Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined ApcMin and KRASV12 mutations

Abstract

Background: Both mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene and activation of the KRAS oncogene are implicated in the pathogenesis of colorectal cancer. Mice harboring a germline ApcMin mutation or intestine-specific expression of the KRASV12 gene have been developed. Both mouse strains develop spontaneous intestinal tumors, including adenoma and carcinoma, though at a different age. The zinc finger transcription factor Krüppel-like factor 5 (KLF5) has previously been shown to promote proliferation of intestinal epithelial cells and modulate intestinal tumorigenesis. Here we investigated the in vivo effect of Klf5 heterozygosity on the propensity of ApcMin/KRASV12 double transgenic mice to develop intestinal tumors.

Results: At 12 weeks of age, ApcMin/KRASV12 mice had three times as many intestinal tumors as ApcMin mice. This increase in tumor number was reduced by 92% in triple transgenic ApcMin/KRASV12/Klf5+/- mice. The reduction in tumor number in ApcMin/KRASV12/Klf5+/- mice was also statistically significant compared to ApcMin mice alone, with a 75% decrease. Compared with ApcMin/KRASV12, tumors from both ApcMin/KRASV12/Klf5+/- and ApcMin mice were smaller. In addition, tumors from ApcMin mice were more distally distributed in the intestine as contrasted by the more proximal distribution in ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both ApcMin and ApcMin/KRASV12 mice but were attenuated in ApcMin/KRASV12/Klf5+/- mice. The levels of beta-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of ApcMin/KRASV12/Klf5+/- mice when compared to ApcMin/KRASV12 mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of ApcMin/KRASV12 mice and modestly reduced in ApcMin/KRASV12/Klf5+/- mice. Tumor tissues displayed higher levels of both Klf5 and beta-catenin, irrespective of the mouse genotype from which tumors were derived.

Conclusions: Results of the current study confirm the cumulative effect of Apc loss and oncogenic KRAS activation on intestinal tumorigenesis. The drastic reduction in tumor number and size due to Klf5 heterozygosity in ApcMin/KRASV12 mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.

Figure 1

The effect of Klf5 heterozygosity on intestinal tumor burden in Apc^Min/KRAS^V12 mutant mice at 12 weeks of age. Tumor burden is plotted as the number of tumors per mouse on the Y-axis. Tumor number from each individual mouse is shown as an open symbol, while the averages of the tumor numbers are represented as black bars. (A) Tumor burden in the small intestine; N = 8 for each genotype and **, P < 0.01. (B) Tumor burden in the colon; N = 8 for each genotype. (C) Overall tumor burden including both the small intestine and colon; N = 8 for each genotype and **, P < 0.01.

Figure 2

Assessment of intestinal tumor size and distribution in mutant mice. Percentages of intestinal tumors are displayed in bar graphs, with black bars representing Apc^Min mice, white bars representing Apc^Min/KRAS^V12 mice and gray bars indicating Apc^Min/KRAS^V12/Klf5^+/- mice. (A) Graph displaying tumor sizes in the small intestine. The tumors are sized based on 4 categories, <1 mm, 1-2 mm, 2-3 mm and >3 mm. Average percentages of tumors are represented on the Y-axis and tumor size categories on the X-axis; N = 8 and groups show a significant trend based on a one-way ANOVA test with P < 0.05. (B) Graph displaying tumor location in the small intestine. Tumor locations in the small intestine are divided into duodenum, jejunum and ileum. The graph is plotted with average percentage of tumors on the Y-axis and tumor locations on the X-axis; N = 8 and groups show a significant trend based on a one-way ANOVA test with P < 0.05.

Figure 3

Quantification of exogenous and endogenous KRAS transcript levels in the small intestine of mutant mice. KRAS transcript levels were measured using quantitative PCR analysis. RNA was extracted from paraffin-embedded intestinal tissue samples. Endogenous (mouse) and exogenous (human) KRAS expression was measured and compared against β-actin. Fold changes were calculated for KRAS levels against β-actin levels using the 2^-ΔΔCt method of relative quantification [60]. (A) Relative fold changes in exogenous (human) KRAS transcript levels in mutant mice compared to the wild type (WT) mice (designated as 1). (B) Relative fold changes in mouse Kras and human KRAS transcript levels in different regions (D = duodenum; J = jejunum; I = ileum) of the mutant mouse intestines.

Figure 4

Immunohistochemical analyses of Klf5 and β-catenin in the normal-appearing small intestines of wild type and mutant mice. The panels are representative sections of normal-appearing small intestinal tissues stained with Klf5 (A-D) or β-catenin antibodies (E-H). Formalin-fixed, paraffin-embedded tissue sections (5 μm in size) were deparaffinized and antigen-retrieved using Citrate buffer (pH 6.0). Sections were stained with appropriate primary and secondary antibodies and developed using DAB chromogen. The resulting brown color is representative of protein expression. The sections were also counter stained with hematoxylin, which stains the nuclei blue. Panels A & E represent Klf5 and β-catenin staining, respectively, in wild type (WT) normal intestinal tissues. Panels B & F show staining in Apc^Min tissues, while panels C & G and panels D & H show representative staining in Apc^Min/KRAS^V12 and Apc^Min/KRAS^V12/Klf5^+/- tissues, respectively.

Figure 5

Nuclear localization of β-catenin in the normal-appearing small intestines of wild type and mutant mice. Panels are magnified immunohistochemical images of representative small intestinal crypts stained with β-catenin antibodies. Red arrowheads in all the panels indicate nuclear β-catenin staining.

Figure 6

Immunohistochemical and Western blot analyses of cyclin D1 in the normal-appearing small intestinal tissues of wild type and mutant mice. (A-D) Immunohistochemical staining of cyclin D1 in the normal-appearing small intestines of wild type (WT) and mutant mice. A small focus of adenomatous tissue is demarcated by the red broken lines in panel D. (E) Quantification of cyclin D1 staining intensities in all fours sections using the Metamorph image analysis software. N = 10; **, P < 0.01. (F) Western blot analyses of Klf5, β-catenin, and cyclin D1 in the small intestines of wild type and mutant mice. Actin serves as a loading control.

Figure 7

Immunohistochemical analyses of Ki67 in the normal-appearing small intestinal tissues of wild type and mutant mice. (A-D) Immunohistochemical staining of Ki67 in the normal-appearing small intestines of wild type (WT) and mutant mice. (E) Quantification of Ki67 cyclin D1 staining intensities in all fours sections using the Metamorph image analysis software. N = 10; **, P < 0.01.

Figure 8

Phosphorylation of MEK and ERK in the normal-appearing small intestinal tissues of wild type and mutant mice. Immunohistochemical analyses were performed with phospho-Mek (pMek; A-D) and Phospho-Erk1/2 (pErk; E-H) antibodies. WT is wild type.

Figure 9

Immunostaining of Klf5 and β-catenin in intestinal adenomas derived from mutant mice. Adenomatous tissues from Apc^Min, Apc^Min/KRAS^V12 and Apc^Min/KRAS^V12/Klf5^+/- mice were formalin-fixed, paraffin-embedded and cut into 5 μm sections. Slides were then stained with Klf5 and β-catenin antibodies after deparaffinization and antigen-retrieval. Protein expression was determined upon secondary antibody treatment and color development using DAB chromogen (brown stain). Nuclei were then counterstained blue using hematoxylin. Panels A & D represent Klf5 and β-catenin staining in Apc^Min tumor tissues. Panels B & E represent comparative staining in Apc^Min/KRAS^V12 tumor tissues, while panels C & F represent staining in Apc^Min/KRAS^V12/Klf5^+/- tumor tissues.