NER Pathway Research Articles

Genetic variability of genes involved in DNA repair influence treatment outcome in osteosarcoma

We conducted a perspective study to investigate the role of ERCC1 (rs11615), ERCC2 (rs13181 and rs1799793), ERCC4 (rs1800067), and ERCC5 (rs17655) in NER pathway in the prognosis of osteosarcoma patients. In total, 146 osteosarcoma patients were recruited between 2008 and 2013. ERCC1 rs11615, ERCC2 rs13181 and rs1799793, ERCC4 rs1800067, and ERCC5 rs17655 gene polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism assay. By multivariate Cox proportional hazards models, we found that carriers of ERCC1 rs11615 TT genotype showed significantly favorable survival compared to wide-type CC genotype, and the adjusted OR (95%CI) was 0.24 (0.08-0.96). Moreover, we found that subjects with ERCC2 rs1799793 AA genotype were associated with decreased hazards of death in multivariate analysis (HR = 0.22, 95%CI = 0.12-0.93). In conclusion, our results suggest that ERCC1 rs11615 and ERCC2 rs1799793 may be useful genetic prognostic markers for osteosarcoma in a Chinese population.

Caught in the Act: R-Loops Are Cleaved by Structure-Specific Endonucleases to Generate DSBs

Unscheduled RNA-DNA hybrids promote the formation DNA double-strand breaks (DSBs) through poorly characterized mechanism. In this issue, Sollier etal. (2014) show that R-loops are processed by XPF and XPG, two structure-specific endonucleases of the NER pathway, to generate DSBs.

Laryngeal cancer risk and common single nucleotide polymorphisms in nucleotide excision repair pathway genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5 and XPA

Because the molecular mechanisms underlying the development of laryngeal cancer are not well understood, we conducted a case-control study to determine the association between eight common SNPs in NER pathway genes and risk of laryngeal cancer, and the association between genetic polymorphisms and environmental factors. A 1:1 matched case-control study of 176 cases and 176 controls was conducted. Laryngeal cancer cases were more likely to smoke and drink (all P values<0.05). Subjects with the ERCC1 rs11615 CC genotype and C allele had an increased risk of laryngeal cancer. Similarly, individuals with the ERCC5 rs17655 GG genotype and G allele had an increased risk of laryngeal cancer. Gene-gene interaction analysis showed that subjects carrying ERCC1 rs11615 C allele and XPG/ERCC5 rs17655 G allele had a greatly increased risk of breast cancer. Stratified analysis revealed that the interaction between polymorphisms of ERCC1 rs11615 and ERCC5 rs17655 and smoking on cancer risk was statistically significant, and ERCC1 rs11615 polymorphisms also had a significant interaction with drinking habit. In conclusion, our study suggests that ERCC1 rs11615 and ERCC5 rs17655 polymorphisms are associated with increased risk of laryngeal cancer, and that they confer more risk among smokers and drinkers.

Abstract C93: Collateral sensitivity to cisplatin of trabectedin-resistant cell lines.

Abstract Introduction: Trabectedin (T) is a marine-derived anticancer drug with a peculiar mechanism of action, ascribed to a covalent modification of DNA by guanine-specific alkylation at the N2 position in the minor groove that induces DNA damage, affects transcription regulation and triggers cell differentiation and death. Trabectedin has been approved in the EU for the second line treatment of soft tissue sarcomas and ovarian carcinomas. In order to investigate the mechanisms of resistance to trabectedin, that generally occurs after prolonged treatment with the drug, two different trabectedin-resistant cell lines such as myxoid liposarcoma 402-91 and ovarian carcinoma A2780 were developed and characterized. Material and Methods: Resistant cell lines (402-91/T and A2780/T) were obtained by stepwise increase in drug concentration using a short exposure (1 hr) applied at 10-20 day intervals. Drug sensitivity was measured by colony assay, DNA content and cell cycle were evaluated by flow cytometric analysis and XPG expression by Western Blotting. Results: 402-91/T and A2780/T cell lines were 16 and 6 fold resistant to trabectedin as compared to parental cells respectively. Resistant cell lines maintained the same DNA content compared with the parental cells, while showed different cell cycle perturbations induced by trabectedin treatment; in fact the characteristic G2/M block was found only in sensitive cell lines. Both 402-91/T and A2780/T cell lines did not express XPG, a protein involved in NER pathway, and were found hypersensitive to UV light. This finding is in keeping with previous observations that NER deficiency is associated to a decreased sensitivity to trabectedin. Being NER activity crucial for the repair of platinum DNA adducts, we tested cisplatin cytotoxicity in 402-91/T and A2780/T cell lines and they were 2.5 and 11 fold more sensitive than the parental cell lines. The collateral sensitivity to cisplatin of trabectedin resistant cell lines is likely related to the important function of NER in the repair of the bulky adducts produced by the drug. Conclusion: The finding that resistance to trabectedin is associated to the loss of NER function, with consequent increased sensitivity to cisplatin, provides the rational to test the sequential combination of trabectedin and platinum complexes in the clinic. This strategy is currently under evaluation in ovarian cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C93. Citation Format: Maurizio D'Incalci, Benedetta Colmegna, Sarah Uboldi, Roberta Frapolli, Simonetta Andrea Licandro, Eugenio Erba, Carlos M. Galmarini, Nadia Badri. Collateral sensitivity to cisplatin of trabectedin-resistant cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C93.

Repair kinetics of acrolein- and (E)-4-hydroxy-2-nonenal-derived DNA adducts in human colon cell extracts

ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) play a role in the pathogenesis of colon cancer. Upon oxidation, PUFAs generate α,β-unsaturated aldehydes or enals, such as acrolein (Acr) and (E)-4-hydroxy-2-nonenal (HNE), which can form cyclic adducts of deoxyguanosine (Acr-dG and HNE-dG, respectively) in DNA. Both Acr-dG and HNE-dG adducts have been detected in human and animal tissues and are potentially mutagenic and carcinogenic. In vivo levels of Acr-dG in DNA are at least two orders of magnitude higher than those of HNE-dG. In addition to the facile reaction with Acr, the higher levels of Acr-dG than HNE-dG in vivo may be due to a lower rate of repair. Previous studies have shown that HNE-dG adducts are repaired by the NER pathway (Choudhury et al. [42]). We hypothesize that Acr-dG adducts are repaired at a slower rate than HNE-dG and that HNE-dG in DNA may influence the repair of Acr-dG. In this study, using a DNA repair synthesis assay and a LC–MS/MS method, we showed that Acr-dG in a plasmid DNA is repaired by NER proteins, but it is repaired at a much slower rate than HNE-dG in human colon cell extracts, and the slow repair of Acr-dG is likely due to poor recognition/excision of the lesions in DNA. Furthermore, using a plasmid DNA containing both adducts we found the repair of Acr-dG is significantly inhibited by HNE-dG, however, the repair of HNE-dG is not much affected by Acr-dG. This study demonstrates that the NER repair efficiencies of the two major structurally-related in vivo cyclic DNA adducts from lipid oxidation vary greatly. More importantly, the repair of Acr-dG can be significantly retarded by the presence of HNE-dG in DNA. Therefore, this study provides a mechanistic explanation for the higher levels of Acr-dG than HNE-dG observed in tissue DNA.

Repressing DNA Repair to Enhance Chemotherapy: Targeting MyD88 in Colon Cancer

Repressing DNA Repair to Enhance Chemotherapy: Targeting MyD88 in Colon Cancer

Abstract 630: ERCC1-202 isoform is responsible for all known functions of ERCC1.

Abstract Background: ERCC1 is a DNA repair protein whose expression is a prognostic and predictive biomarker of chemotherapy effect in NSCLC. Four alternative transcripts of the ERCC1 gene have been described (isoforms 201 to 204). We previously found that only ERCC1-202 isoform is able to remove cisplatin-DNA adducts and to improve survival after cisplatin treatment. Little is known however about the role of the other isoforms. Since the four isoforms are expressed in human samples, we sought to elucidate the contribution of these different ERCC1-isoforms to ERCC1 described functions within and beyond DNA repair. Methods: We used an established ERCC1-deficient A549 cells with selective re-expression of each ERCC1 isoform. We searched for negative dominant functions of the ERCC1-201, -203, and -204 isoforms using proliferation assay (WST-1) and clonogenic assay. Respective cellular localizations of the isoforms were assessed by immunofluorescent (IF) staining and interacting abilities with previously identified ERCC1 partners was estimated by proximity ligation assays (Duolink). We finally investigated their influence on the cellular mitotic process (mitotic spindle shape) by alpha- and gamma-tubulin IF and video microscopy. Results: IF detection of ERCC1 protein isoforms revealed a nuclear localization of ERCC1-201 and -202 isoforms whereas ERCC1-203 and -204 isoforms were mainly detected in the cytoplasm. This alternative cellular localization suggests alternative roles for ERCC1-203 and -204. Only ERCC1-202 formed a stable heterodimer complex with XPF and other interacting partners (XPA, MSH2, FANCG, SLX4 and TRF2). None of the isoforms decreased cisplatin resistance (IC50) associated with ERCC1-202 expression. Interstrand Cross-Link repair (ICL-R) efficiency (mitomycin-C treatment) was also exclusively rescued with ERCC1-202 re-expression. Further, ERCC1 deficient cells displayed mitotic aberrations such as chromosome bridges at anaphase that impaired cytokinesis and generated aneuploidy. Only ERCC1-202 isoform restored chromosome segregation accuracy. Conclusions: Alternative roles for ERCC1 beyond the canonical NER pathway are currently emerging. Our data suggest that all currently known functions of ERCC1 are fulfilled by the same ERCC1 isoform ERCC1-202. Despite their wide expression in human samples, the identification of alternative roles of other isoforms is still under investigation. Our A549 clones, with stable ERCC1 deficiency, provide a promising tool to analyze different functions of ERCC1. Finally, most antibodies used to test ERCC1 status in patients do not distinguish between different isoforms, it is important to develop an antibody (or alternative diagnostic method) able to specifically recognize only the functional ERCC1-202 isoform. Given that ERCC1-202/XPF interaction domains can be produced together it would be realistic to use it as an immunogenic epitope and/or a therapeutic target. Citation Format: Luc Friboulet, Ken André Olaussen, Florence Ponsonnailles, Annabelle Stoclin, Nicolas Dorvault, Julien Adam, Frédéric Commo, Patrick Saulnier, Fabrice André, Jean-Charles Soria. ERCC1-202 isoform is responsible for all known functions of ERCC1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 630. doi:10.1158/1538-7445.AM2013-630

Abstract 4159: Expression of miRNAs associated with DNA repair capacity in women with breast cancer.

Abstract MicroRNAs (miRNA) are endogenous, short (19-24 nucleotides) non-protein-coding RNAs that regulate gene expression at the post-transcriptional level via binding to 3′-untranslated regions of protein-coding transcripts. A low DNA repair capacity (DRC) is an important risk factor for breast cancer (BC). We are currently investigating whether miRNAs expression is associated with dysregulation of DRC in women with BC. To that effect, we obtained plasma from 30 BC cases and 30 controls selected based on their DRC levels. DRC was measured in lymphocytes using a host cell reactivation assay. Samples were analyzed for miRNA expression utilizing protocols from Applied Biosystems (Life Technologies). After QA/QC, 27 BC cases and 29 controls whose DRC data was available from our cohort study were selected and miRNAs isolated from plasma. The miRNA expression profiling was performed utilizing the RT-PCR TaqMan Array Human MicroRNA A Cards v 2.0 (Applied Biosystems). Single-stranded cDNA was synthesized from 200 ng of total RNA in 8 Multiplex RT primer pool reactions containing stem-looped RT primers that were specific to mature miRNAs. U6 snRNA-001973 was selected for normalization based on our own experimental validations. To quantify the association of the miRNA expression the fold change () was estimated for every detector with the p-values based on the t-test. Significant candidate miRNAs were determined with the false discovery rate of 4%. The results showed that twelve miRNAs are underexpressed in patients with BC relative to controls. These candidate miRNAs were associated with the expression of twenty seven DNA repair genes. Two of these genes (RPA3, MMS19L) are part of the NER pathway which has recently been linked to breast carcinogenesis. Our pilot study data suggests that differential expression of some miRNAs might be associated with dysregulation of DRC in women with BC. Supported by grants S06 GM008239-20, MBRS-RISE GM082406 [NIH-NIGMS] and 1SCA157250-2 from the NCI Center to Reduce Health Disparities, and NIH-MBRS Program (NIGMS). Citation Format: Jaime L. Matta, Luisa Morales, Carmen Ortiz, Celestino Zayas, Erick Suarez. Expression of miRNAs associated with DNA repair capacity in women with breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4159. doi:10.1158/1538-7445.AM2013-4159

Cloning and expression of uvrA gene of Corynebacterium pseudotuberculosis to value its role in NER pathway

Caseous lymphadenitis is a infection that affects sheep and goats and caused by Corynebacterium pseudotuberculosis. DNA repair, the target of this study, is essential for survival of any organism, to prevent potential deleterious effects. The objective of this study was to characterize uvrA gene from C.psudotuberculosis using in silico and in vitro techniques. In silico analyzes were performed to check the physical and chemical parameters, search for conserved domains and modeling of 3D structure using bioinformatics programs. The analysis shows that the protein has a molecular weight of about 101.5 kDa and a conserved domain of ABC transporters family and ABC excinuclease. The amplification of uvrA gene was carried out using a Long Range PCR Kit and the insert was cloned into the TOPO PCR 2.1 vector. E. coli strain DH5α was electroporated with recombinant plasmid and after that these were digested with restriction enzymes BamH1 and Xho1 for subsequent cloning into the expression vector pProEX HTB. The constructions were confirmed by sequencing. Financial support: CNPq and FAPEMIG

Single Nucleotide Polymorphisms in the NER Pathway and Clinical Outcome of Patients with Bone Malignant Tumor

The effects of polymorphisms in ERCC5, ERCC6, XPC, CCNH and MMS19L on osteosarcoma response to chemotherapy and the survival of the affected patients were assessed. Genotyping of ERCC5, ERCC6, XPC, CCNH and MMS19L was performed by PCR-RFLP assay. The median PFS was 12.8 months, and the median OS was 18.6 months. Individuals carrying homozygous genotypes of ERCC5 rs17655 and ERCC5 rs1047768 were more like to have good response to treatment, while those carrying homozygous genotypes of MMS19L rs29001322 showed poor response. Osteosarcoma patients carrying TT genotype of ERCC5 rs1047768 showed a significantly longer PFS (16.8 months) and OS (21.4 months) than CC genotype, with HRs(95% CI) of 0.31 (0.10-0.93) and 0.32 (0.06-0.97), respectively. Conversely, those with the TT genotype of MMS19L rs29001322 demonstrated shorter PFS and OS, the HRs (95% CI) being 2.23 (1.08-4.15) and 4.62 (1.45-16.08), respectively. Our findings showed polymorphisms in ERCC5 rs1047768 and MMS19L rs29001322 to be associated with clinical outcome of osteosarcoma patients undergoing chemotherapy.

The Nucleotide Excision Repair System of Borrelia burgdorferi Is the Sole Pathway Involved in Repair of DNA Damage by UV Light

To survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized in Escherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes for Borrelia burgdorferi survival to UV-induced DNA damage was assessed. In contrast to E. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of recombinational repair, transcription-coupled repair, methyl-directed mismatch correction, and repair of arrested replication fork pathways did not decrease survival of B. burgdorferi exposed to UV light. However, the disruption of the B. burgdorferi nucleotide excision repair (NER) pathway (uvrA, uvrB, uvrC, and uvrD) resulted in a 10- to 1,000-fold increase in sensitivity to UV light. A functional NER pathway was also shown to be required for B. burgdorferi resistance to nitrosative damage. Finally, disruption of uvrA, uvrC, and uvrD had only a minor effect upon murine infection by increasing the time required for dissemination.

P-0178 Genetic Analysis of the Dna Repair Gene Xpd (LYS751GLN) and XRCC1 (ARG399GLN) in Romanians with Sporadic Colorectal Cancer

ABSTRACT Introduction Carcinogenesis is a complex process in which genomic instability is affected. The most important processes involved in DNA damage repair are the excision of defective bases - base excision repair (BER pathways) and the excision of the entire defective nucleotide – nucleotide excision repair (NER pathways). Two of the most important proteins involved in the BER and NER, respectively pathway are the X-ray cross-complementation group 1-XRCC1 and the Xeroderma pigmentosum group D protein-XPD Aim: to analyze the distribution of genetic polymorphisms in genes coding for DNA repair enzymes XPD (Lys751Gln) and XRCC1 (Arg399Gln) in Romanian patients with sporadic colorectal cancer (CCR) as compared to control subjects, both females and males; to establish the most frequent haplotype associated with sporadic CCR risk in Romanians; to check for the association with the different stages of carcinomas, as well as with the prognosis of sporadic CCR. Methods 312 samples were collected, of which 150 were sporadic CCR cases (80 female and 70 male patients) and 162 were controls (100 females and 62 males); Genotyped were done by PCR-RFLP analysis. The differences in genotype and allele frequencies between the group of patients with sporadic CCR and the control group were also examined using SPSS® Software version 12 for Windows (SPSS Inc., Chicago Illinois, USA 1989- 2003). Results The risk to develop sporadic CRC was 3.09 (p=0.02) and 3.49 (p=0.001), respectively, in association with the homozygous Gln/Gln-XPD and Gln/Gln-XRCC1 genotype. Also significantly higher risk to develop sporadic CRC was found in females homozygous for Gln/Gln-XPD (OR 3.43, p=0.04) and Gln/Gln-XRCC1 (OR 3.32, p=0.018) genotype and in males homozygous for Gln/Gln-XRCC1 (OR 3.63, p=0.04) genotype. The combined effect of the two polymorphisms on sporadic CRC risk was also evaluated through the construction of haplotypes. A significantly higher risk for sporadic CRC in association with Lys/Gln-Arg/Gln (22% vs. 7.4%, OR 3.52, p Conclusion Genetic variations affecting DNA repair capacity increase the risk for sporadic colorectal cancer in Romanians. The XPD and XRCC1 mutations could be associated with more aggressive forms of sporadic colorectal cancer and positive patients have a worse prognosis compared to negative patients.

Correlation of single nucleotide polymorphisms in NER pathway genes with toxicity in advanced non-small cell lung cancer patients treated with chemotherapy.

e18115 Background: New therapeutic approaches are being developed based on the findings that several genetic abnormalities underlying NSCLC could influence chemosensitivity. We assessed whether polymorphisms in genes of NER pathway, such as ERCC5, ERCC6, MMS19L, CCNH, XPC, RRM1, can affect the tolerability of platinum-based chemotherapy in advanced NSCLC pts. Methods: Eligible pts had histological or cytological confirmed stage IV or IIIB (with malignant pleural effusion) chemo-naïve NSCLC, ECOG PS of 0 to 2 and adequate organ function. Pts received vinorelbine 25 mg/m2 or gemcitabine 1000 mg/m2 on day 1 and 8, or docetaxel 75 mg/m2 or taxel 175 mg/m2 on day 1 plus cisplatin 75 mg/m2 or carboplatin AUC=5 on day 1, every 21 days per cycle, for a maximum of six cycles, unless disease progression or unacceptable toxicity occurred. Hematology and biochemistry analyses were repeated every cycle or before chemotherapy for d8 treatment. Toxicity was classified according to CTC AE 3.0. Venous blood was collected and genomic DNA was isolated. Polymorphisms were assessed. SNPs in ERCC6 (A/G 1413, A/G 1213 and A/G 1097), XPC (T/C 500 and A/C 940), ERCC5 (G/C 1104), RRM1 (−37C/A), CCNH (T/C 270) and MMS19L (G811A) were assessed. Results: Totally 388 pts were included prospectively from Oct 2005 to Mar 2009. Median age was 61 years; 68.8% were male, 36.2% were ECOG PS 1, 33.2% had stage IIIB. The median follow-up time was 18 months (range 8–66 months). Hematological toxicity was the most common AE. SNPs of MMS19L and ERCC6 can predict the AE of chemotherapy in NSCLC,leucopenia (P=0.020), jaundice (P=0.039) by MMS19L. In terms of grade 3-4 AE, SNPs of MMS19L and RRM1 -37C/A have much more predictive effect. SNPs of MMS19L were good at total AE (P=0.042), thrombopenia (P=0.035), SNPs of RRM1 -37C/A were for vomiting (P=0.046) and jaundice (P=0.014). Conclusions: Polymorphisms in ERCC6, MMS19L, RRM1, can predict the tolerability of platinum-based chemotherapy in advanced NSCLC patients. Polymorphisms in other genes of NER pathway, such as ERCC5, CCNH, XPC, have no predictive effect.

Abstract 2866: Exploring the basis for platinum and ionizing radiation combination with R1507 (an anti-IGF1R monoclonal antibody) in a small cell lung cancer (SCLC) model using integrated functional and genomic approaches on both short- and long-term drug exposure reveals adaptive mechanisms

Abstract Introduction: R1507 has single agent activity and remarkable chemo- and radiosensitizing effect in defined SCLC cell lines in vitro. It exhibits a dramatic antitumoral effect when combined to IR and cisplatin in vivo. The molecular mechanisms underlying these effects remain unknown. Materials and methods: Three SCLC cell lines (H69, H146 and H526) were selected according to their specific mutational profile (H146 and H69 cells are mutated for MAP2K4 and both PIK3CA and C-MET, respectively). The effects of R1507 on IGF1R downstream signaling were explored using western blots. Both genomic (HG-U133 plus 2 microarray, Affymetrix) and classical functional analyses were performed on the following conditions: (A) R1507 alone short-term (ST-R1507) (3 hours) vs. long-term (LT-R1507) (4 weeks) exposure, (B) R1507 combined with cisplatin, (C) and with ionizing radiation (IR). Results: (A) ST-R1507 disrupts IGF1R signaling through Akt and MAPK pathways in a dose dependent manner in selected SCLC cell lines. High IGF-1R basal expression is not correlated with sensitivity to R1507. Only cell lines with IGF1R gene amplification (H526 and H146) exhibit sensitivity to R1507. The addition of either MK-2206 (Akt inhibitor) or PHA665752 (c-Met tyrosine kinase inhibitor) to R1507 does not restore the sensitivity to IGF-1R inhibition in H69 cells. The addition of U0126 (MEK inhibitor) to R1507 does not restore the sensitivity to IGF-1R inhibition in H146 cells. LT- vs. ST-R1507 analyses reveal evidence of adaptive mechanisms susceptible to promote tumor escape to R1507 as single agent: (i) activation of cell-cell adhesion, beta-arrestin and MAPK gene expression pathways, (ii) activation of multiple cell surface receptors and intracellular proteins implicated in proliferation, angiogenesis and survival, (iii) transient reduction of phosphorylated IGF-1R staining intensity, in H526 xenografts in vivo. Regarding combination regimens, we identified: (B) the apoptotic mitochondrial and the NER pathways as majorly sensitizing the effects of LT- R1507 to Cisplatin, and (C) the TGF-beta pathway as the key mediator of the sensitization of LT-R1507 to IR. Conclusion: Combined genomic and functional approaches performed under LT-R1507 exposure unravel the activation of multiple alternative signaling pathways that are expected to promote tumor escape. Our study also provides key biological basis for translating the R1507 combined with Cisplatin and IR in the SCLC clinical setting. To analyze both short- and long-term drug exposure appears critical when evaluating new antitumoral regimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2866. doi:1538-7445.AM2012-2866

Abstract 91: The CUL4A ubiquitin ligase, a putative target gene at the 13q34 amplicon and its role in breast cancer pathogenesis &amp; progression

Abstract Breast cancer is a complex and heterogeneous disease and one of the most frequent causes of cancer deaths in developed countries. DNA copy number alterations occur frequently in breast tumours. Regions of DNA amplification are of major interest since often contain oncogenes whose increased expression confers tumour cells a selective advantage and contributes to the carcinogenesis process. In our laboratory prior analysis of familiar and sporadic breast tumours by using array-CGH identified an amplification event at the 13q34 region with an overall frequency of 4.5% that increased to 8.1% in BRCA1-associated tumours. Comprehensive characterization of the amplicon allowed us to define CUL4A as one of the most likely putative oncogenes. CUL4A is an E3 Ubiquitin Ligase which is amplified and/or overexpressed in primary breast tumours and involved in functions such as chromatin regulation, cell cycle regulation or DNA repair through ubiquitylation of NER pathway members. Its deregulation could have implications for oncogenic transformation and treatment response to DNA-damaging agents. One of such compounds is Trabectedin a marine-derived drug that presents antitumor activity in sarcomas and ovarian cancer. To elucidate the possible implication of CUL4A in the initial steps of the tumorigenic process we conducted the stable up-regulation of CUL4A in human mammary epithelial cells (184B5), and in NIH3T3 mouse-derived cell line by lentiviral infection. We also stably silenced CUL4A in the human breast cancer cell lines MDA-MB-157 and HCC1937 that present amplification and/or overexpression of CUL4A. With these modified cell lines we performed functional assays in different in vitro systems such as viability experiments, colony formation both in plastic and in soft agar and 3-D cultures in Matrigel. In order to measure sensitivity to Trabectedin we used silenced CUL4A breast cancer cell lines to perform clonogenic assays. Our preliminary results in CUL4A overexpression in 184B5 model system showed increase in proliferation and colony formation ability. On the other hand, CUL4A silencing in breast cancer cell lines resulted in diminished proliferation and decreased colony-forming abilities in both anchorage dependent and independent conditions. Concerning DNA-repair implication, it seems that high levels of CUL4A could be a good predictor of Trabectedin response. Our results indicate that CUL4A would play a role in promoting oncogenesis and/or in modulating breast cancer progression. Further evaluation of CUL4A amplification/overexpression impact on the maintenance of genome integrity will help elucidate its full potential for therapeutic intervention in breast and other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 91. doi:1538-7445.AM2012-91

Differences in nucleotide excision repair capacity between newly diagnosed colorectal cancer patients and healthy controls

Alteration of DNA integrity is a potential cause of cancer and it is assumed that reduced DNA repair capacity and accumulation of DNA damage may represent intermediate markers in carcinogenesis. In this case-control study, DNA damage and nucleotide excision repair capacity (NER-DRC) were assessed in association with sporadic colorectal cancer (CRC). Both parameters were quantified by comet assay in blood cells of 70 untreated incident patients and 70 age-matched healthy controls. mRNA expression and polymorphisms in relevant NER genes were concurrently analyzed. The aim of this study was to characterize incident CRC patients for NER-DRC and to clarify possible relations between investigated variables. Comet assay and mRNA expression analysis showed that CRC patients differ in repair capacity as compared to controls. Patients had a lower NER-DRC and simultaneously they exhibited higher endogenous DNA damage (for both P < 0.001). Accumulation of DNA damage and decreasing NER-DRC behaved as independent modulating parameters strongly associated with CRC. Expression levels of 6 out of 9 studied genes differed between groups (P ≤ 0.001), but none of them was related to DRC or to any of the studied NER polymorphisms. However, in patients only, XPC Ala499Val modulated expression levels of XPC, XPB and XPD gene, whereas XPC Lys939Gln was associated with XPA expression level in controls (for all P < 0.05). This study provides evidence on altered DRC and DNA damage levels in sporadic CRC and proposes the relevance of the NER pathway in this malignancy. Further, alterations in a complex multigene process like DNA repair may be better characterized by functional quantification of repair capacity than by quantification of individual genes transcripts or gene variants alone.

Titanium dioxide nanoparticles exhibit genotoxicity and impair DNA repair activity in A549 cells

Titanium dioxide nanoparticles (TiO2-NPs) are produced in large quantities, raising concerns about their impact for human health. The aim of this study was to deeply characterize TiO2-NPs genotoxic potential to lung cells, and to link genotoxicity to physicochemical characteristics, e.g., size, specific surface area, crystalline phase. A549 cells were exposed to a panel of TiO2-NPs with diameters ranging from 12 to 140 nm, either anatase or rutile. A set of complementary techniques (comet and micronucleus assays, gamma-H2AX immunostaining, 8-oxoGuanine analysis, H2-DCFDA, glutathione content, antioxidant enzymes activities) allowed us to demonstrate that small and spherical TiO2-NPs, both anatase and rutile, induce single-strand breaks and oxidative lesions to DNA, together with a general oxidative stress. Additionally we show that these NPs impair cell ability to repair DNA, by inactivation of both NER and BER pathways. This study thus confirms the genotoxic potential of TiO2-NPs, which may preclude their mutagenicity and carcinogenicity.

Associated risk of XRCC1 and XPD cross talk and life style factors in progression of head and neck cancer in north Indian population

Effective DNA repair machinery ensures maintenance of genomic integrity. Environmental insults, ageing and replication errors necessitate the need for proper DNA repair systems. Any alteration in DNA repair efficacy would play a dominant role in progression of squamous cell carcinoma of head and neck (SCCHN).Genotypes of XRCC1 gene–Arg194Trp, Arg280His, Arg399Gln and XPD Lys751Gln, by PCR-RFLP were studied in 278 SCCHN patients and an equal number of matched healthy controls residing in north India.In XRCC1 polymorphisms, Arg194Trp and Arg399Gln variants showed a reduced risk, whereas, XPD Lys751Gln variants exhibited ∼2-fold increase in SCCHN risk. With XRCC1-Arg280His variants, there was no association with SCCHN risk. Arg399Gln of XRCC1 appears to have a protective role in people those consume alcohol, while XPD Lys751Gln variants indicated ∼2-fold increased risk of SCCHN in all the co-variate groups.Comparison of gene–gene interaction among XRCC1 Arg280His and XPD Lys751Gln suggested enhanced risk of SCCHN by ∼2.3-fold in group one and ∼6.1-fold in group two. In dichotomized groups of this combination, the risk was ∼2.4 times. Haplotype analysis revealed the frequency of C–G–G–G and C–A–G–G to be significantly associated with an increased risk of SCCHN. On the contrary, T–G–A–A significantly diminished the risk. CART analysis results showed that the terminal node that contains homozygous mutants of XPD Lys751Gln and XRCC1 Arg194Trp, wild type of XRCC1 Arg399Gln and homozygous mutant of XRCC1 Arg280His, represent the highest risk group.Our results demonstrate high degree of gene–gene interaction involving DNA repair genes of NER and BER pathways, namely XRCC1 and XPD. This study amply demonstrates positive association of XPD Arg751Gln polymorphism with an increased risk of SCCHN. Further, XRCC1 Arg280His variant though dormant individually, may also contribute to the development of cancer in combination with XPD Arg751Gln.

Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer

Tobacco smoking is one of the major risk factors in pathogenesis of head and neck squamous cell carcinomas (HNSCC). Many of the chemical compounds present in tobacco are well-known carcinogens which form adducts with DNA. Cells remove these adducts mainly by the nucleotide excision repair pathway (NER). NER also eliminates a broad spectrum of pyrimidine dimers (CPD) and photo-products (6-4PP) induced by UV-radiation or DNA cross-links after cisplatin anti-cancer treatment. In this study DNA damage and repair was examined in peripheral blood lymphocytes obtained from 20 HNSCC patients and 20 healthy controls as well as HTB-43 larynx and SSC-25 tongue cancer cell lines. DNA repair kinetics in the examined cells after cisplatin or UV-radiation treatment were investigated using alkaline comet assay during 240 min of post-treatment incubation. MTT assay was used to analyse cell viability and the Annexin V-FITC kit specific for kinase-3 was employed to determine apoptosis after treating the cells with UV-radiation at dose range from 0.5 to 60 J/m 2. NER capability was assessed in vitro with cell extracts by the use of a bacterial plasmid irradiated with UV-light as a substrate for the repair. The results show that lymphocytes from HNSCC patients and HTB-43 or SSC-25 cancer cells were more sensitive to genotoxic treatment with UV-radiation and displayed impaired DNA repair. Also evidenced was a higher rate of apoptosis induction after UV-radiation treatment of lymphocytes from the HNSCC patients and the HTB-43 cancer cells than after treatment of those from healthy donors. Finally, our results showed that there was a significant decrease in NER capacity in HTB-43 or SSC-25 cancer cells as well as in peripheral blood lymphocytes of HNSCC patients compared to controls. In conclusion, we suggest that the impaired NER pathway might be a critical factor in pathogenesis of head and neck cancer.

TiO2 nanoparticles exhibit genotoxicity and impair both NER and BER DNA repair pathways in A549 cells

TiO2 nanoparticles exhibit genotoxicity and impair both NER and BER DNA repair pathways in A549 cells