H-Ras increases urokinase expression and cell invasion in genetically modified human astrocytes through Ras/Raf/MEK signaling pathway.
Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54-9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas.
Zhao
Yunge
Y
Department of Pathology, University of Virginia, Charlottesville, Virginia 22908, USA. yz5u@virginia.edu
Xiao
Aizhen
A
Dipierro
Charles G
CG
Abdel-Fattah
Rana
R
Amos
Samson
S
Redpath
Gerard T
GT
Carpenter
Joan E
JE
Pieper
Russell O
RO
Hussaini
Isa M
IM
D000704:Analysis of Variance; D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D001932:Brain Neoplasms / Q000473:pathology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection / Q000379:methods; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
D020928:Mitogen-Activated Protein Kinases; D014568:Urokinase-Type Plasminogen Activator; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins