running title: klf4 and colonic tumor suppression · ghaleb et al. 1 1 klf4 suppresses tumor...

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KLF4 Suppresses Tumor Formation in Genetic and Pharmacological Mouse Models of

Colonic Tumorigenesis.

Amr M. Ghaleb1, Enas A. Elkarim1, Agnieszka B. Bialkowska1, and Vincent W. Yang1,2*

1Department of Medicine, Stony Brook University, Stony Brook, NY 11794

2Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794

Running Title: KLF4 and Colonic Tumor Suppression

Keywords: KLF4; mTOR; AOM; Epigenetic; DNA damage repair; Colorectal cancer

This work was supported by grants from the National Cancer Institute (CA084197 and

DK052230) awarded to VWY.

*Vincent W. Yang, Department of Medicine and Department of Physiology and Biophysics,

Stony Brook University School of Medicine, HSC T-16, Room 020, Stony Brook, NY, USA,

Tel.: (631) 444-2066; Fax: (631) 444-3144; E-mail: [email protected]

No conflicts of interest, financial or otherwise, are declared by the authors.

on August 1, 2020. © 2016 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/1541-7786.MCR-15-0410

http://mcr.aacrjournals.org/

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ABSTRACT

The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently down-

regulated in colorectal cancer. Previous studies showed that KLF4 is a tumor suppressor in the

intestinal tract and plays an important role in DNA damage-repair mechanisms. Here the in

vivo effects of Klf4 deletion were examined from the mouse intestinal epithelium (Klf4ΔIS) in a

genetic or pharmacological setting of colonic tumorigenesis: ApcMin/+ mutation or carcinogen

treatment with azoxymethane (AOM), respectively. Klf4ΔIS/ApcMin/+ mice developed significantly

more colonic adenomas with 100% penetrance as compared to ApcMin/+ mice with intact Klf4

(Klf4fl/fl/ApcMin/+). The colonic epithelium of Klf4ΔIS/ApcMin/+ mice showed increased mTOR

pathway activity, together with dysregulated epigenetic mechanism as indicated by altered

expression of HDAC1 and p300. Colonic adenomas from both genotypes stained positive for

γH2AX indicating DNA double strand (DS) breaks. In Klf4fl/fl/ApcMin/+ mice, this was associated

with reduced non-homologous end joining (NHEJ) repair and homologous recombination repair

(HRR) mechanisms as indicated by reduced Ku70 and Rad51 staining, respectively. In a

separate model, following treatment with AOM, Klf4ΔIS mice developed significantly more

colonic tumors than Klf4fl/fl mice, with more Klf4ΔIS mice harboring K-Ras mutations than Klf4fl/fl

mice. Compared to AOM-treated Klf4fl/fl mice, adenomas of treated Klf4ΔIS mice had

suppressed NHEJ and HRR mechanisms as indicated by reduced Ku70 and Rad51 staining.

This study highlights the important role of KLF4 in suppressing the development of colonic

neoplasia under different tumor promoting conditions.

Implications: The study demonstrates that Krüppel-like factor 4 plays a significant role in the

pathogenesis of colorectal neoplasia.




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INTRODUCTION

Colorectal cancer (CRC) is a major cause of cancer mortality in the United States.

Several factors play a role in ultimately causing CRC development. These include; mutations,

epigenetic changes and DNA damage. The majority of colorectal cancers contain mutations in

the adenomatous polyposis coli (APC) tumor suppressor gene (1). APC is found in the normal

intestinal mucosa with an increasing gradient of expression in mature epithelial cells located in

the upper crypt region (2). In addition, APC antagonizes the pro-proliferative Wnt pathway by

negatively regulating the steady-state level of intracellular β-catenin (3, 4). When APC is

inactivated by mutation (which usually leads to a truncated protein), Wnt signaling is

unimpeded, resulting in the nuclear accumulation of β-catenin and subsequent activation of

downstream target genes such as cyclin D1 and c-Myc that promote cell proliferation (5, 6). In

many cancer types, including CRC, accumulation of DNA damage has been linked to cancer,

and genetic deficiencies in DNA damage repair mechanisms are associated with susceptibility

to tumor development (7). Additionally, epigenetic changes that lead to mutations or to

silencing of DNA repair genes may promote tumorigenesis (7).

The nuclear transcription factor Krüppel-like factor 4 (KLF4; also known as gut-enriched

Krüppel-like factor or GKLF) is highly expressed in the terminally differentiated, post-mitotic

intestinal epithelial cells and is an inhibitor of cell proliferation (8, 9). We have previously

shown that in vitro, Klf4 is required for the prevention of genomic instability (10). In the

intestine, the KLF4 promoter has been shown to be regulated by APC in a Cdx2-dependent

manner, the latter being an intestine-specific transcription factor that controls intestinal

development (11). Conversely, KLF4 has been shown to regulate colonic cell growth by

inhibiting β-catenin activity (12, 13). Accordingly, studies have demonstrated a potentially




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causal relationship between KLF4 and several kinds of human cancers. For example,

expression of KLF4 is often reduced in tumors of the gastrointestinal tract (14-16). In addition,

loss of heterozygosity (LOH) and promoter hyper-methylation have been identified as possible

reasons for the reduced expression of KLF4 in a subset of colorectal cancers (16). However,

whether KLF4 plays a causal role in the in vivo development of colonic tumors has not been

definitively established.

In the current study, we investigated the in vivo role of KLF4 in colonic tumorigenesis in

two different systems: the setting of ApcMin mutation and the chemically induced DNA

mutations.




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MATERIALS AND METHODS

Mice. All animal studies were approved by the Stony Brook University Institutional Animal Care

and Use Committee (IACUC). Male C57BL/6J ApcMin/+ founders were originally purchased from

Jackson Laboratories. Mice with floxed Klf4 gene (Klf4fl/fl) and intestine-specific villin-Cre driven

Klf4 deletion (Klf4ΔIS) were previously described (17). ApcMin/+ males were mated with either

Klf4fl/fl or Klf4ΔIS females to obtain ApcMin/+ mice with intact Klf4 allele (Klf4fl/fl/ApcMin/+), or with

intestine-specific Klf4 deletion (Klf4ΔIS/ApcMin/+).

Azoxymethane (AOM) treatment. Mice, Klf4fl/fl and Klf4ΔIS, were injected with AOM

(10mg/kg), i.p., once a week for 4 consecutive weeks. Mice were euthanized at 4 or 12 weeks

after the last injection for tumor development assessment.

Tissue harvesting and tumor assessment. For all groups, following euthanasia with CO2

asphyxiation and cervical dislocation, the entire small intestine and colon were dissected out

and flushed with modified Bouin’s fixative (50% ethanol/5% acetic acid) then cut open

longitudinally. The intestines were examined under a dissecting microscope for the presence

of adenomas. The number and size of adenomas in both the small and large intestine were

recorded as described previously (18).

Tissue preparation and immunostaining. Following macroscopic examination for tumor

formation in harvested tissue, the intestines were Swiss-rolled, fixed in buffered formalin,

embedded in paraffin and 5-mm sections were cut for histological H&E characterization, and

for immunostaining.

For immunostaining, sections were deparaffinized in xylene, rehydrated in ethanol, and

then treated with 10mM Na citrate buffer, pH 6.0, at 120°C for 10min in a pressure cooker. The

histological sections were incubated with blocking buffer (3% bovine serum albumin, and




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0.01% Tween 20 in 1X Tris-buffered PBS (TTBS)) for 1 hour at 37°C. Primary antibodies goat

anti-KLF4 (1:300; R&D), rabbit anti-cyclin D1 (1:200; Biocare Medical), rabbit monoclonal anti-

Ki67 (1:200; Biocare Medical), rabbit anti-γH2AX (1:400; Abcam), rabbit anti-Rad51 (1:200;

Abcam), mouse anti-Ku70 (1:200; Abcam), mouse anti-β-catenin (1:1000; BD), mouse anti-

Cdx2 (1:200; LS Bio), rabbit anti-HDAC1 (1:200; Santa Cruz), rabbit anti-Pp44/42 MAPK (p-

ERK) (1:200; Cell Signaling) and mouse anti-p300 (1:200; Santa Cruz) were added at 4°C

overnight. For IF, appropriate AlexaFluor labeled secondary antibodies (Molecular Probes)

were added at 1:500 dilution in blocking buffer for 30 minutes at 37°C, counterstained with

Hoechst 33258, mounted with Prolong gold (Molecular Probes), and cover-slipped. IHC

detection of cyclin D1, Ki67, and γH2AX was done using goat anti-mouse or anti-rabbit HRP-

labeled (Jackson Immuno Research) secondary antibody at 1:500 dilution in blocking buffer for

30 minutes at 37°C, followed by wash and then DAB color development. For Klf4 and p-ERK

detection by IHC, secondary unconjugated rabbit anti-goat antibody and goat anti-rabbit

antibody (Jackson Immuno Research) was added, respectively, at 1:500 dilution in blocking

buffer for 30 minutes at 37°C. After washing, goat anti-rabbit HRP and donkey anti-goat HRP

labeled tertiary antibodies (Jackson Immuno Research) were then added, respectively, at

1:500 dilution in blocking buffer for 30 minutes at 37°C, followed by DAB color development.

Detection of all other primary antibodies for IHC was carried out using either Mach3 rabbit or

Mach3 mouse HRP-polymer detection as per manufacturer recommendations (Biocare

Medical). Color development in all IHC staining was followed by hematoxylin counterstaining

and mounting.

Anaphase bridging index (ABI) and mitotic index. The ABI was determined as described

before (19). A minimum of 30 anaphases per mouse (4 per group) was scored from H&E




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sections. For Mitotic index, the number of cells undergoing mitosis per crypt (minimum of 60

per mouse) was scored from H&E sections (3 mice per group).

Measurement of loss of heterozygosity (LOH) of the Apc+ locus. DNA was extracted from

paraffin embedded colon tissues of Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice and LOH was

analyzed as described before (18, 20). In brief, five 10μm thick sections per mouse were cut

and collected in a test tube. Tissue was then deparaffinized in 1ml xylenes for 30min at RT.

The xylenes was then discarded and tissue was washed twice in 100% ethanol followed by 2x

wash in 70% ethanol and 2x PBS, then spun down and PBS discarded. DNA was extracted

from pelleted tissue using REDExtract-N-Amp Tissue PCR kit (Sigma), and purified using

QIAmp DNA Micro Kit (Qiagen). Equal amounts of DNA was used for PCR amplification of the

Apc locus followed by HindIII digestion as described before (20). Twenty μl of each HindIII

digestion reaction were electrophoresed through a 2.5% agarose gel. Bands were quantified

using ImageJ software (21). Determination of LOH was carried out as described before (18,

20).

In vitro overexpression or suppression of KLF4. Colon cancer cell line HCT116 was used.

The cell line was originally purchased from American Type Culture Collection (ATCC). Cells

thawed from frozen stock were used, and all experiments were carried out within 6 months of

thawing. We routinely carry out morphology checks on all cell lines and we only passage the

cell lines for three months. In addition, the cell lines were tested by PCR for Mycoplasma

contamination. Furthermore, each experiment had appropriate controls to assure the behavior

of tested cell lines. For Klf4 overexpression, plasmid containing pEGFP-Klf4 that expresses

EGFP-Klf4 fusion protein was used to transfect the cells as described before (10). For KLF4

suppression, KLF4-specific siRNA (Ambion) was used to transfect cells as per manufacturer’s




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recommendations. For all transfection experiments, the cells were harvested 24h post

transfection for immunoblot analysis.

Immunoblotting. Cells were lysed in complete Laemmli buffer, and vortexed for 3-4min for

homogenization. Insoluble material was removed by centrifugation at 12,000 rpm for 5 min,

and the supernatant was collected and heated at 95-100°C for 10min, then cooled down to RT

before loading for gel electrophoresis. For mouse tissues (the entire length of the colon;

normal epithelium and tumors), deparaffinization and hydration was done as described above.

Reversal of crosslinking was then carried out by adding 1ml of 10mM Na citrate buffer, pH6.0,

per sample, and heating at 95°C for 20 min. Tissues were then spun down, citrate buffer

discarded, washed twice in PBS, then spun down and PBS discarded. Pelleted tissue was

then re-suspended in complete Laemmli buffer, and heated at 95-100°C for 10min. Extracted

protein from cells or tissue were resolved using SDS-PAGE gel electrophoresis. For all protein

samples, following protein transfer, the membranes were immunoblotted with the following

primary antibodies: rabbit anti-KLF4 (Santa Cruz), rabbit anti-HDAC1 (Santa Cruz), rabbit ant-

p53 (Santa Cruz), rabbit anti-mTOR (Cell Signal), rabbit anti-phosphorylated mTOR (Cell

Signal), rabbit anti-phosphorylated p70S6K1 (pS371) (Cell Signal), rabbit anti-p27 (Santa

Cruz), mouse anti-p21 (BD), mouse anti-Bax (Santa Cruz), mouse anti-β-actin (Sigma-Aldrich)

and mouse anti-GAPDH (Sigma-Aldrich), overnight at 4°C. Following washing in TTBS, the

blots were then incubated with appropriate horseradish peroxidase-conjugated secondary

antibodies for 1h at RT. The antibody-antigen complex was visualized by ECL

chemiluminescence (Millipore).

PCR and direct DNA sequencing. DNA from AOM-treated Klf4fl/fl and Klf4ΔIS mice was

extracted from paraffin-embedded tissue sections as described above and purified using




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standard phenol/chloroform/isopropanol method. Thus, the DNA extracted represents the

entire length of the colon (normal epithelium and tumors) present in the section collected. PCR

primers for detection of β-catenin and K-Ras mutations were designed to amplify the following:

exon 3 of the Ctnnb1 gene containing the consensus sequence for GSK-3β phosphorylation

and exon 1 of K-Ras (22). PCR amplification conditions were used as described before (22)

using RED-Taq Ready PCR Mix (Sigma). Amplified fragments were then electrophoresed in

1% agarose gel, excised and purified using QIAEX II gel extraction kit (Qiagen). Sequencing

was done at the Genomic Core Facility, Stony Brook University. Thus, the DNA sequenced

represents the entire length of the colon (normal epithelium and tumors) present in the section

collected.

Counting positively stained cells and statistical analysis. For p300, positively stained

colonic epithelial cells were counted in a minimum of 20 crypts per mouse (3 mice/group). For

Ku70 and Rad51 in adenomas, positively stained cells were counted per mouse (3

mice/group), per adenoma, per section, per field at 400x magnification. Four fields were

counted per adenoma. Where applicable, differences in Rad51 IHC staining intensity in

adenomas (2 mice/group) was quantified using ImageJ software (22). Statistical significance

between groups was done using paired 2 tailed student t-test or One way ANOVA. Where

applicable, box plot for data was done using GraphPad Prism version 5.00 for Windows

(GraphPad Software, San Diego, CA).




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RESULTS

Increased adenoma burden and elevated Wnt signaling level in the setting of ApcMin

mutation following intestinal epithelium-specific deletion of Klf4.

We previously showed that haploinsufficiency of Klf4 in ApcMin/+ mice leads to a

significant increase in the number of adenomas formed in the small intestine but with no

significant effect on adenoma development in the colon (18). In the current study we

investigated whether complete deletion of Klf4 in the intestinal epithelium (Klf4ΔIS) in the setting

of ApcMin mutation has an influence on adenoma development in the colon, in addition to the

small intestine. We first compared the number and size of the adenomas that developed in

Klf4fl/fl/ApcMin/+ (which contain intact Klf4 loci) and Klf4ΔIS/ApcMin/+ mice between 16 and 20

weeks of age. Klf4fl/fl/ApcMin/+ mice developed on the average 19.88 ± 14.75 adenomas per

mouse (N = 8) in the small intestine (Supp. Figure 1A). In comparison the average number of

adenomas in the small intestine of Klf4ΔIS/ApcMin/+ mice was 67 ± 27.21 adenomas per mouse

(N = 7) (Supp. Figure 1A). Examining the size of adenomas formed, Klf4ΔIS/ApcMin/+ mice had

higher numbers of adenomas in each of the size categories than the Klf4fl/fl/ApcMin/+ mice, with

significantly more adenomas of 1-2 mm size (Supp. Figure 1B). There was no significant

difference in the distribution of tumor sizes between the two groups of mice.

Since KLF4 has been shown to repress Wnt/β-catenin activity (13), we examined the

levels of β-catenin, Ki67 and cyclin D1 by immunohistochemistry in the normal-appearing

intestinal tissues and in adenomas of age-matched Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice.

Both genotypes showed stronger β-catenin staining in adenomas as compared to their

respective normal-appearing intestinal epithelial cells (Supp. Figures 2A-D). However,

Klf4ΔIS/ApcMin/+ showed an overall stronger β-catenin staining as compared to Klf4fl/fl/ApcMin/+




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mice. A similar pattern of differential staining for Ki67 (Supp. Figures 2E-H) and cyclin D1

(Supp. Figures 2I-L) was observed in both mouse genotypes.

Increased tumor burden and penetrance of colonic polyps following deletion of Klf4 in

the setting of ApcMin mutation.

Analysis of adenoma number in the colon revealed that Klf4ΔIS/ApcMin/+ mice developed

on average 3 adenomas per colon, while the Klf4fl/fl/ApcMin/+ mice had on average less than 1

adenoma (Figure 1A). ApcMin/+ mice are known to develop adenomas mostly in the small

intestine and very few in the colon. Importantly 100% (7/7) of the Klf4ΔIS/ApcMin/+ mice

developed colonic adenomas as compared to about only 50% (4/8) of the Klf4fl/fl/ApcMin/+ mice

(Figure 1A). Also, unlike in the small intestine, deletion of Klf4 from the colonic epithelium

(Figure 1Ba and 1Bb) did not have an overall effect on β-catenin expression in Klf4ΔIS/ApcMin/+

mice as compared to Klf4fl/fl/ApcMin/+ mice in normal tissue (Figure 1Bc and 1Bd). Similar

finding was observed for β-catenin and cyclin D1 staining in adenomas of both mouse

genotypes. However, adenomas of Klf4ΔIS/ApcMin/+ mice tended to have darker staining Ki67

than adenomas of Klf4fl/fl/ApcMin/+ mice (Supp. Figure 3).

Increased loss of heterozygosity (LOH) of wild-type Apc locus and increased anaphase

bridge index (ABI) in colons of Klf4ΔIS/ApcMin/+ mice.

Normal-appearing cells harboring pro-cancer genetic abnormalities are prone to

develop into tumor cells if they are not eliminated, such as the case with ApcMin/+ mice.

Consequently, we focused on examining normal-appearing colonic epithelial cells to determine

the occurrence of pre-existing abnormalities that might help explain the increased penetrance

of colonic adenoma formation in Klf4ΔIS/ApcMin/+ versus Klf4fl/fl/ApcMin/+ mice. As polyps are

initiated by LOH at Apc in ApcMin/+ mice (20), we determined the Apc genotype in the colon of




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Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice, using tissues representing the entire length of the

colon. In all examined, the amplified band for the wild-type Apc allele was weaker than that of

the mutated allele. On the other hand, the gain in the mutated allele was significantly higher in

Klf4ΔIS/ApcMin/+ mice as compared to Klf4fl/fl/ApcMin/+ mice (Figures 2A and 2B). LOH in ApcMin/+

mice is caused by homologous recombination near the centromere (23). Additionally, a

mutation in DNA helicase or telomerase in ApcMin/+ mice increases the number of intestinal

polyps because of high frequency of recombination and chromosomal instability (24). In the

anaphase of cell cycle, such chromosomal instability can be assessed by the frequency of

‘anaphase bridges’, extended chromosome bridging between two spindle poles (24, 25). We

have previously shown that KLF4 suppresses genomic instability (10). To determine the effect

of Klf4 deletion on genomic instability in mice colons, we scored anaphase bridge index (ABI)

in the colonic epithelium (Figure 2C). We found very few anaphase bridges in wild-type mice

while Klf4fl/fl/ApcMin/+ mice had significantly more (ABI mean ± SD = 26.3 ± 3.17%; Figure 2D).

In contrast, the Klf4ΔIS/ApcMin/+ mice had ABI almost twice that of Klf4fl/fl/ApcMin/+ mice (46.5 ±

2.4%; Figure 2D). This result is consistent with previous reports that chromosomal instability is

enhanced by mutations in Apc (26, 27) or by deletion of Klf4 (10).

Deletion of Klf4 in colonic epithelium has no effect on Cdx2 expression in mouse

colons.

Previous studies have shown that increased tumor burden in the colon in a mouse

model with Apc mutation is Cdx2-dependent (19). Additionally Cdx2 was shown to regulate

Klf4 expression in colon cancer lines (11). To determine whether Klf4 deletion has any effect

on Cdx2 expression that may have led to the increase in colonic adenoma incidence in

Klf4ΔIS/ApcMin/+ mice, we stained for Cdx2. As shown in Figures 3A and 3B, we observed no




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differences in staining between Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice, where there is a

decreasing gradient from proximal to distal colon.

Klf4 modulates mTOR pathway activity in colonic epithelium and in colon cancer cell

line HCT116.

Increased colonic polyposis following reduction of Cdx2 in a mouse model with mutated

Apc was linked to activated mTOR kinase-dependent pathway (19). Since KLF4 was

previously shown to be downstream from Cdx2 (11), we tested whether Klf4 deletion in colonic

epithelium of ApcMin/+ mice have any effect on the activity of mTOR pathway. Western blot

analysis of colon tissue lysates showed that, while there is a relative increase in the level of

p70S6K1 (pS371) in Klf4ΔIS/ApcMin/+ mice (Figure 3C, lanes 4-6) as compared to Klf4fl/fl/ApcMin/+

mice (Figure 3C, lanes 1-3). Increased mTOR pathway activity in mouse colon has been

shown to induce cell cycle acceleration which was associated with low levels of p27, which is

an important cell cycle regulator (20). Analysis of the level of p27 in colon tissue lysates

showed that it was relatively decreased in Klf4ΔIS/ApcMin/+ mice (Figure 3C, lanes 4-6) as

compared to Klf4fl/fl/ApcMin/+ mice (Figure 3C, lanes 1-3).

The transcription factor p53 is known to be involved in multiple tumor suppressive

functions (28, 29), and was shown to require KLF4 for the p53-dependent regulatory effects

following DNA damage (30). Also, the ABI observed in Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice

(Figure 2), is a source of DNA damage. Thus we examined the expression level of p53 in the

colonic tissue lysate of both Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice. Compared to

Klf4fl/fl/ApcMin/+ mice, p53 level was elevated in Klf4ΔIS/ApcMin/+ mice (Figure 3C, lanes 1-2 & 4-6

respectively), and this elevation was accompanied by elevated Bax level (Figure 3C, lanes 1-2

& 4-6 respectively).




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The effect of Klf4 expression on the activity of mTOR pathway and on p53-dependent

cell cycle pathway was determined in colon cancer cell line HCT116. Overexpression of Klf4

suppressed the activity of mTOR pathway as indicated by lower levels of phosphorylated

mTOR and p70S6K1 (pS371) as compared to control (Figure 3D, lanes 1 and 2). In contrast,

suppression of KLF4 leads to a relative increase in phosphorylated mTOR and p70S6K1

(pS371) level (Figure 3D, lanes 3 and 4). The effect of Klf4 expression level on p53 expression

was previously shown to be cell-type dependent (31, 32). Consistent with previous reports

(32), p53 level was elevated and suppressed following Klf4 overexpression and suppression,

respectively (Figure 3D, lanes 1 and 2 & 3 and 4). The differences observed for the p53

expression level in response to suppressed KLF4 expression in HCT116 cell line versus

mouse colon lysates could be due to mutations in some regulatory genes harbored by the

colon cancer cell lien HCT116 as compared to those in the mouse colon tissue lysates. While

p21 level was not affected by KLF4 expression level (Figure 3D), there was an inverse

correlation between KLF4 level and Bax level (Figure 3D, lanes 1 and 2 & 3 and 4), consistent

with previous reports (33).

Altered expression of HDAC1 and p300 in colonic epithelium of Klf4ΔIS/ApcMin/+ mice.

Acetyl modifications at histone tails constitute a major epigenetic mechanism that

regulates chromatin structure and gene expression in cancer development (34). Klf4 was

previously shown to suppress genomic instability in vitro (10), and to directly interact with

modifiers of histone acetylation such as p300, a member of the histone acetyltransferase

family (34). To determine whether deletion of Klf4 in the colonic epithelium of ApcMin/+ mice has

any effect on histone acetylation mechanism, we first examined level of histone deacetylase 1

(HDAC1) and of p300, a histone acetyltransferase, by Western blot in colonic tissue lysates.




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While we could not detect p300, HDAC1 level was elevated in Klf4ΔIS/ApcMin/+ mice (Figure 3C,

lanes 4-6) as compared to Klf4fl/fl/ApcMin/+ mice (Figure 3C, lanes 1-3). In HCT116 cells,

HDAC1 level was not affected by Klf4 expression level (Figure 3D), and we could not detect

p300. To confirm the change in HDAC1 as observed by Western blot in colonic tissue lysates,

we stained for HDAC1 and p300. Consistent with Western blot results, Klf4ΔIS/ApcMin/+ mice

showed more colonic epithelium cells staining for HDAC1 as compared to Klf4fl/fl/ApcMin/+ mice

(Figures 3E and 3F). On the other hand, while p300 was detected in Klf4fl/fl/ApcMin/+ mice, it was

significantly lower in Klf4ΔIS/ApcMin/+ mice (Figures 3G, H and I).

Suppressed DNA-damage repair mechanisms in colonic adenomas of Klf4ΔIS/ApcMin/+

mice.

We have previously identified an importance role of Klf4 in DNA damage repair

response and in maintaining genomic stability (10). Cells have evolved various strategies to

promote genome stability through the precise repair of DNA double-strand breaks and other

lesions that are encountered during normal cellular metabolism and from exogenous insults

(35, 36). Given the role of Klf4 in mediating DNA-damage repair, and that Klf4ΔIS/ApcMin/+ mice

have higher markers of genomic instability compared to Klf4fl/fl/ApcMin/+ mice (Figure 2), we

compared the level of γH2AX staining between Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice.

Normal-appearing colonic crypts of both genotypes had few cells staining positive for γH2AX

(Figure 4A and G, respectively). However, adenomas formed in both mouse genotypes stained

strongly for γH2AX (Figure 4D and J, respectively). To determine whether there is any

difference between the two genotypes in the DNA damage repair mechanism, we stained for

Rad51 and Ku70, representing homologous recombination repair (HRR) and non-homologous

end joining (NHEJ) repair mechanisms, respectively (37). Both mouse genotypes stained




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negative for Ku70 in the normal-appearing colonic epithelium (Figure 4B and H, respectively).

On the other hand, adenomas of Klf4fl/fl/ApcMin/+ mice stained positive for Ku70, while

adenomas of Klf4ΔIS/ApcMin/+ mice had significantly lower staining (Figure 4E and K,

respectively, and 4M). For Rad51, both mouse genotypes stained positive in normal looking

colonic epithelium (Figure 4C and I, respectively), with no observed differences between them.

However, adenomas of Klf4fl/fl/ApcMin/+ mice had significantly more positive cells staining for

Rad51 as compared to adenomas of Klf4ΔIS/ApcMin/+ mice (Figure 4F and L, respectively, and

4N). Our results suggest an important role of Klf4 in promoting repair of DNA DSB by

modulating HRR and NHEJ mechanisms.

Susceptibility of Klf4ΔIS mice to AOM-induced colonic tumor formation.

Azoxymethane (AOM) is a colon carcinogen that is commonly used in rodents to study

the pathogenesis of sporadic colorectal cancer. In mice, AOM-induced tumors have been

attributed to mutations in β-catenin, while rare mutations in K-Ras are also observed (22, 38).

To determine whether deletion of Klf4 in colonic epithelium has any effect on the pathogenesis

of AOM-induced CRC, both Klf4fl/fl and Klf4ΔIS mice were treated with AOM (4 injections, once

a week) and then analyzed for tumor development at 4 and 12 weeks post last injection. At 4

weeks post injection, there was no difference between treated Klf4fl/fl and Klf4ΔIS mice when

examined macroscopically. However, H&E staining of the colonic sections revealed the

formation of microadenomas in 25% versus 75% of Klf4fl/fl in comparison to Klf4ΔIS mice (Supp.

Figures 4Aa and 4Ab). The number of microadenomas per section averaged 0.5 ± 0.57 and 2

± 0.82 in Klf4fl/fl and Klf4ΔIS mice, respectively (data not shown). Staining for β-catenin (Supp.

Figures 4Ac and 4Ad) showed slightly lighter staining in normal-appearing colonic epithelium of

Klf4fl/fl mice compared to Klf4ΔIS mice, and darker staining in the microadenomas as compared




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to surrounding normal-appearing colonic epithelium. While there was no observed difference in

cyclin D1 staining at basal level between Klf4fl/fl and Klf4ΔIS mice, there was more intense

staining in the microadenomas as compared to normal-appearing colonic epithelium (Supp.

Figures 4Ae and 4Af).

At 12 weeks post-AOM treatment, Klf4ΔIS mice developed significantly more colonic

adenomas as compared to Klf4fl/fl mice (Figure 5A). Adenomas of both Klf4fl/fl and Klf4ΔIS mice

showed darker β-catenin staining compared to surrounding normal colon epithelium. Though

there was variation in β-catenin staining in adenomas of Klf4ΔIS mice, adenomas of Klf4ΔIS mice

had an overall more intense and nuclear staining as compared to adenomas of Klf4fl/fl mice

(Figures 5Ba and 5Bb). Cyclin D1 showed similar a staining pattern between the two groups as

in β-catenin (Figures 5Bc and 5Bd). We also observed an increase in cyclin D1 staining in

normal-appearing colonic epithelium of both genotypes when compared to mice at 4 weeks

(data not shown). Since AOM treatment was shown to have minimum effect on inducing K-Ras

mutations in mice, we set to determine whether Klf4 deletion would have any effect on the K-

Ras pathway. When stained for p44/42 MAPK (p-ERK), which is a downstream effector of K-

Ras, only 20% (1/5) of Klf4fl/fl mice showed any positive staining for p-ERK in the tumors, while

75% (3/4) of Klf4ΔIS mice showed positive staining for p-ERK (Figures 5Be and 5Bf,

respectively).

Analysis of AOM-induced β-catenin and K-Ras mutations showed no mutations in β-

catenin in either genotype, contrary to what was reported before (22, 38). However, consistent

with previous reports (22, 38), one AOM-treated Klf4fl/fl mouse (1/4) had K-Ras mutations

(Figure 5C), while 3/4 of AOM-treated Klf4ΔIS mice had K-Ras mutations (Figure 5C, and Supp.

Figure 4B). These mutations were detected at 12-weeks-post-injection. Codons affected were




Ghaleb et al. 18

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14, 16-18, 22 and 23 in AOM-treated Klf4fl/fl mouse, and codons 14, 17-19, 21, and 22 in AOM-

treated Klf4ΔIS mice.

We also examined the DSB repair mechanism in the AOM model of colonic

tumorigenesis. Normal-appearing colonic crypts of AOM-treated mice of both genotypes had

few cells/crypt staining positive for γH2AX (Figure 6A and G, respectively). Adenomas formed

in both mouse genotypes stained positive for γH2AX (Figure 6D and J, respectively). Both

mouse genotypes stained negative for Ku70 in normal looking colonic epithelium (Figure 6B

and H, respectively). While adenomas of AOM-treated Klf4fl/fl mice stained positive for Ku70,

adenomas of AOM-treated Klf4ΔIS mice stained significantly lower (Figure 6E and K

respectively, and 6M). For Rad51, both mouse genotypes stained positive in normal looking

colonic epithelium (Figure 6C and I, respectively). On the other hand, adenomas of AOM-

treated Klf4fl/fl mice had Rad 51 positive staining, while adenomas of AOM-treated Klf4ΔIS mice

showed significantly weaker staining for Rad51 (Figure 6F and L, respectively, and 6N). Our

results are consistent with results shown in Figure 4, demonstrating an important role of Klf4 in

promoting repair of DNA DSB by modulating HRR and NHEJ mechanisms. Additionally,

Western blot analysis of p53 level in colonic tissue lysates of AOM-treated Klf4fl/fl and Klf4ΔIS

mice indicated that, compared to Klf4fl/fl mice (Figure 6P, lanes 1-4), Klf4ΔIS mice had higher

p53 level (Figure 6P, lanes 5-8). This result is consistent with our observation on p53 in

Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice (Figure 4).




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DISCUSSION

Many factors such as mutations, epigenetic changes and DNA damage, contribute to

the development of CRC. In humans, familial adenomatous polyposis is a disorder in which

germline mutations of the APC gene lead to florid colonic polyposis early in life (1). The

ApcMin/+ mouse carries a nonsense germline mutation in the murine Apc gene (39). A key

difference between the human disorder and the mouse model is that adenomas mostly occur

in the small intestine than in the colon in ApcMin/+ mice (39), whereas it is the reverse in

humans. We have previously shown that mice with heterozygous whole-body deletion of Klf4

in the setting of ApcMin/+ mutation leads to significant increase in intestinal adenomas as

compared to ApcMin/+ mice (18). In support of our previous finding, our current results show that

complete deletion of Klf4 in the intestinal epithelium of ApcMin/+ mice (Klf4ΔIS/ApcMin/+) leads to a

significant increase in the number of intestinal adenomas but not in size as compared to

ApcMin/+ mice (Klf4fl/fl/ApcMin/+) (Supp. Figure 1). These results indicate that in the small intestine

Klf4 plays a role in adenoma multiplicity (and possibly initiation) rather than their progression.

The Wnt signaling pathway is hyper-activated in the setting of Apc mutation, both in

humans and in mice (40, 41). KLF4 was previously shown to negatively regulate the Wnt

signaling pathway (13, 42). Additionally, we have shown that ApcMin/+ mice with Klf4

haploinsufficiency have higher levels of Wnt signaling components such as β-catenin and

cyclin D1 (18). Consistent with these findings, Klf4ΔIS/ApcMin/+ had higher levels of β-catenin,

proliferation and cyclin D1 in both the normal-appearing intestinal epithelium and adenomas,

as compared to Klf4fl/fl/ApcMin/+ (Supp. Figure 2). These results indicate an important role of

Klf4 in suppressing Wnt signaling pathway in the intestinal epithelium, and thus in adenoma

formation, in the setting of ApcMin/+ mutation.




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Mice with ApcMin/+ mutation develop adenomas mainly in the small intestine, with much

lower incidence and multiplicity in the colon (39, 41). We have previously shown that there was

no significant difference in colonic adenoma incidence and multiplicity between ApcMin/+ mice

and ApcMin/+ mice with Klf4 haploinsufficiency (18). However, analysis of adenoma

development in Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice showed a significant increase in

colonic adenoma number and penetrance in Klf4ΔIS/ApcMin/+ as compared to Klf4fl/fl/ApcMin/+

mice (Figure 1A), suggesting that Klf4 might be a key factor in suppressing colonic

tumorigenesis. In mice, factors that determine tumor development in the small intestine versus

the colon are not very well understood. Analysis of chromosomal instability in the colonic

tissues Klf4ΔIS/ApcMin/+ mice showed significant increase both in LOH of the Apc locus as well

as in aberrant mitosis as indicated by elevated ABI (Figure 2). These in vivo results lend

support to our previous finding about the role of KLF4 in the regulation of chromosomal

instability in vitro (10). It was previously reported that Cdx2, a mouse homolog of Drosophila

melanogaster caudal1 (43) and a key transcription factor for intestinal development and

differentiation (44), is important in regulating colonic tumorigenesis through mTOR-mediated

chromosomal instability (19). KLF4 has been shown to be downstream from Cdx2 (11) and this

was supported by our finding that deletion of Klf4 had no effect on Cdx2 expression in the

colonic tissue (Figures 3A and 3B). However, analysis of the mTOR pathway revealed a

potential role for Klf4 in negatively regulating mTOR signaling since there was an inverse

relationship between the levels of Klf4 and active mTOR pathway in mouse colonic epithelial

cells and in colon cancer cell lines (Figures 3C and 3D). This result is in line with a recent

finding of the inverse relationship between Klf4 and mTOR signaling in mouse embryonic

fibroblasts (45). Several studies have implicated a positive cross interaction between mTOR




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signaling and epigenetic events, such as histone acetylation, regulated by histone deacetylase

1 (HDAC1) (46, 47). Additionally, an inverse interplay between Klf4 and HDAC1 has been

previously reported (48). Our assessment of histone acetylation regulation in normal colonic

epithelial cells of both Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice, indicated elevated HDAC1 and

reduced p300 staining levels in Klf4ΔIS/ApcMin/+ as compared to Klf4fl/fl/ApcMin/+ mice (Figures 3).

Taken together, our findings are strongly suggestive of a role for KLF4 in suppressing colon

tumorigenesis, in an ApcMin/+ setting, through the modulation of the mTOR signaling pathway

and epigenetic changes via the regulation of HDAC1 and p300, and consequently

chromosomal instability.

Carcinogens such as AOM are known to induce colonic tumors in mice by promoting

mutations in β-catenin and to a lesser extent in K-Ras (22, 38). In our model, AOM-treated

C57BL/6 Klf4fl/fl and Klf4ΔIS mice, though both developed colonic tumors with Klf4ΔIS mice

having significantly more than Klf4fl/fl mice, yet neither group had β-catenin mutations, nor did

they have K-Ras mutations in the commonly affected codons 12 and 13 (38). This could either

be due to differences in the mouse strain used in our study versus those used in other studies

(22, 38), or to differences in treatment regimen and tissue collection time point (22, 38). Our

results indicate that the increase in β-catenin expression in tumors of AOM-treated C57BL/6

mice is independent of β-catenin mutations, and in Klf4ΔIS mice corresponds to absence of

Klf4. Additionally, our results show that following exposure to certain carcinogens such as

AOM, Klf4 is important in suppressing mutations in pro-oncogenic genes such as K-Ras.

DNA damage normally occurs in cells as a consequence of both environmental and

endogenous insults. During the normal course of DNA replication or following exposure to DNA

damaging agents, double-strand breaks (DSBs) may arise and are considered one of the most




Ghaleb et al. 22

22

cytotoxic forms of DNA damage (49). Deficiencies in DSB repair can lead to mutations and

chromosomal aberrations that ultimately may result in genomic instability and tumorigenesis

(49). Consequently, cells have effective mechanisms for the accurate and timely repair of

DSBs in DNA (49). In mice, the importance of Klf4 is demonstrated by its ability to modulate

DSB events and regulate DSB repair mechanisms (Figure 4 and 6). Our results indicate that in

the mouse intestinal epithelium, under basal conditions, NHEJ repair mechanism is inactive,

while HRR mechanism is active and is independent of Klf4 expression (Figure 4 and 6).

However in colonic adenomas both NHEJ and HRR mechanisms are active, and are

dependent on Klf4 expression as both mechanisms were suppressed in absence of Klf4. This

differential activation of HRR versus NHEJ in normal epithelium versus adenomas points to a

crucial role for Klf4 in regulating DNA damage repair pathways under pathologic conditions.

Several factors play important roles in regulating DNA damage-repair mechanisms, among

them is the tumor suppressor p53. p53 acts as an important link between upstream signaling

and activation of downstream signaling cascades depending on the extent of DNA damage,

and can activate cell cycle arrest and allow the damage to be repaired or it could transactivate

genes involved in the apoptotic machinery (50). Surprisingly, the primary effect of p53 on DNA

repair mechanisms was shown be the inhibition of HRR and of NHEJ repair mechanisms (50).

Our previous work has shown Klf4 to be a crucial mediator of p53-dependent cell cycle arrest

and DNA damage-repair machinery and to suppress p53-dependent apoptosis (30, 33). Our

data from Klf4ΔIS/ApcMin/+ mice, where there is elevated p53 level and suppressed HRR and

NHEJ mechanisms in the absence of Klf4, are in line with these reports. Elevated p53 level

and suppressed HRR and NHEJ repair mechanisms in absence of Klf4 were also observed in

AOM-treated mice, adding validation to our observation on p53 level in Klf4fl/fl/ApcMin/+ and




Ghaleb et al. 23

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Klf4ΔIS/ApcMin/+ mice. Together, our data hint to a potential mechanism by which Klf4 promotes

damaged DNA repair. It is possible that one way Klf4 promotes DNA damage repair, at least in

the colonic epithelium, is by counteracting the suppressive effects of p53 on HRR and NHEJ

repair mechanisms, thus allowing for the DNA repair to proceed. The exact mechanism by

which Klf4 differentially regulates HRR and/or NHEJ DNA DSB repair pathways remains to be

investigated.

Our findings are summarized in Figure 7, where it shows that Klf4 suppresses colonic

tumor formation in association with ApcMin/+ mutation both by suppressing mTOR pathway and

reducing rates of precancerous epigenetic alterations. In response to the colon carcinogen

AOM, Klf4 suppresses carcinogen-induced K-Ras mutations. In both mouse models, Klf4 is

involved in DNA DSB repair by differential regulation of HRR versus NHEJ in normal versus

tumor tissue. The exact mechanism(s) by which Klf4 suppresses tumorigenesis under these

seemingly different conditions requires further investigation. In conclusion, in the mouse

intestinal mucosa, KLF4 plays an integral role in suppressing tumor formation under conditions

of genetic mutations, epigenetic alterations, aberrant DNA damage repair and carcinogen-

induced mutations. Thus, modulation of KLF4 expression in intestinal epithelium represents a

potential therapeutic approach to prevent colon cancer.




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ACKNOWLEDGEMENT

We thank pathologist Dr. Kenneth R. Shroyer, Department of Pathology; Stony Brook

University, for examining staging adenomas formed.




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FIGURE LEGENDS

Figure 1. Klf4ΔIS/ApcMin/+ mice have increased colonic adenoma burden and

penetrance than Klf4fl/fl/ApcMin/+.

Klf4fl/fl and Klf4ΔIS mice were bred to ApcMin/+ mice. Mice were sacrificed at 16-20 weeks

and the number of adenomas was counted and compared between the two genotypes. (A)

Comparison of the number of adenomas per mouse and of penetrance in the colon between

Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice. Each mark on the graph represents the number of

adenomas per mouse. p < 0.001 by paired 2-tailed t-test between the two genotypes. (B) IHC

staining of normal appearing colonic tissue for Klf4 (a & b) and for β-catenin (c & d). Panels a1,

b1, c1 and d1 are enlarged insets of panels a, b, c and d, respectively.

Figure 2. Normal appearing colonic tissue of Klf4ΔIS/ApcMin/+ mice has increased

LOH of Apc and increased ABI than Klf4fl/fl/ApcMin/+.

(A) Example of LOH result from 3 Klf4fl/fl/ApcMin/+ mice (lanes 1-3) and 3 Klf4ΔIS/ApcMin/+

mice (lanes 4-6). (B) Densitometric analysis comparison of the relative ratio of WT:mutant

band intensity shown in (A). (C) H&E staining showing an example of a mitotic colonic

epithelium cell with normal anaphase (i) and another with anaphase bridging (ii). (D) Graphical

representation of anaphase bridging index (ABI) represented as percent of anaphase cells in

normal appearing colonic tissue that harbors anaphase bridging. For panels B and D, analysis

was done using 2-tailed student t-test and One way ANOVA, respectively. *** p < 0.001.

Figure 3. Altered mTOR and histone-modification pathways in normal appearing

colonic tissue of Klf4ΔIS/ApcMin/+.

IHC staining of normal appearing colonic tissue for Cdx2 in Klf4fl/fl/ApcMin/+ mice (A) and

Klf4ΔIS/ApcMin/+ mice (B). Insets show enlarged sections of the colonic crypts of their respective




Ghaleb et al. 33

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panels. (C) Western blot analysis of some components of the mTOR pathway, HDAC1, p53

and Bax in the colon of Klf4fl/fl/ApcMin/+ mice (lanes 1-3) and Klf4ΔIS/ApcMin/+ mice (lanes 4-6).

(D) Western blot analysis of the effect of KLF4 overexpression or suppression on the

expression level of some components of the mTOR pathway, HDAC1, p53, p21 and Bax in

colon cancer cell line HCT116. Lane 1, cells transfected with EGFP vector. Lane 2, cells

transfected with Klf4-EGFP vector. Lane 3 cells, transfected with scrambled siRNA. Lane 4,

cells transfected with KLF4-specific siRNA. (E and F) IHC staining for HDAC1 in Klf4fl/fl/ApcMin/+

mice and Klf4ΔIS/ApcMin/+ mice, respectively. (G and H) IHC staining for p300 in Klf4fl/fl/ApcMin/+

mice and Klf4ΔIS/ApcMin/+ mice, respectively. (I) Whisker plot analysis of the number of p300

positive cells in colonic crypts of in Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice. At least 20 crypts

were counted per mouse. N = 3 mice in each group, *** p < 0.001. Statistical analysis was

performed using 2-tailed student t-test. For whisker plots the central bar represents the mean

while the box represents the quartile range and whiskers represent the range of the data set.

Figure 4. Klf4 differentially regulates NHEJ and HRR DNA DSB repair

mechanisms in the colonic adenomas of Klf4fl/fl/ApcMin/+ mice versus Klf4ΔIS/ApcMin/+

mice.

IHC staining of γH2AX (A, D G & J), Ku70 (B, E H & K) and Rad51 (C, F, I & L) and in

normal looking colonic crypts of Klf4fl/fl/ApcMin/+ mice (A-C) and Klf4ΔIS/ApcMin/+ mice (G-I) and in

adenomas formed in their respective genotypes (D-F) and (J-L). γH2AX is an indicator DNA

double strand breaks, while Ku70 and Rad51 are indicators of active NHEJ and HRR repair

mechanisms, respectively. (M) Quantification of Ku70 positive cells in adenomas of

Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+. (N) Quantification of Rad51 positive cells in adenomas of




Ghaleb et al. 34

34

Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice. ** p < 0.01 and *** p < 0.001. Statistical analysis was

performed using 2-tailed student t-test. Error bars represent Standard error.

Figure 5. Increased number of colonic tumors and β-catenin and cyclin D1

expression levels and activated K-Ras pathway in Klf4ΔIS mice treated with AOM alone.

(A) Graphical comparison of the number of tumors per mouse in the colon between

AOM-treated Klf4fl/fl and Klf4ΔIS mice. (B) IHC staining for β-catenin (a & b), cyclin D1 (c & d)

and p-ERK (e & f) in colonic tumors of AOM-treated Klf4fl/fl and Klf4ΔIS mice. (C) Consensus

sequence of exon 1 of the mouse K-Ras and mutations detected in the colonic tissue of AOM-

treated Klf4fl/fl (in 1 mouse) and Klf4ΔIS mice (in 3 out of 4 mice).

Figure 6. Klf4 differentially regulates NHEJ and HRR DNA double strand break

repair mechanisms in the colonic adenomas of AOM-treated Klf4fl/fl mice versus Klf4ΔIS

mice.

IHC staining of γH2AX (A, D G & J), Ku70 (B, E H & K) and Rad51 (C, F, I & L) in

normal looking colonic crypts of AOM-treated Klf4fl/fl mice (A-C) and Klf4ΔIS mice (G-I) and in

adenomas formed in their respective genotypes (D-F) and (J-L). γH2AX is an indicator of DNA

double strand breaks, while Ku70 and Rad51 are indicators of active NHEJ and HRR repair

mechanisms, respectively. (M) Quantification of Ku70 positive cells in adenomas of AOM-

treated Klf4fl/fl and Klf4ΔIS mice. (N) Densitometric analysis comparison of Rad51 staining

intensity in adenomas of AOM-treated Klf4fl/fl and Klf4ΔIS mice. (P) Western blot analysis of p53

expression level in colonic tissue lysates of AOM-treated Klf4fl/fl mice (Lanes 1-4) and Klf4ΔIS

mice (Lanes 5-8). *** p < 0.001. Statistical analysis was performed using 2-tailed student t-test.

Error bars represent Standard error.




Ghaleb et al. 35

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Figure 7. Schematic diagram of Klf4 and its proposed targets in three different

models that lead to tumor formation.

In the setting of ApcMin/+ mutation, Klf4 might be playing a role in preventing mTOR

pathway activation and resultant increase in LOH. Additionally, Klf4 may be important in

preventing epigenetic alterations via the regulation of histone acetylation. The broken-line box

outlines the general area of the pathway that is possibly targeted by Klf4. Under conditions

where DNA mutations are induced by exogenous factors, such as AOM, the role of Klf4 is

proposed to be in suppressing gene mutations, such as in K-Ras that may result in tumor

formation. Klf4 is also proposed to play a role in DNA DS break repair through the differential

regulation of NHEJ and HRR DNA repair mechanisms.




Published OnlineFirst February 2, 2016.Mol Cancer Res Amr M. Ghaleb, Enas A. Elkarim, Agnieszka B. Bialkowska, et al. Pharmacological Mouse Models of Colonic TumorigenesisKLF4 Suppresses Tumor Formation in Genetic and

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