A truncated and catalytically inactive isoform of KDM5B histone demethylase accumulates in breast cancer cells and regulates H3K4 tri-methylation and gene expression.

KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 methylation and induces derepression of a gene cluster, including the tumor suppressor Cav1 and several genes involved in the interferon-alpha and -gamma response. We discuss the relevance of this finding considering the hypothesis that KDM5B may possess regulatory roles independent of its catalytic activity.

Corrigendum: The Role of Histone Lysine Methylation in the Response of Mammalian Cells to Ionizing Radiation.

[This corrects the article DOI: 10.3389/fgene.2021.639602.].

The Role of Histone Lysine Methylation in the Response of Mammalian Cells to Ionizing Radiation.

Eukaryotic genomes are wrapped around nucleosomes and organized into different levels of chromatin structure. Chromatin organization has a crucial role in regulating all cellular processes involving DNA-protein interactions, such as DNA transcription, replication, recombination and repair. Histone post-translational modifications (HPTMs) have a prominent role in chromatin regulation, acting as a sophisticated molecular code, which is interpreted by HPTM-specific effectors. Here, we review the role of histone lysine methylation changes in regulating the response to radiation-induced genotoxic damage in mammalian cells. We also discuss the role of histone methyltransferases (HMTs) and histone demethylases (HDMs) and the effects of the modulation of their expression and/or the pharmacological inhibition of their activity on the radio-sensitivity of different cell lines. Finally, we provide a bioinformatic analysis of published datasets showing how the mRNA levels of known HMTs and HDMs are modulated in different cell lines by exposure to different irradiation conditions.

Active microbial ecosystem in Iron-Age tombs of the Etruscan civilization.

Earth's microbial biosphere extends down through the crust and much of the subsurface, including those microbial ecosystems located within cave systems. Here, we elucidate the microbial ecosystems within anthropogenic 'caves'; the Iron-Age, subterranean tombs of central Italy. The interior walls of the rock (calcium-rich macco) were painted ~2500 years ago and are covered with CaCO3 needles (known as moonmilk). The aims of the current study were to: identify biological/geochemical/biophysical determinants of and characterize bacterial communities involved in CaCO3 precipitation; challenge the maxim that biogenic activity necessarily degrades surfaces; locate the bacterial cells that are the source of the CaCO3 precipitate; and gain insight into the kinetics of moonmilk formation. We reveal that this environment hosts communities that consist primarily of bacteria that are mesophilic for temperature and xerotolerance (including Actinobacteria, Bacteroidetes and Proteobacteria); is populated by photosynthetic Cyanobacteria exhibiting heterotrophic nutrition (Calothrix and Chroococcidiopsis); and has CaCO3 precipitating on the rock surfaces (confirmation that this process is biogenic) that acts to preserve rather than damage the painted surface. We also identified that some community members are psychrotolerant (Polaromonas), acidotolerant or acidophilic (members of the Acidobacteria), or resistant to ionizing radiation (Brevundimonas and Truepera); elucidate the ways in which microbiology impacts mineralogy and vice versa; and reveal that biogenic formation of moonmilk can occur rapidly, that is, over a period of 10 to 56 years. We discuss the paradox that these ecosystems, that are for the most part in the dark and lack primary production, are apparently highly active, biodiverse and biomass-rich.

Artemisinin Derivatives with Antimelanoma Activity Show Inhibitory Effect against Human DNA Topoisomerase 1.

Artesunic acid and artemisinin are natural substances with promiscuous anticancer activity against different types of cancer cell lines. The mechanism of action of these compounds is associated with the formation of reactive radical species by cleavage of the sesquiterpene pharmacophore endoperoxide bridge. Here we suggested topoisomerase 1 as a possible molecular target for the improvement of the anticancer activity of these compounds. In this context, we report that novel hybrid and dimer derivatives of artesunic acid and artemisinin, bearing camptothecin and SN38 as side-chain biological effectors, can inhibit growth of yeast cells overexpressing human topoisomerase 1 and its enzymatic activity in vitro. These derivatives showed also anticancer activity in melanoma cell lines higher than camptothecin and paclitaxel. In silico molecular docking calculations highlighted a common binding mode for the novel derivatives, with the sesquiterpene lactone scaffold being located near the traditional recognition site for camptothecin, while the bioactive side-chain effector laid in the camptothecin cleft.

X-ray irradiated cultures of mouse cortical neural stem/progenitor cells recover cell viability and proliferation with dose-dependent kinetics.

Exposure of the developing or adult brain to ionizing radiation (IR) can cause cognitive impairment and/or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical NSPCs are resistant to low/moderate IR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53-related molecular pathways and cell cycle alterations. Irradiated NSPC cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. These results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult NSPC niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to IR.

MiR-146a is over-expressed and controls IL-6 production in cystic fibrosis macrophages.

Cystic fibrosis (CF) is an inherited disease that is characterised by susceptibility to bacterial infections and chronic lung inflammation. Recently, it was suggested that macrophages contribute to impaired host defence and excessive inflammatory responses in CF. Indeed, dysfunction attributed to CF macrophages includes decreased bacterial killing and exaggerated inflammatory responses. However, the mechanisms behind such defects have only been partially defined. MicroRNAs (miRNAs) have emerged as key regulators of several macrophage functions, including their activation, differentiation and polarisation. The goal of this study was to investigate whether miRNA dysregulation underlies the functional abnormalities of CF macrophages. MiRNA profiling of macrophages was performed, with 22 miRNAs identified as differentially expressed between CF and non-CF individuals. Among these, miR-146a was associated with significant enrichment of validated target genes involved in responses to microorganisms and inflammation. As miR-146a dysregulation has been reported in several human inflammatory diseases, we analysed the impact of increased miR-146a expression on inflammatory responses of CF macrophages. These data show that inhibition of miR-146a in lipopolysaccharide-stimulated CF macrophages results in increased interleukin-6 production, which suggests that miR-146a overexpression in CF is functional, to restrict inflammatory responses.

Small Molecule Inhibitors of KDM5 Histone Demethylases Increase the Radiosensitivity of Breast Cancer Cells Overexpressing JARID1B.

Background: KDM5 enzymes are H3K4 specific histone demethylases involved in transcriptional regulation and DNA repair. These proteins are overexpressed in different kinds of cancer, including breast, prostate and bladder carcinomas, with positive effects on cancer proliferation and chemoresistance. For these reasons, these enzymes are potential therapeutic targets. Methods: In the present study, we analyzed the effects of three different inhibitors of KDM5 enzymes in MCF-7 breast cancer cells over-expressing one of them, namely KDM5B/JARID1B. In particular we tested H3K4 demethylation (western blot); radio-sensitivity (cytoxicity and clonogenic assays) and damage accumulation (COMET assay and kinetics of H2AX phosphorylation). Results: we show that all three compounds with completely different chemical structures can selectively inhibit KDM5 enzymes and are capable of increasing sensitivity of breast cancer cells to ionizing radiation and radiation-induced damage. Conclusions: These findings confirm the involvement of H3K4 specific demethylases in the response to DNA damage, show a requirement of the catalytic function and suggest new strategies for the therapeutic use of their inhibitors.

JARID1B expression and its function in DNA damage repair are tightly regulated by miRNAs in breast cancer.

JARID1B/KDM5B histone demethylase's mRNA is markedly overexpressed in breast cancer tissues and cell lines and the protein has been shown to have a prominent role in cancer cell proliferation and DNA repair. However, the mechanism of its post-transcriptional regulation in cancer cells remains elusive. We performed a computational analysis of transcriptomic data from a set of 103 breast cancer patients, which, along with JARID1B upregulation, showed a strong downregulation of 2 microRNAs (miRNAs), mir-381 and mir-486, potentially targeting its mRNA. We showed that both miRNAs can target JARID1B 3'UTR and reduce luciferase's activity in a complementarity-driven repression assay. Moreover, MCF7 breast cancer cells overexpressing JARID1B showed a strong protein reduction when transfected with mir-486. This protein's decrease is accompanied by accumulation of DNA damage, enhanced radiosensitivity and increase of BRCA1 mRNA, 3 features previously correlated with JARID1B silencing. These results enlighten an important role of a miRNA's circuit in regulating JARID1B's activity and suggest new perspectives for epigenetic therapies.

Antitumor Effects of Epidrug/IFNα Combination Driven by Modulated Gene Signatures in Both Colorectal Cancer and Dendritic Cells.

Colorectal cancer results from the progressive accumulation of genetic and epigenetic alterations. IFN signaling defects play an important role in the carcinogenesis process, in which the inability of IFN transcription regulatory factors (IRF) to access regulatory sequences in IFN-stimulated genes (ISG) in tumors and in immune cells may be pivotal. We reported that low-dose combination of two FDA-approved epidrugs, azacytidine (A) and romidepsin (R), with IFNα2 (ARI) hampers the aggressiveness of both colorectal cancer metastatic and stem cells in vivo and triggers immunogenic cell death signals that stimulate dendritic cell (DC) function. Here, we investigated the molecular signals induced by ARI treatment and found that this drug combination increased the accessibility to regulatory sequences of ISGs and IRFs that were epigenetically silenced in both colorectal cancer cells and DCs. Likewise, specific ARI-induced histone methylation and acetylation changes marked epigenetically affected ISG promoters in both metastatic cancer cells and DCs. Analysis by ChIP-seq confirmed such ARI-induced epigenetically regulated IFN signature. The activation of this signal endowed DCs with a marked migratory capability. Our results establish a direct correlation between reexpression of silenced ISGs by epigenetic control and ARI anticancer activity and provide new knowledge for the development of innovative combined therapeutic strategies for colorectal cancer. Cancer Immunol Res; 5(7); 604-16. ©2017 AACR.

Leptin induction following irradiation is a conserved feature in mammalian epithelial cells and tissues.

PURPOSE: Leptin (LEP) is a peptide hormone with multiple physiological functions. Besides its systemic actions, it has important peripheral roles such as a mitogen action on keratinocytes following skin lesions. We previously showed that LEP mRNA is significantly induced in response to neutron irradiation in mouse skin and that the protein increases in the irradiated epidermis and in the related subcutaneous adipose tissue. In this work, we investigated the post-transcriptional regulation of LEP by miRNAs and the conservation of LEP's role in radiation response in human cells. METHODS: We used microarray analysis and real-time polymerase chain reaction (RT-PCR) to analyze modulation of miRNAs potentially targeting LEP in mouse skin following irradiation and bioinformatic analysis of transcriptome of irradiated human cell lines and cancer tissues from radiotherapy-treated patients to evaluate LEP expression. RESULTS AND CONCLUSIONS: We show that a network of miRNAs potentially targeting LEP mRNA is modulated in irradiated mouse skin and that LEP itself is significantly modulated by irradiation in human epithelial cell lines and in breast cancer tissues from radiotherapy-treated patients. These results confirm and extend the previous evidence that LEP has a general and important role in the response of mammalian cells to irradiation.

Yeast as a tool to select inhibitors of the cullin deneddylating enzyme Csn5.

The CSN complex plays a key role in various cellular pathways: through a metalloprotease activity of its Csn5 deneddylating enzyme, it regulates the activity of Cullin-RING ligases (CRLs). Indeed, Csn5 has been found amplified in many tumors, but, due to its pleiotropic effects, it is difficult to dissect its function and the involvement in cancer progression. Moreover, while growing evidences point to the neddylation function as a good target for drug development; specific inhibitors have not yet been developed for the CSN. Here, we propose the yeast Saccharomyces cerevisiae as a model system to screen libraries of small molecules as inhibitors of cullins deneddylation, taking advantage of the unique feature of this organism to survive without a functional CSN5 gene and to accumulate a fully neddylated cullin substrate. By combining molecular modeling and simple genetic tools, we were able to identify two small molecular fragments as selective inhibitors of Csn5 deneddylation function.

Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups.

Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

An high-throughput in vivo screening system to select H3K4-specific histone demethylase inhibitors.

BACKGROUND: Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes. METHODOLOGY/PRINCIPAL FINDINGS: In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity. CONCLUSIONS/SIGNIFICANCE: Our screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases.

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

HMGB1 induces the overexpression of miR-222 and miR-221 and increases growth and motility in papillary thyroid cancer cells.

Experimental and epidemiological studies have revealed that chronic inflammation contributes to cancer progression and even predisposes to cellular transformation. Inflammatory infiltrates in papillary thyroid cancer include lymphocytes, macrophages and cytokines. High-mobility group box 1 protein (HMGB1) is a late inflammatory cytokine that signals danger to the immune system through the receptor for advanced glycation end-products (RAGE) and Toll-like receptor. The activation of the above receptors results in the secretion of growth, chemotactic and angiogenic factors that contribute to chronic inflammation. In this study, we suggest that apart from the activation of signal transduction pathways by the activation of RAGE, the indirect inhibition of cell cycle regulators [such as phosphatase and tensin homolog (PTEN)] may also cause an increase in cell growth and motility. MicroRNAs (miRNAs) have increasingly been implicated in regulating the malignant progression of cancer. MiR-221 and miR-222 have been found to be deregulated in human papillary thyroid carcinomas. They are involved in cell proliferation through the inhibition of the cell cycle regulator, p27kip1, in human papillary carcinomas. In this study, we show that HMGB1 increases the expression of miR-221 and miR-222 in primary cultures of excised papillary lesions and in an established papillary cancer cell line (BC PAP). The overexpression of oncogenic miR-221 and miR-222 caused by HMGB1 is associated with an increase in malignancy scores, namely cell growth and motility.

The transcriptional response of mammalian cancer cells to irradiation is dominated by a cell cycle signature which is strongly attenuated in non-cancer cells and tissues.

PURPOSE: Our goal was to identify genes showing a general transcriptional response to irradiation in mammalian cells and to analyze their response in function of dose, time and quality of irradiation and of cell type. MATERIALS AND METHODS: We used a modified MIAME (Minimal Information About Microarray Experiments) protocol to import microarray data from 177 different irradiation conditions in the Radiation Genes database and performed cut-off-based selections and hierarchical gene clustering. RESULTS: We identified a set of 29 genes which respond to a wide range of irradiation conditions in different cell types and tissues. Functional analysis of the negatively modulated genes revealed a dominant signature of mitotic cell cycle regulation which appears both dose and time-dependent. This signature is prominent in cancer cells and highly proliferating tissues but it is strongly attenuated in non cancer cells. CONCLUSIONS: The transcriptional response of mammalian cancer cells to irradiation is dominated by a mitotic cell cycle signature both dose and time-dependent. This core response, which is present in cancer cells and highly proliferating tissues such as skin, blood and lymph node, is weaker or absent in non-cancer cells and in liver and spleen. CDKN1A (cyclin-dependent kinase inhibitor 1A) appears as the most generally induced mammalian gene and its response (mostly dose- and time-independent) seems to go beyond the typical DNA damage response.

Direct interaction of Gas41 and Myc encoded by amplified genes in nervous system tumours.

In order to understand better the role of the human Tip60 complex component Gas41, we analysed its expression levels in brain tumours and searched for possible interactors. Two-hybrid screening of a human foetal brain library allowed identification of some molecular interactors of Gas41. Among them we found n-Myc transcription factor. The interaction between Gas41 and n-Myc was validated by pull-down experiments. We showed that Gas41 is able to bind both n-Myc and c-Myc proteins, and that the levels of expression of Gas41 and Myc proteins were similar to each other in such brain tumors as neuroblastomas and glioblastomas. Finally, in order to identify which region of Gas41 is involved in the interaction with Myc proteins, we analysed the ability of Gas41 to substitute for its orthologue Yaf9 in yeast; we showed that the N-terminal portions of the two proteins, containing the YEATS domains, are interchangeable, while the C-terminal portions are species-specific. In fact we found that Gas41 C-terminal portion is required for Myc protein interaction in human.

HUVEC respond to radiation by inducing the expression of pro-angiogenic microRNAs.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either repression of translation or RNA degradation. They have been shown to be involved in a variety of biological processes such as development, differentiation and cell cycle control, but little is known about their involvement in the response to irradiation. We showed here that in human umbilical vein endothelial cells (HUVEC) some miRNAs previously shown to have a crucial role in vascular biology are transiently modulated in response to a clinically relevant dose of ionizing radiation. In particular we identified an early transcriptional induction of several members of the microRNA cluster 17-92 and other microRNAs already known to be related to angiogenesis. At the same time we observed a peculiar behavior of the miR-221/222 cluster, suggesting an important role of these microRNAs in HUVEC homeostasis. We observed an increased efficiency in the formation of capillary-like structures in irradiated HUVEC. These results could lead to a new interpretation of the effect of ionizing radiation on endothelial cells and on the response of tumor endothelial bed cells to radiotherapy.