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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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Targeting I2PP2A/SET by FTY720 inhibits tumour growthA. Effects of FTY720 on cell death were measured in A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET cells, in the absence/presence of 20 µM FTY720 or vehicle control for 24 h.B. Cell death in response to FTY720 in A549/sh-I2PP2A/SET cells in the absence/presence of WT-, K209D-, Y122C- or ER-I2PP2A/SET expression was measured by detection of LDH levels compared to controls.C,D. Effects of FTY720 on tumour growth were measured in Balb/c SCID mice bearing A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET xenografts compared to controls for 18 days. The mice were treated daily with 3 mg/kg FTY720 by oral gavage (C). Percent change in tumour volume relative to untreated A549/sh-control mice at Day 18 are shown (D).E. Levels of LDH release to the serum in response to FTY720 treatment compared to untreated controls in SCID mice bearing A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET xenografts were measured by Analytics Inc. Error bars represent s.d. (*p < 0.05, **p < 0.01, ***p < 0.001).
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fig07: Targeting I2PP2A/SET by FTY720 inhibits tumour growthA. Effects of FTY720 on cell death were measured in A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET cells, in the absence/presence of 20 µM FTY720 or vehicle control for 24 h.B. Cell death in response to FTY720 in A549/sh-I2PP2A/SET cells in the absence/presence of WT-, K209D-, Y122C- or ER-I2PP2A/SET expression was measured by detection of LDH levels compared to controls.C,D. Effects of FTY720 on tumour growth were measured in Balb/c SCID mice bearing A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET xenografts compared to controls for 18 days. The mice were treated daily with 3 mg/kg FTY720 by oral gavage (C). Percent change in tumour volume relative to untreated A549/sh-control mice at Day 18 are shown (D).E. Levels of LDH release to the serum in response to FTY720 treatment compared to untreated controls in SCID mice bearing A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET xenografts were measured by Analytics Inc. Error bars represent s.d. (*p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: To validate I2PP2A/SET as a target of FTY720 for its tumour suppressor roles, we determined the effects of FTY720 on cell death and/or tumour suppression in response to shRNA-mediated knockdown of I2PP2A and its reconstitution in A549/sh-I2PP2A/SET versus A549/sh-I2PP2A/SET-WT-I2PP2A/SET cells in situ and in vivo. Treatment with FTY720 or knockdown of I2PP2A/SET alone significantly induced cell death compared to their respective controls (Fig 7A). Importantly, knockdown of I2PP2A/SET prevented FTY720-induced cell death (Fig 7A). Reconstitution of WT-I2PP2A/SET expression in A549/sh-I2PP2A/SET cells restored FTY720-mediated cell death (Fig 7A). These data were consistent with the effects of I2PP2A/SET knockdown and expression of WT-I2PP2A/SET on PP2A activation in the absence/presence of FTY720 in these cells (Fig 6B). Expression of Y122C-I2PP2A/SET-GFP and ER-I2PP2A/SET-GFP restored FTY720-mediated cell death when endogenous I2PP2A/SET was down-regulated compared to controls in A549/sh-I2PP2A/SET cells (Fig 7B). However, expression of K209D-I2PP2A/SET had no significant effect on cell death in response to FTY720 in A549/sh-I2PP2A/SET cells (Fig 7B). I2PP2A/SET-GFP expression in A549/sh-I2PP2A/SET cells was confirmed by Western blotting (Supporting Information Fig S10B).


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)

Targeting I2PP2A/SET by FTY720 inhibits tumour growthA. Effects of FTY720 on cell death were measured in A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET cells, in the absence/presence of 20 µM FTY720 or vehicle control for 24 h.B. Cell death in response to FTY720 in A549/sh-I2PP2A/SET cells in the absence/presence of WT-, K209D-, Y122C- or ER-I2PP2A/SET expression was measured by detection of LDH levels compared to controls.C,D. Effects of FTY720 on tumour growth were measured in Balb/c SCID mice bearing A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET xenografts compared to controls for 18 days. The mice were treated daily with 3 mg/kg FTY720 by oral gavage (C). Percent change in tumour volume relative to untreated A549/sh-control mice at Day 18 are shown (D).E. Levels of LDH release to the serum in response to FTY720 treatment compared to untreated controls in SCID mice bearing A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET xenografts were measured by Analytics Inc. Error bars represent s.d. (*p < 0.05, **p < 0.01, ***p < 0.001).
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fig07: Targeting I2PP2A/SET by FTY720 inhibits tumour growthA. Effects of FTY720 on cell death were measured in A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET cells, in the absence/presence of 20 µM FTY720 or vehicle control for 24 h.B. Cell death in response to FTY720 in A549/sh-I2PP2A/SET cells in the absence/presence of WT-, K209D-, Y122C- or ER-I2PP2A/SET expression was measured by detection of LDH levels compared to controls.C,D. Effects of FTY720 on tumour growth were measured in Balb/c SCID mice bearing A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET xenografts compared to controls for 18 days. The mice were treated daily with 3 mg/kg FTY720 by oral gavage (C). Percent change in tumour volume relative to untreated A549/sh-control mice at Day 18 are shown (D).E. Levels of LDH release to the serum in response to FTY720 treatment compared to untreated controls in SCID mice bearing A549/sh-control, A549/sh-I2PP2A/SET and A549/sh-I2PP2A/SET/WT-I2PP2A/SET xenografts were measured by Analytics Inc. Error bars represent s.d. (*p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: To validate I2PP2A/SET as a target of FTY720 for its tumour suppressor roles, we determined the effects of FTY720 on cell death and/or tumour suppression in response to shRNA-mediated knockdown of I2PP2A and its reconstitution in A549/sh-I2PP2A/SET versus A549/sh-I2PP2A/SET-WT-I2PP2A/SET cells in situ and in vivo. Treatment with FTY720 or knockdown of I2PP2A/SET alone significantly induced cell death compared to their respective controls (Fig 7A). Importantly, knockdown of I2PP2A/SET prevented FTY720-induced cell death (Fig 7A). Reconstitution of WT-I2PP2A/SET expression in A549/sh-I2PP2A/SET cells restored FTY720-mediated cell death (Fig 7A). These data were consistent with the effects of I2PP2A/SET knockdown and expression of WT-I2PP2A/SET on PP2A activation in the absence/presence of FTY720 in these cells (Fig 6B). Expression of Y122C-I2PP2A/SET-GFP and ER-I2PP2A/SET-GFP restored FTY720-mediated cell death when endogenous I2PP2A/SET was down-regulated compared to controls in A549/sh-I2PP2A/SET cells (Fig 7B). However, expression of K209D-I2PP2A/SET had no significant effect on cell death in response to FTY720 in A549/sh-I2PP2A/SET cells (Fig 7B). I2PP2A/SET-GFP expression in A549/sh-I2PP2A/SET cells was confirmed by Western blotting (Supporting Information Fig S10B).

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