miR-214-mediated downregulation of RNF8 induces chromosomal instability in ovarian cancer cells

Defective DNA damage response (DDR) is frequently associated with carcinogenesis. Abrogation of DDR leads to chromosomal instability, a most common characteristic of tumors. However, the molecular mechanisms underlying regulation of DDR are still elusive. The ubiquitin ligase RNF8 mediates the ubiquitination of γH2AX and recruits 53BP1 and BRCA1 to DNA damage sites which promotes DDR and inhibits chromosomal instability. Though RNF8 is a key player involved in DDR, regulation of its expression is still poorly understood. Here, we show that miR-214 could abrogate DDR by repressing RNF8 expression through direct binding to 3′-untranslated region (3′ UTR) of RNF8 mRNA in human ovarian cancer cells. Antagonizing miR-214 by expressing its inhibitors in A2780 cells significantly increased RNF8 expression and thus promoted DNA damage repair. Consistent with the role of miR-214 in regulating RNF8 expression, the impaired DNA repair induced by miR-214 overexpression can be rescued by overexpressing RNF8 mRNA lacking the 3′ UTR. Together, our results indicate that down-regulation of RNF8 mediated by miR-214 impedes DNA damage response to induce chromosomal instability in ovarian cancers, which may facilitate the understanding of mechanisms underlying chromosomal instability.     

The crosstalk between Wnt/β-catenin signaling pathway with DNA damage response and oxidative stress: Implications in cancer therapy

DNA repair is essential for maintaining genomic integrity in cells. The dependence of cancer cell survival on proper DNA repair provides an opportunity to treat defective tumors by DNA damaging agents. Not only Wnt signaling has important functions in controlling gene expression, as well as cell polarity, adhesion and behavior, it also highly interacts with DNA damage response (DDR) in different levels. Furthermore, oxidative stress, which is responsible for majority of DNA lesions, affects Wnt signaling in different ways. A better understanding of the cross-talk between these pathways and events could provide strategies for treatment of cancer cells with deficient DNA repair capacity. As such, we will give a brief overview of the importance of the DNA repair machinery, signaling mechanisms of Wnt/β-catenin pathway, and DDR. We will further review the interactions between Wnt signaling and DDR, and the impact of oxidative stress on Wnt signaling. Finally, Wnt signaling is discussed as a potential treatment strategy for cancer.     

Exome Capture Reveals ZNF423 and CEP164 Mutations, Linking Renal Ciliopathies to DNA Damage Response Signaling

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites?of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR. PAPERFLICK:     

DNA damage responses in cancer stem cells: Implications for cancer therapeutic strategies

The identification of cancer stem cells(CSCs) that are responsible for tumor initiation, growth, metastasis, and therapeutic resistance might lead to a new thinking on cancer treatments. Similar to stem cells,CSCs also display high resistance to radiotherapy and chemotherapy with genotoxic agents. Thus, conventional therapy may shrink the tumor volume but cannot eliminate cancer. Eradiation of CSCs represents a novel therapeutic strategy. CSCs possess a highly efficient DNA damage response(DDR) system, which is considered as a contributor to the resistance of these cells from exposures to DNA damaging agents. Targeting of enhanced DDR in CSCs is thus proposed to facilitate the eradication of CSCs by conventional therapeutics. To achieve this aim, a better understanding of the cellular responses to DNA damage in CSCs is needed. In addition to the protein kinases and enzymes that are involved in DDR, other processes that affect the DDR including chromatin remodeling should also be explored.     

DNA damage accumulation and repair defects in acute myeloid leukemia: implications for pathogenesis,disease progression,and chemotherapy resistance

DNA damage repair mechanisms are vital to maintain genomic integrity. Mutations in genes involved in the DNA damage response (DDR) can increase the risk of developing cancer. In recent years, a variety of polymorphisms in DDR genes have been associated with increased risk of developing acute myeloid leukemia (AML) or of disease relapse. Moreover, a growing body of literature has indicated that epigenetic silencing of DDR genes could contribute to the leukemogenic process. In addition, a variety of AML oncogenes have been shown to induce replication and oxidative stress leading to accumulation of DNA damage, which affects the balance between proliferation and differentiation. Conversely, upregulation of DDR genes can provide AML cells with escape mechanisms to the DDR anticancer barrier and induce chemotherapy resistance. The current review summarizes the DDR pathways in the context of AML and describes how aberrant DNA damage response can affect AML pathogenesis, disease progression, and resistance to standard chemotherapy, and how defects in DDR pathways may provide a new avenue for personalized therapeutic strategies in AML.     

DNA Damage Response as an Anti-Cancer Barrier: Damage Threshold and the Concept of 'Conditional Haploinsufficiency'

DNA damage response (DDR) emerges as a biological tumorigenesis barrier in early stages of cancer development, and a selective pressure that favors outgrowth of malignant clones with defects in the genome maintenance machinery, such as mutations of p53 and other DDR components. Recent studies indicate that the DDR barrier is not alarmed universally among early noninvasive lesions, but rather responds to high-risk tumorigenic threats that occur in high-grade, pre-malignant lesions that are generally more likely to develop into bona fide malignancies. In addition, while the DDR barrier appears to operate in major types of cancer, such as carcinomas of the lung, breast and colon, DDR activation is rare at any stage of progression among testicular germ-cell tumors. Together with observations that several, but not all oncogenic insults are capable of activating the DDR machinery, these new results point to existence of a critical threshold of such oncogene-induced DNA damage. It seems that only cells and lesions that experience DNA replication stress and DNA damage above such threshold activate the cellular senescence or cell death pathways within the DDR machinery. The higher load of DNA damage may also contribute to cancer predisposition in families with inherited heterozygous defects in the DDR barrier, such as in ATM, BRCA1, BRCA2, p53 and other genes. We propose that carriers of such DDR defects may be more prone to malignancy due to ‘conditional haploinsufficiency’: such partial defects may be asymptomatic in normal tissues, yet they may become manifest under conditions of supra-threshold endogenous DNA damage in oncogene-driven pre-malignant lesions.     

SLFN11 inhibits checkpoint maintenance and homologous recombination repair

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA‐damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1‐dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA–ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA‐damaging agents. Finally, we demonstrate that the RPA1‐binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA‐damaging therapeutic agents.     

衰老或肿瘤：癌基因诱导的双向性

在大部分的肿瘤中都发现有癌基因的活化,癌基因的活化被认为是导致肿瘤发生的重要原因．然而,在野生型细胞内,癌基因的活化可以诱导细胞衰老,称为癌基因诱导的细胞衰老(oncogene-induced senescence, OIS),从而抑制进一步的肿瘤发生．因而,癌基因的活化具有诱导衰老或肿瘤的双向性．DNA损伤调控反应(DNA damage checkpoint response, DDR)是细胞应对DNA损伤时感应损伤,从而延迟或阻滞细胞周期进程的一种分子信号传递通路,是诱导细胞衰老的重要机制．癌基因的活化可以引发DNA损伤信号的产生,从而激活DDR,诱导细胞衰老．在DDR异常时,癌基因的激活可引发DNA的过度复制与细胞的过度增殖,并导致基因组不稳定性的积累,最终导致肿瘤发生．DDR的完整性决定了癌基因诱导的双向性．DDR在癌基因诱导中的重要作用,提示了保持和恢复DDR的完整性可以作为肿瘤预防和治疗的新方向．     

Increased RPA1 gene dosage affects genomic stability potentially contributing to 17p13.3 duplication syndrome

A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage response (DDR). Here, we show that cell lines from patients with duplications specifically incorporating RPA1 exhibit a different although characteristic spectrum of DDR defects including abnormal S phase distribution, attenuated DNA double strand break (DSB)-induced RAD51 chromatin retention, elevated genomic instability, and increased sensitivity to DNA damaging agents. Using controlled conditional over-expression of RPA1 in a human model cell system, we also see attenuated DSB-induced RAD51 chromatin retention. Furthermore, we find that transient over-expression of RPA1 can impact on homologous recombination (HR) pathways following DSB formation, favouring engagement in aberrant forms of recombination and repair. Our data identifies unanticipated defects in the DDR associated with duplications in 17p13.3 in humans involving modest RPA1 over-expression.     

Loss of ATRX confers DNA repair defects and PARP inhibitor sensitivity

Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and represents a potential target for cancer therapies. ATRX is known to function as a histone chaperone that helps incorporate histone variant, H3.3, into the genome. Studies have implicated ATRX in key DNA damage response (DDR) pathways but a distinct role in DNA repair has yet to be fully elucidated. To further investigate the function of ATRX in the DDR, we created isogenic wild-type (WT) and ATRX knockout (KO) model cell lines using CRISPR-based gene targeting. These studies revealed that loss of ATRX confers sensitivity to poly(ADP)-ribose polymerase (PARP) inhibitors, which was linked to an increase in replication stress, as detected by increased activation of the ataxia telangiectasia and Rad3-related (ATR) signaling axis. ATRX mutations frequently co-occur with mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2), and the latter mutations also induce HR defects and PARP inhibitor sensitivity. We found that the magnitude of PARP inhibitor sensitivity was equal in the context of each mutation alone, although no further sensitization was observed in combination, suggesting an epistatic interaction. Finally, we observed enhanced synergistic tumor cell killing in ATRX KO cells with ATR and PARP inhibition, which is commonly seen in HR-defective cells. Taken together, these data reveal that ATRX may be used as a molecular marker for DDR defects and PARP inhibitor sensitivity, independent of IDH1/2 mutations. These data highlight the important role of common glioma-associated mutations in the regulation of DDR, and novel avenues for molecularly guided therapeutic intervention.     

Genistein ameliorates cardiac inflammation and oxidative stress in streptozotocin-induced diabetic cardiomyopathy in rats

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.     

Regulation of cell differentiation by the DNA damage response

When faced with DNA double-strand breaks (DSBs), vertebrate cells activate DNA damage response (DDR) programs that preserve genome integrity and suppress malignant transformation. Three established outcomes of the DDR include transient cell cycle arrest coupled with DNA repair, apoptosis, or senescence. However, recent studies in normal and cancer precursor or stem cells suggest that a fourth potential outcome, cell differentiation, is under the influence of DDR programs. Here we review and discuss the emerging evidence that supports the linkage of signaling from DSBs to the regulation of differentiation, including some of the molecular mechanisms driving this under-appreciated DDR outcome. We also consider the physiologic and pathologic consequences of defects in DDR signaling on cell differentiation and malignant transformation.     

Role of deubiquitinases in DNA damage response

DNA damage response (DDR) serves as an integrated cellular network to detect cellular stress and react by activating pathways responsible for halting cell cycle progression, stimulating DNA damage repair, and initiating apoptosis. Efficient DDR protects cells from genomic instability while defective DDR can allow DNA lesions to go unrepaired, causing permanent mutations that will affect future generations of cells and possibly cause disease conditions such as cancer. Therefore, DDR mechanisms must be tightly regulated in order to ensure organismal health and viability. One major way of DDR regulation is ubiquitination, which has been long known to control DDR protein localization, activity, and stability. The reversal of this process, deubiquitination, has more recently come to the forefront of DDR research as an important new angle in ubiquitin-mediated regulation of DDR. As such, deubiquitinases have emerged as key factors in DDR. Importantly, deubiquitinases are attractive small-molecule drug targets due to their well-defined catalytic residues that provide a promising avenue for developing new cancer therapeutics. This review focuses on the emerging roles of deubiquitinases in various DNA repair pathways.     

The MRE11 complex: starting from the ends

The maintenance of genome stability depends on the DNA damage response (DDR), which is a functional network comprising signal transduction, cell cycle regulation and DNA repair. The metabolism of DNA double-strand breaks governed by the DDR is important for preventing genomic alterations and sporadic cancers, and hereditary defects in this response cause debilitating human pathologies, including developmental defects and cancer. The MRE11 complex, composed of the meiotic recombination 11 (MRE11), RAD50 and Nijmegen breakage syndrome 1 (NBS1; also known as nibrin) proteins is central to the DDR, and recent insights into its structure and function have been gained from in vitro structural analysis and studies of animal models in which the DDR response is deficient.     

Selective Induction of Necrotic Cell Death in Cancer Cells by β-Lapachone through Activation of DNA Damage Response Pathway

Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment. However, apoptotic pathway deficiencies are a hallmark of cancer cells. We propose that one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic cell death. Here we show that selective induction of necrotic cell death can be achieved by activation of the DNA damage response pathways. While β-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by β-lapachone in a variety of cancer cells. We found that β-lapachone, unlike DNA damaging chemotherapeutic agents, transiently activates PARP1, a main regulator of the DNA damage response pathway, both in vitro and in vivo. This occurs within minutes of exposure to β-lapachone, resulting in selective necrotic cell death. Inhibition of PAR blocked β-lapachone-induced necrosis. Furthermore, necrotic cell death induced by β-lapachone was significantly reduced in PARP1 knockout cell lines. Our data suggest that selective necrotic cell death can be induced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy     

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.