Abstract 1: Oncogenic extracellular domain mutations of ERBB2 in cancer

Abstract

Abstract The ERBB2 receptor tyrosine kinase gene is frequently amplified and mutated in human cancer. However, mutations characterized to date have been located in the kinase domain of the receptor. Using publically-available sequencing datasets, we have found that extracellular domain mutations of ERBB2 located beneath the dimerization arm recur in lung, breast, and ovarian cancers. We expressed cDNAs harboring these mutations in NIH-3T3 cells and found that the mutants support anchorage-independent proliferation. We furthermore found that substitution of any one of a number of amino acids in this region of the protein is oncogenic. We sought to biochemically characterize the extracellular domain mutations of ERBB2 and found that they cause oncogenic transformation by one of two mechanisms: elevation of C-terminal tail phosphorylation, or reduction-sensitive covalent dimerization unaccompanied by increases in C-terminal phosphorylation. Analysis of tyrosine phosphorylated proteins in the cell by stable isotope labeling followed by mass spectrometry (SILAC) revealed that both classes of mutant receptor phosphorylated the same set of proteins to effect transformation. The most prominent targets were proteins involved in cytoskeletal and membrane dynamics, controlling cell motility. STAT3 was also phosphorylated to a limited extent in ERBB2-transformed cells. Interestingly, STAT3 phosphorylation was mediated solely by a JAK-dependent autocrine loop in cells transformed by the ERBB2 mutants activated by reduction-sensitive dimerization; whereas both ERBB2 and JAK family kinases contributed to STAT3 phosphorylation in cells transformed by ERBB2 mutants exhibiting increased C-terminal phosphorylation. In order to characterize inhibitor sensitivity, we transformed Ba/F3 cells to IL-3 independence with the ERBB2 extracellular domain mutants. All extracellular domain mutants tested were sensitive to irreversible inhibitors of ERBB2, suggesting that treatment with such inhibitors may benefit patients harboring mutations of the extracellular domain of ERBB2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1. doi:1538-7445.AM2012-1