- Open Access
Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined Apc Min and KRASV 12mutations
© Nandan et al; licensee BioMed Central Ltd. 2010
- Received: 3 November 2009
- Accepted: 18 March 2010
- Published: 18 March 2010
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 Apc Min mutation or intestine-specific expression of the KRASV 12gene 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 Apc Min /KRASV 12double transgenic mice to develop intestinal tumors.
At 12 weeks of age, Apc Min /KRASV 12mice had three times as many intestinal tumors as Apc Min mice. This increase in tumor number was reduced by 92% in triple transgenic Apc Min /KRASV 12/Klf5+/- mice. The reduction in tumor number in Apc Min /KRASV 12/Klf5+/- mice was also statistically significant compared to Apc Min mice alone, with a 75% decrease. Compared with Apc Min /KRASV 12, tumors from both Apc Min /KRASV 12/Klf5+/- and Apc Min mice were smaller. In addition, tumors from Apc Min mice were more distally distributed in the intestine as contrasted by the more proximal distribution in Apc Min /KRASV 12and Apc Min /KRASV 12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both Apc Min and Apc Min /KRASV 12mice but were attenuated in Apc Min /KRASV 12/Klf5+/- mice. The levels of β-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of Apc Min /KRASV 12/Klf5+/- mice when compared to Apc Min /KRASV 12mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of Apc Min /KRASV 12mice and modestly reduced in ApcMin/KRASV 12/Klf5+/- mice. Tumor tissues displayed higher levels of both Klf5 and β-catenin, irrespective of the mouse genotype from which tumors were derived.
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 Apc Min /KRASV 12mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.
- KRAS Mutation
- Intestinal Tumor
- Adenomatous Polyposis Coli Gene
- KLF5 Expression
- Intestinal Tumorigenesis
Cancer is the result of deregulated cellular homeostasis and is typically characterized by increased proliferation and/or decreased apoptosis . The mammalian intestinal epithelium is a continuously renewing system that is carefully orchestrated throughout life . 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 . 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 .
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) . Upon activation, RAS becomes attached to GTP and elicits a signaling cascade that induces cell proliferation . 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 . 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 . An autosomal dominant mouse model of multiple intestinal neoplasia (Min) was developed in C57BL/6 mice upon ethylnitrosourea treatment . This mouse strain carries a germline mutation in the mouse Apc gene, resulting in truncation of the protein at amino acid position 850 . As a result, Apc Min mice exhibit a phenotype similar to that of FAP patients .
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 . 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 . Adenomas and carcinomas in mice that express oncogenic KRASV 12from the intestine-specific villin promoter have increased KLF5 expression . In addition, we recently showed that adenoma formation in Apc Min mice was significantly abrogated when Apc Min mice were bred to mice heterozygous for Klf5. We further showed that KLF5 interacts with β-catenin and facilitates the nuclear localization and transcriptional activity of β-catenin . These studies suggest that KLF5 is an essential mediator of intestinal tumorigenesis in the context of Apc Min 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 Apc Min and intestine-specific KRASV 12mutations in the current study.
Klf5 heterozygosity reduces intestinal adenoma formation in Apc Min /KRAS V12 mice
Haploinsufficiency of Klf5 decreases intestinal tumor size in Apc Min /KRAS V12 mice
Change in intestinal tumor localization in mice that possess the KRAS V12 genotype in addition to the Apc Min 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 Apc Min mice were localized in the distal small intestine, predominantly in the ileum (57%) and the jejunum (36%) (Fig. 2B). In contrast, both Apc Min /KRASV 12and Apc Min /KRASV 12/Klf5+/- mice contained a higher percentage of intestinal tumors in the proximal small intestine, duodenum (44% and 64%, respectively) when compared to the Apc Min mice (7%) (Fig. 2B). These differences were found to be statistically significant using the Chi-square test.
Klf5 heterozygosity results in reduced levels of pro-proliferative proteins in the intestines of Apc Min and Apc Min /KRAS V12 mice
The mitogen-activated kinase (MAPK) pathway is activated in the intestinal mucosa of Apc Min /KRAS V12 mice
Intestinal tumors have increased Klf5 and β-catenin expression irrespective of genotype
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 . Inactivation of APC is synonymous with Wnt activation and has been shown to be causal to colorectal carcinogenesis . 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 Apc Min /KRASV 12mice developed more and larger small intestinal tumors than Apc Min mice alone (Figs. 1A and 2A). In comparison, at the same age, KRASV 12mice did not have any tumor, consistent with the previous finding that these mice develop intestinal tumors relatively late in life . 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/KRASV 12double transgenic mice  and the other Apc Min /K-rasD 12double transgenic mice .
While there was a trend for a higher number of colonic tumors in the Apc Min /KRASV 12as compared to Apc Min 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 Apc Min , Apc+/1638, KRASV 12, Apc+/1638/KRASV 12mice 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 Apc Min and Apc Min /KRASV 12mice - 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 KRASV 12transgene from the villin promoter (Fig. 3B). The effect of KRASV 12allele 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 Apc Min mice has been reported .
We recently reported the critical role for Klf5 in tumor initiation in Apc Min mice . Klf5 haploinsufficiency in Apc Min mice resulted in a significant decrease in tumor number and size . Results of the current study demonstrate a similar effect on tumor formation at 12 weeks of age in Apc Min /KRASV 12mice that were heterozygous for the Klf5 alleles, with the intestinal tumor burden reduced by more than 90% in the triple Apc Min /KRASV 12/Klf5+/- transgenic mice when compared to the double Apc Min /KRASV 12transgenic mice (Fig. 1). In addition, the tumors in the Apc Min /KRASV 12/Klf5+/- mice, when formed, were smaller than those from the Apc Min /KRASV 12mice (Fig. 2A). Indeed, Apc Min /KRASV 12mice 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 Apc Min /KRASV 12/Klf5+/- mice survived up to a year without displaying overt morbidity. Taken into consideration that expression of the KRASV 12transgene in the small intestine of Apc Min /KRASV 12/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 Apc Min and KRASV 12mutations is a significant increase in the levels of β-catenin, cyclin D1 and Ki67, in the normal-appearing intestinal tissues in the Apc Min /KRASV 12mice as compared to wild type mice (Figs. 4, 5, 6, 7). This increase is similar to that seen in the intestine from the Apc Min 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 Apc Min /KRASV 12mice 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 Apc Min mutation, rather than KRASV 12over-expression and that the tumor suppressive effect of Klf5 haploinsufficiency in Apc Min /KRASV 12mice is due primarily to the ability of Klf5 to modulate Apc Min 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 Apc Min and Apc Min /KRASV 12mice but was significantly reduced in the crypt cells of Apc Min /KRASV 12/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 . 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 . Moreover, we have shown that KRASV12 increases KLF5 expression in vitro and in vivo. 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 . 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 Apc Min mutation that is further enhanced upon oncogenic KRAS activation (Fig. 8). Upon heterozygous loss of Klf5 in Apc Min /KRASV 12mice, 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 .
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 . Further investigation may prove KLF5 an attractive target for intervention in the prevention or treatment of colorectal cancer.
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 Apc Min /KRASV 12mice. These mice display an increased propensity for developing intestinal tumors at an early age compared to Apc Min mice. Moreover, we were able to significantly reduce tumor burden and size in the compound Apc Min /KRASV 12mice by reducing expression of Klf5 with genetic means. Apc Min /KRASV 12/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 Apc Min and KRASV 12mutations.
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).
All studies involving mice have been approved by the Emory University Institutional Animal Care and Use Committee (IACUC). C57BL/6J mice heterozygous for KRASV 12expressed from a mouse villin promoter were previously generated . Mice double heterozygous for Apc Min and Klf5+/- alleles were generated as previously described . Founder C57BL/6J mice that were heterozygous Apc Min alleles (males) were mated with those that were heterozygous for Klf5+/- alleles (females). The resulting progeny generated double heterozygous Apc Min /Klf5+/- mice. These mice were then mated with the KRASV 12mice 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, Apc Min , Apc Min /KRASV 12and Apc Min /KRASV 12/Klf5+/- mice were used for the study.
Genotype analyses were performed as previously described . 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, Apc Min mutation, and villin-KRAS have been previously described [33, 58, 59].
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-CT1-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 .
Immunohistochemical analysis was performed as previously described . 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 .
Western blot analyses
Western blot analyses were performed as previously described . Proteins were extracted from 20 μm paraffin embedded tissue sections using a previously established protocol . 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.
A one-way ANOVA was used to compare mean numbers of tumors between Apc Min , Apc Min /KRASV 12, and Apc Min /KRASV 12/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 . Proportions of tumors among size categories were compared between Apc Min , Apc Min /KRASV 12, and Apc Min /KRASV 12/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.
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).
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