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Prox1 suppresses the proliferation of neuroblastoma cells via a dual action in p27-Kip1 and Cdc25A

Oncogene volume 32, pages 947–960 (2013)Cite this article

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Abstract

Neuroblastoma is a pediatric tumor that originates from precursor cells of the sympathetic nervous system with less than 40% long-term survival in children diagnosed with high-risk disease. These clinical observations underscore the need for novel insights in the mechanisms of malignant transformation and progression. Accordingly, it was recently reported that Prox1, a homeobox transcription regulator, is expressed in higher levels in human neuroblastoma with favorable prognosis. Consistently, we have recently shown that Prox1 exerts a strong antiproliferative effect on neural precursor cells during embryonic development. Thus, Prox1 is a candidate gene with a critical role in suppressing malignant neuroblastoma transformation. Here, we provide evidence that Prox1 strongly suppresses the proliferation of mouse and human neuroblastoma cell lines and blocks the growth of neuroblastoma tumors in SCID mice. Conversely, short hairpin RNA (shRNA) -mediated knockdown of basal Prox1 expression significantly induces proliferation, genomic instability and the ability of neuroblastoma cells to form tumors. Mechanistically, analysis of an inducible Prox1-overexpressing Neuro2A cell line indicates that Prox1 is sufficient to suppress CyclinD1, CyclinA and CyclinB1, consistent with a role in cell cycle arrest. Surprisingly, Prox1 strongly induces CyclinE1 expression in the same system despite its action on blocking cell cycle progression, which could account for the context dependent oncogenic function of Prox1. Most importantly, Prox1 was sufficient to decrease Cdc25A and induce p27-Kip1, but not p21-Cip1 or p53. By alleviating the Prox1 action in Cdc25A and p27-Kip1 expression, we were able to rescue its effect on cell cycle arrest. Together these data suggest that Prox1 negatively regulates neuroblastoma carcinogenesis through suppression of Cdc25A and induction of p27-Kip1 to counteract CyclinE1 overexpression and block cell cycle progression. Furthermore, these observations render Prox1 a candidate target for the treatment of neuroblastoma tumors.

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References

  1. Maris JM, Hogarty MD, Bagatell R, Cohn SL . Neuroblastoma. Lancet 2007; 369: 2106–2120.

    Article  CAS  Google Scholar 

  2. Pearson AD, Pinkerton CR, Lewis IJ, Imeson J, Ellershaw C, Machin D . High-dose rapid and standard induction chemotherapy for patients aged over 1 year with stage 4 neuroblastoma: a randomised trial. Lancet Oncol 2008; 9: 247–256.

    Article  CAS  Google Scholar 

  3. Becker J, Wang B, Pavlakovic H, Buttler K, Wilting J . Homeobox transcription factor Prox1 in sympathetic ganglia of vertebrate embryos: correlation with human stage 4s neuroblastoma. Pediatr Res 2010; 68: 112–117.

    Article  CAS  Google Scholar 

  4. Wigle JT, Harvey N, Detmar M, Lagutina I, Grosveld G, Gunn MD et al. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J 2002; 21: 1505–1513.

    Article  CAS  Google Scholar 

  5. Kaltezioti V, Kouroupi G, Oikonomaki M, Mantouvalou E, Stergiopoulos A, Charonis A et al. Prox1 regulates the notch1-mediated inhibition of neurogenesis. PLoS Biol 2010; 8: e1000565.

    Article  CAS  Google Scholar 

  6. Chang HH, Lee H, Hu MK, Tsao PN, Juan HF, Huang MC et al. Notch1 expression predicts an unfavorable prognosis and serves as a therapeutic target of patients with neuroblastoma. Clin Cancer Res 2010; 16: 4411–4420.

    Article  CAS  Google Scholar 

  7. Risebro CA, Searles RG, Melville AA, Ehler E, Jina N, Shah S et al. Prox1 maintains muscle structure and growth in the developing heart. Development 2009; 136: 495–505.

    Article  CAS  Google Scholar 

  8. Sosa-Pineda B, Wigle JT, Oliver G . Hepatocyte migration during liver development requires Prox1. Nat Genet 2000; 25: 254–255.

    Article  CAS  Google Scholar 

  9. Wigle JT, Chowdhury K, Gruss P, Oliver G . Prox1 function is crucial for mouse lens-fibre elongation. Nat Genet 1999; 21: 318–322.

    Article  CAS  Google Scholar 

  10. Wigle JT, Oliver G . Prox1 function is required for the development of the murine lymphatic system. Cell 1999; 98: 769–778.

    Article  CAS  Google Scholar 

  11. Schneider M, Buchler P, Giese N, Giese T, Wilting J, Buchler MW et al. Role of lymphangiogenesis and lymphangiogenic factors during pancreatic cancer progression and lymphatic spread. Int J Oncol 2006; 28: 883–890.

    CAS  PubMed  Google Scholar 

  12. Shimoda M, Takahashi M, Yoshimoto T, Kono T, Ikai I, Kubo H . A homeobox protein, prox1, is involved in the differentiation, proliferation, and prognosis in hepatocellular carcinoma. Clin Cancer Res 2006; 12 (20 Pt 1): 6005–6011.

    Article  CAS  Google Scholar 

  13. Nagai H, Li Y, Hatano S, Toshihito O, Yuge M, Ito E et al. Mutations and aberrant DNA methylation of the PROX1 gene in hematologic malignancies. Genes Chromosomes Cancer 2003; 38: 13–21.

    Article  CAS  Google Scholar 

  14. Versmold B, Felsberg J, Mikeska T, Ehrentraut D, Kohler J, Hampl JA et al. Epigenetic silencing of the candidate tumor suppressor gene PROX1 in sporadic breast cancer. Int J Cancer 2007; 121: 547–554.

    Article  CAS  Google Scholar 

  15. Dudas J, Mansuroglu T, Moriconi F, Haller F, Wilting J, Lorf T et al. Altered regulation of Prox1-gene-expression in liver tumors. BMC Cancer 2008; 8: 92.

    Article  Google Scholar 

  16. Takahashi M, Yoshimoto T, Shimoda M, Kono T, Koizumi M, Yazumi S et al. Loss of function of the candidate tumor suppressor prox1 by RNA mutation in human cancer cells. Neoplasia 2006; 8: 1003–1010.

    Article  CAS  Google Scholar 

  17. Petrova TV, Nykanen A, Norrmen C, Ivanov KI, Andersson LC, Haglund C et al. Transcription factor PROX1 induces colon cancer progression by promoting the transition from benign to highly dysplastic phenotype. Cancer Cell 2008; 13: 407–419.

    Article  CAS  Google Scholar 

  18. Louvi A, Artavanis-Tsakonas S . Notch signalling in vertebrate neural development. Nat Rev Neurosci 2006; 7: 93–102.

    Article  CAS  Google Scholar 

  19. Tsarovina K, Schellenberger J, Schneider C, Rohrer H . Progenitor cell maintenance and neurogenesis in sympathetic ganglia involves Notch signaling. Mol Cell Neurosci 2008; 37: 20–31.

    Article  CAS  Google Scholar 

  20. Baxter SA, Cheung DY, Bocangel P, Kim HK, Herbert K, Douville JM et al. Regulation of the lymphatic endothelial cell cycle by the PROX1 homeodomain protein. Biochem Biophys Acta 2011; 1813: 201–212.

    Article  CAS  Google Scholar 

  21. Choksi SP, Southall TD, Bossing T, Edoff K, de Wit E, Fischer BE et al. Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells. Dev Cell 2006; 11: 775–789.

    Article  CAS  Google Scholar 

  22. Li L, Vaessin H . Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis. Genes Dev 2000; 14: 147–151.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Rother K, Kirschner R, Sanger K, Bohlig L, Mossner J, Engeland K . p53 downregulates expression of the G1/S cell cycle phosphatase Cdc25A. Oncogene 2007; 26: 1949–1953.

    Article  CAS  Google Scholar 

  24. Timofeev O, Cizmecioglu O, Settele F, Kempf T, Hoffmann I . Cdc25 phosphatases are required for timely assembly of CDK1-cyclin B at the G2/M transition. J Biol Chem 2010; 285: 16978–16990.

    Article  CAS  Google Scholar 

  25. Itoh Y, Masuyama N, Nakayama K, Nakayama KI, Gotoh Y . The cyclin-dependent kinase inhibitors p57 and p27 regulate neuronal migration in the developing mouse neocortex. J Biol Chem 2007; 282: 390–396.

    Article  CAS  Google Scholar 

  26. Rodriguez-Niedenfuhr M, Papoutsi M, Christ B, Nicolaides KH, von Kaisenberg CS, Tomarev SI et al. Prox1 is a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium and lymphangioblasts. Anat Embryol 2001; 204: 399–406.

    Article  CAS  Google Scholar 

  27. Hope KJ, Cellot S, Ting SB, MacRae T, Mayotte N, Iscove NN et al. An RNAi screen identifies Msi2 and Prox1 as having opposite roles in the regulation of hematopoietic stem cell activity. Cell Stem Cell 2010; 7: 101–113.

    Article  CAS  Google Scholar 

  28. Hope KJ, Sauvageau G . Roles for MSI2 and PROX1 in hematopoietic stem cell activity. Curr Opin Hematol 2011; 18: 203–207.

    Article  CAS  Google Scholar 

  29. Dyer MA, Livesey FJ, Cepko CL, Oliver G . Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina. Nat Genet 2003; 34: 53–58.

    Article  CAS  Google Scholar 

  30. Elkouris M, Balaskas N, Poulou M, Politis PK, Panayiotou E, Malas S et al. Sox1 maintains the undifferentiated state of cortical neural progenitor cells via the suppression of Prox1-mediated cell cycle exit and neurogenesis. Stem Cells 2011; 29: 89–98.

    Article  CAS  Google Scholar 

  31. Akagami M, Kawada K, Kubo H, Kawada M, Takahashi M, Kaganoi J et al. Transcriptional Factor Prox1 Plays an Essential Role in the Antiproliferative Action of Interferon-gamma in Esophageal Cancer Cells. Ann Surg Oncol 2011; 18: 3868–3877.

    Article  Google Scholar 

  32. Laerm A, Helmbold P, Goldberg M, Dammann R, Holzhausen HJ, Ballhausen WG . Prospero-related homeobox 1 (PROX1) is frequently inactivated by genomic deletions and epigenetic silencing in carcinomas of the bilary system. J Hepatol 2007; 46: 89–97.

    Article  CAS  Google Scholar 

  33. Bello B, Reichert H, Hirth F . The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila. Development 2006; 133: 2639–2648.

    Article  CAS  Google Scholar 

  34. Betschinger J, Mechtler K, Knoblich JA . Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells. Cell 2006; 124: 1241–1253.

    Article  CAS  Google Scholar 

  35. Lee CY, Wilkinson BD, Siegrist SE, Wharton RP, Doe CQ . Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. Dev Cell 2006; 10: 441–449.

    Article  CAS  Google Scholar 

  36. Colonques J, Ceron J, Reichert H, Tejedor FJ . A transient expression of Prospero promotes cell cycle exit of Drosophila postembryonic neurons through the regulation of Dacapo. PLoS One 2011; 6: e19342.

    Article  CAS  Google Scholar 

  37. Liu TH, Li L, Vaessin H . Transcription of the Drosophila CKI gene dacapo is regulated by a modular array of cis-regulatory sequences. Mech Dev 2002; 112: 25–36.

    Article  CAS  Google Scholar 

  38. Southall TD, Brand AH . Neural stem cell transcriptional networks highlight genes essential for nervous system development. EMBO J 2009; 28: 3799–3807.

    Article  CAS  Google Scholar 

  39. Kamiya A, Kakinuma S, Onodera M, Miyajima A, Nakauchi H . Prospero-related homeobox 1 and liver receptor homolog 1 coordinately regulate long-term proliferation of murine fetal hepatoblasts. Hepatology 2008; 48: 252–264.

    Article  CAS  Google Scholar 

  40. Petrova TV, Makinen T, Makela TP, Saarela J, Virtanen I, Ferrell RE et al. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J 2002; 21: 4593–4599.

    Article  CAS  Google Scholar 

  41. Griffiths RL, Hidalgo A . Prospero maintains the mitotic potential of glial precursors enabling them to respond to neurons. EMBO J 2004; 23: 2440–2450.

    Article  CAS  Google Scholar 

  42. Airoldi I, Meazza R, Croce M, Di Carlo E, Piazza T, Cocco C et al. Low-dose interferon-gamma-producing human neuroblastoma cells show reduced proliferation and delayed tumorigenicity. Br J Cancer 2004; 90: 2210–2218.

    Article  CAS  Google Scholar 

  43. Ribatti D, Nico B, Pezzolo A, Vacca A, Meazza R, Cinti R et al. Angiogenesis in a human neuroblastoma xenograft model: mechanisms and inhibition by tumour-derived interferon-gamma. Br J Cancer 2006; 94: 1845–1852.

    Article  CAS  Google Scholar 

  44. Kazenwadel J, Michael MZ, Harvey NL . Prox1 expression is negatively regulated by miR-181 in endothelial cells. Blood 2010; 116: 2395–2401.

    Article  CAS  Google Scholar 

  45. Chen Y, Stallings RL . Differential patterns of microRNA expression in neuroblastoma are correlated with prognosis, differentiation, and apoptosis. Cancer Res 2007; 67: 976–983.

    Article  CAS  Google Scholar 

  46. Schulte JH, Marschall T, Martin M, Rosenstiel P, Mestdagh P, Schlierf S et al. Deep sequencing reveals differential expression of microRNAs in favorable versus unfavorable neuroblastoma. Nucleic Acids Res 2010; 38: 5919–5928.

    Article  CAS  Google Scholar 

  47. Georgopoulou N, Hurel C, Politis PK, Gaitanou M, Matsas R, Thomaidou D . BM88 is a dual function molecule inducing cell cycle exit and neuronal differentiation of neuroblastoma cells via cyclin D1 down-regulation and retinoblastoma protein hypophosphorylation. J Biol Chem 2006; 281: 33606–33620.

    Article  CAS  Google Scholar 

  48. Politis PK, Akrivou S, Hurel C, Papadodima O, Matsas R . BM88/Cend1 is involved in histone deacetylase inhibition-mediated growth arrest and differentiation of neuroblastoma cells. FEBS Lett 2008; 582: 741–748.

    Article  CAS  Google Scholar 

  49. Politis PK, Makri G, Thomaidou D, Geissen M, Rohrer H, Matsas R . BM88/CEND1 coordinates cell cycle exit and differentiation of neuronal precursors. Proc Natl Acad Sci USA 2007; 104: 17861–17866.

    Article  CAS  Google Scholar 

  50. Stellas D, Karameris A, Patsavoudi E . Monoclonal antibody 4C5 immunostains human melanomas and inhibits melanoma cell invasion and metastasis. Clin Cancer Res 2007; 13: 1831–1838.

    Article  CAS  Google Scholar 

  51. Hassepass I, Voit R, Hoffmann I . Phosphorylation at serine 75 is required for UV-mediated degradation of human Cdc25A phosphatase at the S-phase checkpoint. J Biol Chem 2003; 278: 29824–29829.

    Article  CAS  Google Scholar 

  52. Nilsson I, Hoffmann I . Cell cycle regulation by the Cdc25 phosphatase family. Prog Cell Cycle Res 2000; 4: 107–114.

    Article  CAS  Google Scholar 

  53. Sexl V, Diehl JA, Sherr CJ, Ashmun R, Beach D, Roussel MF . A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2. Oncogene 1999; 18: 573–582.

    Article  CAS  Google Scholar 

  54. Chu IM, Hengst L, Slingerland JM . The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer 2008; 8: 253–267.

    Article  CAS  Google Scholar 

  55. Grana X, Reddy EP . Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 1995; 11: 211–219.

    CAS  Google Scholar 

  56. Liu S, Yamauchi H . p27-Associated G1 arrest induced by hinokitiol in human malignant melanoma cells is mediated via down-regulation of pRb, Skp2 ubiquitin ligase, and impairment of Cdk2 function. Cancer Lett 2009; 286: 240–249.

    Article  CAS  Google Scholar 

  57. Molinari M . Cell cycle checkpoints and their inactivation in human cancer. Cell Prolif 2000; 33: 261–274.

    Article  CAS  Google Scholar 

  58. Hengst L, Reed SI . Inhibitors of the Cip/Kip family. Curr Top Microbiol Immunol 1998; 227: 25–41.

    CAS  PubMed  Google Scholar 

  59. Sherr CJ, Roberts JM . CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999; 13: 1501–1512.

    Article  CAS  Google Scholar 

  60. Nigg EA . Targets of cyclin-dependent protein kinases. Curr Opin Cell Biol 1993; 5: 187–193.

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to Yukiko Gotoh, Argyris Efstratiadis, Kurt Engeland, Ingrid Hoffman, Aristidis Charonis, Valeria Kaltezioti, Chrisiida Tsimplouli, Kostas Vekrellis, Katerina Melachroinou, Elena Katsantoni, Anthony Gavalas, Dimitra Mangoura, Maria Roubelakis, Eleni Siapati, Stamatis Pagakis, BIU facility of BRAFAA and Addgene for plasmids, reagents, discussions and technical help.

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  1. Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

    I P Foskolou, D Stellas, I Rozani, M D Lavigne & P K Politis

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  1. I P Foskolou
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Foskolou, I., Stellas, D., Rozani, I. et al. Prox1 suppresses the proliferation of neuroblastoma cells via a dual action in p27-Kip1 and Cdc25A. Oncogene 32, 947–960 (2013). https://doi.org/10.1038/onc.2012.129

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