Kinome Sirna Screen Identifies SMG-1 as a Negative Regulator of Hypoxia-inducible Factor-1α in Hypoxia

Abstract

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-1α. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1α-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-1α activity in hypoxia, in part through blocking MAPK activation.

Smg1 haploinsufficiency predisposes to tumor formation and inflammation

“…Here, we examined four assays measuring NMD in a range of tissues or isolated cells; surprisingly, none of these assays showed evidence of any defect in NMD in Smg1 +/gt mice. Similarly, we found no defect in DNA damage responses in Smg1 +/gt mice despite SMG1 having a characterized role in these pathways (1,4,5,(7)(8)(9)(10). These data suggest that expression of Smg1 from a single allele is sufficient for full functionality of these pathways in the situations studied.…”

“…These findings suggest that Smg1 +/gt mice show low-level inflammation, which, with age, can either progress to chronic inflammatory disorders or lead to the development of inflammationenhanced cancers. Although SMG1 does not have a currently described role in regulation of inflammatory responses, it has been suggested to regulate MAPK activation (9). MAPKs are important in regulation of multiple immune regulatory pathways, and studies to determine the role of SMG1 in these pathways are ongoing.…”

“…Depletion of Smg1 in tumor cells markedly increased the extent of cell death induced by TNF-α or granzyme B (7,8). SMG1 kinase activity was also induced in response to hypoxia, where it negatively regulated hypoxia-inducible factor 1α (HIF-1α), in part, by blocking MAPK activation (9). In addition, we recently showed that SMG1 had an essential role in the formation of stress granules (10).…”

SMG-1 suppresses CDK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways

“…11 SMG-1 (suppressor with morphogenetic effect on genitalia) is the most recently characterized member of the PIKK family and was initially found to function as a critical component of the nonsense-mediated mRNA decay (NMD), an evolutionarily conserved RNA surveillance mechanism during which transcripts containing premature termination codons are eliminated. 12,13 In recent years, several other distinct cellular roles have been attributed to SMG-1, including participation in stress responses to IR and UV radiation, 14 as well as hyperoxia and DSBs produced by expression of an endonuclease, 15 telomere integrity, 16 protection against tumor necrosis factor-α-dependent apoptosis, 17,18 responses to hypoxia, 19 lifespan regulation and oxidative stress resistance in Caenorhabditis elegans, 20 regulation of G 1 /S checkpoint during oxidative stress, 21 as well as an essential role in embryogenesis. 22 SMG-1-knockout mice are embryonically lethal, but haploinsufficiency predisposes to a whole range of tumor formation and inflammation, demonstrating that SMG-1 is a tumor suppressor.…”

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay

“…Like ATM, ATR, and the DNA-dependent protein kinase catalytic subunit, Smg1 is linked to DNA damage responses (3,4). Smg1 may also function in telomere maintenance, responses to hypoxia, and TNF-α-induced apoptosis (5)(6)(7). In vitro, Smg1 has a critical role in the degradation of premature termination codon (PTC)-containing transcripts mediated by the nonsense-mediated mRNA decay (NMD) pathway (2,3,8).…”