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. 2009 Apr 14;106(15):6315-20.
doi: 10.1073/pnas.0813221106. Epub 2009 Mar 26.

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer

Verónica Rodilla  1 , Alberto Villanueva, Antonia Obrador-Hevia, Alex Robert-Moreno, Vanessa Fernández-Majada, Andrea Grilli, Nuria López-Bigas, Nicolás Bellora, M Mar Albà, Ferran Torres, Mireia Duñach, Xavier Sanjuan, Sara Gonzalez, Thomas Gridley, Gabriel Capella, Anna Bigas, Lluís Espinosa
Affiliations

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer

Verónica Rodilla et al. Proc Natl Acad Sci U S A. .

Abstract

Notch has been linked to beta-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/beta-catenin (down-regulated when blocking Wnt/beta-catenin) that are directly regulated by Notch (repressed by gamma-secretase inhibitors and up-regulated by active Notch1 in the absence of beta-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through beta-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/beta-catenin pathway in tumors implanted s.c. in nude mice. Crossing APC(Min/+) with Jagged1(+/Delta) mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear beta-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by beta-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Inhibition of Wnt and Notch leads to down-regulation of a common gene program. (A) Gene expression profile from Ls174T/dnTCF4 cells treated for 48 h with doxycyline (to inhibit Wnt signaling) or DAPT (Notch inhibitor) or both compared with untreated cells. Some top significant genes ranked by Student's t test are shown for each group. *, genes further studied in this work. (B) Microarray analysis of 2 doxycycline-treated Ls174T/dnTCF4/N1IC clones (N14, N15) compared with doxycycline-treated Ls174T/dnTCF4. Color codes represent only-Notch targets (blue) and the Notch-targets downstream of Wnt (yellow). (C) ChIP experiment in Ls174T cells and PCR of the indicated promoters. Scheme of the 2-kb proximal promoter showing the position of the primers used, the RBP-binding sites and TSS.
Fig. 2.
Fig. 2.
Notch1 restores tumor growth in the absence of β-catenin/TCF signaling. (A and B) A total of 1.5 × 106 cells were implanted s.c. in nude mice [Ls174T/dnTCF4 (black arrow) and Ls174T/dnTCF4/N1IC (red arrow) clones] untreated or treated with doxycycline for 4 weeks (2 independent experiments, n = 20 mice each). Representative animals for each group (A) and average and SEM of the tumor volume (in cubic millimeters) (B) are shown. P values are based on a nonparametric ANOVA applying a rank transformation on the dependent variable. (C) Alcian blue staining and IHC with α-Ki67 in serial sections of representative tumors. α-flag or α-myc staining shows the expression of dnTCF4 and N1IC (400×).
Fig. 3.
Fig. 3.
Notch is downstream of Wnt/β-catenin through activation of Jagged1. (A) qRT-PCR of different Notch ligands in Ls174T/dnTCF4 cells untreated or treated for 48 h with doxycycline. (B) Western blot with the indicated antibodies of Ls174T/dnTCF4 cells treated with doxycycline for the indicated times. Tubulin is shown as loading control. (C) Recruitment of β-catenin to Jagged1 promoter in Ls174T cell line untreated or treated with DAPT. β-actin gene is shown as a control. (D) Percent transcriptional inhibition in Ls174T cells transfected with siRNA-Jag1 compared with scramble siRNA as determined by qRT-PCR. Down-regulation of Jagged1 by siRNA was determined by Western blot analysis (Lower).
Fig. 4.
Fig. 4.
Jagged1 is essential in APCMin/+ intestinal tumorigenesis. (A) IHC of serial section of wild type intestine and APCMin/+ tumor stained with the indicated antibodies (100×). (B) Stereoscopical image of the methylene blue staining. The average number of visible polyps in the small intestines (>1 mm or ≤1 mm) from the different genotypes at 16 weeks of age is represented. Error bars are SEM. P values are based on a Mann–Whitney U test. (C) (Upper) Immunostaining with α-β-catenin of representative tumors from different genotypes. The average percentage of cells showing nuclear β-catenin is indicated. (Lower) Representative images of α-Ki67 staining and average percentage and SEM of Ki67+ cells per tumor from 5 APCMin/+Jag1+/+ (n = 51) and 4 APCMin/+Jag1+/Δ (n = 27). (D) Sections of normal crypts from different genotypes stained with α-Ki67 antibody (Left). The average and SEM of Ki67+ cells per crypt from 10 APC+/+Jag1+/+, 5 APCMin/+Jag1+/+ and 4 APCMin/+Jag1+/Δ is represented (Right). n, number of crypts counted. P values are based on a Mann–Whitney U test.
Fig. 5.
Fig. 5.
Notch1 and Notch2 are activated in colorectal tumors. (A) Levels of Jagged1 mRNA as measured by qRT-PCR. The red line indicates the average value for each group. (B) Serial sections of colorectal adenoma from a FAP patient stained with the indicated antibodies. Dashed lines indicate the boundary between the normal adjacent (N) and the tumor (T) tissue. Hematoxylin (blue) was used for nuclear staining. (C) Immunoflouresence with α-N1ICv (green) of normal or adenoma tissue from FAP sample. Nuclei were stained with DAPI (blue) (600×). (D) qRT-PCR to determine the levels of the indicated genes in normal tissue compared with FAP samples. The red line indicates the average value for each group. Statistical significance of the differences between the adenoma and normal samples are calculated based on 2-sided Student's t test. P values are: EphB3, P = 0.014; CD44, P = 0.0002; SOX9, P = 0.005; NOX1, P = 0.002; KLF5, P = 0.01; and hes1, not significant.
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