aza following E2 challenge. Expression of PgR is significantly increased in TAM-Wd cells treated with 5-Aza/E2 compared to 5-Aza treated cells (*p<0.001). Figure also shows ICC parallel analysis of PgR protein expression in TAM-Wd cells (10× magnification) (representative of 3 independent experiments). C. Clonal bisulphite sequencing analysis for the pS2 promoter region in TAM-Wd cells ±5-Aza (1×10⁻⁶ M) for 48 hrs. Colours and symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040466#pone-0040466-g003" target="_blank">Figure 3</a>. D. Clonal bisulphite sequencing analysis for the PgR promoter region in TAM-Wd cells ±5-Aza (1×10⁻⁶ M) for 48 hrs. Colours and symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040466#pone-0040466-g003" target="_blank">Figure 3</a>.</p

Tamoxifen-Induced Epigenetic Silencing of Oestrogen-Regulated Genes in Anti-Hormone Resistant Breast Cancer

<div><p>In the present study, we have taken the novel approach of using an <em>in vitro</em> model representative of tamoxifen-withdrawal subsequent to clinical relapse to achieve a greater understanding of the mechanisms that serve to maintain the resistant-cell phenotype, independent of any agonistic impact of tamoxifen, to identify potential novel therapeutic approaches for this disease state. Following tamoxifen withdrawal, tamoxifen-resistant MCF-7 cells conserved both drug resistance and an increased basal rate of proliferation in an oestrogen deprived environment, despite reduced epidermal growth-factor receptor expression and reduced sensitivity to gefitinib challenge. Although tamoxifen-withdrawn cells retained ER expression, a sub-set of ER-responsive genes, including pS2 and progesterone receptor (PgR), were down-regulated by promoter DNA methylation, as confirmed by clonal bisulphite sequencing experiments. Following promoter demethylation with 5-Azacytidine (5-Aza), the co-addition of oestradiol (E2) restored gene expression in these cells. In addition, 5-Aza/E2 co-treatment induced a significant anti-proliferative effect in the tamoxifen-withdrawn cells, in-contrast to either agent used alone. Microarray analysis was undertaken to identify genes specifically up regulated by this co-treatment. Several anti-proliferative gene candidates were identified and their promoters were confirmed as more heavily methylated in the tamoxifen resistant vs sensitive cells. One such gene candidate, growth differentiation factor 15 (GDF15), was carried forward for functional analysis. The addition of 5-Aza/E2 was sufficient to de-methylate and activate GDF15 expression in the tamoxifen resistant cell-lines, whilst in parallel, treatment with recombinant GDF15 protein decreased cell survival. These data provide evidence to support a novel concept that long-term tamoxifen exposure induces epigenetic silencing of a cohort of oestrogen-responsive genes whose function is associated with negative proliferation control. Furthermore, reactivation of such genes using epigenetic drugs could provide a potential therapeutic avenue for the management of tamoxifen-resistant breast cancer.</p> </div

Recombinant GDF15 is anti-proliferative and induces apoptosis in the TAM-Wd cells.

<p>A. Dose response to recombinant GDF-15 protein (1, 3, 10 ng/ml) in MCF-7, TAM-R and TAM-Wd cells. Cell number was assessed after 48 hrs post-treatment. The data shown represent the cell number relative to non-treated control cells. The dose-dependent anti-proliferative effect was statistically significant from 1 ng/ml across all cell-lines (*p<0.001). B. Both the addition of recombinant GDF15 and 5-Aza/E2 caused an increase in apoptotic cells as determined by flow cytometric analysis of M30 bound, TAM-Wd cells. Increases in apoptosis were significant for both treatments (*p<0.001), although 5-Aza/E2 induced a greater response.</p

Genes associated with an anti-proliferative function are silenced by DNA methylation in tamoxifen-resistant cells.

<p>A. MeDIP coupled PCR to determine the methylation status of RASAL1, DUSP7, ATP2B4 and GDF15 in MCF-7, TAM-R and TAM-Wd cells, relative the expression of PEG3 (an imprinted gene). Data is expressed as 2^∧−ΔΔct (n = 3). GAPDH is included as a negative control to demonstrate the successful enrichment of methylated material. The data demonstrate a greater enrichment of methylated DNA in resistant vs tamoxifen-sensitive MCF-7 cells for all four genes (*p<0.05). B. RT-qPCR evaluation of GDF15 expression in MCF-7, TAM-R and TAM-Wd cells +E2 (1×10⁻⁹ M), +5-Aza (1×10⁻⁶ M), 5-Aza + E2 or 5-Aza + E2+ TAM (1×10⁻⁷ M) for 48 hrs. Data shown are normalised to GAPDH and presented relative to the expression calculated for vehicle-treated cells. In the absence of 5-Aza, GDF15 expression is only significantly increased by E2 challenge in MCF-7 cells (*p<0.001). The lower panel shows a western blot of mature, processed GDF15 protein (18 KDa) and β-actin (loading control) (representative of 3 independent experiments). C. Clonal bisulphite sequencing analysis for the GDF15 promoter region in TAM-Wd cells ±5-Aza (1×10⁻⁶ M) for 48 hrs. Colours and symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040466#pone-0040466-g003" target="_blank">Figure 3</a>.</p

Tamoxifen withdrawal from resistant cells reduces EGFR expression and gefitinib sensitivity, but not the rate of proliferation.

<p>A. RT-qPCR evaluation of the basal total EGFR mRNA expression in MCF-7, TAM-R and TAM-Wd cells. Data have been normalised to actin (n = 3) (*p = 0.012). The lower panel shows a western blot of total EGFR protein and β-actin (loading control) (representative of 3 independent experiments). B. MCF-7, TAM-R and TAM-Wd cell concentration response to gefitinib (1×10⁻⁹ M to 1×10⁻⁶ M). Cell number was assessed after 7 days. The data shown represent the cell number relative to non-treated control cells. Cell counts for gefitinib treated (1×10⁻⁶ M) TAM-R cells are significantly lower than vehicle treated controls (*p<0.001). C. Inhibition of cell proliferation in response to gefitinib (1×10⁻⁶ M). Cell counts were taken on days 4, 6, 8 and 11. TAM-R cells were significantly inhibited by gefitinib challenge from day 4 of culture (*p<0.001). The data represent the cell number relative to time-matched non-treated control cells D. Anchorage-dependent proliferation assay of non-treated MCF-7, TAM-R, and TAM-Wd cells (1, 3 and 6 months withdrawn). Data shown represent actual cell number/well recorded over 3 independent experiments. Counts recorded for all resistant cell-lines were greater than MCF-7 cell counts from day 4 of culture (*p<0.001).</p

ELF5 specifies breast cancer subtype.

<p>(A) Sub network of breast cancer subtype gene sets derived from forced ELF5 expression in MCF7 luminal breast cancer cells (inner node color) and knockdown of ELF5 expression in HCC1937 basal breast cancer cells. Node size is proportional to gene set size; thicker green lines indicate greater gene set overlap. Nodes are positioned according to similarity in gene sets. Labels in bold type indicate the functional significance of the four clusters generated, label is plain type is the gene set name. The full network is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001461#pbio.1001461.s016" target="_blank">Figure S16</a>. (B–D) expression signature analysis of the ELF5-induced changes in molecular subtype produced by ELF5 knockdown in HCC1937 cells (B), or forced ELF5 expression in MCF7 cells (C), or T47D cells (D). Bars show the indicated comparisons that produce the associated <i>p</i>-values. BS, borderline significance; NS, not significant.</p

ELF5 suppresses the estrogen-sensitive phenotype.

<p>(A) Western blot showing reduced expression of key genes involved in the response to estrogens following induction of ELF5 expression. (B) Reduced transcriptional activity of reporters of <i>ER</i> and <i>FOXA1</i> (<i>UGT2B17</i> promoter) transcriptional activity following induction of ELF5 in MCF7 cells. Black bars, -DOX, grey bars +DOX 72 h for <i>ERE</i> and 24 h and 48 h for <i>FOXA1</i>. (C) Cell accumulation in MCF7-V5 cell cultures with (+E) or without (−E) 10 nM estrogen treatment, or following expression of ER (+ER) and 10 nM E in the context of induced ELF5. Black bars, -DOX; grey bars +DOX, 72 h and 144 h, respectively. (D) interaction of ELF5-regulated gene sets with estrogen-regulated gene sets. <i>p</i>-Values and odds ratios derived from hypergeometric tests. Number of genes in brackets. (E) Enrichment of gene sets in ELF5 ChIP targets either down (Dn) or Up in response to forced ELF5-V5 expression in T47D cells with DOX. P-Values for hypergeometric tests from GSEA (upper case) or Oncomine (lower case).</p