Abstract
Despite constant advances in the field of pediatric oncology, the survival rate of high-risk neuroblastoma patients remains poor. The molecular and genetic features of neuroblastoma, such as MYCN amplification and stemness status, have established themselves not only as potent prognostic and predictive factors but also as intriguing targets for personalized therapy. Novel thiosemicarbazones target both total level and activity of a number of proteins involved in some of the most important signaling pathways in neuroblastoma. In this study, we found that di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) potently decreases N-MYC in MYCN-amplified and c-MYC in MYCN-nonamplified neuroblastoma cell lines. Furthermore, DpC succeeded in downregulating total EGFR and phosphorylation of its most prominent tyrosine residues through the involvement of NDRG1, a positive prognostic marker in neuroblastoma, which was markedly upregulated after thiosemicarbazone treatment. These findings could provide useful knowledge for the treatment of MYC-driven neuroblastomas that are unresponsive to conventional therapies.
Highlights
Neuroblastoma is a rare solid neuroendocrine tumor arising from neural crest derivates in the developing sympathetic nervous system
We report that di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) markedly decreases the levels of the well-established N-myc downstream regulated gene 1 (NDRG1) suppressor N-MYC [46] and its structural and functional homolog c-MYC [47,48]
This study built upon the previous findings of thiosemicarbazone treatment in neuroblastoma and elucidated its effect on some of the important signaling molecules in this cancer type, namely MYC proteins, EGFR and its downstream targets, or NDRG1, whose exact mechanism of action remains largely elusive
Summary
Neuroblastoma is a rare solid neuroendocrine tumor arising from neural crest derivates in the developing sympathetic nervous system. Following this finding, it was crucial to determine whether the decrease in EGFR levels was mediated by NDRG1 induced after treatment with DpC. Immunofluorescence analysis showed the expected receptor internalization after its stimulation with EGF and confirmed the inhibitory effect of DpC on pY1068-EGFR in cells both unstimulated and stimulated by EGF ligand; a marked decrease in the pEGFR signal was detected, and its localization shifted from predominantly membranous to the form of a single perinuclear cluster, presumably a stress granule (Figure 4B). To examine the differential phosphorylation rate of proteins that might be regulated downstream of RTKs (including EGFR) in response to DpC treatment, the Proteome Profiler Human Phospho-Kinase Array Kit was used (Figure 5C). DppCCrergeugulaltaetsestottoatlalananddpphhoospsphhooryrylalateteddAAKKTTpprorotetieninlelveveelslsininnneueurorobblalastsotommaacecelllllilninees.s. (A(A) I)mImmmunuonbolboltotitntignogfoAfKATKaTndan(dB)(iBm) mimumnoubnlootbtliontgtionfgpoAfKpTA(KS4T7(3S)4p7r3o)tepirnolteevinelsleivnenlseuinronbelausrtoobmlaascimetsSoleoalmsnugttreaarcsetecia(veutdleelpadsit)matwaraeanigartdheetesrp2de(0rluowaμpvtiMi)itvdhaee(n2Sdod0Hpirμnt-eiSMclFYaait5lg(iYvSdu,HeerSeno-KSspSi-Y1ttNiy.5c-DYavBl,eaEdSnl(uKe2snei)-t)sNsoio(mt-dryBeo2Evtwrμ(ay2lnMu)d))e(aoosCtrf(aHdt2haoLμrrwAeeMen-s1h)i(n5Coo,dwfHCetnphHLreAaLens-Aed1ti-5hen2,ne0dCt)meeHDpxeeLpapnACnedr.-±ei2Rmn0Set)DepenDrxnetppsso.CeerrnSm.iotRmaauetleriipvzncreeetesd-. dtaota0ahrevpalruoevsi.d*epd
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