1. Volume 18, Issue 8, Pages 1970-1981 (February 2017)
Targeting DNA Damage Response in Prostate Cancer by Inhibiting Androgen Receptor- CDC6-ATR-Chk1 Signaling 
Styliani Karanika, Theodoros Karantanos, Likun Li, Jianxiang Wang, Sanghee Park, 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, Timothy C. Thompson 
Cell Reports 
Volume 18, Issue 8, Pages (February 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 1970-1981DOI: (10.1016/j.celrep.2017.01.072)
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3. 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 <
(C and D) Protein stability analysis of Cdc6. VCaP (C) and C4-2b (D) cells were transfected with 20 nM Arsi-1 or NCsi for 48 hr prior to the treatment with 100 μg/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.
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4. 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 μM 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 μM KU (KU), an ATM inhibitor; 1 and 3 μM 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) p53 wild-type C4-2b cells were transfected with 20 nM p53si or NCsi for 48 hr prior to western blotting analysis. (J) C4-2b cells were transfected with 20 nM p53si 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.
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5. Figure 3
CDC6 Knockdown Increases the Sensitivity of PCa Cells to Treatment with Chk1/2 Inhibitor AZD through Inhibition of TopBP1-ATR-Chk1 Signaling
VCaP and C4-2b cells were transfected with 20 μM 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.
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6. 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 μM 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.
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7. 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 μM 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 ATM phosphorylation 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 = ) 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 = ) 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 μg of CDC6 plasmid DNA or control empty vector DNA for 24 hr prior to the treatment with DMSO, 1 μM ENZ, 200 nM AZD or ENZ+AZD for 48 hr. (F) CDC6 protein levels markedly increased after enforced CDC6 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.
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8. Figure 6
Combination Treatment with ENZ and AZD7762 Inhibited the Growth of Prostate Tumor Xenografts
VCaP orthotopic xenografts, C4-2b subcutaneous xenografts, and MDA 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 = 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 = , indicated by a pound sign). (C) IHC analysis demonstrated that the combination of ENZ and AZD7762 significantly decreased CDC6 phosphorylation (p = ) and Cdc6 protein levels (p = 0.012) and significantly increased ATM phosphorylation (p = ) 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 = ) and CDC6 protein levels (p = ) and significantly increased ATM phosphorylation (p = ) 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 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 = for tumor volume, and p = for tumor wet weight, indicated by a pound sign in the panels). ∗p < 0.05.
See also Figure S2.
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