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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

SphK1 silencing leads to a decrease in HIF-2 transcriptional activity in A498 and 786-O VHL-defective ccRCC cells. A498 and 786-O cells were treated with 20 nmol/l of siSphK1 or siScr for 72 h then incubated for an additional 16 h under normoxia or hypoxia. (a) HRE reporter gene assay (left) and protein HIF-2α expression (right) in transiently transfected A498 (upper) and 786-O (lower) cells. The y axis shows normalized Firefly luciferase over Renilla luciferase activity relative to the wild-type normoxic response. HIF-2α expression was analyzed by immunoblotting. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of at least four independent experiments; bars, s.e.m. ***P<0.001. Cell lysates were assayed for GLUT-1 (b) and cyclin D1 (c) expression by western blot analysis. Similar results were obtained in three independent experiments, and equal loading was monitored using antibody to tubulin. (d) Cell proliferation and viability was respectively assessed using [3H]-thymidine incorporation assay and MTT assay. Columns, mean of at least four independent experiments; bars, s.e.m. **P<0.01; ***P<0.001.
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fig3: SphK1 silencing leads to a decrease in HIF-2 transcriptional activity in A498 and 786-O VHL-defective ccRCC cells. A498 and 786-O cells were treated with 20 nmol/l of siSphK1 or siScr for 72 h then incubated for an additional 16 h under normoxia or hypoxia. (a) HRE reporter gene assay (left) and protein HIF-2α expression (right) in transiently transfected A498 (upper) and 786-O (lower) cells. The y axis shows normalized Firefly luciferase over Renilla luciferase activity relative to the wild-type normoxic response. HIF-2α expression was analyzed by immunoblotting. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of at least four independent experiments; bars, s.e.m. ***P<0.001. Cell lysates were assayed for GLUT-1 (b) and cyclin D1 (c) expression by western blot analysis. Similar results were obtained in three independent experiments, and equal loading was monitored using antibody to tubulin. (d) Cell proliferation and viability was respectively assessed using [3H]-thymidine incorporation assay and MTT assay. Columns, mean of at least four independent experiments; bars, s.e.m. **P<0.01; ***P<0.001.

Mentions: To establish whether SphK1 silencing was correlated with an inhibition of HIF-2 transcriptional activity, VHL-defective A498 and 786-O cells only known to express HIF-2α (vis-à-vis HIF-1α) under either normoxia and hypoxia38, 39 were used to avoid the confounding role of HIF-1α we previously reported to be regulated by SphK1 activity.32 Accordingly in both VHL-defective A498 and 786-O cells, SphK1 silencing was associated with a decreased HIF-2α protein expression (Figure 3a, right). The role of HIF-2α transcriptional activity was further investigated by a transient-transfection assay with an HRE reporter gene (pHRE-Luc) for HIF-2α. Accordingly, HRE-mediated transcription was remarkably decreased in A498 and 786-O cells treated with siSphK1 (Figure 3a, left) in both normoxia and hypoxia. We next analyzed the level of GLUT-1 and cyclin D1, two well-established specific target proteins of HIF-2α in VHL-defective ccRCC.40, 41 Under normoxia and hypoxia, SphK1 silencing significantly reduced levels of both glucose transporter GLUT-1 (Figure 3b) and cyclin D1 (Figure 3c) in both cell lines. Cyclin D1 is an important regulator of cell-cycle progression, and in vitro and in vivo data have demonstrated that HIF-2α-only-expressing ccRCC cells proliferate faster than their HIF-2α and HIF-1α co-expressing counterparts.42, 43 Therefore, we next assessed cell proliferation in SphK1-silenced VHL-defective ccRCC cells. [3H]Thymidine incorporation assay clearly showed that SphK1-silenced A498 and 786-O cells proliferated much slower than their siScr-silenced counterparts (Figure 3d, left) and showed a survival disadvantage (Figure 3d, right) in both normoxia and hypoxia in line with the findings that HIF-2α likely contributes to tumor cell survival.39


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)

SphK1 silencing leads to a decrease in HIF-2 transcriptional activity in A498 and 786-O VHL-defective ccRCC cells. A498 and 786-O cells were treated with 20 nmol/l of siSphK1 or siScr for 72 h then incubated for an additional 16 h under normoxia or hypoxia. (a) HRE reporter gene assay (left) and protein HIF-2α expression (right) in transiently transfected A498 (upper) and 786-O (lower) cells. The y axis shows normalized Firefly luciferase over Renilla luciferase activity relative to the wild-type normoxic response. HIF-2α expression was analyzed by immunoblotting. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of at least four independent experiments; bars, s.e.m. ***P<0.001. Cell lysates were assayed for GLUT-1 (b) and cyclin D1 (c) expression by western blot analysis. Similar results were obtained in three independent experiments, and equal loading was monitored using antibody to tubulin. (d) Cell proliferation and viability was respectively assessed using [3H]-thymidine incorporation assay and MTT assay. Columns, mean of at least four independent experiments; bars, s.e.m. **P<0.01; ***P<0.001.
© Copyright Policy - open-access
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fig3: SphK1 silencing leads to a decrease in HIF-2 transcriptional activity in A498 and 786-O VHL-defective ccRCC cells. A498 and 786-O cells were treated with 20 nmol/l of siSphK1 or siScr for 72 h then incubated for an additional 16 h under normoxia or hypoxia. (a) HRE reporter gene assay (left) and protein HIF-2α expression (right) in transiently transfected A498 (upper) and 786-O (lower) cells. The y axis shows normalized Firefly luciferase over Renilla luciferase activity relative to the wild-type normoxic response. HIF-2α expression was analyzed by immunoblotting. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of at least four independent experiments; bars, s.e.m. ***P<0.001. Cell lysates were assayed for GLUT-1 (b) and cyclin D1 (c) expression by western blot analysis. Similar results were obtained in three independent experiments, and equal loading was monitored using antibody to tubulin. (d) Cell proliferation and viability was respectively assessed using [3H]-thymidine incorporation assay and MTT assay. Columns, mean of at least four independent experiments; bars, s.e.m. **P<0.01; ***P<0.001.
Mentions: To establish whether SphK1 silencing was correlated with an inhibition of HIF-2 transcriptional activity, VHL-defective A498 and 786-O cells only known to express HIF-2α (vis-à-vis HIF-1α) under either normoxia and hypoxia38, 39 were used to avoid the confounding role of HIF-1α we previously reported to be regulated by SphK1 activity.32 Accordingly in both VHL-defective A498 and 786-O cells, SphK1 silencing was associated with a decreased HIF-2α protein expression (Figure 3a, right). The role of HIF-2α transcriptional activity was further investigated by a transient-transfection assay with an HRE reporter gene (pHRE-Luc) for HIF-2α. Accordingly, HRE-mediated transcription was remarkably decreased in A498 and 786-O cells treated with siSphK1 (Figure 3a, left) in both normoxia and hypoxia. We next analyzed the level of GLUT-1 and cyclin D1, two well-established specific target proteins of HIF-2α in VHL-defective ccRCC.40, 41 Under normoxia and hypoxia, SphK1 silencing significantly reduced levels of both glucose transporter GLUT-1 (Figure 3b) and cyclin D1 (Figure 3c) in both cell lines. Cyclin D1 is an important regulator of cell-cycle progression, and in vitro and in vivo data have demonstrated that HIF-2α-only-expressing ccRCC cells proliferate faster than their HIF-2α and HIF-1α co-expressing counterparts.42, 43 Therefore, we next assessed cell proliferation in SphK1-silenced VHL-defective ccRCC cells. [3H]Thymidine incorporation assay clearly showed that SphK1-silenced A498 and 786-O cells proliferated much slower than their siScr-silenced counterparts (Figure 3d, left) and showed a survival disadvantage (Figure 3d, right) in both normoxia and hypoxia in line with the findings that HIF-2α likely contributes to tumor cell survival.39

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