Knockdown of long non-coding RNA LEF1-AS1 attenuates apoptosis and inflammatory injury of microglia cells following spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is associated with health burden both at personal and societal levels. Recent assessments on the role of lncRNAs in SCI regulation have matured. Therefore, to comprehensively explore the function of lncRNA LEF1-AS1 in SCI, there is an urgent need to understand its occurrence and development. METHODS: Using in vitro experiments, we used lipopolysaccharide (LPS) to treat and establish the SCI model primarily on microglial cells. Gain- and loss of function assays of LEF1-AS1 and miR-222-5p were conducted. Cell viability and apoptosis of microglial cells were assessed via CCK8 assay and flow cytometry, respectively. Adult Sprague-Dawley (SD) rats were randomly divided into four groups: Control, SCI, sh-NC, and sh-LEF-AS1 groups. ELISA test was used to determine the expression of TNF-α and IL-6, whereas the protein level of apoptotic-related markers (Bcl-2, Bax, and cleaved caspase-3) was assessed using Western blot technique. RESULTS: We revealed that LncRNA LEF1-AS1 was distinctly upregulated, whereas miR-222-5p was significantly downregulated in LPS-treated SCI and microglial cells. However, LEF1-AS1 knockdown enhanced cell viability, inhibited apoptosis, as well as inflammation of LPS-mediated microglial cells. On the contrary, miR-222-5p upregulation decreased cell viability, promoted apoptosis, and inflammation of microglial cells. Mechanistically, LEF1-AS1 served as a competitive endogenous RNA (ceRNA) by sponging miR-222-5p, targeting RAMP3. RAMP3 overexpression attenuated LEF1-AS1-mediated protective effects on LPS-mediated microglial cells from apoptosis and inflammation. CONCLUSION: In summary, these findings ascertain that knockdown of LEF1-AS1 impedes SCI progression via the miR-222-5p/RAMP3 axis.

Redundant and additive functions of the four Lef/Tcf transcription factors in lung epithelial progenitors.

In multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multimember paralogs, as exemplified by the four obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the complex genetics involved. Using the developing mouse lung as a model system, we generate two quadruple conditional knockouts, four triple mutants, and various combinations of double mutants, showing that the four Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Prelung-specification, pan-epithelial double knockouts have no lung phenotype; triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas; and the quadruple knockout barely forms a lung, resembling the Ctnnb1 mutant. Postlung-specification deletion of all four Lef/Tcf genes leads to branching defects, down-regulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the corresponding Ctnnb1 mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to reported repressor functions of Lef/Tcf, and represents a thorough in vivo analysis of cell-type-specific genetic redundancy among the four Lef/Tcf paralogs.

The role of CTNNB1 and LEF1 in feather follicles development of Anser cygnoides and Anser anser.

BACKGROUND: Wingless-types/beta-catenin (Wnt/ß-catenin) signaling pathway is one of the most extensively studied transcriptional cascades involved in various types of organogenesis including embryonic and postnatal development. Downy feather quantity is primarily affected by follicular development and gene regulations. OBJECTIVE: This research was aimed to investigate the role of catenin beta-1(CTNNB1) and lymphoid enhancerbinding factor-1 (LEF1) on feather follicles development at different developmental stages. METHODS: Fluorescence quantitative PCR, Western-blot and immunohistochemical methods were used in Anser cygnoides and Anser anser embryos (E12, E13 E18, and E28) and after birth gosling stages (G18, G48, G88) for gene expression analysis. RESULTS: CTNNB1 and LEF1 genes were expressed in Anser cygnoides and Anser anser at different embryonic and after-birth gosling developmental stages and the expression levels were significantly different in different stages (p < 0.05). The mRNA expression of CTNNB1 and LEF1 genes reached the highest level at D88 in Anser cygnoides, while the highest expression levels were at D18 and D88 in Anser anser, and the expression levels of CTNNB1 genes at D88 in all embryonic stages were significantly lower than after-birth stages. CTNNB1 and LEF1 protein expression were the highest at E12 and E28 for Anser cygnoides feather follicles development. While at a similar stage for Anser anser, the expression of CTNNB1 and LEF1 protein was the highest at D48 and D18. Protein expression at embryonic stages was in the epidermis (E) and the hair basal plate (P), the expression site for after-birth stages was in the dermal papilla (DP). CONCLUSION: Our study illustrated that CTNNB1 and LEF1 has an impact on Anser cygnoides and Anser anser feather follicles growth and development.

TCF1 and LEF1 Control Treg Competitive Survival and Tfr Development to Prevent Autoimmune Diseases.

CD4+ Foxp3+ T regulatory (Treg) cells are key players in preventing lethal autoimmunity. Tregs undertake differentiation processes and acquire diverse functional properties. However, how Treg's differentiation and functional specification are regulated remains incompletely understood. Here, we report that gradient expression of TCF1 and LEF1 distinguishes Tregs into three distinct subpopulations, particularly highlighting a subset of activated Treg (aTreg) cells. Treg-specific ablation of TCF1 and LEF1 renders the mice susceptible to systemic autoimmunity. TCF1 and LEF1 are dispensable for Treg's suppressive capacity but essential for maintaining a normal aTreg pool and promoting Treg's competitive survival. As a consequence, the development of T follicular regulatory (Tfr) cells, which are a subset of aTreg, is abolished in TCF1/LEF1-conditional knockout mice, leading to unrestrained T follicular helper (Tfh) and germinal center B cell responses. Thus, TCF1 and LEF1 act redundantly to control the maintenance and functional specification of Treg subsets to prevent autoimmunity.

Mutant Lef1 controls Gata6 in sebaceous gland development and cancer.

Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to carcinogenesis remains unclear. Since Gata6 controls lineage identity in SG, we investigated the link between these two transcription factors. Here, we show that Gata6 is a ß-catenin-independent transcriptional target of mutant Lef1. During epidermal development, Gata6 is expressed in a subset of Sox9-positive Lef1-negative hair follicle progenitors that give rise to the upper SG Overexpression of Gata6 by in utero lentiviral injection is sufficient to induce ectopic sebaceous gland elements. In mice overexpressing mutant Lef1, Gata6 ablation increases the total number of skin tumors yet decreases the proportion of SG tumors. The increased tumor burden correlates with impaired DNA mismatch repair and decreased expression of Mlh1 and Msh2 genes, defects frequently observed in human sebaceous neoplasia. Gata6 specifically marks human SG tumors and also defines tumors with elements of sebaceous differentiation, including a subset of basal cell carcinomas. Our findings reveal that Gata6 controls sebaceous gland development and cancer.

Genome-Wide mRNA-Seq Profiling Reveals that LEF1 and SMAD3 Regulate Epithelial-Mesenchymal Transition Through the Hippo Signaling Pathway During Palatal Fusion.

BACKGROUND: Epithelial-mesenchymal transition (EMT) of the medial edge epithelium (MEE) occurs through fusion of the palatal shelves and is a crucial step in palatogenesis. The key genes, however, and the related signaling pathway of EMT are not yet fully understood. Therefore, the aim of this study was to reveal the key genes and the related signaling pathway of EMT during palatal fusion. MATERIALS AND METHODS: C57BL/6J mice at embryonic gestation day 14.5 (E14.5; n = 6) were used to establish the cleft palate model for mRNA-Seq (HiSeq X Ten). The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for functional annotations of the differentially expressed genes. Quantitative polymerase chain reaction (qPCR) assays were used to validate the RNAseq data. RESULTS: A total of 936 differentially expressed genes, including 558 upregulated and 378 downregulated genes were identified in cases versus controls, respectively. Among these genes, the GO analysis showed that Lymphoid Enhancer-Binding Factor 1 (LEF1) and SMAD Family Member 3 (SMAD3) significantly enriched biological processes, which were EMT related. The KEGG analysis showed that these genes regulated EMT through the Hippo signaling pathway. LEF1 and SMAD3 were downregulated, and the qPCR results corroborated the RNA-seq data. CONCLUSIONS: These results demonstrate that LEF1 and SMAD3 inhibits EMT at the MEE through the Hippo signaling pathway; and that this could contribute to cleft palate formation in embryonic palatal fusion at E 14.5.

Novel lymphoid enhancer-binding factor 1-cytoglobin axis promotes extravasation of osteosarcoma cells into the lungs.

Lung metastasis is a major cause of mortality in patients with osteosarcoma (OS). A better understanding of the molecular mechanism of OS lung metastasis may facilitate development of new therapeutic strategies to prevent the metastasis. We have established high- and low-metastatic sublines (LM8-H and LM8-L, respectively) from Dunn OS cell line LM8 by using in vivo image-guided screening. Among the genes whose expression was significantly increased in LM8-H compared to LM8-L, the transcription factor lymphoid enhancer-binding factor 1 (LEF1) was identified as a factor that promotes LM8-H cell extravasation into the lungs. To identify downstream effectors of LEF1 that are involved in OS lung metastasis, 13 genes were selected based on LM8 microarray data and genomewide meta-analysis of a public database for OS patients. Among them, the cytoglobin (Cygb) gene was identified as a key effector in promoting OS extravasation into the lungs. CYGB overexpression increased the extravasation ability of LM8-L cells, whereas knocking out the Cygb gene in LM8-H cells reduced this ability. Our results showed a novel LEF1-CYGB axis in OS lung metastasis and may provide a new way of developing therapeutic strategies to prevent OS lung metastasis.

EpCAM-Regulated Transcription Exerts Influences on Nanomechanical Properties of Endometrial Cancer Cells That Promote Epithelial-to-Mesenchymal Transition.

Overexpression of epithelial cell adhesion molecule (EpCAM) has been implicated in advanced endometrial cancer, but its roles in this progression remain to be elucidated. In addition to its structural role in modulating cell-surface adhesion, here we demonstrate that EpCAM is a regulatory molecule in which its internalization into the nucleus turns on a transcription program. Activation of EGF/EGFR signal transduction triggered cell-surface cleavage of EpCAM, leading to nuclear internalization of its cytoplasmic domain EpICD. ChIP-seq analysis identified target genes that are coregulated by EpICD and its transcription partner, LEF-1. Network enrichment analysis further uncovered a group of 105 genes encoding functions for tight junction, adherent, and cell migration. Furthermore, nanomechanical analysis by atomic force microscopy revealed increased softness and decreased adhesiveness of EGF-stimulated cancer cells, implicating acquisition of an epithelial-mesenchymal transition (EMT) phenotype. Thus, genome editing of EpCAM could be associated with altering these nanomechanical properties towards a less aggressive phenotype. Using this integrative genomic-biophysical approach, we demonstrate for the first time an intricate relationship between EpCAM-regulated transcription and altered biophysical properties of cells that promote EMT in advanced endometrial cancer. Cancer Res; 76(21); 6171-82. ©2016 AACR.

Transcriptional Analysis of Vitiligo Skin Reveals the Alteration of WNT Pathway: A Promising Target for Repigmenting Vitiligo Patients.

Vitiligo affects 1% of the worldwide population. Halting disease progression and repigmenting the lesional skin represent the two faces of therapeutic challenge in vitiligo. We performed transcriptome analysis on lesional, perilesional, and non-depigmented skin from vitiligo patients and on matched skin from healthy subjects. We found a significant increase in CXCL10 in non-depigmented and perilesional vitiligo skin compared with levels in healthy control skin; however, neither CXCL10 nor other immune factors were deregulated in depigmented vitiligo skin. Interestingly, the WNT pathway, which is involved in melanocyte differentiation, was altered specifically in vitiligo skin. We demonstrated that oxidative stress decreases WNT expression/activation in keratinocytes and melanocytes. We developed an ex vivo skin model and confirmed the decrease activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated ex vivo depigmented skin from vitiligo patients and successfully induced differentiation of resident stem cells into pre-melanocytes. Our results shed light on the previously unrecognized role of decreased WNT activation in the prevention of melanocyte differentiation in depigmented vitiligo skin. Furthermore, these results support further clinical exploration of WNT agonists to repigment vitiligo lesions.

SMADs and YAP compete to control elongation of ß-catenin:LEF-1-recruited RNAPII during hESC differentiation.

The Wnt3a/ß-catenin and Activin/SMAD2,3 signaling pathways synergize to induce endodermal differentiation of human embryonic stem cells; however, the underlying mechanism is not well understood. Using ChIP-seq and GRO-seq analyses, we show here that Wnt3a-induced ß-catenin:LEF-1 enhancers recruit cohesin to direct enhancer-promoter looping and activate mesendodermal (ME) lineage genes. Moreover, we find that LEF-1 and other hESC enhancers recruit RNAPII complexes (eRNAPII) that are highly phosphorylated at Ser5, but not Ser7. Wnt3a signaling further increases Ser5P-RNAPII at LEF-1 sites and ME gene promoters, indicating that elongation remains limiting. However, subsequent Activin/SMAD2,3 signaling selectively increases transcription elongation, P-TEFb occupancy, and Ser7P-RNAPII levels at these genes. Finally, we show that the Hippo regulator, YAP, functions with TEAD to regulate binding of the NELF negative elongation factor and block SMAD2,3 induction of ME genes. Thus, the Wnt3a/ß-catenin and Activin/SMAD2,3 pathways act in concert to counteract YAP repression and upregulate ME genes during early hESC differentiation.

TCF1 and LEF1 act as T-cell intrinsic HTLV-1 antagonists by targeting Tax.

Human T-cell leukemia virus type 1 (HTLV-1) is a delta-type retrovirus that induces malignant and inflammatory diseases during its long persistence in vivo. HTLV-1 can infect various kinds of cells; however, HTLV-1 provirus is predominantly found in peripheral CD4 T cells in vivo. Here we find that TCF1 and LEF1, two Wnt transcription factors that are specifically expressed in T cells, inhibit viral replication through antagonizing Tax functions. TCF1 and LEF1 can each interact with Tax and inhibit Tax-dependent viral expression and activation of NF-κB and AP-1. As a result, HTLV-1 replication is suppressed in the presence of either TCF1 or LEF1. On the other hand, T-cell activation suppresses the expression of both TCF1 and LEF1, and this suppression enables Tax to function as an activator. We analyzed the thymus of a simian T-cell leukemia virus type 1 (STLV-1) infected Japanese macaque, and found a negative correlation between proviral load and TCF1/LEF1 expression in various T-cell subsets, supporting the idea that TCF1 and LEF1 negatively regulate HTLV-1 replication and the proliferation of infected cells. Thus, this study identified TCF1 and LEF1 as Tax antagonistic factors in vivo, a fact which may critically influence the peripheral T-cell tropism of this virus.

TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4(+) T cell fate and interact with Runx3 to silence Cd4 in CD8(+) T cells.

The transcription factors TCF-1 and LEF-1 are essential for early T cell development, but their roles beyond the CD4(+)CD8(+) double-positive (DP) stage are unknown. By specific ablation of these factors in DP thymocytes, we demonstrated that deficiency in TCF-1 and LEF-1 diminished the output of CD4(+) T cells and redirected CD4(+) T cells to a CD8(+) T cell fate. The role of TCF-1 and LEF-1 in the CD4-versus-CD8 lineage 'choice' was mediated in part by direct positive regulation of the transcription factor Th-POK. Furthermore, loss of TCF-1 and LEF-1 unexpectedly caused derepression of CD4 expression in T cells committed to the CD8(+) lineage without affecting the expression of Runx transcription factors. Instead, TCF-1 physically interacted with Runx3 to cooperatively silence Cd4. Thus, TCF-1 and LEF-1 adopted distinct genetic 'wiring' to promote the CD4(+) T cell fate and establish CD8(+) T cell identity.

Physiological role of ß-catenin/TCF signaling in neurons of the adult brain.

Wnt/ß-catenin pathway, the effectors of which are transcription factors of the LEF1/TCF family, is primarily associated with development. Strikingly, however, some of the genes of the pathway are schizophrenia susceptibility genes, and the proteins that are often mutated in neurodegenerative diseases have the ability to regulate ß-catenin levels. If impairment of this pathway indeed leads to these pathologies, then it likely plays a physiological role in the adult brain. This review provides an overview of the current knowledge on this subject. The involvement of ß-catenin and LEF1/TCF factors in adult neurogenesis, synaptic plasticity, and the function of thalamic neurons are discussed. The data are still very preliminary and often based on circumstantial or indirect evidence. Further research might help to understand the etiology of the aforementioned pathologies.

Hematopoietic stem cell fate decisions are regulated by Wnt antagonists: comparisons and current controversies.

Wingless and int (Wnt) proteins are secreted proteins that are important for regulating hematopoietic stem cell self-renewal and differentiation in the bone marrow microenvironment in mice. The mechanisms by which Wnt signaling regulates these hematopoietic cell fate decisions are not fully understood. Secreted Wnt antagonists, which are expressed in bone and bone marrow stromal cells, either bind to Wnt ligands directly or block Wnt receptors and co-receptors to halt Wnt-mediated signal transduction in both osteolineage and hematopoietic cell types. Secreted frizzled related proteins-1 and -2, Wnt inhibitory factor-1, Dickkopf-1, and Sclerostin are Wnt antagonists that influence hematopoietic cell fate decisions in the bone marrow niche. In this review, we compare and contrast the roles of these Wnt antagonists and their effects on hematopoietic development in mice, and also discuss the clinical significance of targeting Wnt antagonists within the context of hematopoietic disease.

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models.

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

Cutting edge: generation of memory precursors and functional memory CD8+ T cells depends on T cell factor-1 and lymphoid enhancer-binding factor-1.

T cell factor (TCF)-1 and lymphoid enhancer-binding factor (LEF)-1 transcription factors have redundant roles in promoting thymocyte maturation. TCF-1 has been recently shown to critically regulate memory CD8+ T cell differentiation and persistence. The complete spectra of regulatory roles for TCF-1 and LEF-1 in CD8+ T cell responses are yet unknown. We conditionally targeted LEF-1, and by combination with germline deletion of TCF-1, we found that loss of both factors completely abrogated the generation of KLR G1(lo)IL-7Rα+ memory precursors in effector CD8+ T cell populations in response to Listeria monocytogenes infection. Whereas CD8+ effectors deficient for TCF-1 and LEF-1 retained the capacity to express IFN-γ, granzyme B, and perforin, they were defective in TNF-α production. In the memory phase, the Ag-specific CD8+ T cells lacking TCF-1 and LEF-1 exhibited an effector phenotype and were severely impaired in secondary expansion upon rechallenge. Thus, TCF-1 and LEF-1 cooperatively regulate generation of memory precursors and protective memory CD8+ T cells.

The role of LEF1 in endometrial gland formation and carcinogenesis.

Endometrial carcinoma is the most common gynecologic cancer, yet the mechanisms underlying this disease process are poorly understood. We hypothesized that Lef1 is required for endometrial gland formation within the uterus and is overexpressed in endometrial cancer. Using Lef1 knockout (KO) mice, we compared uterine gland development to wild-type (WT) controls, with respect to both morphology and expression of the Lef1 targets, cyclin D1 and MMP7. We characterized the dynamics of Lef1 protein expression during gland development and the mouse estrus cycle, by immunostaining and Western blot. Finally, we investigated the roles of cyclin D1 and MMP7 in gland and cancer formation in the mouse, and assessed the relevance of Lef1 to human cancer by comparing expression levels in cancerous and normal endometrial tissues. Lef1 upregulation in mouse endometrium correlates with the proliferative stages of the estrus cycle and gland development during the neonatal period. WT mice endometrial glands began to develop by day 5 and were easily identified by day 9, whereas Lef1 KO mice endometrial glands had not developed by day 9 although the endometrial lining was intact. We found that during gland development cyclin D1 is elevated and localized to the gland buds, and that this requires the presence of Lef1. We also noted that Lef1 protein was expressed at higher levels in endometrial cancers within mice and humans when compared to normal endometrium. Our loss-of-function data indicate that Lef1 is required for the formation of endometrial glands in the mouse uterus. Lef1 protein elevation corresponds to gland formation during development, and varies cyclically with the mouse estrus cycle, in parallel with gland regeneration. Finally, Lef1 is overexpressed in human and mouse endometrial tumors, consistent with it playing a role in gland proliferation.

The balance between two isoforms of LEF-1 regulates colon carcinoma growth.

BACKGROUND: Colon cancer is one of the most aggressive human malignancies, with a very poor prognosis. Although it has been suggested that different isoforms of the lymphoid enhancer factor (LEF-1) have opposing biological activities, the biological outcome of aberrant LEF-1 activation in colon cancer is still unclear. The aim of this study was to evaluate the effect of the different LEF-1 phenotypes on the growth of colon carcinoma cell lines. A deeper understanding of these processes might improve the targeted therapies for colon cancer by regulating the expression of LEF-1. METHODS: The role of different isoforms of LEF-1 on the growth of human colon carcinoma cell lines (SW480 and HT-29) was studied using various in vitro and in vivo assays. In vitro proliferation, migration, adhesion and apoptosis of the cells stably transfected of different isoforms of LEF-1 were monitored by MTT assay, carboxyfluorescein diacetate-succinimidyl ester staining, annexin V staining, ECM adhesion assay and transwell assay, respectively. In nude mice, the formation of neovasculature in the tumors formed by our constructed cells was measured by immunohistochemistry. All the data were analyzed using a t test, and data were treated as significant when p < 0.05. RESULTS: Overexpression of truncated LEF-1 (LEF-1-ΔL) in the colon cell lines, SW480 and HT29, inhibited their growth significantly in vitro and in vivo, but the full-length LEF-1 (LEF-1-FL) promoted the proliferation of HT29. Inactivation of Wnt signaling by LEF-1-ΔL reduced the expression of CXCR4 in colon cell lines, which may lead to a decrease in activities such as migration, adhesion and survival. In nude mice, the formation of neovasculature as well as an increase in tumor volume were inhibited by the short isoform of LEF-1. LEF-1-FL, however, caused an increase in all these parameters compared with controls. CONCLUSIONS: These findings suggest that LEF-1 might play an important role in colon carcinogenesis by acting as a regulator. Enhanced expression of LEF-1-FL, which occurs frequently in colon cancer, may be a new target for clinical therapy.

Aberrant beta-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.