9314

KLF4

EZF|GKLF;Kruppel-like factor 4 (gut);KLF4;Kruppel-like factor 4 (gut)

Krueppel-like factor 4|endothelial Kruppel-like zinc finger protein|epithelial zinc finger protein EZF|gut-enriched krueppel-like factor

9q31

protein-coding

Kruppel-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.

genes that are strongly repressed after B-cell activation are candidates for being inactivated, mutated, or repressed in B-cell malignancies. Kruppel-like factor 4 (Klf4), a gene down-regulated in activated murine B cells, is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this, overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre-B-cell transformation by v-Abl and BCR-ABL, oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death, but not cell-cycle arrest, can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively, our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies.

The Kruppel-like factor 4 (KLF4) transcription factor suppresses tumorigenesis in gastrointestinal epithelium. Thus, its expression is decreased in gastric and colon cancers. Moreover, KLF4 regulates both differentiation and growth that is likely fundamental to its tumor suppressor activity. We dissected the expression of Klf4 in the normal mouse intestinal epithelium along the crypt-villus and cephalo-caudal axes. Klf4 reached its highest level in differentiated cells of the villus, with levels in the duodenum>jejunum>ileum, in inverse relation to the representation of goblet cells in these regions, the lineage previously linked to KLF4. In parallel, in vitro studies using HT29cl.16E and Caco2 colon cancer cell lines clarified that KLF4 increased coincident with differentiation along both the goblet and absorptive cell lineages, respectively, and that KLF4 levels also increased during differentiation induced by the short chain fatty acid butyrate, independently of cell fate. Moreover, we determined that lower levels of KLF4 expression in the proliferative compartment of the intestinal epithelium are regulated by the transcription factors TCF4 and SOX9, an effector and a target, respectively, of beta-catenin/Tcf signaling, and independently of CDX2. Thus, reduced levels of KLF4 tumor suppressor activity in colon tumors may be driven by elevated beta-catenin/Tcf signaling.

The zinc finger-containing transcription factor, Kruppel-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.

Although medulloblastoma is the most common pediatric malignant brain tumor, its molecular underpinnings are largely unknown. We have identified rare, recurrent homozygous deletions of Kruppel-like Factor 4 (KLF4) in medulloblastoma using high-resolution single nucleotide polymorphism arrays, digital karyotyping, and genomic real-time polymerase chain reaction (PCR). Furthermore, we show that there is loss of physiological KLF4 expression in more than 40% of primary medulloblastomas both at the RNA and protein levels. Medulloblastoma cell lines drastically increase the expression of KLF4 in response to the demethylating agent 5-azacytidine and demonstrate dense methylation of the promoter CpG island by bisulfite sequencing. Methylation-specific PCR targeting the KLF4 promoter demonstrates CpG methylation in approximately 16% of primary medulloblastomas. Reexpression of KLF4 in the D283 medulloblastoma cell line results in significant growth suppression both in vitro and in vivo. We conclude that KLF4 is inactivated by either genetic or epigenetic mechanisms in a large subset of medulloblastomas and that it likely functions as a tumor suppressor gene in the pathogenesis of medulloblastoma.

The transcription factor KLF4 may act both as an oncogene and a tumor suppressor in a tissue-depending manner. In T- and pre-B-cell lymphoma, KLF4 was found to act as tumor suppressor. We found the KLF4 promoter methylated in B-cell lymphoma cell lines and in primary cases of B-cell lymphomas, namely, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, and in classic Hodgkin lymphoma (cHL) cases. Promoter hypermethylation was associated with silencing of KLF4 expression. Conditional overexpression of KLF4 in Burkitt lymphoma cell lines moderately retarded proliferation, via cell-cycle arrest in G(0)/G(1). In the cHL cell lines, KLF4 induced massive cell death that could partially be inhibited with Z-VAD.fmk. A quantitative reverse-transcribed polymerase chain reaction array revealed KLF4 target genes, including the proapoptotic gene BAK1. Using an shRNA-mediated knock-down approach, we found that BAK1 is largely responsible for KLF4-induced apoptosis. In addition, we found that KLF4 negatively regulates CXCL10, CD86, and MSC/ABF-1 genes. These genes are specifically up-regulated in HRS cells of cHL and known to be involved in establishing the cHL phenotype. We conclude that epigenetic silencing of KLF4 in B-cell lymphomas and particularly in cHL may favor lymphoma survival by loosening cell-cycle control and protecting from apoptosis.

BACKGROUND: The zinc finger transcription factor Kruppel-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.

Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 laser-microdissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis.

Kruppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays an important role in differentiation and pathogenesis. KLF4 has been suggested to act as an oncogene or tumor suppressor in different tumor types. However, the role of KLF4 in hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrate that forced expression of Klf4 in murine HCC cell lines reduced anchorage-independent growth in soft agar as well as cell migration and invasion activities in vitro. Ectopic Klf4 expression impaired subcutaneous tumor growth and lung colonization in vivo. By contrast, Klf4 knockdown enhanced HCC cell migration. Interestingly, ectopic expression of Klf4 changed the morphology of murine HCC cells to a more epithelial phenotype. Associated with this, we found that expression of Slug, a critical epithelial mesenchymal transition (EMT)-related transcription factor, was significantly down-regulated in Klf4-expressing cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays showed that Klf4 is able to bind and repress the activity of the Slug promoter. Furthermore, ectopic Slug expression partially reverts the Klf4-mediated phenotypes. Consistent with a role as a tumor suppressor in HCC, analysis of the public microarray databases from Oncomine revealed reduced KLF4 expression in human HCC tissues in comparison with normal liver tissues in 3 out of 4 data sets. By quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found reduced KLF4 mRNA in 50% of HCC tissues. Importantly, an inverse correlation between the expression of KLF4 and SLUG was found in HCC tissues. Our data suggest that KLF4 acts as a tumor suppressor in HCC cells, in part by suppressing SLUG transcription.