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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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I2PP2A/SET is overexpressed and C18-ceramide is decreased in primary lung tumour tissuesTen paired samples of fresh frozen lung adenocarcinoma tumours (T) with adjacent, non-cancerous lung tissues (N) were homogenized and Western blotting was done to evaluate I2PP2A/SET. β-actin was used as a control. Full blots can be seen in Supporting Information Fig S15.I2PP2A/SET was detected in N7, N5 versus T7, T5 tissues using IHC (left and right panels, respectively).Ceramides were measured by LC/MS/MS in N1–10 versus T1–10 tissues (normalized to µg of protein), and C18-ceramide levels in N1–10 compared to T1–10 tissues were reported.CerS1 and CerS6 mRNA were measured by Q-PCR in T1–10 compared to N1–10 tissues (normalized to rRNA).I2PP2A/SET was detected by IHC and scored (percentage of I2PP2A/SET expression was calculated using positive staining scored between 1 and 5 and intensity of staining scored between 1 and 3) in TMAs containing lung tumour (1) and non-cancerous lung tissues (2) (n = 48 pairs), and normal lung tissues (n = 4). p < 0.05 was considered significant (calculated using paired t-test).
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fig02: I2PP2A/SET is overexpressed and C18-ceramide is decreased in primary lung tumour tissuesTen paired samples of fresh frozen lung adenocarcinoma tumours (T) with adjacent, non-cancerous lung tissues (N) were homogenized and Western blotting was done to evaluate I2PP2A/SET. β-actin was used as a control. Full blots can be seen in Supporting Information Fig S15.I2PP2A/SET was detected in N7, N5 versus T7, T5 tissues using IHC (left and right panels, respectively).Ceramides were measured by LC/MS/MS in N1–10 versus T1–10 tissues (normalized to µg of protein), and C18-ceramide levels in N1–10 compared to T1–10 tissues were reported.CerS1 and CerS6 mRNA were measured by Q-PCR in T1–10 compared to N1–10 tissues (normalized to rRNA).I2PP2A/SET was detected by IHC and scored (percentage of I2PP2A/SET expression was calculated using positive staining scored between 1 and 5 and intensity of staining scored between 1 and 3) in TMAs containing lung tumour (1) and non-cancerous lung tissues (2) (n = 48 pairs), and normal lung tissues (n = 4). p < 0.05 was considered significant (calculated using paired t-test).

Mentions: Binding of I2PP2A/SET to ceramide in lung cancer cells might have clinical and biological significance. To define the clinical relevance of I2PP2A/SET-C18-ceramide in cancer pathogenesis, we measured I2PP2A/SET and C18-ceramide in tumour versus pathologically non-cancerous adjacent lung tissues obtained from 10 patients with NSCLC using Western blotting and immunohistochemistry (IHC; Fig 2A and B). Data showed that I2PP2A/SET is overexpressed in 70% (7/10, n = 10) of these tumours (Fig 2A), whereas C18-ceramide and CerS1 mRNA levels are decreased (∼50%) in the majority of tumours (8/10, n = 10) compared to non-cancerous lung tissues (Fig 2C and D, respectively). Importantly, at least 4 of 10 patients exhibited overexpression of I2PP2A/SET in combination with down-regulation of C18-ceramide in their lung tumours, suggesting that tumour suppressive ceramide/PP2A signalling is altered in these tumours (Fig 2C and D). However, C16-ceramide was significantly higher (∼1.8-fold, p < 0.01, n = 10) in tumour compared to non-cancerous lung tissues, and there were no significant changes in the levels of other ceramide species (Supporting Information Fig S5).


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)

I2PP2A/SET is overexpressed and C18-ceramide is decreased in primary lung tumour tissuesTen paired samples of fresh frozen lung adenocarcinoma tumours (T) with adjacent, non-cancerous lung tissues (N) were homogenized and Western blotting was done to evaluate I2PP2A/SET. β-actin was used as a control. Full blots can be seen in Supporting Information Fig S15.I2PP2A/SET was detected in N7, N5 versus T7, T5 tissues using IHC (left and right panels, respectively).Ceramides were measured by LC/MS/MS in N1–10 versus T1–10 tissues (normalized to µg of protein), and C18-ceramide levels in N1–10 compared to T1–10 tissues were reported.CerS1 and CerS6 mRNA were measured by Q-PCR in T1–10 compared to N1–10 tissues (normalized to rRNA).I2PP2A/SET was detected by IHC and scored (percentage of I2PP2A/SET expression was calculated using positive staining scored between 1 and 5 and intensity of staining scored between 1 and 3) in TMAs containing lung tumour (1) and non-cancerous lung tissues (2) (n = 48 pairs), and normal lung tissues (n = 4). p < 0.05 was considered significant (calculated using paired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3569657&req=5

fig02: I2PP2A/SET is overexpressed and C18-ceramide is decreased in primary lung tumour tissuesTen paired samples of fresh frozen lung adenocarcinoma tumours (T) with adjacent, non-cancerous lung tissues (N) were homogenized and Western blotting was done to evaluate I2PP2A/SET. β-actin was used as a control. Full blots can be seen in Supporting Information Fig S15.I2PP2A/SET was detected in N7, N5 versus T7, T5 tissues using IHC (left and right panels, respectively).Ceramides were measured by LC/MS/MS in N1–10 versus T1–10 tissues (normalized to µg of protein), and C18-ceramide levels in N1–10 compared to T1–10 tissues were reported.CerS1 and CerS6 mRNA were measured by Q-PCR in T1–10 compared to N1–10 tissues (normalized to rRNA).I2PP2A/SET was detected by IHC and scored (percentage of I2PP2A/SET expression was calculated using positive staining scored between 1 and 5 and intensity of staining scored between 1 and 3) in TMAs containing lung tumour (1) and non-cancerous lung tissues (2) (n = 48 pairs), and normal lung tissues (n = 4). p < 0.05 was considered significant (calculated using paired t-test).
Mentions: Binding of I2PP2A/SET to ceramide in lung cancer cells might have clinical and biological significance. To define the clinical relevance of I2PP2A/SET-C18-ceramide in cancer pathogenesis, we measured I2PP2A/SET and C18-ceramide in tumour versus pathologically non-cancerous adjacent lung tissues obtained from 10 patients with NSCLC using Western blotting and immunohistochemistry (IHC; Fig 2A and B). Data showed that I2PP2A/SET is overexpressed in 70% (7/10, n = 10) of these tumours (Fig 2A), whereas C18-ceramide and CerS1 mRNA levels are decreased (∼50%) in the majority of tumours (8/10, n = 10) compared to non-cancerous lung tissues (Fig 2C and D, respectively). Importantly, at least 4 of 10 patients exhibited overexpression of I2PP2A/SET in combination with down-regulation of C18-ceramide in their lung tumours, suggesting that tumour suppressive ceramide/PP2A signalling is altered in these tumours (Fig 2C and D). However, C16-ceramide was significantly higher (∼1.8-fold, p < 0.01, n = 10) in tumour compared to non-cancerous lung tissues, and there were no significant changes in the levels of other ceramide species (Supporting Information Fig S5).

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