Integrated analysis reveals FOXA1 and Ku70/Ku80 as targets of ivermectin in prostate cancer

Abstract

Ivermectin is a widely used antiparasitic drug and shows promising anticancer activity in various cancer types. Although multiple signaling pathways modulated by ivermectin have been identified in tumor cells, few studies have focused on the exact target of ivermectin. Herein, we report the pharmacological effects and targets of ivermectin in prostate cancer. Ivermectin caused G0/G1 cell cycle arrest, induced cell apoptosis and DNA damage, and decreased androgen receptor (AR) signaling in prostate cancer cells. Further in vivo analysis showed ivermectin could suppress 22RV1 xenograft progression. Using integrated omics profiling, including RNA-seq and thermal proteome profiling, the forkhead box protein A1 (FOXA1) and non-homologous end joining (NHEJ) repair executer Ku70/Ku80 were strongly suggested as direct targets of ivermectin in prostate cancer. The interaction of ivermectin and FOXA1 reduced the chromatin accessibility of AR signaling and the G0/G1 cell cycle regulator E2F1, leading to cell proliferation inhibition. The interaction of ivermectin and Ku70/Ku80 impaired the NHEJ repair ability. Cooperating with the downregulation of homologous recombination repair ability after AR signaling inhibition, ivermectin increased intracellular DNA double-strand breaks and finally triggered cell death. Our findings demonstrate the anticancer effect of ivermectin in prostate cancer, indicating that its use may be a new therapeutic approach for prostate cancer.

Fig. 1. Ivermectin inhibited prostate cancer cell viability.

Cell viability was measured by the MTT assay in AR-positive cells (LNCaP, C4-2, and 22RV1 (A), AR-negative cells (DU145 and PC-3) (B), and BPH-1 or prostate primary cells from BPH patients (C) treated with the indicated concentrations of ivermectin for either 24, 48, or 72 h.

Fig. 2. Ivermectin led to G0/G1 arrest, apoptosis, and DNA damage in prostate cancer.

A The ivermectin arrest cell cycle at G0/G1 was measured by flow cytometry. LNCaP, C4-2, and 22RV1 cells were treated with ivermectin at 4, 8, and 12 μM for 48 h. B Ivermectin induced cell apoptosis detected by PI/Annexin V staining. Cells were treated as in A. The PI + /Annexin V + and PI-/Annexin V + cells were calculated as apoptotic cells. C Western blot analysis of PARP and cleaved-Caspase-3 (c-Caspase-3) in cells treated with ivermectin for 48 h. D Ivermectin increased DNA damage. DNA fragments were shown as comet images in alkaline gel electrophoresis. The tail moment was used to quantify the DNA damage in the treatment of ivermectin for 48 h. E Western blot analysis of γH2A.X in cells treated with the ivermectin for 48 h. F Tumor volume of 22RV1 xenografts after castration treated with vehicle (con) or ivermectin (10 mg/kg, n = 5 for each group). G Representative images of Ki67, γH2A.X, and PSA immunostaining, in 22RV1 tumors treated with vehicle or ivermectin.

Fig. 3. Ivermectin inhibited the FL-AR and AR-V7 signaling activity.

A Western blot analysis of AR and PSA in LNCaP and C4-2 cells treated with ivermectin for 48 h. B RT-qPCR analysis of AR target genes (KLK3, TMPRSS2, and NKX3-1) in LNCaP and C4-2 cells treated with ivermectin for 48 h. C Western blot analysis of FL-AR, ARVs, PSA, and UBE2C in ivermectin-treated 22RV1 cells at 48 h. D RT-qPCR analysis of KLK3 and ARV target genes (UBE2C and CDC20) in 22RV1 cells treated with ivermectin for 48 h. E Western blot analysis of FL-AR, ARVs, PSA, PARP, and γH2A.X in the other two ARV-positive cells lines, LN95 and VCaP, treated with ivermectin for 48 h. F Western blot analysis of AR, PSA, PARP, and γH2A.X in LNCaP and C4-2 cells after the implementation of 4 μM and 8 μM of ivermectin with or without 1 nM R1881. G Ivermectin inhibited the cell cycle at G0/G1 in the presence of R1881. LNCaP and C4-2 cells were treated with ivermectin at 4 and 8 μM for 48 h in the absence or presence of 1 nM R1881. H Cell viability was measured by the MTT assay. LNCaP and C4-2 cells were treated with indicated concentrations of ivermectin for 48 h with or without 5 μM and 10 μM enzalutamide for 48 h.

Fig. 4. Ivermectin repressed E2F targets.

A Normalized-enrichment scores (NES) of GSEA hallmark gene sets for all four comparation in C4-2 and 22RV1 cells. Significant gene sets comparing ivermectin versus vehicle (P value < 0.05) are labeled. B Venn diagram indicating the number of DEGs between C4-2 and 22RV1 cells. C, D The GSEA of C4-2 and 22RV1 concordant altered genes highlighted that hallmark E2F targets (C) and TRANSFAC E2F1 targets (D) were repressed by ivermectin. E, F The protein (E) and mRNA (F) expression of E2F1 decreased in C4-2 and 22RV1 cells treated with ivermectin. G Western blots showing thermostable E2F1 following indicated heat shocks in the presence (+) or absence (−) of 50 μM ivermectin in C4-2 cells.

Fig. 5. Ivermectin interacted with FOXA1 to block pioneer factor activity.

A GSEA showed that genes induced by FOXA1 were inhibited by ivermectin in C4-2 cells. B RT-qPCR analysis of FOXA1 induced genes (CDKN3, CDCA2, and CAMKK2) and FOXA1 repressed EMT-associated genes (MET, MMP7, and SOX9) in C4-2 cells treated with ivermectin for 48 h. C Western blot analysis of FOXA1 and N-cadherin in LNCaP and C4-2 cells treated with ivermectin for 48 h. D ChIP–qPCR analysis for FOXA1 or AR occupancy, and FAIRE–qPCR analysis of chromatin accessibility at a target regulated by AR and FOXA1 (KLK3 and NKX3-1) in C4-2 cells treated with ivermectin. E ChIP–qPCR analysis for FOXA1 and FAIRE-PCR analysis of chromatin accessibility at a target regulated by FOXA1 (E2F1 and MET) in C4-2 cells treated with ivermectin. F FOXA1 knockdown impaired the ivermectin-repressed expression of KLK3 and E2F1 genes. mRNA levels were measured 48 h after the implementation of the ivermectin treatment and siRNA transfection by RT-qPCR in C4-2 cells. G, H Western blots showing thermostable FOXA1 and AR following indicated heat shocks in the presence (+) or absence (−) of 50 μM ivermectin in LNCaP (G) and C4-2 (H) cells. I Western blots showing thermostable FOXA1 following indicated heat shocks in the presence (+) or absence (−) of 50 μM ivermectin in 22RV1 cells. J GSEA showed the inactivation of FOXA1 induced genes in 22RV1 cells after the ivermectin treatment. K RT-qPCR analysis of FL-AR and ARv7 in 22RV1 cells treated with ivermectin for 48 h. L ChIP–qPCR analysis for FOXA1 and FAIRE–qPCR analysis of chromatin accessibility at KLK3 and E2F1 in 22RV1 cells treated with ivermectin.

Fig. 6. Ivermectin targeted to Ku70/Ku80.

A Volcano plot of melting point difference calculated from the ivermectin versus DMSO controls in living 22RV1 cells. Blue circles represent significant melting temperature differences and red circles show all remaining proteins. B KEGG and GO pathways by KOBAS showed the enrichment pathway of the proteins with the melting temperature difference (ΔTm) more than ±3 °C. C Melting curves for Ku70/Ku80 generated from mass spectrum in 22RV1 cells. D, E Western blots showing thermostable Ku70/Ku80 following indicated heat shocks in the presence (+) or absence (−) of 50 μM ivermectin in LNCaP (D) and C4-2 (E) cells.

Fig. 7. Ivermectin inhibited DSBs repair activity.

A GSEA showed that genes associated DNA damage repair were inhibited by ivermectin in C4-2 and 22RV1 cells. B, C Western blot analysis Ku70, Ku80, BRCA1, and Rad51 in whole cell lysate or Ku70, Ku80, and γH2A.X in nuclear and cytoplasmic fractions of C4-2 (B) and 22RV1 (C) cells. Lamin B and GAPDH were probed as nuclear and cytoplasmic loading controls, respectively. D, E The HR and NHEJ repair efficiencies after the ivermectin treatment were analyzed by flow cytometry using reporter constructs digested in vitro with I-SceI endonuclease, and transfected into C4-2 (D) and 22RV1 (E) cells as linear DNA. DS-Red was used for transfection control. Repair rate was normalized to DS-Red.

Fig. 8. A model for mechanisms of ivermectin inhibiting prostate cancer progression.

In PCa, ivermectin could target FOXA1 and Ku70/Ku80. The interaction of ivermectin and FOXA1 reduced the chromatin accessibility of AR signaling and E2F1, leading to cell cycle arrest and inhibiting cell proliferation. The interaction of ivermectin and Ku70/Ku80 block the recruitment of Ku70/Ku80 to DSB sites. Cooperating with the downregulation of AR regulated homologous recombination repair genes, BRCA1 and Rad51, ivermectin increased intracellular DNA damage level and triggered synthetic lethality.