Lysine-specific methyltransferase Set7/9 in stemness, differentiation, and development.

The enzymes performing protein post-translational modifications (PTMs) form a critical post-translational regulatory circuitry that orchestrates literally all cellular processes in the organism. In particular, the balance between cellular stemness and differentiation is crucial for the development of multicellular organisms. Importantly, the fine-tuning of this balance on the genetic level is largely mediated by specific PTMs of histones including lysine methylation. Lysine methylation is carried out by special enzymes (lysine methyltransferases) that transfer the methyl group from S-adenosyl-L-methionine to the lysine residues of protein substrates. Set7/9 is one of the exemplary protein methyltransferases that however, has not been fully studied yet. It was originally discovered as histone H3 lysine 4-specific methyltransferase, which later was shown to methylate a number of non-histone proteins that are crucial regulators of stemness and differentiation, including p53, pRb, YAP, DNMT1, SOX2, FOXO3, and others. In this review we summarize the information available to date on the role of Set7/9 in cellular differentiation and tissue development during embryogenesis and in adult organisms. Finally, we highlight and discuss the role of Set7/9 in pathological processes associated with aberrant cellular differentiation and self-renewal, including the formation of cancer stem cells.

Methyltransferase Set7/9 controls PARP1 expression and regulates cisplatin response of breast cancer cells.

The protein-specific methyltransferase Set7/9 is known for its ability to add methyl groups to lysine residues on many targets, including as histones H1.4, H2A, H2B, H3, and non-histone proteins such as p53, NFκB, E2F1, pRb, Hif1α, ß-catenin, STAT3, and YY1 transcription factors. Set7/9 affects both the landscape of histone modifications and the functionality of the aforementioned TFs, and acts as an essential mediator of vital cellular functions, regulating tumor growth and the neoplastic transformation of normal cells. The number of studies demonstrating the determining role of Set7/9 in cancer is growing. Importantly, the effect of Set7/9 on tumor progression is ambivalent and cancer-type dependent. In this study we analyzed the potential participation of Set7/9 in the essential cellular processes in breast cancer cells and revealed that Set7/9 may be involved in DNA damage signaling and DNA repair processes. We further demonstrated that Set7/9 expression is downregulated in cancerous breast tissues and inversely correlated to PARP1 expression level. Using breast cancer cell lines of HER2-positive and triple negative subtypes we have shown that the attenuation of Set7/9 led to the stabilization of PARP1 on both mRNA and protein levels that in turn resulted in cisplatin resistance acquiring. Finally, we demonstrated that the combination of cisplatin with FDA approved PARP1 inhibitor niraparib (Zejula) has a synergistic effect with cisplatin and thereby allows to overcome cisplatin resistance of Set7/9 deficient breast cancer cells.

Phytochemicals Target Multiple Metabolic Pathways in Cancer.

Cancer metabolic reprogramming is a complex process that provides malignant cells with selective advantages to grow and propagate in the hostile environment created by the immune surveillance of the human organism. This process underpins cancer proliferation, invasion, antioxidant defense, and resistance to anticancer immunity and therapeutics. Perhaps not surprisingly, metabolic rewiring is considered to be one of the "Hallmarks of cancer". Notably, this process often comprises various complementary and overlapping pathways. Today, it is well known that highly selective inhibition of only one of the pathways in a tumor cell often leads to a limited response and, subsequently, to the emergence of resistance. Therefore, to increase the overall effectiveness of antitumor drugs, it is advisable to use multitarget agents that can simultaneously suppress several key processes in the tumor cell. This review is focused on a group of plant-derived natural compounds that simultaneously target different pathways of cancer-associated metabolism, including aerobic glycolysis, respiration, glutaminolysis, one-carbon metabolism, de novo lipogenesis, and ß-oxidation of fatty acids. We discuss only those compounds that display inhibitory activity against several metabolic pathways as well as a number of important signaling pathways in cancer. Information about their pharmacokinetics in animals and humans is also presented. Taken together, a number of known plant-derived compounds may target multiple metabolic and signaling pathways in various malignancies, something that bears great potential for the further improvement of antineoplastic therapy.

20-Hydroxyecdysone Confers Antioxidant and Antineoplastic Properties in Human Non-Small Cell Lung Cancer Cells.

20-Hydroxyecdysone (20E) is an arthropod hormone which is synthesized by some plants as part of their defense mechanism. In humans, 20E has no hormonal activity but possesses a number of beneficial pharmacological properties including anabolic, adaptogenic, hypoglycemic, and antioxidant properties, as well as cardio-, hepato-, and neuroprotective features. Recent studies have shown that 20E may also possess antineoplastic activity. In the present study, we reveal the anticancer properties of 20E in Non-Small Cell Lung Cancer (NSCLC) cell lines. 20E displayed significant antioxidant capacities and induced the expression of antioxidative stress response genes. The RNA-seq analysis of 20E-treated lung cancer cells revealed the attenuation of genes involved in different metabolic processes. Indeed, 20E suppressed several enzymes of glycolysis and one-carbon metabolism, as well as their key transcriptional regulators-c-Myc and ATF4, respectively. Accordingly, using the SeaHorse energy profiling approach, we observed the inhibition of glycolysis and respiration mediated by 20E treatment. Furthermore, 20E sensibilized lung cancer cells to metabolic inhibitors and markedly suppressed the expression of Cancer Stem Cells (CSCs) markers. Thus, in addition to the known beneficial pharmacological activities of 20E, our data uncovered novel antineoplastic properties of 20E in NSCLC cells.

Methyltransferase Set7/9 as a Multifaceted Regulator of ROS Response.

Reactive oxygen species (ROS) induce multiple signaling cascades in the cell and hence play an important role in the regulation of the cell's fate. ROS can cause irreversible damage to DNA and proteins resulting in cell death. Therefore, finely tuned regulatory mechanisms exist in evolutionarily diverse organisms that are aimed at the neutralization of ROS and its consequences with respect to cellular damage. The SET domain-containing lysine methyltransferase Set7/9 (KMT7, SETD7, SET7, SET9) post-translationally modifies several histones and non-histone proteins via monomethylation of the target lysines in a sequence-specific manner. In cellulo, the Set7/9-directed covalent modification of its substrates affects gene expression, cell cycle, energy metabolism, apoptosis, ROS, and DNA damage response. However, the in vivo role of Set7/9 remains enigmatic. In this review, we summarize the currently available information regarding the role of methyltransferase Set7/9 in the regulation of ROS-inducible molecular cascades in response to oxidative stress. We also highlight the in vivo importance of Set7/9 in ROS-related diseases.

The Role of PTEN in Epithelial-Mesenchymal Transition.

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.

Opposing Roles of Wild-type and Mutant p53 in the Process of Epithelial to Mesenchymal Transition.

The central role of an aberrantly activated EMT program in defining the critical features of aggressive carcinomas is well documented and includes cell plasticity, metastatic dissemination, drug resistance, and cancer stem cell-like phenotypes. The p53 tumor suppressor is critical for leashing off all the features mentioned above. On the molecular level, the suppression of these effects is exerted by p53 via regulation of its target genes, whose products are involved in cell cycle, apoptosis, autophagy, DNA repair, and interactions with immune cells. Importantly, a set of specific mutations in the TP53 gene (named Gain-of-Function mutations) converts this tumor suppressor into an oncogene. In this review, we attempted to contrast different regulatory roles of wild-type and mutant p53 in the multi-faceted process of EMT.

How Should the Worldwide Knowledge of Traditional Cancer Healing Be Integrated with Herbs and Mushrooms into Modern Molecular Pharmacology?

Traditional herbal medicine (THM) is a "core" from which modern medicine has evolved over time. Besides this, one third of people worldwide have no access to modern medicine and rely only on traditional medicine. To date, drugs of plant origin, or their derivates (paclitaxel, vinblastine, vincristine, vinorelbine, etoposide, camptothecin, topotecan, irinotecan, and omacetaxine), are very important in the therapy of malignancies and they are included in most chemotherapeutic regimes. To date, 391,000 plant and 14,000 mushroom species exist. Their medical and biochemical capabilities have not been studied in detail. In this review, we systematized the information about plants and mushrooms, as well as their active compounds with antitumor properties. Plants and mushrooms are divided based on the regions where they are used in ethnomedicine to treat malignancies. The majority of their active compounds with antineoplastic properties and mechanisms of action are described. Furthermore, on the basis of the available information, we divided them into two priority groups for research and for their potential of use in antitumor therapy. As there are many prerequisites and some examples how THM helps and strengthens modern medicine, finally, we discuss the positive points of THM and the management required to transform and integrate THM into the modern medicine practice.

Case Report: Primary Leptomeningeal Medulloblastoma in a Child: Clinical Case Report and Literature Review.

Medulloblastoma is one of the most common pediatric central nervous system malignancies worldwide, and it is characterized by frequent leptomeningeal metastasizing. We report a rare case of primary leptomeningeal medulloblastoma of an 11-year-old Caucasian girl with a long-term disease history, non-specific clinical course, and challenges in the diagnosis verification. To date, 4 cases of pediatric primary leptomeningeal medulloblastoma are reported, and all of them are associated with unfavorable outcomes. The approaches of neuroimaging and diagnosis verification are analyzed in the article to provide opportunities for effective diagnosis of this disease in clinical practice. The reported clinical case of the primary leptomeningeal medulloblastoma is characterized by MR images with non-specific changes in the brain and spinal cord and by 18FDG-PET/CT images with diffuse heterogeneous hyperfixation of the radiopharmaceutical along the whole spinal cord. The immunohistochemistry and next-generation sequencing analyses of tumor samples were performed for comprehensive characterization of the reported clinical case.

The Role of E3 Ligase Pirh2 in Disease.

The p53-dependent ubiquitin ligase Pirh2 regulates a number of proteins involved in different cancer-associated processes. Targeting the p53 family proteins, Chk2, p27Kip1, Twist1 and others, Pirh2 participates in such cellular processes as proliferation, cell cycle regulation, apoptosis and cellular migration. Thus, it is not surprising that Pirh2 takes part in the initiation and progression of different diseases and pathologies including but not limited to cancer. In this review, we aimed to summarize the available data on Pirh2 regulation, its protein targets and its role in various diseases and pathological processes, thus making the Pirh2 protein a promising therapeutic target.

The Role of Lysine Methyltransferase SET7/9 in Proliferation and Cell Stress Response.

Lysine-specific methyltransferase 7 (KMT7) SET7/9, aka Set7, Set9, or SetD7, or KMT5 was discovered 20 years ago, yet its biological role remains rather enigmatic. In this review, we analyze the particularities of SET7/9 enzymatic activity and substrate specificity with respect to its biological importance, mostly focusing on its two well-characterized biological functions: cellular proliferation and stress response.

Zeb1-mediated autophagy enhances resistance of breast cancer cells to genotoxic drugs.

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.

p53-Independent Effects of Set7/9 Lysine Methyltransferase on Metabolism of Non-Small Cell Lung Cancer Cells.

Set7/9 is a lysine-specific methyltransferase, which regulates the functioning of both the histone and non-histone substrates, thereby significantly affecting the global gene expression landscape. Using microarray expression profiling, we have identified several key master regulators of metabolic networks, including c-Myc, that were affected by Set7/9 status. Consistent with this observation, c-Myc transcriptional targets-genes encoding the glycolytic enzymes hexokinase (HK2), aldolase (ALDOB), and lactate dehydrogenase (LDHA)-were upregulated upon Set7/9 knockdown (Set7/9KD). Importantly, we showed the short hairpin RNA (shRNA)-mediated attenuation of Set7/9 augmented c-Myc, GLUT1, HK2, ALDOA, and LDHA expression in non-small cell lung cancer (NSCLC) cell lines, not only at the transcriptional but also at the protein level. In line with this observation, Set7/9KD significantly augmented the membrane mitochondrial potential (MMP), glycolysis, respiration, and the proliferation rate of NSCLC cells. Importantly, all these effects of Set7/9 on cell metabolism were p53-independent. Bioinformatic analysis has shown a synergistic impact of Set7/9 together with either GLUT1, HIF1A, HK2, or LDHA on the survival of lung cancer patients. Based on these evidence, we hypothesize that Set7/9 can be an important regulator of energy metabolism in NSCLC.

Set7/9 controls proliferation and genotoxic drug resistance of NSCLC 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.

Regulation of autophagy flux by E3 ubiquitin ligase Pirh2 in lung cancer.

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 RNA-binding protein HuR is a novel target of Pirh2 E3 ubiquitin ligase.

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.

Emerging roles of cancer-testis antigenes, semenogelin 1 and 2, in neoplastic cells.

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.

Linking Metabolic Reprogramming, Plasticity and Tumor Progression.

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.

Strategies to Circumvent the Side-Effects of Immunotherapy Using Allogeneic CAR-T Cells and Boost Its Efficacy: Results of Recent Clinical Trials.

Despite the outstanding results of treatment using autologous chimeric antigen receptor T cells (CAR-T cells) in hematological malignancies, this approach is endowed with several constraints. In particular, profound lymphopenia in some patients and the inability to manufacture products with predefined properties or set of cryopreserved batches of cells directed to different antigens in advance. Allogeneic CAR-T cells have the potential to address these issues but they can cause life-threatening graft-versus-host disease or have shorter persistence due to elimination by the host immune system. Novel strategies to create an "off the shelf" allogeneic product that would circumvent these limitations are an extensive area of research. Here we review CAR-T cell products pioneering an allogeneic approach in clinical trials.

SEMG1/2 augment energy metabolism of tumor cells.

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.