Notch1 mutations drive clonal expansion in normal esophageal epithelium but impair tumor growth
Issuing time:2023-02-06 14:43
Abstract
NOTCH1mutant clones occupy the majority of normal human esophagus by middle age but are comparatively rare in esophageal cancers, suggestingNOTCH1mutations drive clonal expansion but impede carcinogenesis. Here we test this hypothesis. SequencingNOTCH1mutant clones in aging human esophagus reveals frequent biallelic mutations that blockNOTCH1signaling. In mouse esophagus, heterozygousNotch1mutation confers a competitive advantage over wild-type cells, an effect enhanced by loss of the second allele. WidespreadNotch1loss alters transcription but has minimal effects on the epithelial structure and cell dynamics. In a carcinogenesis model,Notch1mutations were less prevalent in tumors than normal epithelium. Deletion ofNotch1reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment.Notch1null tumors showed reduced proliferation. We conclude thatNotch1mutations in normal epithelium are beneficial as wild-typeNotch1favors tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.
Main
Aging tissues accumulate somatic mutations1,2,3,4. Some mutations confer a competitive advantage on progenitor cells, which may form mutant clones that colonize normal tissue. These clonal expansions are often associated with mutations linked to cancer and may represent the first step in malignant transformation4. However, the under-representation ofNOTCH1mutants in esophageal cancer compared with normal aging epithelium suggestsNOTCH1mutations may inhibit malignant transformation2,5.
NOTCH1 is a cell surface receptor composed of an extracellular domain (NEC) and a transmembrane and cytoplasmic subunit (NTM), interacting noncovalently through the negative regulatory region (NRR; Extended data Fig.1a)6,7. The NRR comprises three Lin12-Notch repeats (LNR) and a heterodimerization domain (HD) that inhibits NOTCH1 activation in the absence of ligand8. Ligands bind to conserved epidermal growth factor (EGF) repeats in the NEC. This results in proteolytic cleavage events releasing the intracellular domain (NICD), which translocates to the nucleus and alters target gene transcription8. In the esophagus, NOTCH1 protein is expressed in proliferating cells and regulates both development and adult tissue maintenance (Extended data Fig.1a,b)9.
Different studies have suggested thatNOTCH1is a tumor suppressor or conversely may promote esophageal carcinogenesis10,11,12. Here we investigate howNOTCH1mutants colonize the epithelium, their impact on tissue maintenance and their effect on esophageal carcinogenesis2,4.
NOTCH1 mutant clones in human esophagus
Deep targeted sequencing studies have revealed numerous NOTCH1 mutants in human esophagus but have not visualized clones and resolved which NOTCH1 mutation(s) or copy number alterations they carry2,4. To achieve this, histological sections of normal epithelium from elderly donors were immunostained for NOTCH1 (Fig. 1a). Positive and negative staining areas were microdissected and targeted sequencing for 322 genes associated with cancer was performed (Fig. 1b). A total of 247 protein-altering somatic variants were identified across 86 samples from six donors aged 43–78. The predominant mutant genes were NOTCH1, TP53 and NOTCH2 (refs. 2,4; Supplementary Tables 1–3 and Supplementary Note). Near clonal NOTCH1 mutations with an average variant allele frequency (VAF) of 0.36 were detected in 81% (70/86) of samples (Fig. 1c,d). Ninety-three percent (25/27) of negative staining areas carried nonsense, essential splice mutations or indels in NOTCH1 with copy neutral loss of heterozygosity (CNLOH) of the NOTCH1 locus (human GRCh37—chr9:139,388,896–139,440,238) or a further mutation, likely to disrupt the second NOTCH1 allele (Fig. 1d,e). Fifty-nine percent (35/59) of positively stained samples carried a missense NOTCH1 mutation and most of these had either CNLOH or a second mutation (Fig. 1d–f, Extended data Fig. 1c,d and Supplementary Table 4). Overall, most samples (73%, 51/70) had likely biallelic NOTCH1 alterations (Fig. 1f). To test if the mutations disrupted NOTCH1 function, we stained consecutive sections from additional donors for NOTCH1 protein and NICD1, which is detectable in the nucleus during active signaling (Fig. 1g,h, Extended data Fig. 1a and Supplementary Table 5)13. The proportion of epithelium with active NOTCH1 decreased with age (Kendall’s tau-b = −0.67, P = 0.014). In older donors, in whom NOTCH1 mutations are common, NOTCH1− areas were associated with NICD1 loss. We also found occasional NOTCH1+ NICD1− areas, consistent with the presence of missense mutant proteins that reach the cell membrane but lack signaling activity (Fig. 1g,h). NICD1+ and NICD1− areas were histologically undistinguishable, with no significant differences in tissue thickness, cell density or the expression of the proliferation marker Ki67 (Extended data Fig. 1e–h). We conclude that many NOTCH1 mutant clones in aging human esophagus carry biallelic alterations that disrupt signaling.
Fig. 1: NOTCH1 mutant clones in human esophageal epithelium.
a, Cyrosection of human esophagus. NOTCH1 (green) stains basal and lower suprabasal layer cells, expression is lost in regions of the esophagus. F-actin, magenta; Pa, papillae. Dotted line indicates epithelial submucosal boundary. Image representative of three donors. Scale bar, 100 µm. b, Protocol for c–f. Cryosections were stained for NOTCH1. Contiguous NOTCH1+ and NOTCH1− staining areas were microdissected and sequenced. c, Representative images from b for donor PD40290. NOTCH1 is red, DNA is blue. Upper labels show sample identification (Id) and NOTCH1 staining status (positive, + or negative, −) for each sample. Lower labels show nonsynonymous NOTCH1 mutations and VAF and indicate CNLOH if detected. Only mutations with VAF > 0.1 are displayed. Mutation effects are color coded (indel_splicing, gray; missense, blue; nonsense, red). Dashed lines delineate the epithelium and submucosa (white) and borders of sequenced samples (yellow). Solid lines separate the two images of the adjacent regions. Scale bars, 250 µm. d, Results from b, showing NOTCH1 staining, donor identification, NOTCH1 mutation calling, CNLOH affecting NOTCH1 locus and number of NOTCH1 mutations per sample (n = 86 samples from six donors aged 43–78 years). e, Proportion of missense, nonsense, indel/splicing or intronic/silent NOTCH1 mutations in NOTCH1+ and NOTCH1− samples. Number of NOTCH1 mutations for each group is shown in brackets. f, Proportion of NOTCH1 mutant samples carrying monoallelic or biallelic NOTCH1 alterations in each donor. ‘Biallelic with second mutation’ category includes samples without CNLOH, carrying at least two mutations with VAF ≥ 0.15. Numbers in brackets are total number of NOTCH1 mutated samples per donor. g, NOTCH1 (green, upper panel) and NICD1 (red, lower panel) staining in successive sections of epithelium from an aged donor. ITGA6 (magenta) marks the basal cells. DNA is blue. Inset shows basal and lower suprabasal cells (white rectangles). Dashed lines delineate staining pattern. Images representative of six middle-aged and elderly donors. Scale bar, 100 µm. h, Proportion of tissue positive or negative for NOTCH1 and NICD1 in donors aged 20–78 years (total section length 4774–17988 µm per donor, n = 9 donors). Id, identification. See Supplementary Tables 1–5.
Notch1 mutations increase clonal fitness
To investigate how NOTCH1 mutant clones colonize normal epithelium, we tracked the fate of Notch1 mutant clones in transgenic mice using lineage tracing. Mouse esophageal epithelium consists of layers of keratinocytes. Proliferation is restricted to progenitor cells in the basal layer (Extended data Fig. 2a). Differentiating cells cease dividing, leave the basal layer and migrate toward the epithelial surface where they are shed. Progenitor division is linked to the exit of a nearby differentiating cell from the basal layer, ensuring basal cell density is kept constant14. Dividing progenitors generate either two progenitor daughters, two differentiating daughters or one cell of each type. In wild-type tissue, the probabilities of each progenitor outcome are balanced, generating equal proportions of progenitor and differentiated cells, maintaining cellular homeostasis (Extended data Fig. 2a)15,16. Mutations that alter progenitor fate leading to excessive production of progenitors drive mutant clone growth17,18.
For lineage tracing, we generated AhCreERTRosa26floxedYFPNotch1floxtriple transgenic (YFPCreNotch1) mice. These animals carry a conditional Notch1 allele and a genetic labeling system. An inducible Cre recombinase (AhCreERT) was used to delete one or both conditional Notch1 alleles in Notch1wt/flox or Notch1flox/flox animals and induce a separate conditional yellow fluorescent protein (YFP) reporter allele (Rosa26floxedYFP)15,19. YFP was expressed in recombined epithelial cells and their progeny (Extended data Fig. 2b,c). This model was induced at low dose to recombine scattered single basal cells (clonal induction) or at a higher level to recombine a large proportion of basal cells (high induction) (Extended data Fig. 2c,d).
Excision of the Notch1 allele and expression of the YFP reporter at the Rosa26 locus can occur in combination or separately, resulting in Notch1 mutant or wild-type cells expressing YFP or not (Extended data Fig. 2c,d). We confirmed the recombination status of exon 1 of Notch1 of wild type and fully recombined Notch1+/− and Notch1−/− esophageal epithelium. Notch1 mRNA and protein expression was halved in Notch1+/− and abolished in Notch1−/− cells compared with wild-type keratinocytes (Extended data Fig. 3a–h and Supplementary Table 6). We then performed genetic lineage tracing by inducing recombination in scattered single progenitors in YFPCreNotch1+/+, YFPCreNotch1+/floxor YFPCreNotch1flox/flox mice. YFP-expressing clones were detected by imaging sheets of epithelium stained for YFP and NOTCH1 (Fig. 2a). YFP+Notch1+/− or YFP+Notch1−/− clones were identified from reduced intensity or absence of NOTCH1 immunostaining, respectively, a method validated by detecting Notch1 recombination in microdissected clones (Fig. 2b, Extended data Fig. 3i–n and Supplementary Note).
Fig. 2: Lineage tracing of Notch1 mutant clones.
a, Protocol. YFPCreNotch1+/+, YFPCreNotch1+/flox and YFPCreNotch1flox/flox mice were induced at clonal density. YFP+Notch1 wild type (+/+) and YFP+Notch1 mutant clones (+/− or −/−) were imaged at several time points. b, xy plane basal layer view at 4 weeks p.i. of wild type, Notch1+/− and Notch1−/− clones stained for NOTCH1, magenta, YFP, green and DNA, blue. White dashed lines delineate mutant clones. Scale bars: 30 µm. c,d, Basal (c) and suprabasal (d) cells per clone following induction of Notch1+/− (left panel) or Notch1−/− (right panel) compared to Notch1+/+ clones. Lines show median and quartiles. n mice (clones) for +/+ at 10 d, 2 weeks, 4 weeks, 9 weeks and 13 weeks, respectively: 3 (206)/ 3 (155)/ 3 (143)/ 3 (132)/ 3 (126). n mice (clones) for +/− at 10 d, 4 weeks, 9 weeks and 13 weeks, respectively: 5 (84)/4 (97)/4 (68)/7 (107). n mice (clones) for −/− at 10 d, 2 weeks, 4 weeks, respectively: 6 (68)/ 3 (69)/ 9 (63). Two-tailed Mann–Whitney test of mutant against +/+ at each time point. e, Protocol. YFPCreNotch1+/flox and flox/flox mice were clonally induced, and S phase cells labeled with EdU, 1 h precollection (red). f, EdU+ cells were counted inside clones (green), in wild-type cells adjacent to clones (orange) or distant from clones (beige). g,h, Ratio of EdU+: total basal cells in YFP+Notch1+/− (g) or Notch1−/− (h) mutant clones (YFP+; +/− or −/−), in wild type cells at clone edges (edge +/+) or distant from clones (distant +/+). (Mean ± s.e.m., each dot represents a mouse; g, n = 4830; 1584; 4607 cells in distant +/+; edge +/+; YFP+ +/− clones from four mice; h, n = 3967; 1036; 4279 cells in distant +/+; edge +/+; YFP+ −/− clones from four mice). One-way RM ANOVA; adjusted P values from Tukey’s multiple comparisons test against distant+/+. i, Protocol. Mice were clonally induced and EdU injected 48 h before collection. Labeled cells, red, reveal division outcomes. j, Z plane (side) views of projected confocal z stacks of YFP+Notch1+/− clone 13 weeks p.i. (left), and YFP+Notch1−/− clone 4 weeks (p.i. right) from (i). NOTCH1 (magenta); YFP (green); EdU (gray); DNA (blue). Yellow dashed lines show clone edges. Orange arrow shows differentiating cell adjacent to clone. Images representative of clones in 3 YFPCreNotch1+/flox and 5 YFPCreNotch1flox/flox mice. Scale bars: 30 µm. k,l, Protocol as in i. EdU+ suprabasal/total EdU+ cells in YFP+Notch1+/− (k), YFP+Notch1−/− (l) mutant clones (YFP+; +/− or −/−), in wild type cells at clone edges (edge +/+) or distant from (distant +/+) clones. (Mean ± s.e.m., each dot represents a mouse; k, n = 471; 300; 525 EdU+ cells in distant +/+; edge +/+; YFP+ +/− clones from three mice; l, n = 1304; 723; 1318 EdU+ cells in distant +/+; edge +/+; YFP+ −/− clones from five mice). One-way RM ANOVA; adjusted P values, Tukey’s multiple comparisons test against distant+/+. m,n, Basal cell density in mutant clones (+/− in m, −/− in n) and in respective distant wild-type areas (distant +/+). (Mean ± s.e.m., each dot represents a mouse. n = 3 mice in m, n = 6 mice in n). Two-tailed paired Student’s t-tests. o, Mechanism of Notch1 mutant clone expansion. Mutant cell divisions produce more progenitors than differentiating cells on average. Neighboring wild-type cells stratify at the edge of Notch1−/− mutant clones, allowing accelerated mutant clone expansion. P.i., postinduction. Nb, number. RM, repeated measures; w, weeks. See Supplementary Tables 7 and 8.
The number and location of cells in YFP-expressing clones of each genotype were determined by 3D confocal imaging. The size of YFP+Notch1+/− clones was substantially increased compared to wild-type YFP+Notch1+/+ clones at all time points. YFP+Notch1−/− clones were larger still (Fig. 2b–d, Extended data Fig. 3i,j and Supplementary Table 7). To examine the cellular mechanisms underlying mutant clonal expansion, we used short-term cell tracking by labeling cycling cells with the S phase probe 5-ethynyl-2′-deoxyuridine (EdU).
We first counted the proportion of basal cells positive for EdU at 1 h after labeling, which measures the fraction of cells in S phase (Fig. 2e,f). This value was similar for cells within Notch1+/− clones and wild-type cells distant from clones (Fig. 2g and Supplementary Table 8). Within Notch1−/− mutant clones, the proportion of EdU+ basal cells was marginally lower than in wild-type cells (Fig. 2h). We conclude neither Notch1+/− nor Notch1−/− clonal expansion results from an increase in mutant cell division rate compared with wild-type cells.
A 48 h EdU experiment labeled S phase cells and tracked the fate of the two cells generated by the subsequent mitosis over the following 48 h. The pair of labeled cells may remain in the basal layer, or one or both may differentiate and exit the basal layer (Fig. 2i,j). The ratio of EdU-labeled suprabasal cells to the total EdU-labeled cells reflects the rate of production of differentiating cells in the basal layer and their stratification into the suprabasal layers. In Notch1+/− and Notch1−/− clones, this ratio is decreased, consistent with a tilt in mutant progenitor cell fate, so that more progenitors and fewer differentiating daughters are produced per average cell division (Fig. 2k). Strikingly, adjacent to Notch1−/− clones, there was an increase in the suprabasal EdU+:total EdU+ cell ratio in the wild-type cells at the clone margin compared with wild-type cells further from the mutant clone (Fig. 2j,l). This, along with a small decrease in the proportion of wild-type S phase cells at the clone edge, indicates that wild-type cells adjacent to t
he clone exit the cell cycle, differentiate and exit the basal layer at an increased rate, a phenomenon also reported in previous studies of Notch inhibited keratinocytes interacting with wild type cells (Fig. 2h)18,20.
These observations explain the increased fitness of Notch1−/− over Notch1+/− clones. Cell density was similar in both mutant genotypes and wild-type areas, suggesting that the linkage between cell division and the exit of a nearby differentiating cell from the basal layer is maintained (Fig. 2m,n). Within this constraint, the driving of wild-type cell differentiation and stratification permits Notch1−/− cell division at the clone edge, accelerating clonal expansion (Fig. 2o).
These observations were integrated into a Wright–Fisher style quantitative model in which fit mutant clones expand until they collide with other mutant clones of similar fitness, at which point they revert to neutral competition21. We fitted this model to the clone size data. The inferred fitness for Notch1+/− clones was higher than that of wild-type cells and the inferred fitness of Notch1−/− clones markedly greater than that of heterozygous clones (Extended data Fig. 4a–d, Video 1 and Supplementary Note).
Clones generated by the transgenic deletion of Notch1 alleles may not reflect the behavior of Notch1 mutants that appear during aging. We therefore investigated spontaneous Notch1 mutant clones in control YFPCreNotch1+/+ mice, and the heterozygous epithelium of highly induced YFPCreNotch1+/flox animals. Both strains were aged before immunostaining the epithelium for NOTCH1 (Fig. 3a). The area of epithelium stained negative for NOTCH1 increased progressively to 12% of Notch1+/+ and 78% of Notch1+/− epithelium by 65 weeks (Fig. 3b,c and Supplementary Table 9). Widespread loss of NICD1 staining was seen in aged Notch1+/− tissue (Extended data Fig. 5a,b). These observations suggest that, as in humans, Notch1 mutants colonize the aging mouse esophagus and that selection is enhanced in Notch1+/− epithelium.
a, YFPCreNotch1+/flox and YFPCreNotch1+/+ mice were induced at a high level and aged for 65 weeks. b, Representative NOTCH1 staining in esophageal epithelium of aging YFPCreNotch1+/+ and YFPCreNotch1+/flox mice at the indicated time points. White dashed lines delineate negative areas and solid lines delineate tissue edges. Images representative of three mice per time point. Scale bars: 500 µm c, Percentage of NOTCH1− area increases with age in Notch1+/+ (Kendall’s tau-b correlation = 0.56, P = 0.0062) and Notch1+/− (Kendall’s tau-b correlation = 0.91, P = 8.3 × 10−6) esophagi (Mean ± s.e.m., n = 3 mice per time point). P values shown are from two-sided Welch’s t test. d, Schematic of Notch1+/− cells (purple cells) showing the spontaneous appearance of expanding NOTCH1− cells (black) with aging, possibly caused by genetic events affecting the Notch1 locus. e, Highly induced YFPCreNotch1+/flox mice were aged 54–78 weeks old, when esophageal epithelium was collected and stained for NOTCH1 (magenta), YFP (green) and DNA (blue). Expanding areas devoid or fully stained with YFP appeared distinct from normal-appearing areas marked with a patchwork of small YFP+ clones. Expanded NOTCH1− (yellow) and NOTCH1+ (orange) areas and normal-appearing areas (blue) were isolated for targeted sequencing (n = 246 biopsies from ten mice). Colored circles show the sampled areas. White dashed lines delineate negative areas. Scale bars: 500 µm. f, Proportion of normal appearing, expanded NOTCH1− and expanded NOTCH1+ biopsies with Notch1 mutations or CNLOH. g, Proportion of NOTCH1− and NOTCH1+ areas carrying a secondary missense, nonsense or indel/splicing Notch1 mutation. For f and g, n samples are shown in brackets, redundant samples, defined as biopsies sharing the same mutation and separated by <1 mm were counted once (n = 227 unique biopsies in total). h, Model of colonization by Notch1 clones. Clonal fitness increases from monoallelic and biallelic Notch1 mutation resulting in a selective pressure (blue arrows) for biallelic gene alterations. p.i., postinduction, w.p.i., weeks postinduction. WT, wild type. KO, knock-out allele lacking Notch1 exon 1. Mut, mutation. ND, none detected. See Supplementary Tables 9–11.
To localize potential clones, we stained for NOTCH1 and the YFP reporter. Aging Notch1+/− epithelium contained multiple ovoid areas of homogenous NOTCH1 staining, positive or negative for YFP but far larger than most YFP labeled clones (Fig. 3d,e). These were suggestive of clonal expansion. A total of 246 such ‘expanded’ areas along with typical ‘nonexpanded’ regions were dissected and underwent targeted sequencing for 73 Notch pathway and cancer-related genes (Supplementary Tables 1, 10 and 11). We analyzed for CNLOH and mutations with VAF ≥ 0.2, as below this threshold mutations were considered unlikely to drive clonal expansion. Nintey-seven percent (180/185) of the ‘expanded’ areas had either Notch1 protein-altering mutations with VAF ≥ 0.2 or CNLOH involving the Notch1 locus (GRCm38—chr2:26,457,903-26,503,822). In contrast, only 2 of 61 nonexpanded areas carried Notch1 mutations and none had Notch1 CNLOH (Fig. 3f, Extended data Fig. 5c,d and Supplementary Table 10). Only a few mutations in other genes were found, some may have been passengers within a Notch1 mutant clone. Ninety-four percent (169/180) of expanded areas with Notch1 altering events carried only a single event (about 50% one Notch1 protein-altering mutation and the remainder CNLOH) with an average VAF 0.44, consistent with them being clones carrying spontaneous changes affecting the nonrecombined Notch1 allele (Fig. 3e–g, Supplementary Tables 10, 11 and Extended data Fig. 5c,d). Among clones carrying a Notch1 mutation, 85% of those stained positive for NOTCH1+ harbored missense mutations while NOTCH1 negatively stained clones carried mainly indel/splicing (51%) or nonsense mutations (46%) (Fig. 3g). Overall, these results were consistent with findings in aging human esophagus (Fig. 1).
To test the impact of missense Notch1 mutations, we used an ex vivo functional assay (Extended data Fig. 5e–j and Supplementary Table 10)22. Notch1+/− tissues in Fig. 3e–g were incubated with ethylenediaminetetraacetic acid (EDTA) at 37 °C before fixation. This promotes NOTCH1 cleavage and nuclear migration of NICD without ligand binding (Extended data Fig. 1a)22. Some NOTCH1+ clones displayed nuclear staining, but others did not (Extended data Fig. 5e). Nuclear staining clones were enriched in missense mutations in the ligand binding site, EGF repeats 8–12, whereas non-nuclear staining clones were enriched mutations in the LNR repeats of the NRR domain (Extended data Fig. 5g,h, P = 0.001, Chi-square test). Most of ligand binding domain mutations had highly destabilizing properties, consistent with disrupting ligand binding, a process bypassed in the EDTA assay (Extended data Fig. 5i)23,24. The NRR domain mutants were clustered in the LNR1 and LNR2 domains (Extended data Fig. 5j)25. In contrast, NOTCH1 activating mutations in human T cell acute lymphoblastic leukemia (T-ALL) (https://cancer.sanger.ac.uk/cosmic) cluster in the HD domain of the NRR and promote NEC cleavage without ligand interaction (two-sided Fisher exact test comparing mutation counts in the LNR1-2 and LNR3-HD subregions of the NRR, P = 1.48 × 10−10, Extended data Fig. 5j)26,27. These observations suggest that esophageal NRR domain mutations may prevent the cleavage of NOTCH1. We conclude that in heterozygous epithelium, most spontaneous mutants disrupt NOTCH1 function, conferring a fitness advantage over neighboring cells.
Collectively these observations reveal that haploinsufficiency is key for the normal esophagus to be colonized so effectively by Notch1 mutants. Neutral mutants do not colonize the tissue15,28. Loss of one allele biases mutant progenitor cell fate toward the production of progenitors, increasing the likelihood that mutant clones will expand and persist in the epithelium (Extended data Fig. 4e,f and Video 2). Notch1 inactivated cells have a further increased fitness so that subclonal loss of the second allele within a persisting heterozygous clone will generate cells that outcompete both Notch1+/+ and Notch1+/− neighbors (Fig. 3h). This model explains the high prevalence of clones with NOTCH1 mutation and CNLOH in aging human esophagus.
Notch1−/− epithelium has minimal phenotype
Epithelium lacking functional NOTCH1 might be expected to have a cellular phenotype. To explore the effects of Notch1 loss in the mouse esophagus, we first performed bulk RNA sequencing (RNA-seq) on peeled epithelium from wild type, and highly induced, fully colonized Notch1+/− and Notch1−/− esophagus (Extended data Fig. 3f–h and Extended data Fig. 6a–e). In comparison with wild-type tissue, 20 genes in Notch1+/− and 227 genes in Notch1−/− esophagus were differentially expressed (P adjusted <0.05, Extended data Fig. 6b–d and Supplementary Tables 12,13). These included the Notch1-regulated genes Igfbp3 and Sox9 (Supplementary Table 14)18,29,30. Gene set enrichment analysis (GSEA) showed that transcripts of genes involved in DNA replication were downregulated in Notch1−/− colonized epithelium (Extended data Fig. 6e and Supplementary Table 15).
To phenotype fully colonized Notch1−/− epithelium, we performed single-cell RNA-seq (scRNA-seq) on highly induced YFPCreNotch1flox/flox and uninduced control mouse esophagus (Fig. 4a–i, Extended data 7a–k and Supplementary Table 16). After filtering out poor-quality cells, a total of 13,111 cells remained for analysis, from two biological replicates per genotype (Fig. 4b and Supplementary Note). The proportions of keratinocytes, fibroblasts, immune and endothelial cells were similar in both genotypes, confirmed by staining esophageal sections (Fig. 4c and Extended data Fig. 7b–d)31. Keratinocytes showed no significant difference in density in Uniform Manifold Approximation and Projection (UMAP) space between the two genotypes (Fig.4d and Supplementary Note). The analysis revealed a continuum of keratinocyte cell states, from progenitors expressing Krt14 to differentiating cells expressing Krt4 or Tgm3 to cornified cells expressing Lor (Extended data Fig. 7h–k). We used these markers to discriminate basal and suprabasal cells in UMAP space, finding similar proportions of both populations in control and Notch1−/− epithelium (Fig. 4e and Supplementary Note). In a further analysis, we assigned keratinocytes to cycling basal, resting basal or differentiating cells, finding no substantial differences between genotypes32 (Fig. 4f–i and Supplementary Note).
Fig. 4: Notch1 loss does not alter tissue composition or cell dynamics.
a, YFPCreNotch1flox/flox mice were highly induced and aged for 11 weeks, allowing the mutant cells to completely occupy the esophageal epithelium. Controls were uninduced YFPCreNotch1flox/flox mice (+/+). Esophageal epithelium was dissociated and sequenced. b, UMAP plot shows an overlay of 1,500 cells from each library (n = 2 mice per genotype; +/+1, n = 2,454; +/+2, n = 3,194; −/−1, n = 1,929; −/−2, n = 5,534). c, Left, UMAP plot showing cell types identified via scRNA-seq. Right, stacked bar chart shows the proportion of cell types per library. NA, not available. d, UMAP plot shows an overlay of 1,400 cells annotated as keratinocytes from each library (+/+1, n = 1,555; +/+2, n = 1,932; −/−1, n = 1,403; −/−2, n = 3,919). Milo test shows no significant difference in local cell density through UMAP space (Supplementary Note). e, Left, UMAP plot of keratinocytes. Right, stacked bar chart shows the estimated proportion of keratinocytes per library belonging to the basal or suprabasal layers (Supplementary Note). f, Heat map showing Seurat processed expression values in the keratinocyte population for representative marker genes of basal cells, cell cycle, and differentiation for the 11 clusters shown in g (marker list from ref. 32). Clusters are grouped in three different cell states: cycling basal, resting basal and differentiating cells. g, UMAP plot of keratinocytes representing cell clusters based on Seurat analysis pipeline via the Leiden algorithm. h, UMAP plot of keratinocytes showing cycling basal (orange), resting basal (green) and differentiating (purple) cell states based on clusters and differentiation markers analysis performed in f and g. i, Stacked bar charts show the proportion of keratinocytes per cell state (upper bar) and per cluster (lower bar) in each library. See Supplementary Table 16.
To validate the scRNA-seq findings, we performed a cell-tracking assay. Mice with Notch1−/− esophageal epithelium and littermate controls were injected EdU and 5-bromo-2′-deoxyuridine (BrdU) at 48 h and 1 h, respectively, before collection (Extended data Fig. 7l). Staining for EdU revealed the fate of S phase cells over the following 48 h, BrdU+ cells were currently in S phase. Cells were also stained for phospho-Histone H3 (pHH3), a G2/M phase marker (Extended data Fig. 7m). The ratio of suprabasal EdU+:total EdU+ cells reflecting the generation of differentiating cells and their stratification, the proportion of BrdU+ basal cells and the percentage of pHH3+, BrdU− basal cells were all similar in wild type and Notch1−/− epithelium, consistent with the scRNA-seq findings (Extended data Fig. 7n–r, Supplementary Table 17 and Supplementary Note).
We also examined the epithelium in induced YFPCreNotch1flox/flox mice and control littermates that were aged 52 weeks. Tissue thickness, basal cell density and expression of the differentiation markers KRT14, KRT4 and LOR and the proliferation marker Ki67 were similar in both genotypes, (Fig.5a–d and Supplementary Table 18). Pulse labeling and short-term lineage tracing for 48 h with EdU confirmed no significant difference in the proportion of S phase cells or in the stratification of differentiating cells, respectively, between Notch1−/− and wild-type esophagus (Fig. 5e–h).
Fig. 6: Tumors retain functional Notch1 in carcinogenesis.
a, Uninduced YFPCreNotch1flox/flox mice were treated with DEN and SOR. Tissue was collected 28 weeks after treatment. Tumors were dissected from underlying submucosa and normal epithelium was cut into a gridded array of 2 mm2 samples before targeted sequencing. Scale bar, 1 mm. b, Number of Notch1 mutations per amino acid is plotted by NOTCH1 protein domains in normal gridded biopsies (upper) and tumors (lower) from Notch1 wild type mice (normal, n = 115 biopsies from six mice; tumors, n = 17 biopsies from seven mice). Domains: EGF-like repeats, LNR, HD, TM, transmembrane, RAM, RBP-J-associated module, ANK, ankyrin repeats, TAD, trans-activation domain, PEST, rich in proline, glutamate, serine and threonine. c, dN/dS ratio for Notch1 mutations (top plot) and proportion of Notch1 mutant tissue in normal epithelium (purple bars) (n = 115 biopsies from six mice) and tumors (n = 17 biopsies from seven mice). Two-tailed P value, likelihood ratio test of dN/dS ratios2. d, Representative NOTCH1 (magenta) and KRT14 staining (green) in tumors and surrounding tissue, DNA is blue. Image typical of 10 tumors from six animals. White dashed lines delineate tumor from adjacent normal tissue. Scale bars, 250 µm. e, Proportion of NOTCH1+ staining area in normal epithelium and tumors from the same control animals (each dot represents a mouse, n = 40 tumors from four mice). Two-tailed paired Student’s t test. f, Representative images showing nuclear NICD1 (magenta) in keratinocytes (KRT14, green) inside a tumor in comparison to the normal adjacent tissue. DNA is blue. Image typical of 10 tumors from six animals. Scale bars, 25 µm. g, Proportion of KRT14+ keratinocytes with nuclear NICD1 staining in tumors and surrounding epithelium in the same sections (each dot represents a tumor, n = 10 tumors from six mice). Two-tailed paired Student’s t test. See Supplementary Tables 19–23.
The normal epithelium contained a high density of clones carrying protein-altering mutations. To determine which genes conferred a clonal advantage, we calculated the ratio of silent to protein-altering mutations in each gene, dN/dS3,34. Mutant genes under positive selection with a dN/dS ratio substantially above 1 (q < 0.05) were the Notch pathway genes Notch1, Notch2 and Adam10, plus Fat1, Trp53 and Arid1a, all of which are selected in normal human esophagus along with Ripk4 and Chuk (Supplementary Table 21)2,21.
In tumors, the most prevalent mutant gene was the known mouse esophageal tumor driver Atp2a2, which is not selected in normal epithelium (Extended data Fig. 8a,b and Supplementary Tables 19–22)35,36. Protein-altering Notch1 mutations were under weaker selection and less prevalent in tumors than in the adjacent epithelium (Fig. 6b,c, Extended data Fig. 8a,b and Supplementary Tables 19–22). Immunostaining confirmed more cells stained positive for NOTCH1 and NICD1 in tumors than in normal tissue (Fig. 6d–g and Supplementary Table 23). These findings parallel observations in humans and indicate Notch1 wild-type cells are more likely to contribute to tumors than those carrying Notch1 mutations2,5.
Next, we used a high induction protocol to delete one or both alleles in the entire esophageal epithelium of YFPCreNotch1flox/flox and YFPCreNotch1+/flox mice before DEN and SOR treatment. Uninduced littermates were used as controls (Fig. 7a). The density of tumors was similar in all three genotypes, arguing Notch1 is not required for tumor initiation (Fig. 7b,c and Supplementary Table 24). However, tumors were significantly smaller in Notch1−/− epithelium, in which immunostaining confirmed the loss of Notch1 expression and function (Fig. 7d–f and Supplementary Table 24). Immunostaining for markers of differentiation (LOR, ITGA6 and KRT14) showed multiple layers of undifferentiated keratinocytes in lesions of both genotypes. Markers of apoptosis (cleaved caspase 3), endothelial cells (CD31) and immune cells (CD45) were also similar in tumors from Notch1−/− and Notch1+/+ epithelium (Fig. 7e,f and Extended data Fig. 8d,e). CDH1 loss contributes to tumorigenesis37. Tumors from Notch1+/+, but not Notch1−/−, esophagus displayed focal loss of CDH1 expression (Extended data Fig. 8f–h and Supplementary Table 24).
Fig. 7: Tumor growth is reduced by Notch1 inactivation.
a, Highly induced YFPCreNotch1+/flox (+/−) and YFPCreNotch1flox/flox (−/−) mice or uninduced control (+/+) mice were treated with DEN and SOR and aged for 28 weeks. For b–d, Notch1+/+, n = 11; Notch1+/−n = 10; Notch1−/−, n = 12. b, Representative images of esophagi for each genotype. Scale bar, 1 mm. c, Tumor density per genotype. Mean ± s.e.m., each dot represents a mouse. One-way ANOVA; adjusted P values from Tukey’s multiple comparisons test. d, Tumor areas per genotype. Mean± s.e.m., each dot represents a tumor. Kruskal–Wallis test; adjusted P values from Dunn’s multiple comparisons test. e,f, Tumors from Notch1+/+ (e) and Notch1−/− (f) epithelium were sectioned and stained for H&E (left panel), for keratinocyte progenitor marker Keratin 14 (KRT14, green), and NOTCH1 (magenta) (middle panel) or keratinocyte differentiation marker Loricrin (LOR, magenta) and progenitor markers ITGA6 (gray) and KRT14 (green) (right panel). DNA is blue. Images representative of n = 19 tumors from Notch1+/+ and n = 13 tumors from Notch1−/− epithelium. Scale bars, 250 µm. g, Uninduced YFPCreNotch1flox/flox mice (+/+) were treated with DEN/SOR and aged for 9 weeks. Mice were treated with anti-NOTCH1 NRR1.1E3 or with CTRL for 6 weeks before collection. h, Representative tumors marked by KRT6a staining (red) are shown with white arrowheads in esophageal epithelium from control and anti-NRR1.1E3 treated mice. Scale bars: 100 µm. i, Quantification of tumor area (mean ± s.e.m., each dot represents a tumor, n = 4 mice per group). P values from two-tailed Mann–Whitney test. Data are shown in Supplementary Table 24.
These observations argue that Notch1 favors tumor growth. To test this hypothesis, we treated wild-type mice with a NOTCH1 function blocking antibody (anti-NRR1.1E3)38. The antibody reduced levels of cleaved NOTCH1 in esophageal epithelium, abolished nuclear NICD1 immunostaining and altered levels of multiple transcripts encoding Notch1 loss of function markers (Extended data Fig. 9a–e, Extended data Fig. 6d and Supplementary Table 25). Anti-NRR1.1E3 also reduced the expansion of Notch1−/− clones in clonally induced YFPCreNotch1flox/flox mice by inhibiting NOTCH1 signaling in wild type cells (Extended data Fig. 9f–i and Supplementary Table 25). Wild-type mice were given DEN and SOR, tumors allowed to develop for 9 weeks and anti-NRR1.1E3 or control antibody given for 6 weeks (Fig. 7g). Anti-NRR1.1E3 significantly reduced tumor size compared with control, indicating NOTCH1 signaling favors the growth of established lesions (Fig. 7h, i and Supplementary Table 24).
To understand how Notch1 loss alters tumor growth, we sequenced tumors from Notch1−/− epithelium, finding they share the same driver mutation, Atp2a2, (6/7 tumors), as the tumors from Notch1+/+ epithelium (17/17 tumors) (Extended data Fig. 8a–c and Supplementary Tables 20 and 26)35,36. Comparison of transcriptomes of tumors and adjacent normal tissue showed an upregulation of transcripts encoding genes linked with DNA replication, cell cycle and RNA processing and downregulation of mRNAs associated with lipid metabolism in tumors of both genotypes (Fig. 8a–c, Extended Data Fig. 10a,b and Supplementary Tables 27 and 28). These changes are consistent with the reported effects of Atp2a2 mutation on keratinocytes35,36,39,40. Comparison of tumors from Notch1+/+ and Notch1−/− epithelium revealed DNA replication and cell-cycle-associated transcripts were significantly downregulated in Notch1−/− tumors (Fig. 8d–f, Extended data Fig. 10c,d and Supplementary Tables 29 and 30). Furthermore, the proportion of cycling cells expressing pHH3 and CCNB1 within KRT14+ cells was reduced in tumors from Notch1−/− compared to Notch1+/+ esophagus (Fig. 8g,h, Extended data Fig. 10e,f and Supplementary Table 24). Finally, as RAS/MEK/ERK signaling is activated in Atp2a2 mutant cells, we measured phospho-ERK1/ERK2 and total ERK1/ERK2 staining finding a significant decrease of the former in tumors from Notch1−/− compared to Notch1+/+ epithelium (Fig. 8i,j and Supplementary Table 24)36,40. These findings are consistent with attenuated signaling downstream of mutant Atp2a2 in tumor cells lacking Notch1 (Fig. 8k).
Once an area has been colonized by biallelic Notch1 mutants, the phenotype of mutant cells reverts toward that of wild-type cells. This reversion toward a near-normal cell state explains the normal appearance of aged human esophageal epithelium despite NOTCH1 signaling being disrupted in most of the tissue.
In Atp2a2 mutant tumors, the constraint that links cell division to the exit of differentiating cells from the basal cell layer to maintain cellular homeostasis does not operate. In this context, the faster cells divide, the faster the lesion will expand. As loss of Notch1 slows the cell division rate, Notch1−/− lesions are smaller than wild-type tumors (Fig. 8k).
Might these findings be relevant to humans? Over 90% of human esophageal squamous cell carcinoma (ESCC) retain one or more wild-type copies of NOTCH1 but develop from epithelium where a high proportion of cells have biallelic NOTCH1 disruption, arguing wild-type NOTCH1 favors ESCC development. What of the subset of ESCC that does have biallelic NOTCH1 disruption?5 One possibility is that NOTCH1 loss, in association with multiple other genomic alterations, promotes transformation in these cases. Alternatively, it is plausible that the NOTCH1 alterations in these tumors are ‘passengers’, carried over from normal tissue with the requirement for wild-type NOTCH1 in carcinogenesis bypassed by other genome changes.
Notch1 illustrates how inactivating mutations in the same gene can drive clonal expansion in normal tissue but impair tumor growth. This is due to the differences in cell dynamics between wild-type normal tissue and a mutated tumor. Our results highlight the potential of NOTCH1 blockade in reducing the growth of premalignant tumors. NOTCH1 inhibitors are in clinical development, and investigation of their potential in esophageal neoplasia seems warranted.