Figure 7. FUT1 regulates cancer stemness through a dysregulated AKT/mTOR/4EBP1 signaling axis.

(A) A reversed-phase protein array was performed using lysates of Huh7 HCC cells transfected with shNTC or shFUT1 (clones 544 and 565) or transfected with empty vector control or FUT1 overexpression vector. A heatmap of differentially expressed proteins is shown. (B) GSEA of high-FUT1-expressing HCC (top 50%) and low-FUT1-expressing HCC (bottom 50%) with data extracted from The Cancer Genome Atlas (TCGA) Liver Hepatocellular Carcinoma (LIHC) database showing that high FUT1 expression was significantly correlated with PI3K/AKT/mTOR signaling. (C) Western blot analysis shows enhanced p-AKT, p-mTOR, and p-4EBP1 levels in HCC cells treated with low glucose or when transduced with FUT1-overexpressing plasmid in both Huh7 and CLC13 HCC cells. Decreases in p-AKT, p-mTOR, and p-4EBP1 expression were also observed when Huh7 cells had suppressed FUT1 expression. (D) Western blot analysis shows that treatment of the FUT1-overexpressing cells with 2DGal (10 mM) attenuated FUT1, p-AKT, p-mTOR, and p-4EBP1 expression and reduced UEA-1 expression. The data shown in C and D are representative of 3 independent experiments. EV, empty vector control; HG, high glucose; LG, low glucose; NTC, nontargeting control; OE, overexpression; FDR, false discovery rate; NS, not significant; 2DGal, 2-deoxy-D-galactose.