1. Volume 136, Issue 5, Pages 1680-1688.e7 (May 2009)
TGF-β Receptor Inactivation and Mutant Kras Induce Intestinal Neoplasms in Mice via a β-Catenin-Independent Pathway 
Patty Trobridge, Sue Knoblaugh, M. Kay Washington, Nina M. Munoz, Karen D. Tsuchiya, Andres Rojas, Xiaoling Song, Cornelia M. Ulrich, Takehiko Sasazuki, Senji Shirasawa, William M. Grady 
Gastroenterology 
Volume 136, Issue 5, Pages e7 (May 2009)
DOI: /j.gastro
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2. Figure 1
Photomicrographs of representative examples of distal colon epithelium from the KTT, KVcTwt/wt, and KVcTT mice (age, 20 weeks). (A, C, and E) H&E-stained sections reveal increased goblet cells and crypt length in the KVcTwt/wt and KVcTT mice compared with the KTT mice. (B, D, and F) Ki67 immunostaining of the KTT, KVcTwt/wt, and KVcTT mice reveals more Ki67-labeled cells in the KVcTwt/wt and KVcTT mice compared with the KTT mice (original magnification, 100×).
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3. Figure 2
(A and B) H&E-stained tissue sections from tumors arising in the small intestine (original magnification, 100× [A] and 200× [B]). The tumors display marked desmoplasia and mucinous features, including signet ring cells and mucin lakes (arrow and asterisk, respectively). (C and D) β-catenin immunostaining of the normal epithelium (C, colon; D, small intestine) in the KVcTT mice reveals cytoplasmic membrane distribution, as expected. (E and F) p53 Immunostaining in normal intestinal epithelium (E, colon; F, small intestine) in the KVcTT mice reveals occasional cells with nuclear staining in the base and luminal surface of the epithelium in the colon and small intestine with the majority of cells showing no staining. (G) β-Catenin cellular localization in the neoplasms arising in the KVcTT mice. Immunostaining for β-catenin reveals that <10% of the KVcTT tumors show nuclear β-catenin (compared with 80% of the AVcTT tumors; data not shown) (arrow indicates neoplastic gland with β-catenin expression.). (H) Representative example of p53 immunostaining of KVcTT tumor (original magnification, 100×, respectively). Increased nuclear p53 nuclear immunoreactivity is present in the tumors. The arrow indicates cells that show nuclear p53 immunoreactvity.
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4. Figure 3
Expression of phosphorylated ERK1/2 (pThr202/Tyr204), total ERK1/2, phosphorylated AKT (pSer473), and total AKT in the normal intestinal mucosa of the KTT, KVcTT, and KVcTwt/wt mice and in the intestinal neoplasms of the KVcTT, AVcTT, and ATT mice. Phosphorylated ERK1/2 is present in the adenocarcinomas (n = 5) and mucosa (n = 3) of KVcTT mice as well as in KVcTwt/wt epithelium (n = 3). Phosphorylated ERK1/2 is present in approximately 60%–70% of the neoplasms (n = 9) in AVcTT and ATT mice regardless of whether Tgfbr2 is inactivated. Similarly, AKT phosphorylation is present in 40%–100% of the tumors in the mouse models and is present in the majority of the normal mucosa samples of the KTT, KVcTwt/wt, and KVcTT mice.
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5. Figure 4
Ki67 immunostaining in the KVcTT vs AVcTT neoplasms reveals significantly increased proliferation in the KVcTT tumors compared with the AVcTT tumors. (A and B) Representative examples of tumors from the AVcTT mice (original magnification, 100×). (C and D) Representative examples of tumors from the KVcTT mice (original magnification, 100×).
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6. Figure 5
Immunoblotting of protein lysates extracted from the normal intestinal mucosa from the KTT, KVcTT, and KVcTwt/wt mice and neoplasms from the KVcTT, AVcTT, and ATT mice for p15, p21, cyclin D1, and cdk4. The expression of p15 is reduced in the tumors from the KVcTT mice compared to the normal mucosa and to the AVcTT and ATT tumors. Furthermore, p21 expression is the same or decreased in the KVcTT tumors compared to the normal mucosa and is substantially less than the majority of the AVcTT and ATT tumors. Cyclin D1 and cdk4 expression is increased in all the tumors when compared to the normal mucosa. The “X” indicates that no protein lysate was available from this sample to perform this study. Actin was used to control for protein loading.
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7. Figure 6
Ereg and Erbb1 expression in the intestinal mucosa of the VcTT, KTT, KVcTwt/wt, and KVcTT mice, in the adenocarcinomas from the KVcTT mice, and in human colon cancer cell lines. (A) Epiregulin (Ereg) expression measured by qualitative reverse-transcription polymerase chain reaction (qRT-PCR) is increased in the neoplasms compared with the normal mucosa (**P < .001, ***P < .0001, Mann-Whitney test). (B) Erbb1 (expression measured by qRT-PCR) is increased in the neoplasms compared with the normal mucosa with the statistically significant differences shown. (*P < .05, **P < .01, Mann-Whitney test) REU, relative expression unit. (C) EREG expression in the HCT116 colon cancer cell line is increased by oncogenic KRAS (KRASG13D) and TGFBR2 inactivation. qRT-PCR analysis shows EREG mRNA expression is higher in parental HCT116 (KRASG13D and biallelic TGFBR2 BATRII mutations) than in HCT116 with reconstituted TGFBR2 (HCT116 + TGFBR2 2+8), with only wild-type KRAS (HKe-3), or wild-type KRAS and TGFBR2 reconstitution (HKe-3 + TGFBR2 2+8). The deletion of the mutant KRAS allele has a more pronounced effect on EREG expression than does TGFBR2 reconstitution (*P < .01, Student t test), although the differences between all the groups are statistically significant including between HKe-3 and HKe-3+TGFBR2 2+8 (**P = .03, Student t test). (D) EREG mRNA expression is increased in the SW480 colon cancer cell line (mutant KRAS and wild-type TGFBR2) after treatment with TGF-β RI inhibitor III (300 nmol/L) (616453; Calbiochem).
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8. Figure 7
Metastatic tumors in the Tgfbr2IEKO; LSL-KrasG12D/wt (KVcTT) mouse. (A) Gross appearance of enlarged lymph nodes that were shown to contain metastatic tumor. The enlarged lymph nodes are indicated by arrows. (B) Photomicrograph of H&E-stained lymph node metastasis (original magnification, 100×). (C) Photomicrograph of H&E-stained lung metastasis (original magnification, 100×). (D) Cytokeratin immunostaining of metastatic lesions in the lymph node (original magnification, 100×; insert original magnification, 400×). (E) Results of PCR-based assays demonstrating the recombination of the Tgfbr2E2flx allele in representative lung metastatic tumors (#1 and #2) and mesenteric lymph node (LN #3a and #3b) metastases. The positive and negative control DNA samples are from an AVcTT epithelial cell line and “normal” mesenteric lymph nodes from an age-matched KVcTT mouse that did not have grossly evident metastatic disease.
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9. Supplementary Figure 1
(A, D, and G) H&E and special stains (PAS and Alcian blue) of colon mucosa for KTT, KVcTT, and KVcTwt/wt mice. (B, E, and H) An increased proportion of PAS staining cells/crypt in the KVcTT and KVcTwt/wt mucosa compared with the KTT mucosa can be appreciated. (C, F, and I) There is not a substantial difference in the Alcian blue staining patterns among the different genotypes, which suggests that the mucin-producing cells that are in abundance in the colon epithelium of the KVcTT and KVcTwt/wt mucosa are producing neutral and not acidic mucopolysaccharides.
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10. Supplementary Figure 2
Lysozyme and chromogranin A expression in the small intestinal epithelium of the KTT, KVcTwt/wt, and KVcTT mice. Decreased lysozyme expression is demonstrated in the small intestine of the KVcTwt/wt mice, and near absent lysozyme expression is present in the small intestine of the KVcTT mice. The lack of lysozyme-expressing cells indicates a lack of paneth cells. The chromogranin A expression demonstrates no significant difference in neuroendocrine cell differentiation among the 3 genotypes.
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11. Supplementary Figure 3
(A) COX2 expression in intestinal neoplasms arising in the KVcTT mice is increased. COX2 expression as detected by immunostaining is increased in the tumor epithelium and/or stroma in approximately 70% of tumors. Three different patterns of COX2 expression are displayed from 3 different tumors. The panel on the left demonstrates expression in the tumor epithelium; the middle panel demonstrates strong expression in both the tumor epithelium and stroma; and the panel on the right demonstrates strong expression in the stroma (original magnification, 100×). (B) PGE2 production is also increased in the tumors compared with normal mucosa from the KTT, KVcTwt/wt, or KVcTT mice (*P < .05 comparing the normal mucosa of any genotype to the tumors, **P < .01 comparing the KVcTT normal mucosa to the KVcTT cancers, Mann-Whitney test).
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12. Supplementary Figure 4
Expression of phosphoryated p70S6 kinase detected by immunoblotting of protein lysates from normal intestinal mucosa and neoplasms of the KVcTT mice. No increase in the amount of phosphorylated p70S6kinase in the tumors is observed.
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13. Supplementary Figure 5
Expression of DcR2 and DEC1 in the intestinal mucosa and tumors as detected by immunoblotting. No change in DcR2 in the tumors is observed, and a modest increase in DEC1 is observed. Decreased expression of DcR2 and DEC1 in the tumors, which would indicate escape from senescence, is not observed.
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14. Supplementary Figure 6
Cyclin D1 immunostaining of tumors arising in the KVcTT mice. Cyclin D1 is overexpressed in the intestinal neoplasms of the KVcTT mice. Cyclin D1 immunoreactivity in a representative tumor is shown. These results are consistent with the cyclin D1 immunoblotting studies. Original magnification in A, 100×; in B, 200×.
Gastroenterology  , e7DOI: ( /j.gastro )
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