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. 2017 Jun 23;292(25):10347-10363.
doi: 10.1074/jbc.M117.792192. Epub 2017 May 10.

Pharmacological Targeting of RAD6 Enzyme-Mediated Translesion Synthesis Overcomes Resistance to Platinum-Based Drugs

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Free PMC article

Pharmacological Targeting of RAD6 Enzyme-Mediated Translesion Synthesis Overcomes Resistance to Platinum-Based Drugs

Matthew A Sanders et al. J Biol Chem. .
Free PMC article

Abstract

Platinum drug-induced cross-link repair requires the concerted activities of translesion synthesis (TLS), Fanconi anemia (FA), and homologous recombination repair pathways. The E2 ubiquitin-conjugating enzyme RAD6 is essential for TLS. Here, we show that RAD6 plays a universal role in platinum-based drug tolerance. Using a novel RAD6-selective small-molecule inhibitor (SMI#9) targeting the RAD6 catalytic site, we demonstrate that SMI#9 potentiates the sensitivities of cancer cells with innate or acquired cisplatin or oxaliplatin resistance. 5-Iododeoxyuridine/5-chlorodeoxyuridine pulse-labeling experiments showed that RAD6 is necessary for overcoming cisplatin-induced replication fork stalling, as replication-restart was impaired in both SMI#9-pretreated and RAD6B-silenced cells. Consistent with the role of RAD6/TLS in late-S phase, SMI#9-induced DNA replication inhibition occurred preferentially in mid/late-S phase. The compromised DNA repair and chemosensitization induced by SMI#9 or RAD6B depletion were associated with decreased platinum drug-induced proliferating cell nuclear antigen (PCNA) and FANCD2 monoubiquitinations (surrogate markers of TLS and FA pathway activation, respectively) and with attenuated FANCD2, RAD6, γH2AX, and POL η foci formation and cisplatin-adduct removal. SMI#9 pretreatment synergistically increased cisplatin inhibition of MDA-MB-231 triple-negative breast cancer cell proliferation and tumor growth. Using an isogenic HCT116 colon cancer model of oxaliplatin resistance, we further show that γH2AX and monoubiquitinated PCNA and FANCD2 are constitutively up-regulated in oxaliplatin-resistant HCT116 (HCT116-OxR) cells and that γH2AX, PCNA, and FANCD2 monoubiquitinations are induced by oxaliplatin in parental HCT116 cells. SMI#9 pretreatment sensitized HCT116-OxR cells to oxaliplatin. These data deepen insights into the vital role of RAD6/TLS in platinum drug tolerance and reveal clinical benefits of targeting RAD6 with SMI#9 for managing chemoresistant cancers.

Keywords: DNA damage response; breast cancer; colon cancer; platinum therapy; replication fork arrest; small molecule; tumor growth inhibition; ubiquitin; ubiquitin conjugation.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Inhibition or depletion of RAD6 enhances sensitivities of cancer cells to cisplatin and oxaliplatin. MDA-MB-231 cells were pretreated with SMI#9 (A) or Smartpool RAD6B siRNAs (B and C) or their corresponding controls and then exposed to various concentrations of cisplatin. B, western blot analysis of RAD6 and β-actin expressions in two independent transfections with SMARTpool RAD6B siRNAs or nontarget (NT) siRNA. D, sensitivities of parental HCT116 and isogenic HCT116-OxR cells to oxaliplatin. E, HCT116-OxR cells were pretreated with SMI#9 followed by exposure to the indicated doses of oxaliplatin. Proliferating cells were measured by MTT assay. Data are mean ± S.D. of triplicate experiments. Data sets in A–D were analyzed by one-way ANOVA. F, MDA-MB-231 cells treated with vehicle, SMI#9 (1 μm), cisplatin (0.5 or 1 μm), or a combination of SMI#9 + cisplatin were reseeded at 100 cells per well for colony formation assay (n = 3). Two independent experiments were performed. G, HCT116-OxR cells maintained in 10 μm oxaliplatin in the presence or absence of SMI#9 (1 μm) were reseeded at indicated densities for colony formation. Results in F and G are mean ± S.D. percent colony formation efficiency from three independent experiments and analyzed by Student's t test.
Figure 2.
Figure 2.
RAD6 inhibition or depletion decreases PCNA ubiquitination and FANCD2 steady-state levels. A and B, western blot analysis of the indicated proteins from MDA-MB-231 whole-cell lysates prepared from CDDP-treated cells with or without SMI#9 pretreatment (A) or two independent transfections with SMARTpool RAD6B siRNAs or nontarget (NT) siRNA (B). A replicate blot of PCNA analysis of SMI#9 suppression of CDDP-induced PCNA monoubiquitination is shown in A. C, indicated MDA-MB-231 lysates were immunoprecipitated with anti-PCNA antibody, and the immunoprecipitates and depleted supernatants were western-blotted (WB) with anti-ubiquitin antibody. Efficacy of PCNA immunoprecipitation was verified by reprobing the stripped blots with anti-PCNA antibody. Input lysates were analyzed for PCNA steady-state levels. Arrow indicates monoubiquitinated PCNA, and asterisks indicate the positions of heavy and light chains of IgG. Graph on the right shows the relative levels of monoubiquitinated PCNA in CDDP and SMI#9 + CDDP-treated samples. D, western blot analysis of the indicated proteins in cytoplasmic and nuclear subfractions of MDA-MB-231 cells. E, western blot analysis of the indicated proteins in whole-cell lysates prepared from HCT116 or HCT116-OxR cells exposed to 1 or 10 μm oxaliplatin, respectively. All protein profiles were captured by simultaneous analysis of the indicated proteins from 4 to 20%, 7 to 18% (PCNA blot in A), and 4 to 12% (PCNA immunoprecipitation) gradient gels.
Figure 3.
Figure 3.
SMI#9 inhibits cisplatin (CDDP)-induced FANCD2, RAD6, PCNA, and γH2AX nuclear foci formation in MDA-MB-231 cells. Immunofluorescence staining of FANCD2 (green) and PCNA (red) (A), RAD6 (green) and PCNA (red) (B), and γH2AX (red) (C) counterstained with DAPI to locate the nucleus. D, quantitation of foci-positive (containing more than five foci) cells. Approximately 30–75 cells from three to five fields and two independent experiments were scored. Data were analyzed by two-tailed Student's t test. Representative images are shown. Scale bar, 10 μm.
Figure 4.
Figure 4.
Reinitiation of stalled replication forks is impeded by RAD6 inhibition or silencing. DNA labeling (A), SMI#9 and CDDP treatment protocol for DNA fiber (B), and global DNA replication (D and E) analysis in MDA-MB-231 cells are shown. Cells were pretreated with vehicle or SMI#9 (B and D) or nontarget (NT) or SMARTpool RAD6B siRNAs (E) before labeling with IdU and then treated with CDDP followed by post-labeling with CldU. DNA fibers or cells were immunostained with IdU (red) and CldU (green) antibodies and counterstained with DAPI (blue, D and E). E also shows immunofluorescence staining of RAD6 in NT or RAD6B siRNA-transfected cells. Representative images are shown. Approximately 35–75 individual fibers (B) or cells (D and E) were analyzed for each experiment, and the average of three independent experiments is presented. Quantification of percent of stalled or restarted forks (C) or nuclei labeled with IdU and CldU (F) is shown. C, * and **, p < 0.01 indicates significant decrease or increase in restarted or stalled forks, respectively, between CDDP and SMI#9 or SMI#9 + CDDP groups. Scale bars, 10 μm. G, kinetics of CDDP-DNA adduct removal. MDA-MB-231 cells were treated with 5 μm CDDP for 4 h with or without SMI#9 (5 μm) pretreatment. Cells were rinsed and allowed to recover to facilitate repair of DNA adducts. Genomic DNA isolated from cells at the indicated recovery times were analyzed for cisplatin-DNA adducts by ELISA. Data are expressed mean ± S.D. from two independent experiments.
Figure 5.
Figure 5.
RAD6 inhibitor preferentially inhibits DNA synthesis in mid- to late-S phase cells. Vehicle or SMI#9-pretreated MDA-MB-231 cells were labeled with IdU, then treated with CDDP, and post-labeled with CldU as in Fig. 4A. A, representative images of cells in early-(E) or mid- to late-S (M/L) phases. Replication foci were immunostained with anti-IdU- (red) and anti-CldU (green)-specific antibodies. The mean integrated pixel intensities of CldU and IdU for cells in early- and mid- to late-S phases were measured by ImageJ, and the ratios of CldU to IdU mean pixel intensity are shown for each representative cell in the early- and mid- to late-S phases (A). Scale bar, 10 μm. B, percent of IdU and CldU colabeled populations in each phase; C, percent of cells in each phase. Results are mean ± S.D. of ∼20 cells counted for each treatment group from duplicate experiments.
Figure 6.
Figure 6.
Loss of RAD6 impedes cisplatin-induced recruitment and colocalization of DNA damage-response proteins. MDA-MB-231 cells transiently transfected with NT or SMARTpool RAD6B siRNAs were treated overnight with CDDP, fixed, and immunostained for POL η (green) and PCNA (red) (A), RAD6 (green) (B), or POL η (green) and FANCD2 (red) (C). Representative images from a typical experiment are shown. Scale bars, 10 μm. D, cells with >5 colocalized foci in NT and RAD6B siRNA-transfected cells were scored from ∼30 cells in four to six fields, and data were analyzed by two-tailed Student's t test. E, vehicle or SMI#9 pretreated MDA-MB-231 cells were exposed to CDDP followed by post-labeling with CldU. Cells were fixed and immunostained for CldU (green) and γH2AX (red). Scale bar, 10 μm. F, cells were analyzed by ImageJ for γH2AX/CldU foci colocalization from ∼30 cells in five to nine fields, and data were analyzed by two-tailed Student's t test. Representative images from a typical experiment are shown.
Figure 7.
Figure 7.
SMI#9 treatment enhances sensitivity to cisplatin and inhibits tumor growth. A–D, MDA-MB-231 cells were treated overnight with vehicle, SMI#9, CDDP, or SMI#9 + CDDP, and 5 × 106 viable cells were implanted into the mammary fat pads of female nude mice. A, tumor volumes (mean ± S.E.); C, vertical scatter plots of mass of excised tumors at time of sacrifice. B, H&E analysis of tumors. Inset panels show enlarged images. Arrow in control shows angiogenesis; long and short arrows in SMI#9 and SMI#9 + CDDP indicate mitotic catastrophe and apoptosis, respectively. D, immunohistochemical staining of RAD6 in control (top left panel) and SMI#9 (bottom left panel) groups and PCNA in control (top right panel) and SMI#9 (bottom right panel) groups. Inset panels show enlarged images of RAD6 and PCNA distribution in the control and SMI#9-treated groups. Original magnification, ×40. E–H, MDA-MB-231 cells (5 × 106) were implanted orthotopically and when the tumors reached ∼150 mm3, mice were randomly assigned to the following groups: vehicle control, CDDP (4 mg/kg body weight, once/week, intraperitoneal); SMI#9 (2.5 mg/kg body weight, twice/week, intratumoral); or a combination of SMI#9 and CDDP. E, tumor volumes (mean ± S.E.); F, vertical scatter plots of mass of excised tumors at time of sacrifice at 24 days. G, western blot analysis of the indicated proteins in representative tumor lysates from each treatment group. The position of monoubiquitinated PCNA in CDDP-treated tumors is indicated by an arrow and verified in the adjacent blot. * indicates nonspecific band recognized by anti-mouse antibodies. H, H&E analysis. Arrow in control shows angiogenesis; long and short arrows in SMI#9 and SMI#9 + CDDP indicate apoptosis and multinucleated giant cells, respectively. Original magnification ×40. Data are analyzed by one-way ANOVA and two-tailed Student's t test.

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