Outcomes of cold snare piecemeal EMR for nonampullary small-bowel adenomas larger than 1 cm: a retrospective study.

BACKGROUND AND AIMS: Nonampullary small-bowel adenomas ≥10 mm are typically resected using cautery-based polypectomy, which is associated with significant adverse events. Studies have demonstrated the safety and efficacy of piecemeal cold snare EMR for removing large colon polyps. Our aim was to assess the safety and efficacy of cold snare EMR for removal of large adenomas in the small bowel. METHODS: A retrospective study of patients who underwent lift and piecemeal cold snare EMR of small-bowel adenomas ≥1 cm between January 2014 and March 2019 was conducted at a tertiary care medical center. Polyp characteristics at the time of index and surveillance endoscopy were collected. Primary outcomes were residual or recurrent adenoma (RRA) seen on surveillance endoscopy, polyp eradication rate, and number of endoscopic procedures required for eradication. Adverse events including immediate and delayed bleeding, perforation, stricture, pancreatitis, and postpolypectomy syndrome were assessed. RESULTS: Of 43 patients who underwent piecemeal cold snare EMR, 39 had follow-up endoscopy. Polyps ranged in size from 10 to 70 mm (mean, 26.5 mm). RRA was found in 18 patients (46%), with increased polyp size correlating with higher recurrence (P < .001). Polyp eradication was observed in 35 patients (89%), requiring a median of 2 (range, 1-6) endoscopic procedures. Only 1 patient (2.3%) had immediate postprocedural bleeding. No cases of perforation or postpolypectomy syndrome were seen. CONCLUSIONS: Piecemeal cold snare EMR may be a feasible, safe, and efficacious technique for small-bowel polyps >10 mm. Prospective, randomized studies are needed to assess how outcomes compare with traditional cautery-based polypectomy.

Risk factors associated with adenoma recurrence following cold snare endoscopic mucosal resection of polyps ≥ 20 mm: a retrospective chart review.

Background and study aims Cold snare endoscopic mucosal resection (EMR) is being increasingly utilized for non-pedunculated polyps ≥ 20 mm due to adverse events associated with use of cautery. Larger studies evaluating adenoma recurrence rate (ARR) and risk factors for recurrence following cold snare EMR of large polyps are lacking. The aim of this study was to define ARR for polyps ≥ 20 mm removed by cold snare EMR and to identify risk factors for recurrence. Patients and methods A retrospective chart review of colon cold snare EMR procedures performed between January 2015 and July 2019 at a tertiary care medical center was performed. During this period, 310 non-pedunculated polyps ≥ 20 mm were excised using cold snare EMR with follow-up surveillance colonoscopy. Patient demographic data as well as polyp characteristics at the time of index and surveillance colonoscopy were collected and analyzed. Results A total of 108 of 310 polyps (34.8 %) demonstrated adenoma recurrence at follow-up colonoscopy. Patients with a higher ARR were older ( P  = 0.008), had endoscopic clips placed at index procedure ( P  = 0.017), and were more likely to be Asian and African American ( P  = 0.02). ARR was higher in larger polyps ( P  < 0.001), tubulovillous adenomas ( P  < 0.001), and polyps with high-grade dysplasia ( P  = 0.003). Conclusions Although cold snare EMR remains a feasible alternative to hot snare polypectomy for resection of non-pedunculated polyps ≥ 20 mm, endoscopists must also carefully consider factors associated with increased ARR when utilizing this technique.

Dual oxidases control release of hydrogen peroxide by the gastric epithelium to prevent Helicobacter felis infection and inflammation in mice.

BACKGROUND & AIMS: Dual oxidases (DUOX) are conserved reduced nicotinamide adenine dinucleotide phosphate oxidases that produce H2O2 at the epithelial cell surface. The DUOX enzyme comprises the DUOX and DUOX maturation factor (DUOXA) subunits. Mammalian genomes encode 2 DUOX isoenzymes (DUOX1/DUOXA1 and DUOX2/DUOXA2). Expression of these genes is up-regulated during bacterial infections and chronic inflammatory diseases of the luminal gastrointestinal tract. The roles of DUOX in cellular interactions with microbes have not been determined in higher vertebrates. METHODS: Mice with disruptions of Duoxa1 and Duoxa2 genes (Duoxa(-/-) mice) and control mice were infected with Helicobacter felis to create a model of Helicobacter pylori infection--the most common human chronic infection. RESULTS: Infection with H. felis induced expression of Duox2 and Duoxa2 in the stomachs of wild-type mice, and DUOX protein specifically localized to the apical surface of epithelial cells. H. felis colonized the mucus layer in the stomachs of Duoxa(-/-) mice to a greater extent than in control mice. The increased colonization persisted into the chronic phase of infection and correlated with an increased, yet ineffective, inflammatory response. H. felis colonization also was increased in Duoxa(+/-) mice, compared with controls. We observed reduced expression of the H2O2-inducible katA gene in H. felis that colonized Duoxa(-/-) mice, compared with that found in controls (P = .0002), indicating that Duox causes oxidative stress in these bacteria. In vitro, induction of oxidative defense by H. felis failed to prevent a direct bacteriostatic effect at sustained levels of H2O2 as low as 30 µmol/L. CONCLUSIONS: Based on studies of Duoxa(-/-) mice, the DUOX enzyme complex prevents gastric colonization by H. felis and the inflammatory response. These findings indicate the nonredundant function of epithelial production of H2O2 in restricting microbial colonization.

Regulation of hypoxia-inducible factor 1α (HIF-1α) by lysophosphatidic acid is dependent on interplay between p53 and Krüppel-like factor 5.

Hypoxia-inducible factor 1α (HIF-1α) and p53 are pivotal regulators of tumor growth. Lysophosphatidic acid (LPA) is a lipid mediator that functions as a mitogen by acting through LPA receptors. We have shown previously that LPA stimulates HIF-1α expression in colon cancer cells. To determine the mechanism of HIF-1α induction by LPA, we compared the effect of LPA on HIF-1α in several colon cancer cell lines. LPA transcriptionally induced HIF-1α in colon cancer cells. HIF-1α induction was observed in cells expressing WT p53, where LPA decreased p53 expression. However, LPA failed to induce HIF-1α when the p53 gene was mutated. A decrease in p53 expression was dependent on induction of p53-specific E3 ubiquitin ligase Mdm2 by LPA. Krüppel-like factor 5 (KLF5) is an effector of LPA-induced proliferation of colon cancer cells. Because HIF-1α was necessary for LPA-induced growth of colon cancer cells, we determined the relationship between KLF5 and HIF-1α by a loss-of-function approach. Silencing of KLF5 inhibited LPA-induced HIF-1α induction, suggesting that KLF5 is an upstream regulator of HIF-1α. KLF5 and p53 binding to the Hif1α promoter was assessed by ChIP assay. LPA increased the occupancy of the Hif1α promoter by KLF5, while decreasing p53 binding. Transfection of HCT116 cells with KLF5 or p53 attenuated the binding of the other transcription factor. These results identify KLF5 as a transactivator of HIF-1α and show that LPA regulates HIF-1α by dynamically modulating its interaction with KLF5 and p53.

Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells.

BACKGROUND: Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. METHODS: To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. RESULTS: Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. CONCLUSIONS: Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.

HIF1A C1772T polymorphism leads to HIF-1α mRNA overexpression in prostate cancer patients.

Hypoxia-inducible factor 1α (HIF-1α) gene polymorphisms have been investigated for a possible role in mediating genetic predisposition to cancer. Our previous data show that men homozygous to C1772T polymorphism had 4-fold risk to develop prostate cancer. Therefore, we studied the effects of C1772T polymorphism on HIF-1α expression. HIF-1α mRNA expression levels were significantly higher in peripheral blood leukocytes of prostate cancer patients with the TT genotype compared with the CC genotype. Expression of C1772T HIF-1α in HIF-1α knockout cancer cells showed higher expression levels and stabilization of HIF-1α mRNA compared with the wild-type. Mutated HIF-1α protein half-life was similar to that of the wild-type. Hence, our data provide evidence that C1772T polymorphism causes activation of HIF-1α as a gain-of-function mechanism driven by stabilization of HIF-1α mRNA. These findings may also explain the increased risk of men homozygous to this mutation to develop prostate cancer.

Can we develop effective combination antiangiogenic therapy for patients with hepatocellular carcinoma?

Antiangiogenic therapy has shown promise in the treatment of patients with hepatocellular carcinoma (HCC). Bevacizumab, sorafenib, and sunitinib showed efficacy in patients with HCC; and sorafenib is approved by the FDA for treatment of this cancer. In practice, the clinical benefit of these agents has been heterogeneous; and in patients who do respond, the benefit is modest and/or short-lived. Recent advances in the molecular understanding of tumor angiogenesis along with the rapid development of targeted drug discovery have made it possible to explore novel combination therapy for HCC. We review the clinical trial results, discuss possible molecular mechanisms of resistance, and suggest novel combinations with antiangiogenic therapy.

Combination therapy targeting cancer metabolism.

Cancer cells undergo significant metabolic adaptation. Cellular transformation enhances both glycolysis and mitochondrial respiration efficiency through the induction of HIF-1α and HIF-2α. In this process, energy production and synthesis of macromolecules are maximized with minimal ROS accumulation. Furthermore, a series of antioxidant enzymes are induced to mitigate the damaging effects of ROS. Examination of these metabolic changes provides rationale for a synergistic approach to combination anti-cancer therapy; targeted inhibition of HIF and inhibition of cellular defenses against oxidative stress.

Oncogenic KRAS modulates mitochondrial metabolism in human colon cancer cells by inducing HIF-1α and HIF-2α target genes.

BACKGROUND: Activating KRAS mutations are important for cancer initiation and progression; and have recently been shown to cause primary resistance to therapies targeting the epidermal growth factor receptor. Therefore, strategies are currently in development to overcome treatment resistance due to oncogenic KRAS. The hypoxia-inducible factors-1α and -2α (HIF-1α and HIF-2α) are activated in cancer due to dysregulated ras signaling. METHODS: To understand the individual and combined roles of HIF-1α and HIF-2α in cancer metabolism and oncogenic KRAS signaling, we used targeted homologous recombination to disrupt the oncogenic KRAS, HIF-1α, and HIF-2α gene loci in HCT116 colon cancer cells to generate isogenic HCT116WT KRAS, HCT116HIF-1α-/-, HCT116HIF-2α-/-, and HCT116HIF-1α-/-HIF-2α-/- cell lines. RESULTS: Global gene expression analyses of these cell lines reveal that HIF-1α and HIF-2α work together to modulate cancer metabolism and regulate genes signature overlapping with oncogenic KRAS. Cancer cells with disruption of both HIF-1α and HIF-2α or oncogenic KRAS showed decreased aerobic respiration and ATP production, with increased ROS generation. CONCLUSION: Our findings suggest novel strategies for treating tumors with oncogenic KRAS mutations.

TCF4 and CDX2, major transcription factors for intestinal function, converge on the same cis-regulatory regions.

Surprisingly few pathways signal between cells, raising questions about mechanisms for tissue-specific responses. In particular, Wnt ligands signal in many mammalian tissues, including the intestinal epithelium, where constitutive signaling causes cancer. Genome-wide analysis of DNA cis-regulatory regions bound by the intestine-restricted transcription factor CDX2 in colonic cells uncovered highly significant overrepresentation of sequences that bind TCF4, a transcriptional effector of intestinal Wnt signaling. Chromatin immunoprecipitation confirmed TCF4 occupancy at most such sites and co-occupancy of CDX2 and TCF4 across short distances. A region spanning the single nucleotide polymorphism rs6983267, which lies within a MYC enhancer and confers colorectal cancer risk in humans, represented one of many co-occupied sites. Co-occupancy correlated with intestine-specific gene expression and CDX2 loss reduced TCF4 binding. These results implicate CDX2 in directing TCF4 binding in intestinal cells. Co-occupancy of regulatory regions by signal-effector and tissue-restricted transcription factors may represent a general mechanism for ubiquitous signaling pathways to achieve tissue-specific outcomes.

Targeting both HIF-1 and HIF-2 in human colon cancer cells improves tumor response to sunitinib treatment.

Sunitinib is an oral small-molecule multitargeted receptor tyrosine kinase inhibitor that has recently been shown to have clinical benefit as a single agent in renal cell cancer and gastrointestinal stromal tumors, leading to its Food and Drug Administration approval for treatment of these cancers. However, the benefit is short-lived; and for the majority of cancers, sunitinib single-agent clinical activity is low. Therefore, combination strategies with sunitinib are currently in clinical development. The hypoxia-inducible transcription factors, HIF-1 and HIF-2, induce gene programs important for cancer cell growth and angiogenesis. We hypothesized that inhibiting HIF-1 and HIF-2 would further improve tumor response to sunitinib therapy. To test this hypothesis, HIF-1α and HIF-2α genes were disrupted in colon cancer cells. We found that disruption of HIF-1α, HIF-2α, or both HIF-1α and HIF-2α genes led to improved tumor response to sunitinib. For xenografts in which both HIF-1α and HIF-2α genes were disrupted, there was prolonged complete remission with sunitinib treatment in 50% of mice. This enhanced response was mediated by two potential mechanisms. First, tumor angiogenesis and perfusion were almost completely inhibited by sunitinib when both HIF-1α and HIF-2α genes were disrupted. The enhanced inhibitory effect on tumor angiogenesis was mediated by the inhibition of multiple proangiogenic factors, including vascular endothelial growth factor and angiopoietin-like protein 4, and the induction of the antiangiogenic factor, thrombospondin 1. Second, disruption of HIF-1α, HIF-2α, or both HIF-1α and HIF-2α genes directly inhibited tumor cell proliferation. These preclinical findings have clinical implications and suggest novel clinical trials.

Hypoxia-inducible factor-1 target genes as indicators of tumor vessel response to vascular endothelial growth factor inhibition.

Antiangiogenic therapy improves survival in patients with advanced stage cancers. Currently, there are no reliable predictors or markers for tumor vessel response to antiangiogenic therapy. To model effective antiangiogenic therapy, we disrupted the VEGF gene in three representative cancer cell lines. HCT116 xenografts had low proportions of endothelial tubes covered by pericytes that stained with alpha-smooth muscle actin (SMA) antibody. Upon disruption of VEGF, HCT116(VEGF-/-) xenografts had significantly decreased tumor microvessel perfusion compared with their parental counterparts. Furthermore, HCT116(VEGF-/-) xenografts mounted a tumor-reactive response to hypoxia, characterized by the induction of hypoxia-inducible factor-1 (HIF-1) target genes. One highly induced protein was DPP4, a measurable serum protein that has well-described roles in cancer progression. In contrast, LS174T and MKN45 tumor xenografts had high proportion of endothelial tubes that were covered by SMA+ pericytes. Upon disruption of VEGF, LS174T(VEGF-/-) and MKN45(VEGF-/-) xenografts maintained tumor microvessel perfusion. As such, there were no changes in intratumoral hypoxia or HIF-1 alpha induction. Together, these data show that the extent of tumor vessel response to angiogenic inhibition could be correlated with (a) the preexisting coverage of tumor endothelial tubes with SMA+ pericytes and (b) differential tumor induction of HIF-1 target genes. The data further show that DPP4 is a novel marker of HIF-1 induction. Altogether, these preclinical findings suggest novel clinical trials for predicting and monitoring tumor vessel responses to antiangiogenic therapy.

1alpha,25-dihydroxyvitamin D3 (Calcitriol) inhibits hypoxia-inducible factor-1/vascular endothelial growth factor pathway in human cancer cells.

In vitro and in vivo studies have shown that 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] inhibits angiogenesis in cancer. We now examined whether the antiangiogenic effects of 1,25(OH)(2)D(3) are mediated by the hypoxia-inducible factor (HIF)-1 pathway. Our results showed that 1,25(OH)(2)D(3) reduces the protein expression of both the regulated HIF-1alpha subunit and the vascular endothelial growth factor (VEGF) in various human cancer cells. 1,25(OH)(2)D(3) also inhibited HIF-1 transcriptional activity (measured by reporter gene assay) as well as HIF-1 target genes, including VEGF, ET-1, and Glut-1. We also showed that 1,25(OH)(2)D(3) inhibits cell proliferation under hypoxia. Using HIF-1alpha knockout colon cancer cells, we show that the inhibition of the hypoxia-induced VEGF by 1,25(OH)(2)D(3) is mediated through a HIF-dependent pathway. Because HIF-1 is a major positive contributor in human tumorigenesis and angiogenesis, we believe that its inhibition by 1,25(OH)(2)D(3) strengthens the rationale to use vitamin D and its low-calcemic analogues in cancer chemoprevention and therapy.

Hypoxia-inducible factor-1alpha promotes nonhypoxia-mediated proliferation in colon cancer cells and xenografts.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1alpha is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1alpha can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1alpha is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1alpha in nonhypoxia-mediated cancer cell proliferation remains speculative. We have disrupted HIF-1alpha by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1alpha significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1alpha expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1alpha promoted cell proliferation and survival under hypoxia in vitro. To further test the role of HIF-1alpha within tumor compartments, we generated cells with combined disruptions of both HIF-1alpha and vascular endothelial growth factor (VEGF). In all xenografts, disruption of VEGF led to marked expansion of the hypoxic compartments and growth delay. Nonetheless, the presence or absence of HIF-1alpha did not grossly affect these expanded hypoxic compartments. These data provide compelling evidence that, in a subset of colon cancers, (a) HIF-1alpha is a positive factor for nonhypoxia-mediated cell proliferation in vitro and in vivo and (b) HIF-1alpha is a positive factor for cell proliferation and survival under hypoxic conditions in vitro, but does not grossly contribute to the tumor hypoxic compartments in vivo.

Glutathione S-transferase pi1 promotes tumorigenicity in HCT116 human colon cancer cells.

GSTP1 is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles with glutathione in the process of detoxification. GSTP1 is widely overexpressed in colorectal cancer, from aberrant crypt foci to advanced carcinomas. Increased expression of GSTP1 is associated with multidrug resistance and a worse clinical prognosis. However, GSTP1-null mice have an increased risk of tumor formation. Thus, the biological function of GSTP1 in colorectal cancer biology remains speculative. In an effort to gain further insights into the role of GSTP1 in tumorigenesis, we disrupted the GSTP1 gene in HCT116 human colorectal cancer cells using targeted homologous recombination. We find that loss of GSTP1 resulted in impaired clonogenic survival and proliferation. Specifically, under growth-limiting conditions, (a) GSTP1 protected HCT116 cells from oxidative stress and associated apoptosis and (b) promoted mitogen-activated protein kinase-extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase-mediated G1-S cell cycle progression. In vivo, GSTP1 was critical for engraftment and growth of HCT116 tumor xenografts. These studies directly show that GSTP1 promotes clonogenic survival and proliferation in HCT116 human colon cancer cells.

Overexpression of Krüppel-like factor 4 in the human colon cancer cell line RKO leads to reduced tumorigenecity.

Krüppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor, the expression of which is enriched in the postmitotic cells of the intestinal epithelium. KLF4 is a target gene of the tumor suppressor adenomatous polyposis coli (APC). We sought to determine the role of KLF4 in suppressing the tumorigenecity of RKO colon cancer cells, which do not express KLF4. We utilized an established system in RKO cells, in which an inducible promoter controls expression of KLF4. Four independent assays were used to assess the effects of KLF4 induction on tumor cells. We find that KLF4 overexpression reduces colony formation, cell migration and invasion, and in vivo tumorigenecity. The mechanism of action of KLF4 does not involve apoptosis. These findings, along with our previous findings that KLF4 induces G1/S arrest, suggest that KLF4 is a cell cycle checkpoint protein that can reduce tumorigenecity of colon cancer cells.

Smad4 overexpression in hepatocellular carcinoma is strongly associated with transforming growth factor beta II receptor immunolabeling.

In the normal liver, the transforming growth factor beta (TGF-beta) signaling pathway plays an important role in inhibiting hepatocyte growth. This effect is mediated through Smad4 (or Dpc4), a tumor-suppressor gene that affects gene transcription and controls cell growth. A loss of Smad4 is associated with carcinoma in a number of other organs, including the pancreas and colon. Despite these facts, several recent studies using cDNA microarrays have surprisingly shown overexpression of Smad4 in hepatocellular carcinoma (HCC). Because Smad4 plays a central role in the TGF-beta signaling pathway, we hypothesized that activation of the TGF-beta signaling pathway may explain Smad4 overexpression. To investigate this, 21 surgically resected HCCs were immunostained with antibodies to Smad4 and TGF-beta receptor II. Tumor and normal liver tissues were stained in all cases, and expression in the tumor was scored in comparison to the nonneoplastic liver. Thirteen hepatic adenomas were also immunostained as a control group. The average age at resection was 58 +/- 16 years for the 17 men and 4 women with HCC. TGF-beta receptor II was weakly expressed in the hepatocyte cytoplasm of all normal livers and was overexpressed in 10 of 21 HCCs. Of these 10 HCCs increased Smad4 immunolabeling was also present in 10 of 10 cases. In contrast, of the 11 of HCCs that did not show TGF-beta overexpression, only 1 showed increased Smad4 immunolabeling. Increased TGF-beta receptor II and Smad4 labeling was associated with a worse nuclear grade and increased mitotic activity. For the hepatic adenomas, the 13 women had an average age at resection of 36 +/- 10 years. Whereas 2 adenomas showed over expression of TGF-beta receptor II, there was no Smad4 overexpression in any case. In conclusion, increased Smad4 protein expression in HCC is tightly linked to overexpression of TGF-beta II receptors and is associated with increased mitoses and a worse nuclear grade. Hepatic adenomas only rarely show overexpression of TGF-beta II receptors and did not show increased Smad4 labeling. The results from this study indicate that Smad4 protein overexpression is present in a subset of HCCs and is strongly correlated with immunostaining for TGF-beta II receptor, findings that may represent activation or dysregulation of the TGF-beta signaling pathway.

Opposing effects of Krüppel-like factor 4 (gut-enriched Krüppel-like factor) and Krüppel-like factor 5 (intestinal-enriched Krüppel-like factor) on the promoter of the Krüppel-like factor 4 gene.

KLF4 (Krüppel-like factor 4 or gut-enriched Krüppel-like factor, GKLF) and KLF5 (Krüppel-like factor 5 or intestinal-enriched Krüppel-like factor, IKLF) are two closely related members of the zinc finger-containing Krüppel-like factor family of transcription factors. Although both genes are expressed in the intestinal epithelium, their distributions are different: Klf4 is primarily expressed in the terminally differentiated villus cells while Klf5 is primarily in the proliferating crypt cells. Previous studies show that Klf4 is a negative regulator of cell proliferation and Klf5 is a positive regulator of cell proliferation. In this study, we demonstrate that Klf5 binds to a number of cis-DNA elements that have previously been shown to bind to Klf4. However, while Klf4 activates the promoter of its own gene, Klf5 suppresses the Klf4 promoter. Moreover, Klf5 abrogates the activating effect of Klf4 on the Klf4 promoter and Klf4 abrogates the inhibitory effect of Klf5 on the same promoter. An explanation of this competing effect is due to physical competition of the two proteins for binding to cognate DNA sequence. The complementary tissue localization of expression of Klf4 and Klf5 and the opposing effect of the two Klfs on the Klf4 promoter activity may provide a basis for the coordinated regulation of expression of the Klf4 gene in the intestinal epithelium.