Suppression of Myc oncogenic activity by nucleostemin haploinsufficiency

Nucleostemin (NS), a nucleolar GTPase, is highly expressed in stem/progenitor cells and in most cancer cells. However, little is known about the regulation of its expression. Here, we identify the NS gene as a novel direct transcriptional target of the c-Myc oncoprotein. We show that Myc overexpression enhances NS transcription in cultured cells and in pre-neoplastic B cells from Eμ-myc transgenic mice. Consistent with NS being downstream of Myc, NS expression parallels that of Myc in a large panel of human cancer cell lines. Using chromatin immunoprecipitation we show that c-Myc binds to a well-conserved E-box in the NS promoter. Critically, we show NS haploinsufficiency profoundly delays Myc-induced cancer formation in vivo. NS+/−Eμ-myc transgenic mice have much slower rates of B-cell lymphoma development, with life spans twice that of their wild-type littermates. Moreover, we demonstrate that NS is essential for the proliferation of Myc-overexpressing cells in cultured cells and in vivo: impaired lymphoma development was associated with a drastic decrease of c-Myc-induced proliferation of pre-tumoural B cells. Finally, we provide evidence that in cell culture NS controls cell proliferation independently of p53 and that NS haploinsufficiency significantly delays lymphomagenesis in p53-deficient mice. Together these data indicate that NS functions downstream of Myc as a rate-limiting regulator of cell proliferation and transformation, independently from its putative role within the p53 pathway. Targeting NS is therefore expected to compromise early tumour development irrespectively of the p53 status.

[1]  Michael M. Murphy,et al.  Oncogenic transformation in the absence of Xrcc4 targets peripheral B cells that have undergone editing and switching , 2008, The Journal of experimental medicine.

[2]  Meland,et al.  The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. , 2002, The New England journal of medicine.

[3]  Y. Atlasi,et al.  Differential expression of nucleostemin, a stem cell marker, and its variants in different types of brain tumors , 2010, Molecular carcinogenesis.

[4]  L. Romanova,et al.  Critical Role of Nucleostemin in Pre-rRNA Processing* , 2009, Journal of Biological Chemistry.

[5]  R. Weinberg,et al.  Tumor spectrum analysis in p53-mutant mice , 1994, Current Biology.

[6]  S. McMahon,et al.  Metastasis-associated protein 1 (MTA1) is an essential downstream effector of the c-MYC oncoprotein. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  D. Felsher,et al.  The interaction between Myc and Miz1 is required to antagonize TGFbeta-dependent autocrine signaling during lymphoma formation and maintenance. , 2010, Genes & development.

[8]  L. Penn,et al.  Mechanism for the transcriptional repression by c-Myc on PDGF (β)-receptor , 2001 .

[9]  Jim Yang,et al.  Stat3 and c-Myc Genome-Wide Promoter Occupancy in Embryonic Stem Cells , 2008, PloS one.

[10]  J. Massagué,et al.  Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage. , 2002, Nature.

[11]  W. Gu,et al.  Non-transcriptional control of DNA replication by c-Myc , 2007, Nature.

[12]  M. Henriksson,et al.  The Myc oncoprotein as a therapeutic target for human cancer. , 2006, Seminars in cancer biology.

[13]  J L Cleveland,et al.  Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. , 1998, Genes & development.

[14]  Cai-yun Zhou,et al.  Stem-cell-abundant proteins Nanog, Nucleostemin and Musashi1 are highly expressed in malignant cervical epithelial cells , 2008, BMC Cancer.

[15]  Hua Lu,et al.  Nucleostemin: Another nucleolar "Twister" of the p53-MDM2 loop. , 2010, Cell cycle.

[16]  K. Itahana,et al.  Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23 , 2004, Molecular and Cellular Biology.

[17]  R. Palmiter,et al.  c-myc-induced lymphomagenesis in transgenic mice and the role of the Pvt-1 locus in lymphoid neoplasia. , 1986, Current topics in microbiology and immunology.

[18]  Si-JinLiu,et al.  Role of nucleostemin in growth regulation of gastric cancer,liver cancer and other malignancies , 2004 .

[19]  Olufunmilayo I Olopade,et al.  MYC in breast tumor progression , 2008, Expert review of anticancer therapy.

[20]  Jonghwan Kim,et al.  Global Identification of Myc Target Genes Reveals Its Direct Role in Mitochondrial Biogenesis and Its E-Box Usage In Vivo , 2008, PloS one.

[21]  Wenyi Wei,et al.  Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. , 1997, Science.

[22]  Peggy J. Farnham,et al.  Analysis of Myc Bound Loci Identified by CpG Island Arrays Shows that Max Is Essential for Myc-Dependent Repression , 2003, Current Biology.

[23]  John M Sedivy,et al.  A Large Scale Genetic Analysis of c-Myc-regulated Gene Expression Patterns* 210 , 2003, The Journal of Biological Chemistry.

[24]  L. Donehower,et al.  Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours , 1992, Nature.

[25]  G. Wahl,et al.  c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: a mechanism for oncogene-induced genetic instability. , 2002, Molecular cell.

[26]  Howard Y. Chang,et al.  MYC can induce DNA breaks in vivo and in vitro independent of reactive oxygen species. , 2006, Cancer research.

[27]  Zhiping Weng,et al.  Global mapping of c-Myc binding sites and target gene networks in human B cells , 2006, Proceedings of the National Academy of Sciences.

[28]  Lingjun Meng,et al.  Cancer esearch or and Stem Cell Biology or-Initiating Function of Nucleostemin-Enriched R mary Tumor Cells in , 2010 .

[29]  L. Yajun,et al.  The effect of knocking-down nucleostemin gene expression on the in vitro proliferation and in vivo tumorigenesis of HeLa cells. , 2004, Journal of experimental & clinical cancer research : CR.

[30]  J. Sedivy,et al.  A cell culture model system for genetic analyses of the cell cycle by targeted homologous recombination. , 1993, Oncogene.

[31]  M. Dai,et al.  Ribosomal Protein L23 Activates p53 by Inhibiting MDM2 Function in Response to Ribosomal Perturbation but Not to Translation Inhibition , 2004, Molecular and Cellular Biology.

[32]  P. Pandolfi,et al.  The nucleolus: at the stem of immortality , 2003, Nature Medicine.

[33]  John M. Sedivy,et al.  c-Myc Regulates Cyclin D-Cdk4 and -Cdk6 Activity but Affects Cell Cycle Progression at Multiple Independent Points , 1999, Molecular and Cellular Biology.

[34]  G. Wahl,et al.  MDM2 and MDM4: p53 regulators as targets in anticancer therapy. , 2007, The international journal of biochemistry & cell biology.

[35]  S. De Clercq,et al.  Widespread Overexpression of Epitope-Tagged Mdm4 Does Not Accelerate Tumor Formation In Vivo , 2010, Molecular and Cellular Biology.

[36]  L. Penn,et al.  Mechanism for the transcriptional repression by c-Myc on PDGF beta-receptor. , 2001, Journal of cell science.

[37]  M. Dai,et al.  Aberrant Expression of Nucleostemin Activates p53 and Induces Cell Cycle Arrest via Inhibition of MDM2 , 2008, Molecular and Cellular Biology.

[38]  S. Mai,et al.  Genomic instability in MycER-activated Rat1A-MycER cells , 1996, Chromosome Research.

[39]  Y. Qi,et al.  p19ARF directly and differentially controls the functions of c-Myc independently of p53 , 2004, Nature.

[40]  Clifford A. Meyer,et al.  Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells , 2005, Genome Biology.

[41]  E. Appella,et al.  Phosphorylation of murine p53 at ser-18 regulates the p53 responses to DNA damage. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[42]  Andrea Cocito,et al.  Genomic targets of the human c-Myc protein. , 2003, Genes & development.

[43]  W. Wurst,et al.  Evolutionarily Conserved Role of Nucleostemin: Controlling Proliferation of Stem/Progenitor Cells during Early Vertebrate Development , 2006, Molecular and Cellular Biology.

[44]  G. Mortier,et al.  qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data , 2007, Genome Biology.

[45]  S. Perkins,et al.  Calorie restriction delays spontaneous tumorigenesis in p53-knockout transgenic mice. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[46]  M. Macleod,et al.  Effects of estrogen on global gene expression: identification of novel targets of estrogen action. , 2000, Cancer research.

[47]  J. Massagué,et al.  Repression of p15INK4b expression by Myc through association with Miz-1 , 2001, Nature Cell Biology.

[48]  Michael Q. Zhang,et al.  A global transcriptional regulatory role for c-Myc in Burkitt's lymphoma cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[49]  R. Eisenman,et al.  Myc's broad reach. , 2008, Genes & development.

[50]  W. Hahn,et al.  Abolition of Cyclin-Dependent Kinase Inhibitor p16Ink4a and p21Cip1/Waf1 Functions Permits Ras-Induced Anchorage-Independent Growth in Telomerase-Immortalized Human Fibroblasts , 2003, Molecular and Cellular Biology.

[51]  Davide Ruggero,et al.  Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency , 2008, Nature.

[52]  M. Matin,et al.  Knocking‐down the expression of nucleostemin significantly decreases rate of proliferation of rat bone marrow stromal stem cells in an apparently p53‐independent manner , 2008, Cell proliferation.

[53]  L. Penn,et al.  The Oscar-worthy role of Myc in apoptosis. , 2006, Seminars in cancer biology.

[54]  A. Trumpp,et al.  Genome-wide mapping of Myc binding and gene regulation in serum-stimulated fibroblasts , 2011, Oncogene.

[55]  P. Fernandez,et al.  Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. , 2001, Genes & development.

[56]  B. Mitchell,et al.  Depletion of guanine nucleotides leads to the Mdm2-dependent proteasomal degradation of nucleostemin. , 2009, Cancer research.

[57]  M. Dai,et al.  Inhibition of MDM2-mediated p53 Ubiquitination and Degradation by Ribosomal Protein L5* , 2004, Journal of Biological Chemistry.

[58]  Kathryn A. O’Donnell,et al.  The c-Myc target gene network. , 2006, Seminars in cancer biology.

[59]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[60]  J. Cleveland,et al.  Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. , 1991, Oncogene.

[61]  M. Roussel,et al.  Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis. , 1999, Genes & development.

[62]  J. Wang,et al.  Characterization of differential properties of rabbit tendon stem cells and tenocytes , 2010, BMC musculoskeletal disorders.

[63]  J. Sedivy,et al.  Loss of protooncogene c-Myc function impedes G1 phase progression both before and after the restriction point. , 2003, Molecular biology of the cell.

[64]  Yongchang Chen,et al.  Cloning of the nucleostemin gene and its function in transforming human embryonic bone marrow mesenchymal stem cells into F6 tumor cells. , 2005, International journal of molecular medicine.

[65]  Richard Simon,et al.  Molecular diagnosis of Burkitt's lymphoma. , 2006, The New England journal of medicine.

[66]  L. Staudt,et al.  A library of gene expression signatures to illuminate normal and pathological lymphoid biology , 2006, Immunological reviews.

[67]  R. McKay,et al.  A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. , 2002, Genes & development.

[68]  M. Roussel,et al.  Apoptosis Triggered by Myc-Induced Suppression of Bcl-XL or Bcl-2 Is Bypassed during Lymphomagenesis , 2001, Molecular and Cellular Biology.

[69]  M. Eilers,et al.  Control of cell proliferation by Myc. , 1998, Trends in cell biology.

[70]  Gerard I. Evan,et al.  Induction of apoptosis in fibroblasts by c-myc protein , 1992, Cell.

[71]  M. Eilers,et al.  Transcriptional regulation and transformation by Myc proteins , 2005, Nature Reviews Molecular Cell Biology.

[72]  J. Sedivy,et al.  Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[73]  J. Marine Pharmacological rescue of p53 in cancer therapy: widening the sensitive tumor spectrum by targeting MDMX. , 2010, Cancer cell.