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Abrogating phosphorylation of eIF4B is required for EGFR and mTOR inhibitor synergy in triple-negative breast cancer

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Abstract

Triple-negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive disease. They have a high rate of relapse and often develop resistance to standard chemotherapy. Many TNBCs have elevated epidermal growth factor receptor (EGFR) but are resistant to EGFR inhibitors as monotherapy. In this study, we sought to find a combination therapy that could sensitize TNBC to EGFR inhibitors. Phospho-mass spectrometry was performed on the TNBC cell line, BT20, treated with 0.5 μM gefitinib. Immunoblotting measured protein levels and phosphorylation. Colony formation and growth assays analyzed the treatment on cell proliferation, while MTT assays determined the synergistic effect of inhibitor combination. A Dual-Luciferase reporter gene plasmid measured translation. All statistical analysis was done on CalucuSyn and GraphPad Prism using ANOVAs. Phospho-proteomics identified the mTOR pathway to be of interest in EGFR inhibitor resistance. In our studies, combining gefitinib and temsirolimus decreased cell growth and survival in a synergistic manner. Our data identified eIF4B, as a potentially key fragile point in EGFR and mTOR inhibitor synergy. Decreased eIF4B phosphorylation correlated with drops in growth, viability, clonogenic survival, and cap-dependent translation. Taken together, these data suggest EGFR and mTOR inhibitors abrogate growth, viability, and survival via disruption of eIF4B phosphorylation leading to decreased translation in TNBC cell lines. Further, including an mTOR inhibitor along with an EGFR inhibitor in TNBC with increased EGFR expression should be further explored. Additionally, translational regulation may play an important role in regulating EGFR and mTOR inhibitor synergy and warrant further investigation.

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Acknowledgments

We would like to thank Dr. Daniela Buac for her assistance with the luciferase reporter assay. This work was supported by Susan G. Komen for the Cure Career Catalyst Grant (KG081416;JLB) and National Institutes of Health T32 Training Grant (CA009531;JMM). The Proteomics core is supported, in part, by NIH Center grant P30 CA022453 to the Karmanos Cancer Institute at Wayne State University.

Funding

This work was supported by Susan G. Komen for the Cure Career Catalyst Grant (KG081416; JLB), National Institutes of Health T32 Training Grant (CA009531; JMM) and National Institutes of Health Center grant P30 (CA022453; PS).

Ethical standards

All experiments comply with the current laws of the United States of America.

Conflict of interests

None of the authors declare conflicts of interest with this work.

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Authors and Affiliations

  1. Departments of Oncology, Wayne State University, 4100 John R. St, Detroit, MI, 48201, USA

    Julie M. Madden, Kelly L. Mueller, Aliccia Bollig-Fischer & Julie L. Boerner

  2. Departments of Pharmacology, Wayne State University, 540 E Canfield, Detroit, MI, 48201, USA

    Paul Stemmer & Raymond R. Mattingly

  3. Institute for Environmental Health Sciences, Wayne State University, 540 E Canfield, Detroit, MI, 48201, USA

    Paul Stemmer

  4. Karmanos Cancer Institute, 4100 John R. St, Detroit, MI, 48201, USA

    Raymond R. Mattingly & Julie L. Boerner

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  1. Julie M. Madden
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Correspondence to Julie L. Boerner.

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Madden, J.M., Mueller, K.L., Bollig-Fischer, A. et al. Abrogating phosphorylation of eIF4B is required for EGFR and mTOR inhibitor synergy in triple-negative breast cancer. Breast Cancer Res Treat 147, 283–293 (2014). https://doi.org/10.1007/s10549-014-3102-8

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  • DOI: https://doi.org/10.1007/s10549-014-3102-8

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