ZFP36L2：从应激退出到肠道再生与癌细胞可塑性

Fig. 1 | ZFP36L2 is expressed by ISCs and is associated with the AP-1 gene program. a, Schematic of cell-state plasticity and dynamics during intestinal regeneration (top) and CRC (bottom). b, Dot plot of tumour ISC-like module10 gene expression in normal colon (Normal) and CRC (Tumour) epithelial cells from 25 patients. The dot size indicates the per cent cells (rows) expressing each gene (columns). Colour scale indicates the log2 fold change in average gene expression. Bar plots show the mean expression of each gene across normal colon ISCs. Genes meeting false discovery rate (FDR) < 0.05 using the Wilcoxon rank-sum test (ISC versus all other epithelial subtypes) are shown. c,d, Representative LGR5 and ZFP36L2 RNA FISH (c) and ZFP36L2 IF (d) images of human normal colon crypts. e, Representative LGR5 and ZFP36L2 RNA FISH images of human primary CRC tumours (n = 2), liver metastases (n = 2) and lung metastases (n = 2) from 2 donors. f, Lollipop plot showing ZFP36L2 mutation frequency in the TCGA63, DFCI64 and MSKCC whole-exome sequencing CRC cohorts. n = 96 tumours. g,h, ZFP36L2 autocorrelated gene programs in normal colon (g) and CRC epithelial cells (h). Red, top AP-1 family genes65. n = 12,955 normal colon (24 donors) and 25,317 CRC cells (25 patients). FDR < 0.05. i, GSEA of genes (Supplementary Table 1b–f) ranked by positive ZFP36L2 autocorrelation (Methods), which revealed conserved pathways enriched in normal (white dots) and tumour (black dots) epithelial cells. n = 12,955 normal colon (24 donors) and 25,317 CRC cells (25 patients). FDR < 0.05. j, Violin plots showing AP-1 pathway gene expression in ZFP36L2-high and ZFP36L2-low populations (top and bottom 10 percentile expression). n = 1,806 differentiated, n = 1,326 TA and n = 230 ISC cells from normal colons (24 donors) and 2,622 primary CRC and 2,444 CRC metastasis cells (25 patients). Two-sided Mann–Whitney U-test. Scale bars, 50 µm (c,d) or 200 µm (e).

Fig. 2 | Zfp36l2 is required for injury-induced dedifferentiation during intestinal regeneration. a, Zfp36l2 expression across epithelial cells, as measured by scRNA-seq of colonic crypts (21,000 cells from 11 mice)66. Marker genes for each cell type are indicated by boxes. b, Representative images (left) and quantification (right) of Lgr5 RNA FISH and ZFP36L2 and MUC2 IF in colon tissue from 7-week-old C57BL/6J mice based on the cell position along the crypt base (position 0) to lumen axis. n = 2,275 cells from 31 crypts. c, Representative images of Lgr5 RNA FISH and ZFP36L2 and MUC2 IF in colon tissue collected at the indicated time points from mice treated with 3.5% DSS for 7 days followed by recovery for 7 days. n = 3 mice per time point. Expanded fields of view are provided in Supplementary Figs. 1 and 3. d, Top, schematic of the experiment. Bottom, daily body weight measurements. n = 5 (water) mice; n = 10 (wild-type (WT)) and 14 (IEC) (DSS-treated) mice. Mean (solid line; dots, individual time points) ± s.e.m. (shaded area; dashed lines, s.e.m. boundaries); two-tailed Mann–Whitney U-tests at day 7 and day 14. e, Caecum (top) and colon (bottom) lengths on day 14. n = 3 mice (water); n = 10 (WT) and 12 (IEC) (DSS-treated) mice. Mean ± s.d.; two-tailed Mann–Whitney U-test. NS, not significant. f, Lgr5DTR ablation. Top, schematic of the experiment. Bottom, representative images (n = 2 mice) of colon sections stained by Lgr5 RNA FISH or ZFP36L2 IF at the indicated time points. Expanded fields of view are provided in Supplementary Fig. 5. Controls: Lgr5DTR-negative mice treated with 4 doses of DT over 7 days. g,h, Mice (8–10 weeks old) were administered DT every 2 days for 4 doses. Mice were euthanized and colons were collected 12 days after the first DT dose. g, Images of mouse Lgr5 and Zfp36l2 mRNA FISH on day-12 Swiss-roll colon sections. h, The number of mice showing regeneration of the Lgr5 FISH signal. i, Schematic of the expression and function of ZFP36L2 in injury-induced dedifferentiation to drive regeneration in the mouse colon. Scale bars, 50 µm (b,c,f,g).

Fig. 3 | Loss of ZFP36L2 inhibits CRC metastasis seeding and ISC dedifferentiation but promotes non-canonical differentiation. a–c, Orthotopic caecal xenotransplantation experiments. a, Schematic of the experiment. DOX, doxycycline. b, In vivo abdominal BLI average radiance, normalized to BLI at the time of doxycycline diet initiation. shL2, shZFP36L2. n (left to right) = 8, 5, 7, 8, 8, 14, 10 and 7 animals per group. Mean ± s.e.m.; two- tailed Mann–Whitney U-tests. c, End point ex vivo BLI of MSK107Li samples. Metastasis signals were normalized to orthotopic caecal signals in the same animals. n = 10 (shCtrl) and 7 (shL2) mice. Mean ± s.e.m.; two-tailed Mann– Whitney U-tests. d–g, Orthotopic liver metastasis seeding experiments. d, Schematic of the experiment. e, Representative ex vivo images of MSK107Li liver metastases. f, Average radiance of week 13 metastasis normalized to week  0 BLI. n = 6 (shCtrl) and 5 (shL2) mice. Mean ± s.e.m.; two-tailed Mann–Whitney U-tests. g, Weekly whole-body in vivo liver BLI (mean ± s.e.m.) normalized to week 0. n = 7 (shCtrl) and 5 (shL2) mice. Two-tailed Mann–Whitney U-tests. h, Schematic of the experiment. i. Kernel density estimate contour plots of scRNA-seq data from h, showing the overlap and divergence of cell states in OKG146P organoids transduced with shCtrl or shL2 and cultured in HISC, IGFF or dedifferentiated (Dediff) conditions. j, Violin plots showing expression of the indicated markers. k,l, Representative immunostaining (left) and quantification (right) of LGR5 RNA FISH and KRT20 IF in MSK107Li (k; see also d–g) and CHGB IF in OKG146Li-MS2 liver metastasis (l). The graphs show the per cent LGR5 and KRT20+ cells in each of the 26 regions of interest (ROIs) (252,330 cells; shCtrl) and 8 ROIs (194,530 cells; shL2) or CHGB+ cells in each of the 21 ROIs (254,403 cells; shCtrl) and 20 ROIs (66,338 cells; shL2) from 3 mice per group. Box plots show the interquartile range, with the line indicating the median, and whiskers the minimum and maximum values. Two-tailed Mann– Whitney U-tests. m,n, Representative images (m) and quantification (n) of LGR5 RNA FISH and CK5 and CHGB IF in WT or ZFP36L2 mutated (MUT) paired primary–metastasis CRC samples from patients. n = 8 (ZFP36L2 WT) and 4 (ZFP36L2 MUT) samples. LGR5 RNA FISH: 91 ROIs (1,301,960 cells; ZFP36L2 WT) and 43 ROIs (1,154,616 cells; ZFP36L2 MUT). CHGB and CK5 IF: 113 ROIs (1,814,441 cells; ZFP36L2 WT) and 50 ROIs (1,302,471 cells; ZFP36L2 MUT). Box plots show the interquartile range, with the line indicating the median, and whiskers the minimum and maximum values. Two-tailed Mann–Whitney U-tests. Scale bars, 50 µm (l), 100 µm (k,m) or 1 cm (e).

Fig. 4 | ZFP36L2 forms RNA-dependent, stress-responsive biomolecular condensates. a, Representative endogenous ZFP36L2 and pan-cytokeratin (Pan-CK) IF images of primary CRC and CRC liver metastasis from patient KG146. b, ZFP36L2 IF in MSK107Li organoids transduced with shCtrl (top) or shZFP36L2, demonstrating efficient knockdown and staining specificity. c,d, Top, AlphaFold2-predicted protein structure of human WT ZFP36L2 (c) and CRC patient hotspot mutation Gly144Alafs*43 (d). Bottom, schematic of the protein structures indicating the CCCH zinc fingers (ZFs) and the N-terminal and C-terminal IDRs. Red, ZF domains (amino acids 153–219); grey, IDRs. e, Representative images of maximum-intensity projection of motion-artefact- corrected 3D time-lapse video of OKG146Li organoids co-expressing ZFP36L2– eGFP and H2B–mCherry. Top, condensate positions and trajectories overlaid on the original image. Bottom, corresponding trajectory rendering shown separately. f, Representative images of single z plane confocal live-cell microscopy of MSK107Li H2B–mCherry (red) organoids with doxycycline- inducible expression of eGFP, WT ZFP36L2–eGFP or fsZFP36L2–eGFP. Organoids were plated as single cells on day 0, and doxycycline was added 24 h before imaging. g, Mean number of condensates per cell (top) and number of cells per organoid (bottom) at the indicated time points after single-cell seeding of ZFP36L2–eGFP-expressing MSK107Li H2B–mCherry organoids. Data are the mean ± s.d., and data points represent individual organoids (n = 8). Two-tailed Mann–Whitney U-tests. h, Normalized fluorescence intensity of MSK107Li organoids after photobleaching. Data representative of four replicates (mean ± s.d.). i, Live-cell imaging of ZFP36L2–eGFP-expressing MSK107Li organoids, treated with or without 1 μg μl–1 RNase A, 30 min after treatment. j, Live-cell imaging of OKG146Li organoids expressing doxycycline- inducible eGFP, ZFP36L2–eGFP or ZFP36L2–meGFP. Day 3 organoids were treated with 2 μg ml−1 doxycycline for 24 h before imaging. Insets are shown at higher magnification (bottom). ZFP36L2–meGFP exhibited condensate formation comparable to that of ZFP36L2–eGFP. Scale bars, 10 µm (a,b,e,f (zoom),i,j) or 20 µm (f, non-zoom).

Fig. 5 | ZFP36L2-dependent mRNA binding and decay of stress-associated transcripts. a, Schematic of the HyperTRIBE experiment. b, Features of ZFP36L2-specific ADAR-edited mRNA sequences shared between OKG146Li and MSK107Li organoids. c, ZFP36L2-bound mRNAs (HyperTRIBE, yellow dots) undergoing ZFP36L2-dependent mRNA decay (based on ActD treatment and sequencing). Volcano plots show differential mRNA decay in shL2 versus shCtrl organoids. Wald test (negative binomial), two-sided, Benjamini–Hochberg FDR. HyperTRIBE-identified genes were significantly enriched among transcripts with increased stability after ZFP36L2 knockdown. Fisher’s exact test: MSK107Li, odds ratio = 2.21, P = 2.4 × 10–35; OKG146Li, odds ratio = 2.47, P = 9.2 × 10–75. d, GO biological process enrichment analysis of ZFP36L2- dependent mRNA decay (after ActD treatment and sequencing), showing targets shared between OKG146Li and MSK107Li organoids. n = 379 genes, P < 0.05, fold change > 1 (Supplementary Table 5c). Hypergeometric test (one-sided) with Benjamini–Hochberg correction. e, Scatter plot showing SLAM-seq RNA half-lives in MSK107Li organoids expressing shL2 or shCtrl. Each dot indicates one transcript. One-sided Wilcoxon tests. f, Over-representation analysis of transcripts from SLAM-seq datasets, with significantly increased half-life in shL2 versus shCtrl treatments. Hypergeometric test (one-sided) with Benjamini–Hochberg correction. GO terms are as in d. g,h, Representative images (g) showing multiplexed ZFP36L2 IF and GDF15, GADD45A and HILPDA RNA FISH in OKG146Li-MS2 organoids, and per cent ZFP36L2 puncta positive for the indicated RNA FISH signal (h) in OKG146Li-MS2 organoids. Organoids were cultured in HISC for 7 days, cultured in IGFF for 10 days before switching to HISC for 7 days to induce dedifferentiation (Dediff), or cultured in HISC for 7 days before switching to IGFF (IGFF 1 h) or HISC containing 500 μmol irinotecan (IRI 1 h) for 1 h. Each dot indicates one organoid. n (left to right) = 14, 13, 10, 12 and 13 organoids per condition. Mean ± s.e.m.; two-tailed Mann– Whitney U-test. Scale bars, 1 µm (g, insets) or 10 µm (g).