Long noncoding RNA LEF1-AS1 acts as a microRNA-10a-5p regulator to enhance MSI1 expression and promote chemoresistance in hepatocellular carcinoma cells through activating AKT signaling pathway.

Long noncoding RNAs (lncRNAs) contribute to the development of hepatocellular carcinoma (HCC), which could regulate various HCC biological characteristics. Here, the study seeks to investigate the role of lncRNA LEF1-AS1 in HCC cell chemoresistance by regulating microRNA (miR)-10a-5p and Musashi1 (MSI1). The microarray-based analysis was employed to identify the HCC-related lncRNA-miRNA-gene regulatory network. Expression patterns of LEF1-AS1, miR-10a-5p, and MSI1 in the HCC cell lines, tissues were accessed by means of reverse transcription-quantitative polymerase chain reaction. Next, the interaction among LEF1-AS1, miR-10a-5p, and MSI1 in HCC was accessed by bioinformatics and dual-luciferase reporter gene assay. Then, the cell line resistant to cisplatin was established, which was then treated with sh/oe-lncRNA LEF1-AS1, miR-10a-5p-mimic, and oe/sh-MSI1 vectors alone or in combination. Afterward, the effect of LEF1-AS1, miR-10a-5p, and MSI1 on HCC cell chemoresistance, proliferation, and apoptosis was assessed. At last, in vivo experiments confirmed the role of MSI1 in tumor growth and chemoresistance in HCC. LEF1-AS1 might potentially affect the growth and chemoresistance of HCC cells by regulating miR-10a-5p and MSI1. LEF1-AS1 and MSI1 expression patterns were elevated, while miR-10a-5p was repressed in HCC tissues and cell lines. LEF1-AS1 combined to miR-10a-5p and regulated MSI1, thereby activating the protein kinase B (AKT) signaling pathway. Knockdown of LEF1-AS1 and MSI1 or elevation of miR-10a-5p compromised the proliferation of Huh7 cell line resistant to DDP and promoted its chemosensitivity and apoptosis. At last, these in vitro findings were also confirmed in vivo. Our results unraveled LEF1-AS1 acts as a miR-10a-5p modulator to promote chemoresistance of HCC cells by stimulating MSI1 and activating the AKT signaling pathway, which might provide a novel therapeutic target for HCC.

Optimizations of a novel fluorescence polarization-based high-throughput screening assay for ß-catenin/LEF1 interaction inhibitors.

Aberrant activation of the Wnt/ß-catenin signaling pathway is prominent in the development and metastasis of non-small cell lung cancer (NSCLC). Highly effective inhibition of this pathway highlights a therapeutic avenue against NSCLC. Moreover, ß-catenin/LEF1 interaction regulates ß-catenin nuclear transport as well as the transcriptions of the key oncogenes in Wnt/ß-catenin signaling pathway. Therefore, interruption of this interaction would be a promising therapeutic strategy for NSCLC metastasis. To date, no economical and rapid high-throughput screening (HTS) assay has been reported for the discovery of ß-catenin/LEF1 interaction inhibitors. In this study, we developed a novel fluorescence polarization (FP)-based HTS assay to identify ß-catenin/LEF1 interaction inhibitors. The FITC-LEF1 sequence, incubation time, temperature, and DMSO resistance were optimized, and then a high Z' factor of 0.77 was achieved. A pilot screening of a natural product library via this established FP screening assay identified sanguinarine analogues as potential ß-catenin/LEF1 interaction inhibitors. GST pull-down and surface plasmon resonance (SPR) assay demonstrated that ß-catenin/LEF1 interaction is a potential anticancer target of sanguinarine in vitro. This newly developed FP screening assay will be vital for the rapid discovery of novel Wnt inhibitors targeting ß-catenin/LEF1 interaction.

Cinobufagin Suppresses Melanoma Cell Growth by Inhibiting LEF1.

Constitutive activation of the ß-catenin dependent canonical Wnt signaling pathway, which enhances tumor growth and progression in multiple types of cancer, is commonly observed in melanoma. LEF1 activates ß-catenin/TCF4 transcriptional activity, promoting tumor growth and progression. Although several reports have shown that LEF1 is highly expressed in melanoma, the functional role of LEF1 in melanoma growth is not fully understood. While A375, A2058, and G361 melanoma cells exhibit abnormally high LEF1 expression, lung cancer cells express lower LEF1 levels. A luciferase assay-based high throughput screening (HTS) with a natural compound library showed that cinobufagin suppressed ß-catenin/TCF4 transcriptional activity by inhibiting LEF1 expression. Cinobufagin decreases LEF1 expression in a dose-dependent manner and Wnt/ß-catenin target genes such as Axin-2, cyclin D1, and c-Myc in melanoma cell lines. Cinobufagin sensitively attenuates cell viability and induces apoptosis in LEF1 expressing melanoma cells compared to LEF1-low expressing lung cancer cells. In addition, ectopic LEF1 expression is sufficient to attenuate cinobufagin-induced apoptosis and cell growth retardation in melanoma cells. Thus, we suggest that cinobufagin is a potential anti-melanoma drug that suppresses tumor-promoting Wnt/ß-catenin signaling via LEF1 inhibition.

2,4-Diamino-Quinazoline, a Wnt Signaling Inhibitor, Suppresses Gastric Cancer Progression and Metastasis.

Gastric cancer (GC) is among the most treatment-refractory epithelial malignancies. Aberrant activation of Wnt/ß-catenin-signaling has been implicated in a variety of human cancers, including gastric cancer. Here we report that the elevated expression of lymphoid enhancer binding factor 1 (Lef1) is associated with the TNM (tumor- node-metastasis) stage of gastric cancer. Subsequently, 2,4-diamino-quinazoline (2,4-DAQ), a selective inhibitor of Lef1, was identified to suppress the expression of Wnt/ß-catenin target genes such as AXIN2, MYC and LGR5 and result in the suppression of gastric cancer cell growth through the apoptotic pathway. The 2,4-DAQ also exhibited an inhibitory effect on the migration/invasion of gastric cancer cells. Importantly, the treatment of human gastric tumor xenograft with 2,4-DAQ suppressed tumor growth in a nude mouse model. Furthermore, 2,4-DAQ appears effective on patient-derived organoids (PDOs). Transcriptome sequencing analysis also revealed that 2,4-DAQ are more effective on the gastric cancers that exhibit higher expression levels of Wnt-signaling pathway-related genes than their adjacent normal gastric tissues.

Hepatocyte nuclear factor 1ß suppresses canonical Wnt signaling through transcriptional repression of lymphoid enhancer-binding factor 1.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1ß produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1ß in mIMCD3 renal epithelial cells results in activation of ß-catenin and increased expression of lymphoid enhancer-binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1ß in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1ß binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1ß decreases H3K27 trimethylation repressive marks and increases ß-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1ß recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the ß-catenin-binding domain of LEF1 in HNF-1ß-deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1ß through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1ß regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1ß.

LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis.

Long noncoding RNAs (lncRNAs) play important roles in endothelium development. A lncRNA, LEF1-AS1, is recently emerging as a potent mediator of the proliferation and migration of a number of cells, including smooth muscle cells. However, the effects of LEF1-AS1 in atherosclerosis remains largely unknown. Specimens from patients with coronary artery atherosclerosis were collected. The quantitative real-time polymerase chain reaction was used to analyze levels of LEF1-AS1 and microRNA-544a (miR-544a). Western blot analysis was used to assess PTEN, P-Akt, and T-Akt protein expression. Proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 assay, scratch wound assay, and transwell assay, respectively. The interaction between LEF1-AS1, miR-544a, and PTEN was probed using bioinformatical analysis and dual-luciferase assay. In plasma and tissue of patients with coronary artery atherosclerosis, LEF1-AS1 was upregulated and miR-544a was downregulated. A negative correlation was found between LEF1-AS1 and miR-544a. miR-544a overexpression reversed the inhibition of LEF1-AS1 in smooth muscle cell proliferation and invasion, which were mediated through the PTEN pathway. LEF1-AS1 regulates smooth muscle cell proliferation and migration through the miR-544a/PTEN axis, indicating that LEF1-AS1 may be a potential therapeutic target in atherosclerosis.

LEF-1 drives aberrant ß-catenin nuclear localization in myeloid leukemia cells.

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß- catenin level in human myeloid leukemia.

Effects of microRNA-708 on Epithelial-Mesenchymal Transition, Cell Proliferation and Apoptosis in Melanoma Cells by Targeting LEF1 through the Wnt Signaling Pathway.

This study was conducted in order to elucidate the role microRNA-708 (miR-708) plays between proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) involving melanoma cells by targeting using LEF1 through the Wnt signaling pathway. Male Kunming mice were selected and subsequently divided into normal and model groups to take part in this study. Following cell line selection, the B16 cells with the highest miR-708 expression were selected and assigned into the control, blank, negative control (NC), miR-708 mimic, miR-708 inhibitor, siRNA-LEF1, and miR-708 inhibitor + siRNA-LEF1 groups. A Bioinformatics Web service and dual-luciferase reporter assay were conducted in order to determine the relationship between LEF1 and miR-708. The RT-qPCR method was performed in order to detect the miR-708 expression and mRNA expressions of LEF1, ß-catenin, Wnt3a, N-cadherin, Bcl-2, Bax, Caspase3, E-cadherin, and western blotting was used in order to detect the protein expressions of these genes. MTT assay, scratch test, Transwell assay, and flow cytometry were all conducted in order to detect the cell proliferation, migration, invasion, and cycle/apoptosis, respectively. LEF1 was verified as the target gene of miR-708. In comparison with the normal group, the model group had reduced expressions of miR-708, Bax, Caspase3, and E-cadherin, while showing elevated expressions of LEF1, ß-catenin, Bcl-2, Wnt3a, and N-cadherin. In comparison to the blank and control groups, the miR-708, mimic, and siRNA-LEF1 groups had elevated expressions of Bax, Caspase3, and E-cadherin, while also showing enhanced cell apoptosis. The miR-708, mimic, and siRNA-LEF1 groups also had decreased expressions of LEF1, ß-catenin, Bcl-2, Wnt3a, and N-cadherin, and reduced optical density value 48 h and 72 h after transfection. Besides, these two groups showed declined cell migration and invasion, as well as lengthened G0/G1 phase (increased cell number) and shortened S phase (decreased cell number). Our findings demonstrated that an overexpressed miR-708 inhibits the proliferation, invasion, migration, and EMT, but also promotes the apoptosis of melanoma cells by targeting LEF1 through the suppression of the Wnt signaling pathway.

Inhibition of LEF1-Mediated DCLK1 by Niclosamide Attenuates Colorectal Cancer Stemness.

PURPOSE: Niclosamide, an FDA-approved anthelmintic drug, has been characterized as a potent Wnt inhibitor that can suppress tumor growth and cancer stem-like cell (CSC) populations. However, the underlying molecular mechanisms remain poorly understood. This study aimed to examine how Wnt inhibition by niclosamide preferentially targets CSCs. EXPERIMENTAL DESIGN: The mechanistic role of niclosamide in CSC inhibition was examined in public databases, human colorectal cancer cells, colorectal cancer xenografts, and azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colorectal cancer model. RESULTS: Niclosamide suppresses CSC populations and their self-renewal activities in colorectal cancer cells, and this CSC-targeting effect leads to irreversible disruption of tumor-initiating potential in vivo. Mechanistically, niclosamide downregulates multiple signaling components of the Wnt pathway, specifically lymphoid enhancer-binding factor 1 (LEF1) expression, which is critical for regulating stemness. Subsequently, we identified that the doublecortin-like kinase 1 (DCLK1)-B is a target of LEF1 and upregulates cancer stemness in colorectal cancer cells. We first documented that niclosamide blocks the transcription of DCLK1-B by interrupting the binding of LEF1 to DCLK1-B promoter. DCLK1-B depletion impairs cancer stemness resulting in reduced survival potential and increased apoptosis, thus sensitizing colorectal cancer to chemoradiation. CONCLUSIONS: Disruption of the LEF1/DCLK1-B axis by niclosamide eradicates cancer stemness and elicits therapeutic effects on colorectal cancer initiation, progression, and resistance. These findings provide a preclinical rationale to broaden the clinical evaluation of niclosamide for the treatment of colorectal cancer.

LEF-1 Regulates Tyrosinase Gene Transcription In Vitro.

TYR, DCT and MITF are three important genes involved in maintaining the mature phenotype and producing melanin; they therefore participate in neural crest cell development into melanocytes. Previous studies have revealed that the Wnt signaling factor lymphoid enhancer-binding factor (LEF-1) can enhance DCT and MITF gene expression. However, whether LEF-1 also affects TYR gene expression remains unclear. In the present study, we found that LEF-1 regulated TYR transcription in vitro. LEF-1 overexpression increased TYR gene promoter activity, whereas LEF-1 knockdown by RNA interference significantly decreased TYR expression. Moreover, the core GTTTGAT sequence (-56 to -50) within the TYR promoter is essential for the effect of LEF-1 on TYR expression, and chromatin immunoprecipitation (ChIP) assay indicated that endogenous LEF-1 interacts with the TYR promoter. In addition, we observed a synergistic transactivation of the TYR promoter by LEF-1 and MITF. These data suggest that Wnt signaling plays an important role in regulating melanocyte development and differentiation.

LEF1 regulates glioblastoma cell proliferation, migration, invasion, and cancer stem-like cell self-renewal.

Glioblastoma multiforme (GBM; WHO grade IV) is one of the most common primary tumors of the central nervous system. This disease remains one of the incurable human malignancies because the molecular mechanism driving the GBM development and recurrence is still largely unknown. Here, we show that knockdown of lymphocyte enhancer factor-1 (LEF1), a major transcription factor of Wnt pathway, inhibits U251 cell migration, invasion, and proliferation. Furthermore, downregulation of LEF1 expression inhibits the self-renewal capacity of U251 GBM stem-like cells and decreases the expression level of the GBM stem-like cell (GSC) markers such as CD133 and nestin. Our findings reveal that LEF1 maintains the GBM cell proliferation, migration, and GBM stem-like cell self-renewal. Taken together, these results suggest that LEF1 may be a novel therapeutic target for GBM suppression.

Knockdown of lymphoid enhancer factor 1 inhibits colon cancer progression in vitro and in vivo.

Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.

ERG is a critical regulator of Wnt/LEF1 signaling in prostate cancer.

Chromosomal translocations juxtaposing the androgen-responsive TMPRSS2 promoter with the ETS-family transcription factor ERG result in aberrant ERG upregulation in approximately 50% of prostate cancers. Studies to date have shown important roles of ERG in inducing oncogenic properties of prostate cancer. Its molecular mechanisms of action, however, are yet to be fully understood. Here, we report that ERG activates Wnt/LEF1 signaling cascade through multiple mechanisms. ERG bound to the promoters of various Wnt genes to directly increase ligand expression. Consequently, ERG overexpression increased active ß-catenin level in the cells and enhanced TCF/LEF1 luciferase reporter activity, which could be partially blocked by WNT-3A inhibitor IWP-2. Most importantly, our data defined LEF1 as a direct target of ERG and that LEF1 inhibition fully abolished ERG-induced Wnt signaling and target gene expression. Furthermore, functional assays showed that Wnt/LEF1 activation phenocopied that of ERG in inducing cell growth, epithelial-to-mesenchymal transition, and cell invasion, whereas blockade of Wnt signaling attenuated these effects. Concordantly, LEF1 expression is significantly upregulated in ERG-high human prostate cancers. Overall, this study provides an important mechanism of activation of Wnt signaling in prostate cancer and nominates LEF1 as a critical mediator of ERG-induced tumorigenesis. Wnt/LEF1 pathway might provide novel targets for therapeutic management of patients with fusion-positive prostate cancer.

Dysregulation of TNFα-induced necroptotic signaling in chronic lymphocytic leukemia: suppression of CYLD gene by LEF1.

Impaired cell death program has been noted as one of the hallmarks of chronic lymphocytic leukemia (CLL) and contributes to its accumulation of malignant monoclonal B cells as well as to chemotherapy resistance. A cell can die through the apoptosis or necrosis pathway. Recent investigations suggest that in apoptotic-deficient conditions, such as most types of cancer, a process of programmed necrosis, called necroptosis, prevails. However, the detailed molecular mechanisms underlying this alternative cell death pathway are still not fully understood. Here we demonstrate that CLL cells failed to undergo necroptosis upon stimulation of TNFα combined with pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD). Two core components of necroptotic machine, RIP3 and deubiquitinase cylindromatosis (CYLD), are markedly downregulated in CLL. Moreover, we identified lymphoid enhancer-binding factor 1 (LEF1), a downstream effector of the Wnt/ß-catenin pathway, as a transcription repressor of CYLD in CLL. Knocking down LEF1 sensitizes CLL cells to TNFα/zVAD-induced necroptosis. The present investigation provides the first evidence that CLL cells have defects not only in apoptotic program but also in necroptotic signaling. Targeting the key regulators of necroptotic machine, such as LEF1, to restore this pathway may represent a novel approach for CLL treatment.

Wnt/ß-catenin/LEF-1 signaling in chronic lymphocytic leukemia (CLL): a target for current and potential therapeutic options.

There is a growing body of evidence that Wnt signaling, which is already known to play a critical role in various types of cancer, also has a vital function in B cell neoplasias, particularly in chronic lymphocytic leukemia (CLL). It is known that Wnt proteins are overexpressed in primary CLL cells and several physiological inhibitors are partly inactivated in this disease. Furthermore, ß-catenin is upregulated upon Wnt stimulation and cooperates with the transcription factor lymphoid enhancer binding factor-1 (LEF-1). LEF-1 is excessively overexpressed in CLL cells by more than 3,000-fold compared to normal B cells. Moreover, LEF-1 could be identified as an important regulator of pathophysiologically relevant genes in CLL, and several Wnt/ß-catenin signaling components substantially influence CLL cell survival. In this review we summarize the current state of knowledge about Wnt/ß-catenin/LEF-1 signaling in CLL. Following a short overview of current treatment concepts in CLL, we briefly describe Wnt signaling in human cancers. We then discuss recent progress in understanding regulation of the Wnt/ß-catenin/LEF-1 signaling pathway in this disease. Based on the present scientific evidence we highlight which components of this important signaling pathway could serve as therapeutic targets in CLL. We then present previous results gained from experimental approaches to target different parts of the Wnt/ß-catenin/LEF-1 cascade. Together with potentially promising approaches we also critically reflect on the kind of difficulties and problems that may arise using such strategies.

Small molecule inhibitors of Wnt/beta-catenin/lef-1 signaling induces apoptosis in chronic lymphocytic leukemia cells in vitro and in vivo.

BACKGROUND: Lymphoid enhancer factor-1 (lef-1) is overexpressed in B-cell chronic lymphocytic leukemia (CLL) when compared with normal B cells and transcribes several genes implicated in the pathogenesis of CLL. We therefore hypothesize that antagonism of lef-1 might lead to killing of CLL cells. We used two small molecule inhibitors of Wnt/beta-catenin/lef-1 signaling (CGP049090 and PKF115-584) to test our hypothesis. DESIGN AND METHODS: Enriched CLL cells and healthy B cells were used in this study. Small interfering RNA (siRNA)-mediated knockdown of lef-1 in primary CLL cells was done using nucleofection, and 50% lethal concentration (LC(50)) of two small molecules was assessed using ATP-based cell viability assay. Apoptotic response was investigated in time course experiments with different apoptotic markers. Specificity of the small molecules was demonstrated by coimmunoprecipitation experiments for the lef-1/beta-catenin interaction. In vivo studies were done in JVM-3 subcutaneous xenograft model. RESULTS: Inhibition of lef-1 by siRNA leads to increased apoptosis of CLL cells and inhibited proliferation of JVM-3 cell lines. The two small molecule inhibitors (CGP049090 and PKF115-584) efficiently kill CLL cells (LC(50)<1 microM), whereas normal B cells were not significantly affected. Coimmunoprecipitation showed a selective disruption of beta-catenin/lef-1 interaction. In vivo studies exhibited tumor inhibition of 69% with CGP049090 and 57% with PKF115-584 when compared with vehicle-treated controls, and the intervention was well tolerated. CONCLUSIONS: We have demonstrated that targeting lef-1 is a new and selective therapeutic approach in CLL. CGP049090 or PKF115-584 may be attractive compounds for CLL and other malignancies that deserve further (pre)clinical evaluation.

Human CD1D gene expression is regulated by LEF-1 through distal promoter regulatory elements.

CD1d-expressing cells present lipid Ag to CD1d-restricted NKT cells, which play an important role in immune regulation and tumor rejection. Lymphoid enhancer-binding factor-1 (LEF-1) is one of the regulators of the Wnt signaling pathway, which is a powerful regulator in cellular growth, differentiation, and transformation. There is little evidence connecting Wnt signaling to CD1d expression. In this study, we have identified LEF-1 as a regulator of the expression of the gene encoding the human CD1d molecule (CD1D). We found that LEF-1 binds specifically to the CD1D promoter. Overexpression of LEF-1 in K562 or Jurkat cells suppresses CD1D promoter activity and downregulates endogenous CD1D transcripts, whereas knockdown of LEF-1 using LEF-1-specific small interfering RNA increases CD1D transcripts in K562 and Jurkat cells but there are different levels of surface CD1d on these two cell types. Chromatin immunoprecipitation showed that the endogenous LEF-1 is situated at the CD1D promoter and interacts with histone deacetylase-1 to facilitate the transcriptional repressor activity. Knockdown of LEF-1 using small interfering RNA potentiates an acetylation state of histone H3/H4, supporting the notion that LEF-1 acts as a transcriptional repressor for the CD1D gene. Our finding links LEF-1 to CD1D and suggests a role of Wnt signaling in the regulation of the human CD1D gene.

Transactivation of lifeguard (LFG) by Akt-/LEF-1 pathway in MCF-7 and MDA-MB 231 human breast cancer cells.

Lifeguard (LFG) has been identified as a molecule that uniquely inhibits death mediated by Fas. The molecular function of human LFG and its regulation in carcinogenesis is uncertain. In our study, we investigated the potential regulation of LFG expression by Akt/LEF-1 pathway. The Glycogen synthase kinase-3 (GSK3) can be regulated by different signaling pathways including those mediated by protein kinase Akt. Inhibition of GSK3beta subunits activity results in the stabilisation of the beta-catenin protein and its accumulation in the nucleus, where it associates with members of the TCF/LEF-1 family of transcription factors to mediate gene transcription. In Western blots, RT-PCR and by small interfering RNA directed against LEF-1, we demonstrated that LFG expression correlates with GSK3beta and LEF-1 activation. Moreover, we showed that LFG mRNA was down-regulated after transfection with siRNA against LEF-1 in MDA-MB-231 cells. Our results therefore identify LFG as a target of the Akt/LEF-1 pathway in MDA-MB-231 breast tumour cells, a regulation which could play a key role in breast tumour progression.

The EWS/FLI1 oncogenic protein inhibits expression of the Wnt inhibitor DICKKOPF-1 gene and antagonizes beta-catenin/TCF-mediated transcription.

Tumours of the Ewing family, which comprise Ewing's sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and beta-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and beta-catenin-induced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on beta-catenin/TCF-mediated transcription since it can block transactivation of a consensus beta-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage beta-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores beta-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that beta-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to beta-catenin, which could explain its negative effect on beta-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as beta-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family.

Targeting the WNT/beta-catenin/TCF/LEF1 axis in solid and haematological cancers: Multiplicity of therapeutic options.

Among aberrantly regulated signalling pathways in cancer the WNT/beta-catenin pathway plays an outstanding role, since it was shown to be critically involved in a wide range of neoplasias. While the underlying mechanisms vary, overexpression of WNTs was found to mediate active signalling in some of these diseases. Other cancers show a mutation in pathway members further downstream, such as APC, Axin or beta-catenin, leading to aberrant signalling activation. Another mechanism initiating activation of WNT/beta-catenin signalling is the silencing of expression of negative WNT/beta-catenin regulators, such as DKK and WIF1, by, for example, promoter hypermethylation. All these mechanisms result in a common consequence, the activation of TCF/LEF1 transcription factors and subsequent target gene expression. Several target genes are known to be key players in tumourigenesis, such as c-myc, cyclin D1 or survivin. The variety of possible underlying mechanisms leading to beta-catenin/TCF/LEF1 activation offers multiple options to target the aberrantly activated pathway in order to prevent target gene expression and/or their gene products to exert their tumourigenic function. Here, we summarise the physiological role of WNT/beta-catenin signalling and the consequences of its aberrant activation during tumourigenesis. Furthermore, we discuss the possible strategies to target this pathway and their potential importance in cancer treatment.