Amplification and overexpression of the HER2/ErbB2 receptor tyrosine kinase occur in 25-30% of breast cancer patients and are correlated with poor clinical outcome. The humanized HER2 antibody, trastuzumab, was developed as a targeted therapeutic for HER2-positive breast cancer. Although trastuzumab has clinical activity both as a single agent and in combination with chemotherapy, lack of response to trastuzumab can occur either at the initiation of treatment (innate resistance) or following prolonged exposure (acquired resistance). Understanding the mechanisms underlying response to trastuzumab therapy continues to be an important area of study. BT-474 cells are HER2-amplified, trastuzumab-sensitive breast cancer cells and provide a good model for studying trastuzumab activity both in vitro and in vivo. While adapting BT-474 cells to grow in vivo in the absence of exogenous estrogen supplementation, we discovered that the estrogen-deprived BT-474 line we developed was un-responsive to trastuzumab. This cell line, named BT-474 EEI (exogenous estrogen independent) maintains 3+ HER2 expression (as measured by IHC on xenograft tumors and by FACS or Western analysis on cultured cells) and retains sensitivity to trastuzumab-DM1 immunoconjugate, erlotinib and chemotherapeutic drugs. Expression of ER-α is slightly elevated in BT-474 EEI cells compared to the parental line, likely a consequence of adaptation to low estrogen conditions. In fact, the proliferative response to 17β-estradiol in vitro is greatly enhanced in the BT-474 EEI cells. Both IGF-1R and IRS-1 are up-regulated in BT-474 EEI cells. In addition, the EEI cells show increased expression of HER/ErbB receptor ligands, notably amphiregulin, HB-EGF, epiregulin, and TGF-α. Expression of EGFR, however, is reduced compared to the parental cells. Moreover, basal levels of phosphorylated HER/ErbB receptors (EGFR, HER2 and HER3) are decreased in BT-474 EEI. We also examined signaling through the MAPK and AKT pathways. In contrast to BT-474 cells, which display high basal phospho-AKT, the BT-474 EEI cells show decreased constitutive AKT activation. However, heregulin-induced AKT phosphorylation is equivalent in both cell types. No detectable differences in signaling through the MAPK or mTOR pathways were observed. In summary, the BT-474 EEI cell line displays similar characteristics described in models of acquired trastuzumab resistance (i.e. increased IGF-1R and HER/erbB ligands), yet offers a unique system for studying mechanisms of non-response to trastuzumab . Experiments are currently underway to determine which molecular alterations contribute to the lack of response to trastuzumab observed in BT-474 EEI cells.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA