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

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.