ER-associated degradation ligase HRD1 links ER stress to DNA damage repair by modulating the activity of DNA-PKcs

Abstract

Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.

Keywords: DNA damage; HDAC1; KU70/KU80; TRIM25; endoplasmic reticulum stress.

Fig. 1.

Selective activation of the DNA-PKcs-NHEJ pathway controls cell fate under ER stress. (A) The ER stress–associated DEGs were plotted in a Venn diagram (Gene expression profiles were obtained from GEO datasets GSE200454, GSE1622 56, and GSE169584). The GO enrichment analysis results of DEGs were plotted in the bubble chart. (B) The level of ER stress in tumor and normal adjacent tissue of colon cancer was analyzed (data are obtained from TCGA datasets). (C) The correlation between the pathways response to ER stress pathway and DNA repair in colon cancer (data are obtained from TCGA datasets). (D) The expression levels of URP and DDR proteins were determined by western blots in HCT116 or LoVo cells after TG (1 μg/mL) treatment for the indicated time periods. (E) The γH2AX foci were determined by cell staining in HCT116 or LoVo cells after TG (1 μg/mL) treatment for the indicated time periods. (F) Comet assay used to detect DNA damage in HCT116/LoVo cells after TG (1 μg/mL) treatment for the indicated time periods (Up). The tail moment of comets was calculated using OpenComet software (Bottom). (G) HR or NHEJ efficiencies of ISce-I–induced DSBs in U2OS cells expression EJ5-GFP reporter (NHEJ, Left) or DR-GFP reporter (HR, Right) were determined by measuring GFP-positive cells by flow cytometry (FACS) after TG (1 μg/mL) treatment for the indicated time periods in the presence or absence of KU-57788 (1 μM, 8 h). (H) Representative crystal violet staining images of HCT116 and LoVo cells after TG (1 μg/mL) treatment for the indicated time periods in the presence or absence of KU-57788 (1 μM, 8 h), AZ20 (10 μM, 8 h), or KU-60019 (1 μM, 8 h) (Left). The images were quantified by ImageJ and the data were plotted in the graphs (Right). Data were derived from three independent experiments and represented as mean ± SEM in the bar graph. **P < 0.01, ##P < 0.01 (F–H).

Fig. 2.

Dynamic changes of KU70/KU80 expression determine the activity of DNA-PKcs during ER stress. (A and B) KU70/KU80 foci (A) and their expression levels (B) were determined by cell staining and western blot, receptively, in HCT116 and LoVo cells treated with TG for different time periods. The images were quantified by ImageJ and the data were plotted in the bar graphs. (C) p-DNA-PKcs, KU70, and KU80 protein levels were determined by western blot in HCT116 and LoVo cells after KU70/KU80 OE and/or TG (1 μg/mL) treatment for the indicated time periods. GAPDH was used as a loading control. (D) Representative crystal violet staining images of HCT116 and LoVo cells with the indicated treatments (Up). The images were quantified by ImageJ and the data were plotted in the graphs (Bottom). Data are derived from three independent experiments and represented as mean ± SEM in the bar graph. **P < 0.01, ##P < 0.01 (A, B, and D).

Fig. 3.

ERAD ligase HRD1 regulates KU70/KU80 protein turnover by an indirect mechanism involved HDAC1. (A and B) Representative western blot images of HRD1, KU70, and KU80 protein in HRD1 KD or OE cells (A) in the presence or absence of TG (1 μg/mL) for the indicated time durations (B). (C) The binding between KU70 or KU80 and HRD1 was determined by an in vitro IP assay using purified proteins. (D) Mass spectrum analysis of HRD1 binding proteins following proximity labeling of HRD1 under control and ER-stressed conditions, in the presence of MG132. (E and F) Representative western blot images of HRD1 and HDAC1 protein in HRD1 KD (E) or OE cells (F) in the presence or absence of TG (1 μg/mL) for the indicated time durations. (G) Representative western blot images of HRD1, HDAC1, KU70, and KU80 protein in HDAC1 KD cells or HDAC1 inhibitor SAHA (1 μM) treated cells in the presence or absence of TG (1 μg/mL) for the indicated time durations. β-actin was used as a loading control. (H) Schematic diagram of HRD1-mediated KU70/KU80 protein degradation through binding and inhibiting the protein levels of HDAC1.

Fig. 4.

HRD1 catalyzes HDAC1 ubiquitination and degradation under ER stress. (A and B) The bindings between HRD1 and HDAC1 in living cells or fractioned cell lysates were determined by the PLA (A) and IP assay (B) in the presence or absence of TG (1 μg/mL, 24 h) and/or MG132 (20 μM, 6 h). (C) Analysis of the distribution of HDAC1 and HRD1 by the cell fractionation assay in the presence or absence of TG (1 μg/mL, 24 h) and/or MG132 (20 μM,6 h). (D) The HRD1/HDAC1 interaction and the binding mechanisms were determined by the NanoBiT proximity assay (Bottom). The diagram illustrated the domain structures of HRD1/HDAC1 and the principles of NanoBit proximity assay action (Up); FL, full-length; F, fragment. (E and F) Ub-HDAC1 was determined using IP/western blot assay in HCT116 cells with HRD1 KD (E), WT HRD1 OE or E3 ligase-defective mutant (MT, C291A/C309S/C329S) OE (F). (G) Ub-WT-HDAC1 or Ub-HDCA1 mutants at the predicted ubiquitination sites were determined by the IP/western blot assay in HRD1-V5 OE HCT116 cells. (H) The expression levels of WT HDAC1 or HDCA1 mutants at the predicted ubiquitination sites were determined by the western blot assay in the presence of TG (1 μg/mL, 24 h). (I) Schematic diagram showing that HRD1 binds with HDAC1 at its protein interaction domain (F1) through the C terminus in the cytoplasm and catalyzes HDAC1 polyubiquitination at residue K74 to induce HDAC1 protein degradation. Data are derived from three independent experiments and represented as mean ± SEM in the bar graph. **P < 0.01 (A and D).

Fig. 5.

HDAC1 protects KU70/U80 protein from TRIM25-mediated protein degradation. (A) Representative western blot images of KU70, KU80, and TRIM25 protein in the control or TRIM25 KD cells before and after TG (1 μg/mL) treatment in HCT116 cells. β-actin was used as a loading control. (B) The interactions between KU70 or KU80 and TRIM25 were analyzed by IP assays in HCT116 cells after TG (1 μg/mL) treatment for the indicated time durations. (C) The interaction between KU70 or KU80 and TRIM25 and their binding mechanisms were evaluated by the NanoBiT proximity assay. Data are derived from three independent experiments and represented as mean ± SEM in the bar graph. **P < 0.01. (D) Poly-Ub of WT and MT KU70/KU80 were determined by the IP/western blot assay in the control and/or TRIM25 OE HCT116 cells. (E and F) The expression levels of WT KU70/KU80 and ubiquitination defective mutant (K114/256R KU70 and K195/265/481R KU80) were determined by western blot in TRIM25 KD (E), or TG (1 μg/mL) treated (F) HCT116 cells. β-actin was used as a loading control. (G) Endogenous levels of KU70/KU80 and TRIM25 were determined by western blot in HCT116 cells in the presence or absence of SAHA (1 μM, 24 h) in TRIM25 KD cells. GAPDH was used as a loading control.

Fig. 6.

HDAC1-mediated KU70/KU80 deacetylation impaired KU70 /KU80’s binding ability with TRIM25. (A) The acetylation levels of KU70 (Up) and KU80 (Bottom) were determined by western blot of anti-acetylation (Ac) antibody following IP KU70 or KU80 in the control or HDAC1 OE HCT116 cells with TG (1 μg/mL) treatment for the indicated time periods. (B) Ub-KU70 and Ub-KU80 were determined by IP/western blot in HCT116 cells in the presence or absence of Trim 25 KD and/or SAHA (1 μM, 24 h). (C) The acetylation levels of KU70 (Up) and KU80 (Bottom) were determined by western blot of anti-acetylation (Ac) antibody following IP KU70 or KU80 in the control or Trim 25 KD (B) HCT116 cells with TG (1 μg/mL) treatment for the indicated time periods. (D and E) The acetylation (D) and ubiquitination (E) levels of WT KU70/KU80 and their acetylation defective mutants (K542/544R KU70, K7/126R KU80) were determined by western blot of anti-Ac and anti-Ub antibody, respectively, following IP KU70 or KU80 in HCT116 cells treated with TG (1 μg/mL) for the indicated time periods. (F) The bindings between TRIM25 and WT KU70/KU80, their acetylation defective mutants (K542/544R KU70 and K7/126R KU80) or their acetylation mimic mutants (K542/544Q KU70 and K7/126Q KU80) were compared by IP assays. (G) Proposed model of HDAC1-mediated KU70/KU80 stabilization by catalyzing deacetylation of KU70 at K542/544 and K7/126 at KU80, because the acetylation of KU70/KU80 at these residues is important for E3 ligase TRIM25 binding and the subsequent KU70/KU80 degradation mediated by TRIM25.

Fig. 7.

DNA-PK inhibitor or HDAC1 inhibitor improves the sensitivity of ER stress–induced cancer cell death. (A) Representative crystal violet staining images of the number of shHDAC1 and shnone HCT116 and LoVo cells treated with or without ER stress inducer celecoxib (Left). The images were quantified by ImageJ and the data were plotted in the graphs (Right). (B) HCT116 cells were inoculated into nude mice and then subjected to the celecoxib treatment. (C–E) Tumor volumes at the indicated time (C), tumor images (D), and tumor weight (E) of HCT116 xenografts were presented. (F) Representative immunohistochemistry staining for γH2AX and Ki67 was determined in the indicated xenografts (Left). Quantification of intensity was shown in the bar graph (Right). (Scale bar, 50 μm.) (G) Representative western blot images of HDAC1, KU70, KU80, Lig4, and XRCC4 of the indicated xenografts. Bar graphs represent the mean ± SEM from three independent assays (A and F). The average values of tumor volume and tumor weight are present in the graphs (means ± SD) (n = 5 for each group, C and E). *P < 0.05; **P < 0.01; N.S., not significant (A, C, E, and F).

Fig. 8.

DNA-PK/HDAC1 inhibitors improve the sensitivity of ER stress–induced cancer cell death. (A) Representative crystal violet staining images of the number of HCT116 and LoVo cells treated with celecoxib (10 μM, 24 h), SAHA (1 μM, 24 h), KU-57788 (1 μM, 8 h), or the combinations (Left). The images were quantified by ImageJ and the data were plotted in the graphs (Right). (B) Schematic diagram showed the PDX establishment pipeline with celecoxib, SAHA, KU-57788, or combined treatments. (C–E) PDX-tumor volumes at the indicated time (C), PDX-tumor images (D), and PDX-tumor weight (E) were presented. (F) Representative immunohistochemistry staining for γH2AX and Ki67 were determined in the indicated PDX-tumor (Bottom). Quantification of intensity was shown in the bar graph (Up). (Scale bar, 50 μm.) (G) Representative western blot images of HDAC1, KU70, KU80, Lig4, and XRCC4 of the indicated PDX-tumor. Bar graphs represent the mean ± SEM from three independent assays (A and F). The average values of tumor volume and tumor weight are present in the graphs (C and E) (means ± SD) (n = 5 for each group). **P < 0.01; N.S., not significant (A, C, E, and F).

Fig. 9.

The analysis of HRD1/HDAC1/KU70/KU80 protein expressions in colon cancer tissues. (A and B) Representative WB (A) and quantification (B) of HRD1, HDAC1, TRIM25, KU70, and KU80 protein expression in 41 pairs of human colon cancer tissues (T) and their matched adjacent normal controls (N) are present. β-actin was used as a loading control. (C) The correlation between HRD1, HDAC1, TRIM25, KU70, and KU80 protein levels and tumor stages was shown in the box plot. (D) The mRNA levels of HDAC1, KU70, and KU80 were detected by qRT-PCR assays. (E) The scatter diagram showed the lineal correlation of HDAC1 protein/KU70 protein (P < 0.01) or HDAC1 protein/KU80 protein (P < 0.01) in colon cancer tissues. *P < 0.05; **P < 0.01; N.S., not significant (B–E).