An SNP-guided microRNA map of fifteen common human disorders identifies a consensus disease phenocode aiming at principal components of the nuclear import pathway

Maintaining genome stability is crucial for all cells. The budding yeast Elg1 protein, the major subunit of a replication factor C-like complex, is important for genome stability, since cells lacking Elg1 exhibit increased recombination and chromosomal rearrangements. This genome maintenance function of Elg1 seems to be conserved in higher eukaryotes, since removal of the human Elg1 homolog, encoded by the ATAD5 gene, also causes genome instability leading to tumorigenesis. The fundamental molecular function of the Elg1/ATAD5-replication factor C-like complex (RLC) was, until recently, elusive, although Elg1/ATAD5-RLC was known to interact with the replication sliding clamp PCNA. Two papers have now reported that following DNA replication, the Elg1/ATAD5-RLC is required to remove PCNA from chromatin in yeast and human cells. In this Review, we summarize the evidence that Elg1/ATAD5-RLC acts as a PCNA unloader and discuss the still enigmatic relationship between the function of Elg1/ATAD5-RLC in PCNA unloading and the role of Elg1/ATAD5 in maintaining genomic stability.     

The emerging role of deubiquitinating enzymes in genomic integrity,diseases, and therapeutics

The addition of mono-ubiquitin or poly-ubiquitin chain to signaling proteins in response to DNA damage signal is thought to be a critical event that facilitates the recognition of DNA damage lesion site, the activation of checkpoint function, termination and checkpoint response and the recruitment of DNA repair proteins. Despite the ubiquitin modifiers, removal of ubiquitin from the functional proteins by the deubiquitinating enzymes (DUBs) plays an important role in orchestrating DNA damage response as well as DNA repair processes. Deregulated ubiquitination and deubiquitination could lead to genome instability that in turn causes tumorigenesis. Recent TCGA study has further revealed the connection between mutations in alteration of DUBs and various types of tumors. In addition, emerging drug design based on DUBs provides a new avenue for anti-cancer therapy. In this review, we will summarize the role of deubiquitination and specificity of DUBs, and highlight the recent discoveries of DUBs in the modulation of ubiquitin-mediated DNA damage response and DNA damage repair. We will furthermore discuss the DUBs involved in the tumorigenesis as well as interception of deubiquitination as a novel strategy for anti-cancer therapy.     

The Fbx4 tumor suppressor regulates cyclin D1 accumulation and prevents neoplastic transformation

Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes modulate the accumulation of key cell cycle regulatory proteins. Following the G(1)/S transition, SCF(Fbx4) targets cyclin D1 for proteasomal degradation, a critical event necessary for DNA replication fidelity. Deregulated cyclin D1 drives tumorigenesis, and inactivating mutations in Fbx4 have been identified in human cancer, suggesting that Fbx4 may function as a tumor suppressor. Fbx4(+/-) and Fbx4(-/-) mice succumb to multiple tumor phenotypes, including lymphomas, histiocytic sarcomas and, less frequently, mammary and hepatocellular carcinomas. Tumors and premalignant tissue from Fbx4(+/-) and Fbx4(-/-) mice exhibit elevated cyclin D1, an observation consistent with cyclin D1 as a target of Fbx4. Molecular dissection of the Fbx4 regulatory network in murine embryonic fibroblasts (MEFs) revealed that loss of Fbx4 results in cyclin D1 stabilization and nuclear accumulation throughout cell division. Increased proliferation in early passage primary MEFs is antagonized by DNA damage checkpoint activation, consistent with nuclear cyclin D1-driven genomic instability. Furthermore, Fbx4(-/-) MEFs exhibited increased susceptibility to Ras-dependent transformation in vitro, analogous to tumorigenesis observed in mice. Collectively, these data reveal a requisite role for the SCF(Fbx4) E3 ubiquitin ligase in regulating cyclin D1 accumulation, consistent with tumor suppressive function in vivo.     

Suppression of gross chromosomal rearrangements by a new alternative replication factor C complex

Defects in DNA replication fidelity lead to genomic instability. Gross chromosomal rearrangement (GCR), a type of genomic instability, is highly enhanced by various initial mutations affecting DNA replication. Frequent observations of GCRs in many cancers strongly argue the importance of maintaining high fidelity of DNA replication to suppress carcinogenesis. Recent genome wide screens in Saccharomyces cerevisiae identified a new GCR suppressor gene, ELG1, enhanced level of genome instability gene 1. Its physical interaction with proliferating cell nuclear antigen (PCNA) and complex formation with Rfc2-5p proteins suggest that Elg1 functions to load/unload PCNA onto DNA during a certain DNA metabolism. High level of DNA damage accumulation and enhanced phenotypes with mutations in genes involved in cell cycle checkpoints, homologous recombination (HR), or chromatin assembly in the elg1 strain suggest that Elg1p-Rfc2-5p functions in a fundamental DNA metabolism to suppress genomic instability.     

Poly(ADP-ribose) polymerase: a guardian angel protecting the genome and suppressing tumorigenesis

Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage generated either exogenously or endogenously. This post-translational modification is catalyzed by poly(ADP-ribose) polymerase (PARP, PARP-1, EC 2.4.2.30). It is proposed that this protein plays a multifunctional role in many cellular processes, including DNA repair, recombination, cell proliferation and death, as well as genomic stability. Chemical inhibitors of the enzyme, dominant negative or null mutations of PARP-1 cause a high degree of genomic instability in cells. Inhibition of PARP activity by chemical inhibitors renders mice or rats susceptible to carcinogenic agents in various tumor models, indicating a role for PARP-1 in suppressing tumorigenesis. Despite the above observations, PARP-1 knockout mice are generally not prone to the development of tumors. An enhanced tumor development was observed, however, when the PARP-1 null mutation was introduced into severely compromised immune-deficient mice (a mutation in DNA-dependent protein kinase) or mice lacking other DNA repair or chromosomal guardian molecules, such as p53 or Ku80. These studies indicate that PARP-1 functions as a cofactor to suppress tumorigenesis via its role in stabilization of the genome, and/or by interacting with other DNA strand break-sensing molecules. Studies using PARP-1 mutants and chemical inhibitors have started to shed light on the role of this protein and of the specific protein post-translational modification in the control of genomic stability and hence its involvement in cancer.     

Ambient oxygen promotes tumorigenesis

Oxygen serves as an essential factor for oxidative stress, and it has been shown to be a mutagen in bacteria. While it is well established that ambient oxygen can also cause genomic instability in cultured mammalian cells, its effect on de novo tumorigenesis at the organismal level is unclear. Herein, by decreasing ambient oxygen exposure, we report a ～50% increase in the median tumor-free survival time of p53-/- mice. In the thymus, reducing oxygen exposure decreased the levels of oxidative DNA damage and RAG recombinase, both of which are known to promote lymphomagenesis in p53-/- mice. Oxygen is further shown to be associated with genomic instability in two additional cancer models involving the APC tumor suppressor gene and chemical carcinogenesis. Together, these observations represent the first report directly testing the effect of ambient oxygen on de novo tumorigenesis and provide important physiologic evidence demonstrating its critical role in increasing genomic instability in vivo.     

Human POLB gene is mutated in high percentage of colorectal tumors

Previous small scale sequencing studies have indicated that DNA polymerase β (pol β) variants are present on average in 30% of human tumors of varying tissue origin. Many of these variants have been shown to have aberrant enzyme function in vitro and to induce cellular transformation and/or genomic instability in vivo, suggesting that their presence is associated with tumorigenesis or its progression. In this study, the human POLB gene was sequenced in a collection of 134 human colorectal tumors and was found to contain coding region mutations in 40% of the samples. The variants map to many different sites of the pol β protein and are not clustered. Many variants are nonsynonymous amino acid substitutions predicted to affect enzyme function. A subset of these variants was found to have reduced enzyme activity in vitro and failed to fully rescue pol β-deficient cells from methylmethane sulfonate-induced cytotoxicity. Tumors harboring variants with reduced enzyme activity may have compromised base excision repair function, as evidenced by our methylmethane sulfonate sensitivity studies. Such compromised base excision repair may drive tumorigenesis by leading to an increase in mutagenesis or genomic instability.     

The role of p53-mediated apoptosis as a crucial anti-tumor response to genomic instability: lessons from mouse models

Genomic instability is a major force driving human cancer development. A cellular safeguard against such genetic destabilization, which can ensue from defects in telomere maintenance, DNA repair, and checkpoint function, is activation of the p53 tumor suppressor protein, which commonly responds to these DNA damage signals by inducing apoptosis. If, however, p53 becomes inactivated, as is typical of many tumors and pre-cancerous lesions, then cells with compromised genome integrity pathways survive inappropriately, and the accrual of oncogenic lesions can fuel the carcinogenic process. Studies of mouse models have been instrumental in providing support for this idea. Mouse knockouts in genes important for telomere function, DNA damage checkpoint activation and DNA repair - both non-homologous end joining and homologous recombination - are prone to the development of genomic instability. As a consequence of these DNA damage signals, p53 becomes activated in cells of these mutant mice, leading to the induction of apoptosis, sometimes at the expense of organismal viability. This apoptotic response can be rescued through crosses to p53-deficient mice, but has dire consequences: mice predisposed to genomic instability and lacking p53 are susceptible to tumorigenesis. Thus p53-mediated apoptosis provides a crucial tumor suppressive mechanism to eliminate cells succumbing to genomic instability.     

Mutation at the polymerase active site of mouse DNA polymerase delta increases genomic instability and accelerates tumorigenesis

Mammalian DNA polymerase delta (Pol delta) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol delta. Homozygous Pold1(L604G/L604G) and Pold1(L604K/L604K) mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1(+/L604G) mice did not differ, while that of Pold1(+/L604K) mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1(+/L604K) mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase delta reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.     

The contribution of endogenous sources of DNA damage to the multiple mutations in cancer

There is increasing evidence that most human cancers contain multiple mutations. By the time a tumor is clinically detectable it may have accumulated tens of thousands of mutations. In normal cells, mutations are rare events occurring at a rate of 10(-10) mutations per nucleotide per cell per generation. We have argued that the mutation rates exhibited by normal human cells are insufficient to account for the large number of mutations found in human cancers, and therefore, that an early event in tumorigenesis is the development of a mutator phenotype. In normal cells, spontaneous and induced DNA damage is balanced by multiple pathways for DNA repair, and most DNA damage is repaired without error. However, in tumor cells this balance may be shifted such that damage overwhelms the repair capacity, resulting in the accumulation of multiple mutations. Our hypothesis is that multiple random mutations occur during carcinogenesis. The sequential mutations that are observed in some human tumors result from selective events required for tumor progression. We consider the possibility that endogenous sources of DNA damage, in particular oxidative DNA damage, may contribute to genomic instability and to a mutator phenotype in some tumors. Endogenous and environmental sources of reactive oxygen species (ROS) are abundant. In tumor cells, antioxidant or DNA repair capacity may be insufficient to compensate for the production of ROS, and these endogenous ROS may be capable of damaging DNA and inducing mutations in critical DNA stability genes. The possibility that oxidative DNA damage could be a significant source of the genomic instability characteristic of human cancers is exciting, because it may be feasible to modulate the extent of oxidative damage through antioxidant therapy. The use of antioxidants to reduce the extent of molecular damage by ROS could delay the progression of cancer.     

ATR functions as a gene dosage-dependent tumor suppressor on a mismatch repair-deficient background

The ataxia-telangiectasia mutated and rad3-related (ATR) kinase orchestrates cellular responses to DNA damage and replication stress. Complete loss of ATR function leads to chromosomal instability and cell death. However, heterozygous ATR mutations are found in human cancers with microsatellite instability, suggesting that ATR haploinsufficiency contributes to tumorigenesis. To test this possibility, we generated human cell line and mouse model systems in which a single ATR allele was inactivated on a mismatch repair (MMR)-deficient background. Monoallelic ATR gene targeting in MLH1-deficient HCT 116 colon carcinoma cells resulted in hypersensitivity to genotoxic stress accompanied by dramatic increases in fragile site instability, and chromosomal amplifications and rearrangements. The ATR(+/-) HCT 116 cells also displayed compromised activation of Chk1, an important downstream target for ATR. In complementary studies, we demonstrated that mice bearing the same Atr(+/-)/Mlh1(-/-) genotype were highly prone to both embryonic lethality and early tumor development. These results demonstrate that MMR proteins and ATR functionally interact during the cellular response to genotoxic stress, and that ATR serves as a haploinsufficient tumor suppressor in MMR-deficient cells.     

Combined PTEN Mutation and Protein Expression Associate with Overall and Disease-Free Survival of Glioblastoma Patients

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor commonly inactivated in glioblastoma multiforme (GBM), but the prognostic significance of PTEN remains controversial. Here, we demon- strate significant prognostic value of combined PTEN mutation and expression for the survival of patients with GBM on the basis of analysis of large-scale cancer genomic data. PTEN nonsense mutations associated with sig- nificantly shorter disease-free survival and overexpression of PTEN protein linked to shorter disease-free and overall survival of patients with GBM. PTEN nonsense mutations correlated with decreased p53 and Gata3 protein levels and increased genomic instability in human GBM tissues. Expression of nonsense PTEN mutant decreased p53 and Gata3 levels, producing increased DNA damage both in vitro and in vivo. Mice carrying xenograft tumors with nonsense PTEN mutant displayed significantly shorter survival. Our data demonstrated the prognostic value of combined PTEN mutation and protein expression for patients with GBM and highlighted distinct biologic effects of nonsense and missense mutations of PTEN.     

DNA strand break-sensing molecule poly(ADP-Ribose) polymerase cooperates with p53 in telomere function, chromosome stability, and tumor suppression

Genomic instability is often caused by mutations in genes that are involved in DNA repair and/or cell cycle checkpoints, and it plays an important role in tumorigenesis. Poly(ADP-ribose) polymerase (PARP) is a DNA strand break-sensing molecule that is involved in the response to DNA damage and the maintenance of telomere function and genomic stability. We report here that, compared to single-mutant cells, PARP and p53 double-mutant cells exhibit many severe chromosome aberrations, including a high degree of aneuploidy, fragmentations, and end-to-end fusions, which may be attributable to telomere dysfunction. While PARP(-/-) cells showed telomere shortening and p53(-/-) cells showed normal telomere length, inactivation of PARP in p53(-/-) cells surprisingly resulted in very long and heterogeneous telomeres, suggesting a functional interplay between PARP and p53 at the telomeres. Strikingly, PARP deficiency widens the tumor spectrum in mice deficient in p53, resulting in a high frequency of carcinomas in the mammary gland, lung, prostate, and skin, as well as brain tumors, reminiscent of Li-Fraumeni syndrome in humans. The enhanced tumorigenesis is likely to be caused by PARP deficiency, which facilitates the loss of function of tumor suppressor genes as demonstrated by a high rate of loss of heterozygosity at the p53 locus in these tumors. These results indicate that PARP and p53 interact to maintain genome integrity and identify PARP as a cofactor for suppressing tumorigenesis.     

Role of autophagy in breast cancer

Autophagy is an evolutionarily conserved process of cytoplasm and cellular organelle degradation in lysosomes. Autophagy is a survival pathway required for cellular viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. In neurons, constitutive autophagy limits accumulation of polyubiquitinated proteins and prevents neuronal degeneration. Therefore, autophagy has emerged as a homeostatic mechanism regulating the turnover of long-lived or damaged proteins and organelles, and buffering metabolic stress under conditions of nutrient deprivation by recycling intracellular constituents. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin1(+/-) mice are tumor-prone. We found that allelic loss of beclin1 renders immortalized mouse mammary epithelial cells susceptible to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. Thus, loss of the prosurvival role of autophagy likely contributes to breast cancer progression by promoting genome damage and instability. Exploring the yet unknown relationship between defective autophagy and other breast cancer promoting functions may provide valuable insight into the pathogenesis of breast cancer and may have significant prognostic and therapeutic implications for breast cancer patients.     

Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

ICAD Deficiency in Human Colon Cancer and Predisposition to Colon Tumorigenesis: Linkage to Apoptosis Resistance and Genomic Instability

We previously showed that DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-1-activating DNA breaks. Here we tested the hypothesis that ICAD-deficient colon epithelial cells exhibiting resistance to death stimuli may accumulate additional genetic modifications, leading to a tumorigenic phenotype. We show that ICAD deficiency may be associated with colon malignancy in humans. Indeed, an examination of ICAD expression using immunohistochemistry in an array of both colon cancer and normal tissues revealed that ICAD expression levels were severely compromised in the cancerous tissues. Upon DNA damage caused by a low dose of irradiation, ICAD cells acquire a tumorigenic phenotype. Colon epithelial cells derived from ICAD mice showed a significant resistance to death induced by the colon carcinogen dimethylhydrazine in vitro and in mice. Such resistance was associated with a decrease in PARP-1 activation. In an animal model of dimethylhydrazine-induced colon tumorigenesis, ICAD^−/− mice developed significantly higher numbers of tumors with markedly larger sizes than the wild-type counterparts. Interestingly, the phenotype of the ICAD^−/− mice was not associated with a significant increase in the precancerous aberrant crypt foci suggesting a potential link to tumor progression rather than initiation. More importantly, ICAD deficiency was associated with severe genomic instability as assessed by array comparative genomic hybridization. Such genomic instability consisted most prominently of amplifications but with sizable deletions as compared to the wild-type counterparts affecting several cancer-related genes including RAF-1, GSN, LMO3, and Fzd6 independently of p53. Altogether, our results present a viable case for the involvement of ICAD deficiency in colon carcinogenesis and show that apoptosis and genomic instability may comprise the means by which such deficiency may contribute to the process of increasing susceptibility to carcinogen-induced tumorigenesis.     

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.     

Timely termination of repair DNA synthesis by ATAD5 is important in oxidative DNA damage-induced single-strand break repair

Reactive oxygen species (ROS) generate oxidized bases and single-strand breaks (SSBs), which are fixed by base excision repair (BER) and SSB repair (SSBR), respectively. Although excision and repair of damaged bases have been extensively studied, the function of the sliding clamp, proliferating cell nuclear antigen (PCNA), including loading/unloading, remains unclear. We report that, in addition to PCNA loading by replication factor complex C (RFC), timely PCNA unloading by the ATPase family AAA domain-containing protein 5 (ATAD5)-RFC–like complex is important for the repair of ROS-induced SSBs. We found that PCNA was loaded at hydrogen peroxide (H₂O₂)-generated direct SSBs after the 3′-terminus was converted to the hydroxyl moiety by end-processing enzymes. However, PCNA loading rarely occurred during BER of oxidized or alkylated bases. ATAD5-depleted cells were sensitive to acute H₂O₂ treatment but not methyl methanesulfonate treatment. Unexpectedly, when PCNA remained on DNA as a result of ATAD5 depletion, H₂O₂-induced repair DNA synthesis increased in cancerous and normal cells. Based on higher H₂O₂-induced DNA breakage and SSBR protein enrichment by ATAD5 depletion, we propose that extended repair DNA synthesis increases the likelihood of DNA polymerase stalling, shown by increased PCNA monoubiquitination, and consequently, harmful nick structures are more frequent.