NRDE2 deficiency impairs homologous recombination repair and sensitizes hepatocellular carcinoma to PARP inhibitors

Abstract

To identify novel susceptibility genes for hepatocellular carcinoma (HCC), we performed a rare-variant association study in Chinese populations consisting of 2,750 cases and 4,153 controls. We identified four HCC-associated genes, including NRDE2, RANBP17, RTEL1, and STEAP3. Using NRDE2 (index rs199890497 [p.N377I], p = 1.19 × 10^-9) as an exemplary candidate, we demonstrated that it promotes homologous recombination (HR) repair and suppresses HCC. Mechanistically, NRDE2 binds to the subunits of casein kinase 2 (CK2) and facilitates the assembly and activity of the CK2 holoenzyme. This NRDE2-mediated enhancement of CK2 activity increases the phosphorylation of MDC1 and then facilitates the HR repair. These functions are eliminated almost completely by the NRDE2-p.N377I variant, which sensitizes the HCC cells to poly(ADP-ribose) polymerase (PARP) inhibitors, especially when combined with chemotherapy. Collectively, our findings highlight the relevance of the rare variants to genetic susceptibility to HCC, which would be helpful for the precise treatment of this malignancy.

Keywords: NRDE2; PARP inhibitors; hepatocellular carcinoma; homologous recombination repair; rare variant.

Graphical abstract

Figure 1

The rare variant rs199890497 (p.N377I) in NRDE2 is significantly associated with HCC risk and disrupts the tumor suppressive role of NRDE2 (A) The regional association plot for the risk locus surrounding the index rs199890497 (p.N377I) in NRDE2 in the discovery stage. cM, centi Morgan; LD, linkage disequilibrium. (B) Schematic diagram of the rare nonsynonymous variants (with MAF <1%) in NRDE2. MAF, minor allele frequency. (C) The NRDE2 protein expressions in HCC tissues (Tumor) are significantly lower than those in paired adjacent non-tumor liver tissues (Non-tumor) in tumor microarray (TMA) cohort (n = 84), and lower NRDE2 levels are significantly correlated with decreased overall survival rates of HCC patients. IHC, immunohistochemistry. (D) The promoting effects of stable NRDE2 knockdown on HepG2 cells growth/plate colony formation/migration/invasion were abolished by transiently re-expressing NRDE2 wild-type (NRDE2-WT), but not mutant NRDE2 p.N377I (NRDE2-N377I). OD, optical density. (E) The effects of the enforced expression of NRDE2-WT or NRDE2-N377I in Huh-7 cells on subcutaneous tumor growth in BALB/c nude mice (n = 7/group). The Huh-7 cells stably transfected with Vector, NRDE2-WT, or NRDE2-N377I (1 × 10⁶ cells diluted in 100 μL PBS) were grafted subcutaneously in the left side of the mice back. (F) Measurement of metastases in mice injected with Huh-7 cells with enforced expression of NRDE2 (NRDE2-WT or NRDE2-N377I) via the tail veins (n = 7/group). The Huh-7 cells stably transfected with Vector, NRDE2-WT, or NRDE2-N377I (1 × 10⁶ cells diluted in 250 μL PBS) were injected into the tail vein of the mice. The mice were monitored once a week using bioluminescence imaging. The data are shown as the mean ± standard error of mean (SEM) of three independent experiments and each experiment was done in triplicate except where noted otherwise. ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 by two-tailed unpaired Student’s t test except where noted otherwise.

Figure 2

NRDE2 involves in DNA damage response and promotes DNA double-strand break (DSB) repair (A) Gene set enrichment analysis (GSEA) plot based on RNA sequencing data from the Huh-7 cells stably transfected with Vector or NRDE2-WT. NES, normalized enrichment score. (B) The enrichment maps of Metascape results based on the significantly differentially expressed genes (NRDE2-WT vs. Vector) in Huh-7 cells. (C and D) The effects of knockdown (C) or enforced expression (D) of NRDE2 on the sensitivities of the HepG2 cells to ionizing radiation (IR) or camptothecin (CPT; for 12 h) using clonogenic survival assays. The cell survival rates are counted by calculating the colony numbers. HepG2 cells were stably transfected with non-targeting scrambled shRNA controls (shCtrl) or NRDE2-specific shRNAs (shNRDE2-1 or shNRDE2-2), or stably transfected with Vector, Flag-NRDE2-WT (WT) or Flag-tagged-NRDE2 p.N377I (N377I). WT, wild-type. (E and F) The effects of stable knockdown (E) or stably enforced expression (F) of NRDE2 on apoptosis in HepG2 cells in response to IR (5 Gy) or CPT (10 μM, 12 h). DMSO, dimethyl sulfoxide. (G and H) The effects of stable knockdown (G) or stably enforced expression (H) of NRDE2 on γ-H2AX foci formation in HepG2 cells in response to IR (2 Gy) or CPT (10 μM). NT, no treatment. The γ-H2AX foci of cells in response to IR or CPT are determined using the immunofluorescence assays. (I and J) The neutral comet assays show that the double-strand break (DSB) repair capability is reduced in NRDE2-stably knocked-down (I), or induced in NRDE2-stably enforced-expressed (J) HepG2 cells. HepG2 cells are harvested at the indicated time (NT, and 1 h and 4 h post-treatment) upon IR treatment (8 Gy). The tail moment was analyzed using the CometScore software. The data are shown as the mean ± standard error of mean (SEM) of three independent experiments. n.s., not significant. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 by two-tailed unpaired Student’s t test except where noted otherwise.

Figure 3

NRDE2 promotes homologous recombination (HR)-mediated double-strand break (DSB) repair (A and B) Representative confocal images of endogenous NRDE2 and γ-H2AX in HepG2 cells treated with ionizing radiation (IR, 10 Gy; A) or camptothecin (CPT, 10 μM; B) 4 h later. Co-localization was confirmed with fluorescence-intensity profiles (middle panel). Fluorescence-intensity profiles of NRDE2 and γ-H2AX were obtained using ImageJ software (v1.8.0), along a straight line (white) crossing the nucleus of a representative cell. Histograms represent mean ± standard deviation (SD) of Pearson’s correlation coefficient between NRDE2 and γ-H2AX from 20 randomly selected cells in each group (right panel). NT, no treatment; DMSO, dimethyl sulfoxide. (C) Recruitments of the wild-type NRDE2 (NRDE2-WT) or mutant NRDE2-N377I to the DNA damage sites in U2OS cells by laser micro-irradiation assays. The live-cell imaging of the recruitments of GFP-NRDE2-WT or GFP-NRDE2-N377I to laser damage tracks was generated by laser micro-irradiation in U2OS cells at the indicated time points (seconds). Red arrows indicated the regions damaged by laser micro-irradiation. Intensity quantifications of GFP-NRDE2-WT and GFP-NRDE2-N377I accumulation at laser track sites were performed using ImageJ software (v1.8.0). The intensity values in the micro-irradiated areas were pooled from 10 independent cells and plotted at the indicated time. (D) Chromatin immunoprecipitation assays showing the recruitment of Flag-NRDE2 to I-SceI-induced DSBs in U2OS cells at the indicated time points (h). (E and F) The effects of knockdown (E) or enforced expression (F) of NRDE2 on the HR or non-homologous end-joining (NHEJ) repair efficiency in U2OS cells. HR or NHEJ repair efficiencies are determined using the direct repeat green fluorescent protein (DR-GFP) reporter assays or the EJ5-GFP reporter assays, respectively. (G and H) The effects of stable knockdown (G) or stablely enforced expression (H) of NRDE2 on 53BP1, RPA2 or RAD51 foci formation. HepG2 cells were treated with IR (2 Gy), and were immunostained with the antibodies against 53BP1, RPA2, or RAD51 followed by Alexa Fluor 555-conjugated secondary antibody. Histogram represented the numbers of 53BP1, RPA2, or RAD51 foci per nuclei. The data are shown as the mean ± standard error of mean (SEM) of three independent experiments. n.s., not significant. ^∗∗∗p < 0.001 by two-tailed unpaired Student’s t test except where noted otherwise.

Figure 4

NRDE2 interacts with CK2 complex and facilitates its assembly and activity (A) Two independent IP-MS assays showing the associations of CK2 complex (CK2A1/CK2A2/CK2B) and NRDE2. IP assays were performed in HEK293T cells transiently overexpressing Flag-NRDE2 or empty vector. CK2, casein kinase 2; IP-MS, immunoprecipitation in combination with mass spectrometry; kDa, kilodalton. (B) Co-immunoprecipitation (Co-IP) assays showing the interactions between NRDE2 and the components of CK2 complex (CK2A1/CKA2/CK2B) in HepG2 cells. (C) GST pull-down assays showing the interactions between NRDE2 and CK2A1, CK2A2, and CK2B, respectively. GST-tagged wild-type NRDE2 (GST-N) was expressed and purified from E. coli. His-tagged CK2A1, CK2A2, and CK2B were expressed and purified from HEK293T cells. (D) NRDE2 co-localized with CK2A1/CK2A2/CK2B at nucleus in HepG2 cells with no ionizing radiation (IR) or camptothecin (CPT) treatment by immunofluorescence assays. Fluorescence-intensity profiles were obtained using ImageJ software (v1.8.0), along a straight line (white) crossing the nucleus of a representative cell. (E) The effects of transiently enforced expression of NRDE2-WT or NRDE2-N377I on the assembly of CK2 complex using immunoprecipitation followed by immunoblotting assays in HepG2 cells. (F) LacO/LacR chromatin-targeting protein interaction assays showing the interactions between NRDE2-WT or NRDE2-N377I and CK2A1 in U2OS-lacO cells without IR or CPT treatment. The U2OS-lacO cells were transiently transfected with GFP-CK2A1 and Myc-LacR-NRDE2 (WT or N377I). (G) The interactions between CK2A1 and CK2A2/CK2B assessed by LacO/LacR chromatin-targeting protein interaction assays in U2OS-lacO cells without IR or CPT treatment. The cells were transiently transfected with GFP-LacR-CK2A1, mCherry-CK2B with empty vector, Flag-NRDE2-WT, or Flag-NRDE2-N377I. (H) The effects of endogenous NRDE2 on DNA damage induced recruitments of CK2A1/CK2A2/CK2B to chromatin in HepG2 cells treated with camptothecin (CPT, 10 μM) were investigated by chromatin fractionation assays. Western blotting assays for the total or chromatin fractions from HepG2 cells with stable knockdown of NRDE2 or stably enforced expression of NRDE2 at 4 h after CPT treatment using the indicated antibodies. (I) The effects of knockdown or enforced expression of NRDE2 on cellular CK2 activity in HepG2 cells. We used the phosphorylation levels of CK2 substrates as a readout of cellular CK2 activity. NRDE2 was stably knocked down, and the NRDE2-WT and NRDE2-N377I were transiently expressed in cells. The data are shown as the mean ± standard error of mean (SEM) of three independent experiments. ^∗∗∗p < 0.001 by two-tailed unpaired Student’s t test.

Figure 5

NRDE2-CK2 axis-mediated MDC1 phosphorylation at T378 promotes the homologous recombination (HR) repair (A) Venn plot of candidate CK2 substrates, HR-related factors and known interactors for CK2. (B) MDC1 knockdown abolished NRDE2-WT-mediated enhancement of HR repair efficiency in U2OS cells. U2OS cells transiently transfected with empty vector (Vector), Flag-tagged-NRDE2-WT, or Flag-tagged-NRDE2 p.N377I (NRDE2-N377I) were transiently transfected with siCtrl or pooled MDC1-specific siRNAs. (C) The phosphorylation of endogenous MDC1 following NRDE2 or CK2 transient knockdown in HepG2 cells. The MDC1 immunoprecipitates were subjected to western blotting with the anti-phosphorylated (pan-p-S/T) antibody. (D) Selected LC-MS/MS scan of the MDC1 peptides with phosphorylated T378 and the annotated b- and y-ions. (E) The inhibition effects of NRDE2 knockdown on MDC1 phosphorylation at T378 in HepG2 cells could be abolished by NRDE2-WT, but not NRDE2-N377I. The HepG2 cells were stably transfected with shCtrl and pooled NRDE2-specific shRNAs. Then the HepG2 cells with NRDE2 knockdown were transiently transfected with NRDE2-WT or NRDE2-N377I. The p-T378-MDC1 signal was determined using an antibody specifically recognizing the T378-MDC1 phosphorylation. (F) The effects of knockdown of CK2A1 on the phosphorylation at T378 of endogenous MDC1 in HepG2 cells. The HepG2 cells were transiently transfected with siCtrl or CK2A1-specific siRNAs. (G and H) The effects of T378 phosphorylation on MDC1-mediated recruitments of the MRN complex subunits (NBS1, MRE11, and RAD50) at DSB sites in HepG2 cells treated with IR (2 Gy). Endogenous NRDE2 was stably knocked down by shRNAs, and then the phospho-mimetic (T378D) or -deficient (T378A) mutants of MDC1 were transiently overexpressed in cells as indicated. Then, cells were treated with IR (2 Gy). (I) The effects of NRDE2 on cell cycle progression after DNA damage in HepG2 cells treated with IR (2 Gy). Endogenous NRDE2 was stably knocked down by shRNAs, and the shRNA-resistant NRDE2-WT or NRDE2-N377I was then transiently re-expressed in cells as indicated. Percentage of cell cycle phases was measured by 5-bromodeoxyuridine (BrdU) incorporation with the cells being treated with IR (2 Gy). (J) The effects of NRDE2-mediated T378 phosphorylation on MDC1 induced intra-S-phase checkpoint after DNA damage in HepG2 cells treated with IR (2 Gy). Endogenous NRDE2 was stably knocked down by shRNAs, and then the MDC1-phospho-mimetic (T378D) or -deficient (T378A) mutants of MDC1 were transiently overexpressed in cells as indicated. The data are shown as the mean ± standard error of mean (SEM) of three independent experiments. n.s., not significant. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 by two-tailed unpaired Student’s t test.

Figure 6

NRDE2 deficiency sensitizes HCC cells to PARP1 inhibitor (A) A positive correlation between the expression levels of NRDE2 and PARP1 dependency scores in liver cancer cell lines from the Dependency Map database (https://depmap.org/portal/). (B) A negative correlation between the expression levels of NRDE2 and sensitivity to the PARP1 inhibitor olaparib in multiple HCC cell lines. Cellular viability was assessed in eight types of HCC cell line after treatment with different doses of olaparib for 72 h by CCK-8 assays. IC₅₀, the half-maximal inhibitory concentration. (C) Transiently knockdown of NRDE2 enhances the sensitivity of HepG2 cells (with higher endogenous NRDE2 levels) to the PARP1 inhibitor olaparib. (D) Transiently enforced expression of NRDE2-WT reduces the sensitivity of Huh-7 cells (with lower endogenous NRDE2 levels) to the PARP1 inhibitor olaparib. The percentage of cell viability in HepG2 (C) and Huh-7 (D) cells was assessed after treatment with indicated doses of olaparib for 72 h. (E) Measurements of tumor growth inhibition rate (%TGI) and tumor volume (mm³) in the PDX models with endogenous NRDE2-N377I (HCC-1 and HCC-2) and in the PDX models with NRDE2-WT (HCC-3 and HCC-4). The PDX models were treated with olaparib, oxaliplatin, or the combination (STAR Methods). On the 42^nd day after treatment, the tumors from PDX HCC-1 and HCC-3 are shown in the left panel. The middle and right panels represent the relative tumor volume over time and the %TGI, respectively. (F) Sensitivity of primary HCC cells (from PDX HCC-3 and HCC-1) to olaparib or the combination treatment with oxaliplatin and olaparib. Sanger sequencing showed that HCC-1 cells harbor the heterozygous NRDE2-N377I variant, and the variant allele frequency of the mutant allele was obviously higher than that of the reference allele (>2-fold), and HCC-3 cells harbor the wild-type genotype. The percentage of cell viability in primary HCC cells was assessed after treatment with these drugs for 72 h. NT, no treatment. (G) Model of the function and underlying mechanism that NRDE2 suppresses tumorigenesis and chemosensitivity. This figure was created using BioRender.com. The data are shown as the mean ± standard error of mean (SEM) of three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 by two-tailed unpaired Student’s t test.