Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jul 28;14(15):3684.
doi: 10.3390/cancers14153684.

IKKε Inhibitor Amlexanox Promotes Olaparib Sensitivity through the C/EBP-β-Mediated Transcription of Rad51 in Castrate-Resistant Prostate Cancer

Sophie Gilbert  1 , Benjamin Péant  1 , Anne-Marie Mes-Masson  1   2 , Fred Saad  1   3
Affiliations

IKKε Inhibitor Amlexanox Promotes Olaparib Sensitivity through the C/EBP-β-Mediated Transcription of Rad51 in Castrate-Resistant Prostate Cancer

Sophie Gilbert et al. Cancers (Basel). .

Abstract

The progression of prostate cancer (PC) is often characterized by the development of castrate-resistant PC (CRPC). Patients with CRPC are treated with a variety of agents including new generation hormonal therapies or chemotherapy. However, as the cancer develops more resistance mechanisms, these drugs eventually become less effective and finding new therapeutic approaches is critical to improving patient outcomes. Previously, we have shown that IKKε depletion and IKKε inhibitors, BX795 and Amlexanox, decrease CRPC cell proliferation in vitro and in vivo and that IKKε inhibitors induce a senescence phenotype accompanied by increased DNA damage and genomic instability in CRPC cells. Here, we describe a new role for IKKε in DNA damage repair involving Rad51 and examine the therapeutic potential of Amlexanox combined with the PARP inhibitor Olaparib in CRPC cell lines. Combining Amlexanox with Olaparib decreased CRPC cell proliferation and enhanced DNA damage through the inhibition of Olaparib-induced Rad51 recruitment and expression in CRPC cells or IKKε-depleted PC-3 cells. We demonstrated that Rad51 promoter activity, measured by luciferase assay, was decreased with Amlexanox treatment or IKKε depletion and that Amlexanox treatment decreased the occupancy of transcription factor C/EBP-β on the Rad51 promoter. Our mouse model also showed that Amlexanox combined with Olaparib inhibited tumor growth of CRPC xenografts. Our study highlights a new role for IKKε in DNA damage repair through the regulation of Rad51 transcription and provides a rationale for the combination of Amlexanox and Olaparib in the treatment of patients with CRPC.

Keywords: DNA damage; DNA damage repair; PARP inhibitors; combination therapy; transcription.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Antiproliferative effect of Amlexanox and Olaparib in CRPC cell lines. (A) Impact of Amlexanox and/or Olaparib on CRPC cell proliferation. Untreated or treated cells were incubated in 96-well plates and untreated (control) or treated with indicated concentrations of Amlexanox and/or Olaparib. Cell proliferation was followed by Incucyte live-cell imaging for 4 days. (B) Clonogenic assays were performed on CRPC cells treated with 100 µM Amlexanox and/or 0.5, 10 or 0.25 µM Olaparib, for PC3, DU145 and C4-2B cells, respectively, for 6 days. Cells were fixed and stained with methylene blue. Colonies were counted under a stereomicroscope. (C,D) Representative images (C) and quantification (D) of EdU-positive CRPC cells (red; blue for DAPI) after 24-h pulses of EdU and treatment with 100 µM Amlexanox and/or 0.5, 10 or 0.25 µM Olaparib, for PC3, DU145 and C4-2B cells, respectively. Each experiment was repeated three times. Statistical significance was determined by two-way ANOVA. * p < 0.05; ** p < 0.001; *** p < 0.0001; **** p < 0.00001.
Figure 2
Figure 2
Amlexanox enhances Olaparib-induced DNA damage. Representative images (A) and quantification (B) of γH2AX foci in CRPC cells treated 100 µM Amlexanox and/or 10 µM Olaparib for 48 h. Cells were fixed and γH2AX foci (red) and DAPI (blue) were stained by immunofluorescence. Pictures were taken using a Zeiss microscope and the number of γH2AX foci per nuclei was counted using ImageJ. Each experiment was repeated three times. Statistical significance was determined by two-way ANOVA. ** p < 0.001; *** p < 0.0001; **** p < 0.00001.
Figure 3
Figure 3
Amlexanox inhibits Olaparib-induced Rad51 recruitment. (A–C) Representative images and quantification of Rad51 foci in CRPC cells treated with 100 µM Amlexanox (48 h) and/or 10 µM Olaparib (for 24 h). Cells were fixed and stained for Rad51 foci (red), GMNN (green) and DAPI (blue). Pictures were taken using a Zeiss microscope and the number of Rad51 foci per GMNN-positive cells were counted using ImageJ. (D) PC-3 cells were transfected with lentiviral shIKKε.A or shIKKε.B. Western blots confirmed IKKε depletion with whole cell extracts separated, transferred and probed with IKKε antibody. (E) Representative images and (F) quantification of Rad51 foci in IKKε-depleted PC-3 cells. Each experiment was repeated three times. Statistical significance was determined by two-way ANOVA. * p < 0.05; ** p < 0.001; *** p < 0.0001; **** p < 0.00001. The full Western blots are shown in Figure S5.
Figure 4
Figure 4
Amlexanox decreases Rad51 transcription and expression. (A,B) Rad51 expression in CRPC cells measured by RT-qPCR (A) and Western blot (B) after 48 h of 100 µM Amlexanox (Aml) and/or 10 µM Olaparib (Ola) treatment. (C) Rad51 expression in IKKε-depleted PC-3 cells treated with 10 µM Olaparib for 48 h and measured by RT-qPCR. Statistical significance was determined by two-way ANOVA. ** p < 0.001; *** p < 0.0001; **** p < 0.00001. The full Western blots are shown in Figure S5.
Figure 5
Figure 5
Amlexanox decreases Rad51 transcription by inhibiting C/EBP-β activation. (A,B) Rad51 promoter activity in CRPC cells (A) and in IKKε-depleted PC-3 cells (B) measured by relative light units (RLU) from luciferase assays. (C) Schematic representation of Rad51 promoter with C/EBP-β binding site. (D) CRPC cells were transfected with empty vector or pORF9-C/EBP-β-Flag2 vector. After 48 h, cells were collected and proteins analyzed by Western blot using anti-Flag antibody. (E) Chromatin immunoprecipitation (ChIP) was performed using anti-Flag antibody on chromatin extracts from CRPC cells that were transfected with pORF9-C/EBP-β-Flag2 vector and treated with 100 µM Amlexanox. (F) IKKε-depleted PC-3 cells were transfected with empty vector or pORF9-C/EBP-β-Flag2 vector. (G) Chromatin immunoprecipitation was performed using anti-Flag antibody on chromatin extracts from IKKε-depleted PC-3 cells transfected with pORF9-C/EBP-β-Flag2 vector or control. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. The full Western blot are shown in Figure S5.
Figure 6
Figure 6
Amlexanox-Olaparib combination delays CRPC xenograft tumor growth in mouse model. (A,B) Tumor size ratio between the first and last days of treatment for PC-3 and DU145 xenograft tumors (n = 9 and n = 18, respectively). Amlexanox (25 mg/kg) and/or Olaparib (50 mg/kg) were administered daily by intraperitoneal injection. Tumor size was measured twice a week and all mice were sacrificed at endpoint. (C–F) Representative images (C,D) and quantification (E,F) of percentage of GMNN-positive cells (red) in PC-3 (C,E) and DU145 (D,F) xenografts (n = 5 per condition). Statistical significance was determined by two-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Rodriguez M.I., Majuelos-Melguizo J., Marti Martin-Consuegra J.M., Ruiz de Almodovar M., Lopez-Rivas A., Javier Oliver F. Deciphering the insights of poly(ADP-ribosylation) in tumor progression. Med. Res. Rev. 2015;35:678–697. doi: 10.1002/med.21339. - DOI - PubMed
    1. Wang Y.Q., Wang P.Y., Wang Y.T., Yang G.F., Zhang A., Miao Z.H. An Update on Poly(ADP-ribose)polymerase-1 (PARP-1) Inhibitors: Opportunities and Challenges in Cancer Therapy. J. Med. Chem. 2016;59:9575–9598. doi: 10.1021/acs.jmedchem.6b00055. - DOI - PubMed
    1. Robinson D., Van Allen E.M., Wu Y.M., Schultz N., Lonigro R.J., Mosquera J.M., Montgomery B., Taplin M.E., Pritchard C.C., Attard G., et al. Integrative clinical genomics of advanced prostate cancer. Cell. 2015;161:1215–1228. doi: 10.1016/j.cell.2015.05.001. - DOI - PMC - PubMed
    1. de Bono J., Mateo J., Fizazi K., Saad F., Shore N., Sandhu S., Chi K.N., Sartor O., Agarwal N., Olmos D., et al. Olaparib for Metastatic Castration-Resistant Prostate Cancer. N. Engl. J. Med. 2020;382:2091–2102. doi: 10.1056/NEJMoa1911440. - DOI - PubMed
    1. Hussain M., Mateo J., Fizazi K., Saad F., Shore N., Sandhu S., Chi K.N., Sartor O., Agarwal N., Olmos D., et al. Survival with Olaparib in Metastatic Castration-Resistant Prostate Cancer. N. Engl. J. Med. 2020;383:2345–2357. doi: 10.1056/NEJMoa2022485. - DOI - PubMed

Grants and funding

A.-M.M.-M. and F.S. are researchers of the CRCHUM/ICM, which receives support from the Fonds de recherche du Québec—Santé (FRQS). S.G. is supported by a studentship from the Canderel Fund of the ICM. F.S. holds the Université de Montréal Raymond Garneau Chair for Prostate Cancer Research. This work was supported by the Raymond Garneau Chair in Prostate Cancer research and grants from the Canadian Urologic Oncology Group, Prostate Cancer Canada, ICM and the Cancer Research Society.