Volume 143, Issue 3, Pages 730-740 (September 2012) A Role for the Epidermal Growth Factor Receptor Signaling in Development of Intestinal Serrated Polyps in Mice and Humans  Gerold Bongers, Luciana R. Muniz, Michelle E. Pacer, Alina C. Iuga, Nanthakumar Thirunarayanan, Erik Slinger, Martine J. Smit, E. Premkumar Reddy, Lloyd Mayer, Glaucia C. Furtado, Noam Harpaz, Sergio A. Lira  Gastroenterology  Volume 143, Issue 3, Pages 730-740 (September 2012) DOI: 10.1053/j.gastro.2012.05.034 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Increased EGFR and MAPK phosphorylation in human serrated polyps compared with normal mucosa. (A) In the normal mucosa, EGFR autophosphorylation of residue Y1173 (red, P-EGFR) is found predominantly in the lamina propria (arrow) and occasionally (<1%) in epithelial cells (asterisk). (B) In human serrated polyps, P-EGFR is found in the serrated epithelium. Inset shows higher magnification of the boxed area. (C and D) A subset of (D) serrated polyps expressed HB-EGF (red), whereas the (C) normal tissues were negative. (D) Inset shows higher magnification of the boxed area. (E) In normal mucosa P-ERK1/2 predominantly was detected in inflammatory cells (arrow). Inset shows a higher magnification of the boxed area. (F) In serrated polyps, P-ERK1/2 immunoreactivity is seen in the serrated epithelium extending toward the lumen, but is absent at the crypt base. P-ERK1/2 staining was superimposed over pan-keratin (Pan-Ker, green). For an overview, see Supplementary Table 1. Shown are normal mucosa (A, C, and E: normal), a human hyperplastic polyp (B and D: HPP), and a human sessile serrated adenoma (F: SSA/P). Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (A–F, blue) and pan-keratin (A, B, E, and F, Pan-Ker, green). Scale bars: (A–F) 100 μm, (B, D, and E) 25 μm. Gastroenterology 2012 143, 730-740DOI: (10.1053/j.gastro.2012.05.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Mice expressing HB-EGF in IECs develop serrated polyps. (A and B) Representative pictures of the jejunum of (A) WT and (B) HBGF mice stained with HB-EGF (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). (C) Compared with WT mice, the small intestine of HBGF mice had an increased intestinal diameter (n = 5 – 9), an increased number of IECs (n = 9–16), an equivalent number of proliferating IECs as determined by BrdU incorporation (n = 3–5), and an increase in mRNA expression of the senescence marker p16INK4a (n = 3–4). (D) A polyp in the cecum of an HBGF mouse (marked by dashed line). (E) H&E-stained section of a polyp from an HBGF mouse showing the presence of serrated crypts. Inset shows a higher magnification of the dashed area. *P < .05, ***P < .001 compared with WT. Scale bar: (A, B, E, and inset E) 100 μm. Gastroenterology 2012 143, 730-740DOI: (10.1053/j.gastro.2012.05.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Expression of US28 exacerbates the development of polyps induced by HB-EGF. (A) Increased incidence of serrated polyps in HBUS mice (n = 85) compared with HBGF mice (n = 34). (B) Polyp in the cecum of an HBUS mouse. (C–E) H&E-stained sections of ceca from (C and D) HBUS mice and a (E) human serrated polyp showing the presence of serrated crypts. (D and E) Arrows indicate representative serrated crypts. (F) Focal of dysplasia in a serrated polyp from an HBUS mouse (arrows). Inset shows a higher magnification of a dysplastic crypt (arrows indicate a dysplastic transition). ***P < .001. Scale bars: (C–F) 100 μm. Gastroenterology 2012 143, 730-740DOI: (10.1053/j.gastro.2012.05.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Pattern of cellular proliferation and expression of repair genes in HBUS polyps. (A and B) Compared with (A) WT, (B) serrated polyps from HBUS mice have elongated but symmetric basal-dominant pattern of Ki67 (red). Sections were counterstained with pan-keratin (Pan-Ker, green) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Bars to the right of each panel indicate the length of the proliferative zone (red). (C) Decreased expression of Mgmt mRNA, but not of Mlh1, Msh2, Msh6 in tissue isolated form HBUS polyps compared with normal surrounding tissue (n = 5, #P < .05). Scale bars: (A and B) 100 μm. Gastroenterology 2012 143, 730-740DOI: (10.1053/j.gastro.2012.05.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Modulation of EGFR and ERK1/2 signaling in serrated polyps of HBUS mice. (A) Relative expression (Rel. Exp.) of Egfr and Erbb4 mRNA in IECs isolated from serrated polyps of HBUS mice compared with WT and normal (Norm.) surrounding tissue (n = 6, for each). WT heart tissue was used as a positive control for ERBB4. (B) Western analysis of phosphorylated EGFR (P-EGFR), total EGFR, and β-actin in the ceca of WT or HBUS mice. Densitometric analysis shows that relative immunoreactivity (RI) of P-EGFR is increased significantly in the polyps of HBUS mice compared with WT and normal surrounding cecal tissue (Norm.) (n = 3). (C) Western blot analysis of phosphorylated ERK1/2 and total ERK1/2 (ERK1/2) in the ceca of WT or HBUS mice. Densitometric analysis shows that relative immunoreactivity of P-ERK1/2 is increased significantly in the polyps of HBUS mice compared with WT and normal surrounding cecal tissue (Norm.) (n = 8–12). (D) Phosphorylated EGFR (red) in serrated polyps found in HBUS mice is confined to the serrated epithelium. (E) In serrated polyps from HBUS mice P-ERK1/2 immunoreactivity (red) is seen in the serrated epithelium extending toward the lumen, but is absent at the crypt base (n = 14). To enhance visualization, P-ERK1/2 staining was superimposed over 4′,6-diamidino-2-phenylindole (DAPI; blue) and pan-keratin (Pan-Ker, green). (F) Phosphorylated ERK1/2 (green, MAPK-YT) is found in the same cells as P-EGFR (red) in polyps of HBUS mice (n = 3). (B–D, E) Sections were counterstained with DAPI (blue) and pan-keratin. **P < .01, ***P < .001 compared with WT and normal surrounding tissue. Scale bars: (D) 250 μm, (E, F) 100 μm. Gastroenterology 2012 143, 730-740DOI: (10.1053/j.gastro.2012.05.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Reduced incidence of serrated polyps in HBUS mice treated with the MEK inhibitor PD0325901 (PD) and the EGFR inhibitor gefitinib (Gef). (A) At 7 weeks of age HBUS littermates were assigned randomly to either receive daily oral vehicle (Veh, 1% polysorbate-80, n = 25), gefitinib (75 mg/kg, n = 14), or PD0325901 (12.5 mg/kg, n = 12) for 9 weeks. (B and C) At 16 weeks of age, mice were euthanized and analyzed for the presence of polyps. (B) Treatment with gefitinib (P = .0007) or PD0325901 (P = .038) significantly reduced the incidence of polyps. Size of the serrated lesions was determined by histologic measurement. (C) Gefitinib- and PD0325901-treated HBUS mice had significantly smaller polyps compared with vehicle-treated HBUS mice. *P < .05, **P < .01, ***P < .001 compared with vehicle. Gastroenterology 2012 143, 730-740DOI: (10.1053/j.gastro.2012.05.034) Copyright © 2012 AGA Institute Terms and Conditions