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. 2026 Feb 7;32:49. doi: 10.1186/s10020-026-01429-0

Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

Aashirwad Shahi 1,#, Shengyuan Zhao 2,#, Julia A Goewey Ruiz 2, Dawit Kidane 1,✉
PMCID: PMC13040979  PMID: 41654792

Abstract

Background

One of the most defining features of gastric cancer (GC) is harboring deficiency in DNA repair that subsequently contributes to carcinogenesis. The X-ray repair cross complementing 1 (XRCC1) protein is a key molecular scaffold required for efficient repair of DNA single-strand breaks (SSBs) to maintain genomic stability. However, further investigation is needed to uncover the role of XRCC1 in innate immune signaling and inflammation in GC.

Methods

We evaluated how loss of XRCC1 leads to accumulation of cytosolic DNA using immunofluorescence localization assay and measuring DNA from cytosolic extract. We applied ON-TARGETplus™ SMARTpool siRNAs to knockdown XRCC1 in gastric cell lines and examined the innate immune siganling and inflammation with and without ATM inhibitor treatment. Further, we examined Type I interferon gene expression in various gastric cancer cell lines and assessed its role in cGAS-STING signaling using RT-qPCR, RNA-Seq, and immunoblot analysis. In addition, we generated conditional knockout XRCC1 mice and characterized the innate immune signaling from stomach tissue extract using RT-qPCR, western blot. Further, the DNA damage and histological analysis was done by immunohistochemistry.

Results

In this work, we examined the role of XRCC1 in modulating the innate immune signaling axis via cGAS/STING pathway. We find that XRCC1 deficient gastric cancer cell lines and mouse stomach tissue shows activation of cGAS/STING signaling. Further, ATM inhibition enhances robust cGAS/STING mediate innate immune signaling and PD-L1 expression in XRCC1 deficient gastric cancer cells.

Conclusions

Results from this work demonstrate that XRCC1 is essential to maintain innate immune homeostasis. Further, this work suggest that ATM inhibitors may provide a potential therapeutic strategy to enhance the PD-L1 expression that could increase the efficacy of an immune checkpoint blockade (ICB) in XRCC1 deficient or low expressing GC.

Graphical Abstract

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Supplementary Information

The online version contains supplementary material available at 10.1186/s10020-026-01429-0.

Keywords: XRCC1, Innate immunity, Gastric cancer

Introduction

Gastric cancer (GC) is the fourth leading cause of cancer-related mortality worldwide (Kelley and Duggan 2003; Sung et al. 2021). Epidemiological studies in the united states suggest that racial/ethnic minorities have a 40%−50% increase in risk of GC compared with non-Hispanic whites (Dong et al. 2017). Previous studies have reported on environmental risk factors that influence GC incidence, includes smoking, high salt diet intake, consumption of alcohol, H. pylori, and Epstein-Barr virus infection (Yang et al. 2011; Zhong et al. 2012; Yassibas et al. 2012; Zhang and Zhang 2011). GC cells harbor several thousand mutations in their genomes leading to dysregulation of critical genes, causing a multitude of genetic and epigenetic changes (Vogelstein et al. 2013; Lawrence et al. 2014; Stoler et al. 1999; Weinhold et al. 2014). Several studies have shown that chronic stimulation of the innate immune system contribute to tumorigenesis (Nowarski et al. 2013; Goldszmid and Trinchieri 2012). In particular, DNA repair factors play an essential role in regulating the innate immune signaling and inflammatory response (Bhattacharya et al. 2017). However, the molecular mechanisms that are critical to trigger DNA mediated innate immune signaling to enhance the efficacy of immune based therapy is unknown.

One of the most defining features of GC is harboring deficiency in DNA repair pathways that subsequently contribute to genomic instability and carcinogenesis. Reactive oxygen species (ROS) generated in cells during normal cellular metabolism and in response to exogenous risk factors induces oxidative DNA damage (Cooke et al. 2003). Oxidized DNA base lesions and single stranded DNA breaks (SSBs) are primarily repaired by the base excision repair (BER) pathway (Wilson and Bohr 2007). Abnormal expression of BER genes in GC has been reported (Tao et al. 2004; Kobayashi et al. 2008). Our work, along with other studies, has shown that BER is critical for preventing GC caused by spontaneous or exogenous risk factors (Zhao et al. 2019). A number of germline variants and somatic mutations of BER genes (~ 30%) significantly increase the risk of cancer and influence the efficacy of GC treatment (Zhao et al. 2021). X-ray repair cross-complementing gene 1(XRCC1) is an essential gene for cell survival and protects cells from DNA damage (Thompson and West 2000) and suppress sister chromatid exchange during S phase stage of the cell cycle (Wilson and Thompson 2007). XRCC1 also facilitates the recruitment of DNA repair proteins to sites of DNA damage and is involved in single-strand break repair (SSBR) (Caldecott 2014; Wilson and Kunkel 2000) and double-strand break (DSB) repair pathways (Audebert et al. 2004; Frit et al. 2014; Mladenov and Iliakis 2011). In addition, XRCC1 is also essential for the protein stability of nuclear LIG3 (Caldecott et al. 1995). In mice, deletion of both alleles of XRCC1 leads to early embryonic lethality, apparently because of dysfunctional SSBs repair and consequent apoptosis (Tebbs et al. 1999). Approximately, 77% of stomach tissues derived from patients of gastric cancer harbor low expression of XRCC1 and are associated with poor overall survival (Wang et al. 2010, 2012). However, the molecular mechanisms of how XRCC1 deficiency modulates innate immune signaling and immune based cancer therapy remain unknown.

In this work, we examined whether XRCC1 deficiency, alone or in combination with an ATM inhibitor leads to cytosolic nucleic acid mediate innate immune signaling activation via the stimulator of interferon genes (STING) in GC. STING is a critical component of the cytosolic DNA mediated innate immune response pathway (Xiao and Fitzgerald 2013; Chen et al. 2016; Song et al. 2017). STING can bind to cyclic dinucleotides, such as cyclic di-GMP-AMP, via its globular carboxy-terminal domain, thus facilitating its interaction with the cytosolic kinase (TBK1) and inducing the translocation of transcription factor IRF3 into the nucleus to induce type I interferons cytokines (Wu et al. 2013). Cyclic GMP-AMP synthase (cGAS) and STING signaling is suppressed in GC cells, which enable them to escape from the host immunosurveillance system (Li et al. 2013). Our data shows that XRCC1 deficient cells generate significant chromosomal instability (micronuclei) that may play a major role in triggering innate immune signaling and inflammation. In addition, inhibition of ATM in XRCC1 deficient cancer cells stimulates the cGAS/STING mediated innate immune signaling and increases the expression of programed cell death ligand 1 receptors (PD-L1). Our study fills some of the major knowledge gaps in our understanding on how XRCC1 regulates innate immune signaling in vivo. Further, our result provide new mechanistic insights into how XRCC1 deficiency or low expression alters the vulnerability of the GC cells to enhance the DNA damage response (DDR) targeted therapy and provides an opportunity for future immune checkpoint blockade (ICB) based therapeutics.

Methods

Cell lines and materials

NCI-N87 (ATCC number: CRL-5822) and Hs764T (ATCC number: HTB-135™ ), AGS( ATCC number: CRL-1739) gastric cell lines were obtained from ATCC and were maintained in DMEM media supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin, and 1% L-Glutamine at 37 °C in a 5% CO2 environment. For ATM inhibitor treatment, cells were seeded 1 × 105 before treatment with ATM inhibitor (Ku55933; 1–10 µl at various concentrations for 24 h). The cells were then washed with PBS and used for further analysis.

In vivo mice study

All animal studies were conducted according to protocols approved by the Institutional Animal Care and Usage Committee of Howard University, College of Medicine (protocol # MED-02-23). We obtained XRCC1 flox/flox mice from Jackson Laboratory of strain #:033691, that loxP flanks from exon 4 to 10, to generate XRCC1 deficient mice. We bred XRCC1 flox/flox mice with ERTTM-Cre and treated mice with 75 mg/Kg body weight of tamoxifen administrated via IP for 5 consecutive days to generate Cre+ XRCC1 Δ/Δ.

Immunohistochemical staining

Tissue samples were collected from Cre-XRCC1 flox/flox and Cre+ XRCC1Δ/Δ mice, washed in Phosphate-buffered saline (PBS), and fixed in 10% neutral-buffered formalin solution (pH 7.4) overnight. Tissues were embedded in paraffin and serially sectioned by the MD Anderson Research Histology Core Facility. Immunostaining for γH2AX was performed using rabbit anti-γH2AX (Cell Signaling, Danvers, MA, USA 2577), respectively. Samples were imaged at 40× magnification with bright field using Aperio Scanscope (Nikon Instruments Inc., Melville, NY, USA), Leica Biosystems (Buffalo Grove, IL, USA). Data for γH2AX were scored blindly by two independent lab individuals. To score γH2AX-positive cells of each tissue section, at least 10 fields were used, and the average percentage of γH2AX-positive cells in three mice for every condition was plotted for immunohistochemical analysis as described previously (Thiem et al. 2013). Histopathological scoring of gastric mucosa was performed as described previously (Rogers and Houghton 2009; Rogers et al. 2005).

RNA extraction and illumina RNA-Seq library preparation

Total cellular RNA was isolated from 1.2 × 106 NCI-N87 cells using PureLink™ RNA Mini kit (Invitrogen, Cat#12183018A). The isolated RNA was cleaned with RNA clean & concentrator kit ( Zymo research, cat#R1013). Then the RNA samples were quantified on a spectrophotometer (Multiskan SkyHigh; ThermoScientific). The high quality RNA samples were sent to Novogene corporation Inc. (Sacramento, CA, USA) for library preparation and Next-generation sequencing. The sequencing libraries were generated with TruSeq RNA Sample Prep Kit v2 Set A (Illumina). Briefly, poly(A) containing mRNA molecules were purified in two rounds using oligo(dT) attached magnetic beads from 1 µg of total RNA. After chemical fragmentation, mRNA fragments were reverse-transcribed and converted into double-stranded cDNA molecules. Following end-repair and dA-tailing, paired-end sequencing adaptors were ligated to the ends of the cDNA fragments using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). RNA was isolated and RNA libraries were prepared for sequencing according to vendor (NEB) protocols. Next, we used the R Small RNA Library Prep Set for Illumina R (Multiplex Compatible, Cat #E7330L), according to the protocol established by Podnar, et al. (2014). Analysis of transcript abundance data was performed in R (v4.0.3) following protocols outlined in Bioconductor at University of Texas at Austin, Bioinformatics core facility and Novogene company.

Sample preparation

Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.

Clustering and sequencing

After library quality control, different libraries were pooled based on the effective concentration and targeted data amount, then subjected to Illumina sequencing. The basic principle of sequencing is “Sequencing by Synthesis”, where fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. As each sequencing cluster extends its complementary strand, the addition of each fluorescently labeled dNTP releases a corresponding fluorescence signal. The sequencer captures these fluorescence signals and converts them into sequencing peaks through computer software, thereby obtaining the sequence information of the target fragment.

Bioinformatics analysis pipeline

We performed data quality procedure using raw data (raw reads) of FASTQ format which were firstly processed through fastp software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality. In addition, reads mapping to reference genome were done. Briefly, reference genome and gene model annotation files were downloaded from genome website. Use HISAT2 (2.2.1) to build the index of the reference genome and use HISAT2 to align paired-end clean reads to the reference genome. HISAT2 can use the gene model annotation file to create splice-aware alignments, providing better alignment accuracy compared to other non-splice alignment tools.

Reverse transcription quantitative real-time PCR (RT-qPCR)

To evaluate the expression levels of selected genes by RT-qPCR, 1 µg of DNA-free total RNA isolated from NCI-N87 and Hs174 cells (three biological replicates per condition) was used for first strand cDNA synthesis with High-capacity cDNA Reverse transcription kit (Applied biosytems, cat#4368814) using oligo(dT) and random primers. Quantitative polymerase chain reaction (qPCR) analysis was performed using PowerTrack™ SYBR Green Master Mix (Applied Biosystem, cat#A46109) with three technical replicates for each biological replicate, according to the manufacturer’s recommendation in an Quant studio 3 Real-Time PCR System (Applied Biosystem). Amplification was real-time-monitored and allowed to proceed in the exponential phase, until fluorescent signal reached a significant value (Ct). The fold change was determined using the 2−ΔΔC(t) method (Livak and Schmittgen 2001).

Western blotting

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor (Cat. 25765800, Sigma Aldrich) and a phosphatase inhibitor (Cat. P5726, Sigma Aldrich). After denaturing the samples at 95 °C for 5 min, 30 µg of each protein sample was separated using SDS-PAGE and transferred onto nitrocellulose membranes (Cat. 1620112, Bio-Rad, Hercules, CA). Next, the membranes were blocked with 5% BSA for 1 h, and then incubated with primary antibodies against STING (Cat. 13647 S, Cell Signaling), IRF3 (Cat. 4302 S, Cell Signaling), p-IRF3 (Cat. 4947 S, Cell Signaling), TBK1 (Cat. 3013 S, Cell Signaling), p-TBK1 (Cat. 5483 S, Cell Signaling), PD-L1 (Cat. 13684, cell signaling), β-actin (Cat. 3700 S, Cell Signaling), and Vinculin (Cat. 13901 S, Cell Signaling) overnight at 4 °C. The following day, the membranes were washed with PBST and incubated with anti-mouse (Cat. NXA931, GE Healthcare, Chicago, IL) or anti-rabbit (Cat. NA934V, GE Healthcare) secondary antibody for 2 h before developing with ECL substrates (Cat. 170506, BioRad). The gel images were captured using ChemiDoc XRS+ image acquisition machine (Bio-Rad).

Cytosolic DNA preparation

Cell extract was prepared with 106 cells using two-step centrifugation protocols and collected the supernatant that represent the cytosolic fraction. We further isolated cytosolic DNA from cytosolic fraction and total DNA from total cell extract. Cells were trypsinized and washed with PBS two times before DNA isolation. Whole-cell DNA was isolated using QIAamp DNA Mini Kit (Cat. 51304, Qiagen) according to the manufacturer’s protocol. For cytoplasmic DNA, cells were lysed in hypotonic lysis buffer (10mM HEPES pH 7.4,10mM KCl, 1.5mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1% Triton X- 100) on ice for 5 min before centrifuging at 1700 g for 5 min. The supernatant containing the cytoplasmic fraction was collected and centrifuged at 13,000 g for 10 min to remove other organelles and incompletely lysed cells. Extraction was validated by Western blot with α-tubulin as the cytoplasmic marker and histone 3 as the nuclear marker. DNA concentration was later quantified using PicoGreen dsDNA assay kit (Cat. P7589, ThermoFisher) according to the manufacturer’s protocol.

Transient XRCC1 knockdown using SiRNA

NCI-N87 cells were seeded at a density of 1 × 105 cells/ well in a 6-well plate one day prior to transfection.For transient knockdown of XRCC1, cells were transfected with 25 nM of ON-TARGETplus™ SMARTpool siRNAs (L-009394-00-0005) targeting XRCC1 and a negative control siRNA ON-TARGETplus™ GAPD Control pool (D-001830-10-05) using DharmaFect transfection reagent according to manufacturer’s instructions. Cells were used for ATMi treatment after 48 h of siRNA transfection.

Immunohistology

Gastric tissues from WT (Cre-XRCC1 flox/flox) and Cre+ XRCC1 Δ/Δ mice were collected and fixed in 4% PFA overnight before sending to HistoWiz Inc. for paraffin embedding. Tissues were then sectioned along the longitudinal axis for H&E histological staining and γH2ax staining primary antibodies applied [(Phospho-Histone H2A.X(Ser139) (20E3) Rabbit monoclonal antibody, Cell Signaling #9718)]. Stained slides were then scanned using Scanscope (Leica Biosystem). For each slide, 5 fields were randomly selected, and the number of positively stained nuclei and total nuclei were counted.

Statistical analysis

Three independent experiments were performed for immunofluorescence, and RT-qPCR. Data were statistically analyzed using Student t-test. Data from more than two study groups were analyzed using two way of ANOVA statistical analysis. Furthermore, the expression of interferon gene was calculated using t-test with Graph Pad Prism software. Results were considered significant at P < 0.05.

Results

XRCC1 deficiency triggers accumulation of cytosolic DNA in GC cells

To examine whether XRCC1 deficiency induces accumulation of cytosolic DNA in GC cells as a result of endogenous DNA damage and failure of repair, we performed knockdown (KD) experiments of XRCC1 using ON-TARGET plus siRNA in NCI-N87 and Hs174 GC cells. The KD of XRCC1 was confirmed by Western blot which showed a ~ 79% depletion of the target protein (Fig. 1A). A reduction in XRCC1 protein levels resulted in decreased SSBR capacity as measured by the comet assay (Supplement Fig. 1A &B). In addition treatment with ATM inhibitor induces double strand breaks in XRCC1 deficient gastric cancer cells (Supplement Fig. 1C and D).To determine whether the accumulation of unrepaired DNA damage in XRCC1 deficient cells, either spontaneously or from ATM inhibition, leads to mitotic dysfunction, we treated XRCC1 proficient and deficient cells with an ATM inhibitor (10µM) for 24 h and we examined the presence of micronuclei, which is one of the landmarks of mitotic dysfunction and source of cytosolic DNA. As shown in Fig. 1B, the micronuclei frequency was increased in XRCC1 deficient cells after ATM inhibitor treatment. Interestingly, we observed a significantly pronounced formation of spontaneous micronuclei in XRCC1 deficient cells versus proficient cells. We found that 80% of ATM treated XRCC1 deficient cells harbor micronuclei versus WT (10%) (Fig. 1C, P < 0.001). Results from this experiment highlight the micronuclei formation in ATM inhibitor treated XRCC1 deficient cells which consequently leads to the release of cytosolic DNA. In addition, to determine whether cGAS localization in micronuclei is a general phenomenon in XRCC1 deficient cells, we transfected XRCC1 proficient and deficient XRCC1 cells with pMSCVpuro-GFP-cGAS or stably expressing GFP-cGAS plasmids (generous gift from Dr. Andrew P. Jackson & Dr. Martin A. Reijns, MRC, UK) and examined the colocalization of cGAS at the micronuclei. As seen in Supplement Fig. 2A and B, we found that cGAS strongly colocalized with micronuclei in XRCC1 deficient cells. This result likely suggesting that nuclear DNA (nDNA) released from micronuclei may be an important danger signal to elicit an inflammatory response. To determine if inhibition of ATM increases cytosolic DNA, we prepared a cellular fraction from XRCC1 proficient and deficient cells using a protocol according to the Cell Fraction Kit Abcam (Cat # ab109719). We performed nuclear and cytoplasmic fractionation of cell lysates followed by DNA precipitation and we quantified double-strand (dsDNA) DNA in the cytoplasmic fractions of XRCC1 deficient and WT cells. We found 350 ng/106 cells with cytosolic dsDNA in WT versus 645 ng/106 cells with cytosolic dsDNA in XRCC1 deficient cells treated with ATM inhibitor respectively (Fig. 1D). To further rule out the possibility of ATM inhibitor induced mitochondrial DNA (mtDNA) leakage contributing to an accumulation of cytosolic DNA in XRCC1 deficient cells, we measured the level of ND5 mtDNA using qPCR from the cytosolic extract of cells with and without treatment of ATM inhibitor. We found significantly higher cytosolic mtDNA in ATM inhibitor treated XRCC1 deficient cells versus proficient cells (Fig. 1E). Further, our RNA-Seq analysis demonstrated that the hallmark ROS signaling pathway is enhanced in XRCC1 knockdown GC cells (Fig. 1F). In addition, we validated our results using RT-PCR for ROS associated genes expression and results support our RNA -Seq finding (Supplement Fig. 3). Overall, these results demonstrate that inhibition of ATM enhances the accumulation of cytosolic DNA, from nuclear as well as mitrochdrial DNA, that may activate innate immune signaling in XRCC1 deficient cells.

Fig. 1.

Fig. 1

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ATM inhibitor enhances accumulation of cytosolic DNA in XRCC1 deficient cells. A. Gastric cancer cell line (NCI-N87) were transfected with XRCC1 siRNA (ON TARGET plus siRNA: SMART pool) for 48 hours and XRCC1 knockdown determined by Western blot (β-actin as a loading control). B. ATM inhibitors exacerbate micronuclei formation in XRCC1 deficient cells. XRCC1 proficient and deficient mouse embryonic fibroblast cells (MEFs) treated with ATM inhibitor (Ku55933) for 24 h and nuclear DNA were stained with DAPI and visualized using immunofluorescence assay. C Percent of positive cells with micronuclei in XRCC1 proficient and deficient cells. D Quantification of double stranded DNA from cytosolic fraction and total DNA from total cell extracts using the Cell Fraction Kit (Cat # ab109719, Abcam). E qPCR quantification of cytoplasmic mtDNA from cytosolic fraction normalized with whole extracts using mtDNA specific primers (ND5). F Enrichment of ROS associated genes in XRCC1 proficient and deficient cells. Statistical comparison of the data was analyzed using a Student t-test in GraphPad Prism (** P < 0.01; ****P < 0.0001; ns: represent not significant)

XRCC1 deficient GC cells activate cGAS/STING signaling pathway

Defects in DNA repair pathways and cellular DNA damage response (DDR) affects innate immune responses in various ways. To determine whether inhibition of ATM induces activation of cGAS/STING/TBK1/IRF3 dependent innate immune signaling in XRCC1 deficient cells, we examined TBK1 and IRF3 phosphorylation status using Western blot in XRCC1 proficient and deficient cells treated with an ATM inhibitor. The results showed that pTBK1 and pIRF3 [(phosphorylation of TBK1; pTBK1 (Ser172) and pIRF-3 (Ser396)] increase in ATM treated XRCC1 deficient cells versus proficient cells (Fig. 2A). Further, we analyzed RNA-Seq data to examine whether XRCC1 deficiency promotes the expression of genes involved as cytosolic DNA sensors. We have found that XRCC1 deficiency enhances the enrichment of cytosolic DNA sensor pathway, which is further exacerbated by ATM inhibition (Fig. 2B & C) respectively. The volcano plot and heat map in Fig. 2D and E show there is an elevated expression of genes involved in the cytosolic DNA sensing signaling pathways including STAT1/2, OASL, IRFI1/2/3, ISG15, DDX58, MX1 IRF7/9, and CCL5 in XRCC1 deficient GC cells. In addition, the expression of genes associated with DNA sensor function are validated with RT-qPCR (Supplement Fig. 4). Furthermore, STAT1/STAT2, which are the central transcription factors downstream of interferon signaling, are enriched in XRCC1 defcient cells as well as ATM inhibitior treated gastric cancer cells as compared with wild type cells (Fig. 2E). Results from this part of our study suggest that inhibition of ATM in XRCC1 deficient cells is critical for activation of the cGAS/STING innate immune signaling pathways.

Fig. 2.

Fig. 2

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ATM inhibitors enhance cGAS/STING signal activation. A STING/TBK1/IRF-3 signaling pathway activation detected by Western blot of protein extract from siRNA control (WT) and siRNA-XRCC1 knockdown cells treated with and without ATM inhibitor. Anti-IRF3/ p-IRF3 (ser396); TBK1/P-TBK-1 (Ser172) antibodies were obtained from Cell Signaling. Note that fraction of phosphrylated value calculated obtained by dividing the band intensity of physphrylated proteins relative to the total respective unphosphorylated protein band intensity. All band intensity were measured using Bio-Rad Image lab software. B Enrichment of cytosolic DNA sensing genes in WT and XRCC1 knockout GC cells from HallMark data base in enrichment score Gene Set Enrichment Analysis (GSEA ver. 4.3.2) in GSEA; C Enrichment of cytosolic DNA sensing genes expression in GC cells were treated with 10 µM ATM inhibitor (ATMi, KU55933) for 24 h. D Volcano graph represent the overall gene expression of inflammatory, cytosolic DNA sensing, and inflammatory cell signaling ; E Heat map of set of genes in volved in interferon response in XRCC1 proficient NCI-N87 (N87-WT) and deficient NCI-N87 (N87-KD)

Targeting ATM enhances innate inflammatory response in XRCC1 deficient GC cells

To further examine whether XRCC1 deficiency enhances inflammatory gene expression in GC cells as a consequence of the failure to repair, we performed a knockdown (KD) experiment of XRCC1 using ON-TARGET plus siRNA in NCI-N87 cells. As shown in Fig. 3A, we have found that inflammatory response genes are enriched in the absence of XRCC1. In addition, the expression of interferon genes (e.g. IRF3, IRF7, IRF9, CCL5, IFNB1, and ISG15) is significantly increased in XRCC1 deficient GC cells (Fig. 3B). Next, we checked whether ATM inhibitor treatment exacerbates the expression of innate immune genes in XRCC1 deficient cells, we measured the transcriptional activation of CCL5, CXCL10, and IFNB using RT-qPCR, and found that the mRNA expression of these Type 1 interferon cytokines/chemokines is significantly increased in ATM inhibitor treated XRCC1 deficient cells versus wild type (Fig. 3C, p < 0.001).These cytokines/chemokines are known to be involved in recruiting immune cells (Jinyan Liu et al. 2015; Qiu et al. 2003; Song 2007). Thus, our data suggest that targeting ATM in XRCC1 deficient cancer cells stimulate innate immune response and an inflammatory response.

Fig. 3.

Fig. 3

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Type I interferon response in XRCC1 deficient cells. XRCC1 proficient and deficient NCI-N87 gastric cancer cell line examined for inflammatory response. A Gene expression of inflammatory response change in XRCC1 proficient and deficient NCI-N87 cells from HallMark data base in enrichment score Gene SetEnrichment Analysis (GSEA ver. 4.3.2) in GSEA. B Gene expression of inflammatory response change in XRCC1 proficient and deficient NCI-N87 cells were treated with 10 µM ATM inhibitor (ATMi, KU55933) for 24 h. Preranked mode was performed on data for all contrasts. Each pre-ranked gene list provided consisted of significance-weighted Log2 fold change values for each gene; C mRNA expression of inflammatory cytokines was measured using RT-qPCR. NCI-N87 cells were treated with 10 µM ATM inhibitor (ATMi, KU55933) for 24 h. Statistical comparison of the data was analyzed using a Student t-test in GraphPad Prism (**P < 0.01; ***P < 0.001; ****P < 0.0001)

Loss of XRCC1 induces genomic instability in mice stomach tissue

XRCC1 plays a critical scaffolding role in coordinating repair of DNA base damage and single-strand breaks (Hanssen-Bauer et al. 2012). To determine whether loss of XRCC1 induces spontaneous DSBs in mice, stomach tissue sections were fixed and processed for immunohistochemistry using phosphorylated H2AX (γH2AX) as a DSB marker. We used tamoxifen induced Cre mediated conditional knockout of XRCC1 in stomach tissues and found that Cre+XRCC1 Δ/Δ mice exhibited a significantly increased frequency of DSBs compared with that of Cre-XRCC1 flox/flox mice in the stomach tissue (Fig. 4A). This was confirmed by quantification of the number of positive cells stained for γH2AX (Fig. 4B). As shown in Fig. 2B, XRCC1 deficient mice stomach tissue exhibited a significantly increased frequency of DSBs compared with WT mice (P < 0.0001). Further, protein extracts from stomach tissue of Cre+XRCC1 Δ/Δ shows a significantly increased level of γH2AX by western blot analysis (Fig. 4C). We also examined the efficiency of tamoxifen inducible ERT-Cre driving deletion in stomach tissue of mice using Western blots. Results from this study shows that Cre+ XRCC1 mice exhibit XRCC1 deletion (Supplement Fig. 5A-C). However, we acknowledge that the Western blot from mouse stomach tissue–derived extracts shown in Supplementary Fig. 5A displays a splice-like banding pattern, which may indicate the presence of a modified form of XRCC1. Overall, these data suggest that loss of XRCC1 leads to increased endogenous DNA damage in vivo.

Fig. 4.

Fig. 4

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DNA double strand breaks increased in XRCC1 deficient mouse stomach. A Immunohistochemistry of stomach sections stained with γH2AX (double strand break marker); The black head arrow shows the brown stained cells represent localization of γH2AX. B Quantification of γH2AX-positive cells from Cre-XRCC1 flox/flox and Cre+XRCC1 △/△ stomach tissues. C Western blot of γH2AX cells from Cre-XRCC1 flox/flox and Cre+XRCC1 △/△ stomach tissues. Note that the intensity of band measured from each lane and normalized to loading control and fold chage calculated relative to control (WT: Cre-XRCC1 flox/flox ). All band intensity were measured using Bio-Rad Chemdoc Image analysis software

XRCC1 deficiency promotes cGAS/STING activation and inflammation in mice stomach

To examine whether XRCC1 deficient mice exhibit inflammation in stomach tissue, histological analysis was performed on stomach tissues from 6-month-old mice using H and E staining (Fig. 5A). We found that Cre-XRCC1 flox/flox mice displayed moderate inflammation, whereas Cre+XRCC1 Δ/Δ exhibited strong inflammatory changes (Fig. 5B and C). In addition, we determined whether tamoxifen induces inflammatory response in gastric tissues of mice. Our RT-qPCR data demonstrated that no significant increase of the expression of genes associated with inflammatory response (Supplement Fig. 5D). We also examined whether XRCC1 deficiency activates cGAS/STING mediated innate immune pathway, contributing to inflammation. Western blot analysis reveals a cGAS/STING activation signature including phosphorylation of IRF3 and TBK1 (Fig. 5D). To expand our examination of whether XRCC1 deficiency contributes to increased inflammatory cytokine and chemokine responses, we measured the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL1-β), interferon beta (IFNβ), and IL-6 proinflammatory cytokines and chemokines (chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10)) using RT-qPCR and found a significantly increased level in the Cre+XRCC1 Δ/Δ versus Cre-XRCC1 flox/flox (WT mice; Fig. 5E). This data supports our other findings that XRCC1 deficient mice stomach tissue has an amplified inflammatory response.

Fig. 5.

Fig. 5

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A-E Inflammation-mediated response in XRCC1 deficient mice stomach. A Experimental design of tamoxifen treatment to Cre expression and excised XRCC1 gene segment. B Representative image of gastric histopathology of XRCC1 deficient mice stomach with H and E staining; H & E stain of stomach sections from Cre-XRCC1 flox/flox (WT) and Cre+XRCC1 △/△. C Quantification of inflammatory score between Cre-XRCC1 flox/flox (5 mice) and Cre+XRCC1 Δ/Δmice (5 mice) stomach tissue sections; Low-to-medium-grade inflammation with abundant lymphocytes and neutrophilic granulocytes in XRCC1 deficient mice stomach. Note that inflammation score was done by grading pathological severity as normal (0), mild (Kelley and Duggan 2003), moderate (Sung et al. 2021), severe (Dong et al. 2017); D Western blot analysis of TBK1,p-TBK1(Ser172), IRF3 and p-IRF3 in stomach tissue from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ mice. Note that fraction of phosphrylated value calculated obtained by dividing the band intensity of physphrylated proteins relative to the total respective unphosphorylated protein band intensity (ratio). E mRNA expression of genes of cytokines and chemokines quantified using RT-qPCR from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ

Loss of XRCC1 enhances the expression of PD-L1 in XRCC1 deficient GC cells and in XRCC1 deficient stomach tissue

In this part of our study, we examined whether loss of XRCC1 triggers program cell death ligand-1 expression in GC cells in vitro and in stomach tissues of mouse model. Our data demonstrated that PD-L1 expression is altered in the absence of XRCC1 in gastric tissues derived from Cre+XRCC1 Δ/Δ versus Cre-XRCC1 flox/flox mice (Fig. 6A and B). Further, we extend our study to determine whether inhibiting ATM to do so, we measured the mRNA and protein levels of PD-L1 following inhibition of ATM in XRCC1 deficient GC cells and proficient GC cells and found a significant increase of PD-L1 in the XRCC1 deficient GC cells (Fig. 6C and D). Furthermore, our data shows that STING inhibitor suppressed the expression of PD-L1 by 11% in ATM inhibitor treated XRCC1 deficient gastric cells versus XRCC1 proficient control treated with ATM and STING inhibitor (Supplement Fig. 6). It is noteworthy that we used NCI-N87 gastric cancer (GC) cells, which express XRCC1 at relatively higher levels than AGS cell lines (Supplement Fig. 7) Taken together, this data further support future studies to test whether immune checkpoint blockade (ICB) in combination with ATM inhibitor in XRCC1 deficient gastric cancer.

Fig. 6.

Fig. 6

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Inhibition of ATM enhances the transcriptional expression of PD-L1 in XRCC1 deficient gastric tissues and GC cells. A Western blot analysis of PD-L1 expression in gastric tissue from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ mice. B Quantitative RT-PCR analysis of PD-L1 mRNA expression in gastric tissue from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ mice. C Western blot analysis of PD-L1 expression in NCI-N87 GC cells. Note that the intensity of band measured from each lane and normalized to loading control and fold chage calculated relative to control. All band intensity were measured using Bio-Rad Chemdoc Image analysis software. D XRCC1 proficient NCI-N87(siRNA-control) and deficient (NCI-N87, siRNA-XRCC1) cells treated with ATM inhibitor for 24 h and mRNA expression of PD-L1 measured using RT-qPCR. Statistical comparison of the data was analyzed using a ANOVA-Two way in GraphPad Prism (** P < 0.01; P***<0.001; P****<0.0001; ns: not significant)

Discussion

This study demonstrates that loss of XRCC1 plays a major role in nucleic acids mediated innate immune signaling and inflammation. XRCC1 is a critical scaffold protein that promotes efficient single-strand break repair (SSBR) by coordinating the enzymatic processing of non-conventional 3′ or 5′ DNA termini and SSB gaps (Thompson and West 2000; Caldecott 2003). Using XRCC1 as a representative of SSBR, we assessed the role XRCC1 in cellular protection against the nucleic acids mediated innate immune signaling and inflammation using in vitro and in vivo models. This study found that cells lacking XRCC1 exhbits a significant accumulation of SSBs and subsequent treatment with ATM inhibitor induces DSB. It has been previously reported that an accumulation of oxidative stress related DNA damage eventually causes replication stress in BER deficient cells (Fouquerel et al. 2019). Several work reported that XRCC1 recurited to sites of SSBs following exposure to DNA damaging agents (El-Khamisy et al. 2003; Okano et al. 2003). Low XRCC1 expression levels are also associated with a significant delay in S-phase progression after exposure to DNA damaging agents (Brem and Hall 2005). Our data aligned with previous work that uncovered XRCC1 defceint cells are hypersensitive to a broad range of DNA damage including alkylating agents, ROS or ionizing radiation (Tebbs et al. 1999; Brem and Hall 2005; Thompson et al. 1982, 1990). XRCC1 also carries out other functions associated in the maintenance of replisome stability and required to repair double strand breaks (Brem and Hall 2005; Ying et al. 2016; Eckelmann et al. 2020). At the replication fork, XRCC1 has been found to physically interact with other replication factors (i.e., PCNA, DNA polymerase α-primase, FEN1) (Fan et al. 2004; Clements et al. 2004; Hanzlikova et al. 2018). In addition, in vivo study provides evidence of that loss of XRCC1 increases double strand breaks in mice stomach tissue that may arise from compromised DNA replication. However, in vitro studies demonstrated that XRCC1 depletion did not affect DNA replication progression or fork protection, whereas it did significantly limit the extent of replication fork restart (Eckelmann et al. 2020).

Further, our study highlights that the reduction in XRCC1 expression result in more frequent spontaneous formations of micronuclei and exacerbate by ATM inhibitor treatment. ATM is a critical component of the cellular DDR (Marechal and Zou 2013) and its inhibition impairs the balance between DNA damage and repair. This elevation in micronuclei formation is commonly observed in XRCC1 deficient cells, which likely refelects the accumulation of unrepaired DNA damage, which may be a key factor to drive chromaosmal instability. Additionally, XRCC1 deficient cell lines display increased rates of spontaneous sister-chromatid exchange, chromosomal aberrations and micronuclei (Brem and Hall 2005; Zdzienicka et al. 1992). Collectively, our data supporting that the accumaulation of unrepaired DNA damage in XRCC1 deficient cells may lead to mitotic error and the formation of micronuclei. Interestingly, our data demonstrated that XRCC1 deficiency associated genomic instability leads to activation of cytosolic DNA mediated innate immune signaling. Results from this work shows that GC cells treated with ATM inhibitor significantly increases cytosolic DNA in XRCC1 deficient GC cells, suggesting that genome instability likely contributes to an aberrant inflammatory response. We reasoned that one possible mechanism linking ATM deficiency to cytokine production would be the accumulation of unrepaired DNA damage that ended up moving to the cytoplasm (Ahn et al. 2014). This observation consistent with prior study that demonstrated cytosolic DNA mediated cGAS/STING signaling pathway activation in cancer cells (Kwon and Bakhoum 2020). Additionally, it has been established that most of the downstream phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) induces a Type I interferon response and, thus, produces immunomodulatory cytokines and chemokines (Ding et al. 2018; Pantelidou et al. 2019). Further, Stetson, et al. has shown that an innate immune response is triggered by cytosolic DNA (Stetson and Medzhitov 2006), as STING leads to the activation of IRF3 through phosphorylation and elevates a Type I interferon response (Liu et al. 2015). In this context, activation of cGAS/STING pathway in XRCC1 deficient gastric cancer, along with increase expression of inflammatory cytokines and chemokines, is likely the primary consequence of cytosolic DNA mediated innate immune signaling that drives tissue inflammation in mice (McArthur et al. 2018). Moreover, our in vivo data suggest that activation of innate immune signaling in XRCC1 deficient mice stomach likely enhances the migration of immune cells in tumor microenvironment and regulate an anti-tumor immune response (Harlin et al. 2009; Dangaj et al. 2019).

Additionally, we found a significant augmentation of PD-L1 and MHC class I expression induced by ATM inhibition in GC cells. Several studies have shown that PD-L1 is commonly elevated in cancer cells and is an important mechanism that negatively regulates T-cell activation to prevent anti-tumor immune responses (Pardoll 2012). Some studies have shown that the expression of PD-L1 is epigenetically regulated via promoter methylation (Qiu et al. 2020). PD-L1 expression in cancer cell lines can be stimulated by interferons (IFNs) cytokines and modulate the cytotoxic T-cell lymphocytes (CTLs) in the tumor microenvironment (Tang and Zheng 2018). Immunosuppressive mechanisms within the tumor microenvironment have been associated with the failure of tumor-specific T cells (Schlosser et al. 2014). Others have reported that PD-L1 overexpression in gastric adenocarcinoma tissue exhibit a better response rate to immune checkpoint blockade (ICB) treatment (Ohigashi et al. 2005; Wu et al. 2006). In contrast, GC with low immunogenicity and low PD-L1 expression may contribute towards lower overall response rates to (ICB) therapy. Our data shows that enhanced PD-L1 expression in XRCC1 deficient mice stomach tissue and in ATM inhibitor treated XRCC1 deficient GC cells suggest that XRCC1 modulates PD-L1 expression. It is also possible that activation of cGAS/STING pathways contributed to upregulation of PD-L1 expression in XRCC1 deficient gastric cancer cells. Alternatively, enrichment of STAT1/STAT2 in GC cells may promote downstream activation of interferon signaling pathways, further supporting PD-L1 expression, consistent with other findings (Liu et al. 2020; Canar et al. 2023). Interestingly, we observed a higher molecular weight of PD-L1 in XRCC1 deficient cells suggesting potential post translational modifications or the presence of PD-L1 variants, consistent with the previous studies reporting the presence of PD-L1 variants (Yu et al. 2020; Schulz et al. 2024).

Overall, our data suggest that targeting the DNA damage response pathway exacerbates tumor immunogencity in XRCC1 deficient GC.

Conclusion

In summary, it is worth highlighting that these conclusions may impact selecting an appropriate immune based therapy for tumors harboring deficient or a nonfunctional mutant of XRCC1. Moreover, our study suggests that ATM inhibition not only stimulates inflammatory factors through the cGAS–STING pathway but also augments MHC class I expression, potentially supporting T cell activation. Our findings underscore the significant potenatial of utilizing XRCC1 in modulating DNA mediated innate immune inflammatory response via cGAS/STING signaling pathways, thereby reaveling tumor immunogencity. Additionally, these results provide a preclinical evidence for future to further explore combinatorial therapeutic strategies by combining ATM inhibition with immunotherapy in gastric tumor with aberrant XRCC1 function. Moreover, our approaches align with current therapeutic strategies that utilize PD-L1 and PD-1 blocking antibodies to inhibit immune checkpoints for achiving durable responses across various cancer types (Zou et al. 2016). It is also important to acknowledge that our study has limitations, further experimental investigation are needed to determine whether PD-L1 expression in gastric cancer is solely influenced by XRCC1 mediated cGAS/STING pathway, or if other alternative mechanisms contribute to its regulation.

Supplementary Information

10020_2026_1429_MOESM1_ESM.docx (38.4KB, docx)

Supplementary Material 1. KEY RESOURCES TABLE

Supplementary Material 2. Primers Excel (71.1KB, xlsx)
Supplementary Material 3. Comet Assays (24.2KB, pdf)
10020_2026_1429_MOESM4_ESM.zip (526.4KB, zip)

Supplementary Material 4. Captions of figure and figures

Supplementary Material 5. (1.2MB, pdf)
Supplementary Material 6. (143.9KB, pdf)
Supplementary Material 7. (99.5KB, pdf)

Authors’ contributions

DK: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing. AS: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. SZ: perform immunofluorescence and western blot experiment and participate in data analysis; JAGR: genotyping of mice and collecting of samples.

Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. Research reported in this publication was supported by DeGregorio Family Foundation and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R01AI179899.

Data availability

All RNA-Seq data avalaible and will bedeposited in public available database. Other original data avalible for reanalyze the data reported in this paper avalaiable at the lead contact.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Note applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Aashirwad Shahi and Shengyuan Zhao contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

10020_2026_1429_MOESM1_ESM.docx (38.4KB, docx)

Supplementary Material 1. KEY RESOURCES TABLE

Supplementary Material 2. Primers Excel (71.1KB, xlsx)
Supplementary Material 3. Comet Assays (24.2KB, pdf)
10020_2026_1429_MOESM4_ESM.zip (526.4KB, zip)

Supplementary Material 4. Captions of figure and figures

Supplementary Material 5. (1.2MB, pdf)
Supplementary Material 6. (143.9KB, pdf)
Supplementary Material 7. (99.5KB, pdf)

Data Availability Statement

All RNA-Seq data avalaible and will bedeposited in public available database. Other original data avalible for reanalyze the data reported in this paper avalaiable at the lead contact.

Articles from Molecular Medicine are provided here courtesy of The Feinstein Institute for Medical Research at North Shore LIJ

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