Disruption of cooperation between Ras and MycN in human neuroblastoma cells promotes growth arrest.
OBJECTIVE
Our aim was to examine whether active Ras and MycN cooperation contributes to the malignant phenotype of human neuroblastoma with amplified MycN gene, an aggressive incurable tumor.


METHODS
Human neuroblastoma LAN-1 cells, in which the MycN gene is amplified, were used to examine the impact of the Ras inhibitor farnesylthiosalicylic acid on cell growth, on the levels Ras and MycN proteins, and on profiles of gene expression.


RESULTS
We show that LAN-1 cells express relatively large amounts of MycN and active Ras-GTP. Inhibition of active Ras by farnesylthiosalicylic acid led to attenuation of the Raf-MEK-ERK and phosphoinositide 3-kinase-Akt-glycogen synthase-3 (GSK-3) pathways, to reduction in cyclin D1, phospho-retinoblastoma, and E2F, and to increase in the cyclin-dependent kinase inhibitor p27 and in retinoblastoma-binding protein-1, an inhibitor of E2F transcriptional activity. Ras inhibition by farnesylthiosalicylic acid or by a dominant-negative Ras also led to complete disappearance of MycN protein from the nuclei of LAN-1 cells. This was a result of blocking of Akt inactivation of GSK-3, leading to GSK-3-dependent phosphorylation with consequent proteosomal degradation of MycN. Loss of active Ras and of MycN in LAN-1 cells was manifested in profiles of gene expression that could be expected from the loss of MycN transcriptional activity and of Ras signaling. These changes explain the farnesylthiosalicylic acid-induced inhibition of LAN-1 cell growth.


CONCLUSIONS
Active Ras is needed to block MycN degradation, promoting cooperative Ras- and MycN-dependent cell cycle progression in LAN-1 cells. Ras inhibitors are therefore likely candidates for the treatment of advanced neuroblastoma characterized by high expression of MycN.
Yaari
Shira
S
Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel.
Jacob-Hirsch
Jasmine
J
Amariglio
Ninette
N
Haklai
Ronit
R
Rechavi
Gideon
G
Kloog
Yoel
Y
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015536:Down-Regulation / Q000187:drug effects; D005204:Farnesol / Q000031:analogs & derivatives* / Q000494:pharmacology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D017404:In Situ Hybridization, Fluorescence; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D012459:Salicylates / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D048490:raf Kinases / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D005204:Farnesol; D038362:Glycogen Synthase Kinase 3; D049452:Green Fluorescent Proteins; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016271:Proto-Oncogene Proteins c-myc; D056465:Retinoblastoma-Binding Protein 1; D012459:Salicylates; D025521:Tumor Suppressor Proteins; D048490:raf Kinases; D018631:ras Proteins; C093323:farnesylthiosalicylic acid; C494918:AKT1 protein, human