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Kinome siRNA screen identifies SMG-1 as a negative regulator of hypoxia-inducible factor-1alpha in hypoxia.

Chen RQ, Yang QK, Chen YL, Oliveira VA, Dalton WS, Fearns C, Lee JD - J. Biol. Chem. (2009)

Bottom Line: 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.Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.

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-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.

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SMG-1 negatively regulated the secretion of VEGF and CA-9, as well as the migratory potential of hypoxic tumor cells. A and B, HeLa cells were transfected with SMG-1 or control (Cont) siRNAs. After 48 h, the cells were incubated in 20% oxygen or 1% oxygen at 37 °C overnight. VEGF or CA9 protein in cell culture supernatants was measured using immunoassay kits from Pierce or R&D Systems, respectively, and the resultant data were normalized by cell number determined by MTT. The normalized VEGF or CA9 concentration of control cells under 20% oxygen was taken as 1. C, SKOV-3 cells were mock transfected (Mock) or transfected with control siRNA (Cont) or SMG-1 siRNA (SMG-1). 48 h post transfection, the migratory capacity of the cells in hypoxia was analyzed, and the relative rate of cell migration in these cells was normalized to mock transfected cells whose value was taken as 1.
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Figure 5: SMG-1 negatively regulated the secretion of VEGF and CA-9, as well as the migratory potential of hypoxic tumor cells. A and B, HeLa cells were transfected with SMG-1 or control (Cont) siRNAs. After 48 h, the cells were incubated in 20% oxygen or 1% oxygen at 37 °C overnight. VEGF or CA9 protein in cell culture supernatants was measured using immunoassay kits from Pierce or R&D Systems, respectively, and the resultant data were normalized by cell number determined by MTT. The normalized VEGF or CA9 concentration of control cells under 20% oxygen was taken as 1. C, SKOV-3 cells were mock transfected (Mock) or transfected with control siRNA (Cont) or SMG-1 siRNA (SMG-1). 48 h post transfection, the migratory capacity of the cells in hypoxia was analyzed, and the relative rate of cell migration in these cells was normalized to mock transfected cells whose value was taken as 1.

Mentions: Increased VEGF and CA9 proteins in epithelial cancers are markers of tumor hypoxia and are associated with a poor prognosis, because expression of VEGF and CA9 promotes tumor-associated angiogenesis and the survival capacity of hypoxic tumors, respectively (5, 7). Because both VEGF and CA9 expression are tightly regulated by HIF-1 activity, we suspected that removal of SMG-1, a negative regulator of HIF-1, would substantially enhance the production of VEGF and CA9 in tumor cells under hypoxia. In Fig. 5 (A and B), we demonstrated that in SMG-1-deficient cells the production of VEGF and CA9 was substantially elevated under hypoxia. As hypoxic conditions significantly enhance tumor metastasis partly by boosting the migratory potential of tumor cells, we tested whether depletion of cellular SMG-1 had any effect on hypoxic tumor cell motility. Indeed, we found that SMG-1 depletion significantly promoted the migratory potential of cancer cells in hypoxia (Fig. 5C). These results indicated that SMG-1 repressed a range of malignant responses induced by oxygen deprivation of tumor cells and suggested that human SMG-1 was a tumor suppressor, particularly for hypoxic tumors.


Kinome siRNA screen identifies SMG-1 as a negative regulator of hypoxia-inducible factor-1alpha in hypoxia.

Chen RQ, Yang QK, Chen YL, Oliveira VA, Dalton WS, Fearns C, Lee JD - J. Biol. Chem. (2009)

SMG-1 negatively regulated the secretion of VEGF and CA-9, as well as the migratory potential of hypoxic tumor cells. A and B, HeLa cells were transfected with SMG-1 or control (Cont) siRNAs. After 48 h, the cells were incubated in 20% oxygen or 1% oxygen at 37 °C overnight. VEGF or CA9 protein in cell culture supernatants was measured using immunoassay kits from Pierce or R&D Systems, respectively, and the resultant data were normalized by cell number determined by MTT. The normalized VEGF or CA9 concentration of control cells under 20% oxygen was taken as 1. C, SKOV-3 cells were mock transfected (Mock) or transfected with control siRNA (Cont) or SMG-1 siRNA (SMG-1). 48 h post transfection, the migratory capacity of the cells in hypoxia was analyzed, and the relative rate of cell migration in these cells was normalized to mock transfected cells whose value was taken as 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719310&req=5

Figure 5: SMG-1 negatively regulated the secretion of VEGF and CA-9, as well as the migratory potential of hypoxic tumor cells. A and B, HeLa cells were transfected with SMG-1 or control (Cont) siRNAs. After 48 h, the cells were incubated in 20% oxygen or 1% oxygen at 37 °C overnight. VEGF or CA9 protein in cell culture supernatants was measured using immunoassay kits from Pierce or R&D Systems, respectively, and the resultant data were normalized by cell number determined by MTT. The normalized VEGF or CA9 concentration of control cells under 20% oxygen was taken as 1. C, SKOV-3 cells were mock transfected (Mock) or transfected with control siRNA (Cont) or SMG-1 siRNA (SMG-1). 48 h post transfection, the migratory capacity of the cells in hypoxia was analyzed, and the relative rate of cell migration in these cells was normalized to mock transfected cells whose value was taken as 1.
Mentions: Increased VEGF and CA9 proteins in epithelial cancers are markers of tumor hypoxia and are associated with a poor prognosis, because expression of VEGF and CA9 promotes tumor-associated angiogenesis and the survival capacity of hypoxic tumors, respectively (5, 7). Because both VEGF and CA9 expression are tightly regulated by HIF-1 activity, we suspected that removal of SMG-1, a negative regulator of HIF-1, would substantially enhance the production of VEGF and CA9 in tumor cells under hypoxia. In Fig. 5 (A and B), we demonstrated that in SMG-1-deficient cells the production of VEGF and CA9 was substantially elevated under hypoxia. As hypoxic conditions significantly enhance tumor metastasis partly by boosting the migratory potential of tumor cells, we tested whether depletion of cellular SMG-1 had any effect on hypoxic tumor cell motility. Indeed, we found that SMG-1 depletion significantly promoted the migratory potential of cancer cells in hypoxia (Fig. 5C). These results indicated that SMG-1 repressed a range of malignant responses induced by oxygen deprivation of tumor cells and suggested that human SMG-1 was a tumor suppressor, particularly for hypoxic tumors.

Bottom Line: 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.Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.

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-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.

Show MeSH
Related in: MedlinePlus