Decreased DNA Repair Capacity Research Articles

Decreased DNA repair capacity caused by exposure to metal mixtures is modulated by the PARP1 rs1136410 variant in newborns from a polluted metropolitan area

BackgroundDNA damage caused by exposure to metal mixtures and the potential modulating role of genes involved in DNA repair and the antioxidant response have not been evaluated in newborns. AimThe aim was to evaluate the association between prenatal exposure to metal mixtures and DNA repair capacity (DRC) in newborns from the Metropolitan Area of Mexico City (MAMC), a heavily polluted area, and the impact of variants in genes involved in DNA repair and the antioxidant response on this association. MethodsWe analyzed cord blood samples obtained at delivery from 125 healthy newborns from the MAMC. Twenty-four elements were determined by inductively coupled plasma mass spectrometry (ICP‒MS), but only 12 (Cu, I, Se, Zn, As, Ba, Cs, Mn, Sb, Sr, Pb, and Ti) were quantified in most samples. DRC was assessed by the challenge-comet assay, and OGG1, PARP1, and NFE2L2 genotyping was performed with TaqMan probes. Metal mixtures were identified and analyzed using principal component analysis (PCA) and weighted quantile sum (WQS) regression. Independent adjusted linear regression models were used to evaluate the associations. ResultsA null DRC was observed in 46% of newborns. The metals with the highest concentrations were Mn, Sr, Ti, and Pb. Essential elements showed normal levels. Only the mixture characterized by increased As, Cs, Cu, Se, and Zn levels was inversely associated with DRC. As was the principal contributor (37.8%) in the negative direction in the DRC followed by Ba and Sb, according to the WQS regression. Newborns carrying of the derived (G) allele of the PARP1 rs1136410 variant showed decreased DRC by exposure to some potentially toxic metals (PTMs) (As, Cs, and Ba). ConclusionPrenatal exposure to metal mixtures negatively affected DRC in newborns, and the PARP1 rs1136410 variant had a modulating role in this association.

Androgen signaling stabilizes genomes to counteract senescence by promoting XRCC4 transcription.

Aging is accompanied by a decreased DNA repair capacity, which might contribute to age-associated functional decline in multiple tissues. Disruption in hormone signaling, associated with reproductive organ dysfunction, is an early event of age-related tissue degeneration, but whether it impacts DNA repair in nonreproductive organs remains elusive. Using skin fibroblasts derived from healthy donors with a broad age range, we show here that the downregulation of expression of XRCC4, a factor involved in nonhomologous end-joining (NHEJ) repair, which is the dominant pathway to repair somatic double-strand breaks, is mediated through transcriptional mechanisms. We show that the androgen receptor (AR), whose expression is also reduced during aging, directly binds to and enhances the activity of the XRCC4 promoter, facilitating XRCC4 transcription and thus stabilizing the genome. We also demonstrate that dihydrotestosterone (DHT), a powerful AR agonist, restores XRCC4 expression and stabilizes the genome in different models of cellular aging. Moreover, DHT treatment reverses senescence-associated phenotypes, opening a potential avenue to aging interventions in the future.

Genomic Instability and Epigenetic Changes during Aging.

Aging is considered the deterioration of physiological functions along with an increased mortality rate. This scientific review focuses on the central importance of genomic instability during the aging process, encompassing a range of cellular and molecular changes that occur with advancing age. In particular, this revision addresses the genetic and epigenetic alterations that contribute to genomic instability, such as telomere shortening, DNA damage accumulation, and decreased DNA repair capacity. Furthermore, the review explores the epigenetic changes that occur with aging, including modifications to histones, DNA methylation patterns, and the role of non-coding RNAs. Finally, the review discusses the organization of chromatin and its contribution to genomic instability, including heterochromatin loss, chromatin remodeling, and changes in nucleosome and histone abundance. In conclusion, this review highlights the fundamental role that genomic instability plays in the aging process and underscores the need for continued research into these complex biological mechanisms.

P19-01: Prenatal exposure to mixtures of potentially toxic and essential metals decreased DNA repair capacity in newborns from a polluted area

P19-01: Prenatal exposure to mixtures of potentially toxic and essential metals decreased DNA repair capacity in newborns from a polluted area

Impact of Nuclear De Novo NAD+ Synthesis via Histone Dynamics on DNA Repair during Cellular Senescence To Prevent Tumorigenesis.

NAD+ synthesis is a fundamental process in living cells. The effects of local metabolite production on chromatin influence the epigenetic status of chromatin in DNA metabolism. We have previously shown that K5 acetylation of H2AX by TIP60 is required for the ADP ribosylation activity of PARP-1, for histone H2AX exchange at DNA damage sites. However, the detailed molecular mechanism has remained unclear. Here, we identified de novo NAD synthetase 1 (NAD syn1) as a novel binding partner to H2AX. The enzymatic activity of NAD syn1 is crucial for the ADP ribosylation activity of PARP-1 for the H2AX dynamics at sites of DNA damage. Inhibition of the NAD synthetase activity in the cell nucleus decreased the overall cellular NAD+ concentration, leading to cellular senescence. Accordingly, the acetylation-dependent H2AX dynamics and homologous recombination repair were suppressed, leading to increased tumorigenesis. Our findings have revealed the importance of de novo NAD+ production in the cell nucleus for protection against the decreased DNA repair capacity caused by cellular senescence and thus against tumorigenesis.

Abstract PR-08: Diabetes affects metabolism, redox signaling, DNA repair capacity and mutational burden in breast cancer

Abstract Comorbidities disproportionately affect underserved populations. Diabetes can increase cancer risk and decreases breast cancer survival. Diabetes-associated breast cancer risk impacts African American women more than European Americans. The goal of this study was to understand how diabetes may shape breast tumor biology. We performed an RNA and whole-exome sequencing (WES) analysis, together with global metabolomics, of matched breast tumors from patients with or without diabetes, with the majority being of African American descent. Additionally, as an experimental model, we generated mammary xenografts following orthotopic injection of breast cancer cells (MDA-MB-231, MDA-MB-468, and HS578T) into the mammary fat pad of diabetic mice (NOD.Cg-Rag1 tm1Mom Ins2 Akita Il2rg tm1Wjl/SzJ). After 4-6 weeks we collected the tumors and performed RNAseq and metabolome analysis. Analyzing the metabolome profile of both human breast tumors and xenografts, we observed a distinct metabolite pattern in diabetic breast tumors and diabetic mice. The key observation was the enrichment of food and microbial-derived metabolites under diabetes, indicating that diabetes may promote the accumulation of these metabolites in breast tumors. Next, we analyzed the RNAseq data from the same tumors and xenografts. Here, we applied Gene Set Enrichment Analysis (GSEA) with the transcriptomic data to find specific pathways activated under diabetic conditions. GSEA indicated the enrichment of gene signatures related to mesenchymal and stem cell-like phenotypes both in the patient tumor and xenografts under diabetes. Additionally, we discovered an enrichment of the reactive oxygen species (ROS) pathway in diabetic tumors and xenografts. Consistent with this observation, treating breast cancer cells (MDA-MB-231 and HS578T) with high glucose (25mM) increased superoxide radical production in these cells. Since higher ROS levels could cause DNA damage, we examined DNA damage in breast cancer cells cultured under high glucose. Here we observed an increase in DNA damage under high glucose as indicated by robust nuclear γH2AX and 53BP1 immunostaining. An additional KEGG analysis indicated the inhibition of various DNA repair pathways (e.g. base-excision, nucleotide excision, and mismatch repair) in breast tumors and xenografts. Corroborating this finding with our WES data, we observed a significant enrichment of several mutational signatures (SBS5, SBS15, and SBS30 DNA repair deficiency signatures) in breast tumors of diabetic patients, suggesting that diabetes may interfere with DNA repair pathways in breast tumors. Taken together, this study suggests that diabetes promotes a mesenchymal and stem cell-like phenotype in human breast tumors with higher oxidative stress & DNA damage and a decreased DNA repair capacity. Our data further indicate that breast tumors from diabetic patients may have an increased susceptibility to DNA damage response inhibitors like PARP inhibitors. Citation Format: Gatikrushna Panigrahi, Tiffany Dorsey, Wei Tang, Julian Candia, Amy Zhang, Stefan Ambs. Diabetes affects metabolism, redox signaling, DNA repair capacity and mutational burden in breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR-08.

DNA repair and response to sperm DNA damage in oocytes and embryos, and the potential consequences in ART: a systematic review.

Sperm DNA damage is considered a predictive factor for the clinical outcomes of patients undergoing ART. Laboratory evidence suggests that zygotes and developing embryos have adopted specific response and repair mechanisms to repair DNA damage of paternal origin. We have conducted a systematic review in accordance with guidelines from Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) to identify and review the maternal mechanisms used to respond and repair sperm DNA damage during early embryonic development, how these mechanisms operate and their potential clinical implications. The literature search was conducted in Ovid MEDLINE and Embase databases until May 2021. Out of 6297 articles initially identified, 36 studies were found to be relevant through cross referencing and were fully extracted. The collective evidence in human and animal models indicate that the early embryo has the capacity to repair DNA damage within sperm by activating maternally driven mechanisms throughout embryonic development. However, this capacity is limited and likely declines with age. The link between age and decreased DNA repair capacity could explain decreased oocyte quality in older women, poor reproductive outcomes in idiopathic cases and patients who present high sperm DNA damage. Ultimately, further understanding mechanisms underlying the maternal repair of sperm DNA damage could lead to the development of targeted therapies to decrease sperm DNA damage, improved oocyte quality to combat incoming DNA insults or lead to development of methodologies to identify individual spermatozoa without DNA damage.

Interaction of DNA Repair Gene XPC With Smoking and Betel Quid Chewing Behaviors of Oral Cancer.

Xeroderma pigmentosum complementation group C (XPC) is reported to play important roles in DNA integrity and genomic instability, however, the contribution of XPC to oral carcinogenesis is largely uncertain. Therefore, we aimed at examining the contribution of XPC genotypes to oral cancer. The genotypes of XPC rs2228001 and rs2228000 were examined among 958 oral cancer patients and 958 control subjects by polymerase chain reaction-restriction fragment length polymorphism methodology and corresponding DNA repair capacity was checked. First, the percentages of XPC rs2228001 AC and CC were higher among oral cancer patients than controls. Second, no significant association was observed regarding XPC rs2228000. Third, there was a synergistic influence of smoking and betel quid chewing behaviors and XPC rs2228001 genotype on oral cancer risk. Last, functional experiments showed DNA repair capacity was lower for AC/CC carriers than AA carriers. XPC rs2228001 C allele, which was associated with decreased DNA repair capacity, may interact with smoking and betel quid chewing behaviors on oral cancer risk.

Interaction Between Environmental Risk Factors and Catechol-O-Methyltransferase (COMT) and X-Ray Repair Cross-Complementing Protein 1 (XRCC1) Gene Polymorphisms in Risk of Lung Cancer Among Non-Smoking Chinese Women: A Case-Control Study.

BackgrpoundVarious studies have highlighted the link between polymorphisms in the XRCC1 gene (encoding X-ray repair cross-complementing group 1) with the incidence of decreased DNA repair capacity and an increased predisposition to cancer. Catechol-O-methyltransferase (COMT) plays a crucial role in estrogen-induced cancers. In the present study was analyzed the potential influence of XRCC1 and COMT gene polymorphisms as predisposing factors from a lung cancer perspective, in addition to conducting an investigation into their interaction with environmental risk factors in relation to lung cancer among non-smoking Chinese women.Material/MethodsThe XRCC1 gene T-77C, Arg194Trp, Arg280His, Arg399Gln, COMT gene 186C>T, and Val158Met mutations were evaluated in peripheral blood collected from 261 non-smoking female patients diagnosed with primary lung cancer and 265 female patients with benign lung disease.ResultThe results obtained from this study demonstrated that XRCC1–77TC + CC, XRCC1 399Gln/Gln, COMT 186CT + TT, COMT 158Val/Met genotypes, type of occupation, cooking-oil fumes, and soot exposures were all independent risk factors involved with the occurrence of lung cancer among non-smoking women. Moreover, interactions between environmental exposure factors as well as XRCC1 and COMT gene polymorphisms were determined to play significant contributory roles regarding susceptibility of non-smoking females to lung cancer.ConclusionsTaken together, T-77C and Arg399Gln polymorphisms of the XRCC1 gene, as well as the 186C>T and Val158Met polymorphisms of the COMT gene, increased the risk of lung cancer in non-smoking women, with the factors of occupation type, cooking-oil fumes, and soot exposures representing key contributing factors.

Cytogenetic, DNA-repair and Apoptosis Studies in Hospital Nurses’ Peripheral Blood Lymphocytes Occupationally Exposed to Volatile Anesthetic Gases

Background: Health professionals chronically exposed to anesthetic gases in the operating rooms are at higher risk of lung diseases, hematological, immunological and reproductive alterations. Anesthetic gas exposure often exceeds the safety limits, especially in the case of pediatric anesthetists, or when no proper ventilation has been installed in operating theaters. In the present study we assessed the health risk among anesthetics exposed nurses and measured genotoxicological parameters in the presence or absence of confounding factors such as smoking. Methods: The investigations were carried out in 128 subjects exposed to anesthetic gases from health services. The data were compared to 137 healthy, non-exposed controls. The measured biomarkers were: clinical laboratory routine tests, completed with genotoxicological (chromosome aberrations and sisterchromatid exchange, DNA repair and apoptosis) monitoring system. The exposed group was divided into Halothane and Non-Halothane exposed subgroups to compare the differences within exposures. Results: In the group of health personnel exposed to anesthetic gases, we did not find significant changes in the frequency of chromosome aberrations. Sister-chromatide exchange rates in Halothane exposed and control smokers was increased, compared to nonsmokers. We also found a decrease of apoptosis and inhibition of UV induced DNA repair capacity compared to controls. The exposed individuals had high incidences of malignant diseases (9.3%) mainly breast, skin, and colon cancers. Conclusion: Low level of cytogenetic changes may be related to the decreased apoptotic rate of peripheral blood lymphocytes due to the low risk of misrepair from a decreased DNA-repair capacity. Our results suggest that our biomarkers can be useful in tracking occupational/environmental genotoxic effects among anesthetic gases exposed personnel.

Absence of APE1 (Asp148Glu) gene polymorphism in North-West Indian population: A comparison with world population

This is the first study from North-West India that found absence of APE1 (Asp148Glu; T2197G) gene polymorphism. APE1 (Apurinic/Apyrimidinic endonuclease-1) is the key enzyme of base excision repair (BER) pathway. It is involved in repair of oxidative and alkylative DNA damage caused by exogenous factors. Genetic polymorphism in APE1 gene affects its endonuclease activity leading to decreased DNA repair capacity, which further results in genomic instability mediating various diseases. In this study, we evaluated genotype and allele frequencies of the APE1 (rs 1130409, Asp148Glu; T2197G) gene polymorphism in 450 subjects from North-West India and compared it with other Indian and world populations. Genomic DNA extraction was carried out using standard Phenol-chloroform method and ethanol precipitation. Genotyping was carried out by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and run on 10% PAGE. Genotyping results were confirmed by direct DNA sequencing and running 10% of the samples in duplicate. It was found that APE1 (rs 1130409; Asp148Glu; T2197G) gene polymorphism was absent in all the 450 studied subjects. Our results were concordant with the study from U.S which showed absence of APE1 gene polymorphism. Our study also showed close similarity with a study from Andhra Pradesh, India having very less frequency, of 0.19%. The absence of APE1 (rs 1130409; Asp148Glu) gene polymorphism indicate that APE1 is not a key player for onset of diseases in our population.

Impaired oxidative stress response characterizes HUWE1-promoted X-linked intellectual disability

Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID. Here we showed that the HUWE1 expression is altered and mutation frequency increased in three different XLID individual (HUWE1 p.R2981H, p.R4187C and HUWE1 duplication) cell lines. The effect was most prominent in HUWE1 p.R4187C XLID cells and was accompanied with decreased DNA repair capacity and hypersensitivity to oxidative stress. Analysis of HUWE1 substrates revealed XLID-specific down-regulation of oxidative stress response DNA polymerase (Pol) λ caused by hyperactive HUWE1 p.R4187C. The subsequent restoration of Polλ levels counteracted the oxidative hypersensitivity. The observed alterations in the genome integrity maintenance may be particularly relevant in the cortical progenitor zones of human brain, as suggested by HUWE1 immunofluorescence analysis of cerebral organoids. These results provide evidence that impairments of the fundamental cellular processes, like genome integrity maintenance, characterize HUWE1-promoted XLID.

A low-frequency haplotype spanning SLX4/FANCP constitutes a new risk locus for early-onset breast cancer (

Only a fraction of breast cancer (BC) cases can be yet explained by mutations in genes or genomic variants discovered in linkage, genome-wide association and sequencing studies. The known genes entailing medium or high risk for BC are strongly enriched for a function in DNA double strand repair. Thus, aiming at identifying low frequency variants conferring an intermediate risk, we here investigated 17 variants (MAF: 0.01-0.1) in 10 candidate genes involved in DNA repair or cell cycle control. In an exploration cohort of 437 cases and 1189 controls, we show the variant rs3810813 in the SLX4/FANCP gene to be significantly associated with both BC (≤60 years; OR = 2.6(1.6-3.9), p = 1.6E-05) and decreased DNA repair capacity (≤60 years; beta = 37.8(17.9-57.8), p = 5.3E-4). BC association was confirmed in a verification cohort (N = 2441). Both associations were absent from cases diagnosed >60 years and stronger the earlier the diagnosis. By imputation we show that rs3810813 tags a haplotype with 5 additional variants with the same allele frequency (R2 > 0.9), and a pattern of association very similar for both phenotypes (cases <60 years, p < 0.001, the Bonferroni threshold derived from unlinked variants in the region). In young cases (≤60 years) carrying the risk haplotype, micronucleus test results are predictive for BC (AUC > 0.9). Our findings propose a risk variant with high penetrance on the haplotype spanning SLX4/FANCP to be functionally associated to BC predisposition via decreased repair capacity and suggest this variant is carried by a fraction of these haplotypes that is enriched in early onset BC cases.

Cadmium effects on DNA and protein metabolism in oyster (Crassostrea gigas) revealed by proteomic analyses

Marine molluscs, including oysters, can concentrate high levels of cadmium (Cd) in their soft tissues, but the molecular mechanisms of Cd toxicity remain speculative. In this study, Pacific oysters (Crassostrea gigas) were exposed to Cd for 9 days and their gills were subjected to proteomic analysis, which were further confirmed with transcriptomic analysis. A total of 4,964 proteins was quantified and 515 differentially expressed proteins were identified in response to Cd exposure. Gene Ontology enrichment analysis revealed that excess Cd affected the DNA and protein metabolism. Specifically, Cd toxicity resulted in the inhibition of DNA glycosylase and gap-filling and ligation enzymes expressions in base excision repair pathway, which may have decreased DNA repair capacity. At the protein level, Cd induced the heat shock protein response, initiation of protein refolding as well as degradation by ubiquitin proteasome pathway, among other effects. Excess Cd also induced antioxidant responses, particularly glutathione metabolism, which play important roles in Cd chelation and anti-oxidation. This study provided the first molecular mechanisms of Cd toxicity on DNA and protein metabolism at protein levels, and identified molecular biomarkers for Cd toxicity in oysters.

Abstract LB-029: Negative transcriptional and epigenetic regulation of DNA repair pathways by the heavy metals nickel and arsenic

Abstract Environmental exposure to certain heavy metals, such as nickel and arsenic, has been implicated in a variety of human cancers, including lung, skin, digestive track, and bladder cancers. Importantly, the mechanism underlying the carcinogenicity of nickel and arsenic remains poorly understood as they do not induce direct DNA mutagenesis. However, they do lead to global changes in chromatin structure and transcription, many similar to the effects of hypoxia. Since hypoxia is known to regulate many different DNA repair pathways, we investigated whether nickel and arsenic may similarly affect cellular DNA repair. We discovered that nickel and arsenic can lead to alterations in DNA repair gene expression, stable gene silencing, and decreased DNA repair capacity. First, we measured protein and mRNA levels of different DNA repair genes after NiCl2 or NaAsO2 treatment. We found that both metals induced down-regulation of BRCA1, FANCD2, and MLH1 over 24 to 48 hours at both the protein and mRNA levels. These results were observed in several different cells lines (HeLa, MCF7, BEAS-2B) with one notable exception that high dose arsenic induced up-regulation of BRCA1, FANCD2, and MLH1 in lung cancer-derived cell lines (A549, HCC827, NCI-H460). Next, to study the impact of long-term heavy metal exposure on DNA repair gene expression, we utilized an MLH1 promoter reporter construct that allows selection of cells harboring a silenced MLH1 promoter with ganciclovir. RKO cells stably expressing this construct were grown in the presence of 100 μM NiCl2, 0.5 μM NaAsO2, 1% oxygen, or control conditions. After 3 weeks, we observed that arsenic treatment, like hypoxia, led to a significant increase in promoter silencing compared to control cells, peaking at about 3.7-fold after 4 weeks. Nickel did not increase silencing, which may indicate a different mechanism of gene regulation. Finally, we used a luciferase assay to measure the effect of nickel and arsenic on the two primary DNA double-strand break (DSB) repair mechanisms, homologous recombination (HR) and non-homologous end joining (NHEJ). BEAS-2B cells pretreated with 250 μM NiCl2 or 5 μM NaAsO2 were transfected with a digested, inactive luciferase plasmid and allowed to conduct DSB repair to reactivate luciferase expression. We found that nickel and arsenic led to a 40-50% reduction in cellular HR capacity with no significant effect on NHEJ. To further pursue these results, we are performing chromatin immunoprecipitation studies to identify transcriptional or epigenetic factors mediating nickel and arsenic-induced down-regulation of DNA repair genes. In addition, we are using chromosomal-based assays to further characterize the impact of nickel and arsenic on DNA repair capacity. In conclusion, we have found that nickel and arsenic negatively regulate cellular DNA repair pathways, identifying a novel way in which heavy metals may contribute to carcinogenesis. Citation Format: Susan E. Scanlon, Christine D. Scanlon, Denise C. Hegan, Parker Sulkowski, Peter M. Glazer. Negative transcriptional and epigenetic regulation of DNA repair pathways by the heavy metals nickel and arsenic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-029.

Genetic variant in DNA repair gene GTF2H4 is associated with lung cancer risk: a large-scale analysis of six published GWAS datasets in the TRICL consortium.

DNA repair pathways maintain genomic integrity and stability, and dysfunction of DNA repair leads to cancer. We hypothesize that functional genetic variants in DNA repair genes are associated with risk of lung cancer. We performed a large-scale meta-analysis of 123,371 single nucleotide polymorphisms (SNPs) in 169 DNA repair genes obtained from six previously published genome-wide association studies (GWASs) of 12160 lung cancer cases and 16838 controls. We calculated odds ratios (ORs) with 95% confidence intervals (CIs) using the logistic regression model and used the false discovery rate (FDR) method for correction of multiple testing. As a result, 14 SNPs had a significant odds ratio (OR) for lung cancer risk with P FDR < 0.05, of which rs3115672 in MSH5 (OR = 1.20, 95% CI = 1.14-1.27) and rs114596632 in GTF2H4 (OR = 1.19, 95% CI = 1.12-1.25) at 6q21.33 were the most statistically significant (P combined = 3.99×10(-11) and P combined = 5.40×10(-10), respectively). The MSH5 rs3115672, but not GTF2H4 rs114596632, was strongly correlated with MSH5 rs3131379 in that region (r (2) = 1.000 and r (2) = 0.539, respectively) as reported in a previous GWAS. Importantly, however, the GTF2H4 rs114596632 T, but not MSH5 rs3115672 T, allele was significantly associated with both decreased DNA repair capacity phenotype and decreased mRNA expression levels. These provided evidence that functional genetic variants of GTF2H4 confer susceptibility to lung cancer.

Abstract 4597: A novel variant in DNA repair gene GTF2H4 is associated with lung cancer risk: A reanalysis of GWAS datasets from the TRICL consortium

Abstract DNA repair pathways maintain genomic integrity and stability, and dysfunction of DNA repair is involved in cancer. We hypothesize that genetic variants in DNA repair genes are associated with risk of lung cancer. To test this hypothesis, we performed a large-scale meta-analysis of 123,371 SNPs in 169 DNA repair genes obtained from six datasets of previously published genome-wide association studies (GWASs), consisting of 12,160 lung cancer cases and 16,838 controls. We evaluated functional relevance of the identified, previously unpublished important and significant SNPs. Across the six GWASs, 14 SNPs were found to be significantly associated with lung cancer risk with a false discovery rate (FDR) of P &amp;lt; 0.05, of which rs114596632 in GTF2H4 at 6q31.33 was the most statistically significant SNP (OR = 1.19, 95% CI = 1.12-1.25, Pcombined = 5.40 × 10−10). The SNP rs114596632 was not strongly correlated with the previously reported GWAS hit MSH5 rs3131379 in that region (r2 = 0.383). Further mapping of the human leukocyte antigen (HLA) region identified that rs114596632 was partially tagged by HLA-B*0801 (r2 = 0.592) and HLA-C*0701 (r2 = 0.675). Importantly, the rs114596632 T allele was significantly associated with decreased DNA repair capacity (DRC) measured in peripheral T lymphocytes and decreased mRNA expression levels of GTF2H4 in lymphoblastoid cell lines. We also validated two previously known SNPs in DNA repair genes MSH5 and XRCC4 associated with lung cancer risk. Subsequent pathway analysis with gene relationships across implicated loci (GRAIL) revealed multiple significant connections for GTF2H4, ERCC2, and MMS19. Our results provide evidence that genetic variants of GTF2H4 confer susceptibility to lung cancer and suggest the involvement of GTF2H4 in the etiology of lung cancer. (Supported by U19CA148127, R01CA CA074386, R01CA092824 and the start-up funds from Duke Cancer Institute, Duke University Medical Center) Citation Format: Qingyi Wei, Hongliang Liu, Zhensheng Liu, Christopher I. Amos, Jennifer A. Doherty, Heike Bickeboller, Rayjean J. Hung, Paul Brennan, Richard Houlston, Maria Teresa Landi, Neil E. Caporaso, David Christiani. A novel variant in DNA repair gene GTF2H4 is associated with lung cancer risk: A reanalysis of GWAS datasets from the TRICL consortium. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4597. doi:10.1158/1538-7445.AM2015-4597

Abstract 2915: Identification of the mechanisms of radiosensitization by human papillomavirus (HPV) in head and neck cancer cell lines

Abstract Background: Several clinical studies have shown that HPV+ head and neck squamous cell carcinomas (HNSCC) present a more favorable outcome and greater response to radiotherapy. Although there are some data supporting the hypothesis that HPV-related HNSCC exhibit higher sensitivity to radiotherapy, it is difficult to conclude that the improved outcome of HPV-related HNSCC is only attributable to intrinsic radiosensitivity of the HPV+ cells. We postulate that a complex interaction occurs between intrinsic mechanisms of radio-response and the tumor micro-environment including cells of the immune system stimulated by the presence of HPV. The objectives of this work are to study mechanisms of radio-response in HPV+ cells and to investigate whether irradiated HPV+ cells exhibit increased immunogenic cell death signals including membrane exposure of endoplasmatic reticulum-resident chaperone calreticulin, secretion of High-Mobility-Group-Box 1 protein (HMGB1) and ATP release. Material and Methods: We determined radiosensitivity by clonogenic survival of two HPV+ HNSCC cell lines (UPCI-SCC-154 &amp; UPCI-SCC90) compared to two HPV- cells (SCC61 &amp; SQD9). Cell cycle distribution and G2/M checkpoint were assessed by flow cytometry. DNA damage repair was evaluated by gamma-H2Ax assay, HR and NHEJ reporter gene assay. In addition, cell death (apoptosis, necrosis and mitotic catastrophe) and senscence were measured in the four cell lines together with immunogenic cell death signals, through cytokines, HMGB1, and ATP-release as well as, membrane exposure of calreticulin. Results: The surviving fraction at 2Gy (SF2) for the two HPV+ cell lines was 0,13 and 0,15 for UPCI-SCC-90 and UPCI-SCC-154, respectively while SF2 for SQD9 was 0,49 and 0,16 for SCC-61 a cell line already described as radiosensitive. Cell cycle distribution indicated an important block in G2/M 24h after irradiation for the HPV+ cell lines together with a slower clearance of gamma-H2Ax for HPV+ cells, compared to HPV- cells. Furthermore, HPV+ cells showed decreased DNA repair capacity by NHEJ and HR. Apoptosis rate after irradiation (2Gy) was variable depending on the cell line, but remained overall under 5%. Senescence was not increased after irradiation (2Gy) whereas mitotic catastrophe increased in HPV-negative cell lines after 24h. Cytokine secretion was cell line dependent while HMGB1 and ATP release was increased in HPV+ cell lines compared to HPV- cell lines. Finally, calreticulin membrane exposure after irradiation detected by immunofluorescence was more frequent in HPV+ compared with HPV- cells. Conclusions: Our results confirm an increased radiosenstitivity of HPV+ cells after irradiation together with increased signals recognised as “immunogenic cell death” (calreticulin exposure, HMGB1 and ATP release) providing a rationale to investigate immune responses in HPV+ tumors after radiotherapy. Note: This abstract was not presented at the meeting. Citation Format: Vanesa Bol. Identification of the mechanisms of radiosensitization by human papillomavirus (HPV) in head and neck cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2915. doi:10.1158/1538-7445.AM2015-2915

Abstract 3064: MicroRNA-21 enhances the effect of ionizing radiation via alteration of the DNA damage response

Abstract Micro-RNA 21 (miR-21) is a well-known onco-miR found to be up-regulated in over 18 major cancer types and is linked to radiation and chemotherapy resistance. It is hypothesized that dysregulation of miR-21 may contribute to treatment resistance through multiple DNA damage response pathways by enhancing DNA repair and increasing DNA damage tolerance. Given its involvement in multiple resistance pathways, miR-21 appears to be an attractive target for overcoming resistance with radiation therapy. In vitro, we demonstrated that knockdown of miR-21 revealed an increase in tumor cell death via clonogenic cell survival curves following exposure to ionizing radiation in multiple cancer cell lines including triple negative breast cancer cells, GBM, and lung cancer. A miR-21 knockout (KO) mouse model was then generated based on deletion of a miR-21 upstream promoter element. To determine if mir-21 KO would lead to radiation sensitivity, miR-21 deficient mice were exposed to 6.5Gy total body irradiation (TBI) and compared to control wild type (WT) mice. miR-21 KO mice died at an average of 12 days after TBI vs. over 100 days survival in wild-type mice. Tissue analysis revealed an increase in double strand breaks measured by elevated γ-H2AX in miR-21 deficient mice. An associated decrease in DNA repair genes such as MSH2 and STAG2 was also seen. Therefore, we hypothesize deletion or silencing of miR-21 results in a radiation sensitive phenotype due to a decreased DNA repair capacity, particularly in the repair of double-strand breaks. In silico analysis predicted miR-21 targets involved in the DNA repair response including rad21 and stag2, which are both members of the cohesin complex. The cohesion complex is a ring-shaped protein complex involved in homologous repair and the DNA damage-response. When exposed to ionizing radiation, rad21 and stag2 expression appears inversely correlated with miR-21 levels in vivo and in vitro. Similarly, in the setting of miR-21 knockdown, rad21 and stag2 expression are increased, suggesting dysregulation in the function of the cohesin complex may contribute to genomic instability and a DNA damage-permissive phenotype. In the current study, we propose a novel mechanism for increasing radiation sensitivity through miRNA-mediated pathways controlling cellular DNA-repair. Future studies investigating miR-21 expression in patients may identify subgroups of patients who are at risk for resistance to standard cytotoxic therapies and may open avenues for targeted therapies involving mir-21 mediated DNA repair pathways. Citation Format: Tu Dan, Ajay Palagani, Tiziana DeAngelis, Sunny Han, Lance Liotta, Richard Pestell, Nicole Simone. MicroRNA-21 enhances the effect of ionizing radiation via alteration of the DNA damage response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2015-3064

Ionizing radiation promotes the acquisition of a senescence‐associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosens (665.3)

Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole‐brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ‐irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ‐irradiation (2‐8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ‐irradiation promotes premature senescence (indicated by SA‐β‐galactosidase positivity and upregulation of p16 (INK4a)), which was associated with impaired angiogenic capacity (decreased proliferation and tube‐forming capacity). γ‐Irradiated CMVECs acquired a senescence‐associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL‐6, IL‐1α, and MCP‐1). Collectively, increased vulnerability of γ‐irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence‐associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier).Grant Funding Source: Supported by NIH and the Ellison Medical Foundation