title: Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma.
abstract: Esophageal adenocarcinoma (EAC) is an aggressive malignancy with a poor outcome. Although targeting ERBB2 with trastuzumab has been evaluated in clinical trials, the molecular mechanisms of trastuzumab resistance remain uncharacterized in EAC. The dopamine and cyclic AMP-regulated phosphoprotein of MR 32000 (DARPP-32), also known as PPP1R1B, is located together with ERBB2 at the 17q12-q21 amplicon. We evaluated the expression of a transcript variant of DARPP-32 (t-DARPP) and ERBB2 in 141 primary tumors and investigated the role of t-DARPP in trastuzumab resistance using OE19 and OE33 EAC cell models. Overexpression of t-DARPP mRNA was detected in two-thirds of tumors with a correlation between ERBB2 and t-DARPP overexpression levels (r = 0.58, P = 0.003). Cell viability and clonogenic survival assays showed that t-DARPP increased survival by 40% in response to trastuzumab (P < 0.01). The Annexin-V staining and Western blot analysis indicated that t-DARPP effectively abrogated trastuzumab-induced apoptosis, inhibited cleavage of caspase-3, and blocked trastuzumab-induced dephosphorylation of ERBB2 and AKT proteins. The knockdown of endogenous t-DARPP reversed these effects and sensitized cells to trastuzumab (P < 0.01). The cycloheximide-based protein degradation analysis indicated that t-DARPP extended the half-life of ERBB2, explaining the increase in the basal levels of ERBB2, p-ERBB2(Y1248), and p-AKT(S473). Coimmunoprecipitation and Western blot analysis showed that t-DARPP associated with ERBB2 in a protein complex, and interfered with trastuzumab binding to the ERBB2 receptor. Using EAC-xenografted mouse model, t-DARPP enhanced tumor growth and rendered tumors unresponsive to trastuzumab. This study establishes t-DARPP as a mediator of trastuzumab resistance and underscores its potential importance in clinical trials of EAC.
authors: Department of Surgery, Biostatistics, and Cancer Biology, and Vanderbilt Center for Quantitative Sciences, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors: Jun
authors: J
authors: Hong
authors: Ahmed
authors: A
authors: Katsha
authors: Pengcheng
authors: P
authors: Lu
authors: Yu
authors: Y
authors: Shyr
authors: Abbes
authors: A
authors: Belkhiri
authors: Wael
authors: W
authors: El-Rifai
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000008:administration & dosage / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000235:genetics / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D011485:Protein Binding; D055550:Protein Stability; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000068878:Trastuzumab; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D053148:Caspase 3; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab