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Introduction: Fanconi anemia complementation group E (FANCE) is a Fanconi anemia (FA) pathway gene that regulates DNA repair. We evaluated the clinical relevance of FANCE expression in hepatocellular carcinoma (HCC). Methods: First, the associations between the expression of FA pathway genes including FANCE and clinical outcomes in HCC patients were analyzed in 2 independent cohorts: The Cancer Genome Atlas (TCGA, n = 373) and our patient cohort (n = 53). Localization of FANCE expression in HCC tissues was observed by immunohistochemical staining. Gene set enrichment analysis (GSEA) and gene network analysis (SiGN_BN) were conducted using the TCGA dataset. Next, an in vitro proliferation assay was performed using FANCE-knockdown HCC cell lines (HuH7 and HepG2). The association between mRNA expression of FANCE and that of DNA damage response genes in HCC was analyzed using TCGA and Cancer Cell Line Encyclopedia datasets. Finally, the association between FANCE mRNA expression and overall survival (OS) in various digestive carcinomas was analyzed using TCGA data. Results: FANCE was highly expressed in HCC cells. Multivariate analysis indicated that high FANCE mRNA expression was an independent factor predicting poor OS. GSEA revealed a positive relationship between enhanced FANCE expression and E2F and MYC target gene expression in HCC tissues. FANCE knockdown attenuated the proliferation of HCC cells, as well as reduced cdc25A expression and elevated histone H3 pSer10 expression. SiGN_BN revealed that FANCE mRNA expression was positively correlated with DNA damage response genes (H2A histone family member X and checkpoint kinase 1) in HCC tissues. Significant effects of high FANCE expression on OS were observed in hepatobiliary pancreatic carcinomas, including HCC. Conclusions: FANCE may provide a potential therapeutic target and biomarker of poor prognosis in HCC, possibly by facilitating tumor proliferation, which is mediated partly by cell cycle signaling activation.

Ubiquitin-specific protease 22 (USP22) expresses highly in lung adenocarcinoma (LUAD), which are associated with poor overall survival (OS). Microarray processing was performed to determine gene expression profiling, in which it was found that knocking down USP22 resulted in abnormal expression of a large number of genes. Differentially expressed genes (DEGs)-based protein-protein interaction (PPI) network was organized into 9 functional modules. These functional modules participated significantly in protein modification-related biological process and were involved in cancer-related pathways. The network was constructed to describe the global regulation of USP22-TF/pivot-module-pathway. It suggested that knocking down USP22 may up-regulate the expression of UBC to promote the pathways of cell cycle and ubiquitin-mediated proteolysis in the development of LUAD. More than that, knocking down USP22 can up-regulate STAT1 to activate JAK1-STAT1-caspase pathway, and promote apoptosis of tumor cell. Receiver operating characteristic (ROC) curve analysis suggested that E2F3, H2AFX, TFAP2A, PITX1, IRF7, and FOXM1 may be the potential diagnosis biomarkers for LUAD. On the other hand, BRCA1, FOXM1 and TFAP2A may be prognostic biomarkers of LUAD. In conclusion, we constructed a global regulation network to show that USP22 may promote the development of LUAD through ubiquitination and immunosuppression.

Cancer cells usually depend on the aberrant function of one or few driver genes to initiate and promote their malignancy, an attribute known as oncogene addiction. However, cancer cells might become dependent on the normal cellular functions of certain genes that are not oncogenes but ensure cell survival (non-oncogene addiction). The downregulation of DNA repair genes and the consequent genetic and epigenetic instability is key to promote malignancy, but the activation of the DNA-damage response (DDR) has been shown to become a type of non-oncogene addiction that critically supports tumour survival. While we know that different cancer types can become dependent on specific DDR genes for their survival, a systematic evaluation of DNA repair addiction at the pan-cancer level is missing. In the present study, this systematic evaluation was addressed using data derived from The Cancer Dependency Map and The Cancer Genome Atlas (TCGA). Following this approach, 59 DDR genes were identified as commonly essential in cancer cells with 14 genes being exclusively associated with better overall patient survival and 19 with worse overall survival. Notably, a specific molecular signature among the latter, characterized by DDR genes showing the weakest dependency scores, but significant upregulation was strongly associated with worse survival, supporting the presence and relevance of non-oncogenic addiction to DNA repair in cancer. Particularly, UBE2T, RFC4, POLQ, BRIP1, and H2AFX represent the best predictors of poor overall survival, and some might represent promising therapeutic targets, especially under the synthetic lethality approach.

MicroRNAs(miRNAs) are maladjusted in multifarious malignant tumor and can be considered as both carcinogens and tumor‐inhibiting factor. In the present study, we analyzed the miRNAs expression profiles and clinical information of 481 patients with head and neck squamous cell carcinoma (HNSCC) through the TCGA dataset to identify the prognostic miRNAs signature. A total of 114 significantly differentially expressed miRNAs (SDEMs) were identified, consisting of 60 up‐adjusted and 54 down‐adjusted miRNAs. The Kaplan‐Meier survival method identified the prognostic function of 2 miRNAs (miR‐4652‐5p and miR‐99a‐3P). Univariate and multivariate Cox regression analyses indicated that the 2 miRNAs were significant prognostic elements of HNSCC. Furthermore, bioinformatic analysis was conducted by means of 4 online gene predicted toolkits to recognize the target genes, and enrichment analysis was performed on the target genes by DAVID. The outcomes depicted that target genes were correlated with calcium, as well as cell proliferation, circadian entrainment, EGFR, PI3K‐Akt‐mTOR, and P53 signaling pathways. Finally, the PPI network was conducted in view of STRING database and Cytoscape. Eight hub genes were identified by CytoHubba and MCODE app, respectively, CBL, SKP1, H2AFX, HGF, POLR2F, UBE2I, VAMP2, and GNAI2 genes. As a result, we identified 2 miRNAs signatures, 8 hub genes, and significant signaling pathways for estimating the prognosis of HNSCC. In order to further explore the molecular mechanism of HNSCC occurrence and development, more comprehensive basic and clinical studies are needed., This study provides us with a new insight that multiple miRNAs can be together used for predicting the prognosis of patients with HNSCC. In order to further confirm the prognostic value of the 2 miRNA signatures, our future research may focus on exploring the relationship between miRNA and genic profile of HNSCC.

The aim of the present study is to construct a competitive endogenous RNA (ceRNA) regulatory network by using differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in patients with hepatocellular carcinoma (HCC), and to construct a prognostic model for predicting overall survival (OS) of HCC patients. Differentially expressed lncRNAs, miRNAs, and mRNAs were explored between HCC tissues and normal liver tissues. A prognostic model was built for predicting OS of HCC patients and receiver operating characteristic curves were used to evaluate the performance of the prognostic model. There were 455 differentially expressed lncRNAs, 181 differentially expressed miRNAs, and 5035 differentially expressed mRNAs. A ceRNA regulatory network was constructed based on 43 lncRNAs, 37 miRNAs, and 105 mRNAs. Eight mRNA biomarkers (H2AFX, SQSTM1, ITM2A, PFKP, TPD52L1, ACSL4, STRN3, and CPEB3) were identified as independent risk factors by multivariate Cox regression and were used to develop a prognostic model for OS. The C‐indexes in the model group were 0.776 (95% confidence interval [CI], 0.730‐0.822), 0.745 (95% CI, 0.699‐0.791), and 0.789 (95% CI, 0.743‐0.835) for 1‐, 3‐, and 5‐year OS, respectively. The current study revealed potential molecular biological regulation pathways and prognostic biomarkers by the ceRNA regulatory network. A prognostic model based on prognostic mRNAs in the ceRNA network might be helpful to predict the individual mortality risk for HCC patients. The individual mortality risk calculator can be used by visiting the following URL: https://zhangzhiqiao.shinyapps.io/Smart_cancer_predictive_system_HCC/.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease which arises due to various genetic, epigenetic and environmental factors. DNA methylation is an epigenetic factor that is found to have a role in the development and progression of HNSCC through genetic and epigenetic silencing. Analysis of the methylation data can facilitate us to explore variations in several gene sites and can narrow down our search for curing HNSCC. The aim of this study was to explore and analyze the DNA Methylation data of HNSCC and make intelligent machine learning (ML) models that can predict the expression levels of a particular gene site based on various features. Difference between the gene expression levels of normal and tumor samples obtained from TCGA was calculated and then the genes were classified into hypo-methylated, hyper-methylated and non-methylated, respectively. Moreover, network analysis and functional enrichment analysis was performed to identify the protein-protein interaction (PPI) and involvement in the biological process followed by training logistic regression, support vector machine (SVM) and k-nearest neighbors (KNN) models for prediction. Logistic regression was found to have the highest accuracy of 65% among all the ML models. Furthermore, MYC, POLR2A, ALB, MTOR, H2AFX, SMARCA4, PAX6, GATA3 and MDM2 were identified as the hub genes in the HNSCC network. Whereas, hyper-methylated, hypo-methylated and non-methylated genes were found to be enriched in neuroactive ligand-receptor interaction, neurogenesis, ion transport channels, cell cycle and plasma membrane. In future, more data and features are required for validation and improving the accuracy of the ML models.

Migration and invasion are the initial step in the metastatic process, while metastasis is responsible for the poor prognosis of head and neck squamous cell carcinoma (HNSCC). Since miRNA has been found as an important regulator of gene expression at the post-transcriptional level in various diseases including carcinoma, exploring the role of miRNA in cancer metastasis will facilitate the target therapy of advanced HNSCC. MiR-328-3p has been reported to be an onco-miRNA or a tumor suppressor in several cancers. However, the role of miR-328-3p in HNSCC migration and invasion remains undefined. In this study, we first demonstrated that miR-328-3p enhanced migration and invasion of HNSCC in vitro, accompanying with a promotion of epithelial-mesenchymal transition (EMT) and mTOR activity. Meanwhile, we confirmed that miR-328-3p directly targeted the 3'UTR of H2A histone family, member X (H2AFX), which served as a tumor suppressor in migration and invasion of HNSCC. Moreover, H2AFX could partially reverse the migration and invasion of HNSCC caused by miR-328-3p. Overall, our results indicated that miR-328-3p enhanced migration and invasion of HNSCC through targeting H2AFX and activated the mTOR pathway.

Phosphorylated H2A.X is a critical chromatin marker of DNA damage repair (DDR) in higher eukaryotes. However, H2A.X gene expression remains relatively uncharacterised. Replication-dependent (RD) histone genes generate poly(A)- mRNA encoding new histones to package DNA during replication. In contrast, replication-independent (RI) histone genes synthesise poly(A)+ mRNA throughout the cell cycle, translated into histone variants that confer specific epigenetic patterns on chromatin. Remarkably H2AFX, encoding H2A.X, is a hybrid histone gene, generating both poly(A)+ and poly(A)- mRNA isoforms. Here we report that the selective removal of either mRNA isoform reveals different effects in different cell types. In some cells, RD H2A.X poly(A)- mRNA generates sufficient histone for deposition onto DDR associated chromatin. In contrast, cells making predominantly poly(A)+ mRNA require this isoform for de novo H2A.X synthesis, required for efficient DDR. This highlights the importance of differential H2A.X mRNA 3’-end processing in the maintenance of effective DDR., H2A.X histone variant gene encodes poly(A)+ and poly(A)- mRNA isoforms which are differentially expressed depending on cell lines. Here the authors show that upon DNA damage, cells expressing more poly(A)+ isoform require this isoform for de novo H2A.X synthesis while cells with more poly(A)- isoform have sufficient H2A.X present in chromatin.

Introduction: Chronic obstructive pulmonary disease (COPD) is an independent risk factor of non-small cell lung cancer (NSCLC). This study aimed to analyze the key genes and potential molecular mechanisms that are involved in the development from COPD to NSCLC. Methods: Expression profiles of COPD and NSCLC in GSE106899, GSE12472, and GSE12428 were downloaded from the Gene Expression Omnibus (GEO) database, followed by identification of the differentially expressed genes (DEGs) between COPD and NSCLC. Based on the identified DEGs, functional pathway enrichment and lung carcinogenesis-related networks analyses were performed and further visualized with Cytoscape software. Then, principal component analysis (PCA), cluster analysis, and support vector machines (SVM) verified the ability of the top modular genes to distinguish COPD from NSCLC. Additionally, the corrections between these key genes and clinical staging of NSCLC were studied using the UALCAN and HPA websites. Finally, a prognostic risk model was constructed based on multivariate Cox regression analysis. Kaplan-Meier survival curves of the top modular genes on the training and verification sets were generated. Results: A total of 2350, 1914, and 1850 DEGs were obtained from GSE106899, GSE12472, and GSE12428 datasets, respectively. Following analysis of protein-protein interaction networks, the identified modular gene signatures containing H2AFX, MCM2, MCM3, MCM7, POLD1, and RPA1 were identified as markers for discrimination between COPD and NSCLC. The modular gene signatures were mainly enriched in the processes of DNA replication, cell cycle, mismatch repair, and others. Besides, the expression levels of these genes were significantly higher in NSCLC than in COPD, which was further verified by the immunohistochemistry. In addition, the high expression levels of H2AFX, MCM2, MCM7, and POLD1 correlate with poor prognosis of lung adenocarcinoma (LUAD). The Cox regression prognostic risk model showed the similar results and the predictive ability of this model is independent of other clinical variables. Conclusions: This study revealed several key modules that closely relate to NSCLC with underlying disease COPD, which provide a deeper understanding of the potential mechanisms underlying the malignant development from COPD to NSCLC. This study provides valuable prognostic factors in high-risk lung cancer patients with COPD.

Ovarian cancer is one of the three major gynecologic cancers in the world. The aim of this study is to find the relationship between ovarian cancer and diabetes mellitus by using the genetic screening technique. By GEO database query and related online tools of analysis, we analyzed 185 cases of ovarian cancer and 10 control samples from GSE26712, and a total of 379 different genes were identified, including 104 up-regulated genes and 275 down-regulated genes. The up-regulated genes were mainly enriched in biological processes, including cell adhesion, transcription of nucleic acid and biosynthesis, and negative regulation of cell metabolism. The down-regulated genes were enriched in cell proliferation, migration, angiogenesis and macromolecular metabolism. Protein-protein interaction was analyzed by network diagram and module synthesis analysis. The top ten hub genes (CDC20, H2AFX, ENO1, ACTB, ISG15, KAT2B, HNRNPD, YWHAE, GJA1 and CAV1) were identified, which play important roles in critical signaling pathways that regulate the process of oxidation-reduction reaction and carboxylic acid metabolism. CTD analysis showed that the hub genes were involved in 1,128 distinct diseases (bonferroni-corrected P

DNA damage caused by Helicobacter pylori infection and chronic inflammation or exposure to genotoxic agents is considered an important risk factor of gastric carcinogenesis. In this study, we have evaluated a short-term technique to detect DNA damage response to various chemical carcinogens; it involves visualization of Ser 139-phosphorylated histone H2AX (γ-H2AX) foci by immunohistochemistry and expression analysis of other genes by quantitative RT-PCR. Six-week-old male rats were intragastrically administered N-methyl-N-nitrosourea (MNU), 3,2’-dimethyl-4-aminobiphenyl (DMAB), dimethylnitrosamine (DMN), and 1,2- dimethylhydrazine (DMH) for 5 days/week for 4 weeks, using corn oil as a vehicle. Animals were sacrificed at day 28, and their stomachs were excised. γ-H2AX foci formation, indicating DNA double-strand breaks, was observed in the proliferative zone of both fundic and pyloric glands. The number of positive cells per gland was significantly high in pyloric glands in the MNU group and in fundic glands in the MNU and DMAB groups. A significant increase in p21waf1 mRNA level was observed in the DMN group compared with the control, which was in contrast to the decreasing tendency of the h2afx mRNA level in the MNU and DMN groups. Apoptotic cells positive for γ-H2AX pan or peripheral nuclear staining were observed on the surface layer of the fundic mucosa in the MNU group. The fundic pepsinogen a5 (pga5) mRNA level showed a significant decrease, indicating gland damage. The pyloric pepsinogen c mRNA level showed no change. In conclusion, γ-H2AX in combination with other gene expression analyses could be a useful biomarker in a short-term experiment on gastric chemical genotoxicity.

Purpose: To construct and analyze tumor-infiltrating immune cell and ceRNA (competitive endogenous RNA) networks in metastatic adrenal cortical carcinoma (ACC). Methods: A ceRNA network was established to identify the ceRNAs involved in metastasis of ACC based on 92 samples from TCGA, including 18 cases of metastasis and 74 cases of non-metastatic primary tumors. And the algorithm “cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT)” was used to quantify the proportion of immune cells in ACC. In addition, predictive nomograms based on the types of important immune cells or ceRNAs were constructed to predict ACC prognosis. Moreover, we evaluated the relationships between metastatic ACC-specific immune cells and ceRNA networks to identify the potential immune gene characteristics. Results: Ten prognostic biomarkers were identified as key members of the ceRNA network and three tumor-infiltrating immune cells were identified by CIBERSORT algorithm. Some important co-expression patterns between immune cells and ceRNAs network indicate significant correlation between Macrophages M0 and hsa-miR-130b-3p (P < 0.001), Macrophages M0 and H2AFX (P = 0.003). Conclusions: The present study inferred that the metastasis-related ceRNAs of H2AFX, hsa-miR-130b-3p and Macrophages M0 might play important roles in ACC metastasis.

Accounting for tumor heterogeneity, cancer stem cells (CSC) are involved in tumor metastasis, relapse, and drug resistance. Genes regulating CSC characteristics in lung adenocarcinoma (LUAD) were explored and validated in this study. The mRNA stemness index (mRNAsi) of more than 500 LUAD cases from The Cancer Genome Atlas database were calculated using a one-class logistic regression machine learning algorithm based on the mRNA expression of pluripotent stem cells and their differentiated progeny. mRNAsi-related key genes were identified by weighted correlation network analysis. The expression levels and prognostic roles of key genes were analyzed in Oncomine, PrognoScan, and Kaplan–Meier plotter databases, and validated using data from our center. The mRNAsi was significantly higher in LUAD compared with normal lung tissues. LUAD patients of advanced stage exhibited a higher mRNAsi and worse overall survival (OS). Eight key genes were identified: heat shock 70 kDa protein 4 (HSPA4), cell division cycle associated 7 (CDCA7), cell division cycle 20 (CDC20), cyclin-dependent kinase 1 (CDK1), CAP-GLY domain containing linker protein 1 (CLIP1), cyclin B1 (CCNB1), H2A histone family, member X (H2AFX), and Bloom syndrome, RecQ helicase-like (BLM). These genes were differentially expressed in various types of malignancies and validated in the LUAD cases. LUAD patients with low expression of CDC20, CDK1, CCNB1, H2AFX, or BLM had a significantly better OS, whereas OS was reduced for patients with low expression of CLIP1. In addition, the expression of CDCA7 did not significantly impact the OS of LUAD patients. The protein–protein interaction networks evaluated by STRING demonstrated strong relationships between these key genes, which were validated in our cases. The mRNAsi was significantly higher in LUAD compared with normal samples. Eight mRNAsi-related key genes were associated with prognosis and the cell cycle, and were strongly correlated with each other and differentially expressed in tumor and normal samples. We provide a new strategy for exploring stemness-related genes in LUAD cases.

For several years, studies conducted for discovering tenderness biomarkers have proposed a list of 20 candidates. The aim of the present work was to develop an innovative methodology to select the most predictive among this list. The relative abundance of the proteins was evaluated on five muscles of 10 Holstein cows: gluteobiceps, semimembranosus, semitendinosus, Triceps brachii and Vastus lateralis. To select the most predictive biomarkers, a multi-block model was used: The Data-Driven Sparse Partial Least Square. Semimembranosus and vastus lateralis muscles tenderness could be well predicted (R&sup2, = 0.95 and 0.94 respectively) with a total of 7 out of the 5 times 20 biomarkers analyzed. An original result is that the predictive proteins were the same for these two muscles: &micro, calpain, m-calpain, h2afx and Hsp40 measured in m. gluteobiceps and &micro, calpain, m-calpain and Hsp70-8 measured in m. Triceps brachii. Thus, this method is well adapted to this set of data, making it possible to propose robust candidate biomarkers of tenderness that need to be validated on a larger population.

Background. As the malignant tumor with the highest incidence in teenagers, osteosarcoma has become a major problem in oncology research. In addition to surgical management, the pharmacotherapeutic strategy for osteosarcoma treatment is an attractive way to explore. It has been demonstrated that biochanin A has an antitumor capacity on multiple kinds of solid tumor, including osteosarcoma. But the precise mechanism of biochanin A against osteosarcoma is still needed to be discovered.Objective. To identify the potential therapeutic targets of biochanin A in treating osteosarcoma.Methods. In present study, an integrated approach including network pharmacology and molecular docking technique was conducted, which mainly comprises target prediction, network construction, gene ontology, and pathway enrichment. CCK8 test was employed to evaluate the cell viability of MG63 cells. Western-blot was used to verify the target proteins of biochanin A.Results. Ninety-six and 114 proteins were obtained as the targets of biochanin A and osteosarcoma, respectively. TP53, IGF1, JUN, BGLAP, ATM, MAPK1, ATF3, H2AFX, BAX, CDKN2A, and EGF were identified as the potential targets of biochanin A against osteosarcoma. Based on the western-blot detection, the expression of BGLAP, BAX, and ATF3 in MG63 cell line changed under the treatment of biochanin A.Conclusion. Biochanin A can effectively suppress the proliferation of osteosarcoma and regulate the expression of BGLAP, BAX, and ATF3, which may act as the potential therapeutic targets of osteosarcoma.

Aim: Oxidative stress (OS) is strictly associated with senescence/pathogenesis of biological systems. As putative countermeasure to environmental OS, cerium oxide nanoparticles (nanoceria [NC]) were administered to muscle cells on ground and aboard the International Space Station. Materials & methods: Transcriptional analyses were conducted through microarray technology and hierarchical clustering. Venn diagram and gene ontology analyses were also performed on selected gene lists. Results: Adaptive responses to both NC administration and to permanence in real microgravity conditions occurred. Enrichment in the biological processes related to aging, body fat development and mesodermal tissue proliferation for NC-treated samples were found. Conclusion: Nanotechnology antioxidants promise applications to pathological conditions governed by OS on Earth and in life-hostile environments (low Earth orbit and deep space).

International audience; Tritium (3H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish (Danio rerio). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction (tnnt2d, tnni2a.4, slc6a1a or atp2a1l) and eye opacity (crygm2d9, crygmxl1, mipb or lim2.3) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H2O2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae. © 2018 Elsevier B.V.

The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem–loop (SL) structure. This SL structure interacts with the stem–loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3′hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3′ end of RD-histone mRNAs together with SLBP.

Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), found in cooked meat, is a known food carcinogen that causes several types of cancer, including breast cancer, as PhIP metabolites produce DNA adduct and DNA strand breaks. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity. To date, no study has examined the interaction of PhIP with curcumin in breast epithelial cells. The present study demonstrates the mechanisms by which curcumin inhibits PhIP-induced cytotoxicity in normal breast epithelial cells (MCF-10A). Curcumin significantly inhibited PhIP-induced DNA adduct formation and DNA double stand breaks with a concomitant decrease in reactive oxygen species (ROS) production. The expression of Nrf2, FOXO targets; DNA repair genes BRCA-1, H2AFX and PARP-1; and tumor suppressor P16 was studied to evaluate the influence on these core signaling pathways. PhIP induced the expression of various antioxidant and DNA repair genes. However, co-treatment with curcumin inhibited this expression. PhIP suppressed the expression of the tumor suppressor P16 gene, whereas curcumin co-treatment increased its expression. Caspase-3 and -9 were slightly suppressed by curcumin with a consequent inhibition of cell death. These results suggest that curcumin appears to be an effective anti-PhIP food additive likely acting through multiple molecular targets.

Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder of unclear pathogenesis. Recent studies have identified BRAF(V600E) mutations in most HCL patients, highlighting this abnormality as a molecular hallmark for this disease. Cell lines originating from HCL patients lack BRAF mutations but retain the typical piliferous morphology and the distinctive HCL immunophenotype, thus, constituting suitable tools for identifying alternative tumor genes and leukemic mechanisms in this malignancy. To this end, we integrated genomic and transcriptional profiling of the HCL cell line MLMA. The expression levels of genomically targeted genes were compared to four HCL control cell lines, thus, identifying 91 chromosomally deregulated genes. Gene set enrichment analysis of these indicted apoptosis, proliferation, and DNA damage response as altered processes. Accordingly, prominent target genes overexpressed in this cell line include ATM, BRAF, CDK6, CUTL1/CUX1, H2AFX, and REL. Treatment of MLMA with selective pharmacological inhibitors and specific siRNA-mediated gene knockdowns highlighted a central role for NFkB in their deregulation in HCL. Moreover, relevant expression profiling data from HCL and ABC-DLBCL cell lines display elevated NFkB-pathway activity when compared to GC-DLBCL equivalents. Finally, analysis of HCL patient samples in silico collectively supported the clinical significance of NFkB activation in this disease. In conclusion, we identified deregulated genes and multiple mechanisms underlying aberrantly activated NFkB-pathway in HCL. Therefore, NFkB may represent a B-cell specific hallmark of HCL and a promising novel therapeutic target, most notably in patients lacking BRAF mutations in this entity including variant HCL.

Although the impact of MAPT (Tau) expression has been well documented for neuronal cells in the context of tauopathies and neurodegenerative diseases, the impact and role of Tau expression in cancer, and specifically cancers of neuronal origin, is in its infancy. To determine the correlation between MAPT expression and survival in pediatric neuroblastoma, MAPT gene expression for samples from the TARGET pediatric neuroblastoma dataset was assessed. Initial analyses indicated that increased MAPT expression correlated with increased overall survival in neuroblastoma but not in ovarian cancer. Expression of apoptosis- and proliferation-effector genes in the neuroblastoma samples was consistent with the MAPT related survival result. Furthermore, we determined that higher neuroblastoma expression of APP also associated with neurodegeneration, correlated with better neuroblastoma survival rates. In sum, Gene expression associated with neuronal degenerative diseases was associated with a better neuroblastoma outcome. Abbreviations: ALS: Amyotrophic Lateral Sclerosis; APP: Amyloid Precursor Protein gene; CASP3: Caspase 3 gene; CASP9: Caspase 9 gene; H2AFX: H2A histone family, member X gene; HIST1H2AL: Histone H2A type 1 gene; HIST1H2BK: Histone H2B type 1-K gene; HIST1H3J: Histone H3J gene; HIST1H4B: Histone H4B gene; HIST2H2BE: Histone H2B type 2-E gene; HUGO: human genome organization; KM: Kaplan-Meier survival curve; MAPT: Tau gene; OV: Ovarian cancer; SNCA: alpha-syneculin gene; TARDBP: Transactive response DNA binding protein 43 kDa; TCGA: the cancer genome atlas

A imunossenescência é caracterizada pelo declínio funcional do sistema imune. Envolve mudanças fenotípicas que se traduzem em alterações sistêmicas, tendo importante impacto na sua funcionalidade. A imunossenescência tem sido associada a evolução e desfecho clínico de muitas doenças associadas ao envelhecimento prematuro, como a obesidade. A obesidade é uma condição patológica associada a expansão do tecido adiposo e a uma inflamação crônica de baixo grau que exerce efeitos negativos em múltiplos tecidos. Evidências de senescência celular, como atividade da enzima beta galactosidase (β-gal) aumentada, presença de fenótipo secretor associado a senescência e a expressão aumentada da proteína supressora de tumor TP53 (p53) e CDKN1A (p21) tem sido descritas no tecido adiposo de indivíduos portadores de obesidade. No entanto, é pouco explorado na literatura como esta inflamação sistêmica pode impactar no possível envelhecimento prematuro do sistema imune em indivíduos portadores de obesidade. Assim, o objetivo desta tese foi avaliar alterações associadas a imunossenescência prematura em células mononucleares de sangue periférico (PBMC) expostas ao ambiente pró-inflamatório próprio da obesidade. Para isto, foram utilizadas três abordagens experimentais: (1) análise de parâmetros associados a imunossenescência prematura em PBMC saudáveis incubadas com plasma de indivíduos portadores de obesidade; (2) análise de parâmetros de imunossenescência em macrófagos diferenciados a partir da linhagem celular U-937 e incubados com plasma de indivíduos portadores de obesidade sem comorbidades ou com síndrome metabólica e (3) validação dos parâmetros de imunossenescência em PBMC de indivíduos portadores de obesidade. Nossos resultados mostraram que o plasma de indivíduos portadores de obesidade é capaz de modular o fenótipo de PBMC previamente saudáveis, induzindo aumento na morte celular por apoptose, sinalização comprometida de H2AFX e TP53, disfunção mitocondrial basal e associada a produção de ATP, aumento de linfócitos senescentes T auxiliares CD4+CD28- e citotóxicos CD8+CD28-, secreção de citocinas inflamatórias e ativação de monócitos e macrófagos. Além disso, o plasma de indivíduos portadores de obesidade, independente da presença de comorbidades, induziu aumento da atividade da enzima β-gal associada com a diminuição da viabilidade de macrófagos, aumentando sua capacidade fagocítica. Parte destes dados foram confirmados em PBMC de indivíduos portadores de obesidade, mostrando aumento da taxa de morte celular por apoptose, instabilidade genômica através de uma cinética de reparo de dano ao DNA comprometida, menor sinalização de H2AFX e aumento na frequência de micronúcleos e cariólise. Ainda, foi confirmada a redução da sinalização da via TP53/CDKN2A (p53/p16) e de ERK1/2 e o aumento na secreção de citocinas inflamatórias. Juntos, nossos dados confirmam a hipótese da obesidade como uma patologia associada a imunossenescência prematura e instabilidade genômica. Immunosenescence is characterized by the functional decline of the immune system. It involves phenotypic changes that translate into systemic modifications, having an important impact on its functionality. Immunosenescence has been associated with evolution and clinical outcome of many diseases associated with premature aging, such as obesity. Obesity is a pathological condition associated with the expansion of adipose tissue and chronic low-grade inflammation that exerts its negative effects on multiple tissues. Evidence of cell senescence, such as increased beta-galactosidase (β-gal) enzyme’s activity, presence of the secretory phenotype associated with senescence, and increased expression of the tumor suppressor proteins TP53 (p53) and CDKN1A (p21) have been described in adipose tissue of individuals with obesity. However, it is not understood how this systemic inflammation can impact on premature aging of the immune system in individuals with obesity. Thus, the aim of this thesis is to evaluate changes associated with premature immunosenescence in peripheral blood mononuclear cells (PBMC) exposed to the proinflammatory environment of obesity. We used three different experimental approaches: (1) analysis of parameters associated with premature immunosenescence in healthy PBMC incubated with plasma from individuals with obesity; (2) analysis of immunosenescence parameters in the differentiated U-937 macrophages cell line incubated with plasma from subjects with obesity without comorbidities or with the metabolic syndrome and (3) validation of immunosenescence parameters in PBMC of individuals with obesity. Our results showed that the plasma of individuals with obesity can modulate the phenotype of previously healthy PBMC, inducing increased cell death by apoptosis, compromised signaling of H2AFX and TP53, basal mitochondrial dysfunction associated with ATP production, increased lymphocytes senescent T helper CD4+ CD28- and cytotoxic CD8+CD28-, secretion of inflammatory cytokines and activation of monocytes and macrophages. In addition, the plasma of individuals with obesity, regardless of the presence of comorbidities, induced an increase in the activity of the β-gal enzyme associated with decreased viability of macrophages, associated with increased phagocytic capacity. Some of these data were confirmed in PBMC of individuals with obesity, showing increased rate of cell death by apoptosis, genomic instability through DNA damage repair kinetics, decreased H2AFX signaling and increased micronucleus frequency and karyolysis. Furthermore, we confirmed reduction of TP53/ CDKN2A (TP53/p16) and ERK1/2 pathway signaling and increased secretion of inflammatory cytokines. Taking together, our data confirm the hypothesis of obesity as a pathology associated with premature immunosenescence and genomic instability.

Meiotic synapsis and recombination between homologs permits the formation of cross-overs that are essential for generating chromosomally balanced sperm and eggs. In mammals, surveillance mechanisms eliminate meiotic cells with defective synapsis, thereby minimizing transmission of aneuploidy. One such surveillance mechanism is meiotic silencing, the inactivation of genes located on asynapsed chromosomes, via ATR-dependent serine-139 phosphorylation of histone H2AFX (gamma H2AFX). Stimulation of ATR activity requires direct interaction with an ATR activation domain (AAD)-containing partner. However, which partner facilitates the meiotic silencing properties of ATR is unknown. Focusing on the best-characterized example of meiotic silencing, meiotic sex chromosome inactivation, we reveal this AAD-containing partner to be the DNA damage and checkpoint protein TOPBP1. Conditional TOPBP1 deletion during pachynema causes germ cell elimination associated with defective X chromosome gene silencing and sex chromosome condensation. TOPBP1 is essential for localization to the X chromosome of silencing "sensors," including BRCA1, and effectors, including ATR, gamma H2AFX, and canonical repressive histone marks. We present evidence that persistent DNA double-strand breaks act as silencing initiation sites. Our study identifies TOPBP1 as a critical factor in meiotic sex chromosome silencing

Introduction Histones are basic proteins responsible for DNA packaging in eukaryotic cell nuclei. H2AX is an H2A variant that plays an important role early in the DNA damage response. Although H2AX expression has been investigated, the direct contribution of this abundant chromatin protein in breast cancer is not well understood. The H2AFX gene encodes the same H2AX protein by both a short non-polyadenylated transcript and a longer polyadenylated transcript. We are characterising H2AFX copy number (CN), mRNA expression and protein abundance in breast cancer to discover if this foundation chromatin component contributing to genome stability could play a role in breast cancer. Material and methods H2AFX mRNA abundance was analysed in a panel of breast cell lines by absolute quantitative PCR. The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset was used for bioinformatic analysis. Results and discussions Analysis of the METABRIC dataset shows that H2AFX copy number alterations involving loss of one allele are very common (41%) and correlate with genome instability. No diploid deletions are observed, and only 2% of samples show H2AFX CN amplifications. No mutations were observed in the H2AFX coding region. Although we observed that the H2AFX short transcript is more abundant than the long transcript in the panel of breast cell lines, we found that the ratio of short and long transcript is constant. This validates the relevance of transcriptomic databases using polyA-selected H2AFX mRNAs that only report long transcript abundance. Analysis of H2AFX mRNA levels in METABRIC revealed an increase in H2AFX expression with worsening prognosis, despite the decrease in gene dosage. Furthermore, we found that H2AFX copy number decreases while H2AFX mRNA expression increases during the progression from luminal A to luminal B which are the most common breast cancer subtypes. Conclusion Analysis of primary breast tumour genomic datasets revealed that H2AFX frequently has reduced copy number but is almost never fully deleted, mutated, or amplified. One copy is lost usually as a result of a major deletion in the 11q chromosome arm. Paradoxically, H2AFX expression is frequently increased suggesting that an independent mechanism for regulation exists to drive up-regulation. We are now bringing together H2AFX copy number, mRNA expression and protein data to understand whether H2AFX expression responds to challenges to genome integrity in breast cancer.

Background: In September 2017, the FDA announced re-approval of gemtuzumab ozogamicin (GO), a CD33-directed antibody-drug conjugate, for treatment of newly diagnosed and relapsed/refractory AML setting the pace for a new era of personalized therapeutic options for AML. While the future of GO as a therapy in AML is bright, studies have shown that clinical response to GO is subject to interpatient variability (Gbadamosi 2018). Hitherto, efforts to understand the interpatient variation have focused on profiling CD33 expression levels (Pollard et al. 2016) and more recently on genetic variation in CD33 and ABCB1 predicative of GO response (Chauhan et al. 2019, Rafiee et al. 2019). However, efforts to understand interpatient variability in regards to calicheamicin, the DNA-damaging cytoxin linked to the antibody portion of GO, have been limited. Thus, we hypothesized that interpatient differences in expression levels of genes involved in pharmacodynamic effects of calicheamicin may impact GO response/resistance. Methods: Herein, our group has used the least absolute shrinkage and selection operator (LASSO) regression analysis method to identify critical genes with expression levels predictive of clinical outcomes in response to GO. Using data from the TARGET database, the expression levels of 18 genes, selected for their role in calicheamicin induced DNA-damage response, were extracted for AML patients treated with GO in the AAML03P1 and AAML0531 clinical trials (N=128; Table 1). Using a penalized LASSO regression algorithm (glmnet R-package), a Cox regression model was fit on to the expression levels of the selected gene. A thousand bootstraps of LASSO regression were performed and genes included in greater than 95% of the models were further selected. Average coefficients from the LASSO model were used to generate a gene signature equation designated as the DNA-damage response score (DDRS) given the nature of the genes included. Patients were classified into high (DDRS High) or low (DDRS Low) values for DDRS according to the median value and evaluated using Cox-proportional hazard model for survival data analysis, while the Chi-square test was used to examine the differences between categories, and the Wilcoxon rank-sum test was used to assess the differences in averages where appropriate. Results: The DDRS equation was defined as DDRS = (AKT1*-0.070) + (CASP9*-0.091) + (H2AFX*-0.160) + (XRCC4*0.373) where gene expression levels are multiplied by the coefficients obtained from the LASSO regression (Figure 1). Patients in the DDRS High group had significantly worse event free survival (EFS; HR = 2.22, P < 0.001; Figure 2A), lower complete remission rates (67.7% vs 94.6%, P < 0.001; Figure 3A), and a trend towards worse overall survival (OS; HR = 1.70, P = 0.058) as compared to patients within DDRS Low group. DDRS score was also significantly different amongst patients that were MRD+ve vs. MRD-ve after induction 1 therapy (21% difference, P = 0.038; Figure 3B). Consistent results were seen within standard risk group patients where patients in the DDRS High group had significantly worse EFS (HR = 2.29, P = 0.01; Figure 2B) as compared to those in the DDRS Low group. In preliminary multivariate Cox regression analysis, the DDRS remained a significant predictor of EFS amongst age, risk group, FLT3 status, and WBC (HR 2.42, P < 0.001; Table 2). Conclusion: Our preliminary investigation using LASSO regression model defined DDRS, a gene signature predictive of clinical response in patients treated with GO. The model included four genes well known for their involvement in DNA-damage response: AKT1, a kinase that regulates cell growth and division; CASP9, The initiator caspase involved in apoptosis; H2AFX, a DNA-damage marker that recruits other DNA-damage response proteins to damaged loci; and XRCC4, a ligase for DNA damage repair. Our future work focuses on expanding this investigation in bigger cohorts of patients representing different risk groups of AML as well as in vitro mechanistic studies. Once validated for its sensitivity and specificity to calicheamicin response, our results hold promise towards developing strategies for understanding interpatient variability in GO response and personalizing GO therapy based on diagnostic gene expression signatures. Disclosures No relevant conflicts of interest to declare.

Background Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Although multiple efforts have been made to understand the development of HCC, morbidity, and mortality rates remain high. In this study, we aimed to discover the mRNAs and long non-coding RNAs (lncRNAs) that contribute to the progression of HCC. We constructed a lncRNA-related competitive endogenous RNA (ceRNA) network to elucidate the molecular regulatory mechanism underlying HCC. Methods A microarray dataset (GSE54238) containing information about both mRNAs and lncRNAs was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and lncRNAs (DElncRNAs) in tumor tissues and non-cancerous tissues were identified using the limma package of the R software. The miRNAs that are targeted by DElncRNAs were predicted using miRcode, while the target mRNAs of miRNAs were retrieved from miRDB, miRTarBas, and TargetScan. Functional annotation and pathway enrichment of DEGs were performed using the EnrichNet website. We constructed a protein–protein interaction (PPI) network of DEGs using STRING, and identified the hub genes using Cytoscape. Survival analysis of the hub genes and DElncRNAs was performed using the gene expression profiling interactive analysis database. The expression of molecules with prognostic values was validated on the UALCAN database. The hepatic expression of hub genes was examined using the Human Protein Atlas. The hub genes and DElncRNAs with prognostic values as well as the predictive miRNAs were selected to construct the ceRNA networks. Results We found that 10 hub genes (KPNA2, MCM7, CKS2, KIF23, HMGB2, ZWINT, E2F1, MCM4, H2AFX, and EZH2) and four lncRNAs (FAM182B, SNHG6, SNHG1, and SNHG3) with prognostic values were overexpressed in the hepatic tumor samples. We also constructed a network containing 10 lncRNA–miRNA–mRNA pathways, which might be responsible for regulating the biological mechanisms underlying HCC. Conclusion We found that the 10 significantly overexpressed hub genes and four lncRNAs were negatively correlated with the prognosis of HCC. Further, we suggest that lncRNA SNHG1 and the SNHG3-related ceRNAs can be potential research targets for exploring the molecular mechanisms of HCC.

DNA methylation of tumor suppressor genes (TSGs) represents a frequent and early epigenetic event with potential applications for cancer detection and disease evolution. Our aim was to examine the stratification and prognostic biomarker role of the methylation of a novel panel of TSGs in bladder cancer. The methylation status of 18 TSGs was evaluated in bladder cancer cells (n=14) and paraffin-embedded primary bladder tumors (n=61), using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression, and disease-specific survival were analyzed using univariate and multivariate Cox models. PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF, and CACNA1A were discovered methylated in bladder cancer. The methylation of RUNX3 (p=0.026), TWIST1 (p=0.009), SFRP4 (p=0.002), and CCND2 (p=0.027) correlated to tumor stage. Univariate analyses indicated prognostic associations for recurrence (DLC1, SFRP5, H2AFX, CACNA1G), progression (DLC1, SFRP5, CACNA1G), disease-specific (PRDM2, DLC1, SFRP5, CACNA1G, and TIMP3), and overall survival (SFRP5 and TIMP3). In multivariate analyses, several TSGs remained as independent prognosticators for recurrence (SFRP5, H2AFX), progression (CACNA1G), and disease-specific survival (SFRP5). Thus, a novel set of TSGs was identified, frequently methylated in bladder cancer cells and tumors. TSG methylation allowed histopathologic and outcome stratification using paraffin-embedded tumors. This is clinically relevant by offering a strategy for the management of patients affected with uroepithelial neoplasias in pathology routine laboratories.

Down syndrome (DS) is characterized by mental retardation and the development of Alzheimer?s disease (AD). The reason for this development is not yet clear. Ts65Dn mice were used in this study. We collected their hippocampi and identified protein biomarkers using isobaric tags for relative and absolute quantitation (iTRAQ). We described the biochemical characteristics of proteins and explored their complicated network using GOA, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and network analysis. There were 374 significant differential proteins in the hippocampi of Ts65Dn mice. These were mainly binding proteins, related to single-organism and cellular processes. KEGG pathway analysis revealed that the insulin-signaling pathway was enriched with DS. Using PAJEK, a network of protein interactions was constructed and the top ten hub proteins were FYN, YBX1, VIM, PRKACA, EWSR1, H2AFX, CACNA1A, PTN, TFCP2L1 and CRKL. Through preliminarily discovered differentially expressed proteins by iTRAQ and bioinformatics analysis, we concluded that these proteins could be closely related to the neurological deficits of DS.

© 2017 Elsevier B.V. During colon cancer, epigenetic alterations contribute to the dysregulation of major cellular functions and signaling pathways. Modifications in chromatin signatures such as H3K4me3 and H3K9ac, which are associated with transcriptionally active genes, can lead to genomic instability and perturb the expression of gene sets associated with oncogenic processes. In order to further elucidate early pre-tumorigenic epigenetic molecular events driving CRC, we integrated diverse, genome-wide, epigenetic inputs (by high throughput sequencing of RNA, H3K4me3, and H3K9ac) and compared differentially expressed transcripts (DE) and enriched regions (DER) in an in-vivo rat colon cancer progression model. Carcinogen (AOM) effects were detected genome-wide at the RNA (116 DE genes), K9ac (49 DERs including 24 genes) and K4me3 (7678 DERs including 3792 genes) level. RNA-seq differential expression and pathway analysis indicated that interferon-associated innate immune responses were impacted by AOM exposure. Despite extensive associations between K4me3 DERs and colon tumorigenesis (1210 genes were linked to colorectal carcinoma) including FOXO3, GNAI2, H2AFX, MSH2, NR3C1, PDCD4 and VEGFA, these changes were not reflected at the RNA gene expression level during early cancer progression. Collectively, our results indicate that carcinogen-induced changes in gene K4me3 DERs are harbingers of future transcriptional events, which drive malignant transformation of the colon.

In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.

Background Neuroblastoma (NB) is the most common solid tumor found in children, and deletions within the 11q region are observed in 11% to 48% of these tumors. Notably, such tumors are associated with poor prognosis; however, little is known regarding the molecular targets located in 11q. Methods Genomic alterations of ATM , DNA damage response (DDR)-associated genes located in 11q ( MRE11A, H2AFX , and CHEK1 ), and BRCA1, BARD1, CHEK2, MDM2 , and TP53 were investigated in 45 NB-derived cell lines and 237 fresh tumor samples. PARP (poly [ADP-ribose] polymerase) inhibitor sensitivity of NB was investigated in in vitro and invivo xenograft models. All statistical tests were two-sided. Results Among 237 fresh tumor samples, ATM, MRE11A, H2AFX , and/or CHEK1 loss or imbalance in 11q was detected in 20.7% of NBs, 89.8% of which were stage III or IV. An additional 7.2% contained ATM rare single nucleotide variants (SNVs). Rare SNVs in DDR-associated genes other than ATM were detected in 26.4% and were mutually exclusive. Overall, samples with SNVs and/or copy number alterations in these genes accounted for 48.4%. ATM-defective cells are known to exhibit dysfunctions in homologous recombination repair, suggesting a potential for synthetic lethality by PARP inhibition. Indeed, 83.3% NB-derived cell lines exhibited sensitivity to PARP inhibition. In addition, NB growth was markedly attenuated in the xenograft group receiving PARP inhibitors (sham-treated vs olaprib-treated group; mean [SD] tumor volume of sham-treated vs olaprib-treated groups = 7377 [1451] m 3 vs 298 [312] m 3 , P = .001, n = 4). Conclusions Genomic alterations of DDR-associated genes including ATM, which regulates homologous recombination repair, were observed in almost half of NBs, suggesting that synthetic lethality could be induced by treatment with a PARP inhibitor. Indeed, DDR-defective NB cell lines were sensitive to PARP inhibitors. Thus, PARP inhibitors represent candidate NB therapeutics.

Bisphenol A (BPA) and nonylphenol (NP) are thought to mimic estrogens in their action, and are called endocrine disrupting chemicals. These phenolic chemicals are used in a number of commercial products and have been reported to be weakly estrogenic in previous studies. Their estrogenic activities are mainly dependent on their binding affinity for the estrogen receptors in vitro and in vivo. To identify genes elicited by BPA and NP, we carried out a microarray analysis in MCF-7 cells treated with BPA and NP using human c-DNA microarray including 416 endocrine system related genes. At the minimum foldchange criteria of 1.5, 14 and 29 genes were identified showing significant changes in gene expression resulting from BPA and NP, respectively. Especially, 2 genes were repressed and 6 genes were induced by BPA and NP as 17β-estradiol (E2). To validate the gene expression profiles identified from microarray analyses, the expression patterns of 4 representative genes, Selenoprotein P, plasma, 1 (SEPP1), Matrix Gla protein (MGP), H2A histone family, member X (H2AFX) and breast cancer 1, early onset (BRCA1) were examined by real time RT-PCR. We further examined the expression patterns of phthalic chemicals with estrogenic activity by comparing with those of E2 or phenolic chemicals to evaluate that alterations of these estrogen responsive genes may act as useful biomarkers to the endocrine disrupting chemicals with estrogenic activity.

Purpose: Genomic complexity is present in approximately 15% to 30% of all chronic lymphocytic leukemia (CLL) and has emerged as a strong independent predictor of rapid disease progression and short remission duration in CLL. We conducted this study to advance our understanding of the causes of genomic complexity in CLL. Experimental Design: We have obtained quantitative measurements of radiation-induced apoptosis and radiation-induced ATM autophosphorylation in purified CLL cells from 158 and 140 patients, respectively, and have used multivariate analysis to identify independent contributions of various biological variables on genomic complexity in CLL. Results: Here, we identify a strong independent effect of radiation resistance on elevated genomic complexity in CLL and describe radiation resistance as a predictor for shortened CLL survival. Furthermore, using multivariate analysis, we identify del17p/p53 aberrations, del11q, del13q14 type II (invariably resulting in Rb loss), and CD38 expression as independent predictors of genomic complexity in CLL, with aberrant p53 as a predictor of ∼50% of genomic complexity in CLL. Focusing on del11q, we determined that normalized ATM activity was a modest predictor of genomic complexity but was not independent of del11q. Through single nucleotide polymorphism array–based fine mapping of del11q, we identified frequent monoallelic loss of Mre11 and H2AFX in addition to ATM, indicative of compound del11q–resident gene defects in the DNA double-strand break response. Conclusions: Our quantitative analysis links multiple molecular defects, including for the first time del11q and large 13q14 deletions (type II), to elevated genomic complexity in CLL, thereby suggesting mechanisms for the observed clinical aggressiveness of CLL in patients with unstable genomes. Clin Cancer Res; 16(3); 835–47

The occurrence of a double-strand break in DNA activates a complex series of events that recruit to the break many proteins involved in its repair. A number of these proteins are modified by addition of a small protein, SUMO; this modification is performed SUMO ligases. In this work, Jackson and colleagues show that two such ligases, PIAS1 and PIAS4, add various SUMOs onto DNA repair proteins at double-strand breaks. The PIAS ligases are recruited via their SAP domains, and their activity is required for effective repair. SUMOylation by PIAS1 and PIAS4 is also necessary for the further modification of certain repair factors by ubiquitin, a somewhat larger protein adduct related to SUMO. The successive SUMOylation and ubiquitylation of repair proteins regulates their targeting to, and repair of, DNA breaks. Following the formation of a DNA double-strand break (DSB), cells activate the DNA-damage response and recruit a number of proteins to the lesion. Some of these proteins are modified by the attachment of small ubiquitin-related modifier (SUMO). Here, SUMO1, SUMO2 and SUMO3 are shown to accumulate at DSB sites in mammalian cells. SUMO1 and SUMO2/3 accrual requires the E3 ligase enzymes PIAS4 and PIAS1, which promote DSB repair. DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair1,2. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions3,4,5,6. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage7,8,9,10,11. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.