PMC

Spontaneous gastric tumor development in the antrum in Villin-Cre⁺;Klf4^fl/fl mice. (A) Tumor incidence in the stomach (A1) and other locations (A2) in Villin-Cre⁻;Klf4^fl/fl (Klf4^+/+), Villin-Cre⁺;Klf4^+/fl (Klf4^+/−), and Villin-Cre⁺;Klf4^fl/fl (Klf4^−/−) mice at the ages of 35, 50, and 80 weeks. (B) Representative photographs of macroscopic views of the entire gastric mucosa in 80-week-old Klf4^+/+ and Klf4^−/− mice (B1) and microscopic views of an antral tumor in the Klf4^−/− mouse (B2). (C) Gross morphology of stomachs obtained from 80-week-old Klf4^+/+ (C1) and Klf4^−/− (C2) mice. A visible antral tumor in a Klf4^−/− mouse stomach is indicated by a red arrow.

Lost KLF4 expression and FoxM1 overexpression in gastric tumors. (A) Western blot analyses were performed using total protein lysates extracted from antral mucosa in mice at the ages of 35 (A1), 50 (A2), and 80 (A3) weeks and from antral tumors (A4). KLF4^+/+, Villin-Cre⁻;Klf4^fl/fl; KLF4^+/−, Villin-Cre⁺;Klf4^+/fl; KLF4^−/−, Villin-Cre⁺;Klf4^fl/fl. (B) Immunohistochemical staining for KLF4 (upper panels) and FoxM1 (lower panels) protein. From left to right were: normal, hyperplastic, intramucosal neoplastic, and neoplastic tissue from mice at age of 80 weeks. (C) Immunohistochemical staining of two gastric tumors for KLF4 and FoxM1 protein from mice at age of 80 weeks. The right panels are views of the middle panels at higher magnification.

Induction of gastric tumor development in the antrum in Villin-Cre⁺;Klf4^fl/fl mice. (A) Incidence (A1 & A2) and locations (A3) of gastric tumor formation in Villin-Cre⁻;Klf4^fl/fl (Klf4^+/+), Villin-Cre⁺;Klf4^+/fl (Klf4^+/−), and Villin-Cre⁺;Klf4^fl/fl (Klf4^−/−) mice at the ages of 35 and 50 weeks with or without MNU-based treatment. (B) Gross morphology of visible tumors (white arrows) in the gastric antrum (B1), corpus (B2), or both (“mixed,” B3) in 50-week-old Klf4^−/− mice. (C) Representative photographs of macroscopic views of the entire gastric mucosa in 50-week-old Klf4^+/+ (C1) and Klf4^−/− (C2) mice and a microscopic view of an antral tumor in the Klf4^−/− mouse (C3).

Analysis of the integrity and expression of the Klf4 gene in gastric tumors. (A) DNA was isolated from matched gastric tumor specimens (T) and nontumorous corpus mucosa specimens (N) obtained from Villin-Cre⁻;Klf4^fl/fl (A1) and Villin-Cre⁺;Klf4^fl/fl (A2) mice. PCR analysis was performed for genotyping of Klf4 alleles. (B) An individual antral tumor was obtained immediately after surgery (B1) and divided into two parts: one for primary culture (CC, B2) and one for DNA extraction (TT). DNA was also extracted from a nontumorous corpus mucosa specimen (NT). PCR analysis was performed for genotyping of Klf4 alleles (B3). (C) Klf4 mRNA expression and genetic integrity in corpus tumors. Real-time PCR analysis was performed using total RNA extracted from corpus tumors and adjacent nontumorous tissue specimens from Klf4^+/+ and Klf4^−/− mice. Total RNA obtained from colonic mucosa in Klf4^+/+ and Klf4^−/− mice was used as a control (C1). DNA was extracted from Klf4^−/− corpus tumors, and PCR analysis was performed for genotyping of Klf4 alleles using DNA from TT and CC as a control (C2).

(A) Schematic diagram of the genomic structure of the Klf4 gene with wild-type (Klf4^+/+), floxed (Klf4^fl/fl), and disrupted (Klf4^−/−) alleles. Shown are LoxP sites (red triangles), PCR primer positions (arrows), and expected PCR products using three primers: 172bp for the wild-type allele, 296bp for the floxed allele, and 425bp for the deleted allele. (B) Genotyping using mouse tail DNA at the age of 4 weeks. PCR screening for Klf4 revealed a band of 172bp for the wild-type allele and 296bp for the floxed allele. (C) Detection of Villin-Cre–mediated rearrangement of the Klf4 allele in different tissues in mice at the age of 20 weeks. DNA was extracted from different tissues from a previously genotyped Klf4^−/− mouse. PCR analysis indicated various levels of rearrangement of the Klf4 gene (425bp for the deleted allele). (D) The efficacy of Klf4 deletion in the colon and gastric antrum in Klf4^fl/fl and Klf4^−/− mice was measured using quantitative PCR analysis of colon and antral DNA from those mice at the age of 35 weeks. (E) PAS/Alcian blue staining of gastric corpus and antral mucosa specimens obtained from Villin-Cre⁻;Klf4^fl/fl and Villin-Cre⁺;Klf4^fl/fl mice at the age of 50 weeks. Villin-Cre⁺;Klf4^fl/fl antral mucosa exhibited increases in the number of both PAS- and Alcian blue-positive cells. Hematoxylin and eosin staining of colons obtained from Villin-Cre⁻;Klf4^fl/fl and Villin-Cre⁺;Klf4^fl/fl mice. Goblet cells were nearly absent from Villin-Cre⁺;Klf4^fl/fl colonic mucosa, whereas a normal contour and numerous goblet cells were observed along the crypts and surface epithelium in Villin-Cre⁻;Klf4^fl/fl mice. Positive Klf4 staining in the small intestinal mucosa cells of Villin-Cre⁻;Klf4^fl/fl mice but negative in that of Villin-Cre⁺;Klf4^fl/fl mice.

KLF4 regulation of FoxM1 expression in human gastric tumors. (A) Tissue sections were prepared from 86 formalin-fixed, paraffin-embedded human gastric tumor specimens. Immunostaining of the sections was performed using specific anti-KLF4 and -FoxM1 antibodies. FoxM1 expression levels were inversely correlated with KLF4 expression levels (P<001; χ² test). (B) Representative photographs of two cases showing KLF4 underexpression and FoxM1 overexpression. (C) Deletion mutants of FoxM1 promoter reporters were transfected into SK-GT5 cells in triplicate, and the relative promoter activities were measured 24 hours after transfection. (D) Schematic diagram of the FoxM1 proximal promoter. The nucleotide positions and sequences of the putative KLF4 binding site and PCR forward and reverse primers for ChIP analysis are shown (D1). Chromatin was extracted from SK-GT5 cells, and the ChIP assay was performed using a specific anti-KLF4 antibody and oligonucleotides flanking the FoxM1 promoter region containing the KLF4-binding site (D2). (E) N87 and SK-GT5 cells were transduced with a control Ad-EGFP (Neo) or Ad-KLF4 at an MOI of 10 or 20 for 24 hours. Total protein lysates were harvested from the cell cultures, and the levels of KLF4 (exogenous, as determined using an anti-FLAG antibody) and FoxM1 expression were determined using Western blot analysis (E1). SK-GT5 cells were transduced with Ad-KLF4 or Ad-EGFP at an MOI of 10 for 24 hours. Chromatin fragments were prepared for ChIP analysis using control IgG and an anti-Flag antibody (E2). The pFXM1-360 proximal promoter was transfected into SK-GT5 cells in triplicate with pcDNA3.1, a KLF4 expression vector, nontargeting control siRNA, or FoxM1-siRNA. The relative promoter activities were assessed 24 hours after transfection (E3).