Persistent DNA damage triggers activation of the integrated stress response to promote cell survival under nutrient restriction.

BACKGROUND: Base-excision repair (BER) is a central DNA repair mechanism responsible for the maintenance of genome integrity. Accordingly, BER defects have been implicated in cancer, presumably by precipitating cellular transformation through an increase in the occurrence of mutations. Hence, tight adaptation of BER capacity is essential for DNA stability. However, counterintuitive to this, prolonged exposure of cells to pro-inflammatory molecules or DNA-damaging agents causes a BER deficiency by downregulating the central scaffold protein XRCC1. The rationale for this XRCC1 downregulation in response to persistent DNA damage remains enigmatic. Based on our previous findings that XRCC1 downregulation causes wide-ranging anabolic changes, we hypothesised that BER depletion could enhance cellular survival under stress, such as nutrient restriction. RESULTS: Here, we demonstrate that persistent single-strand breaks (SSBs) caused by XRCC1 downregulation trigger the integrated stress response (ISR) to promote cellular survival under nutrient-restricted conditions. ISR activation depends on DNA damage signalling via ATM, which triggers PERK-mediated eIF2α phosphorylation, increasing translation of the stress-response factor ATF4. Furthermore, we demonstrate that SSBs, induced either through depletion of the transcription factor Sp1, responsible for XRCC1 levels, or through prolonged oxidative stress, trigger ISR-mediated cell survival under nutrient restriction as well. Finally, the ISR pathway can also be initiated by persistent DNA double-strand breaks. CONCLUSIONS: Our results uncover a previously unappreciated connection between persistent DNA damage, caused by a decrease in BER capacity or direct induction of DNA damage, and the ISR pathway that supports cell survival in response to genotoxic stress with implications for tumour biology and beyond.

An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.

BACKGROUND: Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult. METHODS: We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step. RESULTS: We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. CONCLUSIONS: We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.

Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas.

Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as excellent models of human breast carcinomas. Considering the importance of CAS for human cancer, it likely plays a central role in canine tumours as well. So far, however, canine CAS lacks characterisation, and it remains unclear whether the biology between CAS from canine and human tumours is comparable. In this proof-of-principle study, using laser-capture microdissection, we isolated CAS and normal stroma from 13 formalin-fixed paraffin embedded canine simple mammary carcinomas and analysed the expression of seven known human CAS markers by RT-qPCR (Reverse Transcription quantitative PCR) and validated some targets by immunohistochemistry. We found that Col1a1 (Collagen1α1), αSMA (alpha Smooth Muscle Actin), FAP (Fibroblast activation protein), PDGFRß (Platelet-derived growth factor receptor beta), and Caveolin-1 were significantly upregulated in canine CAS, and the expression of CXCL12 (Stromal cell derived factor 1) significantly decreased, whereas MMP2 (Matrix Metalloproteinase 1) and IL6 (Interleukin 6) did not change. Our results suggest strong similarities in CAS biology in canine and human mammary carcinomas but also reveal some differences. To the best of our knowledge, this is the first report to provide a comprehensive expression analysis of the most important CAS markers in canine simple mammary carcinomas and further supports the validity of the dog as model for human cancer.

Measuring DNA Damage Using the Alkaline Comet Assay in Cultured Cells.

Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA-damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic integrity. Given the relevance of DNA damage and its repair in disease, measuring the amount of both aspects is of considerable interest. The comet assay is a widely used method that allows the measurement of both DNA damage and its repair in cells. For this, cells are treated with DNA-damaging agents and embedded into a thin layer of agarose on top of a microscope slide. Subsequent lysis removes all protein and lipid components to leave so-called 'nucleoids' consisting of naked DNA remaining in the agarose. These nucleoids are then subjected to electrophoresis, whereby the negatively charged DNA migrates toward the anode depending on its degree of fragmentation and creates shapes resembling comets, which can be subsequently visualized and analyzed by fluorescence microscopy. The comet assay can be adapted to assess a wide variety of genotoxins and repair kinetics, in addition to both DNA single-strand and double-strand breaks. In this protocol, we describe in detail how to perform the alkaline comet assay to assess single-strand breaks and their repair using cultured human cell lines. We describe the workflow for assessing the amount of DNA damage generated by agents such as hydrogen peroxide (H2O2) and methyl-methanesulfonate (MMS) or present endogenously in cells, and how to assess the repair kinetics after such an insult. The procedure described herein is easy to follow and allows the cost-effective assessment of single-strand breaks and their repair kinetics in cultured cells.

Measurement of DNA Damage Using the Neutral Comet Assay in Cultured Cells.

Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic integrity. Given the relevance of DNA damage and its repair for disease pathogenesis, measuring them is of considerable interest, and the comet assay is a widely used method for this. Cells treated with DNA damaging agents are embedded into a thin layer of agarose on top of a microscope slide. Subsequent lysis removes all protein and lipid components to leave 'nucleoids' consisting of naked DNA remaining in the agarose. These nucleoids are then subjected to electrophoresis, whereby the negatively charged DNA migrates towards the anode depending on its degree of fragmentation, creating shapes resembling comets, which can be visualized and analysed by fluorescence microscopy. The comet assay can be adapted to assess a wide variety of genotoxins and repair kinetics, and both DNA single-strand and double-strand breaks. In this protocol, we describe in detail how to perform the neutral comet assay to assess double-strand breaks and their repair using cultured human cell lines. We describe the workflow for assessing the amount of DNA damage generated by ionizing radiation or present endogenously in the cells, and how to assess the repair kinetics after such an insult. The procedure described herein is easy to follow and cost-effective.

MicroRNA Expression Profiling in the Prefrontal Cortex: Putative Mechanisms for the Cognitive Effects of Adolescent High Fat Feeding.

The medial prefrontal cortex (mPFC), master regulator of higher-order cognitive functions, is the only brain region that matures until late adolescence. During this period, the mPFC is sensitive to stressful events or suboptimal nutrition. For instance, high-fat diet (HFD) feeding during adolescence markedly impairs prefrontal-dependent cognition. It also provokes multiple changes at the cellular and synaptic scales within the mPFC, suggesting that major transcriptional events are elicited by HFD during this maturational period. The nature of this transcriptional reprogramming remains unknown, but may include epigenetic processes, in particular microRNAs, known to directly regulate synaptic functions. We used high-throughput screening in the adolescent mouse mPFC and identified 38 microRNAs differentially regulated by HFD, in particular mir-30e-5p. We used a luciferase assay to confirm the functional effect of mir-30e-5p on a chosen target: Ephrin-A3. Using global pathway analyses of predicted microRNA targets, we identified biological pathways putatively affected by HFD. Axon guidance was the top-1 pathway, validated by identifying gene expression changes of axon guidance molecules following HFD. Our findings delineate major microRNA transcriptional reprogramming within the mPFC induced by adolescent HFD. These results will help understanding the contribution of microRNAs in the emergence of cognitive deficits following early-life environmental events.

Persistent DNA strand breaks induce a CAF-like phenotype in normal fibroblasts.

Cancer-associated fibroblasts (CAFs) are an emerging target for cancer therapy as they promote tumour growth and metastatic potential. However, CAF targeting is complicated by the lack of knowledge-based strategies aiming to selectively eliminate these cells. There is a growing body of evidence suggesting that a pro-inflammatory microenvironment (e.g. ROS and cytokines) promotes CAF formation during tumorigenesis, although the exact mechanisms involved remain unclear. In this study, we reveal that a prolonged pro-inflammatory stimulation causes a de facto deficiency in base excision repair, generating unrepaired DNA strand breaks and thereby triggering an ATF4-dependent reprogramming of normal fibroblasts into CAF-like cells. Based on the phenotype of in vitro-generated CAFs, we demonstrate that midostaurin, a clinically relevant compound, selectively eliminates CAF-like cells deficient in base excision repair and prevents their stimulatory role in cancer cell growth and migration.

GH Dysfunction in Engrailed-2 Knockout Mice, a Model for Autism Spectrum Disorders.

Insulin-like growth factor 1 (IGF-1) signaling promotes brain development and plasticity. Altered IGF-1 expression has been associated to autism spectrum disorders (ASD). IGF-1 levels were found increased in the blood and decreased in the cerebrospinal fluid of ASD children. Accordingly, IGF-1 treatment can rescue behavioral deficits in mouse models of ASD, and IGF-1 trials have been proposed for ASD children. IGF-1 is mainly synthesized in the liver, and its synthesis is dependent on growth hormone (GH) produced in the pituitary gland. GH also modulates cognitive functions, and altered levels of GH have been detected in ASD patients. Here, we analyzed the expression of GH, IGF-1, their receptors, and regulatory hormones in the neuroendocrine system of adult male mice lacking the homeobox transcription factor Engrailed-2 (En2 (-/-) mice). En2 (-/-) mice display ASD-like behaviors (social interactions, defective spatial learning, increased seizure susceptibility) accompanied by relevant neuropathological changes (loss of cerebellar and forebrain inhibitory neurons). Recent studies showed that En2 modulates IGF-1 activity during postnatal cerebellar development. We found that GH mRNA expression was markedly deregulated throughout the neuroendocrine axis in En2 (-/-) mice, as compared to wild-type controls. In mutant mice, GH mRNA levels were significantly increased in the pituitary gland, blood, and liver, whereas decreased levels were detected in the hippocampus. These changes were paralleled by decreased levels of GH protein in the hippocampus but not other tissues of En2 (-/-) mice. IGF-1 mRNA was significantly up-regulated in the liver and down-regulated in the En2 (-/-) hippocampus, but no differences were detected in the levels of IGF-1 protein between the two genotypes. Our data strengthen the notion that altered GH levels in the hippocampus may be involved in learning disabilities associated to ASD.