Glycogen metabolism provides nutritional support to renal cancer cells under conditions of stress and may serve as a marker of response to antiangiogenic therapy with bevacizumab

Abstract

Abstract Purpose: The clear cell type of renal cell carcinoma (ccRCC) derives its histologic appearance from accumulation of glycogen, but the functional significance of this phenomenon is yet unexplored. In physiological conditions, liver and muscle are the main tissues capable of producing and storing glycogen, which is mobilized to produce energy through glycolysis under stress conditions or limited nutrient supply. Tumors frequently experience fluctuations in nutrient availability, uptake and metabolic stress. The purpose of this study is to test the hypothesis that renal tumors express active glycogen synthase (GYS1) to produce glycogen, which may provide with an alternative energy source to support growth and survival under conditions of energetic stress. Agents blocking glycogen utilization (glycogen phosphorylase inhibitors) may contribute to beneficial therapeutic strategies for renal cell carcinoma. In addition, glycogen levels may determine response or resistance to therapeutic strategies that alter nutrient availability (antiangiogenic therapy). Methods: Cultured 786-0 renal cancer cells, clear-cell renal tumors and normal renal cortex samples were subjected to protein extraction or homogenization with sterile water. To detect the levels of GYS1, protein lysates were tested using reverse-phase protein arrays and Western blotting. Lysates in water were used to detect glycogen with the glucoamylase chromogenic method. We tested the functional significance of GYS1 by knocking down its levels with GYS1-specific siRNA, and measured cell numbers and proliferation under normal culture conditions or under metabolic stress mediated by the glycolysis inhibitor 2-deoxyglucose (2DG) or an inhibitor of oxidative phosphorylation (metformin). In order to determine whether GYS1 correlates with responses to antiangiogenic therapy, we tested phospho and total GYS1 expression in renal tumors from bevacizumab-treated patients who participated in a previously published study (Tsavachidou-Fenner et al., Annals of Oncol, 2010). Results: ccRCC tissues and renal cancer cell lines contained glycogen at significantly higher levels than the normal renal cortex. They also expressed higher levels of GYS1. We show that the activation status of GYS1 is controlled by the PI3K/AKT/GSK3α/β cascade via phosphorylation at serine 640, which is considered to be an inactivating event. siRNA-mediated knockdown of GYS1 expression abrogated glycogen synthesis in 786-0 cells. It also suppressed cell growth under conditions of energetic stress induced by 2DG. Blocking glycogen utilization with a glycogen phosphorylase inhibitor had a similar growth suppressing effect on 786-0 cells in vitro, in the presence of metabolic stress mediated by 2DG or metformin. Tumors from patients responding to bevacizumab expressed the inactive form of GYS1 (GYS1-pSer640). In particular, renal tumors from patients with long progression-free survival exhibited higher levels of phospho-GYS1 (p value = 0.00075), consistent with the notion that tumors with lower glycogen production may respond more favorably to anti-angiogenic therapy. Conclusions: Clear-cell renal carcinomas and renal cancer cells produce large amounts of glycogen as a consequence of increased GYS1 levels. Increased glycogen serves as an alternative energy source, enabling cell growth under conditions of metabolic stress. Consequently, glycogen may be responsible for bypassing limitations in nutrient supply imposed by angiogenic therapies, as evidenced by the increased levels of inactive GYS1 in bevacizumab responders. The glycogen pathway may prove a valuable therapeutic target as well as a marker of response to therapies. This talk is also presented as Poster B18.