Prognostic and Biological Role of Neurotrophin- Receptor TrkA and TrkB in Neuroblastoma[1]

Abstract:

Expression of different neurotrophin receptors of the tyrosine kinase (Trk) family plays an important role in the biology and clinical behavior of neuroblastomas (NB). Observations from several independent studies suggest that high expression of TrkA is present in NB with favorable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavorable, aggressive NB with MYCN-amplification. To determine expression of Trk receptors and ligands in primary NB, we developed a reliable semiquantitative duplex RT-PCR protocol, that requires only 1 μg RNA per tumor sample. Activation of TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events and promotes neuronal differentiation in vitro. Activation of TrkB by its ligand brain derived neurotrophic factor (BDNF) has been associated with proliferation and survival of NB cells. To study Trk signal transduction pathways and their biological effects in NB, we stably expressed TrkA and TrkB cDNA in the human NB cell line SH-SY5Y. Introduction of TrkA and TrkB restored responsiveness of SH-SY5Y cells to the ligands NGF and BDNF, respectively, and resulted in morphological differentiation. Expression of TrkA resulted in growth inhibition of the transfectants compared to parental cells, whereas TrkB transfectants demonstrated an increased proliferation rate. Further insight into the differences of TrkA and TrkB signaling may suggest new options for the treatment of NB. As expression of TrkA is a strong prognostic factor especially in MYCN non-amplified NB, a prospective study of Trk receptor expression using RT-PCR should be performed for German neuroblastoma patients.

Abbreviations

Trk (Tyrosine Kinase)

NB (Neuroblastoma)

NGF (Nerve Growth Factor)

BDNF (Brain Derived Neurotrophic Factor)

RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction)

GAPD (Glyceraldehyde-3-phosphate-dehydrogenase)

Die Expression verschiedener Neurotrophinrezeptoren der Tyrosinkinase-(Trk)Familie spielt eine bedeutende Rolle für das biologische und klinische Verhalten von Neuroblastomen (NB). Bisherige Daten verschiedener unabhängiger Studien belegen den Zusammenhang einer hohen Expression von TrkA mit günstigen biologischen Eigenschaften der Tumoren und einer höheren Überlebensrate der Neuroblastompatienten, während TrkB überwiegend von biologisch ungünstigen, aggressiven und MYCN-amplifizierten NB exprimiert wird. Um die Expression von Trk-Rezeptoren und Liganden in primären NB zu bestimmen, haben wir eine zuverlässige, semiquantitative Duplex-RT-PCR-Methode entwickelt, die nur 1 μg RNA pro Tumorprobe erfordert. Aktivierung von TrkA durch den spezifischen Liganden Nerve Growth Factor (NGF) führt über eine Kaskade von Signalelementen zu neuronaler Differenzierung von NB-Zellen in vitro. Aktivierung von TrkB durch den Liganden Brain Derived Neurotrophic Factor (BDNF) wird mit vermehrtem Wachstum und Überleben von NB-Zellen in Verbindung gebracht. Um die Signalwege und biologischen Effekte der Trk-Rezeptoren in Neuroblastomen näher zu untersuchen, transfizierten wir die humane NB-Zellinie SH-SY5Y jeweils mit TrkA und TrkB cDNA. Transfektion von TrkA oder TrkB bewirkte ein wiederhergestelltes Reaktionsvermögen von SH-SY5Y-Zellen auf den jeweiligen Liganden NGF oder BDNF und resultierte in morphologischer Differenzierung beider Zellarten. Expression von TrkA führte zu deutlicher Wachstumsverzögerung der transfizierten Zellen im Vergleich zu parentalen SY5Y-Zellen, während Expression von TrkB zu gesteigerter Proliferationsrate führte. Eine weitere Analyse der Unterschiede TrkA und TrkB aktivierter Signalwege könnte neue Möglichkeiten der Behandlung von Neuroblastomen eröffnen. Da die Expression von TrkA ein starker Prognosefaktor insbesondere für NB mit normaler MYCN Kopienzahl ist, sollte eine prospektive Studie der Trk-Rezeptorexpression an Neuroblastompatienten in Deutschland mittels RT-PCR durchgeführt werden.

Key words:

Neuroblastoma - tyrosine kinase receptors - neurotrophins - proliferation - differentiation

Schlüsselwörter:

Neuroblastom - Tyrosinkinase-Rezeptor - Neurotrophine - Proliferation - Differenzierung

1 This work was supported by Grants from the Deutsche Krebshilfe (A.E.), the National Institutes of Helath Grant NS 34514 (G.M.B.), and the Audrey E. Evans Endowed Chair (G.M.B.)

Literatur

1 This work was supported by Grants from the Deutsche Krebshilfe (A.E.), the National Institutes of Helath Grant NS 34514 (G.M.B.), and the Audrey E. Evans Endowed Chair (G.M.B.)

• 01 Brodeur  G M. TrkA expression in neuroblastomas: a new prognostic marker with biological and clinical sigificance.  J Natl Cancer Inst. 1993;  85 344-345
• 02 Nakagawara  A, Arima-Nakagawara  M, Scavarda  N J, Azar  C G, Cantor  A B, Brodeur  G M. Association between high levels of expression of the Trk gene and favorable outcome in human neuroblastomas.  N Engl J Med. 1993;  328 847-854
• 03 Kogner  P, Barbany  G, Dominici  C, Castello  M, Raschella  G, Persson  H. Coexpression of messenger RNA for Trk protooncogene and low affinity nerve growth factor receptor in neuroblastomas with favorable prognosis.  Cancer Res. 1993;  53 2044-2050
• 04 Brodeur  G M, Nakagawara  A, Yamashiro  D, Ikegaki  N, Liu  X-G, Azar  C G, Lee  C P, Evans  A E. Expression of TrkA, TrkB and TrkC in human neuroblastomas.  J of Neuro-Oncol. 1997;  31 49-55
• 05 Yamashiro  D J, Liu  X-G, Lee  C P, Nakagawara  A, Ikegaki  N, McGregor  L M, Baylin  S B, Brodeur  G M. Expression and function of TrkC in favourable human neuroblastomas.  Eur J of Cancer. 1997;  33 2054-2057
• 06 Nakagawara  A, Arima  M, Azar  C, Scavarda  N, Brodeur  G. Inversion relationship between Trk-expression and N-myc-amplification in human neuroblastomas.  Cancer Res. 1992;  52 1364-1368
• 07 Chelly  J, Kaplan  J C, Gautron  S, Kahn  A. Transcription of the dystrophin gene in human muscle and non-muscle tissues.  Nature. 1988;  333 858-860
• 08 Wang  A M, Doyle  M V, Mark  D F. Quantitation of mRNA by the polymerase chain reaction.  Proc Natl Acad Sci USA. 1989;  86 9717-9721
• 09 Horikoshi  T, Danenberg  K D, Stadlbauer  T, et al. Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphoroase gene expression in human tumors using the polymerase chain reaction.  Cancer Res. 1992;  52 108-116
• 10 Matsushima  H, Bogenmann  E. Expression of TrkA cDNA in neuroblastomas mediates differentiation in vitro and in vivo.  Mol Cell Biol. 1993;  13 7447-7456
• 11 Lavenius  E, Gestblom  C, Johansson  I, Nanberg  E, Pahlmann  S. Transfection of TrkA into human neuroblastoma cells restores their ability to differentiate in response to NGF.  Cell Growth Differ. 1995;  6 727-736
• 12 Poluha  W, Poluha  D K, Ross  A H. TrkA neurogenic receptor regulates differentiation of neuroblastoma cells.  Oncogene. 1995;  10 185-189
• 13 Nakagawara  A, Azar  C G, Scavarda  N J, Brodeur  G M. Expression and function of Trk-B and BDNF in human neuroblastomas.  Mol Cell Biol. 1994;  14 759-767
• 14 Middlemas  D S, Kihl  B K, Zhou  J, Zhu  X. Brain-derived neurotrophic factor promotes survival and chemoprotection of human neuroblastoma cells.  J Biol Chem. 1999;  274 16451-16460
• 15 Azar  C G, Scavarda  N J, Reynolds  C P, Brodeur  G M. Multiple defects of the NGF receptor in human neuroblastomas.  Cell Growth Differ. 1990;  1 421-428
• 16 Biedler  J L, Helson  L, Spengler  B A. Morphology, growth, tumorigenicity and cytogenetics of human neuroblastoma cells in continous culture.  Cancer Res. 1973;  33 2643-2649
• 17 Stephens  R M, Loeb  D M, Copeland  T D, Pawson  T, Greene  L A, Kaplan  D R. Trk receptors use redundant signal transduction pathways involving SHC and PLC-g1 to mediate NGF responses.  Neuron. 1994;  12 1-20
• 18 Loeb  D M, Tsao  H, Lobb  M H, Greene  L A. Nerve growth factor and other growth factors induce an association between Erk1 and the nerve growth factor receptor, gp 140 prototrk.  Neuron. 1992;  9 1053-1065
• 19 Mosmann  T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.  Journal of Immunological Methods. 1983;  65 55-63
• 20 Lavenius  E, Parrow  V, Nanberg  E, Pahlman  S. Basic FGF and IGF1 promote differentiation of human SH-SY5Y neuroblastoma cells in culture.  Growth Factors. 1994;  10 29-39