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t-Darpp Promotes Cancer Cell Survival by Up-regulation of Bcl2 through Akt-Dependent Mechanism

Departments of ¹ Surgery and ² Cancer Biology, and ³ Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee

Requests for reprints: Wael El-Rifai, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, 1255 Light Hall, 2215 Garland Avenue, Nashville, TN 37232. Phone: 615-322-7934; E-mail: wael.el-rifai{at}Vanderbilt.edu.

t-Darpp is a cancer-related truncated isoform of Darpp-32 (dopamine and cyclic-AMP–regulated phosphoprotein of M_r 32,000). We detected overexpression of t-Darpp mRNA in two thirds of gastric cancers compared with normal samples (P = 0.004). Using 20 µmol/L ceramide treatment as a model for induction of apoptosis in AGS cancer cells, we found that expression of t-Darpp led to an increase in Bcl2 protein levels and blocked the activation of caspase-3 and caspase-9. The MitoCapture mitochondrial apoptosis and cytochrome c release assays indicated that t-Darpp expression enforces the mitochondrial transmembrane potential and protects against ceramide-induced apoptosis. Interestingly, the expression of t-Darpp in AGS cells led to 2-fold increase in Akt kinase activity with an increase in protein levels of p-Ser⁴⁷³ Akt and p-Ser⁹ GSK3β. These findings were further confirmed using tetracycline-inducible AGS cells stably expressing t-Darpp. We also showed transcriptional up-regulation of Bcl2 using the luciferase assay with Bcl2 reporter containing P1 full promoter, quantitative reverse transcription-PCR, and t-Darpp small interfering RNA. The Bcl2 promoter contains binding sites for cyclic AMP–responsive element binding protein CREB/ATF1 transcription factors and using the electrophoretic mobility shift assay with a CREB response element, we detected a stronger binding in t-Darpp–expressing cells. The t-Darpp expression led to an increase in expression and phosphorylation of CREB and ATF-1 transcription factors that were required for up-regulating Bcl2 levels. Indeed, knockdown of Akt, CREB, or ATF1 in t-Darpp–expressing cells reduced Bcl2 protein levels. In conclusion, the t-Darpp/Akt axis underscores a novel oncogenic potential of t-Darpp in gastric carcinogenesis and resistance to drug-induced apoptosis. [Cancer Res 2008;68(2):395–403]

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