High DNA Repair Capacity Research Articles

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long-lived and cancer-resistant subterranean wild rodent, Spalax.

The blind mole rat (Spalax) is a wild, long‐lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence‐associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide‐induced senescence (EIS), evidenced by an increased activity of senescence‐associated beta‐galactosidase (SA‐β‐Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well‐known SASP factors: interleukin‐6 (IL6), IL8, IL1α, growth‐related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM‐1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro‐inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA‐MB‐231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF‐κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory‐related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age‐related chronic inflammation supporting healthy aging and longevity.

MicroRNA Expression Changes in Women with Breast Cancer Stratified by DNA Repair Capacity Levels.

Breast cancer (BC) is the most commonly diagnosed cancer in women worldwide and is the leading cause of death among Hispanic women. Previous studies have shown that women with a low DNA repair capacity (DRC), measured through the nucleotide excision repair (NER) pathway, have an increased BC risk. Moreover, we previously reported an association between DRC levels and the expression of the microRNA (miRNA) let-7b in BC patients. MiRNAs can induce genomic instability by affecting the cell's DNA damage response while influencing the cancer pathobiology. The aim of this pilot study is to identify plasma miRNAs related to variations in DRC levels in BC cases. Hypothesis. Our hypothesis consists in testing whether DRC levels can be correlated with miRNA expression levels. Methods. Plasma samples were selected from 56 (27 cases and 29 controls) women recruited as part of our BC cohort. DRC values were measured in lymphocytes using the host-cell reactivation assay. The samples were divided into two categories: low (≤3.8%) and high (>3.8%) DRC levels. MiRNAs were extracted to perform an expression profile analysis. Results. Forty miRNAs were identified to be BC-related (p<0.05, MW), while 18 miRNAs were found to be differentially expressed among BC cases and controls with high and low DRC levels (p<0.05, KW). Among these candidates are miR-299-5p, miR-29b-3p, miR-302c-3p, miR-373-3p, miR-636, miR-331-5p, and miR-597-5p. Correlation analyses revealed that 4 miRNAs were negatively correlated within BC cases with low DRC (p<0.05, Spearman's correlation). Results from multivariate analyses revealed that the clinicopathological characteristics may not have a direct effect on specific miRNA expression. Conclusion. This pilot study provides evidence of four miRNAs that are negatively regulated in BC cases with low DRC levels. Additional studies are needed in order to have a complete framework regarding the overall DRC levels, miRNA expression profiles, and tumor characteristics.

Association between polymorphisms of Xeroderma pigmentosum complementation group C gene (XPC) and susceptibility to schizophrenia

Previous studies revealed that polymorphisms in several DNA repair genes are associated with the risk of schizophrenia. The relationship between three polymorphisms (Ala499Val, PAT, and Lys939Gln) of the XPC (MIM: 613208) and the risk of schizophrenia is investigated. A total of 361 schizophrenia cases and 360 healthy controls were included in the study. Statistical analysis revealed that the Ala/Val genotype (OR = 0.62, P = 0.004) and the carriers of the Val allele (OR = 0.64, P = 0.006) were negatively associated with the risk of schizophrenia. The other two examined polymorphisms did not reveal significant association. The “Val - Lys” haplotype was associated with decrease risk of schizophrenia (OR = 0.71, P = 0.020). It has been showed that the haplotype “Val - Lys” had the lowest DNA damages, indicating that has higher DNA repair capacity. Here we found that this haplotype is associated with lower risk for schizophrenia.

Abstract 3382: Methylation status and microRNA expression changes in women with breast cancer stratifying by DNA repair capacity

Abstract The American Cancer Society estimates 252,710 new breast cancer (BC) cases and 40,610 cancer related-deaths in the US, only in 2017. In addition to the well-established BC risk factors, our studies during the last decade have shown that women having a low DNA repair capacity (DRC) as measured through the nucleotide excision repair pathway is associated with higher BC risk. This finding allows us to study BC etiology based on DRC rather than using the ordinary stratification; for example, we found an association between DRC levels and estrogen receptor status. Since DRC has been associated with different clinical aspects of BC and it is known that it can be regulated through epigenetic mechanisms, more studies regarding the effect of epigenetic changes on DRC in BC are needed. Therefore, the main objective of this study is to identify epigenetic mechanisms that could be influencing DRC levels in women with BC through gene silencing. The main hypothesis consists in testing whether the epigenetic changes such as methylation and microRNA (miRNA) levels can be used to stratify BC patients by DRC levels. Plasma and tumor samples were randomly selected from our BC cohort. DRC values were previously measured in lymphocytes using the host cell reactivation assay. BC cases and controls were divided in two categories: low (≤3.8%) and high (&amp;gt;3.8%) DRC. MicroRNAs were extracted from 56 plasma samples (27 BC cases and 29 controls) to perform an expression profile analysis using the Human MicroRNA A Cards v 2.0 assay. Relative miRNA expression was calculated using the 2-ΔCt method. Methylation status of tumors was obtained through an epigenome-wide DNA methylation analysis stratifying by DRC using the Illumina Human Methylation EPIC 850K array. After unbiased selection and pathologist evaluation, 56 formalin-fixed paraffin-embedded tumors were selected and sectioned for DNA extraction. Based on the DRC, the samples were divided in low (n=24) and high (n=32) using the established cut-off. A significant association between miR-323-3p, miR-29b, and miR-159a expression and the BC outcome was found (p&amp;lt;0.01). Using the DRC categories, all three miRs were found to be differentially expressed when comparing cases and controls with high and low DRC (p&amp;lt;0.05, KW). The methylation data was analyzed using the bump hunting method. The epigenome-wide analysis of the BC methylome revealed 23 regions that were significantly differentially methylated between tumors with low and high DRC (p&amp;lt;0.05). Although DRC levels are low when the BC malignancy is developed, whether these changes occur during carcinogenesis or due to a preexisting epigenetic disposition is still unknown. However, our results lead us to conclude that epigenetic changes such as methylation and miRNA expression can influence the DRC levels in BC. This study was supported by: S06GM008239-20, 9SC1CA182846-04, U54CA163068, 2U54CA163071-06, and R003050/MD007579. Citation Format: Carmen M. Ortiz-Sanchez, Jarline Encarnacion, Rafael Guerrero, Jaime Matta. Methylation status and microRNA expression changes in women with breast cancer stratifying by DNA repair capacity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3382.

Cooperative effect of chidamide and chemotherapeutic drugs induce apoptosis by DNA damage accumulation and repair defects in acute myeloid leukemia stem and progenitor cells

BackgroundMany conventional chemotherapeutic drugs are known to be involved in DNA damage, thus ultimately leading to apoptosis of leukemic cells. However, they fail to completely eliminate leukemia stem cells (LSCs) due to their higher DNA repair capacity of cancer stem cells than that of bulk cancer cells, which becomes the root of drug resistance and leukemia recurrence. A new strategy to eliminate LSCs in acute myeloid leukemia (AML) is therefore urgently needed.ResultsWe report that a low-dose chidamide, a novel orally active benzamide-type histone deacetylase (HDAC) inhibitor, which selectively targets HDACs 1, 2, 3, and 10, could enhance the cytotoxicity of DNA-damaging agents (daunorubicin, idarubicin, and cytarabine) in CD34+CD38− KG1α cells, CD34+CD38− Kasumi cells, and primary refractory or relapsed AML CD34+ cells, reflected by the inhibition of cell proliferation, induction of apoptosis, and increase of cell cycle arrest in vitro. Mechanistically, these events were associated with DNA damage accumulation and repair defects. Co-treatment with chidamide and the DNA-damaging agent IDA gave rise to the production of γH2A.X and inhibited posttranslationally but not transcriptionally the repair gene of ATM, BRCA1, and checkpoint kinase 1 (CHK1) and 2 (CHK2) phosphorylation. Finally, the combination of chidamide and IDA initiated caspase-3 and PARP cleavage, but not caspase-8 and caspase-9, and ultimately induced CD34+CD38− KG1α cell apoptosis. Further analysis of AML patients’ clinical characteristics revealed that the ex vivo efficacy of chidamide in combination with IDA in primary CD34+ samples was significantly correlated to peripheral blood WBC counts at diagnosis, while LDH levels and karyotype status had no effect, indicating that the combination regimen of chidamide and IDA could rapidly diminish tumor burden in patients with R/R AML.ConclusionsThese findings provide preclinical evidence for low-dose chidamide in combination with chemotherapeutic agents in treating recurrent/resistant AML as an alternative salvage regimen, especially those possessing stem and progenitor cells.

Abstract 2049: The role of epigenetic mark profile, cell cycle alteration and DNA repair in resistance of glioblastoma cells to photodynamic therapy

Abstract Glioblastoma is the most aggressive primary brain tumor. Treatment regimens for glioblastoma tumors, such as surgery, radiotherapy, and chemotherapy, are very invasive and can only prolong the median patient life to several months. It was shown that 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) is a promising and less aggressive adjuvant modality for diagnosis and treatment of glioblastoma. Limiting point of PDT outcome is appearing of cells with intrinsic or acquired PDT resistance that finally results in repopulating the tumor and short-term survival of glioma patients. Therefore, our study was designed to determine therapy resistance markers in PDT resistant glioblastoma cell line. Potential markers subsequently can be used for sensitization of glioblastoma to PDT. Glioblastoma (U-87) cell line resistant to PDT (U-87R) was isolated from parental, sensitive line (U-87P) by applying several cycles of ALA-PDT, and further growing of surviving cells. U-87R cells subsequently were characterized from different aspects such as epigenetic markers, cell cycle events, oxidative stress and DNA repair capacity. Assessment of nucleotide modifications and epigenetic marks in PDT resistant glioblastoma and its parental cell line showed significant higher level of two epigenetic marks including 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in U-87R compare to U-87P, before and after PDT. Doubling time of U-87R cells was considerably longer than that of U-87P. Cell cycle analysis showed longer G1 phase in U-87R cells compare to U-87P. Moreover, accumulation in G2 phase following PDT was observed earlier in resistant cells than in parental line. That suggests the role of early activation of DNA damage response in resistant cells. Resistant glioblastoma cells were then sensitized to PDT by applying inhibitor of one of the main kinases of DNA damage response, ATM kinase. In comparison with parental cells, U-87R cells also showed higher activity of some DNA base excision repair (BER) enzymes including glycosylases and AP-endonuclease1 (APE1). Studies of the whole protein profile of PDT resistant and parental cells demonstrated that the level of superoxide dismutase (SOD) was considerably higher in U-87R than in U-87P. That data was then confirmed by detecting lower oxidative stress in U-87R following PDT. PDT resistant glioblastoma cells as a model of resistant cells in glioblastoma tumor population demonstrated the significant role of epigenetic mark profile, cell cycle alteration, higher DNA repair capacity, and antioxidant defense in conferring resistance to photodynamic therapy. This work was supported by the Polish National Science Centre (NCN) grants: DEC-2012/07/B/NZ1/00008, UMO-2014/15/B/NZ5/01444, UMO-2014/13/N/NZ3/00863 Note: This abstract was not presented at the meeting. Citation Format: Somayeh Shahmoradi Ghahe, Karolina Kopania, Agata Ciuba, Marek Foksinski, Ryszard Oliński, Barbara Tudek. The role of epigenetic mark profile, cell cycle alteration and DNA repair in resistance of glioblastoma cells to photodynamic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2049. doi:10.1158/1538-7445.AM2017-2049

Abstract 1424: DNA repair capacity in colon cancer patients - The effect on the response to treatment and long-term survival

Abstract Colorectal carcinoma (CRC) is the third most common cancer worldwide with the highest incidence in Central Europe. It is the fourth leading cause of cancer related deaths mainly due to the late diagnosis and low efficacy of treatment. CRC diagnosed in early stage has a five-year survival rate about 90% which drops to near 12% once distant metastases occur. It is a heterogenous disease with different molecular and clinicopathological features depending on the tumor location. Therefore, different treatment strategies are required. A standard treatment of locally advanced rectal cancer includes neoadjuvant chemoradiotherapy followed by surgery, whereas colon cancer treatment consists of surgical resection of the tumor and/or subsequent adjuvant chemotherapy based on disease characteristics. 5-fluorouracil (5-FU) alone or in combination with other compounds is the most used treatment in CRC. The mechanism of 5-FU on molecular level is either its incorporation into DNA or it imbalances the synthesis of thymidine from uracil resulting in false uracil DNA incorporation. These DNA lesions are repaired by base excision (BER) and mismatch repair (MMR) pathways. An effective DNA damage response (DDR) is essential for the maintenance of genome stability in healthy cells, whereas in malignant cells, the suppression of DNA repair capacity (DRC) would increase the effectiveness of chemotherapy through DNA damage accumulation and consequent apoptosis. In contrary the cells with high DRC may show better survival and therefore patients with these molecular characteristics may contend with poor response, resistance to treatment and decreased survival. The aim of our present study was to investigate DRC of BER and MMR in target tissue as a predictive marker for a treatment strategy and long-term survival. In order to minimize a bias by heterogenous therapy we focused only on patients with newly diagnosed colon cancer. Our set of patients was selected based on the criteria of follow-up at minimum 30 months, subsequent treatment with 5-FU and microsatellite stable tumor tissue characteristics. Tumor and adjacent non-affected tissue samples were obtained from one hundred patients at surgical resection. Protein extracts from tissues were isolated both for protein expression analysis and for measurement of DRC. DNA repair and DDR protein expression levels were tested by Western Blot. Functional assessments of DRC were performed by comet assay-based in vitro DNA repair assay. The analysis is running and the data will be statistically analyzed and compared with clinical data (TNM stage, type and course of treatment, presence of recurrence, patient´s performance, etc.). Understanding DRC effect on the treatment response might contribute to the concept assuming that targeted modulating of DNA repair processes can achieve clinical benefit in cancer treatment. Acknowledgements: GA UK 800216, COST LD14050, GACR P303/15/14789S and AZV 15-27580A. Citation Format: Sona Vodenkova, Michal Kroupa, Katerina Jiraskova, Alessio Naccarati, Alena Opattova, Pavel Vodicka. DNA repair capacity in colon cancer patients - The effect on the response to treatment and long-term survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1424. doi:10.1158/1538-7445.AM2017-1424

Abstract 1899: Let-7b overexpression is associated with higher nucleotide excision repair pathway in women with breast cancer

Abstract Nucleotide Excision Repair (NER) is a critical pathway involved in breast cancer (BC). We have previously published that a low DNA repair capacity (DRC) is associated with a higher risk of BC in Puerto Rican women. In recent years we have focused our investigations on microRNAs (miRNAs) that are differentially associated with a low and a high DRC in women with BC and controls. A discovery experiment with 29 BC cases and 27 controls produced 12 candidate miRNAs associated with DRC including let-7b. The main objective of this study was to elucidate if there is a correlation between let-7b expression and specific DRC levels. Let-7b belongs to a miRNA family with tumor suppressor activity that targets oncogenic genes such as Ras, Myc, and HmgA2. DRC was measured in lymphocytes by means of a host cell reactivation assay with a luciferase reporter gene. We isolated miRNAs from plasma of 145 Puerto Rican women with and without BC (recently diagnosed, untreated cases and controls) using the miRNeasy kit (Qiagen). These women were divided into four groups according to their DRC level: high (&amp;gt;3.8%) and low (&amp;lt;3.8%). The four groups consisted in BC cases with high (n = 32) and low (n = 41) DRC, and controls with high (n = 38) and low (n = 34) DRC. Let-7b expression was measured using TaqMan microRNA assay. In addition, epidemiological data of these women has been collected at their initial BC diagnosis (before treatment) and almost five years after diagnosis. Within the BC group with low DRC, 9 women had presented recurrence. We found no significant difference in let-7b expression between controls without BC when compared to women with BC with both high and low DRC levels. A significant difference in let-7b expression was found in BC cases with high DRC when compared with the remaining groups (p&amp;lt;0.001). The down-regulation of let-7b family miRNAs is associated with poor prognosis in various types of cancers, including breast, lung, and ovarian, among others. Thus, our data reveal a possible role of let-7b involving DRC intrinsically when the malignancy is developed. Our findings support the association previously reported between a poor prognosis in several cancers and low levels of let-7b expression. Our study is innovative because it provides the first evidence that let-7b might play role in DRC (through the NER pathway) regulation in BC. Also, our study suggests that the overexpression of let7-b in women with BC and a high DRC is associated with a better prognosis. Supported by grants from the NCI Center to Reduce Health Disparities and NIH-NIGMS MBRS Program grants #S06 GM008239-20, 9SC1CA182846-04, GM082406, and PSM-MCC Partnership grant #5U54CA163071-04. Citation Format: Jarline Encarnacion, Carmen Ortiz, Ralphdy Vergne, Wanda Vargas, Jaime L. Matta. Let-7b overexpression is associated with higher nucleotide excision repair pathway in women with breast cancer. [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 1899.

ABT-888 and quinacrine induced apoptosis in metastatic breast cancer stem cells by inhibiting base excision repair via adenomatous polyposis coli

PARP inhibitors in combination with other agents are in clinical trial against cancer, but its effect on cancer stem cells (CSCs) is limited. CSCs are responsible for drug resistance, metastasis and cancer relapse due to high DNA repair capacity. Here, we present preclinical effects of Quinacrine (QC) with ABT-888, a PARP inhibitor, on highly metastatic breast cancer stem cells (mBCSCs). An increased level of Adenomatous polyposis coli (APC) was noted after treatment with ABT-888 in QC pre-treated mBCSCs cells. Increased APC physically interacts with PARP-1 and inhibits PARylation causing the non assembly of base excision repair (BER) multiprotein complex, resulting in an irreparable DNA damage and subsequent apoptosis. Knockdown of APC in mBCSCs inhibited DNA damage, increased BER and PARylation, reduces apoptosis while the over-expression of APC in BT20 (APC low expressing) cells reversed the effect. Thus, combination of QC and ABT-888 decreased mBCSCs growth by activating APC and inhibiting BER within the cells.

High DRC Levels Are Associated with Let-7b Overexpression in Women with Breast Cancer.

Nucleotide Excision Repair (NER) is a critical pathway involved in breast cancer (BC). We have previously published that a low DNA repair capacity (DRC) is associated with a higher risk of BC in Puerto Rican women. Let-7b belongs to a miRNA family with tumor suppressor activity that targets oncogenes. We isolated miRNAs from plasma of 153 Puerto Rican women with and without BC. DRC was measured in lymphocytes by means of a host cell reactivation assay. These women were divided into four groups according to their DRC level: High (>3.8%) and low (<3.8%). The four groups consisted of BC patients with high (n = 35) and low (n = 43) DRC and controls with high (n = 39) and low (n = 36) DRC. Epidemiologic data were collected at initial BC diagnosis and almost five years after diagnosis. A significant difference in Let-7b expression was found in BC patients with high DRC versus the remaining groups (p < 0.001). Thus, our data reveal a possible role of Let-7b on DRC during breast carcinogenesis. Our study is innovative because it provides the first evidence that Let-7b may play role in DRC regulation (through the NER repair pathway) in BC.

The ERCC2/XPD Lys751Gln polymorphism affects DNA repair of benzo[a]pyrene induced damage, tested in an in vitro model

Nucleotide excision repair (NER) is an important defense mechanism of the body to exogenous carcinogens and mutagens, such as benzo[a]pyrene (B[a]P). Genetic polymorphisms in ERCC2/XPD, a critical element in NER, are thought to be associated with individual's cancer susceptibility. Although ERCC2/XPD Lys751Gln (rs13181) is the most studied polymorphism, the impact of this polymorphism on DNA repair capacity to carcinogen remains unclear. In the present study, cDNA clones carrying different genotypes of ERCC2/XPD (Lys751Gln) were introduced into an ERCC2/XPD deficient cell line (UV5) in a well-controlled biological system. After B[a]P treatment, cell growth inhibition rates and DNA damage levels in all cells were detected respectively. As expected, we found that the DNA repair capacity in UV5 cells was restored to levels similar to wildtype parent AA8 cells upon introduction of the cDNA clone of ERCC2/XPD (Lys751). Interestingly, after B[a]P treatment, transfected cells expressing variant ERCC2/XPD (751Gln) showed an enhanced cellular sensitivity and a diminished DNA repair capacity. The wildtype genotype AA (Lys) was found to be associated with a higher DNA repair capacity as compared to its polymorphic genotype CC (Gln). These data indicate that ERCC2/XPD Lys751Gln polymorphism affects DNA repair capacity after exposure to environmental carcinogens such as B[a]P in this well-controlled in vitro system and could act as a biomarker to increase the predictive value to develop cancer.

Abstract 3334: Microarray analysis of radioresistant mouse squamous cell carcinoma: comparison of x-ray resistance and carbon-ion beam resistance

Abstract Radiotherapy is an effective locoregional control modality for many types of tumors. However, sometimes treatment failures occur due to radioresistance of cancer cells. To reduce treatment failure, elucidating the epigenetic mechanism of radioresistance is very important. Nucleotides associated with radioresistance could be promising treatment targets or prognostic markers. The cancer stem cells (CSCs) theory helps us to understand the resistance mechanism. CSCs are small population of cells in cancer with stem cell properties such as self-renewal, cell proliferation and multiple-differentiation. CSCs are very important clinical target, because they induce tumor metastasis and recurrence. Targeting CSCs would be promising therapy to reduce treatment failure. X-ray induces DNA damages mostly through reactive oxygen species (ROS). Since CSCs have the low levels of ROS and high DNA repair capacity, and exist under hypoxia condition, CSCs are resistant to X-ray radiotherapy. Therefore, it is difficult to eliminate CSCs with X-ray. Carbon ion beam is known to be effective to cancer cells in hypoxic condition, since it is high linear energy transfer (LET) beam inducing direct damage to DNA rather than mediating ROS. Therefore, carbon ion beam radiotherapy is more effective than x-ray radiotherapy against CSCs. However, even with carbon ion beam, there are treatment failures, indicating that there could be some protection mechanism other than ROS, associated in carbon ion beam resistance. Our purpose is to elucidate the mechanism of radioresistance and to identify the difference between X-ray resistance and carbon-ion beam resistance by nucleotides profiling analysis. We analyzed X-ray resistant (total 60Gy irradiated, 6fraction) and carbon ion beam resistant (total 30Gy irradiated, 6fraction) mouse squamous cell line NR-S1 by micro array. Total 23474 cDNA, 1265 micro RNA (miR) data was obtained and each cell lines expression pattern was totally different. We listed up all the cDNA and miR which specifically expressed in X-ray resistant cells (64 cDNA, 20 miR) and carbon ion beam resistant cells (34 cDNA, 16 miR). This data would be a basis in elucidating the mechanism of radioresistance, and our next step is to identify which cDNA or miR is related with the resistance. Note: This abstract was not presented at the meeting. Citation Format: Sungjae Baek, Hideshi Ishii, Katsutoshi Sato, Naohiro Nishida, Keisuke Tamari, Kazuhiko Hayashi, Yuji Seo, Koichi Kawamoto, Jun Koseki, Masamitsu Konno, Yuichiro Doki, Masaki Mori, Kazuhiko Ogawa. Microarray analysis of radioresistant mouse squamous cell carcinoma: comparison of x-ray resistance and carbon-ion beam resistance. [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 3334. doi:10.1158/1538-7445.AM2015-3334

Abstract 2394: The role of DNA repair in breast cancer risk and recurrence

Abstract BACKGROUND: Breast cancer (BC) is a complex disease that is caused by multiple factors. Deficits in DNA repair capacity (DRC) are known to cause certain familial BC, and dysregulation of DRC develops with progressive carcinogenesis. However, the effect of DRC on carcinogenesis of sporadic breast tumors has not been well characterized_and the factors associated with DRC variability are still poorly understood. DNA repair is integral to maintain genomic integrity, and carcinogenesis occurs when efficient, effective DNA repair is impaired. Our laboratory has just finished the recruitment of nearly 1,200 women in a seven-year molecular epidemiology study focused on the role of DRC as a risk factor for BC. OBJECTIVE: Our aim is to present key findings on the role of DRC as a risk factor for BC and its potential association with BC recurrence. METHODS: A personal interview was conducted for soliciting information on BC risk factors. Among the 493 BC cases and 683 controls the DRC was measured in lymphocytes using a host cell reactivation assay with a luciferase reporter gene. We stratified the women with BC using the median of the DRC level and selected 70 women with a low DRC level (&amp;lt;2.8%) who had been enrolled in the study for at least 5 years in order to assess the BC recurrence. RESULTS: Our 7 year study of DRC of female cohorts in Puerto Rico has shown that women with BC have a DRC that, on average, is 51% lower than women without BC (P &amp;lt; 0.001, Mann Whitney U-test). Women with low DRC have 6.8 (95%CI 5.2, 9.0) times the odds for BC when compared with women with high DRC level. So far, the overall BC recurrence was 17% and the 5-year mortality was 10%. Recurrence was primarily in the lung, liver, bone, or occurred as generalized metastatic disease. CONCLUSIONS: Using a large sample size (n=1,183 participants), we show that women with BC had on an average a 51% lower DRC than controls (P&amp;lt;0.001). We also present the first data on the potential between a low DRC and risk of BC recurrence. With a larger sample size, we will test the hypothesis that low DRC can predict risk of BC recurrence. Supported by grants from the NCI Center to Reduce Health Disparities through the NIH-MBRS Program grants S06 GM008239-20, 9SC1CA182846-04 to PSMHS through JM and MBRS-RISE GM082406 through CO. Citation Format: Jaime L. Matta, Erick Suarez, Wanda Vargas, Carmen Ortiz, Manuel Bayona, Luisa Morales. The role of DNA repair in breast cancer risk and recurrence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2394. doi:10.1158/1538-7445.AM2014-2394

Polymorphisms in the DNA repair gene ERCC2/XPD and breast cancer risk: a HapMap-based case-control study among Han Women in a Chinese less-developed area.

Genetic variations in DNA repair genes may impact repair functions, DNA damage, and breast cancer risk. This study is aimed to assess the associations of genetic polymorphisms in excision repair cross-complementing group 2 (ERCC2) with the risk of developing breast cancer. In total, 101 histopathologically confirmed breast cancer cases and 101 age/region-matched healthy controls were genotyped for rs 3916840, rs 1799793, and rs 238416 in ERCC2 by polymerase chain reaction-restriction fragment length polymorphism. The rs 238416 heterozygous GA genotype combined with the rs 238416 genotypes (GA+AA) showed a significant association with breast cancer susceptibility (corrected p<0.01, odds ratio [OR]=0.29, 95% confidence interval [CI]=0.15-0.54; corrected p<0.01, OR=0.31, 95% CI=0.17-0.56, respectively). The rs 238416 GA genotype carriers had a decreased risk of breast cancer. However, we observed no significant association between the rs 3916840 and rs 1799793 polymorphisms in ERCC2 and breast cancer risk. Moreover, haplotype analysis showed that the ACG haplotype was associated with a significantly decreased risk of breast cancer, whereas the GCG haplotype was associated with a significantly increased risk of breast cancer (corrected p=0.004 and p=0.002, respectively). Multifactor dimensionality reduction analysis demonstrated that the interactions between rs 3916840 and rs 238416 were significantly synergistic. To the best of our knowledge, this study is the first to demonstrate that the rs 238416 heterozygous genotype likely has a higher DNA repair capacity and, thus, can be protective against breast cancer in Chinese Han women.

Women with endometriosis have a higher DNA repair capacity and diminished breast cancer risk.

Breast cancer (BC) and endometriosis are important reproductive health diseases for women. Although endometriosis is not a malignant condition, some of its characteristics mimic that of a malignancy. Endometriosis is associated with increased risk of certain cancers; however, whether it alters BC risk is unclear. This study evaluates the association of endometriosis and BC and explores whether DNA repair capacity (DRC) plays a role in such a relationship. A case-control study of 991 women (385 with BC and 606 controls, all recruited over 5 years) was undertaken in Puerto Rico. Eighty participants with self-reported surgically diagnosed endometriosis were identified, 20 of whom also had a diagnosis of BC. Data from a structured questionnaire and DRC measurements were assessed to determine the association between BC, DRC, and endometriosis. Participants with BC cases were 50% less likely to have history of endometriosis (OR = 0.5 95%CI: 0.3, 0.9, p = 0.038) than women without BC controls. Findings that did not reach statistical significance included the following: women with history of endometriosis had a slightly higher DRC level than those without it; BC cases and history of endometriosis were less likely to have had endometriosis diagnosis before age 38 as compared to controls with endometriosis. Here we report an inverse association between endometriosis and BC, the former possibly conferring a protective effect on the latter. Although the mechanisms involved are unknown they may include protection provided by higher DRC and or hormonal treatments for endometriosis. A larger sample of endometriosis cases is necessary to confirm these results and answer the question of whether a higher DRC capacity may contribute to this potential protection, and to identify other factors at play.

Abstract B48: Targeting chemo-resistance in CCNE1-amplified ovarian cancer

Abstract The putative oncogene CCNE1 (cyclin E1) is frequently amplified in human high-grade serous ovarian cancer. CCNE1-amplified cancers tend to be wildtype for BRCA1 and BRCA2 and are associated with shorter survival and resistance to platinum-based chemotherapy. Cyclin E1 is an activating cofactor for cyclin-dependent kinases (CDK), which stimulate cell cycle progression through phosphorylation of the retinoblastoma (RB) protein and subsequent induction of E2F transcriptional activity (cyclin E1-RB-E2F signaling). Since BRCA1 and BRCA2 are known target genes of E2F transcription factors, we hypothesize that cyclin E1 signaling actively contributes to high DNA repair capacity and chemo-resistance in a BRCA1/2-dependent manner. We predict CCNE1-amplified ovarian cancers to exhibit an “anti-BRCAness” phenotype. In two independent gene expression datasets, generated by TCGA and The Women's Cancer Program at Cedars-Sinai, we found that CCNE1-amplified ovarian cancers maintain higher levels of BRCA1 than CCNE1-wildtype tumors. Moreover, we have employed genetic and pharmacological targeting approaches in order to assess if inhibition of cyclin E1-dependent signaling can induce BRCAness and restore chemo-sensitivity. Here, we show that pharmacological CDK inhibitors (CDKi) effectively shut down E2F-mediated transcription, resulting in downregulation of BRCA1 and BRCA2 in CDKi-treated cells. Long-term exposure of ovarian cancer cell lines to CDKi selected for cells with reduced dependency on cyclin E1, and genomic profiling revealed de novo DNA copy number changes which compensate for loss of cyclin E1 function. Importantly, CDKi-resistant subclones retained lower levels of BRCA1 and were significantly more sensitive to cisplatin than parental cell lines. Collectively, our results suggest that cyclin E1 signaling is required for BRCA1 expression and chemo-resistance. Currently available CDKi such as Dinaciclib, which is in a phase 3 clinical trial, may be useful to specifically sensitize CCNE1-amplified ovarian cancers to cisplatin. Citation Format: Barbie Taylor-Harding, Hasmik Agadjanian, Paul Joseph Aspuria, Takako Mizuno, Dong-Joo Cheon, Sandra Orsulic, Beth Karlan, Christine Walsh, Wolf Ruprecht Wiedemeyer. Targeting chemo-resistance in CCNE1-amplified ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B48.

1128 INVASIVE BLADDER CANCER CELLS ARE DEFICIENT IN DNA REPAIR AND HAVE MUTATOR PHENOTYPES THAT ARE DUE TO SUPPRESSION OF P63 GENE EXPRESSION

You have accessJournal of UrologyBladder Cancer: Basic Research (III)1 Apr 20131128 INVASIVE BLADDER CANCER CELLS ARE DEFICIENT IN DNA REPAIR AND HAVE MUTATOR PHENOTYPES THAT ARE DUE TO SUPPRESSION OF P63 GENE EXPRESSION Hyun-Wook Lee, Hsiang-Tsui Wang, Mao-wen Weng, Josephine Kuo, William C. Huang, Nicholas Donin, Herbert Lepor, Xue-Ru Wu, and Moon-shong Tang Hyun-Wook LeeHyun-Wook Lee New York, NY More articles by this author , Hsiang-Tsui WangHsiang-Tsui Wang New York, NY More articles by this author , Mao-wen WengMao-wen Weng New York, NY More articles by this author , Josephine KuoJosephine Kuo New York, NY More articles by this author , William C. HuangWilliam C. Huang New York, NY More articles by this author , Nicholas DoninNicholas Donin New York, NY More articles by this author , Herbert LeporHerbert Lepor New York, NY More articles by this author , Xue-Ru WuXue-Ru Wu New York, NY More articles by this author , and Moon-shong TangMoon-shong Tang New York, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.743AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Although it is well established that the two distinct clinical phenotypes of human bladder cancer, e.g. the non–invasive low–grade papillary urothelial carcinoma (N–inv–UC) and the high–grade invasive UC (Inv–UC), develop through divergent pathways, the molecular mechanisms underlying the development of these phenotypes remain unclear. We hypothesize that Inv–UC cells have a mutator phenotype because of a low DNA repair capacity and that these tumor cells have a high probability of escaping the immune surveillance system. In contrast, N–inv–UC cells have efficient DNA repair capacity and do not have a mutator phenotype; therefore, these tumor cells have a low probability of accumulating mutations that lead to tumor invasion. METHODS Using host cell reactivation, in vitro DNA damage dependent repair synthesis, and a shuttle vector supF system, we determined 1) DNA repair capacity, 2) ability to carry out DNA damage–dependent mutagenesis, and 3) expression of nucleotide excision repair (NER) genes (XPC & XPA), base excision repair (BER) gene (hOGG1), mismatch repair genes (MLH1, MSH2 & PMS2), and p53 and p63 genes, in cultured human N–inv–UC and Inv–UC cells. RESULTS We found that: 1) N–inv–UC cells have a much higher DNA repair capacity than Inv–UC cells for both oxidative and UV light–induced DNA damage, 2) both oxidative and UV–induced DNA damage elicit significantly more mutations in Inv–UC cells than in N–inv–UC cells, 3) Inv–UC cells do not express nucleotide excision repair gene XPC; base excision repair gene hOGG1, or p63 gene, and 4) the deficiency of DNA repair and the expression of XPC gene in Inv–UC can be partially rescued by transient expression of the p63 gene. CONCLUSIONS Our results provide the first experimental evidence indicating that Inv–UC cells are deficient in both nucleotide excision repair and base excision repair; that these deficiencies cause these cells to have a mutator phenotype which underlies their invasive behavior; and that the deficiency in DNA repair in Inv–UC can be rescued by the restoration of p63 expression. These results provide insight into the molecular underpinnings behind the two major bladder cancer oncogenic pathways, and provide a foundation for further studies to better molecularly differentiate these two distinct phenotypes. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e461 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hyun-Wook Lee New York, NY More articles by this author Hsiang-Tsui Wang New York, NY More articles by this author Mao-wen Weng New York, NY More articles by this author Josephine Kuo New York, NY More articles by this author William C. Huang New York, NY More articles by this author Nicholas Donin New York, NY More articles by this author Herbert Lepor New York, NY More articles by this author Xue-Ru Wu New York, NY More articles by this author Moon-shong Tang New York, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Fragile DNA Repair Mechanism Reduces Ageing in Multicellular Model

DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn increases the amount of unrepaired DNA damage. Despite this vicious circle, we ask, can cells maintain a high DNA repair capacity for some time or is repair capacity bound to continuously decline with age? We here present a simple mathematical model for ageing in multicellular systems where cells subjected to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity, followed by a rapid decline. Furthermore, the time of high functionality increases, and consequently slows down the ageing process, if the DNA repair mechanism itself is vulnerable to DNA damages. Although counterintuitive at first glance, a fragile repair mechanism allows for a faster removal of compromised cells, thus freeing the space for healthy peers. This finding might be a first step toward understanding why a mutation in single DNA repair protein (e.g. Wrn or Blm) is not buffered by other repair proteins and therefore, leads to severe ageing disorders.

Feasibility of a prospective host cell reactivation assay (HCRA) in non-small cell lung cancer (NSCLC) patients.

10586 Background: DNA repair capacity (DRC)—the ability to repair BPDE-induced DNA adducts—measured in peripheral blood lymphocytes (PBLs) by the HCRA has been shown to correlate with survival in patients (p) treated with cisplatin-based chemotherapy. p with high DRC had significantly shorter survival (Bosken et al. JNCI 2002). However, the HCRA of DRC has not been tested prospectively. Methods: Cryopreserved PBLs from 41 NSCLC p were cultured for 96 hours under phytohaemagglutinin stimulation. The pmaxGFP plasmid was nucleoporated in the cells of the host under two different experimental conditions: (i) undamaged (GFP-UND) and (ii) 254nm UV light-damaged plasmid DNA (1,000 J/m2) (GFP-DAM). After transfection, the cells were incubated for 24 hours in culture media to allow, firstly, repair of UV-damaged plasmids, and secondly, reexpression of the GFP reporter protein. By flow cytometry, the dead cells (positively stained with propidium iodide) were excluded and DRC was calculated in alive cells as follows: percentage DRC = (pmaxGFP-DAM/pmaxGFP-UND) * 100, where pmaxGFP-DAM and pmaxGFP-UND correspond to the percentage of alive cells positive for GFP protein. Results: There were no differences in DRC according to gender, season of the year or histology. Median DRC was 8.77 for p with PS 0, 10.49 for p with PS 1, and 21.60 for p with PS 2 (p=0.009). p treated with chemotherapy, with or without surgery and/or radiotherapy, had a higher relative risk (RR) of death associated with increased DRC (p=0.07) (Table). Conclusions: HCRA measured in PBLs is feasible and can be useful in monitoring NSCLC p. Serial measurements to detect increments in DRC can provide a marker of acquired resistance to cisplatin-based chemotherapy. The correlation of DRC with the expression of DNA repair genes in the tumor should be investigated. Cox proportional hazards model of the RR of death associated with increased DRC Unadjusted RR (95%CI) 0.82 (0.66-1.02) p=0.07 Adjusted* RR (95%CI) (for age, sex, cigarettes/day, stage) 0.81 (0.65-1.02) p=0.07 No significant financial relationships to disclose.

Abstract A6: Ethnic differences in risk of lung cancer associated with repair capacity for tobacco carcinogen-induced DNA adduct

Abstract Lung cancer is initiated by tobacco carcinogens-induced DNA damage, such as DNA adducts caused by benzo[a]pyrene diol epoxide (BPDE). These adducts can be removed by an efficient nucleotide excision repair. We have previously reported that DNA repair capacity (DRC) measured by the host-cell reactivation assay is associated with risk of lung cancer in non-Hispanic whites. In the recent analysis with larger sample sizes including minorities, we found that the suboptimal DRC remains associated with increased risk of lung cancer in 1730 cases and 1683 controls of non-Hispanic whites. The mean values of DRC were 8.37% ± 2.8% (SD) in the cases vs. 9.04% ± 3.1% in the controls (P &amp;lt; 0.0001). The odds ratio (OR) was 1.42 (95% confidence interval [CI], 1.24 – 1.64) after adjustment for age, sex, pack-years of smoking, family history of cancer, and other assay-related covariates. The similar results and risk trend have been also found in 127 Mexican-American patients with lung cancer and 176 cancer-free controls with a mean DRC of 8.38% ± 2.7% in the cases vs. 9.04% ± 3.3% in the controls, and an adjusted OR of 1.67 (95% CI, 1.00 – 2.78), although the difference in mean DRC values was borderline significant (P = 0.056) because of the relatively small sample size. However, this difference in DRC for tobacco carcinogen-induced DNA adducts was not significant in 327 African-American patients with lung cancer and 325 cancer-free controls. The mean values of DRC were 8.90% ± 3.1% in the cases vs. 8.66% ± 2.8% in the controls (P = 0.301). The adjusted OR was 0.99 (95% CI, 0.70 – 1.39). The DRC level in African-American controls was non-significantly lower than other ethnic controls (P = 0.144); however, African-American patients had significantly higher DRC than non-Hispanic white patients and Mexican-Americans patients (P = 0.053). The results further confirmed that low DRC is associated with increased risk of lung cancer in non-Hispanic whites and also provided the evidence of this association in Mexican Americans. These findings help us understand the ethnic differences in the development of lung cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A6.