Metformin inhibits growth and enhances radiation response of non-small cell lung cancer (NSCLC) through ATM and AMPK

Background:

We examined the potential of metformin (MET) to enhance non-small cell lung cancer (NSCLC) responses to ionising radiation (IR).

Methods:

Human NSCLC cells, mouse embryonic fibroblasts from wild-type and AMP-activated kinase (AMPK) α1/2-subunit^−/− embryos (AMPKα1/2^−/−-MEFs) and NSCLC tumours grafted into Balb/c-nude mice were treated with IR and MET and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays and immunohistochemistry (IHC).

Results:

Metformin (2.5 μℳ–5 mℳ) inhibited proliferation and radio-sensitised NSCLC cells. Metformin (i) activated the ataxia telengiectasia-mutated (ATM)–AMPK–p53/p21^cip1 and inhibited the Akt–mammalian target of rapamycin (mTOR)–eIF4E-binding protein 1 (4EBP1) pathways, (ii) induced G1 cycle arrest and (iii) enhanced apoptosis. ATM inhibition blocked MET and IR activation of AMPK. Non-small cell lung cancer cells with inhibited AMPK and AMPKα1/2^−/−-MEFs were resistant to the antiproliferative effects of MET and IR. Metformin or IR inhibited xenograft growth and combined treatment enhanced it further than each treatment alone. Ionising radiation and MET induced (i) sustained activation of ATM–AMPK–p53/p21^cip1 and inhibition of Akt–mTOR–4EBP1 pathways in tumours, (ii) reduced expression of angiogenesis and (iii) enhanced expression of apoptosis markers.

Conclusion:

Clinically achievable MET doses inhibit NSCLC cell and tumour growth and sensitise them to IR. Metformin and IR mediate their action through an ATM–AMPK-dependent pathway. Our results suggest that MET can be a clinically useful adjunct to radiotherapy in NSCLC.     

ABC294640, a sphingosine kinase 2 inhibitor,enhances the antitumor effects of TRAIL in non-small cell lung cancer

Evidences suggest that tumor microenvironment may play an important role in cancer drug resistance. Sphingosine kinase 2 (SphK2) is proposed to be the key regulator of sphingolipid signaling. This study is aimed to investigate whether the combination of molecular targeting therapy using a specific inhibitor of SphK2 (ABC294640), with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can enhance the apoptosis of non-small cell lung cancer (NSCLC) cells. Our results revealed that NSCLC cells'' sensitivity to TRAIL is correlated with the level of SphK2. Compared with TRAIL alone, the combination therapy enhanced the apoptosis induced by TRAIL, and knockdown of SphK2 by siRNA presented a similar effect. Combination therapy with ABC294640 increased the activity of caspase-3/8 and up-regulated the expression of death receptors (DR). Additional investigations revealed that translocation of DR4/5 to the cell membrane surface was promoted by adding ABC294640. However, expression of anti-apoptosis proteins such as Bcl^-2 and IAPs was not significantly modified by this SphK2 inhibitor. Overall, this work demonstrates that SphK2 may contribute to the apoptosis resistance in NSCLC, thus indicating a new therapeutic target for resistant NSCLC cells.     

Sequential chemoimmunotherapy for advanced non-small cell lung cancer using cisplatin, etoposide, thymosin-αl and interferon-α2a

A phase II study was performed to evaluate the clinical and immunological effects of a regimen of cisplatin (DDP) and etoposide (VP-16) combined with thymosin-α1 (TA1) and low-dose interferon-α2a (IFN) in the treatment of patients with advanced non-small cell lung cancer (NSCLC). Chemoimmunotherapy cycles were repeated every 3 weeks. There were 24 responses (two complete, 22 partial) among 56 assessable patients. Median survival was 12.6 months. Overall, treatment was well tolerated. Natural killer cell activity and lymphocyte subtypes were depressed by chemotherapy, but this effect was less prominent in patients receiving TA1 and IFN in comparison with a concomitant group of patients treated with DDP and VP-16 only. The combination of DDP and VP-16 and TA1 and IFN is effective in advanced NSCLC with acceptable toxicity. However, the results of this study need to be confirmed in a randomised trial.     

3’untranslated regions (3’UTR) of Gelsolin mRNA displays anticancer effects in non-small cell lung cancer (NSCLC) cells

RNA-based therapeutics has attracted substantial interest from both academics and pharmaceutical companies. In this study, we investigated the function and the underlying mechanism of Gelsolin (GSN) 3’UTR in NSCLC H1299 and A549 cells. We found that transfected Flag-GSN plasmids significantly increased the proliferation, migration and invasion of NSCLC cells, whereas GSN 3’UTR could suppress the promotional effect of GSN protein on the development of NSCLC in vitro. Interestingly, we observed that these in vitro anticancer effects of GSN 3’UTR was independent of the co-expression with GSN coding sequence. Moreover, transfected GSN 3’UTR affected the actin-cytoskeleton remodeling and epithelial-mesenchymal transition (EMT) processes in H1299 and A549 cells, and targeted the co-expressed proteins to the plasma membrane. Subsequently, RNA pull-down assays have been performed to identify Tra2β protein as a GSN 3’UTR binder. We then showed that Tra2β was important for the localized protein expression mediated by GSN 3’UTR. Taken together, our results suggested that GSN 3’UTR may exert anticancer functions in NSCLC cells through regulating the subcellular localized expression of GSN protein mediated by the interaction between GSN 3’UTR-Tra2β.     

TMEPAI/PMEPA1 enhances tumorigenic activities in lung cancer cells

TMEPAI/PMEPA1 is a transmembrane protein that was originally identified as a prostatic RNA, the synthesis of which is induced by testosterone or its derivatives. We have recently identified TMEPAI as a direct target gene of transforming growth factor‐β (TGF‐β)/Smad signaling that participates in negative feedback control of the duration and intensity of TGF‐β/Smad signaling. TMEPAI is constitutively and highly expressed in many types of cancer and is associated with poor prognosis. Here, we report that TMEPAI is highly expressed in the lung adenocarcinoma cell lines Calu3, NCI‐H23, and RERF‐LC‐KJ. Expression of TMEPAI in these cancer cells was significantly suppressed by a TGF‐β receptor kinase antagonist, SB208, and by TGF‐β neutralizing antibodies. These results suggest that constitutive expression of TMEPAI in these cancer cells depends on autocrine TGF‐β stimulation. Knockdown of TMEPAI in Calu3 and NCI‐H23 cells enhanced levels of Smad2 phosphorylation and significantly suppressed cell proliferation in the presence of TGF‐β, indicating that highly expressed TMEPAI suppresses levels of Smad phosphorylation in these cancer cells and reduces the growth inhibitory effects of TGF‐β/Smad signaling. Furthermore, knockdown of TMEPAI in Calu3 and NCI‐H23 cells suppressed sphere formation in vitro and tumor formation in s.c. tissues and in lungs after tail vein injection in NOD‐SCID mice in vivo. Together, these experiments indicate that TMEPAI promotes tumorigenic activities in lung cancer cells.     

Randomized phase 2 study of the cyclin-dependent kinase inhibitor dinaciclib (MK-7965) versus erlotinib in patients with non-small cell lung cancer

Objectives

Dinaciclib (MK-7965, formerly SCH 727965), a novel, small-molecule inhibitor of cyclin-dependent kinases, has been shown to induce apoptosis in preclinical studies of human tumor cell lines, including non-small cell lung cancer (NSCLC) cells. Erlotinib, an epidermal growth factor receptor inhibitor, is approved for the treatment of advanced NSCLC as second- or third-line therapy. This phase 2, randomized, multicenter, open-label study compared dinaciclib with erlotinib in patients with previously treated NSCLC.

Materials and methods

The study was comprised of 2 parts: in part 1, patients were randomized to either intravenous (IV) dinaciclib (50 mg/m²) or oral erlotinib (150 mg) using an adaptive Bayesian design that adjusted the randomization ratio in favor of the more active arm, and in part 2, patients who had progressed on erlotinib were permitted to cross over to receive dinaciclib at the same dosage as in part 1. Patients were followed until disease progression or death, initiation of nonstudy cancer treatment, discontinuation, or withdrawal of consent. The primary efficacy end point was time-to-progression (TTP) in part 1 and objective response rate (ORR) in part 2.

Results

Based on Kaplan–Meier estimates, the median TTP was 1.49 months (95% confidence interval [CI]: 1.31, 2.63) following initial treatment with dinaciclib, compared with 1.58 months (95% CI: 1.38, 2.83) with erlotinib. No objective responses were observed following initial treatment with dinaciclib. Common severe (grade 3 or 4) drug-related adverse effects included neutropenia, leukopenia, vomiting, and diarrhea.

Conclusions

Dinaciclib, administered IV, was well tolerated at the 50 mg/m² dose, but does not have activity as monotherapy in previously treated NSCLC. Evaluation of dinaciclib in combination with other agents for other indications including breast cancer and multiple myeloma is in progress.     

Genomic instability and cellular stress in organ biopsies and peripheral blood lymphocytes from patients with colorectal cancer and predisposing pathologies

Inflammatory bowel disease (IBD) and polyps, are common colorectal pathologies in western society and are risk factors for development of colorectal cancer (CRC). Genomic instability is a cancer hallmark and is connected to changes in chromosomal structure, often caused by double strand break formation (DSB), and aneuploidy. Cellular stress, may contribute to genomic instability. In colorectal biopsies and peripheral blood lymphocytes of patients with IBD, polyps and CRC, we evaluated 1) genomic instability using the γH2AX assay as marker of DSB and micronuclei in mononuclear lymphocytes kept under cytodieresis inhibition, and 2) cellular stress through expression and cellular localization of glutathione-S-transferase omega 1 (GSTO1). Colon biopsies showed γH2AX increase starting from polyps, while lymphocytes already from IBD. Micronuclei frequency began to rise in lymphocytes of subjects with polyps, suggesting a systemic genomic instability condition. Colorectal tissues lost GSTO1 expression but increased nuclear localization with pathology progression. Lymphocytes did not change GSTO1 expression and localization until CRC formation, where enzyme expression was increased. We propose that the growing genomic instability found in our patients is connected with the alteration of cellular environment. Evaluation of genomic damage and cellular stress in colorectal pathologies may facilitate prevention and management of CRC.     

Cyy260, a novel small molecule inhibitor,suppresses non-small cell lung cancer cell growth via JAK2/STAT3 pathway

Non-small cell lung cancer (NSCLC) is a malignant tumor that accounts for the most new cancer cases and cancer-related deaths worldwide, and the proliferation and metastasis of NSCLC are the main reasons for treatment failure and patient death. Traditional chemotherapeutic drugs have low selectivity, which can kill cancer cells and cause damage to normal cells at the same time. Therefore, it is particularly important to study therapies that target cancer cells and to find low-toxicity, high-efficiency anticancer drugs. Cyy260 is a novel small molecule inhibitor that we synthesized for the first time. Here, we investigated the in vitro and in vivo antitumor activities of Cyy260 and explored the underlying mechanisms in NSCLC. Cyy260 had a concentration- and time-dependent inhibitory effect on NSCLC cells, but it was less toxic to normal cells. Cyy260 regulated apoptosis through intracellular and extracellular apoptotic pathways. In addition, Cyy260 could also induce cell cycle arrest, thereby inhibiting cell proliferation. Further analysis of molecular mechanisms showed that the JAK2/STAT3 signaling pathway was involved in the antitumor effect mediated by Cyy260. Analysis of subcutaneously transplanted tumors in mice showed that Cyy260 suppressed tumor growth in vivo. Our results proved that Cyy260 is a novel inhibitor of the JAK2/STAT3 pathway thus may have potential in therapy of NSCLC and other cancers.     

RECK impedes DNA repair by inhibiting the erbB/JAB1/Rad51 signaling axis and enhances chemosensitivity of breast cancer cells

The reversion-inducing cysteine-rich protein with kazal motif (RECK) is an endogenous matrix metalloproteinase (MMP) inhibitor and a tumor suppressor. Its expression is dramatically down-regulated in human cancers. Our recent results suggest a novel MMP-independent anti-cancer activity of RECK by inhibiting the erbB signaling. Activation of the erbB signaling is associated with chemotherapeutic resistance, however, whether RECK could modulate drug sensitivity is still unknown. Here we demonstrated that expression of RECK induced the activation of ATM and ATR pathways, and the formation of γ-H2AX foci in breast cancer cells. RECK inhibited the erbB signaling and attenuated the expression of the downstream molecules Jun activation domain-binding protein 1 (JAB1) and the DNA repair protein RAD51 to impede DNA repair and to increase drug sensitivity. Treatment of epidermal growth factor or over-expression of HER-2 effectively reversed the inhibitory effect of RECK. In addition, ectopic expression of JAB1 counteracted RECK-induced RAD51 reduction and drug sensitization. Our results elucidate a novel function of RECK to modulate DNA damage response and drug resistance by inhibiting the erbB/Jab1/RAD51 signaling axis. Restoration of RECK expression in breast cancer cells may increase sensitivity to chemotherapeutic agents.     

Mutational analysis of the transforming growth factor beta receptor type I gene in primary non-small cell lung cancer

Transforming growth factor-β receptor-dependent signals are critical for cell growth and differentiation and are often disrupted during tumorigenesis. The entire coding region of TGFβRI and flanking intron sequences from 53 primary non-small cell lung cancer (NSCLC) tissues were examined for alterations using SSCP and direct sequencing. No somatic point mutations other than two silent mutations and a polymorphism were found in the TGFβRI gene. The two silent mutations located at codon 344 (AAT to AAC) and codon 406 (TTA to CTA), respectively, and the polymorphism was at the 24th base of intron 7 (G to A). To investigate whether the presence of this polymorphism is associated with NSCLC, we determined its allele distribution in all the 53 carcinomas and 89 normal controls. Interestingly, we found that the subjects with homozygous genotype A/A displayed more than 3-fold increased risk of developing NSCLC than the common wild genotype G/G. As the first report, the present study showed that TGFβRI gene is not a frequent site of spontaneous mutational inactivation while the detected polymorphism is frequent in the pathogenesis of NSCLC.     

CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.     

iR-17在非小细胞肺癌放射抗拒细胞中的表达及作用研究

目的 建立肺癌放射抗拒细胞系H1975R,并探究miR-17在其放射抗拒中的作用。方法 通过梯度剂量连续照射,体外建立肺癌放射抗拒细胞系H1975R。采用克隆形成实验对H1975R和H1975细胞的放射敏感性进行验证。利用RT-PCR检测亲本细胞和放射抗拒细胞的miR-17的表达水平,并通过上调或下调miR-17表达,比较其放射敏感性变化,应用CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡和周期分布。结果 H1975R的放射生物学参数D₀、D_q和SF₂均比亲本H1975大(P＜0.05),显示更强的放射抗拒性。从RT-PCR结果可知,miR-17在H1975R的表达量是H1975的2.67倍(P＜0.01)。上调H1975细胞的miR-17表达后,其放射敏感性降低,而细胞增殖和抗凋亡能力均增强,G₂/M期细胞比例增多(P＜0.05)。下调H1975R细胞的miR-17表达后,其放射敏感性升高,而细胞增殖和抗凋亡能力均降低,G₂/M期细胞比例减少(P＜0.05)。结论 miR-17参与非小细胞肺癌放射抗拒的调控,其可能成为治疗放射抗拒的新靶点。     

Angiogenic activities are increased via upregulation of HIF-1α expression in gefitinib-resistant non-small cell lung carcinoma cells

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have been used to treat patients with non-small cell lung cancer (NSCLC) and activating EGFR mutations; however, the emergence of secondary mutations in EGFR or the acquisition of resistance to EGFR-TKIs can develop and is involved in clinical failure. Since angiogenesis is associated with tumor progression and the blockade of antitumor drugs, inhibition of angiogenesis could be a rational strategy for developing anticancer drugs combined with EGFR-TKIs to treat patients with NSCLC. The signaling pathway mediated by hypoxia-inducible factor-1 (HIF-1) is essential for tumor angiogenesis. The present study aimed to identify the dependence of gefitinib resistance on HIF-1α activity using angiogenesis assays, western blot analysis, colony formation assay, xenograft tumor mouse model and immunohistochemical analysis of tumor tissues. In the NSCLC cell lines, HIF-1α protein expression levels and hypoxia-induced angiogenic activities were found to be increased. In a xenograft mouse tumor model, tumor tissues derived from gefitinib-resistant PC9 cells showed increased protein expression of HIF-1α and angiogenesis within the tumors. Furthermore, inhibition of HIF-1α suppressed resistance to gefitinib, whereas overexpression of HIF-1α increased resistance to gefitinib. The results from the present study provides evidence that HIF-1α was associated with the acquisition of resistance to gefitinib and suggested that inhibiting HIF-1α alleviated gefitinib resistance in NSCLC cell lines.     

Assessment of M867, a selective caspase-3 inhibitor,in an orthotopic mouse model for non-small cell lung carcinoma

Radiation-induced lung injury (RILI) is a significant dose limiting complication of thoracic radiation for lung, breast, and esophageal cancer. Strategies for increasing the therapeutic index of radiation involve the use of radiosensitizing agents. We investigated the potential of M867 to sensitize non-small cell lung cancer (NSCLC) to radiation in vivo, while assessing its protective effects in normal lung parenchyma. H460-Luc2 cells were implanted into the mediastinum of athymic nude mice, which were separated into four treatment groups: control, M867, radiation therapy (RT) or combination. H460-Luc2 cell cultures were treated in parallel. Tumor growth was followed using bioluminescence imaging. Immunohistochemistry staining was used to detect phospho-Smad2/3 and cleaved caspase-3 expression. Western blot was done for the detection of cleaved caspase-3 and phospho-Smad2/3. TUNEL assays were used to measure apoptosis. M867+RT group had significantly increased tumor growth inhibition relative to either treatment alone (p=0.02). M867+RT was associated with increased levels of apoptosis (p<0.01), but combination treatment was associated with a decrease in caspase-dependent apoptosis relative to RT alone (p<0.01). We found that this increase in apoptosis in the M867+RT group was due to caspase-independent cell death. Based on early biomarker analyses of phospho-Smad 2/3 and cleaved caspase-3, M867+RT had a radio-protective effect on normal lung parenchyma. M867 may increase the therapeutic ratio of RT by enhancing the radiosensitivity of NSCLC while mitigating RILI. Further research is warranted to examine the late effects of lung injury and to study differences in the mechanism of action of M867 on lung cancer and normal tissue.     

Effect of a poly(ADP‐ribose) polymerase‐1 inhibitor against esophageal squamous cell carcinoma cell lines

Effective molecular target drugs that improve therapeutic efficacy with fewer adverse effects for esophageal cancer are highly anticipated. Poly(ADP‐ribose) polymerase (PARP) inhibitors have been proposed as low‐toxicity agents to treat double strand break (DSB)‐repair defective tumors. Several findings imply the potential relevance of DSB repair defects in the tumorigenesis of esophageal squamous cell carcinoma (ESCC). We evaluated the effect of a PARP Inhibitor (AZD2281) on the TE‐series ESCC cell lines. Of these eight cell lines, the clonogenic survival of one (TE‐6) was reduced by AZD2281 to the level of DSB repair‐defective Capan‐1 and HCC1937 cells. AZD2281‐induced DNA damage was implied by increases in γ‐H2AX and cell cycle arrest at G2/M phase. The impairment of DSB repair in TE‐6 cells was suggested by a sustained increase in γ‐H2AX levels and the tail moment calculated from a neutral comet assay after X‐ray irradiation. Because the formation of nuclear DSB repair protein foci was impaired in TE‐6 cells, whole‐exome sequencing of these cells was performed to explore the gene mutations that might be responsible. A novel mutation in RNF8, an E3 ligase targeting γ‐H2AX was identified. Consistent with this, polyubiquitination of γ‐H2AX after irradiation was impaired in TE‐6 cells. Thus, AZD2281 induced growth retardation of the DSB repair‐impaired TE‐6 cells. Interestingly, a strong correlation between basal expression levels of γ‐H2AX and sensitivity to AZD2281was observed in the TE‐series cells (R² = 0.5345). Because the assessment of basal DSB status could serve as a biomarker for selecting PARP inhibitor‐tractable tumors, further investigation is warranted.     

The Karyopherin proteins,Crm1 and Karyopherin β1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation

The Karyopherin proteins are involved in nucleo‐cytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest they are important in nuclear envelope component assembly, mitosis and replication. Since these are all critical cellular functions, alterations in the expression of the Karyopherins may have an impact on the biology of cancer cells. In this study, we examined the expression of the Karyopherins, Crm1, Karyopherin β1 (Kpnβ1) and Karyopherin α2 (Kpnα2), in cervical tissue and cell lines. The functional significance of these proteins to cancer cells was investigated using individual siRNAs to inhibit their expression. Microarrays, quantitative RT‐PCR and immunofluorescence revealed significantly higher expression of Crm1, Kpnβ1 and Kpnα2 in cervical cancer compared to normal tissue. Expression levels were similarly elevated in cervical cancer cell lines compared to normal cells, and in transformed epithelial and fibroblast cells. Inhibition of Crm1 and Kpnβ1 in cancer cells significantly reduced cell proliferation, while Kpnα2 inhibition had no effect. Noncancer cells were unaffected by the inhibition of Crm1 and Kpnβ1. The reduction in proliferation of cancer cells was associated with an increase in a subG1 population by cell cycle analysis and Caspase‐3/7 assays revealed increased apoptosis. Crm1 and Kpnβ1 siRNA‐induced apoptosis was accompanied by an increase in the levels of growth inhibitory proteins, p53, p27, p21 and p18. Our results demonstrate that Crm1, Kpnβ1 and Kpnα2 are overexpressed in cervical cancer and that inhibiting the expression of Crm1 and Kpnβ1, not Kpnα2, induces cancer cell death, making Crm1 and Kpnβ1 promising candidates as both biomarkers and potential anticancer therapeutic targets. © 2008 Wiley‐Liss, Inc.     

GPD2在非小细胞肺癌组织中的表达及临床意义

目的:研究GPD2在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达和临床意义,及GPD2基因对裸鼠成瘤能力的影响.方法:选取119例NSCLC肿瘤组织及配对正常组织,行GPD2免疫组化实验,并分析GPD2的表达与NSCLC患者临床病理特征的关系.使用RNA干扰慢病毒载体感染...     

DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first‐line chemotherapy

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM‐Chk2 and ATR‐Chk1‐Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G₁‐S transition (e.g., TP53) and ATM/ATR‐initiated DNA repair (e.g., ARID1A). We hypothesized that DDR‐linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (γ‐H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first‐line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra‐deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (γ‐H2AX^high/pATM^high) was an adverse factor for both progression‐free survival (multivariate Cox: HR 2.23, 95%CI: 1.47–3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20–3.58). The relationship between the γ‐H2AX^high/pATM^high model and progression‐free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild‐type setting. Conversely, this association was no longer observed in an ARID1A‐mutated subgroup. The γ‐H2AX^high/pATM^high model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance.