WNT3a Signaling Inhibits Aromatase Expression in Breast Adipose Fibroblasts-A Possible Mechanism Supporting the Loss of Estrogen Responsiveness of Triple-Negative Breast Cancers.

Estrogen-dependent breast cancers rely on a constant supply of estrogens and expression of estrogen receptors. Local biosynthesis, by aromatase in breast adipose fibroblasts (BAFs), is their most important source for estrogens. Triple-negative breast cancers (TNBC) rely on other growth-promoting signals, including those from the Wnt pathway. In this study, we explored the hypothesis that Wnt signaling alters the proliferation of BAFs, and is involved in regulation of aromatase expression in BAFs. Conditioned medium (CM) from TNBC cells and WNT3a consistently increased BAF growth, and reduced aromatase activity up to 90%, by suppression of the aromatase promoter I.3/II region. Database searches identified three putative Wnt-responsive elements (WREs) in the aromatase promoter I.3/II. In luciferase reporter gene assays, promoter I.3/II activity was inhibited by overexpression of full-length T-cell factor (TCF)-4 in 3T3-L1 preadipocytes, which served as a model for BAFs. Full-length lymphoid enhancer-binding factor (LEF)-1 increased the transcriptional activity. However, TCF-4 binding to WRE1 in the aromatase promoter, was lost after WNT3a stimulation in immunoprecipitation-based in vitro DNA-binding assays, and in chromatin immunoprecipitation (ChIP). In vitro DNA-binding assays, ChIP, and Western blotting revealed a WNT3a-dependent switch of nuclear LEF-1 isoforms towards a truncated variant, whereas ß-catenin levels remained unchanged. This LEF-1 variant revealed dominant negative properties, and most likely recruited enzymes involved in heterochromatin formation. In addition, WNT3a induced the replacement of TCF-4 by the truncated LEF-1 variant, on WRE1 of the aromatase promoter I.3/II. The mechanism described here may be responsible for the loss of aromatase expression predominantly associated with TNBC. Tumors with (strong) expression of Wnt ligands actively suppress aromatase expression in BAFs. Consequently a reduced estrogen supply could favor the growth of estrogen-independent tumor cells, which consequently would make estrogen receptors dispensable. In summary, canonical Wnt signaling within (cancerous) breast tissue may be a major factor controlling local estrogen synthesis and action.

Loss of LEF-1 expression as a diagnostic indicator for extranodal NK/T-cell lymphoma: An immunohistochemical study of 88 cases.

PURPOSE: Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is not a uniform entity but consists of various disease subtypes associated with Epstein-Barr virus (EBV) infection. Lymphoid enhancer binding factor-1 (LEF-1), a member of the T-cell factor/LEF family of transcription factors, plays a significant role in NK-cell and T-cell development. We aimed to explore the expression of LEF-1 in ENKTL and evaluate the applicability of LEF-1 in the diagnosis of ENKTL. METHODS: The expression of LEF-1 was investigated in tissue samples harvested from patients with ENKTL by immunohistochemistry. RESULTS: LEF-1 staining was not observed in 85 of 88 ENKTL cases (97%). Eighty-six of the 88 cases (98%) were positive for CD56, whereas all the tested cases were negative for both CD16 and CD27. Of the cytotoxic T-cell-like features studied, 81 cases (92%) were negative for CD8, 85 of 88 cases (97%) were immunoreactive for the recombinant linker for activation of T cells (LAT), all (100%) were immunoreactive for TIA-1, 85 (97%) were immunoreactive for granzyme B and 65 (74%) were immunoreactive for perforin. CONCLUSION: Loss of LEF-1 expression is a highly specific diagnostic indicator of ENKTL.

Dedicator of cytokinesis protein 2 couples with lymphoid enhancer-binding factor 1 to regulate expression of CD21 and B-cell differentiation.

BACKGROUND: B-cell receptor (BCR) signaling, combined with CD19 and CD21 signals, imparts specific control of B-cell responses. Dedicator of cytokinesis protein 2 (DOCK2) is critical for the migration and motility of lymphocytes. Although absence of DOCK2 leads to lymphopenia, little is known about the signaling mechanisms and physiologic functions of DOCK2 in B cells. OBJECTIVE: We sought to determine the underlying molecular mechanism of how DOCK2 regulates BCR signaling and peripheral B-cell differentiation. METHODS: In this study we used genetic models for DOCK2, Wiskott-Aldrich syndrome protein (WASP), and lymphoid enhancer-binding factor 1 deficiency to study their interplay in BCR signaling and B-cell differentiation. RESULTS: We found that the absence of DOCK2 led to downregulation of proximal and distal BCR signaling molecules, including CD19, but upregulation of SH2-containing inositol 5 phosphatase 1, a negative signaling molecule. Interestingly, DOCK2 deficiency reduced CD19 and CD21 expression at the mRNA and/or protein levels and was associated with reduced numbers of marginal zone B cells. Additionally, loss of DOCK2 reduced activation of WASP and accelerated degradation of WASP, resulting into reduced actin accumulation and early activation of B cells. Mechanistically, the absence of DOCK2 upregulates the expression of lymphoid enhancer-binding factor 1. These differences were associated with altered humoral responses in the absence of DOCK2. CONCLUSIONS: Overall, our study has provided a novel underlying molecular mechanism of how DOCK2 deficiency regulates surface expression of CD21, which leads to downregulation of CD19-mediated BCR signaling and marginal zone B-cell differentiation.

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.

Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis.

Primary Myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by a skewed megakaryopoiesis and an overproduction of proinflammatory and profibrotic mediators that lead to the development of bone marrow (BM) fibrosis. Since we recently uncovered the upregulation of miR-34a-5p in PMF CD34+ hematopoietic progenitor cells (HPCs), in order to elucidate its role in PMF pathogenesis here we unravelled the effects of miR-34a-5p overexpression in HPCs. We showed that enforced expression of miR-34a-5p partially constrains proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF CD34+ cells. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression on HPCs lineage choice, by favouring the megakaryocyte and monocyte/macrophage commitment. Collectively our data unravel the role of miR-34a-5p in HPCs fate decision and suggest that the increased expression of miR-34a-5p in PMF HPCs could be important for the skewing of megakaryopoiesis and the production of monocytes, that are key players in BM fibrosis in PMF patients.

microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest.

microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms--inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis.

Immunohistochemical Investigation into Protein Expression Patterns of FOXO4, IRF8 and LEF1 in Canine Osteosarcoma.

Osteosarcoma (OSA) is the most common type of primary bone malignancy in people and dogs. Our previous molecular comparisons of canine OSA against healthy bone resulted in the identification of differentially expressed protein-expressing genes (forkhead box protein O4 (FOXO4), interferon regulatory factor 8 (IRF8), and lymphoid enhancer binding factor 1 (LEF1)). Immunohistochemistry (IHC) and H-scoring provided semi-quantitative assessment of nuclear and cytoplasmic staining alongside qualitative data to contextualise staining (n = 26 patients). FOXO4 was expressed predominantly in the cytoplasm with significantly lower nuclear H-scores. IRF8 H-scores ranged from 0 to 3 throughout the cohort in the nucleus and cytoplasm. LEF1 was expressed in all patients with significantly lower cytoplasmic staining compared to nuclear. No sex or anatomical location differences were observed. While reduced levels of FOXO4 might indicate malignancy, the weak or absent protein expression limits its primary use as diagnostic tumour marker. IRF8 and LEF1 have more potential for prognostic and diagnostic uses and facilitate further understanding of their roles within their respective molecular pathways, including Wnt/beta-catenin/LEF1 signalling and differential regulation of tumour suppressor genes. Deeper understanding of the mechanisms involved in OSA are essential contributions towards the development of novel diagnostic, prognostic, and treatment options in human and veterinary medicine contexts.

The transcription factor LEF-1 induces an epithelial-mesenchymal transition in MDCK cells independent of ß-catenin.

The epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as the acquisition of migratory and invasive properties. LEF-1 is a member of the lymphoid enhancer-binding factor/T-cell factor (LEF/TCF) family of DNA-binding transcription factors, which interact with nuclear ß-catenin and act as central transcriptional mediators of Wnt signaling. To investigate the role of LEF-1 in EMT, we generated stable LEF-1 transfectants using MDCK cells. The transfectants had a spindle-shaped mesenchymal morphology, and enhanced migration and invasiveness relative to control cells. These EMT changes were accompanied by the downregulation of an epithelial marker protein, E-cadherin, and the upregulation of mesenchymal marker proteins, vimentin and N-cadherin. Consistent with these observations, the mRNA levels of Slug, ZEB1, and ZEB2-EMT-related transcription factors-increased significantly. Although the N-terminally deleted mutant LEF-1 cannot interact with ß-catenin, it retained the ability to induce EMT. Consistent with these observations, neither the expression of a dominant negative ß-catenin/engrailed chimera, nor the expression of a cytoplasmic domain of E-cadherin that sequesters ß-catenin from binding to LEF/TCF, reversed LEF-1-induced EMT. Together, these data indicated that the nuclear function of ß-catenin was not necessary for the induction of Slug, ZEB1, and ZEB2 expression leading to EMT.

Impaired Wnt/beta-catenin and protein patched homolog 1 signaling in extraocular sebaceous carcinoma: A clinical and histopathological study.

Sebaceous carcinoma (SC) is a rare malignant neoplasm with sebaceous differentiation. SC is classified into eyelid and extraocular SC clinically. Most studies have focused on the eyelid SC in terms of pathogenesis, treatment, and prognosis. In skin, Wnt/beta-catenin and hedgehog signaling are two major pathways in sebaceous differentiation. We aimed to characterize the clinical and histopathological features of extraocular SC and to measure the expression of beta-catenin, lymphoid enhancer-binding factor 1 (LEF1), sonic hedgehog (Shh), and protein patched homolog 1 (PTCH) in extraocular SC. Ten cases of extraocular SC were identified from 2007 to 2020. The clinical features, microscopic findings, and prognosis were analyzed. Immunohistochemical stain for beta-catenin, LEF1, Shh, and PTCH were performed in extraocular SC and other benign sebaceous tumors including sebaceous hyperplasia, sebaceous adenoma, and sebaceoma. The male:female ratio was 4:6. The median onset age was 73.5 years (range, 43-88). Seven patients out of 10 were diagnosed after 60 years. Most extraocular SC were located on the head and neck with indurated plaque. Two patients had concurrent internal cancers and three patients showed lymph node metastasis at time of presentation. Five-year overall-survival was 40%. Beta-catenin was expressed membranously in all sebaceous hyperplasia, but was expressed variably in extraocular SC (1/5). While LEF1 was unequivocally expressed in normal hair follicles, LEF1 expression was absent in all extraocular SC and benign sebaceous tumors. Regarding the sonic hedgehog signaling, Shh and PTCH were all expressed in the cytoplasm of sebaceous hyperplasia, sebaceous adenoma, and sebaceoma. In contrast, PTCH was absent in all cases of extraocular SC and only 50% of the extraocular SC expressed cytoplasmic Shh. To conclude, extraocular SC commonly affects facial skin in the elderly. Inactivated Wnt/beta-catenin and aberrant hedgehog pathway may contribute to the carcinogenesis of extraocular SC. Further studies may be required to elucidate the causative mechanism of these pathways in extraocular SC.

[Diagnostic Utility of LEF1 Immunostain in Cytology Specimens of Solid Pseudopapillary Neoplasm of Pancreas].

Solid pseudopapillary neoplasm (SPN) of the pancreas is a rare neoplasm. Diagnosis of SPN requires an integrated approach with aid of radiology, biopsy, cytology, and immunohistochemical stains. Although morphological features in combination with nuclear positivity of ß-catenin IHC have been the gold standard of SPN diagnosis, but overlapping morphology and immunohistochemical findings with other entities in differential diagnoses such as pancreatic neuroendocrine tumors and pancreatic ductal adenocarcinoma make the diagnosis of SPN difficult particularly in limited cytology specimens. Lymphoid enhancer-binding factor 1 (LEF1), a key player in the Wnt signaling pathway, has shown promising diagnostic utility in SPN in recent literatures. METHODS: In this retrospective study, we evaluated the diagnostic utility of LEF1 IHC in SPN in cytology specimens. LEF1 IHC was performed and compared with ß-catenin, synaptophysin, and chromogranin immunostains in 13 SPN and 23 pancreatic neuroendocrine tumors (PanNETs) cytology cases with retrievable cell blocks. RESULTS: LEF1 was positive in 13 of 13 (100%) SPNs and was negative in all PanNETs (0%). CONCLUSION: LEF1 shows 100% sensitivity and specificity in cytology specimens for SPN and can be valuable immuno-stain in the diagnosis of SPN in cytology cell blocks.

Downregulation of Lymphoid enhancer-binding factor 1 (LEF-1) expression (by immunohistochemistry and/ flow cytometry) in chronic Lymphocytic Leukemia with atypical immunophenotypic and cytologic features.

INTRODUCTION: Lymphoid enhancer-binding factor 1 (LEF-1) overexpression has been recently remarkably reported in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and has shown utility in distinguishing CLL/SLL from other B-cell lymphomas. CLL has a well-defined immunophenotype, yet, some cases of CLL demonstrate atypical morphology/ phenotype reflected by low Matutes score (atypical CLL). Till date, LEF1 expression has not been systematically studied in cases of CLL with atypical features. METHODS: In this study, LEF-1 expression was assessed by two different techniques, (immunohistochemistry and flow cytometry), to investigate the expression profile of LEF-1 in cases of CLL/SLL, in comparison with other low-grade B-lymphomas and CLL with atypical features, including atypical immunophenotype and CLL with increased prolymphocytes or morphologically atypical cells. RESULTS: We found that LEF-1 expression is downregulated in CLL with atypical immunophenotype/features compared to classic CLL; Chi-Square P < .0001. The ratio for LEF-1 expression in malignant B-cells/NK (by flow cytometry) in CLL/SLL with classic immunophenotype was higher than atypical CLL and is significantly higher in other small B-cell lymphomas (P < .01). Absence of LEF-1 expression in CLL/SLL is correlated (P < .05) with downregulation of CD5, CD23, CD200, expression of FMC7, brighter expression of CD79b, brighter expression of surface light chain, increased prolymphocytes and lower Matutes score. CONCLUSION: As downregulation of LEF-1 expression is well correlated with atypical CLL, we suggest adding LEF-1 to Matutes score as a beneficial marker to differentiate classic from atypical CLL LEF-1 could also serve as a potential prognostic indicator for CLL clinical course.

FOXP4 promotes laryngeal squamous cell carcinoma progression through directly targeting LEF­1.

Forkhead box (FOX) proteins are multifaceted transcription factors that have been shown to be involved in cell cycle progression, proliferation and metastasis. FOXP4, a member of the FOX family, has been implicated in diverse biological processes in tumor initiation and progression. However, the molecular mechanisms of FOXP4 in laryngeal squamous cell carcinoma (LSCC) remain unknown. In the present study, differentially expressed transcripts in transforming growth factor­ß­treated TU177 cells were screened using microarrays and it was found that FOXP4 was significantly upregulated. The high expression of FOXP4 was detected in LSCC tissues and cells, and predicted poor prognosis. The role of FOXP4 in laryngeal cancer cell proliferation, migration and invasion was determined by gain­ and loss­of­function assays. Besides, FOXP4 was demonstrated to participate in the epithelial­mesenchymal transition process at the mRNA and protein levels. Mechanically, FOXP4 directly bound to the promoter of lymphoid enhancer­binding factor 1 and activated Wnt signaling pathway, which was confirmed via chromatin immunoprecipitation and luciferase reporter assays. Consequently, these findings provided novel mechanisms of FOXP4 in LSCC progression, which may be considered as potential therapeutic and prognostic targets for LSCC.

MiR-34a-5p Inhibits Proliferation, Migration, Invasion and Epithelial-mesenchymal Transition in Esophageal Squamous Cell Carcinoma by Targeting LEF1 and Inactivation of the Hippo-YAP1/TAZ Signaling Pathway.

Background: Our previous studies reported that lymphoid enhancer-binding factor 1 (LEF1) was upregulated in esophageal squamous cell carcinoma (ESCC) and the positive expression of LEF1 was correlated with aberrant clinicopathological characteristics in ESCC patients. However, the upstream mechanism of regulating LEF1 is not clear fully. In this study, we explored the role of miR-34a-5p in ESCC and the possible regulatory mechanism. Methods: In this study, we applied western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), bioinformatics analysis, a luciferase reporter assay, and a series of functional assays to show the potential role of miR-34a-5p in regulating LEF1 in ESCC. Results: By various functional assays, we demonstrated that LEF1 promoted proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in ESCC cells. By bioinformatics analysis and luciferase reporter assay, miR-34a-5p was identified for directly targeting LEF1. Then we investigated the expression of miR-34a-5p and LEF1 in ESCC. As a result, miR-34a-5p was downregulated while LEF1 was upregulated in ESCC tissue and cell lines. Overexpression of miR-34a-5p could inhibit proliferation, migration, invasion and EMT of ESCC cells. The rescue experiment showed that re-expression of LEF1 reversed the suppressive effect caused by miR-34a-5p. At last, we found that miR-34a-5p could suppress Hippo-YAP1/TAZ signaling pathway in ESCC. Conclusion: Our results indicate miR-34a-5p inhibits proliferation, migration, invasion and EMT in ESCC by targeting LEF1 and suppressing the Hippo-YAP1/TAZ signaling pathway, which may provide a new antitumor strategy to delay ESCC progress.

Attenuation of Wnt/ß-catenin signaling in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN) are rare but life-threatening severe cutaneous adverse reactions. Current studies have suggested that the pathobiology of drug-mediated SJS/TEN involves a dysregulation of cellular immunity with overwhelming activation of cytotoxic T lymphocytes. The canonical Wnt signaling pathway plays important roles in T cell development and activation, which may provide potential avenues for alleviating dysregulated immunity in SJS/TEN. In this study, we aimed to assess the implication of Wnt signaling in drug-reactive T cells in SJS/TEN. We showed downregulation of Wnt signaling components, including T cell factor 1 (TCF-1)/lymphoid enhancer binding factor 1 (LEF-1) transcription factors, in SJS/TEN patients, suggesting that canonical Wnt signaling is regulated during cytotoxic T cell responses in SJS/TEN. Further analyses demonstrated that engagement of the T cell receptor by antigen encounter and treatment of a prognostic marker of SJS/TEN, IL-15, in vitro led to the downregulation of LEF-1 and TCF-1 expression in CD8+ T cells. Enhancement of Wnt signaling by adding the Wnt activators attenuated ex vivo activation of drug-specific T cells from SJS/TEN patients, indicating a functional involvement of Wnt signaling in the pathomechanism of SJS/TEN. These findings provide additional insight into the immunopathogenesis and therapeutic intervention of this devastating condition.

miR­300 regulates tumor proliferation and metastasis by targeting lymphoid enhancer­binding factor 1 in hepatocellular carcinoma.

Accumulating evidence indicates that microRNAs (miRNAs) have a critical role in cell proliferation and metastasis in hepatocellular carcinoma (HCC). However, the effect of miR­300 on the development and progression of HCC remains unclear. In the present study, it was observed that miRNA (miR)­300 expression was significantly decreased in HCC cell lines compared with normal liver cells. Furthermore, we detected the effects of miR­300 on cell proliferation and apoptosis, cell cycle, migration and invasion by using MTT, colony formation assay, wound healing, Transwell assay and flow cytometry methods, respectively. The results demonstrated that miR­300 overexpression inhibited proliferation, induced apoptosis and G1/S cell cycle arrest, and suppressed migration and invasion in Huh­7 cells, whereas miR­300 silencing promoted the proliferation, migration and invasion of Hep3B cells. Mechanistically, the transcription factor lymphoid enhancer­binding factor 1 (LEF­1), which was verified as a direct target gene of miR­300, promoted cell proliferation, migration and invasion and mediates the effects of miR­300 on HCC cells. In addition, low expression of miR­300 and high expression of LEF­1 in HCC tissues were found to be associated with poor prognosis of patients with HCC. These findings indicate that miR­300 may be a potential prognostic predictor and therapeutic target for patients with HCC.

MicroRNA-6852 suppresses glioma A172 cell proliferation and invasion by targeting LEF1.

microRNA (miR)-6852 has been demonstrated to suppress the progression of gastric, colorectal and cervical cancer. The mechanism by which miR-6852 regulates glioma cells is yet to be elucidated. In the present study, reverse transcription-quantitative PCR analysis was used and the results demonstrated that miR-6852 expression was reduced in glioma tissues and cells. Cell counting kit-8 and transwell assay analysis indicated that proliferation, migration and invasion of A172 cells in the miR-6852 mimic group were lower than in the miR-NC group. Compared with the Inh-NC group, A172 cells of the Inh-miR-6852 group exhibited higher proliferation, migration and invasion. Additionally, the results indicated that lymphoid enhancer binding factor 1 (LEF1) was directly inhibited by miR-6852 and LEF1 expression was negatively correlated with miR-6852 expression in glioma tissues. Furthermore, the restoration of LEF1 reversed the effects of the miR-6852 mimics. The present findings suggested that miR-6852 inhibited glioma cells proliferation, migration and invasion by targeting the suppression of LEF1.

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.

The transcription factor LEF1 promotes tumorigenicity and activates the TGF-ß signaling pathway in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the most difficult subtype of esophageal cancer to treat due to the paucity of effective targeted therapy. ESCC is believed to arise from cancer stem cells (CSCs) that contribute to metastasis and chemoresistance. Despite advances in diagnosis and treatment, the prognosis of ESCC patients remains poor. METHODS: In this study, we applied western blot, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, RNA-Seq analysis, luciferase reporter assay, Chip-qPCR, bioinformatics analysis, and a series of functional assays to show the potential role of LEF1 in regulating esophageal CSCs. RESULTS: We found that the overexpression of LEF1 was associated with aberrant clinicopathological characteristics and the poor prognosis of ESCC patients. In addition, the elevated expression of LEF1 and OV6 was significantly associated with aberrant clinicopathological features, and poor patient prognosis. Moreover, the overexpression of LEF1 was observed in esophageal CSCs purified by the magnetic sorting of adherent and spheroidal ESCC cells. The increased level of LEF1 in CSCs facilitated the expression of CSC markers, stem cell-like properties, resistance to chemotherapy, and tumorigenicity and increased the percentage of CSCs in ESCC samples. Conversely, the knockdown of LEF1 significantly diminished the self-renewal properties of ESCC. We showed that LEF1 played an important mechanical role in activating the TGF-ß signaling pathway by directly binding to the ID1 gene promoter. A positive association between LEF1 and ID1 expression was also observed in clinical ESCC samples. CONCLUSION: Our results indicate that the overexpression of LEF1 promotes a CSC-like phenotype in and the tumorigenicity of ESCC by activating the TGF-ß signaling pathway. The inhibition of LEF1 might therefore be a novel therapeutic target to inactivate CSCs and inhibit tumor progression.

The usefulness of lymphoid enhancer-binding factor 1 and androgen receptor in diagnosing solid pseudopapillary neoplasm of the pancreas on cytopathology.

BACKGROUND: Solid pseudopapillary neoplasm (SPN) is an uncommon tumor that is challenging to diagnose on cytology due to morphologic overlap with other pancreatic neoplasms. Recently, putative diagnostic markers for SPN have been reported in the surgical pathology literature, with nuclear positivity for lymphoid enhancer-binding factor 1 (LEF1) and androgen receptor (AR) identified in >90% and >80% of cases, respectively. In the current study, the authors sought to evaluate the sensitivity and specificity of LEF1 and AR on SPN cytology specimens and available corresponding surgical resection specimens. METHODS: Immunohistochemistry was performed using monoclonal antibodies against LEF1 and AR on 19 SPN cytology cases and 15 corresponding follow-up surgical resection specimens from 2 institutions. To evaluate specificity, the authors stained 23 non-SPN tumors diagnosed on cytology with corresponding surgical specimens (4 acinar cell carcinomas, 9 pancreatic neuroendocrine tumors, and 10 ductal adenocarcinomas). Positivity for LEF1 and AR was defined as any nuclear staining within neoplastic nuclei. RESULTS: LEF1 was found to be positive in 18 of 19 cytology cases (94.7%) and 15 of 15 corresponding surgical resection specimens (100%). AR was positive in 4 of 16 cytology cases (25.0%) and 4 of 15 corresponding surgical resection specimens (26.7%). Among non-SPN tumors, LEF1 demonstrated a specificity of 87% whereas the specificity for AR was 100%. CONCLUSIONS: LEF1 for SPN on cytology material was found to demonstrate a sensitivity of 94.7% and a specificity of 87%. Although AR was found to have a specificity of 100%, its sensitivity was lower (25%). LEF1 could be a valuable immunostain on cytology cell block material for the diagnosis of SPN. However, the same may not hold true for AR.

Overexpression of MYC binding protein promotes invasion and migration in gastric cancer.

Gastric cancer (GC) is the second leading cause of cancer-associated mortality worldwide. Although the mortality rate of patients with GC has improved, it remains a significant health issue. The MYC proto-oncogene protein serves key roles in cellular proliferation, differentiation, transformation and apoptosis. Previous studies have identified the abnormal expression of MYC-binding protein (MYCBP) during tumorigenesis in multiple types of cancer. Furthermore, evidence demonstrates that the abnormal expression of MYCBP contributes to the invasion and migration of human cancer types, including colon cancer and glioma; however, its influence on GC remains unclear. In the present study, the expression of MYCBP in GC cells and tissues was analyzed by reverse transcription-quantitative polymerase chain reaction. Additionally, GC cell lines were transfected with small interfering RNAs against MYCBP or lymphoid enhancer-binding factor 1 (LEF-1) and assessed by in vitro transwell migration and invasion assays. The results indicated that the expression of MYCBP in GC cells and tissues was markedly increased compared with a normal gastric epithelial cell line and adjacent normal gastric mucosal tissues, respectively. Furthermore, MYCBP downregulation notably inhibited the metastatic capacity of GC cells, and LEF-1 knockdown was found to downregulate the expression of MYCBP. On the basis of the findings of the present study, MYCBP may be a direct target of the ß-catenin/LEF-1 pathway via binding LEF-1, and could potentially be used as a biomarker for the diagnosis and prognosis of GC.