Germline mutational variants of Turkish ovarian cancer patients suspected of Hereditary Breast and Ovarian Cancer (HBOC) by next-generation sequencing.

Hereditary Breast and Ovarian Cancer (HBOC) syndrome is characterized by an increased risk of developing breast cancer (BC) and ovarian cancer (OC) due to inherited genetic mutations. Understanding the genetic variants associated with HBOC is crucial for identifying individuals at high risk and implementing appropriate preventive measures. The study included 630 Turkish OC patients with confirmed diagnostic criteria of The National Comprehensive Cancer Network (NCCN) concerning HBOC. Genomic DNA was extracted from peripheral blood samples, and targeted Next-generation sequencing (NGS) was performed. Bioinformatics analysis and variant interpretation were conducted to identify pathogenic variants (PVs). Our analysis revealed a spectrum of germline pathogenic variants associated with HBOC in Turkish OC patients. Notably, several pathogenic variants in BRCA1, BRCA2, and other DNA repair genes were identified. Specifically, we observed germline PVs in 130 individuals, accounting for 20.63% of the total cohort. 76 distinct PVs in genes, BRCA1 (40 PVs), BRCA2 (29 PVs), ATM (1 PV), CHEK2 (2 PVs), ERCC2 (1 PV), MUTYH (1 PV), RAD51C (1 PV), and TP53 (1PV) and also, two different PVs (i.e., c.135-2 A>G p.? in BRCA1 and c.6466_6469delTCTC in BRCA2) were detected in a 34-year-old OC patient. In conclusion, our study contributes to a better understanding of the genetic variants underlying HBOC in Turkish OC patients. These findings provide valuable insights into the genetic architecture of HBOC in the Turkish population and shed light on the potential contribution of specific germline PVs to the increased risk of OC.

Nucleotide excision repair deficiency is a targetable therapeutic vulnerability in clear cell renal cell carcinoma.

Due to a demonstrated lack of DNA repair deficiencies, clear cell renal cell carcinoma (ccRCC) has not benefitted from targeted synthetic lethality-based therapies. We investigated whether nucleotide excision repair (NER) deficiency is present in an identifiable subset of ccRCC cases that would render those tumors sensitive to therapy targeting this specific DNA repair pathway aberration. We used functional assays that detect UV-induced 6-4 pyrimidine-pyrimidone photoproducts to quantify NER deficiency in ccRCC cell lines. We also measured sensitivity to irofulven, an experimental cancer therapeutic agent that specifically targets cells with inactivated transcription-coupled nucleotide excision repair (TC-NER). In order to detect NER deficiency in clinical biopsies, we assessed whole exome sequencing data for the presence of an NER deficiency associated mutational signature previously identified in ERCC2 mutant bladder cancer. Functional assays showed NER deficiency in ccRCC cells. Some cell lines showed irofulven sensitivity at a concentration that is well tolerated by patients. Prostaglandin reductase 1 (PTGR1), which activates irofulven, was also associated with this sensitivity. Next generation sequencing data of the cell lines showed NER deficiency-associated mutational signatures. A significant subset of ccRCC patients had the same signature and high PTGR1 expression. ccRCC cell line-based analysis showed that NER deficiency is likely present in this cancer type. Approximately 10% of ccRCC patients in the TCGA cohort showed mutational signatures consistent with ERCC2 inactivation associated NER deficiency and also substantial levels of PTGR1 expression. These patients may be responsive to irofulven, a previously abandoned anticancer agent that has minimal activity in NER-proficient cells.

The Curcumin Analog PAC Is a Potential Solution for the Treatment of Triple-Negative Breast Cancer by Modulating the Gene Expression of DNA Repair Pathways.

Breast Cancer (BC) is one of the most common and challenging cancers among females worldwide. Conventional treatments for oral cancer rely on the use of radiology and surgery accompanied by chemotherapy. Chemotherapy presents many side effects, and the cells often develop resistance to this chemotherapy. It will be urgent to adopt alternative or complementary treatment strategies that are new and more effective without these negative effects to improve the well-being of patients. A substantial number of epidemiological and experimental studies reported that many compounds are derived from natural products such as curcumin and their analogs, which have a great deal of beneficial anti-BC activity by inducing apoptosis, inhibiting cell proliferation, migration, and metastasis, modulating cancer-related pathways, and sensitizing cells to radiotherapy and chemotherapy. In the present study, we investigated the effect of the curcumin-analog PAC on DNA repair pathways in MCF-7 and MDA-MB-231 human breast-cancer cell lines. These pathways are crucial for genome maintenance and cancer prevention. MCF-7 and MDA-MB-231 cells were exposed to PAC at 10 µM. MTT and LDH assays were conducted to evaluate the effects of PAC on cell proliferation and cytotoxicity. Apoptosis was assessed in breast cancer cell lines using flow cytometry with annexin/Pi assay. The expression of proapoptotic and antiapoptotic genes was determined by RT-PCR to see if PAC is active in programming cell death. Additionally, DNA repair signaling pathways were analyzed by PCR arrays focusing on genes being related and confirmed by quantitative PCR. PAC significantly inhibited breast-cancer cell proliferation in a time-dependent manner, more on MDA-MB-231 triple-negative breast cancer cells. The flow cytometry results showed an increase in apoptotic activity. These data have been established by the gene expression and indicate that PAC-induced apoptosis by an increased Bax and decreased Bcl-2 expression. Moreover, PAC affected multiple genes involved in the DNA repair pathways occurring in both cell lines (MCF-7 and MDA-MB231). In addition, our results suggest that PAC upregulated more than twice 16 genes (ERCC1, ERCC2, PNKP, POLL, MPG, NEIL2, NTHL1, SMUG1, RAD51D, RAD54L, RFC1, TOP3A, XRCC3, XRCC6BP1, FEN1, and TREX1) in MDA-MB-231, 6 genes (ERCC1, LIG1, PNKP, UNG, MPG, and RAD54L) in MCF-7, and 4 genes (ERCC1, PNKP, MPG, and RAD54L) in the two cell lines. In silico analysis of gene-gene interaction shows that there are common genes between MCF-7 and MDA-MB-321 having direct and indirect effects, among them via coexpression, genetic interactions, pathways, predicted and physical interactions, and shared protein domains with predicted associated genes indicating they are more likely to be functionally related. Our data show that PAC increases involvement of multiple genes in a DNA repair pathway, this certainly can open a new perspective in breast-cancer treatment.

Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

Influence of Morinda citrifolia (Noni) on Expression of DNA Repair Genes in Cervical Cancer Cells.

BACKGROUND: Previous studies have suggested that Morinda citrifolia (Noni) has potential to reduce cancer risk. OBJECTIVE: The purpose of this study was to investigate the effect of Noni, cisplatin, and their combination on DNA repair genes in the SiHa cervical cancer cell line. MATERIALS AND METHODS: SiHa cells were cultured and treated with 10% Noni, 10 µg/dl cisplatin or their combination for 24 hours. Post culturing, the cells were pelleted, RNA extracted, and processed for investigating DNA repair genes by real time PCR. RESULTS: The expression of nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and base excision repair gene XRCC1 was increased 4 fold, 8.9 fold, 4 fold, and 5.5 fold, respectively, on treatment with Noni as compared to untreated controls (p<0.05). In contrast, expression was found to be decreased 22 fold, 13 fold, 16 fold, and 23 fold on treatment with cisplatin (p<0.05). However, the combination of Noni and cisplatin led to an increase of 2 fold, 1.6 fold, 3 fold, 1.2 fold, respectively (p<0.05). CONCLUSIONS: Noni enhanced the expression of DNA repair genes by itself and in combination with cisplatin. However, high expression of DNA repair genes at mRNA level only signifies efficient DNA transcription of the above mentioned genes; further investigations are needed to evaluate the DNA repair protein expression.

MnSOD rs4880 and XPD rs13181 polymorphisms predict the survival of breast cancer patients treated with adjuvant tamoxifen.

UNLABELLED: The enzyme manganese superoxide dismutase (MnSOD) defends against oxidative stress caused by reactive oxygen species (ROS), whereas Xeroderma pigmentosum group D (XPD) protein is involved in DNA repair. Polymorphisms in these genes have previously been associated with the outcome of breast cancer. MATERIAL AND METHODS: Two gene polymorphisms, the MnSOD Val16Ala (rs4880A>G) and the XPD Lys751Gln (rs13181A>C), were analyzed in a cohort of 396 Finnish breast cancer patients by using PCR-RFLP-based methods in a prospective case-control study. The overall survival (OS), breast cancer-specific survival (BCSS), and relapse-free survival (RFS), assessed by using Kaplan-Meier survival analysis and multivariate Cox regression analysis, were evaluated according to the adjuvant treatments and the rs4880 and rs13181 genotypes. RESULTS: In the combined analysis of rs4880 and rs13181 genotypes for patients treated with adjuvant tamoxifen (TAM) an increasing number of low-risk genotypes (rs4880 AA, rs4880 AG, or rs13181 AA) was significantly associated with better RFS, BCSS, and OS (n=64). In addition, there was improved BCSS and RFS among TAM-treated patients carrying the wild-type rs4880 A allele as compared with the other genotypes (n=64). The wild-type rs13181 AA genotype was similarly associated with better RFS and BCSS in the TAM-treated population (n=65). CONCLUSION: This is the first study to show that the MnSOD rs4880 and XPD rs13181 polymorphisms may influence the outcome of breast cancer patients receiving adjuvant TAM monotherapy. Patients carrying the rs4880 A allele or rs13181 AA genotype may have a reduced ability to scavenge ROS and repair the DNA damage generated by TAM treatment.

Predictive value of ERCC1, ERCC2, and XRCC1 overexpression for stage III colorectal cancer patients receiving FOLFOX-4 adjuvant chemotherapy.

OBJECTIVES: To determine the correlation between expression of three DNA repair genes and early failure/clinical outcome of stage III colorectal cancer (CRC) patients administrated with FOLFOX-4, including the excision repair cross-complementation group 1 (ERCC1), the excision repair cross-complementing 2 (ERCC2), and X-ray repair cross-complementing protein 1 (XRCC1). MATERIALS AND METHODS: We retrospectively analyzed clinicopathological features and ERCC1, ERCC2, XRCC1 expressions by immunohistochemical staining in 180 stage III CRC patients undergoing curative resection and treated with FOLFOX-4 chemotherapy to identify predictors of postoperative early failure. RESULTS: Among 180 CRC patients, 44 patients were classified into early failure group, and 136 patients were categorized into non-early failure group. A multivariate logistic regression analysis showed that ERCC1 overexpression (P = 0.005), and high postoperative carcinoembryonic antigen (CEA) levels (P = 0.001) were independent predictors of early failure. Additionally, ERCC1 overexpression was not only a predictor of early failure but also for disease-free survival (P < 0.001) and overall survival (P < 0.001). However, no predictive roles of ERCC2 and XRCC1 expression among these analyzed patients. CONCLUSIONS: ERCC1 overexpression is an important predictor of early failure in patients with stage III CRC administrating FOLFOX-4 adjuvant chemotherapy and this marker may help identify patients who would benefit from intensive follow-up and enhance therapeutic programs.

Cytogenetic effects induced by Prestige oil on human populations: the role of polymorphisms in genes involved in metabolism and DNA repair.

The spill from the oil tanker Prestige (NW Spain, November 2002) was perhaps the biggest ecological disaster that happened worldwide in the last decades. As a consequence of this catastrophe a general concern led to a huge mobilization of human and technical resources. Given that no information was reported in the scientific literature regarding to the chronic repercussions to human health of exposure to oil spills, a pilot study was performed by our group revealing some increased genotoxic effects in the subjects exposed to the oil during cleaning activities. Due to the seriousness of the results, we extended our study comprising a larger population and including an extensive evaluation of the main polymorphic sites in metabolizing and DNA-repair genes. General increases in micronucleus (MN) frequency and decreases in the proliferation index were observed in individuals with longer time of exposure. Age was a significant predictor of MN frequency. CYP1A1 3'-UTR, EPHX1 codons 113 and 139, GSTP1, GSTM1 and GSTT1 metabolic polymorphisms, and XRCC3 codon 241 and XPD codon 751 repair polymorphisms influenced cytogenetic damage levels. In view of these results, it seems essential to pay more attention to the chronic human health effects of exposure to oil and to focus new studies on such a relevant but overlooked public health field that involves a large number of people all over the world.

ERCC2 2251A>C genetic polymorphism was highly correlated with early relapse in high-risk stage II and stage III colorectal cancer patients: a preliminary study.

BACKGROUND: Early relapse in colorectal cancer (CRC) patients is attributed mainly to the higher malignant entity (such as an unfavorable genotype, deeper tumor invasion, lymph node metastasis and advance cancer stage) and poor response to chemotherapy. Several investigations have demonstrated that genetic polymorphisms in drug-targeted genes, metabolizing enzymes, and DNA-repairing enzymes are all strongly correlated with inter-individual differences in the efficacy and toxicity of many treatment regimens. This preliminary study attempts to identify the correlation between genetic polymorphisms and clinicopathological features of CRC, and evaluates the relationship between genetic polymorphisms and chemotherapeutic susceptibility of Taiwanese CRC patients. To our knowledge, this study discusses, for the first time, early cancer relapse and its indication by multiple genes. METHODS: Six gene polymorphisms functional in drug-metabolism - GSTP1 Ile105Val, ABCB1 Ile1145Ile, MTHFR Ala222Val, TYMS double (2R) or triple (3R) tandem repeat - and DNA-repair genes - ERCC2 Lys751Gln and XRCC1 Arg399Gln - were assessed in 201 CRC patients using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) technique and DNA sequencing. Patients were diagnosed as either high-risk stage II (T2 and 3 N0 M0) or III (any T N1 and 2 M0) and were administered adjuvant chemotherapy regimens that included 5-fluorouracil (5FU) and leucovorin (LV). The correlations between genetic polymorphisms and patient clinicopathological features and relapses were investigated. RESULTS: In this study, the distributions of GSTP1 (P = 0.003), ABCB1 (P = 0.001), TYMS (P < 0.0001), ERCC2 (P < 0.0001) and XRCC1 (P = 0.006) genotypes in the Asian population, with the exception of MTHFR (P = 0.081), differed significantly from their distributions in a Caucasian population. However, the unfavorable genotype ERCC2 2251A>C (P = 0.006), tumor invasion depth (P = 0.025), lymph node metastasis (P = 0.011) and cancer stage (P = 0.008) were significantly correlated with early relapse. Patients carrying the ERCC2 2251AC or2251CC genotypes had a significantly increased risk of early relapse (OR = 3.294, 95% CI, 1.272-8.532). CONCLUSION: We suggest that ERCC2 2251A>C alleles may be genetic predictors of early CRC relapse.

Cytogenetic challenge assays for assessment of DNA repair capacities.

Different challenge assays have been used to investigate cellular responses following exposure to DNA damaging agents. Our protocol uses X- or gamma-rays or ultraviolet light to challenge cells to repair the induced damage, and chromosome aberrations as a biomarker to indicate DNA repair proficiency. The assay was used successfully to demonstrate base- and nucleotide-excision repair deficiency in certain polymorphic DNA repair genes, namely XRCC1 751Gln and XPD 312Asn, respectively. In addition, populations with elevated exposure to certain environmental mutagenic agents-cigarette smokers, pesticide sprayers, and residents who lived near uranium mining and milling sites-showed DNA repair deficiency. Because expression of chromosome aberrations is associated with a significantly increased incidence of both cancer morbidity and mortality, the challenge assay may be useful in predicting cancer risk. The protocol for the assay is straightforward and the data have practical applications.