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author:("ghale, Amr M")
1.  Chemoprevention of Familial Adenomatous Polyposis by Bromo-noscapine (EM011) in the ApcMin/+ Mouse Model 
Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age. Although FAP is involved in only 1% of all colon cancer cases, over 80% of sporadic cancers harbor somatic mutations of APC. We show here that bromo-onoscapine (EM011), a rationally-designed synthetic derivative of a natural non-toxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G2/M arrest in wild type mouse embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with siRNA. Furthermore, both β-catenin levels and activity fell to half the original levels with a concomitant reduction of cell proliferation-inducing cyclin D1, c-Myc, and induction of cytostatic protein p21 prior to caspase-3 activation. Additionally, we show a statistically significant reduction in the number of newly emerging intestinal polyps (to 35% compared with untreated mice) as well as the mean size of polyps (to 42% compared with untreated mice) in EM011-treated ApcMin/+ mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of ApcMin/+ mice showed evidence of elevated apoptosis as revealed by immunohistochemistry. We failed to detect any evidence of histopathological and hematological toxicities following EM011 treatment. Taken together, our data are persuasive that a clinical trial of EM011 is possible for the prevention/amelioration of polyposis in FAP patients.
doi:10.1002/ijc.27344
PMCID: PMC3362662  PMID: 22052467
Familial Adenomatous Polyposis (AFP); Colon Cancer; bromo noscapine; EM011; Noscapine; β-catenin
2.  p53 and p21 Determine the Sensitivity of Noscapine-Induced Apoptosis in Colon Cancer Cells 
Cancer research  2007;67(8):3862-3870.
We have previously discovered the naturally occurring antitussive alkaloid noscapine as a tubulin-binding agent that attenuates microtubule dynamics and arrests mammalian cells at mitosis via activation of the c-Jun NH2-terminal kinase pathway. It is well established that the p53 protein plays a crucial role in the control of tumor cell response to chemotherapeutic agents and DNA-damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. In this study, we compared chemosensitivity, cell cycle distribution, and apoptosis on noscapine treatment in four cell lines derived from the colorectal carcinoma HCT116 cells: p53+/+ (p53-wt), p53−/− (p53-null), p21−/− (p21-null), and BAX−/− (BAX-null). Using these isogenic variants, we investigated the roles of p53, BAX, and p21 in the cellular response to treatment with noscapine. Our results show that noscapine treatment increases the expression of p53 over time in cells with wild-type p53 status. This increase in p53 is associated with an increased apoptotic BAX/Bcl-2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. Conversely, loss of p53 and p21 alleles had a counter effect on both BAX and Bcl-2 expression and the p53-null and p21-null cells were significantly resistant to the antiproliferative and apoptotic effects of noscapine. All but the p53-null cells displayed p53 protein accumulation in a time-dependent manner on noscapine treatment. Interestingly, despite increased levels of p53, p21-null cells were resistant to apoptosis, suggesting a proapoptotic role of p21 and implying that p53 is a necessary but not sufficient condition for noscapine-mediated apoptosis.
doi:10.1158/0008-5472.CAN-06-4282
PMCID: PMC3757339  PMID: 17440101
3.  Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts 
Molecular Cancer  2013;12:89.
Background
Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy.
Methods
To determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs null for Klf4 (Klf4−/−), we transfected Klf4−/−MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4+/+) and untransfected or mock-transfected Klf4−/−MEFs.
Results
We show that overexpression of Klf4 in Klf4−/−MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4−/−MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.
Conclusion
Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.
doi:10.1186/1476-4598-12-89
PMCID: PMC3750599  PMID: 23919723
KLF4; Genetic instability; DNA damage responses; Aneuploidy; Centrosome amplification; Mouse embryonic fibroblasts
4.  Notch1 Regulates the Effects of Matrix Metalloproteinase-9 on Colitis-Associated Cancer in Mice 
Gastroenterology  2011;141(4):1381-1392.
Background & Aims
Inflammatory bowel disease increases the risks for colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP9, also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP9 protects against CAC in mice.
Methods
C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor DAPT or DMSO (control) during each DSS cycle; they were sacrificed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP9−/− mice and HCT116 cells that were stably transfected with MMP9.
Results
Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, demonstrated by increased numbers of tumors and level of dysplasia, compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21WAF1/Cip1, Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP9−/− mice, upon γ-radiation–induced damage of DNA.
Conclusion
MMP9 mediates Notch1 signaling via p53 to regulate apoptosis, cell-cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.
doi:10.1053/j.gastro.2011.06.056
PMCID: PMC3186860  PMID: 21723221
colorectal cancer; mouse model; extracellular matrix; IBD; tumor development
5.  Krüppel-like Factor 5 is Important for Maintenance of Crypt Architecture and Barrier Function in Mouse Intestine 
Gastroenterology  2011;141(4):1302-1313.e6.
Background & Aims
Krüppel-like factor 5 (KLF5) is transcription factor that is expressed by dividing epithelial cells of the intestinal epithelium. KLF5 promotes proliferation in vitro and in vivo and is induced by mitogens and various stress stimuli. To study the role of KLF5 in intestinal epithelial homeostasis, we examined the phenotype of mice with conditional deletion of Klf5 in the gut.
Methods
Mice were generated with intestinal-specific deletion of Klf5 (Vil-Cre;Klf5fl/fl).
Morphological changes in the small intestine and colon were examined by immunohistochemistry, immunoblotting, and real-time PCR.
Results
Klf5 mutant mice were born at a normal Mendelian ratio but had high mortality compared to controls. Complete deletion of Klf5 from the intestinal mucosa resulted in neonatal lethality that corresponded with an absence of epithelial proliferation. Variegated intestinal-specific deletion of Klf5 in adult mice resulted in morphological changes that included a regenerative phenotype, impaired barrier function, and inflammation. Adult mutant mice exhibited defects in epithelial differentiation and migration. These changes were associated with reduced expression of Cdx 1, Cdx2, and Eph and ephrin signaling proteins. Concomitantly, Wnt signaling to β-catenin was reduced. Proliferation in regenerative crypts was associated with increased expression of the progenitor cell marker Sox9.
Conclusions
Deletion of Klf5 in the gut epithelium of mice demonstrated that KLF5 maintains epithelial proliferation, differentiation, and cell positioning along the crypt radial axis. Morphological changes that occur with deletion of Klf5 are associated with disruption of canonical Wnt signaling and increased expression of Sox9.
doi:10.1053/j.gastro.2011.06.086
PMCID: PMC3186863  PMID: 21763241
intestinal homeostasis; gastrointestinal development; genetics; GI tract
6.  Altered Intestinal Epithelial Homeostasis in Mice with Intestine-Specific Deletion of the Krüppel-Like Factor 4 Gene 
Developmental biology  2010;349(2):310-320.
The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is expressed in the post-mitotic, differentiated epithelial cells lining the intestinal tract and exhibits a tumor suppressive effect on intestinal tumorigenesis. Here we report a role for KLF4 in maintaining homeostasis of intestinal epithelial cells. Mice with conditional ablation of the Klf4 gene from the intestinal epithelium were viable. However, both the rates of proliferation and migration of epithelial cells were increased in the small intestine of mutant mice. In addition, the brush-border alkaline phosphatase was reduced as was expression of ephrine-B1 in the small intestine, resulting in mispositioning of Paneth cells. In the colon of mutant mice, there was a reduction of the differentiation marker, carbonic anhydrase-1, and failure of differentiation of goblet cells. Mechanistically, deletion of Klf4 from the intestine resulted in a general activation of genes in the Wnt pathway and a global reduction in expression of genes encoding regulators of differentiation. Taken together, these data provide new insights into the function of KLF4 in regulating postnatal proliferation, migration, differentiation, and positioning of intestinal epithelial cells and demonstrate an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis in vivo.
doi:10.1016/j.ydbio.2010.11.001
PMCID: PMC3022386  PMID: 21070761
KLF4; Wnt; Proliferation; Differentiation; Migration; Paneth Cells; Goblet Cells
7.  Expression of the Tumor Suppressor Krüppel-Like Factor 4 as a Prognostic Predictor for Colon Cancer 
Background
The zinc finger transcription factor Krüppel-like factor 4 (KLF4) regulates numerous physiologic processes including proliferation, differentiation, and development. Studies also showed that KLF4 is involved in tumorigenesis and somatic cell reprogramming. Here we aimed to assess whether KLF4 is a prognostic indicator for colon cancer.
Methods
Levels of KLF4 were measured by immunohistochemical analysis of a tissue microarray containing 367 independent colon cancer sections. Univariate data analysis was performed in addition to construction of multivariate models with several clinicopathologic factors to evaluate KLF4 as an independent predictor of survival and cancer recurrence (disease-free survival).
Results
Colon cancer tissues had significantly overall lower KLF4 levels compared to non-cancer tissues (P < 0.0001). Using logistic regression, a trend was noted for decreased odds of KLF4 expression in higher stages of tumors. In both univariate and multivariate analyses, KLF4 was a significant predictor of survival and recurrence.
Conclusions
KLF4 expression is significantly down-regulated in colon cancer and loss of KLF4 is an independent predictor of survival and recurrence.
Impact
These findings suggest that KLF4 may serve as a prognostic biomarker for colon cancer.
doi:10.1158/1055-9965.EPI-10-0677
PMCID: PMC2952064  PMID: 20699379
KLF4; Colon Cancer; Tissue microarray; Survival; Recurrence
8.  Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts 
Background
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4.
Methods
Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels.
Results
One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data.
Conclusions
These data are not only consistent with previous functional studies of KLF4’s role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4’s functions.
PMCID: PMC3165197  PMID: 21892412
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
9.  Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts 
Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.
PMCID: PMC3165197  PMID: 21892412
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
10.  Haploinsufficiency of Krüppel-Like Factor 5 Rescues the Tumor-Initiating Effect of the ApcMin Mutation in the Intestine 
Cancer research  2009;69(10):4125-4133.
Inactivation of the tumor suppressor adenomatous polyposis coli, with the resultant activation of β-catenin, is the initiating event in the development of a majority of colorectal cancers. Krüppel-like factor 5 (KLF5), a proproliferative transcription factor, is highly expressed in the proliferating intestinal crypt epithelial cells. To determine whether KLF5 contributes to intestinal adenoma formation, we examined tumor burdens in ApcMin/+ mice and ApcMin/+/Klf5+/− mice. Compared with ApcMin/+ mice, ApcMin/+/Klf5+/− mice had a 96% reduction in the number of intestinal adenomas. Reduced tumorigenicity in the ApcMin/+/Klf5+/− mice correlated with reduced levels and nuclear localization of β-catenin as well as reduced expression of two β-catenin targets, cyclin D1 and c-Myc. In vitro studies revealed a physical interaction between KLF5 and β-catenin that enhanced the nuclear localization and transcriptional activity of β-catenin. Thus, KLF5 is necessary for the tumor-initiating activity of β-catenin during intestinal adenoma formation in ApcMin/+ mice, and reduced expression of KLF5 offsets the tumor-initiating activity of the ApcMin mutation by reducing the nuclear localization and activity of β-catenin.
doi:10.1158/0008-5472.CAN-08-4402
PMCID: PMC2702486  PMID: 19435907
11.  The absence of LPA2 attenuates tumor formation in an experimental model of colitis-associated cancer 
Gastroenterology  2009;136(5):1711-1720.
Background & Aims
Chronic inflammation is a risk factor for colon cancer (CC). Lysophosphatidic acid (LPA), a naturally produced phospholipid, mediates multiple effects that are vital to disease process, including inflammation and cancer. The expression of LPA receptor 2 (LPA2) is up-regulated in several types of cancer, including ovarian and colon cancer, but the importance of LPA and LPA2 in the development and progression of CC is unclear. In this study, we sought to determine whether LPA and LPA2 regulate the progression of CC in vivo.
Methods
We examined the potential role of LPA in CC progression by administering LPA to ApcMin/+ mice. We determined the loss of LPA2 function in tumorigenesis in the colon by treating mice with genetic deletion of LPA2 (LPA2−/−) with azoxymethane (AOM) and dextran sulfate sodium (DSS).
Results
We found that LPA increased tumor incidence in Apcmin/+ mice. LPA2−/− mice showed reduced mucosal damage and fewer tumors than wild-type (WT) mice. Reduced epithelial cell proliferation and decreases in β-catenin, Krüppel-like factor 5 (KLF5), and cyclooxygenase-2 (COX-2) expression were observed in LPA2−/− mice. Unlike WT mice, induction of monocyte chemoattractant protein-1 (MCP-1) and macrophage migration inhibitory factor (MIF) was significantly attenuated in LPA2−/− mice with reduced infiltration by macrophages.
Conclusion
These results show that LPA is capable of promoting tumorigenesis in the colon. The absence of LPA2 attenuates several effects that contribute to cancer progression in vivo and, hence, the current study identifies LPA2 as an important modulator of CC.
doi:10.1053/j.gastro.2009.01.002
PMCID: PMC2691721  PMID: 19328876
12.  Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined ApcMin and KRASV12 mutations 
Molecular Cancer  2010;9:63.
Background
Both mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene and activation of the KRAS oncogene are implicated in the pathogenesis of colorectal cancer. Mice harboring a germline ApcMin mutation or intestine-specific expression of the KRASV12 gene have been developed. Both mouse strains develop spontaneous intestinal tumors, including adenoma and carcinoma, though at a different age. The zinc finger transcription factor Krüppel-like factor 5 (KLF5) has previously been shown to promote proliferation of intestinal epithelial cells and modulate intestinal tumorigenesis. Here we investigated the in vivo effect of Klf5 heterozygosity on the propensity of ApcMin/KRASV12 double transgenic mice to develop intestinal tumors.
Results
At 12 weeks of age, ApcMin/KRASV12 mice had three times as many intestinal tumors as ApcMin mice. This increase in tumor number was reduced by 92% in triple transgenic ApcMin/KRASV12/Klf5+/- mice. The reduction in tumor number in ApcMin/KRASV12/Klf5+/- mice was also statistically significant compared to ApcMin mice alone, with a 75% decrease. Compared with ApcMin/KRASV12, tumors from both ApcMin/KRASV12/Klf5+/- and ApcMin mice were smaller. In addition, tumors from ApcMin mice were more distally distributed in the intestine as contrasted by the more proximal distribution in ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both ApcMin and ApcMin/KRASV12 mice but were attenuated in ApcMin/KRASV12/Klf5+/- mice. The levels of β-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of ApcMin/KRASV12/Klf5+/- mice when compared to ApcMin/KRASV12 mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of ApcMin/KRASV12 mice and modestly reduced in ApcMin/KRASV12/Klf5+/- mice. Tumor tissues displayed higher levels of both Klf5 and β-catenin, irrespective of the mouse genotype from which tumors were derived.
Conclusions
Results of the current study confirm the cumulative effect of Apc loss and oncogenic KRAS activation on intestinal tumorigenesis. The drastic reduction in tumor number and size due to Klf5 heterozygosity in ApcMin/KRASV12 mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.
doi:10.1186/1476-4598-9-63
PMCID: PMC2856552  PMID: 20298593
13.  Notch Inhibits Expression of the Krüppel-Like Factor 4 Tumor Suppressor in the Intestinal Epithelium 
Molecular cancer research : MCR  2008;6(12):1920-1927.
The zinc finger-containing transcription factor, Krüppel-like factor 4 (KLF4), inhibits cell proliferation. An in vivo tumor suppressive role for KLF4 is demonstrated by the recent finding that Klf4 haploinsufficiency in ApcMin/+ mice promotes intestinal tumorigenesis. Studies also show that KLF4 is required for the terminal differentiation of goblet cells in the mouse intestine. The Notch signaling pathway suppresses goblet cell formation and is up-regulated in intestinal tumors. Here we investigated the relationship between Notch signaling and KLF4 expression in intestinal epithelial cells. The rate of proliferation of HT29 human colon cancer cells was reduced when treated with the γ-secretase inhibitor dibenzazepine (DBZ) to inhibit Notch or siRNA directed against Notch. KLF4 levels were increased in DBZ- or Notch siRNA-treated cells. Conversely, over-expression of Notch in HT29 cells reduced KLF4 levels, suppressed KLF4 promoter activity and increased proliferation rate. Treatment of ApcMin/+ mice with DBZ resulted in a 50% reduction in the number of intestinal adenomas compared to the vehicle-treated group (p < 0.001). Both the normal-appearing intestinal mucosa and adenomas obtained from DBZ-treated ApcMin/+ mice had increased goblet cell numbers and Klf4 staining accompanied by reduced cyclin D1 and Ki67 staining when compared to those from vehicle-treated mice. Results of these studies indicate that Notch signaling suppresses KLF4 expression in intestinal tumors and colorectal cancer cells. Inhibition of Notch signaling increases KLF4 expression and goblet cell differentiation, and reduces proliferation and tumor formation. KLF4 is therefore a potential mediator for the anti-tumor effect of Notch inhibitors such as DBZ.
doi:10.1158/1541-7786.MCR-08-0224
PMCID: PMC2628949  PMID: 19074836
KLF4; goblet cells; γ-secretase inhibitor; ApcMin/+ mouse; adenomas
14.  Krüppel-Like Factor 5 Mediates Cellular Transformation during Oncogenic KRAS-Induced Intestinal Tumorigenesis 
Gastroenterology  2007;134(1):120-130.
Background & Aims
Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that regulates cell proliferation. Oncogenic KRAS mutations are commonly found in colorectal cancers. We aimed to determine whether KLF5 mediates KRAS functions during intestinal tumorigenesis.
Methods
The effects of KLF5 on proliferation and transformation were examined in IEC-6 intestinal epithelial cells stably transfected with an inducible KRASV12G. KLF5 expression was examined in intestinal tumors derived from transgenic mice expressing KRASV12G under a villin promoter and in human colorectal cancers with mutated KRAS.
Results
Induction of KRASV12G in IEC-6 cells resulted in increased expression of KLF5, accompanied by an increased rate of proliferation and anchorage-independent growth. Inhibition of KLF5 expression by MEK inhibitors or KLF5-specific small interfering RNA (siRNA) reduced proliferation and anchorage-independent growth despite KRASV12G induction. Human colorectal cancer cell lines with mutated KRAS contained high levels of KLF5 and reduction of KLF5 by MEK inhibitors or KLF5 siRNA also led to reduced proliferation and transformation. In vivo, both intestinal tumors derived from mice transgenic for villin-KRASV12G and human primary colorectal cancers with mutated KRAS contained high levels of KLF5 and increased staining of the proliferative marker, Ki67.
Conclusions
Elevated levels of KLF5 protein are strongly correlated with activating KRAS mutations in intestinal tumors both in vitro and in vivo. Inhibition of KLF5 expression in these tumor cells resulted in significantly reduced rates of proliferation and transforming activities. We conclude that KLF5 is an important mediator of oncogenic KRAS transforming functions during intestinal tumorigenesis.
doi:10.1053/j.gastro.2007.10.023
PMCID: PMC2194652  PMID: 18054006
15.  Haploinsufficiency of Krüppel-Like Factor 4 Promotes Adenomatous Polyposis Coli–Dependent Intestinal Tumorigenesis 
Cancer research  2007;67(15):7147-7154.
The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently down-regulated in colorectal cancer. Previous studies showed that the expression of KLF4 was activated by the colorectal cancer tumor suppressor adeno-matous polyposis coli (APC) and that KLF4 repressed the Wnt/β-catenin pathway. Here, we examined whether KLF4 plays a role in modulating intestinal tumorigenesis by comparing the tumor burdens in mice heterozygous for the ApcMin allele (ApcMin/+) and those heterozygous for both the ApcMin and Klf4 alleles (Klf4+/−/ApcMin/+). Between 10 and 20 weeks of age, Klf4+/−/ApcMin/+ mice developed, on average, 59% more intestinal adenomas than ApcMin/+ mice (P < 0.0001). Immunohistochemical staining showed that Klf4 protein levels were lower in the normal-appearing intestinal tissues of Klf4+/−/ApcMin/+ mice compared with wild-type, Klf4+/−, or ApcMin/+ mice. In contrast, the levels of β-catenin and cyclin D1 were higher in the normal-appearing intestinal tissues of Klf4+/−/ApcMin/+ mice compared with the other three genotypes. Klf4 levels were further decreased in adenomas from both ApcMin/+ and Klf4+/−/ApcMin/+ mice compared with their corresponding normal-appearing tissues. Reverse transcription-PCR showed an inverse correlation between adenoma size and Klf4 mRNA levels in both Klf4+/−/ApcMin/+ and ApcMin/+ mice. There was also a progressive loss of heterozygosity of the wild-type Apc allele in adenomas with increasing size from Klf4+/−/ApcMin/+ and ApcMin/+ mice. Results from this study show that KLF4 plays an important role in promoting the development of intestinal adenomas in the presence of ApcMin mutation.
doi:10.1158/0008-5472.CAN-07-1302
PMCID: PMC2373271  PMID: 17671182
16.  The Pathobiology of Krüppel-like Factors in Colorectal Cancer 
The Krüppel-like factor (KLF) proteins are zinc finger–containing transcription factors that exert important functions in regulating diverse biologic processes such as growth, proliferation, differentiation, development, inflammation, and apoptosis. Many KLFs have also been shown to play significant roles in tumorigenesis of various organs and tissues. Three in particular—KLF4, KLF5, and KLF6—are often dysregulated in tumors of the gastrointestinal tract, including colorectal cancer. This article reviews the functions of these three KLFs in normal gastrointestinal biology and their pathobiologic roles in colorectal cancer.
PMCID: PMC2394726  PMID: 18504508
17.  Krüppel-like factor 4 prevents centrosome amplification following γ-irradiation-induced DNA damage 
Oncogene  2005;24(25):4017-4025.
Centrosome duplication is a carefully controlled process in the cell cycle. Previous studies indicate that the tumor suppressor, p53, regulates centrosome duplication. Here, we present evidence for the involvement of the mammalian Krüppel-like transcription factor, KLF4, in preventing centrosome amplification following DNA damage caused by γ-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+/+), displayed stable centrosome numbers following γ-irradiation. In contrast, HCT116 cells null for the p53 alleles (HCT116 p53−/−) exhibited centrosome amplification after irradiation. In the latter cell line, KLF4 was not activated following γ-irradiation due to the absence of p53. However, centrosome amplification could be suppressed in irradiated HCT116 p53−/− cells by conditional induction of exogenous KLF4. Conversely, in a HCT116 p53 +/+ cell line stably transfected with small hairpin RNA (shRNA) designed to specifically inhibit KLF4, γ-irradiation induced centrosome amplification. In these cells, the inability of KLF4 to become activated in response to DNA damage was directly associated with an increase in cyclin E level and Cdk2 activity, both essential for regulating centrosome duplication. Cotransfection experiments showed that KLF4 overexpression suppressed the promoter activity of the cyclin E gene. The results of this study demonstrated that KLF4 is both necessary and sufficient in preventing centrosome amplification following γ-radiation-induced DNA damage and does so by transcriptionally suppressing cyclin E expression.
doi:10.1038/sj.onc.1208576
PMCID: PMC1626272  PMID: 15806166
cell cycle; GKLF; p53; Cyclin E; Cdk2; small hairpin RNA (shRNA)
18.  Transcriptional Profiling of the Cell Cycle Checkpoint Gene Krüppel-Like Factor 4 Reveals a Global Inhibitory Function in Macromolecular Biosynthesis 
Gene expression  2006;13(2):85-96.
Krüppel-like factor 4 (KLF4; also known as gut-enriched Krüppel-like factor or GKLF) is known to exhibit checkpoint function during the G1/S and G2/M transitions of the cell cycle. The mechanism by which KLF4 exerts these effects is not fully established. Here we investigated the expression profile of KLF4 in an inducible system over a time course of 24 h. Using oligonucleotide microarrays, we determined that the fold changes relative to control in expression levels of KLF4 exhibited a time-dependent increase from 3- to 20-fold between 4 and 24 h following KLF4 induction. During this period and among a group of 473 cell cycle regulatory genes examined, 96 were positively correlated and 86 were negatively correlated to KLF4's expression profile. Examples of upregulated cell cycle genes include those encoding tumor suppressors such as MCC and FHIT, and cell cycle inhibitors such as CHES1 and CHEK1. Examples of downregulated genes include those that promote the cell cycle including several cyclins and those required for DNA replication. Unexpectedly, several groups of genes involved in macromolecular synthesis, including protein biosynthesis, transcription, and cholesterol biosynthesis, were also significantly inhibited by KLF4. Thus, KLF4 exerts a global inhibitory effect on macromolecular biosynthesis that is beyond its established role as a cell cycle inhibitor.
PMCID: PMC1626270  PMID: 17017123
Cell cycle; Checkpoint; Microarray; Cholesterol; Ribosomal proteins; Transcription
19.  The diverse functions of Krüppel-like factors 4 and 5 in epithelial biology and pathobiology 
Summary
The Krüppel-like factors (KLFs) comprise a family of evolutionarily conserved zinc finger transcription factors that regulate numerous biological processes including proliferation, differentiation, development and apoptosis. KLF4 and KLF5 are two closely related members of this family and are both highly expressed in epithelial tissues. In the intestinal epithelium, KLF4 is expressed in terminally differentiated epithelial cells at the villus borders of the mucosa and inhibits cell growth, while KLF5 is expressed in proliferating epithelial cells at the base of the intestinal crypts and promotes cell growth. KLF4 and KLF5 respond to a myriad of external stress stimuli and are likely involved in restoring cellular homeostasis following exposure to stressors. Confirming their importance in maintaining tissue integrity, KLF4 and KLF5 are both dysregulated in various types of cancer. Here we review the recent advances in defining the physiological and pathobiological roles of KLF4 and KLF5, focusing on their functions in the intestinal epithelium.
doi:10.1002/bies.20581
PMCID: PMC2211634  PMID: 17508399
20.  Krüppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation 
Cell research  2005;15(2):92-96.
Krüppel-like factors (KLFs) are evolutionarily conserved zinc finger-containing transcription factors with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. KLF4 and KLF5 are two closely related members of the KLF family that have a similar tissue distribution in embryos and adults. However, the two KLFs often exhibit opposite effects on regulation of gene transcription, despite binding to similar, if not identical, cis-acting DNA sequences. In addition, KLF4 and 5 exert contrasting effects on cell proliferation in many instances; while KLF4 is an inhibitor of cell growth, KLF5 stimulates proliferation. Here we review the biological properties and biochemical mechanisms of action of the two KLFs in the context of growth regulation.
doi:10.1038/sj.cr.7290271
PMCID: PMC1317089  PMID: 15740636
cancer; cell cycle; KLF; transcription; transformation; zinc fingers; BTE, basic transcription element; BTEB2, basic transcription element binding protein 2; CYP1A1, cytochrome P-450IA1; KLF, Krüppel-like factor; MAPK, mitogen-activated protein kinase

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