Search

Gene expression profiling has identified numerous processes altered in aging, but how these changes arise is largely unknown. Here we combined nascent RNA sequencing and RNA polymerase II chromatin immunoprecipitation followed by sequencing to elucidate the underlying mechanisms triggering gene expression changes in wild-type aged mice. We found that in 2-year-old liver, 40% of elongating RNA polymerases are stalled, lowering productive transcription and skewing transcriptional output in a gene-length-dependent fashion. We demonstrate that this transcriptional stress is caused by endogenous DNA damage and explains the majority of gene expression changes in aging in most mainly postmitotic organs, specifically affecting aging hallmark pathways such as nutrient sensing, autophagy, proteostasis, energy metabolism, immune function and cellular stress resilience. Age-related transcriptional stress is evolutionary conserved from nematodes to humans. Thus, accumulation of stochastic endogenous DNA damage during aging deteriorates basal transcription, which establishes the age-related transcriptome and causes dysfunction of key aging hallmark pathways, disclosing how DNA damage functionally underlies major aspects of normal aging.

Heart failure has reached epidemic proportions in a progressively ageing population. The molecular mechanisms underlying heart failure remain elusive, but evidence indicates that DNA damage is enhanced in failing hearts. Here, we tested the hypothesis that endogenous DNA repair in cardiomyocytes is critical for maintaining normal cardiac function, so that perturbed repair of spontaneous DNA damage drives early onset of heart failure. To increase the burden of spontaneous DNA damage, we knocked out the DNA repair endonucleases xeroderma pigmentosum complementation group G (XPG) and excision repair cross-complementation group 1 (ERCC1), either systemically or cardiomyocyte-restricted, and studied the effects on cardiac function and structure. Loss of DNA repair permitted normal heart development but subsequently caused progressive deterioration of cardiac function, resulting in overt congestive heart failure and premature death within 6 months. Cardiac biopsies revealed increased oxidative stress associated with increased fibrosis and apoptosis. Moreover, gene set enrichment analysis showed enrichment of pathways associated with impaired DNA repair and apoptosis, and identified TP53 as one of the top active upstream transcription regulators. In support of the observed cardiac phenotype in mutant mice, several genetic variants in the ERCC1 and XPG gene in human GWAS data were found to be associated with cardiac remodelling and dysfunction. In conclusion, unrepaired spontaneous DNA damage in differentiated cardiomyocytes drives early onset of cardiac failure. These observations implicate DNA damage as a potential novel therapeutic target and highlight systemic and cardiomyocyte-restricted DNA repair-deficient mouse mutants as bona fide models of heart failure.

Purpose: We investigated whether organoids can be generated from resected tumors of patients who received eight cycles of neoadjuvant FOLFIRINOX chemotherapy before surgery, and evaluated the sensitivity/resistance of these surviving cancer cells to cancer therapy. Experimental Design: We generated a library of 10 pancreatic ductal adenocarcinoma (PDAC) organoid lines: five each from treatment-naïve and FOLFIRINOX-treated patients. We first assessed the histologic, genetic, and transcriptional characteristics of the organoids and their matched primary PDAC tissue. Next, the organoids' response to treatment with single agents—5-FU, irinotecan, and oxaliplatin—of the FOLFIRINOX regimen as well as combined regimen was evaluated. Finally, global mRNA-seq analyses were performed to identify FOLFIRINOX resistance pathways. Results: All 10 patient-derived PDAC organoids recapitulate histologic, genetic, and transcriptional characteristics of their primary tumor tissue. Neoadjuvant FOLFIRINOX-treated organoids display resistance to FOLFIRINOX (5/5), irinotecan (5/5), and oxaliplatin (4/5) when compared with treatment-naïve organoids (FOLFIRINOX: 1/5, irinotecan: 2/5, oxaliplatin: 0/5). 5-Fluorouracil treatment responses between naïve and treated organoids were similar. Comparative global transcriptome analysis of treatment-naïve and FOLFIRINOX samples—in both organoids and corresponding matched tumor tissues—uncovered modulated pathways mainly involved in genomic instability, energy metabolism, and innate immune system. Conclusions: Resistance development in neoadjuvant FOLFIRINOX organoids, recapitulating their primary tumor resistance, suggests continuation of FOLFIRINOX therapy as an adjuvant treatment may not be advantageous for these patients. Gene-expression profiles of PDAC organoids identify targetable pathways involved in chemoresistance development upon neoadjuvant FOLFIRINOX treatment, thus opening up combination therapy possibilities.

Ageing is a complex, multifaceted process leading to widespread functional decline that affects every organ and tissue, but it remains unknown whether ageing has a unifying causal mechanism or is grounded in multiple sources. Phenotypically, the ageing process is associated with a wide variety of features at the molecular, cellular and physiological level—for example, genomic and epigenomic alterations, loss of proteostasis, declining overall cellular and subcellular function and deregulation of signalling systems. However, the relative importance, mechanistic interrelationships and hierarchical order of these features of ageing have not been clarified. Here we synthesize accumulating evidence that DNA damage affects most, if not all, aspects of the ageing phenotype, making it a potentially unifying cause of ageing. Targeting DNA damage and its mechanistic links with the ageing phenotype will provide a logical rationale for developing unified interventions to counteract age-related dysfunction and disease. This Review examines the evidence showing that DNA damage is associated with ageing phenotypes, suggesting that it may have a central role as the cause of ageing.

Organs age differently, causing wide heterogeneity in multimorbidity, but underlying mechanisms are largely elusive. To investigate the basis of organ-specific ageing, we utilized progeroid repair-deficient Ercc1Δ/− mouse mutants and systematically compared at the tissue, stem cell and organoid level two organs representing ageing extremes. Ercc1Δ/− intestine shows hardly any accelerated ageing. Nevertheless, we found apoptosis and reduced numbers of intestinal stem cells (ISCs), but cell loss appears compensated by over-proliferation. ISCs retain their organoid-forming capacity, but organoids perform poorly in culture, compared with WT. Conversely, liver ages dramatically, even causing early death in Ercc1-KO mice. Apoptosis, p21, polyploidization and proliferation of various (stem) cells were prominently elevated in Ercc1Δ/− liver and stem cell populations were either largely unaffected (Sox9+), or expanding (Lgr5+), but were functionally exhausted in organoid formation and development in vitro. Paradoxically, while intestine displays less ageing, repair in WT ISCs appears inferior to liver as shown by enhanced sensitivity to various DNA-damaging agents, and lower lesion removal. Our findings reveal organ-specific anti-ageing strategies. Intestine, with short lifespan limiting time for damage accumulation and repair, favours apoptosis of damaged cells relying on ISC plasticity. Liver with low renewal rates depends more on repair pathways specifically protecting the transcribed compartment of the genome to promote sustained functionality and cell preservation. As shown before, the hematopoietic system with intermediate self-renewal mainly invokes replication-linked mechanisms, apoptosis and senescence. Hence, organs employ different genome maintenance strategies, explaining heterogeneity in organ ageing and the segmental nature of DNA-repair-deficient progerias.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1?/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1?/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1?/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of ?H2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1?/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general. © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Initiation and promoter-proximal pausing are key regulatory steps of RNA Polymerase II (Pol II) transcription. To study the in vivo dynamics of endogenous Pol II during these steps, we generated fully functional GFP-RPB1 knockin cells. GFP-RPB1 photobleaching combined with computational modeling revealed four kinetically distinct Pol II fractions and showed that on average 7% of Pol II are freely diffusing, while 10% are chromatin-bound for 2.4 seconds during initiation, and 23% are promoter-paused for only 42 seconds. This unexpectedly high turnover of Pol II at promoters is most likely caused by premature termination of initiating and promoter-paused Pol II and is in sharp contrast to the 23 minutes that elongating Pol II resides on chromatin. Our live-cell–imaging approach provides insights into Pol II dynamics and suggests that the continuous release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.

Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Tumors deficient in NER are more sensitive to cisplatin treatment. Characterization of the mutational consequences of NER-deficiency may therefore provide important diagnostic opportunities. Here, we analyzed the somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1-/Δ mice, using whole-genome sequencing analysis of clonally derived organoid cultures. Our results indicate that NER-deficiency increases the base substitution load in liver, but not in small intestinal ASCs, which coincides with a tissue-specific aging-pathology observed in these mice. The mutational landscape changes as a result of NER-deficiency in ASCs of both tissues and shows an increased contribution of Signature 8 mutations, which is a pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the acquired base substitutions indicates that deficiency of global-genome NER (GG-NER) is responsible for the altered mutational landscape. In line with this, we observed increased Signature 8 mutations in a GG-NER-deficient human organoid culture in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations may therefore serve as a biomarker for NER-deficiency and could improve personalized cancer treatment strategies.

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. © Kasper WJ Derks, Branislav Misovic, Mirjam CGN van den Hout, Christel EM Kockx, Cesar Payan Gomez, Rutger WW Brouwer, Harry Vrieling, Jan HJ Hoeijmakers, Wilfred FJ van IJcken, and Joris Pothof.

© 2017 Elsevier Inc. The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.

There is a high need to improve the assessment of, especially non-genotoxic, carcinogenic features of chemicals. We therefore explored a toxicogenomics-based approach using genome-wide microRNA and mRNA expression profiles upon short-term exposure in mice. For this, wild-type mice were exposed for seven days to three different classes of chemicals, i.e., four genotoxic carcinogens (GTXC), seven non-genotoxic carcinogens (NGTXC), and five toxic non-carcinogens. Hepatic expression patterns of mRNA and microRNA transcripts were determined after exposure and used to assess the discriminative power of the in vivo transcriptome for GTXC and NGTXC. A final classifier set, discriminative for GTXC and NGTXC, was generated from the transcriptomic data using a tiered approach. This appeared to be a valid approach, since the predictive power of the final classifier set in three different classifier algorithms was very high for the original training set of chemicals. Subsequent validation in an additional set of chemicals revealed that the predictive power for GTXC remained high, in contrast to NGTXC, which appeared to be more troublesome. Our study demonstrated that the in vivo microRNA-ome has less discriminative power to correctly identify (non-)genotoxic carcinogen classes. The results generally indicate that single mRNA transcripts do have the potential to be applied in risk assessment, but that additional (genomic) strategies are necessary to correctly predict the non-genotoxic carcinogenic potential of a chemical.

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. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Mutational characteristics of NER-deficiency may reveal important diagnostic opportunities, as tumors deficient in NER are more sensitive to certain treatments. Here, we analyzed the genome-wide somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1−/Δ mice. Our results indicate that NER-deficiency increases the base substitution load twofold in liver but not in small intestinal ASCs, which coincides with the tissue-specific aging pathology observed in these mice. Moreover, NER-deficient ASCs of both tissues show an increased contribution of Signature 8 mutations, which is a mutational pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the base substitutions indicates that deficiency of global-genome NER (GG-NER) underlies the observed mutational consequences. In line with this, we observe increased Signature 8 mutations in a GG-NER-deficient human organoid culture, in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations could therefore contribute to a predictor of NER-deficiency based on a patient's mutational profile.

Uveal melanoma (UM) is the most frequently found primary intra-ocular tumor in adults. It is a highly aggressive cancer that causes metastasis-related mortality in up to half of the patients. Many independent studies have reported somatic genetic changes associated with high metastatic risk, such as monosomy of chromosome 3 and mutations in BAP1. Still, the mechanisms that drive metastatic spread are largely unknown. This study aimed to elucidate the potential role of microRNAs in the metastasis of UM. Using a next-generation sequencing approach in 26 UM samples we identified thirteen differentially expressed microRNAs between high-risk UM and low/intermediate-risk UM, including the known oncomirs microRNA-17-5p, microRNA-21-5p, and miR-151a-3p. Integration of the differentially expressed microRNAs with expression data of predicted target genes revealed 106 genes likely to be affected by aberrant microRNA expression. These genes were involved in pathways such as cell cycle regulation, EGF signaling and EIF2 signaling. Our findings demonstrate that aberrant microRNA expression in UM may affect the expression of genes in a variety of cancer-related pathways. This implies that some microRNAs can be responsible for UM metastasis and are promising potential targets for future treatment.

Epithelial ovarian cancer is the most lethal gynecological malignancy in the Western world. A major impediment for the successful treatment is the development of drug resistance. The molecular processes that contribute to resistance have been extensively studied; however, there is not much known about regulation by microRNAs (miRNAs). We compared miRNA expression profiles of an isogenic cisplatin-sensitive and -resistant ovarian cancer cell line pair (A2780/A2780 DDP) and found 27 miRNAs to be differentially expressed (2-fold). Five of these, including the family members miR-141/200c, showed a correlation with cisplatin sensitivity in the NCI-60 panel. Overexpression of miR-141 resulted in enhanced resistance to cisplatin in ovarian cancer cell lines. We next correlated the expression level of miR-141 in 132 primary ovarian tumors (108 serous and 24 non-serous) with response to platinum-based chemotherapy. Although no differences were observed in the serous tumors, miR-141 levels were higher in non-serous ovarian tumors that did not respond well to therapy (platinum-free interval

The 3rd US–EU Workshop on systems level understanding of DNA damage responses was held from March 30 to April 1, 2009 in Egmond aan Zee, The Netherlands. Objectives of the workshop were (1) to assess the current science of the DDR, in particular network level responses to chemotherapeutic and environmentally induced DNA damage; and (2) to establish the basis for a reciprocal scientific exchange program between the EU and US in the relevant areas of DDR research. Here, we report the highlights of the meeting program and conclude that this third meeting in 2009 refined the role of DDR networks in human disease.

DNA damage induced by UV irradiation provokes profound changes in gene expression. Both transcriptional regulation and posttranslational modification of proteins have been known for many years, but the involvement of microRNAs in regulation of mRNA translation has been described only recently. This level of gene expression regulation appears to operate at the intermediate time points between fast protein modifications (within minutes) and much slower transcriptional reprogramming (which takes several hours to days to develop). MicroRNAs most clearly contribute to regulation of cell cycle checkpoints and apoptosis, but may also influence other aspects of cellular metabolism, differentiation and proliferation. Interestingly, the RNA silencing machinery redistributes into cytoplasmic RNA granules, termed stress granules (SGs), in cells that go through mitosis after UV irradiation. We discuss the implications of these findings for our understanding of the DNA damage response.

Human syndromes and mouse mutants that exhibit accelerated but bona fide aging in multiple organs and tissues have been invaluable for the identification of nine denominators of aging: telomere attrition, genome instability, epigenetic alterations, mitochondrial dysfunction, deregulated nutrient sensing, altered intercellular communication, loss of proteostasis, cellular senescence and adult stem cell exhaustion. However, whether and how these instigators of aging interrelate or whether they have one root cause is currently largely unknown. Rare human progeroid syndromes and corresponding mouse mutants with resolved genetic defects highlight the dominant importance of genome maintenance for aging. A second class of aging-related disorders reveals a cross connection with metabolism. As genome maintenance and metabolism are closely interconnected, they may constitute the main underlying biology of aging. This review focuses on the role of genome stability in aging, its crosstalk with metabolism, and options for nutritional and/or pharmaceutical interventions that delay age-related pathology.

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors.

SummaryIonizing radiation is extremely harmful for human cells, and DNA double-strand breaks (DSBs) are considered to be the main cytotoxic lesions induced [1]. Improper processing of DSBs contributes to tumorigenesis, and mutations in DSB response genes underlie several inherited disorders characterized by cancer predisposition [2, 3]. Here, we performed a comprehensive screen for genes that protect animal cells against ionizing radiation. A total of 45 C. elegans genes were identified in a genome-wide RNA interference screen for increased sensitivity to ionizing radiation in germ cells. These genes include orthologs of well-known human cancer predisposition genes as well as novel genes, including human disease genes not previously linked to defective DNA-damage responses. Knockdown of eleven genes also impaired radiation-induced cell-cycle arrest, and seven genes were essential for apoptosis upon exposure to irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA-damaging cancer drugs cisplatin and camptothecin. Almost all genes are conserved across animal phylogeny, and their relevance for humans was directly demonstrated by showing that their knockdown in human cells results in radiation sensitivity, indicating that this set of genes is important for future cancer profiling and drug development.

The large extracellular polysaccharide Hyaluronan (HA) and its synthesizing enzymes (Has) have been implicated in regulating the migratory potential of metastatic cancer cells. Here, we analyze the roles of zebrafish Has2 in normal development. Antisense morpholino oligonucleotide (MO)-mediated knockdown of zebrafish Has2 leads to the loss of HA, and severe migratory defects during gastrulation, somite morphogenesis and primordial germ cell migration. During gastrulation, ventrolateral cells of has2 morphant embryos fail to develop lamellipodia and to migrate dorsally, resulting in a blockage of dorsal convergence, whereas extension of the dorsal axis is normal. The effect is cell autonomous, suggesting that HA acts as an autocrine signal to stimulate the migration of HA-generating cells. Upon ectopic expression in axial cells, has2 causes the formation of supernumerary lamellipodia and a blockage of axis extension. Epistasis analyses with constitutively active and dominant-negative versions of the small GTPase Rac1 suggest that HA acts by Rac1 activation, rather than as an essential structural component of the extracellular matrix. Together, our data provide evidence that convergence and extension are separate morphogenetic movements of gastrulation. In addition, they suggest that the same HA pathways are active to auto-stimulate cell migration during tumor invasion and vertebrate embryogenesis.

Recent advances have identified accumulation of DNA damage as a major driver of aging. However, there are numerous kinds of DNA lesions each with their own characteristics and cellular outcome, which highly depends on cellular context: proliferation (cell cycle), differentiation, propensity for survival/death, cell condition and systemic hormonal and immunological parameters. In addition, DNA damage is strongly influenced by cellular metabolism, anti-oxidant status and exogenous factors, consistent with the multi-factorial nature of aging. Notably, DNA lesions interfering with replication have very different outcomes compared to transcription. These considerations provide a conceptual framework in which different types of DNA damage and their setting contribute to the aging process in differential manners.

The emergence of high density technologies monitoring the genome, transcriptome and proteome in relation to genotoxic stress have tremendously enhanced our knowledge on global responses and dynamics in the DNA damage response, including its relation with cancer and aging. Moreover, '-omics' technologies identified many novel factors, their post-translational modifications, pathways and global responses in the cellular response to DNA damage. Based on omics, it is currently estimated that thousands of gene(product)s participate in the DNA damage response, recognizing complex networks that determine cell fate after damage to the most precious cellular molecule, DNA. The development of next generation sequencing technology and associated specialized protocols can quantitatively monitor RNA and DNA at unprecedented single nucleotide resolution. In this review we will discuss the contribution of omics technologies and in particular next generation sequencing to our understanding of the DNA damage response and the future prospective of next generation sequencing, its single cell application and omics dataset integration in unraveling intricate DNA damage signaling networks. (C) 2014 Elsevier B.V. All rights reserved.

Dendritic cell (DC) maturation is a tightly regulated process that requires coordinated and timed developmental cues. Here we investigate whether microRNAs are involved in this process. We identify microRNAs in mouse GM-CSF-generated, monocyte-related DC (GM-DC) that are differentially expressed during both spontaneous and LPS-induced maturation and characterize M-CSF receptor (M-CSFR), encoded by the Csf1r gene, as a key target for microRNA-mediated regulation in the final step toward mature DC. MicroRNA-22, -34a, and -155 are up-regulated in mature MHCII(hi) CD86(hi) DC and mediate Csf1r mRNA and protein down-regulation. Experimental inhibition of Csf1r-targeting microRNAs in vitro results not only in sustained high level M-CSFR protein expression but also in impaired DC maturation upon stimulation by LPS. Accordingly, over-expression of Csf1r in GM-DC inhibits terminal differentiation. Taken together, these results show that developmentally regulated microRNAs control Csf1r expression, supplementing previously identified mechanisms that regulate its transcription and protein surface expression. Furthermore, our data indicate a novel function for Csf1r in mouse monocyte-derived DC, showing that down-regulation of M-CSFR expression is essential for final DC maturation.

MicroRNAs (miRNAs) are evolutionarily conserved non-coding RNAs of similar to 22 nucleotides that regulate gene expression at the level of translation and play vital roles in hippocampal neuron development, function and plasticity. Here, we performed a systematic and in-depth analysis of miRNA expression profiles in cultured hippocampal neurons during development and after induction of neuronal activity. MiRNA profiling of primary hippocampal cultures was carried out using locked nucleic-acid-based miRNA arrays. The expression of 264 different miRNAs was tested in young neurons, at various developmental stages (stage 2-4) and in mature fully differentiated neurons (stage 5) following the induction of neuronal activity using chemical stimulation protocols. We identified 210 miRNAs in mature hippocampal neurons; the expression of most neuronal miRNAs is low at early stages of development and steadily increases during neuronal differentiation. We found a specific subset of 14 miRNAs with reduced expression at stage 3 and showed that sustained expression of these miRNAs stimulates axonal outgrowth. Expression profiling following induction of neuronal activity demonstrates that 51 miRNAs, including miR-134, miR-146, miR-181, miR-185, miR-191 and miR-200a show altered patterns of expression after NMDA receptor-dependent plasticity, and 31 miRNAs, including miR-107, miR-134, miR-470 and miR-546 were upregulated by homeostatic plasticity protocols. Our results indicate that specific miRNA expression profiles correlate with changes in neuronal development and neuronal activity. Identification and characterization of miRNA targets may further elucidate translational control mechanisms involved in hippocampal development, differentiation and activity-depended processes.

Platinum-based chemotherapy (e.g. cisplatin, carboplatin) is standard of care for many types of cancer including ovarian cancer, however, the efficacy of treatment is hampered by the development of therapy resistance. The mechanisms behind platinum resistance are not completely understood. Here, we have investigated the role of the family of p90 Ribosomal S6 kinases (RSK), important downstream mediators of ERK1/2, in the response to cisplatin chemotherapy. Strikingly, whereas treatment with cisplatin did not alter the levels of RSK1 in response to cisplatin treatment, the structurally related RSK2 protein was downregulated in an ovarian cancer cell line (A2780). Furthermore, we found that knockdown of RSK2, in contrast to knockdown of RSK1, gave rise to enhanced cisplatin sensitivity in a cisplatin sensitive as well as a cisplatin-resistant A2780 cell line. These results indicate that RSK2 is regulated in response to cisplatin treatment, and this downregulation may contribute to the cytotoxic action of cisplatin. Since RSK2 is frequently amplified in a growing number of cancers, this may have implications for the sensitivity of these tumours to platinum-based cytotoxics.

Cancer cells that acquire radio-resistance are major problem for treatment outcome. We aimed at identifying mechanisms and networks that control ionizing radiation (IR)-resistance in prostate cancer cells. To this end, we applied to cancer cell lines an identical radiotherapy regiment as performed in the clinic, i.e. a total dose of 78 Gy in steps of 2 Gy per day for 5 consecutive days followed by 2 days rest. We observed that radio-resistance occurs early during treatment as seen in colony survival assays in which 100% of cells that can form colonies are resistant up to 8 Gy. Remarkably, a two-week period of culturing without radiotherapy completely sensitized these resistant cancer cell lines, indicating that resistance is not acquired by permanent mutations or chromosomal rearrangements. Currently, we are characterizing these extreme resistant cancer cell cultures by comparing these to their sensitive parental cancer cell lines and the two-week re-sensitized cancer cell lines. We observe changes in DNA repair pathway utilization, DNA damage checkpoint activation and energy metabolism parameters, but not in PI3-kinase signalling and cancer stem cell markers. We also performed genome-wide gene expression analysis by next generation sequencing and isolated gene signatures that correlate with resistance. Based on our results we applied inhibitors to sensitize these IR-resistant cells. Notably, application of single inhibitors did not lead to sensitivity. Combinations of small molecule inhibitors however, were able to sensitize these cancer cell lines, indicating that IR-resistance is a multi-factorial process. In summary, mapping gene networks and mechanisms will uncover new pathways associated with IR-resistance. Citation Format: Serena T. Bruens, Chiara Milanese, Nicole Verkaik, Pier Mastroberardino, Akos Gyenis, Jiang Chang, Kasper Derks, Erik Wiemer, Dik van Gent, Wytske van Weerden, Guido Jenster, Jan Hoeijmakers, Joris Pothof. Mapping mechanisms of radiotherapy resistance in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1657.

Resistance to therapy is a major limitation towards the cure of irresectable cancer. Cellular senescence, induced by chemotherapy and radiotherapy, can drive therapy resistance in vivo. Senescence is a tumor suppressive mechanism, but senescent cells can secrete a range of proteins that ironically promote tumor growth, migration and metastazation and therapy resistance. Recent evidence has shown that genetic removal of senescent cells indeed causes a strong reduction in tumor growth and metastasis formation. Unfortunately however, therapeutic options to remove senescent cells are currently lacking. Here, we show the development and optimization of a biochemical compound, TASC1, that potently and selectively kills senescent cells in vitro and in vivo and lowers organ toxicity in a mouse model employed to address the off-target effects of cancer therapy. This encouraged us to test the effect of TASC1 against cancer therapy resistance. We focused on Glioblastoma multiforme (GBM), the most aggressive and lethal stage of glioma (III-IV) with a median survival of less than 15 months. Treatment of GBM involves a combination of surgery, radiotherapy and adjuvant chemotherapy (Temozolomide). Response to radiotherapy is generally strong, but resistance occurs rapidly, after which currently no successful follow-up treatment exists. We observed radiation therapy to induce senescence in astrocytes and senescence-like effects in GBM cells, without affecting their progression. Strikingly, while both TASC1 and radiation at various doses independently failed to reduce growth of these GBM cells, the combination of TASC1 with radiation showed a potent synthetic lethal response. Similar effects were seen for other types of cancer and their respective therapies. Thus, TASC1 is a potent adjuvant to (re)sensitize therapy-resistance metastatic cancer cells to treatment. Citation Format: Marjolein P. Baar, Diana A. Putavet, Joris Pothof, Jan H.J. Hoeijmakers, Peter L.J. de Keizer. TASC1, a selective anti-senescence therapeutic which potently and selectively counteract resistance to chemo- and radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2843.

Background MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and play a critical role in development, homeostasis, and disease. Despite their demonstrated roles in age-associated pathologies, little is known about the role of miRNAs in human aging and longevity. Results We employed massively parallel sequencing technology to identify miRNAs expressed in B-cells from Ashkenazi Jewish centenarians, i.e., those living to a hundred and a human model of exceptional longevity, and younger controls without a family history of longevity. With data from 26.7 million reads comprising 9.4 × 108 bp from 3 centenarian and 3 control individuals, we discovered a total of 276 known miRNAs and 8 unknown miRNAs ranging several orders of magnitude in expression levels, a typical characteristics of saturated miRNA-sequencing. A total of 22 miRNAs were found to be significantly upregulated, with only 2 miRNAs downregulated, in centenarians as compared to controls. Gene Ontology analysis of the predicted and validated targets of the 24 differentially expressed miRNAs indicated enrichment of functional pathways involved in cell metabolism, cell cycle, cell signaling, and cell differentiation. A cross sectional expression analysis of the differentially expressed miRNAs in B-cells from Ashkenazi Jewish individuals between the 50th and 100th years of age indicated that expression levels of miR-363* declined significantly with age. Centenarians, however, maintained the youthful expression level. This result suggests that miR-363* may be a candidate longevity-associated miRNA. Conclusion Our comprehensive miRNA data provide a resource for further studies to identify genetic pathways associated with aging and longevity in humans.

MicroRNA-mediated modulation of translation has been recently discovered as a new dimension in gene expression regulation. In this chapter we review how this regulation operates in time between the fast protein modification and degradation steps on the one hand and the slow transcriptional reprogramming associated with more stable changes in gene expression patterns on the other hand. We also discuss the additional layer of complexity associated with spatial redistribution of the RNA silencing machinery in subcellular structures. Various stress conditions induce both a transient change in microRNA expression within the first few hours after exposure and a redistribution of the RNA silencing machinery from P-bodies to Stress Granules, which differ in their function and protein content. Insight into the spatiotemporal aspects of the microRNA response will be indispensable for a full understanding of this level of gene regulation.

MicroRNA-mediated modulation of translation has been recently discovered as a new dimension in gene expression regulation. In this chapter we review how this regulation operates in time between the fast protein modification and degradation steps on the one hand and the slow transcriptional reprogramming associated with more stable changes in gene expression patterns on the other hand. We also discuss the additional layer of complexity associated with spatial redistribution of the RNA silencing machinery in subcellular structures. Various stress conditions induce both a transient change in microRNA expression within the first few hours after exposure and a redistribution of the RNA silencing machinery from P-bodies to Stress Granules, which differ in their function and protein content. Insight into the spatiotemporal aspects of the microRNA response will be indispensable for a full understanding of this level of gene regulation.

Many carcinogenic agents such as ultra-violet light from the sun and various natural and man-made chemicals act by damaging the DNA. To deal with these potentially detrimental effects of DNA damage, cells induce a complex DNA damage response (DDR) that includes DNA repair, cell cycle checkpoints, damage tolerance systems and apoptosis. This DDR is a potent barrier against carcinogenesis and defects within this response are observed in many, if not all, human tumors. DDR defects fuel the evolution of precancerous cells to malignant tumors, but can also induce sensitivity to DNA damaging agents in cancer cells, which can be therapeutically exploited by the use of DNA damaging treatment modalities. Regulation of and coordination between sub-pathways within the DDR is important for maintaining genome stability. Although regulation of the DDR has been extensively studied at the transcriptional and post-translational level, less is known about post-transcriptional gene regulation by microRNAs, the topic of this review. More specifically, we highlight current knowledge about DNA damage responsive microRNAs and microRNAs that regulate DNA damage response genes. We end by discussing the role of DNA damage response microRNAs in cancer etiology and sensitivity to ionizing radiation and other DNA damaging therapeutic agents. (C) 2011 Elsevier B.V. All rights reserved.

DNA damage induced by UV irradiation provokes profound changes in gene expression. Both transcriptional regulation and posttranslational modification of proteins have been known for many years, but the involvement of microRNAs in regulation of mRNA translation has been described only recently. This level of gene expression regulation appears to operate at the intermediate time points between fast protein modifications (within minutes) and much slower transcriptional reprogramming (which takes several hours to days to develop). MicroRNAs most clearly contribute to regulation of cell cycle checkpoints and apoptosis, but may also influence other aspects of cellular metabolism, differentiation and proliferation. Interestingly, the RNA silencing machinery redistributes into cytoplasmic RNA granules, termed stress granules (SGs), in cells that go through mitosis after UV irradiation. We discuss the implications of these findings for our understanding of the DNA damage response.

An RNA interference (RNAi)-based genome-wide screen was performed to detect genes that contribute to genome stability in somatic cells ofCaenorhabditis elegans. We identified 61 such genes; these also affect spontaneous mutagenesis in the germ line. Their sequence suggests a role in DNA repair and/or replication, in chromatin remodeling, or in cell cycle control; there are also many novel genes that are highly conserved from yeast to human. Because known mutator genes are causally involved in many hereditary and sporadic human cancers, it is likely that some of these new mutators are equally relevant in cancer etiology.

Mismatch-repair-deficient mutants were initially recognized as mutation-prone derivatives of bacteria, and later mismatch repair deficiency was found to predispose humans to colon cancers (HNPCC). We generated mismatch-repair-deficient Caenorhabditis elegans by deleting the msh-6 gene and analyzed the fidelity of transmission of genetic information to subsequent generations. msh-6-defective animals show an elevated level of spontaneous mutants in both the male and female germline; also repeated DNA tracts are unstable. To monitor DNA repeat instability in somatic tissue, we developed a sensitive system, making use of heat-shock promoter-driven lacZ transgenes, but with a repeat that puts this reporter gene out of frame. In genetic msh-6-deficient animals lacZ+ patches are observed as a result of somatic repeat instability. RNA interference by feeding wild-type animals dsRNA homologous to msh-2 or msh-6 also resulted in somatic DNA instability, as well as in germline mutagenesis, indicating that one can use C. elegans as a model system to discover genes involved in maintaining DNA stability by large-scale RNAi screens.

The development of drug resistance is still a major impediment for the successful treatment of cancer. This is particularly pressing in the case of advanced ovarian cancer, which has a 5-year survival rate of 30%. Ovarian cancer is treated with paclitaxel and platinum (e.g cisplatin or carboplatin) combination chemotherapy. Despite an initial high chemo-responsiveness, most ovarian cancer patients will relapse and ultimately die of drug-resistant disease. The molecular processes that contribute to resistance have been extensively studied, however, not much is known about the role of microRNAs (miRNAs), small non-coding RNAs that regulate gene expression. We compared miRNA expression profiles of isogenic cisplatin sensitive and resistant cell line pairs. The only miRNA that was consistently downregulated in all three resistant cell lines was miR-634 (FDR Altogether, our findings suggest that miR-634 modulates important cancer relevant targets and that raising miR-634 levels can be a means to resensitize chemotherapy resistant ovarian tumors. Citation Format: Marijn TM van Jaarsveld, Patricia F. van Kuijk, Antonius WM Boersma, Jozien Helleman, Wilfred F. van Ijcken, Ron HJ Mathijssen, Joris Pothof, Els MJJ Berns, Jaap Verweij, Erik AC Wiemer. miR-634 restores drug sensitivity in tumor cells derived from patients with terminal drug-resistant ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 949. doi:10.1158/1538-7445.AM2013-949

Tumor cells are often characterized by a rapid cell division rate and defects in their DNA damage response, which is exploited in the clinic by the use of DNA damaging anticancer agents. Platinum-based chemotherapy (e.g. cisplatin) is used for the treatment of many different cancers including ovarian cancer. Unfortunately, the success of chemotherapy is hampered by the development of drug resistance, and most ovarian tumors eventually become refractory to multiple drugs. The molecular and cellular processes that lead to therapy resistance have been extensively studied at the transcriptional and posttranslational level. Another level of gene regulation is exerted by the recently discovered microRNAs, small non-coding RNAs that interfere with the expression of protein-coding genes. The essential role microRNAs play in the cellular response to genotoxic agents is only just being elucidated. In this project we aim to identify and functionally characterize microRNAs that alter the sensitivity to cisplatin in ovarian cancer cells. LNA™ modified oligonucleotide arrays were used to map the initial microRNA response (up to 24 h) of two human primary epithelial cell lines to cisplatin and γ-irradiation, anticancer therapies that induce different kinds of DNA damage (DNA crosslinks and double strand DNA breaks, respectively). We examined the response to both a low dose and a high dose treatment. Both general DNA damage responsive microRNAs and treatment- and dose-specific microRNAs were identified. Next, we examined whether these microRNAs were also differentially regulated in cisplatin-sensitive and resistant cell lines derived from ovarian cancer, colon cancer and bladder cancer. Indeed, several microRNAs were found to be associated with cisplatin resistance. By ectopically modulating the expression of five microRNAs in ovarian cancer cell lines, we found two microRNAs – not previously linked to cisplatin resistance – for which overexpression resulted either in enhanced sensitivity or enhanced resistance to cisplatin. In search of relevant target genes we discovered genes that were regulated by both microRNAs. SiRNA experiments in which gene expression was knocked down recapitulated the microRNA overexpression results. Interestingly, these target genes belong to major signal transduction routes and have not been previously associated with the response to platinum containing chemotherapy. In summary, we have identified microRNAs that are regulated after DNA damage and are associated with cisplatin resistance. Modulation of the levels of these microRNAs in ovarian cancer cells altered the survival response to cisplatin, suggesting they may sensitize tumors to chemotherapy. Furthermore, we have identified some of the genes through which these microRNAs mediate their effects on cisplatin sensitivity, shedding a new light on the molecular basis of cisplatin resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4952. doi:10.1158/1538-7445.AM2011-4952

Background Drug resistance hampers the efficient treatment of malignancies, including advanced stage ovarian cancer, which has a 5-year survival rate of only 30 %. The molecular processes underlying resistance have been extensively studied, however, not much is known about the involvement of microRNAs. Methods Differentially expressed microRNAs between cisplatin sensitive and resistant cancer cell line pairs were determined using microarrays. Mimics were used to study the role of microRNAs in drug sensitivity of ovarian cancer cell lines and patient derived tumor cells. Luciferase reporter constructs were used to establish regulation of target genes by microRNAs. Results MiR-634 downregulation was associated with cisplatin resistance. Overexpression of miR-634 affected cell cycle progression and enhanced apoptosis in ovarian cancer cells. miR-634 resensitized resistant ovarian cancer cell lines and patient derived drug resistant tumor cells to cisplatin. Similarly, miR-634 enhanced the response to carboplatin and doxorubicin, but not to paclitaxel. The cell cycle regulator CCND1, and Ras-MAPK pathway components GRB2, ERK2 and RSK2 were directly repressed by miR-634 overexpression. Repression of the Ras-MAPK pathway using a MEK inhibitor phenocopied the miR-634 effects on viability and chemosensitivity. Conclusion miR-634 levels determine chemosensitivity in ovarian cancer cells. We identify miR-634 as a therapeutic candidate to resensitize chemotherapy resistant ovarian tumors. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0464-4) contains supplementary material, which is available to authorized users.