Metformin Enhances Trastuzumab Efficacy and Overcomes Resistance in HER2 Type Breast Cancer Cells.

Abstract

Abstract Background: HER2 alterations occur in one-fourth of breast cancers and are associated with an aggressive tumor phenotype. The anti-HER2 agent trastuzumab reduces cell proliferation, angiogenesis, DNA repair and induces antibody-dependent cellular cytotoxicity. Objective response rates and median duration of response for eligible patients given trastuzumab alone is low (12-34% and 9 months respectively), hence, it is administered in combination with other agents. Mechanisms of trastuzumab resistance include: altered receptor antibody interactions, increased signaling through other EGFR type I growth factor receptors, modulation of p27 kip1 and increased insulin-like growth factor 1 receptor (IGF-1) signaling. We have reported that metformin inhibits HER2 expression, tyrosine kinase activity (phosphorylated HER2 at both auto- and Src- phosphorylation sites), AKT/mTOR signaling, Cyclin D1 and E2F1 with G1 arrest in HER2 overexpressing breast cancer cells. We sought to determine if metformin would enhance trastuzumab associated cytotoxicity and modulate acquired resistance in vitro.Methods: We used trastuzumab sensitive (SKBR3, BT474) and resistant cell lines (BT-474/HR20, SKBR3/P2, MCF-7/713 and MCF-7/HER2Δ16) to study the effects of metformin, metformin + trastuzumab and trastuzumab alone. Assays included MTS for proliferation, clonogenicity studies, Western blots, and pull down experiments with Western blots.Results: Trastuzumab sensitive breast cancer cells were less sensitive to metformin alone, compared to trastuzumab resistant HER2 subtype breast cancer cell lines. Trastuzumab sensitive cells showed enhanced growth and clonogenicity inhibition when treated by both metformin and trastuzumab. At the molecular level, these bi-treated cells showed decreases in HER2/pHER2, erbB3/perbB3, and inactivation of AKT and MAPk signaling. Metformin as low as 20mM increased the efficacy of trastuzumab. All HER2 resistant cell lines showed higher levels of IGF1R and HER2/IGF1R complexes, as compared to sensitive parental lines. In these resistant lines Metformin decreased cell proliferation and clonogenicity, HER2/IGF1R complexes (identified through pull down experiments) and protein expression of HER2/pHER2, erbB3/perbB3, IGF1R/pIGF1R as well as downstream signaling via Akt and IGF1 pathways. Metformin overcame trastuzumab resistance as demonstrated by growth and clonogenicity assays.Conclusions: HER2 amplified trastuzumab resistant breast cancer cells showed greater sensitivity to metformin than sensitive parental lines. Metformin reversed trastuzumab resistance and decreased HER2:IGF1R complexes, HER2/pHER2 and IGF1R expression, whereas sensitive parental lines showed no complex formation. These preclinical data suggest a combination of metformin and trastuzumab may have clinical benefit, improving the efficacy and reducing the emergence of or reversing trastuzumab resistance in HER2 positive breast cancer patients.Herceptin graciously provided by Genentech Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1133.