Publications by authors named "Amr M Ghaleb"

35 Publications

Loss of the Krüppel-like factor 4 tumor suppressor is associated with epithelial-mesenchymal transition in colorectal cancer.

J Cancer Metastasis Treat 2019 26;5. Epub 2019 Nov 26.

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

Aim: Colorectal cancer (CRC) is the third leading cancer-related cause of death due to its propensity to metastasize. Epithelial-mesenchymal transition (EMT) is a multistep process important for invasion and metastasis of CRC. Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor highly expressed in differentiated cells of the intestinal epithelium. KLF4 has been shown to play a tumor suppressor role during CRC tumorigenesis - its loss accelerates development and progression of cancer. The present study examined the relationship between KLF4 and markers of EMT in CRC.

Methods: Immunofluorescence staining for KLF4 and EMT markers was performed on archived patient samples after colorectal cancer resection and on colonic tissues of mice with colitis-associated cancer.

Results: We found that KLF4 expression is lost in tumor sections obtained from CRC patients and in those of mouse colon following azoxymethane and dextran sodium sulfate (AOM/DSS) treatment when compared to their respective normal appearing mucosa. Importantly, in CRC patient tumor sections, we observed a negative correlation between KLF4 levels and mesenchymal markers including TWIST, β-catenin, claudin-1, N-cadherin, and vimentin. Similarly, in tumor tissues from AOM/DSS-treated mice, KLF4 levels were negatively correlated with mesenchymal markers including SNAI2, β-catenin, and vimentin and positively correlated with the epithelial marker E-cadherin.

Conclusion: These findings suggest that the loss of KLF4 expression is a potentially significant indicator of EMT in CRC.
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http://dx.doi.org/10.20517/2394-4722.2019.35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304562PMC
November 2019

Kruppel-like factor 4 regulates matrix metalloproteinase and aggrecanase gene expression in chondrocytes.

Cell Tissue Res 2017 12 31;370(3):441-449. Epub 2017 Aug 31.

Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, 1-8 Yamada-oka, Suita, Osaka, 565-0871, Japan.

Kruppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays crucial roles during the development and maintenance of multiple organs. We and others have previously shown that KLF4 is involved in bone modeling and remodeling but roles played by KLF4 during skeletogenesis are still not fully understood. Here, we show that KLF4 is expressed in the epiphyseal growth plate and articular chondrocytes. Most articular chondrocytes expressed KLF4 in embryos but it localized only in a subset of superficial zone cells in postnatal mice. When KLF4 was overexpressed in chondrocytes in vitro, it severely repressed chondrocytic gene expressions. Global gene expression profiling of KLF4-transduced chondrocytes revealed matrix degrading proteinases of the matrix metalloproteinase and disintegrin and metalloproteinase with thrombospondin-1 domain families within the group of upregulated genes. Proteinase induction by KLF4 was alleviated by Trichostatin A treatment suggesting the possible involvement of epigenetic mechanisms on proteinase induction by KLF4. These results indicate the possible involvement of KLF4 in physiological and pathological aspects during cartilage development and maintenance.
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http://dx.doi.org/10.1007/s00441-017-2674-0DOI Listing
December 2017

Krüppel-like factor 4 (KLF4): What we currently know.

Gene 2017 May 22;611:27-37. Epub 2017 Feb 22.

Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA; Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA. Electronic address:

Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell growth, proliferation, and differentiation. Since its discovery in 1996, KLF4 has been gaining a lot of attention, particularly after it was shown in 2006 as one of four factors involved in the induction of pluripotent stem cells (iPSCs). Here we review the current knowledge about the different functions and roles of KLF4 in various tissue and organ systems.
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http://dx.doi.org/10.1016/j.gene.2017.02.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391259PMC
May 2017

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

Krüppel-like Factor 4 Modulates Development of BMI1(+) Intestinal Stem Cell-Derived Lineage Following γ-Radiation-Induced Gut Injury in Mice.

Stem Cell Reports 2016 06 26;6(6):815-824. Epub 2016 May 26.

Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA; Stony Brook University Medical Center, HSC T-16, Room 020, Stony Brook, NY 11794-8160, USA. Electronic address:

In response to ionizing radiation-induced injury, the normally quiescent intestinal stem cells marked by BMI1 participate in the regenerative response. Previously, we established a protective role for Krüppel-like factor 4 (KLF4) in the intestinal epithelium where it reduces senescence, apoptosis, and crypt atrophy following γ-radiation-induced gut injury. We also described a pro-proliferative function for KLF4 during the regenerative phase post irradiation. In the current study, using a mouse model in which Klf4 is deleted from quiescent BMI1(+) intestinal stem cells, we observed increased proliferation from the BMI1(+) lineage during homeostasis. In contrast, following irradiation, Bmi1-specific Klf4 deletion leads to decreased expansion of the BMI1(+) lineage due to a combination of reduced proliferation and increased apoptosis. Our results support a critical role for KLF4 in modulating BMI1(+) intestinal stem cell fate in both homeostasis and the regenerative response to radiation injury.
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http://dx.doi.org/10.1016/j.stemcr.2016.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911500PMC
June 2016

Murine Model for Colitis-Associated Cancer of the Colon.

Methods Mol Biol 2016 ;1438:245-54

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

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), significantly increases the risk for development of colorectal cancer. Specifically, dysplasia and cancer associated with IBD (colitis-associated cancer or CAC) develop as a result of repeated cycles of injury and healing in the intestinal epithelium. Animal models are utilized to examine the mechanisms of CAC, the role of epithelial and immune cells in this process, as well as the development of novel therapeutic targets. These models typically begin with the administration of a carcinogenic compound, and inflammation is caused by repeated cycles of colitis-inducing agents. This review describes a common CAC model that utilizes the pro-carcinogenic compound azoxymethane (AOM) followed by dextran sulfate sodium (DSS) which induces the inflammatory insult.
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http://dx.doi.org/10.1007/978-1-4939-3661-8_14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657253PMC
December 2017

KLF4 Suppresses Tumor Formation in Genetic and Pharmacological Mouse Models of Colonic Tumorigenesis.

Mol Cancer Res 2016 Apr 2;14(4):385-96. Epub 2016 Feb 2.

Department of Medicine, Stony Brook University, Stony Brook, New York. Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York.

Unlabelled: The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently downregulated 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 (Klf4(ΔIS)) in a genetic or pharmacological setting of colonic tumorigenesis:Apc(Min/⁺) mutation or carcinogen treatment with azoxymethane (AOM), respectively.Klf4 (ΔIS)/Apc (Min/⁺) mice developed significantly more colonic adenomas with 100% penetrance as compared with Apc(Min/⁺) mice with intact Klf4 (Klf4(fl/fl)/Apc (Min/⁺)). The colonic epithelium of Klf4 (ΔIS)/Apc (Min/⁺)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 breaks. InKlf4 (ΔIS)/Apc (Min/+) mice, this was associated with reduced nonhomologous 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, Klf4 (ΔIS) mice developed significantly more colonic tumors than Klf4 (fl/fl) mice, with more Klf4 (ΔIS) mice harboring K-Rasmutations than Klf4 (fl/fl)mice. Compared with AOM-treated Klf4 (fl/fl)mice, adenomas of treated Klf4 (ΔIS) 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 KLF4 plays a significant role in the pathogenesis of colorectal neoplasia.
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http://dx.doi.org/10.1158/1541-7786.MCR-15-0410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834227PMC
April 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

IQ Motif-Containing GTPase-Activating Protein 2 (IQGAP2) Is a Novel Regulator of Colonic Inflammation in Mice.

PLoS One 2015 5;10(6):e0129314. Epub 2015 Jun 5.

Department of Medicine, Stony Brook University, Stony Brook, New York, United States of America.

IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca2+/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129314PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457730PMC
April 2016

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

Distinct roles for hematopoietic and extra-hematopoietic sphingosine kinase-1 in inflammatory bowel disease.

PLoS One 2014 2;9(12):e113998. Epub 2014 Dec 2.

Northport Veterans Affairs Medical Center, Northport, New York, United States of America; Department of Medicine, Stony Brook University, Stony Brook, New York, United States of America; Stony Brook Cancer Center, Stony Brook University, Stony Brook, New York, United States of America.

Sphingosine kinase 1 (SK1), one of two SK enzymes, is highly regulated and has been shown to act as a focal point for the action of many growth factors and cytokines. SK1 leads to generation of sphingosine-1-phosphate (S1P) and potentially the activation of S1P receptors to mediate biologic effects. Our previous studies implicated SK1/S1P in the regulation of inflammatory processes, specifically in inflammatory bowel disease (IBD). These studies were conducted using a total body knockout mouse for SK1 and were unable to determine the source of SK1/S1P (hematopoietic or extra-hematopoietic) involved in the inflammatory responses. Therefore, bone marrow transplants were performed with wild-type (WT) and SK1-/- mice and colitis induced with dextran sulfate sodium (DSS). Irrespective of the source of SK1/S1P, bone marrow or tissue, DSS induced colitis in all mice; however, mice lacking SK1 in both hematopoietic and extra-hematopoietic compartments exhibited decreased crypt damage. Systemic inflammation was assessed, and mice with WT bone marrow demonstrated significant neutrophilia in response to DSS. In the local inflammatory response, mice lacking SK1/S1P in either bone marrow or tissue exhibited decreased induction of cytokines and less activation of STAT3 (signal transducer and activator of transcription 3). Interestingly, we determined that extra-hematopoietic SK1 is necessary for the induction of cyclooxygenase 2 (COX2) in colon epithelium in response to DSS-induced colitis. Taken together our data suggest that hematopoietic-derived SK1/S1P regulates specific aspects of the systemic inflammatory response, while extra-hematopoietic SK1 in the colon epithelium is necessary for the autocrine induction of COX2 in DSS-induced colitis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113998PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4252067PMC
August 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

Genetic deletion of Klf4 in the mouse intestinal epithelium ameliorates dextran sodium sulfate-induced colitis by modulating the NF-κB pathway inflammatory response.

Inflamm Bowel Dis 2014 May;20(5):811-20

*Department of Medicine, Stony Brook University, Stony Brook, New York; †Department of Biology and Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia; and ‡Veterans Affairs Medical Center, Decatur, GA.

Background: Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor expressed in the differentiated epithelial cells lining of the intestine. Under physiological conditions, KLF4 inhibits cell proliferation. Conversely, KLF4 mediates proinflammatory signaling in macrophages and its overexpression in the esophageal epithelium activates cytokines, leading to inflammation-mediated esophageal squamous cell cancer formation in mice. Here, we tested whether KLF4 has a proinflammatory activity in experimental colitis in mice.

Methods: Villin-Cre;Klf4 mice with intestine-specific Klf4 deletion (Klf4) and control mice with floxed Klf4 gene (Klf4) were treated or not with 3% dextran sodium sulfate (DSS) for 7 days to induce colitis. Additionally, WT mice were administered or not, nanoparticles loaded with scrambled or Klf4-siRNA, and concomitantly given DSS.

Results: Compared with DSS-treated Klf4 mice, DSS-treated Klf4 mice were significantly less sensitive to DSS-induced colitis. DSS treatment of Klf4 mice induced Klf4 expression in the crypt zone of the colonic epithelium. DSS-treated Klf4 mice had increased proliferation relative to DSS-treated control mice. DSS treatment induced NF-κB signaling pathway in Klf4 mice colon but not Klf4 mice. Additionally, WT mice given DSS and nanoparticle/Klf4-siRNA were less sensitive to colitis and had reduced Klf4 expression and while maintaining the proliferative response in the colonic epithelium.

Conclusions: Our results indicate that Klf4 is an important mediator of DSS-induced colonic inflammation by modulating NF-κB signaling pathway and could be involved in the pathogenesis and/or propagation of inflammatory bowel disease. Thus, Klf4 may represent a novel therapeutic target in inflammatory bowel disease.
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http://dx.doi.org/10.1097/MIB.0000000000000022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091934PMC
May 2014

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

Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts.

Mol Cancer 2013 Aug 6;12:89. Epub 2013 Aug 6.

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

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.
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http://dx.doi.org/10.1186/1476-4598-12-89DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750599PMC
August 2013

Chemoprevention of familial adenomatous polyposis by bromo-noscapine (EM011) in the Apc(Min/+) mouse model.

Int J Cancer 2012 Sep 9;131(6):1435-44. Epub 2012 Feb 9.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

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-noscapine (EM011), a rationally designed synthetic derivative of a natural nontoxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G(2) /M arrest in wild type murine embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with small interfering RNA. 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 before 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 Apc(Min/+) mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of Apc(Min/+) 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.
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http://dx.doi.org/10.1002/ijc.27344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362662PMC
September 2012

Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts.

Am J Cancer Res 2011 Jan;1(1):85-97

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

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165197PMC
January 2011

Krüppel-like factor 5 is important for maintenance of crypt architecture and barrier function in mouse intestine.

Gastroenterology 2011 Oct 18;141(4):1302-13, 1313.e1-6. Epub 2011 Jul 18.

Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.

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). Morphologic changes in the small intestine and colon were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction.

Results: Klf5 mutant mice were born at a normal Mendelian ratio but had high mortality compared with 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 morphologic 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 Caudal type homeobox (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. Morphologic changes that occur with deletion of Klf5 are associated with disruption of canonical Wnt signaling and increased expression of Sox9.
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http://dx.doi.org/10.1053/j.gastro.2011.06.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186863PMC
October 2011

Notch1 regulates the effects of matrix metalloproteinase-9 on colitis-associated cancer in mice.

Gastroenterology 2011 Oct 30;141(4):1381-92. Epub 2011 Jun 30.

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

Background & Aims: Inflammatory bowel disease increases the risks of 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, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 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 difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9-/- mice and HCT116 cells that were stably transfected with MMP-9.

Results: Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown 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 MMP-9-/- mice on γ-radiation-induced damage of DNA.

Conclusions: MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.
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http://dx.doi.org/10.1053/j.gastro.2011.06.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186860PMC
October 2011

Altered intestinal epithelial homeostasis in mice with intestine-specific deletion of the Krüppel-like factor 4 gene.

Dev Biol 2011 Jan 9;349(2):310-20. Epub 2010 Nov 9.

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

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 to the upper crypt region. 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 activation of genes in the Wnt pathway and 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.
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http://dx.doi.org/10.1016/j.ydbio.2010.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022386PMC
January 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

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

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

The absence of LPA2 attenuates tumor formation in an experimental model of colitis-associated cancer.

Gastroenterology 2009 May 9;136(5):1711-20. Epub 2009 Jan 9.

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

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 mice heterozygous for the adenomatous polyposis coli (Apc) allele. We determined the loss of LPA2 function in tumorigenesis in the colon by treating mice with genetic deletion of LPA2 (LPA2-/-) with azoxymethane and dextran sulfate sodium.

Results: We found that LPA increased tumor incidence in Apc(min/+) mice. LPA2-/- mice showed reduced mucosal damage and fewer tumors than wild-type (WT) mice. Reduced epithelial cell proliferation and decreases in beta-catenin, Krüppel-like factor 5, and cyclooxygenase-2 expression were observed in LPA2-/- mice. Unlike WT mice, induction of monocyte chemoattractant protein-1 and macrophage migration inhibitory factor was significantly attenuated in LPA2-/- mice with reduced infiltration by macrophages.

Conclusions: 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.
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http://dx.doi.org/10.1053/j.gastro.2009.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2691721PMC
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

The Pathobiology of Krüppel-like Factors in Colorectal Cancer.

Curr Colorectal Cancer Rep 2008 Apr;4(2):59-64

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2394726PMC
http://dx.doi.org/10.1007/s11888-008-0011-4DOI Listing
April 2008

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