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Early TP53 alterations engage environmental exposures to promote gastric premalignancy in an integrative mouse model

Nature Genetics volume 52, pages 219–230 (2020)Cite this article

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Abstract

Somatic alterations in cancer genes are being detected in normal and premalignant tissue, thus placing greater emphasis on gene–environment interactions that enable disease phenotypes. By combining early genetic alterations with disease-relevant exposures, we developed an integrative mouse model to study gastric premalignancy. Deletion of Trp53 in gastric cells confers a selective advantage and promotes the development of dysplasia in the setting of dietary carcinogens. Organoid derivation from dysplastic lesions facilitated genomic, transcriptional and functional evaluation of gastric premalignancy. Cell cycle regulators, most notably Cdkn2a, were upregulated by p53 inactivation in gastric premalignancy, serving as a barrier to disease progression. Co-deletion of Cdkn2a and Trp53 in dysplastic gastric organoids promoted cancer phenotypes but also induced replication stress, exposing a susceptibility to DNA damage response inhibitors. These findings demonstrate the utility of mouse models that integrate genomic alterations with relevant exposures and highlight the importance of gene–environment interactions in shaping the premalignant state.

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Fig. 1: Lgr5-p53KO promotes dysplastic and metaplastic premalignant lesions in the setting of dietary carcinogen exposure.
Fig. 2: p53KO human non-dysplastic premalignant cells have a selective advantage in the setting of MNU exposure.
Fig. 3: p53KO murine gastric organoids have a competitive advantage in the setting of dietary carcinogenesis.
Fig. 4: Dysplastic and metaplastic Lgr5-p53KO gastric organoids demonstrate genome doubling and capacity to transform.
Fig. 5: Dysregulation of inflammatory, stem cell and cell cycle pathways in dysplastic Lgr5-p53KO gastric organoids.
Fig. 6: CDKN2A/p16 is upregulated in Lgr5-p53KO premalignant gastric lesions and co-altered with TP53 in a subset of human gastric cancers.
Fig. 7: Co-deletion of CDKN2A/p16 and TP53 promotes gastric premalignancy, induces replication stress and sensitizes to DDR pathway blockade.

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Data availability

All requests for raw and analyzed data and materials should be directed to the corresponding authors. Data and materials that can be shared will be released via a material transfer agreement. Raw and processed RNA sequencing data have been deposited online with the accession number GSE141625. Raw genome sequencing data have been deposited online with the accession numbers PRJNA594147 and PRJNA594086. A list of significantly mutated genes from WES is available in Supplementary Table 1. Source data for Figs. 2, 5 and 7 and Extended Data Figs. 3 and 4 are presented with the paper.

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Acknowledgements

We thank A. Sperling, M. Korpal, A. Rustgi and S. Ganesan for insightful discussions and review of the manuscript; H. Singh, D. Micalizzi, D. Liu and A. Nagaraja for insightful discussions; S. Wang and the BWH CytoGenomics Core for assistance with karyotype analysis; A. Gad and L.-H. Ang for assistance with immunohistochemistry and immunofluorescence assays; the Dana-Farber/Harvard Cancer Center in Boston, MA, for the use of the Specialized Histopathology Core, which provided histology and immunohistochemistry services; Harvard Digestive Disease Center and NIH grant P30DK034854 for core services, resources, technology and expertise; the Center for Cancer Genome Discovery for their assistance and expertise in WES of murine tissue; the Dana-Farber/Harvard Cancer Center is supported in part by NCI Cancer Center Support Grant no. NIH 5 P30 CA06516. This work was funded by grants from the National Cancer Institute (P01 CA098101 and U54 CA1630004) to A.J.B. and the American Cancer Society Postdoctoral Fellowship, a KL2/CMERIT Harvard Catalyst Award, the Perry S. Levy Fund for Gastrointestinal Cancer Research, and NIH K08-DK120930 to N.S.S.

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  1. These authors contributed equally: Osamu Kikuchi, Gina Duronio.

Authors and Affiliations

  1. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Nilay S. Sethi, Osamu Kikuchi, Gina N. Duronio, Matthew D. Stachler, Ruben Ferrer-Luna, Yanxi Zhang, Chunyang Bao, Jie-Bin Liu, Keith L. Ligon, Rameen Beroukhim & Adam J. Bass

  2. Gastrointestinal Cancer Treatment Center, Dana-Farber Cancer Institute, Boston, MA, USA

    Nilay S. Sethi & Adam J. Bass

  3. Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA

    Nilay S. Sethi, James M. McFarland, Ruben Ferrer-Luna, Keith L. Ligon, Rameen Beroukhim & Adam J. Bass

  4. Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA

    Matthew D. Stachler & Deepa Patil

  5. Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA, USA

    Matthew D. Stachler, Ewa Sicinska & Keith L. Ligon

  6. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Roderick Bronson

  7. Department of Surgery and Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Francisco Sanchez-Vega

  8. Center for DNA Damage and Repair, Dana-Farber Cancer Institute, Boston, MA, USA

    Jean-Bernard Lazaro

  9. Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Jean-Bernard Lazaro

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Contributions

M.D.S. and J.M.M. contributed equally to the manuscript as joint third authors. N.S.S. and A.J.B conceived and designed the study. N.S.S. and A.J.B. developed the methodology. N.S.S., O.K. and Y.Z. performed the mouse experiments. M.D.S., R. Bronson, D.P. and E.S. reviewed and analyzed histopathology. N.S.S., J.M.M., C.B. and F.S.-V. performed transcriptomic and genomic analyses. R.F.-L., K.L.L. and R. Beroukhim performed and supervised pharmacogenomic analyses. N.S.S., O.K., G.D. and J.-B. Lazaro performed and analyzed DNA damage response and other experiments. J.-B. Liu contributed to resources. N.S.S., O.K., G.D. and A.J.B. performed the formal analysis. N.S.S. and A.J.B. wrote the original draft. N.S.S., O.K., G.D., M.D.S., J.M.M. and A.J.B. edited the manuscipt. N.S.S. and A.J.B. supervised the study. N.S.S. and A.J.B acquired funding for the study.

Corresponding authors

Correspondence to Nilay S. Sethi or Adam J. Bass.

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Competing interests

N.S.S. is a consultant for HVH Precision Analytics. A.J.B. receives funding from Merck, Bayer and Novartis, and is an advisor to Earli and Helix Nano and a co-founder of Signet Therapeutics.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Dietary carcinogen exposure promotes premalignant and malignant gastric lesions.

a Schematic of the central hypothesis: Chronic inflammation and carcinogen exposure collaborate with early genomic alterations (for example TP53 mutations) to enable the development of premalignant gastric lesions and eventual invasive cancer. b, Schematic of p53WT mice treated with DCA, MNU, or DCA+MNU combination for 18 months. c, Dissection microscope images of lower esophagus and stomach flayed open along greater curvature (top panel) and histopathological H&E staining (bottom panel) of gastric antrum. *A subset of mice died before the endpoint of the experiment due to other carcinogen-induced cancers (e.g. thymomas). Scale bar = 125μM. d, Mutation signature analysis shows C→T changes characteristic of alkylating agent associated Signature 11. center line: median, lower hinge: the first quartile (Q1), upper hinge: the third quartile (Q3), extreme of the lower whisker: Q1 – 1.58 * (Q3 – Q1)/sqrt(n), extreme of the upper whisker: Q3 + 1.58 * (Q3 – Q1)/sqrt(n), n = 5. e, Mutation burden and copy number analysis of gastric lesions (n=5).

Extended Data Fig. 2 Lgr5-p53KO cells have a selective advantage in the setting of dietary carcinogen to promote premalignant gastric lesions.

a, Recombination specific PCR of DNA extract from gastric lesions of Lgr5-p53WT and Lgr5-p53KO mice. Data presented as mean ± s.d of three technical replicates. b, Schematic showing Lgr5-p53LSL-R270H experimental design; DCA/MNU treatment and tamoxifen injection schedule during indicated duration depicted below. c, Kaplan-Meier survival curve of Lgr5-p53R270H experiments; table shows frequency of dysplasia in DCA/MNU treated mice of indicated genotype. d, Schematic showing Lgr5-p53KO experimental design; DCA alone, MNU alone, or DCA/MNU combination as well as tamoxifen injection schedule during indicated duration depicted below. Table shows frequency of dysplasia in Lgr5-p53KO mice with indicated treatment after 12 months. *1/6 Cre-negative, tamoxifen-induced control mice treated with DCA/MNU developed dysplasia.

Extended Data Fig. 3 TP53-deleted human premalignant cells have a selective growth advantage in the setting of MNU.

a, Relative proliferation of CP-A cells treated with indicated concentrations of DCA; phase contrast images of CP-A cells treated with DMSO or 100μM DCA. b, Immunoblot showing protein levels of p53 in CP-A cells treated with DMSO or 50μM DCA. This experiment was repeated once with similar results. c, DNA content flow cytometry of CP-A cells expressing control orTP53 shRNA#2 CP-A cells treated with indicated MNU concentrations. This experiment was repeated once with similar results. d, Relative proliferation of CP-A cells treated with DMSO or indicated concentrations of MNU by Celltiter Glo. Data presented as mean ± s.d of three culture replicates; p-value calculated by two-sided Student’s t-test. e, Immunoblot showing protein levels of p53 in genetically modified CP-A cells; top panel shows protein from CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNAs/Cas9 after treatment with MNU 200ng/mL; bottom panel shows protein from CP-A cells expressing a scrambled or two targeting p53 shRNAs treated with DMSO, Doxorubicin, or Nutlin in the presence or absence of doxycycline. This experiment was repeated once. f, Relative proliferation of CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNA/Cas9 (top panel); expressing vector control, scramble control, or two p53 targeting shRNAs. Data presented as mean ± s.d of three culture replicates. g, Quantification of dsDNA breaks in CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNA/Cas9 in the setting of indicated concentrations of MNU by γH2AX immunofluorescence. Data presented as mean ± s.d of four technical and two cell culture replicates; p-value calculated by two-sided Student’s t-test of cell culture replicates. h, FPKM gene counts of TP53 in CP-A cells stably expressing two p53 shRNAs. i, Ratio of TP53 pathway target gene expression levels in CP-A cells expressing indicated p53 shRNA relative to scramble control (cell culture replicates shown).

Source data

Extended Data Fig. 4 p53R270H/+ organoids have a selective growth advantage in the setting of dietary carcinogen exposure.

a, Immunoblot showing expression of mutant p53R270H in whole cell lysates derived from p53LSL-R270H/+ and p53LSL-R270H/R270H gastric organoids with or without AdenoCre induction; immunofluorescent images of p53LSL-R270H/+;mTmG gastric organoids before and after AdenoCre induction. Organoids without induction remain red indicating that recombination has not occurred, whereas those with AdenoCre induction convert to green indicating that recombination has occurred. This experiment was repeated once with similar results. b, Flow cytometry of eGFP+ Mist1-p53R270H/+ and td+ Mist1-p53+/- organoids cultured in DMSO or 50ng/mL MNU. Quantification of eGFP+ Mist1-p53R270H/+ and td+ Mist1-p53+/- organoids at passage 1 (P1) and 3 (P3). c, Phase contrast and immunofluorescent images of eGFP+ Mist1-p53R270H/+ and td+ Mist1-p53+/- organoids cultured in DMSO or 50ng/mL MNU. This experiment was repeated twice with similar results. d, Schematic depicting competitive growth advantage of p53-altered gastric organoids in the setting of MNU.

Source data

Extended Data Fig. 5 Dysplastic Lgr5-p53KO gastric organoids capture properties of premalignant lesions.

a, Recombination specific PCR of DNA extract from premalignant dys-Lgr5-p53WT and dys-Lgr5-p53KO gastric organoids. Data presented as mean ± s.d of n = 3 technical replicates per group. b, Quantification of proliferation by CellTiter-Glo and phase contrast images of nondysplastic and dysplastic Lgr5-p53WT and Lgr5-p53KO gastric organoids in the presence or absence of Nutlin-3 (30 uM for 72 hours). Data presented as mean ± s.d. of n = 6 cell culture replicates. c, Representative images of phase contrast and GFP-immunofluorescent images from dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoid cultures. The experiment was repeated once with similar results. d, Karyotype analysis of premalignant Lgr5-p53KO (n = 2 mice), Lgr5-p53WT (n = 2 mice), and Lgr5-p53WT + AdenoCre gastric organoids; chromosome count of 10 cells per group. Data presented as mean ± s.d.; p-values calculated by Ordinary One-way ANOVA with Sidak’s multiple comparisons adjustment. e, Copy number analysis of a patient specimen of a high-grade dysplastic Barrett’s metaplasia harboring TP53R175H mutation and genome doubling. f, Histopathology of primary tumor xenograft of Lgr5-p53KO gastric organoid showing features of dysplasia. This experiment was repeated twice with similar results. g, DAPI staining and GFP (Lgr5+) immunofluorescence of primary xenograft from Lgr5-p53KO gastric organoids. This experiment was repeated twice with similar results. h, Schematic of low-pass whole genome sequencing (LP-WGS) experiment of cultured and xenograft dys-Lgr5-p53KO gastric organoids. Common and distinct broad somatic copy number alterations that were only found in xenograft tissue are displayed.

Extended Data Fig. 6 Interferon signaling is upregulated in dysplastic Lgr5-p53KO gastric organoids.

a, mRNA expression of Trp53 in p53KO, p53WT, Lgr5-p53KO, Lgr5-p53WT, dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids. Data presented as mean ± s.d. of n = 2 (nondysplastic) and n = 3 (dysplastic) cell culture replicates. b, mRNA expression levels of Csf3, Cxcl10, and Ccl5 in p53KO, p53WT, Lgr5-p53KO, Lgr5-p53WT, dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids by RT-PCR. Data presented as mean ± s.d. of n = 3 cell culture replicates. c, Volcano plot of differential expressed gene-sets in p53KO gastric organoids plotted as normalized enrichment scores (NES) by -log10(p-value), where p-values for gene set enrichment scores were determined by randomly permuting genes (n=100,000 permutations). Individual inflammation gene sets are described with corresponding NES. d, Scatter plot of single sample GSEA (ssGSEA) of interferon pathway and Ccl5 mRNA expression in gastric cancer cell lines annotated by TP53 mutation status (n = 26 for TP53 mutant and n = 10 for TP53 wildtype). Gray shaded region represents 95% confidence interval of the linear regression fit. Pearson’s correlation value R = 0.6 and p-value computed based on a two-sided t-distribution. e, Schematic of proposed vicious feedback cycle in gastric premalignancy: chronic inflammation and carcinogen exposure selects for p53 mutant gastric cells, which in turn stimulate inflammatory cytokines, particularly Csf3, Cxcl10, Ccl5.

Extended Data Fig. 7 WNT pathway is upregulated in dysplastic Lgr5-p53KO gastric organoids.

a, WNT-reporter activity in adherent culture of dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric cells following transient transfection. Data presented as mean ± s.d. of n = 4 cell culture replicates; p-value calculated by two-sided Mann Whitney test. b, Phase contrast images of dys-Lgr5-p53KO and dys-Lgr5-p53WT gastric organoids two days following the first and second passage in WNT independent media (DMEM + 10%FBS). This experiment was repeated twice with similar results. c, Gene network of significantly mutated genes in dysplastic gastric lesions from DCA/MNU-treated Lgr5-p53WT mice (n = 8) using GeNets (adjusted p-value = 0.00065 using Bonferroni correction) (Li et al., 2018).

Extended Data Fig. 8 CDKN2A is upregulated in p53KO premalignant lesions and co-altered with TP53 in human gastric cancer.

a, Scatter plot of single sample GSEA (ssGSEA) of p53 pathway and CDKN2A mRNA expression in gastric cancer cell lines (n = 36). Gray shaded region represents 95% confidence interval of the linear regression fit. Pearson’s correlation value R = −0.36 and p-value computed based on a two-sided t-distribution. b, Heatmap showing mRNA expression of CDKN2B, CDKN2C, CDKN2D, CDKN1B, CDKN1C in CP-A cell line genetically engineered to express two p53 targeting shRNA under doxycycline inducible conditions. c, Oncoporint from cbioportal showing alterations and mRNA expression in CDKN2A and TP53 in human gastric adenocarcinomas from TCGA (n = 478). Deep deletions (solid blue); amplifications (solid red); missense mutations in the COSMIC repository (solid green); nonsense or frameshift mutations (black); elevated mRNA expression > 2.0 (hollow red). Table showing number of cases with double, single, or no alteration in CDKN2A and TP53 in human gastric adenocarcinomas from TCGA (right). d, Oncoprint from cbioportal showing alterations in CDKN2A and TP53 in human esophageal adenocarcinomas from TCGA and (Dulak et al., 2013) combined data-set (n = 337). Deep deletions (solid blue); amplifications (solid red); missense mutations in the COSMIC repository (solid green); nonsense or frameshift mutations (black). Table showing number of cases with double, single, or no alteration in CDKN2A and TP53 in combined esophageal adenocarcinoma data set. e, Table showing number of cases with double, single, or no alteration in CDKN2A and TP53 in combined gastric and esophageal adenocarcinomas from patients treated at DFCI (n = 1299). Q-value determined by one-sided Fischer Exact Test with Benjamin-Hochberg FDR correction.

Extended Data Fig. 9 Co-deletion of CDKN2A and TP53 sensitizes to DNA damage response pathway blockade.

a, Dose-response curves of CHK1 inhibitor AZD7762 of two gastric cancer cell lines wildtype for either TP53 or CDKN2A and two gastric cancer cell lines with co-alteration of TP53 and CDKN2A. b, Relative dependency of 23 gastric cancer cell lines to CHEK1, CHEK2, and WEE1 shRNA-mediated knockdown as shown by CERES dependency score. Black rectangles indicate gastric cancer cell lines with simultaneous disruption of TP53 and CDKN2A. c, Dose-response curve of KE39 (TP53MUT, CDKN2AWT) and GSU (TP53MUT, CDKN2AMUT) gastric cancer cell lines to Prexasertib. Data presented as mean ± s.d. of n = 6 cell culture replicates at each dose; p-value calculated by Comparison of Fits based on differences in IC50. d, Dose-response curve of KE39 (TP53MUT, CDKN2AWT) and GSU (TP53MUT, CDKN2AMUT) gastric cancer cell lines to WEE1 inhibitor AZD1775. Data presented as mean ± s.d. of n = 6 cell culture replicates at each dose; p-value calculated by Comparison of Fits based on differences in IC50. e, Quantification by manual counting of number of organoids in experiment from Fig. 7f, g. f, Quantification of organoid size and number from dys-Lgr5-p53KO-control and dys-Lgr5-p53WT-p16KO gastric organoids treated for 36 hours with DMSO or indicated concentration of ATR inhibitor AZD6738 or WEE1 inhibitor AZD1775. Data presented as mean ± s.d. of n = 6–8 technical replicates at each dose. g, Representative phase contrast images from experiment (f).

Supplementary information

Supplementary Information

Supplementary Notes 1–8 and Tables 2 and 3

Reporting Summary

Supplementary Table 1

A list of significantly mutated genes determined by WES of the indicated gastric lesions.

Source data

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 5

Unprocessed western blots.

Source Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 4

Unprocessed western blots.

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Sethi, N.S., Kikuchi, O., Duronio, G.N. et al. Early TP53 alterations engage environmental exposures to promote gastric premalignancy in an integrative mouse model. Nat Genet 52, 219–230 (2020). https://doi.org/10.1038/s41588-019-0574-9

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