Notch Signaling Mediates Differentiation in Barrett's Esophagus and Promotes Progression to Adenocarcinoma

Abstract

Background & aims: Studies are needed to determine the mechanism by which Barrett's esophagus (BE) progresses to esophageal adenocarcinoma (EAC). Notch signaling maintains stem cells in the gastrointestinal tract and is dysregulated during carcinogenesis. We explored the relationship between Notch signaling and goblet cell maturation, a feature of BE, during EAC pathogenesis.

Methods: We measured goblet cell density and levels of Notch messenger RNAs in BE tissues from 164 patients, with and without dysplasia or EAC, enrolled in a multicenter study. We analyzed the effects of conditional expression of an activated form of NOTCH2 (pL2.Lgr5.N2IC), conditional deletion of NOTCH2 (pL2.Lgr5.N2fl/fl), or loss of nuclear factor κB (NF-κB) (pL2.Lgr5.p65fl/fl), in Lgr5⁺ (progenitor) cells in L2-IL1B mice (which overexpress interleukin 1 beta in esophagus and squamous forestomach and are used as a model of BE). We collected esophageal and stomach tissues and performed histology, immunohistochemistry, flow cytometry, transcriptome, and real-time polymerase chain reaction analyses. Cardia and forestomach tissues from mice were cultured as organoids and incubated with inhibitors of Notch or NF-kB.

Results: Progression of BE to EAC was associated with a significant reduction in goblet cell density comparing nondysplastic regions of tissues from patients; there was an inverse correlation between goblet cell density and levels of NOTCH3 and JAG2 messenger RNA. In mice, expression of the activated intracellular form of NOTCH2 in Lgr5⁺ cells reduced goblet-like cell maturation, increased crypt fission, and accelerated the development of tumors in the squamocolumnar junction. Mice with deletion of NOTCH2 from Lgr5⁺ cells had increased maturation of goblet-like cells, reduced crypt fission, and developed fewer tumors. Esophageal tissues from in pL2.Lgr5.N2IC mice had increased levels of RelA (which encodes the p65 unit of NF-κB) compared to tissues from L2-IL1B mice, and we found evidence of increased NF-κB activity in Lgr5⁺ cells. Esophageal tissues from pL2.Lgr5.p65fl/fl mice had lower inflammation and metaplasia scores than pL2.Lgr5.N2IC mice. In organoids derived from pL2-IL1B mice, the NF-κB inhibitor JSH-23 reduced cell survival and proliferation.

Conclusions: Notch signaling contributes to activation of NF-κB and regulates differentiation of gastric cardia progenitor cells in a mouse model of BE. In human esophageal tissues, progression of BE to EAC was associated with reduced goblet cell density and increased levels of Notch expression. Strategies to block this pathway might be developed to prevent EAC in patients with BE.

Keywords: Barrett’s Esophagus; Carcinogenesis; Esophageal Adenocarcinoma; Goblet Cells.

Figure 1.

There was a significant decrease in GC density from non-dysplastic regions of BE epithelium with neoplastic progression in patients without dysplasia, low grade dysplasia (LGD), high grade dysplasia (HGD), and adenocarcinoma (EAC), measured for each subject as proportion of evaluable grids with any GCs (A) and proportion of evaluable grids with >3 GCs (B). Expression of TFF3 (C) and MUC2 (D) were also correlated with GC density Correlogram of the expression of TFF3 and MUC2 and NOTCH1-4 (E). Significant inverse correlation between NOTCH3 and GC density (F). Representative IHC images (G-I) demonstrating that glands with high Notch1 and Notch3 expression on IHC contained few GCs and also showed high levels of proliferation (e.g. for Ki67, “high” and “low” refer to regions of high and low expression, respectively, GCs are labeled with an arrow). IHC analyses demonstrated significant correlation between Notch1 and Notch3 with Ki67 (J, K).

Figure 2.

A BE mouse model with engineered Notch2IC expression in gastric Lgr5+ stem cells lead to dysplasia, increased Notch-IC staining and decreased survival rates. (A) GSEA comparing L2-IL1B versus WT mice with a set of Notch dependent genes. (B) single gene expression analysis of Notch1 and Notch2. (C) GSEA of a Notch signature comparing tumor bearing mice and L2-IL1B mice that were previously treated with bile acid. (D) Generating mouse models with the conditional knockout of Notch2 and overexpression of activated Notch2-IC in Lgr5+ cells on top of the inflammatory background of L2-IL1B mice. (E) qRT-PCR validates Notch2 expression levels in genetically modified L2-IL1B mice. (F) differential gene expression pattern of human donors (HGD/EAC vs. non-dysplastic BE) and our mouse models (mice overexpressing IL1B and NOTCH2 in Lgr5 cells vs. mice only overexpressing IL1B). (G) IHC staining of Notch-IC. Shown are representative examples at 13 months after induction. (H) Lineage tracing experiments of our mouse strains that were crossed to Rosa26-LacZ mice indicating the presence of Lgr5 expressing progenitor cells in dysplastic tissue. (I) In situ hybridization (ISH) for Lgr5 depicting Lgr5⁺ progenitor cells at the SCJ in areas of BE. (J) Intranuclear Notch-IC reactivity in the three mouse strains at indicated time points as positive cells in the BE region in 10 high-power fields. (K) Statistical quantification of Lgr5⁺ cells per 10 high -power fields as shown as representative images in (I). (L) Kaplan-Meier curves showing decreased survival of pL2.Lgr5.N2IC mice compared to the other two strains. Data is presented as means ± standard deviation. Statistical analysis were performed using one-way ANOVA and Tukey’s multiple comparison test; *p<.05.

Figure 3.

Overexpression of NOTCH2 in Lgr5+ progenitor cells accelerates the development of metaplasia, dysplasia, and esophageal tumors in L2-IL1B mice. (A) (upper panel) Macroscopic images of the distal esophagus and gastric cardia, sliced along the sagittal plane, and (lower panel) representative H&E staining of pL2.Lgr5 control mice, pL2.Lgr5N2fl/fl, pL2.Lgr5.N2IC mice at indicated time points. Statistical analysis of the (B) macroscopic tumor score, and histopathological scoring for (C) metaplasia, and (D) dysplasia. Data is presented as means ± standard deviation. Statistical analysis were performed using one-way ANOVA and Tukeys multiple comparison test.*p<.05.

Figure 4.

Notch2 overexpression in Lgr5+ cells leads to increased crypt fission and impedes the differentiation of mucus producing cells. (A) Representative images for goblet-like cell maturation and crypt fission. (B) Statistical summary of PAS staining indicating goblet-like cell maturation and (C) goblet-like cells frequencies indicated as GC ratio. (D) Linear regression of pathological progression correlating the macroscopic and dysplasia score with GC ratio. (E) Cell proliferation was evaluated by Ki67 reactivity and (F) crypt fission events. Analysis was performed in the BE region in 10 high-power fields. Data is presented as means ± standard deviation. Statistical analysis were performed using one-way ANOVA and Tukey’s multiple comparison test. *p<.05.

Figure 5.

Notch activity in progenitor cells determines cell fate and growth rates of ex vivo cultured organoids. (A) IHC results of 3D cultured organoids. Organoids were isolated from indicated mouse strains and analysed for Notch-IC, Ki67 and PAS reactivity with representative images at passages 3-5 after isolation. (B) Differential gene expression of organoid cells was performed via qRT-PCR. (C) Organoid survival rates and (D) organoid wall thickness (relative to the organoid diameter) that serves an indicator of goblet-like maturation. Representative examples are displayed (right). (E-G) pL2.Lgr5.N2IC or pL2.Lgr5 mice derived organoids were treated with the gamma-secretase inhibitor of Notch signaling, DAPT (50μM). (E) Summarized IHC results of 3D cultured organoids. (F) Flow cytometry of singularized organoid cells derived from mouse (ms; L2-IL1B) and human (hu, BE) samples. The representative histogram indicates the range of Muc2+ cells for vehicle (dark, 19.4%) and inhibitor (50μM; light grey, 56.8%) treated conditions quantified via flow cytometry after enzymatic cell separation of organoids 3D culture. Data is presented as means ± standard deviation of at least three independent experiments. Statistical analysis was performed using Student's T-Test, *p<.05 (G) qRT-PCR of pL2.Lgr5.N2IC derived organoids. At least three independent experiments were performed; Data are presented as mean standard deviation. (A-E, G) Data is presented as means ± standard deviation. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test or student t-test. *p<.05.

Figure 6.

Aberrant Notch expression results in increased NF-κB in progenitor and progenitor derived cells. (A) GSEA display an enrichment of NF-κB associated gene sets in mice with overexpressed Notch2 versus mice with less Notch signalling. (B) qRT-PCR of RelA expression levels of indicated mouse strains. (C) Representative IHC images of pIKK (left) and a combinatory staining of pIKK and Alcain Blue (right) derived from mice at 7 months. (D) Exemplary immunofluorescence images of pIKK (red) and anti-GFP (Lgr5; green) staining derived from mice at 10 months. (E) Corresponding statistically summarized results in the BE region as number of positive cells in 10 high-power fields. Data is presented as means ± standard deviation. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test. *p<.05.

Figure 7.

A RelA knockout reduces the pathology observed in mice with induced inflammation and Notch signaling. A) Representative IHC images of RelA KO mice that are depicted next to L2-IL1B and pL2.Lgr5.N2IC mice. (B-H) Histopathological evaluations for metaplasia, dysplasia, inflammation, GC ratio, and numbers of pIKK+, Ki67+ and PAS+ cells in the BE region in 10 high-power fields. Statistical analysis was performed using one-way ANOVA and Tukeys multiple comparison test, *p<.05, or the Kruskal-Wallis test #p<.05. All indicated IHC based results were derived from mice ranging from 10 to 12 months of age. Only mice with the same age were compared. (I-L) L2-IL1B mice derived organoid cultures were treated with the NF-κB inhibitor, JSH-23 (10μM), or vehicle control. (I) qRT-PCR of L2-IL1B mice derived organoids. (J) Cell activity was measured via MTT assay and (K) organoid growth was determined microscopically. (L) Flow cytometry of organoid cells after enzymatic disaggregation to determine the proportion of Muc2+ cells, derived from (ms; L2-IL1B) and human (hu, BE) samples. The representative histogram indicates the range of MUC/Muc2⁺ cells for vehicle (dark, 22.5%) and inhibitor (light grey, 41.1%) treated conditions. Data is presented as means ± standard deviation of at least three independent experiments. Statistical analysis was performed using Student's t-test, *p<.05