Fig 1: Destruction of the PHGDH/cMyc axis hampered neutrophil/macrophage recruitment by liver cancer cells and impaired liver cancer progression. A) Cell proliferation was evaluated by spectrofluorometer at wavelength OD450 in PHGDH-depleted PLC/PRF/5 or Hep3B cells rescued with Vec, rPHGDH-WT, rPHGDH-NES, and rPHGDH-dACT. Cells expressing shNT rescued with Vec were used as control (mean ± SD, two-way ANOVA). B) Sorafenib inhibition was evaluated by trypan blue staining in PHGDH-depleted PLC/PRF/5 or Hep3B cells rescued with Vec, rPHGDH-WT, rPHGDH-NES, and rPHGDH-dACT. Cells expressing shNT rescued with Vec were used as control (mean ± SD, two-way ANOVA). C) Neutrophil recruitment was evaluated by cell migration, which was performed by placing the medium from culturing PHGDH-depleted PLC/PRF/5 or Hep3B cells in the lower well and neutrophil cells in the upper transwell chamber. Scale bars: 20 µm (mean ± SD, one-way ANOVA followed by Tukey's multiple comparisons test, n = 3 per group). D) Macroscopic images and H&E staining of PhgdhLKO mouse livers under different AAV treatments (Phgdh-WT with or without neutralized antibodies against Cxcl1/Il8 and Phgdh-dACT) at W9 after hydrodynamic injection of MET/CAT constructs and the SB transposase. The tumor region is outlined with a white dashed line. Scale bars: 100 µm (upper); 1 cm (lower). The tumor nodule/liver weight ratios were measured (mean ± SD, one-way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). E) Liver/body weight ratios of MET/CAT-transfected mice under different AAV treatments (Phgdh-WT with or without neutralizing antibodies (nAb) against Cxcl1/Il8 and Phgdh-dACT) at W9 were measured (mean ± SD, one-way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). F,H,J) IHC or IF staining of Ki67, Ly6G/CD11b, and F4/80 in mouse liver sections in (D). The relative number of Ki67+ cells (F), tumor-associated neutrophil (TAN) (H), or tumor-associated macrophage (TAM) (J) was pointed out by white arrows and calculated. Scale bars: 100 µm (F), 50 µm (H,J) (mean ± SD, one-way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). G,I) Using fresh liver tumors in (D), representative flow cytometry data of neutrophils (CD11b+ Ly6G+) and TAMs (F4/80+) from indicated livers (mean ± SD, one-way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). K) Kaplan–Meier plot showing the survival of MET/CAT-transfected mice under different AAV treatments (Phgdh-WT with or without neutralized antibodies against Cxcl1/Il8 and Phgdh-dACT). (Log-rank test, n = 5 per group). L) Using fresh liver tumors from the mice group Phgdh-WT with or without neutralized antibodies against Ly6G, representative flow cytometry data of neutrophils (CD11b+ Ly6G+) from indicated livers (mean ± SD, one-way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). M) Kaplan–Meier plot showing the survival of MET/CAT-transfected mice under different treatments (Phgdh-WT with or without neutralized antibodies against Ly6G). (Log-rank test, n = 5 per group).
Fig 2: PHGDH/cMyc axis drives CXCL1/IL8 expression. A–D) RNA‐sequencing analyses were performed using PHGDH‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Gene Ontology (GO) enrichment analyses of the differentially expressed genes were presented (A). GSEA enrichment plot of the KEGG pathway NOD‐like receptor‐related gene was shown in (B). The correlation of all NOD‐like receptor‐related gene expression with the phgdh expression status was displayed by the ranking metric score. A positive score indicates a correlation with the rPHGDH‐dACT and a negative score indicates a correlation with rPHGDH‐WT; The red indicates a gene that contributes most to the enrichment result and the blue indicates a gene that contributes less (C). The total differentially expressed genes (FC>2 or FC<0.5; p value <0.05) were displayed using a volcano plot. FC, fold change of rPHGDH‐dACT compared to rPHGDH‐WT (D). E) In PHGDH‐depleted PLC/PRF/5 cells with rPHGDH‐WT or rPHGDH‐dACT or further depletion of c‐Myc, qRT‐PCR validated the top up‐regulated genes from (C) (mean ± SD, one‐way ANOVA followed by Dunnett's multiple comparisons test, n = 3). F) ELISA examined the concentration of CXCL1/IL8 and IL1B in the medium culturing PHGDH‐depleted PLC/PRF/5 cells, which were rescued with rPHGDH‐WT or rPHGDH‐dACT (mean ± SD, two‐tailed Student's t‐test, n = 3). G) Immunoblotting analysis of CXCL1/IL8, PHGDH, and cMyc was performed using the indicated cells and antibodies. H) Co‐IP analysis of cMyc, p300, and PHGDH was performed using PLC/PRF/5 cells expressing WT or K148R mutant Myc. Antibody against cMyc was used to enrich the cMyc‐associated complex. Immunoblotting analysis of PHGDH, p300, cMyc, and AF9 was performed using the indicated antibodies. I) qRT‐PCR validated CXCL1/IL8 and IL1B genes using PLC/PRF/5 cells expressing WT or K148R mutant Myc (mean ± SD, two‐tailed Student's t‐test, n = 3). J) ELISA examined the concentration of CXCL1/IL8 and IL1B in the medium culturing PLC/PRF/5 cells expressing WT or K148R mutant Myc (mean ± SD, two‐tailed Student's t‐test, n = 3). K) Immunoblotting analysis of CXCL1/IL8, PHGDH, and cMyc was performed using the indicated cells and antibodies. L) ChIP analysis of PHGDH, cMyc, p300, RNA Pol II (Pol 2), AF9, and H3Kac on CXCL1 gene promoter was performed using indicated cells. IgG was used as a blank control (mean ± SD, two‐tailed Student's t‐test, n = 3).
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