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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 inhibits A549-derived xenograft tumour growth in SCID miceEffects of FTY720 (3 mg/kg/day via oral gavage for 15 days) on A549-derived xenograft tumour growth was evaluated in Balb/c SCID mice (n = 12). Tumour sizes were measured at every 2–3 days.PP2A (total), P-PP2A (inactive) and cMyc (total) were detected by IHC in tumour tissues obtained from mice treated with FTY720 compared to vehicle-treated controls.Electron microscopy of control and FTY720 treated tumours. Arrows indicate areas of mitochondria damage and cytoplasmic swelling.FTY720 and P-FTY720 were measured by LC/MS/MS in tumour and serum samples obtained from mice treated with FTY720 compared to vehicle treated controls. Error bars show s.d. (*p < 0.05, **p < 0.01).
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fig04: FTY720 inhibits A549-derived xenograft tumour growth in SCID miceEffects of FTY720 (3 mg/kg/day via oral gavage for 15 days) on A549-derived xenograft tumour growth was evaluated in Balb/c SCID mice (n = 12). Tumour sizes were measured at every 2–3 days.PP2A (total), P-PP2A (inactive) and cMyc (total) were detected by IHC in tumour tissues obtained from mice treated with FTY720 compared to vehicle-treated controls.Electron microscopy of control and FTY720 treated tumours. Arrows indicate areas of mitochondria damage and cytoplasmic swelling.FTY720 and P-FTY720 were measured by LC/MS/MS in tumour and serum samples obtained from mice treated with FTY720 compared to vehicle treated controls. Error bars show s.d. (*p < 0.05, **p < 0.01).

Mentions: Because FTY720 directly bound I2PP2A/SET, we explored its ability to inhibit tumour growth via activation of PP2A tumour suppressor signalling. The therapeutic potential of FTY720 against A549-xenografts generated in the flanks of SCID mice was determined after oral administration of FTY720 (3 mg/kg/day for 15 days, n = 12) (LaMontagne et al, 2006). FTY720 significantly (p < 0.05, n = 12) inhibited the growth of A549-xenograft-derived tumours compared to controls (Fig 4A). To examine whether FTY720-mediated tumour suppression was linked to PP2A activation, we examined P-PP2A (inactive) and PP2A (total) in tumours obtained from control versus FTY720-treated animals. The data showed that FTY720 treatment decreased inactive P-PP2A (Y307) without affecting total PP2A compared to controls (Fig 4B, upper and middle right/left panels, respectively), suggesting PP2A activation. Because activation of PP2A results in c-Myc degradation (Mukhopadhyay et al, 2009), leading to inhibition of human telomerase reverse transcriptase (hTERT) transcription, we also confirmed FTY720-mediated PP2A activation by measuring c-Myc and hTERT in these tumour tissues. Activation of PP2A by FTY720 was consistent with decreased c-Myc (as detected using IHC or Western blotting) and hTERT mRNA (measured by Q-PCR) in these tumours compared to controls (Fig 4B, lower panel, or Supporting Information Fig S8A and B). In addition, examination of the tumours by transmission electron microscopy (TEM) revealed that ∼40% of tumours (n = 20) obtained from mice treated with oral FTY720 had damaged/swollen mitochondria consistent with cellular damage leading to inhibition of tumour growth (Fig 4C).


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 inhibits A549-derived xenograft tumour growth in SCID miceEffects of FTY720 (3 mg/kg/day via oral gavage for 15 days) on A549-derived xenograft tumour growth was evaluated in Balb/c SCID mice (n = 12). Tumour sizes were measured at every 2–3 days.PP2A (total), P-PP2A (inactive) and cMyc (total) were detected by IHC in tumour tissues obtained from mice treated with FTY720 compared to vehicle-treated controls.Electron microscopy of control and FTY720 treated tumours. Arrows indicate areas of mitochondria damage and cytoplasmic swelling.FTY720 and P-FTY720 were measured by LC/MS/MS in tumour and serum samples obtained from mice treated with FTY720 compared to vehicle treated controls. Error bars show s.d. (*p < 0.05, **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569657&req=5

fig04: FTY720 inhibits A549-derived xenograft tumour growth in SCID miceEffects of FTY720 (3 mg/kg/day via oral gavage for 15 days) on A549-derived xenograft tumour growth was evaluated in Balb/c SCID mice (n = 12). Tumour sizes were measured at every 2–3 days.PP2A (total), P-PP2A (inactive) and cMyc (total) were detected by IHC in tumour tissues obtained from mice treated with FTY720 compared to vehicle-treated controls.Electron microscopy of control and FTY720 treated tumours. Arrows indicate areas of mitochondria damage and cytoplasmic swelling.FTY720 and P-FTY720 were measured by LC/MS/MS in tumour and serum samples obtained from mice treated with FTY720 compared to vehicle treated controls. Error bars show s.d. (*p < 0.05, **p < 0.01).
Mentions: Because FTY720 directly bound I2PP2A/SET, we explored its ability to inhibit tumour growth via activation of PP2A tumour suppressor signalling. The therapeutic potential of FTY720 against A549-xenografts generated in the flanks of SCID mice was determined after oral administration of FTY720 (3 mg/kg/day for 15 days, n = 12) (LaMontagne et al, 2006). FTY720 significantly (p < 0.05, n = 12) inhibited the growth of A549-xenograft-derived tumours compared to controls (Fig 4A). To examine whether FTY720-mediated tumour suppression was linked to PP2A activation, we examined P-PP2A (inactive) and PP2A (total) in tumours obtained from control versus FTY720-treated animals. The data showed that FTY720 treatment decreased inactive P-PP2A (Y307) without affecting total PP2A compared to controls (Fig 4B, upper and middle right/left panels, respectively), suggesting PP2A activation. Because activation of PP2A results in c-Myc degradation (Mukhopadhyay et al, 2009), leading to inhibition of human telomerase reverse transcriptase (hTERT) transcription, we also confirmed FTY720-mediated PP2A activation by measuring c-Myc and hTERT in these tumour tissues. Activation of PP2A by FTY720 was consistent with decreased c-Myc (as detected using IHC or Western blotting) and hTERT mRNA (measured by Q-PCR) in these tumours compared to controls (Fig 4B, lower panel, or Supporting Information Fig S8A and B). In addition, examination of the tumours by transmission electron microscopy (TEM) revealed that ∼40% of tumours (n = 20) obtained from mice treated with oral FTY720 had damaged/swollen mitochondria consistent with cellular damage leading to inhibition of tumour growth (Fig 4C).

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