Volume 139, Issue 3, Pages 929-941 (September 2010) F-box and WD Repeat Domain-Containing 7 Regulates Intestinal Cell Lineage Commitment and Is a Haploinsufficient Tumor Suppressor  Rocio Sancho, Anett Jandke, Hayley Davis, Markus E. Diefenbacher, Ian Tomlinson, Axel Behrens  Gastroenterology  Volume 139, Issue 3, Pages 929-941 (September 2010) DOI: 10.1053/j.gastro.2010.05.078 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Fbw7 deletion in the gut impairs intestinal cell fate decisions. (A) Deletion of PCR analysis of DNA extracted from different mouse tissues (sb, small bowel; lb, large bowel; li, liver; and ta, tail). (B) Q-PCR analysis for fbw7 mRNA levels in isolated crypt-enriched and villous-enriched fractions from fbw7f/f and fbw7ΔG intestines (data are mean ± SEM, n = 3 different mice crypt/villous preps). (C) fbw7 ISH and Ki67 IHC in fbw7f/f intestinal sections. Scale bar, 50 μm (low magnification) and 10 μm (high magnification). (D) H&E, AB-PAS, chromogranin, and lysozyme staining in representative sections from fbw7f/f and fbw7ΔG intestines. Scale bar represents 50 μm. (E) Quantification of goblet (AB-PAS+), enteroendocrine (chromogranin+), and Paneth (lysozyme+) cells in fbw7f/f and fbw7ΔG (data are presented as mean ± SEM, n > 3 for each genotype). (*P ≤ .05 and **P ≤ .01 in Student t test, n.s, not significant.) Gastroenterology 2010 139, 929-941DOI: (10.1053/j.gastro.2010.05.078) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Accumulation of progenitor cells in fbw7ΔG crypts. (A) H&E staining in representative crypts from fbw7f/f, fbw7ΔG/+, and fbw7ΔG intestines. Scale bar represents 25 μm. (B) Immunohistochemistry for BrdU on representative crypts of fbw7f/f, fbw7ΔG/+, and fbw7ΔG intestines. Black arrowheads represent BrdU+ proliferative progenitors. Scale bar represents 50 μm. (C) Quantification of BrdU+ is expressed as percentage of positive cells per crypt ± SEM, considering fbw7f/f as 100% (n > 3 for each genotype). (D) Immunohistochemistry for MCM6 and staining for alkaline phosphatase on representative sections of fbw7f/f and fbw7ΔG intestine. Scale bar represents 50 μm (low magnification) and 15 μm (high magnification). (E) Q-PCR analysis of different intestinal stem cell and progenitors markers mRNA transcripts in fbw7ΔG intestines compared with fbw7f/f intestines. The data are normalized to β-actin and represented as fold induction over fbw7f/f mice (mean ± SEM, n ≥ 3 for each genotype). (F) Immunofluorescence staining for Ngn3 on representative crypts from fbw7f/f and fbw7ΔG intestines. (*P ≤ .05 in Student t test, n.s, not significant.) Gastroenterology 2010 139, 929-941DOI: (10.1053/j.gastro.2010.05.078) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Fbw7 homozygous deletion increases c-Jun and active-Notch1 (A) Western analysis of protein lysates from fbw7f/f and fbw7ΔG intestines. (B) Q-PCR analysis of fbw7, hes1, hes6, hes5, c-jun, and cyclind1 transcripts in fbw7ΔG intestines compared with fbw7f/f intestines. (Mean fold induction ± SEM, n ≥ 3 for each genotype.) (C) Immunohistochemistry for phosphorylated c-Jun (p-c-Jun) p-c-Junser73 and NICD1 on representative crypts from fbw7f/f and fbw7ΔG intestines. Arrows indicate p-c-Junser73 or NICD1-positive cells. Scale bar represents 50 μm. (D) Coimmunofluorescence for Ngn3 and Hes5 on representative crypts from fbw7f/f and fbw7ΔG intestines. Yellow arrows indicate Ngn3+ cells, and white arrows indicate Hes5+ cells. Scale bar represents 50 μm. (*P ≤ .05 in Student t test, n.s, not significant.) Gastroenterology 2010 139, 929-941DOI: (10.1053/j.gastro.2010.05.078) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Fbw7 behaves as haploinsufficient tumor suppressor in APCmin/+ induced intestinal tumorigenesis. (A, left) Quantification of number of APCmin/+ induced tumors in different intestinal regions from fbw7f/f (n = 5), fbw7ΔG/+ (n = 6), and fbw7ΔG (n = 3) mice (D, duodenum; J, jejunum; I, ileum; C, colon). (A, right) Quantification of Ki67+ cells in APCmin/+ induced tumors in fbw7f/f, fbw7ΔG/+, and fbw7ΔG mice intestines. Data are represented as number or Ki67+ cells/μm2 (×10-3) mean ± SEM (n ≥ 6 tumors for each genotype). (B) Western analysis of protein lysates from APCmin/+ induced tumors in fbw7f/f, fbw7ΔG, and fbw7ΔG/+ mice for NICD1, c-Jun, c-Junser73, and β-Actin. (C) Q-PCR analysis of fbw7, c-jun, hes1, hes5 transcripts in APCmin/+ induced tumors in fbw7f/f, fbw7ΔG, and fbw7ΔG/+ mice. The data are normalized to β-actin and represented as fold induction over APCmin/+ induced tumors in control littermates (mean ± SEM, n ≥ 3 pool of tumors for each genotype) (D) AB-PAS staining in representative sections of APCmin/+ induced tumors in fbw7f/f and fbw7ΔG. Scale bar represents 50 μm. (E) Q-PCR analysis of fbw7, lgr5, prom1, ngn3, neurod1, and insm1 transcripts in APCmin/+ induced tumors in fbw7f/f and fbw7ΔG/+. The data are normalized to β-actin and represented as fold induction over APCmin/+ induced tumors in control littermates (mean ± SEM, n ≥ 3 pool of tumors for each genotype). (F) Immunofluorescence for Ngn3 in representative sections of APCmin/+ induced tumors in fbw7f/f and fbw7ΔG mice. Yellow dashed line indicates tumor area (T). Scale bar represents 50 μm. (*P ≤ .05 in Student t test, n.s, not significant.) Gastroenterology 2010 139, 929-941DOI: (10.1053/j.gastro.2010.05.078) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 c-Jun deletion in fbw7ΔG mice restores proliferation in the crypts, whereas it has no effect on fbw7ΔG-induced defect in differentiation. (A) AB-PAS, BrdU+, and Ngn3 staining in representative sections from fbw7f/f, fbw7ΔG, and fbw7ΔG;c-junΔG intestines. Scale bar represents 50 μm. (B) Quantification of goblet (AB-PAS+), BrdU+, and Ngn3+ cells in fbw7f/f, fbw7ΔG, and fbw7ΔG; c-JunΔG intestines (data are presented as mean ± SEM, n = 3 for each genotype). (*P ≤ .05 and **P ≤ .01 in Student t test.) Gastroenterology 2010 139, 929-941DOI: (10.1053/j.gastro.2010.05.078) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 c-jun deletion in fbw7ΔG; APCmin/+ mice reduces tumor progression but not tumor initiation. (A) Quantification of number of APCmin/+ induced tumors in fbw7f/f (n = 5), fbw7ΔG/+ (n = 6), fbw7ΔG (n = 3), and fbw7ΔG;c-junΔG (n = 3) mice intestines. (B) Quantification of tumor area in APCmin/+ induced tumors in fbw7f/f, fbw7ΔG/+, fbw7ΔG, and fbw7ΔG;c-junΔG mice intestines. Data are represented as the mean of μm2 ± SEM (n ≥6 tumors for each genotype). (C) Immunohistochemistry for NICD1 in representative sections of APCmin/+ induced tumors in fbw7ΔG and fbw7ΔG;c-junΔG mice. Scale bar represents 50 μm. (D) Immunohistochemistry for c-Jun in representative sections of APCmin/+ induced tumors in fbw7ΔG and fbw7ΔG;c-junΔG mice. Scale bar represents 50 μm. (E) Western analysis of protein lysates from APCmin/+ induced tumors in fbw7f/f and fbw7ΔG;c-junΔG for active-Notch1, c-Jun, and β-actin as a control. (F) Q-PCR analysis of fbw7, c-jun, hes1, hes5, lgr5, and ngn3 transcripts in APCmin/+ induced tumors in fbw7f/f and fbw7ΔG;c-junΔG mice. The data are normalized to β-actin and represented as fold induction over APCmin/+ induced tumors in control littermates. (Mean ± SEM, n ≥3 pool of tumors for each genotype.) (*P ≤ .05 in Student t test.) Gastroenterology 2010 139, 929-941DOI: (10.1053/j.gastro.2010.05.078) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Increased Hes5 mRNA levels in FBW7mut human colorectal tumors. (A) Summary table of the tumors used for the ISH and IHC analysis. Sample Id, specimen type, FBW7 mutation status, FBW7 loss of heterozygosity (LOH), and the mean ± standard deviation of the score recorded for c-JUN IHC and HES5 ISH are presented. n.a, non applicable. (B) H&E, IHC for c-JUN, and ISH for HES5 in representative sections of FBW7wt and FBW7mut colorectal carcinomas. Dashed line squares indicate high magnification. (C) Score quantification of c-JUN IHC performed in FBW7wt and FBW7mut tumors. Data are represented as the mean ± standard deviation. (D) Score quantification of Hes5 ISH performed in FBW7wt and FBW7mut tumors. Data are represented as the mean ± standard deviation. (*P ≤ .05 in Student t test, n.s, not significant.) Gastroenterology 2010 139, 929-941DOI: (10.1053/j.gastro.2010.05.078) Copyright © 2010 AGA Institute Terms and Conditions