Autophagy is emerging as a new mechanism of cellular escape from different forms of cellular stress including the cytotoxic effects of chemotherapeutic agents. There is preclinical evidence that this mechanism may explain tumor resistance to chemotherapy as well as to biologic agents. In this study, we investigated the effects of the combination of inhibitors of autophagy and inhibitors of the HER family of receptors in breast cancer cell lines. Chloroquine (CQ) is a 4-aminoquinolone with antimalarial, immunosuppressive, and anti-autophagy activity. It raises the intra-lysosomal pH thereby impairing protein degradation and causing accumulation of autophagosomes. Erlotinib (E) is an inhibitor of the EGFR tyrosine kinase and trastuzumab (T) is a monoclonal antibody against the HER2 receptor.

We used the SK-BR-3 cell line (high ErbB1 and ErbB2 expression), and the MCF-7 cell line (low ErbB1 and ErbB2 expression) and studied the effect of T alone, E alone, and in combination with CQ on cell growth inhibition using the MTT assay. Our results indicate that combinations of CQ and T or E for 7 days cause synergistic cell growth inhibition in SK-BR-3 cells (combination index 0.17-0.31) but have no effect on cell growth inhibition in MCF-7 cells.

Flow cytometry analysis revealed that in SK-BR-3 cells exposure to T or E induces G1-phase accumulation and S-phase blockade. However, combinations of T or E with CQ did not further inhibit cell cycle progression. Analysis of apoptosis using the TUNEL reaction assay or by FACS analysis indicated that the combined treatment increases apoptotic cell death associated with increased caspase-3 activity as compared with the treatment with T or E alone, suggesting that activation of apoptotic signal pathways may be involved in the synergistic effect.

Finally, we determined the effect of the combination of CQ and T or E on receptor activation as well as downstream signaling and found that combined treatment enhanced the inhibition of ErbB2 as well as Akt phosphorylation, but had no effect on Erk activation as compared with T or E alone in SK-BR-3 cells.

Conclusion: The combination of CQ with T or E in breast cancer cell lines with high ErbB1 and ErbB2 expression results in synergistic growth inhibition, enhanced apoptosis, and increased inhibition of ErbB signaling via the PI-3K/AKT pathway. This enhanced cytotoxic activity and its potential clinical utility should be explored further. This work was supported in part by NIH grant CA84119 and CA96515.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1253.