title: Phenotype-assisted transcriptome analysis identifies FOXM1 downstream from Ras-MKK3-p38 to regulate in vitro cellular invasion.
abstract: The Ras oncogene is known to activate three major MAPK pathways, ERK, JNK, p38 and exert distinct cellular phenotypes, that is, apoptosis and invasion through the Ras-MKK3-p38-signaling cascade. We attempted to identify the molecular targets of this pathway that selectively govern the invasive phenotype. Stable transfection of NIH3T3 fibroblasts with MKK3(act) cDNA construct revealed similar p38-dependent in vitro characteristics observed in Ha-Ras(EJ)-transformed NIH3T3 cells, including enhanced invasiveness and anchorage-independent growth correlating with p38 phosphorylation status. To identify the consensus downstream targets of the Ras-MKK3-p38 cascade involved in invasion, in vitro invasion assays were used to isolate highly invasive cells from both, MKK3 and Ha-Ras(EJ) transgenic cell lines. Subsequently a genome-wide transcriptome analysis was employed to investigate differentially regulated genes in invasive Ha-Ras(EJ)- and MKK3(act)-transfected NIH3T3 fibroblasts. Using this phenotype-assisted approach combined with system level protein-interaction network analysis, we identified FOXM1, PLK1 and CDK1 to be differentially regulated in invasive Ha-Ras(EJ)-NIH3T3 and MKK3(act)-NIH3T3 cells. Finally, a FOXM1 RNA-knockdown approach revealed its requirement for both invasion and anchorage-independent growth of Ha-Ras(EJ)- and MKK3(act)-NIH3T3 cells. Together, we identified FOXM1 as a key downstream target of Ras and MKK3-induced cellular in vitro invasion and anchorage-independent growth signaling.
authors.affiliation: Cancer Vaccine, Ludwig Institute for Cancer Research Ltd, Melbourne Centre for Clinical Sciences, Heidelberg, VIC, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Behren
authors.forename: S
authors.initials: S
authors.lastname: Mühlen
authors.forename: G A
authors.initials: GA
authors.lastname: Acuna Sanhueza
authors.forename: C
authors.initials: C
authors.lastname: Schwager
authors.forename: P K
authors.initials: PK
authors.lastname: Plinkert
authors.forename: P E
authors.initials: PE
authors.lastname: Huber
authors.forename: A
authors.initials: A
authors.lastname: Abdollahi
authors.forename: C
authors.initials: C
authors.lastname: Simon
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion; D002465:Cell Movement; D005419:Flavonoids / Q000494:pharmacology; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D007093:Imidazoles / Q000494:pharmacology; D048371:MAP Kinase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D020411:Oligonucleotide Array Sequence Analysis; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C107585:FOXM1 protein, human; D005419:Flavonoids; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors; D007093:Imidazoles; D011725:Pyridines; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; C482530:MAP2K3 protein, human; D018631:ras Proteins; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one