Kinome siRNA screen identifies SMG-1 as a negative regulator of hypoxia-inducible factor-1alpha in hypoxia.
Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1alpha-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1alpha activity in hypoxia, in part through blocking MAPK activation.
Chen
Run-Qiang
RQ
From the Departments of Immunology and Microbial Science, La Jolla, California 92037.
Yang
Qing-Kai
QK
From the Departments of Immunology and Microbial Science, La Jolla, California 92037.
Chen
Yan-Ling
YL
From the Departments of Immunology and Microbial Science, La Jolla, California 92037.
Oliveira
Vasco A
VA
Chemistry, The Scripps Research Institute, La Jolla, California 92037.
Dalton
William S
WS
Chemistry, The Scripps Research Institute, La Jolla, California 92037.
Fearns
Colleen
C
Department of Experimental Therapeutics and Interdisciplinary Oncology, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida, Tampa, Florida 33612.
Lee
Jiing-Dwan
JD
From the Departments of Immunology and Microbial Science, La Jolla, California 92037. Electronic address: jdlee@scripps.edu.
D000951:Antigens, Neoplasm / Q000096:biosynthesis; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000096:biosynthesis; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics* / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D040081:Protein Array Analysis; D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D034741:RNA, Small Interfering / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis
D000951:Antigens, Neoplasm; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C497442:HIF1A protein, human; C467484:VEGFA protein, human; C514704:SMG1 protein, human; C089226:CA9 protein, human