Androgen Receptor Inhibitor Enhances the Antitumor Effect Of

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. 2018 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 2018 American Association for Cancer Research. the treatment of breast cancer in patients with a germline BRCA

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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 (1 104 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 explored siRNA were purchased from Genolutions. Lipofectamine the mechanisms responsible for the effects of AZD3514/olaparib 2000 (Invitrogen) was used for the transfection according treatment, especially with respect to the link between AR inhibi- to the manufacturer's instructions. The sequence of the AR- tion and DDR activity in breast cancer cells. Finally, we attempted specificsiRNAwas50-AAGAAGGCCAGUUGUAUGGAC-30,the to identify a marker of sensitivity to AZD3514/olaparib treatment. sequence of the TOPORS-specificsiRNAwas50- CAAGGAGC- This is the first study to demonstrate the underlying mechanisms CUGUCUAGUAAUU-30, and the sequence of the nonspecific of AR inhibition, especially on DDR capacity. In addition, we control siRNA was 50-AATTCTCCGAACGTGTCACG-30. suggest that AR and PARP inhibitor combination therapy could be þ effective by inducing the HRD phenotype in patients with AR RT-PCR and real-time PCR breast cancer. Total RNA was isolated using TRI reagent (Molecular Research Center) as described previously (12). cDNA was Materials and Methods synthesized by RT-PCR using ImProm-II reverse transcriptase (Promega). qRT-PCR was performed using an iCycler iQ Detec- Reagents tion System (Bio-Rad Laboratories, Inc.) and SYBR Green. The AZD3514 and olaparib were kindly provided by AstraZeneca. mRNA expression levels of ATM were normalized relative to actin cDNA and fold changes in expression were calculated Cell lines versus controls. The sequences of the ATM-specificprimerwere Six human breast cancer cell lines (MDA-MB-157, -231, BT- 50-AGCACAGAAGTGCCTCCAAT-30 (forward) and 50-GCCAA- 549, HCC70, HCC1143, and Hs578T) authenticated by short TACTGGACTGGTGCT-30 (reverse). tandem repeat analysis, were purchased from the ATCC. Another 6 breast cancer cells (MDA-MB-453, -468, BT-474, MCF7, T47D, Comet assays and SKBr3) verified by DNA fingerprinting analysis, were pur- Degree of DNA damage was assessed using an alkaline comet chased from the Korean Cell Line Bank (Seoul, Korea). All cell assay using the Trevigen Comet Assay Kit (Trevigen). Tail lines were banked, cultured as described previously (20), and moments were measured using the Comet Assay IV Program passaged for less than 6 months before use. (Andor Technology).

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AZD3514 Enhances the Antitumor Effects of Olaparib in Breast Cancer

Immunofluorescence assay In vivo study RAD51 and gH2AX foci formation were evaluated following In vivo experimentation was conducted in the animal facility of AZD3514 and/or olaparib treatment as described previously (12). Seoul National University (Seoul, South Korea) in accord with Foci were visualized using a Nikon A1 confocal laser scanning institutional guidelines after obtaining prior approval from the microscope (Nikon). At least 100 cells were counted and the Institutional Animal Care and Use Committee (IACUC) commit- percentages of cells with more than five RAD51 or gH2AX foci tee (SNU-140422-13). Female Balb/c athymic nude mice 6 weeks were calculated. old were tested to measure the in vivo activities of AZD3514 and/or olaparib. MDA-MB-468 cells (1 107) were subcutaneously IHC and TUNEL assay transplanted into each mouse, and drugs were administered when IHC was performed using anti-rabbit antibodies against Ki-67 tumor volumes reached 200 mm3. AZD3514 (50 mg/kg) and/or (GeneTex) and NKX3.1 (Abcam) at a dilution of 1:100. The olaparib (30 mg/kg) were administered via oral gavage once daily terminal deoxynucleotidyl transferase–mediated dUTP nick end for 28 consecutive days. Tumor volumes was calculated using 2 fi labeling (TUNEL) assay was conducted using the ApopTag In situ [(width) (height)]/2, and mice were sacri ced with CO2 at the Apoptosis Detection Kit (Chemicon International) as described end of the observation period. Tumors were excised and preserved previously (12). for further analysis as described previously (20).

A Control DHT 1 mmol/L of AZD3514 1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2 Figure 1. Fold change in ATM mRNA expression mRNA ATM Fold change in AR inhibition downregulates DNA 0.0 repair molecules. A, The expressions HCC1143 MDA-MB-453 MDA-MB-468 MCF7 of ATM mRNA in breast cancer cells were analyzed by qRT-PCR after DHT or AZD3514 treatment. ATM B expression levels were normalized HCC1143 MDA-MB-453 MDA-MB-468 MCF7 versus actin and renormalized by value at untreated controls, and are p-ATM presented in the bar graph with SEs (n ¼ 3). B, DDR protein expressions ATM were assessed by Western blotting after treating cells with increasing concentrations of AZD3514 for 5 days. p-ATR

ATR

p-chk1

chk1

p-chk2

chk2

Actin

AZD3514 C CCC 0.1 1 5 (µmol/L) 0.1 1 5 (µmol/L) 0.1 1 5 (µmol/L) 0.1 1 5 (µmol/L)

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75 Statistical analysis All experiments were repeated at least three times. Results are presented as mean SEs. The analysis was conducted using 3.14 1.46 2.25 2.78 5.75 0.63 0.39 0.24 0.49 SigmaPlot version 9.0, and the two-sided Student t test was used for group comparisons. Statistical signiﬁcance was accept- Combination index (CI value at ED ed for P < 0.01. 50 SD) Results AZD3514 suppressed DDR activation in breast cancer cells Because AR is a potential therapeutic target in breast cancer, we 0.134 0.007 0.017 0.155

observed AR protein expression levels in 12 breast cancer cell lines 0.01 0.05

mol/L, mean (Supplementary Fig. S1). All cell lines including hormone recep- 10 10 10 m > 2 3.1 8.4 3.14 > > 3.64 9.822 1:1 Combination IC ( tor–positive, HER2-positive and TNBC cell lines expressed AR. Although AZD3514 at concentrations of <10 mmol/L reduced cell survival by 50% in some breast cancer cell lines, it had mild SD)

antiproliferative effects on most of the breast cancer cell lines, irrespective of AR expression level (Supplementary Fig. S2). 50 Antiproliferative effect through AR inhibition by AZD3514 0.009

0.02 was not prominent in breast cancer cells as monotherapy. How-

mol/L, mean ever, AZD3514 downregulated the expression of DDR molecules 10 10 10 10 10 10 10 m > > 4.52 > 5.8 > > > Olaparib IC > ( (p-ATM, p-chk1, and p-chk2) in a number of breast cancer cell lines. ATM mRNA expression levels were signiﬁcantly suppressed

SD) in MDA-MB-468 and MCF7 cells by AZD3514, but no change was

observed in MDA-MB-453 or HCC1143 cells (Fig. 1A). Concor- – ﬁ 50 dantly, activation of the ATM chk2 axis was signi cantly suppressed in MDA-MB-468 and MCF7 cells but not in MDA-MB-453 0.54 0.101 0.1 0.024 and HCC1143 by AZD3514 (Fig. 1B). These results support the mol/L, mean 10 10 10 10 10

m roles of AR in DDR activation in breast cancer cells. > > 7.3 > > 8.08 > 8.25 AZD3514 IC (

AZD3514 enhanced the antitumor effect of olaparib in four of the nine breast cancer cell lines þ þ þ þþ þþ þþ Our data revealed that AR inhibition suppressed the expression þþþ and activation of ATM–chk2 axis, and AR signaling has been Weakly positive Weakly positive 7.72 reported to promote DDR by upregulating DDR gene expression (17). Accordingly, we hypothesized that AR inhibition by AZD3514 might enhance the antitumor effect of PARP inhibition by compromising DNA repair activity. To determine whether AR

ed ed inhibition increases the antitumor effect of PARP inhibitor in fi fi breast cancer cells, the growth-inhibitory effects of AZD3514 Normal Normal Normal Normal Normal Ampli Ampli Normal Normal with/without olaparib was evaluated using an MTT assay. Breast cancer cell lines exhibited various levels of response to AZD3514/ olaparib treatment irrespective of subtype or AR expression level (Table 1). On the basis of the results obtained, MDA-MB-453 and MDA-MB-468 cells were chosen for further experiments; the antiproliferative effect of AZD3514/olaparib was confirmed using a CFA. AZD3514/olaparib treatment suppressed the proliferation of MDA-MB-468 but not that of MDA-MB-453 cells (Fig. 2A). MCF7 cells showed AZD3514 and olaparib acted synergistically, þþ þþ þ while HCC1143 cells showed they acted in an antagonistic manner (Supplementary Fig. S3A). The effects of AZD3514/olaparib treatment on cell-cycle progression were determined by a cell-cycle analysis. The results obtained showed that AZD3514/ –

þ þ olaparib synergistically induced G2 M cell-cycle arrest and apoptosis in MDA-MB-468 and MCF7 cells (Fig. 2B; Supplementary Luminal Luminal Fig. S3B). However, MDA-MB-453 and HCC1143 cells with CI values of >1 were unaffected by AZD3514/olaparib. Consistent with cell-cycle data, combination treatment induced the expres- Combination effect of AZD3514 and olaparib in breast cancer cell lines sions of G2–M cell-cycle molecules, including cyclin B1 and cdc2, in only MDA-MB-468 cells. Increases in PARP and caspase-3 Hs578T TNBC BT-549 TNBC MDA-MB-231 TNBC MDA-MB-468 TNBC HCC1143 TNBC MDA-MB-453 HER2 BT-474 HER2 T47D Table 1. Cell line Subtype ER Expression PR Expression HER2 Expression AR Expression MCF7 cleavage were also clearly detected in MDA-MB-468 cells treated

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AZD3514 Enhances the Antitumor Effects of Olaparib in Breast Cancer

A MDA-MB-453 MDA-MB-468 2.5 mmol/L of AZD3514 −++− 120 120 0.1 mmol/L of Olaparib −+− + 100 100

80 80

60 60 MDA-MB-453

40 40

Cell viability (%) 20 20 Cell viability (%)

0 0 MDA-MB-468 Control Control 2.5 mmol/L of AZD3514 2.5 mmol/L of AZD3514 0.1 mmol/L of Olaparib 0.1 mmol/L of Olaparib 2.5 mmol/L of AZD3514 + 0.1 mmol/L of Olaparib 2.5 mmol/L of AZD3514 + 0.1 mmol/L of Olaparib

B MDA-MB-453 MDA-MB-468 m DMSO 1 mol/L of AZD3514 DMSO 1 mmol/L of AZD3514 1 mmol/L of Olaparib 1 mmol/L of AZD3514 + 1 mmol/L of Olaparib 1 mmol/L of AZD3514 + 80 1 mmol/L of Olaparib 80 1 mmol/L of Olaparib

60 60

40 40

20 20 % Cell-cycle phases % Cell-cycle phases

0 0 Sub-G1 G1 G2–M Sub-G1 G1 G2–M

Sub-G1 phase (%) 0.9 2.8 3.3 2.3 Sub-G1 phase (%) 1.7 2.9 4.2 9.1

G1 phase (%) 72.3 62.4 63.7 68.8 G1 phase (%) 59.0 62.0 48.0 43.4

G2–M phase (%) 11.2 17.7 17.0 12.2 G2–M phase (%) 13.7 19.4 20.9 30.2

C MDA-MB-453 MDA-MB-468

AZD3514 (mmol/L) 0 1 0 1 0 1 0 1 Olaparib (mmol/L) 0 0 1 1 0 0 1 1

PARP

Cyclin B1

cdc2

Caspase-3

Actin

Figure 2.

AZD3514 plus olaparib induces G2–M cell-cycle arrest and cell death in MDA-MB-468 cells. A, The growth-inhibitory effects of AZD3514/olaparib were evaluated using a CFA. The cells were incubated with the indicated concentrations of AZD3514 and/or olaparib for 14 days, and percentages of surviving cells were calculated. Results are presented with SEs (n ¼ 3; , P < 0.001). B, Cells were exposed to AZD3514 and/or olaparib at the indicated doses for 5 days, and percentages of cells in the G1, G2–M phase, and percentages of apoptotic cells were calculated. Results are presented with SEs (n ¼ 3; , P < 0.005). C, The expression levels of cell-cycle–related proteins, PARP, and caspase-3 cleavages were determined by Western blotting.

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with AZD3514/olaparib (Fig. 2C). These results show that com- NKX3.1 regulation is a direct effect of AR-directed therapy. To bination treatment increased the antitumor effect by inducing determine whether TOPORS silencing adversely affects the activ- G2–M cell-cycle arrest and the apoptosis of MDA-MB-468 cells. ity of AZD3514/olaparib by modulating ATM activation, the viabilities of TOPORS siRNA knocked down MDA-MB-468 cells AZD3514/olaparib impaired DNA damage repair capacity. AR treated with AZD3514 with/without olaparib were measured suppression by AZD3514 significantly downregulated DDR gene using the CFA. TOPORS depletion increased resistance to expression, which could explain the sensitivity of breast cancer AZD3514/olaparib in MDA-MB-468 cells (Fig. 4F). Furthermore, cells to PARP inhibitors (Fig. 1). In MDA-MB-468 cells, AZD3514/ similar results were observed for MCF7 cells (Supplementary Fig. olaparib treatment decreased ATM–chk2 axis activity, the expres- S4). Our data indicate that posttranslational regulation of NKX3.1 sions of DDR proteins (e.g., RAD51 and ERCC1; Fig. 3A), led to a via the modulation of TOPORS expression by AZD3514 induced dramatic reduction in RAD51 foci formation, and increased the ATM inactivation, and that this increased sensitivity to olaparib in number of gH2AX foci (Fig. 3B). Furthermore, AZD3514/olaparib AR-positive, TOPORS-expressing breast cancer cells. treatment led to an accumulation of DNA damage in these cells (Fig. 3C). The same experiments were also performed in MCF7 Coadministration of AZD3514 and olaparib significantly impeded and HCC1143 cells. In MCF7 cells, AZD3514/olaparib sup- tumor growth in an in vivo mouse xenograft model. Treatment with pressed DDR proteins and increased DNA damage levels, whereas AZD3514 plus olaparib significantly reduced tumor growth dur- this was not observed in HCC1143 cells (Supplementary Fig. S3C ing and after treatment (Fig. 5A). As shown in Fig. 5B, cotreatment and S3D). These results indicate that the mechanism responsible with AZD3514 and olaparib was well tolerated. Consistent with for the enhancement of cellular sensitivity to olaparib by our in vitro findings, tumor tissues from mice treated with AZD3514 involves abrogation of the HRR pathway in some breast AZD3514/olaparib showed lower Ki-67 expression that those of cancer cells. mice treated with vehicle, AZD3514, or olaparib, and a TUNEL assay showed increased apoptosis (Fig. 5C). The protein levels of AZD3514 inhibited ATM activation by downregulating NKX3.1 NKX3.1 in tumor tissues from mice treated with AZD3514/ola- expression negatively associated with TOPORS expression. To deter- parib were dramatically lower than in other treatment groups, mine how AR inhibition regulates ATM activation in MDA-MB- whereas TOPORS expression levels were significantly increased in 468 cells, we focused on molecules directly targeted by AR that are tissues from AZD3514 alone or in combination with olaparib known to regulate DDR activation. Bowen and colleagues dem- (Fig. 5C). Furthermore, levels of proteins related to proliferation onstrated NKX3.1 enhances DDR by increasing ATM activation in (e.g., AKT and ERK) were reduced by AZD3514/olaparib treat- – prostate cancer cells (23 26). Therefore, we hypothesized that ment and PARP and caspase-3 cleavage were elevated (Fig. 5D). In NKX3.1 expression may affect the antiproliferative effects of addition, AZD3514/olaparib treatment suppressed the expres- AZD3514/olaparib in MDA-MB-468 cells by inactivating DDR sion of DNA damage repair proteins associated with decreased € induced by ATM. Interestingly, although both treatment-na ve levels of NKX3.1 induced by TOPORS induction in tumor tissues, MDA-MB-453 and MDA-MB-468 cells expressed NKX3.1, which resulted in increased gH2AX levels. These findings dem- AZD3514 downregulated NKX3.1 expression in only MDA-MB- onstrate that the antitumor effect of AZD3514/olaparib involves 468 cells (Fig. 4A). Furthermore, this downregulation of NKX3.1 compromising DDR in breast cancer cell lines and in our xenograft – expression cooccurred with suppressed activation of the ATM model. chk2 axis. Because NKX3.1 expression was downregulated in MDA-MB-468 cells only after AR inhibition, we focused on the posttranslational modification of NKX3.1, which can be regulated Discussion by TOPORS, an E3 ubiquitin ligase (27). To determine whether In this study, we aimed to investigate a new treatment strategy different levels of NKX3.1 suppression after AZD3514 treatment using a novel AR inhibitor AZD3514 in breast cancer. Our in vitro were the result of TOPORS induction by AZD3514 and a subse- and in vivo results from this study aid understanding of the impact quent increase in NKX3.1 degradation, we measured TOPORS of AR signaling on DDR in breast cancer. This article is the first to levels by Western blotting. As was expected, TOPORS levels show that a link exists between AR and DDR activity in breast differed in MDA-MB-453 and MDA-MB-468 cells, and AZD3514 cancer. Furthermore, it suggests combinational AR/PARP inhib- increased TOPORS expression only in MDA-MB-468 cells, which itor strategies be considered in breast cancer. We believe this expressed TOPORS in the basal state (Fig. 4B). To determine preclinical research study provides fundamental data for future whether the downregulation of NKX3.1 by TOPORS occurs clinical trials of AR inhibition. mechanically via ubiquitination, MDA-MB-468 cells were In phase II single-arm trial (NCT01889238) of an AR inhibitor cotreated with MG132 (a proteasomal inhibitor) and AZD3514. enzalutamide showed clinical benefit in 47% of patients with An immunoprecipitation assay was then conducted to confirm TNBC with AR expression and an androgen-related gene signature whether NKX3.1 ubiquitination influenced ATM activity. The (28, 29). On the basis of the promising results of phase II trial and results showed that MG132 cotreatment inhibited AZD3514- preclinical data, phase III trial of enzalutamide in combination induced NKX3.1 degradation and led to maintenance of ATM with paclitaxel or as monotherapy in TNBC (NCT02929576) is activation (Fig. 4C). In addition, TOPORS knockdown stabilized ongoing, and these studies suggested that enzalutamide might be NKX3.1 expression levels and maintained ATM–chk2 axis activa- a novel therapeutic strategy for TNBC. However, the identification tion in MDA-MB-468 cells treated with AZD3514 (Fig. 4D). These of androgen-related gene expression profiles is a complex and modulations of DDR activities by AZD3514-induced TOPORS costly process (3, 30), and AR-related subtyping of breast cancer is upregulation and resultant increases in NKX3.1 were also not clearly classified from other subtypes of breast cancer, thus observed when AR expression was depleted by AR-specific siRNA targeting AR to treat breast cancer presents many hurdle in treatment (Fig. 4E). Thus, DDR modulation through TOPORS– standard clinical practice. According to our results, targeting AR

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Figure 3. AZD3514 enhances olaparib-induced DNA damage accumulation by compromising DNA damage repair response. A, Cells were treated with AZD3514 and/or olaparib for 5 days, and Western blotting was conducted to assess the expression levels of DDR proteins. B, An immunoﬂuorescence assay was conducted to evaluate

RAD51 and gH2AX foci formation after AZD3514 and/or olaparib treatment. At least 100 nuclei were assessed per experiment. The percentages of cells containing more than 5 RAD51 and gH2AX foci in three experiments are presented as a bar graph with SEs, , P < 0.005; , P < 0.001. C, Degree of DNA damage accumulation was assessed using an alkaline comet assay. Cells were treated with AZD3514 and/or olaparib for 5 days, and degrees of DNA damage accumulation were analyzed using the alkaline comet assay. Mean tail moments were calculated for three independent experiments and are represented as a bar graph. Column entries are the means of three independent experiments; bars represent SEs (, P < 0.005). www.aacrjournals.org Mol Cancer Ther; 17(12) December 2018 2513

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Figure 4. TOPORS expression increases the combination effects of AZD3514 and olaparib by inducing ATM inactivation via the posttranslational suppression of NKX3.1. A, Cells were incubated with different doses of AZD3514 for 5 days, and the levels of NKX3.1, ATM, and chk2 proteins were analyzed by Western blotting. B, TOPORS expression was assessed by Western blotting following AZD3514 treatment for 5 days. C, Posttranslation modification of NKX3.1 protein by ubiquitination in MDA-MB-468 cells was analyzed by treating 10 mmol/L of MG132 with/without AZD3514 for 5 days. D, MDA-MB-468 cells were transfected with nonspecific control or TOPORS-specific siRNA, and exposed to AZD3514 for 5 days. Protein levels changed in a TOPORS protein level–dependent manner after AZD3514 treatment. E, Cells were transfected with AR-specific or nonspecific siRNA and the levels of TOPORS, NKX3.1, and DDR proteins were then examined by Western blotting. F, TOPORS silencing using TOPORS-specific siRNA decreased cellular sensitivity to AZD3514/olaparib inhibition. CFA was conducted using cells transfected with siRNA targeting TOPORS or nonspecific control siRNA for 3 days and then treated with AZD3514 and/or olaparib for 14 days. Cell viability percentages were calculated and are presented in a bar graph with SEs (n ¼ 3; , P < 0.001).

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AZD3514 Enhances the Antitumor Effects of Olaparib in Breast Cancer

is not effective enough to suppress cell viability even in cells made using AR inhibitors in combination with other targeted expressing AR, and thus, new therapeutic strategies are required to therapeutic agents, such as, aromatase inhibitor and NYP-BEZ235 extend the use of AR inhibitors in AR-expressing patients with (a PI3K and mTOR dual inhibitor; refs. 31–33). However, these breast cancer. To fulﬁll this unmet need, some attempts have been approaches are also limited in context- and subtype-speciﬁc cases.

Figure 5. AZD3514/olaparib treatment exerts antitumor effects in an MDA-MB-468 xenograft model. A, An MDA-MB-468 xenograft mouse model was established and mice were treated with 50 mg/kg AZD3514 (n ¼ 8), 30 mg/kg olaparib (n ¼ 8), or 50 mg/kg AZD3514 plus 30 mg/kg olaparib (n ¼ 8), or vehicle only (n ¼ 8) daily for 28 days. Tumor volumes were assessed every other day and are presented in the graph with SEs. B, Mouse body weights were measured to assess treatment toxicities. C, IHC staining for Ki-67, hematoxylin and eosin staining (H&E), and TUNEL assays were performed. The expression levels of NKX3.1 and TOPORS were examined in tumor tissues. Representative images are presented (400 original magniﬁcation). Scale bars represent 50 mm. D, The expression levels of proteins associated with proliferation, apoptosis, and DNA damage response were evaluated by Western blotting.

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Min et al.

Figure 6. Proposed model for the action mechanism of AZD3514 on DDR modulation.

AZD3514 inhibits AR signaling through androgen-depen- it has been well established that loss of NKX3.1 is associated with dent and -independent mechanisms (34–36). It was developed prostate carcinoma progression (38–40). Some previous studies by AstraZeneca as a second-generation anti-androgen, and demonstrated that NKX3.1 may also play a broader role in cellular some reports have described the action mechanisms of response to DNA damage (24–26, 40). Furthermore, depletion of AZD3514 in castration-resistant prostate cancer (CRPC) cells. NKX3.1 in prostate cancer cells was found to be associated with a Enzalutamide (Medivation) inhibits AR nuclear translocation, significant decrease in p-ATM levels (25), and Bowen and collea- DNA binding, and coactivator recruitment by binding with gues found NKX3.1 binds to ATM and accelerates ATM activation high affinity to the ligand-binding domain of AR, whereas (23). In this study, protein levels of NKX3.1 were suppressed by AZD3514 inhibits AR nuclear translocation and transcriptional AZD3514 treatment in sensitive MDA-MB-468 and MCF7 cells, activity, and downregulates AR levels (35). In a phase I clinical and these suppressions corresponded to significant decreases in trial on patients with CRPC, AZD3514 reduced PSA activity in the levels of p-ATM and p-chk2. These observations indicate AR 17%–25% of patients, although its development was later inhibition modulates DDR activity in an ATM–chk2 activation discontinued due to tolerability issues (35, 37). However, the dependent manner by downregulating NKX3.1 protein, a direct modulation of DDR activities by AZD3514-induced AR inhi- target of AR signaling. bition assessed in this study was also observed after AR deple- Unlike prostate cancer, less than 1% of patients with breast tion using siRNA (Fig. 4E). Thus, the DDR-modulating effect of cancer have a genetic alteration in NKX3.1. In fact, all nine breast AZD3514 is not additional effect, but rather a cellular mech- cancer cell lines using in this study expressed NKX3.1 protein, and anism of AR. Therefore, dual targeting of AR and PARP in breast no genetic alteration was found in NKX3.1 (Supplementary Fig. cancer based on downregulation of DDR offers a promising S5). Thus, the genetic and protein expressional statuses of NKX3.1 treatment strategy for patients with breast cancer. do not appear to determine the combinatorial effects of AR plus NKX3.1 is a haploinsufficient androgen-regulated tumor sup- PARP inhibitor via the modulation of ATM–chk2 dependent DDR pressor gene that is downregulated in prostate carcinoma. The activity. Interestingly, we found NKX3.1 protein levels were down- Cancer Genome Atlas reported 64 of 333 patients with prostate regulated by AZD3514 in MDA-MB-468 and MCF7 cells, which cancer (19%) showed a genetic alteration in the NKX3.1 gene, and exhibited synergistic sensitivity to AZD3514/olaparib, whereas

2516 Mol Cancer Ther; 17(12) December 2018 Molecular Cancer Therapeutics

AZD3514 Enhances the Antitumor Effects of Olaparib in Breast Cancer

NKX3.1 protein levels were not downregulated by AZD3514 in reduced levels of NKX3.1 led to the loss of DDR activity, and MDA-MB-453 and HCC1143. To identify the different mechan- finally increased sensitivity to PARP inhibitor. isms responsible for downregulating NKX3.1 protein expression This study shows how AR inhibition effects DDR activity in after AR inhibition, we assessed the possibility of the posttrans- breast cancer, in addition its findings suggests TOPORS baseline lational modification–based regulation of NKX3.1. TOPORS, a expression might be useful for predicting response to the com- robust E3 ubiquitin ligase is known to ubiquitinate NKX3.1 in bined inhibitions of AR and PARP in breast cancer. Hopefully, this prostate cancer (25, 27, 41). Guan and colleagues demonstrat- new strategy will result in the use of combined AR and PARP ed overexpression of TOPORS leads to NKX3.1 ubiquitination, inhibitors for the treatment of breast cancer, and encourage others and that knockdown of TOPORS leads to a higher steady-state to undertake clinical trials to explore this potential strategy. level of NKX3.1 and extends its half-life (27). We found NKX3.1 protein levels were maintained by inhibiting protea- Disclosure of Potential Conflicts of Interest somal degradation even in the presence of AZD3514, which S.-A. Im reports receiving a commercial research support from suggests NKX3.1 protein was mediated by posttranslational Research Fund from AstraZeneca and is also a consultant/advisory modification induced by AR inhibition. Furthermore, NKX3.1 board member for AstraZeneca, Roche, Hanmi, Novartis, Pfizer, protein and TOPORS protein levels were found to be inversely and Spectrum. No potential conflicts of interest were disclosed by related. These observations suggest that TOPORS functioning as the other authors. a negative regulator of NKX3.1 may be involved in regulation of DDR activity by controlling NKX3.1 protein expression (Fig. 6). Authors' Contributions In fact, MDA-MB-453 and HCC1143 cells exhibited an antagonistic effect between AZD3514 and olaparib have no protein Conception and design: A. Min, K.-H. Lee, M.J. O'Connor, S.-A. Im expression of TOPORS, which can sustain DDR activation via Development of methodology: A. Min Acquisition of data (provided animals, acquired and managed patients, retaining NXK3.1 protein expression. Subsequently, TOPORS provided facilities, etc.): A. Min deficiency contributes to decreased sensitivity to PARP inhib- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, itor, olaparib. These findings indicate TOPORS is a key mol- computational analysis): A. Min, K.-H. Lee, Y. Yang, M.J. O'Connor, S.-A. Im ecule in terms of the synergism shown by combinatorial AR and Writing, review, and/or revision of the manuscript: A. Min, K.-H. Lee, PARP inhibitor treatment, and suggest that TOPORS might D.K. Kim, K.J. Suh, Y. Yang, S.-A. Im serve as a predictive marker to select patients likely to benefit Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Min, H. Jang, S. Kim, K.-H. Lee, D.K. Kim from an AR/PARP inhibitor treatment strategy. Study supervision: S.-A. Im In this study, we evaluated the antitumor activities of AZD3514 in breast cancer cell lines and in a xenograft model. Acknowledgments AR inhibition by AZD3514 alone did not effectively suppress This research was supported by Basic Science Research Program through the cell proliferation, but increased levels of DNA damage accu- National Research Foundation of Korea (NRF) funded by the Ministry of mulation due to the inactivation of DDR molecules, especially Science, ICT and future Planning (2015R1A2A2A01004655) and also sup- of ATM and chk2. AZD3514 treatment led to HRD in cells and ported by Doosan Yonkang Foundation (30-2013-0140; to S.A. Im). This enhanced sensitivity to PARP inhibitor. Interestingly, AR- research was partly supported by the SNUH Research Fund (03-2016-0030; to expressing breast cancer cells exhibited different responses to S.A. Im). AZD3514/olaparib that depended on the downregulation of The costs of publication of this article were defrayed in part by the payment of NKX3.1 protein expression by AR inhibition. In TOPORS- page charges. This article must therefore be hereby marked advertisement in expressing breast cancer cells, NKX3.1 protein levels were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. suppressed by TOPORS expression–induced AR inhibition. Thus, AR inhibition induced TOPORS expression, and led to Received March 2, 2018; revised July 24, 2018; accepted September 13, 2018; the proteosomal degradation of NKX3.1. Subsequently, published first September 19, 2018.

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2518 Mol Cancer Ther; 17(12) December 2018 Molecular Cancer Therapeutics

Androgen Receptor Inhibitor Enhances the Antitumor Effect of PARP Inhibitor in Breast Cancer Cells by Modulating DNA Damage Response

Ahrum Min, Hyemin Jang, Seongyeong Kim, et al.

Mol Cancer Ther 2018;17:2507-2518. Published OnlineFirst September 19, 2018.

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