Background: Triple-negative breast cancer (TNBC) has a poor clinical prognosis and is characterized by a lack of druggable targets and a hypoxic tumor microenvironment. Hypoxia-induced glycogen accumulation and utilization are involved in cancer proliferation and therapy resistance, making modulation of glycogen metabolism of therapeutic interest. Therefore, we studied expression of glycogen synthase 1 (GYS1, the key regulator of glycogen synthesis) and glycogen stores in publicly available expression data and human breast tumors including TNBC. Also, we studied downregulation of GYS1 in preclinical breast cancer models, focusing on TNBC.
Methods: GYS1 mRNA expression and correlations with survival per clinical subtype were assessed in the METABRIC dataset. A tissue micro-array was constructed from primary tumors of 396 breast cancer patients with long-term follow-up, including normal breast control tissue. Triplicate tissue cores were stained immunohistochemically for GYS1, glycogen and the hypoxic marker carbonic anhydrase 9, and with periodic acid-Schiff staining for glycogen. In four TNBC cell lines and an MDA-MB-231 xenograft model, GYS1 protein expression, glycogen content and cell proliferation in normoxia and hypoxia were evaluated +/- GYS1 knockdown by siRNA or shRNA. Sensitivity of shGYS1 cell lines to drugs targeting mitochondria was tested.
Results: In the METABRIC dataset (n = 1904), overexpression of GYS1 mRNA was associated with poor overall survival (HR 1.20 [95% CI 1.05 - 1.38]), which was mainly driven by the TNBC patients (n = 299, HR 1.52 [95% CI 1.09 - 2.14]). Immunohistochemically, most primary breast tumors had elevated GYS1 levels and glycogen content compared to normal breast tissue, with subtype specific analyses ongoing. In all breast cancer cell lines, hypoxia induced GYS1 protein expression and increased glycogen content. Acute siRNA-mediated GYS1 knockdown decreased proliferation of (TN)BC cell lines and MDA-MB-231 spheroids in both hypoxia and normoxia. shGYS1 MDA-MB-231 cells had decreased glycogen levels and shGYS1 MDA-MB-231 xenograft growth was impaired, especially in the first six weeks after inoculation. In control xenografts, immunohistochemical GYS1 expression was most pronounced adjacent to the necrotic tumor core, whereas shGYS1 xenografts lacked GYS1 expression. Finally, shGYS1 MDA-MB-231 cells were more sensitive to inhibitors of mitochondrial protein homeostasis, suggesting a potential synergistic approach to overcome eventual metabolic adaptation.
Conclusions: GYS1 is overexpressed in primary breast tumors and high mRNA levels correlate with poor survival in TNBC. GYS1 downregulation impairs TNBC proliferation in vitro and in vivo, highlighting glycogen synthesis as potential novel therapeutic target in TNBC.
Citation Format: Ellen C. De Heer, Christos E. Zois, Esther Bridges, Mieke C. Zwager, Tineke van der Sluis, Bert van der Vegt, Carolien P. Schröder, Steven de Jong, Adrian L. Harris, Mathilde Jalving. Glycogen synthesis as potential novel target in triple negative breast cancer: Glycogen synthase 1 expression in human breast cancers and the impact of downregulation on proliferation of preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5813.