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. 2010 Mar 18;9:63. doi: 10.1186/1476-4598-9-63

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

Mandayam O Nandan 1, Amr M Ghaleb 1, Beth B McConnell 1, Nilesh V Patel 1, Sylvie Robine 2, Vincent W Yang 1,3,✉
PMCID: PMC2856552  PMID: 20298593

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 β-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 β-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.

Background

Cancer is the result of deregulated cellular homeostasis and is typically characterized by increased proliferation and/or decreased apoptosis [1]. The mammalian intestinal epithelium is a continuously renewing system that is carefully orchestrated throughout life [2]. Several important signaling pathways are involved in maintaining intestinal epithelial homeostasis and include the Wnt, Notch, Eph/Ephrin, Hedgehog and bone morphogenetic protein (BMP) pathways [2]. It is well established that genetic perturbations in proliferation or differentiation of intestinal epithelial cells can lead to physiological changes which may aid in the development of colorectal cancer [3].

Specific mutations have been associated with colorectal carcinogenesis. RAS genes are one of the most frequently mutated oncogenes in human tumors and occur in approximately 50% of colon cancers [4,5]. There are three isoforms of the RAS gene, KRAS, HRAS and NRAS - however, a majority of human tumors possess mutations in the KRAS gene [3-8]. RAS is a membrane-bound protein that is activated by growth factors including epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) [9]. Upon activation, RAS becomes attached to GTP and elicits a signaling cascade that induces cell proliferation [10]. KRAS gene is indispensible for normal embryonic survival - targeted homozygous deletion of the mouse K-ras gene resulted in embryonic lethality between E12.5 and term [11,12]. In contrast, homozygous deletions in mouse H-r as or N-ras gene did not result in any significant phenotypic or viability changes [12-14].

Loss of heterozygosity (LOH) with consequent inactivation of tumor suppressor genes has been causally implicated in colon cancer formation [8]. One of the best-characterized tumor suppressor genes in colon cancer is the adenomatous polyposis coli (APC) gene. APC is part of the Wnt signaling pathway that regulates intestinal epithelial cell proliferation. Inactivation of APC causes nuclear translocation of normally membrane-bound β-catenin and subsequent activation of the β-catenin/TCF4 complex with resultant increased proliferation [15-17]. Patients with familial adenomatous polyposis (FAP) harbor heritable mutations in the APC gene and spontaneously develop adenomatous polyps throughout their intestinal tracts at an early age [18,19]. The APC gene is also inactivated in greater than 80% of sporadic colorectal cancer [20]. An autosomal dominant mouse model of multiple intestinal neoplasia (Min) was developed in C57BL/6 mice upon ethylnitrosourea treatment [21]. This mouse strain carries a germline mutation in the mouse Apc gene, resulting in truncation of the protein at amino acid position 850 [22]. As a result, ApcMin mice exhibit a phenotype similar to that of FAP patients [22].

Krüppel-like factors (KLFs) are zinc finger-containing, Sp1-like transcription factors that are involved in diverse physiological processes including proliferation, differentiation and embryonic development [23,24]. In the intestine, Krüppel-like factor 5 (KLF5) is predominantly expressed in the proliferating crypt epithelial cells [25,26]. KLF5 is important for embryonic development since homozygous deletion of Klf5 in mice is embryonic lethal [27]. We previously demonstrated that KLF5 has a pro-proliferative effect in cultured cells and does so by activating cell cycle regulatory proteins such as cyclin D1, cyclin B1 and Cdc2 [28,29]. In addition, KLF5 has been shown to be an important mediator of the HRAS and KRAS oncogenic pathways [28,30] as well as the Wnt pathway [31]. Adenomas and carcinomas in mice that express oncogenic KRASV12 from the intestine-specific villin promoter have increased KLF5 expression [30]. In addition, we recently showed that adenoma formation in ApcMin mice was significantly abrogated when ApcMin mice were bred to mice heterozygous for Klf5 [32]. We further showed that KLF5 interacts with β-catenin and facilitates the nuclear localization and transcriptional activity of β-catenin [32]. These studies suggest that KLF5 is an essential mediator of intestinal tumorigenesis in the context of ApcMin mutation.

Since KLF5 has been shown to mediate the function of both APC and RAS, and mutations in APC and KRAS are common events in colorectal cancer, we examined the role of KLF5 in mediating intestinal tumor formation in mice compound for ApcMin and intestine-specific KRASV12 mutations in the current study.

Results

Klf5 heterozygosity reduces intestinal adenoma formation in ApcMin/KRASV12 mice

To determine the effect of Klf5 heterozygosity on intestinal adenoma formation in mice that harbor both ApcMin and KRASV12 mutations, we crossed mice that were heterozygous for the ApcMin and Klf5 genes with those that were heterozygous for the KRASV12 gene directed by the intestine-specific villin promoter [33]. Intestines from the resulting progeny were assessed for tumor number and size at 12 weeks of age. Tumors were observed in mice from three genotypes of the resulting progeny (ApcMin, ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/-) but not in ApcMin/Klf5+/- or KRASV12mice. The mice with the compound ApcMin/KRASV12 genotype had a greater propensity for developing tumors in the small intestine than the ApcMin mice (Fig. 1A). The latter had an average of 71 small intestinal tumors per mouse while ApcMin/KRASV12 mice had an average of 226 tumors. The deletion of one of the Klf5 alleles in ApcMin/KRASV12 mice reduced the average tumor number to 19 per mouse - a 92% reduction (Fig. 1A). In the colon, the number of tumors per mouse was much fewer compared to the small intestine, with no significant differences in numbers of tumors between the three genotypes (Fig. 1B). Fig. 1C shows the combined tumor burden in both the small intestines and colons of the three different strains of mice.

Figure 1.

Figure 1

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The effect of Klf5 heterozygosity on intestinal tumor burden in ApcMin/KRASV12 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.

Haploinsufficiency of Klf5 decreases intestinal tumor size in ApcMin/KRASV12 mice

In addition to tumor number, we measured the tumor size from the mice described above. The majority of the tumors, irrespective of genotype, were less than 1 mm in size (Fig. 2A). However, the percentage of tumors that were smaller than 1 mm in ApcMin/KRASV12 mice (49% overall) was lower than either ApcMin (69% overall) or ApcMin/KRASV12/Klf5+/- (62% overall) mice. In contrast, ApcMin/KRASV12 mice had a higher percentage of tumors that were 1-2 mm in size (39%) when compared to ApcMin/KRASV12/Klf5+/- mice (33%) or ApcMin mice (28%) (Fig. 2A). Similarly, ApcMin/KRASV12 mice also displayed a greater number of tumors that were 2-3 mm or greater than 3 mm when compared to the other two genotypes. These differences in tumor size showed a statistically significant trend when analyzed by the Chi-square test.

Figure 2.

Figure 2

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Assessment of intestinal tumor size and distribution in mutant mice. Percentages of intestinal tumors are displayed in bar graphs, with black bars representing ApcMin mice, white bars representing ApcMin/KRASV12 mice and gray bars indicating ApcMin/KRASV12/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.

Change in intestinal tumor localization in mice that possess the KRASV12 genotype in addition to the ApcMin genotype

An interesting observation when comparing intestinal tumors among the different genotypes concerned the localization of the tumors. We observed that a larger percentage of tumors in ApcMin mice were localized in the distal small intestine, predominantly in the ileum (57%) and the jejunum (36%) (Fig. 2B). In contrast, both ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- mice contained a higher percentage of intestinal tumors in the proximal small intestine, duodenum (44% and 64%, respectively) when compared to the ApcMin mice (7%) (Fig. 2B). These differences were found to be statistically significant using the Chi-square test.

We then determined the level of KRAS transcripts in intestinal tissues from mice with the different genotypes using quantitative PCR. Both ApcMin/KRASV12 mice and ApcMin/KRASV12/Klf5+/- mice contained high levels of exogenous (human) KRAS mRNA in the intestine while wild type and ApcMin mice had only background expression (Fig. 3A). Since uneven KRAS expression could potentially contribute to the altered regional localization in the intestines of mice harboring KRASV12, we measured both endogenous (mouse) and exogenous (human) KRAS transcript levels in different segments of the intestine. We found that levels of exogenous KRAS transcripts were highly elevated in all three segments of the intestine of ApcMin/KRASV12 mice, with no significant regional differences (Fig. 3B). Similarly, no regional differences in the levels of endogenous Kras were found in the intestines of either ApcMin or ApcMin/KRASV12 mice (Fig. 3B).

Figure 3.

Figure 3

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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.

Klf5 heterozygosity results in reduced levels of pro-proliferative proteins in the intestines of ApcMin and ApcMin/KRASV12 mice

We previously showed that KLF5 is pro-proliferative in the normal intestinal epithelial cells [30,34] and is increased in tumors from mice that contain the ApcMin allele [32] or the KRASV12 allele [30]. Here we observed increased levels of Klf5 protein in the normal-appearing small intestinal tissues of both ApcMin and ApcMin/KRASV12 mice when compared to that of wild type mice (Fig. 4A-C). The introduction of a mutant Klf5 allele into ApcMin/KRASV12 mice resulted in a reduction in Klf5 (Fig. 4D) to a level that was more similar to the wild type intestine (Fig. 4A). Similarly, the levels of β-catenin were increased in the normal-appearing intestinal tissues of ApcMin and ApcMin/KRASV12 mice when compared to wild type mice (Fig. 4E-G). Again, this increase in β-catenin was attenuated in the ApcMin/KRASV12/Klf5+/- mice (Fig. 4H). Moreover, an increase in nuclear localized β-catenin was noted in the crypt epithelial cells of ApcMin and ApcMin/KRASV12 mice compared to wild type mice (Fig. 5A-C). Similar to total β-catenin, the number of crypt epithelial cells containing nuclear β-catenin was reduced in ApcMin/KRASV12/Klf5+/- mice relative to ApcMin and ApcMin/KRASV12 mice (Fig. 5D). These results indicate that Klf5 modulates both steady-state β-catenin levels and cellular localization of β-catenin in intestinal epithelial cells secondary to the ApcMin mutation.

Figure 4.

Figure 4

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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 ApcMin tissues, while panels C & G and panels D & H show representative staining in ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- tissues, respectively.

Figure 5.

Figure 5

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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.

We then performed immunohistochemical analyses on cyclin D1, a shared target between KLF5 and β-catenin [32]. Similar to the expression patterns of Klf5 and β-catenin, there was an increase in cyclin D1 levels in the intestine of both ApcMin and ApcMin/KRASV12 mice when compared to that of wild type mice (Fig. 6A-C). Cyclin D1 staining in the normal-appearing intestinal epithelium in ApcMin/KRASV12/Klf5+/- mice was reduced when compared to ApcMin and ApcMin/KRASV12 mice, except for a small focus of adenomatous tissue where cyclin D1 remained high (Fig. 6D). We also quantified cyclin D1 levels by quantitative image analysis (Fig. 6E) and Western blot analysis (Fig. 6F). As seen, both measurements confirmed the trend of cyclin D1 levels in the intestine from mice of the four genotypes as revealed by immunohistochemical staining. Similar trends in the levels of Klf5 and β-catenin were also documented by Western blot analysis (Fig. 6F). Lastly, levels of the proliferation marker, Ki67, in the normal-appearing intestinal tissues of the four strains of mice closely paralleled the levels of Klf5, β-catenin and cyclin D1, by immunohistochemical staining (Fig. 7A-D) and image quantification (Fig. 7E).

Figure 6.

Figure 6

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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.

Figure 7

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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.

The mitogen-activated kinase (MAPK) pathway is activated in the intestinal mucosa of ApcMin/KRASV12 mice

We previously established that MAPK pathway, as reflected by ERK phosphorylation, was an important intermediate in oncogenic KRAS-mediated induction of KLF5 [28,30]. Hence, we immunostained samples of small intestinal tissues for phospho-MEK and phospho-ERK proteins. We found that staining intensities for pMek were increased in normal-appearing small intestinal epithelial cells from both ApcMin and ApcMin/KRASV12 mice when compared to wild type mice (Fig. 8A-C). A moderate reduction in pMek staining was noted in the intestine of ApcMin/KRASV12/Klf5+/- mice compared to that of ApcMin/KRASV12 mice (Fig. 8C &8D). A similar pattern was also observed when pErk1/2 staining was performed (Fig. 8E-H). These results indicate that the MAPK pathway is activated in the intestine of ApcMin/KRASV12 mice and that Klf5 heterozygosity modestly reduces this activation.

Figure 8.

Figure 8

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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.

Intestinal tumors have increased Klf5 and β-catenin expression irrespective of genotype

We also stained intestinal tumors derived from ApcMin, ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- mice for Klf5 and β-catenin. As seen in Fig. 9, the levels of both Klf5 and β-catenin were elevated in the adenomatous tissues of all three strains compared to the normal-appearing intestinal tissues. These results indicate that despite the differences in expression among proliferative markers in the normal intestinal epithelia of the mutant mice, expression patterns of these markers are similar in tumor tissues irrespective of genotype.

Figure 9.

Figure 9

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Immunostaining of Klf5 and β-catenin in intestinal adenomas derived from mutant mice. Adenomatous tissues from ApcMin, ApcMin/KRASV12 and ApcMin/KRASV12/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 ApcMin tumor tissues. Panels B & E represent comparative staining in ApcMin/KRASV12 tumor tissues, while panels C & F represent staining in ApcMin/KRASV12/Klf5+/- tumor tissues.

Discussion

Colorectal cancer is the result of cumulative mutations in genes involved in regulating proliferation or apoptosis. APC is an integral part of the Wnt signaling pathway that regulates intestinal epithelial homeostasis [35]. Inactivation of APC is synonymous with Wnt activation and has been shown to be causal to colorectal carcinogenesis [35]. Also, among the frequently mutated genes in colorectal cancer is KRAS, specifically in codons 12, 13 and 61 [36-39]. It was shown that mutations in APC and KRAS occur in approximately 80% and 50%, respectively, of sporadic colorectal cancer [4,5,20]. Recent studies aimed at comprehensive sequencing of genes mutated in colorectal cancer confirmed that APC and KRAS mutations are among the most common mutations found in colorectal cancer [40,41].

Results of our study confirmed the cooperative effect of activated Wnt and RAS signaling in mice. At 12 weeks of age, compound heterozygous ApcMin/KRASV12 mice developed more and larger small intestinal tumors than ApcMin mice alone (Figs. 1A and 2A). In comparison, at the same age, KRASV12 mice did not have any tumor, consistent with the previous finding that these mice develop intestinal tumors relatively late in life [33]. This cooperative nature between Apc and KRAS mutations in leading to increased tumor formation is similar to that observed in two previous studies, one involving Apc+/1638/KRASV12 double transgenic mice [42] and the other ApcMin/K-rasD12 double transgenic mice [43].

While there was a trend for a higher number of colonic tumors in the ApcMin/KRASV12 as compared to ApcMin mice alone in our study (Fig. 1B), the difference did not reach statistical significance, due to the relatively small number of tumors in this region. The propensity for the ApcMin, Apc+/1638, KRASV12, Apc+/1638/KRASV12 mice to develop tumors in the small intestine rather than the colon has been reported [21,33,42]. It is of interest to note that there is a difference in regional distribution of small bowel tumors between ApcMin and ApcMin/KRASV12 mice - tumors in the former mice were more distally distributed while those in the latter were more proximally distributed (Fig. 2B). This difference in tumor distribution does not appear to be due to regional variations in expression of the KRASV12 transgene from the villin promoter (Fig. 3B). The effect of KRASV12 allele introduction on the shift in tumor distribution more proximally is therefore not clear at this time. A similar trend toward distribution of small bowel tumors in the ApcMin mice has been reported [44].

We recently reported the critical role for Klf5 in tumor initiation in ApcMin mice [32]. Klf5 haploinsufficiency in ApcMin mice resulted in a significant decrease in tumor number and size [32]. Results of the current study demonstrate a similar effect on tumor formation at 12 weeks of age in ApcMin/KRASV12 mice that were heterozygous for the Klf5 alleles, with the intestinal tumor burden reduced by more than 90% in the triple ApcMin/KRASV12/Klf5+/- transgenic mice when compared to the double ApcMin/KRASV12 transgenic mice (Fig. 1). In addition, the tumors in the ApcMin/KRASV12/Klf5+/- mice, when formed, were smaller than those from the ApcMin/KRASV12 mice (Fig. 2A). Indeed, ApcMin/KRASV12 mice had to be euthanized by 12 weeks of age, due to the presence of rectal prolapse from the large tumor burden. In contrast, the majority of ApcMin/KRASV12/Klf5+/- mice survived up to a year without displaying overt morbidity. Taken into consideration that expression of the KRASV12 transgene in the small intestine of ApcMin/KRASV12/Klf5+/- mice remains robust (Fig. 3A), our study suggests that haploinsufficiency of Klf5 attenuates the cumulative effect of Apc inactivation and oncogenic KRAS activation.

Our results show that a combined effect of ApcMin and KRASV12 mutations is a significant increase in the levels of β-catenin, cyclin D1 and Ki67, in the normal-appearing intestinal tissues in the ApcMin/KRASV12 mice as compared to wild type mice (Figs. 4, 5, 6, 7). This increase is similar to that seen in the intestine from the ApcMin mice (Figs. 4, 5, 6, 7). Haploinsufficiency of Klf5 attenuated the increase in the levels of these three proteins in the normal-appearing intestine of ApcMin/KRASV12 mice to levels that resembled the wild type intestine (Figs. 4, 5, 6, 7). These results indicate that the increase in β-catenin and cyclin D1 levels in the intestine of mutant mice is primarily a consequence of ApcMin mutation, rather than KRASV12 over-expression and that the tumor suppressive effect of Klf5 haploinsufficiency in ApcMin/KRASV12 mice is due primarily to the ability of Klf5 to modulate ApcMin signaling. These notions are supported by the observation that increased nuclear localization of β-catenin is observed in the normal-appearing intestinal crypt epithelial cells of both ApcMin and ApcMin/KRASV12 mice but was significantly reduced in the crypt cells of ApcMin/KRASV12/Klf5+/- mice (Fig. 5). The se findings are consistent with our previous observation that Klf5 both stabilizes β-catenin and facilitates nuclear import of β-catenin [32]. However, it should be noted that a recent report showed that activated KRAS also facilitates nuclear translocation of β-catenin following loss of Apc in zebrafish [45]. Moreover, we have shown that KRASV12 increases KLF5 expression in vitro and in vivo [30]. Combining the results of these studies, it is highly plausible that KLF5 is a common mediator for the increased β-catenin activity due to both APC loss and KRAS activation.

MEK and ERK phosphorylation are hallmarks of activation of the RAS signaling pathway which stimulates cell proliferation [46]. We previously reported that MEK/ERK phosphorylation is essential for mediating oncogenic RAS-induced KLF5 expression in vitro [28,30]. Previous studies have documented enhanced MEK/ERK protein phosphorylation in mice containing both oncogenic KRAS mutations and Apc inactivation [47,48]. Results of the current study showed a similar increase in MEK/ERK phosphorylation in the normal-appearing intestines of mice with ApcMin mutation that is further enhanced upon oncogenic KRAS activation (Fig. 8). Upon heterozygous loss of Klf5 in ApcMin/KRASV12 mice, MEK/ERK phosphorylation levels are only modestly reduced. These results suggest that RAS activation of MEK/ERK phosphorylation is upstream of KLF5 induction, although KLF5 could potentially regulate MEK/ERK phosphorylation through a feedback mechanism, as previously proposed [49].

Our study adds to a growing list of literature demonstrating the combined effect of Apc and KRAS mutation on intestinal tumorigenesis in mice [42,43,50,51]. In the setting of Apc mutation, inhibition of intestinal tumor formation has been documented secondary to deletion of several genes crucial for tumorigenesis [32,52-56]. However, ours is the first in which to show a critical role of Klf5 in mediating the tumorigenic effect of combined Apc and KRAS mutations, a commonly encountered scenario in colorectal cancer in humans. This suggests that therapies targeted to KLF5 may have potential therapeutic benefit to patients with colorectal cancer. Indeed, a recent screen for small molecule inhibitors of KLF5 expression has yielded several potent compounds that inhibit proliferation of colorectal cancer cells [57]. Further investigation may prove KLF5 an attractive target for intervention in the prevention or treatment of colorectal cancer.

Conclusions

Loss of tumor suppressor genes and activation of oncogenes are hallmarks of cancers. In the case of colorectal cancer, loss of APC and activation of KRAS are common. Here, we present a robust mouse model of intestinal tumorigenesis with the generation of ApcMin/KRASV12 mice. These mice display an increased propensity for developing intestinal tumors at an early age compared to ApcMin mice. Moreover, we were able to significantly reduce tumor burden and size in the compound ApcMin/KRASV12 mice by reducing expression of Klf5 with genetic means. ApcMin/KRASV12/Klf5+/- mice display reduced levels of Klf5 protein as well as β-catenin, cyclin D1 and Ki67, all known markers of proliferation and transformation. We conclude that Klf5 is a crucial mediator of initiation and progression of intestinal tumors resulted from ApcMin and KRASV12 mutations.

Methods

Reagents

Antibodies used in the experiments were previously described [30,32]. Antibodies against KLF5 were generated against a synthetic KLF5 peptide in rabbits (Strategic Diagnostics, Newark, DE). Anti-KLF5 antibody was used at a dilution of 1:15,000 for immunohistochemistry and at 1:4,000 for Western blot analysis. Mouse monoclonal antibody against total β-catenin was purchased from Invitrogen (Carlsbad, CA) and used at a dilution of 1:1,000 for Western blot analyses. For immunohistochemical analysis, total β-catenin antibodies purchased from BD Biosciences (San Jose, CA) were used at 1:250 dilutions. Rabbit monoclonal cyclin D1 antibodies were purchased from Biocare Medical (Concord, CA) and used at 1:200 dilutions in immunohistochemical analyses and 1:2,500 dilutions for Western blot analysis. Anti-Ki67 antibodies were purchased from Novocastra (Leica Microsystems, Bannockburn, IL) and used at 1:500 dilutions. Anti-Phospho-MEK1 and anti-Phospho-ERK1/2 antibodies, used at 1:100 dilutions, were purchased from Cell Signaling Technology (Danvers, MA).

Mice

All studies involving mice have been approved by the Emory University Institutional Animal Care and Use Committee (IACUC). C57BL/6J mice heterozygous for KRASV12 expressed from a mouse villin promoter were previously generated [33]. Mice double heterozygous for ApcMin and Klf5+/- alleles were generated as previously described [32]. Founder C57BL/6J mice that were heterozygous ApcMin alleles (males) were mated with those that were heterozygous for Klf5+/- alleles (females). The resulting progeny generated double heterozygous ApcMin/Klf5+/- mice. These mice were then mated with the KRASV12 mice to generate the triple transgenic mice used in this study. Littermates of the crosses consisted of mice wild type for all alleles, mice that were heterozygous for only one of the three alleles, mice with two heterozygous alleles and mice with all three heterozygous alleles. Out of this progeny wild type, ApcMin, ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- mice were used for the study.

Genotype analyses

Genotype analyses were performed as previously described [58]. Tail-tips from newly weaned mice were collected and processed using the Red Extract-N-Amp kit as per protocol (Sigma Aldrich, St. Louis, MO). Allele-specific PCR analyses were performed using 2 μl of mouse DNA and appropriate primers for genotypic analyses. Primers to identify KLF5, ApcMin mutation, and villin-KRAS have been previously described [33,58,59].

Tumor assessment

Mice were sacrificed at 12 weeks of age by CO2 asphyxiation, as per IACUC guidelines. The mice were dissected and the small intestine and colon removed. The intestinal tissues were cleaned with phosphate-buffered saline (PBS) and cut open. Using a dissecting microscope, the intestinal tissues were examined in a blinded fashion, for the presence and size measurements of tumors. The adenomas found were counted and measured according to <1 mm, 1-2 mm, 2-3 mm and >3 mm size groups.

RNA purification and quantitative PCR

RNA was extracted from formalin-fixed paraffin-embedded tissue samples using the RT2 FFPE RNA extraction kit (SA Biosciences, Frederick, MD). Sixty μm tissue sections were cut from paraffin sample blocks and digested with Proteinase K for 30 minutes. Samples were then boiled and centrifuged to remove paraffin. RNA was extracted from the liquid samples using Trizol LS reagent (Invitrogen, Carlsbad, CA) and subsequently purified using a spin column. RNA was quantified and used (100 ng/sample) in quantitative PCR. Specific primers against mouse KRas, human KRAS and mouse β-actin were purchased from SA Biosciences (Frederick, MD) and Qiagen (Valencia, CA) respectively. Quantitative PCR was performed using the Power SYBR Green RNA-to-CT 1-Step kit (Invitrogen, Carlsbad, CA) as per protocol. Observed CT levels were then used to calculate fold change using the 2-ΔΔCt method of relative quantification [60].

Immunohistochemistry

Immunohistochemical analysis was performed as previously described [30]. Intestinal tissues dissected from mice were fixed overnight with 10% formalin buffer (Thermo Fisher Scientific, Fair Lawn, NJ). The tissues were then paraffinized using a tissue paraffinizer (Shandon Excelsior and Histocenter, Thermo Scientific, NJ). The paraffinized tissues were embedded onto paraffin blocks and cut into 5 μm sections using a microtome (Microm, Thermo Scientific, NJ). The sections were then dried onto charged slides and used for staining. The slides containing paraffin-embedded tissue sections were deparaffinized by baking in a 60°C oven for 1 hr and subsequent incubation in a xylene bath. Sections were incubated in a 5% hydrogen peroxide bath to block endogenous tissue peroxidases. The sections were then hydrated by incubation in a decreasing alcohol bath series (100%, 95%, 70%) followed by antigen retrieval in citrate buffer solution (10 mM Sodium citrate, 0.05% Tween-20, pH 6.0) at 125°C for 10 min using a decloaking chamber (Biocare Medical, CA). Tissue sections were then incubated with blocking buffer containing avidin (2% milk, 0.05% Tween-20, 5% normal serum) for 30 min at 37°C. Subsequently, antibodies, with Biotin, were added to the blocking buffer at appropriate concentrations and incubated with tissue sections for 1 hr at 37°C. Sections were washed and incubated with secondary antibodies at the appropriate concentration for 30 min at 37°C. Vectorstain ABC solution (Vector Labs, Burlingame, CA) and Betazoid DAB (Biocare Medical, CA) were used to reveal staining in tissues. The sections were then incubated in Gill's Hematoxylin (Vector Labs, CA), dehydrated and cover-slipped for observation. Slides were observed under a Zeiss Axioskop (Carl Zeiss MicroImaging, Thornwood, NY) and representative pictures taken.

Quantification of immunohistochemical staining intensity

Staining intensities for immunohistochemical analyses were quantifies using Metamorph image analysis software (Version 7.1.1) (Molecular Devices, Downington, PA). Individual images were specifically quantified as previously described [30].

Western blot analyses

Western blot analyses were performed as previously described [28]. Proteins were extracted from 20 μm paraffin embedded tissue sections using a previously established protocol [61]. Tissue sections were deparaffinized using xylene with the addition of 7.5% methanol. Samples were then centrifuged and the pellet dried in a fume hood for 3 min. The pellets were then resuspended in 20 mM Tris-HCl (pH 7.5) containing 2% SDS and the suspension heated in a 100°C heat block for 20 min. Subsequently, the samples were incubated in a 60°C oven for 2 hr. Protein content was measured and equal amounts of samples were loaded onto Bis-Tris gels (Invitrogen, CA). Proteins were transferred to nitrocellulose membranes (BioRad, Hercules, CA) and probed with appropriate primary antibodies. Blots were then washed and secondary antibodies applied at appropriate concentrations. Protein bands were then visualized on film upon chemiluminescent detection.

Statistical analysis

A one-way ANOVA was used to compare mean numbers of tumors between ApcMin, ApcMin/KRASV12, and ApcMin/KRASV12/Klf5+/- mice given independence of samples, equality of variances as tested by Levene's test, and a Gaussian distribution of the data. Multiple pair wise comparisons were made among groups using Tukey's test. Tumors were categorized based on size into 4 ordinal categories (<1 mm, 1-2 mm, 2-3 mm, and greater than 3 mm) using previously published measurement protocols [30]. Proportions of tumors among size categories were compared between ApcMin, ApcMin/KRASV12, and ApcMin/KRASV12/Klf5+/- mice using a Chi-square test for homogeneity. P < 0.05 was considered indicative of statistical significance. Similar methods were used to ascertain statistical significance in relation to tumor location. The statistical software package SAS 9.2 was used for statistical analysis.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MON and VWY conceived the design of the study and participated in drafting the manuscript. MON performed the immunohistochemical and Western blot analyses. AMG and MON were involved in the assessment of tumor burden and sizing from mice. BBM and AMG helped in providing transgenic mice and with the setup of immunohistochemical analyses. NVP performed statistical analyses on mice data. SR provided critical reagents and advised on the study design. All authors read and approved the final manuscript.

Contributor Information

Mandayam O Nandan, Email: mnandan@emory.edu.

Amr M Ghaleb, Email: aghaleb@emory.edu.

Beth B McConnell, Email: bmccon2@emory.edu.

Nilesh V Patel, Email: nvpatel@learnlink.emory.edu.

Sylvie Robine, Email: Sylvie.Robine@curie.fr.

Vincent W Yang, Email: vyang@emory.edu.

Acknowledgements

This work was in part supported by grants from the National Institutes of Health (DK52230, DK64399, and CA84197 to VWY; CA130138 to AMG; and DK76742 to BBM) and the Atlanta Clinical and Translational Science Institute (RR25010 and RR25008 to NVP).

References

  1. Green DR, Evan GI. A matter of life and death. Cancer Cell. 2002;1:19–30. doi: 10.1016/S1535-6108(02)00024-7. [DOI] [PubMed] [Google Scholar]
  2. Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet. 2006;7:349–359. doi: 10.1038/nrg1840. [DOI] [PubMed] [Google Scholar]
  3. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759–767. doi: 10.1016/0092-8674(90)90186-I. [DOI] [PubMed] [Google Scholar]
  4. Bos JL, Fearon ER, Hamilton SR, Verlaan-de Vries M, van Boom JH, Eb AJ van der, Vogelstein B. Prevalence of ras gene mutations in human colorectal cancers. Nature. 1987;327:293–297. doi: 10.1038/327293a0. [DOI] [PubMed] [Google Scholar]
  5. Forrester K, Almoguera C, Han K, Grizzle WE, Perucho M. Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. Nature. 1987;327:298–303. doi: 10.1038/327298a0. [DOI] [PubMed] [Google Scholar]
  6. Rodenhuis S, Wetering ML van de, Mooi WJ, Evers SG, van Zandwijk N, Bos JL. Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung. N Engl J Med. 1987;317:929–935. doi: 10.1056/NEJM198710083171504. [DOI] [PubMed] [Google Scholar]
  7. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell. 1988;53:549–554. doi: 10.1016/0092-8674(88)90571-5. [DOI] [PubMed] [Google Scholar]
  8. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;319:525–532. doi: 10.1056/NEJM198809013190901. [DOI] [PubMed] [Google Scholar]
  9. Luttrell LM, Daaka Y, Lefkowitz RJ. Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol. 1999;11:177–183. doi: 10.1016/S0955-0674(99)80023-4. [DOI] [PubMed] [Google Scholar]
  10. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003;3:459–465. doi: 10.1038/nrc1097. [DOI] [PubMed] [Google Scholar]
  11. Koera K, Nakamura K, Nakao K, Miyoshi J, Toyoshima K, Hatta T, Otani H, Aiba A, Katsuki M. K-ras is essential for the development of the mouse embryo. Oncogene. 1997;15:1151–1159. doi: 10.1038/sj.onc.1201284. [DOI] [PubMed] [Google Scholar]
  12. Esteban LM, Vicario-Abejon C, Fernandez-Salguero P, Fernandez-Medarde A, Swaminathan N, Yienger K, Lopez E, Malumbres M, McKay R, Ward JM. Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development. Mol Cell Biol. 2001;21:1444–1452. doi: 10.1128/MCB.21.5.1444-1452.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Umanoff H, Edelmann W, Pellicer A, Kucherlapati R. The murine N-ras gene is not essential for growth and development. Proc Natl Acad Sci USA. 1995;92:1709–1713. doi: 10.1073/pnas.92.5.1709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ise K, Nakamura K, Nakao K, Shimizu S, Harada H, Ichise T, Miyoshi J, Gondo Y, Ishikawa T, Aiba A, Katsuki M. Targeted deletion of the H-ras gene decreases tumor formation in mouse skin carcinogenesis. Oncogene. 2000;19:2951–2956. doi: 10.1038/sj.onc.1203600. [DOI] [PubMed] [Google Scholar]
  15. Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science. 1996;272:1023–1026. doi: 10.1126/science.272.5264.1023. [DOI] [PubMed] [Google Scholar]
  16. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science. 1997;275:1784–1787. doi: 10.1126/science.275.5307.1784. [DOI] [PubMed] [Google Scholar]
  17. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275:1787–1790. doi: 10.1126/science.275.5307.1787. [DOI] [PubMed] [Google Scholar]
  18. Bodmer WF, Bailey CJ, Bodmer J, Bussey HJ, Ellis A, Gorman P, Lucibello FC, Murday VA, Rider SH, Scambler P. Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature. 1987;328:614–616. doi: 10.1038/328614a0. [DOI] [PubMed] [Google Scholar]
  19. Leppert M, Dobbs M, Scambler P, O'Connell P, Nakamura Y, Stauffer D, Woodward S, Burt R, Hughes J, Gardner E. The gene for familial polyposis coli maps to the long arm of chromosome 5. Science. 1987;238:1411–1413. doi: 10.1126/science.3479843. [DOI] [PubMed] [Google Scholar]
  20. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell. 1996;87:159–170. doi: 10.1016/S0092-8674(00)81333-1. [DOI] [PubMed] [Google Scholar]
  21. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science. 1990;247:322–324. doi: 10.1126/science.2296722. [DOI] [PubMed] [Google Scholar]
  22. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C, Gould KA, Dove WF. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science. 1992;256:668–670. doi: 10.1126/science.1350108. [DOI] [PubMed] [Google Scholar]
  23. Dang DT, Pevsner J, Yang VW. The biology of the mammalian Kruppel-like family of transcription factors. Int J Biochem Cell Biol. 2000;32:1103–1121. doi: 10.1016/S1357-2725(00)00059-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nandan MO, Yang VW. The role of Kruppel-like factors in the reprogramming of somatic cells to induced pluripotent stem cells. Histol Histopathol. 2009;24:1343–1355. doi: 10.14670/hh-24.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ghaleb AM, Nandan MO, Chanchevalap S, Dalton WB, Hisamuddin IM, Yang VW. Kruppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation. Cell Res. 2005;15:92–96. doi: 10.1038/sj.cr.7290271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McConnell BB, Ghaleb AM, Nandan MO, Yang VW. The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology. Bioessays. 2007;29:549–557. doi: 10.1002/bies.20581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shindo T, Manabe I, Fukushima Y, Tobe K, Aizawa K, Miyamoto S, Kawai-Kowase K, Moriyama N, Imai Y, Kawakami H. Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling. Nat Med. 2002;8:856–863. doi: 10.1038/nm738. [DOI] [PubMed] [Google Scholar]
  28. Nandan MO, Yoon HS, Zhao W, Ouko LA, Chanchevalap S, Yang VW. Kruppel-like factor 5 mediates the transforming activity of oncogenic H-Ras. Oncogene. 2004;23:3404–3413. doi: 10.1038/sj.onc.1207397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nandan MO, Chanchevalap S, Dalton WB, Yang VW. Kruppel-like factor 5 promotes mitosis by activating the cyclin B1/Cdc2 complex during oncogenic Ras-mediated transformation. FEBS Lett. 2005;579:4757–4762. doi: 10.1016/j.febslet.2005.07.053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nandan MO, McConnell BB, Ghaleb AM, Bialkowska AB, Sheng H, Shao J, Babbin BA, Robine S, Yang VW. Kruppel-like factor 5 mediates cellular transformation during oncogenic KRAS-induced intestinal tumorigenesis. Gastroenterology. 2008;134:120–130. doi: 10.1053/j.gastro.2007.10.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ziemer LT, Pennica D, Levine AJ. Identification of a mouse homolog of the human BTEB2 transcription factor as a beta-catenin-independent Wnt-1-responsive gene. Mol Cell Biol. 2001;21:562–574. doi: 10.1128/MCB.21.2.562-574.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McConnell BB, Bialkowska AB, Nandan MO, Ghaleb AM, Gordon FJ, Yang VW. Haploinsufficiency of Kruppel-like factor 5 rescues the tumor-initiating effect of the Apc(Min) mutation in the intestine. Cancer Res. 2009;69:4125–4133. doi: 10.1158/0008-5472.CAN-08-4402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Janssen KP, el-Marjou F, Pinto D, Sastre X, Rouillard D, Fouquet C, Soussi T, Louvard D, Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice. Gastroenterology. 2002;123:492–504. doi: 10.1053/gast.2002.34786. [DOI] [PubMed] [Google Scholar]
  34. Chanchevalap S, Nandan MO, McConnell BB, Charrier L, Merlin D, Katz JP, Yang VW. Kruppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells. Nucleic Acids Res. 2006;34:1216–1223. doi: 10.1093/nar/gkl014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell. 2000;103:311–320. doi: 10.1016/S0092-8674(00)00122-7. [DOI] [PubMed] [Google Scholar]
  36. Capon DJ, Seeburg PH, McGrath JP, Hayflick JS, Edman U, Levinson AD, Goeddel DV. Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations. Nature. 1983;304:507–513. doi: 10.1038/304507a0. [DOI] [PubMed] [Google Scholar]
  37. McCoy MS, Toole JJ, Cunningham JM, Chang EH, Lowy DR, Weinberg RA. Characterization of a human colon/lung carcinoma oncogene. Nature. 1983;302:79–81. doi: 10.1038/302079a0. [DOI] [PubMed] [Google Scholar]
  38. Bos JL, Toksoz D, Marshall CJ, Verlaan-de Vries M, Veeneman GH, Eb AJ van der, van Boom JH, Janssen JW, Steenvoorden AC. Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia. Nature. 1985;315:726–730. doi: 10.1038/315726a0. [DOI] [PubMed] [Google Scholar]
  39. Srivastava SK, Yuasa Y, Reynolds SH, Aaronson SA. Effects of two major activating lesions on the structure and conformation of human ras oncogene products. Proc Natl Acad Sci USA. 1985;82:38–42. doi: 10.1073/pnas.82.1.38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N. The consensus coding sequences of human breast and colorectal cancers. Science. 2006;314:268–274. doi: 10.1126/science.1133427. [DOI] [PubMed] [Google Scholar]
  41. Wood LD, Parsons DW, Jones S, Lin J, Sjoblom T, Leary RJ, Shen D, Boca SM, Barber T, Ptak J. The genomic landscapes of human breast and colorectal cancers. Science. 2007;318:1108–1113. doi: 10.1126/science.1145720. [DOI] [PubMed] [Google Scholar]
  42. Janssen KP, Alberici P, Fsihi H, Gaspar C, Breukel C, Franken P, Rosty C, Abal M, El Marjou F, Smits R. APC and oncogenic KRAS are synergistic in enhancing Wnt signaling in intestinal tumor formation and progression. Gastroenterology. 2006;131:1096–1109. doi: 10.1053/j.gastro.2006.08.011. [DOI] [PubMed] [Google Scholar]
  43. Luo F, Brooks DG, Ye H, Hamoudi R, Poulogiannis G, Patek CE, Winton DJ, Arends MJ. Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways. Int J Exp Pathol. 2009;90:558–574. doi: 10.1111/j.1365-2613.2009.00667.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Haigis KM, Hoff PD, White A, Shoemaker AR, Halberg RB, Dove WF. Tumor regionality in the mouse intestine reflects the mechanism of loss of Apc function. Proc Natl Acad Sci USA. 2004;101:9769–9773. doi: 10.1073/pnas.0403338101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Phelps RA, Chidester S, Dehghanizadeh S, Phelps J, Sandoval IT, Rai K, Broadbent T, Sarkar S, Burt RW, Jones DA. A two-step model for colon adenoma initiation and progression caused by APC loss. Cell. 2009;137:623–634. doi: 10.1016/j.cell.2009.02.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol. 1997;9:180–186. doi: 10.1016/S0955-0674(97)80061-0. [DOI] [PubMed] [Google Scholar]
  47. Haigis KM, Kendall KR, Wang Y, Cheung A, Haigis MC, Glickman JN, Niwa-Kawakita M, Sweet-Cordero A, Sebolt-Leopold J, Shannon KM. Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. Nat Genet. 2008;40:600–608. doi: 10.1038/ng.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Trobridge P, Knoblaugh S, Washington MK, Munoz NM, Tsuchiya KD, Rojas A, Song X, Ulrich CM, Sasazuki T, Shirasawa S, Grady WM. TGF-beta receptor inactivation and mutant Kras induce intestinal neoplasms in mice via a beta-catenin-independent pathway. Gastroenterology. 2009;136:1680–1688. doi: 10.1053/j.gastro.2009.01.066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yang Y, Goldstein BG, Nakagawa H, Katz JP. Kruppel-like factor 5 activates MEK/ERK signaling via EGFR in primary squamous epithelial cells. FASEB J. 2007;21:543–550. doi: 10.1096/fj.06-6694com. [DOI] [PubMed] [Google Scholar]
  50. Sansom OJ, Meniel V, Wilkins JA, Cole AM, Oien KA, Marsh V, Jamieson TJ, Guerra C, Ashton GH, Barbacid M, Clarke AR. Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo. Proc Natl Acad Sci USA. 2006;103:14122–14127. doi: 10.1073/pnas.0604130103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Hung KE, Maricevich MA, Richard LG, Chen WY, Richardson MP, Kunin A, Bronson RT, Mahmood U, Kucherlapati R. Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment. Proc Natl Acad Sci USA. 2010;107:1565–1570. doi: 10.1073/pnas.0908682107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Rakoff-Nahoum S, Medzhitov R. Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88. Science. 2007;317:124–127. doi: 10.1126/science.1140488. [DOI] [PubMed] [Google Scholar]
  53. Yekkala K, Baudino TA. Inhibition of intestinal polyposis with reduced angiogenesis in ApcMin/+ mice due to decreases in c-Myc expression. Mol Cancer Res. 2007;5:1296–1303. doi: 10.1158/1541-7786.MCR-07-0232. [DOI] [PubMed] [Google Scholar]
  54. Ramocki NM, Wilkins HR, Magness ST, Simmons JG, Scull BP, Lee GH, McNaughton KK, Lund PK. Insulin receptor substrate-1 deficiency promotes apoptosis in the putative intestinal crypt stem cell region, limits Apcmin/+ tumors, and regulates Sox9. Endocrinology. 2008;149:261–267. doi: 10.1210/en.2007-0869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wilkins JA, Sansom OJ. C-Myc is a critical mediator of the phenotypes of Apc loss in the intestine. Cancer Res. 2008;68:4963–4966. doi: 10.1158/0008-5472.CAN-07-5558. [DOI] [PubMed] [Google Scholar]
  56. Zeilstra J, Joosten SP, Dokter M, Verwiel E, Spaargaren M, Pals ST. Deletion of the WNT target and cancer stem cell marker CD44 in Apc(Min/+) mice attenuates intestinal tumorigenesis. Cancer Res. 2008;68:3655–3661. doi: 10.1158/0008-5472.CAN-07-2940. [DOI] [PubMed] [Google Scholar]
  57. Bialkowska AB, Du Y, Fu H, Yang VW. Identification of novel small-molecule compounds that inhibit the proproliferative Kruppel-like factor 5 in colorectal cancer cells by high-throughput screening. Mol Cancer Ther. 2009;8:563–570. doi: 10.1158/1535-7163.MCT-08-0767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. McConnell BB, Klapproth JM, Sasaki M, Nandan MO, Yang VW. Kruppel-like factor 5 mediates transmissible murine colonic hyperplasia caused by Citrobacter rodentium infection. Gastroenterology. 2008;134:1007–1016. doi: 10.1053/j.gastro.2008.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Dietrich WF, Lander ES, Smith JS, Moser AR, Gould KA, Luongo C, Borenstein N, Dove W. Genetic identification of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse. Cell. 1993;75:631–639. doi: 10.1016/0092-8674(93)90484-8. [DOI] [PubMed] [Google Scholar]
  60. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  61. Shi SR, Liu C, Balgley BM, Lee C, Taylor CR. Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry. J Histochem Cytochem. 2006;54:739–743. doi: 10.1369/jhc.5B6851.2006. [DOI] [PubMed] [Google Scholar]

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