Expression of lymphoid enhancer-binding factor 1 in breast fibroepithelial lesions.

Phyllodes tumors (PTs) are rare epithelial-mesenchymal tumors of the breast with malignant potential. Here, we evaluate the nuclear expression of lymphoid enhancer-binding factor 1 (LEF-1), a transcription factor downstream of Wnt/ß-catenin signaling, in fibroepithelial lesions of the breast. Excised fibroepithelial lesions of the breast were retrospectively reviewed, blinded to the original diagnosis, and classified according to World Health Organization (WHO) criteria. A tissue microarray (TMA) was composed with two representative cores from each case, including 24 benign lesions, 11 borderline phyllodes, and 8 malignant PTs. ß-Catenin, LEF-1, p120, and E-cadherin immunohistochemistry was performed on the TMA, and staining was quantified. The malignant/borderline PTs showed higher stromal LEF-1 expression than benign tumors (P < 0.001). Stromal cells expressed LEF-1 in 100% (16/16 of core TMA) of malignant phyllodes, compared with 73% (16/22) borderline and 27% (13/48) benign tumors. The average LEF-1 H-score was 24.9, 6.1, and 1.5 for malignant, borderline, and benign tumors, respectively. Nuclear expression of ß-catenin in the stromal component was more often seen in malignant than in borderline and benign tumors (44% versus 32% and 23%, respectively). Nine TMA cores of malignant tumors without nuclear ß-catenin staining demonstrated LEF-1 expression. Both LEF-1 and nuclear ß-catenin showed expression in the majority of borderline/malignant PTs suggesting a biological progression of Wnt/ß-catenin pathway activation in the stromal component from benign to malignant tumors. Inhibitors for the Wnt/ß-catenin pathway may provide alternative treatment options in the future for malignant or metastatic PTs.

Lymphoid enhancer binding factor 1 (LEF1) expression is significantly higher in Hodgkin lymphoma associated with Richter syndrome relative to de novo classic Hodgkin lymphoma.

Lymphoid enhancer binding factor 1 (LEF1) is consistently upregulated in chronic lymphocytic leukemia (CLL) and in a subset of large B cell lymphoma. Knowledge of LEF1 expression in Hodgkin lymphoma is limited. In this study, we used immunohistochemistry to survey LEF1 expression in various subsets of Hodgkin lymphoma, de novo classic Hodgkin lymphoma (CHL) (n = 43), Hodgkin lymphoma associated with Richter syndrome (HL-RS) (n = 20), and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) (n = 9). LEF1 expression was significantly higher in HL-RS compared with de novo CHL (12/20, 60% vs. 12/43, 28%; p = 0.0248). Only a single case (1/9; 11%) of NLPHL showed LEF1 expression. Epstein-Barr virus encoded RNA (EBER) was detected in 17 (40%) cases of de novo CHL and 14 (70%) HL-RS. Notably, we identified a correlation between LEF1 expression and EBER positivity (p = 0.0488). We concluded that LEF1 is commonly positive in CHL but not in NLPHL, and such a distinction may be helpful in this differential diagnosis. The higher frequency of LEF1 upregulation in HL-RS relative to de novo CHL suggests that these neoplasms might have different underlying pathogenic mechanisms and warrants further investigation.

Diagnostic Utility of LEF1 Immunohistochemistry in Differentiating Deep Penetrating Nevi From Histologic Mimics.

Deep penetrating nevi (DPNs) are intermediate grade lesions which have the capacity to recur, metastasize, or progress to melanoma. Differentiating DPN from other melanocytic lesions including blue and cellular blue nevi can be diagnostically challenging, and markers to distinguish these entities can be useful. Mutations of the ß-catenin and mitogen-activated protein kinase pathways have recently been elucidated as distinctive of DPN. This pathway can subsequently activate lymphoid enhancer-binding factor 1 (LEF1), a transcription factor shown to facilitate the epithelial-mesenchymal transition to propagate tumorigenesis. Seventy-two cases in total were examined on hematoxylin and eosin sections and with ß-catenin and LEF1 immunohistochemistry. This included: DPN (14), cellular blue nevi (19), blue nevi (15), congenital melanocytic nevi (12), and melanoma (12). Nuclear expression of LEF1, present throughout the entire depth of the lesion, was noted in 13/14 (93%) of DPN, 0/19 (0%) of cellular blue nevi, 0/15 (0%) of blue nevi, 1/12 (8%) of congenital melanocytic nevi, and 9/12 (75%) of melanoma cases. Nuclear expression of ß-catenin, present throughout the entire depth of the lesion, was noted in 14/14 (100%) of DPN, 0/18 (0%) of cellular blue nevi, 0/15 (0%) of blue nevi, 1/12 (8%) of congenital melanocytic nevi, and 1/12 (8%) of melanoma cases. A majority of congenital melanocytic nevi demonstrated a gradient of LEF1 and ß-catenin expression with more intense staining superficially and loss of staining with increasing depth. Deep, uniform nuclear LEF1 combined with ß-catenin immunohistochemical staining can be useful in distinguishing DPN from histologic mimics.

The expression of KI-67 and LEF-1 in patients after breast cancer resection and its effects on patients' prognosis.

PURPOSE: To investigate the expression of KI-67 and LEF-1 in patients after breast cancer resection and its effects on patients' prognosis. METHODS: A total of 89 breast cancer patients admitted to the first affiliated Hospital of Shantou University Medical College from January 2010 to February 2013 were enrolled as the study group, and 76 healthy individuals were enrolled as the control group. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of KI-67 and LEF-1 in the serum. The relationship of the two indexes and clinicopathological data of the breast cancer patients were analyzed. In addition, the diagnostic value of KI-67 and LEF-1 in breast cancer patients was analyzed by receiver operating characteristic (ROC) curves, and their diagnostic value in the postoperative 5-year recurrence was also analyzed. Furthermore, the expression of KI-67 and LEF-1 in patients with postoperative recurrent breast cancer within 5 years was evaluated. RESULTS: The expression of KI-67 and LEF-1 in the study group was higher than in the control group (p<0.05), and the expression of KI-67 and LEF-1 was significantly related to the tumor size and lymph node metastasis (both p<0.05). ROC curve showed that the area under the curve (AUC) of the diagnostic value of KI-67 and LEF-1 for breast cancer patients was 0.860 and 0.858 respectively, and that of the diagnostic value KI-67 combined with LEF-1 for breast cancer patients was 0.924. In addition, the AUC of the diagnostic value of KI-67 and LEF-1 for the recurrence of breast cancer within 5 years was 0.699 and 0.651, respectively, and that of diagnostic value of KI-67 combined with LEF-1 for the recurrence of breast cancer within 5 years was 0.758. The expression of KI-67 and LEF-1 in patients with recurrent disease within 5 years after operation was higher than in patients without recurrence. CONCLUSION: The expression of KI-67 and LEF-1 in breast cancer patients is significantly higher than in healthy individuals, which has certain diagnostic value in breast cancer. The expression of the two indexes is related to tumor size and lymph node metastasis, and the survival of patients with high expression of KI-67 and LEF-1 is worse.

Concurrent Wnt pathway component expression in breast and colorectal cancer.

Wnt signaling pathway regulates important cell functions such as proliferation and migration and is frequently deregulated in colorectal and breast cancer. Thus, it constitutes an attractive therapeutic target with many drugs being investigated in clinical trials. Eighty-two breast and 102 colorectal carcinomas were analyzed for: relative mRNA expression levels of Wnt pathway components namely Wnt3 ligand, Frizzled 7 receptor and LEF1 transcriptional factor, their concurrent expression patterns and their correlation with clinicopathological features. Regarding breast carcinomas, increased relative mRNA expression levels of WNT3 were found in 54 % of cases whereas decreased relative mRNA expression levels were observed in FZD7 and LEF1 in 82 % and 43 % of cases, respectively. Expression levels of WNT3 were significantly correlated with tumour grade (p = 0.021) in breast cancer. As far as colorectal carcinomas are concerned, increased relative mRNA expression levels of WNT3, FZD7 and LEF1 were found in 60 %, 37 % and 48 % of cases respectively. A statistically significant correlation emerged between LEF1expression levels and pT-category (p = 0.027), suggesting a possible association with tumour aggressiveness in colorectal carcinomas. Statistically significant linear correlations were observed between the expression of WNT3/LEF1 (R = 0.233, p = 0.035) and FZD7/LEF1 (R = 0.359, p = 0.001) in breast carcinomas as well as in colorectal carcinomas (R = 0.536, p < 0.01 and R = 0.210, p = 0.034) respectively. Our results demonstrate a possible clinical significance of Wnt pathway gene expression levels in both tumour types. The distinct expression patterns and simultaneous expression of the investigated genes underscore the complexity of this pathway in breast and colorectal carcinogenesis and highlights the necessity of patient selection with regard to the effectiveness of Wnt pathway inhibitors.

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.

Transforming growth factor-ß1 enhances proliferative and metastatic potential by up-regulating lymphoid enhancer-binding factor 1/integrin αMß2 in human renal cell carcinoma.

Renal cell carcinoma (RCC) is a kind of malignant tumor with high recurrence, and it is urgent to find molecular markers for diagnosis and prognosis of RCC. Our study investigated the expression and function of integrin αMß2 in RCC cells, aiming to understand the role of integrin αMß2 in RCC and develop new therapeutic target for RCC. Overexpression and knockdown of lymphoid enhancer-binding factor 1 (LEF1) were performed using vector containing full-length cDNA and via siRNA technology, respectively. The expressions of mRNA and protein were detected by RT-PCR and Western blot, respectively. Proliferation of RCC cell was analyzed using WST-1 assay, and metastasis of RCC cell was evaluated using the transwell system. Our results demonstrated that LEF1 and integrin αMß2 were up-regulated in RCC cells via TGF-ß1-dependent mechanism, and LEF1 together with ß-catenin directly increased integrin αMß2 level. On the other hand, TGF-ß1-induced proliferation, migration and invasion were suppressed by function-blocking antibody against integrin αMß2 in RCC cells. In addition, integrin αMß2 is crucial for LEF1 mediated cell invasion by regulating MMP-2, MMP-9 and calpain-2 secretion in RCC cells. LEF1/integrin αMß2 expression was regulated by TGF-ß1, and LEF1/integrin αMß2 was involved in TGF-ß1's improvement effects on the proliferation and metastasis of RCC. Blocking integrin αMß2 activity could be a therapeutic option for patients with advanced RCC.

Consistent LEF-1 and MYB Immunohistochemical Expression in Human Papillomavirus-Related Multiphenotypic Sinonasal Carcinoma: A Potential Diagnostic Pitfall.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is a distinct, newly-described sinonasal tract neoplasm characterized by a salivary gland tumor-like appearance with myoepithelial and ductal cells, frequent surface squamous dysplasia, and relatively indolent behavior. When considering a diagnosis of HMSC, aggressive high-grade salivary gland carcinomas, particularly those with a basaloid morphology such as basal cell adenocarcinoma and adenoid cystic carcinoma, enter the differential diagnosis. The full morphologic and immunophenotypic profile of HMSC continues to be unraveled. In this series of ten cases, we demonstrate that this tumor has consistent, strong immunohistochemical expression of LEF-1 yet lacks nuclear expression of ß-catenin, and also has consistent yet variable expression of MYB protein. While LEF-1 expression may be a useful diagnostic adjunct, it can also be a pitfall, as other salivary tumors such as basal cell adenocarcinoma have been previously shown to express LEF-1. Additionally, MYB protein expression is not a discriminatory marker when trying to separate HMSC from adenoid cystic carcinoma.

Tissue engineering of human hair follicles using a biomimetic developmental approach.

Human skin constructs (HSCs) have the potential to provide an effective therapy for patients with significant skin injuries and to enable human-relevant drug screening for skin diseases; however, the incorporation of engineered skin appendages, such as hair follicles (HFs), into HSCs remains a major challenge. Here, we demonstrate a biomimetic approach for generation of human HFs within HSCs by recapitulating the physiological 3D organization of cells in the HF microenvironment using 3D-printed molds. Overexpression of Lef-1 in dermal papilla cells (DPC) restores the intact DPC transcriptional signature and significantly enhances the efficiency of HF differentiation in HSCs. Furthermore, vascularization of hair-bearing HSCs prior to engraftment allows for efficient human hair growth in immunodeficient mice. The ability to regenerate an entire HF from cultured human cells will have a transformative impact on the medical management of different types of alopecia, as well as chronic wounds, which represent major unmet medical needs.

Repression of WT1-Mediated LEF1 Transcription by Mangiferin Governs ß-Catenin-Independent Wnt Signalling Inactivation in Hepatocellular Carcinoma.

BACKGROUND/AIMS: The development of hepatocellular carcinoma (HCC) is a complex process which involves deregulation of multiple signalling pathways. The hyper-activation of Wnt signalling promotes sustained expansion, invasion, and neovascularization of HCC. Mangiferin, a natural small molecule present in Mangifera indica L. has been shown to inactivate ß-catenin, which is an indispensable regulator in Wnt pathway. Our study aimed to determine whether mangiferin has any inhibitory effect on HCC and examine how it modulates Wnt signalling. METHODS: The tumour inhibitory effect of mangiferin was examined by in vitro cellular models and an in vivo orthotopic HCC implantation model. The genes responsible for mangiferin-mediated anti-HCC were delineated by polymerase chain reaction (PCR) microarray. The expression of target genes was further determined by quantitative PCR and immuno-blotting assays. The binding capacity of Wilms' tumour 1 (WT1) to the lymphoid enhancer-binding factor 1 (LEF1) promoter was confirmed by chromatin immunoprecipitation-qPCR. RESULTS: Oral administration of mangiferin inhibited orthotopic tumour growth. Cellular investigations confirmed the dose-dependent inhibition of mangiferin on HCC expansion and invasion. PCR array combined with Gene Ontology analysis revealed that the Wnt pathway was the predominant target of mangiferin and LEF1 was the most reduced gene in the Wnt pathway. Overexpression of LEF1 diminished repression of Wnt signalling and reduced proliferation activity in mangiferin-treated HCC cells. The mangiferin-mediated down-regulation of LEF1 was independent of ß-catenin but associated with WT1 protein. WT1 knock-in in HCC cells further enhanced LEF1 expression. Chromatin immunoprecipitation assays revealed that the mangiferin induced repression of LEF1 was associated with decreased occupancy of WT1 on the LEF1 promoter. CONCLUSION: Our study identifies a novel mechanism of hepatocellular carcinoma inhibition through ß-catenin-independent Wnt signalling, which is regulated by WT1-associated LEF1 repression. The study also highlights mangiferin as a promising Wnt inhibitor for HCC treatment.

Gene expression profiling of low-grade endometrial stromal sarcoma indicates fusion protein-mediated activation of the Wnt signaling pathway.

OBJECTIVE: Low-grade endometrial stromal sarcomas (LGESS) harbor chromosomal translocations that affect proteins associated with chromatin remodeling Polycomb Repressive Complex 2 (PRC2), including SUZ12, PHF1 and EPC1. Roughly half of LGESS also demonstrate nuclear accumulation of ß-catenin, which is a hallmark of Wnt signaling activation. However, the targets affected by the fusion proteins and the role of Wnt signaling in the pathogenesis of these tumors remain largely unknown. METHODS: Here we report the results of a meta-analysis of three independent gene expression profiling studies on LGESS and immunohistochemical evaluation of nuclear expression of ß-catenin and Lef1 in 112 uterine sarcoma specimens obtained from 20 LGESS and 89 LMS patients. RESULTS: Our results demonstrate that 143 out of 310 genes overexpressed in LGESS are known to be directly regulated by SUZ12. In addition, our gene expression meta-analysis shows activation of multiple genes implicated in Wnt signaling. We further emphasize the role of the Wnt signaling pathway by demonstrating concordant nuclear expression of ß-catenin and Lef1 in 7/16 LGESS. CONCLUSIONS: Based on our findings, we suggest that LGESS-specific fusion proteins disrupt the repressive function of the PRC2 complex similar to the mechanism seen in synovial sarcoma, where the SS18-SSX fusion proteins disrupt the mSWI/SNF (BAF) chromatin remodeling complex. We propose that these fusion proteins in LGESS contribute to overexpression of Wnt ligands with subsequent activation of Wnt signaling pathway and formation of an active ß-catenin/Lef1 transcriptional complex. These observations could lead to novel therapeutic approaches that focus on the Wnt pathway in LGESS.

CDX2 and LEF-1 expression in pilomatrical tumors and their utility in the diagnosis of pilomatrical carcinoma.

BACKGROUND: The Wnt signaling pathway has been implicated in the pathogenesis of pilomatrical tumors. Lymphoid enhancer-binding factor 1 (LEF-1) is a downstream component of this pathway, and Caudal-related homeobox transcription factor 2 (CDX2) has been postulated to regulate it, but little is known about expression of these transcription factors in pilomatrical tumors. METHODS: Immunohistochemistry for CDX2, ß-catenin, LEF-1, CK19, CK5, Special AT-rich sequence- binding protein 2 (SATB2), cadherin 17 and androgen receptor was performed on pilomatricomas (PMs) (N = 12), pilomatrical carcinomas (PMCAs) (N = 12) and non-pilomatrical cutaneous tumors (N = 18). RESULTS: PMs and PMCAs were positive for CDX2 (9/12 PMs, sensitivity = 75%, specificity = 100%; 11/12 PMCAs, sensitivity = 92%, specificity = 100%; P < 0.01), ß-catenin (12/12 PMs, sensitivity = 100%, specificity = 94%; 10/12 PMCAs, sensitivity = 83%, specificity = 94%; P < 0.01) and LEF-1 (12/12 PMs, sensitivity = 100%, specificity = 56%; 12/12 PMCAs, sensitivity = 100%, specificity = 56%; P < 0.01). CDX2 expression was commonly focal, within a discrete subpopulation of squamoid cells. The LEF-1 expression pattern was different and discernable between pilomatrical tumors (strong, diffuse) and non-pilomatrical tumors (weak, patchy). CONCLUSION: This study reaffirms the importance of the Wnt signaling pathway in the tumorigenesis of pilomatrical tumors, and this introduces CDX2 as a possible regulator and marker of pilomatrical tumorigenesis. LEF-1 and CDX2 performed at least as well as ß-catenin, if not better when taking into account expression pattern, as a diagnostic marker for PMCA, and should be considered in the workup of ambiguous primitive-appearing cutaneous tumors.

MiR-557 works as a tumor suppressor in human lung cancers by negatively regulating LEF1 expression.

MicroRNAs play an important role in regulating post-transcriptional gene expression in the progression of various human cancers. In this study, we investigated the role of microRNA-557 in human lung cancer cells. The molecular mechanism of microRNA-557 was also clarified in the proliferation and invasion of human lung cancer cells. Our results showed microRNA-557 levels were obviously decreased in clinical lung cancer specimens and lung cancer cell lines. Cell viability of A549 and NCI-H460 cells transfected with microRNA-557 mimics was significantly decreased than those transfected with negative control mimics. MicroRNA-557 promoted cell death of A549 and NCI-H460 but did not affect the cell apoptosis of lung cancer cells. Overexpression of microRNA-557 inhibited cell invasion of A549 and NCI-H460 cells. TargetScan analysis showed that microRNA-557 might target 3' untranslated region of lymphocyte enhancement factor 1, and the western blotting results showed that transfection of microRNA-557 mimics significantly decreased the levels of lymphocyte enhancement factor 1 in A549 and H460 cells. MicroRNA-557 might work as a tumor suppressor by negatively regulating the expression of lymphocyte enhancement factor 1 in lung cancer cells.

Diagnostic Utility of Lymphoid Enhancer Binding Factor 1 Immunohistochemistry in Small B-Cell Lymphomas.

OBJECTIVES: Recent studies have shown that lymphoid enhancer binding factor 1 (LEF1) is a useful marker for chronic lymphocytic B-cell leukemia (CLL)/small lymphocytic lymphoma. Yet, it is not still being widely used in a diagnostic setting. In this study, we document the experience with LEF1 immunohistochemistry during routine diagnostics. METHODS: In total, 191 B-cell lymphoma cases from Hammersmith Hospital, Imperial College NHS Healthcare Trust (London, UK) were investigated by immunohistochemistry for LEF1 during routine diagnostic workup. These cases included both bone marrow trephines and lymph node biopsy specimens. The monoclonal antibody clone EPR2029Y was used. RESULTS: LEF1 expression was strong and diffuse (>70% of cells) in most cases. Few CLL cases showed a staining in proliferation centers only. Seventy-seven of 80 CLL cases expressed LEF1. Other entities expressing LEF1 included one of 38 follicular lymphomas, two of 33 marginal zone lymphomas, and one diffuse large B-cell lymphoma with a background of follicular lymphoma grade 3B. Sensitivity for LEF1 for the diagnosis of CLL was 0.96, and specificity was 0.93. CONCLUSIONS: In this study, we could demonstrate the diagnostic utility of LEF1. LEF1 is a sensitive and specific marker for CLL and is helpful in the diagnosis of diagnostically challenging small B-cell lymphomas.

Expression and colocalization of ß-catenin and lymphoid enhancing factor-1 in prostate cancer progression.

The purpose of this study was to objectively investigate ß-catenin and LEF1 abundance, subcellular localization, and colocalization across benign and staged prostate cancer (PCa) specimens. A tissue microarray containing tumor-adjacent histologically benign prostate tissue (BPT; n = 48 patients), high-grade prostatic intraepithelial neoplasia (HGPIN; n = 25), localized PCa (n = 42), aggressive PCa (n = 31), and metastases (n = 22) was stained using multiplexed immunohistochemistry with antibodies toward E-cadherin, ß-catenin, and LEF1. Multispectral imaging was used for quantitation, and protein expression and colocalization was evaluated across PCa progression. Stromal nuclear ß-catenin abundance was greater in HGPIN and PCa compared with BPT (P < .05 for both), and epithelial nuclear ß-catenin abundance was lower in metastatic PCa than in BPT (P < .05 for both). Epithelial and stromal nuclear LEF1 abundance was greater in HGPIN compared with BPT, whereas epithelial nuclear LEF1 was also greater in metastases. The proportion of epithelial and stromal nuclear double-positive ß-catenin(+)/LEF1(+) cells was greater in HGPIN compared with BPT. In addition, the proportion of epithelial ß-catenin(+)/LEF1(+) cells was greater in localized PCa and metastases compared with BPT. A significant amount of stromal cells were positive for LEF1 but not ß-catenin. ß-Catenin and LEF1 abundance were negatively correlated in the epithelium (P < .0001) but not the stroma (P > .05). We conclude that ß-catenin and LEF1 colocalization is increased in HGPIN and metastasis relative to BPT, suggesting a role for ß-catenin/LEF1-mediated transcription in both malignant transformation and metastasis of PCa. Furthermore, our results suggest that LEF1 abundance alone is not a reliable readout for ß-catenin activity in prostate tissues.

Conditional deletion of Sox17 reveals complex effects on uterine adenogenesis and function.

The importance of canonical Wnt signaling to murine uterine development is well established. Mouse models in which uterine-specific Wnt ligands, ß-catenin, or Lef1 are disrupted result in failure of postnatal endometrial gland development. Sox17 is a transcription factor characterized in numerous tissues as an antagonist of Wnt signaling. Thus, we hypothesized that conditional ablation of Sox17 would lead to hyperproliferation of endometrial glands in mice. Contrary to our prediction, disruption of Sox17 in epithelial and stromal compartments led to inhibition of endometrial adenogenesis and a loss of reproductive capacity. Epithelium-specific Sox17 disruption resulted in normal adenogenesis although reproductive capacity remained impaired. These findings suggest that non-epithelial, Sox17-positive cells are necessary for adenogenesis and that glands require Sox17 to properly function. To our knowledge, these findings are the first to implicate Sox17 in endometrial gland formation and reproductive success. The data presented herein underscore the importance of studying Sox17 in uterine homeostasis and function.

Lgr4 controls specialization of female gonads in mice.

Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is a type of membrane receptor with a seven-transmembrane structure. LGR4 is homologous to gonadotropin receptors, such as follicle-stimulating hormone receptor (Fshr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr). Recently, it has been reported that Lgr4 is a membrane receptor for R-spondin ligands, which mediate Wnt/beta-catenin signaling. Defects of R-spondin homolog (Rspo1) and wingless-type MMTV integration site family, member 4 (Wnt4) cause masculinization of female gonads. We observed that Lgr4(-/-) female mice show abnormal development of the Wolffian ducts and somatic cells similar to that in the male gonads. Lgr4(-/-) female mice exhibited masculinization similar to that observed in Rspo1-deficient mice. In Lgr4(-/-) ovarian somatic cells, the expression levels of lymphoid enhancer-binding factor 1 (Lefl) and Axin2 (Axin2), which are target genes of Wnt/beta-catenin signaling, were lower than they were in wild-type mice. This study suggests that Lgr4 is critical for ovarian somatic cell specialization via the cooperative signaling of Rspo1 and Wnt/beta-catenin.

Splicing of a non-coding antisense transcript controls LEF1 gene expression.

In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.

Flow cytometric analysis of lymphoid enhancer-binding factor 1 in diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma.

OBJECTIVES: Nuclear overexpression of lymphoid enhancer-binding factor 1 (LEF1) assessed by immunohistochemistry has been shown to be highly associated with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) among small B-cell lymphomas. The purpose of this study was to evaluate the utility of flow cytometric analysis of LEF1 in the diagnosis of CLL/SLL. METHODS: Normal peripheral blood was used to validate the test. Flow cytometric analysis of LEF1 was performed in 64 patient samples qualitatively and quantitatively by comparing the staining intensity and the ratios of the median fluorescence intensities (MFIs) of LEF1 in B cells of interest to the internal reference cell populations. The results were correlated with the pathologic diagnosis. RESULTS: Proper sample processing ensured sufficient separation of positive LEF1 staining in T cells from negative staining in normal B and natural killer (NK) cells. Qualitative analysis of patient samples showed that all 25 cases of CLL/SLL but none of the other small B-cell lymphomas were positive for LEF1. Using a B/NK MFI ratio of 1.5 and B/T MFI ratio of 0.45 separated CLL/SLL cases from non-CLL lymphomas. CONCLUSIONS: Flow cytometric analysis of LEF1 is sufficient to differentiate CLL/SLL from other small B-cell lymphomas and may serve as a useful tool in the diagnosis of CLL/SLL.

MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway.

Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting ß-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators ß-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify ß-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging.