ИСТИНА

Neuroblastoma is an undifferentiated malignant tumor of the sympathetic nervous system, it is common in children and is treated by retinoic acid to induce differentiation. Expression of receptor tyrosine kinase EGFR is up-regulated in malignant neuroblastoma, while inhibition of EGFR-dependent signaling blocks neuroblastoma differentiation. It was shown in vitro that phosphorylation of EGFR and activation of EGFR-dependent MAPK-kinases (Erk1/2 and Akt) is dramatically increased in neuroblastoma upon serum withdrawal. Urokinase (uPA), present in the serum, and its specific receptor (uPAR) facilitates tumor growth and invasion. uPA and uPAR mediate the activation of extracellular proteolysis and intracellular signaling through a number of receptors that interact laterally with uPAR. We studied the differentiation and intracellular signaling effects upon uPAR expression and activation in mouse neuroblastoma cell line (Neuro2a). The time of serum deprivation correlated positively with the increase in uPAR expression. After 72 hours uPAR was increased by 50% compared to control. uPAR co-localized with phosphorylated EGFR on the plasma membrane and uPAR overexpression increased co-localization. uPAR overexpression didn't affect the total amount of EGFR or NeuN (neuronal specific nuclear protein), while blocking of uPAR activity by specific antibody resulted in a rapid (5 min) reduction in phospho-EGFR (Y1068) level in Neuro2a. uPAR blocking resulted in the activation of EGFR-dependent kinases p38 (T180/Y182), Akt (S473) and c-Src (Y412) and in the decrease of Erk1/2 phosphorylation (T202/Y204). It is known that in neuronal cells such signaling decrease cell viability and promote apoptosis. Durable uPAR blocking led to 4-fold reduction in N2a proliferation, DNA fragmentation and generation of apoptotic PARP-1 fragments. Thus, for the first time we show that expression and activity of uPAR regulates differentiation and survival pathways in neuroblastoma, suggesting that uPAR can be considered as a therapeutic target in the treatment of neuroblastomas. Source of funding: Grant of the Russian Science Foundation (№14-24-00086).