Fig 2: GD-induced D5P downregulation increases MYH9/LATS1 aggregation and LATS1 degradation to activate YAP. A, B Immunoblotting analysis of phosphorylated YAP (serine 127) and YAP in HCT116 cells cultured with normal medium, glucose-free medium or glucose-free medium supplemented with G (A) or D5P (B) for the indicated times. GAPDH or α-Tubulin was used as the loading control. C Immunoblotting analysis of LATS1 in the supernatants (Sup) and pellets of the HCT116 cell lysate treated with or without GD (0 mM, 4 h). GAPDH was used as a loading control. D Immunoblotting analysis of LATS1, YAP, LC3A/B and P21 expression in HCT116 cells under normal conditions or GD (0 mM, 4 h) with or without MG132 or 3MA. α-Tubulin was used as a loading control. E Guanosine supplementation diminished GD-induced LATS1 insolubilization. HCT116 cells were cultured with normal medium, glucose-free medium or glucose-free medium supplemented with G for 4 h. Tubulin was used as a loading control. F Coomassie blue staining and MS analysis of GD-induced LATS1-interacting proteins in HCT116 cells. The bands marked in the red boxes (left) were sent for MS analysis. G The effects of GD and G/GMP supplementation on the interaction between MYH9 and LATS1 in HCT116 cells were examined using Co-IP assay. H HCT116 cells were cultured with normal medium (CTRL), glucose-free medium or glucose-free medium supplemented with 5 mM G for indicated time before immunoblotting analysis of MYH9 and LATS1. G3BP1 was used as a loading control for the pellets. I Guanosine supplementation diminished KL-11743-induced LATS1 insolubilization. HCT116 cells were cultured under normal conditions, treated with or without KL-11743, or KL-11743 plus G supplementation for 4 h. PNP or actin was used as a loading control. J Immunoblotting analysis of the recruitment of LATS1 to actin in SW480 cells under normal conditions, GD (0 mM) or GD supplemented with G. K Representative fluorescence images of LATS1 aggregation and F-actin in LATS1-GFP overexpressing HCT116 cells under normal conditions, GD (0 mM) or GD supplemented with G for 3 h. DAPI served as the nuclear stain. L Representative immunofluorescence images and quantitative analysis of YAP nuclear localization in HCT116 NC, shMYH9 and shMYH10 cells treated with GD (0 mM) for 4 h. DAPI served as the nuclear stain. M Immunoblotting analysis of LATS1 in the supernatants (Sup) and pellets of HCT116 NC, shMYH9, and shMYH10 cells under normal conditions or GD (4 h). G3BP1 was used as a loading control. N Immunoblotting analysis of MYH9, LATS1, phosphorylated YAP (serine 127) and phosphorylated JNK in the supernatants (Sup) and pellets of HCT116 NC and shPGM2 cells treated under normal conditions, GD (4 h) or GD supplemented with G (4 h). GAPDH was used as a loading control. O Immunoblotting analysis of LATS1 and YAP in the supernatants (Sup) and pellets of HCT116 cells treated under normal conditions, GD (0 mM, 4 h) or GD supplemented with D5P. GAPDH was used as a loading control. P Schematic diagram depicting that D5P disrupts the interaction between LATS1 and MYH9/F-actin and recovers the inhibition of LATS1 on YAP. The experiments in (A–E), (G–J), and (M–O) were repeated twice independently. In (L), data are the mean ± S.D.; P values were calculated using a two-tailed unpaired Student’s t-test. ***, P < 0.001

Fig 3: Dysregulation of YAP activity reprograms purine nucleotide metabolism and cellular response to GD. A Set enrichment of polar metabolites from HCT116-shYAP and HCT116-NC cells as determined by 600MRM analysis. B Relative levels of metabolites involved in glucose metabolism in HCT116-NC and HCT116-shYAP cells. C Relative levels of metabolites involved in purine (adenine and guanine) nucleotide metabolism in HCT116-NC and HCT116-shYAP cells. D Relative levels of metabolites involved in purine (adenine and guanine) nucleotide metabolism in HCT116-EV- and HCT116-YAP-5SA-overexpressing cells. E Cluster analysis of polar metabolites of HCT116-NC and HCT116-shYAP cells under normal culture or GD conditions (0 mM, 6 h). F Analysis of PNP enzyme activity in HCT116-NC and HCT116-shYAP cells treated under GD. G Analysis of PNP enzyme activity in HCT116 cells treated under GD with or without D5P supplementation. H Schematic diagram depicting that GD-induced D5P downregulation triggers YAP activation, promoting PNP activity to recover D5P level. I Immunoblotting analysis of LATS1, YAP and phosphorylated JNK in the supernatants (Sup) or LATS1 in the pellets of HCT116-NC and HCT116-shYAP cells under the indicated treatment. G3BP1 was used as a loading control. J Immunoblotting analysis of LATS1, YAP and phosphorylated JNK in the supernatants of HCT116-EV- and YAP-5SA-overexpressing cells with the indicated treatment. Tubulin was used as a loading control. The experiments in (I), (J) were repeated twice independently. In (B–D) and (F–G), data are the mean ± S.D.; P values were calculated using a two-tailed unpaired Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance

Fig 4: Oral administration of D5P attenuates CRC development in APC mutant mice. A Kaplan–Meier survival analysis of colon adenocarcinoma patients stratified by PNP expression. Data from GEPIA database. B Rectal prolapse observation showing that the onset of CRC in APCmin/+ mice treated with D5P was delayed. C Survival probability analysis of APCmin/+ mice treated with or without D5P (4 mM) in drinking water. D Representative colon images of the APCmin/+ mice treated with or without D5P (4 mM). E The quantification of tumor number presented in (D). Colon tumor number of APCmin/+ mice treated with or without D5P (4 mM). F Representative H&E staining of colon tumor from mice treated with or without D5P (4 mM). G Large field view of YAP staining in colon sections of mice treated with or without D5P (4 mM). The numeric labeled regions were zoom in and showed in (H) and (I). H A representative image of non-cancerous area in (G). YAP staining of the non-cancerous area of colon from mice treated with or without D5P (4 mM). I A representative image of cancerous area in (G) and quantitative results of YAP staining in the cancerous area of colon from mice treated with or without D5P (4 mM). J Schematic diagram showing the anti-cancer function of D5P via the MYH9/LATS1-YAP-PNP-D5P negative feedback loop. *, P < 0.05; **, P < 0.01