t-Darpp promotes cancer cell survival by up-regulation of Bcl2 through Akt-dependent mechanism.
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 micromol/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 >or=2-fold increase in Akt kinase activity with an increase in protein levels of p-Ser(473) Akt and p-Ser(9) GSK3 beta. 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.
Belkhiri
Abbes
A
Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Dar
Altaf A
AA
Zaika
Alexander
A
Kelley
Mark
M
El-Rifai
Wael
W
D051697:Activating Transcription Factor 1; D002277:Carcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D045304:Cytochromes c / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D019254:Genes, bcl-2; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D009687:Nuclear Proteins / Q000502:physiology; D051058:Oncogene Protein v-akt / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D015398:Signal Transduction / Q000235:genetics; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
C531110:ATF1 protein, human; D051697:Activating Transcription Factor 1; C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D009687:Nuclear Proteins; C497928:PPP1R1B protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D045304:Cytochromes c; D051058:Oncogene Protein v-akt; D053148:Caspase 3; D053453:Caspase 9