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. 2012 Mar;10(3):369-77.
doi: 10.1158/1541-7786.MCR-11-0497. Epub 2012 Jan 4.

Inhibition of the Nedd8 system sensitizes cells to DNA interstrand cross-linking agents

Younghoon Kee  1 , Min Huang, Sophia Chang, Lisa A Moreau, Eunmi Park, Peter G Smith, Alan D D'Andrea
Affiliations

Inhibition of the Nedd8 system sensitizes cells to DNA interstrand cross-linking agents

Younghoon Kee et al. Mol Cancer Res. 2012 Mar.

Abstract

The Fanconi anemia pathway is required for repair of DNA interstrand cross-links (ICL). Fanconi anemia pathway-deficient cells are hypersensitive to DNA ICL-inducing drugs such as cisplatin. Conversely, hyperactivation of the Fanconi anemia pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the Fanconi anemia pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8 conjugation system increases cellular sensitivity to DNA ICL-inducing agents. Mechanistically, the Nedd8 inhibition, either by siRNA-mediated knockdown of Nedd8-conjugating enzymes or treatment with a Nedd8-activating enzyme inhibitor MLN4924, suppressed DNA damage-induced FANCD2 monoubiquitination and CHK1 phosphorylation. Our data indicate that inhibition of the Fanconi anemia pathway is largely responsible for the heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combination of Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistant cancer cells.

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Figures

Figure 1
Figure 1. Knockdown of Nedd8 conjugation system disrupts FANCD2 monoubiquitination and CHK1 phosphorylation
A. Representative western blot for the candidate siRNA screens. HeLa cells were treated with siRNAs against various E3 ligase components. Forty eight to sixty hours later cells were treated with UV (30J/m2), then the cells were harvested and lysed for the western blot analysis. B. HeLa cells were treated with UBA3 siRNA#1, followed by UV treatment. Cells were harvested at indicated time points. C. HeLa cells were transfected with the siRNAs and 48 hours later cells were incubated with 10μM BrdU for 40 miutes. Integrated BrdU was visualized using FITC-conjugated BrdU antibody and quantified by FACS analysis. D. HCT116 cells were treated with two different UBE2M siRNAs, followed by UV (30J/m2) treatment before harvested for western blot analysis. E. HCT116 cells were treated with UBE2M#1 siRNA, followed by PUVA (Psoralen+UVA) treatment, then harvested at indicated time points for western blot analysis. F and G. HeLa cells were treated with UBE2M siRNAs followed by UV (30J/m2) and PUVA treatment, respectively, for western blot analysis.
Figure 2
Figure 2. Knockdown of UBE2M abrogates damage-inducible FANCD2 foci formation
HeLa cells were treated with control or UBE2M#1 siRNA for 48 hours, followed by HU (2mM) treatment for 12hours for analyzing damage-inducible foci formation.
Figure 3
Figure 3. MLN4924 suppresses damage-inducible CHK1 phosphorylation and FANCD2 monoubiquitination
A. HeLa cells were treated with 1μM of MLN4924 or DMSO vehicle for 20 hours, followed by Psoralen+UVA (PUVA) treatment, and cells were harvested at indicated time points for western blottings. B. HCT116 cells were treated with 3μM of MLN4924 or DMSO vehicle for 18 hours, followed by UV (30J/m2) treatment, and cells were harvested at indicated time points for western blottings. C. HeLa cells were treated with Cisplatin (10μM) for 16 hours, followed by treatment with MLN4924 (2μM) or DMSO for indicated times before harvested for western blottings. D. HeLa cells were treated with indicated concentrations of MLN4924 for 12 hours followed by 12 hour-treatment of 10μM Cisplatint. E. HeLa cells were treated with MLN4924 (1μM) or DMSO for 16 hours, followed by HU (2mM) treatment for 20 hours before fixation for the immunofluoresence assay. Experiments were performed in triplicate and the average numbers of foci were plotted as graphs. F. HeLa cells were synchronized in S phase using double thymidine block, then the cells were released into fresh medium containing nocodazole. During the last 3 hours of incubation cells were treated with DMSO, MG132, or MLN4924 before harvested. The lysates were analyzed by western blottings. Protein band intensities were measured and plotted as graphs.
Figure 4
Figure 4. Inhibition of the Nedd8 conjugation system sensitizes cells to DNA crosslinking agents and PARP1 inhibitor
A. HeLa cells were treated with siRNAs against control, FANCM, or UBE2M for 48 hours, followed by MMC treatment, and the cells were incubated for 7-10 days before fixation (see Methods for detail). B. HCT116 cells were treated with 20nM of MLN4924 or DMSO control, and next day MMC was added for further incubation of 7 days before fixation. C. Left panel. 2008 ovarian cancer cells harboring vector (2008) or FANCF cDNA (2008+F) were treated with 20nM of MLN4924, followed by treatment with indicated amount of Cisplatin. After 5 days, viability of cells was measured as described in Methods. Right panel. The 2008 and 2008+FANCF cells were treated with 2μM of MLN4924 for 20 hours, followed by UV (30J/m2) irradiation, and the cells were harvested at indicated time points. D and E. Chromosomal aberrations were measured in 293T cells that were treated with 100nM of MLN4924 (D) or transfected with siRNAs (E), followed by MMC treatment for 3 days.
Figure 5
Figure 5. A protein neddylation event is required upstream of the FA pathway
A schematic model for the Nedd8-mediated activation of the DNA damage-inducible FANCD2 monoubiquitination. While interruption of the Nedd8 conjugation system suppresses the ATR-mediated signaling pathways, particularly CHK1 phosphorylation and FANCD2 monoubiquitination, ATM-mediated pathways or PCNA ubiquitination remain unaffected. We hypothesize that specific protein neddylation events, most likely the activation of specific CRL E3 ligases, is required for damage-inducible CHK1 phosphorylation and FANCD2 monoubiquitination. We suggest that disruption in the CRL-mediated destruction of FANCM is one mechanism leading to the abrogation of the FA pathway.
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