Nickolai A. Barlev

miR-16 and miR-26a target checkpoint kinases Wee1 and Chk1 in response to p53 activation by genotoxic stress

The tumour suppressor p53 is a crucial regulator of cell cycle arrest and apoptosis by acting as a transcription factor to regulate a variety of genes. At least in part, this control is exerted by p53 via regulating expression of numerous microRNAs. We identified two abundantly expressed microRNAs, miR-16 and miR-26a, whose expression is regulated by p53 during the checkpoint arrest induced by the genotoxic drug, doxorubicin. Importantly, among the targets of these miRs are two critical checkpoint kinases, Chk1 and Wee1. The p53-dependent augmentation of miR-16 and miR-26a expression levels led to the cell cycle arrest of tumour cells in G1/S and increased apoptosis. Strikingly, the bioinformatics analysis of survival times for patients with breast and prostate cancers has revealed that co-expression of mir-16 and miR-26a correlated with a better survival outcome. Collectively, our data provide a novel mechanism whereby p53 represses Chk1 and Wee1 expression, at least partially, via upregulation of miR-16 and miR-26a and thus sensitizes tumour cells to genotoxic therapies.

Characterization of novel markers of senescence and their prognostic potential in cancer.

Cellular senescence is a terminal differentiation state that has been proposed to have a role in both tumour suppression and ageing. This view is supported by the fact that accumulation of senescent cells can be observed in response to oncogenic stress as well as a result of normal organismal ageing. Thus, identifying senescent cells in in vivo and in vitro has an important diagnostic and therapeutic potential. The molecular pathways involved in triggering and/or maintaining the senescent phenotype are not fully understood. As a consequence, the markers currently utilized to detect senescent cells are limited and lack specificity. In order to address this issue, we screened for plasma membrane-associated proteins that are preferentially expressed in senescent cells. We identified 107 proteins that could be potential markers of senescence and validated 10 of them (DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A and PLD3). We demonstrated that a combination of these proteins can be used to specifically recognize senescent cells in culture and in tissue samples and we developed a straightforward fluorescence-activated cell sorting-based detection approach using two of them (DEP1 and B2MG). Of note, we found that expression of several of these markers correlated with increased survival in different tumours, especially in breast cancer. Thus, our results could facilitate the study of senescence, define potential new effectors and modulators of this cellular mechanism and provide potential diagnostic and prognostic tools to be used clinically.

The 26S proteasome is a multifaceted target for anti-cancer therapies

Proteasomes play a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins. Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target. Currently, the vast majority of inhibitors are aimed at blunting the proteolytic activities of proteasomes. However, recent achievements in solving structures of proteasomes at very high resolution provided opportunities to design new classes of small molecules that target other physiologically-important enzymatic activities of proteasomes, including the de-ubiquitinating one. This review attempts to catalog the information available to date about novel classes of proteasome inhibitors that may have important pharmacological ramifications.

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

26S proteasome is a large multi-subunit protein complex involved in proteolytic degradation of proteins. In addition to its canonical proteolytic activity, the proteasome is also associated with recently characterized endoribonuclease (endo-RNAse) activity. However, neither functional significance, nor the mechanisms of its regulation are currently known. In this report, we show that 26S proteasome is able to hydrolyze various cellular RNAs, including AU-rich mRNA of c-myc and c-fos. The endonucleolytic degradation of these mRNAs is exerted by one of the 26S proteasome subunits, PSMA5 (α5). The RNAse activity of 26S proteasome is differentially affected by various extra-cellular signals. Moreover, this activity contributes to the process of degradation of c-myc mRNA during induced differentiation of K562 cells, and may be controlled by phosphorylation of the adjacent subunits, PSMA1 (α6) and PSMA3 (α7). Collectively, the data presented in this report suggest a causal link between cell signalling pathways, endo-RNAse activity of the 26S proteasome complex and metabolism of cellular RNAs.

Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism

The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.

BTK modulates p53 activity to enhance apoptotic and senescent responses

p53 is a tumor suppressor that prevents the emergence of transformed cells by inducing apoptosis or senescence, among other responses. Its functions are regulated tightly by posttranslational modifications. Here we show that Brutons tyrosine kinase (BTK) is a novel modulator of p53. We found that BTK is induced in response to DNA damage and p53 activation. BTK induction leads to p53 phosphorylation, which constitutes a positive feedback loop that increases p53 protein levels and enhances the transactivation of its target genes in response to stress. Inhibiting BTK reduced both p53-dependent senescence and apoptosis. Further, BTK expression also upregulated DNA damage signals and apoptosis. We conclude that despite being involved in oncogenic signals in blood malignancies, BTK has antineoplastic properties in other contexts, such as the enhancement of p53s tumor suppressor responses. Along with evidence that BTK expression correlates with good prognosis in some epithelial tumors, our findings may encourage a reevaluation of the clinical uses of BTK inhibitors in cancer therapy. Cancer Res; 76(18); 5405-14. ©2016 AACR.

BTK blocks the inhibitory effects of MDM2 on p53 activity

p53 is a tumour suppressor that is activated in response to various types of stress. It is regulated by a complex pattern of over 50 different post-translational modifications, including ubiquitination by the E3 ligase MDM2, which leads to its proteasomal degradation. We have previously reported that expression of Bruton’s Tyrosine Kinase (BTK) induces phosphorylation of p53 at the N-terminus, including Serine 15, and increases its protein levels and activity. The mechanisms involved in this process are not completely understood. Here, we show that BTK also increases MDM2 and is necessary for MDM2 upregulation after DNA damage, consistent with what we have shown for other p53 target genes. Moreover, we found that BTK binds to MDM2 on its PH domain and induces its phosphorylation. This suggested a negative regulation of MDM2 functions by BTK, supported by the fact BTK expression rescued the inhibitory effects of MDM2 on p53 transcriptional activity. Indeed, we observed that BTK mediated the loss of the ubiquitination activity of MDM2, a process that was dependent on the phosphorylation functions of BTK. Our data together shows that the kinase activity of BTK plays an important role in disrupting the MDM2-p53 negative feedback loop by acting at different levels, including binding to and inactivation of MDM2. This study provides a potential mechanism to explain how BTK modulates p53 functions.

One-carbon metabolism and nucleotide biosynthesis as attractive targets for anticancer therapy

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.

Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits.

The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS-PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells.

The 2nd International Workshop 'Novel Therapeutic Strategies in Cancer' in St-Petersburg

The 2nd workshop ‘Novel Therapeutic Strategies in Cancer’ sponsored by the Russian Government program, supporting the research in Russian universities under the guidance of the world-leading scientists was held in September 2012. Presentations reviewed recent findings of the leading scientists from Russia, Europe, US and China on various aspects of cell death regulation and aging in cancer, cancer metabolism and stem cell research. The opening keynote lecture by Douglas R Green (Memphis, USA) was focused on elucidating complex mechanisms of cell death in embryonic development via the opposing functions of the pro-apoptotic protease, caspase-8. Caspase8 is known to promote cell death but also mediates cell survival, preventing the TNF-induced necrosis. The latter function is essential for proper development of mouse embryos after triggering the TNF signaling. The lethality of caspase-8-deficient embryos was rescued by knockout of receptor interacting protein kinase-3, which mediates programmed necrosis, or ‘necroptosis’. The knockout experiments in mice showed that caspase-8 prevented necrosis by formation of the proteolytically active complex that contained FLICE-like inhibitory protein long. Another aspect of cell proliferation control during the aging of brain was discussed in the report of Grigori Enikolopov (Cold Spring Harbor, USA; Moscow, Russia). The process of neuronal self-renewal takes place in hippocampus of the adult brain and is ensued by differentiation of neural stem cells into astrocytes. Aging is associated with a continuous decline in the number of new neurons. He presented recent findings on the mechanisms, which control division and differentiation of neural stem cells during aging. It turned out that the main mechanism driving the age-related decrease in adult hippocampal neurogenesis was the disappearance of neural stem cells via their conversion into mature astrocytes. Therefore, age-related deficit may be compensated by increasing the number of stem cells or number of neurons produced.