Search

Autophagy is a highly conserved process of cellular self-digestion that involves the formation of autophagosomes for the delivery of intracellular components and dysfunctional organelles to lysosomes. This process is induced by different signals including starvation, mitochondrial dysfunction, and DNA damage. The molecular link between autophagy and DNA damage is not well understood yet. Importantly, tumor cells utilize the mechanism of autophagy to cope with genotoxic anti-cancer drug therapy. Another mechanism of drug resistance is provided to cancer cells via the execution of the EMT program. One of the critical transcription factors of EMT is Zeb1. Here we demonstrate that Zeb1 is involved in the regulation of autophagy in several breast cancer cell models. On the molecular level, Zeb1 likely facilitates autophagy through the regulation of autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to several genotoxic drugs. The attenuation of Zeb1 expression in TNBC cells led to the opposite effect. Consequently, we propose that Zeb1 augments the resistance of breast cancer cells to genotoxic drugs, at least partially, via autophagy. Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells.

The SET domain containing lysine-specific methyltransferase, Set7/9, covalently attaches methyl moieties to a variety of histone and non-histone substrates. Among the substrates of Set7/9 are: p53, NF-kB, PARP1, E2F1, and other transcription factors that regulate many vital processes in the cell. Through the post-translational regulation of these critical master-regulators Set7/9 is involved in regulation of cell proliferation, cancer progression, and DNA damage response. Noteworthy, the role of Set7/9 in tumorigenesis is contradictory and apparently depends on the cellular context. In this study, we investigated the effect of Set7/9 on tumorigenic characteristics of lung cancer cells. We showed that CRISPR/Cas9-mediated knock-out of Set7/9 in A549 and its shRNA-mediated knock-down in H1299 NSCLC cell lines both augment the proliferation rate of tumor cells compared to the matching wild-type cells. Mechanistically, ablation of Set7/9 increased the expression of cyclin A2 and D1 genes thereby promoting the accumulation of cells in S phase. Furthermore, knockout of Set7/9 decreased the expression of E-cadherin, whose product is critical for cell-cell interactions. Accordingly, this led to the increased migration of lung cancer cells. Finally, both ablation or pharmacological inhibition of Set7/9 enzymatic methyltransferase activity by the selective inhibitor (R)-PFI-2 sensitized NSCLC cells to genotoxic drug, doxorubicin. This effect was also recapitulated on patients-derived NSCLC cell lines. Taken together, our results suggest that Set7/9 plays anti-proliferative and DNA damage-protective roles in NSCLC cells and hence represents an attractive target for anti-cancer chemotherapy.

Autophagy is a special catabolic cellular program that is induced in response to deprivation of nutrients and energy starvation. During the execution of this program, cellular components, including aggregates, as well as damaged organelles and some proteins are encapsulated in special vesicles known as autophagosomes and subsequently are degraded after fusion of autophagosomes with lysosomes. Importantly, at late stages of tumorigenesis cancer cells employ autophagy to sustain proliferation in unfavorable conditions, including anti-cancer drug therapy. E3 ubiquitin ligases play an important role in controlling autophagy. Here we demonstrate that the E3 ligase, a p53-induced RING-H2 protein (Pirh2), is involved in the regulation of autophagy in non-small cell lung cancer cells. Knockdown of Pirh2 decreased the expression of genes involved in all steps of autophagy. Concomitantly, Pirh2 knockdown cell lines exhibited much less of the processed form of LC3 compared to the respective cell lines with normal levels of Pirh2. These results were confirmed by the immune fluorescence microscopy using LC3 antibody and the LysoTracker dye. In agreement with the protective role of autophagy, cells with attenuated expression of Pirh2 were more sensitive to the treatment with doxorubicin. Collectively, we have uncovered a novel function of Pirh2 in the regulation of autophagy in lung cancer cells.

The RING-finger protein Pirh2 is a p53 family-specific E3 ubiquitin ligase. Pirh2 also ubiquitinates several other important cellular factors and is involved in carcinogenesis. However, its functional role in other cellular processes is poorly understood. To address this question, we performed a proteomic search for novel interacting partners of Pirh2. Using the GST-pulldown approach combined with LC-MS/MS, we revealed 225 proteins that interacted with Pirh2. We found that, according to the GO description, a large group of Pirh2-associated proteins belonged to the RNA metabolism group. Importantly, one of the identified proteins from that group was an RNA-binding protein ELAVL1 (HuR), which is involved in the regulation of splicing and protein stability of several oncogenic proteins. We demonstrated that Pirh2 ubiquitinated the HuR protein facilitating its proteasome-mediated degradation in cells. Importantly, the Pirh2-mediated degradation of HuR occurred in response to heat shock, thereby affecting the survival rate of HeLa cells under elevated temperature. Functionally, Pirh2-mediated degradation of HuR augmented the level of c-Myc expression, whose RNA level is otherwise attenuated by HuR. Taken together, our data indicate that HuR is a new target of Pirh2 and this functional interaction contributes to the heat-shock response of cancer cells affecting their survival.

Pentacyclic triterpenoid acids bearing a polyhydroxylated A ring have been found in many plants and are of interest for their biological properties, including antitumor activity. The present article reports on the design and synthesis of a series of conjugates of a cationic mitochondria-targeted compound, 4-(1-H-indol-3-ylvinyl)-N-methylpyridinium iodide (F16), with maslinic, corosolic, and asiatic acids and with a tri-hydroxylated analogue of betulinic acid. In the new hybrid compounds, the triterpenoids bearing a polyhydroxylated ring A were linked to the cationic fragment F16 in the C-28 side chain through a butyl spacer. The results of in vitro bioscreening in two human tumor cell lines (H1299 and A549) and noncancer fibroblasts (MEFs) revealed that all the studied conjugates demonstrated a significantly stronger antitumor effect than the original natural triterpenoid acids. In particular, the antitumor activity of the corosolic acid conjugate against A549 and H1299 cell lines was 36 and 24 times higher than the activity of ursolic acid. Conjugates of oxygenated triterpenoids containing hydroxyl or acetyl groups at the C-2 atom or C-1/C-2 atoms of the triterpene core exhibited a slight decrease in antitumor activity compared to the corresponding F16-derivatives of betulinic, ursolic and oleanolic acids. Among the studied compounds, acceptable selectivity in relation to tumor and non-malignant cell lines was shown only by the F16 derivatives of the maslinic and asiatic acids, since they were not the active agents against fibroblasts MEFs within the assay.

Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN) is one of the critical tumor suppressor genes and the main negative regulator of the PI3K pathway. PTEN is frequently found to be inactivated, either partially or fully, in various malignancies. The PI3K/AKT pathway is considered to be one of the main signaling cues that drives the proliferation of cells. Perhaps it is not surprising, then, that this pathway is hyperactivated in highly proliferative tumors. Importantly, the PI3K/AKT pathway also coordinates the epithelial–mesenchymal transition (EMT), which is pivotal for the initiation of metastases and hence is regarded as an attractive target for the treatment of metastatic cancer. It was shown that PTEN suppresses EMT, although the exact mechanism of this effect is still not fully understood. This review is an attempt to systematize the published information on the role of PTEN in the development of malignant tumors, with a main focus on the regulation of the PI3K/AKT pathway in EMT.

Simple Summary In the present review, we discuss the role of metabolic reprogramming which occurs in malignant cells. The process of metabolic reprogramming is also known as one of the “hallmarks of cancer”. Due to several reasons, including the origin of cancer, tumor microenvironment, and the tumor progression stage, metabolic reprogramming can be heterogeneous and dynamic. In this review, we provide evidence that the usage of metabolic drugs is a promising approach to treat cancer. However, because these drugs can damage not only malignant cells but also normal rapidly dividing cells, it is important to understand the exact metabolic changes which are elicited by particular drivers in concrete tissue and are specific for each stage of cancer development, including metastases. Finally, the review highlights new promising targets for the development of new metabolic drugs. Abstract The specific molecular features of cancer cells that distinguish them from the normal ones are denoted as “hallmarks of cancer”. One of the critical hallmarks of cancer is an altered metabolism which provides tumor cells with energy and structural resources necessary for rapid proliferation. The key feature of a cancer-reprogrammed metabolism is its plasticity, allowing cancer cells to better adapt to various conditions and to oppose different therapies. Furthermore, the alterations of metabolic pathways in malignant cells are heterogeneous and are defined by several factors including the tissue of origin, driving mutations, and microenvironment. In the present review, we discuss the key features of metabolic reprogramming and plasticity associated with different stages of tumor, from primary tumors to metastases. We also provide evidence of the successful usage of metabolic drugs in anticancer therapy. Finally, we highlight new promising targets for the development of new metabolic drugs.

Сancer-testis antigens (CTAs) comprise proteins which are aberrantly expressed in various malignancies, yet under normal situation are restricted to only germ cells. Semenogelins 1 and 2 (SEMG1 and 2, respectively) belong to the family of non-X-linked (autosomal) cancer-testis antigens. They are the major protein ingredients of human semen and share 78% of similarity between them on the gene level. SEMG1/2 gene products regulate the motility and fertility of sperm, as well as provide sperm the antibacterial defense. Besides, SEMG1 and SEMG2 were detected in various malignancies including small cell lung cancer (SCLC). However, the biological role of both SEMG1 and 2 proteins in tumorigenesis has not been fully understood. We demonstrate here that SEMG1 and SEMG2 (SEMGs) exhibit different patterns of expression and sub-cellular localization in non-small cell lung cancer (NSCLC) cell lines. To elucidate the biological properties of SEMGs in NSCLC, we established H1299 cell lines that were stably transduced with either SEMGs-overexpressing or knockdown vectors, respectively. Using fluorescence-based dihydroethidium (DHE) assay we showed that both SEMGs augmented the production of reactive oxygen species (ROS) up to 2 times. Moreover, SEMGs (especially SEMG1) strongly increased the number of Annexin V–positive apoptotic cells manifesting an increased sensitivity to genotoxic drugs including doxorubicin, etoposide, and cisplatin. Taken our results together, SEMGs may arguably play a positive role in tumorigenesis by sensitizing NSCLCs to genotoxic therapy.

Breast cancer is one of the world’s leading causes of oncological disease-related death. It is characterized by a high degree of heterogeneity on the clinical, morphological, and molecular levels. Based on molecular profiling breast carcinomas are divided into several subtypes depending on the expression of a number of cell surface receptors, e.g., ER, PR, and HER2. The Her2-positive subtype occurs in ~10–15% of all cases of breast cancer, and is characterized by a worse prognosis of patient survival. This is due to a high and early relapse rate, as well as an increased level of metastases. Several FDA-approved drugs for the treatment of Her2-positive tumors have been developed, although eventually cancer cells develop drug resistance. These drugs target either the homo- or heterodimerization of Her2 receptors or the receptors’ RTK activity, both of them being critical for the proliferation of cancer cells. Notably, Her2-positive cancers also frequently harbor mutations in the TP53 tumor suppressor gene, which exacerbates the unfavorable prognosis. In this review, we describe the molecular mechanisms of RTK-specific drugs and discuss new perspectives of combinatorial treatment of Her2-positive cancers through inhibition of the mutant form of p53.

Lysine-specific methyltransferase Set7/9 (KMT7) belongs to the SET domain family of proteins. Besides the SET domain, Set7/9 also contains a so-called MORN (Membrane Occupation and Recognition Nexus) domain whose function in high eukaryotes is largely unknown. Set7/9 has been shown to specifically methylate both histones H1 and H3 as well as a number of non-histone substrates, including p53, E2F1, RelA, AR, and other important transcription factors. However, despite the ever growing list of potential substrates of Set7/9, the question of its substrate specificity is still debatable. To gain a better understanding of the Set7/9 substrate specificity and to clarify the importance of structural domains of Set7/9 for protein-protein interactions (PPIs) we determined interactomes for both MORN and SET domains of Set7/9 by pull-down assay coupled with mass-spectrometry. Importantly, we demonstrated that most of PPIs of Set7/9 are mediated via its MORN domain. The latter has preference towards positively charged amino acids that are often found in RNA-binding proteins. One of the Set7/9-interacting proteins was identified as Sam68, an RNA splicing protein with a KH (heterogeneous nuclear ribonucleoprotein K (hnRNP K) homology) domain. Importantly, the RG-rich domain of Sam68 that is also present in many splicing factors was found to interact with Set7/9. We revealed that Set7/9 not only co-immunoprecipitated with Sam68, but also methylated the latter on K208. Functionally, knockout of Set7/9 decreased the protein level of Sam68 in cells resulting in altered regulation of cell cycle and apoptosis. Finally, the bioinformatics analysis established a correlation between the high levels of Sam68/Set7/9 co-expression and better survival rates of patients with colon cancer.

International audience; The p53 protein is a key tumor suppressor in mammals. In response to various forms of genotoxic stress p53 stimulates expression of genes whose products induce cell cycle arrest and/or apoptosis. An E3-ubiquitin ligase, Mdm2 (mouse-double-minute 2) and its human ortholog Hdm2, physically interact with the amino-terminus of p53 to mediate its ubiquitin-mediated degradation via the proteasome. Thus, pharmacological inhibition of the p53-Mdm2 interaction leads to overall stabilization of p53 and stimulation of its anti-tumorigenic activity. In this study we characterize the biological effects of a novel class of non-genotoxic isatin Schiff and Mannich base derivatives (ISMBDs) that stabilize p53 on the protein level. The likely mechanism behind their positive effect on p53 is mediated via the competitive interaction with Mdm2. Importantly, unlike Nutlin, these compounds selectively promoted p53-mediated cell death. These novel pharmacological activators of p53 can serve as valuable molecular tools for probing p53-positive tumors and set up the stage for development of new anti-cancer drugs.

Ca2+ -mediated signaling is widely spread in nature and plays critical role in the individual development of various organisms ranging from microorganisms to mammals. In vertebrates, Ca2+ is involved in important developmental events: fertilization, body plan establishment, and organogenesis. The two later events are defined by embryonic stem cells (ESCs). ESCs are capable of self-renewal and are pluripotent by nature, that is, can give rise to all types of cells that make up the body. Given the paramount importance of Ca2+ signalization in the development, it is therefore not surprising this process also plays role in the biology of stem cells. In this review, we scrutinize the published experimental data on the role of Ca2+ ions in embryonic stem cells self-renewal and pluripotency. In line with this, we also discuss possible mechanisms of p53 inhibition as a major hindrance to self-renewal of ESCs. Finally, we argue about the role of G-protein-coupled receptors (GPCRs), the largest family of heteromeric transmembrane receptors, and GPCR-mediated signalization in stem cells, and propose the role for the GPCR-G-protein-PLC-Ca2+ -downstream signaling pathway in the regulation of pluripotency of both mouse and human ESCs.

As an anticancer therapeutic, Interferon-alpha (IFNα) is used to treat a number of malignancies. However, the application of IFNα is restricted mostly due to its high toxicity. Therefore, novel combination therapeutic regimens are required to decrease the toxicity of IFNα and enhance its efficacy. Here we show that the treatment of p53-deficient human non-small lung carcinoma H1299 cells with IFNα in combination with an inhibitor of MDM2, Nutlin-3a, synergistically affects the proliferation of cancer cells. Importantly, Nutlin-3a was able to reduce the effective dose of IFNα about 3.4 times. Strikingly, this phenomenon is p53-independent, because H1299 cells lack p53, but is highly dependent on MDM2 because its ablation makes tumor cells completely insensitive to IFNα alone or in combination with Nutlin-3a. On the contrary, overexpression of MDM2 makes H1299 cells more susceptible to both IFNα and IFNα/Nutlin-3a treatments. Mechanistically, treatment with combination of IFNα and Nutlin-3a attenuates cyclin D1/CDK4 on the protein level and hence blocks cell cycle progression. This mechanism may be responsible, at least in part, for the anti-proliferative effects on H1299 cells observed. Our data suggest that the expression of MDM2 confers sensitivity of cancer cells to IFNα/Nutlin-3a treatment. Moreover, our data also confirm positive effect of Nutlin even on p53-deficient neoplasms.

Cancer-related metabolism has recently emerged as one of the "hallmarks of cancer". It has several important features, including altered metabolism of glucose and glutamine. Importantly, altered cancer metabolism connects different biochemical pathways into the one fine-tuned metabolic network, which stimulates high proliferation rates and plasticity to malignant cells. Among the keystones of cancer metabolism are one-carbon metabolism and nucleotide biosynthesis, which provide building blocks to anabolic reactions. Accordingly, the importance of these metabolic pathways for anticancer therapy has well been documented by more than fifty years of clinical use of specific metabolic inhibitors - methotrexate and nucleotides analogs. In this review we discuss one-carbon metabolism and nucleotide biosynthesis as common and specific features of many, if not all, tumors. The key enzymes involved in these pathways also represent promising anti-cancer therapeutic targets. We review different aspects of these metabolic pathways including their biochemistry, compartmentalization and expression of the key enzymes and their regulation at different levels. We also discuss the effects of known inhibitors of these pathways as well as the recent data on other enzymes of the same pathways as perspective pharmacological targets.

Currently, immunotherapy is attracting a lot of attention and may potentially become a leading approach in the treatment of cancer. One emerging therapeutic, the chimeric-antigen receptor T-cell adoptive immunotherapy (CAR-T) is showing remarkable efficacy in the treatment of several B-cell malignancies. The popularity of CAR-T has been founded on two CAR T-cell products recently approved by FDA (during 2017) in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia and B-cell lymphoma. However, their toxicities observed in clinical trials were extremely significant and in some cases even fatal with no approved algorithms for toxicity prediction being available to date. A deeper understanding of the biological basis of such complications is the key to prompt and comprehensive clinical management. Here we review the wide spectrum of effects associated with CAR T cell therapy with a major focus on the pathogenesis of cytokine release syndrome and neurotoxicity as the most common, potentially life-threatening effects of this treatment. We discuss the basis of clinical management and the existing models that predict the severity of toxicity, as well as the key factors that modulate this event. Finally, we will summarize the literature detailing universal allogenic CAR T-cells and their toxicity profile.

// Larissa Lezina 1, * , Vasilisa Aksenova 1, * , Olga Fedorova 1 , Daria Malikova 1 , Oleg Shuvalov 1 , Alexey V. Antonov 2 , Dmitri Tentler 1 , Alexander V. Garabadgiu 3 , Gerry Melino 2, 3 , Nikolai A. Barlev 1, 3 1 Gene Expression Laboratory, Institute of Cytology, Saint-Petersburg, 194064, Russia 2 MRC Toxicology Unit, Leicester, LE1 9HN, UK 3 Molecular Pharmacology Laboratory, Saint-Petersburg Institute of Technology, Saint-Petersburg, 190013, Russia * These authors have contributed equally to this work Correspondence to: Nikolai A. Barlev, e-mail: nick.a.barlev@gmail.com Keywords: Set7/9, genotoxic stress, operetta, Mdm2, DNA damage Received: July 23, 2014 Accepted: July 20, 2015 Published: August 01, 2015 ABSTRACT 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.

Ca

Major tumor suppressor and transcription factor p53 coordinates expression of many genes hence affecting critical cellular functions including cell cycle, senescence, and apoptosis. The NR4A family of orphan receptors (NR4A1-3) belongs to the superfamily of nuclear receptors. They regulate genes involved in proliferation, cell migration, and apoptosis. In this study, we report an identification of NR4A3 as a direct transcriptional target of p53. Using various techniques, we showed that p53 directly bound the promoter of NR4A3 gene and induced its transcription. Functionally, over-expression of NR4A3 attenuated proliferation of cancer cells and promoted apoptosis by augmenting the expression of pro-apoptotic genes, PUMA and Bax. Knockdown of NR4A3 reversed these phenotypes. Importantly, NR4A3 exhibited tumor suppressive functions both in p53-dependent and independent manner. In addition, NR4A3 physically interacted with an anti-apoptotic Bcl-2 protein hence sequestering it from blunting apoptosis. These observations were corroborated by the bioinformatics analysis, which demonstrated a correlation between high levels of NR4A3 expression and better survival of breast and lung cancer patients. Collectively, our studies revealed a novel transcriptional target of p53, NR4A3, which triggers apoptosis and thus likely has a tumor suppressive role in breast and lung cancers.

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.

Potato cyst nematodes (PCN) collected in six localities in the Leningrad region of North West Russia were identified as Globodera rostochiensis pathotype Ro1 and were used for subsequent resistance tests. Seventy-nine accessions of cultivated and closely related wild potato species from the VIR collection in Russia were screened on resistance to G. rostochiensis pathotype Ro1 and on the presence of molecular markers for H1 and Gro1-4 resistance genes. No associations were detected between the resistance level of diploid and tetraploid Andean and tetraploid Chilean potato landraces (indigenous cultivars) and their related wild species and their geographical distribution or presence of PCR-based markers that are associated with the H1 and Gro1-4 genes. At the same time, all susceptible genotypes lacked such markers. New sources of resistance were found and could be used in breeding.

With 7 figures and 1 table Abstract The causal agent of potato wart (Synchytrium endobioticum) is an obligate parasitic chytrid fungus. It is included as a quarantine pathogen in 55 countries, with losses in susceptible cultivars reaching 50–100%. The aim of our study was to characterize the resistance to S. endobioticum pathotype 1 in cultivated potatoes from a well-characterized subset of the Vavilov Institute of Plant Industry collection and to determine whether this resistance is associated with cultivated potato species taxonomy, with ploidy, with geographic distance or with a molecular marker Nl25-1400 proposed for molecular screening for resistance to pathotype 1 of S. endobioticum. Within the diversity of 52 landrace genotypes, our work shows a lack of such predictive associations with wart resistance. High intraspecific variation of wart diseases resistance allows the selection of extremely resistant and susceptible genotypes available for future genetic and breeding studies.

In 2004, COP1 was characterized as p53 E3-specific human ubiquitin ligase according to in vitro studies. Later, oncogenic activity of COP1 and correlation of its overexpression with level of p53 protein in clinical tumor samples and cell lines were shown. High expression level of COP1 was described in 25 of 32 cases of breast carcinomas and in 76 out of 171 cases of ovarian carcinomas. High expression level of COP1 was described in hepatocellular carcinoma cell lines (PLC, Hep3B and HepG2, Huh7). In 40 of 55 cases of pancreatic cancer, overexpression of COP1 was also noted. Some types of leukemia, melanoma, breast, lung and prostate cancer contain focal deletions of COP1. COP1 overexpression was also found in non- oncological diseases like Duchenne muscular dystrophy, ischemic cardiomyopathy and juvenile dermatomyositis. Similar to other p53-related E3 -ubiquitin ligases COP1 has different substrates. The selection of target for ubiquitination depends on cell type or differentiation stage. Among the substrates of COP1, besides p53 are: c-Jun, ETV1, ACC, TORC2, FOXO1 and C/EBPα, FIP200. To identify novel proteins that interact with COP1 we applied proteomics. Proteins co-immunoprecipitated with COP1 were analyzed by MALDI -TOF mass spectrometry. To achieve this, COP1–3xFlag expression vector was generated and transfected into HEK 293T cells by calcium phosphate transfection method. Protein complexes were immunoprecipitated with anti-FLAG beads. Bound proteins were then separated by 1D SDS–PAGE gel electrophoresis and analyzed by means of ESI-LC/MS/MS mass spectrometry. Subsequent bioinformatics analysis of the interacting proteins was employed. About 25% of identified proteins were attributed to the cytoskeleton proteins, almost 20% of proteins had known role in metabolism. According to the literature data, it was not surprising to find several proteins involved in the lipid metabolism. Finally, a significant groups of interactants play role in transcription, cell adhesion, apoptosis, protein modification and transport. It is important to note that several COP1-bound proteins can be strong regulators of cell cycle in G2/M transition. Our data can be used for the subsequent studies of previously unknown roles of COP1 in different diseases. This work was funded by Grants from Russian Scientific Foundation – Russia (No. 114-15-00816 ) and Grant of Molecular and Cellular Biology of the Presidium of the Russian Academy of Sciences.

Gene therapy suggests a promising approach to treat genetic diseases by applying genes as pharmaceuticals. Cancer is a complex disease, which strongly depends on a particular genetic make-up and hence can be treated with gene therapy. From about 2,000 clinical trials carried out so far, more than 60% were cancer targeted. Development of precise and effective gene therapy approaches is intimately connected with achievements in the molecular biology techniques. The field of gene therapy was recently revolutionized by the introduction of "programmable" nucleases, including ZFNs, TALENs, and CRISPR, which target specific genomic loci with high efficacy and precision. Furthermore, when combined with DNA transposons for the delivery purposes into cells, these programmable nucleases represent a promising alternative to the conventional viral-mediated gene delivery. In addition to "programmable" nucleases, a new class of TALE- and CRISPR-based "artificial transcription effectors" has been developed to mediate precise regulation of specific genes. In sum, these new molecular tools may be used in a wide plethora of gene therapy strategies. This review highlights the current status of novel genome editing tools and discusses their suitability and perspectives in respect to cancer gene therapy studies.

Human pluripotent stem cells provide a versatile platform for regenerative studies, drug testing and disease modeling. That the expression of only four transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), is sufficient for generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells has revolutionized the field and also highlighted the importance of OKSM as targets for genome editing. A number of novel genome-editing systems have been developed recently. In this review, we focus on successful applications of several such systems for generation of iPSCs. In particular, we discuss genome-editing systems based on zinc-finger fusion proteins (ZFs), transcription activator-like effectors (TALEs) and an RNA-guided DNA-specific nuclease, Cas9, derived from the bacterial defense system against viruses that utilizes clustered regularly interspaced short palindromic repeats (CRISPR).