Volume 64, Issue 3, Pages 493-506 (November 2016) Polo-like Kinase-1 Regulates Myc Stabilization and Activates a Feedforward Circuit Promoting Tumor Cell Survival  Daibiao Xiao, Ming Yue, Hexiu Su, Ping Ren, Jue Jiang, Feng Li, Yufeng Hu, Haining Du, Hudan Liu, Guoliang Qing  Molecular Cell  Volume 64, Issue 3, Pages 493-506 (November 2016) DOI: 10.1016/j.molcel.2016.09.016 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 64, 493-506DOI: (10.1016/j.molcel.2016.09.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 PLK1 Inhibition Decreases N-Myc Expression and Myc Transcriptional Activity (A) PLK1 depletion by specific shRNAs in MYCN-amplified Kelly and BE-2C cells. Relative PLK1 mRNA levels were quantitated by real-time qPCR. Data shown represent the means (±SEM) of triplicates. (B) PLK1, N-Myc, and c-caspase-3 protein levels were analyzed by immunoblot, with actin as a loading control. (C) Immunoblot detection of N-Myc protein levels in Kelly cells after 24 hr of treatment with BI2536 or BI6727 (20 nM). Actin was used as a loading control. (D) Time-course analysis of N-Myc protein levels by immunoblot upon BI2536 (20 nM) treatment, with actin as a loading control. (E–G) Immunoblot detection of N-Myc levels in the presence of different PLK1 inhibitors. MYCN-amplified cells from neuroblastoma and small cell lung carcinoma were treated with 20 nM BI2536 (E), BI6727 (F), or GSK461364 (G) for 24 hr. Actin was used as a loading control. (H) Real-time qPCR analysis of representative Myc targets in Kelly cells upon BI2536 (20 nM) treatment for 24 hr. Data shown represent the means (±SEM) of triplicates. ∗p < 0.05; ∗∗p < 0.01. Molecular Cell 2016 64, 493-506DOI: (10.1016/j.molcel.2016.09.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 PLK1 Enhances N-Myc Protein Stability (A and B) Real-time qPCR analysis of MYCN expression upon PLK1 shRNA knockdown (A) or pharmacological inhibition (B) in Kelly and BE-2C cells. Data shown represent the means (±SEM) of triplicates. ∗p < 0.05. (C and D) MG132 rescued N-Myc loss resulted from PLK1 inhibition. Kelly cells were infected with control shRNA or two specific PLK1 shRNAs for 36 hr (C) or treated with BI2536 (20 nM) for 24 hr (D). Before harvest, cells were treated with MG132 (10 μM) for 6 hr as indicated. N-Myc and PLK1 levels were analyzed by immunoblot, with actin as a loading control. (E) Kelly cells were subjected to BI2536 (20 nM) or DMSO (control solvent) for 2 hr as indicated, treated with cycloheximide (CHX, 20 μg/mL), and then harvested at the indicated time points. N-Myc levels were analyzed by immunoblot, with actin as a loading control. (F) Quantitation of N-Myc stability assays. N-Myc band density was normalized to actin and then normalized to t = 0 controls. See also Figure S1. Molecular Cell 2016 64, 493-506DOI: (10.1016/j.molcel.2016.09.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 PLK1 Stabilizes N-Myc by Counteracting Fbw7-Mediated Degradation (A) Time-course analysis of T50 phosphorylation within N-Myc and T58 phosphorylation within c-Myc by immunoblot upon BI2536 (20 nM) treatment in Kelly and HeLa cells. Actin was used as a loading control. (B) In vitro kinase analysis of bacterially expressed GST-N-Myc and GST-c-Myc. Kinase used in the assays was constitutively active human PLK1 (T210D). Loading controls were shown as Coomassie blue staining in the bottom panels. GST and casein were used as negative and positive controls for kinase assays, respectively. (C) Fbw7 depletion rescued N-Myc loss resulting from PLK1 inhibition. Kelly or BE-2C cells were infected with the control shRNA or validated shRNAs targeting Fbw7 or Huwe1 for 24 hr and then treated with or without BI2536 (20 nM) for 24 hr. N-Myc levels were analyzed by immunoblot, with actin as a loading control. (D) PLK1 kinase activity was essential for counteracting Fbw7-mediated N-Myc degradation. The 293T cells were transfected with plasmids encoding N-Myc, Fbw7γ, and PLK1 WT or kinase mutant (K82R) as shown. Where indicated, cells were treated with MG132 (10 μM) for 6 hr before harvest. Protein levels were analyzed by immunoblot, with actin as a loading control. (E) 293T cells were transfected with plasmids encoding N-Myc, Fbw7γ, and PLK1 as shown. Then, 24 hr later, cells were treated with CHX (20 μg/mL) and harvested at the indicated time points. Protein levels were analyzed by immunoblot, with actin as a loading control. The asterisk denotes a non-specific band detected with hemagglutinin antibody. (F) Quantitation of N-Myc stability assays. N-Myc band density was normalized to actin and then normalized to t = 0 controls. See also Figure S2. Molecular Cell 2016 64, 493-506DOI: (10.1016/j.molcel.2016.09.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 PLK1 Impairs Fbw7 Protein Stability (A) Real-time qPCR analysis of FBW7 expression upon PLK1 shRNA knockdown. Data shown represent the means (±SEM) of triplicates. (B) Protein levels of Fbw7 with or without PLK1 shRNA knockdown. Kelly or BE-2C cells were infected with the control shRNA or validated shRNAs targeting PLK1 for 48 hr. Cell lysates were immunoprecipitated with specific Fbw7 antibodies, and Fbw7 protein levels were analyzed by immunoblot. PLK1 and N-Myc levels were directly analyzed by immunoblot, with actin as a loading control. (C) 293T cells transfected with plasmids encoding Fbw7γ in the presence or absence of PLK1 as indicated. Then, 24 hr later, cells were treated with CHX (20 μg/mL) and harvested at the indicated time points. Fbw7γ and PLK1 levels were analyzed by immunoblot, with actin as a loading control. (D) Quantitation of Fbw7γ stability assays. Fbw7 band density was normalized to actin and then normalized to t = 0 controls. (E and F) MYCN-amplified cells from neuroblastoma and small cell lung carcinoma were treated with or without 20 nM BI2536 (E) or BI6727 (F) for 24 hr. Where indicated, cells were treated with MG132 (10 μM) for 6 hr before harvest. Cell lysates were immunoprecipitated with specific Fbw7 antibodies, and Fbw7 protein levels were analyzed by immunoblot. N-Myc, cyclin E, and Mcl1 protein levels were directly analyzed by immunoblot, with actin as a loading control. See also Figure S3. Molecular Cell 2016 64, 493-506DOI: (10.1016/j.molcel.2016.09.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 PLK1 Promotes Fbw7 Phosphorylation and Autopolyubiquitination (A) Endogenous interaction between Fbw7 with PLK1. Lysates from Kelly or BE-2C cells were subjected to immunoprecipitation using anti-PLK1 antibody, and the co-precipitated Fbw7 was detected by immunoblot using anti-Fbw7 antibody. (B) In vivo Fbw7 polyubiquitination in the presence or absence of PLK1 mutants. The 293T cells were transfected with plasmids expressing ubiquitin, Fbw7γ, and increasing amounts of PLK1 mutants as indicated, followed by lysis in RIPA buffer containing 6 M urea. Ubiquitin-conjugated Fbw7 proteins were immunoprecipitated with FLAG tag antibody and subjected to immunoblot assay with ubiquitin antibody. (C) 293T cells were transfected with plasmids expressing Fbw7γ WT, S58/T284A, or S58/T284D mutant as indicated. Then, 24 hr later, cells were treated with CHX (20 μg/mL) and harvested at the indicated time points. Fbw7γ levels were analyzed by immunoblot, with actin as a loading control. (D) Quantitation of Fbw7γ stability assays. Fbw7 band density was normalized to actin and then normalized to t = 0 controls. (E) In vitro kinase analysis of bacterially expressed GST-Fbw7γ. Kinases used in the assays were constitutively active human PLK1 (T210D) or a kinase-defective mutant of PLK1 (K82R). Loading controls were shown as Coomassie blue staining in the bottom panels. GST and casein were used as negative and positive controls for kinase assays, respectively. (F) Bacterially expressed GST-Fbw7γ WT or its S58/T284A mutant was subjected to in vitro kinase assays as performed in (E). (G) PLK1 promoted Fbw7 self-polyubiquitination in vivo. The 293T cells were transfected with plasmids expressing ubiquitin, Fbw7γ or its ΔF mutant, and increasing amounts of PLK1 as indicated, followed by detection procedures as shown in (B). (H) In vivo polyubiquitination of Fbw7 WT and its mutants. The 293T cells were transfected with plasmids expressing Fbw7γ WT, S58/T284A, or S58/T284D mutant as indicated, followed by detection procedures as shown in (B). See also Figures S4 and S5. Molecular Cell 2016 64, 493-506DOI: (10.1016/j.molcel.2016.09.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 N-Myc Directly Activates PLK1 Transcription (A) Real-time qPCR analysis of PLK1 expression upon MYCN shRNA knockdown in Kelly cells. Data shown represent the means (±SEM) of triplicates. (B) Immunoblot detection of PLK1 levels after 48 hr of MYCN shRNA knockdown, with actin as a loading control. (C) Schematic representation of Myc response element (Myc RE) in the PLK1 promoter and its mutant (REmut). The +1 denotes the transcription initiation site. (D) Luciferase assays performed using control, Myc RE, and REmut constructs in the presence or absence of exogenous N-Myc. Data shown represent the means (±SEM) of triplicates. (E) Binding of N-Myc to the PLK1 and nucleolin promoters analyzed by ChIP assays in Kelly cells. Results shown presented averages (±SEM) of fold changes between N-Myc ChIP and isotype IgG control in triplicates. (F) Relative expression of PLK1 in 643 primary neuroblastoma tumors. Non-Amp, MYCN-nonamplified tumors (n = 550); Amp, MYCN-amplified tumors (n = 93). (G) Correlation between mRNA levels of PLK1 and MYCN in 643 primary neuroblastoma tumors. (H) Representative N-Myc and PLK1 immunochemistry staining in MYCN-amplified neuroblastoma tumors; sections from 20641 (a MYCN non-amplified, low-stage neuroblastoma tumor) were used as a negative control. The scale bar represents 50 μm. (I) Kaplan-Meier survival curves of neuroblastoma patients based on PLK1 expression. Data were generated from the dataset accessible at http://r2.amc.nl. ∗p < 0.05; ∗∗p < 0.01. See also Figure S6 and Table S1. Molecular Cell 2016 64, 493-506DOI: (10.1016/j.molcel.2016.09.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Combined PLK1 and Bcl2 Inhibition Synergistically Impairs Viabilities of MYC-Overexpressing Cells In Vitro and Inhibits MYCN-Amplified Xenograft Tumor Growth In Vivo (A) Viabilities of P493 cells were examined by phosphatidylinositol (PI)-Annexin V staining after 24 hr of treatment with BI2536 or BI6727 (20 nM) in the presence or absence of Tet (200 nM). Data shown represent the means (±SEM) of triplicates. (B) Immunoblot analysis of Myc levels in the indicated tumor cells, with actin as a loading control. (C) Viabilities of indicated tumor cells were examined by PI-Annexin V staining after 24 hr of treatment with BI6727 (10 nM), ABT199 (5 μM), and a BI6727/ABT199 combination. P493 cells had been pre-treated with or without Tet (200 nM) for 24 hr before administration of BI6727 and/or ABT199. Data shown represent the means (±SEM) of triplicates. (D) Viabilities of Kelly cells upon Fbw7 siRNA knockdown were examined by PI-Annexin V staining after 24 hr of treatment with BI6727 (10 nM), ABT199 (5 μM), and a BI6727/ABT199 combination. Data shown represent the means (±SEM) of triplicates. (E–G) Xenograft tumor growth (E) and tumor weight (F) of Kelly and body weight (G) of mouse after 3 weeks of treatment with vehicle (DMSO), BI6727 (10 mg/kg), ABT199 (20 mg/kg), and a BI6727/ABT199 combination. Six tumors were analyzed for each group. (H–J) Xenograft tumor growth (H) and tumor weight (I) of NCI-H526 and body weight (J) of mouse after 3 weeks of treatment as performed in (E). (K) N-Myc and c-caspase-3 immunochemistry staining in tumor sections derived from (F). Representative staining micrographs were shown in the upper panel. The lower panel presented quantification of N-Myc and c-caspase-3 immunostaining. Six tumors were analyzed each group. Scale bars represent 50 μm. (L) Model depicting the PLK1-Myc signaling circuit in promotion of tumorigenesis. PLK1 and Myc create a positive, feedforward activation loop in MYC-overexpressing cells that is essential for sustaining mutual, high expression, leading to amplification of Myc-regulated oncogenic programs. See text for more details. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005. See also Figure S7. Molecular Cell 2016 64, 493-506DOI: (10.1016/j.molcel.2016.09.016) Copyright © 2016 Elsevier Inc. Terms and Conditions