Kruppel-like Transcription Factor Research Articles

KLFII Epigenetically Regulates Glycodelin-A, a Marker of Endometrial Biology Via Histone-Modifying Chromatin Mechanisms

Endometrial biology is characterized by programmed proliferation and differentiation that is synchronous with ovarian folliculogenesis to maximize the chance of pregnancy. Glycodelin-A, an endometrial secretory protein, promotes pregnancy mostly through immunomodulatory mechanisms. Glycodelin-A is repressed during the proliferative and early secretory phase and activated thereafter. Progesterone activates glycodelin via the Sp1 (Specificity Protein 1) transactivator. We identify a novel role for Kruppel-like transcription factor 11 (KLF11) as a glycodelin-A repressor. Although KLF11 bound 2 distinct regulatory elements, it regulated glycodelin promoter activity differentially through each element. Whereas KLF11 weakly activated glycodelin promoter activity via a region that also bound Sp1, the dominant effect of KLF11 was repression of promoter activity, messenger RNA (mRNA), and protein expression via a novel, specific binding element. KLF11 mediated this repression by recruiting the SIN3/histone deacetylase (HDAC) corepressor complex to the glycodelin promoter. KLF11 may solely, or by competing with Sp1, repress glycodelin-A levels and thereby influence its role in the endometrium.

Integrative genomic and functional profiling of the pancreatic cancer genome

BackgroundPancreatic cancer is a deadly disease with a five-year survival of less than 5%. A better understanding of the underlying biology may suggest novel therapeutic targets. Recent surveys of the pancreatic cancer genome have uncovered numerous new alterations; yet systematic functional characterization of candidate cancer genes has lagged behind. To address this challenge, here we have devised a highly-parallel RNA interference-based functional screen to evaluate many genomically-nominated candidate pancreatic cancer genes simultaneously.ResultsFor 185 candidate pancreatic cancer genes, selected from recurrently altered genomic loci, we performed a pooled shRNA library screen of cell growth/viability across 10 different cell lines. Knockdown-associated effects on cell growth were assessed by enrichment or depletion of shRNA hairpins, by hybridization to barcode microarrays. A novel analytical approach (COrrelated Phenotypes for On-Target Effects; COPOTE) was used to discern probable on-target knockdown, based on identifying different shRNAs targeting the same gene and displaying concordant phenotypes across cell lines. Knockdown data were integrated with genomic architecture and gene-expression profiles, and selected findings validated using individual shRNAs and/or independent siRNAs. The pooled shRNA library design delivered reproducible data. In all, COPOTE analysis identified 52 probable on-target gene-knockdowns. Knockdown of known oncogenes (KRAS, MYC, SMURF1 and CCNE1) and a tumor suppressor (CDKN2A) showed the expected contrasting effects on cell growth. In addition, the screen corroborated purported roles of PLEKHG2 and MED29 as 19q13 amplicon drivers. Most notably, the analysis also revealed novel possible oncogenic functions of nucleoporin NUP153 (ostensibly by modulating TGFβ signaling) and Kruppel-like transcription factor KLF5 in pancreatic cancer.ConclusionsBy integrating physical and functional genomic data, we were able to simultaneously evaluate many candidate pancreatic cancer genes. Our findings uncover new facets of pancreatic cancer biology, with possible therapeutic implications. More broadly, our study provides a general strategy for the efficient characterization of candidate genes emerging from cancer genome studies.

TNF-α Induces Phenotypic Modulation in Cerebral Vascular Smooth Muscle Cells: Implications for Cerebral Aneurysm Pathology

Little is known about vascular smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. Tumor necrosis factor-alpha (TNF-α) has been associated with aneurysms, but potential mechanisms are unclear. Cultured rat cerebral SMCs overexpressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, smooth muscle myosin heavy chain), while dominant-negative cells suppressed expression. Tumor necrosis factor-alpha treatment inhibited this contractile phenotype and induced pro-inflammatory/matrix-remodeling genes (monocyte chemoattractant protein-1, matrix metalloproteinase-3, matrix metalloproteinase-9, vascular cell adhesion molecule-1, interleukin-1 beta). Tumor necrosis factor-alpha increased expression of KLF4, a known regulator of SMC differentiation. Kruppel-like transcription factor 4 (KLF4) small interfering RNA abrogated TNF-α activation of inflammatory genes and suppression of contractile genes. These mechanisms were confirmed in vivo after exposure of rat carotid arteries to TNF-α and early on in a model of cerebral aneurysm formation. Treatment with the synthesized TNF-α inhibitor 3,6-dithiothalidomide reversed pathologic vessel wall alterations after induced hypertension and hemodynamic stress. Chromatin immunoprecipitation assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4-dependent alterations in promoter regions of myocardin, SMCs, and inflammatory genes. In conclusion, TNF-α induces phenotypic modulation of cerebral SMCs through myocardin and KLF4-regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory/matrix-remodeling phenotype, which has important implications for the mechanisms behind intracranial aneurysm formation.

Aberrant expression of BCL11B in mycosis fungoides and its potential role in interferon‐induced apoptosis

BCL11B is a Kruppel-like C2H2-type zinc finger transcription factor, which has been associated with several human malignancies. Recent evidence showed that overexpressed BCL11B conferred chemoresistance to malignant T cells and that inhibiting BCL11B led to increased apoptosis, suggesting its potential pathogenic relevance in cutaneous T-cell lymphomas (CTCL), which were characterized by the resistance to chemotherapy-induced apoptosis. To investigate the expression pattern of BCL11B in different stages of mycosis fungoides (MF), quantitative reverse transcription polymerase chain reaction and immunohistochemistry were performed to compare the mRNA and protein expression among different stages of MF and benign inflammatory dermatoses (BID), respectively. BCL11B demonstrated significant upregulation in all stages of MF, compared with BID, in both mRNA expression level and protein level. In addition, BCL11B expression increased with advancing lesion tumor stage and overall disease stage. Further, to evaluate the dynamic expression of BCL11B under CTCL-directed treatment, BCL11B expression and cell apoptosis were evaluated after interferon (IFN)-α-2b and methotrexate treatment on CTCL cell line Hut78 cells. IFN-α-2b, but not methotrexate, induced BCL11B inhibition and cell apoptosis, suggesting that BCL11B may play important roles in the anti-CTCL effect of IFN-α-2b. In conclusion, our study demonstrated the overexpression of BCL11B in MF lesions and its potential relevance to disease progression. In addition, we provided evidence for BCL11B inhibitory approaches as a potential treatment to target chemoresistant tumor cells in advanced MF.

1168 BL-8030: A NOVEL, POTENT, SELECTIVE, ORALLY AVAILABLE INHIBITOR OF HEPATITIS C VIRUS NS3/4A PROTEASE

essentially hepatotropic, recent studies suggest that it can also infect peripheral blood mononuclear cells (PBMC). HCV infection has been clinically associated with serum lipid abnormalities. The aim of this study is to analyze the possible roles of genes involved in HCV-mediated lipid metabolism in PBMC from HCV infected patients. Materials and Methods: Using The Human Adipogenesis RT ProfilerTM PCR Array, we have analyzed the expression profile of 84 genes in chronic HCV PBMCs patients normalized with PBMCs from heathy donors. To compared the results obtained from HCV+ PBMCs with the expression in the hepatocytes, we have used a JFH1/HCV in vitro system after 48h post infection normalized with uninfected Huh 7.5.1 cell line. Results: Both in vivo and in vitro models HCV was able to upregulate the same genes (14% in both of them) involved in lipid metabolism such as SREBP1c, FASN, FABP4, ACACB and in signal transduction (29% and 27% respectively) resulted modulated both in HCV PBMCs and J6/JFH1-infection system. In both models the Kruppel-like transcription factor 15 (KLF15). KLF15 results significantly upregulated in HCV+ PBMCs as well as the J6/JFH1 in vitro infection. In addition, in PBMCs, KLF15 upregulation is statistically and positively correlated to the Body Weight, the BMI and the ALT levels. Furthermore, in PBMCs from HCV+ donors, KLF15 expression correlates with the HCV Viral Load and the antibody titer (anti-NS3 anti-NS4 and anti-capsid), suggesting a possible intriguing role of this factor in HCV infection. Conclusions: Our results confirm the ability of HCV to affect lipid metabolism; we have highlighted a similar modulation both in HCV patients lymphoid cells and in J6/JFH1 HCV infection model. In particular, the virus-related KLF15 enhanced expression, in both in vivo and in vitro models, suggests this protein as new possible marker of HCV infection and as a potential therapeutic target useful to counteract HCV infection.

Characteristics of pericardial interstitial cells and their implications in pericardial fibrocalcification

Pericardial fibrocalcification (PF) is a prominent feature of human pericardial pathology, including constrictive pericarditis and, to a lesser extent, degenerated autologous pericardial substitutes. However, the role of pericardial interstitial cells (PICs) in the pathogenesis of PF has yet to be established. Using a combination of histology and immunohistochemistry, we showed that the critical cellular event in PF in situ was the transdifferentiation of PICs into myofibroblasts/osteoblasts and that the percentage of myofibroblasts/osteoblasts correlated positively with the severity of PF. In vitro studies demonstrated that PICs, similar to mesenchymal stem cells, had the potential to differentiate along adipogenic, osteogenic, chondrogenic or myogenic lineages. However, PICs exhibited a more limited self-renewal capacity and a lower expression of Oct4 (POU5F1) and Kruppel-like transcription factor Klf4, underwent earlier senescence and spontaneously transdifferentiated into myofibroblasts/osteoblasts. Quantitative-real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) confirmed that the mRNA levels of α-smooth muscle actin (α-SMA), alkaline phosphatase (ALP), core-binding factor α1/runt-related transcription factor2 (Cbfa1/Runx2), transforming growth factor (TGF)-β1 and bone morphogenetic protein (BMP)-2 were upregulated as the passage number increased. The mRNA level of platelet-derived growth factor (PDGF)-AA was also significantly upregulated with higher levels at passage 3. Ectopic expression of Oct4 and Klf4 enhanced the colony formation of PICs and selectively impaired induction of genes involved in transdifferentiation into myofibroblasts/osteoblasts (α-SMA, ALP, Cbfa1/Runx2, PDGF-AA and BMP-2). These data, while offering new insights into the biology of PICs, reinforce the central role of these cells in cell-mediated PF and may assist in future strategies to treat fibrocalcific pericardial diseases.

Expression of Microphthalmia-associated Transcription Factor (MITF), Which Is Critical for Melanoma Progression, Is Inhibited by Both Transcription Factor GLI2 and Transforming Growth Factor-β

The melanocyte-specific transcription factor M-MITF is involved in numerous aspects of melanoblast lineage biology including pigmentation, survival, and migration. It plays complex roles at all stages of melanoma progression and metastasis. We established previously that GLI2, a Kruppel-like transcription factor that acts downstream of Hedgehog signaling, is a direct transcriptional target of the TGF-β/SMAD pathway and contributes to melanoma progression, exerting antagonistic activities against M-MITF to control melanoma cell invasiveness. Herein, we dissected the molecular mechanisms underlying both TGF-β and GLI2-driven M-MITF gene repression. Using transient cell transfection experiments with M-MITF promoter constructs, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified a GLI2 binding site within the -334/-296 region of the M-MITF promoter, critical for GLI2-driven transcriptional repression. This region is, however, not needed for inhibition of M-MITF promoter activity by TGF-β. We determined that TGF-β rapidly repressed protein kinase A activity, thus reducing both phospho-cAMP-response element-binding protein (CREB) levels and CREB-dependent transcription of the M-MITF promoter. Increased GLI2 binding to its cognate cis-element, associated with reduced CREB-dependent transcription, allowed maximal inhibition of the M-MITF promoter via two distinct mechanisms.

Sequence-specific Recruitment of Heterochromatin Protein 1 via Interaction with Krüppel-like Factor 11, a Human Transcription Factor Involved in Tumor Suppression and Metabolic Diseases

Heterochromatin protein 1 (HP1) proteins are "gatekeepers" of epigenetic gene silencing that is mediated by lysine 9 of histone H3 methylation (H3K9me). Current knowledge supports a paradigm whereby HP1 proteins achieve repression by binding to H3K9me marks and interacting to H3K9 histone methyltransferases (HMTs), such as SUV39H1, which methylate this residue on adjacent nucleosomes thereby compacting chromatin and silencing gene expression. However, the mechanism underlying the recruitment of this epigenetic regulator to target gene promoters remains poorly characterized. In the current study, we reveal for the first time a mechanism whereby HP1 is recruited to promoters by a well characterized Krüppel-like transcription factor (KLF), in a sequence-specific manner, to mediate complex biological phenomena. A PXVXL HP1-interacting domain identified at position 487-491 of KLF11 mediates the binding of HP1α and KLF11 in vitro and in cultured cells. KLF11 also recruits HP1α and its histone methyltransferase, SUV39H1, to promoters to limit KLF11-mediated gene activation. Indeed, a KLF11ΔHP1 mutant derepresses KLF11-regulated cancer genes, by inhibiting HP1-SUV39H1 recruitment, decreasing H3K9me3, while increasing activation-associated marks. Biologically, impairment of KLF11-mediated HP1-HMT recruitment abolishes tumor suppression, providing direct evidence that HP1-HMTs act in a sequence-specific manner to achieve this function rather than its well characterized binding to methylated chromatin without intermediary. Collectively, these studies reveal a novel role for HP1 as a cofactor in tumor suppression, expand our mechanistic understanding of a KLF associated to human disease, and outline cellular and biochemical mechanisms underlying this phenomenon, increasing the specificity of targeting HP1-HMT complexes to gene promoters.

Molecular mechanism of barrel cortex development controlled by thalamocortical axon innervation

During thymic development, thymocytes expressing a T cell receptor consisting of an alpha and beta chain (TCRαβ), commit to either the cytotoxic- or T helper-lineage fate. This lineage dichotomy is controlled by key transcription factors, including the T helper (Th) lineage master regulator, the Th-inducing BTB/POZ domain-containing Kruppel-like zinc-finger transcription factor, ThPOK, (formally cKrox or Zfp67; encoded by Zbtb7b), which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4+ thymocytes and the Runt related transcription factor 3 (Runx3), which counteracts ThPOK in MHC class I restricted precursor cells and promotes the lineage commitment of CD8αβ+ cytolytic T lymphocytes (CTL). ThPOK continues to repress the CTL gene program in mature CD4+ T cells, even as they differentiate into effector Th cell subsets. The Th cell fate however is not fixed and two recent studies showed that mature, antigen-stimulated CD4+ T cells have the flexibility to terminate the expression of ThPOK and functionally reprogram to cytotoxic effector cells. This unexpected plasticity of CD4+ T cells results in the post-thymic termination of the Th lineage fate and the functional differentiation of distinct MHC class II-restricted CD4+ CTL. The recognition of CD4 CTL as a defined separate subset of effector cells and the identification of the mechanisms and factors that drive their reprogramming finally create new opportunities to explore the physiological relevance of these effector cells in vivo and to determine their pivotal roles in both, protective immunity as well as in immune-related pathology.

Protein Kinase C (PKC) δ Suppresses Keratinocyte Proliferation by Increasing p21Cip1 Level by a KLF4 Transcription Factor-dependent Mechanism

PKCδ increases keratinocyte differentiation and suppresses keratinocyte proliferation and survival. However, the mechanism of proliferation suppression is not well understood. The present studies show that PKCδ overexpression increases p21(Cip1) mRNA and protein level and promoter activity and that treatment with dominant-negative PKCδ, PKCδ-siRNA, or rottlerin inhibits promoter activation. Analysis of the p21(Cip1) promoter upstream regulatory region reveals three DNA segments that mediate PKCδ-dependent promoter activation. The PKCδ response element most proximal to the transcription start site encodes six GC-rich DNA elements. Mutation of these sites results in a loss of PKCδ-dependent promoter activation. Gel mobility supershift and chromatin immunoprecipitation reveal that these DNA elements bind the Kruppel-like transcription factor KLF4. PKCδ increases KLF4 mRNA and protein level and KLF4 binding to the GC-rich elements in the p21(Cip1) proximal promoter. In addition, KLF4-siRNA inhibits PKCδ-dependent p21(Cip1) promoter activity. PKCδ increases KLF4 expression leading to enhanced KLF4 interaction with the GC-rich elements in the p21(Cip1) promoter to activate transcription.

Kruppel-like factor 5 is required for formation and differentiation of the bladder urothelium

Kruppel-like transcription factor 5 (Klf5) was detected in the developing and mature murine bladder urothelium. Herein we report a critical role of KLF5 in the formation and terminal differentiation of the urothelium. The ShhGfpCre transgene was used to delete the Klf5floxed alleles from bladder epithelial cells causing prenatal hydronephrosis, hydroureter, and vesicoureteric reflux. The bladder urothelium failed to stratify and did not express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and the uroplakins. The effects of Klf5 deletion were unique to the developing bladder epithelium since maturation of the epithelium comprising the bladder neck and urethra was unaffected by the lack of KLF5. mRNA analysis identified reductions in Pparγ, Grhl3, Elf3, and Ovol1expression in Klf5 deficient fetal bladders supporting their participation in a transcriptional network regulating bladder urothelial differentiation. KLF5 regulated expression of the mGrhl3 promoter in transient transfection assays. The absence of urothelial Klf5 altered epithelial–mesenchymal signaling leading to the formation of an ectopic alpha smooth muscle actin positive layer of cells subjacent to the epithelium and a thinner detrusor muscle that was not attributable to disruption of SHH signaling, a known mediator of detrusor morphogenesis. Deletion of Klf5 from the developing bladder urothelium blocked epithelial cell differentiation, impaired bladder morphogenesis and function causing hydroureter and hydronephrosis at birth.

Noncanonical K27-Linked Polyubiquitination of TIEG1 Regulates Foxp3 Expression and Tumor Growth

Earlier, we demonstrated the essential role of Kruppel-like transcription factor, TIEG1, in TGF-β-induced regulatory T cell (Treg) development. In this article, we demonstrate that IL-6, which promotes Th17 development, abrogated TIEG1 nuclear translocation and inhibited TGF-β-induced Treg development. Tyrosine kinase Tyk2-mediated phosphorylation of TIEG1 at Tyr179 promoted noncanonical K-27-linked polyubiquitination, which inhibited TIEG1 nuclear translocation. To test the role of TIEG1-regulated Treg/Th17 development in antitumor immunity, we analyzed TRAMP-C2 tumor growth in TIEG1(-/-) mice. The defective Treg development and elevated Th17 response resulted in enhanced immune reactivity in the tumor and inhibition of TRAMP-C2 tumor growth in TIEG1(-/-) mice. Thus, our results uncovered a novel regulatory mechanism that modulates Tregs and may regulate tumor progression.

The Myeloid Transcription Factor KLF2 Regulates the Host Response to Polymicrobial Infection and Endotoxic Shock

Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation invivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia inducible factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NF-κB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation invivo and an essential regulator of the innate immune system.

Emerging Roles of Kruppel‐Like Factor 6 and Kruppel‐Like Factor 6 Splice Variant 1 in Ovarian Cancer Progression and Treatment

Epithelial ovarian cancer is one of the most lethal gynecologic cancers and the fifth most frequent cause of female cancer deaths in the United States. Despite dramatic treatment successes in other cancers through the use of molecular agents targeted against genetically defined events driving cancer development and progression, very few insights into epithelial ovarian cancer have been translated from the laboratory to the clinic. If advances are to be made in the early diagnosis, prevention, and treatment of this disease, it will be critical to characterize the common and private (personalized) genetic defects underlying the development and spread of epithelial ovarian cancer. The tumor suppressor Kruppel-like factor 6 and its alternatively spliced, oncogenic isoform, Kruppel-like factor 6 splice variant 1, are members of the Kruppel-like zinc finger transcription factor family of proteins, which have diverse roles in cellular differentiation, development, proliferation, growth-related signal transduction, and apoptosis. Inactivation of Kruppel-like factor 6 and overexpression of Kruppel-like factor 6 splice variant 1 have been associated with the progression of a number of human cancers and even with patient survival. This article summarizes our recent findings demonstrating that a majority of epithelial ovarian cancer tumors have Kruppel-like factor 6 allelic loss and decreased expression coupled with increased expression of Kruppel-like factor 6 splice variant 1. The targeted reduction of Kruppel-like factor 6 in ovarian cancer cell lines results in marked increases in cell proliferation, invasion, tumor growth, angiogenesis, and intraperitoneal dissemination in vivo. In contrast, the inhibition of Kruppel-like factor 6 splice variant 1 decreases cellular proliferation, invasion, angiogenesis, and tumorigenicity; this provides the rationale for its potential therapeutic application. These results and our recent demonstration that the inhibition of Kruppel-like factor 6 splice variant 1 can dramatically prolong survival in a preclinical mouse model of ovarian cancer are reviewed and discussed.

Abstract 5685: TGF-beta Signaling and Kruppel-like Factor 10 Regulate Bone Marrow-derived EPC Differentiation, Function, and Neovascularization

Background: Endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of contributing to cardiovascular regeneration. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Hematopoiesis consists of a cascade by which hematopoietic stem cells (HSCs) give rise to either a common myeloid progenitor (CMPs) or common lymphoid progenitor (CLPs). The CMP cells can give rise to either granulocyte-monocyte progenitors (GMPs) or megakaryocyte-erythrocyte progenitors (MEPs). Methods and Results: Herein, we identify that CMPs and GMPs preferentially differentiate into CD34+KDR+ EPCs as measured by FACS, uptake of DiIacLDL and U.lex Lectin, matrigel studies, and endothelial markers. Co-culture studies in matrigel demonstrate increased network formation and incorporation when HUVECs were co-incubated with CMPs and GMPs only. Treatment of CMPs or GMPs with IFN-g blocked EPC differentiation, whereas treatment with TGF-b1 potently induced EPC differentiation. Conversely, EPC formation was markedly impaired using blocking antibodies or TGF-b1 deficient CMPs or GMPs, whereas IFN-g deficient mice have increased EPC differentiation. Functionally, EPCs derived from CMPs and GMPs more potently adhered to fibronectin-coated plates, migrated faster in transwell Boyden chambers, and released VEGF nearly ~2 to 2.5-fold higher than EPCs from MEPs and HSCs. Finally, we identified that a TGF-b1-responsive Kruppel-like transcription factor, KLF10, as robustly expressed ~60-fold in EPCs derived from CMPs and GMPs, compared to hematopoietic progenitors lacking EPC markers. Consistently, KLF10−/− mice have marked defects in EPC differentiation (−23.6% vs. WT, P<0.002), TGF-b1 responsiveness (completedly abolished), and impaired blood flow recovery in response to hindlimb ischemia (−23.8% vs. WT, P<0.008), an effect associated with reduced levels of circulating EPCs (−28.2% vs. WT, P<0.05). Conclusion: Among bone marrow progenitors, a hierarchy exists for EPC differentiation. Targeting KLF10 may alter EPC differentiation from bone marrow-derived CMPs and GMPs and influence cardiovascular repair.

The Krüppel traffic report: Cooperative signals direct KLF8 nuclear transport

The Krüppel traffic report: Cooperative signals direct KLF8 nuclear transport

Activation of Protein Kinase C by Phorbol 12-Myristate 13-Acetate suppresses the growth of lung cancer cells through KLF6 induction

Phorbol 12-myristate 13-acetate (PMA) modulates cell proliferation and survival by activating several intracellular signaling pathways. Protein kinase C (PKC) plays a key role in PMA-induced growth arrest of non-small cell lung cancer (NSCLC) cells. Kruppel-like transcription factor 6 (KLF6), which is associated with negative control of cell proliferation, is down-regulated in many cancers, including NSCLC. In this study, we found that KLF6 is down- regulated in 17 lung cancer cell lines and in cells representing early stages of lung cancer development. Moreover, PMA induced cell growth arrest through KLF6 induction in H358 NSCLC cells. The increase in KLF6 by PMA was associated with up-regulation of the cyclin- dependent kinase inhibitors (CDKIs) p21WAF1/CIP1 and p27KIP1. In addition, inhibition of PKC or JNK activation decreased PMA-induced KLF6 induction and activation of PKC alone by Bryostatin-1 and Thymeleatoxin increased KLF6 levels. Moreover, siRNA-mediated knockdown of KLF6 reduced PMA-induced cell growth inhibition concomitantly with decreased expression of both p21WAF1/CIP1 and p27KIP1, and in accordance, over-expression of KLF6 alone up-regulated both CDKIs protein levels. Our results demonstrate the induction of the tumor suppressor KLF6 following PKC activation and its importance for PMA-mediated cancer cell growth arrest.

Kruppel-like transcription factor 5 promotes breast cell survival through pERK-mediated MKP-1 protein stabilization.

Abstract Abstract #3019 Kruppel-like Factor 5 (KLF5) is a transcription factor promoting cell survival and tumorigenesis in multiple cancers. High level of KLF5 mRNA is associated with a shorter survival for breast cancer patients. However, the role of KLF5 and mechanism of KLF5 actions in breast cancer remain unclear. In this study, we found that KLF5 knockdown by siRNA in two basal type breast cell lines MCF10A and BT20 induces apoptosis, as indicated by loss of cell viability and cleaved PARP and caspase 3. Interestingly, a survival phosphotase, MKP-1, is downregulated at protein level by KLF5 ablation. Consistently, KLF5 over-expression increases the MKP-1 protein expression. However, MKP-1 is not a KLF5 direct target gene because the MKP-1 mRNA level is not regulated by KLF5. By the cycloheximide chase assays, we found that KLF5 decreases the MKP-1 protein degradation. However, KLF5 does not decrease the MKP-1 E3 ubiquitin ligase SKP2 expression. The ERK inhibitor U0126 specifically blocks the KLF5 induced MKP-1, suggesting that KLF5 up-regulates MKP-1 through activating ERK signaling. Finally, we demonstrated that MKP-1 over-expression blocks KLF5 knockdown induced apoptosis in MCF10A cells. These findings suggest that KLF5 is a survival factor which promotes breast cell survival partially through pERK-mediated MKP-1 stabilization. The KLF5-pERK-MKP-1 signaling axis may provide new therapeutic targets for invasive breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3019.

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp -308 to -188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp -65 to -56) and GC-box 2 (bp -18 to -9), the latter of which is also bound by FBI-1. We found that FRE3 (bp -244 to -236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression.

Identification of different Ikaros cDNA transcripts in Philadelphia-positive adult acute lymphoblastic leukemia by a high-throughput capillary electrophoresis sizing method

Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA. In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts. We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus. Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia.