Fig 2: PEPT1 knockdown inhibited HCC metastasis in vitro and in vivo. A) Huh7 and PLC/PRF/5 cells were infected with lentiviral particles expressing shRNA targeting PEPT1. The knockdown efficacy was detected by Western blot analysis. B) Representative images and quantification of the indicated cells in the wound‐healing migration assays. Scale bar, 100 µm. C,D) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. E,F) Representative images and quantification of the luciferase expression in pulmonary metastatic lesions. G,H) Representative HE staining and IHC results of PEPT1 in lungs metastatic tumor resected from tail vein injection metastasis mouse model. Scale bar, 500 µm, 50 µm. The number of lung metastatic tumors in each group were determined. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig 3: PEPT1‐mediated dipeptides transport was essential for activating MAP4K4/G3BP2 axis. A) Endogenous interaction between PEPT1 and MAP4K4 was determined using co‐IP with anti‐MAP4K4 antibodies in Huh7 and PLC/PRF/5 cells. B) Metabolomics analysis identified dipeptides downregulated in Huh7 cells with stably PEPT1 knockdown. C) The uptake of Pro‐Gly in HCC cells with PEPT1 overexpressing and knockdown. D) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. E) Huh7 and PLC/PRF/5 cells were incubated with the indicated concentrations of Ile‐Ala or Gln‐Tyr for 24 hours, and the protein expression of MAP4K4, G3BP2, EMT‐associated proteins were detected by Western blot. Error bars indicate means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig 4: PEPT1 was upregulated in HCC and predicted poor clinical outcome. A) The protein expression levels of PEPT1 in normal hepatocytes and several HCC cell lines were detected by Western blot. B,C) PEPT1 protein levels in fresh HCC and adjacent nontumor tissues detection by Western blot (n = 12). D) Representative IHC images of PEPT1 in HCC tissue (n = 90) and corresponding normal tissue (n = 90). Scale bar, 200 µm. E) The corresponding IHC scores (H score) was compared on the right. F) Proportion of different expression levels of PEPT1 in HCC patients at different stages. G,H) Kaplan–Meier analysis of overall survival (OS) or disease‐free survival (DFS) curves after having assigned HCC patients to high/low of PEPT1 expression subgroups. I,J) Kaplan–Meier analysis of overall survival (OS) data from GSE20140 (I) and ICJC (LIRI‐JP) liver cancer database. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig 5: PEPT1/MAP4K4/G3BP2 signaling axis promoted HCC metastasis. A,C) Western blot analysis showed that the knockdown efficacy of MAP4K4 or G3BP2 in Bel7405 and HCCLM3 cells with PEPT1 overexpressing. B,D) Transwell assays revealed that migration and invasion ability was abrogated in Bel7405 and HCCLM3 cells with MAP4K4 or G3BP2 knockdown compared with the control group. Scale bar, 250 µm. E,G) Western blot analysis showed that the overexpression efficacy of G3BP2 (or PEPT1) in Huh7 cells with MAP4K4 (or G3BP2) knockdown. F,H) Inhibited migration and invasion of Huh7 cells with MAP4K4 (or G3BP2) knocked down was rescued by the overexpression of G3BP2 (or PEPT1). Left, representative images of Transwell assays, scale bar, 250 µm. Right, statistical analysis of Transwell assays. I) A schematic diagram of the PEPT1/MAP4K4/G3BP2 regulatory signaling axis that facilitates HCC metastasis.