Publications by authors named "Mandayam O Nandan"

28 Publications

  • Page 1 of 1

KLF5 mediates the hyper-proliferative phenotype of the intestinal epithelium in mice with intestine-specific endogenous K-Ras expression.

Am J Cancer Res 2018 1;8(4):723-731. Epub 2018 Apr 1.

Department of Medicine, School of Medicine, Stony Brook University, Stony Brook NY 11794, USA.

Oncogenic activation is a common mutational event in colorectal cancer. We previously showed that transcription factor, Krüppel-like factor 5 (KLF5), contributes to intestinal polyposis in mice with activation. At 14 months of age, mice developed small intestinal and colonic hyperplastic polyps while had none. The intestinal crypts of mice contained a higher number of mitotic figures and increased crypt heights compared to controls. The intestinal epithelium of mice showed prolific KLF5 expression throughout and above the elongated crypts. In contrast, KLF5 expression was limited to the upper crypt region in the controls. The levels of K-Ras effectors were significantly increased in as compared to controls. The mice showed decreased survival upon treatment with azoxymethane (AOM) as compared to controls. Furthermore, loss of one of alleles reduced levels of K-Ras effector proteins and prevented mortality of mice upon AOM treatment. The mice spontaneously develop hyperplastic intestinal polyps and display a hyper-proliferative intestinal phenotype with elongated crypts, increased numbers of mitotic figures, elevated expression of KLF5, and other pro-proliferative targets. Induction of colonic tumorigenesis with AOM is detrimental to mice that is in part dependent of KLF5.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934562PMC
April 2018

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses.

J Vis Exp 2016 07 13(113). Epub 2016 Jul 13.

Department of Medicine, Stony Brook University School of Medicine; Department of Physiology & Biophysics, Stony Brook University School of Medicine;

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.
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http://dx.doi.org/10.3791/54161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993444PMC
July 2016

Role of Krüppel-like factor 5 in the maintenance of the stem cell niche in the intestinal crypt.

Stem Cell Transl Investig 2015;2(2)

Department of Medicine, Stony Brook University School of Medicine, HSC-T16 Room 020, Stony Brook, NY 11794, United States ; Department of Physiology and Biophysics, Stony Brook University School of Medicine, HSC-T16 Room 020, Stony Brook, NY 11794, United States.

The intestinal epithelium is a tissue that undergoes continuous self-renewal initiated at the bottom of the crypts, which harbor the intestinal stem cell (ISC) pool. The ISC pool is sub-divided into crypt base columnar (CBC) cells at the crypt bottom and label retention cells (LRC) at position +4 from the crypt bottom. CBC cells are marked by Leucine-rich repeat-containing G-protein coupled receptor (Lgr5) while LRC cells are identified by several markers including Bmi1, mTert, Hopx, Lrig1, and Sox9. Krüppel-like factors (KLFs) belong to a family of transcription factors that exert important physiological function in various tissues. In the intestine, KLF4 is predominantly expressed in the terminally differentiated, non-proliferating cells lining the villus. Its deletion in the adult mouse intestine results in perturbed homeostasis. In contrast, KLF5 is expressed in actively proliferating cells of the intestinal crypt, including CBC cells and transit amplifying (TA) cells. We recently investigated the effect of deletion specifically from the Lgr5-expressing CBC cells in adult mouse intestine using an inducible Cre recombinase system. Shortly (3-5 days) after Cre induction, proliferation of both CBC and TA cells ceased, which was accompanied by an increase in apoptosis in the crypt. Beginning at two weeks following Cre induction, both Klf5 expression and proliferation re-appeared but without the re-emergence of Lgr5-positive CBC cells, which were eventually depleted by four months following induction. These findings indicate that KLF5 plays an important role in regulating proliferation and survival of CBC stem cells in the intestine.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474380PMC
January 2015

Krüppel-like factor 6 regulates mitochondrial function in the kidney.

J Clin Invest 2015 Mar 17;125(3):1347-61. Epub 2015 Feb 17.

Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.
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http://dx.doi.org/10.1172/JCI77084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362257PMC
March 2015

Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells.

Stem Cell Res 2015 Jan 6;14(1):10-9. Epub 2014 Nov 6.

Department of Medicine, Stony Brook University School of Medicine, HSC-T16 Room 020, Stony Brook, NY 11794, United States. Electronic address:

Krüppel-like factor 5 (KLF5) is a pro-proliferative transcription factor that is expressed in dividing epithelial cells of the intestinal crypt. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) has been identified as a stem cell marker in both small intestinal and colonic epithelial cells. To determine whether KLF5 regulates proliferation of intestinal stem cells, we investigated the effects of Klf5 deletion specifically from the intestinal stem cells in adult mice. Mice with inducible intestinal stem cell-specific deletion of Klf5 (Lgr5-Klf5(fl/fl)) were injected with tamoxifen for 5 consecutive days to induce Lgr5-driven Cre expression. Intestinal and colonic tissues were examined by immunohistochemistry at various time points up to 112days following start of tamoxifen treatment. Klf5 is co-localized in the crypt-based columnar (CBC) cells that express Lgr5. By 11days following the start of tamoxifen treatment, Lgr5-positive crypts from which Klf5 was deleted exhibited a loss of proliferation that was accompanied by an increase in apoptosis. Beginning at 14days following the start of tamoxifen treatment, both Klf5 expression and proliferation were re-established in the transit-amplifying epithelial cells but not in the Lgr5-positive CBC cells. By 112days post-treatment, up to 90% of the Lgr5-positive cells from which Klf5 was deleted were lost from the intestinal crypts. These results indicate a critical role for KLF5 in the survival and maintenance of intestinal stem cells.
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http://dx.doi.org/10.1016/j.scr.2014.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310780PMC
January 2015

Krüppel-like factor 4 is a radioprotective factor for the intestine following γ-radiation-induced gut injury in mice.

Am J Physiol Gastrointest Liver Physiol 2015 Jan 20;308(2):G121-38. Epub 2014 Nov 20.

Department of Medicine, Stony Brook University, Stony Brook, New York; and

Gut radiation-induced injury is a concern during treatment of patients with cancer. Krüppel-like factor 4 (KLF4) is expressed in differentiated villous epithelial cells of the small intestine. We previously showed that KLF4 protects cells from apoptosis following γ-irradiation in vitro. We sought to determine whether KLF4 mediates the small intestinal response to γ-irradiation in vivo. Mice with intestinal epithelium-specific deletion of Klf4 (Klf4(ΔIS)) and control (Klf4(fl/fl)) mice were irradiated with total-body γ-radiation. Following irradiation, the Klf4(ΔIS) mice had significantly increased mortality compared with irradiated Klf4(fl/fl) mice. Immunohistochemistry and immunofluorescence staining were used to assess the morphological changes, levels of proliferation, and apoptosis in the intestinal epithelium. At 96 h following irradiation, there was a regenerative response manifested by an expansion of the proliferative zone in both mouse groups, with the control mice having a higher proliferative activity than the Klf4(ΔIS) group. In addition, there was a significant increase in the number of Klf4/Ki67-copositive cells in the irradiated control mice compared with unirradiated mice. Also, the irradiated Klf4(ΔIS) mice had a significantly higher number of crypt cells positive for apoptosis, p53, and p21 compared with irradiated Klf4(fl/fl) mice. Taken together, our data suggest that Klf4 may function as a radioprotective factor against gastrointestinal syndrome in mice following γ-irradiation by inhibiting apoptosis in the acute response to irradiation and contributing to crypt regeneration.
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http://dx.doi.org/10.1152/ajpgi.00080.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297857PMC
January 2015

Inducible intestine-specific deletion of Krüppel-like factor 5 is characterized by a regenerative response in adult mouse colon.

Dev Biol 2014 Mar 15;387(2):191-202. Epub 2014 Jan 15.

Department of Medicine, Stony Brook University School of Medicine, HSC-T16 Room 020, Stony Brook, NY 11794, USA. Electronic address:

Krüppel-like factor 5 (KLF5) is a pro-proliferative transcriptional regulator primarily expressed in the intestinal crypt epithelial cells. Constitutive intestine-specific deletion of Klf5 is neonatal lethal suggesting a crucial role for KLF5 in intestinal development and homeostasis. We have previously shown Klf5 to play an active role regulating intestinal tumorigenesis. Here we examine the effect of inducible intestine-specific deletion of Klf5 in adult mice. Klf5 is lost from the intestine beginning at day 3 after the start of a 5-day treatment with the inducer tamoxifen. Although the mice have no significant weight loss or lethality, the colonic tissue shows signs of epithelial distress starting at day 3 following induction. Accompanying the morphological changes is a significant loss of proliferative crypt epithelial cells as revealed by BrdU or Ki67 staining at days 3 and 5 after start of tamoxifen. We also observed a loss of goblet cells from the colon and Paneth cells from the small intestine upon induced deletion of Klf5. In addition, loss of Klf5 from the colonic epithelium is accompanied by a regenerative response that coincides with an expansion in the zone of Sox9 expression along the crypt axis. At day 11, both proliferation and Sox9 expression return to baseline levels. Microarray and quantitative PCR analyses reveal an up-regulation of several regeneration-associated genes (Reg1A, Reg3G and Reg3B) and down-regulation of many Klf5 targets (Ki-67, cyclin B, Cdc2 and cyclin D1). Sox9 and Reg1A protein levels are also increased upon Klf5 loss. Lentiviral-mediated knockdown of KLF5 and exogenous expression of KLF5 in colorectal cancer cell lines confirm that Sox9 expression is negatively regulated by KLF5. Furthermore, ChIP assays reveal a direct association of KLF5 with both the Sox9 and Reg1A promoters. We have shown that disruption of epithelial homeostasis due to Klf5 loss from the adult colon is followed by a regenerative response led by Sox9 and the Reg family of proteins. Our study demonstrates that adult mouse colonic tissue undergoes acute physiological changes to accommodate the loss of Klf5 withstanding epithelial damage further signifying importance of Klf5 in colonic homeostasis.
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http://dx.doi.org/10.1016/j.ydbio.2014.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949508PMC
March 2014

A colon cancer-derived mutant of Krüppel-like factor 5 (KLF5) is resistant to degradation by glycogen synthase kinase 3β (GSK3β) and the E3 ubiquitin ligase F-box and WD repeat domain-containing 7α (FBW7α).

J Biol Chem 2014 Feb 7;289(9):5997-6005. Epub 2014 Jan 7.

From the Departments of Medicine and.

Krüppel-like factor 5 (KLF5) is a zinc finger transcription factor that is highly expressed in the crypt epithelial cells of the intestine and plays a critical role in regulating proliferation of both normal intestinal epithelial cells and colorectal cancer cells. Stability of the KLF5 is mediated by proteasomal degradation via phosphorylation by glycogen synthase kinase 3β (GSK3β) and recognition by F-box and WD repeat domain-containing 7 (FBW7) of a phosphodegron sequence surrounding serine 303 in KLF5. A genomic analysis of colorectal cancer tissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence. We hypothesized that due to its close proximity to the phosphodegron sequence, the P301S mutation may affect signaling that is involved in proper KLF5 degradation. We demonstrated that the P301S KLF5 mutant has a longer half-life than wild type (WT) KLF5. Furthermore, P301S KLF5 has a higher transcriptional activity than WT KLF5 as demonstrated by luciferase assays using cyclin D1 and CDC2 promoter constructs. In contrast to WT KLF5, P301S KLF5 does not physically interact with FBW7α. Concomitantly, the P301S KLF5 mutant displays reduced levels of phosphorylation at serine 303 in comparison with WT KLF5. These results of our study indicate that amino acid residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence by FBW7α and that the P301S mutation inhibits this recognition, leading to a degradation-resistant protein with elevated levels and enhanced transcriptional activity. These findings raise a potentially oncogenic role for the P301S KLF5 mutant in colorectal cancer.
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http://dx.doi.org/10.1074/jbc.M113.508549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937667PMC
February 2014

The E3 ubiquitin ligase SMAD ubiquitination regulatory factor 2 negatively regulates Krüppel-like factor 5 protein.

J Biol Chem 2011 Nov 27;286(46):40354-64. Epub 2011 Sep 27.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

The zinc finger transcription factor Krüppel-like factor 5 (KLF5) is regulated posttranslationally. We identified SMAD ubiquitination regulatory factor 2 (SMURF2), an E3 ubiquitin ligase, as an interacting protein of KLF5 by yeast two-hybrid screen, coimmunoprecipitation, and indirect immunofluorescence studies. The SMURF2-interacting domains in KLF5 were mapped to its carboxyl terminus, including the PY motif of KLF5 and its zinc finger DNA-binding domain. KLF5 protein levels were reduced significantly upon overexpression of SMURF2 but not catalytically inactive SMURF2-C716A mutant or SMURF1. SMURF2 alone reduced the protein stability of KLF5 as shown by cycloheximide chase assay, indicating that SMURF2 specifically destabilizes KLF5. In contrast, KLF5(1-165), a KLF5 amino-terminal construct that lacks the PY motif and DNA binding domain, was not degraded by SMURF2. The degradation of KLF5 by SMURF2 was blocked by the proteasome inhibitor MG132, and SMURF2 efficiently ubiquitinated both overexpressed and endogenous KLF5. In contrast, knocking down SMURF2 by siRNAs significantly enhanced KLF5 protein levels, reduced ubiquitination of KLF5, and increased the expression of cyclin D1 and PDGF-A, two established KLF5 target genes. In consistence, SMURF2, but not the E3 ligase mutant SMURF2-C716A, significantly inhibited the transcriptional activity of KLF5, as demonstrated by dual luciferase assay using the PDGF-A promoter, and suppressed the ability of KLF5 to stimulate cell proliferation as measured by BrdU incorporation. Hence, SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by targeting it for proteasomal degradation.
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http://dx.doi.org/10.1074/jbc.M111.258707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220560PMC
November 2011

An Update on the Biology of RAS/RAF Mutations in Colorectal Cancer.

Curr Colorectal Cancer Rep 2011 Jun;7(2):113-120

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

Deaths caused by colorectal cancer (CRC) are among the leading causes of cancer-related death in the United States and around the world. Approximately 150,000 Americans are diagnosed with CRC each year and around 50,000 will die from it. Mutations in many key genes have been identified that are important to the pathogenesis of CRC. Among the genes mutated in CRC, RAS and RAF mutations are common events. Both RAS and RAF are critical mediators of the mitogen-activated protein kinase (MAPK) pathway that is involved in regulating cellular homeostasis, including proliferation, survival, and differentiation. In this review, we provide a historical perspective and update on RAS/RAF mutations as related to colorectal cancer. Additionally, we will review recent mouse models of RAS and RAF mutations that have an impact on CRC research.
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http://dx.doi.org/10.1007/s11888-011-0086-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3103074PMC
June 2011

Expression of the tumor suppressor Krüppel-like factor 4 as a prognostic predictor for colon cancer.

Cancer Epidemiol Biomarkers Prev 2010 Oct 10;19(10):2631-8. Epub 2010 Aug 10.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.

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 done 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 with noncancer tissues (P < 0.0001). Using logistic regression, a trend was noted for decreased odds of KLF4 expression in higher stages of tumors. In univariate and multivariate analyses, KLF4 was a significant predictor of survival and recurrence.

Conclusions: KLF4 expression is significantly downregulated 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.
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http://dx.doi.org/10.1158/1055-9965.EPI-10-0677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952064PMC
October 2010

Genetic and Chemical Models of Colorectal Cancer in Mice.

Curr Colorectal Cancer Rep 2010 Mar;6(2):51-59

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 201 Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, GA 30322, USA.

Colorectal cancer (CRC) is a significant health concern because of its associated mortality. Most CRCs exhibit dysregulation of the Wnt signaling pathway, caused by mutational inactivation of the adenomatous polyposis coli tumor suppressor gene (APC) or mutational activation of β-catenin. Disease progression is accompanied by additional mutations in the KRAS oncogene and p53 tumor suppressor gene. Other CRCs are microsatellite unstable because of mutational inactivation or epigenetic silencing of key molecules responsible for DNA mismatch repair. This review focuses on several common mouse models of CRC, highlighting the consequences of germline mutation of the aforementioned tumor suppressor genes or proto-oncogenes. This article also discusses chemical carcinogens that adversely affect the intestinal tissues with formation of colorectal neoplasia in mice. These mouse models have significantly contributed to the understanding of the mechanisms responsible for CRC pathogenesis and also may serve as potential vehicles for therapeutic intervention.
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http://dx.doi.org/10.1007/s11888-010-0046-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2850068PMC
March 2010

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

Mol Cancer 2010 Mar 18;9:63. Epub 2010 Mar 18.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 201 Whitehead Research Building, 615 Michael Street, Atlanta, GA 30322, USA.

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 beta-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 beta-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.
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http://dx.doi.org/10.1186/1476-4598-9-63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2856552PMC
March 2010

The role of Krüppel-like factors in the reprogramming of somatic cells to induced pluripotent stem cells.

Histol Histopathol 2009 10;24(10):1343-55

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.

The potential for clinical application of pluripotent embryonic stem cells is immense but hampered by moral and ethical complications. Recent advances in the reprogramming of somatic cells by defined factors to a state that resemble embryonic stem cells have created tremendous excitement in the field. Four factors, Sox2, Oct4, Klf4 and c-Myc, when exogenously introduced into somatic cells, can lead to the formation of induced pluripotent stem (iPS) cells that have the capacity for self-renewal and differentiation into tissues of all three germ layers. In this review, we focus on the role of Krüppel-like factors (KLFs) in regulating somatic cell reprogramming. KLFs are zinc finger-containing transcription factors with diverse biological functions. We first provide an overview of the KLF family of regulatory proteins, paying special attention to the established biological and biochemical functions of KLF4 and KLF5. We then review the role of KLFs in somatic cell reprogramming and delineate the putative mechanism by which KLFs participates the establishment and self-renewal of iPS cells. Further research is likely to provide additional insight into the mechanisms of somatic cell reprogramming and refinement of the technique with which to generate clinically relevant iPS cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753264PMC
http://dx.doi.org/10.14670/HH-24.1343DOI Listing
October 2009

Haploinsufficiency of Krüppel-like factor 5 rescues the tumor-initiating effect of the Apc(Min) mutation in the intestine.

Cancer Res 2009 May 12;69(10):4125-33. Epub 2009 May 12.

Division of Digestive Diseases, Department of Medicine, Department of Pharmacology, and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA.

Inactivation of the tumor suppressor adenomatous polyposis coli, with the resultant activation of beta-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 Apc(Min/+) mice and Apc(Min/+)/Klf5(+/-) mice. Compared with Apc(Min/+) mice, Apc(Min/+)/Klf5(+/-) mice had a 96% reduction in the number of intestinal adenomas. Reduced tumorigenicity in the Apc(Min/+)/Klf5(+/-) mice correlated with reduced levels and nuclear localization of beta-catenin as well as reduced expression of two beta-catenin targets, cyclin D1 and c-Myc. In vitro studies revealed a physical interaction between KLF5 and beta-catenin that enhanced the nuclear localization and transcriptional activity of beta-catenin. Thus, KLF5 is necessary for the tumor-initiating activity of beta-catenin during intestinal adenoma formation in Apc(Min/+) mice, and reduced expression of KLF5 offsets the tumor-initiating activity of the Apc(Min) mutation by reducing the nuclear localization and activity of beta-catenin.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-4402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2702486PMC
May 2009

Notch inhibits expression of the Krüppel-like factor 4 tumor suppressor in the intestinal epithelium.

Mol Cancer Res 2008 Dec;6(12):1920-7

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA.

The zinc finger-containing transcription factor, Krüppel-like factor 4 (KLF4), inhibits cell proliferation. An in vivo tumor-suppressive role for KLF4 is shown by the recent finding that Klf4 haploinsufficiency in Apc(Min/+) 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 gamma-secretase inhibitor dibenzazepine to inhibit Notch signaling or small interfering RNA directed against Notch. KLF4 levels were increased in dibenzazepine-treated or Notch small interfering RNA-treated cells. Conversely, overexpression of Notch in HT29 cells reduced KLF4 levels, suppressed KLF4 promoter activity, and increased proliferation rate. Treatment of Apc(Min/+) mice with dibenzazepine resulted in a 50% reduction in the number of intestinal adenomas compared with the vehicle-treated group (P < 0.001). Both the normal-appearing intestinal mucosa and adenomas obtained from dibenzazepine-treated Apc(Min/+) mice had increased goblet cell numbers and Klf4 staining accompanied by reduced cyclin D1 and Ki-67 staining when compared with 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 antitumor effect of Notch inhibitors such as dibenzazepine.
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http://dx.doi.org/10.1158/1541-7786.MCR-08-0224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628949PMC
December 2008

Krüppel-like factor 5 mediates transmissible murine colonic hyperplasia caused by Citrobacter rodentium infection.

Gastroenterology 2008 Apr 11;134(4):1007-16. Epub 2008 Jan 11.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.

Background & Aims: Krüppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in proliferating crypt cells of the intestinal epithelium. KLF5 has a pro-proliferative effect in vitro and is induced by mitogenic and stress stimuli. To determine whether KLF5 is involved in mediating proliferative responses to intestinal stressors in vivo, we examined its function in a mouse model of transmissible murine colonic hyperplasia triggered by colonization of the mouse colon by the bacteria Citrobacter rodentium.

Methods: Heterozygous Klf5 knockout (Klf5(+/-)) mice were generated from embryonic stem cells carrying an insertional disruption of the Klf5 gene. Klf5(+/-) mice or wild-type (WT) littermates were infected with C rodentium by oral gavage. At various time points postinfection, mice were killed and distal colons were harvested. Colonic crypt heights were determined morphometrically from sections stained with H&E. Frozen tissues were stained by immunofluorescence using antibodies against Klf5 and the proliferation marker, Ki67, to determine Klf5 expression and numbers of proliferating cells per crypt.

Results: Infection of WT mice with C rodentium resulted in a 2-fold increase in colonic crypt heights at 14 days postinfection and was accompanied by a 1.7-fold increase in Klf5 expression. Infection of Klf5(+/-) mice showed an attenuated induction of Klf5 expression, and hyperproliferative responses to C rodentium were reduced in the Klf5(+/-) animals as compared with WT littermates.

Conclusion: Our study shows that Klf5 is a key mediator of crypt cell proliferation in the colon in response to pathogenic bacterial infection.
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http://dx.doi.org/10.1053/j.gastro.2008.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2336106PMC
April 2008

Human cancer cells commonly acquire DNA damage during mitotic arrest.

Cancer Res 2007 Dec;67(24):11487-92

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

The mitotic checkpoint is a mechanism that arrests the progression to anaphase until all chromosomes have achieved proper attachment to mitotic spindles. In cancer cells, satisfaction of this checkpoint is frequently delayed or prevented by various defects, some of which have been causally implicated in tumorigenesis. At the same time, deliberate induction of mitotic arrest has proved clinically useful, as antimitotic drugs that interfere with proper chromosome-spindle interactions are effective anticancer agents. However, how mitotic arrest contributes to tumorigenesis or antimitotic drug toxicity is not well defined. Here, we report that mitotic chromosomes can acquire DNA breaks during both pharmacologic and genetic induction of mitotic arrest in human cancer cells. These breaks activate a DNA damage response, occur independently of cell death, and subsequently manifest as karyotype alterations. Such breaks can also occur spontaneously, particularly in cancer cells containing mitotic spindle abnormalities. Moreover, we observed evidence of some breakage in primary human cells. Our findings thus describe a novel source of DNA damage in human cells. They also suggest that mitotic arrest may promote tumorigenesis and antimitotic toxicity by provoking DNA damage.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-5162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2248235PMC
December 2007

Krüppel-like factor 5 mediates cellular transformation during oncogenic KRAS-induced intestinal tumorigenesis.

Gastroenterology 2008 Jan 17;134(1):120-30. Epub 2007 Oct 17.

Division of Digestive Diseases, Department of Medicine, Emory University, Atlanta, Georgia, USA.

Background & Aims: Krüppel-like factor 5 (KLF5) is a zinc finger-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 inducible KRAS(V12G). KLF5 expression was examined in intestinal tumors derived from transgenic mice expressing KRAS(V12G) under villin promoter and in human colorectal cancers with mutated KRAS.

Results: Induction of KRAS(V12G) in IEC-6 cells resulted in increased expression of KLF5, accompanied by increased rates of proliferation and anchorage-independent growth. Inhibition of KLF5 expression by mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors or KLF5-specific small interfering RNA reduced proliferation and anchorage-independent growth despite KRAS(V12G) induction. Human colorectal cancer cell lines with mutated KRAS contained high levels of KLF5 and reduction of KLF5 by MEK inhibitors or KLF5 small interfering RNA also led to reduced proliferation and transformation. In vivo, both intestinal tumors derived from mice transgenic for villin-KRAS(V12G) 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 in vitro and in vivo. Inhibition of KLF5 expression in 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.
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http://dx.doi.org/10.1053/j.gastro.2007.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2194652PMC
January 2008

Haploinsufficiency of Krüppel-like factor 4 promotes adenomatous polyposis coli dependent intestinal tumorigenesis.

Cancer Res 2007 Aug;67(15):7147-54

Division of Digestive Diseases, Department of Medicine, and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30033, USA.

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 adenomatous polyposis coli (APC) and that KLF4 repressed the Wnt/beta-catenin pathway. Here, we examined whether KLF4 plays a role in modulating intestinal tumorigenesis by comparing the tumor burdens in mice heterozygous for the Apc(Min) allele (Apc(Min/+)) and those heterozygous for both the Apc(Min) and Klf4 alleles (Klf4(+/-)/Apc(Min/+)). Between 10 and 20 weeks of age, Klf4(+/-)/Apc(Min/+) mice developed, on average, 59% more intestinal adenomas than Apc(Min/+) mice (P < 0.0001). Immunohistochemical staining showed that Klf4 protein levels were lower in the normal-appearing intestinal tissues of Klf4(+/-)/Apc(Min/+) mice compared with wild-type, Klf4(+/-), or Apc(Min/+) mice. In contrast, the levels of beta-catenin and cyclin D1 were higher in the normal-appearing intestinal tissues of Klf4(+/-)/Apc(Min/+) mice compared with the other three genotypes. Klf4 levels were further decreased in adenomas from both Apc(Min/+) and Klf4(+/-)/Apc(Min/+) 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(+/-)/Apc(Min/+) and Apc(Min/+) mice. There was also a progressive loss of heterozygosity of the wild-type Apc allele in adenomas with increasing size from Klf4(+/-)/Apc(Min/+) and Apc(Min/+) mice. Results from this study show that KLF4 plays an important role in promoting the development of intestinal adenomas in the presence of Apc(Min) mutation.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-1302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373271PMC
August 2007

The diverse functions of Krüppel-like factors 4 and 5 in epithelial biology and pathobiology.

Bioessays 2007 Jun;29(6):549-57

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA.

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.
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http://dx.doi.org/10.1002/bies.20581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211634PMC
June 2007

Kruppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells.

Nucleic Acids Res 2006 25;34(4):1216-23. Epub 2006 Feb 25.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine Atlanta, GA, USA.

Lipopolysaccharide (LPS) is a bacterially-derived endotoxin that elicits a strong proinflammatory response in intestinal epithelial cells. It is well established that LPS activates this response through NF-kappaB. In addition, LPS signals through the mitogen-activated protein kinase (MAPK) pathway. We previously demonstrated that the Krüppel-like factor 5 [KLF5; also known as intestine-enriched Krüppel-like factor (IKLF)] is activated by the MAPK. In the current study, we examined whether KLF5 mediates the signaling cascade elicited by LPS. Treatment of the intestinal epithelial cell line, IEC6, with LPS resulted in a dose- and time-dependent increase in KLF5 messenger RNA (mRNA) and protein levels. Concurrently, mRNA levels of the p50 and p65 subunits of NF-kappaB were increased by LPS treatment. Pretreatment with the MAPK inhibitor, U0126, or the LPS antagonist, polymyxin B, resulted in an attenuation of KLF5, p50 and p65 NF-kappaB subunit mRNA levels from LPS treatment. Importantly, suppression of KLF5 by small interfering RNA (siRNA) resulted in a reduction in p50 and p65 subunit mRNA levels and NF-kappaB DNA binding activity in response to LPS. LPS treatment also led to an increase in secretion of TNF-alpha and IL-6 from IEC6, both of which were reduced by siRNA inhibition of KLF5. In addition, intercellular adhesion molecule-1 (ICAM-1) levels were increased in LPS-treated IEC6 cells and this increase was associated with increased adhesion of Jurkat lymphocytes to IEC6. The induction of ICAM-1 expression and T cell adhesion to IEC6 by LPS were both abrogated by siRNA inhibition of KLF5. These results indicate that KLF5 is an important mediator for the proinflammatory response elicited by LPS in intestinal epithelial cells.
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http://dx.doi.org/10.1093/nar/gkl014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1383625PMC
March 2006

Krüppel-like factor 5 promotes mitosis by activating the cyclin B1/Cdc2 complex during oncogenic Ras-mediated transformation.

FEBS Lett 2005 Aug;579(21):4757-62

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 201 Whitehead Research Building, 615 Michael Street, Atlanta, GA 30322, USA.

We previously showed that the zinc finger-containing transcription factor Krüppel-like factor 5 (KLF5) is important in mediating transformation by oncogenic H-Ras through induction of cyclin D1 expression and acceleration of the G1/S transition of the cell cycle. Here we present evidence of a role for KLF5 in accelerating mitotic entry in H-Ras-transformed NIH3T3 fibroblasts. When compared with non-transformed parental NIH3T3 cells, H-Ras-transformed fibroblasts exhibit an increase in mitotic index, levels of cyclin B1 and Cdc2, and cyclin B1/Cdc2 kinase activity. Inhibition of KLF5 expression in H-Ras-transformed cells with KLF5-specific small interfering RNA (siRNA) results in a decrease in each of the aforementioned parameters, with a concomitant reduction in the transforming potential of the cells. Conversely, over-expression of KLF5 in NIH3T3 cells leads to an increase in the promoter activity of the genes encoding cyclin B1 and Cdc2. These results indicate that KLF5 accelerates mitotic entry in H-Ras-transformed cells by transcriptionally activating cyclin B1 and Cdc2, which leads to an increase in cyclin B1/Cdc2 kinase activity. Extending our previous observation that KLF5 activates cyclin D1 transcription to promote G1/S transition, our current results further support a crucial function for KLF5 in mediating cellular transformation caused by oncogenic H-Ras.
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http://dx.doi.org/10.1016/j.febslet.2005.07.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1626271PMC
August 2005

Krüppel-like factor 4 prevents centrosome amplification following gamma-irradiation-induced DNA damage.

Oncogene 2005 Jun;24(25):4017-25

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

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 gamma-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+/+), displayed stable centrosome numbers following gamma-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 gamma-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, gamma-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 gamma-radiation-induced DNA damage and does so by transcriptionally suppressing cyclin E expression.
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http://dx.doi.org/10.1038/sj.onc.1208576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1626272PMC
June 2005

Krüppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation.

Cell Res 2005 Feb;15(2):92-6

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.

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.
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http://dx.doi.org/10.1038/sj.cr.7290271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317089PMC
February 2005

All-trans retinoic acid inhibits proliferation of intestinal epithelial cells by inhibiting expression of the gene encoding Kruppel-like factor 5.

FEBS Lett 2004 Dec;578(1-2):99-105

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 201 Whitehead Research Building, 615 Michael Street, Atlanta, GA, USA.

Retinoids are known inhibitors of epithelial cell proliferation. Previous studies indicate that Kruppel-like factor 5 (KLF5) is a pro-proliferative transcription factor. Here, we examined the effect of all-trans retinoid acid (ATRA) on proliferation of the intestinal epithelial cell line, IEC6. Treatment of IEC6 cells with ATRA inhibited their proliferation due to G1 cell cycle arrest. This inhibition was correlated with a decrease in the levels of KLF5 mRNA and promoter activity. In contrast, constitutive expression of KLF5 in stably transfected IEC6 cells with a KLF5-expressing plasmid driven by a viral promoter abrogated the growth inhibitory effect of ATRA. Moreover, ATRA inhibited proliferation of several human colon cancer cell lines with high levels of KLF5 expression but not those with low levels of KLF5 expression. Our results indicate that KLF5 is a potential mediator for the inhibitory effect of ATRA on intestinal epithelial cell proliferation.
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http://dx.doi.org/10.1016/j.febslet.2004.10.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1599793PMC
December 2004

Krüppel-like factor 5 mediates the transforming activity of oncogenic H-Ras.

Oncogene 2004 Apr;23(19):3404-13

Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA.

Previous studies indicate that Krüppel-like factor 5 (KLF5), also known as intestinal-enriched Krüppel-like factor (IKLF), is a positive regulator of cell proliferation and gives rise to a transformed phenotype when overexpressed. Here we demonstrate that levels of KLF5 transcript and protein are significantly elevated in oncogenic H-Ras-transformed NIH3T3 cells. These cells display an accelerated rate of proliferation in both serum-containing and serum-deprived media and form anchorage-independent colonies in soft agar assays. H-Ras-transformed cells also contain elevated mitogen-activated protein kinase (MAPK) activity. When treated with inhibitors of MEK (MAPK kinase), H-Ras-transformed cells lose their growth advantage and no longer form colonies. Significantly, levels of KLF5 transcript and protein are substantially reduced in H-Ras-transformed cells treated with MEK inhibitors. Moreover, inhibition of KLF5 expression in H-Ras-transformed cells with KLF5-specific small interfering RNA (siRNA) leads to a decreased rate of proliferation and a significant reduction in colony formation. H-Ras-transformed cells also contain elevated levels of Egr1 that are diminished by MEK inhibitors. Inhibition of Egr1 by siRNA results in a reduced level of KLF5, indicating that Egr1 mediates the inductive action of MAPK on KLF5. Lastly, KLF5 activates expression of cyclin D1. These findings indicate that the increased expression of KLF5 in H-Ras-transformed cells is secondary to increased MAPK activity from H-Ras overexpression and that the elevated level of KLF5 is in part responsible for the proproliferative and transforming activities of oncogenic H-Ras.
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http://dx.doi.org/10.1038/sj.onc.1207397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1351030PMC
April 2004

Identification of Krüppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer.

Oncogene 2004 Jan;23(2):395-402

Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA.

Krüppel-like factor 4 (KLF4 or GKLF) is an inhibitor of the cell cycle. The gene encoding KLF4 is localized on chromosome 9q, previously shown to exhibit allelic loss in colorectal cancer (CRC). In this study, we show that the mean level of KLF4 mRNA in a panel of 30 CRC was 52% that of paired normal colonic tissues. Similarly, the levels of KLF4 mRNA and protein in a panel of six established CRC cell lines were significantly lower than those of an untransformed colonic epithelial cell line. Using highly polymorphic DNA markers that flank the KLF4 locus, we found evidence for loss of heterozygosity (LOH) in two of eight surgically resected CRC specimens. In addition, LOH was observed in five of six CRC cell lines with one additional cell line exhibiting hemizygous deletion in the KLF4 gene. We also found that the 5'-untranslated region of KLF4 was hypermethylated in a subset of resected CRC specimens and cell lines. Lastly, the open-reading frame of KLF4 in two of three CRC cell lines examined contained several point mutations that resulted in a diminished ability to activate the p21(WAF1/Cip1) promoter. These findings indicate that KLF4 is a potential tumor suppressor gene in CRC.
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http://dx.doi.org/10.1038/sj.onc.1207067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1351029PMC
January 2004
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