9314

KLF4

The Kruppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. Although KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition. Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing, while chromatin immunoprecipitation identified endogenous KLF4 binding to the GC-rich/E-box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent epithelial-to-mesenchymal transition in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.

The molecular basis for the resistance of tumor cells to cell-mediated cytotoxicity remains poorly understood and thus poses a major challenge for cancer immunotherapy. The present study was designed to determine whether the WNT1-inducible signaling pathway protein 2 (WISP2, also referred to as CCN5), a key regulator of tumor cell plasticity, interferes with tumor susceptibility to cytotoxic T-lymphocyte (CTL)-mediated lysis. We found that silencing WISP2 signaling in human breast adenocarcinoma MCF7 cells impairs CTL-mediated cell killing by a mechanism involving stem cell marker Kruppel-like factor-4 (KLF-4) induction and microRNA-7 (miR-7) downregulation. Inhibition of transforming growth factor beta (TGF-beta) signaling using the A83-01 inhibitor in MCF7-shWISP2 cells resulted in a significant reversal of the epithelial-to-mesenchymal-transitioned (EMT) phenotype, the expression of KLF-4 and a partial recovery of target susceptibility to CTLs. More importantly, we showed that silencing KLF-4 was accompanied by a reduction in MCF7-shWISP2 resistance to CTLs. Using human breast cancer tissues, we demonstrated the coexpression of KLF-4 with EMT markers and TGF-beta pathway signaling components. More importantly, we found that KLF-4 expression was accompanied by miR-7 inhibition, which is partly responsible for impairing CTL-mediated lysis. Thus, our data indicate that WISP2 has a role in regulating tumor cell susceptibility through EMT by inducing the TGF-beta signaling pathway, KLF-4 expression and miR-7 inhibition. These studies indicate for the first time that WISP2 acts as an activator of CTL-induced killing and suggests that the loss of its function promotes evasion of immunosurveillance and the ensuing progression of the tumor.

Ovarian cancer presents therapeutic challenges due to its typically late detection, aggressive metastasis, and therapeutic resistance. The transcription factor Kruppel-like factor 4 (KLF4) has been implicated in human cancers as a tumor suppressor or oncogene, although its role depends greatly on the cellular context. The role of KLF4 in ovarian cancer has not been elucidated in mechanistic detail. In this study, we investigated the role of KLF4 in ovarian cancer cells by transducing the ovarian cancer cell lines SKOV3 and OVCAR3 with a doxycycline-inducible KLF4 lentiviral vector. Overexpression of KLF4 reduced cell proliferation, migration, and invasion. The epithelial cell marker gene E-cadherin was significantly upregulated, whereas the mesenchymal cell marker genes vimentin, twist1 and snail2 (slug) were downregulated in both KLF4-expressing SKOV3 and OVCAR3 cells. KLF4 inhibited the transforming growth factor beta (TGFbeta)-induced epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Taken together, our data demonstrate that KLF4 functions as a tumor suppressor gene in ovarian cancer cells by inhibiting TGFbeta-induced EMT.

Kruppel-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)-beta1. However, treatment with 5-aza-2'-deoxycytidine attenuated the TGF-beta1-induced downregulation of KLF4 and E-cadherin and the upregulation of alpha-smooth muscle actin (alpha-SMA) in the HK-2 cells. DNA methyltransferase 1 (Dnmt1) participated in the TGF-beta1-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 alpha-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.

A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic beta-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Kruppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to beta-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome.

Hepatocellular carcinoma (HCC) is one of the most malignant and lethal human cancers. epithelial-mesenchymal transition (EMT) enhances the carcinogenesis of HCC, and therapies targeting EMT appear to be promising treatments. We have previously shown that Kruppel-like factor 4 (KLF4) suppressed EMT of HCC cells through downregulating EMT-associated proteins. Here, we examined the roles of microRNAs (miRNAs) in KLF4-regulated EMT in HCC cells. KLF4 induced expression of 3 miRNAs (miR-153, miR-506 and miR-200b) that targeted 3'-UTR of Snail1, Slug and ZEB1 mRNAs, respectively, to inhibit protein translation in HCC cells, which was confirmed by promoter luciferase assay. expression of either miRNA significantly inhibited HCC cell growth and invasiveness, while the effect of combined expression of all 3 miRNAs was more pronounced. Furthermore, overexpression of antisense of all 3 miRNAs abolished the inhibitory effect of KLF4 on HCC cell growth and invasiveness. Together, our data suggest that KLF4 inhibits EMT-enhanced HCC growth and invasion, possibly through reducing EMT-related proteins Snail1, Slug and ZEB1 via increasing miR-153, miR-506 and miR-200b.

In order to improve therapeutic efficacy, it is a current emergency to better know the mechanisms underlying cisplatin resistance in lung cancer cells. In this study, we aim to investigate the role of Kruppel-like factor 4 (KLF4) in cisplatin-resistant lung cancer cells. We developed cisplatin-resistant lung cancer cell line A549/DDP, and then a battery of experiments was used to analyze the effects of KLF4 in cisplatin resistance of lung cancer. We found that KLF4 was significantly downregulated in cisplatin-resistant A549 cells and forced KLF4 expression inhibited cell growth and induced apoptosis. Further, we found that overexpression of KLF4 was able to inhibit cell migration and invasion, to inhibit the expression of Slug, Twist, and vimentin, and to increase the expression of E-cadherin and subsequent inhibition of the EMT process. Thus, overexpression of KLF4 may be a potential strategy for lung cancer treatment, especially for cisplatin-resistant cases.

BACKGROUND: Kruppel-like factor 4 (KLF4) exerts tumor suppressive or oncogenic functions in a cell-type-dependent manner, but its prognostic role in urothelial carcinoma of bladder (UCB) remains unclear. We aimed to determine how KLF4 regulates epithelial-mesenchymal transition (EMT) and predicts patient survival in UCB. PATIENTS AND METHODS: The roles of KLF4 and other EMT regulators in cancer progression were studied in UCB specimens of 398 patients, UCB cell lines. The results were validated by open-access The cancer Genome Atlas dataset. RESULTS: Over a median follow-up of 46.5 months, tissue microarray demonstrated that strong KLF4 expression was associated with higher risk toward metastasis and death (P<0.001). KLF4 expression positively correlated with TWIST1 and vimentin, and inversely correlated with E-cadherin expression. Metastasis-free survival was poorest in KLF4/TWIST1 coexpression group, followed by KLF4 or TWIST1 expression-alone group, and no-expression group (P<0.001). Multivariate analysis substantiated that KLF4/TWIST1 coexpression independently predicted overall mortality and metastasis risk with hazard ratios of 2.43 (95% CI: 1.65-3.64) and 7.54 (CI: 4.03-12.10). The cancer Genome Atlas dataset of bladder cancer also revealed a trend toward decreased overall survival in the high KLF4 expression group as compared to the low KLF4 group. In vitro, KLF4 is accompanied with decreased E-cadherin and beta-catenin expressions, increased vimentin and fibronectin expressions, and enhanced migration/invasion. KLF4 knockdown suppressed TWIST1 expression and inhibited EMT, migration and invasion, whereas enforced KLF4 overexpression activated TWIST1 expression and restored EMT and metastatic phenotype. Furthermore, TWIST1 knockdown abolished KLF4-faciliated EMT and metastatic feature without affecting KLF4 expression. CONCLUSIONS: KLF4 promotes TWIST1-mediated EMT and may represent a novel prognostic predictor in UCB.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

miR-21 induces epithelial-mesenchymal transition (EMT) of human cholangiocarcinoma (CCA) cells. However, the mechanism by which this occurs remains unclear. In the present study, high throughput platform was employed to detect the genes that are differential expressed in QBC939 cells transfected with a hsa-miR-21 antagomir or control vectors. The EMT-related Kruppel-like factor 4 (KLF4) gene was downregulated after miR-21 was knocked down. Overexpression of miR-21 upregulated KLF4, Akt, ERK and mesenchymal cell markers (N-cadherin and vimentin), downregulated the expression of epithelial cell marker E-cadherin and reduced cell migration and invasion. Immunohistochemistry showed that KLF4, pAkt and pERK were upregulated in tumor xenografts transfected with miR-21 mimics. Inhibitors of the PI3K-Akt and ERK1/2 pathways, LY294002 and U0126, significantly suppressed the EMT phenotype. The present data demonstrated that overexpression of miR-21, accompanied with KLF4, augmented the EMT via inactivation of Akt and ERK1/2 pathways. In conclusion, we have identified a novel mechanism that may be targeted in an attempt to relieve the malignant biological behavior of CCA cells.

BACKGROUND: epithelial-to-mesenchymal transition (EMT) contributes to the invasion and metastasis of epithelial tumors. Sirtuin 6 (SIRT6), an NAD-dependent deacetylase, is known to promote metastasis of non-small cell lung cancer (NSCLC). METHODS: In this work, we determined the role of SIRT6 in the EMT of NSCLC cells and identified the key EMT-related genes involved in the oncogenic activity of SIRT6. RESULTS: We report that depletion of SIRT6 inhibits transforming growth factor-beta1 (TGF-beta1)-induced EMT in A549 and H1299 NSCLC cells, which is rescued by ectopic expression of SIRT6. Knockdown of SIRT6 leads to a reduction in Snail protein without affecting the mRNA level. Immunoprecipitation experiments demonstrate a physical association between SIRT6 and Snail. SIRT6 deacetylates Snail and prevents its proteasomal degradation. Silencing of Snail blunts SIRT6-induced NSCLC cell migration and invasion, while overexpression of Snail restores the invasion and EMT in SIRT6-depleted NSCLC cells. SIRT6 depletion leads to an upregulation of kruppel-like factor 4 (KLF4) and reduced Snail binding to the promoter of Klf4 in NSCLC cells. Knockdown of KLF4 rescues the invasive capacity in SIRT6-depleted NSCLC cells. Conversely, co-expression of KLF4 impairs SIRT6-induced aggressive behavior. In vivo data further demonstrate that SIRT6-induced NSCLC metastasis is antagonized by overexpression of KLF4. CONCLUSIONS: These findings provide mechanistic insights into the pro-metastatic activity of SIRT6 and highlight the role of the SIRT6/Snail/KLF4 axis in regulating EMT and invasion of NSCLC cells.

epithelial-mesenchymal transition (EMT) is implicated in various pathological processes within the prostate, including benign prostate hyperplasia (BPH) and prostate cancer progression. However, an ordered sequence of signaling events initiating carcinoma-associated EMT has not been established. In a model of transforming growth factor beta (TGFbeta)-induced prostatic EMT, SLUG is the dominant regulator of EMT initiation in vitro and in vivo, as demonstrated by the inhibition of EMT following Slug depletion. In contrast, SNAIL depletion was significantly less rate limiting. TGFbeta-stimulated KLF4 degradation is required for SLUG induction. expression of a degradation-resistant KLF4 mutant inhibited EMT, and furthermore, depletion of Klf4 was sufficient to initiate SLUG-dependent EMT. We show that KLF4 and another epithelial determinant, FOXA1, are direct transcriptional inhibitors of SLUG expression in mouse and human prostate cancer cells. Furthermore, self-reinforcing regulatory loops for SLUG-KLF4 and SLUG-FOXA1 lead to SLUG-dependent binding of polycomb repressive complexes to the Klf4 and Foxa1 promoters, silencing transcription and consolidating mesenchymal commitment. Analysis of tissue arrays demonstrated decreased KLF4 and increased SLUG expression in advanced-stage primary prostate cancer, substantiating the involvement of the EMT signaling events described in model systems.

Transforming growth factor-beta (TGF-beta) signaling and TGF-beta-promoted epithelial-to-mesenchymal transition (EMT) have been postulated to be the common pathway causing pulmonary fibrosis. However, the up- or downstreaming markers of TGF-beta-induced EMT still need to be further recognized. In the present study, we investigated the regulation on Kruppel-like factor 4 (KLF-4) and plasminogen activator inhibitor-1 (PAI-1) by TGF-beta in the murine lung epithelial LA-4 cells and then examined the regulation of both markers in the TGF-beta-induced EMT by the PAI-1 knockdown or the KLF-4 overexpression. Our study indicated that TGF-beta induced EMT in mouse LA-4 lung epithelial cells via reducing E-cadherin, while promoting Collagen I and alpha-SMA. And PAI-1 was upregulated, whereas KLF-4 was downregulated in the TGF-beta-induced EMT model in LA-4 cells. Moreover, the siRNA-mediated PAI-1 knockdown inhibited the TGF-beta-induced EMT, whereas the adenovirus-medicated KLF-4 overexpression markedly reduced the PAI-1 expression and inhibited the TGF-beta-induced EMT in LA-4 cells. In conclusion, our study confirmed the downregulation of KLF-4 in the TGF-beta-induced EMT in LA-4 cells. And the KLF-4 overexpression significantly reduced the TGF-beta-induced PAI-1 and thus inhibited the TGF-beta-induced EMT in mouse lung epithelial LA-4 cells. It implies that KLF-4 might be a promising target for effective control of the pulmonary fibrosis.