Cell fate after DNA damage

Het is essentieel voor een cel om zijn genetische informatie onveranderd en intact door te geven aan zijn nageslacht. Toch ontstaan in elke cel per dag enkele tienduizenden beschadigingen aan het DNA. Om te voorkomen dat deze schade leidt tot blijvende veranderingen in het DNA, genaamd: “mutaties”, hebben cellen verschillende mechanismen ontwikkeld die samen ‘de DNA-schaderespons’ (DSR) wordt genoemd. De DSR zorgt er onder andere voor dat schade opgemerkt wordt, dat lopende processen in de cel tijdelijk stilgelegd worden, en de schade vervolgens gerepareerd wordt. Erfelijke mutaties in DNA-reparatiegenen BRCA1 en BRCA2, onderstrepen het belang van een functionele DNA-reparatiemachinerie. Vrouwen met een erfelijke mutatie in één van deze reparatiegenen hebben tot 70% kans op het ontwikkelen van borstkanker voor hun 70-ste levensjaar. Naast het feit dat defecten in DNA-reparatiegenen kanker kunnen veroorzaken, biedt de DSR ook mogelijkheden voor doelgerichte behandeling van tumorcellen. Wanneer een tumorcel bijvoorbeeld niet genoeg tijd krijgt om DNA-schade te repareren, kan de hoeveelheid schade zo groot worden dat de tumorcel een programma activeert om zichzelf uit te schakelen. Het doel van dit promotieonderzoek was om factoren en mechanismen te identificeren die het lot van kankercellen na DNA-schade bepalen. Specifiek werd onderzocht hoe cellen het verlies van een essentieel DNA-reparatiegen (BRCA2) kunnen overleven. Hiernaast is onderzocht op welke manieren tumorcellen celdood kunnen ontlopen na DNA-schade geïnduceerd door chemotherapie of remmers van de DSR. Er is gebleken dat er verschillende manieren zijn hoe tumorcellen DNA-schade overleven. Deze manieren vormen veel mogelijkheden voor doelgerichte therapieën, maar maken het voorspellen van de respons op therapieën moeilijk

Modeling of Cisplatin-Induced Signaling Dynamics in Triple-Negative Breast Cancer Cells Reveals Mediators of Sensitivity

Triple-negative breast cancers (TNBCs) display great diversity in cisplatin sensitivity that cannot be explained solely by cancer-associated DNA repair defects. Differential activation of the DNA damage response (DDR) to cisplatin has been proposed to underlie the observed differential sensitivity, but it has not been investigated systematically. Systems-level analysis-using quantitative time-resolved signaling data and phenotypic responses, in combination with mathematical modeling-identifies that the activation status of cell-cycle checkpoints determines cisplatin sensitivity in TNBC cell lines. Specifically, inactivation of the cell-cycle checkpoint regulator MK2 or G3BP2 sensitizes cisplatin-resistant TNBC cell lines to cisplatin. Dynamic signaling data of five cell cycle-related signals predicts cisplatin sensitivity of TNBC cell lines. We provide a time-resolved map of cisplatin-induced signaling that uncovers determinants of chemo-sensitivity, underscores the impact of cell-cycle checkpoints on cisplatin sensitivity, and offers starting points to optimize treatment efficacy

BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity

Loss of BRCA2 affects genome stability and is deleterious for cellular survival. Using a genome-wide genetic screen in near-haploid KBM-7 cells, we show that tumor necrosis factor-alpha (TNF alpha) signaling is a determinant of cell survival upon BRCA2 inactivation. Specifically, inactivation of the TNF receptor (TNFR1) or its downstream effector SAM68 rescues cell death induced by BRCA2 inactivation. BRCA2 inactivation leads to proinflammatory cytokine production, including TNF alpha, and increases sensitivity to TNF alpha. Enhanced TNF alpha sensitivity is not restricted to BRCA2 inactivation, as BRCA1 or FANCD2 inactivation, or hydroxyurea treatment also sensitizes cells to TNF alpha. Mechanistically, BRCA2 inactivation leads to cGAS-positive micronuclei and results in a cell-intrinsic interferon response, as assessed by quantitative mass-spectrometry and gene expression profiling, and requires ASK1 and JNK signaling. Combined, our data reveals that micronuclei induced by loss of BRCA2 instigate a cGAS/STING-mediated interferon response, which encompasses rewired TNF alpha signaling and enhances TNF alpha sensitivity

The DNA damage response during mitosis

<p>Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence.</p><p>The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit.</p><p>Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. (C) 2013 Elsevier B.V. All rights reserved.</p>

Analysis of 16,172 patient-derived tumor samples indicate TPX2 as being essential for survival of genomically instable cancer cells

Mutations in homologous recombination (HR) genes, including BRCA1 and BRCA2, compromise DNA repair and lead to genomic instability (GI). GI is lethal to normal cells but is a characteristic of many cancers. Apparently, these cancers are somehow re-wired to survive high levels of GI. Identification of the genetic alterations that allow viability of genomically instable tumor cells may uncover novel therapeutic targets. To elucidate how these tumor cells are rewired, we analyzed publically available mRNA expression data of 16,172 human cancer samples. Functional genomic mRNA profiling (FGmRNA-profiling) was applied on these samples to infer levels of GI and to capture the downstream effects of somatic copy number alterations on gene expression. A genome-wide association analysis was subsequently performed to assess the correlation between FGmRNA signals of individual genes with the degree of GI. From the top 250 genes with strong positive correlation with GI, 11 genes were prioritized based on a co-functionality network in which genes are co-regulated and share similar predicted biological function. The 11 genes that were identified in this cluster were: BIRC5, UBE2C, CENPA, CDCA3, DEK, SKP2, TPX2, KIF2C, RAD21, MYBL2 and WDR67. To validate these findings in genetically-defined models, we engineered a panel of 5 triple negative breast cancer (TNBC) cell lines with doxycycline-inducible shRNAs targeting BRCA2. BRCA2 depletion resulted in a failure of RAD51 foci to localize to DNA double strand breaks which generated isogenic cell line pairs proficient and deficient of HR repair. First, we depleted each of the identified 11 genes using RNA interference in BT-549 cells and observed that depletion of TPX2, a microtubule-associated protein, led to largest differential levels of cell death when comparing the BRCA2-deficient with the BRCA2-proficient context (86.6% vs 32.9% cell death in BRCA2-depleted vs controlled depleted cells respectively). Subsequently, we could replicate this decreased survival with TPX2 depletion in a BRCA2-deficient context in an additional 2 out of 4 other TNBC cell lines. Furthermore, we investigated whether BRCA2-depleted cells were also more sensitive to depletion of Aurora kinase A, a substrate of TPX2. For this purpose, mouse mammary tumor cell lines, derived from Tp53-/- or Brca2-/-;Tp53-/- mice, or a Brca2-reconstituted version thereof were treated with an Aurora A inhibitor, Alisertib. Again, we found that the BRCA2-deficient cell line was more sensitive to Aurora A inhibition than the two BRCA2-proficient cell lines. In conclusion, FGmRNA-profiling of mRNA expression data of human cancer samples identified TPX2 as an essential gene for survival of BRCA2-deficient breast cancer cells, when compared to BRCA2-proficient cells. Thus, targeting the TPX2/AURKA axis could potentially act as a novel therapeutic target in the treatment of genomically instable cancers

Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

Crosstalk and complexity within signaling pathways and their perturbation by oncogenes limit component-by-component approaches to understanding human disease. Network analysis of how normal and oncogenic signaling can be rewired by drugs may provide opportunities to target tumors with high specificity and efficacy. Using targeted inhibition of oncogenic signaling pathways, combined with DNA-damaging chemotherapy, we report that time-staggered EGFR inhibition, but not simultaneous coadministration, dramatically sensitizes a subset of triple-negative breast cancer cells to genotoxic drugs. Systems-level analysis—using high-density time-dependent measurements of signaling networks, gene expression profiles, and cell phenotypic responses in combination with mathematical modeling—revealed an approach for altering the intrinsic state of the cell through dynamic rewiring of oncogenic signaling pathways. This process converts these cells to a less tumorigenic state that is more susceptible to DNA damage-induced cell death by reactivation of an extrinsic apoptotic pathway whose function is suppressed in the oncogene-addicted state.National Institutes of Health (U.S.) (Grant CA112967)National Institutes of Health (U.S.) (Grant GM68762)National Institutes of Health (U.S.) (Grant ES015339)United States. Dept. of Defense (Fellowship BC097884

RIF1 promotes replication fork protection and efficient restart to maintain genome stability

textabstractHomologous recombination (HR) and Fanconi Anemia (FA) pathway proteins in addition to their DNA repair functions, limit nuclease-mediated processing of stalled replication forks. However, the mechanism by which replication fork degradation results in genome instability is poorly understood. Here, we identify RIF1, a non-homologous end joining (NHEJ) factor, to be enriched at stalled replication forks. Rif1 knockout cells are proficient for recombination, but displayed degradation of reversed forks, which depends on DNA2 nuclease activity. Notably, RIF1-mediated protection of replication forks is independent of its function in NHEJ, but depends on its interaction with Protein Phosphatase 1. RIF1 deficiency delays fork restart and results in exposure of under-replicated DNA, which is the precursor of subsequent genomic instability. Our data implicate RIF1 to be an essential factor for replication fork protection, and uncover the mechanisms by which unprotected DNA replication forks can lead to genome instability in recombination-proficient conditions