ARID1A regulates DNA repair through chromatin organization and its deficiency triggers DNA damage-mediated anti-tumor immune response.

AT-rich interaction domain protein 1A (ARID1A), a SWI/SNF chromatin remodeling complex subunit, is frequently mutated across various cancer entities. Loss of ARID1A leads to DNA repair defects. Here, we show that ARID1A plays epigenetic roles to promote both DNA double-strand breaks (DSBs) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). ARID1A is accumulated at DSBs after DNA damage and regulates chromatin loops formation by recruiting RAD21 and CTCF to DSBs. Simultaneously, ARID1A facilitates transcription silencing at DSBs in transcriptionally active chromatin by recruiting HDAC1 and RSF1 to control the distribution of activating histone marks, chromatin accessibility, and eviction of RNAPII. ARID1A depletion resulted in enhanced accumulation of micronuclei, activation of cGAS-STING pathway, and an increased expression of immunomodulatory cytokines upon ionizing radiation. Furthermore, low ARID1A expression in cancer patients receiving radiotherapy was associated with higher infiltration of several immune cells. The high mutation rate of ARID1A in various cancer types highlights its clinical relevance as a promising biomarker that correlates with the level of immune regulatory cytokines and estimates the levels of tumor-infiltrating immune cells, which can predict the response to the combination of radio- and immunotherapy.

Deposition of onco-histone H3.3-G34W leads to DNA repair deficiency and activates cGAS/STING-mediated immune responses.

Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.

ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition.

The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.

Selective inhibitors of bromodomain BD1 and BD2 of BET proteins modulate radiation-induced profibrotic fibroblast responses.

Radiotherapy can induce various adverse effects including fibrosis in cancer patients. Radiation-induced aberrant expression of profibrotic genes has been associated with dysregulated epigenetic mechanisms. Pan-BET (bromodomain and extraterminal domain) inhibitors, such as JQ1 and I-BET151, have been reported to attenuate the profibrotic response after irradiation. Despite their profound preclinical efficacy, the clinical utility of pan-inhibitors is limited due to observed cytotoxicicities. Recently, inhibitors were developed that selectively target the first (BD1) and second (BD2) bromodomain of the BET proteins (iBET-BD1 [GSK778] and iBET-BD2 [GSK046]). Here, their potential to attenuate radiation-induced fibroblast activation with low-toxicity was investigated. Our results indicated that cell proliferation and cell cycle progression in fibroblasts from BJ cells and six donors were reduced when treated with I-BET151 and iBET-BD1, but not with iBET-BD2. After irradiation, induction of DGKA and profibrotic markers, especially COL1A1 and ACTA2, was attenuated with all BET inhibitors. H3K27ac enrichment was similar at the DGKA enhancer region after I-BET151 treatment and irradiation, but was reduced at the COL1A1 transcription start site and the ACTA2 enhancer site. iBET-BD2 did not change H3K27ac levels in these regions. BRD4 occupancy at these regions was not altered by any of the compounds. Cell migration activity was measured as a characteristic independent of extracellular matrix production and was unchanged in fibroblasts after irradiation and BET inhibitor-treatment. In conclusion, iBET-BD2 efficiently suppressed radiation-induced expression of DGKA and profibrotic markers without showing cytotoxicity. Thus BD2-selective targeting is a promising new therapeutic avenue for further investigations to prevent or attenuate radiotherapy-induced fibrosis.

Epigenome-Wide Analysis of Methylation Changes in the Sequence of Gallstone Disease, Dysplasia, and Gallbladder Cancer.

BACKGROUND AND AIMS: Gallbladder cancer (GBC) is a highly aggressive malignancy of the biliary tract. Most cases of GBC are diagnosed in low-income and middle-income countries, and research into this disease has long been limited. In this study we therefore investigate the epigenetic changes along the model of GBC carcinogenesis represented by the sequence gallstone disease → dysplasia → GBC in Chile, the country with the highest incidence of GBC worldwide. APPROACH AND RESULTS: To perform epigenome-wide methylation profiling, genomic DNA extracted from sections of formalin-fixed, paraffin-embedded gallbladder tissue was analyzed using Illumina Infinium MethylationEPIC BeadChips. Preprocessed, quality-controlled data from 82 samples (gallstones n = 32, low-grade dysplasia n = 13, high-grade dysplasia n = 9, GBC n = 28) were available to identify differentially methylated markers, regions, and pathways as well as changes in copy number variations (CNVs). The number and magnitude of epigenetic changes increased with disease development and predominantly involved the hypermethylation of cytosine-guanine dinucleotide islands and gene promoter regions. The methylation of genes implicated in Wnt signaling, Hedgehog signaling, and tumor suppression increased with tumor grade. CNVs also increased with GBC development and affected cyclin-dependent kinase inhibitor 2A, MDM2 proto-oncogene, tumor protein P53, and cyclin D1 genes. Gains in the targetable Erb-B2 receptor tyrosine kinase 2 gene were detected in 14% of GBC samples. CONCLUSIONS: Our results indicate that GBC carcinogenesis comprises three main methylation stages: early (gallstone disease and low-grade dysplasia), intermediate (high-grade dysplasia), and late (GBC). The identified gradual changes in methylation and CNVs may help to enhance our understanding of the mechanisms underlying this aggressive disease and eventually lead to improved treatment and early diagnosis of GBC.

LOC283731 promoter hypermethylation prognosticates survival after radiochemotherapy in IDH1 wild-type glioblastoma patients.

MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild-type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy-associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl-CpG-immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long-term (LTS; >36 months) and 15 short-term (STS; 6-10 months) surviving GBM patients. Even after exclusion of the G-CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change ± 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n = 62) of non-G-CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p = 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n = 89), and was independent of gender, performance (KPS) and MGMT status (p = 0.0485, HR = 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild-type/non-G-CIMP GBMs.

Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer.

Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

A polymorphism in the base excision repair gene PARP2 is associated with differential prognosis by chemotherapy among postmenopausal breast cancer patients.

BACKGROUND: Personalized therapy considering clinical and genetic patient characteristics will further improve breast cancer survival. Two widely used treatments, chemotherapy and radiotherapy, can induce oxidative DNA damage and, if not repaired, cell death. Since base excision repair (BER) activity is specific for oxidative DNA damage, we hypothesized that germline genetic variation in this pathway will affect breast cancer-specific survival depending on treatment. METHODS: We assessed in 1,408 postmenopausal breast cancer patients from the German MARIE study whether cancer specific survival after adjuvant chemotherapy, anthracycline chemotherapy, and radiotherapy is modulated by 127 Single Nucleotide Polymorphisms (SNPs) in 21 BER genes. For SNPs with interaction terms showing p<0.1 (likelihood ratio test) using multivariable Cox proportional hazard analyses, replication in 6,392 patients from nine studies of the Breast Cancer Association Consortium (BCAC) was performed. RESULTS: rs878156 in PARP2 showed a differential effect by chemotherapy (p=0.093) and was replicated in BCAC studies (p=0.009; combined analysis p=0.002). Compared to non-carriers, carriers of the variant G allele (minor allele frequency=0.07) showed better survival after chemotherapy (combined allelic hazard ratio (HR)=0.75, 95% 0.53-1.07) and poorer survival when not treated with chemotherapy (HR=1.42, 95% 1.08-1.85). A similar effect modification by rs878156 was observed for anthracycline-based chemotherapy in both MARIE and BCAC, with improved survival in carriers (combined allelic HR=0.73, 95% CI 0.40-1.32). None of the SNPs showed significant differential effects by radiotherapy. CONCLUSIONS: Our data suggest for the first time that a SNP in PARP2, rs878156, may together with other genetic variants modulate cancer specific survival in breast cancer patients depending on chemotherapy. These germline SNPs could contribute towards the design of predictive tests for breast cancer patients.

Reduced promoter methylation and increased expression of CSPG4 negatively influences survival of HNSCC patients.

Proteoglycans are often overexpressed in tumors and can be found on several normal and neoplastic stem cells. In this study, we analyzed in-depth the role of CSPG4 in head and neck squamous cell carcinomas (HNSCC). Analysis of CSPG4 in a homogeneous study sample of HPV-negative stage IVa HNSCCs revealed overexpression of protein and mRNA levels in a subgroup of HNSCC tumors and a significant association of high CSPG4 protein levels with poor survival. This could be validated in three publicly available microarray datasets. As a potential cause for upregulated CSPG4 expression, we identified DNA hypomethylation in a CpG-island of the promoter region. Accordingly, we found an inverse correlation of methylation and patient outcome. Finally, CSPG4 re-expression was achieved by demethylating treatment of highly methylated HNSCC cell lines establishing a direct link between methylation and CSPG4 expression. In conclusion, we identified CSPG4 as a novel biomarker in HNSCC on several biological levels and established a causative link between DNA methylation and CSPG4 protein and mRNA expression.

Germline variants of base excision repair genes and breast cancer: A polymorphism in DNA polymerase gamma modifies gene expression and breast cancer risk.

Base excision repair (BER) removes DNA damage induced by endogenous reactive oxygen species or ionizing radiation, important breast cancer risk factors. Genetic variation associated with impaired BER might thus increase breast cancer risk. Therefore, we assessed risk associations of 123 common single nucleotide polymorphisms (SNPs) in 19 BER genes in 1,639 postmenopausal breast cancer cases and 1,967 controls from the German population-based case-control study MARIE. SNPs were tagging SNPs representing genetic variation across the gene together with potentially functional SNPs. Risk associations were assessed using conditional logistic regression, adjusted for potential breast cancer risk factors. Significant associations between polymorphisms and breast cancer risk were found for one SNP in PARP2 and three SNPs in the mitochondrial DNA polymerase gamma, POLG. A SNP in the promoter region of POLG (rs2856268, A>G) showed a protective effect for homozygous GG carriers (odds ratio 0.81, 95% confidence intervals 0.65-1.00). Joint analysis of an enlarged sample set and haplotype analysis supported the results for POLG. Quantification of POLG mRNA expression in lymphocytes of 148 breast cancer patients revealed higher mRNA levels for rs2856268 GG carriers (p value = 0.038). A luciferase promoter assay showed significant differences between constructs harboring the respective alleles. Taken together, our results suggest that genetic variation in the POLG promoter region affects DNA polymerase gamma levels in mitochondria. This could contribute to the reported increase in mitochondrial mutation frequency resulting in dysfunction and altered breast cancer risk. Risk effects and the functional impact of the POLG promoter variant require further confirmation.

Epigenetically mediated downregulation of the differentiation-promoting chaperon protein CRABP2 in astrocytic gliomas.

Impairment of endogenous differentiation pathways like retinoic acid (RA) signaling seems to be a central pathogenetic event in astrocytic gliomas. Among others, expression of the differentiation-promoting RA chaperon protein cellular retinoic acid binding protein 2 (CRABP2) is extenuated in high-grade gliomas. Against this background, we aimed at identifying potential pathomechanisms underlying reduced CRABP2 expression in these tumors. Using MassARRAY methylation analysis, we detected extensive CpG methylation upstream of the CRABP2 gene locus in a study sample comprising 100 astrocytic gliomas of WHO Grade II to IV. Compared to nontumorous control samples, tumors revealed increased CpG methylation and methylation levels were inversely correlated to CRABP2 mRNA expression. Substantiating our in situ findings, CRABP2 mRNA levels increased in glioma cell lines after exposure to the demethylating agent 5-aza-2'-deoxycytidine. Finally, a distinct CpG methylation signature distinguished between primary glioblastoma on the one hand and the group of astrocytoma WHO II-III and secondary glioblastoma on the other hand. Altogether, our observations suggest that epigenetic silencing of CRABP2 might contribute to an immature phenotype in glioma cells.

The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells.

Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC(50) values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of >1000-fold in resistance between normal and NER-deficient cells (IC(50) values for cells with deficiency in ERCC6: 0.15µM, XPC: 0.18µM, and normal cells: >180µM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes.

Polymorphisms in oxidative stress-related genes and postmenopausal breast cancer risk.

Breast cancer is the most frequent cancer type among women in western countries. In addition to established risk factors like hormone replacement therapy, oxidative stress may play a role in carcinogenesis through an unbalanced generation of reactive oxygen species that leads to genetic instability. The aim of this study is to assess the influence of common single nucleotide polymorphisms (SNPs) in candidate genes related to oxidative stress on postmenopausal breast cancer risk. We genotyped 109 polymorphisms (mainly tagging SNPs) in 22 candidate genes in 1,639 postmenopausal breast cancer cases and 1,967 controls (set 1) from the German population-based case-control study "MARIE". SNPs showing association in set 1 were tested in further 863 cases and 2,863 controls from MARIE (set 2) using a joint analysis strategy. Six polymorphisms evaluated in the combined set showed significantly modified breast cancer risk per allele in the joint analysis, including SNPs in CYBA (encoding a subunit of the NADPH oxidase: rs3794624), MT2A (metallothionein 2A: rs1580833), TXN (thioredoxin: rs2301241), and in TXN2 (thioredoxin 2: rs2267337, rs2281082, rs4821494). Associations with the CYBA rs3794624 (OR per allele: 0.93, 95% CI 0.87-0.99) and TXN rs2301241 variants (OR per allele: 1.05, 95% CI 1.00-1.10) were confirmed in the summary risk estimate analysis using up to three additional studies. We found some evidence for association of polymorphisms in genes of the thioredoxin system, CYBA, and MT2A with postmenopausal breast cancer risk. Summary evidence including independent datasets indicated moderate effects in CYBA and TXN that warrant confirmation in large independent studies.

Down-regulation of tumor suppressor A kinase anchor protein 12 in human hepatocarcinogenesis by epigenetic mechanisms.

UNLABELLED: The A kinase anchor protein 12 (AKAP12) is a central mediator of protein kinase A and protein kinase C signaling. Although AKAP12 has been described to act as a tumor suppressor and its expression is frequently down-regulated in several human malignancies, the underlying molecular mechanisms responsible for the AKAP12 reduction are poorly understood. We therefore analyzed the expression of AKAP12 and its genetic and epigenetic regulatory mechanisms in human hepatocarcinogenesis. Based on tissue microarray analyses (n = 388) and western immunoblotting, we observed a significant reduction of AKAP12 in cirrhotic liver (CL), premalignant lesions (DN), and hepatocellular carcinomas (HCCs) compared to histologically normal liver specimens (NL). Analyses of array comparative genomic hybridization data (aCGH) from human HCCs revealed chromosomal losses of AKAP12 in 36% of cases but suggested additional mechanisms underlying the observed reduction of AKAP12 expression in hepatocarcinogenesis. Quantitative methylation analysis by MassARRAY of NL, CL, DN, and HCC tissues, as well as of various tumorigenic and nontumorigenic liver cell lines revealed specific hypermethylation of the AKAP12α promoter but not of the AKAP12ß promoter in HCC specimens and in HCC cell lines. Consequently, restoration experiments performed with 5-aza-2'deoxycytidine drastically increased AKAP12α mRNA levels in a HCC cell line (AKN1) paralleled by AKAP12α promoter demethylation. As hypermethylation is not observed in CL and DN, we investigated microRNA-mediated posttranscriptional regulation as an additional mechanism to explain reduced AKAP12 expression. We found that miR-183 and miR-186 are up-regulated in CL and DN and are able to target AKAP12. CONCLUSION: In addition to genetic alterations, epigenetic mechanisms are responsible for the reduction of the tumor suppressor gene AKAP12 in human hepatocarcinogenesis.

Epigenetic Modulation of Radiation-Induced Diacylglycerol Kinase Alpha Expression Prevents Pro-Fibrotic Fibroblast Response.

Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.

Diacylglycerol Kinase Alpha in Radiation-Induced Fibrosis: Potential as a Predictive Marker or Therapeutic Target.

Radiotherapy is an efficient tool in cancer treatment, but it brings along the risk of side effects such as fibrosis in the irradiated healthy tissue thus limiting tumor control and impairing quality of life of cancer survivors. Knowledge on radiation-related fibrosis risk and therapeutic options is still limited and requires further research. Recent studies demonstrated that epigenetic regulation of diacylglycerol kinase alpha (DGKA) is associated with radiation-induced fibrosis. However, the specific mechanisms are still unknown. In this review, we scrutinized the role of DGKA in the radiation response and in further cellular functions to show the potential of DGKA as a predictive marker or a novel target in fibrosis treatment. DGKA was reported to participate in immune response, lipid signaling, exosome production, and migration as well as cell proliferation, all processes which are suggested to be critical steps in fibrogenesis. Most of these functions are based on the conversion of diacylglycerol (DAG) to phosphatidic acid (PA) at plasma membranes, but DGKA might have also other, yet not well-known functions in the nucleus. Current evidence summarized here underlines that DGKA activation may play a central role in fibrosis formation post-irradiation and shows a potential of direct DGKA inhibitors or epigenetic modulators to attenuate pro-fibrotic reactions, thus providing novel therapeutic choices.

Laryngeal cancer risk associated with smoking and alcohol consumption is modified by genetic polymorphisms in ERCC5, ERCC6 and RAD23B but not by polymorphisms in five other nucleotide excision repair genes.

Laryngeal cancer is known to be associated with smoking and high alcohol consumption. Nucleotide excision repair (NER) plays a key role in repairing DNA damage induced by these exposures and might affect laryngeal cancer susceptibility. In a population-based case-control study including 248 cases and 647 controls, the association of laryngeal cancer with 14 single nucleotide polymorphisms (SNPs) in 8 NER genes (XPC, XPA, ERCC1, ERCC2, ERCC4, ERCC5, ERCC6 and RAD23B) was analyzed with respect to smoking and alcohol exposure. For genotyping, sequence specific hybridization probes were used. Data were evaluated by conditional logistic regression analysis, stratified for age and gender, and adjusted for smoking, alcohol consumption and education. Pro-carriers of ERCC6 Arg1230Pro showed a decreased risk for laryngeal cancer (OR = 0.53, 95% CI 0.34-0.85), strongest in heavy smokers and high alcohol consumers. ERCC5 Asp1104His was associated with risk in heavy smokers (OR = 1.70, 95% CI 1.1-2.5). Val-carriers of RAD23B Ala249Val had an increased cancer risk in heavy smokers (OR = 1.6, 95% CI 1.1-2.5) and high alcohol consumers (OR = 2.0, 95% CI 1.1-3.4). The combined effect of smoking and alcohol intake affected risk, at high exposure level, for ERCC6 1230Pro carriers (OR = 0.47, 95% CI 0.22-0.98) and RAD23B 249Val carriers (OR = 2.6, 95% CI 1.3-4.9). When tested for gene-gene interaction, presence of 3 risk alleles in the XPC-RAD23B complex increased the risk 2.1-fold. SNPs in the other genes did not show a significant association with laryngeal cancer risk. We conclude that common genetic variations in NER genes can significantly modify laryngeal cancer risk.

Genetic variation in normal tissue toxicity induced by ionizing radiation.

Radiotherapy is an important weapon in the treatment of cancer, but adverse reactions developing in the co-irradiated normal tissue can be a threat for patients. Early reactions might disturb the usual application schedule and limit the radiation dose. Late appearing and degenerative reactions might reduce or destroy normal tissue function. Genetic markers conferring the ability to identify hyper-sensitive patients in advance would considerably improve therapy. Association studies on genetic variation and occurrence of side effects should help to identify such markers. This survey includes published studies and novel data from our own laboratory. It illustrates the presence of candidate polymorphisms in genes involved in the cellular response to irradiation which could be used as predictive markers for radiosensitivity in breast or prostate cancer patients. For other tumor types such as head and neck cancers or brain tumors, the available data are much more limited. In any case, further validation of these markers is needed in large patient cohorts with systematically recorded data on side effects and patient characteristics. Genetic variation contributing to radiosensitivity should be screened on a broader basis using newly developed, more comprehensive approaches such as genome-wide association studies.

DNA methylation profiling to explore colorectal tumor differences according to menopausal hormone therapy use in women.

Aim: Use of menopausal hormone therapy (MHT) has been associated with a reduced risk for colorectal cancer, but mechanisms underlying this relationship are not well understood. In the colon, MHT appears to act through estrogen receptor ß (ERß) which may influence DNA methylation by binding to DNA. Using genome-wide methylation profiling data, we aimed to identify genes that may be differentially methylated according to MHT use. Materials & methods: DNA methylation was measured using Illumina HumanMethylation450k arrays in two independent tumor sample sets of colorectal cancer patients. Differential methylation was determined using R/limma. Results: In the discovery analysis, two CpG sites showed differential DNA methylation according to MHT use, both were not replicated. In stratified analyses, 342 CpG sites were associated with current MHT use only in ERß-positive tumors. Conclusion: The suggestive findings of differential methylation according to current MHT use in ERß-positive tumors warrant further investigation.

Genome-wide DNA methylation differences according to oestrogen receptor beta status in colorectal cancer.

Involvement of sex hormones in colorectal cancer (CRC) development has been linked to oestrogen receptor ß (ERß). Expression of ERß is found reduced in tumour tissue and inversely related to mortality. However, mechanisms are not well understood. Our study aimed to detect differentially methylated genes associated with ERß expression, which could point to mechanisms by which ERß could influence risk and prognosis of CRC. Epigenome-wide DNA methylation profiling was performed using Illumina HumanMethylation450k BeadChip arrays in two independent tumour sample sets of CRC patients recruited in 2003-2010 by the German DACHS study (discovery cohort n = 917, replication cohort n = 907). ERß expression was measured using immunohistochemistry and scored as negative, moderate and high. Differentially methylated CpG sites and genomic regions were determined using limma in the R-package RnBeads. For the comparison of tumours with moderate/high ERß versus negative expression, differentially methylated CpG sites were identified but not confirmed by replication. Comparing tumours of high with tumours of negative ERß expression revealed 2,904 differentially methylated CpG sites of which 403 were replicated (FDR adjusted p-value<0.05). Replicated CpGs were annotated to genes such as CD36, HK1 or LRP5. A survival analysis indicates that 30 of the replicated CpGs are also associated with overall survival (FDR-adjusted p-value<0.05). The regional analysis identified 60 differentially methylated promotor regions. The epigenome-wide analysis identified both novel genes as well as genes already implicated in CRC. Follow-up mechanistic studies to better understand the regulatory role of ERß could inform potential targets for improving treatment or prevention of CRC.