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Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer.

Bouquerel P, Gstalder C, Müller D, Laurent J, Brizuela L, Sabbadini RA, Malavaud B, Pyronnet S, Martineau Y, Ader I, Cuvillier O - Oncogenesis (2016)

Bottom Line: Importantly, downregulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC.Taking advantage of a monoclonal antibody neutralizing extracellular S1P, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 or the S1P receptor 1 (S1P1) is silenced.These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.

ABSTRACT
The sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway has been reported to modulate the expression of the canonical transcription factor hypoxia-inducible HIF-1α in multiple cell lineages. HIF-2α is also frequently overexpressed in solid tumors but its role has been mostly studied in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, where HIF-2α has been established as a driver of a more aggressive disease. In this study, the role of SphK1/S1P signaling with regard to HIF-2α was investigated in various cancer cell models including ccRCC cells. Under hypoxic conditions or in ccRCC lacking a functional von Hippel-Lindau (VHL) gene and expressing high levels of HIF-2α, SphK1 activity controls HIF-2α expression and transcriptional activity through a phospholipase D (PLD)-driven mechanism. SphK1 silencing promotes a VHL-independent HIF-2α loss of expression and activity and reduces cell proliferation in ccRCC. Importantly, downregulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC. Taking advantage of a monoclonal antibody neutralizing extracellular S1P, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 or the S1P receptor 1 (S1P1) is silenced. Here, we report the first evidence that the SphK1/S1P signaling pathway regulates the transcription factor hypoxia-inducible HIF-2α in diverse cancer cell lineages notably ccRCC, where HIF-2α has been established as a driver of a more aggressive disease. These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

No MeSH data available.


Related in: MedlinePlus

S1P1 mediates the effect of S1P on HIF-1α and HIF-2α protein content in CAKI-1 and A498 ccRCC cells. (a) The relative mRNA expression of S1P1–5 in A498 and CAKI-1 was measured after 1 h of incubation under normoxic (black) or hypoxic (white) conditions. Columns, mean of at least five independent experiments; bars, s.e.m. **P<0.01. (b) A498 and CAKI-1 cells were treated with W146 (5 μm) or ethanol (control), then incubated under normoxia (Nx) or hypoxia for 6 h. HIF-1α and HIF-2α expression was analyzed by immunoblotting. (c) A498 and CAKI-1 cells were transfected with 50 nmol/l of siS1P1 or siScr for 72 h, then incubated under normoxia (Nx) or hypoxia for an additional 6 h. Cell lysates were assayed for HIF-1α and HIF-2α expression by immunoblotting. (d) CAKI-1 shS1P1 and CAKI-1 shCtrl cell lines were incubated under normoxia (Nx) or hypoxia (Hx) for 6 h, and HIF-1α and HIF-2α expression was analyzed by immunoblotting. For all experiments, similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin.
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fig7: S1P1 mediates the effect of S1P on HIF-1α and HIF-2α protein content in CAKI-1 and A498 ccRCC cells. (a) The relative mRNA expression of S1P1–5 in A498 and CAKI-1 was measured after 1 h of incubation under normoxic (black) or hypoxic (white) conditions. Columns, mean of at least five independent experiments; bars, s.e.m. **P<0.01. (b) A498 and CAKI-1 cells were treated with W146 (5 μm) or ethanol (control), then incubated under normoxia (Nx) or hypoxia for 6 h. HIF-1α and HIF-2α expression was analyzed by immunoblotting. (c) A498 and CAKI-1 cells were transfected with 50 nmol/l of siS1P1 or siScr for 72 h, then incubated under normoxia (Nx) or hypoxia for an additional 6 h. Cell lysates were assayed for HIF-1α and HIF-2α expression by immunoblotting. (d) CAKI-1 shS1P1 and CAKI-1 shCtrl cell lines were incubated under normoxia (Nx) or hypoxia (Hx) for 6 h, and HIF-1α and HIF-2α expression was analyzed by immunoblotting. For all experiments, similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin.

Mentions: Exogenous S1P is a ligand for five high-affinity G protein-coupled receptors (S1P1–5), with specific effects depending on the suite of S1P receptor subtypes expressed.5 Representative CAKI-1 and A498 ccRCC cells express all S1P receptors, we evaluated their involvement in both HIF-1α and HIF-2α regulation. We show that S1P1 mRNA is increased after 60 min of hypoxia, while S1P2–5 mRNA expression is unchanged (Figure 7a). To investigate the contribution of S1P1 in the regulation of HIF-1α and HIF-2α in our models, the targeting of S1P1 was achieved with the specific antagonist W146. HIF-2α expression was markedly reduced in both A498 and CAKI-1 cells (Figure 7b). Similarly, the downregulation of S1P1 by siRNA strategy in A498 and CAKI-1 cells or shRNA in CAKI-1 cells (Supplementary Figure 5) was accompanied by a strong decrease in HIF-2α expression (Figures 7c and d). We also observed a significant reduction in HIF-1α accumulation under hypoxia in CAKI-1 cells that can produce both HIF-1α and HIF-2α (Figures 7b and c). Similar findings were found in prostate PC-3 and glioblastoma U87 cells (Supplementary Figure 6), suggesting a potential exclusive contribution of S1P1 receptor subtype in mediating the effect of S1P on both HIF-1α and HIF-2α content in cancer cells.


Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer.

Bouquerel P, Gstalder C, Müller D, Laurent J, Brizuela L, Sabbadini RA, Malavaud B, Pyronnet S, Martineau Y, Ader I, Cuvillier O - Oncogenesis (2016)

S1P1 mediates the effect of S1P on HIF-1α and HIF-2α protein content in CAKI-1 and A498 ccRCC cells. (a) The relative mRNA expression of S1P1–5 in A498 and CAKI-1 was measured after 1 h of incubation under normoxic (black) or hypoxic (white) conditions. Columns, mean of at least five independent experiments; bars, s.e.m. **P<0.01. (b) A498 and CAKI-1 cells were treated with W146 (5 μm) or ethanol (control), then incubated under normoxia (Nx) or hypoxia for 6 h. HIF-1α and HIF-2α expression was analyzed by immunoblotting. (c) A498 and CAKI-1 cells were transfected with 50 nmol/l of siS1P1 or siScr for 72 h, then incubated under normoxia (Nx) or hypoxia for an additional 6 h. Cell lysates were assayed for HIF-1α and HIF-2α expression by immunoblotting. (d) CAKI-1 shS1P1 and CAKI-1 shCtrl cell lines were incubated under normoxia (Nx) or hypoxia (Hx) for 6 h, and HIF-1α and HIF-2α expression was analyzed by immunoblotting. For all experiments, similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4815047&req=5

fig7: S1P1 mediates the effect of S1P on HIF-1α and HIF-2α protein content in CAKI-1 and A498 ccRCC cells. (a) The relative mRNA expression of S1P1–5 in A498 and CAKI-1 was measured after 1 h of incubation under normoxic (black) or hypoxic (white) conditions. Columns, mean of at least five independent experiments; bars, s.e.m. **P<0.01. (b) A498 and CAKI-1 cells were treated with W146 (5 μm) or ethanol (control), then incubated under normoxia (Nx) or hypoxia for 6 h. HIF-1α and HIF-2α expression was analyzed by immunoblotting. (c) A498 and CAKI-1 cells were transfected with 50 nmol/l of siS1P1 or siScr for 72 h, then incubated under normoxia (Nx) or hypoxia for an additional 6 h. Cell lysates were assayed for HIF-1α and HIF-2α expression by immunoblotting. (d) CAKI-1 shS1P1 and CAKI-1 shCtrl cell lines were incubated under normoxia (Nx) or hypoxia (Hx) for 6 h, and HIF-1α and HIF-2α expression was analyzed by immunoblotting. For all experiments, similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin.
Mentions: Exogenous S1P is a ligand for five high-affinity G protein-coupled receptors (S1P1–5), with specific effects depending on the suite of S1P receptor subtypes expressed.5 Representative CAKI-1 and A498 ccRCC cells express all S1P receptors, we evaluated their involvement in both HIF-1α and HIF-2α regulation. We show that S1P1 mRNA is increased after 60 min of hypoxia, while S1P2–5 mRNA expression is unchanged (Figure 7a). To investigate the contribution of S1P1 in the regulation of HIF-1α and HIF-2α in our models, the targeting of S1P1 was achieved with the specific antagonist W146. HIF-2α expression was markedly reduced in both A498 and CAKI-1 cells (Figure 7b). Similarly, the downregulation of S1P1 by siRNA strategy in A498 and CAKI-1 cells or shRNA in CAKI-1 cells (Supplementary Figure 5) was accompanied by a strong decrease in HIF-2α expression (Figures 7c and d). We also observed a significant reduction in HIF-1α accumulation under hypoxia in CAKI-1 cells that can produce both HIF-1α and HIF-2α (Figures 7b and c). Similar findings were found in prostate PC-3 and glioblastoma U87 cells (Supplementary Figure 6), suggesting a potential exclusive contribution of S1P1 receptor subtype in mediating the effect of S1P on both HIF-1α and HIF-2α content in cancer cells.

Bottom Line: Importantly, downregulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC.Taking advantage of a monoclonal antibody neutralizing extracellular S1P, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 or the S1P receptor 1 (S1P1) is silenced.These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.

ABSTRACT
The sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway has been reported to modulate the expression of the canonical transcription factor hypoxia-inducible HIF-1α in multiple cell lineages. HIF-2α is also frequently overexpressed in solid tumors but its role has been mostly studied in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, where HIF-2α has been established as a driver of a more aggressive disease. In this study, the role of SphK1/S1P signaling with regard to HIF-2α was investigated in various cancer cell models including ccRCC cells. Under hypoxic conditions or in ccRCC lacking a functional von Hippel-Lindau (VHL) gene and expressing high levels of HIF-2α, SphK1 activity controls HIF-2α expression and transcriptional activity through a phospholipase D (PLD)-driven mechanism. SphK1 silencing promotes a VHL-independent HIF-2α loss of expression and activity and reduces cell proliferation in ccRCC. Importantly, downregulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC. Taking advantage of a monoclonal antibody neutralizing extracellular S1P, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 or the S1P receptor 1 (S1P1) is silenced. Here, we report the first evidence that the SphK1/S1P signaling pathway regulates the transcription factor hypoxia-inducible HIF-2α in diverse cancer cell lineages notably ccRCC, where HIF-2α has been established as a driver of a more aggressive disease. These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

No MeSH data available.


Related in: MedlinePlus