Background: Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), is a novel cancer gene overexpressed in 2/3 of gastric cancer patients. DARPP-32 promotes cancer cell survival, drug resistance, and invasion. Activation of STAT3, signal transducer and activator of transcription 3, is important for tumorigenesis and drug resistance. Among factors that regulate STAT3, SOCS3 acts as a negative regulator of STAT3 to prevent its uncontrolled oncogenic activation. In this study, we aimed to investigate if DARPP-32 regulates STAT3 and determine the underlying molecular mechanisms.
Methods: 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. Luciferase reporter and immunohistochemistry stain were used in this study.
Results: Overexpression of DARPP-32 in AGS gastric cancer cells increased phosphorylation of STAT3 (Y705) and activation of STAT3 luciferase reporter (P<0.01), and decreased SOCS3 protein expression. DARPP-32 expression increased phosphorylation and ubiquitination of the SOCS3 protein, and subsequently shortened the SOCS3 protein half-life. We provide evidence showing that DARPP-32 promotes activation of IGF1R-SRC axis, critical for phosphorylation and degradation of SOCS3, a negative regulator of STAT3 activity. Proximity ligation and co-immunoprecipitation assays indicated that DARPP-32 and IGF1R co-exist in the same protein complex where DARPP-32 binding to IGF1R promotes its phosphorylation with activation of downstream SRC and STAT3. We obtained double knockouts mice (Darpp32KO/Tff1KO) to determine the histological changes at 3 age groups (3, 6, 9- 12 month). Using the different age groups, we found that the double knockout mouse model have significantly lower incidence of LGD at the age of 3 month, as compared to TFF1 KO (P<0.05) mice. The Darpp32KO/Tff1KO gastric mucosa demonstrated lack of phosphorylation and nuclear localization of STAT3 with significant delay in the onset of tumor lesions. Immunohistochemistry analysis confirmed overexpression of DARPP-32 and nuclear localization of STAT3 in human gastric cancers.
Conclusion: The in vitro studies indicate that DARPP-32 plays a critical role in activation of STAT3 signaling through regulation of IGF1R and SRC, leading to SOCS3 degradation. In vivo results suggest that lack of DARPP-32 delays the development of tumors in the TFF1 knockout mouse model but is not sufficient alone to abrogate the full carcinogenesis cascade. These novel findings add to the oncogenic functions of DARPP-32 and highlight its potential role in the pro-inflammatory gastric carcinogenesis cascade.
Citation Format: Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael El-Rifai. Overexpression of DARPP-32 promotes activation of STAT3 through IGF1R-SRC axis in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5447.