Upregulated Muc4/sialomucin complex interacts with ErbB2 and modulates phosphorylation of the receptor tyrosine kinase in gallbladder carcinoma of BK5.ErbB2 transgenic mice—A potential mechanism for gallbladder carcinogenesis

5520 Background: Gallbladder carcinoma (GBCa) is associated with a dismal prognosis, because most GBCa are in an advanced stage at the time of diagnosis. BK5.ErbB2 transgenic mice (Tg) overexpressing erbB2 (Cancer Res 2001), coupled with the induction of COX-2, develop GB carcinoma (GBCa) at a high incidence. However, the mechanism for activation of erbB2 and the subsequent erbB2 signaling pathways important for the development of GBCa in BK5.erbB2 mice has not been fully investigated. Recently, Muc4 (sialomucin complex) has been shown to influence erbB2 (tyrosine kinase) activity through direct interaction with the receptor and to potentiate tumor growth in mammalian carcinoma cells. Aim: To study the expression level of Muc4 and its interaction with erbB2 in GBCa to develop a better understanding of the mechanisms involved in gallbladder carcinogenesis. Methods: The mucin gene expression in Tg GBCa was determined by competitive PCR and immunoblot. The tissue distribution of Muc4 protein and mRNA was evaluated by immunofluorescence staining using CSLM (confocal scanning laser microscope) and in situ hybridization, respectively. The complex formation of Muc4 with erbB2 and the phosphorylaton status of erbB2 were assessed by immunoprecipitation and immunoblot. Results: In the GBCa that developed in BK5.erbB2 transgenic mice, the Muc4 mRNA level was highly increased (200% that of non-Tg mice), whereas the Muc1 and Muc3 levels were decreased. Both Muc4 mRNA and protein were strongly expressed in the cancerous epithelia of the GBCa, whereas they were detected in only trace amounts in the normal GB. Little or no Muc4 protein was found in the trachea, forestomach or esophagus, other tissues in which the expression of erbB2 is driven by the K5 promoter that overexpresses erbB2. In the CSLM images, Muc4 was observed to co-localize with erbB2 in the GBCa. The results from immunoprecipitation experiments revealed a direct interaction between Muc4 and hyperphosphorylated erbB2 in GBCa from Tg mice. The erbB2 hyperphosphorylation was not observed in either non-Tg GB or other tissues overexpressing erbB2. Conclusions: In BK5.erbB2 Tg mice, upregulation of Muc4 and its interaction with erbB2 may be involved in the process of gallbladder carcinogenesis through modulation of phosphorylation of the receptor tyrosine kinase. Supported by Pharmacia LS2002-6879, ES 07784, CA 16672 and CA 099526.