The histone demethylase enzyme KDM3A is a key estrogen receptor regulator in breast cancer

Abstract

Endocrine therapy has successfully been used to treat estrogen receptor (ER)-positive breast cancer, but this invariably fails with cancers becoming refractory to treatment. Emerging evidence has suggested that fluctuations in ER co-regulatory protein expression may facilitate resistance to therapy and be involved in breast cancer progression. To date, a small number of enzymes that control methylation status of histones have been identified as co-regulators of ER signalling. We have identified the histone H3 lysine 9 mono- and di-methyl demethylase enzyme KDM3A as a positive regulator of ER activity. Here, we demonstrate that depletion of KDM3A by RNAi abrogates the recruitment of the ER to cis-regulatory elements within target gene promoters, thereby inhibiting estrogen-induced gene expression changes. Global gene expression analysis of KDM3A-depleted cells identified gene clusters associated with cell growth. Consistent with this, we show that knockdown of KDM3A reduces ER-positive cell proliferation and demonstrate that KDM3A is required for growth in a model of endocrine therapy-resistant disease. Crucially, we show that KDM3A catalytic activity is required for both ER-target gene expression and cell growth, demonstrating that developing compounds which target demethylase enzymatic activity may be efficacious in treating both ER-positive and endocrine therapy-resistant disease.

Figure 1.

KDM3A is required for ER signalling. (A) MCF-7 and T47D cells were transiently transfected with a control siRNA (siSCR) or two independent KDM3A targeting siRNAs (siKDM3A-B and siKDM3A-C) and grown for 72 h prior to western analysis using antibodies specific to KDM3A and α-tubulin. α-Tubulin expression was used to confirm equal amounts of total protein between samples. (B) MCF-7 and T47D cell lines were subject to transient transfection with either siSCR or siKDM3A-B siRNAs and grown in steroid-depleted medium for 48 h prior to 4 h treatment with vehicle or 10 nM E₂ and subsequent RNA extraction. Resultant cDNA was analysed for pS2 and GREB1 expression by qPCR. Data are the average of three independent experiments ± SEM. Gene expression is shown relative to that measured in vehicle treated siSCR transfected cells. P-values were determined by Student's t-test (* denotes P < 0.05). (C) MCF-7 cells were treated as in (B) prior to RNA extraction and hybridization to an Illumina HT12.2 Bead CHIP array. The number of ER-regulated genes (defined as genes significantly (P < 0.05) up- or down-regulated 1.5-fold by E₂ stimulation in siSCR transfected cells (n = 534)), and the number of KDM3A-regulated genes (defined as genes significantly up- or down-regulated 1.5-fold by KDM3A knockdown compared to siSCR controls in E₂ stimulated conditions (n = 3426)), was determined. E₂-stimulated genes down-regulated by KDM3A knockdown or E₂-repressed genes up-regulated by KDM3A knockdown were classified as E₂-KDM3A-regulated genes (n = 222).

Figure 2.

KDM3A regulates H3K9me1/2 demethylation and ER recruitment to target genes. (A) MCF-7 and T47D cells were transiently transfected with siSCR, siKDM3A-B or siKDM3A-C and grown for 48 h prior to western analysis using antibodies specific to H3K9me2 and histone H3. Histone H3 expression was used to confirm equal amounts of total histone protein between samples. (B–E) MCF-7 cells were transiently transfected with either siSCR or siKDM3A-B and grown for 24 h prior to treatment with vehicle or 10 nM E₂ for 45 min followed by ChIP analysis using antibodies specific to KDM3A (B), H3K9me2 (C), H3K9me1 (D), ER (E) and isotype controls (IgG). Recruitment to ERE2 within the pS2 promoter and an ERE within the GREB1 promoter was assessed by qPCR. Data are an average of at least 3 independent experiments ± SEM and is expressed relative to the level of recruitment measured in vehicle treated siSCR transfected cells. P-values were determined by Turkey's multiple comparison test (* denotes P < 0.05).

Figure 3.

KDM3A catalytic activity is required for ER signalling. (A) HEK293 cells were transiently transfected with 0.5 μg of either V5-tagged wild-type KDM3A or demethylase-inactive KDM3A_{H1120G/D1122N}–expressing plasmids and grown for 48 h prior to immunofluorescence using V5 (green), H3K9me1 and H3K9me2 (red) specific antibodies. Ectopic expression of wild type KDM3A (left panel) and KDM3A_{H1120G/D1122N} (right panel) was detected by V5 staining. Cells were counterstained using DAPI. The broken circles in DAPI and H3K9me1/2 fluorescent cell images indicate the position of cells ectopically expressing KDM3A. (B–E) MCF-7 cells were transiently transfected with siKDM3A-B and incubated for 2 h prior to transduction with lentivirus expressing either wild-type KDM3A or KDM3A_{H1120G/D1122N}. Parallel control experiments were also performed in which MCF-7 cells were transfected with siSCR or siKDM3A-B. Cells were grown for 65 h prior to RNA extraction and resultant cDNA was analysed for KDM3A (B), pS2 (C), GREB1 (D) and CCND1 (E) expression by qPCR. Data shown is for all siKDM3A transfected cells and is the average of at least three independent experiments ± SEM. Gene expression is shown relative to expression measured in siSCR control cells. P-values were determined by Turkey's multiple comparison test (* denotes P < 0.05).

Figure 4.

KDM3A is required for ER positive BCa cell growth. (A) Gene expression analysis of cell cycle regulatory genes (CCND1, CCNA2, CDK2, CDK4, CDK1 and CDC25A) between siSCR and siKDM3A-B-transfected E₂-treated MCF-7 cells. Data are the average of three independent experiments ± SEM. Gene expression is shown relative to that measured in siSCR-transfected cells. (B) MCF-7 cells were transiently transfected with either siSCR, siKDM3A-B or siKDM3A-C and grown in steroid-depleted media containing 10 nM E₂ for 54 h in the Incucyte Zoom live cell imager. Cell confluence was measured every 6 h. Data were normalized for each sample to the cell confluence measured at 0 h. Data are the average of three independent experiments ± SEM. (C) MCF-7 cells were treated as in (B) except grown for 96 h prior to cell counting. Data are the average of three independent experiments ± SEM and is expressed relative to the number of cells counted in siSCR transfected cells. (D) MCF-7 cells were treated as in (C) prior to analysis using a BrdU ELISA assay. Data are the average of three independent experiments ± SEM and is expressed relative to the absorbance for BrdU staining measured in siSCR transfected cells. (E) MCF-7 cells were treated as in (C) prior to harvesting for cell cycle analysis by propidium iodide flow cytometry. Data shows the % of cells in each phase of the cell cycle and is the average of three independent experiments ± SEM. (F) RNAi rescue experiment in MCF-7 cells (as described in Figure 3B–E) grown for 156 h prior to cell counting. Data shown is for all siKDM3A transfected cells and is the average of three independent experiments ± SEM. P-values were determined by Student's t-test (* denotes P < 0.05).

Figure 5.

KDM3A is required for ER-target gene expression and cell growth in Tamoxifen-resistant cells. (A) MMU2 cells were transiently transfected with siSCR and siKDM3A-B and grown for 72 h prior to RNA extraction. Resultant cDNA was analysed for KDM3A, pS2, GREB1, CCND1 and PIM1 expression by qPCR. Data are the average of three independent experiments ± SEM. Gene expression is shown relative to that measured in siSCR transfected cells. (B) MMU2 cells were transiently transfected with siSCR, siKDM3A-B and siKDM3A-C and grown for 60 h in the Incucyte Zoom live cell imager. Cell confluence was measured every 6 hours. Data were normalized to cell confluence measured at 0 h. Data are the average of five independent experiments ± SEM. P-values were determined by Student's t-test (* denotes P < 0.05). (C) MMU2 cells were treated as in (B) except grown for 72 h prior to cell counting. Data are the average of three independent experiments ± SEM and is expressed relative to the number of cells counted in siSCR transfected cells.