X-ray Repair Research Articles

The NRF2-dependent transcriptional axis, XRCC5/hTERT drives tumor progression and 5-Fu insensitivity in hepatocellular carcinoma

Human telomerase reverse transcriptase (hTERT) is highly expressed in many tumors and is essential for tumorigenesis and metastasis in multiple cancers. However, the molecular mechanisms underlying its high expression level in hepatocellular carcinoma (HCC) remain unclear. In this study, we identified X-ray repair cross-complementing 5 (XRCC5), a novel hTERT promoter-binding protein in HCC cells, using biotin-streptavidin-agarose pull-down assay. We found that XRCC5 was highly expressed in HCC cells, in which it transcriptionally upregulated hTERT. Functionally, the transgenic expression of XRCC5 promoted HCC progression and 5-fluorouracil resistance, whereas short hairpin RNA knockdown of XRCC5 had converse effects in vitro and in vivo. Moreover, hTERT overexpression reversed XRCC5 knockdown- or 5-fluorouracil (5-Fu)-mediated HCC inhibition. Mechanistically, nuclear-factor-erythroid-2-related factor 2 (NRF2) interacted with XRCC5, which in turn upregulated hTERT. However, the upregulation was insignificant when NRF2 was reduced, suggesting that the XRCC5-mediated hTERT expression was NRF2 dependent. The HCC patients with high expression levels of XRCC5 and hTERT had shorter overall survival times compared with those with low XRCC5 and hTERT levels in their tumor tissues. Collectively, our study demonstrates the molecular mechanisms of the XRCC5/NRF2/hTERT signaling in HCC metastasis, which will aid in the identification of novel strategies for the diagnosis and treatment of HCC.

A meta-analysis of XRCC1 single nucleotide polymorphism and susceptibility to gynecological malignancies.

Background:Gynecological malignant tumor is a serious threat to women's health, cervical cancer, endometrial cancer and ovarian cancer are the most common. The eponymous protein encoded by the XRCC1 (X-ray repair cross complementation 1) gene is an important functional protein in the process of single-stranded DNA damage. Non-synonymous mutations of XRCC1 gene cause amino acid sequence changes that affect protein function and DNA repair ability, and may affect the interaction with other DNA repair proteins, leading to increased risk of tumor development. Many studies have assessed the association between XRCC1 gene polymorphism and the risk of cancer in the female reproductive system, but the results have been inconclusive. In this study, the relationship between XRCC1 Arg399Gln, Arg194Trp, Arg280His single nucleotide polymorphisms and susceptibility to gynecological malignancies was further explored by meta-analysis.Methods:English database: Pubmed, Medline, Excerpta Medica Database, Cochrance, etc; Chinese database: China national knowledge infrastructure, Wanfang Database, etc. STATA14 was used for statistical analysis, such as odd ratio (OR) value, subgroup analysis, heterogeneity test, sensitivity analysis, and publication bias.Results:In gynecologic cancers, the allele frequency difference of Arg399Gln case control group was statistically significant (GvsA: P = .007). There was no significant difference in allele frequency in the Arg194Trp and Arg280His case control groups (P = .065, 0.198). In different gene models, Arg399Gln was significantly correlated with gynecologic cancers susceptibility (GGvs AA: OR 0.91; 95% confidence interval [CI], 0.85 0.98); Arg194Trp was significantly correlated with gynecologic cancers susceptibility (CCvs TT: OR 0.94; 95% CI 0.88,1.00; CCvs CT: OR 0.97; 95% CI 0.90, 1.05); Arg280His was significantly correlated with gynecologic cancers susceptibility (GGvs AA: OR 0.98; 95% CI 0.94, 1.02; GGvs GA: OR 1.00;95% CI 0.97, 1.04). In the subgroup analysis, Arg399Gln and Arg194Trp were significantly correlated with gynecologic cancers susceptibility in the Asian race (P = .000, 0.049). In the analysis of different cancer subgroups, Arg399Gln and cervical cancer susceptibility were statistically significant (P = .039). Arg194Trp and endometrial cancer susceptibility were statistically significant (P = .033, 0.001).Conclusions:XRCC1 Arg399Gln, Arg194Trp, Arg280His single nucleotide polymorphisms were associated with gynecologic cancer susceptibility. Arg399Gln genotype was statistically significant in relation to cervical cancer susceptibility. Arg194Trp genotype was statistically significant in relation to endometrial cancer susceptibility.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

X-ray repair cross-complementing protein 1 (XRCC1) loss promotes \u03b2-lapachone \u2013induced apoptosis in pancreatic cancer cells

Backgroundβ-lapachone (β-lap), the NQO1 bioactivatable drug, is thought to be a promising anticancer agent. However, the toxic side effects of β-lap limit the drug use, highlighting the need for a thorough understanding of β-lap’s mechanism of action. β-lap undergoes NQO1-dependent futile redox cycling, generating massive ROS and oxidative DNA lesions, leading to cell death. Thus, base excision repair (BER) pathway is an important resistance factor. XRCC1, a scaffolding component, plays a critical role in BER.MethodsWe knocked down XRCC1 expression by using pLVX-shXRCC1 in the MiaPaCa2 cells and BxPC3 cells and evaluated β-lap-induced DNA lesions by γH2AX foci formation and alkaline comet assay. The cell death induced by XRCC1 knockdown + β-lap treatment was analysed by relative survival, flow cytometry and Western blotting analysis.ResultsWe found that knockdown of XRCC1 significantly increased β-lap-induced DNA double-strand breaks, comet tail lengths and cell death in PDA cells. Furthermore, we observed combining XRCC1 knockdown with β-lap treatment switched programmed necrosis with β-lap monotherapy to caspase-dependent apoptosis.ConclusionsThese results indicate that XRCC1 is involved in the repair of β-lap-induced DNA damage, and XRCC1 loss amplifies sensitivity to β-lap, suggesting targeting key components in BER pathways may have the potential to expand use and efficacy of β-lap for gene-based therapy.

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells.

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (1O2) only at telomeres or mitochondria in Jurkat T cells. We found that targeted 1O2 production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted 1O2 formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet 1O2 formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.

Loss of the Base Excision Repair Gene Apex1 Leads to Dysfunctional Adult Hematopoietic Stem and Progenitor Cells

Postnatal hematopoietic stem (and progenitor) cells (HS(P)Cs) are especially vulnerable to oxidative stress, leading to early hematopoietic senescence and/or malignant transformation. Elevated intracellular reactive oxygen species (ROS) can, among others, oxidize nucleotides, and thus can result in genotoxicity and mutagenesis if left unrepaired. Oxidized bases, as well as other spontaneous single base modifications, are recognized and repaired by the base excision repair (BER) pathway. Hence, the BER pathway is crucial to maintain genome integrity. In contrast to other DNA repair pathways however, the role of BER in maintaining HSPC functionality remains enigmatic, chiefly because knockout (KO) of BER genes is in many cases embryonic lethal.BER is a complex multi-step repair process. After initial removal and excision of the damaged base, the apurinic/apyrimidinic (AP) site is processed by the AP endonuclease (APEX1) enzyme. At this point, the BER pathway branches into 2 sub-pathways, namely the short-patch (SP-BER; wherein DNA polymerase beta (Polβ), Ligase III (Lig3) together with X-ray repair cross-complementing protein 1 (Xrcc1) are active) and the long-patch BER (LP-BER; wherein Lig1, Flap Structure-Specific Endonuclease 1 (Fen1), and sometimes Polβ are active) for the repair synthesis and the gap filling steps.In this study we wished to address the role of BER in adult hematopoiesis. Therefore, we used CRISPR-Cas9 to KO different BER genes in adult bone marrow (BM) HS(P)Cs, including two genes common to the BER (sub-)pathway(s) (Apex1 and Polβ) as well as one gene in the SP-BER (Xrcc1) and one gene in the LP-BER (Lig1) pathway. The effect thereof was evaluated on HS(P)C repopulation in vivo as well as on HS(P)C expansion during long-term in vitro culture (using the culture medium described by Wilkinson et al., Nature 2019). All CRISPR-Cas9 experiments were validated using a second sgRNA targeting the selected BER genes.Lig1-KO caused in vivo HSPC dysfunction: at 20 weeks post-transplantation, significantly less Lig1 KO cells were observed in the committed progenitor (HPC) and lineage committed (Lin +) BM compartments. By contrast, KO of Xrcc1 had only minor effects on HS(P)C repopulation, but we observed increased HSC expansion and myeloid biased differentiation in some recipient mice, which might correspond to clonal hematopoiesis and is consistent with the finding of XRCC1 loss-of-function mutation in myelodysplastic patients (Joshi et al, Ann Hematol 2016). Knockout of Polβ did not affect hematopoiesis in vivo or in vitro. The most severe phenotype was observed when we knocked out Apex1, as Apex1-KO HS(P)Cs failed to repopulate irradiated recipient mice. Already after 2 weeks, significantly less Apex1 deficient cells were detected in the different blood lineages and nearly no CRISPR-Cas9 KO cells could be detected from 4 weeks onwards. This was confirmed in vitro, where reduced expansion of Apex1 KO BM cells was observed.APEX1 has two major functional activities, namely its nuclease activity, involved in BER, and its redox activity (also called Ref-1 function) important in reducing oxidized transcription factors and therefore implicated in transcriptional regulation. However, little is known regarding the nuclease and Ref-1 function(s) in primary adult hematopoietic cells. We therefore cultured BM HS(P)Cs for 1 week in the continuous presence of 2 distinct chemicals blocking the APEX1 nucleases, or 2 different chemicals inhibiting specifically the Ref-1 function. We demonstrated that both APEX1 functions are essential for hematopoiesis, even if the 2 functions appear to support the survival, expansion and maintenance of HS(P)Cs through different mechanisms. While the Ref-1 function was essential for proliferation (as both Ref-1 inhibitors cause cell cycle arrest) of all the lineages (including the Lin + cells), both inhibitors of the nuclease function affected more the expansion/survival of the less committed HS(P)Cs without leading to any cell cycle arrest.In conclusion, this study demonstrates for the first time the important role of BER genes in adult hematopoiesis, often deregulated in cancer, including hematopoietic malignancies. We observed a particularly severe phenotype upon loss of Apex1 in adult HSPCs, and ongoing studies (such as RNA sequencing analysis) should provide novel insights in underlying mechanisms of APEX1 deficiencies in HS(P)Cs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Associations of rs1799794 and rs1799796 polymorphisms with risk of breast cancer: A meta-analysis.

The aim of this meta-analysis was to investigate the rs1799794 and rs1799796 polymorphisms of X-ray repair cross-complementing group 3 (XRCC3) in relation to breast cancer susceptibility. PubMed, Embase, the Cochrane Library, Web of Science, and Scopus were searched for eligible studies published until June 24, 2019. All analyses were carried out using Stata 14.0 software. Subgroup analyses were performed according to cancer types, ethnicity, source of controls, and method. Our meta-analysis included articles reporting 13 studies of SNP rs1799794 and seven articles reporting 10 studies of SNP rs1799796. Overall, significant associations were observed between the XRCC3 rs1799794 polymorphism and breast cancer risk in the dominant model and heterozygote model (GG + AG vs. AA: odds ratio [OR] =1.06, 95% confidence interval [CI]: 1.00-1.11, P = 0.037, I2 = 47%; AG vs. AA: OR = 1.08, 95% CI: 1.02-1.13, P = 0.006, I2 = 42.3%) and between the XRCC3 rs1799796 polymorphism and breast cancer risk in the homozygote model (GG vs. AA: OR = 0.91, 95% CI: 0.84-0.99, P = 0.021, I2 = 33.3%). The results of this meta-analysis suggest that the variant G allele of the XRCC3 rs1799794 polymorphism is a low-penetrant risk factor for developing breast cancer, whereas the variant G allele of the XRCC3 rs1799796 polymorphism has a protective effect against breast cancer development.

CircANTXR1 Contributes to the Malignant Progression of Hepatocellular Carcinoma by Promoting Proliferation and Metastasis.

BackgroundCircular RNA (circRNA) is a key regulator for the malignant progression of cancer. However, the role of circRNA anthrax toxin receptor 1 (circANTXR1) in hepatocellular carcinoma (HCC) is still unclear.MethodsQuantitative real-time PCR was performed to detect RNA expression. Cell proliferation, migration and invasion were determined using MTT assay, EdU staining, colony formation assay, wound healing assay and transwell assay. The protein levels of metastasis markers, x-ray repair cross complementing 5 (XRCC5) and exosome markers were examined using Western blot analysis. Xenograft tumor models were built to investigate the role of circANTXR1 in HCC tumorigenesis. The relationship between microRNA (miR)-532-5p and circANTXR1 or XRCC5 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. The identification of exosomes were performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).ResultsCircANTXR1 was a stable and highly expressed circRNA in HCC. Silenced circANTXR1 inhibited the proliferation, migration and invasion of HCC cells in vitro, and suppressed HCC tumor growth in vivo. MiR-532-5p could be sponged by circANTXR1, and its inhibitor could reverse the inhibition of circANTXR1 silencing on HCC cells progression. In addition, we discovered that XRCC5 was a target of miR-532-5p. Furthermore, XRCC5 overexpression could reverse the suppressive effect of miR-532-5p overexpression on HCC cell proliferation, migration and invasion. Exosome was involved in the transport of circANTXR1 in HCC cells. Exosome circANTXR1 might be a potential serum biomarker for HCC patients.ConclusionCircANTXR1 promotes the progression of HCC through the miR-532-5p/XRCC5 axis, which might be a potential serum biomarker and therapeutic target of HCC.

Methylmercury chronic exposure affects the expression of DNA single-strand break repair genes, induces oxidative stress, and chromosomal abnormalities in young dyslipidemic APOE knockout mice

Mercury (Hg) is one of the most toxic environmental pollutants, especially when methylated, forming methylmercury (MeHg). MeHg affects DNA repair, increases oxidative stress, and predisposes to cancer. MeHg neurotoxicity is well-known, but recently MeHg-associated cardiovascular effects were recognized. This study evaluated circulating lipids, oxidative stress, and genotoxicity after MeHg-chronic exposure (20 mg/L in drinking water) in C57BL/6J wild-type and APOE knockout (ko) mice, the latter, being spontaneously dyslipidemic. Experimental mice were assigned to four groups: non-intoxicated and MeHg-intoxicated wild-type mice and non-intoxicated and MeHg-intoxicated APOE ko mice. Plasma levels of triglycerides, total cholesterol (TC), HDL, and LDL were analyzed. Liver lipid peroxidation and splenic gene expression of xeroderma pigmentosum complementation groups A, C, D, and G (XPA, XPC, XPD, and XPG), X-ray repair cross-complementing protein 1 (XRCC1), and telomerase reverse transcriptase (TERT) were measured. Fur Hg levels confirmed chronic MeHg intoxication. MeHg exposure raises TC levels both in wild-type and APOE ko mice. HDL and LDL-cholesterol levels were increased only in the MeHg-challenged APOE ko mice. MeHg increased liver lipid peroxidation, regardless of the genetic background. Unintoxicated APOE ko mice showed higher expression of TERT than all other groups. APOE deficiency increases XPA expression, regardless of MeHg intoxication. Furthermore, MeHg-intoxicated mice had more cytogenetic abnormalities, effect which was independent of APOE deficiency. More studies are needed to dissect the interactions between circulating lipids, MeHg intoxication, and DNA-repair pathways even at young age, interactions that likely play critical roles in cell senescence and the risk for chronic disorders later in life.

Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella

Key messageIn Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency.Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA–DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA–DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p < 0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.

XRCC2 Arg188His polymorphism and colorectal cancer risk: a meta-analysis.

ObjectiveTo investigate the potential correlation between the Arg188His (rs3218536) polymorphism of X-ray repair cross-complementing 2 (XRCC2) and colorectal cancer (CRC) risk, as the association remains unclear.MethodsThe CNKI, PubMed, EMBASE and Cochrane library databases were systematically searched for relevant studies published up to July 2021. Data were extracted from included studies, and analysed for pooled or subgroup odds ratios (ORs) with 95% confidence intervals (CIs) using STATA 12.0 software.ResultsSeven published studies were included. Pooled analysis revealed that the XRCC2 Arg188His polymorphism was associated with increased CRC risk (His versus Arg: OR 1.14, 95% CI 1.01, 1.29). Trial Sequential Analysis to test the power of the results showed that they were unreliable and the meta-analysis required additional studies.ConclusionThe current meta-analysis suggests that the XRCC2 Arg188His polymorphism may be a risk factor for CRC.

Clinical Impact of X-Ray Repair Cross-Complementary 1 (XRCC1) and the Immune Environment in Colorectal Adenoma-Carcinoma Pathway Progression.

PurposeColorectal cancer (CRC) can develop via a hypermutagenic pathway characterized by frequent somatic DNA base-pair mutations. Alternatively, the immunogenicity of tumor cells themselves may influence the anticancer activity of the immune effector cells. Impaired DNA repair mechanisms drive mutagenicity, which then increase the neoantigen load and immunogenicity. However, no studies have analyzed immune checkpoint protein expression, particularly programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1), in adenoma–carcinoma progression and its relationship with the emergence of other DNA repair gene mutation.Materials and MethodsWe investigated mutations of 10 genes involved in DNA repair function: XRCC1, TP53, MLH1, MSH, KRAS, GSTP, UMP, MTHF, DPYD, and ABCC2. We performed sequencing to determine mutations and immunohistochemistry of immune checkpoints in clinical samples and determined changes in XRCC1 expression during progression through the adenoma–carcinoma pathway. We further investigated the prognostic associations of gene XRCC1 according to the expression, mutational profile, and immune profile using The Cancer Genome Atlas-colon adenocarcinoma (TCGA-COAD) dataset.ResultsFrom clinical samples, XRCC1 mutation demonstrated the strongest association with adenomas with a mutation frequency of 56.2% in adenomas and 34% in CRCs (p =0.016). XRCC1 was abnormally expressed and altered by mutations contributing to adenoma carcinogenesis. High expression of XRCC1, CD4, FOXP3, and PD-1/PD-L1 showed an overall upward trend with increased lesion severity (all p < 0.01). PD-1/PD-L1 expression and CD4+ intraepithelial lymphocytes (IELs) correlated with cytological dysplasia progression, specifically in patients with wild-type XRCC1 (all p < 0.01), whereas FOXP3 expression was independently associated with adenoma–carcinoma progression. From TCGA-COAD analysis, XRCC1 expression was associated with patients survival, tumor-infiltrating lymphocytes and immune marker expression.ConclusionIncreased IEL density and PD-1/PD-L1 expression correlate with cytological dysplasia progression and specifically with the XRCC1 mutation status in CRC. Our findings support a stepwise dysplasia-carcinoma sequence of adenoma carcinogenesis and an XRCC1 hypermutated phenotypic mechanism of lesions.

The roles of risk model based on the 3-XRCC genes in lung adenocarcinoma progression.

BackgroundThe abnormal expression of deoxyribonucleic acid (DNA) repair genes might be the cause of tumor development and resistance of malignant cells to chemotherapeutic drugs. A risk model based on the X-ray repair of cross-complementary (XRCC) genes was constructed to improve the diagnosis and treatment of lung adenocarcinoma (LUAD) patients.MethodsThe expression levels, diagnostic values, and prognostic values of XRCC genes were identified, and the roles and regulatory mechanisms of the risk model based on the XRCC4/5/6 in LUAD progression was explored via The Cancer Genome Atlas (TCGA) and Oncomine databases.ResultsXRCC1/2/3/4/5/6, XRCC7 (PRKDC), and XRCC9 (FANCG) were overexpressed, and had diagnostic value for LUAD. The XRCC genes were involved in DNA repair, and participated in the regulation of non-homologous end-joining, homologous recombination, etc. The overall survival (OS), tumor (T) stage, and survival status of patients were significantly different between the Cluster1 and Cluster2 groups. XRCC4/5/6 were independent risk factors affecting the prognosis of LUAD patients. The risk score was related to the prognosis, sex, clinical stage, T, lymph node (N), and metastasis (M) stage, as well as the survival status of LUAD patients. The clinical stage and risk score were independent risk factors for poor prognosis in LUAD patients. The risk model was involved in RNA degradation, cell cycle, basal transcription factors, DNA replication etc. The risk scores were significantly correlated with the expression levels of TGFBR1, CD160, TNFSF4, TNFRSF14, IL6R, CXCL16, TNFRSF25, TAPBP, CCL16, and CCL14.ConclusionsThe risk model based on the XRCC4/5/6 genes could predict the progression of LUAD patients.

Ethnicity-stratified analysis of the association between XRCC3 Thr241Met polymorphism and leukemia: an updated meta-analysis

BackgroundPresently, whether X-ray repair cross complementing group 3 (XRCC3) Thr241Met polymorphism is correlated to leukemia risk remains controversial. Because of this reason, the objective of current study is to explore whether XRCC3 Thr241Met polymorphism confers risk to leukemia.MethodsTwo independent authors systematically and comprehensively searched Pubmed, Embase, the Cochrane library, Google academic, China National Knowledge Infrastructure (CNKI). Search time is from database foundation to March 2021.ResultsOverall, significant associations between leukemia risk and XRCC3 Thr241Met polymorphism were found in Caucasian population by allele contrast (T vs. C: OR 1.20, 95% CI 1.02–1.40), homozygote comparison (TT vs. CC: OR 1.35, 95% CI 1.05–1.73), and recessive genetic model (TT vs. TC/CC: OR 1.31, 95% CI 1.04–1.64).ConclusionsThe present meta-analysis suggests that the XRCC3 Thr241Met polymorphism may be a risk factor for leukemia in Caucasian population.

Associations of polycyclic aromatic hydrocarbons exposure and its interaction with XRCC1 genetic polymorphism with lung cancer: A case-control study

Humans are extensively exposed to polycyclic aromatic hydrocarbons (PAHs) daily via multiple pathways. Epidemiological studies have demonstrated that occupational exposure to PAHs increases the risk of lung cancer, but related studies in the general population are limited. Hence, we conducted a case-control study among the Chinese general population to investigate the associations between PAHs exposure and lung cancer risk and analyze the modifications of genetic polymorphisms in DNA repair genes. In this study, we enrolled 122 lung cancer cases and 244 healthy controls in Wuhan, China. Urinary PAHs metabolites were determined by gas chromatography-mass spectrometry, and rs25487 in X-ray repair cross-complementation 1 (XRCC1) gene was genotyped by the Agena Bioscience MassARRAY System. Then, multivariable logistic regression models were performed to estimate the potential associations. We found that urinary hydroxynaphthalene (OH-Nap), hydroxyphenanthrene (OH-Phe) and the sum of hydroxy PAHs (∑OH-PAHs) levels were significantly higher in lung cancer cases than those in controls. After adjusting for gender, age, BMI, smoking status, smoking pack-years, drinking status and family history, urinary ∑OH-Nap and ∑OH-Phe levels were positively associated with lung cancer risk, with dose-response relationships. Compared with those in the lowest tertiles, individuals in the highest tertiles of ∑OH-Nap and ∑OH-Phe had a 2.13-fold (95% CI: 1.10, 4.09) and 2.45-fold (95% CI: 1.23, 4.87) increased risk of lung cancer, respectively. Effects of gender, age, smoking status and smoking pack-years on the associations of PAHs exposure with lung cancer risk were shown in the subgroup analysis. Furthermore, associations of urinary ∑OH-Nap and ∑OH-PAHs levels with lung cancer risk were modified by XRCC1 rs25487 (Pinteraction ≤ 0.025), and were more pronounced in wild-types of rs25487. These findings suggest that environmental exposure to naphthalene and phenanthrene is associated with increased lung cancer risk, and polymorphism of XRCC1 rs25487 might modify the naphthalene exposure-related lung cancer effect.

Impact of XRCC1 genetic variants on its tissue expression and breast cancer risk: A case-control study.

X-ray repair cross-complementing group 1 (XRCC1), a coordinator protein of the DNA repair complex, is thought to be involved in cancer progression. This case-control study aimed to investigate the association of two biallelic single-nucleotide polymorphisms (SNPs; Arg399Gln, Arg194Trp) of the XRCC1 gene with its tissue expression level and breast cancer (BC) risk in Egyptian women. This study included 100 BC female patients (case group 1) and 100 healthy females (control group 2). The XRCC1 tissue expression was assessed by immunohistochemistry (IHC). Genotyping of the two XRCC1 SNPs (Arg399Gln, Arg194Trp) using real-time polymerase chain reaction (PCR) was also conducted. The XRCC1 expression level was significantly lower in cancerous tissues than adjacent non-cancerous tissues (p < .001). The XRCC1 399Gln/Gln genotype, 399Gln allele, the dominant, and recessive models were significantly associated with lower XRCC1 expression in breast cancerous tissues and increased risk for BC (3.390-, 1.965-, 2.241-, and 2.429-folds, respectively). The XRCC1 399Gln/Gln genotype was associated with lower incidence of advanced tumor grade (OR: 0.06; 95%CI: 0.01-0.74; p=.028). Conversely, the XRCC1 Arg194Trp polymorphism did not show any significant association with either XRCC1 expression in breast cancer tissues or BC risk in all genetic models. The XRCC1 haplotypes, 399Gln/194Arg and 399Gln/194Trp, were associated with 1.800- and 1.675-folds risk for BC, respectively. The XRCC1 gene polymorphism (Arg399Gln) is associated with reduced XRCC1 tissue expression and enhanced BC risk with a well-differentiated nature in Egyptian women. Moreover, XRCC1 haplotypes, 399Gln/194Arg and 399Gln/194Trp, were associated with increased BC risk.

GA Genotype of the Arg280His Polymorphism on The XRCC1 Gene: Genetic Susceptibility Genotype in Differentiated Thyroid Carcinomas?

Differentiated thyroid carcinomas (DTC) are the most common form of endocrine malignancies. The role of genetic variations in the development of papillary thyroid carcinoma (PTC) is approximately 60.0-70.0%. The X-ray repair cross-complementing group 1 (XRCC1) protein has an important role in DNA repair mechanisms and genomic polymorphisms of XRCC1 gene affect the function of the protein. In the present case-control study, we aimed to compare the genotype frequency distributions of XRCC1 single nucleotide polymorphisms (SNPs) in terms of the presence of other risk factors (Hashimoto’s thyroiditis, smoking, obesity, radiation exposure) in patients with thyroid nodules who had fine-needle aspiration biopsy (FNAB) and/or thyroid surgery due to thyroid cancer. The genotype frequency distributions of three common XRCC1 SNPs (Arg194Trp, Arg399Gln, Arg280His) were compared to those with DTC (n = 228), benign thyroid nodules (BTN, n = 100) and healthy controls (n = 93) in terms of certain pre defined risk factors such as the presence of Hashimoto’s thyroiditis, smoking, obesity, a family history of thyroid cancer and radiation exposure. The frequency of the GA genotype of Arg280His in DTC cases was found to be higher than in those with BTN and the healthy control group (p <0.001). The DTC group had the lowest frequency of AA genotype of Arg280His (35.5%, p <0.001). Among those with a family history of thyroid cancer, 78.9% had a GA genotype and 21.1% had the AA genotype of Arg280His (p = 0.004). The Arg280His GA genotype was more common in DTC than in cancer-free controls. The GA genotype frequency was also high in DTC cases with a family history of thyroid cancer.

Nonhomologous end joining repair pathway molecules as predictive biomarkers for patients with oral squamous cell carcinoma

Nonhomologous end-joining (NHEJ) is critical for the repair of either pathologic double-strand breaks (DSBs) and/or for the repair of physiologic DSBs created during radiotherapy to kill the tumor cell. Therefore, patients with higher expression of NHEJ repair proteins might develop resistance to ionizing radiation, allowing the disease to recur. As cancer of the oral cavity is a serious health problem globally, the present study aimed to examine the expression of Ku70/80, X-ray repair cross-complementing protein 4 (XRCC4) and DNA ligase IV-core molecules of the NHEJ pathway in patients with oral cancer. Protein expression of Ku70/80, XRCC4, and DNA ligase IV were studied by Immunohistochemistry and mRNA expression of Ku70 and Ku80 were studied using reverse transcription polymerase chain reaction. Data were analyzed statistically using SPSS. A univariate survival analysis revealed an association of Ku70 mRNA with shorter overall survival (OS). While protein expression of XRCC4 showed an association with reduced relapse-free survival and shorter OS. Multivariate survival analysis demonstrated that XRCC4 and DNA ligase IV are independent prognosticators for predicting adverse disease outcomes. Strong expression of repair proteins - XRCC4 and DNA ligase IV is associated with unfavorable disease outcome in patients with oral squamous cell carcinoma.

Concurrent Treatment with Pim Kinase Inhibitor Downregulates Alternative Non-Homologous End-Joining Repair and Decreases Genomic Instability in FLT3-ITD Cells Treated with Topoisomerase 2 Inhibitor Chemotherapy Drugs

Internal tandem duplication of fms -like tyrosine like kinase 3 (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML) cases, causing constitutive FLT3 signaling. While FLT3-ITD AML patients generally achieve remission following combination chemotherapy including cytarabine and a topoisomerase (topo) 2 inhibitor, they relapse rapidly, and FLT3-ITD AML cells frequently have new structural chromosome changes at relapse. Genomic instability in FLT3-ITD AML appears to result from increased generation of reactive oxygen species, which cause DNA double-strand breaks (DSBs), and upregulated DNA DSB repair by an error-prone alternative non-homologous end-joining (Alt-NHEJ) pathway.Pim-1 is an oncogenic serine/threonine kinase that is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to the proliferative and anti-apoptotic effects of FLT3-ITD, and also phosphorylates and stabilizes FLT3, creating a positive feedback loop in FLT3-ITD cells. Pim kinase inhibitors are in clinical trials. We previously showed that concurrent treatment with a Pim kinase inhibitor enhances apoptosis induction by topo 2 inhibitors in FLT3-ITD cells. Here, we tested the effect of concurrent Pim kinase inhibitor treatment on Alt-NHEJ repair of topo 2 inhibitor-induced DNA DSBs and on genomic instability.Functional changes in DSB repair activities induced by treatment with the topo 2 inhibitor daunorubicin (DNR) in the absence and presence of the pan-Pim kinase inhibitor AZD1208 were studied in Ba/F3-ITD cells, with FLT3-ITD, using stably integrated green fluorescent protein (GFP)-based DSB repair reporter constructs DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical (C-)NHEJ and Alt-NHEJ, respectively. DNR, which induced DNA DSBs, did not induce HR or C-NHEJ repair activity, but induced a 50% increase in Alt-NHEJ activity at 36 hours. Concurrent treatment with AZD1208, which increased DNR-induced DNA DSBs more than two-fold, completely abrogated the DNR-induced increase in Alt-NHEJ repair activity. Treatment of Ba/F3-ITD cells with the topo 2 inhibitors mitoxantrone (MXR) or etoposide (VP-16) also induced Alt-NHEJ repair activity, which was abrogated by concurrent treatment with AZD1208.We next studied mechanisms underlying Pim kinase inhibitor downregulation of Alt-NHEJ. The effects of DNR, MXR or VP-16 on cellular and nuclear expression of the Alt-NHEJ proteins poly (ADP-ribose) polymerase (PARP) 1, DNA polymerase θ, DNA ligase 3 and co-factor X-ray repair cross-complementing protein 1 (XRCC1) were measured in the FLT3-ITD cell lines Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 in the absence and presence of AZD1208. Cellular and nuclear expression of XRCC1 and DNA ligase 3, but not PARP or DNA polymerase θ, decreased in cells co-treated with AZD1208 and topo 2 inhibitors, compared to topo 2 inhibitors alone. XRCC1 was identified as a potential direct Pim-1 substrate by global profiling using the reverse in-gel kinase assay, and was confirmed as a robust Pim substrate in a reverse in-gel kinase assay. This provides a potential mechanistic link between Pim-1 kinase and ALT NHEJ.Alt-NHEJ repair is associated with genomic instability characterized by increased chromosome deletions, insertions and translocations. We studied induction of chromosome changes by topo 2 inhibitor treatment with and without Pim kinase inhibitor in FLT3-ITD cells. Ba/F3-ITD and 32D-ITD cells were incubated with DNR and/or AZD1208, or DMSO control, for 36 hours, then prepared for cytogenetic analysis.For each treatment, 20 consecutive metaphases were analyzed for induction of chromatid breaks and exchanges. No chromosome changes were seen in either cell line with DMSO or AZD1208 treatment. In Ba/F3-ITD cells, chromosome abnormalities were seen in 17 of 20 metaphases after DNR treatment, but only 8 of 20 after combination treatment (p=0.008). In 32D-ITD cells, chromosome abnormalities were seen in 8 of 20 metaphases with DNR, and 0 metaphases with combination (p=0.003).Thus concurrent treatment with Pim kinase inhibitor abrogates induction of Alt-NHEJ repair by topo 2 inhibitor treatment in FLT3-ITD cells and decreases the genomic instability that may contribute to rapid relapse of FLT3-ITD AML after chemotherapy. The data support further preclinical and clinical testing of Pim kinase inhibitors with chemotherapy in AML with FLT3-ITD. DisclosuresTron:AstraZeneca: Employment, Equity Ownership.

Identification of an IFN-β-associated gene signature for the prediction of overall survival among glioblastoma patients.

BackgroundBrain glioblastoma multiforme (GBM) is the most common primary malignant intracranial tumor. The prognosis of this disease is extremely poor. While the introduction of β-interferon (IFN-β) regimen in the treatment of gliomas has significantly improved the outcome of patients; The mechanism by which IFN-β induces increased TMZ sensitivity has not been described. Therefore, the main objective of the study was to elucidate the molecular mechanisms responsible for the beneficial effect of IFNβ in GBM.MethodsMessenger RNA expression profiles and clinicopathological data were downloaded from The Cancer Genome Atlas (TCGA) GBM and GSE83300 dataset from the Gene Expression Omnibus. Univariate Cox regression analysis and lasso Cox regression model established a novel 4-gene IFN-β signature (peroxiredoxin 1, Sec61 subunit beta, X-ray repair cross-complementing 5, and Bcl-2-like protein 2) for GBM prognosis prediction. Further, GBM samples (n=50) and normal brain tissues (n=50) were then used for real-time polymerase chain reaction experiments. Gene set enrichment analysis (GSEA) was performed to further understand the underlying molecular mechanisms. Pearson correlation was applied to calculate the correlation between the long non-coding RNAs (lncRNAs) and IFN-β-associated genes. An lncRNA with a correlation coefficient |R2|>0.3 and P<0.05 was considered to be an IFN-β-associated lncRNA.ResultsPatients in the high-risk group had significantly poorer survival than patients in the low-risk group. The signature was found to be an independent prognostic factor for GBM survival. Furthermore, GSEA revealed several significantly enriched pathways, which might help explain the underlying mechanisms. Our study identified a novel robust 4-gene IFN-β signature for GBM prognosis prediction. The signature might contain potential biomarkers for metabolic therapy and treatment response prediction for GBM patients.ConclusionsIn the present study, we established a novel IFN-β-associated gene signature to predict the overall survival of GBM patients, which may help in clinical decision making for individual treatment.