Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc.
The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis. Hypoxia is a potent stimulator of VEGF expression, and hypoxia-inducible factor-1 (HIF-1) is considered to be critical for this induction. However, we have previously demonstrated that induction of VEGF by hypoxia was preserved when HIF-1alpha was silenced. We sought to better define the molecular basis of this HIF-1-independent regulation. In colon cancer cells, hypoxia stimulated multiple K-ras effector pathways including phosphatidylinositol 3-kinase. VEGF promoter deletion studies identified a novel promoter region between -418 and -223 bp that was responsive to hypoxia in a PI3K/Rho/ROCK-dependent manner. Electrophoretic mobility shift assays identified a fragment between -300 and -251 bp that demonstrated a unique shift only in hypoxic conditions. Inhibition of PI3K or ROCK blocked the formation of this complex. A binding site for c-Myc, a target of ROCK, was identified at -271 bp. A role for c-Myc in the hypoxic induction of VEGF was demonstrated by site-directed mutagenesis of the VEGF promoter and silencing of c-Myc by small interfering RNA. Collectively, these findings suggest an alternative mechanism for the hypoxic induction of VEGF in colon cancer that does not depend upon HIF-1alpha but instead requires the activation of PI3K/Rho/ROCK and c-Myc.
Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Yusuke
Y
Mizukami
Kotoyo
K
Fujiki
Eva-Maria
EM
Duerr
Manish
M
Gala
Won-Seok
WS
Jo
Xiaobo
X
Zhang
Daniel C
DC
Chung
D001483:Base Sequence; D003110:Colonic Neoplasms / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000860:Hypoxia; D047908:Intracellular Signaling Peptides and Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D016271:Proto-Oncogene Proteins c-myc; D042461:Vascular Endothelial Growth Factor A; D054460:rho-Associated Kinases