USP10/XAB2/ANXA2 axis promotes DNA damage repair to enhance chemoresistance to oxaliplatin in colorectal cancer

Abstract

Background: Oxaliplatin-based chemotherapy is the first-line treatment for colorectal cancer (CRC). However, oxaliplatin resistance remains a major challenge contributing to treatment failure and poor prognosis. An increased capacity for DNA damage repair is a key mechanism underlying oxaliplatin resistance. Although XPA binding protein 2 (XAB2) is implicated in various DNA damage repair mechanisms, its specific role in mediating oxaliplatin resistance remains unclear.

Methods: XAB2 was identified through analysis of public datasets. Western blot analysis and immunohistochemistry were performed to evaluate XAB2 expression, while survival analysis was performed to assess its clinical significance in CRC. Functional experiments were then conducted to assess the impact of XAB2 on proliferation, DNA damage repair, and oxaliplatin resistance in CRC. RNA sequencing (RNA-seq) and Chromatin immunoprecipitation-sequencing (ChIP-seq) were used to identify XAB2 target genes. Co-immunoprecipitation (Co-IP) and mass spectrometry were used to identify the proteins interacting with XAB2. Dual-luciferase reporter assays, ChIP-qPCR, Co-IP, ubiquitination site mass spectrometry, and ubiquitin assays were used to analyse the interactions and potential mechanisms involving XAB2, Annexin A2 (ANXA2), and ubiquitin-specific protease 10 (USP10).

Results: XAB2 was found to be expressed in CRC and was associated with poor prognosis in patients with CRC. XAB2 promoted CRC cell proliferation and enhanced oxaliplatin resistance by promoting DNA damage repair. Mechanistically, CRC cells treated with oxaliplatin exhibited increased USP10 nuclear expression. USP10 bound to XAB2 and deubiquitinated XAB2 K48-linked polyubiquitination at K593, thereby stabilising XAB2 by reducing its degradation via the ubiquitin-proteasome pathway. XAB2 upregulates ANXA2 expression at the transcriptional level by binding to the ANXA2 promoter, thereby promoting DNA damage repair, mitigating oxaliplatin-induced DNA damage, and enhancing oxaliplatin resistance.

Conclusions: In summary, this study demonstrates that the USP10/XAB2/ANXA2 axis promotes proliferation, DNA damage repair, and oxaliplatin resistance in CRC. These findings uncover a novel mechanism of oxaliplatin resistance in CRC and suggest potential therapeutic targets for improving the efficacy of oxaliplatin in CRC treatment.

Keywords: Colorectal cancer; DNA damage repair; Deubiquitination; Oxaliplatin resistance; XAB2.

Fig. 1

XAB2 is highly expressed in CRC and is associated with poor prognosis in patients with CRC. A Overlap of differentially expressed (adjusted P value < 0.05 and|logFC|≥1) oncogenes from six datasets and expression levels of XAB2 in six datasets. B-C Expression levels of XAB2 in unpaired and paired COADREAD cohorts from TCGA. D Expression levels of XAB2 in unpaired samples grouped by cancer type from TCGA. E qRT-PCR (left) and western blot (right) analysis of XAB2 expression in the normal human colonic epithelial cell line NCM460 and CRC cell lines. F Western blot analysis of XAB2 expression in eight pairs of fresh CRC and adjacent non-neoplastic tissues. G Representative XAB2 IHC staining images in CRC and adjacent non-neoplastic tissues. H Statistical analysis of XAB2 expression in CRC and adjacent non-neoplastic tissues. I Kaplan–Meier analysis showing OS curves of patients with CRC stratified by high versus low XAB2 expression from TCGA. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

XAB2 promotes CRC cell proliferation in vitro and in vivo. A Knockdown of XAB2 in SW480 and HCT116 cells confirmed by western blot analysis. B Overexpression of XAB2 in HT29 and RKO cells confirmed by western blot analysis. C-F Proliferative ability of cells with XAB2 knockdown or overexpression determined by CCK8 assay (C-D) and colony formation assay (E-F). G-H Transplanted xenografts derived from cells with sh-NC and sh-XAB2 were established in BALB/c nude mice (n = 5). Tumor volume and weight were measured. Data are presented as mean ± SD (**P < 0.01, ***P < 0.001)

Fig. 3

XAB2 increases the resistance of CRC cells to oxaliplatin. A GSEA performed using TCGA database, showing XAB2-related enrichment plots. B HCT116 and HCT116/L-OHP cells treated with varying concentrations of oxaliplatin for 48 h. Cell viability was analysed using CCK8 assay, and IC50 values were presented. The resistance index was calculated by dividing the IC50 for HCT116/L-OHP cells by that of HCT116 cells. C Western blot (left) analysis and qRT-PCR (right) of XAB2 expression in CRC cell line HCT116 and oxaliplatin-resistant CRC cell line HCT116/L-OHP. D-E CRC cells treated with varying concentrations of oxaliplatin for 48 h. Cell viability was analysed using CCK8 assay, and IC50 values were presented. F-G Flow cytometry assays assessing the effect of XAB2 on apoptosis of cells treated with or without oxaliplatin (7.5 µM) for 48 h. Data are presented as mean ± SD (**P < 0.01, ***P < 0.001)

Fig. 4

XAB2 enhances DNA damage repair of CRC cells. A-B Distribution of γH2AX in CRC cells treated with oxaliplatin (7.5 µM) for 24 h analysed via IF. γH2AX is stained green, and the nucleus is stained blue. Scale bar = 20 μm. C-D Expression levels of γH2AX in CRC cells treated with oxaliplatin (7.5 µM) for 24 h analysed using western blot analysis. E-F Representative images (left) and bar charts (right) from alkaline comet assays of control cells, XAB2-overexpressing cells, and XAB2 knockdown cells treated with oxaliplatin (7.5 µM) for 48 h. Scale bar = 100 μm. Data are presented as mean ± SD (**P < 0.01, ***P < 0.001)

Fig. 5

XAB2 binds to the ANXA2 promoter to activate its transcription. A Venn diagram illustrating the intersection analysis of RNA-seq and ChIP-seq data. B Genome-wide distribution of peaks identified in ChIP-seq data. C Chromosomal distribution of reads from ChIP-Seq data. D qRT-PCR results showing RNA levels in cells with XAB2 knockdown or overexpression. E ChIP-seq peaks showing XAB2 enrichment at the ANXA2 promoter. F Five predicted XAB2-binding motifs with the most significant differences among peaks. G ChIP-qPCR analysis showing XAB2 enrichment of XAB2 on the ANXA2 promoter relative to control IgG-treated SW480 and HCT116 cell. H Putative wild-type and mutant binding sites between XAB2 and the ANXA2 promoter. I Dual-luciferase reporter assay using firefly luciferase reporter vectors and Renilla luciferase as an internal control. J-K Western blot analysis of ANXA2 protein levels in XAB2 overexpressing and knockdown cells. L XAB2 plasmid transfected into ANXA2-downregulated CRC cells, with ANXA2 expression confirmed using western blot analysis. Data are presented as mean ± SD (**P < 0.01, ***P < 0.001)

Fig. 6

ANXA2 mediates XAB2-induced proliferation and oxaliplatin resistance in CRC cells. A-C XAB2 plasmid transfected into ANXA2-downregulated CRC cells, with the proliferative ability of cells determined by CCK8 assay (A) and colony formation assay (B-C). D Transplanted xenografts derived from cells with oe-NC + sh-NC, oe-XAB2 + sh-NC, oe-NC + sh-ANXA2, and oe-XAB2 + sh-ANXA2 were established in BALB/c nude mice (n = 5). Tumor volume and weight were measured. E Viability of HT29 and RKO cells analysed using CCK8 assay after treatment with various concentrations of oxaliplatin for 48 h, with IC50 values displayed. F Apoptosis of cells treated with or without oxaliplatin (7.5 µM) for 48 h detected by flow cytometry assays. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 7

ANXA2 mediates XAB2-induced DNA damage repair in CRC cells. A Expression level of γH2AX in CRC cells treated with oxaliplatin (7.5 µM) for 24 h analysed using western blot analysis. B γH2AX distribution in CRC cells treated with oxaliplatin (7.5 µM) for 24 h analysed using IF. γH2AX is stained green, and the nucleus is stained blue. Scale bar = 20 μm. C Representative images (left) and bar charts (right) from alkaline comet assays of oe-NC + sh-NC, oe-XAB2 + sh-NC, oe-NC + sh-ANXA2, and oe-XAB2 + sh-ANXA2 cells treated with oxaliplatin (7.5 µM) for 48 h. Scale bar = 100 μm. Data are presented as mean ± SD (**P < 0.01, ***P < 0.001)

Fig. 8

USP10 interacts with XAB2. A Distinct bands stained with silver and identified by MS. B Lysates from SW480 and HCT116 cells immunoprecipitated with the IgG control, anti-XAB2 antibody, or anti-USP10 antibody followed by immunoblotting with USP10 and XAB2 antibodies. C Interaction between Flag-USP10 and Myc-XAB2 confirmed by Co-IP in HEK293T cells. D Increase in nuclear expression of USP10 in CRC cells verified by IF after treatment with oxaliplatin (7.5 µM) for 24 h, which can be inhibited by the ATM inhibitor Ku55933 (20mM). E Increase in nuclear expression of USP10 in CRC cells verified by western blot analysis after treatment with oxaliplatin (7.5 µM) for 24 h. F Schematic representation of Flag-tagged full-length (FL) USP10 with its various deletion mutants. G Co-IP confirming the interaction between Myc-XAB2 and Flag-tagged FL USP10 or its indicated mutants in HEK293T cells

Fig. 9

USP10 stabilises XAB2 expression. A qRT-PCR analysis indicated that USP10 does not regulate XAB2 at the mRNA level. B Western blot analysis demonstrated a gradual increase in XAB2 protein levels with increasing amounts of Flag-USP10 plasmids transfected. C Effect of USP10-siRNAs on XAB2 expression 48 h post-transfection. D Western blot analysis of XAB2 expression with or without spautin-1 treatment for 24 h at various concentrations. E-F XAB2 expression levels after varying durations of CHX (50 µg/ml) administration, with or without MG132 (10µM) treatment for 8 h. G-H XAB2 expression levels after varying durations of CHX (50 µg/ml) administration in cells 48 h post-transfection with Flag vector, Flag-USP10 WT, or Flag-USP10 C424A mutant plasmids. I-J XAB2 expression levels after varying durations of CHX (50 µg/ml) administration, with or without stable USP10 knockdown. K-L XAB2 expression levels after varying durations of CHX (50 µg/ml) administration, with or without spautin-1 (1µM) pretreatment. Data are presented as mean ± SD (**P < 0.01, ***P < 0.001)

Fig. 10

USP10 deubiquitinates XAB2 K48-linked polyubiquitination at K593. A SW480 cells lysed 48 h post-transfection with or without Flag-USP10, immunoprecipitated with an anti-XAB2 antibody or IgG control, and subjected to immunoblotting with a ubiquitin (Ub) antibody. B Lysates of SW480 cells with or without stable USP10 knockdown immunoprecipitated with an anti-XAB2 antibody or IgG control, followed by immunoblotting with a Ub antibody. C The lysates of SW480 cells with or without spautin-1 (1µM) pretreatment for 24 h were immunoprecipitated with the anti-XAB2 antibody or IgG control, followed by immunoblotting with the Ub antibody. D SW480 cells lysed 48 h post-transfection with Myc-XAB2, HA-Ub, and Flag-vector/Flag-USP10 WT/Flag-USP10 C424A, respectively, then immunoprecipitated with a Myc antibody and immunoblotted with an HA antibody. E Ubiquitination site MS of the XAB2 peptide in SW480 cells with stable USP10 knockdown. F-G Lysates from SW480 cells lysed 48 h after transfection with Myc-XAB2 WT/Myc-XAB2 K590R/Myc-XAB2 K593R, HA-Ub, and Flag-vector/Flag-USP10, immunoprecipitated using Myc antibody, and immunoblotted with an HA antibody. H SW480 cells lysed 48 h after transfection with Myc-XAB2, Flag-USP10, and HA-Ub WT/K0/K48/K63, respectively, then immunoprecipitated with a Myc antibody and immunoblotted with an HA antibody. I Western blot analysis indicated the positive XAB2 protein regulation by USP10 depended on the K593 of XAB2

Fig. 11

USP10 promotes CRC proliferation and oxaliplatin resistance through XAB2. A-C XAB2 plasmid transfected into USP10-downregulated CRC cells, with the proliferative ability of cells assessed using CCK8 assay (A) and colony formation assay (B-C). D Transplanted xenografts derived from cells with sh-NC + oe-NC, sh-USP10 + oe-NC, sh-NC + oe-XAB2, and sh-USP10 + oe-XAB2 were established in BALB/c nude mice (n = 5). Tumor volume and weight were measured. E Viability of HT29 and RKO cells analysed using CCK8 assay after treatment with various concentrations of oxaliplatin for 48 h, with IC50 values displayed. F Apoptosis of cells treated with or without oxaliplatin (7.5 µM) for 48 h detected by flow cytometry assays. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 12

USP10 promotes DNA damage repair in CRC cells through XAB2. A Expression level of γH2AX in CRC cells treated with oxaliplatin (7.5 µM) for 24 h analysed using western blot analysis. B γH2AX distribution in CRC cells treated with oxaliplatin (7.5 µM) for 24 h analysed using IF. γH2AX is stained green, and the nucleus is stained blue. Scale bar = 20 μm. C Representative images (left) and bar charts (right) of alkaline comet assays of sh-NC + oe-NC, sh-USP10 + oe-NC, sh-NC + oe-XAB2, and sh-USP10 + oe-XAB2 cells treated with oxaliplatin (7.5 µM) for 48 h. Scale bar = 100 μm. Data are presented as mean ± SD (**P < 0.01, ***P < 0.001)