Abstract 1662: REDD1 silencing in A549 lung cancer cells as a potential target for the treatment of anoxic tumor cells
Authors: Ides Johan , Lardon Filip , Wouters An , Ponsaerts Peter , Pattyn Greet G.O. , Lambrechts Hilde A.J. , Pauwels Patrick , Peeters Marc , Pauwels Bea 
Published: June 2012

Abstract
Introduction: Cellular stress responses are essential for normal cellular growth and development while deregulation of one of these processes can play a role in tumorigenesis. Mammalian target of rapamycin (mTOR) is regulated by a lot of these stress responses and regulates cell growth and cell cycle progression. REDD1 is a response gene that can alter the mTOR status in response to DNA damage, hypoxia and glucose deprivation. Hypoxia has become a big issue in solid tumors due to its adverse effects on tumor behavior. mTOR will be rapidly inhibited by activation of REDD1 under anoxia. REDD1 silencing could therefore establish a continuous translation under anoxic conditions and might resensitize anoxic cells to certain cytostatic drugs and/or radiotherapy that target cellular growth and proliferation.
Material & Methods: Human A549 lung carcinoma cells were transfected with short hairpin RNA targeted against REDD1 to constitutively silence REDD1 translation (A549shREDD1). After puromycin selection, REDD1 protein expression was determined by western blot analysis. A549 and A549shREDD1 cells were incubated under anoxic conditions (&lt;0.1% O2) in a Bactron anaerobic IV chamber and harvested at different time points (24-96h). Vindelov staining was used to analyze cell cycle distribution by flow cytometry. The IC50 value of gemcitabine under normoxic and anoxic conditions was determined using the sulforhodamine B test. Cells were treated with gemcitabine (0 – 100nM) for 24h. 2h prior to treatment, cells were incubated under anoxic conditions to induce REDD1 upregulation.
Results: An increase of REDD1 protein in A549 cells could already be detected after 2h incubation under anoxia. REDD1 downregulation was confirmed in the A549shREDD1 cells. After a 72h incubation period under anoxia, cell cycle analysis showed an increase of the percentage G0/1 cells in A549 cells compared to A549shREDD1 cells (A549: 80.68 ± 15.06%; A549shREDD1: 65.28 ± 11.23%). Interestingly, the cell fraction in the S phase turned out to be larger in anoxic A549shREDD1 compared to anoxic A549 cells (A549: 14.33 ± 12.96% A549shREDD1: 23.34 ± 6.23%). REDD1 might play a potential role in anoxia-mediated cell cycle arrest. The difference in REDD1 expression had no influence on the IC50 values of gemcitabine of the different cell lines (IC50 normoxia: A549: 17.34 ± 9.21; A549shREDD1: 19.49 ± 2.97; IC50 anoxia: A549: 17.47 ± 5.79; A549shREDD1: 23.83 ± 1.03). Moreover, REDD1 inhibition did not cause a sensitization of anoxic cells.
Conclusion: Inhibiting REDD1 translation seems to overcome anoxia-induced G0/1 arrest. This could be an interesting approach to resensitize anoxic cells to conventional cancer treatments that target dividing cells. Although gemcitabine typically interacts with the cell cycle, no difference in IC50 values could be observed between the wild type A549 and REDD1 silenced A549 cells, neither under normoxia nor under anoxia.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2011-1662