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Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined ApcMinand KRASV 12mutations
Molecular Cancervolume 9, Article number: 63 (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 ApcMinmutation 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 ApcMin/KRASV 12double transgenic mice to develop intestinal tumors.
At 12 weeks of age, ApcMin/KRASV 12mice had three times as many intestinal tumors as ApcMinmice. This increase in tumor number was reduced by 92% in triple transgenic ApcMin/KRASV 12/Klf5+/- mice. The reduction in tumor number in ApcMin/KRASV 12/Klf5+/- mice was also statistically significant compared to ApcMinmice alone, with a 75% decrease. Compared with ApcMin/KRASV 12, tumors from both ApcMin/KRASV 12/Klf5+/- and ApcMinmice were smaller. In addition, tumors from ApcMinmice were more distally distributed in the intestine as contrasted by the more proximal distribution in ApcMin/KRASV 12and ApcMin/KRASV 12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both ApcMinand ApcMin/KRASV 12mice but were attenuated in ApcMin/KRASV 12/Klf5+/- mice. The levels of β-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of ApcMin/KRASV 12/Klf5+/- mice when compared to ApcMin/KRASV 12mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of ApcMin/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 ApcMin/KRASV 12mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.
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, ApcMinmice 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 ApcMinmice was significantly abrogated when ApcMinmice 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 ApcMinmutation.
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 ApcMinand intestine-specific KRASV 12mutations in the current study.
Klf5 heterozygosity reduces intestinal adenoma formation in ApcMin/KRASV12mice
To determine the effect of Klf5 heterozygosity on intestinal adenoma formation in mice that harbor both ApcMinand KRASV 12mutations, we crossed mice that were heterozygous for the ApcMinand Klf5 genes with those that were heterozygous for the KRASV 12gene directed by the intestine-specific villin promoter . 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/KRASV 12and ApcMin/KRASV 12/Klf5+/-) but not in ApcMin/Klf5+/- or KRASV 12mice. The mice with the compound ApcMin/KRASV 12genotype had a greater propensity for developing tumors in the small intestine than the ApcMinmice (Fig. 1A). The latter had an average of 71 small intestinal tumors per mouse while ApcMin/KRASV 12mice had an average of 226 tumors. The deletion of one of the Klf5 alleles in ApcMin/KRASV 12mice 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.
Haploinsufficiency of Klf5 decreases intestinal tumor size in ApcMin/KRASV12mice
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/KRASV 12mice (49% overall) was lower than either ApcMin(69% overall) or ApcMin/KRASV 12/Klf5+/- (62% overall) mice. In contrast, ApcMin/KRASV 12mice had a higher percentage of tumors that were 1-2 mm in size (39%) when compared to ApcMin/KRASV 12/Klf5+/- mice (33%) or ApcMinmice (28%) (Fig. 2A). Similarly, ApcMin/KRASV 12mice 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.
Change in intestinal tumor localization in mice that possess the KRASV12genotype in addition to the ApcMingenotype
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 ApcMinmice were localized in the distal small intestine, predominantly in the ileum (57%) and the jejunum (36%) (Fig. 2B). In contrast, both ApcMin/KRASV 12and ApcMin/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 ApcMinmice (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/KRASV 12mice and ApcMin/KRASV 12/Klf5+/- mice contained high levels of exogenous (human) KRAS mRNA in the intestine while wild type and ApcMinmice had only background expression (Fig. 3A). Since uneven KRAS expression could potentially contribute to the altered regional localization in the intestines of mice harboring KRASV 12, 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/KRASV 12mice, with no significant regional differences (Fig. 3B). Similarly, no regional differences in the levels of endogenous Kras were found in the intestines of either ApcMinor ApcMin/KRASV 12mice (Fig. 3B).
Klf5 heterozygosity results in reduced levels of pro-proliferative proteins in the intestines of ApcMinand ApcMin/KRASV12mice
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 ApcMinallele  or the KRASV 12allele . Here we observed increased levels of Klf5 protein in the normal-appearing small intestinal tissues of both ApcMinand ApcMin/KRASV 12mice when compared to that of wild type mice (Fig. 4A-C). The introduction of a mutant Klf5 allele into ApcMin/KRASV 12mice 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 ApcMinand ApcMin/KRASV 12mice when compared to wild type mice (Fig. 4E-G). Again, this increase in β-catenin was attenuated in the ApcMin/KRASV 12/Klf5+/- mice (Fig. 4H). Moreover, an increase in nuclear localized β-catenin was noted in the crypt epithelial cells of ApcMinand ApcMin/KRASV 12mice 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/KRASV 12/Klf5+/- mice relative to ApcMinand ApcMin/KRASV 12mice (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 ApcMinmutation.
We then performed immunohistochemical analyses on cyclin D1, a shared target between KLF5 and β-catenin . Similar to the expression patterns of Klf5 and β-catenin, there was an increase in cyclin D1 levels in the intestine of both ApcMinand ApcMin/KRASV 12mice when compared to that of wild type mice (Fig. 6A-C). Cyclin D1 staining in the normal-appearing intestinal epithelium in ApcMin/KRASV 12/Klf5+/- mice was reduced when compared to ApcMinand ApcMin/KRASV 12mice, 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).
The mitogen-activated kinase (MAPK) pathway is activated in the intestinal mucosa of ApcMin/KRASV12mice
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 ApcMinand ApcMin/KRASV 12mice 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/KRASV 12mice (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/KRASV 12mice and that Klf5 heterozygosity modestly reduces this activation.
Intestinal tumors have increased Klf5 and β-catenin expression irrespective of genotype
We also stained intestinal tumors derived from ApcMin, ApcMin/KRASV 12and ApcMin/KRASV 12/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.
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 ApcMin/KRASV 12mice developed more and larger small intestinal tumors than ApcMinmice 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 ApcMin/K-rasD 12double transgenic mice .
While there was a trend for a higher number of colonic tumors in the ApcMin/KRASV 12as compared to ApcMinmice 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, 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 ApcMinand ApcMin/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 ApcMinmice has been reported .
We recently reported the critical role for Klf5 in tumor initiation in ApcMinmice . Klf5 haploinsufficiency in ApcMinmice 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 ApcMin/KRASV 12mice that were heterozygous for the Klf5 alleles, with the intestinal tumor burden reduced by more than 90% in the triple ApcMin/KRASV 12/Klf5+/- transgenic mice when compared to the double ApcMin/KRASV 12transgenic mice (Fig. 1). In addition, the tumors in the ApcMin/KRASV 12/Klf5+/- mice, when formed, were smaller than those from the ApcMin/KRASV 12mice (Fig. 2A). Indeed, ApcMin/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 ApcMin/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 ApcMin/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 ApcMinand KRASV 12mutations is a significant increase in the levels of β-catenin, cyclin D1 and Ki67, in the normal-appearing intestinal tissues in the ApcMin/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 ApcMinmice (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/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 ApcMinmutation, rather than KRASV 12over-expression and that the tumor suppressive effect of Klf5 haploinsufficiency in ApcMin/KRASV 12mice is due primarily to the ability of Klf5 to modulate ApcMinsignaling. 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 ApcMinand ApcMin/KRASV 12mice but was significantly reduced in the crypt cells of ApcMin/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 ApcMinmutation that is further enhanced upon oncogenic KRAS activation (Fig. 8). Upon heterozygous loss of Klf5 in ApcMin/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 ApcMin/KRASV 12mice. These mice display an increased propensity for developing intestinal tumors at an early age compared to ApcMinmice. Moreover, we were able to significantly reduce tumor burden and size in the compound ApcMin/KRASV 12mice by reducing expression of Klf5 with genetic means. ApcMin/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 ApcMinand 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 ApcMinand Klf5+/- alleles were generated as previously described . Founder C57BL/6J mice that were heterozygous ApcMinalleles (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 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, ApcMin, ApcMin/KRASV 12and ApcMin/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, ApcMinmutation, 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 ApcMin, ApcMin/KRASV 12, and ApcMin/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 ApcMin, ApcMin/KRASV 12, and ApcMin/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.
Green DR, Evan GI: A matter of life and death. Cancer Cell. 2002, 1: 19-30. 10.1016/S1535-6108(02)00024-7
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. 10.1038/nrg1840
Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell. 1990, 61: 759-767. 10.1016/0092-8674(90)90186-I
Bos JL, Fearon ER, Hamilton SR, Verlaan-de Vries M, van Boom JH, Eb van der AJ, Vogelstein B: Prevalence of ras gene mutations in human colorectal cancers. Nature. 1987, 327: 293-297. 10.1038/327293a0
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. 10.1038/327298a0
Rodenhuis S, Wetering van de ML, 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.
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. 10.1016/0092-8674(88)90571-5
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.
Luttrell LM, Daaka Y, Lefkowitz RJ: Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol. 1999, 11: 177-183. 10.1016/S0955-0674(99)80023-4
Malumbres M, Barbacid M: RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003, 3: 459-465. 10.1038/nrc1097
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. 10.1038/sj.onc.1201284
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. 10.1128/MCB.21.5.1444-1452.2001
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. 10.1073/pnas.92.5.1709
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. 10.1038/sj.onc.1203600
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. 10.1126/science.272.5264.1023
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. 10.1126/science.275.5307.1784
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. 10.1126/science.275.5307.1787
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. 10.1038/328614a0
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. 10.1126/science.3479843
Kinzler KW, Vogelstein B: Lessons from hereditary colorectal cancer. Cell. 1996, 87: 159-170. 10.1016/S0092-8674(00)81333-1
Moser AR, Pitot HC, Dove WF: A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science. 1990, 247: 322-324. 10.1126/science.2296722
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. 10.1126/science.1350108
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. 10.1016/S1357-2725(00)00059-5
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.
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. 10.1038/sj.cr.7290271
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. 10.1002/bies.20581
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.
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. 10.1038/sj.onc.1207397
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. 10.1016/j.febslet.2005.07.053
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. 10.1053/j.gastro.2007.10.023
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. 10.1128/MCB.21.2.562-574.2001
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. 10.1158/0008-5472.CAN-08-4402
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. 10.1053/gast.2002.34786
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. 10.1093/nar/gkl014
Bienz M, Clevers H: Linking colorectal cancer to Wnt signaling. Cell. 2000, 103: 311-320. 10.1016/S0092-8674(00)00122-7
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. 10.1038/304507a0
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. 10.1038/302079a0
Bos JL, Toksoz D, Marshall CJ, Verlaan-de Vries M, Veeneman GH, Eb van der AJ, 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. 10.1038/315726a0
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. 10.1073/pnas.82.1.38
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. 10.1126/science.1133427
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. 10.1126/science.1145720
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. 10.1053/j.gastro.2006.08.011
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. 10.1111/j.1365-2613.2009.00667.x
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. 10.1073/pnas.0403338101
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. 10.1016/j.cell.2009.02.037
Robinson MJ, Cobb MH: Mitogen-activated protein kinase pathways. Curr Opin Cell Biol. 1997, 9: 180-186. 10.1016/S0955-0674(97)80061-0
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. 10.1038/ng.115
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. 10.1053/j.gastro.2009.01.066
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. 10.1096/fj.06-6694com
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. 10.1073/pnas.0604130103
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. 10.1073/pnas.0908682107
Rakoff-Nahoum S, Medzhitov R: Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88. Science. 2007, 317: 124-127. 10.1126/science.1140488
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. 10.1158/1541-7786.MCR-07-0232
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. 10.1210/en.2007-0869
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. 10.1158/0008-5472.CAN-07-5558
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. 10.1158/0008-5472.CAN-07-2940
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. 10.1158/1535-7163.MCT-08-0767
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. 10.1053/j.gastro.2008.01.013
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. 10.1016/0092-8674(93)90484-8
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. 10.1006/meth.2001.1262
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. 10.1369/jhc.5B6851.2006
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).
The authors declare that they have no competing interests.
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.
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