DNA replication stress mediates APOBEC3 family mutagenesis in breast cancer

BACKGROUND: The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. RESULTS: HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo. HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo. Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro. APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. CONCLUSION: These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically

Arena3D: visualizing time-driven phenotypic differences in biological systems

<p>Abstract</p> <p>Background</p> <p>Elucidating the genotype-phenotype connection is one of the big challenges of modern molecular biology. To fully understand this connection, it is necessary to consider the underlying networks and the time factor. In this context of data deluge and heterogeneous information, visualization plays an essential role in interpreting complex and dynamic topologies. Thus, software that is able to bring the network, phenotypic and temporal information together is needed. Arena3D has been previously introduced as a tool that facilitates link discovery between processes. It uses a layered display to separate different levels of information while emphasizing the connections between them. We present novel developments of the tool for the visualization and analysis of dynamic genotype-phenotype landscapes.</p> <p>Results</p> <p>Version 2.0 introduces novel features that allow handling time course data in a phenotypic context. Gene expression levels or other measures can be loaded and visualized at different time points and phenotypic comparison is facilitated through clustering and correlation display or highlighting of impacting changes through time. Similarity scoring allows the identification of global patterns in dynamic heterogeneous data. In this paper we demonstrate the utility of the tool on two distinct biological problems of different scales. First, we analyze a medium scale dataset that looks at perturbation effects of the pluripotency regulator Nanog in murine embryonic stem cells. Dynamic cluster analysis suggests alternative indirect links between Nanog and other proteins in the core stem cell network. Moreover, recurrent correlations from the epigenetic to the translational level are identified. Second, we investigate a large scale dataset consisting of genome-wide knockdown screens for human genes essential in the mitotic process. Here, a potential new role for the gene <it>lsm14a </it>in cytokinesis is suggested. We also show how phenotypic patterning allows for extensive comparison and identification of high impact knockdown targets.</p> <p>Conclusions</p> <p>We present a new visualization approach for perturbation screens with multiple phenotypic outcomes. The novel functionality implemented in Arena3D enables effective understanding and comparison of temporal patterns within morphological layers, to help with the system-wide analysis of dynamic processes. Arena3D is available free of charge for academics as a downloadable standalone application from: <url>http://arena3d.org/</url>.</p

The National Lung Matrix Trial of personalized therapy in lung cancer

The majority of targeted therapies for non-small-cell lung cancer (NSCLC) are directed against oncogenic drivers that are more prevalent in patients with light exposure to tobacco smoke1,2,3. As this group represents around 20% of all patients with lung cancer, the discovery of stratified medicine options for tobacco-associated NSCLC is a high priority. Umbrella trials seek to streamline the investigation of genotype-based treatments by screening tumours for multiple genomic alterations and triaging patients to one of several genotype-matched therapeutic agents. Here we report the current outcomes of 19 drug–biomarker cohorts from the ongoing National Lung Matrix Trial, the largest umbrella trial in NSCLC. We use next-generation sequencing to match patients to appropriate targeted therapies on the basis of their tumour genotype. The Bayesian trial design enables outcome data from open cohorts that are still recruiting to be reported alongside data from closed cohorts. Of the 5,467 patients that were screened, 2,007 were molecularly eligible for entry into the trial, and 302 entered the trial to receive genotype-matched therapy—including 14 that re-registered to the trial for a sequential trial drug. Despite pre-clinical data supporting the drug–biomarker combinations, current evidence shows that a limited number of combinations demonstrate clinically relevant benefits, which remain concentrated in patients with lung cancers that are associated with minimal exposure to tobacco smoke

Telomerase activity and telomere length in primary and metastatic tumors from pediatric bone cancer patients

The presence of telomerase activity has been analyzed in almost all tumor types and tumor-derived cell lines. However, there are very few studies that focus on the presence of telomerase activity in bone tumors, and most of them report analysis on very few samples or bone-derived cell lines. The objective of this study was to analyze the telomere length and telomerase activity in primary tumors and metastatic lesions from pediatric osteosarcoma and Ewing's sarcoma patients. The presence of telomerase activity was analyzed by the telomeric repeat amplification protocol assay, and the telomere length was measured by Southern blot. Results were related to survival and clinical outcome. Telomerase activity was detected in 85% of the bone tumor metastases (100% Ewing's sarcomas and 75% osteosarcomas) but only in 12% of the primary tumors (11.1% osteosarcomas and 12.5% Ewing's sarcomas). Bone tumor tissues with telomerase activity had mean telomere lengths 3 kb shorter than those with no detectable telomerase activity (p = 0.041). The presence of telomerase activity was associated with survival (p = 0.009), and longer event-free survival periods were found in patients who lacked telomerase activity compared with those who had detectable telomerase activity levels in their tumor tissues (p = 0.037). The presence of longer telomeres in primary pediatric bone tumors than in metastases could be indicative of alternative mechanisms of lengthening of telomeres for their telomere maintenance rather than telomerase activity. Nevertheless, the activation of telomerase seems to be a crucial step in the malignant progression and acquisition of invasive capability of bone tumors

Signaling through the TRAIL receptor DR5/FADD pathway plays a role in the apoptosis associated with skeletal myoblast differentiation

Apoptosis rather than differentiation is a physiological process during myogenesis and muscle regeneration. When cultured myoblasts were induced to differentiate, we detected an increase in caspase 8 activity. Pharmacological inhibition of caspase 8 activity decreased apoptosis. Expression of a dominant-negative mutant of the adapter protein FADD also abrogated apoptosis, implicating a death ligand pathway. Treatment with TRAIL, but not Fas, induced apoptosis in these myoblasts. Accordingly, treatment with a soluble TRAIL decoy receptor or expression of a dominant-negative mutant of the TRAIL receptor DR5 abrogated apoptosis. While TRAIL expression levels remained unaltered in apoptotic myoblasts, DR5 expression levels increased. Finally, we also detected a reduction in FLIP, a death-receptor effector protein and caspase 8 competitive inhibitor, to undetectable levels in apoptotic myoblasts. Thus, our data demonstrate an important role for the TRAIL/DR5/FADD/caspase 8 pathway in the apoptosis associated with skeletal myoblast differentiation. Identifying the functional apoptotic pathways in skeletal myoblasts may prove useful in minimizing the myoblast apoptosis that contributes pathologically to a variety of diseases and in minimizing the apoptosis of transplanted myoblasts to treat these and other disease states

The Epstein–Barr virus nuclear antigen-1 promotes telomere dysfunction via induction of oxidative stress

The Epstein–Barr virus (EBV) nuclear antigen (EBNA)-1 promotes the accumulation of chromosomal aberrations in malignant B cells by inducing oxidative stress. Here we report that this phenotype is associated with telomere dysfunction. Stable or conditional expression of EBNA1 induced telomere abnormalities including loss or gain of telomere signals, telomere fusion and heterogeneous length of telomeres. This was accompanied by the accumulation of extrachromosomal telomeres, telomere dysfunction-induced foci (TIFs) containing phosphorylated histone H2AX and the DNA damage response protein 53BP1, telomere-associated promyelocytic leukemia nuclear bodies (APBs), telomeric-sister chromatid exchanges and displacement of the shelterin protein TRF2. The induction of TIFs and APBs was inhibited by treatment with scavengers of reactive oxygen species (ROS) that also promoted the relocalization of TRF2 at telomeres. These findings highlight a novel mechanism by which EBNA1 may promote malignant transformation and tumor progression

Cell Proliferation in the Presence of Telomerase

BACKGROUND: Telomerase, which is active early in development and later in stem and germline cells, is also active in the majority of human cancers. One of the known functions of telomerase is to extend the ends of linear chromosomes, countering their gradual shortening at each cell division due to the end replication problem and postreplication processing. Telomerase concentration levels vary between different cell types as well as between different tumors. In addition variable telomerase concentrations will exist in different cells in the same tumor when telomerase inhibitors are used, because of limitations of drug delivery in tissue. Telomerase extends short telomeres more frequently than long telomeres and the relation between the extension frequency and the telomere length is nonlinear. METHODOLOGY/PRINCIPAL FINDINGS: Here, the biological data of the nonlinear telomerase-telomere dynamics is incorporated in a mathematical theory to relate the proliferative potential of a cell to the telomerase concentration in that cell. The main result of the paper is that the proliferative capacity of a cell grows exponentially with the telomerase concentration. CONCLUSIONS/SIGNIFICANCE: The theory presented here suggests that long term telomerase inhibition in every cancer progenitor or cancer stem cell is needed for successful telomere targeted cancer treatment. This theory also can be used to plan and assess the results of clinical trials targeting telomerase

Expression of NES-hTERT in Cancer Cells Delays Cell Cycle Progression and Increases Sensitivity to Genotoxic Stress

Telomerase is a reverse transcriptase associated with cellular immortality through telomere maintenance. This enzyme is activated in 90% of human cancers, and inhibitors of telomerase are currently in clinical trials to counteract tumor growth. Many aspects of telomerase biology have been investigated for therapy, particularly inhibition of the enzyme, but little was done regarding its subcellular shuttling. We have recently shown that mutations in the nuclear export signal of hTERT, the catalytic component of telomerase, led to a mutant (NES-hTERT) that failed to immortalize cells despite nuclear localization and catalytic activity. Expression of NES-hTERT in primary fibroblast resulted in telomere-based premature senescence and mitochondrial dysfunction. Here we show that expression of NES-hTERT in LNCaP, SQ20B and HeLa cells rapidly and significantly decreases their proliferation rate and ability to form colonies in soft agar while not interfering with endogenous telomerase activity. The cancer cells showed increased DNA damage at telomeric and extra-telomeric sites, and became sensitive to ionizing radiation and hydrogen peroxide exposures. Our data show that expression of NES-hTERT efficiently counteracts cancer cell growth in vitro in at least two different ways, and suggest manipulation with the NES of hTERT or its subcellular shuttling as a new strategy for cancer treatment

siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells.</p> <p>Methods</p> <p>siRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied <it>in vivo </it>by injection of the siRNA-transfected breast cancer cells into nude mice.</p> <p>The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined <it>in vitro </it>by MTT assay, FACS and SA-β-galactosidase staining.</p> <p>Results</p> <p>The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. <it>In vivo</it>, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 μM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 μM doxorubicin killed twice as many cancer cells, showing a cumulative effect of the two treatments.</p> <p>Conclusion</p> <p>The study demonstrated the potential of telomerase inhibition as an effective treatment for breast cancer. When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.</p