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Small-molecule inhibitor targeting orphan nuclear receptor COUP-TFII for prostate cancer treatment

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Science Advances  29 Apr 2020:
Vol. 6, no. 18, eaaz8031
DOI: 10.1126/sciadv.aaz8031
  • Fig. 1 Identification of COUP-TFII small-molecule inhibitor through high-throughput screening.

    (A) Screening strategy. 293T cells were transfected with COUP-TFII and NGFIA-Luc (or Gal4-VP16 and TK-Luc as negative control). Luciferase activity was detected 18 hours after compound treatment. (B) Schematic of three rounds of screening. (C) Structure of COUP-TFII inhibitor CIA1 and CIA2. (D) Dose-dependent inhibition of NGFIA reporter expression by the inhibitor. 293T cells that transfected with vectors were treated with CIA1 or CIA2 for 18 hours. n = 3 per group. (E) LNCaP cells were transfected with siCOUP-TFII for 24 hours and then treated with CIA1 or CIA2 for 72 hours. Cell viability was measured. n = 3 per group. (F) CIA1 and CAI2 function in a COUP-TFII–dependent manner. LNCaP cells were transfected with siCOUP-TFII (siCII) or control small interfering RNA (siRNA) (siCon) for 48 hours and then treated with CIA1 or CIA2 for 18 hours. Target gene expression was measured by quantitative polymerase chain reaction (qPCR). n = 3 per group. (G) GSEA showed that CIA1 reduced COUP-TFII–induced genes and increased COUP-TFII–repressed genes. NES, normalized enrichment score; FDR, false discovery rate.

  • Fig. 2 Direct interaction between the inhibitor and COUP-TFII protein.

    (A) CETSA was performed using LNCaP cells. COUP-TFII overexpressed 293T cells (B) or LNCaP cells (C) were used for biotinylated inhibitor pulldown assay. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation. Twenty micromolar CIA1 was used as competitor. (D) Biotinylated inhibitor pulldown assay using COUP-TFII fragments overexpressed 293T cells. Flag-CII-C (C-terminal 147 to 414 amino acids) and Flag-CII-N (N-terminal 1 to 182 amino acids). (E) Biotinylated inhibitor pulldown assay using purified glutathione S-transferase–tagged COUP-TFII ligand-binding domain (GST-LBD; 172 to 414 amino acids). GST antibody was used for Western blot assay. (F) Biotinylated inhibitor pulldown assay using overexpressed COUP-TFII with amino acid mutation in 293T cells. WT, wild type. (G) 293T cells that transfected with NGFIA-Luc and mutated COUP-TFII were treated with CIA1 for 18 hours. Luciferase assay was performed. n = 3 per group. (H) Biotinylated inhibitor pulldown assay using overexpressed nuclear receptors in 293T cells. Twenty micromolar CIA1 was used as competitor. HA, hemagglutinin.

  • Fig. 3 Reduced prostate cancer cell growth, invasion, and angiogenesis by COUP-TFII inhibitor.

    (A) CIA1 and CIA2 reduced cell growth of prostate cancer cells. Cell viability was measured 96 hours after CIA1 or CIA2 treatment. n = 3 per group. (B) CIA1 and CIA2 reduced colony formation ability of prostate cancer cells. PC3 cells were treated with inhibitor for 12 days. n = 3 per group. Two-way analysis of variance (ANOVA). (C) CIA1 and CIA2 reduced prostate cancer cell invasion. PC3 cells were treated with 1 μM CIA1 or CIA2 for 48 hours. Invasion was measured by transwell assay. n = 3 per group. One-way ANOVA. DMSO, dimethyl sulfoxide. (D) Angiogenesis was measured by human umbilical cord endothelial cell sprouting assay. n = 3 per group. One-way ANOVA. ***P < 0.001.

  • Fig. 4 Inhibition of prostate cancer tumor growth by COUP-TFII inhibitor.

    (A) CIA1 treatment has antitumor activity in mice bearing LNCaP xenograft tumors. CIA1 (2.6 mg/kg) was given daily by intraperitoneal injection. Tumor growth curve was shown. n = 5 per group. Two-way ANOVA. Tumor weight was shown in (B). n = 5 per group. t test. (C) Ki67 staining in tumor tissues treated with DMSO and CIA1. n = 6 per group. t test. (D) Indicated protein levels in collected tumor tissue were measured by Western blot assay. (E) CD31 staining in tumor tissues treated with DMSO and CIA1. n = 6 per group. t test. **P < 0.01 and ***P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 5 Inhibition of prostate cancer tumor growth by COUP-TFII inhibitor.

    CIA1 treatment reduced tumor growth in the mice that bear PC3 xenograft (A), LNCaP-abl xenograft (C), 22Rv1 xenograft (E), and PDX tumor (G). CIA1 (2.6 mg/kg) was given daily by intraperitoneal injection. n = 5 to 6 per group. Two-way ANOVA. The corresponding tumor weight was shown in (B), (D), (F), and (H). n = 5 to 6 per group. t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Disrupted COUP-TFII binding to FOXA1 by inhibitor.

    (A) MS analysis of CIA1 reduced COUP-TFII–binding proteins. Cells were treated with 4 μM CIA1 for 3 hours and then with biotin for 4 hours. IBAQ, intensity-based absolute quantification. (B) LNCaP cells with flag-COUP-TFII overexpression were treated with 4 μM CIA1 for 7 hours. IP was performed. (C) LNCaP cell growth was measured 4 days after with siRNA transfection. n = 3 per group. One-way ANOVA. (D) GSEA of FOXA1 signature with CIA1 regulated genes. FOXA1 signature was generated by GSEA37314. (E) LNCaP cells were transfected with siRNA for 72 hours. Western blot was performed, and qPCR was performed in (F). n = 3 per group. Two-way ANOVA. (G) LNCaP cells were treated with 4 μM CIA1 for 12 hours. ChIP-qPCR was performed. n = 3 per group. Two-way ANOVA. IgG, immunoglobulin G. (H) LNCaP cells were transfected with siRNA for 48 hours and then treated with 2 μM CIA1 for 18 hours. Western blot was performed, and qPCR was performed in (I). n = 3 per group. Two-way ANOVA. ns, P > 0.05; **P < 0.01 and ***P < 0.001.

Supplementary Materials

  • Supplementary Materials

    Small-molecule inhibitor targeting orphan nuclear receptor COUP-TFII for prostate cancer treatment

    Leiming Wang, Chiang-Min Cheng, Jun Qin, Mafei Xu, Chung-Yang Kao, Jingjing Shi, Erli You, Wanchun Gong, Laura Pedro Rosa, Peter Chase, Louis Scampavia, Franck Madoux, Timothy Spicer, Peter Hodder, H. Eric Xu, Sophia Y. Tsai, Ming-Jer Tsai

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