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Posttranslational regulation of FOXA1 by Polycomb and BUB3/USP7 deubiquitin complex in prostate cancer

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Science Advances  07 Apr 2021:
Vol. 7, no. 15, eabe2261
DOI: 10.1126/sciadv.abe2261
  • Fig. 1 EZH2 increases FOXA1 protein stability through physical interaction.

    (A and B) PCa cells were infected with control (shCtrl) or two independent shEZH2 lentiviruses (shEZH2-C and shEZH2-3′) for 72 hours and subjected to WB. (C) LNCaP cells were infected with the indicated EZH2 lentivirus for 96 hours and analyzed by WB. (D) LNCaP cells were treated with protein synthesis inhibitor CHX (150 μg/ml) for 0, 2, 4, and 6 hours before they were collected for WB. (E and F) 293T cells were transfected with indicated plasmids for 48 hours. Coimmunoprecipitation (co-IP) was performed using a hemagglutinin (HA) (E) or FLAG antibody (F), followed by WB. (G) Co-IP with FOXA1, EZH2, or immunoglobulin G (IgG) antibody in LNCaP nuclear lysates followed by WB. (H) Purified recombinant GST–green fluorescent protein (GFP) or GST-EZH2 coupled to GST beads were used to pull-down purified MBP-FOXA1, which was analyzed by WB using an MBP antibody. (I) Nuclear extracts from LNCaP cells were fractionated and subjected to WB. (J) Co-IP using LNCaP nuclear lysates was performed with FOXA1, SUZ12, or IgG antibody. (K and L) EZH2 was cotransfected into 293T cells along with various FLAG-tagged FOXA1 domain constructs (K). Co-IP assay was performed with anti-FLAG M2 beads followed by WB using anti-EZH2 (L). EZH2-binding domains are highlighted in yellow.

  • Fig. 2 EZH2 attenuates FOXA1 ubiquitination and degradation requiring its MTase activities.

    (A) LNCaP cells were treated with EZH2 inhibitors at indicated doses for 7 days before WB analysis. (B and C) LNCaP cells were treated with 2 μM EZH2 inhibitors for 96 hours and CHX (150 μg/ml) over indicated time course before WB (B). FOXA1 protein levels were plotted in (C) and half-lives estimated. Error bars, means ± SEM, n = 3. (D) LNCaP with stable control or EZH2 KD was treated with 20 μM MG132 for 6 hours before WB. (E) LNCaP cells were treated with either DMSO or 20 μM MG132. Co-IP was performed with IgG or FOXA1 antibody, followed by WB using anti-Ub. (F) LNCaP with stable control or EZH2 KD were treated with 20 μM MG132. Co-IP was performed with FOXA1 antibody and WB by anti-Ub. (G) LNCaP cells expressing indicated plasmids were treated with 20 μM MG132 and then subjected to co-IP by anti-FOXA1 and WB by anti-HA (Ub). (H) LNCaP cells were treated with 2 μM EZH2 inhibitors for 6 days before Co-IP. (I) 293T cells were transfected with indicated plasmids. Co-IP was performed with a pan-methyl lysine antibody and WB by anti-FOXA1. (J) LNCaP cells were treated with EZH2 inhibitors at the indicated doses for 72 hours, followed by 20 μM MG132 treatment before co-IP.

  • Fig. 3 EZH2 methylates FOXA1 protein at K295.

    (A) Amino acid sequences surrounding FOXA1 K295 from different species comparing to human H3K27 site. TAD, transactivation domain. (B) 293T cells were transfected with control, FLAG-FOXA1, FLAG-K295A, or FLAG-K295R. Co-IP was performed with anti-FLAG M2 beads. (C) LNCaP cells stably expressing FLAG-FOXA1 or FLAG-K295A were treated with CHX (150 μg/ml) for indicated time course and collected for WB. FLAG (FOXA1) protein levels were measured and half-lives estimated. Error bars, means ± SEM, n = 3. (D) LNCaP cells were transfected with control, FLAG-FOXA1, or FLAG-K295A and treated with 20 μM MG132 for 16 hours before co-IP. (E) FOXA1-KD LNCaP cells were infected with control, HA-FOXA1, or HA-K295A lentiviruses and subjected to RNA-seq. Heatmaps show genes that were up- or down-regulated by WT FOXA1 reexpression. (F) Nitrocellulose membrane blotted with nonmethylated (K295NM) or K295-methylated peptides at different picomoles were subjected to WB with the K295me1 antibody. (G) Co-IP using LNCaP nuclear lysates was performed with either IgG or K295me1 antibody. (H) 293T cells were transfected with control, FLAG-FOXA1, FLAG-K295A, or FLAG-K295R before co-IP by anti-FLAG. (I) 293T cells were transfected with the indicated plasmids. Co-IP was performed with anti-FLAG M2 beads.

  • Fig. 4 Deubiquitinase USP7 interacts with methylated FOXA1 to remove FOXA1 protein ubiquitination.

    (A) LNCaP cells were infected with either shCtrl or two independent shUSP7 lentiviruses (shUSP7-1 and shUSP7-2) and subjected to WB. (B) 293T cells were transfected with FLAG-USP7, either alone or together with FOXA1. Co-IP was performed with a FOXA1 antibody. (C) 293T cells were transfected with the indicated plasmids and treated with 20 μM MG132 for 16 hours before co-IP by anti-FOXA1 and WB by anti-HA (Ub). (D) LNCaP cells stably expressing the indicated proteins were transfected with HA-Ub for 72 hours, treated with 20 μM MG132 for 16 hours, and then subjected to co-IP. (E) 293T cells were transfected with control, FLAG-FOXA1, FLAG-K295A, or FLAG-K295R. Co-IP was performed with anti-FLAG M2 beads. (F) 293T cells were transfected with FLAG-FOXA1 along with different HA-EZH2 constructs. Co-IP was performed using anti-FLAG M2 beads. (G) Co-IP using LNCaP nuclear lysates was performed with IgG, FOXA1, or K295me1 antibody.

  • Fig. 5 BUB3 recruits USP7 to methylated FOXA1 for cell cycle gene regulation.

    (A) Co-IP in 293T cells transfected with indicated plasmids was performed using anti-FLAG M2 beads as shown in Fig. 4E, followed by WB. (B and C) Co-IP was performed in LNCaP cells using FOXA1, USP7, or IgG antibody (B) or FOXA1, EZH2, or IgG antibody (C). (D) LNCaP cells were infected with indicated lentivirus and treated with 20 μM MG132 for 16 hours. Co-IP was performed with IgG or FOXA1 antibody. (E) LNCaP cells expressing indicated plasmids were treated with 20 μM MG132 before co-IP with a FOXA1 antibody. (F) A working model showing that PRC2 methylates (me) FOXA1, which recruits BUB3 and USP7 to remove ubiquitination (Ub), thus preventing FOXA1 degradation by the proteasome system (barrel shape structure). (G) Venn diagram showing overlap between USP7-, BUB3-, and FOXA1-induced gene sets derived from RNA-seq (adjusted P < 0.05 and fold change > 3). (H) Gene ontology (GO) analysis of USP7/BUB3/FOXA1-coinduced genes. (I) Heatmap of USP7/BUB3/FOXA1-coinduced genes in LNCaP cells with indicated gene KD. (J and K) Genome browser view of two cell cycle genes showing FOXA1 ChIP-seq (top tracks), RNA-seq in siCtrl or EZH2-KD (green tracks), and RNA-seq in shCtrl, USP7-KD, BUB3-KD, and FOXA1-KD (bottom 4) LNCaP cells.

  • Fig. 6 EZH2 and FOXA1 induce cell cycle progression and PCa growth.

    (A) PCa tumor sections from 14-week-old double knockout (DKO) (PBCre4:Ptenf/f:Rb1f/f), DKO E/+ (PBCre4:Ptenf/f:Rb1f/f:Ezh2+/f), and DKO E/E mice (PBCre4:Ptenf/f:Rb1f/f:Ezh2f/f) were stained with Ezh2 or Foxa1 antibodies. (B) IHC was performed in TMA of primary prostate tumors (n = 123). Shown are representative images (top) of FOXA1 and H3K27me3 staining with IHC scores of 1 or 3 and overall correlation (bottom) between FOXA1 and H3K27me3 immunostaining intensities. (C) Venn diagram showing overlap between EZH2-induced, EPZ-6438–repressed, and FOXA1-induced gene sets derived from previous studies (3, 37). (D) GO analysis of EZH2/FOXA1-induced genes. (E) Heatmap showing the levels of EZH2/FOXA1-induced genes in the The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) samples (66) sorted by EZH2. (F) Kaplan-Meier curves showing biochemical recurrence (BCR)–free survival of patients in the TCGA-PRAD dataset with high versus low expression of EZH2/FOXA1-induced genes. Log-rank test, P < 0.05. (G to K) LNCaP cells stably overexpressing control or FOXA1 were treated with 2 μM GSK-126 or EPZ-6438 for 5 days before WB (G) and cell cycle analysis (H). Cell growths were measured with WST-1 reagent every 2 days (I and J). Data were normalized to day 0. Error bars, means ± SEM, n = 3. Cells were analyzed for colony formation assay for 2 weeks (K). n = 3.

  • Fig. 7 Therapeutic targeting of FOXA1-driven PCa using EZH2 and/or USP7 inhibitors.

    (A) USP7 and BUB3 levels in benign adjacent (N) and localized PCa (T). (B) Kaplan-Meier analyses of patients in the TCGA-PRAD dataset stratified by USP7 and BUB3 expressions. (C) IHC of FOXA1, BUB3, and USP7 was performed in adjacent sections of TMA of primary prostate tumors (n = 110). Shown are representative images with IHC scores of 1 or 3. (D) WST-1 growth assay of PCa cells treated with EZH2 or USP7 inhibitors. Error bars, means ± SEM, n = 3. (E) Colony formation assay of PCa cells treated with DMSO, EPZ-6438 (1 μM in LNCaP and 0.5 μM in C4-2B and 22Rv1), P5091 (3 μM in LNCaP and 2 μM in C4-2B and 22Rv1), or their combination for 2 weeks. (F) Representative images of mitogen- and stress-activated protein kinase (MSK)–PCa2 organoids after 10 days of treatment by DMSO, 10 μM EPZ-6438, 5 μM P5091, or combination. (G to J) C4-2B xenograft growth in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice received vehicle, EPZ-6438 (200 mg/kg) (twice a day; oral gavage), P5091 (10 mg/kg) (twice a week; intravenous), or both for 30 days (G). Tumor weights were measured at end point (H and I), and sections were stained with indicated antibodies (J). Error bars, means ± SEM, n = 6, and *P < 0.05 by analysis of variance (ANOVA). (K) A model depicting the therapeutic strategies to target FOXA1-driven PCa.

Supplementary Materials

  • Supplementary Materials

    Posttranslational regulation of FOXA1 by Polycomb and BUB3/USP7 deubiquitin complex in prostate cancer

    Su H. Park, Ka-wing Fong, Jung Kim, Fang Wang, Xiaodong Lu, Yongik Lee, Lourdes T. Brea, Kristine Wadosky, Chunming Guo, Sarki A. Abdulkadir, John D. Crispino, Deyu Fang, Panagiotis Ntziachristos, Xin Liu, Xue Li, Yong Wan, David W. Goodrich, Jonathan C. Zhao, Jindan Yu

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