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c-Rel is a critical mediator of NF-κB-dependent TRAIL resistance of pancreatic cancer cells.

Geismann C, Grohmann F, Sebens S, Wirths G, Dreher A, Häsler R, Rosenstiel P, Hauser C, Egberts JH, Trauzold A, Schneider G, Sipos B, Zeissig S, Schreiber S, Schäfer H, Arlt A - Cell Death Dis (2014)

Bottom Line: In line, siRNA targeting c-Rel strongly reduced TRAIL-induced NFATc2 activity in TRAIL-resistant PDAC cells.Finally, TRAIL-induced expression of COX-2 was diminished through siRNA targeting c-Rel or NFATc2 and pharmacologic inhibition of COX-2 with celecoxib or siRNA targeting COX-2, enhanced TRAIL apoptosis.In conclusion, we were able to delineate a novel c-Rel-, NFATc2- and COX-2-dependent antiapoptotic signalling pathway in PDAC with broad clinical implications for pharmaceutical intervention strategies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gastroenterology and Hepatology, 1st Department of Internal Medicine I, University Hospital Schleswig-Holstein, Kiel, Germany.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies with an overall life expectancy of 6 months despite current therapies. NF-κB signalling has been shown to be critical for this profound cell-autonomous resistance against chemotherapeutic drugs and death receptor-induced apoptosis, but little is known about the role of the c-Rel subunit in solid cancer and PDAC apoptosis control. In the present study, by analysis of genome-wide patterns of c-Rel-dependent gene expression, we were able to establish c-Rel as a critical regulator of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in PDAC. TRAIL-resistant cells exhibited a strong TRAIL-inducible NF-κB activity, whereas TRAIL-sensitive cells displayed only a small increase in NF-κB-binding activity. Transfection with siRNA against c-Rel sensitized the TRAIL-resistant cells in a manner comparable to siRNA targeting the p65/RelA subunit. Gel-shift analysis revealed that c-Rel is part of the TRAIL-inducible NF-κB complex in PDAC. Array analysis identified NFATc2 as a c-Rel target gene among the 12 strongest TRAIL-inducible genes in apoptosis-resistant cells. In line, siRNA targeting c-Rel strongly reduced TRAIL-induced NFATc2 activity in TRAIL-resistant PDAC cells. Furthermore, siRNA targeting NFATc2 sensitized these PDAC cells against TRAIL-induced apoptosis. Finally, TRAIL-induced expression of COX-2 was diminished through siRNA targeting c-Rel or NFATc2 and pharmacologic inhibition of COX-2 with celecoxib or siRNA targeting COX-2, enhanced TRAIL apoptosis. In conclusion, we were able to delineate a novel c-Rel-, NFATc2- and COX-2-dependent antiapoptotic signalling pathway in PDAC with broad clinical implications for pharmaceutical intervention strategies.

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NFATc2 and COX-2 are downstream mediators of c-Rel in TRAIL resistance. (a and b) Cells were transfected with control or NFATc2 siRNA for 48 h and treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content (a) or caspase-3/-7 activity (b). Data are presented as % of sub-G1 content over basal activity (untreated cells) or expressed as n-fold caspase-3/-7-activity normalized to the cellular protein content and represent the mean value±S.D. from six independent experiments. (c and d) Panc1 (c) and Patu8998t (d) cells were transfected with the indicated siRNA for 48 h. Then, cells were left untreated or treated with 10 ng/ml TRAIL for 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting COX-2. For normalization, beta-actin was analysed as control. Data are expressed as normalized mRNA level and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (e and f) Cells were pretreated with 75 μM celecoxib (Cele) for 1/2 h and then treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content (e) or caspase-3/-7 activity (f). Data are presented as % of sub-G1 content over basal activity (untreated cells) or expressed as n-fold caspase-3/-7 activity normalized to the cellular protein content and represent the mean value±S.D. from six independent experiments. *P-values <0.05. (g) Patu8998t cells were transfected with control or COX-2 siRNA for 48 h. Then, cells were left untreated or treated with 10 ng/ml TRAIL for 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting COX-2. For normalization, beta-actin was analysed as control. Data are expressed % induction of COX-2 mRNA with control siRNA-transfected cells set as 100% and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (h) Cells were transfected with control or COX-2 siRNA for 48 h and treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content. Data are presented as % of sub-G1 content over basal activity (untreated cells) and represent the mean value±S.D. from six independent experiments
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fig6: NFATc2 and COX-2 are downstream mediators of c-Rel in TRAIL resistance. (a and b) Cells were transfected with control or NFATc2 siRNA for 48 h and treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content (a) or caspase-3/-7 activity (b). Data are presented as % of sub-G1 content over basal activity (untreated cells) or expressed as n-fold caspase-3/-7-activity normalized to the cellular protein content and represent the mean value±S.D. from six independent experiments. (c and d) Panc1 (c) and Patu8998t (d) cells were transfected with the indicated siRNA for 48 h. Then, cells were left untreated or treated with 10 ng/ml TRAIL for 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting COX-2. For normalization, beta-actin was analysed as control. Data are expressed as normalized mRNA level and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (e and f) Cells were pretreated with 75 μM celecoxib (Cele) for 1/2 h and then treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content (e) or caspase-3/-7 activity (f). Data are presented as % of sub-G1 content over basal activity (untreated cells) or expressed as n-fold caspase-3/-7 activity normalized to the cellular protein content and represent the mean value±S.D. from six independent experiments. *P-values <0.05. (g) Patu8998t cells were transfected with control or COX-2 siRNA for 48 h. Then, cells were left untreated or treated with 10 ng/ml TRAIL for 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting COX-2. For normalization, beta-actin was analysed as control. Data are expressed % induction of COX-2 mRNA with control siRNA-transfected cells set as 100% and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (h) Cells were transfected with control or COX-2 siRNA for 48 h and treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content. Data are presented as % of sub-G1 content over basal activity (untreated cells) and represent the mean value±S.D. from six independent experiments

Mentions: To investigate whether c-Rel induced NFATc2 expression is involved in TRAIL resistance, we analysed the effect of siRNA-mediated knockdown of NFATc2 in TRAIL-induced PDAC apoptosis. Sub-G1 analysis (Figure 6a) and caspase-3/-7 assays (Figure 6b) showed that siRNA against NFATc2 strongly enhanced the apoptotic response to TRAIL in both cell lines.


c-Rel is a critical mediator of NF-κB-dependent TRAIL resistance of pancreatic cancer cells.

Geismann C, Grohmann F, Sebens S, Wirths G, Dreher A, Häsler R, Rosenstiel P, Hauser C, Egberts JH, Trauzold A, Schneider G, Sipos B, Zeissig S, Schreiber S, Schäfer H, Arlt A - Cell Death Dis (2014)

NFATc2 and COX-2 are downstream mediators of c-Rel in TRAIL resistance. (a and b) Cells were transfected with control or NFATc2 siRNA for 48 h and treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content (a) or caspase-3/-7 activity (b). Data are presented as % of sub-G1 content over basal activity (untreated cells) or expressed as n-fold caspase-3/-7-activity normalized to the cellular protein content and represent the mean value±S.D. from six independent experiments. (c and d) Panc1 (c) and Patu8998t (d) cells were transfected with the indicated siRNA for 48 h. Then, cells were left untreated or treated with 10 ng/ml TRAIL for 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting COX-2. For normalization, beta-actin was analysed as control. Data are expressed as normalized mRNA level and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (e and f) Cells were pretreated with 75 μM celecoxib (Cele) for 1/2 h and then treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content (e) or caspase-3/-7 activity (f). Data are presented as % of sub-G1 content over basal activity (untreated cells) or expressed as n-fold caspase-3/-7 activity normalized to the cellular protein content and represent the mean value±S.D. from six independent experiments. *P-values <0.05. (g) Patu8998t cells were transfected with control or COX-2 siRNA for 48 h. Then, cells were left untreated or treated with 10 ng/ml TRAIL for 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting COX-2. For normalization, beta-actin was analysed as control. Data are expressed % induction of COX-2 mRNA with control siRNA-transfected cells set as 100% and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (h) Cells were transfected with control or COX-2 siRNA for 48 h and treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content. Data are presented as % of sub-G1 content over basal activity (untreated cells) and represent the mean value±S.D. from six independent experiments
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fig6: NFATc2 and COX-2 are downstream mediators of c-Rel in TRAIL resistance. (a and b) Cells were transfected with control or NFATc2 siRNA for 48 h and treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content (a) or caspase-3/-7 activity (b). Data are presented as % of sub-G1 content over basal activity (untreated cells) or expressed as n-fold caspase-3/-7-activity normalized to the cellular protein content and represent the mean value±S.D. from six independent experiments. (c and d) Panc1 (c) and Patu8998t (d) cells were transfected with the indicated siRNA for 48 h. Then, cells were left untreated or treated with 10 ng/ml TRAIL for 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting COX-2. For normalization, beta-actin was analysed as control. Data are expressed as normalized mRNA level and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (e and f) Cells were pretreated with 75 μM celecoxib (Cele) for 1/2 h and then treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content (e) or caspase-3/-7 activity (f). Data are presented as % of sub-G1 content over basal activity (untreated cells) or expressed as n-fold caspase-3/-7 activity normalized to the cellular protein content and represent the mean value±S.D. from six independent experiments. *P-values <0.05. (g) Patu8998t cells were transfected with control or COX-2 siRNA for 48 h. Then, cells were left untreated or treated with 10 ng/ml TRAIL for 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting COX-2. For normalization, beta-actin was analysed as control. Data are expressed % induction of COX-2 mRNA with control siRNA-transfected cells set as 100% and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (h) Cells were transfected with control or COX-2 siRNA for 48 h and treated with 10 ng/ml TRAIL for 24 and 5 h, respectively. Apoptosis was determined by analysing sub-G1 content. Data are presented as % of sub-G1 content over basal activity (untreated cells) and represent the mean value±S.D. from six independent experiments
Mentions: To investigate whether c-Rel induced NFATc2 expression is involved in TRAIL resistance, we analysed the effect of siRNA-mediated knockdown of NFATc2 in TRAIL-induced PDAC apoptosis. Sub-G1 analysis (Figure 6a) and caspase-3/-7 assays (Figure 6b) showed that siRNA against NFATc2 strongly enhanced the apoptotic response to TRAIL in both cell lines.

Bottom Line: In line, siRNA targeting c-Rel strongly reduced TRAIL-induced NFATc2 activity in TRAIL-resistant PDAC cells.Finally, TRAIL-induced expression of COX-2 was diminished through siRNA targeting c-Rel or NFATc2 and pharmacologic inhibition of COX-2 with celecoxib or siRNA targeting COX-2, enhanced TRAIL apoptosis.In conclusion, we were able to delineate a novel c-Rel-, NFATc2- and COX-2-dependent antiapoptotic signalling pathway in PDAC with broad clinical implications for pharmaceutical intervention strategies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gastroenterology and Hepatology, 1st Department of Internal Medicine I, University Hospital Schleswig-Holstein, Kiel, Germany.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies with an overall life expectancy of 6 months despite current therapies. NF-κB signalling has been shown to be critical for this profound cell-autonomous resistance against chemotherapeutic drugs and death receptor-induced apoptosis, but little is known about the role of the c-Rel subunit in solid cancer and PDAC apoptosis control. In the present study, by analysis of genome-wide patterns of c-Rel-dependent gene expression, we were able to establish c-Rel as a critical regulator of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in PDAC. TRAIL-resistant cells exhibited a strong TRAIL-inducible NF-κB activity, whereas TRAIL-sensitive cells displayed only a small increase in NF-κB-binding activity. Transfection with siRNA against c-Rel sensitized the TRAIL-resistant cells in a manner comparable to siRNA targeting the p65/RelA subunit. Gel-shift analysis revealed that c-Rel is part of the TRAIL-inducible NF-κB complex in PDAC. Array analysis identified NFATc2 as a c-Rel target gene among the 12 strongest TRAIL-inducible genes in apoptosis-resistant cells. In line, siRNA targeting c-Rel strongly reduced TRAIL-induced NFATc2 activity in TRAIL-resistant PDAC cells. Furthermore, siRNA targeting NFATc2 sensitized these PDAC cells against TRAIL-induced apoptosis. Finally, TRAIL-induced expression of COX-2 was diminished through siRNA targeting c-Rel or NFATc2 and pharmacologic inhibition of COX-2 with celecoxib or siRNA targeting COX-2, enhanced TRAIL apoptosis. In conclusion, we were able to delineate a novel c-Rel-, NFATc2- and COX-2-dependent antiapoptotic signalling pathway in PDAC with broad clinical implications for pharmaceutical intervention strategies.

Show MeSH
Related in: MedlinePlus