XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species

Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPC(KD)) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers.     

Novel Oncogenic Mutations of CBL in Human Acute Myeloid Leukemia That Activate Growth and Survival Pathways Depend on Increased Metabolism

Acute myeloid leukemia (AML) is characterized by multiple mutagenic events that affect proliferation, survival, as well as differentiation. Recently, gain-of-function mutations in the α helical structure within the linker sequence of the E3 ubiquitin ligase CBL have been associated with AML. We identified four novel CBL mutations, including a point mutation (Y371H) and a putative splice site mutation in AML specimens. Characterization of these two CBL mutants revealed that coexpression with the receptor tyrosine kinases FLT3 (Fms-like tyrosine kinase 3) or KIT-induced ligand independent growth or ligand hyperresponsiveness, respectively. Growth of cells expressing mutant CBL required expression and kinase activity of FLT3. In addition to the CBL-dependent phosphorylation of FLT3 and CBL itself, transformation was associated with activation of Akt and STAT5 and required functional expression of the small GTPases Rho, Rac, and Cdc42. Furthermore, the mutations led to constitutively elevated intracellular reactive oxygen species levels, which is commonly linked to increased glucose metabolism in cancer cells. Inhibition of hexokinase with 2-deoxyglucose blocked the transforming activity of CBL mutants and reduced activation of signaling mechanisms. Overall, our data demonstrate that mutations of CBL alter cellular biology at multiple levels and require not only the activation of receptor proximal signaling events but also an increase in cellular glucose metabolism. Pathways that are activated by CBL gain-of-function mutations can be efficiently targeted by small molecule drugs.     

Aneuploidy, the primary cause of the multilateral genomic instability of neoplastic and preneoplastic cells

Cancers have a clonal origin, yet their chromosomes and genes are non-clonal or heterogeneous due to an inherent genomic instability. However, the cause of this genomic instability is still debated. One theory postulates that mutations in genes that are involved in DNA repair and in chromosome segregation are the primary causes of this instability. But there are neither consistent correlations nor is there functional proof for the mutation theory. Here we propose aneuploidy, an abnormal number of chromosomes, as the primary cause of the genomic instability of neoplastic and preneoplastic cells. Aneuploidy destabilizes the karyotype and thus the species, independent of mutation, because it corrupts highly conserved teams of proteins that segregate, synthesize and repair chromosomes. Likewise it destabilizes genes. The theory explains 12 of 12 specific features of genomic instability: (1) Mutagenic and non-mutagenic carcinogens induce genomic instability via aneuploidy. (2) Aneuploidy coincides and segregates with preneoplastic and neoplastic genomic instability. (3) Phenotypes of genomically unstable cells change and even revert at high rates, compared to those of diploid cells, via aneuploidy-catalyzed chromosome rearrangements. (4) Idiosyncratic features of cancers, like immortality and drug-resistance, derive from subspecies within the 'polyphyletic' diversity of individual cancers. (5) Instability is proportional to the degree of aneuploidy. (6) Multilateral chromosomal and genetic instabilities typically coincide, because aneuploidy corrupts multiple targets simultaneously. (7) Gene mutation is common, but neither consistent nor clonal in cancer cells as predicted by the aneuploidy theory. (8) Cancers fall into a near-diploid (2 N) class of low instability, a near 1.5 N class of high instability, or a near 3 N class of very high instability, because aneuploid fitness is maximized either by minimally unstable karyotypes or by maximally unstable, but adaptable karyotypes. (9) Dominant phenotypes, because of aneuploid genotypes. (10) Uncertain developmental phenotypes of Down and other aneuploidy syndromes, because supply-sensitive, diploid programs are destabilized by products from aneuploid genes supplied at abnormal concentrations; the maternal age-bias for Down's would reflect age-dependent defects of the spindle apparatus of oocytes. (11) Non-selective phenotypes, e.g., metastasis, because of linkage with selective phenotypes on the same chromosomes. (12) The target, induction of genomic instability, is several 1000-fold bigger than gene mutation, because it is entire chromosomes. The mutation theory explains only a few of these features. We conclude that the transition of stable diploid to unstable aneuploid cell species is the primary cause of preneoplastic and neoplastic genomic instability and of cancer, and that mutations are secondary.     

The Ever Expanding Role for c-Myc in Promoting Genomic Instability

Genomic instability (GI) is a hallmark of many cancers. GI is believed toconfer upon impending neoplastic cells the ability to accumulate all of therequisite mutations for transformation within the relatively short time-frame of anorganism’s lifespan. Recently described properties of the c-Myc oncoproteinshow that, in addition to its directly transforming role, it can mediate GI via theinduction of reactive oxygen species and by promoting whole chromosomeinstability leading to tetraploidy and aneuploidy. Mediators of both propertieshave been identified and have begun to provide a framework within which tounderstand not only how c-Myc alters the genome but how it might alsocooperate with its more traditional transforming activities. These genome-alteringproperties of c-Myc suggest that they provide the protein with the ability to confera “mutator phenotype” to cells in which its expression is de-regulated.     

Genomic instability induced by mutant succinate dehydrogenase subunit D (SDHD) is mediated by O2(-•) and H2O2

SDHD mutations are associated with human cancers but the mechanisms that may contribute to transformation are unknown. The hypothesis that mutations in SDHD increase levels of superoxide leading to genomic instability was tested using site-directed mutagenesis to generate a truncated SDHD cDNA that was expressed in Chinese hamster fibroblasts. Stable expression of mutant SDHD resulted in 2-fold increases in steady-state levels of superoxide that were accompanied by a significantly increased mutation rate as well as a 70-fold increase in mutation frequency at the hprt locus. Overexpression of MnSOD or treatment with polyethylene glycol conjugated (PEG)-catalase suppressed mutation frequency in SDHD mutant cells by 50% (P<0.05). Simultaneous treatment with PEG-catalase and PEG-SOD suppressed mutation frequency in SDHD mutant cells by 90% (P<0.0005). Finally, 95% depletion of glutathione using l-buthionine-[S,R]-sulfoximine (BSO) in SDHD mutant cells caused a 4-fold increase in mutation frequency (P<0.05). These results demonstrate that mutations in SDHD cause increased steady-state levels of superoxide which significantly contributed to increases in mutation rates and frequency mediated by superoxide and hydrogen peroxide. These results support the hypothesis that mutations in SDHD may contribute to carcinogenesis by increasing genomic instability mediated by increased steady-state levels of reactive oxygen species.     

Transmembrane domain is crucial to the subcellular localization and function of Myc target 1

Deregulation of c‐MYC occurs in a variety of human cancers. Overexpression of c‐MYC promotes cell growth, proliferation, apoptosis, transformation and genomic instability. MYC target 1 (MYCT1) is a direct target gene of c‐MYC, and its murine homologue MT‐MC1 recapitulated multiple c‐Myc‐related phenotypes. However, the molecular mechanism of MYCT1 remains unclear. Here, we identified the transmembrane (TM) domain of MYCT1, not the nuclear localization sequence, is indispensable to the vesicle‐associated localization of MYCT1 protein in the cytoplasmic membrane vesicle. Overexpression of MYCT1, not MYCT1 (ΔTM), decreased cell viability under serum deprivation and increased tumour cell migration ability. We further identified CKAP4 interacted with MYCT1 and contributed to the function of MYCT1. In addition, we found that a mutation, A88D, which is observed in patient sample, changed the localization, and abolished the effect on cell viability and cell migration, suggesting that the TM domain is critical to MYCT1.     

Proliferation and tissue remodeling in cancer: the hallmarks revisited

Although cancers are highly heterogeneous at the genomic level, they can manifest common patterns of gene expression. Here, we use gene expression signatures to interrogate two major processes in cancer, proliferation and tissue remodeling. We demonstrate that proliferation and remodeling signatures are partially independent and result in four distinctive cancer subtypes. Cancers with the proliferation signature are characterized by signatures of p53 and PTEN inactivation and concomitant Myc activation. In contrast, remodeling correlates with RAS, HIF-1α and NFκB activation. From the metabolic point of view, proliferation is associated with upregulation of glycolysis and serine/glycine metabolism, whereas remodeling is characterized by a downregulation of oxidative phosphorylation. Notably, the proliferation signature correlates with poor outcome in lung, prostate, breast and brain cancer, whereas remodeling increases mortality rates in colorectal and ovarian cancer.     

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.     

Opposite Roles for p38MAPK-Driven Responses and Reactive Oxygen Species in the Persistence and Resolution of Radiation-Induced Genomic Instability

We report the functional and temporal relationship between cellular phenotypes such as oxidative stress, p38MAPK-dependent responses and genomic instability persisting in the progeny of cells exposed to sparsely ionizing low-Linear Energy Transfer (LET) radiation such as X-rays or high-charge and high-energy (HZE) particle high-LET radiation such as ⁵⁶Fe ions. We found that exposure to low and high-LET radiation increased reactive oxygen species (ROS) levels as a threshold-like response induced independently of radiation quality and dose. This response was sustained for two weeks, which is the period of time when genomic instability is evidenced by increased micronucleus formation frequency and DNA damage associated foci. Indicators for another persisting response sharing phenotypes with stress-induced senescence, including beta galactosidase induction, increased nuclear size, p38MAPK activation and IL-8 production, were induced in the absence of cell proliferation arrest during the first, but not the second week following exposure to high-LET radiation. This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous ROS levels with a catalase inhibitor reduced the level of genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization.     

ATM deficiency and oxidative stress: a new dimension of defective response to DNA damage

ATM is one of the sentries at the gate of genome stability. This multifunctional protein kinase orchestrates the intricate array of cellular responses to DNA double-strand breaks. Absence or inactivation of ATM leads to the pleiotropic genetic disorder ataxia-telangiectasia (A-T), whose hallmarks are neuronal degeneration, immunodeficiency, genomic instability, premature aging and cancer predisposition. Several features of the complex clinical and cellular phenotype of A-T are reminiscent of other syndromes involving neurodegeneration, premature aging or genomic instability. A common denominator of many of these conditions is the perturbation of the cellular balance of reactive oxygen species, which leads to constant oxidative stress. Of these disorders, ATM deficiency is one of the most extensively studied with regard to the genome instability-oxidative stress connection. This connection may provide new insights into the phenotypes associated with genetic deficiencies of DNA damage responses, and point to new strategies to alleviate some of their clinical symptoms.     

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.     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.