Search

Abbreviations: AO: acridine orange; ATM: ATM serine/threonine kinase; CHEK1: checkpoint kinase 1; CHEK2: checkpoint kinase 2; CI: combination index; DMSO: dimethyl sulfoxide; DSBs: double-strand breaks; GBM: glioblastoma; HR: homologous recombination; H2AX: H2A.X variant histone; IHC: immunohistochemistry; LAPTM4B: lysosomal protein transmembrane 4 beta; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PARP: poly(ADP-ribose) polymerase; RAD51: RAD51 recombinase; SQSTM1: sequestosome 1; SSBs: single-strand breaks; RNF168: ring finger protein 168; XPO1: exportin 1.

Gastric cancer (GC) is the fifth most prevalent form of cancer worldwide. CagA - positive Helicobacter pylori infects more than 60% of the human population. Moreover, chronic infection of CagA-positive H. pylori can directly affect GC incidence. In the current study, we have repurposed FDA-approved antibiotics that are viable alternatives to current regimens and can potentially be used as combination therapy against the CagA of H. pylori . The 100 FDA-approved gram negative antibiotics were screened against CagA protein using the AutoDock 4.2 tool. Further, top nine compounds were selected based on higher binding affinity with CagA. The trajectory analysis of MD simulations reflected that binding of these drugs with CagA stabilizes the system. Nonetheless, atomic density map and principal component analysis also support the notion of stable binding of antibiotics to the protein. The residues ASP96, GLN100, PRO184, and THR185 of compound cefpiramide, doxycycline, delafloxacin, metacycline, oxytetracycline, and ertapenem were involved in the binding with CagA protein. These residues are crucial for the CagA that aids in entry or pathogenesis of the bacterium. The screened FDA-approved antibiotics have a potential druggability to inhibit CagA and reduce the progression of H. pylori borne diseases.

Circular RNAs (circRNAs), a new class of non-coding RNAs, have been recently confirmed to regulate cell development, functions and certain types of pathological responses. In addition, it has been proved that circ_0000190 can serve as a tumor suppressor in several cancers. However, the underlying mechanism and biological functions of it in cervical cancer (CC) remain to be revealed. In our study, relative expression of indicated molecules was detected by RT-qPCR analysis. Loss-of-function and gain-of-function experiments were conducted to detect cell functions. Mechanism experiments including RIP assay, luciferase reporter assay and pull down assay were applied to verify the interaction among the indicated molecules. Overexpressed circ_0000190 attenuated CC progression in vitro and in vivo . Circ_0000190 functioned through the modulation of miR-1252-5p/EIF4EBP2 axis. Rescue experiments found that miR-1252-5p overexpression or EIF4EBP2 knockdown could reverse the influence on CC cells caused by circ_0000190 overexpression. Interestingly, it was found that EIF4EBP2 could bind to proto-oncogene eIF4E and prevent eIF4E from forming into complex and functioning. Circ_0000190 served as a tumor suppressor in CC and down-regulated circ_0000190 expression could weaken the binding ability of EIF4EBP2 to eIF4E thus leading to CC tumorigenesis. In our investigation, a novel tumor suppressive gene circ_0000190 was recognized, which could be treated as a promising biomarker for the diagnosis of CC.

Previous studies have explored the association between protein-coding genes and microRNAs (miRNAs) in lung adenocarcinoma (LUAD). However, the influence of the miR-199a-3p/anterior gradient 2 (AGR2) axis in LUAD has not yet been fully explored. Therefore, this study aimed to examine the underlying roles of AGR2 and miR-199a-3p in the development of LUAD. The expression levels of miR-199a-3p and AGR2 in LUAD tissues and cells were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A luciferase assay was also performed to identify the interaction between AGR2 and miR-199a-3p. Moreover, the cell counting kit 8 (CCK-8), 5'-bromo-2'-deoxyuridine (BrdU), and adhesion assays were used along with flow cytometry to verify the malignancy of LUAD in vitro, while a xenograft tumor assay was performed to confirm the tumor growth in vitro. The findings showed a decrease in the expression of miR-199a-3p in LUAD. Additionally, miR-199a-3p overexpression inhibited the growth of LUAD cells in vitro and in vivo, while elevating the apoptosis rate of the cells. AGR2 knockdown had the same effect in the cells as that of miR-199a-3p overexpression. It was also found that miR-199a-3p directly targeted AGR2 in LUAD cells to suppress tumorigenesis. In conclusion, this study suggests that miR-199a-3p plays an anti-tumorigenic role in LUAD by targeting AGR2. Moreover, our study provides insights into the development of novel therapeutic targets for the treatment of LUAD.

As a malignant disease, lung cancer has a high morbidity and mortality rate. Baicalin is derived from Radix Scutellariae and has anti-tumor effects, however, its role in lung cancer remains unknown. Here, functional assays suggested baicalin suppressed in vitro lung cancer phenotypes. We used micro (mi)RNA array analysis to explore baicalin effects on miRNA expression. We observed baicalin increased miR-340-5p expression, whereas inhibition of this expression abolished anti-tumor effects of baicalin. Furthermore, neuroepithelial cell transforming 1 (NET1) functioned as a miR-340-5p target, and acted in a baicalin-dependent manner to regulate lung cancer progression. Thus, baicalin elicited antitumor activities by affecting the miR-340-5p/NET1 axis, suggesting a new approach to lung cancer clinical management.

Esophageal squamous cell carcinoma (ESCC), a major form of esophageal cancer, is a serious threat to human health. This study was conducted to investigate the pathogenesis of ESCC and find effective therapies to improve it. Protein expression of transfected plasmids was detected by RT-qPCR and western blot. Co-immunoprecipitation assay was performed to verify the binding of LETM1 and KIF14. CCK-8, wound healing and transwell assays were used to assess the proliferation, invasion and migration of ESCC cells. Finally, the angiogenesis was assessed using tubule formation assay. The co-immunoprecipitation results showed that LETM1 could bind to KIF14. The cytological and protein results demonstrated that interference with LETM1 caused downregulation of KIF14 expression, which led to inhibition of proliferation, invasion, migration and angiogenesis in ESCC cells. Taken together, interfering with LETM1 to downregulate KIF14 may become a new target for ESCC treatment.

Lung squamous cell carcinoma (LUSC) is a prevalent subtype of nonsmall cell lung cancer (NSCLC). Dysregulated long noncoding RNAs (lncRNAs) are increasingly identified as pivotal modulators in cancer progression. NCK1 divergent transcript (NCK1-AS1) is a lncRNA that has been proven to be oncogenic in different types of human cancers. However, whether it exerts similar functions in LUSC remains to be elusive. The present study focused on investigating the influence of NCK1-AS1 on the cellular process in LUSC and exploring its underlying mechanism. Through online bioinformatics analysis, we obtained a high NCK1-AS1 level in LUSC tissues. Meanwhile, we confirmed that NCK1-AS1 was upregulated in LUSC cells. Gain- or loss-of-function assays suggested that NCK1-AS1 prompted cell proliferation and migration, whilst impeded cell apoptosis in LUSC. Mechanistically, we revealed that NCK1-AS1 induced the upregulation of its nearby gene NCK adaptor protein 1 (NCK1) at the transcriptional level by interacting with the transcription factor MYC proto-oncogene (MYC). Rescue assays indicated that NCK1 participated in the regulation of NCK1-AS1 on LUSC progression. In conclusion, we firstly demonstrated the oncogenic role of NCK1-AS1 in LUSC and illustrated its downstream molecular mechanism.

Jnks are mitogen activated protein kinases that are best known for regulating transcription and apoptotic signaling. However, they also play important roles in controlling cell motility and invasion by phosphorylating many actin and microtubule regulatory proteins. These mechanisms have important implications for normal cell motility as well as cancer metastasis. Jnks are activated by growth factors and cytokines that stimulate cell motility, and this often requires upstream activation of Rho GTPases. Our recent work indicates that Jnks may also regulate Rho GTPase activation. Specifically, we found that Jnk-dependent phosphorylation of the RhoA guanine nucleotide exchange factor (RhoGEF) Net1A promotes its cytosolic accumulation to drive RhoA activation and actin cytoskeletal reorganization. Net1A is unusual among RhoGEFs in that it is sequestered in the nucleus to prevent aberrant RhoA activation. Importantly, Jnk-stimulated cytosolic localization of Net1A is sufficient to stimulate cell motility and extracellular matrix invasion in non-invasive breast cancer cells. Since Net1A expression is critical for cancer cell motility and invasion in vitro , and breast cancer metastasis in vivo , these data uncover a previously unappreciated regulatory mechanism that may contribute to metastasis in multiple types of cancer.

The polycomb repressive complex 2 (PRC2) is an important developmental regulator responsible for the methylation of histone 3 lysine 27 (H3K27). Here, we show that the PRC2 complex regulates the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. Depletions of the catalytic subunit of the PRC2 complex, EZH2, have shown that EZH2 is required for cell viability, suggesting that EZH2 promotes proliferation. We found that EZH2 directly represses both positive and negative cell cycle genes, thus enabling the PRC2 complex to tightly control the cell cycle. We show that modest inhibition or depletion of EZH2 leads to enhanced proliferation and an accumulation of cells in S phase. This effect is mediated by direct repression of cyclin D1 ( Ccnd1 ) and cyclin E1 ( Ccne1 ) by the PRC2 complex. Our results show that PRC2 has pleiotropic effects on proliferation as it serves to restrain cell growth, yet clearly has a function required for cell viability as well. Intriguingly, we also find that the retinoblastoma protein gene ( Rb1 ) is a direct target of the PRC2 complex. However, modest depletion of EZH2 is not sufficient to maintain Rb1 expression, indicating that the PRC2 dependent upregulation of cyclin D1 is sufficient to inhibit Rb1 expression. Taken together, our results show that the PRC2 complex regulates skeletal muscle proliferation in a complex manner that involves the repression of Ccnd1 and Ccne1 , thus restraining proliferation, and the repression of Rb1 , which is required for mitotic exit and terminal differentiation.

Macrophages are innate immune cells that constantly patrol an organism to fulfill protective and homeostatic roles. Previous studies have shown that Rho GTPase activity is required for macrophage mobility, yet the roles of upstream regulatory proteins controlling Rho GTPase function in these cells are not well defined. Previously we have shown that the RhoA GEF Net1 is required for human breast cancer cell motility and extracellular matrix invasion. To assess the role of Net1 in macrophage motility, we isolated bone marrow macrophage (BMM) precursors from wild type and Net1 knockout mice. Loss of Net1 did not affect the ability of BMM precursors to differentiate into mature macrophages in vitro , as measured by CD68 and F4/80 staining. However, Net1 deletion significantly reduced RhoA activation, F-actin accumulation, adhesion, and motility in these cells. Nevertheless, similar to RhoA/RhoB double knockout macrophages, Net1 deletion did not impair macrophage recruitment to the peritoneum in a mouse model of sterile inflammation. These data demonstrate that Net1 is an important regulator of RhoA signaling and motility in mouse macrophages in vitro , but that its function may be dispensable for macrophage recruitment to inflammatory sites in vivo .

The LMO2/LDB1 macromolecular complex is critical in hematopoietic stem and progenitor cell specification and in the development of acute leukemia. This complex is comprised of core subunits of LMO2 and LDB1 as well as s ingle- s tranded DNA- b inding p rotein (SSBP) cofactors and DNA-binding b asic h elix- l oop- h elix (bHLH) and GATA transcription factors. We analyzed the steady-state abundance and kinetic stability of LMO2 and its partners via Halo protein tagging in conjunction with variant proteins deficient in binding their respective direct protein partners. We discovered a hierarchy of protein stabilities (with half-lives in descending order) as follows: LDB1 > SSBP > LMO2 > TAL1. Importantly, LDB1 is a remarkably stable protein that confers enhanced stability upon direct and indirect partners, thereby nucleating the formation of the multisubunit protein complex. The data imply that free subunits are more rapidly degraded than those incorporated within the LMO2/LDB1 complex. Our studies provided significant insights into LMO2/LDB1 macromolecular protein complex assembly and stability, which has implications for understanding its role in blood cell formation and for therapeutically targeting this complex in human leukemias., (Copyright © 2020 American Society for Microbiology.)

The most prominent cancer-associated fibroblasts (CAFs) in tumor stroma is known to form a protective structure to support tumor growth. Anterior gradient-2 (AGR2), a tumor secretory protein is believed to play a pivotal role during tumor microenvironment (TME) development. Here, we report that extracellular AGR2 enhances fibroblasts elongation and migration significantly. The early stimulation of RhoA showed the association of AGR2 by upregulation of G1-S phase-regulatory protein cyclin D1 and FAK phosphorylation through fibroblasts growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR). Our finding indicates that secretory AGR2 alters fibroblasts elongation, migration, and organization suggesting the secretory AGR2 as a potential molecular target that might be responsible to alter fibroblasts infiltration to support tumor growth.

Senescence is activated in response to chemotherapy to prevent the propagation of cancer cells. In transformed cells, recent studies have shown that this response is not always definitive and that persistent populations can use senescence as an adaptive pathway to restart proliferation and become more aggressive. Here we discuss the results showing that an incomplete and heterogeneous senescence response plays a key role in chemotherapy resistance. Surviving to successive chemotherapy regimens, chronically existing senescent cells can create a survival niche through paracrine cooperations with neighboring cells. This favors chemotherapy escape of premalignant clones but might also allow the survival of adjacent clones presenting a lower fitness. A better characterization of senescence heterogeneity in transformed cells is therefore necessary. This will help us to understand this incomplete response to therapy and how it could generate clones with increased tumor capacity leading to disease relapse.

Human beta-defensin-1 (hBD-1) is one of a number of small cationic host-defense peptides. Besides its well-known broad-spectrum antimicrobial function, hBD-1 has recently been identified as a chromosome 8p tumor-suppressor gene. The role of hBD-1 in modulating the host immune response to oncogenesis, associated with cell signaling and potential therapeutic applications, has become increasingly appreciated over time. In this study, multiple approaches were used to illustrate hBD-1 anti-tumor activities. Results demonstrate that hBD-1 peptide alters human epidermal growth factor receptor 2 (HER2) signal transduction and represses retroviral-mediated transgene expression in cancer cells. Loss of orthologous murine defense-1 (mBD1) in mice enhances nickel sulfate-induced leiomyosarcoma and causes mouse kidney cells to exhibit increased susceptibility to HPV-16 E6/7-induced neoplastic transformation. Furthermore, for the first time, a novel function of the urine-derived hBD-1 peptide was discovered to suppress bladder cancer growth and this may lead to future applications in the treatment of malignancy.

Oncoproteins play a vital role in the pathogenesis of myeloid leukemia. Most targeted therapies for myeloid leukemia are small molecules or monoclonal antibodies that inhibit the activity of the oncoproteins. However, leukemia cells often develop resistance to these drugs through overexpression of the target protein and/or by obtaining new mutations in the target protein to render them resistant to the drug. Oncoproteins degradation induced by small molecules through ubiquitin or autophagy pathway is considered a better way to avoid drug resistance. Here, we describe the latest advances in the use of small molecules to degrade oncoproteins. We first discuss examples of existing cancer drugs and candidate drugs that act by degrading oncoproteins, and then review the latest development of rational design of small molecules that induce selective degradation of target proteins. Furthermore, small-molecule-based proteolysis-targeting chimeras (PROTACs) have demonstrated that this technology can effectively degrade target proteins.

Numb is a protein whose asymmetric segregation during cell division determines cell fate and has numerous functions relevant to multiple fields of study, including developmental neurobiology and cancer biology. Little is known about the role of Numb in esophageal squamous cell carcinoma (ESCC), the predominant histological esophageal carcinoma in Asian populations. In this study, we focused on the expression and biologic functions of Numb in the context of ESCC. From analysis of tissue microarrays with 212 patients, it was found that Numb was significantly upregulated in ESCC tissues compared with corresponding non-cancerous tissues. Kaplan-Meier survival analysis suggests that higher expression of Numb was significantly associated with a high tumor recurrence (p = 0.015) and poor overall post-surgical survival (p = 0.016). Using multiple Cox regression, the expression of Numb was determined to be an independent predictor of poor prognosis. When siRNA was used to knockdown Numb in ESCC cell lines, there was a consistent increase in caspase-3 dependent apoptosis and inhibition of cellular proliferation, as well as downregulation of expression of the cancer stem cell markers Oct-4, SOX-2 and Nanog. In addition, downregulated Numb expression was not significantly associated with the migration of ESCC cells. These results indicate that Numb acts as an oncoprotein and has potential as a novel prognostic biomarker and therapeutic target in ESCC patients.

An accumulated evidence supports that MicroRNAs (miRNAs) have shown a prominent role in pathological processes and different tumor onset. However, to date, the potential functional roles and molecular mechanisms by how microRNA-424-5p(miR-424-5p) affects cancer cell proliferation are greatly unclear, especially in epithelial ovarian cancer(EOC).In this study, we demonstrated that miR-424-5p was significantly down-regulated in EOC tissues and cell lines. The level of miR-424-5p was negatively correlated with tumor size, TNM stage, pathological grade, lymphatic metastasis of EOC. Restoring miR-424-5p expression in EOC cells dramatically suppressed cell proliferation and caused an accumulation of cells in G1 phase, and thus contributed to better prognosis of EOC patients. Mechanistically, miR-424-5p inhibits CCNE1 expression through targeting CCNE1 3'UTR, and subsequent arrest cell cycle in G1/G0 phase by inhibiting E2F1-pRb pathway. This study revealed functional and mechanistic links between miR-424-5p and CCNE1 in the progression of EOC and provide an important insight into that miR-424-5p may serve as a therapeutic target in EOC.

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3β activity., Abbreviations: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III β-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium.

While lentiviral expression vectors are widely used in many facets of molecular biology, due to their ability to stably express heterologous genes in both dividing and non-dividing cells, they suffer from the disadvantage that introns inserted into the vector genome are generally rapidly lost by splicing in packaging cell lines. The presence of an intron, if achievable, has the potential to facilitate the expression of transgene cDNAs, as splicing has been extensively shown to facilitate mRNA biogenesis and function. Moreover, if a stable intron could be introduced into a lentiviral vector, this could greatly facilitate the expression of microRNAs (miRNAs), and especially miRNA clusters, as the introduction of pri-miRNA stems into the exonic region of a lentiviral vector can strongly reduce both vector titer and the expression of any miRNA-linked indicator gene due to cleavage of the vector RNA genome by cellular Drosha. Here, we describe a novel lentiviral vector design in which transgenes and/or miRNAs are expressed using an antisense-orientated, inducible promoter driving an expression cassette bearing a functional intron. We demonstrate that this lentiviral vector, called pTREX, is able to express higher levels of both transgenes and pri-miRNA clusters when compared with a closely similar conventional lentiviral vector.

The regulation of adipose tissue expansion by adipocyte hypertrophy and/or hyperplasia is the topic of extensive investigations given the potential differential contribution of the 2 processes to the development of numerous chronic diseases associated with obesity. We recently discovered that the loss-of-function of the Src homology domain-containing protein Nck2 in mice promotes adiposity accompanied with adipocyte hypertrophy and impaired function, and enhanced adipocyte differentiation in vitro. Moreover, in severely-obese human's adipose tissue, we found that Nck2 expression is markedly downregulated. In this commentary, our goal is to expand upon additional findings providing further evidence for a unique Nck2-dependent mechanism regulating adipogenesis. We propose that Nck2 should be further investigated as a regulator of the reliance of white adipose tissue on hyperplasia versus hypertrophy during adipose tissue expansion, and hence, as a potential novel molecular target in obesity.

Cyclin D1 and cyclin E1, as vital regulatory factors of G1-S phase cell cycle progression, are frequently constitutive expressed and associated with pathogenesis and tumorigenesis in most human cancers and they have been regarded as promising targets for cancer therapy. In this study, we established NVP-BEZ235, a potent dual kinase inhibitor, could induce neuroblastoma cells proliferation inhibition without apoptosis activation. Moreover, we showed NVP-BEZ235 could induce neuroblastoma cells arrested at G0/G1 phase accompanied with significant reduction of the cyclin D1 and E1 proteins in a dose dependent manner at nanomole concentration. Additionally we found that GSK3β was dephosphorylated and activated by NVP-BEZ235 and then triggered cyclin D1 and cyclin E1 degradation through ubiquitination proteasome pathway, based on the evidences that NVP-BEZ235 induced downregulation of cyclin D1 and cyclin E1 were obviously recovered by proteasome inhibitor and the blockade of GSK3β contributed to remarkable rescue of cyclin D1 and cyclin E1. Analogous results about its anti-proliferation effects and molecular mechanism were observed on neuroblastoma xenograft mouse model in vivo. Therefore, these results indicate that NVP-BEZ235-induced cyclin D1 and cyclin E1 degradation, which happened through activating GSK3β, and GSK3β-dependent down-regulation of cyclin D1 and cyclin E1 should be available for anticancer therapeutics.

Mucin1 (MUC1) is a transmembrane oncogenic protein that plays a central role in malignant transformation and disease evolution, including cell proliferation, survival, self-renewal, and metastatic invasion. MUC1 has been shown to interact with diverse effectors such as β-catenin, receptor tyrosine kinases, and c-Abl, which are of importance in the pathogenesis of various hematological malignancies. In myeloid leukemia, MUC1 has been shown to have an essential role in leukemia stem-cell function, the induction of reactive oxygen species (ROS), and the promotion of terminal myeloid differentiation. As such, MUC1 is an attractive therapeutic target in hematologic malignancies. Targeting MUC1 has been shown to be an effective approach for inducing cell death in tumor in in vivo and in vitro models. Furthermore, MUC1 inhibition is synergistic with other therapeutic agents in the treatment of hematologic disorders. This review will explore the role of MUC1 in hematological malignancies, and strategies for targeting this oncoprotein.

ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.

The classification of a gene as an oncogene or a tumor suppressor has been a staple of cancer biology for decades. However, as we delve deeper into the biology of these genes, this simple classification has become increasingly difficult for some. In the case of heterogeneous nuclear ribonuclear protein K (hnRNP K), its role as a tumor suppressor has recently been described in acute myeloid leukemia and demonstrated in a haploinsufficient mouse model. In contrast, data from other clinical correlation studies suggest that hnRNP K may be more fittingly described as an oncogene, due to its increased levels in a variety of malignancies. hnRNP K is a multifunctional protein that can regulate both oncogenic and tumor suppressive pathways through a bevy of chromatin-, DNA-, RNA-, and protein-mediated activates, suggesting its aberrant expression may have broad-reaching cellular impacts. In this review, we highlight our current understanding of hnRNP K, with particular emphasis on its apparently dichotomous roles in tumorigenesis.

Never in Mitosis (NIMA) Related Kinase 2 (NEK2) plays a key role in regulating mitotic processes, including centrosome duplication and separation, microtubule stabilization, kinetochore attachment and spindle assembly checkpoint. NEK2 is aberrantly overexpressed in a wide variety of human cancers and has been implicated in various aspects of malignant transformation, including tumorigenesis, drug resistance and tumor progression. The close relationship between NEK2 and cancer has made it an attractive target for anticancer therapeutic development; however, the mechanisms of how NEK2 coordinates altered signaling to malignant transformation remains unclear. In this paper, we discuss the functional roles of NEK2 in cancer development; highlight some of the significant NEK2 signaling in cancer, and summarize recent advances in the development of NEK2 inhibitors.

Understanding how transcriptional regulators are themselves controlled is important in attaining a complete picture of the intracellular effects that follow signaling cascades during early development and cell-restricted differentiation. We have addressed this issue by focusing on the regulation of EKLF/KLF1, a zinc finger transcription factor that plays a necessary role in the global regulation of erythroid gene expression. Using biochemical affinity purification, we have identified the DEK oncoprotein as a critical factor that interacts with an essential upstream enhancer element of the EKLF promoter and exerts a positive effect on EKLF levels. This element also binds a core set of erythroid transcription factors, suggesting that DEK is part of a tissue-restricted enhanceosome that contains BMP4-dependent and -independent components. Together with local enrichment of properly coded histones and an open chromatin domain, optimal transcriptional activation of the EKLF locus can be established., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Development of the cardiovascular system is critically dependent on the ability of endothelial cells (ECs) to reorganize their intracellular actin architecture to facilitate migration, adhesion, and morphogenesis. Nck family cytoskeletal adaptors function as key mediators of actin dynamics in numerous cell types, though their role in EC biology remains largely unexplored. Here, we demonstrate an essential requirement for Nck within ECs. Mouse embryos lacking endothelial Nck1/2 expression develop extensive angiogenic defects that result in lethality at about embryonic day 10. Mutant embryos show immature vascular networks, with decreased vessel branching, aberrant perivascular cell recruitment, and reduced cardiac trabeculation. Strikingly, embryos deficient in endothelial Nck also fail to undergo the endothelial-to-mesenchymal transition (EnMT) required for cardiac valve morphogenesis, with loss of Nck disrupting expression of major EnMT markers, as well as suppressing mesenchymal outgrowth. Furthermore, we show that Nck-null ECs are unable to migrate downstream of vascular endothelial growth factor and angiopoietin-1, and they exhibit profound perturbations in cytoskeletal patterning, with disorganized cellular projections, impaired focal adhesion turnover, and disrupted actin-based signaling. Our collective findings thereby reveal a crucial role for Nck as a master regulator within the endothelium to control actin cytoskeleton organization, vascular network remodeling, and EnMT during cardiovascular development., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Epstein-Barr virus (EBV)-associated diffuse large B-cell lymphoma (DLBCL) of the elderly constitutes a provisional clinicopathological entity in the current World Health Organization (WHO) classification and its genomic features remain sparsely characterized. We investigated a cohort of 26 cases of untreated de novo EBV-positive DLBCL of the elderly by high-resolution array-based comparative genomic profiling and fluorescence in situ hybridization (FISH). Moreover, we screened for activating mutations affecting nuclear factor (NF)-κB pathway signaling and chromatin remodeling (EZH2, CD79B, CARD11 and MYD88) due to their impact of gene expression signatures and postulated upcoming therapeutic targetability. We identified an overlap between genomic aberrations previously described to be exclusive features of plasmablastic lymphoma (PL), post-transplant lymphoproliferative disorder (PTLD) and DLBCL, respectively, indicating a close cytogenetic relationship between these entities. Few mutations affecting CD79B and CARD11 and no MYD88 mutations were detectable, hinting at EBV-mediated activation of NF-κB as an alternative to pathologically enforced B-cell receptor signaling in this rare entity.

Objectives: DJ-1 is a multifunctional protein implicated in redox dependent cell fate decisions. The aim of our study was to determine the pleural fluid (PF) levels of DJ-1 in malignant pleural effusions (MPEs) secondary to lung cancer. Additionally, we opted to assess potential correlations of DJ-1 PF levels with the PF levels of superoxide dismutase-1 (SOD1) and 8-isoprostane that are known antioxidant enzymes and have been previously reported in MPEs., Methods: Forty lung cancer patients with cytological proof of MPE were enrolled in this study. The PF levels of DJ-1, SOD1, and 8-isoprostane were measured by means of enzyme-linked immunosorbent assay., Results: The median PF levels of DJ-1 were 826 ng/mL (interquartile range, IQR: 482-1010 ng/mL). DJ-1 PF levels significantly correlated with PF Cu/Zn-SOD1 and PF 8-isoprostane levels (Spearman's rho, r; r = -0.476, P = 0.002 and r = -0.264, P = 0.033, respectively), PF lactate dehydrogenase (r = -0.497, P = 0.001) and total PF cell counts (r = -0.325, P = 0.041). Finally, in patients aged over 65 the PF DJ-1 levels were significantly higher than patients aged less than 65 (875 ng/mL vs. 607 ng/mL, respectively, P = 0.037)., Discussion: To our knowledge, this is the first report to determine DJ-1's levels in MPEs due to lung cancer. The negative correlations between DJ-1, SOD1, and 8-isorpostane warrant further investigation regarding the altered redox regulation associated with MPEs.

The Zuotin-related factor 1, ZRF1, has recently been identified as an epigenetic regulator of gene transcription in stem cells and cancer. During differentiation of human teratocarcinoma cells, ZRF1 promotes transcriptional induction of developmental genes that are repressed by Polycomb complexes. Importantly, ZRF1 has recently been shown to be required for both neural differentiation of embryonic stem cells (ESCs) and for maintenance of neural progenitor cell (NPC) identity. Moreover, a dual role has now emerged for ZRF1 in cancer: on the one hand, ZRF1 plays a crucial role in oncogene-induced senescence (OIS) by activating the INK4/ARF locus, thus working as a tumor suppressor; on the other hand, ZRF1 promotes leukemogenesis in acute myeloid leukemia (AML) in a Polycomb-independent fashion. Therefore, increasing evidence points to ZRF1 as a novel target for therapy of neurodegenerative diseases and cancer.

The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation.

Neuroblastoma is one of the most common solid tumors in childhood and usually accompanied with poor prognosis and rapid tumor progression when diagnosed with amplification of the proto-oncogene N-Myc. The amplification of N-Myc has major influence on the maintenance of aerobic glycolysis, also known as the Warburg effect. This specific switch in the conversion of pyruvate to lactate instead of the conversion of pyruvate to acetyl-coenzyme A even in the presence of oxygen has important benefits for the tumor, e.g. increased production of enzymes and enzyme substrates that are involved in tumor progression, angiogenesis and inhibition of apoptosis. The antiprotozoal drug nifurtimox, which is generally used for the treatment of infections with the parasitic protozoan Trypanosoma cruzi, has been reported to have cytotoxic properties in the therapy of neuroblastoma. However, its action of mechanism has not been described in detail yet. The presented in vitro study on the neuroblastoma cell lines LA-N-1, IMR-32, LS and SK-N-SH shows an increased production of oxidative stress, a reduced lactate dehydrogenase enzyme activity and reduced lactate production after nifurtimox treatment. Furthermore, nifurtimox leads to reduced mRNA and protein levels of the proto-oncogene protein N-Myc. Thus, the current work gives new insights into the effect of nifurtimox on tumor metabolism revealing a shifted glucose metabolism from production of lactate to oxidative phosphorylation and a reduced expression of the major molecular prognostic factor in neuroblastoma N-Myc, presenting nifurtimox as a possible adjuvant therapeutic agent against (high risk) neuroblastoma.

Wnt/β-catenin and Hedgehog/Gli signalings play key roles in multiple biogenesis such as embryonic development and tissue homeostasis. Dysregulations of these 2 pathways are frequently found in most cancers, particularly in colon cancer. Their crosstalk has been increasingly appreciated as an important mechanism in regulating colon cancer progression. Our studies into the link between Wnt/β-catenin and Hedgehog/Gli signalings in colon cancer revealed several possible crosstalk points and suggested potential therapeutic strategies for colon cancer.

Members of the ETS family of transcription factors are involved in several developmental processes including endothelial cell specification and blood vessel formation, but their exact roles remain unclear. The family member Erg is highly expressed in endothelial cells as compared to other developing cell types including chondrocytes, hematopoietic cells and mesodermal cells. To study the specific roles ERG plays in endothelial cell specification and function during early embryogenesis, we conditionally ablated it by mating ErgloxP/loxP and Tie2-Cre mice. We found that mutant embryos died by mid-gestation and that angiogenesis and vascular integrity were highly compromised. Our study reveals that ERG has essential and cell autonomous roles in endothelial cell development and blood vessel maintenance.

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that plays a significant role in mitotic progression and cellular responses to DNA damage. While traditionally viewed as a tumor suppressor, inhibition of PP2A has recently come to attention as a novel therapeutic means of driving senescent cancer cells into mitosis and promoting cell death via mitotic catastrophe. These findings have been corroborated in numerous studies utilizing naturally produced compounds that selectively inhibit PP2A. To overcome the known human toxicities associated with these compounds, a water-soluble small molecule inhibitor, LB100, was recently developed to competitively inhibit the PP2A protein. This review summarizes the pre-clinical studies to date that have demonstrated the anti-cancer activity of LB100 via its chemo- and radio-sensitizing properties. These studies demonstrate the tremendous therapeutic potential of LB100 in a variety of cancer types. The results of an ongoing phase 1 trial are eagerly anticipated.

Mutations in DJ-1 are a cause of recessive, early-onset Parkinson's disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin β2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The role of latent Epstein-Barr virus (EBV) infection in the pathogenesis of low-grade B cell non-Hodgkin lymphoma (B-NHL) has not been studied. We therefore investigated the incidence of latent EBV infection in a group of patients with leukemic low-grade B-NHL, as well as the incidence of viral latent membrane protein 1 (LMP1) oncoprotein expression in the same patient group. Furthermore, in an attempt to elucidate the role of this viral oncoprotein in non-EBV-related lymphomas, we correlated the expression of LMP1 with the level of oxidative stress, a parameter related to apoptosis. In the present study we detected lower levels of oxidative stress in the sera of LMP1-positive patients. This possibly implies an anti-apoptotic role of this viral oncoprotein in low-grade B cell lymphomas. However, LMP1 expression status did not affect expression of the major anti-apoptotic gene BCL-2.

Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area.

Deregulated cell cycle progression can often be traced to intrinsic defects in specific regulatory proteins in cancer cells. Knowledge of these primary defects has led to targeted approaches that exploit the defects and spare normal cells. However, the success of such targeted approaches is still hit-or-miss. Genetic and epigenetic variability inherent in most tumors often results in phenotypic heterogeneity that, in turn, results in de novo or acquired resistance to therapeutic agents. The ability of cells to compensate and adapt to the inhibition of a specific cell cycle mediator is not remarkable. What is novel and of great potential importance is that the ability of cells to exhibit such adaptability varies markedly. "Phenotypic stability factors" that restrict the ability of cells to undergo epithelial-mesenchymal transitions (EMT) may dictate the success or failure of targeted therapies by interfering with compensatory changes such as deregulation of CDK2 activity. Identification of existing and new agents that induce and maintain phenotypic stability factors will inform and enable synergistic approaches to the eradication of even the most aggressive tumors.

Aberrant activation of SHH pathway is a major cause of medulloblastoma (MB), the most frequent brain malignancy of the childhood. A few Hedgehog inhibitors, all antagonizing the membrane transducer Smo, have been approved or are under clinical trials for the treatment of human MB. However, the efficacy of these drugs is limited by the occurrence of novel mutations or by activation of downstream or non-canonical Hedgehog components. Thus, the identification of novel druggable downstream pathways represents a critical step to overcome this problem. In the present work we demonstrate that aerobic glycolysis is a valuable HH-dependent downstream target, since its inhibition significantly counteracts the HH-mediated growth of normal and tumor cells. Hedgehog activation induces transcription of hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2), two key gatekeepers of glycolysis. The process is mediated by the canonical activation of the Gli transcription factors and causes a robust increase of extracellular lactate concentration. We show that inhibition of glycolysis at different levels blocks the Hedgehog-induced proliferation of granule cell progenitors (GCPs), the cells from which medulloblastoma arises. Remarkably, we demonstrate that this glycolytic transcriptional program is also upregulated in SHH-dependent tumors and that pharmacological targeting with the pyruvate kinase inhibitor dichloroacetate (DCA) efficiently represses MB growth in vitro and in vivo. Together, these data illustrate a previously uncharacterized pharmacological strategy to target Hedgehog dependent growth, which can be exploited for the treatment of medulloblastoma patients.

ATM activation following DNA damage is a critical event which is required for efficient DNA repair and cell survival, yet signaling mechanisms controlling its activation are incompletely understood. The RhoGEF Net1 has previously been reported to control Rho GTPase activation and downstream cell survival outcomes following double strand DNA damage. However the role of Net1 isoforms in controlling ATM-dependent cell signaling has not been assessed. In the present work we show that expression of the Net1A isoform is specifically required for efficient activation of ATM but not the related kinase DNA-PK after ionizing radiation. Surprisingly Net1A overexpression also potently suppresses ATM activation and phosphorylation of its substrate H2AX. This effect does not require catalytic activity towards RhoA or RhoB, and neither Rho GTPase affects ATM activation, on its own. Consistent with a role in controlling ATM activation, Net1A knockdown also impairs DNA repair and cell survival. Taken together these data indicate that Net1A plays a plays a previously unrecognized, Rho GTPase-independent role in controlling ATM activity and downstream signaling after DNA damage to impact cell survival.

The condensin complex is required for chromosome condensation during mitosis; however, the role of this complex during interphase is unclear. Neuroblastoma is the most common extracranial solid tumor of childhood, and it is often lethal. In human neuroblastoma, MYCN gene amplification is correlated with poor prognosis. This study demonstrates that the gene encoding the condensin complex subunit SMC2 is transcriptionally regulated by MYCN. SMC2 also transcriptionally regulates DNA damage response genes in cooperation with MYCN. Downregulation of SMC2 induced DNA damage and showed a synergistic lethal response in MYCN-amplified/overexpression cells, leading to apoptosis in human neuroblastoma cells. Finally, this study found that patients bearing MYCN-amplified tumors showed improved survival when SMC2 expression was low. These results identify novel functions of SMC2 in DNA damage response, and we propose that SMC2 (or the condensin complex) is a novel molecular target for the treatment of MYCN-amplified neuroblastoma.

Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.

A precise balance between quiescence and proliferation is crucial for the lifelong function of hematopoietic stem cells (HSCs). Cyclins E1 and E2 regulate exit from quiescence in fibroblasts, but their role in HSCs remains unknown. Here, we report a non-redundant role for cyclin E1 in mouse HSCs. A long-term culture-initiating cell (LTC-IC) assay indicated that the loss of cyclin E1, but not E2, compromised the colony-forming activity of primitive hematopoietic progenitors. Ccne1(-/-) mice showed normal hematopoiesis in vivo under homeostatic conditions but a severe impairment following myeloablative stress induced by 5-fluorouracil (5-FU). Under these conditions, Ccne1(-/-) HSCs were less efficient in entering the cell cycle, resulting in decreased hematopoiesis and reduced survival of mutant mice upon weekly 5-FU treatment. The role of cyclin E1 in homeostatic conditions became apparent in aged mice, where HSC quiescence was increased in Ccne1(-/-) animals. On the other hand, loss of cyclin E1 provided HSCs with a competitive advantage in bone marrow serial transplantation assays, suggesting that a partial impairment of cell cycle entry may exert a protective role by preventing premature depletion of the HSC compartment. Our data support a role for cyclin E1 in controlling the exit from quiescence in HSCs. This activity, depending on the physiological context, can either jeopardize or protect the maintenance of hematopoiesis.

In recent years, several genetic and epigenetic alterations have been identified and linked with deregulated signaling pathways that promote growth and survival of lymphoma cells. These discoveries have raised hopes that a new era of targeted therapy will eventually improve treatment outcome of lymphoma. In this focused review, we summarize emerging preclinical and clinical data supporting the development of novel agents targeting B cell receptor signaling, phosphoinositol-3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) oncogenic pathways. Furthermore, we discuss new data on targeting chromatin modulating mechanisms.

Mixed lineage leukemia 1 (MLL1) is a gene that is disrupted by chromosomal translocation characteristically in a large proportion of infant leukemia and also in a fraction of childhood and adult leukemia. MLL1 encodes a chromatin regulatory protein related to the Drosophila Trithorax protein, a well-studied epigenetic factor that functions during development to maintain expression of its target genes. Although tremendous progress has been made understanding the downstream targets of MLL1 fusion oncoproteins and how manipulation of those targets impacts leukemogenesis, very little is known regarding how the initial expression of an MLL1 fusion protein impacts on that cell's behavior, particularly how the cell cycle is affected. Here, we focused on the function of endogenous MLL1 in the stem and progenitor cell types that are likely to be transformed upon MLL1 translocation. Our studies reveal a differential response of stem or progenitor populations to acute loss of MLL1 on proliferation and survival. These data suggest that the effects of MLL1 fusion oncoproteins will initiate the leukemogenic process differentially depending on the differentiation state of the cell type in which the translocation occurs.