Use of DARPP-32 to increase interactions between EGFR and ERBB3 and to promote gastric cancer resistance to gefitinib.

Abstract

18 Background: Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP−32), is overexpressed during gastric carcinogenesis. Gefitinib is a specific and effective EGFR inhibitor that has shown antitumor activity in clinical trials against gastrointestinal cancers. Gastric tumors can become resistant to gefitinib. However, molecular mechanisms that mediate resistance to gefitinib remain poorly understood. Methods: The expression of DARPP−32 in the multi-step carcinogenesis cascade was examined using IHC analysis. Cell survival was determined by clonogenic survival and ATP−Glo assays. Apoptosis was assessed by Annexin-V and immunoblot analyses. The association between DARPP−32 and EGFR was evaluated by immunofluorescence and co-IP assays. Results: The composite expression score progressively increased significantly from normal or gastritis to adenocarcinoma (p<0.001). Overexpression of DARPP−32 in MKN-28 cells blocked gefitinib-induced apoptosis and increased the drug’s IC50 10−fold, compared to that of control cells (P<0.01). And DARPP-32 expressing MKN−28 cells had activated the PI3K-AKT pathway as compared to control cells. The three−way co-IP experiments demonstrated the existence of DARPP-32, EGFR, and ERBB3 in the same protein complex. DARPP-32 enhanced EGFR-ERBB3 heterodimerization promoting phosphorylation of ERBB3, and then activated the PI3K-AKT pathway. Following treatment with gefitinib (25 μM) overnight, MKN−28 cells expressing DARPP-32 displayed stable protein levels of EGFR. The knockdown of endogenous DARPP-32 by DARPP-32 shRNA in SNU-16 cell lines reversed these signaling effects and increased sensitivity to gefitinib (p<0.01). Conclusions: Our results suggest that DARPP-32 overexpression may participate in the progression to neoplasia. DARPP-32 also plays a role in increasing the stability of EGFR protein; DARPP-32 promotes resistance of gastric cancer cells to gefitinib by promoting interaction between EGFR and ERBB3 and activating PI3K–AKT signaling. DARPP-32 may have potential as a predictive biomarker in gastric cancer prognosis and clinical response to treatment.