Suppressor Components Research Articles

An Advanced Solvent for the Caustic-Side Solvent Extraction of Cesium from Nuclear Waste: Comparing Lipophilic Guanidines for Improved Hydrolytic Stability

ABSTRACT An advanced solvent has been developed for application in the extraction of cesium from alkaline nuclear waste. Four lipophilic guanidines have been compared to arrive at a more hydrolysis-resistant suppressor component of the solvent. Plans call for deployment of the Next-Generation Caustic Side-Solvent Extraction (NG-CSSX) process at industrial scale for separation of radioactive Cs-137 from the legacy salt waste stored at the US Department of Energy Savannah River Site (SRS). In the solvent used in NG-CSSX, an alkyl guanidine “suppressor” component facilitates efficient stripping of the loaded cesium from the solvent. However, as typical of guanidine compounds, the suppressor previously used in the NG-CSSX solvent, N,N‘,N’’-tri(3,7-dimethyloctyl)guanidine (TiDG), suffers from hydrolytic degradation under process conditions. Recently, the sterically hindered alkyl guanidine N,N’-dicyclohexyl-N’’-(10-nonadecyl)guanidine (DCNDG) has been found to offer 8 to 44 times greater hydrolysis resistance than TiDG. Here, the process performance characteristics of the NG-CSSX solvent incorporating DCNDG are determined in comparison with TiDG and two other guanidine suppressors. The solvent has been adjusted in density to accommodate use in the SRS Salt Waste Processing Facility and tested under aggressive bench-scale conditions intended to increase plant throughput. Experiments include the effect of ageing at normal and off-normal temperatures of the distribution of Cs+ through the NG-CSSX process, the fate and effects of guanidine degradation products, suppressor capacity, coalescence times for aqueous-solvent dispersions, tendency for emulsification, third-phase formation, and degree of protonation of the guanidine in stripping. While the chosen structures of the four compared guanidines mainly effect differences in stability, subtle differences in other system properties such as emulsion formation and suppressor capacity provide insight into the function of this important solvent component. In comparison with TiDG and two other guanidines, DCNDG provides greatly increased stability while not compromising the excellent functional properties of the NG-CSSX solvent.

Integrated genomic sequencing in myeloid blast crisis chronic myeloid leukemia (MBC-CML), identified potentially important findings in the context of leukemogenesis model

Chronic myeloid leukemia (CML) is a model of leukemogenesis in which the exact molecular mechanisms underlying blast crisis still remained unexplored. The current study identified multiple common and rare important findings in myeloid blast crisis CML (MBC-CML) using integrated genomic sequencing, covering all classes of genes implicated in the leukemogenesis model. Integrated genomic sequencing via Whole Exome Sequencing (WES), Chromosome-seq and RNA-sequencing were conducted on the peripheral blood samples of three CML patients in the myeloid blast crisis. An in-house filtering pipeline was applied to assess important variants in cancer-related genes. Standard variant interpretation guidelines were used for the interpretation of potentially important findings (PIFs) and potentially actionable findings (PAFs). Single nucleotide variation (SNV) and small InDel analysis by WES detected sixteen PIFs affecting all five known classes of leukemogenic genes in myeloid malignancies including signaling pathway components (ABL1, PIK3CB, PTPN11), transcription factors (GATA2, PHF6, IKZF1, WT1), epigenetic regulators (ASXL1), tumor suppressor and DNA repair genes (BRCA2, ATM, CHEK2) and components of spliceosome (PRPF8). These variants affect genes involved in leukemia stem cell proliferation, self-renewal, and differentiation. Both patients No.1 and No.2 had actionable known missense variants on ABL1 (p.Y272H, p.F359V) and frameshift variants on ASXL1 (p.A627Gfs*8, p.G646Wfs*12). The GATA2-L359S in patient No.1, PTPN11-G503V and IKZF1-R208Q variants in the patient No.3 were also PAFs. RNA-sequencing was used to confirm all of the identified variants. In the patient No. 3, chromosome sequencing revealed multiple pathogenic deletions in the short and long arms of chromosome 7, affecting at least three critical leukemogenic genes (IKZF1, EZH2, and CUX1). The large deletion discovered on the short arm of chromosome 17 in patient No. 2 resulted in the deletion of TP53 gene as well. Integrated genomic sequencing combined with RNA-sequencing can successfully discover and confirm a wide range of variants, from SNVs to CNVs. This strategy may be an effective method for identifying actionable findings and understanding the pathophysiological mechanisms underlying MBC-CML, as well as providing further insights into the genetic basis of MBC-CML and its management in the future.

Local and Targeted Delivery of Immune Checkpoint Blockade Therapeutics

Immune checkpoint blockade (ICB) therapy elicits antitumor response by inhibiting immune suppressor components, including programmed cell death protein 1 and its ligand (PD-1/PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Despite improved therapeutic efficacy, the clinical response rate is still unsatisfactory as revealed by the fact that only a minority of patients experience durable benefits. Additionally, "off-target" effects after systemic administration remain challenging for ICB treatment. To this end, the local and targeted delivery of ICB agents instead could be a potential solution to maximize the therapeutic outcomes while minimizing the side effects.In this Account, our recent studies directed at the development of different strategies for the local and targeted delivery of ICB agents are discussed. For example, transdermal microneedle patches loaded with anti-programmed death-1 antibody (aPD1) and anti-CTLA4 were developed to facilitate sustained release of ICB agents at the diseased sites. Triggered release could also be achieved by various stimuli within the tumor microenvironment, including low pH and abnormally expressed enzymes. Recently, the combination of an anti-programmed death-ligand 1 antibody (aPD-L1) loaded hollow-structured microneedle patch with cold atmospheric plasma (CAP) therapy was also reported. Microneedles provided microchannels to facilitate the transdermal transport of CAP and further induce immunogenic tumor cell death, which could be synergized by the local release of aPD-L1. In addition, in situ formed injectable or sprayable hydrogels were tailored to deliver immunomodulatory antibodies to the surgical bed to inhibit tumor recurrence after primary tumor resection. In paralell, inspired by the unique targeting ability of platelets toward the inflammatory sites, we engineered natural platelets decorated with aPD-L1 for targeted delivery after tumor resection to inhibit tumor recurrence. We further constructed a cell-cell combination delivery platform based on conjugates of platelets and hematopoietic stem cells (HSCs) for leukemia treatment. With the homing ability of HSCs to the bone marrow, the HSC-platelet-aPD1 assembly could effectively deliver aPD1 in an acute myeloid leukemia mouse model. Besides living cells, we also leveraged HEK293T-derived vesicles with PD1 receptors on their surfaces to disrupt the PD-1/PD-L1 immune inhibitory pathway. Moreover, the inner space of the vesicles allowed the packaging of an indoleamine 2,3-dioxygenase inhibitor, further reinforcing the therapeutic efficacy. A similar approach has also been demonstrated by genetically engineering platelets overexpressing PD1 receptor for postsurgical treatment. We hope the local and targeted ICB agent delivery methods introduced in this collection would further inspire the development of advanced drug delivery strategies to improve the efficiency of cancer treatment while alleviating side effects.

Therapeutic Potential of Chemically Modified miR-489 in Triple-Negative Breast Cancers.

Triple-negative breast cancers (TNBCs) lack ER, PR and her2 receptors that are targets of common breast cancer therapies with poor prognosis due to their high rates of metastasis and chemoresistance. Based on our previous studies that epigenetic silencing of a potential metastasis suppressor, arrestin domain-containing 3 (ARRDC3), is linked to the aggressive nature of TNBCs, we identified a sub-group of tumor suppressing miRNAs whose expressions were significantly up-regulated by ARRDC3 over-expression in TNBC cells. Among these tumor suppressing miRs, we found that miR-489 is most anti-proliferative in TNBC cells. miR-489 also blocked DNA damaging responses (DDRs) in TNBC cells. To define the mechanism by which miR-489 inhibits TNBC cell functions, we screened the potential target genes of miR-489 and identified MDC-1 and SUZ-12 as novel target genes of miR-489 in TNBC cells. To further exploit the therapeutic potentials of miR-489 in TNBC models, we chemically modified the guide strand of miR-489 (CMM489) by replacing Uracil with 5-fluorouracil (5-FU) so that tumor suppressor (miR-489) and DNA damaging (5-FU) components are combined into a single agent as a novel drug candidate for TNBCs. Our studies demonstrated that CMM489 shows superior effects over miR-489 or 5-FU in inhibition of TNBC cell proliferation and tumor progression, suggesting its therapeutic efficacy in TNBC models.

Immune landscape in Burkitt lymphoma reveals M2-macrophage polarization and correlation between PD-L1 expression and non-canonical EBV latency program

BackgroundThe Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood.MethodsIn this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis.ResultsOur results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway.ConclusionIn conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.

Effect of naive and cancer-educated fibroblasts on colon cancer cell circadian growth rhythm

Opportunistic modification of the tumour microenvironment by cancer cells enhances tumour expansion and consequently eliminates tumour suppressor components. We studied the effect of fibroblasts on the circadian rhythm of growth and protein expression in colon cancer HCT116 cells and found diminished oscillation in the proliferation of HCT116 cells co-cultured with naive fibroblasts, compared with those co-cultured with tumour-associated fibroblasts (TAFs) or those cultured alone, suggesting that TAFs may have lost or gained factors that regulate circadian phenotypes. Based on the fibroblast paracrine factor analysis, we tested IL6, which diminished HCT116 cell growth oscillation, inhibited early phase cell proliferation, increased early phase expression of the differentiation markers CEA and CDX2, and decreased early phase ERK5 phosphorylation. In conclusion, our data demonstrate how the cancer education of naive fibroblasts influences the circadian parameters of neighbouring cancer cells and highlights a putative role for IL6 as a novel candidate for preoperative treatments.

PLK1 targets NOTCH1 during DNA damage and mitotic progression

Notch signaling plays a complex role in carcinogenesis, and its signaling pathway has both tumor suppressor and oncogenic components. To identify regulators that might control this dual activity of NOTCH1, we screened a chemical library targeting kinases and identified Polo-like kinase 1 (PLK1) as one of the kinases involved in arsenite-induced NOTCH1 down-modulation. As PLK1 activity drives mitotic entry but also is inhibited after DNA damage, we investigated the PLK1-NOTCH1 interplay in the G2 phase of the cell cycle and in response to DNA damage. Here, we found that PLK1 regulates NOTCH1 expression at G2/M transition. However, when cells in G2 phase are challenged with DNA damage, PLK1 is inhibited to prevent entry into mitosis. Interestingly, we found that the interaction between NOTCH1 and PLK1 is functionally important during the DNA damage response, as we found that whereas PLK1 activity is inhibited, NOTCH1 expression is maintained during DNA damage response. During genotoxic stress, cellular transformation requires that promitotic activity must override DNA damage checkpoint signaling to drive proliferation. Interestingly, we found that arsenite-induced genotoxic stress causes a PLK1-dependent signaling response that antagonizes the involvement of NOTCH1 in the DNA damage checkpoint. Taken together, our data provide evidence that Notch signaling is altered but not abolished in SCC cells. Thus, it is also important to recognize that Notch plasticity might be modulated and could represent a key determinant to switch on/off either the oncogenic or tumor suppressor function of Notch signaling in a single type of tumor.

Role of Mitochondria in Ferroptosis

Ferroptosis is a regulated necrosis process driven by iron-dependent lipid peroxidation. Although ferroptosis and cellular metabolism interplay with one another, whether mitochondria are involved in ferroptosis is under debate. Here, we demonstrate that mitochondria play a crucial role in cysteine-deprivation-induced ferroptosis but not in that induced by inhibiting glutathione peroxidase-4 (GPX4), the most downstream component of the ferroptosis pathway. Mechanistically, cysteine deprivation leads to mitochondrial membrane potential hyperpolarization and lipid peroxide accumulation. Inhibition of mitochondrial TCA cycle or electron transfer chain (ETC) mitigated mitochondrial membrane potential hyperpolarization, lipid peroxide accumulation, and ferroptosis. Blockage of glutaminolysis had the same inhibitory effect, which was counteracted by supplying downstream TCA cycle intermediates. Importantly, loss of function of fumarate hydratase, a tumor suppressor and TCA cycle component, confers resistance to cysteine-deprivation-induced ferroptosis. Collectively, this work demonstrates the crucial role of mitochondria in cysteine-deprivation-induced ferroptosis and implicates ferroptosis in tumor suppression.

Identification and Functional Characterization of Anti-metastasis and Anti-angiogenic Activities of Triethylene Glycol Derivatives.

We had previously reported anticancer activity in the water extract (WEX) of Ashwagandha leaves, and identified Triethylene glycol (TEG) as an active tumor suppressor component. In this study, we investigated anti-migratory and anti-angiogenesis activities of WEX and TEG. We conducted in vitro and in vivo experiments using TEG, and its two derivatives, Triethyleneglycol dimethacrylate (TD-10), and Tetraethyleneglycol dimethacrylate (TD-11). The data revealed strong anticancer and anti-metastasis potentials in the derivatives. Non-toxic, anti-migratory doses of the derivatives showed inhibition of canonical Wnt/β-catenin axis and consequent downregulation of EMT-signaling proteins (Vimentin, MMPs and VEGF). These results endorse that the TD-10 and TD-11 have potential to safely put a check on the aggressiveness of the metastatic cells and therefore represent promising candidates for the treatment of metastatic cancers.

Peripheral Immunotype Correlates with Minimal Residual Disease Status and Is Modulated by Immunomodulatory Drugs in Multiple Myeloma

Data indicate reversal of immune dysfunction with active treatment; however, the precise contribution of specific immune effector and immune suppressor components to achieve a minimal residual disease (MRD) state and immunomodulatory drug–mediated immunomodulatory effects in multiple myeloma (MM) patients remains poorly understood. In this prospective proof-of-principle study we sought to determine the dynamic alterations in natural killer (NK), NK-T, and T cells, including maturation and activating/inhibitory repertoire associated with MRDpos versus MRDneg status after autologous stem cell transplantation (ASCT) and during lenalidomide-based maintenance therapy. Of the 46MM patients enrolled, 36 had bone marrow MRD assessment 60+ days post-ASCT, 30 had longitudinal blood immunotyping during maintenance (pretherapy and after cycles 1, 3, and 6), and 20 had both MRD assessment and longitudinal immunotyping. Multicolor flow cytometry was used for MRD and immunotyping. Although the absolute number of NK cells was significantly lower in patients with MRDpos response, phenotypically NK cells in these patients displayed higher expression of activating receptors KIRDS4 and decreased expression of inhibitory molecules NKG2A compared with the MRDneg group. Furthermore, we observed significantly lower frequencies of T cells displaying KIR3DL1 in MRDpos versus MRDneg patients. Longitudinal immunotyping during lenalidomide maintenance showed loss of mature NK effector function, augmentation of NK-T effector function, and acquisition of PD1 independent anergic state. Our findings also suggest skewing of T cells toward an exhausted state during the maintenance phase in MRDpos patients. Put together, these observations provide a distinctive signature for MRDneg and MRDpos groups. These data support exploration of immune profiling in prospective clinical trials according to MRD-defined responses to identify patients that may benefit from maintenance intensification/modification or maintenance withdrawal.

Potential of Central, Eastern and Western Africa Medicinal Plants for Cancer Therapy: Spotlight on Resistant Cells and Molecular Targets.

Cancer remains a major health hurdle worldwide and has moved from the third leading cause of death in the year 1990 to second place after cardiovascular disease since 2013. Chemotherapy is one of the most widely used treatment modes; however, its efficiency is limited due to the resistance of cancer cells to cytotoxic agents. The present overview deals with the potential of the flora of Central, Eastern and Western African (CEWA) regions as resource for anticancer drug discovery. It also reviews the molecular targets of phytochemicals of these plants such as ABC transporters, namely P-glycoprotein (P-gp), multi drug-resistance-related proteins (MRPs), breast cancer resistance protein (BCRP, ABCG2) as well as the epidermal growth factor receptor (EGFR/ErbB-1/HER1), human tumor suppressor protein p53, caspases, mitochondria, angiogenesis, and components of MAP kinase signaling pathways. Plants with the ability to preferentially kills resistant cancer cells were also reported. Data compiled in the present document were retrieved from scientific websites such as PubMed, Scopus, Sciencedirect, Web-of-Science, and Scholar Google. In summary, plant extracts from CEWA and isolated compounds thereof exert cytotoxic effects by several modes of action including caspases activation, alteration of mitochondrial membrane potential (MMP), induction of reactive oxygen species (ROS) in cancer cells and inhibition of angiogenesis. Ten strongest cytotoxic plants from CEWA recorded following in vitro screening assays are: Beilschmiedia acuta Kosterm, Echinops giganteus var. lelyi (C. D. Adams) A. Rich., Erythrina sigmoidea Hua (Fabaceae), Imperata cylindrical Beauv. var. koenigii Durand et Schinz, Nauclea pobeguinii (Pobég. ex Pellegr.) Merr. ex E.M.A., Piper capense L.f., Polyscias fulva (Hiern) Harms., Uapaca togoensis Pax., Vepris soyauxii Engl. and Xylopia aethiopica (Dunal) A. Rich. Prominent antiproliferative compounds include: isoquinoline alkaloid isotetrandrine (51), two benzophenones: guttiferone E (26) and isoxanthochymol (30), the isoflavonoid 6α-hydroxyphaseollidin (9), the naphthyl butenone guieranone A (25), two naphthoquinones: 2-acetylfuro-1,4-naphthoquinone (4) and plumbagin (37) and xanthone V1 (46). However, only few research activities in the African continent focus on cytotoxic drug discovery from botanicals. The present review is expected to stimulate further scientific efforts to better valorize the African flora.

Targeting the RB-E2F pathway in breast cancer.

Mutations of the retinoblastoma tumor suppressor gene (RB1) or components regulating the CDK-RB-E2F pathway have been identified in nearly every human malignancy. Re-establishing cell cycle control through CDK inhibition has therefore emerged as an attractive option in the development of targeted cancer therapy. The most successful example of this today is the use of the CDK4/6 inhibitor palbociclib combined with aromatase inhibitors for the treatment of estrogen receptor-positive breast cancers. Multiple studies have demonstrated that the CDK-RB-E2F pathway is critical for the control of cell proliferation. More recently, studies have highlighted additional roles of this pathway, especially E2F transcription factors themselves, in tumor progression, angiogenesis and metastasis. Specific E2Fs also have prognostic value in breast cancer, independent of clinical parameters. We discuss here recent advances in understanding of the RB-E2F pathway in breast cancer. We also discuss the application of genome-wide genetic screening efforts to gain insight into synthetic lethal interactions of CDK4/6 inhibitors in breast cancer for the development of more effective combination therapies.

The tumor suppressor SHIP1 colocalizes in nucleolar cavities with p53 and components of PML nuclear bodies

The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in haematopoietic cells. By converting PI(3,4,5)P3 to PtdIns(3,4)P2 at the plasma membrane, SHIP1 modifies PI3-kinase mediated signaling. We have recently demonstrated that SHIP1 is a nucleo-cytoplasmic shuttling protein and SHIP1 nuclear puncta partially colocalize with FLASH, a component of nuclear bodies. In this study, we demonstrate that endogenous SHIP1 localizes to intranucleolar regions of both normal and leukemic haematopoietic cells. In addition, we report that ectopically expressed SHIP1 accumulates in nucleolar cavities and colocalizes with the tumor suppressor protein p53 and components of PML nuclear bodies (e.g. SP100, SUMO-1 and CK2). Moreover, SHIP1 also colocalizes in nucleolar cavities with components of the ubiquitin-proteasome pathway. By using confocal microscopy data, we generated 3D-models revealing the enormous extent of the SHIP1 aggresomes in the nucleolus. Furthermore, treatment of cells with the proteasome inhibitor MG132 causes an enlargement of nucleolar SHIP1 containing structures. Unexpectedly, this accumulation can be partially prevented by treatment with the inhibitor of nuclear protein export Leptomycin B. In recent years, several proteins aggregating in nucleolar cavities were shown to be key factors of neurodegenerative diseases and cancerogenesis. Our findings support current relevance of nuclear localized SHIP1.

The Hippo signaling pathway in liver regeneration and tumorigenesis

The Hippo signaling pathway is an evolutionarily conserved signaling module that plays critical roles in liver size control and tumorigenesis. The Hippo pathway consists of a core kinase cascade in which the mammalian Ste20-like kinases (Mst1/2, orthologs of Drosophila Hippo) and their cofactor Salvador (Sav1) form a complex to phosphorylate and activate the large tumor suppressor (Lats1/2). Lats1/2 kinases in turn phosphorylate and inhibit the transcription co-activators, the Yes-associated protein (YAP) and the transcriptional co-activator with PDZ-binding motif (TAZ), two major downstream effectors of the Hippo pathway. Losses of the Hippo pathway components induce aberrant hepatomegaly and tumorigenesis, in which YAP coordinates regulation of cell proliferation and apoptosis and plays an essential role. This review summarizes the current findings of the regulation of Hippo signaling in liver regeneration and tumorigenesis, focusing on how the loss of tumor suppressor components of the Hippo pathway results in liver cancers and discussing the molecular mechanisms that regulate the expression and activation of its downstream effector YAP in liver tumorigenesis.

Low Doses of Cisplatin can Help Dendritic Cell Vaccines to Reduce Immunosuppression in Tumor Microenvironment

ABSTRACT Introduction Lack of effectiveness of most immunotherapy methods requires new approaches. One of the most promising approaches, intensively investigated worldwide, is combined therapy based on dendritic cells (DC) and low-dose chemotherapy. The using of low-dose chemotherapy reduces the tumor's suppressor component, which in turn decreases the suppression of the immune system of cancer patients. The aim: to investigate the effect of chemo-immunotherapy on the suppression component of the immune system in murine sarcoma-37 (S37) model. Methods In experimental investigation 80 CBA mice have been involved. S37 was injected intramuscularly at lethal dose (2*106 cells per animal). Cisplatin was administered intraperitoneally five times according to the two schemes: 0.2 or 2 mg/kg on the 7th day after tumor transplantation with interval of 1 day and 3 days, respectively. DC vaccines were administered intravenously 3 times on 4 day after the chemotherapy with 3 days interval. Results We have found that both of the proposed chemoimmunotherapy schemes had a significant antitumor and immunomodulation effect. The most pronounced effect was observed when used DC-vaccine and cisplatin at dose of 2 mg/kg in combination. Combination of DC vaccine and 2 mg/kg cisplatin had a synergistic effect: significantly decreasing of primary tumor volume in animals compared to the control (p = 0.001) and DC-vaccine (p = 0.007) groups have been found. We have shown that administration of DC vaccine and 2 mg/kg cispatin decreases the TGF-b mRNA expression level in the tumor microenvironment (р = 0.0013) compared with the control group. Moreover, the administration of this combined therapy decrease VEGF mRNA expression level in tumor microenvironment in 2 times (р = 0.005) compared with the control group. Conclusions Low-dose cisplatin in combination with DC-vaccine reduces immunosuppression and neoangiogenesis in the tumor microenvironment. Disclosure All authors have declared no conflicts of interest.

Alcohol Interacts with Genetic Alteration of the Hippo Tumor Suppressor Pathway to Modulate Tissue Growth in Drosophila

Alcohol-mediated cancers represent more than 3.5% of cancer-related deaths, yet how alcohol promotes cancer is a major open question. Using Drosophila, we identified novel interactions between dietary ethanol and loss of tumor suppressor components of the Hippo Pathway. The Hippo Pathway suppresses tumors in flies and mammals by inactivating transcriptional co-activator Yorkie, and the spectrum of cancers associated with impaired Hippo signaling overlaps strikingly with those associated with alcohol. Therefore, our findings may implicate loss of Hippo Pathway tumor suppression in alcohol-mediated cancers. Ethanol enhanced overgrowth from loss of the expanded, hippo, or warts tumor suppressors but, surprisingly, not from over-expressing the yorkie oncogene. We propose that in parallel to Yorkie-dependent overgrowth, impairing Hippo signaling in the presence of alcohol may promote overgrowth via additional alcohol-relevant targets. We also identified interactions between alcohol and Hippo Pathway over-activation. We propose that exceeding certain thresholds of alcohol exposure activates Hippo signaling to maintain proper growth control and prevent alcohol-mediated mis-patterning and tissue overgrowth.

MiR-221/222 is the regulator of Cx43 expression in human glioblastoma cells

The miR-221/222 cluster is significantly upregulated in malignant glioma cells and regulates the expression of multiple genes associated with glioma cell proliferation, invasion and apoptosis, which was shown in our previous studies. Cx43 has been identified as a tumor suppressor and major component for the establishment of gap junction intercellular communication (GJIC) in glial cells, which is frequently reduced or deleted in high-grade gliomas. According to bioinformatic analysis, connexin 43 (Cx43) may be one of the target genes of miR-221/222. The aim of the present study was to validate Cx43 as a target gene of miR-221/222 and to determine whether overexpression of miR-221/222 is one of the molecular mechanisms for the reduced expression of Cx43 in malignant gliomas. We transfected miR-221/222 antisense oligonucleotides (AS-miR-221/222) into U251 human glioblastoma cells using a lipofectamine method. Northern blot analysis was conducted to detect the expression of the miR-221/222 cluster. Luciferase reporter assays were exploited to confirm Cx43 as a target gene of miR-221/222. Cx43 expression was assessed by western blotting and immunofluorescence staining. Scrape loading and dye transfer (SLDT) assays were used for examination of GJIC. Proliferation and invasion of U251 cells were evaluated by MTT and transwell assays, respectively. Cell cycle kinetics and apoptosis were determined with flow cytometry. We found that expression of the miR-221/222 cluster was significantly reduced while Cx43 expression was upregulated in U251 cells transfected with AS-miR-221/222, and the GJIC deficiency in parental U251 cells was re-established. Moreover, the luciferase activity determined by the luciferase reporter assay was enhanced in AS-miR-221/222-treated cells, and cell proliferation and invasion were suppressed while apoptosis was induced. We conclude that miR-221/222 function as oncogenic microRNAs in human gliomas, at least in part, by targeting Cx43.

Cbk1 kinase and Bck2 control MAP kinase activation and inactivation during heat shock

Saccharomyces cerevisiae Cbk1 kinase is a LATS/NDR tumor suppressor orthologue and component of the Regulation of Ace2 and Morphogenesis signaling network. Cbk1 was previously implicated in regulating polarized morphogenesis, gene expression, and cell integrity. Here we establish that Cbk1 is critical for heat shock and cell wall stress signaling via Bck2, a protein associated with the Pkc1-Mpk1 cell integrity pathway. We demonstrate that cbk1 and bck2 loss-of-function mutations prevent Mpk1 kinase activation and Mpk1-dependent gene expression but do not disrupt Mpk1 Thr-190/Tyr-192 phosphorylation. Bck2 overexpression partially restores Mpk1-dependent Rlm1 transcription factor activity in cbk1 mutants, suggesting that Bck2 functions downstream of Cbk1. We demonstrate that Bck2 precisely colocalizes with the mitogen-activated protein kinase (MAPK) phosphatase Sdp1. During heat shock, Bck2 and Sdp1 transiently redistribute from nuclei and the cytosol to mitochondria and other cytoplasmic puncta before returning to their pre-stressed localization patterns. Significantly, Cbk1 inhibition delays the return of Bck2 and Sdp1 to their pre-stressed localization patterns and delays Mpk1 Thr-190/Tyr-192 dephosphorylation upon heat shock adaptation. We conclude that Cbk1 and Bck2 are required for Mpk1 activation during heat shock and cell wall stress and for Mpk1 dephosphorylation during heat shock adaptation. These data provide the first evidence that Cbk1 kinase regulates MAPK-dependent stress signaling and provide mechanistic insight into Sdp1 phosphatase regulation.

Silencing of TESTIN by dense biallelic promoter methylation is the most common molecular event in childhood acute lymphoblastic leukaemia

BackgroundAberrant promoter DNA methylation has been reported in childhood acute lymphoblastic leukaemia (ALL) and has the potential to contribute to its onset and outcome. However, few reports demonstrate consistent, prevalent and dense promoter methylation, associated with tumour-specific gene silencing. By screening candidate genes, we have detected frequent and dense methylation of the TESTIN (TES) promoter.ResultsBisulfite sequencing showed that 100% of the ALL samples (n = 20) were methylated at the TES promoter, whereas the matched remission (n = 5), normal bone marrow (n = 6) and normal PBL (n = 5) samples were unmethylated. Expression of TES in hyperdiploid, TEL-AML+, BCR-ABL+, and E2A-PBX+ subtypes of B lineage ALL was markedly reduced compared to that in normal bone marrow progenitor cells and in B cells. In addition TES methylation and silencing was demonstrated in nine out of ten independent B ALL propagated as xenografts in NOD/SCID mice.ConclusionIn total, 93% of B ALL samples (93 of 100) demonstrated methylation with silencing or reduced expression of the TES gene. Thus, TES is the most frequently methylated and silenced gene yet reported in ALL. TES, a LIM domain-containing tumour suppressor gene and component of the focal adhesion complex, is involved in adhesion, motility, cell-to-cell interactions and cell signalling. Our data implicate TES methylation in ALL and provide additional evidence for the involvement of LIM domain proteins in leukaemogenesis.

Emerging roles of E2Fs in cancer: an exit from cell cycle control

Mutations of the retinoblastoma tumour suppressor gene (RB1) or components regulating the RB pathway have been identified in almost every human malignancy. The E2F transcription factors function in cell cycle control and are intimately regulated by RB. Studies of model organisms have revealed conserved functions for E2Fs during development, suggesting that the cancer-related proliferative roles of E2F family members represent a recent evolutionary adaptation. However, given that some human tumours have concurrent RB1 inactivation and E2F amplification and overexpression, we propose that there are alternative tumour-promoting activities for the E2F family, which are independent of cell cycle regulation.