c-Myc can induce DNA damage,increase reactive oxygen species,and mitigate p53 function: a mechanism for oncogene-induced genetic instability

Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. We determined whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlated with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disabled the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduced ROS, decreased DNA damage and p53 activation, and improved survival. We propose that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability.     

Mutagenesis induced by the tumor microenvironment

Genomic instability is a commonly observed feature of tumors. Most investigations addressing the mechanism of tumor progression have focused on the genetic factors that may play a role. Growing evidence now suggests that, in addition to these endogenous factors, the exogenous environment within solid tumors may by itself be mutagenic and constitute a significant source of genetic instability. The tumor microenvironment is characterized by regions of fluctuating hypoxia, low pH, and nutrient deprivation. Each of these microenvironmental factors has been shown to cause severe disturbance in cell metabolism and physiology. Both in vivo and in vitro data demonstrate that exposure of tumor cells to adverse conditions can directly cause mutations, contributing to genetic instability. In this review, we will reexamine the current body of evidence on the role of the tumor microenvironment in inducing mutagenesis and consequent tumor progression.     

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.     

Non-heme dioxygenases in tumor hypoxia: They’re all bound with the same fate

Tumor tissues are known to harbor hypoxic areas. The hypoxic microenvironment promotes angiogenesis. Hypoxic tumor cells also manifest genome instability. DNA damage repair pathways, such as double-strand break repair, mismatch repair and base excision repair are known to be altered during hypoxia. This review is focused on the non-heme Fe(II) and 2-oxoglutarate-dependent dioxygenases which are involved in repair of DNA alkylation adducts. Activities of these DNA repair enzymes are completely oxygen-dependent and little information is available about inhibition of these enzymes during hypoxia. While impairment of function of non-heme dioxygenase during tumor hypoxia has been implicated in different studies, the possible outcomes with respect to mutagenesis and genomic instability are explored here.     

Hypoxia relieves X-ray-induced delayed effects in normal human embryo cells

We studied the effect of hypoxia on X-ray-induced delayed effects in normal human embryo cells to elucidate the role of oxidative stress in the susceptibility of cells to induction of genetic instability by radiation. We examined X-ray-induced delayed cell death, giant cell formation, and chromosome aberrations under normally oxygenated (20%) and hypoxic (2%) conditions at 28-38 population doublings postirradiation. The results revealed that hypoxia reduced the X-ray-induced delayed effects, suggesting that radiation enhances cellular oxidative stress, which plays a significant role in determining the susceptibility of irradiated cells to genetic instability. The present study emphasizes the biological significance of epigenetic effects, such as oxygen tension, as well as direct DNA damage in the induction of genetic instability by radiation.     

Dietary phytochemicals, HDAC inhibition, and DNA damage/repair defects in cancer cells

Genomic instability is a common feature of cancer etiology. This provides an avenue for therapeutic intervention, since cancer cells are more susceptible than normal cells to DNA damaging agents. However, there is growing evidence that the epigenetic mechanisms that impact DNA methylation and histone status also contribute to genomic instability. The DNA damage response, for example, is modulated by the acetylation status of histone and non-histone proteins, and by the opposing activities of histone acetyltransferase and histone deacetylase (HDAC) enzymes. Many HDACs overexpressed in cancer cells have been implicated in protecting such cells from genotoxic insults. Thus, HDAC inhibitors, in addition to unsilencing tumor suppressor genes, also can silence DNA repair pathways, inactivate non-histone proteins that are required for DNA stability, and induce reactive oxygen species and DNA double-strand breaks. This review summarizes how dietary phytochemicals that affect the epigenome also can trigger DNA damage and repair mechanisms. Where such data is available, examples are cited from studies in vitro and in vivo of polyphenols, organosulfur/organoselenium compounds, indoles, sesquiterpene lactones, and miscellaneous agents such as anacardic acid. Finally, by virtue of their genetic and epigenetic mechanisms, cancer chemopreventive agents are being redefined as chemo- or radio-sensitizers. A sustained DNA damage response coupled with insufficient repair may be a pivotal mechanism for apoptosis induction in cancer cells exposed to dietary phytochemicals. Future research, including appropriate clinical investigation, should clarify these emerging concepts in the context of both genetic and epigenetic mechanisms dysregulated in cancer, and the pros and cons of specific dietary intervention strategies.     

The antioxidant function of the p53 tumor suppressor

It is widely accepted that the p53 tumor suppressor restricts abnormal cells by induction of growth arrest or by triggering apoptosis. Here we show that, in addition, p53 protects the genome from oxidation by reactive oxygen species (ROS), a major cause of DNA damage and genetic instability. In the absence of severe stresses, relatively low levels of p53 are sufficient for upregulation of several genes with antioxidant products, which is associated with a decrease in intracellular ROS. Downregulation of p53 results in excessive oxidation of DNA, increased mutation rate and karyotype instability, which are prevented by incubation with the antioxidant N-acetylcysteine (NAC). Dietary supplementation with NAC prevented frequent lymphomas characteristic of Trp53-knockout mice, and slowed the growth of lung cancer xenografts deficient in p53. Our results provide a new paradigm for a nonrestrictive tumor suppressor function of p53 and highlight the potential importance of antioxidants in the prophylaxis and treatment of cancer.     

Cigarette smoke metabolically promotes cancer,via autophagy and premature aging in the host stromal microenvironment

Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells.     

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.     

Distinct functions for WRN and TP53 in a shared pathway of cellular response to 1-beta-D-arabinofuranosylcytosine and bleomycin

Mutations in the WRN or the TP53 genes lead to spontaneous genetic instability, an elevated risk of tumor formation, and sensitivity to compounds that interfere with DNA replication, such as camptothecin and DNA interstrand cross-linking drugs. We investigated the hypothesis that WRN and TP53 are involved in cellular responses to DNA replication-blocking lesions by exposing WRN deficient and TP53 mutant lymphoblastoid cell lines (LCLs) to 1-beta-d-arabinofuranosylcytosine (AraC) and bleomycin. Loss of WRN or TP53 function resulted in induction of apoptosis and lesser proliferative survival in response to AraC and bleomycin. WRN and TP53 operate in a shared DNA damage response pathway, since in cells in which TP53 was inactivated by SV-40 transformation, no difference in AraC and bleomycin sensitivity was found regardless of WRN status. In contrast to TP53 mutant LCLs, WRN-deficient cells showed unaffected cell cycle arrest after AraC and bleomycin exposure, which indicates that WRN is not involved in DNA damage-activated cell cycle arrest. Neither WRN nor TP53 deficiency affected cellular recovery from exposure to AraC and bleomycin, which disagrees with a direct role in repair of these DNA lesions. Our results indicate that WRN and TP53 perform different functions in a shared DNA damage response pathway.     

A p53-dependent checkpoint pathway prevents rereplication

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.     

Delayed activation of DNA damage checkpoint and radiation-induced genomic instability

Ionizing radiation induces genomic instability, transmitted over many generations through the progeny of surviving cells. It is manifested as the expression of delayed effects such as delayed cell death, delayed chromosomal instability and delayed mutagenesis. Induced genomic instability exerts its delayed effects for prolonged periods of time, suggesting the presence of a mechanism by which the initial DNA damage in the surviving cells is memorized. Our recent studies have shown that transmitted memory causes delayed DNA breakage, which in turn activates DNA damage checkpoint, and is involved in delayed manifestation of genomic instability. Although the mechanism(s) involved in DNA damage memory remain to be determined, we suggest that ionizing radiation-induced mega-base deletion destabilizes chromatin structure, which can be transmitted many generations through the progeny, and is involved in initiation and perpetuation of genomic instability. The possible involvement of delayed activation of a DNA damage checkpoint in the delayed induction of genomic instability in bystander cells is also discussed.     

Total-genome analysis of BRCA1/2-related invasive carcinomas of the breast identifies tumor stroma as potential landscaper for neoplastic initiation

We have shown that the tumor microenvironment of sporadic breast cancer is diverse in genetic alterations and contributes to the cancer phenotype. The dynamic morphology of the mammary gland might be of special interest in hereditary breast/ovarian cancer syndrome (HBOC). We hypothesized that hotspots of loss of heterozygosity or allelic imbalance (LOH/AI) within the tumor stroma of BRCA1/2-related breast cancers provide an impaired mammary stroma that could facilitate later malignant transformation of the breast epithelium. We conducted a total genome LOH/AI scan of DNA derived from the epithelium and stroma of 51 BRCA1/2-related breast cancers, using 372 microsatellite markers. We compared these data with those from a set of 134 sporadic breast cancers. HBOC-related breast cancers accumulated significantly more genetic alterations than did sporadic breast cancers. BRCA1/2-related breast cancer stroma showed LOH/AI at 59.7% of all loci analyzed, similar to the average frequency of LOH/AI observed in the epithelium (66.2%). This is remarkably different from sporadic breast cancers, for which the average epithelial LOH/AI frequency (36.7%) far exceeds the average stromal LOH/AI frequency (28.4%) (P=.03). We identified 11 hotspot loci of LOH/AI in the BRCA1/2 stroma, encompassing genes such as POLD1, which functions in DNA replication, and SDHB. In a subset of samples, enriched for BRCA1 cases, we found 45.0% overall LOH/AI in the stroma, which was significantly higher than the 41.8% LOH/AI observed in corresponding epithelium (P=.04). Together, our data indicate that, in HBOC-related breast cancers, the accumulation of genomic instability in the cancer stroma coincides with that in the neoplastic epithelium, and we postulate that such a genetically unstable stroma might facilitate a microenvironment that functions as a landscaper that promotes genomic instability in the epithelium and, subsequently, neoplastic transformation.     

细胞周期检控点

细胞周期是高度有组织的时序调控过程,受到DNA损伤检控点、DNA复制检控点和纺锤体检控点等细胞周期检控点的精确调控。细胞周期检控点的作用主要是调节细胞周期的时序转换,以确保DNA复制、染色体分离等细胞重要生命活动的高度精确性,并对DNA损伤、DNA复制受阻、纺锤体组装和染色体分离异常等细胞损伤及时做出反应,以防止突变和遗传不稳定的发生。细胞周期检控点的功能缺陷,将导致细胞基因组的不稳定,与细胞癌变密切相关。因此细胞周期检控点对于维持细胞遗传信息的稳定性和完整性以及防止细胞癌变和遗传疾病的发生起着至关重要的作用。     

DNA damage-mediated induction of a chemoresistant niche

While numerous cell-intrinsic processes are known to play decisive roles in chemotherapeutic response, relatively little is known about the impact of the tumor microenvironment on therapeutic outcome. Here, we use a well-established mouse model of Burkitt's lymphoma to show that paracrine factors in the tumor microenvironment modulate lymphoma cell survival following the administration of genotoxic chemotherapy. Specifically, IL-6 and Timp-1 are released in the thymus in response to DNA damage, creating a "chemo-resistant niche" that promotes the survival of a minimal residual tumor burden and serves as a reservoir for eventual tumor relapse. Notably, IL-6 is released acutely from thymic endothelial cells in a p38-dependent manner following genotoxic stress, and this acute secretory response precedes the gradual induction of senescence in tumor-associated stromal cells. Thus, conventional chemotherapies can induce tumor regression while simultaneously eliciting stress responses that protect subsets of tumor cells in select anatomical locations from drug action.     

Role of induced genetic instability in the mutagenic effects of chemicals and radiation

Recent studies have demonstrated that cells exposed to ionizing radiation or alkylating agents can develop prolonged genetic instability. Induced genetic instability is manisested in multiple ways, including delayed reproductive death, an increased rate of point mutations, and an increased rate of chromosome rearrangements. In many respects these changes are similar to the genetic instability associated with cancer and some human genetic diseases. Therefore, as with cancer cells, multiple mechanisms may be involved, some occuring in the early stages and some in the later stages. The high percentage of cells that develop induced genetic instability after exposure to stress, and the prolonged period over which the instability occurs, indicates that the instability is not in response to residual damage in the DNA or mutations in specific genes. Instead, changes affecting most of the exposed cells, such as epigenetic alterations in gene expression or chain reactions of chromosome rearrangements, are a more likely explanation. Learning more about the mechanisms involved in this process is essential for understanding the consequences of exposure of cells to ionizing radiation or alkylating agents.     

Dysregulation of lysophospholipid signaling by p53 in malignant cells and the tumor microenvironment

The TP53 gene has been widely studied for its roles in cell cycle control, maintaining genome stability, activating repair mechanisms upon DNA damage, and initiating apoptosis should repair mechanisms fail. Thus, it is not surprising that mutations of p53 are the most common genetic alterations found in human cancer. Emerging evidence indicates that dysregulation of lipid metabolism by p53 can have a profound impact not only on cancer cells but also cells of the tumor microenvironment (TME). In particular, intermediates of the sphingolipid and lysophospholipid pathways regulate many cellular responses common to p53 such as cell survival, migration, DNA damage repair and apoptosis. The majority of these cellular events become dysregulated in cancer as well as cell senescence. In this review, we will provide an account on the seminal contributions of Prof. Lina Obeid, who deciphered the crosstalk between p53 and the sphingolipid pathway particularly in modulating DNA damage repair and apoptosis in non-transformed as well as transformed cells. We will also provide insights on the integrative role of p53 with the lysophosphatidic acid (LPA) signaling pathway in cancer progression and TME regulation.