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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.

This report presents novel results on the involvement of epithelial to mesenchymal transition (EMT) in the process of autophagy. 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, forced expression of Zeb1 in otherwise epithelial cells likely facilitates autophagy through the regulation of several autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to genotoxic drugs. On the contrary, 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, by promoting autophagy. Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells. Citation Format: Nick A. Barlev, Olga Fedorova, Alexandra Daks, Eugene M. Tulchinsky. Zeb1-mediated autophagy enhances resistance of breast cancer cells to genotoxic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1237.

Cancer testis antigens SEMG1 and SEMG2 (semenogelins 1 and 2) are the main proteins of human seminal fluid and are also expressed in malignant neoplasms of various origins. The biological role of semenogelins in the reproductive process is well understood, but almost nothing is known about their functions in tumor cells. In the present study, we used Mia-Paca2 cell line with overexpression of SEMG1 and SEMG2a as a cell model of human pancreatic cancer. Mia-Paca2 cells transduced with the corresponding vector served as control. Using flow cytometry and dihydroethidium (DHE) fluorescent agent detecting predominantly superoxide anion, we have shown that overexpression of SEMG1 and SEMG2 increases the ROS level. In addition, both semenogelins increased the susceptibility of tumor cells to the widely used chemotherapeutic genotoxic drugs doxorubicin and cisplatin. These findings showed that oncosuppressive properties are possessed by SEMG1 and SEMG2 in the cell model of pancreatic cancer.

Background. Cytokines are considered as important factors that enhance the efficacy of CAR-T cell therapy. Besides, they are key elements of the pathogenesis of cytokine release syndrome and neurotoxicity in applying the CAR-T technology. However, cytokine effects in the context of CAR-T therapy have not yet been properly studied. Aim. To quantitatively assess cytokine secretion using multiplex assay with co-incubation of anti-CD19 CAR-T lymphocytes with epithelial HeLa and A431 cell lines expressing CD19 on their surface. Materials & Methods. T-lymphocytes were transduced with the lentiviral vector containing anti-CD19-CAR gene. CAR expression was tested based on GFP reporter using flow cytometry. To confirm a specific CAR-T cell activation response to tumor antigen, the levels of interleukin-2, interferon-γ, and tumor necrosis factor-α were measured by means of immunoassay. Cytotoxic activity of CAR-T lymphocytes obtained was examined with their direct co-culturing with target cells. The levels of cytokines isolated prior to and after incubation of targets with CAR-T cells were compared using multiplex assay. Results. The level of some proinflammatory cytokines (interleukin-6, interleukin-1β, interferon-γ) (P < 0.01) increased. The difference in the levels of anti-inflammatory cytokines (interleukin-4, interleukin-10) was inconsiderable, and in the HeLa cell line experiment it was insignificant (p > 0.05). The concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) was many times higher after incubation with CAR-T lymphocytes (p < 0.01). Conclusion. The trial revealed multiple enhancement of GM-CSF, one of the key elements of the pathogenesis of cy tokine release syndrome and CAR-T-associated neurotoxicity. The results of further studies of GM-CSF can contribute to improving the efficacy of CAR-T therapy with considerably lower toxicity.

Aim. To compare different methods of lentivirus concentration in order to select the best way of providing high-level transduction for generating laboratory CAR-T cells. Materials & Methods. Concentration of lentiviral supernatant was carried out by 4 methods: ultrafiltration, ultracentrifugation, polyethylene glycol (PEG), water-soluble non-ionic polymer, precipitation method, and ion-exchange chromatography. Functional viral titer was determined by mCherry reporter protein expression in the transduced HeLa cell line as well as by rapid immunochromatographic (IC) tests. Physical titer was determined by ELISA. Transduction efficiency of healthy donor’s T-lymphocytes was assessed by flow cytometry with respect to signal intensity of reporter protein FusionRed. Functional activity of generated anti-CD19 CAR-T was evaluated by microscopy after co-cultivation with CD19-HeLa cell line as well as subsequent cytokine testing. Results. Lentivirus purification and concentration by ultrafiltration provided the greatest number of transduced cells, i.e. 84.7 %. Methods of ultracentrifugation, PEG precipitation, and ion-exchange chromatography yielded 56.08 %, 74.22 %, and 21.05 % of T-cell transduction, respectively. Results of rapid IC tests were comparable (г = 0.91) with cell line titer data. The mean T-cell transduction efficiency was 59.55 % ± 2.94 %, and its maximum reached 76.26 %. Conclusion. The focus was laid on optimization of CAR-T cell production during the generation of lentiviral vectors and their purification. Ultrafiltration was selected as the best method of lentiviral supernatant concentration to efficiently transduce T-lymphocytes and to generate functional CAR-T cell population.

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.

Cancer-testicular Antigens (CTAs) belong to a group of proteins that under normal conditions are strictly expressed in a male’s reproductive tissues. However, upon malignisation, they are frequently re-expressed in neoplastic tissues of various origin. A number of studies have shown that different CTAs affect growth, migration and invasion of tumor cells and favor cancer development and metastasis. Two members of the CTA group, Semenogelin 1 and 2 (SEMG1 and SEMG2, or SEMGs) represent the major component of human seminal fluid. They regulate the motility and capacitation of sperm. They are often re-expressed in different malignancies including breast cancer. However, there is almost no information about the functional properties of SEMGs in cancer cells. In this review, we highlight the role of SEMGs in the reproductive system and also summarize the data on their expression and functions in malignant cells of various origins.

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.

SEMG1 and SEMG2 genes belong to the family of cancer-testis antigens (CTAs), whose expression normally is restricted to male germ cells but is often restored in various malignancies. High levels of SEMG1 and SEMG2 expression are detected in prostate, renal, and lung cancer as well as hemoblastosis. However, the functional importance of both SEMGs proteins in human neoplasms is still largely unknown. In this study, by using a combination of the bioinformatics and various cellular and molecular assays, we have demonstrated that SEMG1 and SEMG2 are frequently expressed in lung cancer clinical samples and cancer cell lines of different origins and are negatively associated with the survival rate of cancer patients. Using the pull-down assay followed by LC-MS/MS mass-spectrometry, we have identified 119 proteins associated with SEMG1 and SEMG2. Among the SEMGs interacting proteins we noticed two critical glycolytic enzymes-pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Importantly, we showed that SEMGs increased the protein level and activity of both PKM2 and LDHA. Further, both SEMGs increased the membrane mitochondrial potential (MMP), glycolysis, respiration, and ROS production in several cancer cell lines. Taken together, these data provide first evidence that SEMGs can up-regulate the energy metabolism of cancer cells, exemplifying their oncogenic features.

HER2 (Human Epidermal Growth Factor Receptor 2), also known as ERBB2, CD340, and Neu protooncogene, is a member of the epidermal growth factor receptor (EGRF) family. Members of the ERBB family, including HER2, activate molecular cascades that stimulate proliferation and migration of cancer cells, as well as their resistance to the anticancer therapy. These proteins are often overexpressed and/or mutated in various cancer types and represent promising targets for the anti-cancer therapy. Currently, anti-HER2 drugs have been approved for the treatment of several types of solid tumors. HER2-specific therapy includes monoclonal antibodies and low-molecular weight inhibitors of tyrosine kinase receptors, such as lapatinib, neratinib, and pyrotinib. In addition to the activation of molecular pathways responsible for cell proliferation and survival under stress conditions, HER2 directly regulates programmed cell death. Here, we review the studies focused on the involvement of HER2 in various signaling pathways and its role in the regulation of apoptosis.

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.

The epithelial–mesenchymal transition (EMT) plays a pivotal role in the differentiation of vertebrates and is critically important in tumorigenesis. Using this evolutionarily conserved mechanism, cancer cells become drug-resistant and acquire the ability to escape the cytotoxic effect of anti-cancer drugs. In addition, these cells gain invasive features and increased mobility thereby promoting metastases. In this respect, the process of EMT is critical for dissemination of solid tumors including breast cancer. It has been shown that miRNAs are instrumental for the regulation of EMT, where they play both positive and negative roles often as a part of a feed-back loop. Recent studies have highlighted a novel association of p53 and EMT where the mutation status of p53 is critically important for the outcome of this process. Interestingly, p53 has been shown to mediate its effects via the miRNA-dependent mechanism that targets master-regulators of EMT, such as Zeb1/2, Snail, Slug, and Twist1. This regulation often involves interactions of miRNAs with lncRNAs. In this review, we present a detailed overview of miRNA/lncRNA-dependent mechanisms that control interplay between p53 and master-regulators of EMT and their importance for breast cancer.

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.

Mycoplasmas are bacteria lacking the cell wall, which is the major characteristic of this taxonomic class (Mollicutes). Among bacteria, mycoplasmas possess the smallest genome known for free-living organisms. This feature limits the autonomy of bacteria and makes them increasingly susceptible to changes in the host organism. Many mycoplasmas themselves cause pathological changes in the host organism, often complicated by immune disorders. Infection with certain strains of mycoplasma results in the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells, which is the major mediator of the inflammatory response. Furthermore, mycoplasmas can inhibit p53-mediated checkpoint control of cell cycle and apoptosis. Collectively, these properties indicate that mycoplasmas might act as cancer-promoting factors. In this review, we summarize the information known to date on the role of mycoplasmas in the regulation of the host immune response and their functional interactions with p53.

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.

Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, is over-expressed in many tumors, including almost half of triple-negative breast cancers. The latter belong to a very-aggressive and drug-resistant form of malignancy. Although humanized anti-EGFR antibodies can work efficiently against these cancers both as monotherapy and in combination with genotoxic drugs, instability and high production costs are some of their known drawbacks in clinical use. In addition, the development of antibodies to target membrane proteins is a very challenging task. Accordingly, the main focus of the present work is the design of supramolecular agents for the targeting of membrane proteins in cancer cells and, hence, more-specific drug delivery. These were produced using a novel double-imprinting approach based on the solid-phase method for preparation of molecularly imprinted polymer nanoparticles (nanoMIPs), which were loaded with doxorubicin and targeted toward a linear epitope of EGFR. Additionally, upon binding, doxorubicin-loaded anti-EGFR nanoMIPs elicited cytotoxicity and apoptosis only in those cells that over-expressed EGFR. Thus, this approach can provide a plausible alternative to conventional antibodies and sets up a new paradigm for the therapeutic application of this class of materials against clinically relevant targets. Furthermore, nanoMIPs can promote the development of cell imaging tools against difficult targets such as membrane proteins.

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.

Tumor suppressor protein p63 is a transcription factor of the p53 protein family. It is known that the proteins of this family, namely, p53, p63, and p73, perform a number of common tumor suppressor functions related to cell cycle regulation, apoptosis, and malignant transformation of cells, in addition to specific functions in the development of tissues and organs. All the proteins of the p53 family are regulated at post-translational level due to ubiquitination by E3 ligases and subsequent proteasomal degradation. The expression of the majority of E3 ubiquitin ligases, which modify the proteins of the p53 family, is regulated by the same proteins, thus forming negative feedback with them. In the present study, we first showed that the expression of the PIRH2 (RCHY1) gene, the product of which ubiquitinates all three members of the p53 family, is also regulated by p63. Regulation of the PIRH2 expression occurs at the transcription level by only fulllength TAp63 isoform, but not ΔNp63 isoform, which lacks a transactivation domain.

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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.

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.