MiR-34a Expression Has an Effect for Lower Risk of Metastasis and Associates with Expression Patterns Predicting Clinical Outcome in Breast Cancer

Peer reviewe

NQO1 expression correlates inversely with NFκB activation in human breast cancer

n/

Investigating chromosomal instability in long-term survivors with glioblastoma and grade 4 astrocytoma

Background Only a small group of patients with glioblastoma multiforme (GBM) survives more than 36 months, so-called long-term survivors. Recent studies have shown that chromosomal instability (CIN) plays a prognostic and predictive role among different cancer types. Here, we compared histological (chromosome missegregation) and bioinformatic metrics (CIN signatures) of CIN in tumors of GBM typical survivors (<= 36 months overall survival), GBM long-term survivors and isocitrate dehydrogenase (IDH)-mutant grade 4 astrocytomas.Methods Tumor sections of all gliomas were examined for anaphases and chromosome missegregation. Further CIN signature activity analysis in the The Cancer Genome Atlas (TCGA)-GBM cohort was performed.Results Our data show that chromosome missegregation is pervasive in high grade gliomas and is not different between the 3 groups. We find only limited evidence of altered CIN levels in tumors of GBM long-term survivors relative to the other groups, since a significant depletion in CIN signature 11 relative to GBM typical survivors was the only alteration detected. In contrast, within IDH-mutant grade 4 astrocytomas we detected a significant enrichment of CIN signature 5 and 10 activities and a depletion of CIN signature 1 activity relative to tumors of GBM typical survivors.Conclusions Our data suggest that CIN is pervasive in high grade gliomas, however this is unlikely to be a major contributor to the phenomenon of long-term survivorship in GBM. Nevertheless, further evaluation of specific types of CIN (signatures) could have prognostic value in patients suffering from grade 4 gliomas

Investigating chromosomal instability in long-term survivors with glioblastoma and grade 4 astrocytoma

Background Only a small group of patients with glioblastoma multiforme (GBM) survives more than 36 months, so-called long-term survivors. Recent studies have shown that chromosomal instability (CIN) plays a prognostic and predictive role among different cancer types. Here, we compared histological (chromosome missegregation) and bioinformatic metrics (CIN signatures) of CIN in tumors of GBM typical survivors (<= 36 months overall survival), GBM long-term survivors and isocitrate dehydrogenase (IDH)-mutant grade 4 astrocytomas.Methods Tumor sections of all gliomas were examined for anaphases and chromosome missegregation. Further CIN signature activity analysis in the The Cancer Genome Atlas (TCGA)-GBM cohort was performed.Results Our data show that chromosome missegregation is pervasive in high grade gliomas and is not different between the 3 groups. We find only limited evidence of altered CIN levels in tumors of GBM long-term survivors relative to the other groups, since a significant depletion in CIN signature 11 relative to GBM typical survivors was the only alteration detected. In contrast, within IDH-mutant grade 4 astrocytomas we detected a significant enrichment of CIN signature 5 and 10 activities and a depletion of CIN signature 1 activity relative to tumors of GBM typical survivors.Conclusions Our data suggest that CIN is pervasive in high grade gliomas, however this is unlikely to be a major contributor to the phenomenon of long-term survivorship in GBM. Nevertheless, further evaluation of specific types of CIN (signatures) could have prognostic value in patients suffering from grade 4 gliomas

Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

We would like to thank M.Oren (Weizmann Institute of Science) for kindly providing the MDM2 antibodies, the core facility for Bioinformatics and Expression Analysis (BEA, Karolinska, Huddinge) for assisting in massive parallel sequencing and computational infrastructure, as well as E Dratkiewicz, AS Nilsson, and JF Martinez for excellent technical assistance.Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.This work was funded by the following grants: the Swedish Cancer Society (grant number: 170176), the Swedish Research Council (VR-MH 2014-46602-117891-30), Novo Nordisk Foundation (NNF20OC0060590), Danish National Research Foundation (project CARD, DNRF 125), the Danish Cancer Society (R204-A12617-B153), DFF 1026-00241B (all granted to JB), and the Grant agency of the Czech Republic: GACR 20-28685S (granted to ZS and MM). Open access funding provided by Karolinska Institute.S

TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase–mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1’s role in HR, with potential clinical implications for cancer treatment

DNA damage-induced dynamic changes in abundance and cytosol-nuclear translocation of proteins involved in translational processes, metabolism, and autophagy

Ionizing radiation (IR) causes DNA double-strand breaks (DSBs) and activates a versatile cellular response regulating DNA repair, cell-cycle progression, transcription, DNA replication and other processes. In recent years proteomics has emerged as a powerful tool deepening our understanding of this multifaceted response. In this study we use SILAC-based proteomics to specifically investigate dynamic changes in cytoplasmic protein abundance after ionizing radiation; we present in-depth bioinformatics analysis and show that levels of proteins involved in autophagy (cathepsins and other lysosomal proteins), proteasomal degradation (Ubiquitin-related proteins), energy metabolism (mitochondrial proteins) and particularly translation (ribosomal proteins and translation factors) are regulated after cellular exposure to ionizing radiation. Downregulation of no less than 68 ribosomal proteins shows rapid changes in the translation pattern after IR. Additionally, we provide evidence of compartmental cytosol-nuclear translocation of numerous DNA damage related proteins using protein correlation profiling. In conclusion, these results highlight unexpected cytoplasmic processes actively orchestrated after genotoxic insults and protein translocation from the cytoplasm to the nucleus as a fundamental regulatory mechanism employed to aid cell survival and preservation of genome integrity.</p

Macrophages, Nitric Oxide and microRNAs Are Associated with DNA Damage Response Pathway and Senescence in Inflammatory Bowel Disease

Background: Cellular senescence can be a functional barrier to carcinogenesis. We hypothesized that inflammation modulates carcinogenesis through senescence and DNA damage response (DDR). We examined the association between senescence and DDR with macrophage levels in inflammatory bowel disease (IBD). In vitro experiments tested the ability of macrophages to induce senescence in primary cells. Inflammation modulating microRNAs were identified in senescence colon tissue for further investigation. Methodology/Principal Findings: Quantitative immunohistochemistry identified protein expression by colon cell type. Increased cellular senescence (HP1γ; P = 0.01) or DDR (γH2A.X; P = 0.031, phospho-Chk2, P = 0.014) was associated with high macrophage infiltration in UC. Co-culture with macrophages (ANA-1) induced senescence in >80% of primary cells (fibroblasts MRC5, WI38), illustrating that macrophages induce senescence. Interestingly, macrophage-induced senescence was partly dependent on nitric oxide synthase, and clinically relevant NO• levels alone induced senescence. NO• induced DDR in vitro, as detected by immunofluorescence. In contrast to UC, we noted in Crohn’s disease (CD) that senescence (HP1γ; P<0.001) and DDR (γH2A.X; P<0.05, phospho-Chk2; P<0.001) were higher, and macrophages were not associated with senescence. We hypothesize that nitric oxide may modulate senescence in CD; epithelial cells of CD had higher levels of NOS2 expression than in UC (P = 0.001). Microarrays and quantitative-PCR identified miR-21 expression associated with macrophage infiltration and NOS2 expression. Conclusions: Senescence was observed in IBD with senescence-associated β-galactosidase and HP1γ. Macrophages were associated with senescence and DDR in UC, and in vitro experiments with primary human cells showed that macrophages induce senescence, partly through NO•, and that NO• can induce DDR associated with senescence. Future experiments will investigate the role of NO• and miR-21 in senescence. This is the first study to implicate macrophages and nitrosative stress in a direct effect on senescence and DDR, which is relevant to many diseases of inflammation, cancer, and aging.Cancer Research Institute (New York, N.Y.) (Intramural Research Program)National Cancer Institute (U.S.) (Cancer Research Training Award Fellowship)Danish Cancer SocietyDanish National Research FoundationEuropean Commission (projects: Infla-Care, Biomedreg and DDResponse

FANCM c.5101C > T mutation associates with breast cancer survival and treatment outcome

Breast cancer (BC) is a heterogeneous disease, and different tumor characteristics and genetic variation may affect the clinical outcome. The FANCM c.5101C> T nonsense mutation in the Finnish population associates with increased risk of breast cancer, especially for triple-negative breast cancer patients. To investigate the association of the mutation with disease prognosis, we studied tumor phenotype, treatment outcome, and patient survival in 3,933 invasive breast cancer patients, including 101 FANCM c.5101C> T mutation carriers and 3,832 non-carriers. We also examined association of the mutation with nuclear immunohistochemical staining of DNA repair markers in 1,240 breast tumors. The FANCM c.5101C>T mutation associated with poor 10-year breast cancer-specific survival (hazard ratio (HR) 51.66, 95% confidence interval (CI) 1.09-2.52, p=0.018), with a more pronounced survival effect among familial cases (HR=2.93, 95% CI 1.5-5.76, p=1.80 x 10 23). Poor disease outcome of the carriers was also found among the estrogen receptor (ER) positive subgroup of patients (HR=1.8, 95% CI 1.09-2.98, p=0.021). Reduced survival was seen especially among patients who had not received radiotherapy (HR=3.43, 95% CI 1.6-7.34, p=1.50x10(-3)) but not among radiotherapy treated patients (HR=1.35, 95% CI 0.82-2.23, p=0.237). Significant interaction was found between the mutation and radiotherapy (p=0.040). Immunohistochemical analyses show that c.5101C> T carriers have reduced PAR-activity. Our results suggest that FANCM c.5101C>T nonsense mutation carriers have a reduced breast cancer survival but postoperative radiotherapy may diminish this survival disadvantage.Peer reviewe

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway(1,2). Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis