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.