Anti-Proliferative and Cytoprotective Activity of Aryl Carbamate and Aryl Urea Derivatives with Alkyl Groups and Chlorine as Substituents.

Natural cytokinines are a promising group of cytoprotective and anti-tumor agents. In this research, we synthesized a set of aryl carbamate, pyridyl urea, and aryl urea cytokinine analogs with alkyl and chlorine substitutions and tested their antiproliferative activity in MDA-MB-231, A-375, and U-87 MG cell lines, and cytoprotective properties in H2O2 and CoCl2 models. Aryl carbamates with the oxamate moiety were selectively anti-proliferative for the cancer cell lines tested, while the aryl ureas were inactive. In the cytoprotection studies, the same aryl carbamates were able to counteract the CoCl2 cytotoxicity by 3-8%. The possible molecular targets of the aryl carbamates during the anti-proliferative action were the adenosine A2 receptor and CDK2. The obtained results are promising for the development of novel anti-cancer therapeutics.

New immunological potential markers for triple negative breast cancer: IL18R1, CD53, TRIM, Jaw1, LTB, PTPRCAP.

Breast cancer (BC) is the second leading cause of cancer death in women worldwide, and settings of specific prognostic factors and efficacious therapies are made difficult by phenotypic heterogeneity of BC subtypes. Therefore, there is a current urgent need to define novel predictive genetic predictors that may be useful for stratifying patients with distinct prognostic outcomes. Here, we looked for novel molecular signatures for triple negative breast cancers (TNBCs). By a bioinformatic approach, we identified a panel of genes, whose expression was positively correlated with disease-free survival in TNBC patients, namely IL18R1, CD53, TRIM, Jaw1, LTB, and PTPRCAP, showing specific immune expression profiles linked to survival prediction; most of these genes are indeed expressed in immune cells and are required for productive lymphocyte activation. According to our hypothesis, these genes were not, or poorly, expressed in different TNBC cell lines, derived from either primary breast tumours or metastatic pleural effusions. This conclusion was further supported in vivo, as immuno-histochemical analysis on biopsies of TNBC invasive ductal carcinomas highlighted differential expression of these six genes in cancer cells, as well as in intra- and peri-tumoral infiltrating lymphocytes. Our data open to the possibility that inter-tumour heterogeneity of immune markers might have predictive value; further investigations are recommended in order to establish the real power of cancer-related immune profiles as prognostic factors.

p53 mutants cooperate with HIF-1 in transcriptional regulation of extracellular matrix components to promote tumor progression.

Mutations in the TP53 gene and microenvironmentally driven activation of hypoxia-inducible factor-1 (HIF-1) typically occur in later stages of tumorigenesis. An ongoing challenge is the identification of molecular determinants of advanced cancer pathogenesis to design alternative last-line therapeutic options. Here, we report that p53 mutants influence the tumor microenvironment by cooperating with HIF-1 to promote cancer progression. We demonstrate that in non-small cell lung cancer (NSCLC), p53 mutants exert a gain-of-function (GOF) effect on HIF-1, thus regulating a selective gene expression signature involved in protumorigenic functions. Hypoxia-mediated activation of HIF-1 leads to the formation of a p53 mutant/HIF-1 complex that physically binds the SWI/SNF chromatin remodeling complex, promoting expression of a selective subset of hypoxia-responsive genes. Depletion of p53 mutants impairs the HIF-mediated up-regulation of extracellular matrix (ECM) components, including type VIIa1 collagen and laminin-γ2, thus affecting tumorigenic potential of NSCLC cells in vitro and in mouse models in vivo. Analysis of surgically resected human NSCLC revealed that expression of this ECM gene signature was highly correlated with hypoxic tumors exclusively in patients carrying p53 mutations and was associated with poor prognosis. Our data reveal a GOF effect of p53 mutants in hypoxic tumors and suggest synergistic activities of p53 and HIF-1. These findings have important implications for cancer progression and might provide innovative last-line treatment options for advanced NSCLC.

ZNF281 inhibits neuronal differentiation and is a prognostic marker for neuroblastoma.

Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB.

Alterations in neuronal metabolism contribute to the pathogenesis of prion disease.

Neurodegenerative conditions are characterised by a progressive loss of neurons, which is believed to be initiated by misfolded protein aggregations. During this time period, many physiological and metabolomic alterations and changes in gene expression contribute to the decline in neuronal function. However, these pathological effects have not been fully characterised. In this study, we utilised a metabolomic approach to investigate the metabolic changes occurring in the hippocampus and cortex of mice infected with misfolded prion protein. In order to identify these changes, the samples were analysed by ultrahigh-performance liquid chromatography-tandem mass spectroscopy. The present dataset comprises a total of 498 compounds of known identity, named biochemicals, which have undergone principal component analysis and supervised machine learning. The results generated are consistent with the prion-inoculated mice having significantly altered metabolic profiles. In particular, we highlight the alterations associated with the metabolism of glucose, neuropeptides, fatty acids, L-arginine/nitric oxide and prostaglandins, all of which undergo significant changes during the disease. These data provide possibilities for future studies targeting and investigating specific pathways to better understand the processes involved in neuronal dysfunction in neurodegenerative diseases.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Polypharmacology of Approved Anticancer Drugs.

The major drug discovery efforts in oncology have been concentrated on the development of selective molecules that are supposed to act specifically on one anticancer mechanism by modulating a single or several closely related drug targets. However, a bird's eye view on data from multiple available bioassays implies that most approved anticancer agents do, in fact, target many more proteins with different functions. Here we will review and systematize currently available information on the targets of several anticancer drugs along with revision of their potential mechanisms of action. Polypharmacology of the current antineoplastic agents suggests that drug clinical efficacy in oncology can be achieved only via modulation of multiple cellular mechanisms.

Automated Detection of Heterogeneity in Medical Tactile Images.

Artificial tactile sensing is a capability important for many applications and, in particular, for endoscopic surgery. A recently developed Medical Tactile Endosurgical Complex (MTEC) that is a certified and commercially available product is an efficient tool that provides such a capability. Currently the analysis of intraoperative tactile images that are registered and visualized by MTEC is performed manually by a surgeon. We show that heterogeneity detection - a key constituent of intraoperative tactile images analysis - can be efficiently automated. Such automation essentially reduces the requirement of attention retaining during the MTEC-based palpation.

p73 promotes glioblastoma cell invasion by directly activating POSTN (periostin) expression.

Glioblastoma Multiforme is one of the most highly metastatic cancers and constitutes 70% of all gliomas. Despite aggressive treatments these tumours have an exceptionally bad prognosis, mainly due to therapy resistance and tumour recurrence. Here we show that the transcription factor p73 confers an invasive phenotype by directly activating expression of POSTN (periostin, HGNC:16953) in glioblastoma cells. Knock down of endogenous p73 reduces invasiveness and chemo-resistance, and promotes differentiation in vitro. Using chromatin immunoprecipitation and reporter assays we demonstrate that POSTN, an integrin binding protein that has recently been shown to play a major role in metastasis, is a transcriptional target of TAp73. We further show that POSTN overexpression is sufficient to rescue the invasive phenotype of glioblastoma cells after p73 knock down. Additionally, bioinformatics analysis revealed that an intact p73/ POSTN axis, where POSTN and p73 expression is correlated, predicts bad prognosis in several cancer types. Taken together, our results support a novel role of TAp73 in controlling glioblastoma cell invasion by regulating the expression of the matricellular protein POSTN.

E3 ubiquitin ligase Pirh2 enhances tumorigenic properties of human non-small cell lung carcinoma cells.

The product of RCHY1 human gene, Pirh2, is a RING-finger containing E3 ligase that modifies p53 with ubiquitin residues resulting in its subsequent degradation in proteasomes. Transcription of RCHY1 is regulated by p53 itself thus forming a negative regulatory feedback loop. Functionally, by eliminating p53, Pirh2 facilitates tumorigenesis. However, the role of Pirh2 in cancer cells lacking p53 is yet not well understood. Therefore, we decided to elucidate the role of Pirh2 in p53-negative human non-small cell lung carcinoma cells, H1299. We found that ectopic expression of Pirh2 enhanced cell proliferation, resistance to doxorubicin, and increased migration potential. Ablation of Pirh2 by specific shRNA reversed these phenotypes. Mechanistically, Pirh2 increased mRNA and protein levels of the c-Myc oncogene. The bioinformatics data indicate that co-expression of both c-Myc and Pirh2 strongly correlated with poor survival of lung cancer patients. Collectively, our results suggest that Pirh2 can be considered as a potential pharmacological target for developing anticancer therapies to treat p53-negative cancers.

Exploration of individuality in drug metabolism by high-throughput metabolomics: The fast line for personalized medicine.

In many cases, individuality in metabolism of a drug is a reliable predictor of the drug efficacy/safety. Modern high-throughput metabolomics is an ideal instrument to track drug metabolism in an individual after treatment. Productivity and low cost of the metabolomics are sufficient to analyse a large cohort of patients to explore individual variations in drug metabolism and to discover drug metabolic biomarkers indicative of drug efficacy/safety. The only potential disadvantage of metabolomics becoming a routine clinical procedure is a need to treat the patient once before making a prognosis. However, in many clinical applications this would not be a limitation. Here, we explore current opportunities and challenges for translating high-throughput metabolomics into the platform for personalized medicine.

KMT Set7/9 affects genotoxic stress response via the Mdm2 axis.

Genotoxic stress inflicted by anti-cancer drugs causes DNA breaks and genome instability. DNA double strand breaks induced by irradiation or pharmacological inhibition of Topoisomerase II activate ATM (ataxia-telangiectasia-mutated) kinase signalling pathway that in turn triggers cell cycle arrest and DNA repair. ATM-dependent gamma-phosphorylation of histone H2Ax and other histone modifications, including ubiquitnylation, promote exchange of histones and recruitment of DNA damage response (DDR) and repair proteins. Signal transduction pathways, besides DDR itself, also control expression of genes whose products cause cell cycle arrest and/or apoptosis thus ultimately affecting the sensitivity of cells to genotoxic stress. In this study, using a number of experimental approaches we provide evidence that lysine-specific methyltransferase (KMT) Set7/9 affects DDR and DNA repair, at least in part, by regulating the expression of an E3 ubiquitin ligase, Mdm2. Furthermore, we show that Set7/9 physically interacts with Mdm2. Several cancer cell lines with inverse expression of Set7/9 and Mdm2 displayed diminished survival in response to genotoxic stress. These findings are signified by our bioinformatics studies suggesting that the unleashed expression of Mdm2 in cancer patients with diminished expression of Set7/9 is associated with poor survival outcome.

Polypharmacology of small molecules targeting the ubiquitin-proteasome and ubiquitin-like systems.

Targeting the ubiquitin-proteasome system (UPS) and ubiquitin-like signalling systems (UBL) has been considered a promising therapeutic strategy to treat cancer, neurodegenerative and immunological disorders. There have been multiple efforts recently to identify novel compounds that efficiently modulate the activities of different disease-specific components of the UPS-UBL. However, it is evident that polypharmacology (the ability to affect multiple independent protein targets) is a basic property of small molecules and even highly potent molecules would have a number of "off target" effects. Here we have explored publicly available high-throughput screening data covering a wide spectrum of currently accepted drug targets in order to understand polypharmacology of small molecules targeting different components of the UPS-UBL. We have demonstrated that molecules targeting a given UPS-UBL protein also have high odds to target a given off target spectrum. Moreover, the off target spectrum differs significantly between different components of UPS-UBL. This information can be utilized further in drug discovery efforts, to improve drug efficiency and to reduce the risk of potential side effects of the prospective drugs designed to target specific UPS-UBL components.

Perspective on multi-target antiplatelet therapies: high content phenotypic screening as an unbiased source of novel polypharmacological strategies.

Platelets play an important role in cardiovascular thrombosis as well as in many other pathological conditions such as inflammation, atherosclerosis and cancer. While multi-target strategies to treat complex diseases are gaining considerable attention, current development of antiplatelet therapies is mostly oriented towards several single targets, arising from our present understanding of the regulation of platelet activation. Limited efforts to develop multi-target agents or multidrug therapies are mostly due to a lack of a systematic basis to define target combinations with synergistic effects. Here we discuss the perspective to use high content phenotypic screening of in vitro models as a potential source for inference of synergetic multi-target strategies to control platelet activation.

p63 Sustains self-renewal of mammary cancer stem cells through regulation of Sonic Hedgehog signaling.

The predominant p63 isoform, ΔNp63, is a master regulator of normal epithelial stem cell (SC) maintenance. However, in vivo evidence of the regulation of cancer stem cell (CSC) properties by p63 is still limited. Here, we exploit the transgenic MMTV-ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) mouse model of carcinogenesis to dissect the role of p63 in the regulation of mammary CSC self-renewal and breast tumorigenesis. ErbB2 tumor cells enriched for SC-like properties display increased levels of ΔNp63 expression compared with normal mammary progenitors. Down-regulation of p63 in ErbB2 mammospheres markedly restricts self-renewal and expansion of CSCs, and this action is fully independent of p53. Furthermore, transplantation of ErbB2 progenitors expressing shRNAs against p63 into the mammary fat pads of syngeneic mice delays tumor growth in vivo. p63 knockdown in ErbB2 progenitors diminishes the expression of genes encoding components of the Sonic Hedgehog (Hh) signaling pathway, a driver of mammary SC self-renewal. Remarkably, p63 regulates the expression of Sonic Hedgehog (Shh), GLI family zinc finger 2 (Gli2), and Patched1 (Ptch1) genes by directly binding to their gene regulatory regions, and eventually contributes to pathway activation. Collectively, these studies highlight the importance of p63 in maintaining the self-renewal potential of mammary CSCs via a positive modulation of the Hh signaling pathway.

TAp73 promotes anabolism.

Metabolic adaptation has emerged as a hallmark of cancer and a promising therapeutic target, as rapidly proliferating cancer cells adapt their metabolism increasing nutrient uptake and reorganizing metabolic fluxes to support biosynthesis. The transcription factor p73 belongs to the p53-family and regulates tumorigenesis via its two N-terminal isoforms, with (TAp73) or without (ΔNp73) a transactivation domain. TAp73 acts as tumor suppressor, at least partially through induction of cell cycle arrest and apoptosis and through regulation of genomic stability. Here, we sought to investigate whether TAp73 also affects metabolic profiling of cancer cells. Using high throughput metabolomics, we unveil a thorough and unexpected role for TAp73 in promoting Warburg effect and cellular metabolism. TAp73-expressing cells show increased rate of glycolysis, higher amino acid uptake and increased levels and biosynthesis of acetyl-CoA. Moreover, we report an extensive TAp73-mediated upregulation of several anabolic pathways including polyamine and synthesis of membrane phospholipids. TAp73 expression also increases cellular methyl-donor S-adenosylmethionine (SAM), possibly influencing methylation and epigenetics, and promotes arginine metabolism, suggestive of a role in extracellular matrix (ECM) modeling. In summary, our data indicate that TAp73 regulates multiple metabolic pathways that impinge on numerous cellular functions, but that, overall, converge to sustain cell growth and proliferation.

Bioinformatics analysis of the serine and glycine pathway in cancer cells.

Serine and glycine are amino acids that provide the essential precursors for the synthesis of proteins, nucleic acids and lipids. Employing 3 subsequent enzymes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine phosphatase (PSPH), phosphoserine aminotransferase 1 (PSAT1), 3-phosphoglycerate from glycolysis can be converted in serine, which in turn can by converted in glycine by serine methyl transferase (SHMT). Besides proving precursors for macromolecules, serine/glycine biosynthesis is also required for the maintenance of cellular redox state. Therefore, this metabolic pathway has a pivotal role in proliferating cells, including cancer cells. In the last few years an emerging literature provides genetic and functional evidences that hyperactivation of serine/glycine biosynthetic pathway drives tumorigenesis. Here, we extend these observations performing a bioinformatics analysis using public cancer datasets. Our analysis highlighted the relevance of PHGDH and SHMT2 expression as prognostic factor for breast cancer, revealing a substantial ability of these enzymes to predict patient survival outcome. However analyzing patient datasets of lung cancer our analysis reveled that some other enzymes of the pathways, rather than PHGDH, might be associated to prognosis. Although these observations require further investigations they might suggest a selective requirement of some enzymes in specific cancer types, recommending more cautions in the development of novel translational opportunities and biomarker identification of human cancers.

Metabolic effect of TAp63α: enhanced glycolysis and pentose phosphate pathway, resulting in increased antioxidant defense.

TAp63α is a member of the p53 family, which plays a central role in epithelial cancers. Recently, a role has emerged for p53 family members in cancer metabolic modulation. In order to assess whether TAp63α plays a role in cancer metabolism, we exploited p53-null osteosarcoma Tet-On Saos-2 cells, in which the expression of TAp63α was dependent on doxycycline supplementation to the medium. Metabolomics labeling experiments were performed by incubating the cells in 13C-glucose or 13C15N-glutamine-labeled culture media, as to monitor metabolic fluxes upon induced expression of TAp63α. Induced expression of TAp63α resulted in cell cycle arrest at the G1 phase. From a metabolic standpoint, expression of Tap63α promoted glycolysis and the pentose phosphate pathway, which was uncoupled from nucleotide biosynthesis, albeit prevented oxidative stress in the form of oxidized glutathione. Double 13C-glucose and 13C15N-glutamine metabolic labeling confirmed that induced expression of TAp63α corresponded to a decreased flux of pyruvate to the Krebs cycle and decreased utilization of glutamine for catabolic purposes in the TCA cycle. Results were not conclusive in relation to anabolic utilization of labeled glutamine, since it is unclear to what extent the observed minor TAp63α-dependent increases of glutamine-derived labeling in palmitate could be tied to increased rates of reductive carboxylation and de novo synthesis of fatty acids. Finally, bioinformatics elaborations highlighted a link between patient survival rates and the co-expression of p63 and rate limiting enzymes of the pentose phosphate pathway, G6PD and PGD.

DNA damage modulates interactions between microRNAs and the 26S proteasome.

26S proteasomes are known as major non-lysosomal cellular machines for coordinated and specific destruction of ubiquitinylated proteins. The proteolytic activities of proteasomes are controlled by various post-translational modifications in response to environmental cues, including DNA damage. Besides proteolysis, proteasomes also associate with RNA hydrolysis and splicing. Here, we extend the functional diversity of proteasomes by showing that they also dynamically associate with microRNAs (miRNAs) both in the nucleus and cytoplasm of cells. Moreover, DNA damage induced by an anti-cancer drug, doxorubicin, alters the repertoire of proteasome-associated miRNAs, enriching the population of miRNAs that target cell cycle checkpoint regulators and DNA repair proteins. Collectively, these data uncover yet another potential mode of action for proteasomes in the cell via their dynamic association with microRNAs.

AERS spider: an online interactive tool to mine statistical associations in Adverse Event Reporting System.

BACKGROUND: Exploration of the Adverse Event Reporting System (AERS) data by a wide scientific community is limited due to several factors. First, AERS data must be intensively preprocessed to be converted into analyzable format. Second, application of the currently accepted disproportional reporting measures results in false positive signals. METHODS: We proposed a data mining strategy to improve hypothesis generation with respect to potential associations. RESULTS: By numerous examples, we illustrate that our strategy controls the false positive signals. We implemented a free online tool, AERS spider (www.chemoprofiling.org/AERS). CONCLUSIONS: We believe that AERS spider would be a valuable tool for drug safety experts.