Resistance to TRAIL is mediated by DARPP-32 in gastric cancer

Abbes Belkhiri, Shoumin Zhu, Zheng Chen, Mohammed Soutto, Wael El-Rifai

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Purpose: Dopamine and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32), is overexpressed during the gastric carcinogenesis cascade. Here, we investigated the role of DARPP-32 in promoting resistance to treatment with TRAIL. Experimental Design: In vitro cell models including stable expression and knockdown of DARPP-32 were used. The role of DARPP-32 in regulating TRAIL-dependent apoptosis was evaluated by clonogenic survival assay, Annexin V staining, immunofluorescence, quantitative reverse transcriptase PCR, Western blot, and luciferase reporter assays. Results: Stable expression of DARPP-32 in MKN-28 cells enhanced cell survival and suppressed TRAIL-induced cytochrome c release and activation of caspase-8, -9, and -3. Conversely, short hairpin RNA-mediated knockdown of endogenous DARPP-32 sensitized the resistant MKN-45 cells to TRAIL-induced apoptosis and enhanced TRAIL-mediated activation of caspase-8, -9, and -3. DARPP-32 induced BCL-xL expression through activation of Src/STAT3 signaling, and treatment with the Src-specific inhibitor PP1 abrogated DARPP-32-dependent BCL-xL upregulation and cell survival in MKN-28 cells. The TRAIL treatment induced caspase-dependent cleavage of NF-κBp65 protein; this cleavage was prevented by DARPP-32, thus maintaining NF-κB activity and the expression of its target, FLIP(S) protein. This suggests that upregulation of BCL-xL could play a possible role in blocking the mitochondria intrinsic apoptosis pathway, whereas the DARPP-32 effect on the NF-κB/FLIP(S) axis could serve as an additional negative feedback loop that blocks TRAIL-induced activation of caspase-8. Conclusion: Our findings uncover a novel mechanism of TRAIL resistance mediated by DARPP-32, whereby it inhibits the intrinsic apoptosis pathway through upregulation of BCL-xL, and the extrinsic apoptosis pathway through the NF-κB/FLIP(S) axis.

Original languageEnglish (US)
Pages (from-to)3889-3900
Number of pages12
JournalClinical Cancer Research
Volume18
Issue number14
DOIs
StatePublished - Jul 15 2012
Externally publishedYes

Fingerprint

Dopamine and cAMP-Regulated Phosphoprotein 32
Stomach Neoplasms
CASP8 and FADD-Like Apoptosis Regulating Protein
Caspase 8
Apoptosis
Up-Regulation
Caspase 9
Cell Survival
Annexin A5
Caspases
Cytochromes c
Luciferases
Reverse Transcriptase Polymerase Chain Reaction
Small Interfering RNA

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Resistance to TRAIL is mediated by DARPP-32 in gastric cancer. / Belkhiri, Abbes; Zhu, Shoumin; Chen, Zheng; Soutto, Mohammed; El-Rifai, Wael.

In: Clinical Cancer Research, Vol. 18, No. 14, 15.07.2012, p. 3889-3900.

Research output: Contribution to journalArticle

Belkhiri, Abbes ; Zhu, Shoumin ; Chen, Zheng ; Soutto, Mohammed ; El-Rifai, Wael. / Resistance to TRAIL is mediated by DARPP-32 in gastric cancer. In: Clinical Cancer Research. 2012 ; Vol. 18, No. 14. pp. 3889-3900.
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abstract = "Purpose: Dopamine and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32), is overexpressed during the gastric carcinogenesis cascade. Here, we investigated the role of DARPP-32 in promoting resistance to treatment with TRAIL. Experimental Design: In vitro cell models including stable expression and knockdown of DARPP-32 were used. The role of DARPP-32 in regulating TRAIL-dependent apoptosis was evaluated by clonogenic survival assay, Annexin V staining, immunofluorescence, quantitative reverse transcriptase PCR, Western blot, and luciferase reporter assays. Results: Stable expression of DARPP-32 in MKN-28 cells enhanced cell survival and suppressed TRAIL-induced cytochrome c release and activation of caspase-8, -9, and -3. Conversely, short hairpin RNA-mediated knockdown of endogenous DARPP-32 sensitized the resistant MKN-45 cells to TRAIL-induced apoptosis and enhanced TRAIL-mediated activation of caspase-8, -9, and -3. DARPP-32 induced BCL-xL expression through activation of Src/STAT3 signaling, and treatment with the Src-specific inhibitor PP1 abrogated DARPP-32-dependent BCL-xL upregulation and cell survival in MKN-28 cells. The TRAIL treatment induced caspase-dependent cleavage of NF-κBp65 protein; this cleavage was prevented by DARPP-32, thus maintaining NF-κB activity and the expression of its target, FLIP(S) protein. This suggests that upregulation of BCL-xL could play a possible role in blocking the mitochondria intrinsic apoptosis pathway, whereas the DARPP-32 effect on the NF-κB/FLIP(S) axis could serve as an additional negative feedback loop that blocks TRAIL-induced activation of caspase-8. Conclusion: Our findings uncover a novel mechanism of TRAIL resistance mediated by DARPP-32, whereby it inhibits the intrinsic apoptosis pathway through upregulation of BCL-xL, and the extrinsic apoptosis pathway through the NF-κB/FLIP(S) axis.",
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T1 - Resistance to TRAIL is mediated by DARPP-32 in gastric cancer

AU - Belkhiri, Abbes

AU - Zhu, Shoumin

AU - Chen, Zheng

AU - Soutto, Mohammed

AU - El-Rifai, Wael

PY - 2012/7/15

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N2 - Purpose: Dopamine and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32), is overexpressed during the gastric carcinogenesis cascade. Here, we investigated the role of DARPP-32 in promoting resistance to treatment with TRAIL. Experimental Design: In vitro cell models including stable expression and knockdown of DARPP-32 were used. The role of DARPP-32 in regulating TRAIL-dependent apoptosis was evaluated by clonogenic survival assay, Annexin V staining, immunofluorescence, quantitative reverse transcriptase PCR, Western blot, and luciferase reporter assays. Results: Stable expression of DARPP-32 in MKN-28 cells enhanced cell survival and suppressed TRAIL-induced cytochrome c release and activation of caspase-8, -9, and -3. Conversely, short hairpin RNA-mediated knockdown of endogenous DARPP-32 sensitized the resistant MKN-45 cells to TRAIL-induced apoptosis and enhanced TRAIL-mediated activation of caspase-8, -9, and -3. DARPP-32 induced BCL-xL expression through activation of Src/STAT3 signaling, and treatment with the Src-specific inhibitor PP1 abrogated DARPP-32-dependent BCL-xL upregulation and cell survival in MKN-28 cells. The TRAIL treatment induced caspase-dependent cleavage of NF-κBp65 protein; this cleavage was prevented by DARPP-32, thus maintaining NF-κB activity and the expression of its target, FLIP(S) protein. This suggests that upregulation of BCL-xL could play a possible role in blocking the mitochondria intrinsic apoptosis pathway, whereas the DARPP-32 effect on the NF-κB/FLIP(S) axis could serve as an additional negative feedback loop that blocks TRAIL-induced activation of caspase-8. Conclusion: Our findings uncover a novel mechanism of TRAIL resistance mediated by DARPP-32, whereby it inhibits the intrinsic apoptosis pathway through upregulation of BCL-xL, and the extrinsic apoptosis pathway through the NF-κB/FLIP(S) axis.

AB - Purpose: Dopamine and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32), is overexpressed during the gastric carcinogenesis cascade. Here, we investigated the role of DARPP-32 in promoting resistance to treatment with TRAIL. Experimental Design: In vitro cell models including stable expression and knockdown of DARPP-32 were used. The role of DARPP-32 in regulating TRAIL-dependent apoptosis was evaluated by clonogenic survival assay, Annexin V staining, immunofluorescence, quantitative reverse transcriptase PCR, Western blot, and luciferase reporter assays. Results: Stable expression of DARPP-32 in MKN-28 cells enhanced cell survival and suppressed TRAIL-induced cytochrome c release and activation of caspase-8, -9, and -3. Conversely, short hairpin RNA-mediated knockdown of endogenous DARPP-32 sensitized the resistant MKN-45 cells to TRAIL-induced apoptosis and enhanced TRAIL-mediated activation of caspase-8, -9, and -3. DARPP-32 induced BCL-xL expression through activation of Src/STAT3 signaling, and treatment with the Src-specific inhibitor PP1 abrogated DARPP-32-dependent BCL-xL upregulation and cell survival in MKN-28 cells. The TRAIL treatment induced caspase-dependent cleavage of NF-κBp65 protein; this cleavage was prevented by DARPP-32, thus maintaining NF-κB activity and the expression of its target, FLIP(S) protein. This suggests that upregulation of BCL-xL could play a possible role in blocking the mitochondria intrinsic apoptosis pathway, whereas the DARPP-32 effect on the NF-κB/FLIP(S) axis could serve as an additional negative feedback loop that blocks TRAIL-induced activation of caspase-8. Conclusion: Our findings uncover a novel mechanism of TRAIL resistance mediated by DARPP-32, whereby it inhibits the intrinsic apoptosis pathway through upregulation of BCL-xL, and the extrinsic apoptosis pathway through the NF-κB/FLIP(S) axis.

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