CRL4 ubiquitin ligase stimulates Fanconi anemia pathway-induced single-stranded DNA-RPA signaling
DNA-crosslinking agents like cisplatin and mitomycin C (MMC) are indispensible for the treatment of many solid malignancies. These anticancer drugs generate DNA interstrand crosslinks (ICLs) that cause cell death by blocking replication forks. Many factors counteracting ICL-induced DNA replication stress, including the Fanconi anemia (FA) pathway, are regulated by ubiquitination and, therefore, ubiquitin ligases are potential targets for the sensitization of cancer cells to crosslinking agents. In this study, we investigated the function of the CRL4 ubiquitin ligase in modulating the response of cancer cells to ICL induction.
The two cullin paralogs CUL4A and CUL4B, which form the CRL4 ligase scaffold, were depleted in cancer cells by small interfering RNA followed by analysis of the cellular and biochemical responses to ICLs elicited upon cisplatin or MMC treatment.
We report that the combined depletion of CUL4A and CUL4B weakens an FA pathway-dependent S phase checkpoint response. CRL4 positively stimulates the monoubiquitination of FANCD2 required for the recruitment of XPF-ERCC1, a structure-specific endonuclease that, in turn, contributes to the display of single-stranded DNA (ssDNA) at ICLs. After CRL4 down regulation, the missing ssDNA results in reduced recruitment of RPA, thereby dampening activation of ATR and CHK1 checkpoint kinases and allowing for S phase progression despite ICL induction.
Our findings indicate that CRL4 promotes cell survival by potentiating an FA pathway-dependent ssDNA-RPA signaling platform installed at ICLs. The anticancer efficacy of crosslinking agents may, therefore, be enhanced by down regulating CRL4 activity.
KeywordsChemotherapy Cisplatin Crosslink - CUL4 - Fanconi anemia ssDNA
Ataxia telangiectasia-mutated and Rad3-related
Checkpoint kinase 1
Cullin-RING ubiquitin ligases
Damaged DNA-binding 1
DNA damage response
Replication protein A
Platinum- and mitomycin-based drugs are used against solid malignancies including lung, bladder, esophageal, testicular, ovarian and cervical cancer . The mechanism of action of cis-diamminedichloroplatinum (II) (cisplatin) and mitomycin C (MMC) involves the formation of DNA interstrand crosslinks (ICLs), which lead to cell death primarily by interfering with DNA replication . A common cause of treatment failure is the emergence of resistance developing in most patients even after an initially favorable response. Cancer cells avoid ICL-induced cytotoxicity by eliciting the DNA damage response (DDR), which coordinates cell cycle progression with DNA repair [3, 4]. A universal DDR trigger is DNA replication stress involving persistent stretches of single-stranded DNA (ssDNA) at stalled replication forks. The locally arising ssDNA is rapidly coated by replication protein A (RPA), thus forming ssDNA-RPA complexes that provide a platform for engagement of the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase. This serine/threonine kinase phosphorylates RPA, as well as signaling intermediates like checkpoint kinase 1 (CHK1) and histone H2AX, to trigger cell cycle checkpoints [5, 6]. The efficiency of checkpoint activation determines how cancer cells respond to chemotherapy [7, 8] and, accordingly, RPA hyperphosphorylation has been linked to increased cisplatin resistance .
The DDR cascade is driven by posttranslational modifications involving, besides phosphorylation, polypeptide modifiers like ubiquitin [10, 11]. Cullin-RING ubiquitin ligases (CRLs) contain a cullin scaffold (CUL1 to 5, CUL7 or CUL9) that recruits substrate receptors to target proteins for ubiquitination [12, 13, 14, 15]. CRL activation may require modification of cullin subunits by the ubiquitin-like modifier NEDD8 . MLN4924 (pevonedistat) is a small-molecule antagonist of this neddylation reaction, thereby inhibiting CRLs and preventing the ubiquitination and subsequent degradation of proteins . A prominent target of CRL-mediated degradation under replication stress is the replication-licensing factor CDT1, whose function is to initiate replication forks. Normally, only one round of DNA synthesis is allowed during each cell cycle [14, 18]. However, by preventing the ubiquitination and proteasomal degradation of CDT1, MLN4924 induces the superfluous initiation of extra replication forks, causing aberrant DNA re-replication [15, 19, 20].
Previous reports demonstrated that MLN4924 also sensitizes cancer cells to the cytotoxic action of cisplatin and MMC [21, 22, 23], implying that CRL inhibitors may mitigate resistance against crosslinking agents. However, the mechanism of this synergy between MLN4924 and crosslinking drugs remained unclear. It was not known which of the many possible CRLs susceptible to inhibition by MLN4924 are implicated in the response to DNA-crosslinking agents and, in particular, it was not known how CRLs affect the detection or signaling of DNA damage inflicted by these drugs. Here, we identified CRL4 as an additional player modulating the cellular sensitivity to cisplatin and MMC, and found that the cullin paralogs CUL4A and CUL4B display redundant functions in regulating cell survival after treatment with crosslinking agents. The concomitant down regulation of these exchangeable CUL4 scaffolds diminishes the Fanconi anemia (FA) pathway-dependent recruitment of XPF-ERCC1, which as part of a nuclease complex contributing to the formation of ssDNA at ICL sites. Accordingly, this CRL4 depletion interferes with the assembly of ssDNA-RPA intermediates upon cisplatin or MMC treatment, such that activation of ATR and the phosphorylation of RPA, CHK1 and H2AX are reduced. Our results indicate that CRL4 activity protects from cancer cell death after treatment with crosslinking agents by stimulating an FA pathway-induced S phase checkpoint.
Cell lines and treatment
HeLa (catalog designation CCL-2) and SKOV3 cells (catalog designation HTB-77) were purchased from ATCC and cultured in low-glucose Dulbecco’s modified Eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium, respectivel. Cell culture media (obtained from Gibco) were supplemented with 10% (v/v) fetal calf serum and 100 U/ml penicillin-streptomycin. All cells were recently tested negative for mycoplasma contamination and authenticated by short tandem repeat profiling (Microsynth). Cells were incubated at 37 °C in a humidified atmosphere under 5% CO2. The cisplatin (Sigma) solutions were prepared freshly each time in DMEM. MMC (Sigma) was dissolved as a 1.5-mM stock solution in phosphate-buffered saline (PBS) and MLN4924 (ApexBio) as a 50-mM stock solution in dimethyl sulfoxide (DMSO) and further diluted in cell culture medium. Cells were treated with crosslinking agents 3 days after siRNA transfections, except for the viability assays where the drugs were applied 2 days after transfections.
Transfections were performed with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. All siRNA sequences are shown in the Additional file 1: Table S1. The siRNA concentrations were 24 nM except for siDDB1, which was used at a concentration of 8 nM.
Resazurin was purchased from Alfa Aesar and viability measured according to the manufacturer’s instruction. Briefly, 2000 cells per well were seeded into a 96-well plate and 24 h later treated with the indicated drug concentrations. Following 2 days, resazurin was added to the cells and fluorescence measured after 3 h (LS55 luminescence Spectrometer; Perking Elmer). Cell viability was expressed as the percentage of controls obtained in the absence of cisplatin and IC50 values were calculated using GraphPad Prism.
Cell death was measured using the LDH Cytotoxicity Assay Kit (Pierce). Briefly, 5000 cells per well were seeded into a 96-well plate. After 24 h, cells were treated with increasing concentrations of cisplatin for 2 days and the released LDH was measured in the supernatant according to the manufacturer’s instruction. Results are calculated as the ratio of released LDH in relation to maximal LDH activity in each condition, and expressed as the percentage of the ratios detected with untreated controls.
Cell survival was performed as described . Briefly, cells were treated with increasing concentrations of cisplatin for 2 h, extensively washed with PBS and further incubated in fresh media without drug for 10 days. Colonies were fixed and stained with 0.25% (w/v) crystal violet solved in 80% (v/v) ethanol. Colonies composed of at least 50 cells were counted and surviving fractions were normalized to untreated controls.
Cells were treated as indicated, washed once with PBS and lysed in RIPA buffer [50 mM Tris-HCl, pH 7.5, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 150 mM NaCl, 2 mM EDTA] complemented with 1 mM N-ethylmaleimide (NEM, Sigma), 1 mM phenylmethylsulfonyl fluoride, PhosStop (Roche) and Complete Protease Inhibitor cocktail (Roche) for 10 min on ice. After sonication for 5 cycles (30 s on, 30 s off) at 4 °C (Biorupture Plus; Diagenode), protein concentration was determined by the BCA protein assay (Pierce) according to the manufacturer’s instruction. Laemmli buffer was added and boiled for 5 min at 98 °C; 10 μg of protein were separated on 4–20% Criterion TGX stain-free precast gels and transferred to nitrocellulose membranes using a Turbo transfer device (Bio-Rad). Membranes were incubated with primary antibodies (Additional file 1: Table S2) over night at 4 °C followed by incubation with fluorescence-labelled secondary antibodies for 30 min. Membranes were developed using the Odyssey CLx Imaging System and quantification of protein expression was performed using the Image Studio Lite Software (Li-Core Biosciences).
Cell cycle analysis
Replicative cells were labelled for 3 h with 5-ethynyl-2′-deoxyuridine (EdU, Sigma) and fixed in 1% (w/v) paraformaldehyde for 10 min. Coupling of Alexa Fluor 488 was performed using the Click-iT EdU Flow Cytometry Assay Kit (Invitrogen) according to the manufacturer’s instruction. DNA contents were quantified by 4′,6-diamidino-2-phenylindole (DAPI) staining. Mitotic cells were visualized by incubation with the phospho-histone H3 (pH 3) antibody (Additional file 1: Table S2) for 2 h, followed by a 1-h secondary antibody incubation using anti-mouse Alexa 647. Approximately 10,000 and 50,000 cells per sample were analyzed for EdU and pH 3, respectively, using a Fortessa LSR ll flow cytometer followed by data analysis using the FlowJo software.
Cells were grown on glass coverslips and treated as indicated 3 days after siRNA transfections. Following the indicated incubation periods, cells were washed with PBS and pre-extraction buffer [25 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose, 0.5% (v/v) Triton X-100] was added for 2 min . Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 10 min, followed by incubation with PBS containing 0.2% (v/v) Triton X-100 and 3% (w/v) bovine serum albumin (BSA) for 10 min. Coverslips were then washed with 1% BSA in PBS and incubated with primary antibodies (Addional file 1, Table S2) diluted with 1% BSA in PBS. Secondary antibodies, diluted with 1% BSA in PBS and containing DAPI were added for 30 min at 37 °C after washing three times for 10 min with 1% BSA in PBS. Images of immunostained cells were taken with an SP8 confocal microscope (Leica) and analysed with the ImageJ software.
Monitoring of ssDNA
To detect ssDNA, cells were labelled with 25 μM 5-iodo-2′-deoxyuridine (IdU, Sigma) 30 h prior to anticancer drug treatment as described . The ssDNA was detected using an anti-IdU antibody (BD Biosciences) under native conditions. To quantify the totally incorporated IdU, DNA was denaturated with 2 M HCl in 0.5% (v/v) Tween 20 for 40 min and washed twice with 0.1 M Na-borate buffer, pH 9.0, prior to antibody staining. Images taken with an SP8 confocal microscope (Leica) were analysed using the ImageJ software.
Quantitative reverse transcriptase-PCR (qRT-PCR)
To determine the knock down efficiency, mRNA was extracted 3 days after siRNA transfection using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Thereafter, cDNA was synthesized from 500 ng mRNA with the iScript cDNA synthesis kit from Bio-Rad. Gene specific primers were designed using the NCBI Primer-BLAST  and GAPDH served as the internal control (Additional file 1: Table S1). Quantitative PCR was performed using the KAPA SYBR Fast qPCR Master Mix (2x) kit (KAPA Biosystems) according to the manufacturer’s instructions. The amplification conditions in the Bio-Rad CFX instrument consisted of an initial step of 3 min at 95 °C followed by 40 cycles of 3 s at 95 °C and 40 s at 60 °C. The delta-delta ct method was used to determine relative mRNA expression levels between siRNA-transfected samples and control samples transfected with non-coding siRNA .
In vitro protein dephosphorylation
HeLa cells were harvested 3 days after siRNA transfections and lysed for 30 min on ice under mild lysis conditions [1% (wt/vol) NP-40, 0.5% (wt/vol) SDS, complete protease inhibitor cocktail, EDTA-free (Roche)] followed by sonication for 10 cycles (30 s on, 30 s off) at 4 °C (Biorupture Plus, Diagenode). Cell lysates were then diluted in CIP buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM DTT, complete protease inhibitor cocktail - EDTA-free) and complemented with calf intestinal alkaline phosphatase (2 U/μg of protein, Sigma), and / or PhosStop (Roche) and / or 1 mM N-ethylmaleimide (NEM, an inhibitor of deubiquitinases) . Reactions were incubated for 2 h at 37 °C, boiled in Laemmli buffer for 5 min and subjected to Western blot analysis.
GraphPad Prism 5 was used to perform statistical analyses. The data presented were acquired from a minimum of two independent experiments. The Student’s t-test (unpaired, two-tailed) was used to analyze immunoblot and flow cytometry assays and all data are shown as the mean ± SEM. Immunofluorescence microscopy experiments were analyzed using 1-way ANOVA according to Kruskal-Wallis. Median values were presented as horizontal lines, boxes show the upper and lower quartiles and whiskers the 10-90th percentiles. P values of *P < 0.05, **P < 0.01 and ***P < 0.001 were considered to indicate statistical significance.
CUL4A/B depletion potentiates the cytotoxicity of crosslinking agents
Next, we depleted different cullins by siRNA transfections to understand which of the possible cullin targets of neddylation modulates this vulnerability to DNA-crosslinking agents. Cell viability assays, carried out in the presence of 5 μM cisplatin, confirmed a potentiation of cisplatin toxicity upon down regulation of CUL3 as reported before for SKOV3 and ES2 ovarian carcinoma cells . The new finding of this screen is that a sensitization to cisplatin cytotoxicity is also detected upon simultaneous down regulation of the two scaffold paralogs of CRL4, i.e., CUL4A and CUL4B (Fig. 1b). Dose dependence experiments showed that this co-depletion of CUL4A and CUL4B mimics to a considerable extent the sensitizing effect of MLN4924 when cells are treated with cisplatin or MMC for 48 h (Fig. 1c and d). Nearly the same increase of sensitivity to cisplatin was achieved upon depletion of the CRL4 adaptor protein Damaged DNA-binding 1 (DDB1) instead of the CUL4A/B scaffold. Instead, no sensitization was elicited upon individual depletion of only one of the cullins, CUL4A or CUL4B, indicating that the two interchangeable scaffolds have a redundant function. These results were confirmed using distinct combinations of siRNA sequences targeting CUL4A and CUL4B to exclude off-target effects (Additional file 1: Figure S1c and S1d). The efficiency of protein down regulation upon siRNA transfections is documented in Additional file 1: Figure S2.
Further assays measuring the release of lactate dehydrogenase as a marker of membrane disruption (Fig. 1e) confirmed that the CUL4A/B depletion enhances cisplatin-induced cell death. Finally, the increased cytotoxicity of cisplatin upon a combined CUL4A/B depletion, but not after down regulation of only one of the cullins individually, was confirmed in a long-term colony-forming assay (Fig. 1f).
CUL4A/B depletion reduces H2AX/RPA phosphorylation upon ICL induction
CRL4 deficiency impairs interstrand crosslink-dependent assembly of ssDNA-RPA complexes
In view of the observation that the cisplatin-dependent RPA phosphorylation is reduced in CRL4 deficient cells, we next tested whether CRL4 is required for the recruitment of RPA to sites of cisplatin damage. By immunofluorescence analysis, some increase of RPA2 foci in chromatin was observed in CUL4A/B-deficient cells even without any genotoxic treatment (Fig. 3a and b). This response is expected from the loss of CRL4-dependent regulation of CDT1 described in previous reports (see Discussion). The replication licensing factor CDT1 is nearly completely degraded within 24 h after genotoxic stress caused by cisplatin in CRL4-proficient cells (Additional file 1: Figure S4a and S4b). Instead, the CUL4A/B depletion results in a pronounced stabilization of CDT1, such that the cells maintain high CDT1 levels despite cisplatin exposure. This results in uncoupling of the minichromosome maintenance (MCM) helicase activity and an uncontrolled re-replication that triggers RPA recruitment and other ATR-dependent signaling reactions . However, this RPA recruitment to chromatin was not or only slightly further increased by cisplatin treatment of CRL4-deficient cells. As a consequence, CUL4A/B-depleted cells exposed to cisplatin display significantly lower levels of RPA foci when compared to CRL4-proficient controls treated with the same cisplatin concentrations (Fig. 3a and b).
Next, we tested whether RPA recruitment to chromatin in response to cisplatin damage is related to the ssDNA formation. For that purpose, ssDNA induction was assessed using a well-established method based on the incorporation of 5-iodo-2′-deoxyuridine (IdU), which allows for the probing of cells with an antibody that binds to IdU only in the ssDNA conformation. Using this same approach, Huang et al.  did not detect ssDNA intermediates after 4- to 6-h treatments with MMC or psoralen (plus UV-A radiation). In agreement with this earlier report, we also observed that ssDNA as well as pS33 remain below the detection threshold within the first 6 h of cisplatin exposure (Additional file 1: Figure S4c-e). However, a longer incubation time of 24 h revealed the formation of clearly detectable ssDNA foci in control cells (Fig. 3a). Again, an increase of ssDNA was observed in CUL4A/B-deficient cells even without genotoxic treatment, as expected from the loss of CRL4-dependent licensing regulation and the notion that the display of ssDNA provides an initial signal for ATR-mediated S phase checkpoint activation upon uncontrolled re-replication . Consistent with the differential recruitment of RPA, ssDNA foci were substantially reduced in CUL4A/B-depleted cells compared to CRL4-proficient controls exposed to the same cisplatin concentrations (Fig. 3a and c). It is important to ascertain in these experiments that IdU is equally incorporated into DNA under the different experimental conditions, as shown by immunofluorescence after DNA denaturation (Additional file 1: Figure S4f-g).
To demonstrate the general relevance of the above-described findings, we also assessed the appearance of ssDNA and consequent RPA phosphorylation in MMC-exposed HeLa and SKOV3 cells (Fig. 3d and e). Immunofluorescence quantifications confirmed that the CUL4A/B depletion counteracts partially the ICL-dependent display of ssDNA and pS33 upon exposure to the crosslinking agent (Fig. 3f and g). Decreased pS33 and pS4/8 levels upon CUL4A/B depletion were also found in immunoblots following MMC treatment of both HeLa and SKOV3 cells (Additional file 1: Figure S4 h). These results indicate that CRL4 activity is required for the generation of a ssDNA-RPA signaling platform essential for the DDR mitigating the cytotoxicity of crosslinking agents.
FANCD2-dependent ssDNA formation upon cisplatin treatment
Additionally, RPA2 phosphorylation on serine 33 was assessed (Fig. 4a and c) to prove that depletion of FANCD2 not only suppresses the formation of ssDNA but also the consequent foci of phosphorylated RPA2 in cisplatin-exposed cells. This result was confirmed by immunoblots using two distinct siRNA sequences to deplete FANCD2 (Additional file 1: Figure S5a and S5b). In all cases, the lack of FANCD2 reduced markedly the level of pS33 in cisplatin-treated cells. We concluded that the experimental conditions of our study verify the involvement of FANCD2 in the induction of ssDNA serving as a hub for the initiation of RPA signaling at ICL sites.
CUL4A/B depletion impedes recruitment of FANCD2 and XPF-ERCC1
As stated above, monoubiquitination of FANCD2 is a key prerequisite for recruitment of the downstream nuclease complex including XPF-ERCC1 to ICL sites . We therefore tested whether CRL4 might influence the FANCD2 ubiquitination in response to crosslinking agents. Upon analysis in immunoblots, the monoubiquitination of FANCD2 is indeed impaired in CUL4A/B-depleted cells relative to CRL4-proficient controls (Fig. 5e). Quantifications are presented in Fig. 5f as the ratio of ubiquitinated and unmodified FANCD2 for each condition over three independent experiments. After treatment with 5-μM cisplatin, ~ 50% of FANCD2 is ubiquitinated in control cells. Instead, in CUL4A/B-depleted cells, only ~ 30% of FANCD2 molecules appear in this modified form after the same cisplatin exposure. These results indicate that CRL4 activity stimulates the monoubiquitination of FANCD2 and, accordingly, the FANCD2-dependent recruitment of downstream nucleases.
CUL4A/B depletion suppresses the S phase checkpoint after exposure to crosslinking agents
This elevated DNA synthesis is in agreement with the dampened activation of the intra-S checkpoint transducer CHK1, a direct target of ATR. An increase of CHK1 phosphorylation (generating pCHK1) was observed in CRL4-proficient HeLa cells treated with cisplatin lasting at least 24 h after initiation of drug exposure (Fig. 6c and d). Although there was an initial increase of pCHK1 in CRL4-deficient cells, at later timepoints pCHK1 levels were significantly lower. In MMC-exposed cells, CUL4A/B depletion also impedes CHK1 phosphorylation, albeit partially (Additional file 1: Figure S6a). Although CHK1 had been identified as a possible CRL4 substrate , we did not observe any overall changes of CHK1 protein level. One may argue that the reduced phosphorylation of CHK1 in CUL4A/B-depleted cells exposed to crosslinking agents results from a compromised viability. However, over three independent experiments the combined CUL4A/B deficiency had no statistically significant influence on the phosphorylation of CHK2 protein in cisplatin-treated cells (Additional file 1: Figure S6b and S6c). These findings indicate that CRL4 activity stimulates mainly the ATR/CHK1 signaling pathway in cells treated with crosslinking agents.
An identical response with stimulation of DNA synthesis in CRL4-deficient relative to CRL4-proficient counterparts was detected after exposure to MMC (Additional file 1: Figure S6d and S6e). These findings confirm that the cells respond to cisplatin and MMC treatment with an effective S phase checkpoint that suppresses DNA synthesis. However, this cell cycle checkpoint is at least in part abrogated by concomitant depletion of the CRL4 scaffold proteins CUL4A and CUL4B, such that cisplatin- or MMC-exposed and CUL4A/B-depleted cells display higher rates of DNA synthesis than control cells treated with these same crosslinking drug. Cell cycle analyses established that a depletion of FANCD2 or ERCC1 results in a defective S phase checkpoint and elevated DNA synthesis in cisplatin-treated cells (Additional file 1: Figure S6f and S6 g) exactly as observed after CUL4A/B depletion (Fig. 6a). This identical checkpoint defect is consistent with the notion that CRL4 activity positively regulates the observed ICL –> FANCD2 –> XPF-ERCC1 –> ssDNA-RPA –> ATR/CHK1 pathway.
We next tested whether the weakened S phase checkpoint, translating to an accelerated S phase progression, leads to an increase of the M phase population. For that purpose, cisplatin-treated cells were stained for DNA content and histone H3 phosphorylation at position Ser10 (pH3), a well-established marker of mitosis, and subsequently analyzed by flow cytometry (Fig. 6e). The proportion of cells reaching M phase was reduced upon cisplatin exposure in a dose-dependent manner. However, the CUL4A/B depletion doubled the fraction of M phase cells relative to the non-coding siRNA controls (Fig. 6f). Because the tested cisplatin concentration of 5 μM is toxic in CUL4A/B-depleted cells, we concluded that the CUL4A/B deficiency allows for entering mitosis despite irreparable DNA damage, thereby causing mitotic catastrophe.
Inhibition of Neddylation recapitulates the effects of CUL4A/B depletion
This study was instigated by the surprising observation that, in the presence of the DNA-crosslinking agents cisplatin and MMC, a CRL4 deficiency causes the same cell cycle checkpoint defect as a depletion of FANCD2 or ERCC1. Therefore, our findings indicate that CRL4 positively regulates a FANCD2- and ERCC1-dependent checkpoint response that depends on the local deployment of ssDNA. This involvement of the FA pathway, which is dedicated to the repair of ICLs [8, 31, 32], implies that the ICL lesions induced by cisplatin and MMC constitute the actual trigger for the observed ssDNA induction. Cisplatin, MMC and other crosslinking agents are highly cytotoxic because the resulting ICLs interfere with processes requiring separation of the two DNA strands and thereby block DNA replication . Although it is well known that the repair of ICLs during DNA replication requires the FA pathway, the mechanism by which crosslinking agents induce the S phase checkpoint is less well understood. A previous study, describing the role of the FA pathway after 4- to 6-h treatments with MMC or psoralen (plus ultraviolet irradiation), did not detect any ssDNA intermediates. The authors of that earlier study reported that, during this time window of 4–6 h, the FA pathway members FANCM and FAAP24 are able to initiate an RPA-dependent checkpoint response to ICLs without generating ssDNA intermediates . In agreement with this earlier report, we also observed that ssDNA levels remain below the detection threshold within 6 h of cisplatin or MMC exposure. However, abundant ssDNA foci, which depend on FANCD2 for the recruitment of a nuclease complex comprising ERCC1, are detected after 24-h treatments with these same crosslinking agents. The ssDNA foci emerging in a time-dependent manner ultimately reinforce an S phase checkpoint by recruitment of RPA and subsequent engagement and activation of the ATR and CHK1 protein kinases. We conclude that, in addition to the previously discovered ssDNA-independent and short-term checkpoint response to ICLs, there is a ssDNA-dependent and sustained response to the same lesions, together culminating in cell cycle arrest. We unexpectedly observed that cells lacking CLR4 activity are impaired in this ssDNA-dependent signaling response to ICLs and the functional consequence of our finding is that a CLR4 deficiency potentiates the cytotoxicity of cisplatin and MMC.
Our report establishes an unforeseen functional link between two distinct ubiquitination systems, i.e., the FA pathway and CRL4 complexes. CRL4 ubiquitin ligases are formed by assembly of one of two closely related scaffold proteins (CUL4A or CUL4B) with the adaptor protein DDB1, which associates with substrate receptors. The scaffold subunit also binds to the RING finger protein RBX1 mediating the association with ubiquitin-delivering enzymes. The two paralogs CUL4A and CUL4B share high sequence similarity. Importantly, these CUL4A/B paralogs are amplified or overexpressed in human carcinomas and provide negative prognostic markers for survival . CRL4 complexes employ a variety of substrate receptors that target specific proteins for ubiquitination [12, 13]. For example, the CRL4CDT2 ubiquitin ligase promotes degradation of the replication licensing factor CDT1 after replication origin firing to ensure that DNA is replicated only once per cell cycle [18, 48]. Exposure to DNA-damaging agents also induces rapid CDT1 proteolysis through CRL4-mediated ubiquitination [49, 50], whereas ectopic CDT1 expression promotes DNA re-replication [18, 20]. Accordingly, CRL4-deficient cells display higher constitutive levels of CDT1 and the resulting deleterious re-replication has been shown to activate DDR signaling [20, 35, 36]. A cursory interpretation of our findings might, therefore, implicate the aberrant stabilization of cell cycle factors like CDT1 and p21, as described to explain the efficacy of pevonedistat against melanoma cells , together with deregulated MCM activity , as the cause of an increased sensitivity of CRL4-deficient cells to crosslinking agents. However, the lack of CRL4 increases ssDNA levels only in undamaged cells and this slightly higher ssDNA content, seen in comparison to CRL4-proficient counterparts, is not further enhanced by exposure to cisplatin or MMC. On the contrary, in the presence of such crosslinking agents the extent of ssDNA remains significantly lower in CRL4-deficient cells compared to the CRL4-proficient controls. This observation points to an additional, fundamentally different effect of CRL4 down regulation that suppresses the ICL-induced ssDNA formation, thus limiting ssDNA-RPA signaling and the consequent activation of checkpoint kinases. Consequently, the ICL-triggered phosphorylation of RPA2 (at position Ser33) and the phosphorylation of downstream effectors like H2AX is reduced in cells lacking CRL4 activity compared to CRL4-proficient controls.
Taken together, at least a subset of cancer cells is vulnerable to the combination of a crosslinking agent and CRL4 inhibition. One future challenge is to develop selective CRL4 inhibitors to avoid side effects due to the unnecessary blockage of other cullin-type ubiquitin ligases. Also, it is necessary to discover and validate biomarkers for the identification of those cancer subsets that are most susceptible to a combined treatment of crosslinking agents and CRL4 inhibitors. Our study provides insight into a novel function of CRL4 in mitigating the cytotoxicity of ICLs through stimulation an FA pathway-dependent checkpoint response. Consequently, CRL4 is a potential new therapeutic target to improve the anticancer efficacy of ICL-inducing drugs.
Supplementary information is available online
Supplementary information accompanies this paper.
T.C. designed and performed most experiments; D.T. performed part of the cell survival assays; T.C. and H.N. planned the research and wrote the manuscript. All authors have read and approved the manuscript.
This work was supported by the Swiss National Science Foundation (Grant 31003A_170111/1) and the Velux Foundation (Project 753). These funding bodies had no role in the study and collection, analysis and interpretation of data, and in writing the manuscript.
Ethics approval and consent to participate
None of the cell lines used in this study required ethics approval.
Consent for publication
All authors have read and approved the manuscript.
The authors declare that they have no competing interests.
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- 51.Benamar M, Guessous F, Du K, Corbett P, Obeid J, Gioeli D, et al. Inactivation of the CRL4-CDT2-SET8/p21 ubiquitylation and degradation axis underlies the therapeutic efficacy of pevonedistat in melanoma. EBioMedicine. 2016; cited 2016 Jul 15]; Available from: http://linkinghub.elsevier.com/retrieve/pii/S2352396416302791.
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