Abstract 1454: The role of AMPK in the radiosensitization of breast tumor cells by Vitamin D

Previous studies in our laboratory have demonstrated that the active form of vitamin D, 1,25-di hydroxy vitamin D3 (1,25 D3) can confer enhanced sensitivity to irradiation (IR). Studies using ZR-75-1 breast tumor cells indicated that radiosensitization was likely to be occurring through the promotion of autophagy. Interestingly, our experimental system demonstrated that autophagy can actually have dual roles. Inhibition of autophagy both enhanced sensitivity to radiation and attenuated radiation sensitization by 1,25D3. The current work was designed to distinguish the molecular mechanisms for cytoprotective autophagy and cytotoxic autophagy. In vitro studies assessed the effects of 100nM 1,25 D3 alone, radiation (4 Gy IR) and the combination treatment of 100nM 1,25 D3 concurrently with radiation. Cell viability was assessed over a period of 6 days by trypan blue exclusion. Sensitization to radiation was confirmed by clonogenic survival. Autophagy induction was measured by RFP-LC3 redistribution, formation of puncta by acridine orange staining, while autophagic flux was based on degradation of p62 by western blotting and the colocalization of RFP-LC3 and lysotracker green. Autophagy was inhibited pharmacologically using 200nM Bafilomycin A1 or 5uM Chloroquine and genetically using ATG5 and ATG 7 knockdown. AMPK activation was monitored by flow cytometry for phopho-AMPK. The impact of AMPK inhibition on cell viability was monitored over a period of 3 days by trypan blue exclusion using the pharmacological inhibitor, Compound C (5uM). The effects of AMPK inhibition on autophagy induction were monitored by the redistribution of RFP-LC3.1,25D3 sensitized ZR-75-1 cells to radiation based on reduced clonogenic survival and markedly greater suppression of cell viability by trypan blue exclusion. Acridine orange staining, RFP-LC3 redistribution and p62 degradation were indicative of autophagy with either radiation alone or 1,25 D3 + IR. Autophagy blockade with Chloroquine, Bafilomycin A1 or knockdown of Atg5 and ATG 7 increased sensitivity to radiation alone while attenuating the impact of 1,25 D3 + radiation. AMPK phosphorylation was observed with 1,25D3+IR but not with IR alone. Inhibition of AMPK by Compound C diminished autophagy induction by 1,25D3+IR and attenuated the radiosensitization.This experimental model appears to provide a useful tool to distinguish the factors involved in cytoprotective and cytotoxic autophagy. AMPK appears to play a significant role in the radiation sensitization of breast tumor cells by 1,25D3 as well as in the cytotoxic function of autophagy, but not in cytoprotective autophagy. However, the involvement of AMPK will have to be confirmed through genetic silencing of AMPK as well as its downstream effectors such as mTOR and ULK. In addition, analysis of differential gene expression and protein profiles should shed light on the factors that differentially regulate these pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1454. doi:1538-7445.AM2012-1454