Truncated p95erbB2 Isoforms Are Capable of Transforming Human Mammary Epithelial Cells.

Abstract

Abstract Background:Objective clinical response to trastuzumab monotherapy in erbB2-amplified first line metastatic breast cancer is 34% (Vogel, et al., JCO 20: 719-26, 2002). Amongst patients who respond, most develop resistance (defined by disease progression on trastuzumab). One proposed mechanism of trastuzumab resistance is proteolytic cleavage of erbB2 receptor from its full-length (p185) form into truncated, constitutively active p95. Increased expression of p95erbB2 correlates with increased nodal involvement and poor clinical outcome. Because p95erbB2 lacks the trastuzumab binding epitope, expression may designate patients who would be suitable for treatment with erbB2 kinase inhibitors.Materials and Methods:Recombinant p185erbB2 and p95erbB2 constructs were stably expressed in several cell types via retroviral vector. Additionally, an intracellular form of p95erbB2 that arises via alternative translation and an intracellular p95erbB2 construct containing two copies of a nuclear localization sequence were also expressed. Expression and proper subcellular localization of constructs were confirmed by cell fractionation, western blot analysis and confocal microscopy. Transformation of human mammary epithelial (HMEC) and NIH3T3 cells by p185erbB2 and p95erbB2 isoforms was evaluated by anchorage independent growth using a quantitative fluorescent soft agar assay, and effects on migration and invasion of these cells were investigated by wound-healing and transwell assays. Cells transfected with oncogenic Ras or empty vector were used as positive and negative controls in these experiments.Results and Discussion:Recombinant p185erbB2 and p95erbB2 constructs were stably expressed in HMEC and NIH3T3 cells, and were correctly directed to the cell membrane; nuclear targeted intracellular p95erbB2 was correctly localized to the cell nucleus. Both p185 erbB2 and membrane-bound truncated p95erbB2 were sufficient to transform HMEC cells as compared to empty vector control transfected cells [mean fluorescence intensity empty vector control 2480 ± 464 (1 standard deviation); mean fluorescence intensity p185erbB2 9208 ± 2528, p= 0.0106; mean fluorescence intensity p95erbB2 6615 ± 1588, p= 0.0124)] as was the positive control oncogenic Ras (mean fluorescence intensity 4350 ± 433, p=0.0069). Interestingly, nuclear-targeted p95erbB2 was also sufficient to transform HMEC cells (mean fluorescence intensity 6492 ± 818, p=0.0018). These data support the hypothesis that truncated p95erbB2 species may be major pathogenic drivers in erbB2-amplified cancers. P95erbB2 therefore represents an attractive target for diagnosis and treatment of erbB2+ breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3136.