Abstract 356: DARPP-32, a bridge between pro-inflammatory NF-kB and STAT3 signaling in gastric cancer
Authors: Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael El-Rifai
Journal: Cancer Research (2017)

Abstract Background: Gastric cancer is one of the most frequently diagnosed malignancies with poor prognosis in the world. We have previously shown that Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is a novel cancer gene, which is overexpressed in 2/3 of gastric cancers patients; and associated with increased cancer cell survival, drug resistance, and invasion. However, the mechanisms of how DARPP-32 promotes gastric carcinogenesis remain unclear. Methods: DARPP-32 promoter activity and stat3 activity were measured by luciferase. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. The association between DARPP-32 and STAT3 was evaluated by immunofluorescence. SOCS3 degradation was analyzed by Ubiquitination Assay. The interaction between DARPP-32 and IGF1R was evaluated by co-immunoprecipitation assays. Results: Promoter analysis of DARPP-32 indicated the presence of NF-κB transcription factor putative binding sites in the DARPP-32 promoter region. We cloned the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). TNF-α treatment induced DARPP-32 reporter activity (P &amp;lt; 0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence (-996 to -1008 bp), containing putative NF-κB binding sites, is the most active region. We confirmed that activation of NF-kB by TNF-α and H.pylori infection leads to induction of DARPP-32 mRNA and protein level. Interestingly, we found that DARPP-32 induction leads to increased phosphorylation of STAT3 and activation of STAT3 luciferase reporter (P&amp;lt;0.01). We detected a decrease in the protein level of SOC3, a negative regulator of STAT3. DARPP-32 shortened the SOCS3 protein half-life by mediating an increase in phosphorylation and ubiquitination of the SOCS3 protein following treatment with MG132 and IL6. Furthermore, we detected an increase in SRC and IGF1R phosphorylation in DARPP-32-overexpressing cells. SRC inhibitor (Dasatinib) and SRC-siRNA blocked DARPP-32-induced activation of STAT3. We further examined our hypothesis that DARPP-32 could interact with IGF1R and enhance IGF1R autophosphorylation. Using co-immunoprecipitation, we found that DARPP-32 and IGF1R co-exist in the same protein complex. Conclusion: We demonstrated, for the first time, the role of NF-κB in DARPP-32 transcriptional regulation. The in vitro studies indicate that DARPP-32 plays a role in activation of STAT3 signaling through inhibition of SOCS3 and activation of IGF1R and SRC. DARPP-32 interacted with IGF1R, which increasing the phosphorylation of IGF1R and SRC, and induced SOCS3 phosphorylation and degradation. This cascade of events leads to STAT3 activation in gastric cancer cells. We propose DARPP-32 as an important signaling bridge between NF-kB and STAT3 in response to pro-inflammatory signaling in gastric carcinogenesis. Citation Format: Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael El-Rifai. DARPP-32, a bridge between pro-inflammatory NF-kB and STAT3 signaling in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 356. doi:10.1158/1538-7445.AM2017-356