64 results on '"Nikiforov MA"'
Search Results
2. PP2A-B56α controls oncogene-induced senescence in normal and tumor human melanocytic cells
- Author
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Mannava, S, Omilian, AR, Wawrzyniak, JA, Fink, EE, Zhuang, D, Miecznikowski, JC, Marshall, JR, Soengas, MS, Sears, RC, Morrison, CD, and Nikiforov, MA
- Published
- 2012
- Full Text
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3. Cell-Based Methods for the Identification of MYC-Inhibitory Small Molecules
- Author
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Soucek, L, Sodir, NM, Norris, MD, Burkhart, CA, Haber, M, Gudkov, AV, Nikiforov, MA, Soucek, L, Sodir, NM, Norris, MD, Burkhart, CA, Haber, M, Gudkov, AV, and Nikiforov, MA
- Abstract
In 'The Myc Gene: Methods and Protocols',experts in the field summarize the standard and novel techniques that allow the studying of Myc mechanism of action in normal and cancer cells, in vitro and in vivo.
- Published
- 2013
4. Distinct inflammatory Th17 subsets emerge in autoimmunity and infection.
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Bouch RJ, Zhang J, Miller BC, Robbins CJ, Mosher TH, Li W, Krupenko SA, Nagpal R, Zhao J, Bloomfeld RS, Lu Y, Nikiforov MA, Song Q, and He Z
- Subjects
- Humans, Autoimmunity, Th17 Cells, Inflammation, Autoimmune Diseases, Colitis
- Abstract
Th17 cells play a critical role in both tissue homeostasis and inflammation during clearance of infections as well as autoimmune and inflammatory disorders. Despite numerous efforts to distinguish the homeostatic and inflammatory roles of Th17 cells, the mechanism underlying the divergent functions of inflammatory Th17 cells remains poorly understood. In this study, we demonstrate that the inflammatory Th17 cells involved in autoimmune colitis and those activated during colitogenic infection are distinguishable populations characterized by their differential responses to the pharmacological molecule, clofazimine (CLF). Unlike existing Th17 inhibitors, CLF selectively inhibits proautoimmune Th17 cells while preserving the functional state of infection-elicited Th17 cells partially by reducing the enzyme ALDH1L2. Overall, our study identifies two distinct subsets within the inflammatory Th17 compartment with distinct regulatory mechanisms. Furthermore, we highlight the feasibility to develop disease-promoting Th17 selective inhibitor for treating autoimmune diseases., (© 2023 Bouch et al.)
- Published
- 2023
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5. Compartmentalization and regulation of GTP in control of cellular phenotypes.
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Wolff DW, Bianchi-Smiraglia A, and Nikiforov MA
- Subjects
- Humans, Phenotype, Guanosine Triphosphate
- Abstract
Genetic or pharmacological inhibition of enzymes involved in GTP biosynthesis has substantial biological effects, underlining the need to better understand the function of GTP levels in regulation of cellular processes and the significance of targeting GTP biosynthesis enzymes for therapeutic intervention. Our current understanding of spatiotemporal regulation of GTP metabolism and its role in physiological and pathological cellular processes is far from complete. Novel methodologies such as genetically encoded sensors of free GTP offered insights into intracellular distribution and function of GTP molecules. In the current Review, we provide analysis of recent discoveries in the field of GTP metabolism and evaluate the key enzymes as molecular targets., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
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- 2022
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6. Phosphorylation of guanosine monophosphate reductase triggers a GTP-dependent switch from pro- to anti-oncogenic function of EPHA4.
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Wolff DW, Deng Z, Bianchi-Smiraglia A, Foley CE, Han Z, Wang X, Shen S, Rosenberg MM, Moparthy S, Yun DH, Chen J, Baker BK, Roll MV, Magiera AJ, Li J, Hurley E, Feltri ML, Cox AO, Lee J, Furdui CM, Liu L, Bshara W, LaConte LEW, Kandel ES, Pasquale EB, Qu J, Hedstrom L, and Nikiforov MA
- Subjects
- GMP Reductase genetics, GMP Reductase metabolism, Guanosine Triphosphate metabolism, Humans, Nucleotides metabolism, Phosphorylation, Melanoma metabolism, Receptor, EphA4 metabolism
- Abstract
Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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7. Assessment of Intracellular GTP Levels Using Genetically Encoded Fluorescent Sensors.
- Author
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Bianchi-Smiraglia A and Nikiforov MA
- Subjects
- Cell Movement physiology, Guanosine Triphosphate metabolism, Fluorescent Dyes chemistry, rhoA GTP-Binding Protein metabolism
- Abstract
Changes in intracellular GTP levels, even incremental ones, profoundly affect the activity of several GTP-binding proteins ultimately resulting in alteration of several basal cellular phenotypes including cell motility, invasion, and tumorigenesis. However, until recently, no tools were available for GTP quantification in live cells. Therefore, in the current chapter, we describe the methodology for the quantitative assessment of spatiotemporal changes in GTP levels in the cells using genetically encoded fluorescent ratiometric GTP sensors termed GEVALs for GTP evaluators., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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8. Author Correction: Regulation of local GTP availability controls RAC1 activity and cell invasion.
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Bianchi-Smiraglia A, Wolff DW, Marston DJ, Deng Z, Han Z, Moparthy S, Wombacher RM, Mussell AL, Shen S, Chen J, Yun DH, O'Brien Cox A, Furdui CM, Hurley E, Feltri ML, Qu J, Hollis T, Kengne JBN, Fongang B, Sousa RJ, Kandel ME, Kandel ES, Hahn KM, and Nikiforov MA
- Published
- 2021
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9. Regulation of local GTP availability controls RAC1 activity and cell invasion.
- Author
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Bianchi-Smiraglia A, Wolff DW, Marston DJ, Deng Z, Han Z, Moparthy S, Wombacher RM, Mussell AL, Shen S, Chen J, Yun DH, O'Brien Cox A, Furdui CM, Hurley E, Feltri ML, Qu J, Hollis T, Kengne JBN, Fongang B, Sousa RJ, Kandel ME, Kandel ES, Hahn KM, and Nikiforov MA
- Subjects
- Cell Membrane metabolism, Cell Movement, Guanosine Triphosphate chemistry, HEK293 Cells, Humans, IMP Dehydrogenase genetics, IMP Dehydrogenase metabolism, Kinetics, Protein Binding, rac1 GTP-Binding Protein chemistry, rac1 GTP-Binding Protein genetics, Guanosine Triphosphate metabolism, rac1 GTP-Binding Protein metabolism
- Abstract
Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells., (© 2021. The Author(s).)
- Published
- 2021
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10. PHF10 subunit of PBAF complex mediates transcriptional activation by MYC.
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Soshnikova NV, Tatarskiy EV, Tatarskiy VV, Klimenko NS, Shtil AA, Nikiforov MA, and Georgieva SG
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- Cell Line, Tumor, Cellular Senescence, Chromatin Assembly and Disassembly, Disease Progression, G1 Phase Cell Cycle Checkpoints, Gene Expression Regulation, Neoplastic, Homeodomain Proteins metabolism, Humans, Melanoma metabolism, Neoplasm Metastasis, Neoplasm Proteins metabolism, Promoter Regions, Genetic, Proto-Oncogene Proteins c-myc metabolism, Sequence Analysis, RNA, Transcriptional Activation, Up-Regulation, Gene Expression Profiling methods, Gene Regulatory Networks, Homeodomain Proteins genetics, Melanoma genetics, Neoplasm Proteins genetics, Proto-Oncogene Proteins c-myc genetics
- Abstract
The PBAF complex, a member of SWI/SNF family of chromatin remodelers, plays an essential role in transcriptional regulation. We revealed a disease progression associated elevation of PHF10 subunit of PBAF in clinical melanoma samples. In melanoma cell lines, PHF10 interacts with MYC and facilitates the recruitment of PBAF complex to target gene promoters, therefore, augmenting MYC transcriptional activation of genes involved in the cell cycle progression. Depletion of either PHF10 or MYC induced G1 accumulation and a senescence-like phenotype. Our data identify PHF10 as a pro-oncogenic mechanism and an essential novel link between chromatin remodeling and MYC-dependent gene transcription., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2021
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11. The fatty acid elongase ELOVL6 regulates bortezomib resistance in multiple myeloma.
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Lipchick BC, Utley A, Han Z, Moparthy S, Yun DH, Bianchi-Smiraglia A, Wolff DW, Fink E, Liu L, Furdui CM, Lee J, Lee KP, and Nikiforov MA
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- Animals, Bortezomib pharmacology, Cell Line, Tumor, Drug Resistance, Neoplasm, Fatty Acid Elongases, Humans, Mice, Multiple Myeloma drug therapy, Multiple Myeloma genetics
- Abstract
Resistance to the proteasome inhibitor bortezomib (BTZ) represents a major obstacle in the treatment of multiple myeloma (MM). The contribution of lipid metabolism in the resistance of MM cells to BTZ is mostly unknown. Here we report that levels of fatty acid elongase 6 (ELOVL6) were lower in MM cells from BTZ-nonresponsive vs BTZ-responsive patients and in cultured MM cells selected for BTZ resistance compared with parental counterparts. Accordingly, depletion of ELOVL6 in parental MM cells suppressed BTZ-induced endoplasmic reticulum (ER) stress and cytotoxicity, whereas restoration of ELOVL6 levels in BTZ-resistant MM cells sensitized them to BTZ in tissue culture settings and, as xenografts, in a plasmacytoma mouse model. Furthermore, for the first time, we identified changes in the BTZ-induced lipidome between parental and BTZ-resistant MM cell lines underlying a functional difference in their response to BTZ. We demonstrated that restoration of ELOVL6 levels in BTZ-resistant MM cells resensitized them to BTZ largely via upregulation of ELOVL6-dependent ceramide species, which was a prerequisite for BTZ-induced ER stress and cell death in these cells. Our data characterize ELOVL6 as a major clinically relevant regulator of MM cell resistance to BTZ, which can emerge from the impaired ability of these cells to alter ceramide composition in response to BTZ., (© 2021 by The American Society of Hematology.)
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- 2021
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12. Cell-Based Methods for the Identification of Myc-Inhibitory Small Molecules.
- Author
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Burkhart CA, Haber M, Norris MD, Gudkov AV, and Nikiforov MA
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- Cell Line, Tumor, Genes, myc genetics, Genes, myc physiology, Humans, Oncogene Proteins drug effects, Oncogene Proteins metabolism, Proto-Oncogene Proteins c-myc genetics, Proto-Oncogene Proteins c-myc metabolism, Small Molecule Libraries pharmacology, Transcription Factors drug effects, Transcription Factors metabolism, Drug Screening Assays, Antitumor methods, Genes, myc drug effects, Proto-Oncogene Proteins c-myc antagonists & inhibitors
- Abstract
Oncoproteins encoded by dominant oncogenes have long been considered as targets for chemotherapeutic intervention. However, oncogenic transcription factors have often been dismissed as "undruggable." Members of the Myc family of transcription factors have been identified as promising targets for cancer chemotherapy in multiple publications reporting the requirement of Myc proteins for maintenance of almost every type of tumor. Here, we describe cell-based approaches to identify c-Myc small molecule inhibitors by screening complex libraries of diverse small molecules based on Myc functionality and specificity.
- Published
- 2021
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13. Novel combination therapy for melanoma induces apoptosis via a gap junction positive feedback mechanism.
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Bagati A, Hutcherson TC, Koch Z, Pechette J, Dianat H, Higley C, Chiu L, Song Y, Shah J, Chazen E, Nicolais A, Casey P, Thompson K, Burke K, Nikiforov MA, Zirnheld J, and Zucker SN
- Abstract
Metastatic melanoma cells overexpressing gap junctions were assayed for their ability to propagate cell death by a novel combination therapy that generates reactive oxygen species (ROS) by both 1) non-thermal plasma (NTP) and 2) tirapazamine (TPZ) under hypoxic conditions. Results demonstrate additive-to-synergistic effects of combination therapy compared to each agent individually. NTP induces highly localized cell death in target areas whereas TPZ partially reduces viability over the total surface area. However, when high gap junction expression was induced in melanoma cells, effects of combination NTP+TPZ therapy was augmented, spreading cell death across the entire plate. Similarly, in vivo studies of human metastatic melanoma in a mouse tumor model demonstrate that the combined effect of NTP+TPZ causes a 90% reduction in tumor volume, specifically in the model expressing gap junctions. Treatment with NTP+TPZ increases gene expression in the apoptotic pathway and oxidative stress while decreasing genes related to cell migration. Immune response was also elicited through differential regulation of cytokines and chemokines, suggesting potential for this therapy to induce a cytotoxic immune response with fewer side effects than current therapies. Interestingly, the gap junction protein, Cx26 was upregulated following treatment with NTP+TPZ and these gap junctions were shown to maintain functionality during the onset of treatment. Therefore, we propose that gap junctions both increase the efficacy of NTP+TPZ and perpetuate a positive feedback mechanism of gap junction expression and tumoricidal activity. Our unique approach to ROS induction in tumor cells with NTP+TPZ shows potential as a novel cancer treatment., Competing Interests: CONFLICTS OF INTEREST The patent described in this manuscript (number 9,586,056) is the property of Haniva, LLC, of which SNZ is the President and holds equity. TCH holds equity and is the Secretary of Haniva Technology, LLC, a subsidiary of Haniva, LLC. All remaining authors declare no competing interests.
- Published
- 2020
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14. Targeting Multiple Myeloma through the Biology of Long-Lived Plasma Cells.
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Utley A, Lipchick B, Lee KP, and Nikiforov MA
- Abstract
Multiple myeloma (MM) is a hematological malignancy of terminally differentiated bone marrow (BM) resident B lymphocytes known as plasma cells (PC). PC that reside in the bone marrow include a distinct population of long-lived plasma cells (LLPC) that have the capacity to live for very long periods of time (decades in the human population). LLPC biology is critical for understanding MM disease induction and progression because MM shares many of the same extrinsic and intrinsic survival programs as LLPC. Extrinsic survival signals required for LLPC survival include soluble factors and cellular partners in the bone marrow microenvironment. Intrinsic programs that enhance cellular fidelity are also required for LLPC survival including increased autophagy, metabolic fitness, the unfolded protein response (UPR), and enhanced responsiveness to endoplasmic reticulum (ER) stress. Targeting LLPC cell survival mechanisms have led to standard of care treatments for MM including proteasome inhibition (Bortezomib), steroids (Dexamethasone), and immunomodulatory drugs (Lenalidomide). MM patients that relapse often do so by circumventing LLPC survival pathways targeted by treatment. Understanding the mechanisms by which LLPC are able to survive can allow us insight into the treatment of MM, which allows for the enhancement of therapeutic strategies in MM both at diagnosis and upon patient relapse.
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- 2020
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15. Pharmacological polyamine catabolism upregulation with methionine salvage pathway inhibition as an effective prostate cancer therapy.
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Affronti HC, Rowsam AM, Pellerite AJ, Rosario SR, Long MD, Jacobi JJ, Bianchi-Smiraglia A, Boerlin CS, Gillard BM, Karasik E, Foster BA, Moser M, Wilton JH, Attwood K, Nikiforov MA, Azabdaftari G, Pili R, Phillips JG, Casero RA Jr, and Smiraglia DJ
- Subjects
- Acetyltransferases genetics, Acetyltransferases metabolism, Adenine administration & dosage, Adenine analogs & derivatives, Animals, Apoptosis, Cell Line, Tumor, Drug Therapy, Combination, Humans, Male, Mice, Mice, Inbred BALB C, Prostatic Neoplasms enzymology, Prostatic Neoplasms genetics, Prostatic Neoplasms metabolism, Purine-Nucleoside Phosphorylase genetics, Purine-Nucleoside Phosphorylase metabolism, Pyrrolidines administration & dosage, Salvage Therapy, Spermine administration & dosage, Spermine analogs & derivatives, Spermine metabolism, Methionine metabolism, Polyamines metabolism, Prostatic Neoplasms drug therapy
- Abstract
Prostatic luminal epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen, sensitizing them to perturbations of connected metabolic pathways. Enhanced flux is driven by spermidine/spermine N1-acetyltransferase (SSAT) activity, which acetylates polyamines leading to their secretion and drives biosynthetic demand. The methionine salvage pathway recycles one-carbon units lost to polyamine biosynthesis to the methionine cycle to overcome stress. Prostate cancer (CaP) relies on methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme, to relieve strain. Here, we show that inhibition of MTAP alongside SSAT upregulation is synergistic in androgen sensitive and castration recurrent CaP models in vitro and in vivo. The combination treatment increases apoptosis in radical prostatectomy ex vivo explant samples. This unique high metabolic flux through polyamine biosynthesis and connected one carbon metabolism in CaP creates a metabolic dependency. Enhancing this flux while simultaneously targeting this dependency in prostate cancer results in an effective therapeutic approach potentially translatable to the clinic.
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- 2020
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16. The role of polo-like kinase 3 in the response of BRAF-mutant cells to targeted anticancer therapies.
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Babagana M, Kichina JV, Slabodkin H, Johnson S, Maslov A, Brown L, Attwood K, Nikiforov MA, and Kandel ES
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- Animals, Antineoplastic Agents therapeutic use, Cell Line, Tumor, Gene Expression Regulation, Neoplastic drug effects, Humans, Melanoma genetics, Mice, SCID, Molecular Targeted Therapy, Mutation drug effects, Protein Kinase Inhibitors therapeutic use, Proto-Oncogene Proteins B-raf genetics, Tumor Suppressor Proteins, Vemurafenib therapeutic use, Antineoplastic Agents pharmacology, Melanoma drug therapy, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases genetics, Proto-Oncogene Proteins B-raf antagonists & inhibitors, Vemurafenib pharmacology
- Abstract
The activation of oncogenic mitogen-activated protein kinase cascade via mutations in BRAF is often observed in human melanomas. Targeted inhibitors of BRAF (BRAFi), alone or as a part of a combination therapy, offer a significant benefit to such patients. Unfortunately, some cases are initially nonresponsive to these drugs, while others become refractory in the course of treatment, underscoring the need to understand and mitigate the underlying resistance mechanisms. We report that interference with polo-like kinase 3 (PLK3) reduces the tolerance of BRAF-mutant melanoma cells to BRAFi, while increased PLK3 expression has the opposite effect. Accordingly, PLK3 expression correlates with tolerance to BRAFi in a panel of BRAF-mutant cell lines and is elevated in a subset of recurring BRAFi-resistant melanomas. In PLK3-expressing cells, R406, a kinase inhibitor whose targets include PLK3, recapitulates the sensitizing effects of genetic PLK3 inhibitors. The findings support a role for PLK3 as a predictor of BRAFi efficacy and suggest suppression of PLK3 as a way to improve the efficacy of targeted therapy., (© 2019 Wiley Periodicals, Inc.)
- Published
- 2020
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17. KLF9-dependent ROS regulate melanoma progression in stage-specific manner.
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Bagati A, Moparthy S, Fink EE, Bianchi-Smiraglia A, Yun DH, Kolesnikova M, Udartseva OO, Wolff DW, Roll MV, Lipchick BC, Han Z, Kozlova NI, Jowdy P, Berman AE, Box NF, Rodriguez C, Bshara W, Kandel ES, Soengas MS, Paragh G, and Nikiforov MA
- Subjects
- Acetylcysteine adverse effects, Adult, Aged, Aged, 80 and over, Animals, Humans, Kruppel-Like Transcription Factors genetics, Melanocytes drug effects, Melanocytes metabolism, Melanocytes pathology, Melanoma genetics, Melanoma metabolism, Melanoma, Experimental chemically induced, Melanoma, Experimental metabolism, Melanoma, Experimental pathology, Mice, Knockout, Middle Aged, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins B-raf metabolism, Skin Neoplasms metabolism, Kruppel-Like Transcription Factors metabolism, Melanoma pathology, Reactive Oxygen Species metabolism, Skin Neoplasms pathology
- Abstract
Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: Braf
CA mice, where BrafV600E causes premalignant melanocytic hyperplasia, and BrafCA /Pten-/- mice, where BrafV600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in BrafCA mice but did not affect formation and growth of BrafCA /Pten-/- primary melanomas. It also, as expected, promoted BrafCA /Pten-/- metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in BrafCA and BrafCA /Pten-/- melanocytic cells. Mechanistically, we demonstrated that BRAFV600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAFV600E . PTEN depletion in BRAFV600E -melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAFV600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.- Published
- 2019
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18. XBP1-KLF9 Axis Acts as a Molecular Rheostat to Control the Transition from Adaptive to Cytotoxic Unfolded Protein Response.
- Author
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Fink EE, Moparthy S, Bagati A, Bianchi-Smiraglia A, Lipchick BC, Wolff DW, Roll MV, Wang J, Liu S, Bakin AV, Kandel ES, Lee AH, and Nikiforov MA
- Subjects
- Animals, Endoplasmic Reticulum Stress, Female, HCT116 Cells, HEK293 Cells, Humans, Kruppel-Like Transcription Factors genetics, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, RNA, Messenger genetics, RNA, Messenger metabolism, Up-Regulation, X-Box Binding Protein 1 genetics, Kruppel-Like Transcription Factors metabolism, Unfolded Protein Response physiology, X-Box Binding Protein 1 metabolism
- Abstract
Transcription factor XBP1s, activated by endoplasmic reticulum (ER) stress in a dose-dependent manner, plays a central role in adaptive unfolded protein response (UPR) via direct activation of multiple genes controlling protein refolding. Here, we report that elevation of ER stress above a critical threshold causes accumulation of XBP1s protein sufficient for binding to the promoter and activation of a gene encoding a transcription factor KLF9. In comparison to other XBP1s targets, KLF9 promoter contains an evolutionary conserved lower-affinity binding site that requires higher amounts of XBP1s for activation. In turn, KLF9 induces expression of two regulators of ER calcium storage, TMEM38B and ITPR1, facilitating additional calcium release from ER, exacerbation of ER stress, and cell death. Accordingly, Klf9 deficiency attenuates tunicamycin-induced ER stress in mouse liver. These data reveal a role for XBP1s in cytotoxic UPR and provide insights into mechanisms of life-or-death decisions in cells under ER stress., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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19. Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma.
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Bianchi-Smiraglia A, Bagati A, Fink EE, Affronti HC, Lipchick BC, Moparthy S, Long MD, Rosario SR, Lightman SM, Moparthy K, Wolff DW, Yun DH, Han Z, Polechetti A, Roll MV, Gitlin II, Leonova KI, Rowsam AM, Kandel ES, Gudkov AV, Bergsagel PL, Lee KP, Smiraglia DJ, and Nikiforov MA
- Subjects
- Animals, Cell Line, Tumor, HEK293 Cells, Humans, Mice, Receptors, Aryl Hydrocarbon genetics, Receptors, Aryl Hydrocarbon metabolism, Biogenic Polyamines biosynthesis, Clofazimine pharmacology, Multiple Myeloma drug therapy, Multiple Myeloma genetics, Multiple Myeloma metabolism, Multiple Myeloma pathology, Neoplasm Proteins antagonists & inhibitors, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Neoplasms, Experimental drug therapy, Neoplasms, Experimental genetics, Neoplasms, Experimental metabolism, Neoplasms, Experimental pathology, Receptors, Aryl Hydrocarbon antagonists & inhibitors
- Abstract
Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.
- Published
- 2018
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20. FOXQ1 controls the induced differentiation of melanocytic cells.
- Author
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Bagati A, Bianchi-Smiraglia A, Moparthy S, Kolesnikova K, Fink EE, Kolesnikova M, Roll MV, Jowdy P, Wolff DW, Polechetti A, Yun DH, Lipchick BC, Paul LM, Wrazen B, Moparthy K, Mudambi S, Morozevich GE, Georgieva SG, Wang J, Shafirstein G, Liu S, Kandel ES, Berman AE, Box NF, Paragh G, and Nikiforov MA
- Subjects
- Animals, Cell Line, Tumor, Forkhead Transcription Factors genetics, Melanocytes pathology, Melanoma genetics, Melanoma pathology, Mice, Mice, Knockout, Microphthalmia-Associated Transcription Factor genetics, Microphthalmia-Associated Transcription Factor metabolism, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins B-raf metabolism, Skin Neoplasms genetics, Skin Neoplasms pathology, Forkhead Transcription Factors metabolism, Melanocytes metabolism, Melanoma metabolism, Signal Transduction, Skin Neoplasms metabolism
- Abstract
Oncogenic transcription factor FOXQ1 has been implicated in promotion of multiple transformed phenotypes in carcinoma cells. Recently, we have characterized FOXQ1 as a melanoma tumor suppressor that acts via repression of N-cadherin gene, and invasion and metastasis. Here we report that FOXQ1 induces differentiation in normal and transformed melanocytic cells at least partially via direct transcriptional activation of MITF gene, melanocytic lineage-specific regulator of differentiation. Importantly, we demonstrate that pigmentation induced in cultured melanocytic cells and in mice by activation of cAMP/CREB1 pathway depends in large part on FOXQ1. Moreover, our data reveal that FOXQ1 acts as a critical mediator of BRAF
V600E -dependent regulation of MITF levels, thus providing a novel link between two major signal transduction pathways controlling MITF and differentiation in melanocytic cells.- Published
- 2018
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21. Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution.
- Author
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Bianchi-Smiraglia A, Rana MS, Foley CE, Paul LM, Lipchick BC, Moparthy S, Moparthy K, Fink EE, Bagati A, Hurley E, Affronti HC, Bakin AV, Kandel ES, Smiraglia DJ, Feltri ML, Sousa R, and Nikiforov MA
- Subjects
- Animals, Bacterial Proteins genetics, Cell Line, Tumor, Guanosine Triphosphate genetics, Humans, Hydrogen-Ion Concentration, Luminescent Proteins genetics, Mutation, Bacterial Proteins metabolism, Biosensing Techniques, Guanosine Triphosphate metabolism, Luminescent Proteins metabolism
- Abstract
GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.
- Published
- 2017
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22. Melanoma Suppressor Functions of the Carcinoma Oncogene FOXQ1.
- Author
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Bagati A, Bianchi-Smiraglia A, Moparthy S, Kolesnikova K, Fink EE, Lipchick BC, Kolesnikova M, Jowdy P, Polechetti A, Mahpour A, Ross J, Wawrzyniak JA, Yun DH, Paragh G, Kozlova NI, Berman AE, Wang J, Liu S, Nemeth MJ, and Nikiforov MA
- Subjects
- Animals, Antigens, CD metabolism, Cadherins metabolism, Carcinogenesis genetics, Carcinogenesis pathology, Cell Line, Tumor, Cell Transformation, Neoplastic pathology, Disease Progression, Forkhead Transcription Factors metabolism, Gene Expression Regulation, Neoplastic, HEK293 Cells, Humans, Mice, SCID, Microphthalmia-Associated Transcription Factor metabolism, Neoplasm Invasiveness, Neoplasm Metastasis, Phenotype, beta Catenin metabolism, Forkhead Transcription Factors genetics, Melanoma genetics, Melanoma pathology, Oncogenes, Skin Neoplasms genetics, Skin Neoplasms pathology
- Abstract
Lineage-specific regulation of tumor progression by the same transcription factor is understudied. We find that levels of the FOXQ1 transcription factor, an oncogene in carcinomas, are decreased during melanoma progression. Moreover, in contrast to carcinomas, FOXQ1 suppresses epithelial-to-mesenchymal transition, invasion, and metastasis in melanoma cells. We find that these lineage-specific functions of FOXQ1 largely depend on its ability to activate (in carcinomas) or repress (in melanoma) transcription of the N-cadherin gene (CDH2). We demonstrate that FOXQ1 interacts with nuclear β-catenin and TLE proteins, and the β-catenin/TLE ratio, which is higher in carcinoma than melanoma cells, determines the effect of FOXQ1 on CDH2 transcription. Accordingly, other FOXQ1-dependent phenotypes can be manipulated by altering nuclear β-catenin or TLE proteins levels. Our data identify FOXQ1 as a melanoma suppressor and establish a mechanism underlying its inverse lineage-specific transcriptional regulation of transformed phenotypes., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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23. Microphthalmia-associated transcription factor suppresses invasion by reducing intracellular GTP pools.
- Author
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Bianchi-Smiraglia A, Bagati A, Fink EE, Moparthy S, Wawrzyniak JA, Marvin EK, Battaglia S, Jowdy P, Kolesnikova M, Foley CE, Berman AE, Kozlova NI, Lipchick BC, Paul-Rosner LM, Bshara W, Ackroyd JJ, Shewach DS, and Nikiforov MA
- Subjects
- Animals, Cell Line, Tumor, Disease Models, Animal, Disease Progression, Ectopic Gene Expression, Extracellular Matrix metabolism, Female, GMP Reductase genetics, GMP Reductase metabolism, Gene Expression Regulation, Neoplastic, Heterografts, Humans, Intracellular Space metabolism, Melanocytes metabolism, Melanoma metabolism, Melanoma pathology, Melanoma, Experimental, Mice, Neoplasm Invasiveness, Neoplasm Metastasis, Neoplasms genetics, rho GTP-Binding Proteins metabolism, Guanosine Triphosphate metabolism, Microphthalmia-Associated Transcription Factor metabolism, Neoplasms metabolism, Neoplasms pathology
- Abstract
Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells; however, little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAF
V600E -melanoma cells and underlies the increased invasion in these cells. Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.- Published
- 2017
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24. The Immortal Senescence.
- Author
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Bianchi-Smiraglia A, Lipchick BC, and Nikiforov MA
- Subjects
- Animals, Biomarkers, Disease Susceptibility, Energy Metabolism, Gene Expression Regulation, Humans, Oncogenes, Signal Transduction, beta-Galactosidase metabolism, Cellular Senescence physiology
- Abstract
Activation of oncogenic signaling paradoxically results in the permanent withdrawal from cell cycle and induction of senescence (oncogene-induced senescence (OIS)). OIS is a fail-safe mechanism used by the cells to prevent uncontrolled tumor growth, and, as such, it is considered as the first barrier against cancer. In order to progress, tumor cells thus need to first overcome the senescent phenotype. Despite the increasing attention gained by OIS in the past 20 years, this field is still rather young due to continuous emergence of novel pathways and processes involved in OIS. Among the many factors contributing to incomplete understanding of OIS are the lack of unequivocal markers for senescence and the complexity of the phenotypes revealed by senescent cells in vivo and in vitro. OIS has been shown to play major roles at both the cellular and organismal levels in biological processes ranging from embryonic development to barrier to cancer progression. Here we will briefly outline major advances in methodologies that are being utilized for induction, identification, and characterization of molecular processes in cells undergoing oncogene-induced senescence. The full description of such methodologies is provided in the corresponding chapters of the book.
- Published
- 2017
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25. Detection of Nucleotide Disbalance in Cells Undergoing Oncogene-Induced Senescence.
- Author
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Nikiforov MA and Shewach DS
- Subjects
- Cell Line, Chromatography, High Pressure Liquid, DNA Damage, Deoxyribonucleotides chemistry, Deoxyribonucleotides metabolism, Fibroblasts metabolism, Gene Expression, Genes, myc, Humans, Nucleotides chemistry, Ribonucleotide Reductases metabolism, Thymidylate Synthase metabolism, Cellular Senescence genetics, Nucleotides metabolism, Oncogenes genetics
- Abstract
DNA damage response has been characterized as an important mediator of senescence phenotypes induced by activated oncogenes in normal human cells. Depletion of intracellular deoxyribonucleotide pools has been recently recognized as one of the major causes for DNA damage in these cells. Cells undergoing oncogene-induced senescence display decreased expression of several rate-limiting enzymes involved in the biosynthesis of deoxyribonucleotides, including thymidylate synthase (TS) and ribonucleotide reductase (RR). Individual depletion of these enzymes leads to premature senescence. Reciprocally, ectopic expression of TS and RR or addition of deoxyribonucleosides resulted in suppression of senescence phenotypes in normal or tumor cells caused by overexpression of activated HRAS or depletion of C-MYC, respectively. Therefore, in the current chapter, we will describe a methodology for the quantitative measurement of nucleotide pools in senescent cells.
- Published
- 2017
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26. Oxidative stress and proteasome inhibitors in multiple myeloma.
- Author
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Lipchick BC, Fink EE, and Nikiforov MA
- Subjects
- Animals, Antineoplastic Agents pharmacology, Humans, Multiple Myeloma metabolism, Multiple Myeloma pathology, Proteasome Inhibitors pharmacology, Reactive Oxygen Species metabolism, Antineoplastic Agents therapeutic use, Multiple Myeloma drug therapy, Oxidative Stress drug effects, Proteasome Inhibitors therapeutic use
- Abstract
Multiple myeloma is a form of plasma cell neoplasm that accounts for approximately 10% of all hematological malignancies. Recently, several novel drugs have been discovered that almost doubled the overall survival of multiple myeloma patients. One of these drugs, the first-in-class proteasome inhibitor bortezomib (Velcade) has demonstrated remarkable response rates in multiple myeloma patients, and yet, currently this disease remains incurable. The major factor undermining the success of multiple myeloma treatment is a rapidly emerging resistance to the available therapy. Thus, the development of stand-alone or adjuvant anti-myeloma agents becomes of paramount importance. Overproduction of intracellular reactive oxygen species (ROS) often accompanies malignant transformation due to oncogene activation and/or enhanced metabolism in tumor cells. As a result, these cells possess higher levels of ROS and lower levels of antioxidant molecules compared to their normal counterparts. Unbalanced production of ROS leads to oxidative stress which, if left unchecked, could be toxic for the cell. In multiple myeloma cells where high rates of immunoglobulin synthesis is an additional factor contributing to overproduction of ROS, further induction of oxidative stress can be an effective strategy to cope with this disease. Here we will review the available data on the role of oxidative stress in the cytotoxicity of proteasome inhibitors and the use of ROS-inducing compounds as anti-myeloma agents., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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27. Slowing down the G rand T ouring P rototype speed of cancer cells.
- Author
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Bianchi-Smiraglia A and Nikiforov MA
- Abstract
Enzymes involved in de novo production of guanosine triphosphate (GTP) have been recently revealed as integral components of melanoma progression through modulation of the activity of small GTPases. Here, we discuss the biology and therapeutic implications of these findings.
- Published
- 2016
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28. Mitochondrial thioredoxin reductase regulates major cytotoxicity pathways of proteasome inhibitors in multiple myeloma cells.
- Author
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Fink EE, Mannava S, Bagati A, Bianchi-Smiraglia A, Nair JR, Moparthy K, Lipchick BC, Drokov M, Utley A, Ross J, Mendeleeva LP, Savchenko VG, Lee KP, and Nikiforov MA
- Subjects
- Animals, Apoptosis drug effects, Bortezomib pharmacology, Cell Line, Tumor, Endoplasmic Reticulum Stress drug effects, Female, Humans, Mice, Multiple Myeloma enzymology, Multiple Myeloma pathology, Oxidative Stress, Reactive Oxygen Species metabolism, Multiple Myeloma drug therapy, Proteasome Inhibitors therapeutic use, Thioredoxin Reductase 2 physiology
- Abstract
It is generally accepted that intracellular oxidative stress induced by proteasome inhibitors is a byproduct of endoplasmic reticulum (ER) stress. Here we report a mechanism underlying the ability of proteasome inhibitors bortezomib (BTZ) and carfilzomib (CFZ) to directly induce oxidative and ER stresses in multiple myeloma (MM) cells via transcriptional repression of a gene encoding mitochondrial thioredoxin reductase (TXNRD2). TXNRD2 is critical for maintenance of intracellular red-ox status and detoxification of reactive oxygen species. Depletion of TXNRD2 to the levels detected in BTZ- or CFZ-treated cells causes oxidative stress, ER stress and death similar to those induced by proteasome inhibitors. Reciprocally, restoration of near-wildtype TXNRD2 amounts in MM cells treated with proteasome inhibitors reduces oxidative stress, ER stress and cell death by ~46%, ~35% and ~50%, respectively, compared with cells with unrestored TXNRD2 levels. Moreover, cells from three MM cell lines selected for resistance to BTZ demonstrate elevated levels of TXNRD2, indirectly confirming its functional role in BTZ resistance. Accordingly, ectopic expression of TXNRD2 in MM cell xenografts in immunocompromised mice blunts therapeutic effects of BTZ. Our data identify TXNRD2 as a potentially clinically relevant target, inhibition of which is critical for proteasome inhibitor-dependent cytotoxicity, oxidative stress and ER stress.
- Published
- 2016
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29. Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity.
- Author
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Bianchi-Smiraglia A, Wawrzyniak JA, Bagati A, Marvin EK, Ackroyd J, Moparthy S, Bshara W, Fink EE, Foley CE, Morozevich GE, Berman AE, Shewach DS, and Nikiforov MA
- Subjects
- Adenosine analogs & derivatives, Adenosine pharmacology, Animals, Cell Line, Tumor, Enzyme Inhibitors pharmacology, Female, Humans, Immunoblotting, Immunohistochemistry, Melanoma pathology, Mice, Mice, SCID, Nucleotidyltransferases antagonists & inhibitors, Nucleotidyltransferases genetics, Skin Neoplasms, Melanoma, Cutaneous Malignant, Guanosine Monophosphate metabolism, Melanoma enzymology, Nucleotidyltransferases metabolism
- Abstract
Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF(V600E) or NRAS(Q61R) human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.
- Published
- 2015
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30. Nrf2 amplifies oxidative stress via induction of Klf9.
- Author
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Zucker SN, Fink EE, Bagati A, Mannava S, Bianchi-Smiraglia A, Bogner PN, Wawrzyniak JA, Foley C, Leonova KI, Grimm MJ, Moparthy K, Ionov Y, Wang J, Liu S, Sexton S, Kandel ES, Bakin AV, Zhang Y, Kaminski N, Segal BH, and Nikiforov MA
- Subjects
- Animals, Binding Sites, Bleomycin, Cell Line, Tumor, Genes, Reporter, Humans, Kruppel-Like Transcription Factors metabolism, Luciferases genetics, Luciferases metabolism, Lung metabolism, Lung pathology, Mice, NF-E2-Related Factor 2 metabolism, NIH 3T3 Cells, Promoter Regions, Genetic, Protein Binding, Pulmonary Fibrosis chemically induced, Pulmonary Fibrosis metabolism, Pulmonary Fibrosis pathology, Reactive Oxygen Species, Signal Transduction, Gene Expression Regulation, Kruppel-Like Transcription Factors genetics, NF-E2-Related Factor 2 genetics, Oxidative Stress, Pulmonary Fibrosis genetics
- Abstract
Reactive oxygen species (ROS) activate NF-E2-related transcription factor 2 (Nrf2), a key transcriptional regulator driving antioxidant gene expression and protection from oxidant injury. Here, we report that in response to elevation of intracellular ROS above a critical threshold, Nrf2 stimulates expression of transcription Kruppel-like factor 9 (Klf9), resulting in further Klf9-dependent increases in ROS and subsequent cell death. We demonstrated that Klf9 independently causes increased ROS levels in various types of cultured cells and in mouse tissues and is required for pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Mechanistically, Klf9 binds to the promoters and alters the expression of several genes involved in the metabolism of ROS, including suppression of thioredoxin reductase 2, an enzyme participating in ROS clearance. Our data reveal an Nrf2-dependent feedforward regulation of ROS and identify Klf9 as a ubiquitous regulator of oxidative stress and lung injury., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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31. A purine nucleotide biosynthesis enzyme guanosine monophosphate reductase is a suppressor of melanoma invasion.
- Author
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Wawrzyniak JA, Bianchi-Smiraglia A, Bshara W, Mannava S, Ackroyd J, Bagati A, Omilian AR, Im M, Fedtsova N, Miecznikowski JC, Moparthy KC, Zucker SN, Zhu Q, Kozlova NI, Berman AE, Hoek KS, Gudkov AV, Shewach DS, Morrison CD, and Nikiforov MA
- Subjects
- Animals, Cell Line, Tumor, Cell Movement, Extracellular Matrix metabolism, GMP Reductase antagonists & inhibitors, GMP Reductase genetics, Guanosine Triphosphate metabolism, HCT116 Cells, Humans, IMP Dehydrogenase metabolism, Melanoma metabolism, Melanoma pathology, Mice, Phenotype, RNA Interference, RNA, Small Interfering metabolism, Transplantation, Heterologous, rac1 GTP-Binding Protein genetics, rac1 GTP-Binding Protein metabolism, rho GTP-Binding Proteins metabolism, GMP Reductase metabolism, Melanoma enzymology, Purine Nucleosides biosynthesis
- Abstract
Melanoma is one of the most aggressive types of human cancers, and the mechanisms underlying melanoma invasive phenotype are not completely understood. Here, we report that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in de novo biosynthesis of purine nucleotides, was downregulated in the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR downregulates the amounts of several GTP-bound (active) Rho-GTPases and suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade in vitro, and grow as tumor xenografts in vivo. Mechanistically, we demonstrated that GMPR partially depletes intracellular GTP pools. Pharmacological inhibition of de novo GTP biosynthesis suppressed whereas addition of exogenous guanosine increased invasion of melanoma cells as well as cells from other cancer types. Our data identify GMPR as a melanoma invasion suppressor and establish a link between guanosine metabolism and Rho-GTPase-dependent melanoma cell invasion., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
32. Cell-based methods for the identification of MYC-inhibitory small molecules.
- Author
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Burkhart CA, Haber M, Norris MD, Gudkov AV, and Nikiforov MA
- Subjects
- Cell Line, Tumor, Gene Expression, Genes, Reporter, Humans, Phenotype, Small Molecule Libraries, Antineoplastic Agents pharmacology, Drug Screening Assays, Antitumor methods, Proto-Oncogene Proteins c-myc antagonists & inhibitors
- Abstract
Oncoproteins encoded by dominant oncogenes have long been considered as targets for chemotherapeutic intervention. However, oncogenic transcription factors have often been dismissed as "undruggable." Members of Myc family of transcription factors have been identified as promising targets for cancer chemotherapy in multiple publications reporting the requirement of Myc proteins for maintenance of almost every type of tumor. Here, we describe cell-based approaches to identify c-Myc small molecule inhibitors by screening complex libraries of diverse small molecules based on Myc functionality and specificity.
- Published
- 2013
- Full Text
- View/download PDF
33. Depletion of deoxyribonucleotide pools is an endogenous source of DNA damage in cells undergoing oncogene-induced senescence.
- Author
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Mannava S, Moparthy KC, Wheeler LJ, Natarajan V, Zucker SN, Fink EE, Im M, Flanagan S, Burhans WC, Zeitouni NC, Shewach DS, Mathews CK, and Nikiforov MA
- Subjects
- Cell Proliferation, Cells, Cultured, Cellular Senescence physiology, DNA Replication genetics, Deoxyribonucleotides genetics, Fibroblasts metabolism, Fibroblasts physiology, Humans, Proto-Oncogene Proteins p21(ras) physiology, Ribonucleotide Reductases biosynthesis, Ribonucleotide Reductases physiology, Thymidylate Synthase biosynthesis, Thymidylate Synthase physiology, Cellular Senescence genetics, DNA Damage genetics, Deoxyribonucleotides metabolism, Oncogenes physiology
- Abstract
In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
34. Recent progress in genetics of aging, senescence and longevity: focusing on cancer-related genes.
- Author
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Berman AE, Leontieva OV, Natarajan V, McCubrey JA, Demidenko ZN, and Nikiforov MA
- Subjects
- Aging metabolism, Animals, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic metabolism, DNA Damage, Genetic Predisposition to Disease, Humans, Longevity genetics, Models, Genetic, Neoplasms metabolism, Neoplasms pathology, Phenotype, Aging genetics, Cellular Senescence genetics, Neoplasms genetics, Signal Transduction genetics
- Abstract
It is widely believed that aging results from the accumulation of molecular damage, including damage of DNA and mitochondria and accumulation of molecular garbage both inside and outside of the cell. Recently, this paradigm is being replaced by the "hyperfunction theory", which postulates that aging is caused by activation of signal transduction pathways such as TOR (Target of Rapamycin). These pathways consist of different enzymes, mostly kinases, but also phosphatases, deacetylases, GTPases, and some other molecules that cause overactivation of normal cellular functions. Overactivation of these sensory signal transduction pathways can cause cellular senescence, age-related diseases, including cancer, and shorten life span. Here we review some of the numerous very recent publications on the role of signal transduction molecules in aging and age-related diseases. As was emphasized by the author of the "hyperfunction model", many (or actually all) of them also play roles in cancer. So these "participants" in pro-aging signaling pathways are actually very well acquainted to cancer researchers. A cancer-related journal such as Oncotarget is the perfect place for publication of such experimental studies, reviews and perspectives, as it can bridge the gap between cancer and aging researchers.
- Published
- 2012
- Full Text
- View/download PDF
35. Short hairpin RNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization.
- Author
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Flanagan SA, Cooper KS, Mannava S, Nikiforov MA, and Shewach DS
- Subjects
- Adenosine Triphosphate metabolism, Cell Line, Tumor, Checkpoint Kinase 1, Cytidine Triphosphate metabolism, Enzyme Activation drug effects, Guanosine Triphosphate metabolism, HT29 Cells, Humans, Phosphorylation, Protein Kinases metabolism, Tumor Stem Cell Assay methods, Antimetabolites, Antineoplastic pharmacology, DNA Mismatch Repair, Floxuridine pharmacology, RNA, Small Interfering pharmacology, Radiation Tolerance genetics, Thymidylate Synthase antagonists & inhibitors
- Abstract
Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur., Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2'-deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively., Results: TS shRNA produced profound (≥ 90%) and prolonged (≥ 8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations., Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA mismatches underlying radiosensitization. Importantly, shRNA suppression of TS avoids FP-mediated TS elevation and its negative prognostic role. These studies support the further exploration of TS suppression as a novel radiosensitizing strategy., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
36. Ribonucleotide reductase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C-MYC depletion.
- Author
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Mannava S, Moparthy KC, Wheeler LJ, Leonova KI, Wawrzyniak JA, Bianchi-Smiraglia A, Berman AE, Flanagan S, Shewach DS, Zeitouni NC, Gudkov AV, Mathews CK, and Nikiforov MA
- Subjects
- Cell Line, Tumor, Down-Regulation, Gene Expression Regulation, Neoplastic, Genotype, Humans, Melanoma genetics, Melanoma pathology, Phenotype, Proto-Oncogene Proteins c-myc genetics, RNA Interference, Ribonucleoside Diphosphate Reductase metabolism, Ribonucleotide Reductases genetics, Skin Neoplasms genetics, Skin Neoplasms pathology, Thymidylate Synthase genetics, Time Factors, Transfection, Tumor Suppressor Proteins metabolism, Cellular Senescence drug effects, DNA Damage drug effects, Deoxyribonucleosides pharmacology, Melanoma enzymology, Proto-Oncogene Proteins c-myc metabolism, Ribonucleotide Reductases metabolism, Skin Neoplasms enzymology, Thymidylate Synthase metabolism
- Abstract
The down-regulation of dominant oncogenes, including C-MYC, in tumor cells often leads to the induction of senescence via mechanisms that are not completely identified. In the current study, we demonstrate that MYC-depleted melanoma cells undergo extensive DNA damage that is caused by the underexpression of thymidylate synthase (TS) and ribonucleotide reductase (RR) and subsequent depletion of deoxyribonucleoside triphosphate pools. Simultaneous genetic inhibition of TS and RR in melanoma cells induced DNA damage and senescence phenotypes very similar to the ones caused by MYC-depletion. Reciprocally, overexpression of TS and RR in melanoma cells or addition of deoxyribo-nucleosides to culture media substantially inhibited DNA damage and senescence-associated phenotypes caused by C-MYC depletion. Our data demonstrate the essential role of TS and RR in C-MYC-dependent suppression of senescence in melanoma cells.
- Published
- 2012
- Full Text
- View/download PDF
37. Controversial aspects of oncogene-induced senescence.
- Author
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Bianchi-Smiraglia A and Nikiforov MA
- Subjects
- DNA Damage, DNA Repair, Humans, Tumor Suppressor Proteins metabolism, raf Kinases genetics, raf Kinases metabolism, ras Proteins genetics, ras Proteins metabolism, Cellular Senescence, Oncogenes
- Abstract
Oncogene-induced senescence (OIS) is a fail-safe mechanism that is developed to suppress cell proliferation caused by aberrant activation of oncoproteins in normal cells. Most of the available literature considers senescence to be caused by activated RAS or RAF proteins. In the current review, we will discuss some of the controversial aspects of RAS- or RAF-induced senescence in different types of normal cells: are tumor suppressors important for OIS? What is the role of DNA damage in OIS? Are there different types of OIS?
- Published
- 2012
- Full Text
- View/download PDF
38. Krüppel-like factor 9 and progesterone receptor coregulation of decidualizing endometrial stromal cells: implications for the pathogenesis of endometriosis.
- Author
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Pabona JM, Simmen FA, Nikiforov MA, Zhuang D, Shankar K, Velarde MC, Zelenko Z, Giudice LC, and Simmen RC
- Subjects
- Adult, Endometriosis etiology, Endometriosis genetics, Endometriosis pathology, Endometrium pathology, Female, Gene Expression, Humans, Kruppel-Like Transcription Factors genetics, Promoter Regions, Genetic, Receptors, Progesterone genetics, Signal Transduction physiology, Stromal Cells pathology, Wnt Signaling Pathway physiology, Endometriosis metabolism, Endometrium metabolism, Kruppel-Like Transcription Factors metabolism, Receptors, Progesterone metabolism, Stromal Cells metabolism
- Abstract
Context: Endometriosis is characterized by progesterone resistance and associated with infertility. Krüppel-like factor 9 (KLF9) is a progesterone receptor (PGR)-interacting protein, and mice null for Klf9 are subfertile. Whether loss of KLF9 expression contributes to progesterone resistance of eutopic endometrium of women with endometriosis is unknown., Objective: The aims were to investigate 1) KLF9 expression in eutopic endometrium of women with and without endometriosis, 2) effects of attenuated KLF9 expression on WNT-signaling component expression and on WNT inhibitor Dickkopf-1 promoter activity in human endometrial stromal cells (HESC), and 3) PGR and KLF9 coregulation of the stromal transcriptome network., Methods: Transcript levels of KLF9, PGR, and WNT signaling components were measured in eutopic endometrium of women with and without endometriosis. Transcript and protein levels of WNT signaling components in HESC transfected with KLF9 and/or PGR small interfering RNA were analyzed by quantitative RT-PCR and Western blot. KLF9 and PGR coregulation of Dickkopf-1 promoter activity was evaluated using human Dickkopf-1-luciferase promoter/reporter constructs and by chromatin immunoprecipitation. KLF9 and PGR signaling networks were analyzed by gene expression array profiling., Results: Eutopic endometrium from women with endometriosis had reduced expression of KLF9 mRNA together with those of PGR-B, WNT4, WNT2, and DKK1. KLF9 and PGR were recruited to the DKK1 promoter and modified each other's transactivity. In HESC, KLF9 and PGR coregulated components of the WNT, cytokine, and IGF gene networks that are implicated in endometriosis and infertility., Conclusion: Loss of KLF9 coregulation of endometrial stromal PGR-responsive gene networks may underlie progesterone resistance in endometriosis.
- Published
- 2012
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- View/download PDF
39. KLF9 is a novel transcriptional regulator of bortezomib- and LBH589-induced apoptosis in multiple myeloma cells.
- Author
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Mannava S, Zhuang D, Nair JR, Bansal R, Wawrzyniak JA, Zucker SN, Fink EE, Moparthy KC, Hu Q, Liu S, Boise LH, Lee KP, and Nikiforov MA
- Subjects
- Antineoplastic Agents pharmacology, Blotting, Western, Bortezomib, Cell Line, Tumor, Cell Survival drug effects, Gene Expression Profiling, Gene Expression Regulation, Neoplastic drug effects, Humans, Indoles, Kruppel-Like Transcription Factors metabolism, Multiple Myeloma metabolism, Multiple Myeloma pathology, Oligonucleotide Array Sequence Analysis, Panobinostat, Promoter Regions, Genetic genetics, Protein Binding, Proto-Oncogene Proteins c-bcl-2 genetics, Proto-Oncogene Proteins c-bcl-2 metabolism, RNA Interference, Reverse Transcriptase Polymerase Chain Reaction, Transcription Factors genetics, Transcription Factors metabolism, Apoptosis drug effects, Boronic Acids pharmacology, Hydroxamic Acids pharmacology, Kruppel-Like Transcription Factors genetics, Multiple Myeloma genetics, Pyrazines pharmacology
- Abstract
Bortezomib, a therapeutic agent for multiple myeloma (MM) and mantle cell lymphoma, suppresses proteosomal degradation leading to substantial changes in cellular transcriptional programs and ultimately resulting in apoptosis. Transcriptional regulators required for bortezomib-induced apoptosis in MM cells are largely unknown. Using gene expression profiling, we identified 36 transcription factors that displayed altered expression in MM cells treated with bortezomib. Analysis of a publically available database identified Kruppel-like family factor 9 (KLF9) as the only transcription factor with significantly higher basal expression in MM cells from patients who responded to bortezomib compared with nonresponders. We demonstrated that KLF9 in cultured MM cells was up-regulated by bortezomib; however, it was not through the induction of endoplasmic reticulum stress. Instead, KLF9 levels correlated with bortezomib-dependent inhibition of histone deacetylases (HDAC) and were increased by the HDAC inhibitor LBH589 (panobinostat). Furthermore, bortezomib induced binding of endogenous KLF9 to the promoter of the proapoptotic gene NOXA. Importantly, KLF9 knockdown impaired NOXA up-regulation and apoptosis caused by bortezomib, LBH589, or a combination of theses drugs, whereas KLF9 overexpression induced apoptosis that was partially NOXA-dependent. Our data identify KLF9 as a novel and potentially clinically relevant transcriptional regulator of drug-induced apoptosis in MM cells.
- Published
- 2012
- Full Text
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40. Transcriptional Regulation of CRD-BP by c-myc: Implications for c-myc Functions.
- Author
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Noubissi FK, Nikiforov MA, Colburn N, and Spiegelman VS
- Abstract
The coding region determinant binding protein, CRD-BP, is a multifunctional RNA binding protein involved in different processes such as mRNA turnover, translation control, and localization. It is mostly expressed in fetal and neonatal tissues, where it regulates many transcripts essential for normal embryonic development. CRD-BP is scarce or absent in normal adult tissues but reactivated and/or overexpressed in various neoplastic and preneoplastic tumors and in most cell lines. Its expression has been associated with the most aggressive form of some cancers. CRD-BP is an important regulator of different genes including a variety of oncogenes or proto-oncogenes (c-myc, β-TrCP1, GLI1, etc.). Regulation of CRD-BP expression is critical for proper control of its targets as its overexpression may play an important role in abnormal cell proliferation, suppression of apoptosis, invasion, and metastasis. Molecular bases of the regulatory mechanisms governing CRD-BP expression are still not completely elucidated. In this article, we have identified c-myc as a novel transcriptional regulator of CRD-BP. We show that c-myc binds to CRD-BP promoter and induces its transcription. This induction of CRD-BP expression contributes to the role of c-myc in the regulation of translation, increase in cell size, and acceleration of cell cycle progression via a mechanism involving upregulation of β-TrCP1 levels and activities and accelerated degradation of PDCD4.
- Published
- 2010
- Full Text
- View/download PDF
41. Pathways of oncogene-induced senescence in human melanocytic cells.
- Author
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Bansal R and Nikiforov MA
- Subjects
- Humans, Melanoma metabolism, Proto-Oncogene Proteins B-raf metabolism, ras Proteins metabolism, Cellular Senescence, Melanocytes metabolism, Oncogenes
- Published
- 2010
- Full Text
- View/download PDF
42. C-MYC overexpression is required for continuous suppression of oncogene-induced senescence in melanoma cells.
- Author
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Zhuang D, Mannava S, Grachtchouk V, Tang WH, Patil S, Wawrzyniak JA, Berman AE, Giordano TJ, Prochownik EV, Soengas MS, and Nikiforov MA
- Subjects
- Cell Line, Tumor, Cyclin-Dependent Kinase Inhibitor p16 genetics, Cyclin-Dependent Kinase Inhibitor p16 metabolism, Disease Progression, Gene Deletion, Humans, Melanocytes metabolism, Melanoma enzymology, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinase Kinases metabolism, Neoplasm Metastasis genetics, Neoplasm Metastasis pathology, Phenotype, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins B-raf metabolism, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism, ras Proteins genetics, ras Proteins metabolism, Cellular Senescence drug effects, Gene Expression, Melanoma genetics, Melanoma pathology, Proto-Oncogene Proteins c-myc genetics, Proto-Oncogene Proteins c-myc metabolism
- Abstract
Malignant melanomas often harbor activating mutations in BRAF (V600E) or, less frequently, in NRAS (Q61R). Intriguingly, the same mutations have been detected at higher incidences in benign nevi, which are largely composed of senescent melanocytes. Overexpression of BRAF(V600E) or NRAS(Q61R) in human melanocytes in vitro has been shown to induce senescence, although via different mechanisms. How oncogene-induced senescence is overcome during melanoma progression remains unclear. Here, we report that in the majority of analysed BRAF(V600E)- or NRAS(Q61R)-expressing melanoma cells, C-MYC depletion induced different yet overlapping sets of senescence phenotypes that are characteristic of normal melanocytes undergoing senescence due to overexpression of BRAF(V600E) or NRAS(Q61R), respectively. These senescence phenotypes were p16(INK4A)- or p53-independent, however, several of them were suppressed by genetic or pharmacological inhibition of BRAF(V600E) or phosphoinositide 3-kinase pathways, including rapamycin-mediated inhibition of mTOR-raptor in NRAS(Q61R)-expressing melanoma cells. Reciprocally, overexpression of C-MYC in normal melanocytes suppressed BRAF(V600E)-induced senescence more efficiently than NRAS(Q61R)-induced senescence, which agrees with the generally higher rates of activating mutations in BRAF than NRAS gene in human cutaneous melanomas. Our data suggest that one of the major functions of C-MYC overexpression in melanoma progression is to continuous suppress BRAF(V600E)- or NRAS(Q61R)-dependent senescence programs.
- Published
- 2008
- Full Text
- View/download PDF
43. MLH1 deficiency enhances radiosensitization with 5-fluorodeoxyuridine by increasing DNA mismatches.
- Author
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Flanagan SA, Krokosky CM, Mannava S, Nikiforov MA, and Shewach DS
- Subjects
- Base Pair Mismatch radiation effects, Cell Cycle drug effects, Cell Cycle radiation effects, Cell Death drug effects, Cell Death radiation effects, HCT116 Cells, Humans, MutL Protein Homolog 1, Mutation genetics, Nucleotides metabolism, Plasmids genetics, RNA, Small Interfering metabolism, Radiation Tolerance radiation effects, Radiation, Ionizing, Adaptor Proteins, Signal Transducing deficiency, Base Pair Mismatch drug effects, Floxuridine pharmacology, Nuclear Proteins deficiency, Radiation Tolerance drug effects
- Abstract
The antitumor drug 5-fluoro-2'-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. Although radiosensitization with FdUrd requires dTTP depletion and S-phase arrest, the exact mechanism by which these events produce radiosensitization remains unknown. We hypothesized that the depletion of dTTP produces DNA mismatches that, if not repaired before irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0-1 cells that lack the expression of the required MMR protein MLH1 (inactive MLH1), and in MMR-proficient (wild-type MLH1) HCT116 1-2 cells. Although HCT116 0-1 cells were less sensitive to FdUrd (IC(50) = 3.5 microM) versus HCT116 1-2 cells (IC(50) = 0.75 microM), when irradiation followed FdUrd (IC(50)) the MLH1-inactivated cells exhibited greater radiosensitization compared with MMR-wild-type cells [radiation enhancement ratio (RER) = 1.8 +/- 0.28 versus 1.1 +/- 0.1, respectively] and an increase (> or =8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1-2 MLH1-wild-type cells, FdUrd (IC(50)) did not produce radiosensitization nor did it increase the mutation frequency, but after short hairpin RNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER = 1.6 +/- 0.10 and 1.5 +/- 0.06, respectively) and an increase in nucleotide misincorporations (8-fold and 6-fold, respectively). Incubation with higher concentrations of FdUrd (IC(90)) after suppression of MLH1 produced a further increase in ionizing radiation sensitivity in both SW620 and HCT116 1-2 cells (RER = 1.8 +/- 0.03 and 1.7 +/- 0.13, respectively) and nucleotide misincorporations (>10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.
- Published
- 2008
- Full Text
- View/download PDF
44. Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells.
- Author
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Mannava S, Grachtchouk V, Wheeler LJ, Im M, Zhuang D, Slavina EG, Mathews CK, Shewach DS, and Nikiforov MA
- Subjects
- Gene Expression Regulation, Enzymologic drug effects, Gene Expression Regulation, Neoplastic drug effects, Humans, IMP Dehydrogenase genetics, IMP Dehydrogenase metabolism, IMP Dehydrogenase physiology, Melanocytes metabolism, Melanoma genetics, Promoter Regions, Genetic, Protein Binding, Proto-Oncogene Proteins c-myc antagonists & inhibitors, Proto-Oncogene Proteins c-myc genetics, Proto-Oncogene Proteins c-myc metabolism, RNA, Small Interfering pharmacology, Ribose-Phosphate Pyrophosphokinase genetics, Ribose-Phosphate Pyrophosphokinase metabolism, Ribose-Phosphate Pyrophosphokinase physiology, Thymidylate Synthase genetics, Thymidylate Synthase metabolism, Thymidylate Synthase physiology, Transfection, Tumor Cells, Cultured, Cell Proliferation drug effects, Melanoma metabolism, Melanoma pathology, Nucleotides biosynthesis, Proto-Oncogene Proteins c-myc physiology
- Abstract
To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.
- Published
- 2008
- Full Text
- View/download PDF
45. c-Myc depletion inhibits proliferation of human tumor cells at various stages of the cell cycle.
- Author
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Wang H, Mannava S, Grachtchouk V, Zhuang D, Soengas MS, Gudkov AV, Prochownik EV, and Nikiforov MA
- Subjects
- Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p21 genetics, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Fibroblasts cytology, Fibroblasts metabolism, Flow Cytometry, Green Fluorescent Proteins metabolism, Humans, Immunoblotting, Keratinocytes cytology, Keratinocytes metabolism, Melanocytes cytology, Melanocytes metabolism, Neoplasms genetics, Proto-Oncogene Proteins c-myc genetics, RNA, Small Interfering pharmacology, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism, Cell Cycle physiology, Cell Proliferation, Neoplasms metabolism, Neoplasms pathology, Proto-Oncogene Proteins c-myc deficiency
- Abstract
A major role for c-Myc in the proliferation of normal cells is attributed to its ability to promote progression through G(1) and into S phase of the cell cycle. The absolute requirement of c-Myc for cell cycle progression in human tumor cells has not been comprehensively addressed. In the present work, we used a lentiviral-based short hairpin RNA (shRNA) expression vector to stably reduce c-Myc expression in a large number of human tumor cell lines and in three different types of normal human cells. In all cases, cell proliferation was severely inhibited, with normal cells ultimately undergoing G(0)/G(1) growth arrest. In contrast, tumor cells demonstrated a much more variable cell cycle response with cells from several lines accumulating in S or G(2)/M phases. Moreover, in some tumor lines, the phase of cell cycle arrest caused by inhibition of c-Myc could be altered by depleting tumor suppressor protein p53 or its transcriptional target p21(CIP/WAF). Our data suggest that, as in the case of normal cells, c-Myc is essential for sustaining proliferation of human tumor cells. However its rate-limiting role in cell cycle control is variable and is reliant upon the status of other cell cycle regulators.
- Published
- 2008
- Full Text
- View/download PDF
46. Tumor cell-selective regulation of NOXA by c-MYC in response to proteasome inhibition.
- Author
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Nikiforov MA, Riblett M, Tang WH, Gratchouck V, Zhuang D, Fernandez Y, Verhaegen M, Varambally S, Chinnaiyan AM, Jakubowiak AJ, and Soengas MS
- Subjects
- Apoptosis genetics, Binding Sites, Bortezomib, Drug Design, E2F1 Transcription Factor metabolism, Humans, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Melanocytes drug effects, Melanocytes metabolism, Promoter Regions, Genetic, Proto-Oncogene Proteins c-bcl-2 genetics, Proto-Oncogene Proteins c-myc genetics, RNA Interference, RNA, Messenger analysis, RNA, Messenger metabolism, Transcriptional Activation, Tumor Suppressor Protein p53 metabolism, Antineoplastic Agents pharmacology, Boronic Acids pharmacology, Melanoma enzymology, Protease Inhibitors pharmacology, Proteasome Inhibitors, Proto-Oncogene Proteins c-bcl-2 metabolism, Proto-Oncogene Proteins c-myc metabolism, Pyrazines pharmacology, Skin Neoplasms enzymology
- Abstract
The proteasome controls a plethora of survival factors in all mammalian cells analyzed to date. Therefore, it is puzzling that proteasome inhibitors such as bortezomib can display a preferential toxicity toward malignant cells. In fact, proteasome inhibitors have the salient feature of promoting a dramatic induction of the proapoptotic protein NOXA in a tumor cell-restricted manner. However, the molecular determinants that control this specific regulation of NOXA are unknown. Here, we show that the induction of NOXA by bortezomib is directly dependent on the oncogene c-MYC. This requirement for c-MYC was found in a variety of tumor cell types, in marked contrast with dispensable roles of p53, HIF-1alpha, and E2F-1 (classical proteasomal targets that can regulate NOXA mRNA under stress). Conserved MYC-binding sites identified at the NOXA promoter were validated by ChIP and reporter assays. Down-regulation of the endogenous levels of c-MYC abrogated the induction of NOXA in proteasome-defective tumor cells. Conversely, forced expression of c-MYC enabled normal cells to accumulate NOXA and subsequently activate cell death programs in response to proteasome blockage. c-MYC is itself a proteasomal target whose levels or function are invariably up-regulated during tumor progression. Our data provide an unexpected function of c-MYC in the control of the apoptotic machinery, and reveal a long sought-after oncogenic event conferring sensitivity to proteasome inhibition.
- Published
- 2007
- Full Text
- View/download PDF
47. Anti-oncogenic role of the endoplasmic reticulum differentially activated by mutations in the MAPK pathway.
- Author
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Denoyelle C, Abou-Rjaily G, Bezrookove V, Verhaegen M, Johnson TM, Fullen DR, Pointer JN, Gruber SB, Su LD, Nikiforov MA, Kaufman RJ, Bastian BC, and Soengas MS
- Subjects
- Cell Cycle, Cell Proliferation, Cellular Senescence, Cyclin-Dependent Kinase Inhibitor p16 genetics, Cyclin-Dependent Kinase Inhibitor p16 metabolism, DNA Damage, Fibroblasts metabolism, Heterochromatin genetics, Heterochromatin metabolism, Humans, Infant, Melanocytes pathology, Melanoma metabolism, Melanoma pathology, Mitogen-Activated Protein Kinases metabolism, Mutation, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins B-raf metabolism, Signal Transduction, Endoplasmic Reticulum metabolism, Genes, ras genetics, Melanoma genetics, Mitogen-Activated Protein Kinases genetics
- Abstract
Dysfunction of the endoplasmic reticulum (ER) has been reported in a variety of human pathologies, including cancer. However, the contribution of the ER to the early stages of normal cell transformation is largely unknown. Using primary human melanocytes and biopsies of human naevi (moles), we show that the extent of ER stress induced by cellular oncogenes may define the mechanism of activation of premature senescence. Specifically, we found that oncogenic forms of HRAS (HRAS(G12V)) but not its downstream target BRAF (BRAF(V600E)), engaged a rapid cell-cycle arrest that was associated with massive vacuolization and expansion of the ER. However, neither p53, p16(INK4a) nor classical senescence markers--such as foci of heterochromatin or DNA damage--were able to account for the specific response of melanocytes to HRAS(G12V). Instead, HRAS(G12V)-driven senescence was mediated by the ER-associated unfolded protein response (UPR). The impact of HRAS on the UPR was selective, as it was poorly induced by activated NRAS (more frequently mutated in melanoma than HRAS). These results argue against premature senescence as a converging mechanism of response to activating oncogenes and support a direct role of the ER as a gatekeeper of tumour control.
- Published
- 2006
- Full Text
- View/download PDF
48. Transcriptional activation by the Myc oncoprotein.
- Author
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Cole MD and Nikiforov MA
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, CREB-Binding Protein metabolism, Chromatin genetics, Chromatin metabolism, Genes, myc, Humans, Models, Biological, Mutation, Neoplasms genetics, Neoplasms metabolism, Nuclear Proteins metabolism, Protein Structure, Tertiary, Proto-Oncogene Proteins c-myc antagonists & inhibitors, Proto-Oncogene Proteins c-myc chemistry, Signal Transduction, Ubiquitin metabolism, Proto-Oncogene Proteins c-myc metabolism, Transcriptional Activation
- Abstract
The Myc transcription factor functions as a downstream effector of most mitogenic signals. Myc is synthesized rapidly in response to extracellular mitogenic signals, and blocking Myc induction abolishes or at least severely attenuates any mitogenic response. Furthermore, ectopic Myc expression can often bypass the requirement for extracellular signals for entry into S phase. Thus, the Myc transcription factor is both necessary and in many ways sufficient to promote the growth of diverse cell types. Given this potent biological activity, it is not surprising that mutations in the myc gene are among the most frequent in human and animal cancers. Understanding the molecular basis of Myc function has been a central issue in the fields of cancer biology and signal transduction for 20 years.
- Published
- 2006
- Full Text
- View/download PDF
49. The Mad and Myc basic domains are functionally equivalent.
- Author
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Nikiforov MA, Popov N, Kotenko I, Henriksson M, and Cole MD
- Subjects
- Amino Acid Sequence, Apoptosis physiology, Base Sequence, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, DNA Primers, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, HL-60 Cells, Humans, Molecular Sequence Data, Protein Binding, Proto-Oncogene Proteins c-myc chemistry, Proto-Oncogene Proteins c-myc metabolism, DNA-Binding Proteins physiology, Proto-Oncogene Proteins c-myc physiology, Repressor Proteins
- Abstract
The Myc/Max/Mad family of transcription factors plays a fundamental role in the regulation of cell proliferation, oncogenic transformation, and cell differentiation. However, it remains unclear whether different heterodimers, such as Myc/Max and Mad/Max, recognize the same or different target genes in vivo. We show by chromatin immunoprecipitation that Myc target genes are also recognized by Mad1 in differentiated HL60 cells. We also substituted the complete basic region of Myc for the corresponding region of Mad. Wild-type c-Myc was then compared with c-Myc(Mad-BR) in oncogenic transformation, regulation of cell proliferation, induction of apoptosis, activation of chromosomal gene expression, and direct binding to chromosomal sites by chromatin immunoprecipitation. We find that the wild-type c-Myc and c-Myc/MadBR proteins have indistinguishable biological activity and target gene recognition in vivo. These data are consistent with a model in which Myc and Mad regulate a common set of target genes.
- Published
- 2003
- Full Text
- View/download PDF
50. A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism.
- Author
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Nikiforov MA, Chandriani S, O'Connell B, Petrenko O, Kotenko I, Beavis A, Sedivy JM, and Cole MD
- Subjects
- Animals, Cell Separation, Cells, Cultured, Fibroblasts physiology, Flow Cytometry, Gene Library, Genes, myc, Glycine Hydroxymethyltransferase genetics, Humans, Mitochondria enzymology, Promoter Regions, Genetic, Proto-Oncogene Proteins c-myc genetics, Rats, Rats, Mutant Strains, Carbon metabolism, Cell Physiological Phenomena, Glycine Hydroxymethyltransferase metabolism, Proto-Oncogene Proteins c-myc metabolism
- Abstract
A cDNA library enriched with Myc-responsive cDNAs but depleted of myc cDNAs was used in a functional screen for growth enhancement in c-myc-null cells. A cDNA clone for mitochondrial serine hydroxymethyltransferase (mSHMT) that was capable of partial complementation of the growth defects of c-myc-null cells was identified. Expression analysis and chromatin immunoprecipitation demonstrated that mSHMT is a direct Myc target gene. Furthermore, a separate gene encoding the cytoplasmic isoform of the same enzyme is also a direct target of Myc regulation. SHMT enzymes are the major source of the one-carbon unit required for folate metabolism and for the biosynthesis of nucleotides and amino acids. Our data establish a novel functional link between Myc and the regulation of cellular metabolism.
- Published
- 2002
- Full Text
- View/download PDF
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