targeting dna damage response in prostate ......et al., 2003; yoshida et al., 2010), which inhibits...
TRANSCRIPT
Article
Targeting DNA Damage Re
sponse in ProstateCancer by Inhibiting AndrogenReceptor-CDC6-ATR-Chk1 SignalingGraphical Abstract
Highlights
d CDC6 expression is increased during prostate cancer (PCa)
progression
d AR or CDC6 knockdown, together with AZD7762, suppresses
TopBP1-ATR-Chk1 signaling
d AR or CDC6 knockdown sensitizes PCa cells to AZD7762
d Enzalutamide and AZD7762 combination treatment
generates synergistic therapeutic effects
Karanika et al., 2017, Cell Reports 18, 1970–1981February 21, 2017 ª 2017 The Author(s).http://dx.doi.org/10.1016/j.celrep.2017.01.072
Authors
Styliani Karanika, Theodoros Karantanos,
Likun Li, ..., Wei Zhang, Shuhua Li,
Timothy C. Thompson
In Brief
CDC6 is an androgen receptor (AR) target
gene and an essential regulator of DNA
replication and checkpoint activation.
Karanika et al. show that combined
inhibition of the AR and Chk1 signaling
promotes DNA damage accumulation in
prostate cancer cells to induce cell death,
regardless of p53 status.
Cell Reports
Article
Targeting DNA Damage Response in ProstateCancer by Inhibiting AndrogenReceptor-CDC6-ATR-Chk1 SignalingStyliani Karanika,1,4 Theodoros Karantanos,1,4 Likun Li,1,4 Jianxiang Wang,1 Sanghee Park,1 Guang Yang,1 Xuemei Zuo,1
Jian H. Song,1 Sankar N. Maity,1 Ganiraju C. Manyam,2 Bradley Broom,2 Ana M. Aparicio,1 Gary E. Gallick,1
Patricia Troncoso,3 Paul G. Corn,1 Nora Navone,1 Wei Zhang,1 Shuhua Li,1 and Timothy C. Thompson1,5,*1Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA2Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA3Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA4Co-first author5Lead Contact
*Correspondence: [email protected]://dx.doi.org/10.1016/j.celrep.2017.01.072
SUMMARY
Cell division cycle 6 (CDC6), an androgen receptor(AR) target gene, is implicated in regulating DNAreplication and checkpoint mechanisms. CDC6expression is increased during prostate cancer(PCa) progression and positively correlates with ARin PCa tissues. AR or CDC6 knockdown, togetherwithAZD7762, aChk1/2 inhibitor, results indecreasedTopBP1-ATR-Chk1signaling andmarkedly increasedataxia-telangiectasia-mutated (ATM) phosphoryla-tion, a biomarker of DNA damage, and synergisticallyincreases treatment efficacy. Combination treatmentwith the AR signaling inhibitor enzalutamide (ENZ)and the Chk1/2 inhibitor AZD7762 demonstratessynergy 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 overexpressionsignificantly reduced ENZ- and AZD7762-inducedapoptosis. Additive or synergistic therapeutic activ-ities are demonstrated in AR-positive animal xeno-graft models. These findings have important clinicalimplications, since they introduce a therapeutic strat-egy for AR-positive, metastatic, castration-resistantPCa, 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 and
1970 Cell Reports 18, 1970–1981, February 21, 2017 ª 2017 The AutThis is an open access article under the CC BY-NC-ND license (http://
novel 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 upstreammediators 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 Mendez, 2008). Furthermore, CDC6 overexpression during
G2 phase blocks mitotic entry by activating Chk1 (Clay-Farrace
hor(s).creativecommons.org/licenses/by-nc-nd/4.0/).
Figure 1. CDC6 Is Induced during PCa Progression and Is Positively Correlated with AR Expression
(A) Immunohistochemical analysis of AR, CDC6, and P-CDC6 (S54) in normal prostate, primary prostate tumor, and bone metastases. NL, normal prostate; PCa,
prostate cancer; Bone met, bone metastasis. Treatment information of patient with metastasis is available in Table S1.
(B) qRT-PCR analysis of CDC6 mRNA levels. VCaP and C4-2b cells were transfected with 20 nM ARsi or NCsi for 48 hr. **p < 0.01; ***p < 0.0001.
(C and D) Protein stability analysis of Cdc6. VCaP (C) and C4-2b (D) cells were transfected with 20 nMArsi-1 or NCsi for 48 hr prior to the treatment with 100 mg/mL
cycloheximide (CHX) for indicated time. Left panels: western blotting analysis; right panels: densitometry analysis for Cdc6 protein stability in ARsi (red) and
NCsi (blue) transfected cells.
See also Table S1.
et 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 aimof the present studywas 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
CDC6 Is Induced during PCa Progression and IsPositively Correlated with AR ExpressionWe 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). It
Cell Reports 18, 1970–1981, February 21, 2017 1971
was 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 twomol-
ecules. To determine themechanism 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 orCDC6Knockdown Increases the Sensitivity of PCaCells to theChk1/2 Inhibitor AZD7762 through Inhibitionof 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 increased
1972 Cell Reports 18, 1970–1981, February 21, 2017
sub-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, p53wild-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 p53wild-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,
Figure 2. Effect of AR Knockdown and Chk1/2 Inhibitor AZD7762 on TopBP1-ATR-Chk1 Signaling and PCa Cell Survival
(A–J) In (A), (B), (E), and (H), VCaP and C4-2b cells were transfected with 20 mM ARsi or NCsi 24 hr prior to the treatment with 200 nM AZD7762 (AZD) for 48 hr.
(A and B) Western blotting analysis of Cdc6 and TopBP1-ATR-Chk1 signaling molecules in ARsi-, AZD-, and ARsi+AZD-treated VCaP (A) and C4-2b (B) cells.
(C and D) Western blotting analysis of P-Chk1 (S317) in VCaP and C4-2b cells that were treated with 200 nM AZD; 5 and 10 mMKU-60019 (KU), an ATM inhibitor;
1 and 3 mM VE-821 (VE), an ATR inhibitor; or a combination of AZD and KU or VE for 48 hr. (E and F) Cell-cycle analyses of ARsi-, AZD-, and ARsi+AZD-treated
VCaP (E) and C4-2b (F) cells. (G and H) DNA fragmentation analyses of ARsi-, AZD-, and ARsi+AZD-treated VCaP (G) and C4-2b (H) cells. (I) p53wild-type C4-2b
cells were transfected with 20 nMp53si or NCsi for 48 hr prior to western blotting analysis. (J) C4-2b cells were transfected with 20 nMp53si or NCsi for 48 hr prior
to the treatment with 200 nM AZD for 48 hr, followed by cell-cycle analysis.
*p < 0.05, statistically significant in sub-G1 cell distribution (E and F) or in DNA fragmentation (G and H) comparing the combination of ARsi and AZD to ARsi or
AZD alone; in G0-G1 and S cell distribution comparing p53si + DMSO to NCsi + DMSO (J); or in sub-G1, G0-G1, S, and G2-M cell distribution comparing p53si +
AZD to NCsi + AZD (J). Detailed statistical information on (J) is available in Table S2.
See also Table S2.
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 DNADamage 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 with
ENZ 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 DNAdamage 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
Cell Reports 18, 1970–1981, February 21, 2017 1973
Figure 3. CDC6Knockdown Increases the Sensitivity of PCaCells to Treatmentwith Chk1/2 Inhibitor AZD through Inhibition of TopBP1-ATR-
Chk1 Signaling
VCaP and C4-2b cells were transfected with 20 mM CDC6si or NCsi 24 hr prior to the treatment with 200 nM AZD for 48 hr.
(A and B) WB analysis of TopBP1-ATR-Chk1 signaling proteins in VCaP (A) and C4-2b (B) cells.
(C and D) Flow cytometry analysis of CDC6si7- and AZD-treated VCaP (C) and C4-2b (D) cells. Top panels: representative cell-cycle profiles. Bottom panels:
quantitative analysis of cell-cycle distribution. Red arrows point to sub-G1, and blue arrows point to S phase.
(E and F) DNA fragmentation analysis of CDC6si7- and AZD-treated VCaP (E) and C4-2b (F) cells.
*p < 0.05, statistically significant in sub-G1 cell distribution (B and E) or in DNA fragmentation (C and F) when comparing the combination of ARsi and AZD7762 to
ARsi or AZD7762 alone.
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 tested
1974 Cell Reports 18, 1970–1981, February 21, 2017
whether 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).
Figure 4. TOPBP1 Knockdown Increases the Sensitivity of PCa Cells to Treatment with Chk1/2 Inhibitor AZD
VCaP and C4-2b cells were transfected with 20 mM TOPBP1si or NCsi 24 hr prior to the treatment with 200 nM AZD for 48 hr.
(A and B) WB analysis of TopBP1, Chk1, P-Chk1 (S317), Cdc25C, and P-Cdc25C after TOPBP1 knockdown and treatment of AZD7762 in VCaP (A) and C4-2b
(B) cells.
(C and D) Flow cytometry analysis for sub-G1 cell distribution in TOPBP1si- and AZD-treated VCaP (C) and C4-2b (D) cells.
(E and F) DNA fragmentation analysis of TOPBP1si- and AZD-treated VCaP (E) and C4-2b (F) cells.
*p < 0.05, statistically significant in sub-G1 cell distribution (C and D) or in DNA fragmentation (E and F) when comparing the combination of TOPBP1si and
AZD7762 to TOPBP1si or AZD alone.
Combination Treatment with ENZ and AZD7762 Inhibitsthe Growth of Prostate Tumor XenograftsTo 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
amissensep53mutation (Leeet al., 2011).Weadministered 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 luciferinmetabolism 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 produced
Cell Reports 18, 1970–1981, February 21, 2017 1975
Figure 5. Combination Treatment with ENZ and AZD Inhibits CDC6-TopBP1-ATR-Chk1 Signaling, Promoting DNA Damage and Apoptosis in
PCa Cells
(A–E) VCaP and C4-2b cells were transfected with DMSO, 1 mM ENZ, and 200 nM AZD or ENZ+AZD for 48 hr. (A) ENZ and AZD combination treatment reduced
phosphorylation and protein levels of CDC6; TopBP1 protein levels; and phosphorylations of ATR, Chk1, and Cdc25C, leading to increased ATMphosphorylation
in both VCaP and C4-2b cells. (B and C) Flow cytometric analysis. ENZ and AZD combination treatment increased the percentage of apoptotic (sub-G1) cells in
VCaP (B) more than treatment with ENZ (p = 0.0053) or AZD7762 (p = 0.012) alone did, and it also increased the percentage of apoptotic (sub-G1) cells in C4-2b
(C) more than treatment with ENZ (p = 0.009) or AZD7762 (p = 0.008) alone did. (D and E) DNA fragmentation assays. The combination treatment increased
apoptosis in VCaP cells (D)more than treatment with ENZ alone (p < 0.001) or AZD7762 (p < 0.001) alone did, and it also increased apoptosis in C4-b cells (E) more
than treatment with ENZ (p = 0.018) or AZD7762 (p = 0.0046) alone did.
(F–H) Overexpression of CDC6 reduces ENZ- and/or AZD-induced apoptotic cell death. VCaP and C4-2b cells were transfected with 1 mg of CDC6 plasmid DNA
or control empty vector DNA for 24 hr prior to the treatment with DMSO, 1 mMENZ, 200 nMAZDor ENZ+AZD for 48 hr. (F) CDC6protein levelsmarkedly increased
after enforcedCDC6 expression in VCaP and C4-2b cells. (G) Flow cytometric analysis for apoptotic cell death (Sub-G1 cells). (H) DNA fragmentation assay. Data
in (H) are presented as fold of DMSO control. *p < 0.05, statistically significant when comparing combination treatment to single-agent treatment (B–E) and
comparing CDC6 overexpression to empty vector (G and H).
(I) Proposed signaling schema.
significantly 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).
1976 Cell Reports 18, 1970–1981, February 21, 2017
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
Figure 6. Combination Treatment with ENZ and AZD7762 Inhibited the Growth of Prostate Tumor Xenografts
VCaP orthotopic xenografts, C4-2b subcutaneous xenografts, and MDA-133-4 PDX model were treated with vehicle control (C), enzalutamide (E), AZD7762 (A),
or enzalutamide + AZD7762 (E+A) for 35, 21, and 28 days, respectively.
(A–C) VCaP xenografts. (A) ENZ alone and combined with AZD7762 reduced tumor growth more than the control treatment (p = 0.05 and p = 0.02, respectively)
via IVIS measurements. (B) Treatment with ENZ and AZD7762 as single agents had significant effects (p = 0.009 and p < 0.001, respectively) on tumor wet
weights, and mice given the combination had tumors with significantly lower wet weight than mice given ENZ (p < 0.001) or AZD7762 (p = 0.008) alone did. The
combination treatment had synergistic effects on wet weights as determined using two-way ANOVA (p = 0.0097, indicated by a pound sign). (C) IHC analysis
demonstrated that the combination of ENZ and AZD7762 significantly decreased CDC6 phosphorylation (p = 0.01208) and Cdc6 protein levels (p = 0.012) and
significantly increased ATM phosphorylation (p = 0.01208) compared to the control treatment in VCaP xenografts. Full IHC analysis results, including comparison
of combination treatment with single-agent treatment, and quantitative analysis results can be found in Figure S2.
(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) The
combination 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 control
treatment 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 treatment
Cell Reports 18, 1970–1981, February 21, 2017 1977
synergistically 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), and
27 (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 canpro-
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 treatmentwith 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
1978 Cell Reports 18, 1970–1981, February 21, 2017
increased the apoptotic response toDNAdamage inC4-2b cells.
Although it was not a major focus of this study, we infer that, in
p53 wild-type C4-2b cells, the combination ofCDC6 knockdown
andAZD7762 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 andAZD7762 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 othermodels, likely owing to its p53wild-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 mCRPC.
EXPERIMENTAL PROCEDURES
Cell Lines and Reagents
The human PCa cell line VCaP was validated as described previously (Li
et al., 2014), and LNCaP C4-2b (C4-2b) was recently validated in the MD
Anderson Characterized Cell Line Core Facility using the same method.
Cycloheximide (239763) was purchased from Calbiochem; and AZD7762,
enzalutamide (MDV3100), KU-60019, and VE-821 were purchased from
SelleckChem.
RNAi
CDC6si6 (SI04218389), CDC6si7 (SI04254782), TOPBP1si3 (SI00749553),
TP53si_7 (SI026233764), TP53si_8 (SI02623754), TP53si_9 (SI02655170),
TP53si_13 (SI4384079), and NCsi (1022076) were purchased from QIAGEN;
ARsi1 (s1538) and ARsi2 (s1540) were purchased from Life Technologies;
and TOPBP1si_sc (sc-41068) was purchased from Santa Cruz Biotechnology.
Gene knockdown experiments were performed using the Lipofectamine
RNAiMax transfection reagent (Life Technologies). PCa cells were seeded at
desired densities (VCaP: 1.0 3 106 per well; and C4-2b: 5 3 105 per well in
six-well plates, 1/5 or 1/30 of these cell numbers in 24- or 96-well plates,
respectively). Cells were transfected with 20 nM siRNA in the following day.
24 hr later, VCaP and C4-2b cells were treated with DMSO or AZD772, either
for 24 hr for WB analysis or for 48 hr for DNA fragmentation assay or flow cy-
tometric analysis.
WB Analysis
For WB analysis of the effects of CDC6si, ARsi, TOPBP1si, AZD7762, and
combinations of them, cells were transfected with siRNA for 24 hr and then
treated with DMSO or AZD7762 for 24 hr. Afterward, cells were treated with
a serum-free medium overnight and then with full serum for 4 hr (synchroniza-
tion) before protein extract preparation. For WB analysis of ENZ and AZD7762
effects, cells were treatedwith DMSOor ENZ for 24 hr. DMSOor AZD7762was
then added for 24 hr. Synchronization was achieved as described earlier.
Antibodies against CDC6 (3387), ATR (2790), P-ATR Ser428 (2853), Chk1
(2360), P-Chk1Ser317 (12302), Cdc25C (4688), P-Cdc25C Ser216 (9528),
ATM (2873), and P-ATM Ser1981 (13050) were purchased from Cell Signaling
Technology. Antibodies against P-CDC6 Ser54 (ab75809), P-Chk1ser296
(ab79758), and TopBp1 (ab2402) were purchased from Abcam. Antibodies
against GAPDH (365062) and AR (816) were purchased from Santa Cruz
Biotechnology. When indicated, densitometric analysis was performed, and
quantification of integrated density was assessed using the NIS-Elements-
AR software program (version 3.0; Nikon), followed by GAPDH normalization.
DNA Fragmentation Assay
A DNA fragmentation assay was performed for apoptotic evaluation of siRNA
and/drug effects. Cells were transfected with CDC6si, ARsi, TOPBP1si, or
NCsi; treated with DMSO or ENZ for 24 hr; and then treated with DMSO,
AZD7762, or ENZ+AZD7762 for 48 hr. DNA fragmentation analysis was per-
formed according to manufacturer’s instructions. Data are presented as
mean ± SE.
Flow Cytometry Analysis
Cells were treated as described earlier and were prepared for flow cyto-
metric analysis as described previously (Li et al., 2014). Data are presented
as mean ± SD.
Protein Stability Assay
VCaP and C4-2b cells were treated with NCsi or ARsi for 48 hr and then
incubated with 100 mg/mL cycloheximide for the indicated time periods. Cell
extracts were obtained via lysis in a modified RIPA buffer.
Animal Studies
All animal experiments were conducted in accordance with accepted stan-
dards of humane animal care approved by MDACC IACUC. Data are pre-
sented as mean ± SE.
Orthotopic VCaP Xenografts
VCaP xenografts were generated in mice as described previously (Li et al.,
2014). The experimental groups received DMSO, ENZ (10 mg/kg daily),
AZD7762 (25 mg/kg, twice daily every third day) or a combination of ENZ
and AZD7762 for 35 days. Tumor size was monitored weekly according to
luminescence signal, using the IVIS 200 Imaging System (PerkinElmer). The
mice were sacrificed, and their tumors were collected.
Subcutaneous C4-2b Xenografts
Aliquots of 6 3 106 C4-2b cells in 100 mL of 10% fetal calf serum (FCS) and
T medium + 50% Matrigel were injected subcutaneously into the right flanks
of athymic nude male mice (Taconic) to induce subcutaneous tumors. Tumors
were allowed to grow for 24 days before treatment. The experimental groups
received treatment for 21 days in doses as described earlier. Tumor size was
monitored by measuring three dimensions and using the following formula:
length/2 3 width/2 3 height/2 3 p 3 4/3. The mice were sacrificed, and their
tumors were collected.
MDA-PCa-133-4 PDXs
Subcutaneous PDXs of MDA-PCa-133-4 were generated by implanting
0.125-cm3 tumor fragment into the left flanks of previously castrated severe
combined immunodeficiency mice (SCID) mice (Charles River Laboratories).
RNA sequencing (RNA-seq) analysis showed that this tumor harbors a
missense p53 mutation (P72R). Tumors were allowed to grow until they
reached a volume of 50 mm3. The experimental groups received treatment
for 28 days in doses as described earlier. Tumor size was monitored as
described for the C4-2b model. The mice were sacrificed, and tumors were
collected.
Patient-Derived Xenografts
The MD Anderson Cancer Center PCa PDXs were developed, as described
previously (Li et al., 2014).
Immunohistochemical Analysis
Eleven human normal prostate specimens, 28 human primary PCa specimens
(not previously treated) obtained after radical prostatectomy, and 9 metastatic
PCa specimens (submitted to various previous treatments; Table S1) were
obtained after patients provided informed consent and were used to analyze
phosphorylated CDC6 (P-CDC6) and CDC6 expression. Antibodies against
AR (816), P-CDC6 S54 (12920), and CDC6 (8341) obtained from Santa Cruz
Biotechnology were used for IHC. CDC6 and AR immunostainings were also
performed and scored on tissue microarray slides composed of 34 tumor
xenografts generated from different histologic types of PCa (Table S2). For
IHC analysis of tumor xenografts, specimens were prepared as described
previously (Li et al., 2014). Antibodies against P-CDC6 S54 and CDC6 (Santa
Cruz Biotechnology), P-ATM S1981 (32420), and ATM (1292; Abcam) were
used. Immunostaining scoring was performed as described previously (Li
et al., 2014).
Statistical Analysis
The results are presented as the mean ± SE or mean ± SD from at least
three independent experiments. Comparisons of groups were appropriately
analyzed using the Student’s t test, the Mann-Whitney U test, Spearman’s
rho, or the Kruskal-Wallis rank test. p values less than 0.05 were considered
statistically significant, and all tests were two-tailed. Synergism was deter-
mined using two-way ANOVA (Slinker, 1998; Li et al., 2014).
Cell Reports 18, 1970–1981, February 21, 2017 1979
SUPPLEMENTAL INFORMATION
Supplemental Information includes three figures and three tables can be found
with this article online at http://dx.doi.org/10.1016/j.celrep.2017.01.072.
AUTHOR CONTRIBUTIONS
T.C.T., S.K., T.K., and L.L. conceived and designed the study and wrote the
paper. In vitro studies, including siRNA and drug treatments, WB analysis,
flow cytometric assays, DNA fragmentation experiments, protein stability as-
says, statistic and synergy analysis: L.L., S.K., T.K., J.W., X.Z., W.Z., and
S.L.; IHC and tissue microarray analysis: G.Y., S.K., and T.K.; xenograft model
studies: S.P., S.K., T.K., J.W., J.H.S., and G.G. P.G.C. contributed to manu-
script preparation. S.N.M., A.M.A., P.T., and N.N. contributed to pathological
analysis of human samples and the establishment of PDX lines. B.B and
G.C.M. performed bioinformatics analyses of RNA-seq data.
ACKNOWLEDGMENTS
We thank XinhaiWan and Jun Yang for guiding in animal experiments and Don-
ald Norwood and Linda Bohannon for editing the manuscript. Funding: this
work was supported in part by National Cancer Institute grant R0150588 (to
T.C.T.); National Cancer Institute grant P50140388; the Prostate Cancer
Specialized Program of Research Excellence at The University of Texas MD
Anderson Cancer Center; National Cancer Institute grant CA16672; MD An-
derson Cancer Center Support Grant; and Tony’s Prostate Cancer Research
Foundation.
Received: January 20, 2015
Revised: November 11, 2016
Accepted: January 26, 2017
Published: February 21, 2017
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Cell Reports, Volume 18
Supplemental Information
Targeting DNA Damage Response in Prostate
Cancer by Inhibiting Androgen
Receptor-CDC6-ATR-Chk1 Signaling
Styliani Karanika, Theodoros Karantanos, Likun Li, Jianxiang Wang, SangheePark, Guang Yang, Xuemei Zuo, Jian H. Song, Sankar N. Maity, Ganiraju C.Manyam, Bradley Broom, Ana M. Aparicio, Gary E. Gallick, Patricia Troncoso, Paul G.Corn, Nora Navone, Wei Zhang, Shuhua Li, and Timothy C. Thompson
Fig. S1
P-Cdc6 S54 P-Cdc6 S54 pre-absorbed with a P-Cdc6 S54 peptide
Fig. S1. Verification of the specificity of P-Cdc6 (Ser54) antibody (related to Figure 1). The specificity of P-Cdc6 (Ser54) antibody was verified by incubating human bone met tissue sections with P-Cdc6 (Ser54) antibody (1 µg/ml) (left panel) or with the same antibody pre-absorbed with a phosphor-Cdc6 (Ser54) peptide (100 µg/ml) (right panel). Both antibody and peptide were from Santa Cruz Biotech. The staining in the tissue slide incubated with the pre-absorbrd antibody was mostly abolished as compared with that incubated with the specific P-Cdc6 (Ser54) antibody.
A B
C E
A E+A
Fig. S2
C E
A E+A
Figure S2. Combination treatment with enzalutamide and AZD7762 inhibited the growth of prostate tumor xenografts (Related to Figure 6). A. Representative IVIS images showing that in VCaP xenografts, combination treatment reduced tumor growth more than the control treatment, enzalutamide or AZD7762 treatment alone. B. Representative images showing that in C4-2b subcutaneous model, combination treatment reduced tumor growth more than the control treatment, enzalutamide or AZD7762 treatment alone. C, control; E, enzalutamide; A, AZD7762, E+A, enzalutamide+AZD7762.
Fig. S3
Figure S3. Combination treatment with enzalutamide and AZD7762 decreased the expression and phosphorylation of CDC6 and increased the phosphorylation of ATM in prostate tumor xenografts (Related to Figure 6). A. IHC analysis demonstrated that the combination of enzalutamide and AZD7762 significantly decreased CDC6 phosphorylation more so than the control treatment (P=0.01208), enzalutamide (P=0.03662) and AZD7762 (P=0.03662) did in VCaP xenografts. Furthermore, the combination of two drugs reduced CDC6 protein levels more so than the control treatment (P=0.012), enzalutamide (P=0.021) and AZD7762 (P=0.021) did in these xenografts. B. The combination of enzalutamide and AZD7762 significantly increased ATM phosphorylation more so than did the control treatment (P=0.01208), enzalutamide (P=0.03662) and AZD7762 (P=0.03662) in VCaP xenografts. No significant differences in ATM protein levels with the different therapies where observed in VCaP xenografts. C. The combination of enzalutamide and AZD7762 significantly decreased s CDC6 phosphorylation more so than the control treatment (P=0.01208), enzalutamide (P=0.02144) and AZD7762 (P=0.02144) did in C4-2b xenografts. In addition, the combination treatment decreased CDC6 protein levels more so than did control treatment (P=0.01208), enzalutamide (P=0.03662) and AZD7762 (P=0.01208) in these xenografts. D. The combination of enzalutamide and AZD7762 significantly increased ATM phosphorylation more so than control treatment (P=0.01208), enzalutamide (P=0.03662) and AZD7762 (P=0.03662) did in C4-2b xenografts.
Table S1. Treatment information for patients with metastatic disease.
(Related to Figure 1)
Sample ID# ADT-yes/no ADT therapy received Date on Date off
TR08-16-40 yes Casodex Aug.,1999 7/28/2000
Lupron 10/1/1999 10/14/2001
DES 10/14/2001 4/18/2003
TR08-16-42 yes Lupron 5/26/2004 11/16/2004
Xopondex 10/12/2004 5/9/2005
Casodex 4/13/2004 5/26/2004
DES 3/20/2005 5/9/2005
TR12-10-42 yes Casodex 10/26/2000 unk
Lupron 10/16/2000
TR08-16-44 yes Flutamide 10/16/2000 unk
Lupron 10/16/2000 1/26/2006
TR08-16-45 yes Casodex 6/29/2004 unk 2004
Lupron 6/29/2004 unk 2004
Casodex Jul. 2000 Apr. 2001
Lupron Jul. 2000 Apr. 2001
TR08-16-46 yes Casodex 6/24/2004 3/1/2005
Lupron 7/6/2004 12/21/2004
TR08-16-47 yes Lupron unk 1999 1/30/2003
Casodex unk 1999 4/18/2001
TR12-10-45 yes Lupron Jun. 2000 unk 2001
Casodex Jun. 1999 unk 2001
TR08-16-49 yes Lupron unk 1999 unk 2006
Flutamide unk unk 2005
Casodex Nov. 2005 unk 2006
Table S2. Cell cycle analysis (Related Figure 2E-F, Figure 3C-D and Figure 5B-C). VCaP and C4-2b Cells were treated with negative control siRNA (NCsi), ARsi, or CDC6si 24 h prior to AZD7762 (AZD) treatment for 48 h, or pretreated with 1 µM enzalutamide (ENZ) or vehicle control DMSO, followed by treatment with DMSO, ENZ, AZD or ENZ+AZD for 48 h. Cells were stained with propidium iodide, and analyzed on a FACS Canto II flow cytometer (BD Biosciences). Cell cycle profiles and quantitative data were obtained using FlowJo software (Tree Star Inc.)
VCaP C4-2b Sub-G1 G0-G1 S G2-M
NCsi+DMSO NCsi+AZD ARsi+DMSO ARsi+AZD NCsi+DMSO NCsi+AZD ARsi+DMSO ARsi+AZD 3.77 10.65 10.20 15.56 65.04 60.43 79.36 71.24 7.89 7.48 2.30 2.87 16.70 15.15 6.51 8.30
4.51 5.11 4.54 8.44 63.81 67.02 74.86 69.24 7.89 7.65 2.98 2.45 16.90 18.12 10.23 9.63
Sub-G1 G0-G1 S G2-M
NCsi+DMSO NCsi+AZD CDC6si+DMSO CDC6si+AZD NCsi+DMSO NCsi+AZD CDC6si+DMSO CDC6si+AZD 3.78 16.13 6.86 26.53 69.47 50.97 64.40 48.53 3.60 5.37 4.72 3.64 16.53 21.37 17.63 15.47
4.38 4.76 8.13 20.83 65.53 67.97 70.23 62.83 8.75 8.44 3.77 3.40 16.97 18.60 13.67 10.86
Sub-G1 G0-G1 S G2-M
DMSO ENZ AZD ENZ+AZD DMSO ENZ AZD ENZ+AZD 5.70 10.69 11.77 18.70 72.53 71.67 66.87 65.63 3.62 2.94 7.56 3.36 15.00 12.23 9.67 9.87
4.82 6.49 6.08 12.11 63.93 66.70 67.17 62.70 7.98 4.02 6.03 3.41 16.93 14.50 19.00 13.80
Table S3. Statistical analysis of cell cycle data in p53si and AZD treated C4-2b cells (Related to figure 2J). C4-2b Cells were treated with 20 nM negative control siRNA (NCsi), or p53si 24 h prior to AZD7762 (AZD) treatment for 48 h. Cells were stained with propidium iodide, and analyzed on a FACS Canto II flow cytometer (BD Biosciences). Cell cycle profiles and quantitative data were obtained using FlowJo software (Tree Star Inc.). Statistical analysis was performed using two-tailed Student t-test.
Condition Sub-G1 (%) G0-G1 (%) S (%) G2-M (%)
NCsi+DMSO
NCsi+AZD
P53si_7+DMSO
P53si_7+AZD
P53si_9+DMSO
P53si_9+AZD
7.07 55.81 9.81 17.06 8.93 57.00 9.09 16.52 8.47 47. 84 7.78 17.94 25.8 40.83 5.98 13.72 9.17 47.57 7.94 17.56 24.3 39.06 5.87 13.76
Comparison p-value
NCsi+DMSO: p53si_7+DMSO
NCsi+DMSO: p53si_9+DMSO
NCsi+AZD:p53si_7+AZD
NCsi+AZD:p53si_9+AZD
Sub-G1 G0-G1 S G2-M
0.6615 0.0296 0.0041 0.2821
0.4304 0.0429 0.0094 0.4605
0.0008 0.0023 0.0096 0.0251
0.0141 0.0022 0.0108 0.0021