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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

PLD regulates SphK1-dependent HIF-2α expression in CAKI-1 and A498 ccRCC cells. (a), CAKI-1 (left) and A498 (right) cells were incubated under hypoxia for the indicated times and then tested for PLD and SphK1 enzymatic activities. Points, mean of at least three experiments; bars, s.e.m. **P<0.01; ***P<0.001. Inset, HIF-2α expression in CAKI-1 cells exposed to hypoxia for the indicated times. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to α-tubulin. (b, c), CAKI-1 (left) and A498 (right) cells were untreated or treated with 1-butanol (1-ButOH) or tert-butanol (t-ButOH) as control (0.8%). SphK1 activity (b) and HIF-2α expression (c) were determined in normoxia or after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001. (d, e) CAKI-1 (left) and A498 (right) cells were transfected with siPLD1 (50 nmol/L), siPLD2 (50 nmol/L) or siPLD1 (50 nmol/L) and siPLD2 (50 nmol/L) or siScr (50 nmol/L) for 72 h then incubated under normoxia or hypoxia. SphK1 activity (d) and HIF-2α expression (e) were determined after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001.
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fig2: PLD regulates SphK1-dependent HIF-2α expression in CAKI-1 and A498 ccRCC cells. (a), CAKI-1 (left) and A498 (right) cells were incubated under hypoxia for the indicated times and then tested for PLD and SphK1 enzymatic activities. Points, mean of at least three experiments; bars, s.e.m. **P<0.01; ***P<0.001. Inset, HIF-2α expression in CAKI-1 cells exposed to hypoxia for the indicated times. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to α-tubulin. (b, c), CAKI-1 (left) and A498 (right) cells were untreated or treated with 1-butanol (1-ButOH) or tert-butanol (t-ButOH) as control (0.8%). SphK1 activity (b) and HIF-2α expression (c) were determined in normoxia or after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001. (d, e) CAKI-1 (left) and A498 (right) cells were transfected with siPLD1 (50 nmol/L), siPLD2 (50 nmol/L) or siPLD1 (50 nmol/L) and siPLD2 (50 nmol/L) or siScr (50 nmol/L) for 72 h then incubated under normoxia or hypoxia. SphK1 activity (d) and HIF-2α expression (e) were determined after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001.

Mentions: Because phospholipase D (PLD) activity has been involved in the control of HIF-2α expression in ccRCC35 and is an upstream regulator of SphK1 in a different physiological context,36 we next examined the interactions between PLD and SphK1 signaling with regard to HIF-2α expression. In hypoxic CAKI-1, A498 cells (Figure 2a) and 786-O cells (Supplementary Figure 1A), an early transient increase in PLD activity (peaking at 15–30 min) followed by activation of SphK1 (peaking at 60 min) was observed. Accordingly, accumulation of HIF-2α did not occur before 2–3 h of hypoxia in CAKI-1 cells (Figure 2a, inset). Confirming that SphK1 activation was a consequence of PLD stimulation, butan-1-ol (1-ButOH), a potent inhibitor of PLD activation, markedly inhibited SphK1 activity in all ccRCC cell lines (Figure 2b and Supplementary Figure 1B). To rule out any possible non-specific effect of the alcohols on SphK1 activity, cells were treated in the presence of t-butanol (t-ButOH), a tertiary alcohol, which is not a substrate for PLD inhibition. As expected, t-ButOH did not alter SphK1 activity in ccRCC cell lines (Figure 2b and Supplementary Figure 1B). Unlike t-ButOH, 1-ButOH markedly inhibited HIF-2α expression in CAKI-1 cells (Figure 2c, left), as well as in normoxic and hypoxic A498 cells (Figure 2c, right) and in 786-0 cells (Supplementary Figure 1c). To investigate which PLD isozyme could be involved in the PLD/SphK1/HIF-2α signaling sequence, siRNAs directed to PLD1 and PLD2 isoforms were used.37 A roughly 50% PLD knock-down in CAKI-1, A498 and in 786-0 cells was achieved without additive effect when both siRNAs were combined (Supplementary Figure 2). Both PLD1 and PLD2 siRNAs significantly reduced—although not to the same extent—SphK1 activity in CAKI-1 (Figure 2d, left), A498 (Figure 2d, right) and 786-0 cells (Supplementary Figure 1D). Accordingly, the downregulation of SphK1 activity was accompanied by a marked reduction in HIF-2α expression in hypoxic CAKI-1 cells (Figure 2e, left), as well as in normoxic and hypoxic A498 (Figure 2e, right) and 786-O cells (Supplementary Figure 1E). These data suggest that both PLD1 and PLD2 isozymes are likely required for regulation of HIF-2α through SphK1 signaling in ccRCC cell lines.


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)

PLD regulates SphK1-dependent HIF-2α expression in CAKI-1 and A498 ccRCC cells. (a), CAKI-1 (left) and A498 (right) cells were incubated under hypoxia for the indicated times and then tested for PLD and SphK1 enzymatic activities. Points, mean of at least three experiments; bars, s.e.m. **P<0.01; ***P<0.001. Inset, HIF-2α expression in CAKI-1 cells exposed to hypoxia for the indicated times. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to α-tubulin. (b, c), CAKI-1 (left) and A498 (right) cells were untreated or treated with 1-butanol (1-ButOH) or tert-butanol (t-ButOH) as control (0.8%). SphK1 activity (b) and HIF-2α expression (c) were determined in normoxia or after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001. (d, e) CAKI-1 (left) and A498 (right) cells were transfected with siPLD1 (50 nmol/L), siPLD2 (50 nmol/L) or siPLD1 (50 nmol/L) and siPLD2 (50 nmol/L) or siScr (50 nmol/L) for 72 h then incubated under normoxia or hypoxia. SphK1 activity (d) and HIF-2α expression (e) were determined after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001.
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fig2: PLD regulates SphK1-dependent HIF-2α expression in CAKI-1 and A498 ccRCC cells. (a), CAKI-1 (left) and A498 (right) cells were incubated under hypoxia for the indicated times and then tested for PLD and SphK1 enzymatic activities. Points, mean of at least three experiments; bars, s.e.m. **P<0.01; ***P<0.001. Inset, HIF-2α expression in CAKI-1 cells exposed to hypoxia for the indicated times. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to α-tubulin. (b, c), CAKI-1 (left) and A498 (right) cells were untreated or treated with 1-butanol (1-ButOH) or tert-butanol (t-ButOH) as control (0.8%). SphK1 activity (b) and HIF-2α expression (c) were determined in normoxia or after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001. (d, e) CAKI-1 (left) and A498 (right) cells were transfected with siPLD1 (50 nmol/L), siPLD2 (50 nmol/L) or siPLD1 (50 nmol/L) and siPLD2 (50 nmol/L) or siScr (50 nmol/L) for 72 h then incubated under normoxia or hypoxia. SphK1 activity (d) and HIF-2α expression (e) were determined after 1 h and 6 h of hypoxia, respectively. Similar results were obtained in at least three independent experiments, and equal loading was monitored using antibody to tubulin. Columns, mean of three independent experiments; bars, s.e.m. The two-tailed P-values between the means of normoxic or hypoxic cells are ns, not significant; **P<0.01; ***P<0.001.
Mentions: Because phospholipase D (PLD) activity has been involved in the control of HIF-2α expression in ccRCC35 and is an upstream regulator of SphK1 in a different physiological context,36 we next examined the interactions between PLD and SphK1 signaling with regard to HIF-2α expression. In hypoxic CAKI-1, A498 cells (Figure 2a) and 786-O cells (Supplementary Figure 1A), an early transient increase in PLD activity (peaking at 15–30 min) followed by activation of SphK1 (peaking at 60 min) was observed. Accordingly, accumulation of HIF-2α did not occur before 2–3 h of hypoxia in CAKI-1 cells (Figure 2a, inset). Confirming that SphK1 activation was a consequence of PLD stimulation, butan-1-ol (1-ButOH), a potent inhibitor of PLD activation, markedly inhibited SphK1 activity in all ccRCC cell lines (Figure 2b and Supplementary Figure 1B). To rule out any possible non-specific effect of the alcohols on SphK1 activity, cells were treated in the presence of t-butanol (t-ButOH), a tertiary alcohol, which is not a substrate for PLD inhibition. As expected, t-ButOH did not alter SphK1 activity in ccRCC cell lines (Figure 2b and Supplementary Figure 1B). Unlike t-ButOH, 1-ButOH markedly inhibited HIF-2α expression in CAKI-1 cells (Figure 2c, left), as well as in normoxic and hypoxic A498 cells (Figure 2c, right) and in 786-0 cells (Supplementary Figure 1c). To investigate which PLD isozyme could be involved in the PLD/SphK1/HIF-2α signaling sequence, siRNAs directed to PLD1 and PLD2 isoforms were used.37 A roughly 50% PLD knock-down in CAKI-1, A498 and in 786-0 cells was achieved without additive effect when both siRNAs were combined (Supplementary Figure 2). Both PLD1 and PLD2 siRNAs significantly reduced—although not to the same extent—SphK1 activity in CAKI-1 (Figure 2d, left), A498 (Figure 2d, right) and 786-0 cells (Supplementary Figure 1D). Accordingly, the downregulation of SphK1 activity was accompanied by a marked reduction in HIF-2α expression in hypoxic CAKI-1 cells (Figure 2e, left), as well as in normoxic and hypoxic A498 (Figure 2e, right) and 786-O cells (Supplementary Figure 1E). These data suggest that both PLD1 and PLD2 isozymes are likely required for regulation of HIF-2α through SphK1 signaling in ccRCC cell lines.

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