Lymphoid Enhancer factor/T-cell Factor Research Articles

Diverse LEF/TCF Expression in Human Colorectal Cancer Correlates with Altered Wnt-Regulated Transcriptome in a Meta-Analysis of Patient Biopsies.

Aberrantly activated Wnt signaling causes cellular transformation that can lead to human colorectal cancer. Wnt signaling is mediated by Lymphoid Enhancer Factor/T-Cell Factor (LEF/TCF) DNA-binding factors. Here we investigate whether altered LEF/TCF expression is conserved in human colorectal tumor sample and may potentially be correlated with indicators of cancer progression. We carried out a meta-analysis of carefully selected publicly available gene expression data sets with paired tumor biopsy and adjacent matched normal tissues from colorectal cancer patients. Our meta-analysis confirms that among the four human LEF/TCF genes, LEF1 and TCF7 are preferentially expressed in tumor biopsies, while TCF7L2 and TCF7L1 in normal control tissue. We also confirm positive correlation of LEF1 and TCF7 expression with hallmarks of active Wnt signaling (i.e., AXIN2 and LGR5). We are able to correlate differential LEF/TCF gene expression with distinct transcriptomes associated with cell adhesion, extracellular matrix organization, and Wnt receptor feedback regulation. We demonstrate here in human colorectal tumor sample correlation of altered LEF/TCF gene expression with quantitatively and qualitatively different transcriptomes, suggesting LEF/TCF-specific transcriptional regulation of Wnt target genes relevant for cancer progression and survival. This bioinformatics analysis provides a foundation for future more detailed, functional, and molecular analyses aimed at dissecting such functional differences.

3D-microenvironments initiate TCF4 expression rescuing nuclear β-catenin activity in MCF-7 breast cancer cells

Mechanical cues sensed by tumor cells in their microenvironment can influence important mechanisms including adhesion, invasion and proliferation. However, a common mechanosensitive protein and/or pathway can be regulated in different ways among diverse types of tumors. Of particular interest are human breast epithelial cancers, which markedly exhibit a heterogeneous pattern of nuclear β-catenin localization, a protein known to be involved in both mechanotransduction and tumorigenesis. β-catenin can be aberrantly accumulated in the nucleus wherein it binds to and activates lymphoid enhancer factor/T cell factor (LEF/TCF) transcription factors. At present, little is known about how mechanical cues are integrated into breast cancer cells harboring impaired mechanisms of β-catenin's nuclear uptake and/or retention. This prompted us to investigate the influence of mechanical cues on MCF-7 human breast cancer cells which are known to fail in relocating β-catenin into the nucleus due to very low baseline levels of LEF/TCFs. Exploiting three-dimensional (3D) microscaffolds realized by two-photon lithography, we show that surrounding MCF-7 cells have not only a nuclear pool of β-catenin, but also rescue from their defective expression of TCF4 and boost invasiveness. Together with heightened amounts of vimentin, a β-catenin/TCF-target gene regulator of proliferation and invasiveness, such 3D-elicited changes indicate an epithelial-to-mesenchymal phenotypic switch of MCF-7 cells. This is also consistent with an increased in situ MCF-7 cell proliferation that can be abrogated by blocking β-catenin/TCF-transcription activity. Collectively, these data suggest that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of β-catenin nuclear activity in MCF-7 cells. The employed methodology could, therefore, provide a mechanism-based rationale to dissect further aspects of mechanotranscription in breast cancerogenesis, somewhat independent of β-catenin's nuclear accumulation. More importantly, by considering the heterogeneity of β-catenin signaling pathway in breast cancer patients, these data may open alternative avenues for personalized disease management and prevention. Statement of significanceMechanical cues play a critical role in cancer pathogenesis. Little is known about their influence in breast cancer cells harboring impaired mechanisms of β-catenin's nuclear uptake and/or retention, involved in both mechanotransduction and tumorigenesis. We engineered 3D scaffold, by two-photon lithography, to study the influence of mechanical cues on MCF-7 cells which are known to fail in relocating β-catenin into the nucleus. We found that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of β-catenin nuclear activity that boost cell proliferation and invasiveness. Thus, let us suggest that our system could provide a mechanism-based rationale to further dissect key aspects of mechanotranscription in breast cancerogenesis and progression, somewhat independent of β-catenin's nuclear accumulation.

Characterization of a beta-catenin nuclear localization defect in MCF-7 breast cancer cells

Beta-catenin plays a key role in transducing Wnt signals from the plasma membrane to the nucleus. Here we characterize an unusual subcellular distribution of beta-catenin in MCF-7 breast cancer cells, wherein beta-catenin localizes to the cytoplasm and membrane but atypically did not relocate to the nucleus after Wnt treatment. The inability of Wnt or the Wnt agonist LiCl to induce nuclear localization of beta-catenin was not due to defective nuclear transport, as the transport machinery was intact and ectopic GFP-beta-catenin displayed rapid nuclear entry in living cells. The mislocalization is explained by a shift in the retention of beta-catenin from nucleus to cytoplasm. The reduced nuclear retention is caused by unusually low expression of lymphoid enhancer factor/T-cell factor (LEF/TCF) transcription factors. The reconstitution of LEF-1 or TCF4 expression rescued nuclear localization of beta-catenin in Wnt treated cells. In the cytoplasm, beta-catenin accumulated in recycling endosomes, golgi and beta-COP-positive coatomer complexes. The peripheral association with endosomes diminished after Wnt treatment, potentially releasing β-catenin into the cytoplasm for nuclear entry. We propose that in MCF-7 and perhaps other breast cancer cells, beta-catenin may contribute to cytoplasmic functions such as ER-golgi transport, in addition to its transactivation role in the nucleus.

β-catenin promotes the type I IFN synthesis and the IFN-dependent signaling response but is suppressed by influenza A virus-induced RIG-I/NF-κB signaling.

BackgroundThe replication cycle of most pathogens, including influenza viruses, is perfectly adapted to the metabolism and signal transduction pathways of host cells. After infection, influenza viruses activate several cellular signaling cascades that support their propagation but suppress those that interfere with viral replication. Accumulation of viral RNA plays thereby a central role. Its sensing by the pattern recognition receptors of the host cells leads to the activation of several signal transduction waves that result in induction of genes, responsible for the cellular innate immune response. Type I interferon (IFN) genes and interferon-stimulated genes (ISG) coding for antiviral-acting proteins, such as MxA, OAS-1 or PKR, are primary targets of these signaling cascades. β- and γ-catenin are closely related armadillo repeat-containing proteins with dual roles. At the cell membrane they serve as adapter molecules linking cell-cell contacts to microfilaments. In the cytosol and nucleus, the proteins form a transcriptional complex with the lymphoid enhancer factor/T-cell factor (LEF/TCF), regulating the transcription of many genes, thereby controlling different cellular functions such as cell cycle progression and differentiation.ResultsIn this study, we demonstrate that β- and γ-catenin are important regulators of the innate cellular immune response to influenza A virus (IAV) infections. They inhibit viral replication in lung epithelial cells by enhancing the virus-dependent induction of the IFNB1 gene and interferon-stimulated genes. Simultaneously, the prolonged infection counteracts the antiviral effect of β- and γ-catenin. Influenza viruses suppress β-catenin-dependent transcription by misusing the RIG-I/NF-κB signaling cascade that is induced in the course of infection by viral RNA.ConclusionWe identified β- and γ-catenin as novel antiviral-acting proteins. While these factors support the induction of common target genes of the cellular innate immune response, their functional activity is suppressed by pathogen evasion.

Aberrant β-Catenin and Lef1 Expression May Predict the Clinical Outcome for Patients with Oropharyngeal Cancer

Beta-catenin, normally expressed on the epithelial cell surface, plays a crucial role in cadherin-mediated cell adhesion. Recent evidence suggests that beta-catenin is also involved in other functions such as intracellular signaling via the Wnt pathway by creating a nuclear complex with members of the Lymphoid-Enhancer-Factor/T-Cell-Factor (LEF/TCF) family of transcription factors, and gene regulation that it is implicated in the development of several tumors. Little information is available on beta-catenin expression and its main partner in the Wnt signaling pathway, LEF1, in oropharyngeal squamous cell carcinomas (OP-SCCs). The aim of this study is to investigate the expression of beta-catenin and LEF1 expression in human primary OP-SCCs and to evaluate their clinical and prognostic significance. OP-SCCs and normal peritumoral areas were analyzed by immunohistochemistry, Western-blot and RT-PCR. Beta-catenin was overexpressed in tumors in comparison to normal peritumoral areas and displayed predominantly intracellular (cytosolic/nuclear) localization in 62% of the tumors. Immunoreactivity was correlated with clinicopathological parameters and long-term follow-up, and a significant association was found between protein expression and development of local recurrences (P =0.03). The OP-SCCs with poor clinical outcome, which displayed intracellular beta-catenin expression, were also strongly positive for LEF1, with their co-expression statistically significant (P = 0.040). All (100%) advanced (stages 3+4) SCCs, 66.7% of the SCCs with positive lymph nodes and 80% of the SSCs that developed local recurrences were LEF1 positive. Cox regression analysis confirmed a poorer overall survival in cases with high expression of beta-catenin and LEF1. Our results suggest that assessing intracellular beta-catenin and LEF1 expression might help in patient risk stratification and outcome prediction, and serve as novel therapeutic targets in advanced OP-SCC.

Axing Wnt signals

Wnt signaling is a highly conserved and context-dependent signal transduction pathway that belongs to the so-called morphogens. Wnt signals are instrumental during development, but also play a central role in tissue homeostasis. For instance, in the intestine Wnt signaling is crucial for both stem cell maintenance and proliferation in the transient amplifying compartment 1. This Wnt-dependent stem cell regulatory role is observed in diverse adult tissues, but is not a general feature as Wnt signals can also drive differentiation of cells, such as mesenchymal stem cells (MSCs) 2. Wnt signaling can be divided into a canonical and a non-canonical pathway. The non-canonical pathway mainly mediates migration, while the canonical signals are mostly conveyed by β-catenin, a transcription factor that is actively repressed in the cytoplasm in the absence of Wnt signals. β-catenin levels are kept under control by a multi-protein complex composed of the tumor suppressor adenomatous polyposis coli (APC); two kinases, casein kinase 1 (CK1) and glycogen synthase kinase 3 beta (GSK3-β); and Axin2 that serves as a scaffold to hold the complex together. This complex allows CK1 and GSK3-β to phosphorylate β-catenin at specific serine and threonine residues, priming its recognition by the U3 ubiquitin ligase β-transducin repeat-containing protein (β-TRCP). Consequently, β-catenin is ubiquitinated and targeted for proteosomal degradation (Figure 1A). However, in the presence of extracellular Wnt ligands, β-catenin degradation is halted. Wnt binds the membrane-bound receptor complex formed by frizzled (Fzd) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6). As a result, the destruction complex is dissolved by a still poorly understood mechanism. This allows β-catenin to accumulate in the cytosol and, subsequently, translocate into the nucleus. There, it associates with the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors, converting them from repressors to activators of transcription. These steps are followed by additional nuclear events that ultimately trigger a complex transcriptional program depending on the cellular context that will direct cell fate, cell proliferation, and stem cell maintenance or cellular differentiation (Figure 1B) (See review by MacDonald et al., 2009) 3.

A short upstream promoter region mediates transcriptional regulation of the mouse doublecortin gene in differentiating neurons

BackgroundDoublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced Dcx expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling Dcx gene expression by studying its transcriptional regulation during neuronal differentiation.ResultsTo determine and analyze important regulatory sequences of the Dcx promoter, we studied a putative regulatory region upstream from the mouse Dcx coding region (pdcx2kb) and several deletions thereof. These different fragments were used in vitro and in vivo to drive reporter gene expression. We demonstrated, using transient expression experiments, that pdcx2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing brain (E14.5). We determined the temporal profile of Dcx promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the Dcx gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of Dcx promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family and NF-Y/CAAT (Nuclear Factor-Y).ConclusionsStudies of Dcx gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the Dcx coding sequence are sufficient to induce specific Dcx expression in vitro: in heterogeneous differentiated neurons from mESC, in primary mouse cerebellar neurons (PND3) and in organotypic slice cultures. Furthermore, a region in the 3'-end region of the Dcx promoter is highly conserved across several species and exerts positive control on Dcx transcriptional activation. Together, these results indicate that the proximal 3'-end region of the mouse Dcx regulatory sequence is essential for Dcx gene expression during differentiation of neuronal precursors.

Wnt/β-Catenin and Retinoic Acid Receptor Signaling Pathways Interact to Regulate Chondrocyte Function and Matrix Turnover

Activation of the Wnt/beta-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/beta-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/beta-catenin reporter activity and beta-catenin nuclear accumulation. This stimulation was accompanied by increased gene expression of Wnt proteins and receptors and was inhibited by co-treatment with Dickkopf-related protein-1, an extracellular inhibitor of Wnt/beta-catenin signaling, suggesting that RA modulates Wnt signaling at Wnt cell surface receptor level. RA also enhanced matrix loss triggered by Wnt/beta-catenin signaling, whereas treatment with a retinoid antagonist reduced it. Interestingly, overexpression of retinoic acid receptor gamma (RARgamma) strongly inhibited Wnt/beta-catenin signaling in retinoid-free cultures, whereas small interfering RNA-mediated silencing of endogenous RARgamma expression strongly increased it. Small interfering RNA-mediated silencing of RARalpha or RARbeta had minimal effects. Co-immunoprecipitation and two-hybrid assays indicated that RARgamma interacts with beta-catenin and induces dissociation of beta-catenin from lymphoid enhancer factor in retinoid-free cultures. The N-terminal domain (AF-1) of RARgamma but not the C-terminal domain (AF-2) was required for association with beta-catenin, whereas both AF-1 and AF-2 were necessary for inhibition of beta-catenin transcriptional activity. Taken together, our data indicate that the Wnt and retinoid signaling pathways do interact in chondrocytes, and their cross-talks and cross-regulation play important roles in the regulation of cartilage matrix homeostasis.

Acetylation regulates subcellular localization of the Wnt signaling nuclear effector POP-1.

Lymphoid enhancer factor/T-cell factor (LEF/TCF) are transcription factors that mediate the Wnt signaling pathway, and have crucial roles during embryonic development in various organisms. Here we report that acetylation enhances nuclear retention of POP-1, the Caenorhabditis elegans LEF/TCF homolog, through increasing nuclear import and blocking nuclear export. We identify three lysines that are acetylated in vivo, and demonstrate their essential requirement for proper nuclear localization and biological activity of POP-1 during C. elegans embryogenesis. The conservation of these lysines among other LEF/TCF family members suggests that acetylation may be an important, evolutionarily conserved mechanism regulating subcellular distribution of LEF/TCF factors.

Functional Diversity of Xenopus Lymphoid Enhancer Factor/T-cell Factor Transcription Factors Relies on Combinations of Activating and Repressing Elements

Lymphoid enhancer factor/T-cell factor (LEF/TCF) high mobility group box transcription factors are the nuclear transducers of the Wnt/beta-catenin signaling cascade. In Xenopus, three members of the LEF/TCF family, XLEF-1, XTCF-3, and XTCF-4, with distinct but partially overlapping expression patterns have been identified. The individual Xenopus LEF/TCF family members differ extremely in their properties of target gene regulation. We observed that in contrast to LEF-1, neither XTCF-3 nor XTCF-4 can induce secondary axis formation upon ventral overexpression in Xenopus embryos. To identify functional motifs within the LEF/TCF transcription factors responsible for target gene activation or repression, we created various mutants and a set of XLEF-1/XTCF-3 chimeras. In overexpression studies, we asked whether these constructs can mimic an activated Wnt/beta-catenin pathway and lead to the formation of a secondary body axis. In addition, we examined their capacity to rescue a loss-of-function phenotype given by dominant negative LEF-1 expression. We further analyzed their ability to directly activate target genes in reporter gene assays using the LEF/TCF target promoters, siamois and fibronectin. We found that a region homologous to exon IVa of human TCF-1 is an activating element. This is flanked by two small repressing motifs, LVPQ and SXXSS. Our findings implicate that the motifs identified here play an essential role in determining cell type-specific activity of LEF/TCF transcription factors.

Expression of lymphoid enhancer factor/T-cell factor proteins in colon cancer

Molecular genetic analysis of colon cancers has established that the Wnt signaling pathway is involved in early tumor development. Mutation of midstream components can activate the pathway, making it independent of Wnt ligands and maintaining constant pressure to change target gene expression. The transcription factors that connect the pathway to target genes are members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family. The genes for two members of this family, TCF 7 and LEF 1, produce full-length forms that mediate Wnt signals and truncated dominant negative forms that limit Wnt signals and may function as growth suppressors. Results from studies of their expression in colon cancer suggests that because Wnt-linked cancers progress to malignancy, there may be a strengthening of the Wnt signal by selective expression of the activating forms of LEF/TCFs and a bias against suppressing, truncated forms.

Regulation and possible function of beta-catenin in human monocytes.

In this study, we demonstrate that adherence factors, serum constituents, LPS, and zymosan are capable of inducing a cellular accumulation of beta-catenin in human monocytes. Whereas adherence-dependent accumulation of beta-catenin can be blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, accumulation induced by the remaining stimuli cannot be prevented by inhibition of phosphatidylinositol 3-kinase, implying the involvement of beta-catenin in other not yet described signal transduction pathways. A role of beta-catenin in adherence-dependent processes by interacting with classical cadherins can be excluded as we could not detect cadherins in monocytes. To test whether it is possible that beta-catenin interacts with LEF/TCF (lymphoid enhancer factor/T cell factor) transcription factors, we studied the expression of this protein family. TCF-4 was identified as the LEF/TCF transcription factor present in human monocytes. However, neither cellular induction of beta-catenin nor cotransfection experiments with beta-catenin conducted in the monocytic cell line THP-1 resulted in the activation of a LEF/TCF-dependent promoter, suggesting the requirement of additional signals. Concurrent with this suggestion, we found that LPS and zymosan, two physiological inducers of beta-catenin, caused an increase in the expression of genes that are positively regulated by beta-catenin.