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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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FTY720-mediated lung tumour suppression involves the kinase domain of RIPK1Roles of siRNA-mediated knockdown of RIPK1 on cell death in the absence/presence of ectopic PP2Ac-HA expression were measured by detection of LDH release compared to controls.Roles of ectopic expression of WT-, DDD-, or DKD-RIPK1 in the regulation of cell death were measured by detection of LDH release in RIPK1−/− MEFs. Error bars represent s.d. (*p < 0.05, **p < 0.01).Our novel data suggest that FTY720 directly binds and targets I2PP2A/SET oncoprotein, mimicking sphingosine/ceramide, which results in PP2A activation, subsequently leading to RIPK1-dependent necroptosis, and lung tumour suppression.
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fig09: FTY720-mediated lung tumour suppression involves the kinase domain of RIPK1Roles of siRNA-mediated knockdown of RIPK1 on cell death in the absence/presence of ectopic PP2Ac-HA expression were measured by detection of LDH release compared to controls.Roles of ectopic expression of WT-, DDD-, or DKD-RIPK1 in the regulation of cell death were measured by detection of LDH release in RIPK1−/− MEFs. Error bars represent s.d. (*p < 0.05, **p < 0.01).Our novel data suggest that FTY720 directly binds and targets I2PP2A/SET oncoprotein, mimicking sphingosine/ceramide, which results in PP2A activation, subsequently leading to RIPK1-dependent necroptosis, and lung tumour suppression.

Mentions: Because I2PP2A/SET-FTY720 binding activated PP2A, we explored the role of PP2A on RIPK1-induced necroptosis. Ectopic PP2Ac (catalytic domain) expression alone or in combination with FTY720 induced necroptosis, however, siRNA-mediated knockdown of RIPK1 prevented necroptosis in response to PP2Ac (Fig 9A). Induction of PP2A-dependent necroptosis by FTY720 was also detected in H157 (K-Ras mutant) and H827 (EGFR mutant) human lung cancer cells, in which inhibition of RIPK1 or PP2A using necrostastin or OA and knockdown of RIPK1 or PP2Ac prevented FTY720-mediated cell death compared to controls (Supporting Information Fig S12A–C). Interestingly, ectopic PP2Ac had no effect on FTY720-mediated cell death in RIPK1−/− MEFs compared to vector-transfected controls (Supporting Information Fig S13A). Interestingly, siRNA-mediated knockdown of RIPK3, a known inducer of necroptosis via MKLN activation (Sun et al, 2012), or Drp1, a downstream target of PGAM5, which is a recently identified necroptosis inducer (Wang et al, 2012), did not prevent FTY720-mediated A549 cell death (Supporting Information Fig S13B–D). In fact, knockdown of RIPK3 increased FTY720-mediated LDH release around twofold compared to controls (Supporting Information Fig S13D). Thus, these data indicate that PP2A activation plays an upstream role in selective RIPK1-dependent necroptosis by FTY720 in these cells.


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)

FTY720-mediated lung tumour suppression involves the kinase domain of RIPK1Roles of siRNA-mediated knockdown of RIPK1 on cell death in the absence/presence of ectopic PP2Ac-HA expression were measured by detection of LDH release compared to controls.Roles of ectopic expression of WT-, DDD-, or DKD-RIPK1 in the regulation of cell death were measured by detection of LDH release in RIPK1−/− MEFs. Error bars represent s.d. (*p < 0.05, **p < 0.01).Our novel data suggest that FTY720 directly binds and targets I2PP2A/SET oncoprotein, mimicking sphingosine/ceramide, which results in PP2A activation, subsequently leading to RIPK1-dependent necroptosis, and lung tumour suppression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569657&req=5

fig09: FTY720-mediated lung tumour suppression involves the kinase domain of RIPK1Roles of siRNA-mediated knockdown of RIPK1 on cell death in the absence/presence of ectopic PP2Ac-HA expression were measured by detection of LDH release compared to controls.Roles of ectopic expression of WT-, DDD-, or DKD-RIPK1 in the regulation of cell death were measured by detection of LDH release in RIPK1−/− MEFs. Error bars represent s.d. (*p < 0.05, **p < 0.01).Our novel data suggest that FTY720 directly binds and targets I2PP2A/SET oncoprotein, mimicking sphingosine/ceramide, which results in PP2A activation, subsequently leading to RIPK1-dependent necroptosis, and lung tumour suppression.
Mentions: Because I2PP2A/SET-FTY720 binding activated PP2A, we explored the role of PP2A on RIPK1-induced necroptosis. Ectopic PP2Ac (catalytic domain) expression alone or in combination with FTY720 induced necroptosis, however, siRNA-mediated knockdown of RIPK1 prevented necroptosis in response to PP2Ac (Fig 9A). Induction of PP2A-dependent necroptosis by FTY720 was also detected in H157 (K-Ras mutant) and H827 (EGFR mutant) human lung cancer cells, in which inhibition of RIPK1 or PP2A using necrostastin or OA and knockdown of RIPK1 or PP2Ac prevented FTY720-mediated cell death compared to controls (Supporting Information Fig S12A–C). Interestingly, ectopic PP2Ac had no effect on FTY720-mediated cell death in RIPK1−/− MEFs compared to vector-transfected controls (Supporting Information Fig S13A). Interestingly, siRNA-mediated knockdown of RIPK3, a known inducer of necroptosis via MKLN activation (Sun et al, 2012), or Drp1, a downstream target of PGAM5, which is a recently identified necroptosis inducer (Wang et al, 2012), did not prevent FTY720-mediated A549 cell death (Supporting Information Fig S13B–D). In fact, knockdown of RIPK3 increased FTY720-mediated LDH release around twofold compared to controls (Supporting Information Fig S13D). Thus, these data indicate that PP2A activation plays an upstream role in selective RIPK1-dependent necroptosis by FTY720 in these cells.

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