Published OnlineFirst September 19, 2018; DOI: 10.1158/1535-7163.MCT-18-0234 Small Molecule Therapeutics Molecular Cancer Therapeutics Androgen Receptor Inhibitor Enhances the Antitumor Effect of PARP Inhibitor in Breast Cancer Cells by Modulating DNA Damage Response Ahrum Min1,2, Hyemin Jang1, Seongyeong Kim1, Kyung-Hun Lee1,2,3,4, Debora Keunyoung Kim5, Koung Jin Suh1,4,6, Yaewon Yang1,4,7, Paul Elvin8, Mark J. O'Connor9, and Seock-Ah Im1,2,3,4 Abstract The androgen receptor (AR) is expressed in 60%–70% of MDA-MB-468 cells by AZD3514 occurred in parallel breast cancers regardless of estrogen receptor status, and has with the suppression of ATM–chk2 axis activation, and the been proposed as a therapeutic target in breast cancers that suppression of NKX3.1 by AZD3514 was found to result retain AR. In this study, the authors aimed to investigate a from AZD3514-induced TOPORS upregulation and a resul- new treatment strategy using a novel AR inhibitor AZD3514 tant increase in NKX3.1 degradation. The study shows in breast cancer. AZD3514 alone had a minimal antiproli- posttranslational regulation of NKX3.1 via TOPORS upre- ferative effect on most breast cancer cell lines irrespective of gulation by AZD3514-induced ATM inactivation–increased AR expression level, but it downregulated the expressions of olaparib sensitivity in AR-positive and TOPORS-expressing DNA damage response (DDR) molecules, including ATM breast cancer cells, and suggests the antitumor effect of and chk2, which resulted in the accumulation of damaged AZD3514/olaparib cotreatment is caused by compromised DNA in some breast cancer cells. Furthermore, AZD3514 DDR activity in breast cancer cell lines and in a xenograft enhanced cellular sensitivity to a PARP inhibitor olaparib model. These results provide a rationale for future clinical by blocking the DDR pathway in breast cancer cells. Fur- trials of olaparib/AR inhibitor combination treatment in thermore, the downregulation of NKX3.1 expression in breast cancer. Mol Cancer Ther; 17(12); 2507–18. Ó2018 AACR. Introduction dated. About 50%–80% of invasive breast cancers (regardless of ER status) express AR and recent studies indicate AR expression In patients with breast cancer, endocrine therapies targeting is positively associated with a good prognosis. In addition, estrogen and estrogen receptor (ER) signaling pathways are recent studies showed that AR-expressing triple-negative considered as crucial. However, over a quarter of patients with breast cancer (TNBC) is dependent on AR signaling; thus, breast cancer do not express ER and exhibit resistance to endo- targeting AR seems to improve outcomes in TNBC (1–7). crine therapy. Although sex steroid hormone receptors, such as, Despite the prevalence and clinical significance of AR expression ER and progesterone receptor, are critical for the development in breast cancer, preclinical evidence supporting the use of AR- and progression of breast cancer, the potential role of androgen targeting agents and potential biomarkers of response to receptor (AR) in breast cancer has not been thoroughly eluci- AR inhibitors in breast cancer are lacking (4). Although clinical trials using enzalutamide (NCT01889238, NCT02091960, and NCT02929576), bicalutamide (NCT00468715 and 1 2 Cancer Research Institute, Seoul National University, Seoul, Korea. Biomedical NCT02605486), darolutamide (NCT03383679), enobosarm Research Institute, Seoul National University Hospital, Seoul, Korea. (NCT02971761), and other AR inhibitors (seviteronel, CR1447, 3Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea. 4Translational Medicine, Seoul National University College of and others) are ongoing, because of a lack of preclinical under- Medicine, Seoul, Korea. 5Rice University, Houston, Texas. 6Department of standing, AR antagonists are not currently used in standard Internal Medicine, Seoul National University Bundang Hospital, Seoul, Korea. clinical practice. Nonetheless, more research is required before 7Department of Internal Medicine, Chungbuk University Hospital, Cheong-Ju, AR modulation–based strategies are accepted clinically for the 8 Korea. Oncology IMED, AstraZeneca UK Ltd., Cambridge, United Kingdom. treatmentofbreastcancer. 9 Bioscience, Oncology, IMED Biotech Unit, AstraZeneca UK Ltd., Cambridge, Some breast cancers are associated with homologous recom- United Kingdom. bination deficiency (HRD), which has been shown to be Note: Supplementary data for this article are available at Molecular Cancer particularly sensitive to DNA-damaging agents and PARP inhi- Therapeutics Online (http://mct.aacrjournals.org/). bitors (8–10). Although PARP inhibitors have produced prom- Corresponding Author: Seock-Ah Im, Seoul National University, 101 Daehak-ro, ising results in patients with breast cancer with compromised Jongno-gu, Seoul 03080, Republic of Korea (South). Phone: 822-2072-0850; HR repair (HRR) activities (11–14), only BRCA deficiencies or Fax: 822-762-9662; E-mail: [email protected] BRCAness are considered viable PARP inhibitor targets. In fact, doi: 10.1158/1535-7163.MCT-18-0234 olaparib is the first PARP inhibitor with confirmed efficacy for Ó2018 American Association for Cancer Research. the treatment of breast cancer in patients with a germline BRCA www.aacrjournals.org 2507 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst September 19, 2018; DOI: 10.1158/1535-7163.MCT-18-0234 Min et al. mutation as indicated by the results of a phase III randomized Cell growth inhibition assay trial, the OlympiAD study (15). The data from the OlympiAD The cell viabilities at the early time point (120 hours) were trial resulted in olaparib being approved by the FDA as a single determined using an MTT assay as described previously (12). Cells agentfortreatmentofmetastaticbreastcancerwithBRCA1/2 were exposed to AZD3514 or olaparib alone or in combination at germline mutation. However, only 5%–10% of the patient various concentrations for 5 days. Combination index (CI) was population with breast cancer is thought to be BRCAness, and calculated using CalcuSyn software (Biosoft). Drug synergism was no other HRD marker has been demonstrated to predict sen- defined as a CI value at ED75 of <1 as determined by the Chou– sitivity to PARP inhibitors (16). Therefore, a strategy for extend- Talalay method (21). The long-term efficacies of AZD3514, ola- ing the usage of PARP inhibitors in breast cancer is required to parib, or AZD3514/olaparib were assessed using a colony forma- meet the unmet needs of patients. tion assay (CFA). Cells were treated with specific concentrations of Recent studies have demonstrated that AR signaling is a key each drug, and then cultured until colonies formed (14 days). regulator of DNA damage response (DDR) in prostate cancer, in Colonies were counted using a GELCOUN Tumor Colony Count- which AR is the main therapeutic target. AR modulates the er (Oxford Optronix Ltd.). transcriptions of a network of DDR genes in prostate cancer (17, 18). Polkinghorn and colleagues showed androgen depletion Western blot analysis causes the downregulation of DDR genes and impaired DNA Total and phosphorylated (p) protein expression levels after 5 repair, and that AR activation promoted resistance to radiation via days of treatment and protein expression levels were determined rapid repair of IR-induced DNA damage (17). Another group by Western blotting as described previously (22). The following also reported a link between AR and the DNA repair circuit in primary antibodies were purchased from Cell Signaling Technol- response to a genotoxic insult (18). These reports suggest AR ogy; antibodies against HER2, caspase 3, ATR, PR, AKT, p-AKT, is involved in DDR activity via the regulation of DDR compo- ERK, p-ERK, p-chk1, p-chk2, p-ATM, and p-ATR. Antibodies nents. We supposed AR inhibition impedes DDR activity and against AR, ATM, chk1, chk2, RAD51, TOPORS, ERa, cyclin B1, increases cellular sensitivity to PARP inhibitors, which selectively and cdc2 were obtained from Santa Cruz Biotechnology. Anti- target cancer cells with defective DDR. Thus, AR inhibition con- ERCC1 antibody was obtained from Thermo Fisher Scientific, tributes to the cells that may create HRD phenotype, resulting in anti-NKX3.1 antibody from Abcam, anti-p-histone H2A.X anti- sensitive to PARP inhibitor. Supporting our hypothesis, Li and body (clone JBW301) from Millipore, and anti-PARP antibody colleagues demonstrated that combination of enzalutamide and from BD Biosciences. Anti-a-tubulin and anti-actin antibodies olaparib downregulated expression of DDR genes resulting in (Sigma Aldrich) were used as controls. þ reduced HR efficiency in AR prostate cancer cells (19). It provides new insights into the possibility of an expanded BRCAness Cell-cycle analysis concept in clinical application of olaparib, but it is unclear how The DNA contents of cells (1 Â 104 cells) treated with AZD3514 AR inhibition influences on DDR efficiency in breast cancer. and/or olaparib were determined using a FACSCalibur flow In this study, we evaluated the effect of AZD3514, a selective cytometer (BD Biosciences) after propidium iodide staining. androgen receptor downregulator, as a monotherapy and in combination with olaparib in breast cancer cells, in the hope siRNA transfection that the information gained would help extend usage of PARP and siRNA specific for TOPORS and nonspecificcontrol AR inhibitors in breast cancer treatment. In addition, we