Autophagy for Cancer Therapy through Inhibition of Pro-apoptotic Proteins and Mammalian Target of Rapamycin Signaling

Carolyn Cao,Jeffrey M. Albert,Robert W. Mutter,Kwang Woon Kim,Michael Freeman,Bo Lu,Dennis E. Hallahan

doi:10.1074/jbc.m607094200

Abstract

Autophagy is an alternative cell death pathway that is induced by mammalian target of rapamycin (mTOR) inhibitors and up-regulated when apoptosis is defective. We investigated radiation-induced autophagy in the presence or absence of Bax/Bak with or without an mTOR inhibitor, Rad001. Two isogenic cell lines, wild type (WT) and Bak/Bak(-/-) mouse embryonic fibroblasts and tumor cell lines were used for this study. Irradiated Bak/Bak(-/-) cells had a decrease of Akt/mTOR signaling and a significant increase of pro-autophagic proteins ATG5-ATG12 COMPLEX and Beclin-1. These molecular events resulted in an up-regulation of autophagy. Bax/Bak(-/-) cells were defective in undergoing apoptosis but were more radiosensitive than the WT cells in autophagy. Both autophagy and sensitization of Bak/Bax(-/-) cells were further enhanced in the presence of Rad001. In contrast, inhibitors of autophagy rendered the Bak/Bax(-/-) cells radioresistant, whereas overexpression of ATG5 and Beclin-1 made the WT cells radiosensitive. When this novel concept of radiosensitization was tested in cancer models, small interfering RNAs against Bak/Bax also led to increased autophagy and sensitization of human breast and lung cancer cells to gamma radiation, which was further enhanced by Rad001. This is the first report to demonstrate that inhibition of pro-apoptotic proteins and induction of autophagy sensitizes cancer cells to therapy. Therapeutically targeting this novel pathway may yield significant benefits for cancer patients.

Highlights

Tory data, showing that overexpression of the antiapoptotic protein Bcl2 makes tumor xenografts more sensitive to ionizing radiation [8]
Using the Bax/BakϪ/Ϫ double knock-out (DKO) mouse embryonic fibroblast (MEF) and cancer cell models, we showed that the radiosensitization in the absence of Bak and Bax results from increased autophagy, which is blocked by an autophagic inhibitor, 3-MA, or small interfering RNAs against pro-autophagic proteins such as ATG5 and Beclin-1
Radiation Sensitization When the Bax/Bak-mediated Apoptosis Is Attenuated—We used DKO MEF cells that were deficient for Bak and Bax to determine the extent of apoptosis and radiation sensitivity as a consequence of lacking Bak and Bax

Summary

Introduction

Tory data, showing that overexpression of the antiapoptotic protein Bcl2 makes tumor xenografts more sensitive to ionizing radiation [8]. Using the Bax/BakϪ/Ϫ DKO MEF and cancer cell models, we showed that the radiosensitization in the absence of Bak and Bax results from increased autophagy, which is blocked by an autophagic inhibitor, 3-MA, or small interfering RNAs (siRNAs) against pro-autophagic proteins such as ATG5 and Beclin-1.

Results

Conclusion