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Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis.

Saddoughi SA, Gencer S, Peterson YK, Ward KE, Mukhopadhyay A, Oaks J, Bielawski J, Szulc ZM, Thomas RJ, Selvam SP, Senkal CE, Garrett-Mayer E, De Palma RM, Fedarovich D, Liu A, Habib AA, Stahelin RV, Perrotti D, Ogretmen B - EMBO Mol Med (2012)

Bottom Line: Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis.The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720.Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1(-/-) MEFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

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Phosphorylation of FTY720 by SK-2 is dispensable for I2PP2A/SET binding and tumour suppressionA. Binding of B-FTY720 to purified I2PP2A/SET (elution) was measured by avidin column pull-down followed by Western blotting in the absence/presence of unlabeled C18-ceramide, FTY720 or P-FTY720 (10 µM/each), used as competitors. Structure of P-FTY720 is shown.B. Effects of FTY720 and P-FTY720 on c-Myc expression in A549 cells were examined by Western blotting. Actin was used as a loading control. Relative c-Myc levels are shown blow the first panel.C. Effects of FTY720 on ectopically expressed c-Myc-V5 in WT compared to SK-1−/− and SK-2−/− MEFs were measured by Western blotting. Actin was used as a loading control. Relative c-Myc levels are shown blow the first panel. Full blots can be seen in Supporting Information Fig S15.D,E. Roles of FTY720 in tumour suppression was measured in WT (C57B7/6) and SK2−/− mice, bearing Lewis lung adenocarcinoma-derived allografts (D), and % change in tumour volume relative to WT and SK-2−/− mice in response to FTY720 compared to controls at Day 13 are shown (E).F,G. FTY720 and P-FTY720 accumulation in serum (F) versus tumour tissues (G) grown in WT versus SK-2−/− mice in the absence/presence of FTY720 were measured using LC/MS/MS (normalized to Pi). Error bars represent s.d. (**p < 0.01, ***p < 0.001).
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fig05: Phosphorylation of FTY720 by SK-2 is dispensable for I2PP2A/SET binding and tumour suppressionA. Binding of B-FTY720 to purified I2PP2A/SET (elution) was measured by avidin column pull-down followed by Western blotting in the absence/presence of unlabeled C18-ceramide, FTY720 or P-FTY720 (10 µM/each), used as competitors. Structure of P-FTY720 is shown.B. Effects of FTY720 and P-FTY720 on c-Myc expression in A549 cells were examined by Western blotting. Actin was used as a loading control. Relative c-Myc levels are shown blow the first panel.C. Effects of FTY720 on ectopically expressed c-Myc-V5 in WT compared to SK-1−/− and SK-2−/− MEFs were measured by Western blotting. Actin was used as a loading control. Relative c-Myc levels are shown blow the first panel. Full blots can be seen in Supporting Information Fig S15.D,E. Roles of FTY720 in tumour suppression was measured in WT (C57B7/6) and SK2−/− mice, bearing Lewis lung adenocarcinoma-derived allografts (D), and % change in tumour volume relative to WT and SK-2−/− mice in response to FTY720 compared to controls at Day 13 are shown (E).F,G. FTY720 and P-FTY720 accumulation in serum (F) versus tumour tissues (G) grown in WT versus SK-2−/− mice in the absence/presence of FTY720 were measured using LC/MS/MS (normalized to Pi). Error bars represent s.d. (**p < 0.01, ***p < 0.001).

Mentions: Phosphorylation of FTY720 is catalysed by nuclear SK-2, and it is required for its immune-suppressor and anti-MS activities (Billich et al, 2003; Paugh et al, 2003). However, whether P-FTY720 generation plays a role in I2PP2A/SET binding or tumour suppression is unknown. Therefore, we first examined whether P-FTY720 is necessary for its I2PP2A/SET binding in A549 cells. To achieve this, purified I2PP2A/SET was incubated with B-FTY720 in the absence/presence of unlabeled C18-Pyr-Cer, FTY720 or P-FTY720 as competitors. Incubation with unlabeled C18-ceramide or FTY720, but not P-FTY720, competed significantly with B-FTY720-I2PP2A/SET binding (Fig 5A). These data suggest that P-FTY720 is not as efficient as FTY720 to bind I2PP2A/SET, supporting our molecular modelling (Fig 3B) and in vitro SPR studies (Fig 3E and F), which suggested that presence of a primary hydroxyl group in the FTY720 structure is important for I2PP2A/SET binding and that I2PP2A/SET has a lower affinity to P-FTY720 compared to C18-ceramide and FTY720, respectively.


Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis.

Saddoughi SA, Gencer S, Peterson YK, Ward KE, Mukhopadhyay A, Oaks J, Bielawski J, Szulc ZM, Thomas RJ, Selvam SP, Senkal CE, Garrett-Mayer E, De Palma RM, Fedarovich D, Liu A, Habib AA, Stahelin RV, Perrotti D, Ogretmen B - EMBO Mol Med (2012)

Phosphorylation of FTY720 by SK-2 is dispensable for I2PP2A/SET binding and tumour suppressionA. Binding of B-FTY720 to purified I2PP2A/SET (elution) was measured by avidin column pull-down followed by Western blotting in the absence/presence of unlabeled C18-ceramide, FTY720 or P-FTY720 (10 µM/each), used as competitors. Structure of P-FTY720 is shown.B. Effects of FTY720 and P-FTY720 on c-Myc expression in A549 cells were examined by Western blotting. Actin was used as a loading control. Relative c-Myc levels are shown blow the first panel.C. Effects of FTY720 on ectopically expressed c-Myc-V5 in WT compared to SK-1−/− and SK-2−/− MEFs were measured by Western blotting. Actin was used as a loading control. Relative c-Myc levels are shown blow the first panel. Full blots can be seen in Supporting Information Fig S15.D,E. Roles of FTY720 in tumour suppression was measured in WT (C57B7/6) and SK2−/− mice, bearing Lewis lung adenocarcinoma-derived allografts (D), and % change in tumour volume relative to WT and SK-2−/− mice in response to FTY720 compared to controls at Day 13 are shown (E).F,G. FTY720 and P-FTY720 accumulation in serum (F) versus tumour tissues (G) grown in WT versus SK-2−/− mice in the absence/presence of FTY720 were measured using LC/MS/MS (normalized to Pi). Error bars represent s.d. (**p < 0.01, ***p < 0.001).
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fig05: Phosphorylation of FTY720 by SK-2 is dispensable for I2PP2A/SET binding and tumour suppressionA. Binding of B-FTY720 to purified I2PP2A/SET (elution) was measured by avidin column pull-down followed by Western blotting in the absence/presence of unlabeled C18-ceramide, FTY720 or P-FTY720 (10 µM/each), used as competitors. Structure of P-FTY720 is shown.B. Effects of FTY720 and P-FTY720 on c-Myc expression in A549 cells were examined by Western blotting. Actin was used as a loading control. Relative c-Myc levels are shown blow the first panel.C. Effects of FTY720 on ectopically expressed c-Myc-V5 in WT compared to SK-1−/− and SK-2−/− MEFs were measured by Western blotting. Actin was used as a loading control. Relative c-Myc levels are shown blow the first panel. Full blots can be seen in Supporting Information Fig S15.D,E. Roles of FTY720 in tumour suppression was measured in WT (C57B7/6) and SK2−/− mice, bearing Lewis lung adenocarcinoma-derived allografts (D), and % change in tumour volume relative to WT and SK-2−/− mice in response to FTY720 compared to controls at Day 13 are shown (E).F,G. FTY720 and P-FTY720 accumulation in serum (F) versus tumour tissues (G) grown in WT versus SK-2−/− mice in the absence/presence of FTY720 were measured using LC/MS/MS (normalized to Pi). Error bars represent s.d. (**p < 0.01, ***p < 0.001).
Mentions: Phosphorylation of FTY720 is catalysed by nuclear SK-2, and it is required for its immune-suppressor and anti-MS activities (Billich et al, 2003; Paugh et al, 2003). However, whether P-FTY720 generation plays a role in I2PP2A/SET binding or tumour suppression is unknown. Therefore, we first examined whether P-FTY720 is necessary for its I2PP2A/SET binding in A549 cells. To achieve this, purified I2PP2A/SET was incubated with B-FTY720 in the absence/presence of unlabeled C18-Pyr-Cer, FTY720 or P-FTY720 as competitors. Incubation with unlabeled C18-ceramide or FTY720, but not P-FTY720, competed significantly with B-FTY720-I2PP2A/SET binding (Fig 5A). These data suggest that P-FTY720 is not as efficient as FTY720 to bind I2PP2A/SET, supporting our molecular modelling (Fig 3B) and in vitro SPR studies (Fig 3E and F), which suggested that presence of a primary hydroxyl group in the FTY720 structure is important for I2PP2A/SET binding and that I2PP2A/SET has a lower affinity to P-FTY720 compared to C18-ceramide and FTY720, respectively.

Bottom Line: Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis.The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720.Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1(-/-) MEFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

Show MeSH
Related in: MedlinePlus