Abstract
Lung cancer is the leading cause of cancer deaths worldwide1. Mutations in the tumour suppressor gene TP53 occur in 50% of lung adenocarcinomas (LUADs) and are linked to poor prognosis1,2,3,4, but how p53 suppresses LUAD development remains enigmatic. We show here that p53 suppresses LUAD by governing cell state, specifically by promoting alveolar type 1 (AT1) differentiation. Using mice that express oncogenic Kras and null, wild-type or hypermorphic Trp53 alleles in alveolar type 2 (AT2) cells, we observed graded effects of p53 on LUAD initiation and progression. RNA sequencing and ATAC sequencing of LUAD cells uncovered a p53-induced AT1 differentiation programme during tumour suppression in vivo through direct DNA binding, chromatin remodelling and induction of genes characteristic of AT1 cells. Single-cell transcriptomics analyses revealed that during LUAD evolution, p53 promotes AT1 differentiation through action in a transitional cell state analogous to a transient intermediary seen during AT2-to-AT1 cell differentiation in alveolar injury repair. Notably, p53 inactivation results in the inappropriate persistence of these transitional cancer cells accompanied by upregulated growth signalling and divergence from lung lineage identity, characteristics associated with LUAD progression. Analysis of Trp53 wild-type and Trp53-null mice showed that p53 also directs alveolar regeneration after injury by regulating AT2 cell self-renewal and promoting transitional cell differentiation into AT1 cells. Collectively, these findings illuminate mechanisms of p53-mediated LUAD suppression, in which p53 governs alveolar differentiation, and suggest that tumour suppression reflects a fundamental role of p53 in orchestrating tissue repair after injury.
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Data availability
Data from in vivo LUAD RNA-seq, in vivo LUAD ATAC–seq, in vitro LUAD cell line RNA-seq, in vivo single-cell multiomics (RNA-seq and ATAC–seq) and in vivo lung injury scRNA-seq experiments are available from the GEO under the accession number GSE231681. The mouse LUAD microarray data have been previously published30 and are available at the GEO (GSE23875). The mouse p53 ChIP–seq datasets in mouse embryonic fibroblasts, mouse embryonic stem cells and mouse transitional cells after bleomycin injury have been previously published14,31,32 and are available at the GEO (GSE46240, GSE26361 and GSE141635, respectively). The mouse LUAD scRNA-seq data have been previously published29 and available at the GEO (GSE152607). Expression data for the human TGCA-LUAD cohort1 are available through the NCI GDC Data Portal (https://portal.gdc.cancer.gov/projects/TCGA-LUAD). Expression data for the human GSE13213 LUAD cohort68 is available at the GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse13213). Expression data for the human E-MTAB-8516 LUSC cohort69 is available at ArrayExpress (https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-8615?query=E-MTAB-8615). The MSigDB database (https://gsea-msigdb.org/gsea/msigdb/)40 was used to perform GSEA52 analyses. All other data supporting the findings of this study are in the article, supplementary information or are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank P. Chu for processing and embedding all paraffin-embedded mouse tissue samples; staff at the Stanford Functional Genomics Facility for help with the library preparation and sequencing of LUAD cell lines; K. Nuno and A. Spencley for technical advice regarding the ATAC–seq experiments; and T. Desai, M. Winslow, J. Sage, S. Ferreria, L. Cong, D. Bhattacharya and A. Boutelle for advice and critical reading of the manuscript. Research in this article was supported by NCI PHS grant CA009302, NCI R35 grant CA197591 (to L.D.A.) and the NSF (GRFP, to A.M.K.). A.M.K was also supported by the Stanford Graduate Fellowship.
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Contributions
A.M.K. designed and performed experiments, interpreted and analysed data and wrote the manuscript. A.M.K. performed all mouse tumour studies, lung injury studies, in vivo sequencing experiments and the subsequent analyses for these experiments. A.M.K., A.G. and K.J.H. analysed and interpreted the previously published29 scRNA-seq data analyses. A.G. performed hierarchical clustering to identify clusters 5A, 5B and 5C, defined gene expression programmes within cluster 5, performed CytoTRACE, pseudotime and CellRank analyses, performed copy number variation analyses, and identified transitional cell markers across cancer cell clusters. K.J.H. and M.W. sorted TIGIT+ LUAD cells. R.L.Z. performed IF, ChIP–qPCR and LUAD cell line reactivation experiments, and assisted with BHT lung injury experiments. N.R. performed ChIP–qPCR experiments. J.A.S. performed all human tumour genomics analyses. K.T.B.-R. generated LUAD cell lines and performed RNA-seq on them. M.W. generated the A549 TP53 knockout cell lines, performed p53 reactivation experiments in vivo and performed IF experiments. I.L., W.L.T., D.Z.L., J.B.S. and S.K.P. collected and curated the human LUAD samples. A.M.N. assisted with trajectory analyses, and C.V.R. provided support for scRNA-seq analyses. L.D.A. designed experiments, interpreted data and wrote the manuscript with input from all authors.
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Extended data figures and tables
Extended Data Fig. 1 p5353,54 suppresses late-stage lung adenocarcinoma better than wild-type p53.
(a) Representative Western blot for p53 and GAPDH in p53LSL-wt/p53LSL-wt (n = 3) and p53LSL-53,54/LSL-53,54 (n = 3) LUAD cell lines infected with adenoviral (Ad)-Empty or Ad-Cre for 48 h. Samples were run on the same gel. For gel source data, see Supplementary Figure 1. LSL, lox-stop-lox. (b) RT-qPCR of a panel of p53 target genes in p53LSL-wt/p53LSL-wt (n = 3) and p53LSL-53,54/LSL-53,54 (n = 3) cell lines infected with Ad-Empty or Ad-Cre for 48 h. (c) Schematic for the 24-week lung cancer study. Histology cohort: KFT (n = 6), KT (n = 5), and KPT (n = 5) mice. (d) Representative histological H&E images of 24-week lungs from KFT (n = 6) and KT (n = 5) mice. Scale bar, 100 μm. (e) Quantification of 24-week lung tumor burden, number, and size (n = 112, 268 and 1,429 tumors for KFT, KT, and KPT cohorts, respectively). (f) Histopathological analysis of tumor patterns in 24-week KFT (n = 8), KT (n = 8), and KPT (n = 5) mice. Chart displays the percentage of mice that displayed tumors of a given pattern. NE, neuroendocrine; AAH, atypical adenomatous hyperplasia. (g) Histopathological analysis of tumor pleomorphism (see Methods) in 24-week KFT (n = 8), KT (n = 8), and KPT (n = 5) mice. The chart displays the percentage of mice that displayed tumors of a given score. (h) Histopathological analysis of mitotic activity in the most advanced lesion in 24-week KFT (n = 8), KT (n = 8), and KPT (n = 5) mice. HPF, high powered field. (i) Representative Ki67 IHC in tumors from 24-week KFT (n = 4) and KT (n = 4) mice. Scale bar, 50 μm. (j) Percentage Ki67-positive nuclei in (n = 20) tumors in 24-week KFT (n = 4) and KT (n = 4) mice. All data are mean ± s.d. P values were calculated by two-tailed Student’s t-test (b, j) and Kruskal-Wallis test with multiple comparisons (two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli) (e, h).
Extended Data Fig. 2 p5353,54-mediated tumor suppression is dependent upon its expression in AT2 cells.
(a) Schematic for the 10-week AT2 cell-of-origin tumor study. (b) Representative histological H&E images of lungs from 10-week KFT (n = 8), KT (n = 8), and KPT (n = 5) mice. Images below are a higher magnification view of the image above. Scale bars: top, 5 mm; middle, 500 μm; bottom, 50 μm. (c) Representative Ki67 and phosphohistone H3 (pHH3) IHC in tumors from 10-week KFT (n = 4), KT (n = 4), and KPT (n = 4) mice. Scale bar, 50 μm. (d) Percentage Ki67-positive nuclei in (n = 20) tumors in 10-week KFT (n = 4), KT (n = 4), and KPT (n = 4) mice. (e) Percentage pHH3-positive nuclei in (n = 20) tumors in 10-week KFT (n = 4), KT (n = 4), and KPT (n = 4) mice. (f) Schematic for the 10-week cancer cell autonomy study. Histology cohort: KT;p53LSL-53,54/+ (KFT-LSL) (n = 8) and KT;p53LSL-wt/+ (KT-LSL) (n = 8) mice. The results of this study demonstrate that p5353,54 expression in AT2 cells results in enhanced tumor suppression. (g) Quantification of lung tumor burden, number, and size (n = 83 and 213 tumors for KFT-LSL and KT-LSL cohorts, respectively). (h) Representative Ki67 IHC in tumors from KFT-LSL (n = 4) and KT-LSL (n = 4) mice. Scale bar, 50 μm. (i) Percentage Ki67-positive nuclei in (n = 20) tumors from KFT-LSL (n = 4) and KT-LSL (n = 4) mice. (j) Representative IF of HOPX or PDPN and tdTomato (tdT) in KFT-LSL (n = 3) mice and KT-LSL (n = 3) mice. Scale bar, 20 μm. (k) HOPX and PDPN quantification in (n = 50 [KFT-LSL] and 100 [KT-LSL]) tumors in KFT-LSL (n = 3) mice and KT-LSL (n = 3) mice (see Methods). Tumors were binned based on the percentage AT1 marker positivity of tdTomato+ cells. Low = 0–5%, medium = 5–20%, high = 20–100%. All data are mean ± s.d. P values were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test (d-e), two-tailed Mann Whitney U test (g), and two-tailed Student’s t-test (i).
Extended Data Fig. 3 p5353,54 and wild-type p53 induce transcriptional and epigenetic profiles distinct from p53-deficient programs.
(a) FACS-sorting and library preparation schematic (see Methods). RIN, RNA integrity number. (b) Unsupervised hierarchical clustering of RNA-sequencing samples from KFT (n = 4), KT (n = 3), and KPT (n = 4) mice. (c) Heatmap of the 5,065 differentially expressed genes (DEGs, P-adjusted ≤ 0.05) between p53-proficient (KFT and KT) and p53-deficient (KPT) cells. DEGs identified in the analysis of KFT vs. KPT cells and KT vs. KPT cells are shown. (d) Significantly enriched gene ontology (GO) biological process terms in p5353,54 hyperactivated genes, identified by PANTHER56. (e) GO term analysis by PANTHER56 of genes upregulated in KT or KPT cells. (f) GSEA shows enrichment of the indicated GO biological process term in KT cells relative to KPT cells. NES, normalized enrichment score. (g) Percentage of all called peaks that are differentially accessible in the ATAC-sequencing experiments. (h) Principal component analysis of the KT (n = 4) vs. KPT (n = 4) ATAC-sequencing samples. (i) Peak annotation of differentially accessible regions in KT vs. KPT ATAC-sequencing experiment. UTR, untranslated region. (j) Principal component analysis of the KFT (n = 2) vs. KT (n = 2) ATAC-sequencing samples. (k) Peak annotation of differentially accessible regions in KFT vs. KT ATAC-sequencing experiment. P values were calculated by GSEA52 (f).
Extended Data Fig. 4 p5353,54 and wild-type p53 activate AT1 cell transcriptional and epigenetic programs.
(a) Expanded version of GSEA shown in Fig. 2a of alveolar epithelial cell signatures (Supplementary Table 6). Normalized enrichment score (NES) > 0 indicates the signature is enriched in KFT or KT cells relative to KPT cells. *Indicates gene sets significantly enriched (P ≤ 0.05). (b) GSEA shows enrichment of the indicated AT1 signatures in specific genotypes. (c) AT1 cells (HOPX+) as a ratio of all alveolar epithelial cells (NKX2-1+) (n = 600) at homeostasis in p5353,54/53,54 (n = 3), p53+/+ (n = 3), and p53−/− (n = 3) mice (no oncogenic Kras). (d) Proliferating (BrdU+) cells as a ratio of all alveolar epithelial cells (NKX2-1+) (n = 600) at homeostasis in p5353,54/53,54 (n = 3), p53+/+ (n = 3), and p53−/− (n = 3) mice (no oncogenic Kras). (e) GSEA shows enrichment of non-alveolar cell signatures (Supplementary Table 6) in KPT cells. NES < 0 indicates the signature is enriched in KPT cells. *Indicates gene sets significantly enriched (P ≤ 0.05). (f) (Top) Plot from Fig. 1j showing differentially accessible regions (P-adjusted ≤ 0.05) at AT1-associated gene promoters (± 1kb from the transcription start site, Angelidis_Mouse_AT1 signature, denoted by red/blue dots). (Bottom) Plot from Fig. 1k showing differentially accessible regions (P-adjusted ≤ 0.05) at AT1-associated gene promoters (Angelidis_Mouse_AT1 signature). (g) Counts per million (CPM) indicating AT1 gene expression in KT (n = 3) and KPT (n = 4) RNA-sequencing samples. All data are mean ± s.d. P values were calculated by GSEA52 (a, b, e), ordinary one-way ANOVA with Tukey’s multiple comparisons test (c, d) and using the DESeq2 R package (two-tailed Wald test adjusted for multiple testing using the procedure of Benjamini and Hochberg) (g).
Extended Data Fig. 5 Single-cell analyses show that p53 promotes AT1 cancer cell formation.
(a) Uniform Manifold Approximation and Projection (UMAP) plot of single-cell multiome (scRNA and scATAC) profiles of cancer cells (n = 6,929) collected from 10-week KFT (n = 2), KT (n = 2), and KPT (n = 2) mice. (b) Dot plot showing the RNA expression of alveolar epithelial cell signatures (Supplementary Table 6, AT1 and AT2 signatures are derived from the Mouse Cell Atlas) and markers in all cells across clusters. Sig = signature, Trans. sig. = transitional cell signature. (c) Dot plot showing the computed gene activity from ATAC data of alveolar epithelial cell signatures (Supplementary Table 6, AT1 and AT2 signatures are derived from the Mouse Cell Atlas) and markers in all cells across clusters. (d) Heatmap showing expression of AT2 and AT1 markers (log-transformed transcripts per million rescaled across all cells) in all cells. (e) Feature plots show the RNA expression/computed gene activity of (top) AT2 cell signatures and markers and (bottom) AT1 cell signatures and markers in all cells (Supplementary Table 6, AT1 and AT2 signatures are derived from Mouse Cell Atlas). (f) Feature plot shows the RNA expression of GO lung differentiation term genes (Supplementary Table 6) in all cells. (g) ATAC-sequencing tracks showing chromatin accessibility at Sftpc (AT2 gene), Ager (AT1 gene), and Cldn4 (transitional cell gene) in all cells split by clusters. (h) UMAP plot from (a) split by KFT (n = 1,200), KT (n = 4,318), and KPT (n = 1,411) cells. (i) Relative proportions of all clusters split by genotype. (j) Relative proportions of AT1-like cancer cells in each genotype. KFT was set to 1.0.
Extended Data Fig. 6 p53 promotes an AT1 differentiation program in LUAD.
(a) Representative IF of tdTomato (tdT) and AGERhigh/medium/low, HOPXhigh/medium/low, and SPChigh/low tumors. Scale bar, 20 μm. (b) Uncropped images from Fig. 2b of AGER and tdTomato in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bar, 200 μm. (c) Expanded view of images from Fig. 2b of AGER and tdTomato in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bars, 20 μm. (d) Percentage tdTomato+ cancer cells positive for AGER in individual (n = 100 [KFT], 100 [KT], and 200 [KPT]) tumors in KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Red lines; break points for high, medium, and low staining. (e) Expanded view of images from Fig. 2b of HOPX and tdTomato in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bars, 20 μm. (f) Representative IF of PDPN and tdTomato in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bars, 20 μm. (g) Representative IF of SPC and tdTomato in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bar, 20 μm. (h) PDPN quantification in (n = 100 [KFT], 100 [KT], and 200 [KPT]) tumors in KFT (n = 4), KT (n = 3), and KPT (n = 3) mice (see Methods). The graph shows the percentage of tumors that are classified as AT1 marker high, medium, and low. Low = 0–5%, medium = 5–20%, high = 20–100%. (i) SPC quantification in (n = 100 [KFT], 100 [KT], and 200 [KPT]) tumors in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice (see Methods). The graph shows the percentage of tumors that are classified as AT2 marker high and low. Low = 0–70%, high = 70–100%. (j) Percentage tdTomato+ cancer cells positive for SPC in individual (n = 100 [KFT], 100 [KT], and 200 [KPT]) tumors in KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Red lines; break points for high and low staining. (k) Representative IF of AGER, SPC, and tdTomato in 10-week KFT (n = 3), KT (n = 3), and KPT (n = 3) mice. Arrowheads indicate AGER+ cells in KPT mice that co-stain with SPC. Scale bar, 10 μm. (l) Quantification of SPC-positivity of AGER+ cancer cells (n = 150) in 10-week KFT (n = 3), KT (n = 3), and KPT (n = 3) mice. The percentage of AGER+tdTomato+ cells that co-stained with SPC were quantified. All data are mean ± s.d. P values were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test (d, j).
Extended Data Fig. 7 p53 directly promotes AT1 identity in vitro and in vivo.
(a) PDPN quantification as a percentage of p53+ (n = 600) cells from p5353,54 (n = 3) and p53wt (n = 3) LUAD cell lines or as a percent of DAPI+ (n = 600) cells in p53null (n = 3) LUAD cell lines. (b) Representative IF of PDPN and p53 from p5353,54 (n = 3), p53wt (n = 3), and p53null (n = 3) LUAD lines. Scale bar, 20 μm. (c) GSEA shows enrichment of AT1 cell signatures (Supplementary Table 6) in p53wt LUAD cells. NES > 0 indicates the signature is enriched in p53wt cells. *Indicates gene set is significantly enriched (P ≤ 0.05). (d) GSEA shows enrichment of alveolar signatures in specific genotypes. (e) Schematic for in vivo p53 restoration experiments. (f) Representative IF of PDPN and p53 from p5353,54-restored (n = 3) and p53wt-restored (n = 3) mice. Scale bar, 20 μm. (g) PDPN quantification as a percentage of p53+CYTOKERATIN (CK)+ tumors (n = 150) from p5353,54-restored (n = 3) and p53wt-restored (n = 3) mice or as a percent of CK+ tumors (n = 150) from p53null (n = 3) mice. (h) Representative IF of PDPN, p53, and CK in tumors from p5353,54-restored (n = 3), p53wt-restored (n = 3), and p53null (n = 3) mice. Scale bar, 20 μm. (i) GSEA shows p53 restoration induces AT1-associated gene expression profiles (Supplementary Table 6). Gene expression data30 comprised p53null (n = 8) and p53restored (n = 10) lung tumor samples. NES > 0 indicates signature enrichment in p53restored cells. *Indicates gene sets significantly enriched (P ≤ 0.05). (j) Percentage of genes in each AT1 signature that fulfill the stated conditions. The P values indicate the enrichment of p53 bound genes31 in each signature. (k) ChIP-seq31 tracks show p53 binding at AT1-associated genes. All data are mean ± s.d. P values were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test (a, g), by GSEA52 (c, d, i), and ChIP enrichment analysis57 (j).
Extended Data Fig. 8 p53 promotes AT1-like identity in human LUAD.
(a) (Left) AT1 marker expression in human (left) TP53WT (n = 249) and TP53MUT (n = 280) LUADs (TCGA), (middle) TP53WT (n = 78) and TP53MUT (n = 38) LUADs (GSE13213), and (right) TP53WT (n = 34) and TP53MUT (n = 73) lung squamous cell carcinomas (LUSC, E-MTAB-8615). Center line, median; boxes, interquartile range (IQR); whiskers, 25% quantile – 1.5*IQR & 75% quantile + 1.5*IQR; points, outliers. (b) Representative IF of AGER, HT1-56, and SPC in (n = 3) normal human lung samples. Scale bar, 50 μm. (c) Representative IF of HT1-56 and CYTOKERATIN (CK, cancer cells) in TP53WT (n = 8) and TP53MUT (n = 5) human LUADs. Scale bar, 5 μm. (d) Percentage of CK+ (n = 1,300) cells positive for HT1-56 in TP53WT (n = 8) and TP53MUT (n = 5) human LUADs. (e) Representative IF of SPC and CK in TP53WT (n = 5) and TP53MUT (n = 5) human LUADs. Scale bar, 5 μm. (f) Percentage of CK+ (n = 1,000) cells positive for SPC in TP53WT (n = 5) and TP53MUT (n = 5) human LUADs. (g) Representative IF of AGER, HT1-56, and CK in TP53WT (n = 8) and TP53MUT (n = 5) human LUADs. Scale bar, 20 μm. (h) Representative IF of CLDN4 and CK in TP53WT (n = 5) and TP53MUT (n = 5) human LUADs. Scale bar, 5 μm. (i) Percentage CK+ (n = 1,000) cells positive for CLDN4+ in TP53WT (n = 5) and TP53MUT (n = 5) human LUADs. (j) Representative IF of AGER and p53 in TP53knockout (KO) A549 cells transfected with an empty vector or a P53 expression vector (n = 3). Scale bar, 20 μm. (k) AGER quantification as a percentage of p53+ (n = 300) cells in (n = 3) A549 lines transfected with P53 or as a percentage of DAPI+ (n = 300) cells in (n = 3) A549 lines transfected with empty vector. All bar graphs are mean ± s.d. P values were calculated by two-way ANOVA test (a, see Methods) and two-tailed Student’s t-test (d, f, i, k).
Extended Data Fig. 9 p53 is activated in transitional and AT1-like cells.
(a) t-distributed stochastic neighbor embedding (tSNE) plot of (n = 3,891) cells29 across all clusters across all genotypes (KT [n = 1,071], KPT [n = 2,614], and normal AT2 [T, n = 206] cells) and all timepoints. (b) t-SNE plots of cancer cells split by both genotype and timepoint. (c) Proportion of cells in each cluster split by genotype and timepoint. (d) Heatmap showing expression of AT2, transitional (cluster 5), and AT1 cell markers (log-transformed TPMs rescaled across all cells) in all cells in (a). (e) (left) t-SNE plot of 2-week KT cells from the scRNA-sequencing dataset29 color-coded by cluster shows the predominance of clusters 1 and 2, which resemble AT2 cells. (Right) Feature plots showing the expression of AT2 markers (Sftpc and Sftpd), p53, and a panel of p53 target genes in 2-week KT cells. (f) Dot plot showing the expression of transitional (cluster 5) and AT1 cell markers in all cells in (a) split by genotype (T, KT, and KPT). (g) Relative proportions of cluster 3 and 5 cells in KT and KPT tumors. (h) Representative IF of CLDN4 and tdTomato in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bars, 50 μm (top) & 20 μm (bottom). (i) Quantification of CLDN4+ (n = 100 [KFT], 100 [KT], and 200 [KPT]) tumors (see Methods) in 10-week and 24-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Data are mean ± s.d. (j) t-SNE plots split by genotype shown in Fig. 3a with the cluster 5 sub-clusters color-coded. (k) Relative composition of subclusters 5A, 5B, and 5C split by genotype and timepoint. (l) GSEA shows enrichment of an injury-induced transitional cell signature (Supplementary Table 6) in KPT mice. P values were calculated by GSEA52 (l).
Extended Data Fig. 10 p53 restricts transitional cancer cell formation and promotes their differentiation into AT1-like cells.
(a) Expanded view from Fig. 3g of p19ARF, CLDN4 and tdTomato in tumors from 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bars, 20 μm. (b) Quantification of p19ARF staining in (n = 100 [KFT], 100 [KT], and 200 [KPT]) CLDN4+ cancer cells in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. (c) Expanded view from Fig. 3g of HNF4A and CLDN4 in tumors from 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bars, 20 μm. (d) Quantification of HNF4A staining in (n = 100 [KFT], 100 [KT], and 200 [KPT]) CLDN4+ cancer cells in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. (e) Expanded view from Fig. 3g of phosphorylated-ERK (pERK) and CLDN4 in tumors from 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. Scale bars, 20 μm. (f) Quantification of pERK staining in (n = 100 [KFT], 100 [KT], and 200 [KPT]) CLDN4+ cancer cells in 10-week KFT (n = 4), KT (n = 3), and KPT (n = 3) mice. (g) Representative IF of CLDN4, p19ARF, HNF4A, and pERK in tumors from 10-week KPT (n = 3) mice. Scale bar, 20 μm. (h) Copy number variations (CNVs) (red, amplifications; blue, deletions) inferred from the scRNA-sequencing data. (i) (Left) Frequency of CNVs across tumor progression or (right) across clusters. (j) Partition based graph abstraction of indicated clusters. (k) Cell-cell transitions estimated by CellRank based on pseudotime. (l) Scatter plots displaying the expression trend of programs identified in Fig. 3d. (m) Expression of cluster 3 markers29 in cluster 5 cells. Center line is the median with the upper and lower quartiles shown. Bar graphs are mean ± s.d. P values were calculated by two-tailed Pearson correlation test (df = 851) (l) and two-tailed Mann Whitney U test (m).
Extended Data Fig. 11 p53 promotes AT1 differentiation after lung injury.
(a) (Left) Representative IF of AGER and p53 in p53LSL-wt/LSL-wt transitional (TIGIT+)29 LUAD cells 1 and 3 days after Ad-Empty (p53null) or Ad-Cre (p53wt) treatment. Scale bar, 20 μm. (Right) AGER quantification as a percent of DAPI+ (n = 200) p53null cells or p53+ (n = 200) p53wt cells 1 (top) and 3 (bottom) days post-adenoviral treatment. (b) Plot from Fig. 3h depicting an expression score of transitional cell markers12. (c) Transitional cell marker12 induction across cancer clusters. (d) Enriched gene sets in cluster 5 markers29 identified by Enrichr58,59,60. (e) (Left) tdTomato+ (n = 600) cells that are SPC+ in control SpcT (n = 3) and SpcPT (n = 3) mice. (Right) SPC+ (n = 600) cells that are tdTomato+ in the same cohort. (f) Representative IF of SPC and tdTomato in control SpcT (n = 3) and SpcPT (n = 3) mice. Scale bar, 20 μm. (g) Expanded view of Fig. 4c, d of AGER/tdTomato or CLDN4/tdTomato in 4-day BHT-treated SpcFT (n = 3), SpcT (n = 3), and SpcPT (n = 3) mice. Scale bar, 20 μm. (h) Representative IF of BrdU and SPC in 4-day BHT-treated SpcFT (n = 3), SpcT (n = 3), and SpcPT (n = 3) mice. Scale bar, 20 μm. (i) tdTomato+ (n = 600) cells that are (left) AGER+, (left-middle) CLDN4+, or (right-middle) SPC+ in 2-day BHT-treated SpcT (n = 3) and SpcPT (n = 3) mice. (Right) Proliferating (BrdU+) SPC+ (n = 600) cells in the same cohort. (j) tdTomato+ (n = 600) cells that are SPC+ in 4-day BHT-treated SpcFT (n = 3), SpcT (n = 3), and SpcPT (n = 3) mice. (k) Representative IF of SPC and tdTomato in 4-day BHT-treated SpcFT (n = 3), SpcT (n = 3), and SpcPT (n = 3) mice. Scale bar, 20 μm. (l) ChIP-sequencing14 tracks of p53 binding at AT1 genes in transitional cells after injury. Data are mean ± s.d. P values were calculated by two-tailed Student’s t-test (e, i) and ordinary one-way ANOVA with Tukey’s multiple comparisons test (j).
Extended Data Fig. 12 p53 promotes alveolar lineage fidelity during lung injury repair.
(a) Expression of the top 100 markers (by P-adjusted value) for each cluster. (Right) Representative genes. (b) Expression of indicated marker/signatures (Supplementary Table 6) across clusters. (c) UMAP from Fig. 4g colored by pseudotime. (d) UMAP from Fig. 4g colored by CytoTRACE score. (e) p53 pathway signature expression (Supplementary Table 6) across clusters. (f) (Left) UMAP from Fig. 4g split by genotype. (Middle) p53 expression across clusters. (Right) p53 pathway signature expression (Supplementary Table 6). (g) Overlap of p53 and p53 target gene expression from (f). (h) (Top-left) Expression of Dclk1 or (right, bottom) liver and LUAD cluster (cluster 5B/C, 10, 11, or 12) programs (Supplementary Table 6) in SpcT (n = 332) and SpcPT (n = 189) transitional cells from BHT injury scRNA-sequencing data. (i) Top enriched Hallmark gene sets58,59,60 in genes upregulated in SpcPT compared to SpcT transitional cells. (j) CLDN4+tdTomato+ (n = 100) cells that are VIMENTIN-positive in 4-day BHT-treated SpcT (n = 3) and SpcPT (n = 3) mice. (k) Expanded view from Fig. 4k of CLDN4, VIMENTIN, and tdTomato in 4-day BHT-treated SpcT (n = 3) and SpcPT (n = 3) mice. Scale bar, 20 μm. (l) SpcPT transitional cell signature (Supplementary Table 6) expression in scRNA-sequencing data29 from KT (n = 1,071) and KPT (n = 2,614) LUAD cells. (m) Representative IF of CLDN4 and VIMENTIN in tumors from 10-week KT (n = 3) and KPT (n = 3) mice. Scale bar, 5 μm. (n) Expanded view of (m). Scale bar, 20 μm. (o) CLDN4-positive (n = 100) tumor cells that are VIMENTIN-positive in 10-week KT (n = 3) and KPT (n = 3) mice. Bar graphs are mean ± s.d. Box plots: center line, median; box limits, upper/lower quartiles; whiskers, 2.5–97.5 percentile; points, outliers. P values were calculated by two-tailed non-parametric Wilcoxon rank sum test (h, bar graph) and two-tailed Student’s t-test (h [box plots], j, l, o).
Supplementary information
Supplementary Figures
This file contains two supplementary figures. Supplementary Fig. 1 has three panels showing uncropped western blot gels and a legend. Supplementary Fig. 2 has six panels showing FACS plots and a legend.
Supplementary Table 1
Differentially expressed genes between KT cells and KPT cells (sheet 1) and between KFT cells and KPT cells (sheet 2).
Supplementary Table 2
Functional enrichment analysis showing the GO Biological Process terms enriched in genes hyperactivated in KFT cells.
Supplementary Table 3
Functional enrichment analysis showing the GO Biological Process terms enriched in KT upregulated genes.
Supplementary Table 4
Functional enrichment analysis showing the GO Biological Process terms enriched in KPT upregulated genes.
Supplementary Table 5
Differentially accessible regions in KT cells and KPT cells (sheet 1) and KT cells and KFT cells (sheet 2).
Supplementary Table 6
Custom gene signatures used in GSEA and other analyses.
Supplementary Table 7
Human LUAD sample characterizations.
Supplementary Table 8
Genes comprising each signalling programme in cluster 5 (sheets 1–3).
Supplementary Table 9
Lung injury scoring parameters (sheet 1) and histology scoring of SpcT and SpcPT mice (sheet 2).
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Kaiser, A.M., Gatto, A., Hanson, K.J. et al. p53 governs an AT1 differentiation programme in lung cancer suppression. Nature 619, 851–859 (2023). https://doi.org/10.1038/s41586-023-06253-8
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DOI: https://doi.org/10.1038/s41586-023-06253-8
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