Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: EZH2 methylates p38α, and EZH2 phosphorylation at T367 is critical for p38α methylation and phosphorylation in TNBC (A) Co-immunoprecipitation (co-IP) and immunoblots of methylated p38α in a panel of breast cancer cells. EZH2 shRNA knockdown decreased methylated p38α. (B) Lysine N-methyltransferase (KMTase) activity assay in whole-cell lysates of MDA-MB-231 cells at the indicated conditions. KMTase activity was assessed using 0.1 mg/mL of human recombinant p38α and measuring S-adenosyl homocysteine (SAH) production detected by bioluminescence. shEZH2 and EPZ significantly reduced methylated p38α compared to control (lanes 1–3), which was rescued by WT-EZH2 (lane 4). EPZ reduced methylated p38α compared to WT-EZH2 (lanes 4–5). Bars depict mean ± SEM, ∗p ≤ 0.05. (C) KMTase activity assay in whole-cell lysates of T4 and Vari068 patient-derived TNBC cells control and treated with EPZ as in (B). Bars show mean ± SEM, ∗p ≤ 0.05. (D) IP and immunoblots of methylated p38α in MDA-MB-231 cells transduced with scrambled shRNA (control) or 3′ UTR EZH2-targeting shRNA (shEZH2) rescued with Myc-tagged WT-EZH2, T367A-EZH2, or vector (pBabe). (E) Immunoblot of cells in (D).

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Methylation, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, shRNA, Activity Assay, Recombinant, Derivative Assay, Transduction, Plasmid Preparation

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: EZH2 methylates p38α protein at lysine 139 and lysine 165 leading to enhanced p38α protein stability (A) Schematic representation of p38 protein indicating the position of its functional domains and methylation sites (red dots). The amino acid positions of each domain are indicated below the structures. The table summarizes the unique sites of methylation identified by LC-MS-MS analyses of methylated proteins. Recombinant histone H3 and GST-p38α were incubated with or without recombinant PRC2 complex (EZH2/EED/SUZ12/RbAp48/AEBP2) and S-adenosyl methionine methyl donor in sodium phosphate-buffered HMTase buffer solution for one hour at 37°C. Samples were subsequently run on a gel and digested in-gel using trypsin or Arg-C and analyzed for monomethylation, dimethylation, and trimethylation by LC-MS/MS. The presence of methylation was confirmed by β- and γ-ion. (B) Pulse-chase analysis of MDA-MB-231 shVector and shEZH2 treated with 100 μg/mL of cycloheximide (CHX) at the indicated time points. Cell extracts were immunoblotted with anti-EZH2 and anti-p38α. α-Tubulin was used as the loading control. (C) Pulse-chase analysis for MDA-MB-231 treated with vehicle (control) or GSK-343 (1 μM) for 48 h and treated with 100 μg/mL of cycloheximide (CHX) at the indicated time points. Cell extracts were immunoblotted with anti-EZH2 and anti-p38α. α-Tubulin was used as the loading control. (D) HA-tagged p38α wild-type and p38α mutants K139A, K165A, and K139A/K165A were transduced into MDA-MB-231 cells and subjected to IP and WB using anti-p38α and anti-pan methyl-K antibody. Actin was used as the loading control. (E) CHX pulse-chase assay of cells in (D). (F) Ubiquitination assay. Indicated cells were treated with the proteasome inhibitor MG-132 (50 μM) for 5 h or vehicle. Whole-cell extracts were subsequently immunoprecipitated by anti-magnetics A beads followed by immunoblot using antibodies against ubiquitin and p38α. (G) Invasion assay of MDA-MB-231 cells transduced with HA-tagged p38α wild-type and p38α mutants K139A, K165A, and K139A/K165A. Scale bar, 10 μm. Bars show mean ± SEM, ∗p ≤ 0.05.

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Functional Assay, Methylation, Liquid Chromatography with Mass Spectroscopy, Recombinant, Incubation, Pulse Chase, Ubiquitin Assay, Immunoprecipitation, Western Blot, Invasion Assay, Transduction

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: Combined pharmacological blockade of EZH2 and p38 enzymatic activities reduces neoplastic functions (A) Immunoblots of MDA-MB-231 cells treated with GSK-343 (3 μM for 48 h), SB202190 (p38i, 20 μM for 48 h), or the combination. (B) Cells in A were subjected to growth assays. (C) Synergistic effect of EZH2 inhibitor and p38 inhibitor in MDA-MB-231 cells incubated with various doses of GSK-343 and SB202190 for 4 d. A matrix for synergy score was calculated ( Ianevski et al., 2017 ). (D and E) Wound healing assay to quantify cell migration (D) and reconstituted Boyden basement membrane-invasive chamber assay of MDA-MB-231 cells treated as in (A). Representative chambers after crystal violet staining are shown above bars. Data for B-E are from at least three independent experiments carried out in at least triplicate. Data for B, (D and E) are presented as mean ± SEM. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.005; ∗∗∗∗p ≤ 0.0001.

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Western Blot, Incubation, Wound Healing Assay, Migration, Boyden Chamber Assay, Staining

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: Combined targeting of EZH2 and p38 enzymatic activities reduces primary breast cancer growth and metastasis (A) Primary tumor growth curves of NOD/SCID mice orthotopically implanted with MDA-MB-231 cells. When primary tumors reached 100 mm 3 , mice were treated intraperitoneally with EPZ-6438 (10 mg/kg/day), SB202190 (p38i, 1 mg/kg/day), combination, or control (4% DMSO-30% PEG 300-5% Tween 80), 5 days/week for 56 days (n = 10/group). Primary tumor growth as assessed by caliper measurements, shown as mean ± SEM. (B) Quantification of tumor volume at day 56 shown as mean ± SEM. (C) MDA-MB-231 cells were injected intracardially in nude mice (n = 10/group) and treated as in (A), for 3 weeks. Bars show the number of metastases per mouse in each group on day 21 after heart inoculation ±SEM. (D) Representative H&E-stained sections of lung metastases. Magnification 600x. Scale bar 50 μm. (E) Co-immunoprecipitation and immunoblots of methylated p38α in whole-cell lysates of primary MDA-MB-231 xenograft tumors derived from (A). (F) Immunoblots for the indicated proteins in lysates obtained from the MDA-MB-231 primary orthotopic xenografts in (A). For A-C, ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.005; ∗∗∗∗p ≤ 0.0001.

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Injection, Staining, Immunoprecipitation, Western Blot, Methylation, Derivative Assay

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: Combined inhibition of EZH2 methyltransferase and p38 kinase activities reduce AKT signaling in vivo (A) RNA sequencing studies of mammary tumors treated with EPZ-6438 (10 mg/kg/day), SB202190 (p38i, 1 mg/kg/day), combination, or control (4% DMSO-30% PEG 300-5% Tween 80) and excised at day 56. The graph shows significantly deregulated pathways by the combination EPZ/p38i vs. control. (B) Immunoblot of primary xenografts treated as indicated in (A). Combined EPZ/p38i reduces p -AKT compared to single inhibitors. (C) Immunoblots for pAKT and total AKT in MDA-MB-231 cells transduced with HA-tagged WT-p38α, K139A-p38α, K165A-p38α, and K139A/K165A-p38α. P38α mutants display reduced pAKT levels compared to WT-p38α. (D) Immunoblots of MDA-MB-231 EZH2 KD rescued with Myc-tagged WT-EZH2, T367A-EZH2, or vector (pBabe) show that T367 phosphorylation is necessary to upregulate pAKT1 without changes in total AKT1. (E) Representative images of human primary invasive carcinomas. Case 1 shows an invasive high-grade ductal carcinoma with concordant high cyto-pEZH2-T367 and high pAKT, while case 2 shows an intermediate-grade invasive carcinoma with low expression of both proteins. Bars, 50 μm. (F) Schematic illustrating our working model of EZH2 function in breast cancer through H3K27me3-dependent and independent activities. P and Me represent phosphorylation and methylation, respectively.

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Inhibition, In Vivo, RNA Sequencing Assay, Western Blot, Transduction, Plasmid Preparation, Expressing, Methylation

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet:

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Plasmid Preparation, Derivative Assay, Recombinant, In Situ, Staining, RNA Sequencing Assay, ChIP-sequencing, shRNA, Software

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: PRC2 protein EED and EZH2 interact with AR. (a) Immunoprecipitation of VCaP cell lysates with the indicated mouse monoclonal anti-EED antibody (05–1,320, Millipore), rabbit polyclonal anti-EED antibody (09–774, Millipore), control IgG and anti-AR antibody was followed by immunoblot analysis. Representative graph from at least three independent experiments is shown. (b) Immunoprecipitation of 22Rv1,C4–2, LNCaP and VCaP cell lysates with anti-EZH2, anti-AR antibody and control IgG was followed by immunoblot analysis. (c) HEK293T cells transfected with Halo-AR (full length), Halo-DBD, Halo-LBD, Halo-NTD plasmids and empty vector were lysed and subjected to pull-down assay using HaloLink resin (Promega), followed by immunoblot analysis. (d) Purified EZH2 and EED were respectively mixed with AR (full length) and pulled down with anti-AR antibody and protein A beads. RING1B served as a negative control. (e) Purified EED was mixed with AR N-terminal fragment and pulled down with anti-AR antibody and protein A beads. RING1B served as a negative control.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Pull Down Assay, Purification, Negative Control

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: EZH2 and EED knockdown decreases AR and downstream targets. (a) EZH2 was depleted by shRNA in C4–2 cells. After 48 hr, cells were lysed and blotted by EZH2, EED (rabbit polyclonal anti-EED antibody, 09–774, Millipore), AR, PSA and GAPDH. (b) EED was depleted by shRNA in C4–2 cells. After 48 hr, cells were lysed and blotted by EZH2, EED (rabbit polyclonal anti-EED antibody, 09–774, Millipore), AR, PSA and GAPDH. (c) LNCaP and VCaP cells were subjected to cotransfection of PSA or TMPRSS2 firefly luciferase reporter constructs and pRL-TK (Renilla luciferase). Lentivirus packaged with two distinct shRNAs of EZH2 or EED were added 24 hr after the cotransfection to knockdown EZH2 or EED. The luciferase activity was normalized using Renilla bioluminescence.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: shRNA, Cotransfection, Luciferase, Construct, Activity Assay

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: EZH2 knockdown and astemizole treatment demonstrate similar inhibition patterns of AR signaling blockage.(a) EZH2 knockdown and astemizole-treated samples cluster together based on log expression of 1,571 (top 10%) high variation genes. (b) Heat maps for the expression level of genes down- or up-regulated by EZH2 knockdown, GSK126 and astemizole treatment. (c) The number of overlapped differential genes in each paired group is significantly larger than the number of genes overlapped by chance. (d) 426 AR-induced genes were compared and the expression is similar between EZH2 knockdown and astemizole-treated samples. (e) Comparison of PSA gene track between groups. (f) Comparison of PSA gene track between groups. (g) GSEA shows that AR target genes are significantly enriched (Q value = 0.0429) in downregulated genes due to EZH2 knockdown. (h) GSEA shows that AR target genes are significantly enriched (Q value = 0.0413) in downregulated genes due to astemizole treatment.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Inhibition, Expressing

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: Astemizole has potent therapeutic effects on prostate cancer. (a) Astemizole critically thwarts cell proliferation in C4–2 and other AR-positive prostate cancer cell lines. (b) The wound healing assay indicates that astemizole compromises the migration of C4–2 cells. (c) Astemizole decreases the invasive abilities of C4–2 cells compared to vehicle treatment. Cell count was analyzed and the difference was statistically significant. (d)C4–2 cells were treated with 2.5, 5 and 7.5 μM of astemizole. Cells were lysed 48 hr after treatment and blotted with anti-LC3-A/B antibody. The ratio of LC3-A/B-II/I to GAPDH was elevated as dose increased, which indicates that astemizole induces autophagy in prostate cancer cells. (e) Castration-resistant VCaP xenograft mouse models were generated. Castrated mice bearing CPRC xenografts received vehicle or astemizole treatment (50 mg kg−1) daily (5 days per week). Caliper measurements were taken every 4 days to determine tumor volume. Mean tumor volume SEM, *p < 0.05, **p < 0.01 vs. vehicle was marked. (f) Kaplan–Meier survival plot compares progression-free survival. (g) Upper panel: Proteins were blotted and quantitated to compare the protein levels of EZH2 and AR in astemizole-treated group (n = 8) compared to vehicle-treated group (n = 12). Lower panel: The expression of EZH2 and AR was decreased in response to astemizole treatment. (h) The proportion of the cells stained with EZH2/AR/PSA in astemizole-treated group (n = 6) were significantly lower than that in vehicle-treated group (n = 6).

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Wound Healing Assay, Migration, Cell Counting, Generated, Expressing, Staining