Figure 7.

Autophagy-impaired liver cancers are more sensitive to glycolysis inhibition. (A) MTT assay showing SMMC7721 cells silenced with ATG5 shRNA were more sensitive to glycolysis inhibition in vitro. SMMC7721 cells transfected with control or ATG5 shRNA were then treated with the indicated doses of lonidamine, 2-DG and 3-BrPA for 24 h. Results are mean±SEM for experiments performed in triplicate. (B) The glycolysis inhibitor 3-BrPA inhibits the growth of autophagy-deficient xenograft tumors. SMMC7721 cells with stable knockdown of control or ATG5 were injected subcutaneously into the right flanks of nude mice. Two wk after the cells were injected, the mice in 2 groups were randomly separated into 4 groups. The mice were treated with either saline or 3-BrPA (5 mg/kg) by intraperitoneal administration every 3 d. When the tumor volume reached 450 mm³, the tumors were excised and photographed. The values are presented as the means ± SEM (n = 7–12). *p<0.05, **p<0.01. (C) Tumor volume curves on the indicated days. Tumor diameters were measured at fixed timepoints, and the tumor volumes were calculated. The values are presented as the means ± SEM, n = 7–12, *p<0.05 (Student t test). (D) Representative immunohistochemical staining results for MKI67 from tumor xenografts in nude mice. Scale bar: 50 μm. (E) A model depicting how autophagy might suppress glycolysis through the selective degradation of HK2. HK2 is subjected to K63-linked ubiquitination by TRAF6 at the K41 residue. Under high autophagic flux, SQSTM1 binds to and forms aggregates with the ubiquitinated HK2, then SQSTM1 recognizes MAP1LC3B and leads the aggregates to phagophores. Lysosomes fuse with the completed autophagosome and subsequently HK2 is degraded. In contrast, under low autophagic flux, the formation of a complex containing ubiquitinated HK2, SQSTM1, and MAP1LC3B is limited, and HK2 fails to be degraded in autolysosomes.