Fig 2: Combination of DMBA treatment with PP2A deficient in Lgr5+ drives intestinal neoplasia in both the small intestine and colon. (A) H&E staining and (B) Beta-catenin IHC were performed on the serial sections. Multiple beta-cateninhigh adenomas were observed throughout the colon 36 days after induction. (C) Quantitative analysis of nucleus beta-catenin H-score from (B). (D) High level expression of beta-catenin was apparent in the transformed stem cell compartment (Peyer’s patches are stained blue). Multiple beta-cateninhigh transformed cells were observed throughout the intestinal 36 day after induction (magnified at right panel, denoted by arrows). Original magnifications: (A) left, 4×; (A) right, 10×; (B) left, 20×; (B) right, 40×; (D) center, 20×; (D) right, 40×; Scale bar, 100 µm. **, p < 0.01 as determined with one-way ANOVA.

Fig 3: Ppp2r1a knockdown triggers neoantigen generation and sensitises to ICB therapies in MSS tumours.Ppp2r1a knockdown and PD-1 blockade synergistically elicit tumour rejection in a CD8+ T cell-dependent manner. a The flowchart of animal experiments. BALB/c mice were inoculated with 0.5 × 106 CT26 cells transfected with scramble (scr) or Ppp2r1a shRNA (shppp2r1a) subcutaneously in the flank. In all experiments, polyclonal hamster IgG and Anti-PD-1 (clone: RMP1-14, BioXcell) were administered using doses of 200 µg for the initial injection and 100 µg for subsequent injections. Mice were then intraperitoneally injected with isotype control IgG or anti-PD1 antibodies (200 µg, per mouse) on days 5, 8, and 11 following tumour injection. b–e The indicated CT26 clones were injected into syngeneic BALB/c mice. Tumour growth curves were compared and tumour samples were harvested for further analysis. Three independent experiments were done. Data of representative experiments are shown as mean ± s.e.m. (n = 5). P value was determined by two-way ANOVA. f (Left) Immunofluorescence of CD8 and CD4 was performed on CT26-scr and CT26-shppp2r1a tumours to assess CD8+ and CD4+ T cell infiltration at the indicated time points. Bar = 25 µm. f (Right) Quantification of CD8+ density. Three independent experiments were done. Data of representative experiments are shown as mean ± s.e.m. (n = 5). P = 0.0046 was determined by two-sided one-way ANOVA. g A Venn diagram showing a total of 220 genes, including 270 missense transcripts, shared by three CT26-shppp2r1a tumours but not found in the CT26-scr tumour. h Distribution of the 20 most frequent TCR rearrangements identified in peripheral blood from mice (n = 3 for each group) injected with the indicated CT26 clones. The width of the violins is proportional to the number of TCR templates in each level of the y axis; the bars inside the violins show the quartiles of the 20 templates; bars span the first to third quartiles; the horizontal lines inside the bars represent the median; the grey zones represent all samples; the white circle shows the median value. TCR analysis was performed on blood samples obtained 13 days after injection of the tumour cells, as described in the “Methods” section. Source data are provided as a Source data file.

Fig 4: PP2A loss or inhibition enhances cytotoxic T cell infiltration in colorectal cancer.a, b Ppp2r1a-loss colon tumours were induced in Lgr5-EGFP-CreERT2; Ppp2r1aflox/flox by treatment with DMBA and tamoxifen for 36 days. Control colon tissues (Control) and Ppp2r1a-loss colon tumours were harvested for analysis. a Representative images of immunofluorescence showing increased CD8+ and CD20+ and decreased Foxp3+ infiltration in murine Ppp2r1a-loss colon tumours compared to controls. Arrows indicate positive signals. Images are representative of three biological independent samples for each group. Bar = 25 µm. b Gene set enrichment analysis (GSEA) for cytokine, chemokine, IFN-gamma response, and JAK-STAT signalling pathways in murine Ppp2r1a-loss colon tumours compared to controls. c TCGA-COAD (n = 461) and d TCGA-READ (n = 172) omics analysis revealed that CIP2A and SET levels were significantly positively correlated with CD8+, CD4+, and B cell infiltration levels and significantly negatively correlated with Treg infiltration, while PPP2R1A levels were negatively correlated with CD8+ infiltration but positively correlated with Treg infiltration. Results are determined by the Spearman correlation. Source data are provided as a Source data file.

Fig 5: MLH1 loss caused by DNMT3A/B upregulation is mediated through the PP2A-Rb-E2F pathway.a Immunoprecipitation performed with Capturem Protein A columns. Organoid lysates from WT mice were incubated with 1 µg of PPP2R1A antibody for 10 min. The antibody–lysate complex was applied to equilibrate Protein A spin columns. The eluted fraction was then subjected to SDS-PAGE to confirm the presence of PP2A B subunit antigen (52 kDa, detected by Cell Signaling #2290). Images are representative of one biological independent sample for each group. b Volcano plot for the comparison of RNA-seq data of Lgr5-EGFP-CreERT2; Ppp2r1aflox/flox intestinal organoids treated with and without DMBA and tamoxifen for 50 days. Differently expressed genes (fold change >2 and FDR < 0.05) are denoted in red or green. Plot is representative of three biological independent samples for each group. c Intersection of ppp2r1a interaction protein in a and differently expressed genes in b data (IP protein data are available in Supplementary Data 1 and RNA-seq data are available in GSE120241 with NIH/NCBI at GEO dataset). d The top 3 significant gene ontology categories from the 58 interacted proteins identified by GSEA (whole gene ontology category is listed in Supplementary Table 4). e Venn diagram showing overlap (gene numbers) among genes associated with regulation of TP53 activity, cell cycle, and RNA polymerase II transcription. The intersection contained four genes, including hdac2, ppp2cb, akt1, and rb1. Western blot analysis of f CT26 transfected with the indicated shRNAs, and treated with g vehicle control or 5-azacytidine (AZA, 1 µM for 1 days). Blots are representative of three biological independent samples for each group. h Human tissue array containing MSI and MSS colorectal tumours was assayed for p-Rb (n = 141, P = 0.0132) and p-HDAC2 (n = 138, P = 0.02) levels by immunohistochemistry. (Left) Representative pictures showing increased p-Rb and p-HDAC2 levels in MSI compared to MSS. Arrows indicate positive signals. Bar = 25 µm. (Right) Quantitative data are shown. P value was determined by two-sided Mann–Whitney U-test. Source data are provided as a Source data file.