oa Regulation of ERBB2 signaling and response to Trastuzumab by DARPP-32 in esophageal adenocarcinoma

Background: Esophageal adenocarcinoma (EAC) is characterized by a poor response to therapy with less than 10% five-year survival rates. Although Herceptin, a humanized monoclonal anti-ERBB2 antibody, has been successfully used for treatment of ERBB2-positive metastatic breast and gastroesophageal junction adenocarcinoma, a significant proportion of patients either fail to achieve initial response or develop acquired resistance. Clinical trials using Herceptin for the treatment of esophageal and gastroesophageal adenocarcinomas are ongoing in several Western countries. Understanding mechanisms that could hamper the response to Herceptin could influence the therapeutic decision making options and the management of these patients. Methods and Results: Truncated DARPP-32 (t-DARPP) (dopamine and cyclic AMP-regulated phosphoprotein of Mr 32,000), located at the 17q21 region, is a commonly amplified and overexpressed gene in upper gastrointestinal malignancies. The CellTiter cell viability, clonogenic survival, and Annexin V assays results indicated that stable or transient t-DARPP expression in OE19 cells significantly promoted cell survival and blocked apoptosis after treatment with 20 μg/ml Herceptin (p<0.01). The western blot results showed that t-DARPP sustained p-ERBB2 (Y1248) and p-AKT (S473) protein levels in response to Herceptin. Conversely, knockdown of endogenous t-DARPP in OE33 cells significantly decreased cell survival and p-ERBB2 (Y1248) and p-AKT (S473) protein levels in response to Herceptin (p<0.01). Western blot results showed that transient and stable t-DARPP expression without Herceptin increased protein levels of ERBB2, p-ERBB2 (Y1248), and p-AKT (S473). We hypothesized that t-DARPP enhances ERBB2 protein stability, thus blocking ERBB2 receptor steady-state down-regulation. The cycloheximide-based chase assay results showed that stable expression of t-DARPP increased the half-life of ERBB2 protein to 43.6 hours as opposed to 29.6 hours in control cells. We next investigated the effect of t-DARPP on Herceptin binding to ERBB2 receptor. Using co-immunoprecipitation, we have also shown that t-DARPP interacts with ERBB2 protein, hence, interfering with Herceptin binding to ERBB2 receptor, blocking Herceptin-induced down-regulation of downstream signaling. We also developed a Herceptin-resistant OE19 cell model, generated after selection with Herceptin for six months. The acquired resistance cell model demonstrated upregulation of endogenous t-DARPP and recapitulated the signaling events of Herceptin resistance. Conclusions: Our data demonstrate, for the first time, that t-DARPP enhances AKT signaling and is a key contributor to Herceptin resistance in EAC. In addition, t-DARPP mediates resistance to Herceptin through blocking ERBB2 receptor drug accessibility in EAC. The studies using in vivo models are ongoing.