DNA Damage and Repair in Cancers

Brain tumours kill more children and adults under 40 than any other cancer, with approximately half of primary brain tumours being diagnosed as high-grade malignancies known as glioblastomas. Despite de-bulking surgery combined with chemo-/radiotherapy regimens, the mean survival for these patients is only around 15 months, with less than 10% surviving over 5 years. This dismal prognosis highlights the urgent need to develop novel agents to improve the treatment of these tumours. To address this need, we carried out a human kinome siRNA screen to identify potential drug targets that augment the effectiveness of temozolomide (TMZ)—the standard-of-care chemotherapeutic agent used to treat glioblastoma. From this we identified ERK5/MAPK7, which we subsequently validated using a range of siRNA and small molecule inhibitors within a panel of glioma cells. Mechanistically, we find that ERK5 promotes efficient repair of TMZ-induced DNA lesions to confer cell survival and clonogenic capacity. Finally, using several glioblastoma patient cohorts we provide target validation data for ERK5 as a novel drug target, revealing that heightened ERK5 expression at both the mRNA and protein level is associated with increased tumour grade and poorer patient survival. Collectively, these findings provide a foundation to develop clinically effective ERK5 targeting strategies in glioblastomas and establish much-needed enhancement of the therapeutic repertoire used to treat this currently incurable disease. Full article

Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related deaths worldwide. In this study, a total of 749 HCC patients from 5 cohorts were studied to examine the relationships between enhancement of DNA repair and cancer aggressiveness, tumor immune microenvironment, and patient survival in HCC, utilizing a DNA repair pathway score. Our findings suggest that the DNA repair pathway was not only enhanced by the stepwise carcinogenic process of HCC, but also significantly enhanced in grade 3 HCC compared with grade 1 and 2 tumors. DNA repair high HCC was associated with worse survival, elevated intratumor heterogeneity, and mutation load, but not with the fraction of immune cell infiltration nor immune response. HCC tumors with a DNA repair high score enriched the cell proliferation- and other cancer aggressiveness-related gene sets. Interestingly, these features were more pronounced in grade 1 and 2 HCC compared to grade 3 HCC. To our knowledge, this is the first study to use DNA repair pathway-related gene set expression data to examine and validate the clinical relevance of DNA repair pathway activity in HCC. The DNA repair score may be used to better understand and predict prognosis in HCC. Full article

PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. Methods: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. Results: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. Conclusions: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs. Full article

It was recently shown that the 5’ to 3’ exoribonuclease XRN2 is involved in the DNA damage response. Importantly, loss of XRN2 abrogates DNA double stranded break repair via the non-homologous end-joining pathway. However, the mechanistic details of how XRN2 functions in the non-homologous end-joining repair process are unknown. In this study, we elucidated that XRN2-mediated RNA:DNA hybrid resolution is required to allow Ku70 binding to DNA ends. These data suggest that XRN2 is required for the initiation of non-homologous end-joining repair. Interestingly, we uncovered a role for XRN2 in the homologous recombination repair pathway. Loss of XRN2 lead to a decrease in the repair of double strand breaks by homologous recombination. Strikingly, when we removed RNA:DNA hybrids by RNaseH1 over-expression, homologous recombination was not restored. We found RNA:DNA hybrid formation at and downstream of the DSB site, suggesting that unregulated transcription inhibits homologous recombination repair. In summary, our results indicate a relation between RNA:DNA hybrid resolution and double strand break repair pathway choice. Full article

The response of head and neck squamous cell carcinoma (HNSCC) to radiotherapy depends on human papillomavirus type 16 (HPV) status, and where improved outcome and survival is observed in HPV-positive disease. However, strategies to further radiosensitise the tumours, particularly relatively radioresistant HPV-negative HNSCC, are actively being sought. The impact of targeting the major protein kinases involved in the signaling of DNA double-strand break (DSB) repair, namely ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the catalytic subunit of DNA-dependent protein kinase (DNA-Pkcs), on the radiosensitisation of HNSCC cells was examined. The response to both conventional photon radiotherapy, but also proton beam therapy, was analysed by clonogenic assays and 3D spheroid growth. We observed that inhibition of ATM, ATR, and particularly DNA-Pkcs, caused a significant reduction in HNSCC cell survival post-irradiation with both photons and protons, with less of an impact on the most radiosensitive HPV-positive cell line. The inhibition of DNA-Pkcs and, to a lesser extent ATM, in combination with radiation was also more effective at inhibiting the growth of 3D spheroids derived from relatively radioresistant HPV-negative HNSCC. Similar effects of the inhibitors were observed comparing photon and proton irradiation, demonstrating the potential for targeting DSB repair as an effective combination treatment for HNSCC. Full article

The aberrant hypermethylation of BRCA1 promoter CpG islands induces the decreased expression of BRCA1 (Breast Cancer 1) protein. It can be detected in sporadic breast cancer without BRCA1 pathogenic variants, particularly in triple-negative breast cancers (TNBC). We investigated BRCA1 hypermethylation status (by methylation-specific polymerase chain reaction (MS-PCR) and MassARRAY^® assays), and BRCA1 protein expression using immunohistochemistry (IHC), and their clinicopathological significance in 248 chemotherapy-naïve TNBC samples. Fifty-five tumors (22%) exhibited BRCA1 promoter hypermethylation, with a high concordance rate between MS-PCR and MassARRAY^® results. Promoter hypermethylation was associated with reduced IHC BRCA1 protein expression (p = 0.005), and expression of Programmed death-ligand 1 protein (PD-L1) by tumor and immune cells (p = 0.03 and 0.011, respectively). A trend was found between promoter hypermethylation and basal marker staining (p = 0.058), and between BRCA1 expression and a basal-like phenotype. In multivariate analysis, relapse-free survival was significantly associated with N stage, adjuvant chemotherapy, and histological subtype. Overall survival was significantly associated with T and N stage, histology, and adjuvant chemotherapy. In addition, patients with tumors harboring BRCA1 promoter hypermethylation derived the most benefit from adjuvant chemotherapy. In conclusion, BRCA1 promoter hypermethylation is associated with TNBC sensitivity to adjuvant chemotherapy, basal-like features and PD-L1 expression. BRCA1 IHC expression is not a good surrogate marker for promoter hypermethylation and is not independently associated with prognosis. Association between promoter hypermethylation and sensitivity to Poly(ADP-ribose) polymerase PARP inhibitors needs to be evaluated in a specific series of patients. Full article

Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. Methods: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. Results: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. Conclusion: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL. Full article

Glioblastoma (GBM) patients have an estimated survival of ~15 months with treatment, and the standard of care only modestly enhances patient survival. Identifying biomarkers representing vulnerabilities may allow for the selection of efficacious chemotherapy options to address personalized variations in GBM tumors. Irinotecan targets topoisomerase I (TOP1) by forming a ternary DNA–TOP1 cleavage complex (TOP1cc), inducing apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair enzyme that may reduce the effectiveness of irinotecan. We treated GBM cell lines with increasing concentrations of irinotecan and compared the IC₅₀ values. We found that the TDP1/TOP1 activity ratio had the strongest correlation (Pearson correlation coefficient R = 0.972, based on the average from three sets of experiments) with IC₅₀ values following irinotecan treatment. Increasing the TDP1/TOP1 activity ratio by the ectopic expression of wild-type TDP1 increased in irinotecan IC₅₀, while the expression of the TDP1 catalytic-null mutant did not alter the susceptibility to irinotecan. The TDP1/TOP1 activity ratio may be a new predictive indicator for GBM vulnerability to irinotecan, allowing for the selection of individual patients for irinotecan treatment based on risk–benefit. Moreover, TDP1 inhibitors may be a novel combination treatment with irinotecan to improve GBM patient responsiveness to genotoxic chemotherapies. Full article

DNA damage and the generation of reactive oxygen species (ROS) are key mechanisms of apoptotic cell death by commonly used genotoxic drugs. However, the complex cellular response to these pharmacologic agents remains yet to be fully characterized. Several studies have described the role of transcription factor octamer-1 (Oct-1)/Pit-1, Oct-1/2, and Unc-86 shared domain class 2 homeobox 1 (POU2F1) in the regulation of the genes important for cellular response to genotoxic stress. Evaluating the possible involvement of other POU family transcription factors in these pathways, we revealed the inducible expression of Oct-6/POU3F1, a regulator of neural morphogenesis and epidermal differentiation, in cancer cells by genotoxic drugs. The induction of Oct-6 occurs at the transcriptional level via reactive oxygen species (ROS) and ataxia telangiectasia mutated- and Rad3-related (ATR)-dependent mechanisms, but in a p53 independent manner. Moreover, we provide evidence that Oct-6 may play a role in the regulation of cellular response to DNA damaging agents. Indeed, by using the shRNA approach, we demonstrate that in doxorubicin-treated H460 non-small-cell lung carcinoma (NSCLC) cells, Oct-6 depletion leads to a reduced G2-cell cycle arrest and senescence, but also to increased levels of intracellular ROS and DNA damage. In addition, we could identify p21 and catalase as Oct-6 target genes possibly mediating these effects. These results demonstrate that Oct-6 is expressed in cancer cells after genotoxic stress, and suggests its possible role in the control of ROS, DNA damage response (DDR), and senescence. Full article

A reduced ability to properly repair DNA is linked to a variety of human diseases, which in almost all cases is associated with an increased probability of the development of cellular transformation and cancer. DNA damage, that ultimately can lead to mutations and genomic instability, is due to many factors, such as oxidative stress, metabolic disorders, viral and microbial pathogens, excess cellular proliferation and chemical factors. In this review, we examine the evidence connecting DNA damage and the mechanisms that viruses and bacteria have evolved to hamper the pathways dedicated to maintaining the integrity of genetic information, thus affecting the ability of their hosts to repair the damage(s). Uncovering new links between these important aspects of cancer biology might lead to the development of new targeted therapies in DNA-repair deficient cancers and improving the efficacy of existing therapies. Here we provide a comprehensive summary detailing the major mechanisms that viruses and bacteria associated with cancer employ to interfere with mechanisms of DNA repair. Comparing these mechanisms could ultimately help provide a common framework to better understand how certain microorganisms are involved in cellular transformation. Full article

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key component of the DNA-PK complex that has a well-characterized function in the non-homologous end-joining repair of DNA double-strand breaks. Since its identification, a large body of evidence has demonstrated that DNA-PKcs is frequently overexpressed in cancer, plays a critical role in tumor development and progression, and is associated with poor prognosis of cancer patients. Intriguingly, recent studies have suggested novel functions beyond the canonical role of DNA-PKcs, which has transformed the paradigm of DNA-PKcs in tumorigenesis and has reinvigorated the interest to target DNA-PKcs for cancer treatment. In this review, we update recent advances in DNA-PKcs, in particular the emerging roles in tumor metastasis, metabolic dysregulation, and immune escape. We further discuss the possible molecular basis that underpins the pleiotropism of DNA-PKcs in cancer. Finally, we outline the biomarkers that may predict the therapeutic response to DNA-PKcs inhibitor therapy. Understanding the functional repertoire of DNA-PKcs will provide mechanistic insights of DNA-PKcs in malignancy and, more importantly, may revolutionize the design and utility of DNA-PKcs-based precision cancer therapy. Full article

Ionizing radiation may be of both artificial and natural origin and causes cellular damage in living organisms. Radioactive isotopes have been used significantly in cancer therapy for many years. The formation of DNA double-strand breaks (DSBs) is the most dangerous effect of ionizing radiation on the cellular level. After irradiation, cells activate a DNA damage response, the molecular path that determines the fate of the cell. As an important element of this, homologous recombination repair is a crucial pathway for the error-free repair of DNA lesions. All components of DNA damage response are regulated by specific microRNAs. MicroRNAs are single-stranded short noncoding RNAs of 20–25 nt in length. They are directly involved in the regulation of gene expression by repressing translation or by cleaving target mRNA. In the present review, we analyze the biological mechanisms by which miRNAs regulate cell response to ionizing radiation-induced double-stranded breaks with an emphasis on DNA repair by homologous recombination, and its main component, the RAD51 recombinase. On the other hand, we discuss the ability of DNA damage response proteins to launch particular miRNA expression and modulate the course of this process. A full understanding of cell response processes to radiation-induced DNA damage will allow us to develop new and more effective methods of ionizing radiation therapy for cancers, and may help to develop methods for preventing the harmful effects of ionizing radiation on healthy organisms. Full article

In recent years, a considerable correlation has emerged between autophagy and genome integrity. A range of mechanisms appear to be involved where autophagy participates in preventing genomic instability, as well as in DNA damage response and cell fate decision. These initial findings have attracted particular attention in the context of malignancy; however, the crosstalk between autophagy and DNA damage response is just beginning to be explored and key questions remain that need to be addressed, to move this area of research forward and illuminate the overall consequence of targeting this process in human therapies. Here we present current knowledge on the complex crosstalk between autophagy and genome integrity and discuss its implications for cancer cell survival and response to therapy. Full article

DNA damage is well recognized as a critical factor in cancer development and progression. DNA lesions create an abnormal nucleotide or nucleotide fragment, causing a break in one or both chains of the DNA strand. When DNA damage occurs, the possibility of generated mutations increases. Genomic instability is one of the most important factors that lead to cancer development. DNA repair pathways perform the essential role of correcting the DNA lesions that occur from DNA damaging agents or carcinogens, thus maintaining genomic stability. Inefficient DNA repair is a critical driving force behind cancer establishment, progression and evolution. A thorough understanding of DNA repair mechanisms in cancer will allow for better therapeutic intervention. In this review we will discuss the relationship between DNA damage/repair mechanisms and cancer, and how we can target these pathways. Full article