Abstract
Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis whose expression suppresses tumor growth in vivo. Like many angiogenesis-related genes, TSP-1 expression is tightly controlled by various mechanisms, but there is little data regarding the contribution of post-transcriptional processing to this regulation. NOL7 is a novel tumor suppressor that induces an antiangiogenic phenotype and suppresses tumor growth, in part through upregulation of TSP-1. Here we demonstrate that NOL7 is an mRNA-binding protein that must localize to the nucleoplasm to exert its antiangiogenic and tumor suppressive effects. There, it associates with the RNA-processing machinery and specifically interacts with TSP-1 mRNA through its 3′UTR. Reintroduction of NOL7 into SiHa cells increases luciferase expression through interaction with the TSP-1 3′UTR at both the mRNA and protein levels. NOL7 also increases endogenous TSP-1 mRNA half-life. Further, NOL7 post-transcriptional stabilization is observed in a subset of angiogenesis-related mRNAs, suggesting that the stabilization of TSP-1 may be part of a larger novel mechanism. These data demonstrate that NOL7 significantly alters TSP-1 expression and may be a master regulator that coordinates the post-transcriptional expression of key signaling factors critical for the regulation of the angiogenic phenotype.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Hanahan D, Weinberg RA . The hallmarks of cancer. Cell 2000; 100: 57–70.
Hanahan D, Weinberg RA . Hallmarks of cancer: the next generation. Cell (Research Support, NIH, Extramural Review) 2011; 144: 646–674.
Carmeliet P, Jain RK . Angiogenesis in cancer and other diseases. Nature 2000; 407: 249–257.
Folkman J . Role of angiogenesis in tumor growth and metastasis. Semin Oncol 2002; 29 (6 Suppl 16): 15–18.
Carmeliet P . Angiogenesis in health and disease. Nat Med 2003; 9: 653–660.
Bergers G, Hanahan D . Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 2008; 8: 592–603.
Ferrara N, Kerbel RS . Angiogenesis as a therapeutic target. Nature 2005; 438: 967–974.
Heath VL, Bicknell R . Anticancer strategies involving the vasculature. Nat Rev Clin Oncol 2009; 6: 395–404.
Quesada AR, Medina MA, Alba E . Playing only one instrument may be not enough: limitations and future of the antiangiogenic treatment of cancer. Bioessays 2007; 29: 1159–1168.
Nowak DG, Woolard J, Amin EM, Konopatskaya O, Saleem MA, Churchill AJ et al. Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors. J Cell Sci 2008; 121 (Pt 20): 3487–3495.
Rennel E, Waine E, Guan H, Schuler Y, Leenders W, Woolard J et al. The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice. Br J Cancer 2008; 98: 1250–1257.
He Y, Smith SK, Day KA, Clark DE, Licence DR, Charnock-Jones DS . Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity. Mol Endocrinol 1999; 13: 537–545.
Robinson CJ, Stringer SE . The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 2001; 114 (Pt 5): 853–865.
Harper SJ, Bates DO . VEGF-A splicing: the key to anti-angiogenic therapeutics? Nat Rev Cancer 2008; 8: 880–887.
Ferrara N, Davis-Smyth T . The biology of vascular endothelial growth factor. Endocr Rev 1997; 18: 4–25.
Yamazaki Y, Morita T . Molecular and functional diversity of vascular endothelial growth factors. Mol Divers 2006; 10: 515–527.
Hall-Pogar T, Zhang H, Tian B, Lutz CS . Alternative polyadenylation of cyclooxygenase-2. Nucleic Acids Res 2005; 33: 2565–2579.
Lukiw WJ, Bazan NG . Cyclooxygenase 2 RNA message abundance, stability, and hypervariability in sporadic Alzheimer neocortex. J Neurosci Res 1997; 50: 937–945.
Hargrove JL, Schmidt FH . The role of mRNA and protein stability in gene expression. FASEB J 1989; 3: 2360–2370.
Rastinejad F, Polverini PJ, Bouck NP . Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 1989; 56: 345–355.
Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA 1990; 87: 6624–6628.
Volpert OV . Modulation of endothelial cell survival by an inhibitor of angiogenesis thrombospondin-1: a dynamic balance. Cancer Metastasis Rev 2000; 19: 87–92.
Ren B, Yee KO, Lawler J, Khosravi-Far R . Regulation of tumor angiogenesis by thrombospondin-1. Biochim Biophys Acta 2006; 1765: 178–188.
Lawler J, Detmar M . Tumor progression: the effects of thrombospondin-1 and -2. Int J Biochem Cell Biol 2004; 36: 1038–1045.
Volpert OV, Stellmach V, Bouck N . The modulation of thrombospondin and other naturally occurring inhibitors of angiogenesis during tumor progression. Breast Cancer Res Treat 1995; 36: 119–126.
Bleuel K, Popp S, Fusenig NE, Stanbridge EJ, Boukamp P . Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc Natl Acad Sci USA 1999; 96: 2065–2070.
Streit M, Velasco P, Brown LF, Skobe M, Richard L, Riccardi L et al. Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am J Pathol 1999; 155: 441–452.
Weinstat-Saslow DL, Zabrenetzky VS, VanHoutte K, Frazier WA, Roberts DD, Steeg PS . Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res 1994; 54: 6504–6511.
Kang JH, Kim SA, Hong KJ . Induction of TSP1 gene expression by heat shock is mediated via an increase in mRNA stability. FEBS Lett 2006; 580: 510–516.
Okamoto M, Ono M, Uchiumi T, Ueno H, Kohno K, Sugimachi K et al. Up-regulation of thrombospondin-1 gene by epidermal growth factor and transforming growth factor beta in human cancer cells--transcriptional activation and messenger RNA stabilization. Biochim Biophys Acta 2002; 1574: 24–34.
Phelan MW, Forman LW, Perrine SP, Faller DV . Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells. J Lab Clin Med 1998; 132: 519–529.
Janz A, Sevignani C, Kenyon K, Ngo CV, Thomas-Tikhonenko A . Activation of the myc oncoprotein leads to increased turnover of thrombospondin-1 mRNA. Nucleic Acids Res 2000; 28: 2268–2275.
Laderoute KR, Alarcon RM, Brody MD, Calaoagan JM, Chen EY, Knapp AM et al. Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor. Clin Cancer Res 2000; 6: 2941–2950.
Penttinen RP, Kobayashi S, Bornstein P . Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proc Natl Acad Sci USA 1988; 85: 1105–1108.
Levy NS, Chung S, Furneaux H, Levy AP . Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J Biol Chem 1998; 273: 6417–6423.
Sengupta S, Jang BC, Wu MT, Paik JH, Furneaux H, Hla T . The RNA-binding protein HuR regulates the expression of cyclooxygenase-2. J Biol Chem 2003; 278: 25227–25233.
Sheflin LG, Zou AP, Spaulding SW . Androgens regulate the binding of endogenous HuR to the AU-rich 3'UTRs of HIF-1alpha and EGF mRNA. Biochem Biophys Res Commun 2004; 322: 644–651.
Dixon DA, Tolley ND, King PH, Nabors LB, McIntyre TM, Zimmerman GA et al. Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells. J Clin Invest 2001; 108: 1657–1665.
Mazan-Mamczarz K, Hagner PR, Corl S, Srikantan S, Wood WH, Becker KG et al. Post-transcriptional gene regulation by HuR promotes a more tumorigenic phenotype. Oncogene 2008; 27: 6151–6163.
Collart MA, Timmers HT . The eukaryotic Ccr4-not complex: a regulatory platform integrating mRNA metabolism with cellular signaling pathways? Prog Nucleic Acid Res Mol Biol 2004; 77: 289–322.
Denis CL, Chen J . The CCR4-NOT complex plays diverse roles in mRNA metabolism. Prog Nucleic Acid Res Mol Biol 2003; 73: 221–250.
Azzouz N, Panasenko OO, Colau G, Collart MA . The CCR4-NOT complex physically and functionally interacts with TRAMP and the nuclear exosome. PLoS ONE 2009; 4: e6760.
Garneau NL, Wilusz J, Wilusz CJ . The highways and byways of mRNA decay. Nat Rev Mol Cell Biol 2007; 8: 113–126.
West S, Gromak N, Proudfoot NJ . Human 5' --> 3' exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature 2004; 432: 522–525.
Teixeira A, Tahiri-Alaoui A, West S, Thomas B, Ramadass A, Martianov I et al. Autocatalytic RNA cleavage in the human beta-globin pre-mRNA promotes transcription termination. Nature 2004; 432: 526–530.
Kim M, Krogan NJ, Vasiljeva L, Rando OJ, Nedea E, Greenblatt JF et al. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 2004; 432: 517–522.
Kawauchi J, Mischo H, Braglia P, Rondon A, Proudfoot NJ . Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination. Genes Dev 2008; 22: 1082–1092.
El Hage A, Koper M, Kufel J, Tollervey D . Efficient termination of transcription by RNA polymerase I requires the 5' exonuclease Rat1 in yeast. Genes Dev 2008; 22: 1069–1081.
Richard P, Manley JL . Transcription termination by nuclear RNA polymerases. Genes Dev 2009; 23: 1247–1269.
Hilleren P, McCarthy T, Rosbash M, Parker R, Jensen TH . Quality control of mRNA 3'-end processing is linked to the nuclear exosome. Nature 2001; 413: 538–542.
de Almeida SF, Garcia-Sacristan A, Custodio N, Carmo-Fonseca M . A link between nuclear RNA surveillance, the human exosome and RNA polymerase II transcriptional termination. Nucleic Acids Res 2010; 38: 8015–8026.
Anderson JT, Wang X . Nuclear RNA surveillance: no sign of substrates tailing off. Crit Rev Biochem Mol Biol 2009; 44: 16–24.
Glaunsinger BA, Lee YJ . How tails define the ending: divergent roles for polyadenylation in RNA stability and gene expression. RNA BIOL 2010; 7: 13–17.
Mukherjee D, Gao M, O’Connor JP, Raijmakers R, Pruijn G, Lutz CS et al. The mammalian exosome mediates the efficient degradation of mRNAs that contain AU-rich elements. EMBO J 2002; 21: 165–174.
Houseley J, LaCava J, Tollervey D . RNA quality control by the exosome. Nat Rev Mol Cell Biol 2006; 7: 529–539.
Moore MJ . Nuclear RNA turnover. Cell 2002; 108: 431–434.
Vasudevan S, Peltz SW . Nuclear mRNA surveillance. Curr Opin Cell Biol 2003; 15: 332–337.
Brennan CM, Gallouzi IE, Steitz JA . Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J Cell Biol 2000; 151: 1–14.
Gallouzi IE, Brennan CM, Stenberg MG, Swanson MS, Eversole A, Maizels N et al. HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc Natl Acad Sci USA 2000; 97: 3073–3078.
Hasina R, Pontier AL, Fekete MJ, Martin LE, Qi XM, Brigaudeau C et al. NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype. Oncogene 2006; 25: 588–598.
Zhou G, Doçi CL, Lingen MW . Identification and functional analysis of NOL7 nuclear and nucleolar localization signals. BMC Cell Biol (Research Support, NIH, Extramural Research Support, Non-US Govt) 2010; 11: 74.
Doçi CL, Mankame TP, Langerman A, Ostler KR, Kanteti R, Best T et al. Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer. Int J Gynecol Pathol (Research Support, NIH, Extramural Research Support, Non-US Govt) 2012; 31: 15–24.
Neugebauer KM . On the importance of being co-transcriptional. J Cell Sci 2002; 115 (Pt 20): 3865–3871.
Zorio DA, Bentley DL . The link between mRNA processing and transcription: communication works both ways. Exp Cell Res 2004; 296: 91–97.
Lau NC, Kolkman A, van Schaik FM, Mulder KW, Pijnappel WW, Heck AJ et al. Human Ccr4-Not complexes contain variable deadenylase subunits. Biochem J 2009; 422: 443–453.
Ladomery MR, Harper SJ, Bates DO . Alternative splicing in angiogenesis: the vascular endothelial growth factor paradigm. Cancer Lett 2007; 249: 133–142.
Qiu Y, Hoareau-Aveilla C, Oltean S, Harper SJ, Bates DO . The anti-angiogenic isoforms of VEGF in health and disease. Biochem Soc Trans 2009; 37 (Pt 6): 1207–1213.
Dibrov A, Kashour T, Amara FM . The role of transforming growth factor beta signaling in messenger RNA stability. Growth Factors 2006; 24: 1–11.
Hashimoto-Uoshima M, Yan YZ, Schneider G, Aukhil I . The alternatively spliced domains EIIIB and EIIIA of human fibronectin affect cell adhesion and spreading. J Cell Sci 1997; 110: Pt 18 2271–2280.
Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C et al. Alternative isoform regulation in human tissue transcriptomes. Nature 2008; 456: 470–476.
Touriol C, Roussigne M, Gensac MC, Prats H, Prats AC . Alternative translation initiation of human fibroblast growth factor 2 mRNA controlled by its 3'-untranslated region involves a Poly(A) switch and a translational enhancer. J Biol Chem 2000; 275: 19361–19367.
Claffey KP, Shih SC, Mullen A, Dziennis S, Cusick JL, Abrams KR et al. Identification of a human VPF/VEGF 3' untranslated region mediating hypoxia-induced mRNA stability. Mol Biol Cell 1998; 9: 469–481.
Levy AP, Levy NS, Goldberg MA . Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996; 271: 2746–2753.
Levy AP, Levy NS, Wegner S, Goldberg MA . Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem 1995; 270: 13333–13340.
Levy NS, Goldberg MA, Levy AP . Sequencing of the human vascular endothelial growth factor (VEGF) 3' untranslated region (UTR): conservation of five hypoxia-inducible RNA-protein binding sites. Biochim Biophys Acta 1997; 1352: 167–173.
Kanies CL, Smith JJ, Kis C, Schmidt C, Levy S, Khabar KS et al. Oncogenic Ras and transforming growth factor-beta synergistically regulate AU-rich element-containing mRNAs during epithelial to mesenchymal transition. Mol Cancer Res 2008; 6: 1124–1136.
Chung JH, Rho JK, Xu X, Lee JS, Yoon HI, Lee CT et al. Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs. Lung Cancer 2011; 73: 176–182.
Thiery JP, Acloque H, Huang RY, Nieto MA . Epithelial-mesenchymal transitions in development and disease. Cell 2009; 139: 871–890.
Lingen MW . Endothelial cell migration assay. A quantitative assay for prediction of in vivo biology. Methods Mol Med (Research Support, US Govt, PHS) 2003; 78: 337–347.
Acknowledgements
We wish to thank Drs Ira Wool and Yuen-Ling Chan for their assistance in sucrose gradient ultracentrifugation. This work was supported in part by Illinois Department of Public Health Penny Severns Cancer Research Fund (MWL).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies the paper on the Oncogene website
Rights and permissions
About this article
Cite this article
Doçi, C., Zhou, G. & Lingen, M. The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression. Oncogene 32, 4377–4386 (2013). https://doi.org/10.1038/onc.2012.464
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/onc.2012.464