Growth inhibitory effects of trastuzumab, erlotinib, and bevacizumab are proportional to average cellular levels of HER2 expression in breast cancer cell lines, and the effects of combinations of the three are additive in cell lines with high HER2 expression

1229 MCF-7 cells were transfected with HER2 and cell sublines were selected for graded levels of HER2 overexpression. Dose response curves for trastuzumab, erlotinib, and bevacizumab were obtained in each of these sublines, and in established breast cancer cell lines expressing low levels (parental MCF-7, MDA-231), and high levels (SKBR-3, BT474, NH27) of HER2, using tumor growth inhibition as an endpoint. At all drug concentrations tested, growth inhibition by each drug was proportional to the average levels of HER2 expression in the different cell lines. Using drug concentrations estimated to be in the clinically relevant range (1 mg/ml for trastuzumab and bevacizumab, and 500nM for erlotinib) each targeted agent inhibited the high HER2-expressing cell lines by 30-50%. All two-agent combinations inhibited these cell lines by 50-70%, and the three-agent combination inhibited these cell lines by 70-85%. Serial flow cytometry studies showed that the triple combination inhibited >20% of cells in G1, and led to delayed S phase progression in an additional ∼30% of cells. By comparison, the correlation of response with HER2 expression level was not observed with anti-IGF1 receptor therapy. Rb dysfunctional tumors (MDA-468, BT549, DU4475, and MDA-436) which do not overexpress HER2, were unresponsive to trastuzumab, erlotinib, and bevacizumab, singly or in any combination, except for EGFR-amplified MDA-468 cells, which exhibited ∼25% growth inhibition with erlotinib and erlotinib-containing combinations. Drug effect in MDA-468 cells was not associated with G1 arrest or S phase progression delay. These findings indicate that the combination of trastuzumab, erlotinib, and bevacizumab is more potent than any of these targeted agents individually, and that its growth inhibitory effects are highly selective for tumor cells with high levels of HER2 expression.