Targeting DNA Damage Response in Prostate Cancer by Inhibiting Androgen Receptor-CDC6-ATR- Chk1 Signaling

SUMMARY
Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 expression is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown, together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia-mutated (ATM) phosphoryla- tion, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment with the AR signaling inhibitor enzalutamide (ENZ) and the Chk1/2 inhibitor AZD7762 demonstrates synergy with regard to inhibition of AR-CDC6-ATR- Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP- C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ- and AZD7762-induced apoptosis. Additive or synergistic therapeutic activ- ities are demonstrated in AR-positive animal xeno- graft models. These findings have important clinical implications, since they introduce a therapeutic strat- egy for AR-positive, metastatic, castration-resistant PCa, regardless of p53 status, through targeting AR- CDC6-ATR-Chk1 signaling.

INTRODUCTION
Metastatic prostate cancer remains an incurable disease with variable prognosis (Wu et al., 2014). After an initial period of response to systemic hormone therapy, the disease inexorably progresses to a state known as metastatic, castration-resistant prostate cancer (mCRPC) (Karanika et al., 2014). The therapeutic armamentarium for mCRPC is limited to chemotherapy andnovel inhibitors of androgen receptor (AR) signaling, such as abiraterone acetate and enzalutamide (ENZ), which provide only moderate survival benefits (Ryan et al., 2013; Scher et al., 2012).DNA damage response (DDR) refers to coordinated cellular mechanisms that prevent DNA damage accumulation and main- tain genomic integrity (Karanika et al., 2014), and it plays a cen- tral role in prostate cancer (PCa) initiation, development, and progression (Tapia-Laliena et al., 2014). AR signaling in PCa cells has been associated with numerous aspects of DDR pathways, including regulation of ATM-Chk2 signaling for the initiation of DDR (Ide et al., 2012), poly(ADP-ribose) polymerase function (Schiewer et al., 2012), and non-homologous end joining recom- bination (Polkinghorn et al., 2013). AR was reported to regulate TopBP1-ATR-Chk1 signaling (Li et al., 2014), whereas ENZ decreases CHEK1 expression in PCa cells (L.L., S.K., G.Y., J.W., S.P., B.B., G.C.M., J.H.S., G.E.G., T.K., P.G.C., P.T.,X.Z., T.C.T., unpublished data).One of the main types of DNA damage is DNA strand breakage, which activates a cascade of intracellular events that promote cell-cycle arrest and DDR, ensuring genomic integ- rity, which can be particularly critical for cell survival in patients with aggressive malignancies accumulating a myriad of genetic errors (Karanika et al., 2014).

DNA strand breaks activate ATR via upstream mediators such as TopBP1, leading to Chk1-medi- ated checkpoint activation and cell-cycle arrest. Cells are thereby able to repair DNA damage, alleviating replication stress and genomic instability. Chk1 pathway inhibition results in DNA damage accumulation and, thus, increased ATM auto-phos- phorylation, which mediates apoptosis of cells with incompletely replicated DNA (d’Adda di Fagagna, 2008; Sarmento et al., 2015). These findings demonstrate that ATR-Chk1 signaling is crucial for the prevention of DNA-damage-induced cell death associated with increased ATM phosphorylation/activation.CDC6 is an essential regulator of DNA replication in eukaryotic cells, and its best characterized function is pre-replicative com- plex assembly at origins of replication during G1 phase (Borlado and Me´ ndez, 2008). Furthermore, CDC6 overexpression during G2 phase blocks mitotic entry by activating Chk1 (Clay-Farraceet al., 2003; Yoshida et al., 2010), which inhibits G2/M progres- sion. CDC6 is required for Chk1 activation upon replication inhi- bition (Oehlmann et al., 2004), and human CDC6 interacts with ATR, promoting activation of the replication checkpoint (Yoshida et al., 2010). Consequently, CDC6 decreases genomic insta- bility, which is vital for cancer cell survival. CDC6 can also man- ifest oncogenic activities via regulation of DNA replication and repression of tumor suppressors (Gonzalez et al., 2006). Target- ing ATR-Chk1 signaling increases the sensitivity to treatment with DNA-damaging agents (Bartucci et al., 2012), making this approach particularly attractive for the development of cancer therapies.

Notably, CHEK1 knockdown increases the sensitivity of PCa stem cells to radiotherapy through increased DNA dam- age (Fokas et al., 2012). AZD7762, a Chk1/2 inhibitor, synergizes with DNA-damaging agents and radiation to induce apoptosis via DNA double-strand breaks mediated by ATM activation in many cell types (Mitchell et al., 2010; Sausville et al., 2014). Furthermore, Brooks et al. found that Chk1 inhibition can selec- tively induce apoptosis in melanoma cells in proportion to the level of endogenous DNA damage related to replicative stress without further induction of DNA damage by chemotherapy (Brooks et al., 2013).Taking the results of these studies into account, a potential approach to treatment of mCRPC is the inhibition of more than one level of a specific DDR signaling cascade, with the goal of completely abolishing a specific signaling pathway. This approach would take advantage of the fact that cancer cells eventually accumulate more DNA damage than normal cells do, eluding adverse effects of chemotherapy. Targeting the ATR-Chk1 pathway at multiple levels to inhibit the repair of DNA dam- age induced by replication stress in cancer cells may represent an effective strategy for more complete DDR pathway inhibition. Because CDC6 is an AR target gene (Jin and Fondell, 2009; Bai et al., 2005) and is also involved with ATR-Chk1 signaling, this is a particularly intriguing strategy for AR-positive PCa. In addition, this approach may be effective under conditions of wild-type p53, which is involved in multiple DDR pathways and can miti- gate the response to some DNA-damaging agents (Ma et al., 2012).The aim of the present study was to determine the role of CDC6 in regulation of ATR-Chk1 signaling and to test the combined in- hibition of AR and Chk1 signaling as a therapeutic approach for AR-positive mCRPC. Through this study, we also aimed to intro- duce a therapeutic approach that effectively targets a DDR pathway that promotes sufficient DNA damage accumulation in PCa cells to induce cell death, regardless of p53 status.

RESULTS
We first evaluated the CDC6 expression and phosphorylation and analyzed its correlation with AR expression in normal human prostates and in primary and metastatic prostate tumor specimens. In our immunohistochemistry (IHC) analysis, we found that AR expression (p = 0.0057) and CDC6 expression (p < 0.001) and phosphorylation (p = 0.0083) increased signifi- cantly during PCa development and progression (Figure 1A). Itwas reported that AR regulates CDC6 expression (Jin and Fon- dell, 2009; Bai et al., 2005) and protein stability (Bai et al., 2005), indicating functional associations between these two mol- ecules. To determine the mechanism of AR regulation of CDC6 in our experimental models, we performed qRT-PCR and protein stability analysis for CDC6 using AR siRNA (ARsi)- and nega- tive-control siRNA (NCsi)-transfected VCaP and LNCaP C4-2b (C4-2b) cells. Our results demonstrated that AR regulation of CDC6 occurs at the mRNA level and not through regulation of protein stability (Figures 1B–1D).AR or CDC6 Knockdown Increases the Sensitivity of PCa Cells to the Chk1/2 Inhibitor AZD7762 through Inhibition of TopBP1-ATR-Chk1 SignalingTargeting Chk1/2 with AZD7762 increases the efficacy of DNA- damaging modalities such as chemotherapy and radiation ther- apy in patients with multiple malignancies (Mitchell et al., 2010; Sausville et al., 2014). Thus, we tested our hypothesis that the combination treatment with AR or CDC6 downregulation and AZD7762 results in synergistic activities in VCaP and C4-2b. The combination of ARsi and AZD7762 reduced protein levels of CDC6, TopBP1, ATR, and Chk1, and it reduced phosphoryla- tion of ATR and Cdc25C in VCaP and C4-2b (Figures 2A and 2B).

These signaling effects were accompanied by synergistically increased ATM S1981 phosphorylation, a marker of DNA dam- age. Phosphorylation of Chk1 S317, the site phosphorylated by ATR or ATM (Canman, 2001; Kastan and Lim, 2000; Gatei et al., 2003), was markedly elevated by AZD7762. Interestingly, Chk1 S317 phosphorylation was positively correlated with AZD7762-mediated, DNA-damage-induced phosphorylation of ATM S1981, yet phosphorylation of Cdc25C S216, a Chk1 target, was substantially reduced (Figures 2A and 2B). To further evaluate the contribution of ATM and ATR to the phosphorylation of Chk1 at S317 in these specific cell contexts, we treated VCaP and C4-2b cells with ATM inhibitor KU-60019 or ATR inhibitor VE-821 in the absence and presence of AZD7762. The results showed that both ATM inhibitor and ATR inhibitor can reduce basal and AZD7762-induced phosphorylation of Chk1 S317 (Figures 2C and 2D). Since ATR and phosphorylated ATR (P-ATR) were unchanged or reduced, it is unlikely that ATR activities are related to the substantially increased Chk1 S317 phosphorylation in response to AZD7762; instead, the increased Chk1 phosphorylation at S317 is most likely due to DNA-dam- age-induced ATM phosphorylation of Chk1 S317.Flow cytometric analysis demonstrated that the combination treatment with AR knockdown and AZD7762 resulted in a greater apoptotic effect than did AR knockdown (p < 0.001 in both cell lines) or AZD7762 alone (VCaP, p = 0.01; C4-2b, p < 0.001) (Figures 2E and 2F). DNA fragmentation analysis demonstrated that ARsi and AZD7762 combination increased the rate of apoptosis over AR knockdown (VCaP, p < 0.001; C4-2b, p < 0.001) and AZD7762 (VCaP, p = 0.013; C4-2b,p < 0.001) alone (Figures 2G and 2H). The differing responses of VCaP and C4-2b cells to AZD7762 treatment are notable. In addition to higher treatment efficacy from the combination of ARsi and AZD7762, inhibition of AR with ARsi caused G1 arrest and reduction of cells in S phase in both cell lines; however, inhibition of Chk1 by AZD7762 led to significantly increasedsub-G1 cells and DNA fragmentation in VCaP but had very little effect on C4-2b (Figures 2E–2H), which may be largely due to different p53 status in the two cell lines (VCaP, p53 mutant; C4-2b, p53 wild-type).

It was reported that p53-mediated G1 ar- rest in response to DNA damage can spare cells from AZD7762 action that predominantly occurs during G2 (Castedo et al., 2004; Zhou and Bartek, 2004; Benada and Macurek, 2015). To address this possibility, we knocked down p53 using small inter- fering RNA (siRNA) in p53 wild-type C4-2b cells and analyzed the effect on cell-cycle distribution. As expected, knockdown of p53 significantly reduced the G0-G1 cell fraction and the combina- tion of p53 knockdown and AZD7762 resulted in significantly reduced G0-G1, S, and G2-M cells and significantly increased sub-G1 cells (Figures 2I and 2J; Table S3).Interestingly, we found that CDC6 knockdown and combina- tion treatment with AZD7762 also markedly reduced TopBP1 protein levels and ATR S428 and Cdc25C S216 phosphorylation in both cell lines (Figures 3A and 3B). We also found that the combination treatment resulted in markedly greater ATM auto- phosphorylation than did treatment with both agents alone, suggesting a synergistic increase in DNA damage (Figures 3A and 3B). Additionally, the combination treatment increased Chk1 S317 phosphorylation in both cell lines (Figures 3A and 3B). To determine the biological effects of combination treatment with CDC6si and AZD7762, we examined apoptotic activities using flow cytometry and a DNA fragmentation assay. Combina- tion treatment increased the percentage of sub-G1 (apoptotic) cells more than CDC6 knockdown did (VCaP, p = 0.01; C4-2b, p < 0.001) or AZD7762 (VCaP, p = 0.0048; C4-2b, p < 0.001) alone(Figures 3C and 3D). The combination treatment also resulted in greater DNA fragmentation/apoptosis than CDC6 knockdown did (VCaP, p < 0.001; C4-2b, p = 0.004) and AZD7762 (VCaP,p = 0.03; C4-2b, p = 0.03) alone (Figures 3E and 3F).TopBp1 is an essential activator for the ATR-Chk1 signaling pathway (Cimprich and Cortez, 2008; Wardlaw et al., 2014). Combination of AR knockdown or CDC6 knockdown with AZD7762 led to markedly reduced TopBP1 protein levels (Fig- ures 2A, 2B, 3A, and 3B).

These observations prompt us to test whether knockdown of TOPBP1 could also synergize with AZD7762 in the induction of PCa apoptosis and cell death. West- ern blotting (WB) analysis showed that TOPBP1 knockdown reduced Chk1 S317 phosphorylation, Cdc25C expression, and Cdc25C S216 phosphorylation and that TOPBP1 knockdown, together with AZD7762, further reduced Cdc25C S216 phos- phorylation (Figures 4A and 4B). Flow cytometric analysis demonstrated that the combination treatment with TOPBP1 knockdown and AZD7762 also resulted in a greater apoptotic effect than TOPBP1 knockdown did (in VCaP: TOPBP1si_sc, p = 0.0023; and TOPBP1si_3 p = 0.0066; in C4-2b: TOPBP1si_ sc, p = 0.0142; and TOPBP1si_3, p = 0.0037) and AZD7762(in VCaP: TOPBP1si_sc, p = 0.0014; and TOPBP1si_3, p = 0.0063; in C4-2b: TOPBP1si_sc, p = 0.0006; and TOPBP1si_3, p = 0.0002) alone (Figures 4C and 4D). DNA fragmentation analysis demonstrated that this combination increased the rate of apoptosis over TOPBP1 knockdown (in VCaP: TOPBP1si_sc, p < 0.0001; and TOPBP1si_3, p = 0.0047; in C4-2b: TOPBP1si_sc, p = 0.0028; and TOPBP1si_3, p = 0.0368) and AZD7762 (in VCaP: TOPBP1si_sc,p < 0.0001; and TOPBP1si_3, p = 0.0027; in C4-2b: TOPBP1si_sc, p = 0.0003; and TOPBP1si_3, p = 0.0011) alone (Figures 4E and 4F).Treatment with ENZ and AZD7762 Inhibits CDC6- TopBP1-ATR-Chk1 Signaling and Promotes DNA Damage and Apoptosis in PCa CellsENZ is a potent AR signaling inhibitor approved for treatment of mCRPC (Scher et al., 2012), and by combining it with AZD7762, we can translate our findings into a viable therapeutic approach for prostate cancer. We initially treated VCaP and C4-2b withENZ and/or AZD7762. We found that the combination treatment markedly reduced Cdc6 phosphorylation and protein levels, TopBP1 protein levels, and ATR and Chk1 phosphorylation levels (Figure 5A).

Importantly, the combination treatment syner- gistically increased ATM phosphorylation and reduced Cdc25C phosphorylation, markers for DNA damage and the abrogation of G2/M checkpoint, respectively (Figure 5A).Next, we examined the apoptotic effect of combination treat- ment with ENZ and AZD7762 using flow cytometry and a DNA fragmentation assay. The results of flow cytometric analysis demonstrated that the combination treatment significantly increased the percentage of apoptotic (sub-G1) cells over that resulting from ENZ (VCaP, p = 0.0053; C4-2b, p = 0.009) and AZD7762 (VCaP, p = 0.012; C4-2b, p = 0.008) alone (Figures 5B and 5C). The results of DNA fragmentation analysis demon- strated that the combination treatment increased the apoptotic effect over that induced by ENZ (VCaP, p < 0.001; C4-2b, p = 0.018) and AZD7762 (VCaP, p < 0.001; C4-2b, p = 0.0046)alone (Figures 5D and 5E).To strengthen our finding regarding the role of CDC6 in the combination therapy using ENZ and AZD7762, we testedwhether overexpression of CDC6 can overcome ENZ- and AZD7762-induced PCa cell death. Our data demonstrated that overexpression of CDC6 (Figure 5F) significantly reduced sub-G1 cells (Figure 5G) and apoptotic DNA fragmentation (Fig- ure 5H) in both VCaP and C4-2b.Overall, our data demonstrated that combination treatment with ENZ and AZD7762 downregulated CDC6 and TopBP1- ATR-Chk1 signaling, released Cdc25C from inactivating phos- phorylation by Chk1, and abolished G2/M checkpoint, resulting in increased DNA damage and apoptosis (Figure 5I).To test our hypothesis that combination treatment with ENZ and AZD7762 is a potential therapeutic approach for mCRPC, we used three different animal models: VCaP, C4-2b xenografts, and patient-derived xenograft (PDX) MDA-133-4, which harbors a missense p53 mutation (Lee et al., 2011).

We administered treat- ment to mice with orthotopic VCaP xenografts and monitored tu- mor progression. Treatment with ENZ alone and the combination of ENZ and AZD7762 reduced tumor growth compared to control mice (p = 0.05 and p = 0.02, respectively), but the differences be- tween combination and single-agent treatment were not statisti- cally significant, according to assessment using an in vitro imag- ing system (IVIS) (Figures 6A and S2A). However, ENZ is an inducer of CYP450; it increases luciferin metabolism and, through this activity, reduces bioluminescence. In comparison, AZD7762 is an ATP-competitive Chk1/2 inhibitor; it binds to their respective ATP-binding sites and increases the availability of ATP, which is free to react with D-luciferin to produce light and, through this activity, increase bioluminescence, leading to false increased signal. When we evaluated tumor wet weights, we found that sin- gle-agent treatment reduced tumor growth significantly (ENZ, p = 0.009; AZD7762, p < 0.001), whereas the combination treat- ment significantly reduced weights more than that resulting from treatment with ENZ (p < 0.001) or AZD7762 (p = 0.008) alone. Remarkably, the combination treatment was synergistic with re- gard to tumor wet weight (p = 0.0097) (Figure 6B), according to two-way ANOVA (Slinker, 1998).To further validate our in vivo data and establish associations with our WB data, we analyzed CDC6 and ATM expression and phosphorylation in mice. Our in vitro studies demonstrated that combination treatment with ENZ and AZD7762 significantly reduced CDC6 expression and phosphorylation and increased ATM phosphorylation. IHC analysis of VCaP xenografts demon- strated that ENZ reduced CDC6 phosphorylation significantly, compared to control (p = 0.036), whereas AZD7762 did not produce significant effects (p = 0.06) (Figure S3A). However, the combination treatment significantly reduced CDC6 phos- phorylation compared to control (p = 0.012), ENZ-treated (p = 0.0366), and AZD776-treated (p = 0.036) mice (Figures 6C and S3A). Similarly, CDC6 expression was reduced to a greater extent in ENZ-treated mice than in control mice (p = 0.036), whereas the difference in AZD7762-treated and control mice was not statistically significant (p = 0.4) (Figure S3A).

The combination treatment reduced CDC6 expression significantly, compared to the control (p = 0.012), ENZ (p = 0.0214), and AZD7762 (p = 0.0214). Treatment with ENZ or AZD7762 alone did not have a significant effect on ATM phosphorylation, whereas the combination treatment significantly increased this phosphorylation over that in control (p = 0.021), ENZ-treated (p = 0.036), and AZD7762-treated (p = 0.036) mice (Figures 6C and S3A).In subcutaneous C4-2b xenografts, we found that ENZ and AZD7762, as single agents, did not significantly affect tumor vol- ume or wet weight, compared to the control treatment (Figures 6D and 6E). In contrast, the combination treatment producedsignificantly lower tumor volumes at 8 days than in control (p = 0.004) and ENZ-treated (p = 0.047) mice and significantly lower tumor volumes at 11 days than in AZD7762-treated mice (p = 0.04). The differences continued to be statistically significant throughout the 21-day treatment period (Figure 6D). Moreover, the combination treatment produced significantly lower tumor wet weights than those in the control (p < 0.001), ENZ-treated (p = 0.02), and AZD7762-treated (p = 0.022) mice (Figures 6E and S2B).IHC analysis of C4-2b xenograft tumors showed that single- agent treatment did not significantly reduce CDC6 phosphoryla- tion, whereas the combination treatment significantly reduced it to a greater extent than that in control (p = 0.012), ENZ-treated (p = 0.021), and AZD7762-treated (p = 0.021) mice (Figures 6F and S3B). CDC6 protein expression was reduced by single- agent treatment, but differences did not reach statistical signifi- cance compared to control mice (p = 0.4). In comparison, the combination treatment significantly reduced CDC6 expression(D–F) C4-2b xenografts. Neither of the single agents had a significant effect on tumor volume, whereas the combination treatment resulted in significantly lower tumor volumes at 8 days than the control treatment (p = 0.004) and enzalutamide (p = 0.047) and at 11 days than AZD7762 (p = 0.04). (D)

These differences continued to be statistically significant over 21 days. (E) Neither of the single agents had a significant effect on tumor wet weights, but the combination treatment produced significantly lower tumor wet weights than the control treatment (p < 0.001), ENZ (p = 0.02), or AZD7762 (p = 0.022) did. (F) IHC analysis demonstrated that the combination of ENZ and AZD7762 significantly decreased CDC6 phosphorylation (p = 0.01208) and CDC6 protein levels (p = 0.01208) and significantly increased ATM phosphorylation (p = 0.01208) compared to the control treatment. Full IHC analysis results, including comparison of combination treatment with single-agent treatment and quantitative analysis results can be found in Figure S2.(G and H) The subcutaneous MDA-133-4 PDX model. (G) The combination treatment had a greater effect on tumor volume than ENZ alone at 21 (p = 0.016), 24 (p = 0.004), and 27 (p = 0.015) days or AZD7762 alone at 9 (p = 0.04), 14 (p = 0.014), 17 (p = 0.005), 21 (p = 0.009), 24 (p = 0.006), and 27 (p < 0.001) days. (H) Thecombination treatment also had a greater effect on tumor wet weights than did ENZ (p = 0.036) and AZD7762 (p = 0.034) alone did.Synergism was evaluated using ANOVA (p = 0.0004 for tumor volume, and p = 0.0107 for tumor wet weight, indicated by a pound sign in the panels). *p < 0.05. See also Figure S2.compared to the control (p = 0.012), ENZ-treated (p = 0.036), and AZD7762-treated (p = 0.012) mice (Figures 6F and S3B). Moreover, we found that neither of the single-agent treatments had a greater effect on ATM phosphorylation than the controltreatment did. However, the combination treatment signifi- cantly increased ATM phosphorylation over that with control (p = 0.012), ENZ treatment (p = 0.036), and AZD7762 treatment (p = 0.036) (Figure 6F), indicating that this combination treatmentsynergistically increases the incidence of DNA damage in PCa cells.

We also used the MDA-133-4 PDX, which was shown to harbor a frameshift mutation of p53 that results in a truncated protein, as an additional model (Lee et al., 2011; data not shown). We found that the combination treatment had a greater effect than ENZ did at 21 (p = 0.016), 24 (p = 0.004), and 28 (p = 0.015) days; and than AZD7762 did at 9 (p = 0.04), 14 (p = 0.014), 17 (p = 0.005), 21 (p = 0.009), 24 (p = 0.006), and27 (p < 0.001) days (Figure 6G).
Similar to the results for mice with VCaP xenografts, single-agent treatment had a greater ef- fect on tumor wet weight than the control treatment did, but the combination treatment further reduced wet weights more than ENZ (p = 0.036) and AZD7762 (p = 0.034) alone did (Fig- ure 6H). These data suggested that ENZ and AZD7762 can syn- ergistically inhibit tumor growth in a mCRPC model such as MDA-133-4 PDX, which we confirmed via two-way ANOVA (tumor volume, p = 0.0004; and wet weight, p = 0.0107). No sig- nificant differences in body weight were found between drug- treated and vehicle-control-treated mice in all three models.

DISCUSSION
For this study, we hypothesized that the targeting of ATR-Chk1 signaling in PCa cells is an effective approach. To maximize the therapy effect, we evaluated the impact of targeting CDC6 on ATR-Chk1 signaling. Gonzalez at al. claimed that CDC6 exerts oncogenic activity though repression of the INK4/ARF locus (Gonzalez et al., 2006). Additionally, Sideridou et al. showed that not only p14, p15, and p16 but also E-cadherin is downregu- lated at the transcriptional level by increased CDC6 (Sideridou et al., 2011). Interestingly, p14ARF can repress AR trans- activation in prostate cancer cells (Lu et al., 2013). CDC6 can pro- tect genomic integrity via activation of DDR (Clay-Farrace et al., 2003; Yoshida et al., 2010; Oehlmann et al., 2004), yet deregu- lated overexpression of CDC6 can lead to rereplication, a form of replication stress that can result in genomic instability (Liontos et al., 2007). Within this context, the role of CDC6 in PCa is partic- ularly intriguing, given that CDC6 is a direct AR target gene (Jin and Fondell, 2009; Bai et al., 2005) and that AR upregulates the expression of genes involved in DNA repair and DDR (Polking- horn et al., 2013; Li et al., 2014). Indeed, we found that CDC6 was upregulated during PCa progression and CDC6 downregu- lation synergized with the dual Chk1/2 inhibitor, AZD7762, to inhibit TopBP1-ATR-Chk1 signaling in VCaP and C4-2b cells and to increase its cytotoxic effects. It was notable that, in marked contrast to treatment with single agents, the combination of CDC6 knockdown and AZD7762 markedly affected the expression of TopBP1, ATR (Figures 3A and 3B), and down- stream biological effects (Figures 3C–3F). To monitor increased DNA damage, we utilized the ATM S1981 phosphorylation as a DNA damage marker, which is increased under Chk1 inhibi- tion-mediated accumulation of DNA damage (Sarmento et al., 2015).

Experimentally, we demonstrated that ATM S1981 phos- phorylation was increased following CDC6 knockdown and further increased by a combination treatment of CDC6si together with AZD7762 (Figures 3A and 3B). Importantly, CDC6 knockdown and AZD7762 combination treatment significantly increased the apoptotic response to DNA damage in C4-2b cells. Although it was not a major focus of this study, we infer that, in p53 wild-type C4-2b cells, the combination of CDC6 knockdown and AZD7762 treatment will increase ATM-dependent p53 phos- phorylation/activation, leading to increased Bax and p21 protein expressions and subsequent p53-dependent G1 arrest, which may spare C4-2b cells from AZD7762-mediated DNA-damage- induced cell death. We used p53 knockdown to analyze the role of p53 in C4-2b cells following AZD7762 treatment and demonstrated that reduction of p53 levels significantly reduced G0-G1 and S cell fractions and significantly increased sub-G1 cells in AZD7762-treated cells (Figures 2I and 2J; Table S3).Previous publications have shown that TopBp1 plays an important role in the activation of the ATR-Chk1 pathway (Cim- prich and Cortez, 2008; Wardlaw et al., 2014; Li et al., 2014). Our results show that AR or CDC6 knockdown combined with AZD7762 treatment coordinately downregulated TopBP1 in both VCaP and C4-2b models and that, similar to AR or CDC6 knockdown, the knockdown of TOPBP1 synergizes with AZD7762 in the induction of apoptotic cell death in VCaP and C4-2b PCa cells. These data, together with the results of ENZ and AZD7762 combination experiments, demonstrated that a synergistic therapeutic effect can be reached by targeting AR and Chk1 simultaneously through inhibition/downregulation of TopBP1-ATR-Chk1 signaling.

To validate our in vitro results, we selected VCaP and C4-2b xenograft and human MDA-133-4 PDX AR-positive models for our studies on the basis of their phenotypic characteristics, which allow us to evaluate specific drug activities within the context of androgen dependence and variable p53 status. We found that ENZ and AZD7762 synergistically inhibit tumor growth in VCaP xenografts compared to single agents. Regarding C4-2b xenografts, we found that AZD7762 was not as effective as it was in the other models, likely owing to its p53 wild-type sta- tus (Ma et al., 2012). Reduced response to ENZ and to the ENZ and AZD7762 combination treatment was anticipated, since this model is AR positive but androgen independent (Nguyen et al., 2014). However, even though this model is androgen indepen- dent, the combination of AR and Chk1/2 inhibition exhibited marked activity. This suggests that PCa that is AR positive yet fails to respond to ENZ treatment alone (Nguyen et al., 2014) will respond to this combination therapeutic approach. Similar to VCaP, in the MDA-PCa-133-4 model, combination treatment of ENZ and AZD7762 synergistically inhibited tumor growth in this model, despite the marked activities of single-agent treat- ment. IHC analysis demonstrated that this combination reduced CDC6 phosphorylation and expression and increased the levels of ATM phosphorylation, a DNA damage biomarker, in VCaP and C4-2b xenografts.

In summary, we demonstrated that CDC6 is upregulated during progression of PCa and is positively associated with AR expression. Our results indicated that targeting of AR-CDC6 via gene knockdown or ENZ, together with inhibition of Chk1/2 signaling by AZD7762, resulted in maximal suppression of TopBP1-ATR-Chk1 signaling and the induction of DNA damage and apoptosis in vitro. Furthermore, this therapeutic strategy ex- hibited additive or synergistic therapeutic activities in xenograft and PDX models in vivo. Importantly, one of the models we used, C4-2b, is androgen positive but androgen independent and wild-type for p53. Its marked response to the combination treatment indicates that the combined inhibition of androgen signaling and Chk1/2 can be effective in the absence of intact AR signaling and the presence of wild-type p53. Additional studies are required to confirm the efficacy of this approach and evaluate alternative methods of targeting DDR using AR signaling inhibitors combined with DDR-targeted agents in patients with AZD7762 mCRPC.