Function Of KLF4 Research Articles

KLF4 Induces Colorectal Cancer by Promoting EMT via STAT3 Activation.

Krüppel-like factor 4 (KLF4) has been demonstrated to exert a pro-carcinogenic effect in solid tissues. However, the precise biological function and underlying mechanisms in colorectal cancer (CRC) remains elucidated. To investigate whether KLF4 participates in the proliferation and invasion of CRC. The expression of KLF4 was investigated using immunohistochemistry and immunoblotting. The clinical significance of KLF4 was evaluated. Furthermore, the effect of inhibiting or overexpressing KLF4 on tumor was examined. Immunoblotting and qPCR were used to detect Epithelial-mesenchymal transition-related proteins levels. Additionally, the molecular function of KLF4 is related to the STAT3 signaling pathway and was determined through JASPAR, GSEA analysis, and in vitro experiments. KLF4 exhibits down-regulated expression in CRC and is part of the vessel invasion, TNM stage, and worse prognosis. In vitro studies have shown that KLF4 promotes cellular proliferation and invasion, as well as EMT processes. Xenograft tumor models confirmed the oncogenic role of KLF4 in nude mice. Furthermore, GSEA and JASPAR databases analysis reveal that the binding of KLF4 to the signal transducer and activator of transcription 3 (STAT3) promoter site induces activation of p-STAT3 signaling. Subsequent targeting of STAT3 confirmed its pivotal role in mediating the oncogenic effects exerted by KLF4. The study suggests that KLF4 activates STAT3 signaling, inducing epithelial-mesenchymal transition, thereby promoting CRC progression.

A Study of Dual Function and Expression Pattern of Klf4 between Two Different Processes of Differentiation and Maintenance of Pluripotency in Spermatogonial Stem Cells

Krüppel-like factor 4 (Klf4) is a transcription factor involved in proliferation, self-renewal, differentiation, apoptosis, and somatic cell reprogramming; thus, it has a critical role in accurate spermatogenesis. In this study, we examine the different protein localizations of Klf4 in the mouse testis and in two populations of spermatogonial stem cells (SSCs), including differentiated and undifferentiated cell types. We show that in contrast to its function during spermatogenesis in the adult testis, in vitro Klf4 is a factor in maintaining stemness in different SSC populations. Furthermore, we suggest that its functions could be age-dependent. Bioinformatics analysis in the Cytoscape platform was conducted to highlight pathways and networks of Klf4 interactions. Our results support the different functions of Klf4 dependent on the age and differentiation state of SSCs.

Selective Inhibition of mTORC1 Signaling Supports the Development and Maintenance of Pluripotency.

Insight into the molecular mechanisms governing the development and maintenance of pluripotency is important for understanding early development and the use of stem cells in regenerative medicine. We demonstrate the selective inhibition of mTORC1 signaling is important for developing the inner cell mass (ICM) and the self-renewal of human embryonic stem cells. S6K suppressed the expression and function of pluripotency-related transcription factors (PTFs) OCT4, SOX2, and KLF4 through phosphorylation and ubiquitin proteasome-mediated protein degradation, indicating that S6K inhibition is required for pluripotency. PTFs inhibited mTOR signaling. The phosphorylation of S6 was decreased in PTF-positive cells of the ICM in embryos. Activation of mTORC1 signaling blocked ICM formation and the selective inhibition of S6K by rapamycin increased the ICM size in mouse blastocysts. Thus, selective inhibition of mTORC1 signaling supports the development and maintenance of pluripotency.

Divergent Roles of KLF4 During Primordial Germ Cell Fate Induction from Human Embryonic Stem Cells.

As a core transcriptional factor regulating pluripotency, Krüppel-like factor 4 (KLF4) has gained much attention in the field of stem cells during the past decades. However, few research have focused on the function of KLF4 during human primordial germ cell (PGC) specification. Here, we induced human PGC-like cells (hPGCLCs) from human embryonic stem cells (hESCs) and the derived hPGCLCs upregulated PGC-related genes, like SOX17, BLIMP1, TFAP2C, NANOS3, and the naïve pluripotency gene KLF4. The KLF4-knockout hESCs formed typical multicellular colonies with clear borders, expressed pluripotency genes, such as NANOG, OCT4, and SOX2, and exhibited no differences in proliferation capacity compared with wild type hESCs. Notably, KLF4 deletion in hESCs did not influence the induction of PGCLCs in vitro. In contrast, overexpression of KLF4 during PGC induction process inhibited the efficiency of PGCLC formation from hESCs in vitro. Overexpression of KLF4 may regenerate the naïve ground state in hESCs and results in repression for PGC specification. Thus, KLF4 could be a downstream target of human PGC program and the upregulation of KLF4 is prepared for late stage of germline development.

Krüppel-like Factors 4 and 5 in Colorectal Tumorigenesis.

Krüppel-like factors (KLFs) are transcription factors regulating various biological processes such as proliferation, differentiation, migration, invasion, and homeostasis. Importantly, they participate in disease development and progression. KLFs are expressed in multiple tissues, and their role is tissue- and context-dependent. KLF4 and KLF5 are two fascinating members of this family that regulate crucial stages of cellular identity from embryogenesis through differentiation and, finally, during tumorigenesis. They maintain homeostasis of various tissues and regulate inflammation, response to injury, regeneration, and development and progression of multiple cancers such as colorectal, breast, ovarian, pancreatic, lung, and prostate, to name a few. Recent studies broaden our understanding of their function and demonstrate their opposing roles in regulating gene expression, cellular function, and tumorigenesis. This review will focus on the roles KLF4 and KLF5 play in colorectal cancer. Understanding the context-dependent functions of KLF4 and KLF5 and the mechanisms through which they exert their effects will be extremely helpful in developing targeted cancer therapy.

Decreased Krüppel-like factor 4 in adenomyosis impairs decidualization by repressing autophagy in human endometrial stromal cells

BackgroundPoor decidualization and abnormal autophagy conditions in the endometria of adenomyosis patients have been reported previously. However, the specific regulatory mechanism of decidualization in adenomyosis and its relationship with autophagy levels have not been clarified.MethodsEndometrial tissues from adenomyosis patients and uteri from an adenomyosis mouse model were collected for the detection of different expression patterns of KLF4 and autophagy markers (LC3-B/LC3-A and Beclin-1) compared with control groups. Human endometrial stromal cells (hESCs) isolated from adenomyosis and control endometrial tissues were employed to elucidate the biological functions of KLF4 in autophagy and decidualization. Gene expression regulation was examined by quantitative real-time PCR (qRT-PCR), western blotting and luciferase reporter assays. In addition, DNA promoter-protein interactions were examined by chromatin immunoprecipitation (ChIP)/PCR assay and avidin–biotin conjugate DNA precipitation (ABCD) assay.ResultsKLF4 expression was decreased in endometrial tissues from adenomyosis patients compared with those from fertile controls, especially in stromal compartments. The opposite results were observed for autophagy marker (LC3-B/LC3-A and Beclin-1) expression. At the same time, KLF4 reversed the poor decidualization of hESCs from adenomyosis patients. In addition, KLF4 could induce hESC decidualization by promoting the autophagy level. Mechanistically, KLF4 bound to a conserved site in the autophagy-related 5 (ATG5) promoter region and promoted ATG5 expression. Similar expression patterns of KLF4 and autophagy markers were detected in adenomyotic mice.ConclusionsKLF4 overexpression increases the autophagy level of hESCs by transcriptionally promoting ATG5 expression, and abnormally decreased KLF4 in adenomyosis impairs hESC decidualization by repressing autophagy.

KLF4 suppresses the proliferation and metastasis of NSCLC cells via inhibition of MSI2 and regulation of the JAK/STAT3 signaling pathway

BackgroundNon-small cell lung cancer (NSCLC) remains an aggresive tumor with poor survival rates. Krüppel-like factor 4 (KLF4) is known to be involved in progression of NSCLC; however, the detailed mechanism by which KLF4 regulates the progression of NSCLC remains unclear. MethodsIn order to investigate the function of KLF4 in NSCLC, cell proliferation was measured by MTT and colony formation assays. The migration and invasion of NSCLC cells were detected via wound healing and Transwell assays, respectively. Then, the interaction between KLF4 and MSI2 was confirmed using a dual-luciferase reporter assay, and the mechanism by which KLF4 regulates the tumorigenesis of NSCLC was assessed by RT-qPCR and Western blotting. ResultsThe results showed that KLF4 was downregulated, while MSI2 was upregulated in NSCLC. Additionally, KLF4 could inhibit transcription of MSI2, and overexpression of KLF4 or knockdown of MSI2 could inhibit the proliferation, migration and invasion of NSCLC cells. Moreover, KLF4 could inhibit JAK2/STAT3 signalling pathway. ConclusionsIn conclusion, KLF4 significantly inhibited the proliferation, invasion and migration of NSCLC cells via inactivation of MSI2/JAK2/STAT3 signalling pathway. Thereby, our finding might shed new lights on exploring the new strategies against NSCLC.

The Partial Role of KLF4 and KLF5 in Gastrointestinal Tumors.

Background KLF4 and KLF5 are members of the KLF transcription factor family, which play an important role in many gastrointestinal tumors. To gain a deeper insight into its function and role, bioinformatics was used to analyze the function and role of KLF4 and KLF5 in gastrointestinal tumors. Methods Data were collected from several online databases. Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN database analysis, Kaplan-Meier Plotter analysis, LOGpc system, the Pathology Atlas, and the STRING website were used to analyze the data. We download relevant data from TCGA and then perform GO enrichment and KEGG enrichment analysis. The effects of KLF5 on gastric cancer cell proliferation were measured by CCK-8 assay. The effect of KLF5 on the expression of CyclinD1 and MMP9 was detected by Western blot. Results KLF4 and KLF5 were differentially expressed in normal and tumor tissues of the gastrointestinal tract, and their differential expression is related to several genes or pathways. KEGG analysis showed that KLF5 was coexpressed with endocytosis-related genes. KLF5 promotes the proliferation of gastric cancer cells and the expression of metastasis-related molecules. Conclusion KLF4 and KLF5 are of great significance for developing gastrointestinal tumors and can be used as therapeutic targets.

Mechanism of KLF4 in the development and progression of hepatocellular carcinoma

The pathogenesis of hepatocellular carcinoma is complex and has not been fully clarified. KLF4, also known as gut-enriched Kruppel-like factor (GKLF), plays the dual regulatory role of tumor suppressor and tumor promoter. KLF4 plays an anti-cancer role in liver cancer. This article introduces the biological function and regulatory mechanism of KLF4 in the progression of liver cancer, and research on the mechanism of action of KLF4 in liver cancer is expected to provide new ideas for targeted therapy and prognosis monitoring of liver cancer.

Transcriptome analysis of sevoflurane exposure effects at the different brain regions.

Sevoflurane is a most frequently used volatile anesthetics, but its molecular mechanisms of action remain unclear. We hypothesized that specific genes play regulatory roles in brain exposed to sevoflurane. Thus, we aimed to evaluate the effects of sevoflurane inhalation and identify potential regulatory genes by RNA-seq analysis. Eight-week old mice were exposed to sevoflurane. RNA from medial prefrontal cortex, striatum, hypothalamus, and hippocampus were analysed using RNA-seq. Differently expressed genes were extracted and their gene ontology terms were analysed using Metascape. These our anesthetized mouse data and the transcriptome array data of the cerebral cortex of sleeping mice were compared. Finally, the activities of transcription factors were evaluated using a weighted parametric gene set analysis (wPGSA). JASPAR was used to confirm the existence of binding motifs in the upstream sequences of the differently expressed genes. The gene ontology term enrichment analysis result suggests that sevoflurane inhalation upregulated angiogenesis and downregulated neural differentiation in each region of brain. The comparison with the brains of sleeping mice showed that the gene expression changes were specific to anesthetized mice. Focusing on individual genes, sevoflurane induced Klf4 upregulation in all sampled parts of brain. wPGSA supported the function of KLF4 as a transcription factor, and KLF4-binding motifs were present in many regulatory regions of the differentially expressed genes. Klf4 was upregulated by sevoflurane inhalation in the mouse brain. The roles of KLF4 might be key to elucidating the mechanisms of sevoflurane induced functional modification in the brain.

Sox2 and Klf4 as the Functional Core in Pluripotency Induction without Exogenous Oct4

Ectopic expression of Oct4, Sox2, Klf4, and c-Myc can reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs). For years, Oct4 has been considered the key reprogramming factor core of the four factors. Here, we challenge this view by reporting a core function of Sox2 and Klf4 in reprogramming. We found that polycistronic expression of Sox2 and Klf4 was sufficient to induce pluripotency in the absence of exogenous Oct4, and the stoichiometry of Sox2 and Klf4 was essential. Sox2 and Klf4 cooperatively bound across the genome, leading to epigenetic remodeling of their targets, including pluripotency genes and gradual activation of the pluripotency network. Interestingly, cells of different germ layer origins, fibroblasts (mesoderm) and neural progenitor cells (ectoderm), showed convergent reprogramming trajectories and similar efficiency. This work demonstrates a core function of Sox2 and Klf4 in pluripotency induction and shows that this mechanism is independent of germ layer origin.

Abstract 4651: Polo-like kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma

Abstract INTRODUCTION Advanced nasopharyngeal carcinoma (NPC) is an aggressive disease with no targeted therapies and poor outcomes. New innovative targets are urgently needed. Krüppel-like factor 4 (klf4, gklf) is a zinc-finger transcription factor involved in a wide variety of cellular processes, including apoptosis, cell cycle progression and stem cell renewal. Emerging data indicate that klf4 dysregulation either facilitates or impedes tumor progression. The function of klf4 in NPC remains elusive. METHODS we examined the expression of klf4 in human NPC (n=152) with a tissue immunohistochemistry (IHC) assay, and performed klf4 knockdown stable cell line with two different small hairpin RNAs (shRNAs) to evaluate the impact of klf4 on NPC. Global gene expression experiments were performed to explore the molecular mechanisms underlying klf4-dependent tumorigenesis. Small-molecule kinase inhibitor screening was performed to identify potential upstream kinases of klf4. Co-immunoprecipitation assay used to test the interaction between plk1 and klf4. Mass spectrometry analysis was performed to determine the specific site at which klf4 is phosphorylated by plk1. Statistical analyses were conducted using GraphPad Prism, SPSS software and JavaGSEA Desktop Application. A p value of <0.05 was considered to indicate statistical significance. RESULTS Our investigation showed that high expression of klf4 was correlated with poor prognosis in NPC. Moreover, genome-wide profiling revealed that klf4 directly activated oncogenic programmes, including gene sets associated with KRAS, VEGF, and MYC signalling. We further found that inhibition of polo-like kinase 1 could downregulate the expression of klf4 and that plk1 directly phosphorylated klf4 at Ser234. Notably, phosphorylation of klf4 by plk1 caused the recruitment and binding of the E3 ligase traf6, which resulted in klf4 K32 K63-linked ubiquitination and klf4 stabilization. Moreover, klf4 could enhance traf6 expression at the transcriptional level, thus initiating a klf4-traf6 feed-forward loop. Treatment with the plk1 inhibitor volasertib (BI6727) significantly inhibited tumor growth in nude mice. CONCLUSION In this study, we revealed that klf4 is upregulated in NPC and is correlated with poor prognoses. Our study demonstrated for the first time that plk1 can directly interact with klf4 and phosphorylate Ser234, alter the affinity of klf4 for the E3 ligase traf6, and promote the K63-linked ubiquitination of klf4 K32. In addition, klf4 can enhance traf6 expression at the transcriptional level and further promote klf4 expression. The resulting increase in klf4 ubiquitination leads to stabilization and upregulation of klf4, which leads to tumorigenesis in NPC. These results expand our understanding of the role of KLF4 in NPC and validate PLK1 inhibitors as potential therapeutic agents for NPC. Citation Format: Jia Mai, Zhuo-Yan Zhong, Xiu-Xing Chen, Yan-Qun Xiang, Xuan Li, Hai-Liang Zhang, Rong Deng, Xiao-Feng Zhu. Polo-like kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4651.

KLF4 as a rheostat of osteolysis and osteogenesis in prostate tumors in the bone.

We previously showed that KLF4, a gene highly expressed in murine prostate stem cells, blocks the progression of indolent intraepithelial prostatic lesions into aggressive and rapidly growing tumors. Here we show that the anti-tumorigenic effect of KLF4 extends to PC3 human prostate cancer cells growing in the bone. We compared KLF4 null cells to cells transduced with a DOX-inducible KLF4 expression system, and find KLF4 function inhibits PC3 growth in monolayer and soft agar cultures. Furthermore, KLF4 null cells proliferate rapidly, forming large, invasive and osteolytic tumors when injected into mouse femurs whereas KLF4 re-expression immediately after their intra-femoral inoculation blocks tumor development and preserves a normal bone architecture. KLF4 re-expression in established KLF4 null bone tumors inhibits their osteolytic effects, preventing bone fractures and inducing an osteogenic response with new bone formation. In addition to these profound biological changes, KLF4 also induces a transcriptional shift from an osteolytic program in KLF4 null cells to an osteogenic program. Importantly, bioinformatic analysis shows that genes regulated by KLF4 overlap significantly with those expressed in metastatic prostate cancer patients and in three individual cohorts with bone metastases, strengthening the clinical relevance of the findings in our xenograft model.

Concise Review: Regulation of Self-Renewal in Normal and Malignant Hematopoietic Stem Cells by Krüppel-Like Factor 4.

Pluripotent and tissue‐specific stem cells, such as blood‐forming stem cells, are maintained through a balance of quiescence, self‐renewal, and differentiation. Self‐renewal is a specialized cell division that generates daughter cells with the same features as the parental stem cell. Although many factors are involved in the regulation of self‐renewal, perhaps the most well‐known factors are members of the Krüppel‐like factor (KLF) family, especially KLF4, because of the landmark discovery that this protein is required to reprogram somatic cells into induced pluripotent stem cells. Because KLF4 regulates gene expression through transcriptional activation or repression via either DNA binding or protein‐to‐protein interactions, the outcome of KLF4‐mediated regulation largely depends on the cellular context, cell cycle regulation, chromatin structure, and the presence of oncogenic drivers. This study first summarizes the current understanding of the regulation of self‐renewal by KLF proteins in embryonic stem cells through a KLF circuitry and then delves into the potential function of KLF4 in normal hematopoietic stem cells and its emerging role in leukemia‐initiating cells from pediatric patients with T‐cell acute lymphoblastic leukemia via repression of the mitogen‐activated protein kinase 7 pathway. stem cells translational medicine 2019;8:568–574

Abstract A46: KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway in breast cancer

Abstract Krüppel-like factor 4 (KLF4) has critical roles in breast cancer development and progression and several solid tumors. Tamoxifen (TAM) resistance represents a daunting challenge to the successful treatment of breast cancer. KLF4 expression, function, and regulation in the efficacy of TAM therapy in breast cancer have yet to be demonstrated. Here, we investigated the clinical significance and biologic effects of KLF4 in breast cancer. Firstly, higher expression of KLF4 was correlated with increased TAM sensitivity in breast cells, and analysis of GEO datasets indicated that KLF4 expression was positively correlated with ERa and enhanced expression of KLF4 sensitized breast cancer patients to endocrine therapy. Knockdown of KLF4 in MCF-7 and BCAP37 cells led to increased TAM resistance, while ectopic KLF4 expression promoted the responsiveness of MCF-7/TAM and T47D cells to TAM. Secondly, ectopic KLF4 overexpression suppressed MCF-7/TAM cell growth, invasion, and migration ability. Besides, KLF4 expression was downregulated in breast cancer tumor tissues and high expression of KLF4 linked to favorable outcome. Mechanistically, KLF4 may enhance the responsiveness of breast cancer cells to TAM through suppressing mitogen-activated protein kinase (MAPK) signaling pathway. We found that ERK and P38 were relatively activated in MCF-7/TAM compared with MCF-7, and treatment with MAPK-specific inhibitors can significantly suppress cell viability. Knockdown of KLF4 activated ERK and P38 and drove MCF-7 cells to become resistant to TAM. Conversely, overexpression of KLF4 in MCF-7/TAM cells suppressed ERK and P38 signaling and resulted in enhanced sensitivity to TAM. Therefore, our findings suggest that KLF4 contributes to TAM efficiency in breast cancer via phosphorylation modification of ERK and P38 signaling. Collectively, this study highlighted the significance of KLF4/MAPK signal interaction in regulating TAM resistance of breast cancer, and suggested that targeting the KLF4/MAPK signaling may be a potential therapeutic strategy for breast cancer treatment, especially for TAM-resistant patients. Note: This abstract was not presented at the conference. Citation Format: Yunlu Jia, Wang Linbo. KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A46.

Peripheral Chemoreceptor Ablation Modulates Renal MiR‐155/KLF4 Expression in Chronic Heart Failure

Background and SignificanceRecent evidence suggests aberrant carotid body chemoreceptor (CBC) function contributes to tonic elevation of renal nerve activity and reductions in renal blood flow in chronic heart failure (CHF). These changes combined with anemia and capillary rarefaction likely contribute to tissue hypoxia, and may drive renal fibrosis and attendant dysfunction. Previous work indicates that stable expression of hypoxia‐inducible factor 1‐α (HIF1α) and down regulation of MiR‐155 drive gene programs that mediate epithelial‐mesenchymal transition (EMT) in renal tubular epithelial cells, including increases in Krüppel‐like factor 4 (KLF4) and matrix metalloproteinase 9 (MMP9). To date, no studies have examined this signaling pathway in CHF, thus our objective was to determine the influence of CBC on renal cortical expression of HIF1α, KLF4, MiR‐155, MMP9, and α‐smooth muscle actin (SMA) in CHF.HypothesisWe hypothesize that CBC‐mediated reductions in renal blood flow activate EMT pathways in CHF by exacerbating tissue hypoxia, leading to sustained accumulation of HIF1α, downregulation of MiR‐155, and KLF4/MMP9/SMA induction.MethodsTo address this hypothesis, we measured expression of HIF1α, MiR‐155, KLF4, MMP9, and SMA in renal cortical tissue from sham, and CHF animals (n=4–8 per group) with and without ablation of the CBC (CBA). CHF was induced in rats by coronary artery ligation (CAL) and CBA was performed (4 weeks post‐CAL) by cryogenic ablation. CHF was confirmed via echocardiography under isoflurane anesthesia (1–1.5%). At 8‐weeks post‐CAL, rats were humanely euthanized and renal cortical tissue was collected and analyzed for HIF1α, KLF4, MMP9, and SMA expression via western blot and MiR‐155 expression by RT‐qPCR.ResultsEjection fraction was 72±2% in sham animals, 34±2% in CHF animals, and 32±2% in CHF‐CBA animals (p<0.05 sham vs. CHF and CHF‐CBA). Expression of HIF1α was 1.37±0.33 in CHF rats relative to sham, and 1.25±0.15 in CHF‐CBA relative to sham. KLF4 was 1.64±0.25 in CHF rats relative to sham, and this effect was attenuated in CHF‐CBA (1.06±0.16 relative to sham, p<0.05 vs. CHF). MMP9 expression in CHF rats was 1.79±0.43 relative to sham and 1.092±0.23 in CHF‐CBA relative to sham. SMA expression in CHF rats was 0.58±0.17 relative to sham and 0.83±0.15 relative to sham in CHF‐CBA. MiR‐155 expression in CHF rats was 0.48±0.11 relative to sham, and this effect was attenuated by CBA (0.73±0.12 relative to sham, p<0.05 vs. CHF).ConclusionThese results suggest a novel relationship between CBC function and expression of renal cortical MiR‐155 and KLF4 in CHF, and that CBA has the potential to influence EMT pathways in the kidney in this pathophysiological setting.Support or Funding InformationThis work was supported by a grant from NHLBI (1 R15 HL138600‐01)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of KLF4 expression by small activating RNA promotes migration and invasion in colorectal epithelial cells.

RNA activation mediated by small double-stranded RNAs targeting promoter sequence named small activating RNAs (saRNAs) is one of the mechanisms for gene activation. Artificial regulation of gene expression through RNA activation does not affect the alteration of the genomic DNA sequences or exogenous plasmid DNA, therefore it is a relative manageable approach for gene perturbation. KLF4 is a member of zinc-finger transcription factors and its functions in colorectal cells are still controversial. In order to elucidate the functions of KLF4, we synthesized saRNAs that target the promoter regions of KLF4 and transfected into varied colorectal epithelial cell lines. We found the KLF4 gene expression is specifically increased in the human normal epithelial cell NCM460 and colorectal epithelial cancer cell Caco-2 and HCT116, but not in other human colorectal epithelial cell lines. In addition, we observed that saRNAs induced overexpression of KLF4 could promote cell migration/invasion in NCM460 and HCT116 cell lines. This effect is mediated partly by inducing EMT and facilitating nuclear translocation of β-catenin.

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.

Effects of Klf4 and c-Myc Knockdown on Pluripotency Maintenance in Porcine Induced Pluripotent Stem Cell.

Objective The importance of Oct4 and Sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions of Klf4 and c-Myc has not been fully investigated. In the present study, we attempted to determine the roles of Klf4 and c-Myc on pluripotency maintenance of porcine induced pluripotent stem (piPS) cells. Materials and Methods In this experimental study, we performed short hairpin RNA (shRNA) to knock down the Klf4 and c-Myc functions of piPS cells and examined pluripotency markers and teratoma formation to evaluate piPS cell pluripotency. The shRNA-Klf4 and shRNA-c-Myc vectors containing a reporter gene, TagFP635, were transfected into piPS cells by lentivirus infection. The piPS cells fully expressing infrared fluorescence were selected to confirm gene knockdown of Klf4 and c-Myc reverse transcription-polymerase chain reaction (RT-PCR). Next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by piPS cell transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. Results Our findings indicated that Klf4 and c-Myc functions of piPS cells were knocked down by shRNA transfection, and knockdown of Klf4 and c-Myc functions impaired expression of pluripotency markers such as Oct4, AP, SSEA-3, SSEA-4, TRA-1-6, and TRA-1-81. Furthermore, piPS cells without Klf4 and c-Myc expression failed to form teratomas. ConclusionThe pluripotency of piPS cells are crucially dependent upon Klf4 and c-Myc expression. These findings, suggesting potential mechanisms of Klf4 and c-Myc contribution to piPS cell formation, have important implications for application, regulation, and tumorigenesis of piPS cells.

Klf4 reduces stemness phenotype, triggers mesenchymal-epithelial transition (MET)-like molecular changes, and prevents tumor progression in nasopharygeal carcinoma.

The reprogramming factor Krüppel-like factor 4 (Klf4), one of the Yamanaka's reprogramming factors, plays an essential role in reprogramming somatic cells into induced pluripotent stem cells (iPSCs). Klf4 is dysregulated and displays divergent functions in multiple malignancies, but the biological roles of Klf4 in nasopharyngeal carcinoma (NPC) remain unknown. The present study revealed that Klf4 downregulation in a cohort of human NPC biopsies is significantly associated with invasive and metastatic phenotypes of NPC. Our results showed exogenous expression of Klf4 significantly inhibited cell proliferation, decreased stemness, triggered mesenchymal-epithelial transition (MET)-like molecular changes, and suppressed migration and invasion of NPC cells, whereas depletion of endogeneous Klf4 by RNAi reversed the aforementioned biological behaviors and characheristics. Klf4 silencing significantly enhanced the metastatic ability of NPC cells in vivo. In addition, CHIP assay confirmed that E-cadherin is a transcriptional target of Klf4 in NPC cells. Additional studies demonstrated that Klf4-induced MET-like cellular marker alterations, and reduced motility and invasion of NPC cells were mediated by E-cadherin. This study revealed the clinical correlation between Klf4 expression and epithelial-mesenchymal transition (EMT) biomarkers (including its target gene E-cadherin) in a cohort of NPC biopsies. Taken together, our findings suggest, for what we believe is the first time, that Klf4 functions as a tumor suppressor in NPC to decrease stemness phenotype, inhibit EMT and prevent tumor progression, suggesting that restoring Klf4 function may provide therapeutic benefits in NPC.