Human Breast Cancer Cells Selected for Resistance to Trastuzumab In vivo Overexpress Epidermal Growth Factor Receptor and ErbB Ligands and Remain Dependent on the ErbB Receptor Network

Abstract

Purpose: We have investigated mechanisms of acquired resistance to the HER2 antibody trastuzumab in BT-474 human breast cancer cells. Experimental Design: BT-474 xenografts established in athymic nude mice were eliminated by trastuzumab. Continuous cell lines (HR for Herceptin resistant) were generated from tumors that recurred in the presence of continuous antibody therapy. Results:The isolated cells behaved resistant to trastuzumab in culture as well as when reinjected into nude mice. They retained HER2 gene amplification and trastuzumab binding and were exquisitely sensitive to peripheral blood mononuclear cells ex vivo in the presence of the antibody. The HR cells exhibited higher levels of phosphorylated epidermal growth factor receptor (EGFR) and EGFR/HER2 heterodimers. Phosphorylation of HER2 in HR cells was inhibited by the EGFR tyrosine kinase inhibitors erlotinib and gefitinib. Gefitinib also inhibited the basal association of p85 with phosphorylated HER3 in HR cells. Both inhibitors as well as the dual EGFR/HER2 inhibitor, lapatinib, induced apoptosis of the HR cells in culture. Growth of established HR5 xenografts was inhibited by erlotinib in vivo. In addition, the HR cells overexpressed EGFR, transforming growth factor a, heparin-binding EGF, and heregulin RNAs compared with the parental trastuzumabsensitive cells. Conclusions: These results are consistent with the inability of trastuzumab to block the heterodimerization of HER2 and suggest that amplification of ligand-induced activation of ErbB receptors is a plausible mechanism of acquired resistance to trastuzumab that should be investigated in primary mammary cancers.HER2/neu (ErbB2) is a member of the ErbB family of transmembrane receptor tyrosine kinases, which also includes the epidermal growth factor (EGF) receptor (EGFR; ErbB1), HER3 (ErbB3), and HER4 (ErbB4). Binding of ligands to the extracellular domain of EGFR, HER3, and HER4 induces the formation of kinase-active homodimers and heterodimers to which activated HER2 is recruited as a preferred partner (1). Although HER2 does not bind any of the ErbB ligands directly, its catalytic activity can potently amplify signaling by ErbBcontaining heterodimers via increasing ligand binding affinity and/or receptor recycling and stability (2 -5).Amplification of the HER2/neu (ERBB2) gene occurs in f25% of invasive breast cancers and is associated with poor patient outcome (6). Trastuzumab (Herceptin), a humanized monoclonal IgG1 that binds to the ectodomain of HER2, induces clinical responses in HER2-overexpressing breast cancers and prolongs patient survival when combined with chemotherapy (7 -13). The clinical efficacy of trastuzumab seems limited to breast cancers that overexpress HER2 as measured by intense membrane staining in the majority of tumor cells with HER2 antibodies (3+ by immunohistochemistry) or excess copies of the HER2 gene determined by fluorescence in situ hybridization. Therefore, HER2 overexpression by immunohistochemistry and/or fluorescence in situ hybridization is...

Feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors

“…Indeed, breast cancer cells with HER2 amplification are particularly sensitive to apoptosis induced by PI3K inhibitors (14). In addition, this association of HER2/HER3 dimer with p85 has been found to be essential for the viability of HER2-dependent cells (22,26,27).…”

“…In breast cancer cells with HER2 gene amplification, HER2 is the main kinase that phosphorylates HER3 (19,22). As XL147 does not affect the catalytic activity of HER2 (Fig.…”

“…In these cells, the main tyrosine phosphorylated protein precipitated with p85 antibodies is pHER3. This HER3 and p85 association depends on the catalytic activity of HER2 as it is disrupted by HER2 tyrosine kinase inhibitors (TKIs) (21,22). Thus, we examined if, upon inhibition of PI3K, there was maintenance or recovery of the HER3/p85 association.…”

MKNK1 is a YB-1 target gene responsible for imparting trastuzumab resistance and can be blocked by RSK inhibition

“…As described above, HR5 and HR6 cells were derived in vivo by exposing mice harboring BT474 tumors to trastuzumab until resistant clones arose (Ritter et al, 2007). This model of acquired resistance led us to discover a possible role for MNK1 in mediating trastuzumab resistance.…”

“…In order to understand the mechanisms of trastuzumab resistance further, use of physiologically relevant models is essential. Ritter et al (2007) established resistant cell lines from BT474 xenografts in mice that initially responded to trastuzumab but eventually recurred (HR5 and HR6 clones) (Ritter et al, 2007). This model is valuable as it recapitulates the scenario commonly seen in clinical development of trastuzumab resistance.…”