Patterns of DNA damage response in intracranial germ cell tumors versus glioblastomas reflect cell of origin rather than brain environment: Implications for the anti‐tumor barrier concept and treatment

The DNA damage response (DDR) machinery becomes commonly activated in response to oncogenes and during early stages of development of solid malignancies, with an exception of testicular germ cell tumors (TGCTs). The active DDR signaling evokes cell death or senescence but this anti‐tumor barrier can be breached by defects in DDR factors, such as the ATM‐Chk2‐p53 pathway, thereby allowing tumor progression. The DDR barrier is strongly activated in brain tumors, particularly gliomas, due to oxidative damage and replication stress. Here, we took advantage of rare human primary intracranial germ cell tumors (PIGCTs), to address the roles of cell‐intrinsic factors including cell of origin, versus local tissue environment, in the constitutive DDR activation in vivo. Immunohistochemical analysis of 7 biomarkers on a series of 21 PIGCTs (germinomas and other subtypes), 20 normal brain specimens and 20 glioblastomas, revealed the following: i) The overall DDR signaling (γH2AX) and activation of the ATM‐Chk2‐p53 pathway were very low among the PIGCTs, reminiscent of TGCTs, and contrasting sharply with strong DDR activation in glioblastomas; ii) Except for one case of embryonal carcinoma, there were no clear aberrations in the ATM‐Chk2‐p53 pathway components among the PIGCT cohort; iii) Subsets of PIGCTs showed unusual cytosolic localization of Chk2 and/or ATM. Collectively, these results show that PIGCTs mimic the DDR activation patterns of their gonadal germ cell tumor counterparts, rather than the brain tumors with which they share the tissue environment. Hence cell‐intrinsic factors and cell of origin dictate the extent of DDR barrier activation and also the ensuing pressure to select for DDR defects. Our data provide conceptually important insights into the role of DNA damage checkpoints in intracranial tumorigenesis, with implications for the differential biological responses of diverse tumor types to endogenous stress as well as to genotoxic treatments such as ionizing radiation or chemotherapy.     

Constitutive overexpression of CDC25A in primary human mammary epithelial cells results in both defective DNA damage response and chromosomal breaks at fragile sites

CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. There are several lines of evidence that indicate a role for CDC25A in cancer development, consistent with the fact that its overexpression is detected in human cancers. In particular we previously reported that CDC25A is overexpressed also in early breast carcinoma. Recent data suggest that oncogene activation during early stages of tumor development causes DNA replication stress resulting in the induction of DNA damage response (DDR) and that the selection of cells defecting in their DDR could lead to malignant progression. To address how CDC25A overexpression contributes to breast cancer development we established a cell model in which CDC25A was constitutively overexpressed in hTERT-immortalized primary human mammary epithelial cells. At the earliest passages following CDC25A transduction we observed DDR signs associated with unscheduled DNA replication origins. In the latest passages DDR was significantly impaired and, even after ionizing radiation exposition, cells failed to induce G1 and G2 checkpoints; moreover DNA replication stress conditions, such as aphidicolin treatment, highlighted increased fragile site breakages and destabilized chromosomes just in these latest passages cells. Our data suggest that CDC25A overexpression, pushing the cell through the cell cycle transitions, induces DDR alterations that might enhance genomic instability.     

Oncogene- and tumor suppressor gene-mediated suppression of cellular senescence

Data accumulating during the last two decades suggest that tumorigenesis is held in check by two major intrinsic failsafe mechanisms; apoptosis and cellular senescence. While apoptosis is a programmed cell death process, cellular senescence, which is the focus of this article, is defined as irreversible cell cycle arrest. This process is triggered either by telomere erosion or by acute stress signals including oncogenic stress induced by overactive oncogenes or underactive tumor suppressor genes. The outcome of this is often replication overload and oxidative stress resulting in DNA damage. Oncogenic stress induces at least three intrinsic pathways, p16/pRb-, Arf/p53/p21- and the DNA damage response (DDR)-pathways, that induce premature senescence if the stress exceeds a threshold level. Oncogene-induced senescence (OIS) is frequently observed in premalignant lesions both in animal tumor models and in human patients but is essentially absent in advanced cancers, suggesting that malignant tumor cells have found ways to bypass or escape senescence. This review focuses on cell-autonomous mechanism by which certain oncogenes, tumor suppressor genes and components of the DDR/DNA-repair machinery suppress senescence – mechanisms that are exploited by tumor cells to evade senescence and continue to multiply. In this way, tumor cells become addicted to the continuous activity of senescence suppressor proteins. However, some senescence pathways, although under suppression, may remain intact and can be re-established if senescence suppressor proteins are inactivated or if senescence inducers are reactivated. This can hopefully form the basis for a “pro-senescence therapy” strategy to combat cancer in the future.     

ATM- and ATR-mediated response to DNA damage induced by a novel camptothecin,ST1968

DNA damage response and checkpoint activation are expected to influence the sensitivity to DNA-damaging agents. This study was designed to investigate the DNA damage response to the novel camptothecin, ST1968, in two tumor cell lines with a different biological background (A2780 and KB), which underwent distinct cell cycle perturbations and cell death modalities. Following treatment with the camptothecin or ionizing radiation, both inducing double-strand DNA breaks, the ovarian carcinoma A2780 cells exhibited activation of the ATM-Chk2 pathway and early induction of apoptosis. In contrast, the squamous carcinoma KB cells exhibited activation of ATR-Chk1 pathway, a persistent G₂/M-phase arrest, cellular senescence, mitotic catastrophe and delayed apoptosis, suggesting a defective ATM pathway. The cellular response to UV-induced DNA damage, which activates ATR-Chk1 pathway, was similar in the two cell lines exhibiting early apoptosis induction. Inhibition of ATM in A2780 cells, resulting in reduced phosphorylation of Chk2, enhanced ST1968-induced apoptosis, but had no effect in KB cells. The susceptibility to camptothecin-induced apoptosis of A2780 cells was likely p53-dependent but not related to the activation of the ATM pathway. In contrast, the inhibition of Chk1 enhanced apoptosis response in KB cell but not in A2780. Thus, depending on the biological context, the camptothecin activated ATM-Chk2 or ATR-Chk1 pathways, both having a protective role. In conclusion, our results are consistent with the interpretation that the modality of cell death response is not the critical determinant of sensitivity to camptothecins, and support the interest of inhibition of checkpoint kinases to improve the efficacy of camptothecins.     

Retinoblastoma pathway defects show differential ability to activate the constitutive DNA damage response in human tumorigenesis

Loss of G(1)-S control and aberrations of the p16(Ink4a)-cyclin D1/cyclin-dependent kinase (CDK) 4(6)-pRb-E2F-cyclin E/CDK2 pathway are common in human cancer. Previous studies showed that oncogene-induced aberrant proliferation, such as on cyclin E overexpression, causes DNA damage and checkpoint activation. Here, we show that, in a series of human colorectal adenomas, those with deregulation of cyclin D1 and/or p16(Ink4a) showed little evidence of constitutive DNA damage response (DDR), contrary to cyclin E-overexpressing higher-grade cases. These observations were consistent with diverse cell culture models with differential defects of retinoblastoma pathway components, as overexpression of cyclin D1 or lack of p16(Ink4a), either alone or combined, did not elicit detectable DDR. In contrast, inactivation of pRb, the key component of the pathway, activated the DDR in cultured human or mouse cells, analogous to elevated cyclin E. These results highlight differential effect of diverse oncogenic events on driving the 'cancer cell cycles' and their ability to deregulate the replication-driving CDK2 kinase and to alarm the DDR as a potential anticancer barrier in accordance with their hierarchical positions along the retinoblastoma pathway. Our data provide new insights into oncogene-evoked DDR in human tumorigenesis, with potential implications for individualized management of tumors with elevated cyclin D1 versus cyclin E, due to their distinct clinical variables and biological behavior.     

Chemotherapy induced DNA damage response: Convergence of drugs and pathways

Chemotherapeutics target rapidly dividing cancer cells by directly or indirectly inducing DNA damage. Upon recognizing DNA damage, cells initiate a variety of signaling pathways collectively referred to as the DNA damage response (DDR). Interestingly, the pathways used to elicit this response are as varied as the types of DNA damage induced. However, the activation of these various pathways has similar results including DNA repair, suppression of global general translation, cell cycle arrest and, ultimately, either cell survival or cell death. This review will focus on a series of chemotherapy-induced DNA lesions and highlight recent advances in our understanding of the DDR, the DNA repair pathways it activates and the cellular consequences of these converging pathways.     

Targeting ATR in DNA damage response and cancer therapeutics

The ataxia telangiectasia and Rad3-related (ATR) plays an important role in maintaining genome integrity during DNA replication through the phosphorylation and activation of Chk1 and regulation of the DNA damage response. Preclinical studies have shown that disruption of ATR pathway can exacerbate the levels of replication stress in oncogene-driven murine tumors to promote cell killing. Additionally, inhibition of ATR can sensitise tumor cells to radiation or chemotherapy. Accumulating evidence suggests that targeting ATR can selectively sensitize cancer cells but not normal cells to DNA damage. Furthermore, in hypoxic conditions, ATR blockade results in overloading replication stress and DNA damage response causing cell death. Despite the attractiveness of ATR inhibition in the treatment of cancer, specific ATR inhibitors have remained elusive. In the last two years however, selective ATR inhibitors suitable for in vitro and – most recently – in vivo studies have been identified. In this article, we will review the literature on ATR function, its role in DDR and the potential of ATR inhibition to enhance the efficacy of radiation and chemotherapy.     

REST‐dependent expression of TRF2 renders non‐neuronal cancer cells resistant to DNA damage during oxidative stress

REST is a neuronal gene silencing factor ubiquitously expressed in non‐neuronal tissues. REST is additionally believed to serve as a tumor suppressor in non‐neuronal cancers. Conversely, recent findings on REST‐dependent tumorigenesis in non‐neuronal cells consistently suggest a potential role of REST as a tumor promoter. Here, we have uncovered for the first time the mechanism by which REST contributes to cancer cell survival in non‐neuronal cancers. We observed abundant expression of REST in various types of non‐neuronal cancer cells compared to normal tissues. The delicate roles of REST were further evaluated in HCT116 and HeLa, non‐neuronal cancer cell lines expressing REST. REST silencing resulted in decreased cell survival and activation of the DNA damage response (DDR) through a decrease in the level of TRF2, a telomere‐binding protein. These responses were correlated with reduced colony formation ability and accelerated telomere shortening in cancer cells upon the stable knockdown of REST. Interestingly, REST was down‐regulated under oxidative stress conditions via ubiquitin proteasome system, suggesting that sustainability of REST expression is critical to determine cell survival during oxidative stress in a tumor microenvironment. Our results collectively indicate that REST‐dependent TRF2 expression renders cancer cells resistant to DNA damage during oxidative stress, and mechanisms to overcome oxidative stress, such as high levels of REST or the stress‐resistant REST mutants found in specific human cancers, may account for REST‐dependent tumorigenesis.     

DNA damage response mediators MDC1 and 53BP1: constitutive activation and aberrant loss in breast and lung cancer, but not in testicular germ cell tumours

MDC1 and 53BP1 are critical components of the DNA damage response (DDR) machinery that protects genome integrity and guards against cancer, yet the tissue expression patterns and involvement of these two DDR adaptors/mediators in human tumours remain largely unknown. Here we optimized immunohistochemical analyses of human 53BP1 and MDC1 proteins in situ and identified their virtually ubiquitous expression, both in proliferating and quiescent, differentiated tissues. Focus formation by 53BP1 and/or MDC1 in human spermatogenesis and subsets of breast and lung carcinomas indicated physiological and 'pathological' activation of the DDR, respectively. Furthermore, aberrant reduction or lack of either protein in significant proportions of carcinomas supported the candidacy of 53BP1 and MDC1 for tumour suppressors. Contrary to carcinomas, almost no activation or loss of MDC1 or 53BP1 were found among testicular germ-cell tumours (TGCTs), a tumour type with unique biology and exceptionally low incidence of p53 mutations. Such concomitant presence (in carcinomas) or absence (in TGCTs) of DDR activation and DDR aberrations supports the roles of MDC1 and 53BP1 within the ATM/ATR-regulated checkpoint network which, when activated, provides an early anti-cancer barrier the pressure of which selects for DDR defects such as p53 mutations or loss of 53BP1/MDC1 during cancer progression.     

BRCA1 and BRCA2: different roles in a common pathway of genome protection

The proteins encoded by the two major breast cancer susceptibility genes, BRCA1 and BRCA2, work in a common pathway of genome protection. However, the two proteins work at different stages in the DNA damage response (DDR) and in DNA repair. BRCA1 is a pleiotropic DDR protein that functions in both checkpoint activation and DNA repair, whereas BRCA2 is a mediator of the core mechanism of homologous recombination. The links between the two proteins are not well understood, but they must exist to explain the marked similarity of human cancer susceptibility that arises with germline mutations in these genes. As discussed here, the proteins work in concert to protect the genome from double-strand DNA damage during DNA replication.     

DNA-Schadensantwort und ihre pharmakologische Beeinflussung

Chemical carcinogens, ionizing radiation and genotoxic anti-cancer drugs target DNA and DNA damage triggers genotoxicity and cell death. The elucidation of DNA damage-triggered signaling pathways is crucial for understanding the action of carcinogens and cancer initiation and progression as well as the action of genotoxic anti-cancer drugs. Potentially lethal DNA lesions for cells are DNA double-strand breaks and damage which blocks DNA replication. Cells are equipped with sensor systems which recognize the lesions and transduce the signals via kinases to downstream players, which inhibit cell cycle progression and stimulate DNA repair or, alternatively, activate apoptotic pathways. Key players of the DNA damage response (DDR) are the MRN complex and ATM, ATR and DNA-PK, which recognize DNA breaks and phosphorylate a large number of substrates, including CHK proteins, p53 and BRCA1/2. Pharmacological inhibition of DDR aimed at inhibiting the activation of DNA repair functions selectively kills cancer cells that exhibit genetic defects such as BRCA mutations (synthetic letality) and thas ameliorates the effects of anti-cancer drugs on human cells.     

DNA damage responsive microRNAs misexpressed in human cancer modulate therapy sensitivity

The DNA damage response (DDR) is activated upon DNA damage and prevents accumulation of mutations and chromosomal rearrangements, both driving carcinogenesis. Tumor cells often have defects in the DDR, which in combination with continuous cell proliferation are exploited by genotoxic cancer therapies. Most cancers, overcome initial sensitivity and develop drug resistance, e.g. by modulation of the DDR. Not much is known, however, about DNA damage responsive microRNAs in cancer therapy resistance. Therefore, we mapped temporal microRNA expression changes in primary breast epithelial cells upon low and high dose exposure to the DNA damaging agents ionizing radiation and cisplatin. A third of all DDR microRNAs commonly regulated across all treatments was also misexpressed in breast cancer, indicating a DDR defect. We repeated this approach in primary lung epithelial cells and non‐small cell lung cancer samples and found that more than 40% of all DDR microRNAs was deregulated in non‐small cell lung cancer. Strikingly, the microRNA response upon genotoxic stress in primary breast and lung epithelial cells was markedly different, although the biological outcome of DNA damage signaling (cell death/senescence or survival) was similar. Several DDR microRNAs deregulated in cancer modulated sensitivity to anti‐cancer agents. In addition we were able to distinguish between microRNAs that induced resistance by potentially inducing quiescence (miR‐296‐5p and miR‐382) or enhancing DNA repair or increased DNA damage tolerance (miR‐21). In conclusion, we provide evidence that DNA damage responsive microRNAs are frequently misexpressed in human cancer and can modulate chemotherapy sensitivity.     

Complex engagement of DNA damage response pathways in human cancer and in lung tumor progression

Tumor initiation and progression provide a multitude of occasions for the generation of DNA damage and the consequent activation of the DNA damage response (DDR) pathway. DDR signaling involves the engagement of key factors such as ATM, CHK2, 53BP1 and the phosphorylation of histone H2AX (gamma-H2AX). The systematic study of DDR in human tumors and normal tissues by high-throughput tissue microarrays revealed that ATM and gamma-H2AX were engaged in cancer but the extent of their activation was strongly affected by the organ and cell type involved, whereas 53BP1 loss was the most consistent feature among the tumor studied. Unexpectedly, we also observed activated DDR markers in morphologically normal tissues, also in association with inflammation. Analysis of the dynamic engagement of DDR along the different stages of lung tumorigenesis showed that 53BP1 loss occurs early at the transition from normal to dysplastic change whereas the activated forms of ATM and CHK2, but not gamma-H2AX, initially accumulate in pre-invasive lesions and are then lost during tumor progression. In individual lung tumors, the activation of ATM, CHK2 and the presence of 53BP1 were consistently correlated, whereas gamma-H2AX did not correlate with activated ATM. Finally, the study of associations between critical clinicopathological parameters and activated DDR factors highlighted a statistically meaningful correlation between reduced local tumor extension and the phosphorylation of ATM, CHK2 and the presence of 53BP1, whereas no significant correlations with parameters such as survival or relapse of early-stage lung carcinomas were found.     

DNA Repair: Translation to the Clinic

It has been well established that an accumulation of mutations in DNA, whether caused by external sources (e.g. ultraviolet light, radioactivity) or internal sources (e.g. metabolic by-products, such as reactive oxygen species), has the potential to cause a cell to undergo carcinogenesis and increase the risk for the development of cancer. Therefore, it is critically important for a cell to have the capacity to properly respond to and repair DNA damage as it occurs. The DNA damage response (DDR) describes a collection of DNA repair pathways that aid in the protection of genomic integrity by detecting myriad types of DNA damage and initiating the correct DNA repair pathway. In many instances, a deficiency in the DDR, whether inherited or spontaneously assumed, can increase the risk of carcinogenesis and ultimately tumorigenesis through the accumulation of mutations that fail to be properly repaired. Interestingly, although disruption of the DDR can lead to the initial genomic instability that can ultimately cause carcinogenesis, the DDR has also proven to be an invaluable target for anticancer drugs and therapies. Making matters more complicated, the DDR is also involved in the resistance to first-line cancer therapy. In this review, we will consider therapies already in use in the clinic and ongoing research into other avenues of treatment that target DNA repair pathways in cancer.     

Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic instability

SSX cancer/testis antigens are frequently expressed in melanoma tumors and represent attractive targets for immunotherapy, but their role in melanoma tumorigenesis has remained elusive. Here, we investigated the cellular effects of SSX2 expression. In A375 melanoma cells, SSX2 expression resulted in an increased DNA content and enlargement of cell nuclei, suggestive of replication aberrations. The cells further displayed signs of DNA damage and genomic instability, associated with p53‐mediated G1 cell cycle arrest and a late apoptotic response. These results suggest a model wherein SSX2‐mediated replication stress translates into mitotic defects and genomic instability. Arrest of cell growth and induction of DNA double‐strand breaks was also observed in MCF7 breast cancer cells in response to SSX2 expression. Additionally, MCF7 cells with ectopic SSX2 expression demonstrated typical signs of senescence (i.e. an irregular and enlarged cell shape, enhanced β‐galactosidase activity and DNA double‐strand breaks). Since replication defects, DNA damage and senescence are interconnected and well‐documented effects of oncogene expression, we tested the oncogenic potential of SSX2. Importantly, knockdown of SSX2 expression in melanoma cell lines demonstrated that SSX2 supports the growth of melanoma cells. Our results reveal two important phenotypes of ectopic SSX2 expression that may drive/support tumorigenesis: First, immediate induction of genomic instability, and second, long‐term support of tumor cell growth.     

Convergence of mitogenic and DNA damage signaling in the G1 phase of the cell cycle

Research into the molecular basis of cancer has a central tenet. Cancer arises from genetic alterations that disconnect growth and differentiation signaling pathways from the machinery that regulates cellular proliferation. In multi-cellular eukaryotes, proliferation is regulated by external signals, such as the availability of growth factors and nutrients and by internal signals, such as those sensing cellular integrity. Cellular stress created either by lack of mitogens or damage to cellular components, such as DNA, stimulates responses that enforce temporal or permanent withdrawal from the cell cycle. Although these stress responses stem from different sources and activate distinct pathways, they converge on the same components of the cell cycle machinery in the G1 phase of the cell cycle. This review will highlight and compare aspects of the G1 arrest in response to stress generated either by lack of mitogens or damage to DNA.     

SWI/SNF complexes are required for full activation of the DNA-damage response

SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited. To investigate the role of SWI/SNF complexes in the DNA-damage response (DDR), we used shRNAs to deplete BRG1 and BRM and then exposed these cells to a panel of 6 genotoxic agents. Compared to controls, the shRNA knockdown cells were hypersensitive to certain genotoxic agents that cause double-strand breaks (DSBs) associated with stalled/collapsed replication forks but not to ionizing radiation-induced DSBs that arise independently of DNA replication. These findings were supported by our analysis of DDR kinases, which demonstrated a more prominent role for SWI/SNF in the activation of the ATR-Chk1 pathway than the ATM-Chk2 pathway. Surprisingly, γH2AX induction was attenuated in shRNA knockdown cells exposed to a topoisomerase II inhibitor (etoposide) but not to other genotoxic agents including IR. However, this finding is compatible with recent studies linking SWI/SNF with TOP2A and TOP2BP1. Depletion of BRG1 and BRM did not result in genomic instability in a tumor-derived cell line but did result in nucleoplasmic bridges in normal human fibroblasts. Taken together, these results suggest that SWI/SNF tumor-suppressor activity involves a role in the DDR to attenuate replicative stress and genomic instability. These results may also help to inform the selection of chemotherapeutics for tumors deficient for SWI/SNF function.