Role Of Krüppel-like Factor 4 Research Articles

3,3’-Diindolylmethane Enhances Paclitaxel Sensitivity by Suppressing DNMT1-Mediated KLF4 Methylation in Breast Cancer

Paclitaxel (PTX) is a first-line chemotherapeutic drug for the treatment of breast cancer, but drug resistance seriously limits its clinical use. The aim of the present work was to explore the effect of 3,3’-diindolylmethane (DIM) on PTX sensitivity and its possible mechanism in breast cancer. The expression of Krüppel-like factor 4 (KLF4) and DNA-methyltransferase 1 (DNMT1) in breast cancer tissues were assessed by immunohistochemistry and Western blotting. The methylation of KLF4 was evaluated by the MassARRAY platform. The lentivirus carrying KLF4 and DNMT1 gene or shRNA targeting DNMT1 were used to overexpress KLF4 or knockdown DNMT1 in MCF-7 and T47D breast cancer cells and the role of KLF4 and DNMT1 in regulation of PTX sensitivity was investigated. The effect of PTX on inhibiting the proliferation of MCF-7 and T47D cells was measured by CCK-8 assay. Flow cytometry was used to examine cell apoptosis. The expression of mRNA and protein was evaluated by qRT-PCR and Western blotting analysis, respectively. Our data showed that the expression of DNMT1 was increased, and the methylation level of CpG sites (−148 bp) in the KLF4 promoter was increased while the KLF4 expression was significantly decreased in breast cancer tissues. Overexpression of KLF4 increased the sensitivity of MCF-7 and T47D cells to PTX. DNMT1 increased the methylation of the KLF4 promoter and decrease the expression of KLF4. Knockdown of DNMT1 increased the sensitivity of MCF-7 and T47D cells to PTX. DIM enhanced the PTX sensitivity of MCF-7 and T47D cells, decreased the expression of DNMT1 and the methylation level of KLF4 promoter, thus increasing the level of KLF4. Furthermore, overexpression of DNMT1 attenuated the effect of DIM on the regulation of PTX sensitivity. Collectively, our data indicated that DNMT1-mediated hypermethylation of KLF4 promoter leads to downregulation of KLF4 in breast cancer. The level of KLF4 is correlated with the sensitivity of MCF-7 and T47D cells to PTX. DIM could enhance the antitumor efficacy of PTX on MCF-7 and T47D cells by regulating DNMT1 and KLF4.

Roles of KLF4 and AMPK in the inhibition of glycolysis by pulsatile shear stress in endothelial cells

Vascular endothelial cells (ECs) sense and respond to hemodynamic forces such as pulsatile shear stress (PS) and oscillatory shear stress (OS). Among the metabolic pathways, glycolysis is differentially regulated by atheroprone OS and atheroprotective PS. Studying the molecular mechanisms by which PS suppresses glycolytic flux at the epigenetic, transcriptomic, and kinomic levels, we have demonstrated that glucokinase regulatory protein (GCKR) was markedly induced by PS in vitro and in vivo, although PS down-regulates other glycolysis enzymes such as hexokinase (HK1). Using next-generation sequencing data, we identified the binding of PS-induced Krüppel-like factor 4 (KLF4), which functions as a pioneer transcription factor, binding to the GCKR promoter to change the chromatin structure for transactivation of GCKR. At the posttranslational level, PS-activated AMP-activated protein kinase (AMPK) phosphorylates GCKR at Ser-481, thereby enhancing the interaction between GCKR and HK1 in ECs. In vivo, the level of phosphorylated GCKR Ser-481 and the interaction between GCKR and HK1 were increased in the thoracic aorta of wild-type AMPKα2+/+ mice in comparison with littermates with EC ablation of AMPKα2 (AMPKα2-/-). In addition, the level of GCKR was elevated in the aortas of mice with a high level of voluntary wheel running. The underlying mechanisms for the PS induction of GCKR involve regulation at the epigenetic level by KLF4 and at the posttranslational level by AMPK.

The association between KLF4 as a tumor suppressor and the prognosis of hepatocellular carcinoma after curative resection.

Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor in klfs family, is known for its crucial role in regulating cell growth, proliferation, and differentiation. This research aimed to explore the prognostic significance of KLF4 in hepatocellular carcinoma’s (HCC) patients after curative resection and the role of KLF4 in HCC progression. There were 185 HCC patients who had hepatectomy from July 2010 to July 2011 included in this study. KLF4 expression was detected by microarray immunohistochemical technique, western blot, and qRT-PCR. Then, the correlation between the prognosis of patients and KLF4 expression was evaluated based on patients’ follow-up data. The research found KLF4 expression was significantly downregulated in HCC tissues compared to para-tumorous tissues. More importantly, the overall survival rate (OS) and recurrence-free survival rate (RFS) of HCC patients with low KLF4 expression were both significantly decreased compared to those with a high level of KLF4. Further function and mechanism analysis showed that KLF4 could inhibit the proliferation, migration, invasion and epithelial-mesenchymal transition of HCC cells. The study revealed that KLF4 was not only a tumor suppressor in HCC but also can be regarded as a valuable prognostic factor and potential biological target for diagnosis and treatment in HCC patients.

KLF4 suppresses the tumor activity of cutaneous squamous cell carcinoma (SCC) cells via the regulation of SMAD signaling and SOX2 expression

Kruppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays a role in terminal differentiation of epidermal keratinocytes. There are conflicting reports regarding the role of KLF4 in tumor development, with both the tumor suppressive and/or oncogenic properties depending on different conditions and cell types. In this study, we investigated the functional importance of KLF4 in cutaneous squamous cell carcinoma (SCC). Immunohistochemistry showed that KLF4 expression was relatively low in SCC lesion compared to normal epidermis. To examine the effects of KFL4, we transduced SCC lines (SCC12 and SCC13 cells) with the KLF4-expressing recombinant adenovirus. Overexpression of KLF4 significantly decreased cell proliferation and colony forming activity. In addition, overexpression of KLF4 markedly reduced invasive potential, along with the downregulation of epithelial-mesenchymal transition (EMT)-related molecules. In a mechanistic study, KLF4 inhibited SOX2, of which expression is critical for tumor initiation and growth of SCC. Further investigations indicated that SOX2 expression is induced by TGF-β/SMAD signaling, and that overexpression of KLF4 inhibited SMAD signaling via upregulation of SMAD7, an important inhibitory SMAD molecule. Based on these data, KLF4 plays a tumor suppressive role in cutaneous SCC cells.

Abstract 17171: Kruppel-Like Factor 4 Deletion Regulates Lymphatic Endothelial Cell Homeostasis

Introduction and Hypothesis: Lymphatic vasculature exquisitely controls the clearance of metabolic end products, removal of bacterial endotoxins, and trafficking of immune cells. Signaling pathways that converge into endothelial cell kruppel-like factor 4 (KLF4) provide an atheroprotective role; however, the role of lymphatic endothelial cell (LEC)-KLF4 in these processes has not been clarified. Thus, we examined whether LEC-specific deletion of Klf4 alleles LECs. Methods and Results: After several generations of breeding, we transferred ROSA mT/mG (dual-fluorescent reporter allele mT/mG) to Prox1 CreERT2 (tamoxifen, TAM) inducible mice in C57 background to generate ROSA mT/mG ::Prox1 CreERT2 mice. In the next breeding scheme, ROSA mT/mG ::Prox1 CreERT2 female mice were crossed with male Klf4 fl/wt (Klf4 floxed allele) mice to generate heterozygous ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt offspring (50%). Thereafter, we bred male ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt with female ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt to produce ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/fl genotype (25%). These mice (12-14 weeks old) received TAM for five consecutive days, and on 21 st or 28 th day all mice were sacrificed. To confirm LEC Cre activation after TAM administration, we prepared cryosections of lung and the heart tissues. Mice receiving TAM showed lively and clear separation of green fluorescent protein (GFP) and tomato-Red colors, showing LEC specificity of Prox1 CreERT2 transgenic methodology. Cryosectioning combined with low- and high-resolution microscopy, and morphometric analyses of H&E sections showed disruption of LEC function and integrity in these mice. Biochemical experiments underway will address the role of KLF4 in lymphatic vessels homeostasis. Conclusion: These results indicate a key role for Klf4 in the regulation of lymphatic vessels.

Kruppel-like factor 4 (KLF-4) plays a crucial role in simvastatin (SVT)-induced differentiation of rabbit articular chondrocytes

Simvastatin is a cholesterol-lowing reagent that is derived synthetically from the fermentation of Aspergillus terreus. Recently, SVT has been shown to possess a protective effect of chondrocytes.Kruppel-like factor 4 (KLF-4) is a zinc finger transcription factor that plays crucial roles during the development and maintenance of multiple organs. However, the roles of KLF-4 in chondrocytes have not been well unknown. Here, we investigated whether KLF-4 regulates SVT-caused differentiated phenotype of chondrocytes. A KLF-4 cDNA or KLF-4 siRNA was transfected into SVT-treated chondrocytes. Western blot analysis, RT-PCR and immunofluorescence staining analyzed expression of type II collagen and SOX-9, marker proteins of differentiation. The results showed overexpression of KLF-4 accelerates SVT-induced type II collagen expression, as determined by western blot analysis and causes sulfated-proteoglycan synthesis, as detected by Alcian blue staining. RT-PCR revealed that ectopic expression of KLF-4 induces SVT-caused SOX-9, a transcription factor of type II collagen, expression. Transfection of KLF-4 siRNA reversed SVT-caused type II collagen and SOX-9 expression and inhibited SVT-induced sulfated proteoglycan production. This study indicates that KLF-4 plays critical role in SVT-caused chondrocytes differentiation.

Krüppel-like factor 4 mediates cellular migration and invasion by altering RhoA activity.

Kru¨ppel like factor 4 (KLF4) is a transcription factor that regulates genes related to differentiation and proliferation. KLF4 also plays a role in metastasis via epithelial to mesenchymal transition. Here, we investigate the function of Klf4 in migration and invasion using mouse embryonic fibroblasts and the RKO human colon cancer cell line. Compared to wild-type, cells lacking Klf4 exhibited increased migration-associated phenotypes. In addition, overexpression of Klf4 in Klf4-/- MEFs attenuated the presence of stress fibers to wild-type levels. An invasion assay suggested that lack of Klf4 resulted in increased invasive capacity. Finally, analysis of RhoA showed elevated RhoA activity in both RKO and MEF cells. Taken together, our results strongly support the novel role of KLF4 in a post-translational regulatory mechanism where KLF4 indirectly modulates the actin cytoskeleton morphology via activity of RhoA in order to inhibit cellular migration and invasion.

Deficiency of KLF4 compromises the lung function in an acute mouse model of allergic asthma

Asthma is a chronic inflammatory disease of the airways and the mechanisms are not fully understood. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of monocytes, granulocyte and myeloid cells at early stage of differentiation. They possess phenotypic plasticity and regulate airway inflammation. We recently reported that Kruppel-like factor 4 (KLF4) regulates MDSC differentiation into fibrocytes, emerging effectors in chronic inflammation. However, the role of KLF4 in asthma is not known. Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine and a key initiator of allergic airway inflammation. Given the fact that TSLP promotes Th2 cytokine production that increases MDSC differentiation into fibrocytes, we postulate that KLF4 regulates asthma in a TSLP-dependent manner. In this study, we utilized a model of allergic asthma with ovalbumin challenge (OVA). We found that upon OVA treatment the wild type mice had increased MDSC infiltration into the lung, up-regulation of KLF4 and TSLP gene expression, and higher levels of Th2 cytokines including IL4 and IL13. Consistently, lack of KLF4 expression in monocytes and lung epithelial cells resulted in decreased TSLP expression and lower levels of Th2 cytokines in mice, and fibrocyte generation was compromised. KLF4 deficiency in these cells also led to decreased airway hyperresponsiveness (AHR), a cardinal feature of asthma, as assessed by whole body plethysmography. Moreover, lung fibrosis as measured by trichome staining was attenuated and the population of CD45 + COL1A1+ fibrocytes was diminished in this setting. Together, our results suggest that KLF4 regulates asthma development in a TSLP- and fibrocyte-dependent manner.

Abstract 12521: Intracellular Fatty Acid Balance Regulated by Elovl6 Plays a Notable Phenotypic Response of Vascular Smooth Muscle Cells via AMPK-KLF4-Dependent Signaling

Introduction: Krüppel-like factor 4 (KLF4) is a transcription factor controlling differentiation and proliferation of vascular smooth muscle cells (VSMC). We recently showed that disruption of elongation of long chain fatty acids 6 (Elovl6), rate-limiting enzyme catalyzing the elongation of saturated and monounsaturated, suppressed VSMC proliferation and differentiation via AMP-activated protein kinase (AMPK) activation. However, little is known about the role of KLF4 in passing metabolic stress on to the regulating VSMC proliferation and differentiation. Hypothesis: We examine whether KLF4 is involved in the suppression of Elovl6-disrupted VSMC proliferation. Methods and Results: Immunohistochemical staining of aorta revealed that KLF4 expression was markedly increased in Elovl6-null (Elovl6 -/- ) mice compared with wild-type mice. Western blot analysis also showed that the KLF4 expression was markedly induced in human aortic smooth muscle cell (HASMC) with siRNA-mediated Elovl6 depletion and in Elovl6 -/- mouse aorta. Previous study demonstrated that Elovl6 depletion in VSMC altered long chain fatty acid (LCFA) composition in which palmitate was increased and oleate was decreased. Accordingly, palmitate increased and oleate decreased KLF4 expression. To clarify the effects of KLF4 on cell-cycle regulatory gene expressions and cell proliferation, we examined a siRNA-mediated knockdown of KLF4 in HASMC. The expression of SMα-actin and SM22α genes were decreased in siKLF4-introduced HASMC, and siElovl6-triggered inhibition of cell proliferation was partially recovered by concomitant treatment of siKLF4. Elovl6 depletion in HASMC markedly induced p53 phosphorylation and p21 expression, whereas depletion of KLF4 abrogated the induction of these proteins. We further identified the treatment with AMPK activator (AICAR) significantly induced KLF4 expression, whereas AMPK inhibitor (compound C) markedly inhibited the AICAR-induced or Elovl6-knockdown-induced KLF4 expression. Conclusions: Overall, our results indicate that KLF4 is activated by metabolic stress triggered by perturbed intracellular LCFA composition and AMPK-KLF4 axis plays an indispensable role in controlling VSMC proliferation and differentiation

KLF4 Knockdown Attenuates TBI-Induced Neuronal Damage through p53 and JAK-STAT3 Signaling.

Traumatic brain injury (TBI) is induced by complex primary and secondary mechanisms that give rise to cell death, inflammation, and neurological dysfunction. Understanding the mechanisms that drive neurological damage as well as those that promote repair can guide the development of therapeutic drugs for TBI. Kruppel-like factor 4 (KLF4) has been reported to negatively regulate axon regeneration of injured retinal ganglion cells (RGCs) through inhibition of JAK-STAT3 signaling. However, the role of KLF4 in TBI remains unreported. Reactive oxygen species (ROS)-induced neuronal death is a pathophysiological hallmark of TBI. In this study, we used H2 O2 -treated RGCs in vitro and the optic nerve crush model in vivo to simulate neuronal damage in TBI. The function of KLF4 in RGC survival and axon regeneration in these models was investigated. In addition, the effects of KLF4 knockdown on neuronal damage after a brain impact that mimics moderate TBI were studied. The results show that H2 O2 induces p53-dependent apoptosis of RGCs in vitro through upregulation of KLF4. Additionally, KLF4 knockdown in vivo significantly enhances CNTF-induced axon regeneration of RGCs after optic nerve crush, and more importantly, prevents neuronal damage after a moderate brain impact in rats. Our Western blot analysis and immunoprecipitation assay results indicate that these effects of KLF4 knockdown are mediated by the p53 and JAK-STAT3 pathways. These findings provide evidence that KLF4 plays an important role in the pathophysiology of TBI. Blocking KLF4 may be a potential therapeutic strategy for the treatment of TBI, either alone or in combination with agents that target complementary mechanisms.

Krüppel-like factor 4: A new potential biomarker of lung cancer.

Lung cancer is most prevalent human cancer worldwide. However, no molecular markers are currently available for predicting lung cancer prognosis. Therefore, identifying novel biomarkers may be useful for improving clinical diagnosis and patient stratification. Krüppel-like factor 4 (KLF4) is a transcription factor with opposing roles in different human cancers. Its overexpression in several cancers is correlated with a poor prognosis. However, the expression and role of KLF4 in lung cancer remains to be elucidated. The aim of this study was to determine the profile of KLF4 expression in different types of lung cancer. The KLF4 protein expression level was tested and evaluated by immunohistochemical analysis in 47 lung tumors and normal tissues, and then correlated with clinical characteristics. A differential expression of KLF4 was observed between normal tissue and each of the lung cancer types. A significant decrease in KLF4 expression was observed in non-small-cell lung cancer (NSCLC) compared with that in normal tissue, while significant overexpression was detected in small-cell lung cancer. Furthermore, a higher rate of expression was observed in stage II, III and IV disease compared with stage I disease in NSCLC tissues. KLF4 expression was not found to be associated with age or gender. Our results suggested that the KLF4 protein level may be a potential biomarker in patients with advanced lung cancer.

Kruppel-like factor 4 (KLF-4) inhibits the epithelial-to-mesenchymal transition and proliferation of human endometrial carcinoma cells

Kruppel-like factors (KLFs) are a group of transcriptional regulators, being tumor-suppressive in various types of cancers, but not clear in human endometrial carcinoma (EC). We investigated the KLF-4 expression in both mRNA and protein levels in 29 EC specimens with RT-qPCR and Western blotting methods, and then to determine its promotion to Epithelial-to-mesenchymal transition (EMT) and proliferation of EC Ishikawa cells, via analyzing EMT-associated markers and via CCK-8 and colony forming assay. We found the downregulation of KLF-4 in the 29 EC specimens, correlating with the EC malignance. Moreover, we confirmed reduced levels of EMT and cell proliferation of Ishikawa cells post-KLF-4 overexpression. In conclusion, the significantly reduced KLF-4 correlated with the EC malignance. And the overexpressed KLF-4 promoted the EMT and proliferation of EC cells in vitro. The present study recognized the tumor suppressive role of KLF-4 in EC.

Krüppel-like factor 4 promotes human osteosarcoma growth and metastasis via regulating CRYAB expression.

Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, is an essential regulator in many cellular processes, including differentiation, proliferation, inflammation, pluripotency, and apoptosis. Along with these roles in normal cells and tissues, KLF4 has been reported as a tumor suppressor or an oncogene in many cancers. However, the role of KLF4 in osteosarcoma is largely unknown. Here we found the expression of KLF4 was significantly increased in human osteosarcoma tissues compared with the normal tissues. Elevated KLF4 promoted human osteosarcoma cell proliferation and metastasis. Subsequently, mechanistic studies revealed KLF4 specifically bound the promoter of CRYAB and upregulated CRYAB expression in human osteosarcoma cells. Moreover, we found that KLF4 enhanced osteosarcoma cell proliferation and migration via upregulating CRYAB. Therefore, our studies suggested KLF4 may be a potential target for human osteosarcoma therapy.

The Janus-faced roles of Krüppel-like factor 4 in oral squamous cell carcinoma cells

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that regulates many essential processes, including development and cell differentiation, proliferation, and apoptosis. Along with these roles in normal cells and tissues, KLF4 has important tumor suppressive and oncogenic functions in some malignancies. However, the roles of KLF4 in oral squamous cell carcinoma remain unclear. This study investigated the epigenetic alterations and possible roles of KLF4 in oral cancer carcinogenesis. Notably, KLF4 expression was significantly decreased in human oral cancer tissues compared with healthy controls, and KLF4 promoter hypermethylation contributed to the suppression of KLF4 expression. KLF4 expression was associated with tumor grade. Its expression was much lower in poorly differentiated oral cancers than in well-differentiated cancer cells. KLF4 exerted its antitumor activity in vitro and/or in vivo by inhibiting cell proliferation, cell cycle progression, cell colony formation and by inducing apoptosis. In addition, KLF4 over-expression promoted oral cancer cell migration and invasion in vitro. Knockdown of KLF4 promoted oral cancer cells growth and colony formation, and simultaneously inhibited cell migration and invasion. Mechanistic studies revealed that MMP-9 might contribute to KLF4-mediated cell migration and invasion. These results provide evidence that KLF4 might play Janus-faced roles in oral cancer carcinogenesis, acting both as a tumor suppressor and as an oncogene.

Epigenetic repression of Krüppel-like factor 4 through Dnmt1 contributes to EMT in renal fibrosis.

Krüppel-like factor 4 (KLF4) is a transcription factor which plays divergent roles in a number of physiological or pathological process. However, the expression and role of KLF4 in renal fibrosis remain undetermined. The aim of the present study was to determine the epigenetic alterations of KLF4 and its potential role and mechanisms of action in epithelial-to-mesenchymal transition (EMT) in renal fibrosis. The hypermethylation of the KLF4 promoter accompanied by a decrease in KLF4 expression were observed in mice subjected to unilateral ureteral obstruction (UUO) and in HK-2 cells stimulated with transforming growth factor (TGF)-β1. However, treatment with 5-aza-2′-deoxycytidine attenuated the TGF-β1-induced downregulation of KLF4 and E-cadherin and the upregulation of α-smooth muscle actin (α-SMA) in the HK-2 cells. DNA methyltransferase 1 (Dnmt1) participated in the TGF-β1-mediated hypermethylation of the KLF4 promoter in the HK-2 cells. In addition, functional analysis demonstrated that the overexpression of KLF4 led to an increase in the expression of E-cadherin and zonula occludens-l (ZO-1), and a decrease in the expression of α-SMA and fibroblast-specific protein 1 (FSP-1), thus reversing the effects of the suppression of KLF4. These data suggest that KLF4 inhibits the progression of EMT in renal epithelial cells. In conclusion, our findings demonstrate that KLF4 is downregulated during EMT in renal fibrosis in vivo and in vitro; thus, KLF4 functions as a suppressor of renal fibrogenesis. The hypermethylation of KLF4 directly mediated by Dnmt1 contributes to the progression of EMT in renal epithelial cells. KLF4 promoter methylation may thus be a promising diagnostic marker or therapeutic target in renal fibrosis.

Downregulation of the KLF4 transcription factor inhibits the proliferation and migration of canine mammary tumor cells

Canine mammary tumor (CMT) is the most common neoplasm in female dogs, and over 50% of CMTs are diagnosed as malignant. Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and is associated with cell proliferation, differentiation, migration, and apoptosis. Although the role of KLF4 is still controversial in various human cancers, KLF4 has been identified as an oncogene in human breast cancer. Moreover, high KLF4 expression is correlated with an aggressive phenotype in CMT. Therefore, investigating the function of KLF4 may help better understand the pathogenesis of CMT.In this study, partial sequences of canine KLF4 and KLF4 expression were identified in various normal canine tissues, as well as CMT cells and Madin–Darby canine kidney (MDCK) cells. Kenpaullone, a small molecule inhibitor of KLF4, downregulated KLF4 expression in CMT cells and reduced CMT cell proliferation, migration, and colony formation in soft agar. Kenpaullone treatment induced S and G2/M phase arrest in CMT and MDCK cells, and induced death in CMT cells, but not in MDCK cells. It was concluded that KLF4 is expressed in various normal canine tissues, and downregulation of KLF4 inhibited CMT cell proliferation and migration, and induced cell death. The results of this study suggest that KLF4 may represent a suitable therapeutic target for CMT therapy.

KLF4 Regulates Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Repressing Gbl Expression and Altering mTORC2 Activity

Abstract Tyrosine kinase inhibitors (TKIs) are the standard treatment for eradicating BCR-ABL-positive progenitor cells in chronic myeloid leukemia (CML); however, disease often relapses upon drug discontinuation because TKIs do not effectively eliminate leukemic stem cells (LSC). The development of novel strategies aimed at eradicating LSC without harming normal hematopoietic stem cells (HSC) is essential for the cure of CML patients. The generation of LSC-directed therapy relies on the identification of novel molecular pathways that selectively regulate LSC function independent of BCR-ABL. The Krüppel-like factor 4(KLF4) is a transcription factor that can either activate or repress gene transcription acting as an oncogene or a tumor suppressor depending on the cellular context. Analysis of a published dataset from chronic phase CML patients revealed elevated levels of KLF4 in LSC compared to progenitor cells indicating that KLF4 is likely implicated in LSC regulation. To study the role of KLF4 in LSC function, we used a CML mouse model combining somatic deletion of the Klf4 gene and retroviral transduction and transplantation of HSC. In contrast to mice receiving BCR-ABL-transduced Klf4fl/fl HSC that developed and succumbed to CML, mice transplanted with BCR-ABL-transduced Klf4Δ/Δ (Klf4fl/fl Vav-iCre+) HSC showed a progressive loss of leukemia despite an initial expansion of myeloid leukemic cells, which led to increased overall survival. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSC in bone marrow and the spleen. Furthermore, deletion of KLF4 impaired the ability of LSC to recapitulate leukemia in secondary recipients suggesting a loss of self-renewal capacity. In contrast to LSC, KLF4 deletion led to increased self-renewal of normal HSC assessed by serial competitive transplantation. To identify KLF4 target genes involved in LSC self-renewal, we performed a global gene expression analysis using Klf4Δ/Δ LSC purified by cell sorting from leukemic mice. Analysis of gene expression in Klf4Δ/Δ LSC revealed significant upregulation of GβL, a component of mTOR complexes. Finally, we identified that KLF4 binds to GβL promoter by Chip-Seq analysis and that silencing resulted in inhibition of mTORC2 but not mTORC1 activity in 32D-BCR-ABL-positive CML cells. Our findings suggest that KLF4 transcriptionally represses GβL expression in LSC and that mTORC2 inhibition has the potential to completely eradicate LSC and induce treatment-free remission. Disclosures No relevant conflicts of interest to declare.

High cytoplasmic expression of Krüppel-like factor 4 is an independent prognostic factor of better survival in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality in the world. Hepatocarcinogenesis is complex, with an extraordinary molecular heterogeneity. Krüppel-like factor 4 (KLF4) plays an important role in cell proliferation and differentiation, and it can function as a tumor suppressor or an oncoprotein, depending on tissue type. The role of KLF4 in HCC remains controversial. The aim of this study was to explore the clinical significance of KLF4 expression in HCC. The study included 205 patients with surgical resection. We performed immunostaining for KLF4 and Ki-67 to investigate the correlations of the clinicopathological parameters of HCC and to examine the proliferative index. KLF4 staining was observed in the cytoplasm of non-tumorous hepatocytes and tumor cells. We subdivided the immunohistological staining results for KLF4 into low expression (Staining 0 and 1+) and high expression (Staining 2+ and 3+) subgroups. The expression of KLF4 was significantly correlated with tumor differentiation (p = 0.001). The Ki-67 proliferative index was significantly lower in well-differentiated HCCs (0.781% ± 1.02% vs. 2.16% ± 3.14%, p = 0.012), but not significantly different between low-KLF4 expression and high-KLF4 expression (1.87% ± 2.93% vs. 2.51% ± 3.28%, p = 0.32). Kaplan–Meier analysis showed that a high expression of KLF4 was significantly correlated with a longer disease-specific survival (p = 0.019). Univariate and multivariate analyses showed that high KLF4 expression was an independent predictor of a better disease-specific survival (p = 0.017; hazard ratio = 0.398; 95% confidence interval: 0.19–0.85). High cytoplasmic expression of KLF4 was associated with better disease-specific survival and was an independently favorable prognostic factor in hepatocellular carcinoma. These promising results suggest that KLF4 may play an anti-oncogenic role in hepatocarcinogenesis.

The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis

BackgroundKrüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied.ResultsEndothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos.ConclusionsThis study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.

Epigenetic alterations of Krüppel-like factor 4 and its tumor suppressor function in renal cell carcinoma

Krüppel-like factor 4 (KLF4) is a transcription factor that can have divergent functions in different malignancies. The expression and role of KLF4 in renal cell cancer remain unclear. The purpose of this study is to determine epigenetic alterations and possible roles of KLF4 in renal cell carcinoma. The KLF4 expression in primary renal cell cancer tissues and case-matched normal renal tissues was determined by protein and messenger RNA analyses. The epigenetic alterations were detected by methylation-specific PCR and Sequenom MassARRAY. Kaplan-Meier curves and the log-rank test were used for the survival analysis. The effects of KLF4 on cell growth and epithelial-to-mesenchymal transition (EMT) were determined in renal cancer cell lines after viral-based and RNA activation-mediated overexpression of KLF4. In vivo antitumor activity of KLF4 was evaluated by using stably KLF4-transfected renal cancer cells. KLF4 expression was dramatically decreased in various pathological types of renal cancer and associated with poor survival after nephrectomy. Hypermethylation of KLF4 promoter mainly contributed to its expression suppression. In vitro assays indicated that KLF4 overexpression inhibited renal cancer cell growth and survival. KLF4 overexpression also suppressed renal cancer cell migration and invasion by altering the EMT-related factors. In vivo assay showed that ectopic expression of KLF4 also inhibited tumorigenicity and metastasis of renal cancer. Our results suggest that KLF4 is a putative tumor suppressor gene epigenetically silenced in renal cell cancers by promoter CpG methylation and that it has prognostic value for renal cell progression.