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.

Neuroblastoma - tyrosine kinase receptors - neurotrophins - proliferation - differentiation

Neuroblastom - Tyrosinkinase-Rezeptor - Neurotrophine - Proliferation - Differenzierung

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.)

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