Lymphoid Enhancer Factor 1 Expression Research Articles

Utility of LEF1 to differentiate desmoid fibromatosis from its histologic mimics.

Diagnosis of desmoid-type fibromatosis (DF) may be challenging on biopsy due to morphologic overlap with reactive fibrosis (scar) and other uniform spindle cell neoplasms. Evaluation of nuclear β-catenin, a surrogate of Wnt pathway activation, is often difficult in DF due to weak nuclear expression and high background membranous/cytoplasmic staining. Lymphoid enhancer-factor 1 (LEF1) is a recently characterized effector partner of β-catenin which activates the transcription of target genes. We investigated the performance of LEF1 and β-catenin immunohistochemistry in a retrospective series of 156 soft tissue tumors, including 35 DF, 3 superficial fibromatosis, and 121 histologic mimics (19 soft tissue perineurioma, 8 colorectal perineurioma, 4 intraneural perineurioma, 26 scars, 23 nodular fasciitis, 6 low-grade fibromyxoid sarcomas, 6 angioleiomyomas, 5 neurofibromas, 5 dermatofibrosarcoma protuberans, 3 low-grade myofibroblastic sarcomas, 3 synovial sarcomas, 3 inflammatory myofibroblastic tumors, 2 schwannomas, and 1 each of Gardner-associated fibroma, radiation-associated spindle cell sarcoma, sclerotic fibroma, dermatofibroma, and glomus tumor). LEF1 expression was not only seen in 33/35 (94%) of DF but also observed in 19/23 (82%) nodular fasciitis, 7/19 (37%) soft tissue perineurioma, 2/3 (66%) synovial sarcoma, and 6/26 (23%) scar, as well as in 1 radiation-associated spindle cell sarcoma. The sensitivity and specificity of LEF1 IHC for diagnosis of DF were 94% and 70%, respectively. By comparison, β-catenin offered similar sensitivity, 94%, but 88% specificity. Positivity for LEF1 and β-catenin in combination showed sensitivity of 89%, lower than the sensitivity of β-catenin alone (94%); however, the combination of both LEF1 and β-catenin improved specificity (96%) compared to the specificity of β-catenin alone (88%). Although LEF1 has imperfect specificity in isolation, this stain has diagnostic utility when used in combination with β-catenin.

Prognostic evidence of LEF1 isoforms in childhood acute lymphoblastic leukemia.

The lymphoid enhancer factor 1 (LEF1) is a DNA-binding transcription factor that functions in the Wnt signaling pathway. Increased LEF1 activity is associated with progression of several types of cancer including leukemia. Here, we investigated LEF1 isoform expression and genomic variations in acute lymphoblastic leukemia (ALL). LEF1 isoform expression was evaluated by quantitative real-time PCR in 87 newly diagnosed childhood ALL patients and controls. Moreover, Western blot analysis was performed for detection of LEF1 expression and the hotspot region of LEF1 was screened by deep sequencing. The LEF1 mRNA expression of B cell ALL patients was higher than the controls (LEF1-total P=.011, LEF1-long P=.026). Moreover, B-ALL samples showing higher total LEF1 expression had significantly shorter relapse-free survival (P=.008) and overall survival (P=.011). Although full-length LEF1 expression was similar to the controls in T-ALL, 50% (n=15) of the ALL patients had increased full-length LEF1 protein expression. Imbalance between short- and full-length LEF1 isoforms may lead to cell survival in ALL. Beside the LEF1 activation, LEF1 gene variations were rarely observed in our cohort. The results indicate that the Wnt pathway may have a pathogenic function in a group of ALL patients and high LEF1-total expression might be a marker for shorter relapse-free survival time in B cell ALL.

Prognostic Impact of Lymphoid Enhancer Factor 1 Expression and Serum Galectin.3 in Egyptian AML Patients

Background Deregulation of the Wnt signaling pathway had a role in haematological malignancies. Previous studies reported that lymphoid enhancer factor 1 (LEF1) expression and serum Galectin-3 level could affect clinical parameters and outcome in acute myeloid leukemia patients, but as far as we know, no study has addressed their combined effect on AML patients. Aim We studied the expression of LEF1 by real-time qPCR and measured serum level of Gal.3 by ELISA technique in peripheral blood of 69 AML patients and correlated it with different clinicopathological criteria of patients, response, PFS and OS. Results We found high expression (LEF1high) was associated with better OS (p = 0.02) and EFS (p = 0.019) compared to LEF1low, low serum Gal.3 level had better OS (p = 0.014) and EFS (p = 0.02) compared to high serum Gal.3 level. LEF1high less likely to carry a FLT3-ITD (p = 0.047) compared to LEF1low patient, also LEF1high characterized by favorable risk (p = 0.02) than LEF1low patients. While patients with higher Gal-3 levels characterized by poor risk (p = 0.02) than lower Gal.3 lels, also more likely to carry a FLT3-ITD with borderline significance (p = 0.054). Combined LEF1high/Gal.3 low patients had lower baseline blast percentages (p = 0.02), favorable risk (p = 0.01), less likely to carry FLT3-ITD (p = 0.02), higher CR rate (p = 0.055), shorter time to CR (0.001) than other groups. Among high Gal.3 level group, LEF1highexpression improved OS and EFS (20 and 15 months respectively) vs LEF1low expression (13 and 8 months respectively). Conclusion We conclude that high LEF1 expression was a favorable prognostic marker which can define AML patient risk and outcome independent from assessing the serum galectin.3 level.

The osteogenic differentiation of human adipose-derived stem cells is regulated through the let-7i-3p/LEF1/\u03b2-catenin axis under cyclic strain

BackgroundThe Wnt/β-catenin pathway is involved in the osteogenic differentiation of human adipose-derived stem cells (hASCs) under cyclic strain. Very little is known about the role of microRNAs in these events.MethodsCells were obtained using enzyme digestion methods, and proliferation was detected using Cell Counting Kit 8. Cell cycles and immunophenotypes were detected by flow cytometry. The multilineage potential of hASCs was induced by induction media. Cyclic strain was applied to hASCs (0.5 Hz, 2 h/day, 6 days) to induce osteogenic differentiation and miRNA changes. Bioinformatic and dual-luciferase analyses confirmed lymphoid enhancer factor 1 (LEF1) as a potential target of let-7i-3p. The effect of let-7i-3p on LEF1 in hASCs transfected with a let-7i-3p mimic and inhibitor was analyzed by immunofluorescence. hASCs were transfected with a let-7i-3p mimic, inhibitor, or small interfering RNA (siRNA) against LEF1 and β-catenin. Quantitative real-time PCR (qPCR) and western blotting were performed to examine the osteogenic markers and Wnt/β-catenin pathway at the mRNA and protein levels, respectively. Immunofluorescence and western blotting were performed to confirm the activation of the Wnt/β-catenin pathway.ResultsFlow cytometry showed that 82.12% ± 5.83% of the cells were in G1 phase and 17.88% ± 2.59% of the cells were in S/G2 phase; hASCs were positive for CD29, CD90, and CD105. hASCs could have the potential for osteogenic, chondrogenic, and adipogenic differentiation. MicroRNA screening via microarray showed that let-7i-3p expression was decreased under cyclic strain. Bioinformatic and dual-luciferase analyses confirmed that LEF1 in the Wnt/β-catenin pathway was the target of let-7i-3p. Under cyclic strain, the osteogenic differentiation of hASCs was promoted by overexpression of LEF1and β-catenin and inhibited by overexpression of let-7i-3p. hASCs were transfected with let-7i-3p mimics and inhibitor. Gain- or loss-of-function analyses of let-7i-3p showed that the osteogenic differentiation of hASCs was promoted by decreased let-7i-3p expression and inhibited by increased let-7i-3p expression. Furthermore, high LEF1 expression inactivated the Wnt/β-catenin pathway in let-7i-3p-enhanced hASCs. In contrast, let-7i-3p inhibition activated the Wnt/β-catenin pathway.ConclusionsLet-7i-3p, acting as a negative regulator of the Wnt/β-catenin pathway by targeting LEF1, inhibits the osteogenic differentiation of hASCs under cyclic strain in vitro.

Lymphoid Enhancer Factor 1 Contributes to Hepatocellular Carcinoma Progression Through Transcriptional Regulation of Epithelial-Mesenchymal Transition Regulators and Stemness Genes.

Lymphoid enhancer factor 1 (LEF1) activity is associated with progression of several types of cancers. The role of LEF1 in progression of hepatocellular carcinoma (HCC) remains poorly known. We investigated LEF1 expression in HCC and its interactions with epithelial‐mesenchymal transition (EMT) regulators (e.g., Snail, Slug, Twist) and stemness genes (e.g., octamer‐binding transcription factor 4 [Oct4], sex determining region Y‐box 2 [Sox2], Nanog homeobox [Nanog]). Microarray analysis was performed on resected tumor samples from patients with HCC with or without postoperative recurrence. LEF1 expression was associated with postoperative recurrence as validated by immunohistochemical staining in another HCC cohort. Among 74 patients, 44 displayed a relatively high percentage of LEF1 staining (>30% of HCC cells), which was associated with a reduced recurrence‐free interval (P < 0.001) and overall survival (P = 0.009). In multivariate analysis, a high percentage of LEF1 staining was significantly associated with low albumin level (P = 0.035), Twist overexpression (P = 0.018), Snail overexpression (P = 0.064), co‐expression of Twist and Snail (P = 0.054), and multinodular tumors (P = 0.025). Down‐regulation of LEF1 by short hairpin RNA decreased tumor sphere formation, soft agar colony formation, and transwell invasiveness of HCC cell lines Mahlavu and PLC. Xenotransplant and tail vein injection experiments revealed that LEF1 down‐regulation in Mahlavu reduced tumor size and metastasis. LEF1 up‐regulation in Huh7 increased sphere formation, soft agar colony formation, and transwell invasiveness. LEF1 was shown to physically interact with and transcriptionally activate promoter regions of Oct4, Snail, Slug, and Twist. Furthermore, Oct4, Snail, and Twist transactivated LEF1 to form a regulatory positive‐feedback loop. Conclusion: LEF1 plays a pivotal role in HCC progression through transcriptional regulation of Oct4 and EMT regulators.

Knockdown of Lymphoid Enhancer factor 1 Inhibits Colon Cancer Progression In Vitro and In Vivo

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

Increased LEF1 Expression and Decreased Notch2 Expression Are Strong Predictors of Poor Outcomes in Colorectal Cancer Patients

Background/Objective. We aimed to examine the expression of lymphoid enhancer factor 1 (LEF1) and Notch2 in colorectal cancer (CRC) and their association with clinicopathologic variables and CRC patients' prognosis. Methods. Immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis were performed to assess the expression of LEF1 and Notch2 in 184 patients with CRC. Results. We observed a strong negative correlation between LEF1 expression and Notch2 expression (P < 0.001). Both LEF1 mRNA and protein expression increased while the Notch2 mRNA and protein expression decreased in tumor specimens compared with the matched paratumorous normal tissue (P < 0.001). An increase in LEF1 protein expression was significantly associated with lymph node metastases, distant metastasis, advanced TNM (tumor-node-metastasis) stage, and shorter overall survival. A decrease in Notch2 protein expression was associated with poorly differentiated tumors, lymph node metastases, distant metastasis, advanced TNM stage, and shorter overall survival of patients. In the multivariate Cox regression analysis, the LEF1 protein expression (P < 0.001), Notch2 protein expression (P < 0.001), TNM stage (P < 0.001), and the combination of increased LEF1 protein coexpression and decreased Notch2 protein coexpression (P < 0.001) were found to be independent prognostic indicators in CRC. Conclusion. Our results suggest that increased LEF1 coexpression and decreased Notch2 coexpression represent a risk factor for poor overall survival of CRC patients.

Abstract 4276: PGE2 induces LEF-1 expression in breast cancer cells, but is not required for the nuclear localization of beta-catenin

Abstract We have previously demonstrated that poorly differentiated MDA-MB-231 cells displayed delayed tumor onset, reduced frequency of occurrence and loss of their capacity for extrapulmonary colonization when transfected stably with a potent and specific cyclooxygenase (COX) −2 shRNA sequence. We attributed the reduction in tumor growth and extrapulmonary colonization to reduced invasive capabilities, reduced angiogenesis, altered metabolism and differences in the stabilization of hypoxia-inducible factor-1α. We also observed loss of expression of oncogenes, receptors and degradative enzymes associated with tumor growth and metastasis including matrix metalloproteinase-1, CXC Receptor-4, roundabout-1, hyaluronan synthase-2, and monocarboxylate transporter 2. In addition to these previously reported transcriptome changes, we observed that the most highly downregulated transcription factor was Lymphoid-Enhancer Factor-1 (LEF-1) a result validated by qPCR showing an 8-fold loss of LEF-1 in COX-2-silenced cells. Here, we present evidence demonstrating that LEF-1 accumulation in the nucleus of COX-2-silenced cells is reduced compared to COX-2 containing parental cells. Induction of COX-2 by phorbol esters resulted in nuclear accumulation of LEF-1 in COX-2 containing, but not in COX-2-silenced, cells. Addition of physiological concentrations of PGE2 to COX-2-silenced cells partially restored LEF-1 expression in the nucleus of these cells. LEF-1 is a member of the T-cell specific factor (TCF) family of transcription factors that direct transcription of downstream target genes upon nuclear translocation of β-catenin from the cytoplasm. β-catenin translocation to the nucleus occurs in response to canonical signaling of the ligand Wnt to its receptor Frizzled. An earlier study showed that the COX-2 product prostaglandin E2 promoted the translocation of β-catenin to the nucleus of colon cancer cells via G-protein coupled receptor signaling. As expected, immunofluorescence of COX-2 containing MDA-MB-231 breast cancer cells showed nuclear expression of β-catenin. However, the nuclear localization of β-catenin persisted even in the absence of PGE2, as observed in COX-2-silenced cells, suggesting that PGE2 expression is not necessary for the nuclear localization of β-catenin in MDA-MB-231 cells. Taken together, our results demonstrate that while PGE2 is not necessary for the nuclear localization of β-catenin in MDA-MB-231 cells, it can promote the nuclear accumulation of the TCF family member LEF-1, thereby further promoting the non-canonical activation of the Wnt pathway in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4276. doi:1538-7445.AM2012-4276

A role for YY1 in repression of dominant negative LEF-1 expression in colon cancer

Lymphoid enhancer factor 1 (LEF-1) mediates Wnt signaling via recruitment of β-catenin to target genes. The LEF1 gene is aberrantly transcribed in colon cancers because promoter 1 (P1) is a Wnt target gene and is activated by TCF–β-catenin complexes. A second promoter in intron 2 (P2) produces dominant negative LEF-1 isoforms (dnLEF-1), but P2 is silent because it is repressed by an upstream distal repressor element. In this study we identify Yin Yang 1 (YY1) transcription factor as the P2-specific factor necessary for repression. Site-directed mutagenesis and EMSA were used to identify a YY1-binding site at +25 in P2, and chromatin immunoprecipitation assays detected YY1 binding to endogenous LEF1 P2. Mutation of this site relieves P2 repression in transient transfections, and knockdown of endogenous YY1 relieves repression of integrated P2 reporter constructs and decreases the H3K9me3 epigenetic marks. YY1 is responsible for repressor specificity because introduction of a single YY1-binding site into the P1 promoter makes it sensitive to the distal repressor. We also show that induced expression of dnLEF-1 in colon cancer cells slows their rate of proliferation. We propose that YY1 plays an important role in preventing dnLEF-1 expression and growth inhibition in colon cancer.

Bcr-Abl Regulates IRES-Dependent Translation of Lymphoid Enhancer Factor-1 in Chronic Myelogenous Leukemia.

The constitutive tyrosine kinase activity of Bcr-Abl leads to aberrant expression of multiple genes by several mechanisms, including dysregulation of transcription. Recently though, increasing attention has been focused on the effect of Bcr-Abl in dysregulating translation. Our group has previously documented the effects of Bcr-Abl on key regulators of cap-dependent translation and the role that this mechanism plays in transformation (Ly et al, Cancer Research, 2003; Prabhu et al., Oncogene, in press). Here we describe a novel form of translational control by Bcr-Abl. Specifically, we show how Bcr-Abl regulates cap-independent translation of Lymphoid Enhancer Factor-1 (LEF-1) via a bona fide internal ribosome entry site (IRES) in the 5′ untranslated region (UTR) of LEF1. Lymphoid Enhancer Factor-1 (LEF-1), a transcription factor that mediates Wnt signals via interaction with β-catenin, is often expressed in cancers derived from aberrant Wnt signaling. Lately, it has been reported that LEF1 transcripts are elevated in CML. We examined LEF-1 expression in primary CML cells and cell lines (K562 and Ba/F3-Bcr-Abl) and show that LEF-1 protein is detected in all patient-derived cells. Treatment of these cells with the Bcr-Abl imatinib mesylate (imatinib) inhibits LEF-1 expression in imatinib-sensitive cancers, but not in cancers that exhibit clinical resistance even though such cancers express imatinib-sensitive Bcr-Abl. For those cancers that are sensitive, inhibition of Bcr-Abl has a partial effect on LEF1 mRNA levels, and a significant effect on LEF-1 protein levels. LEF-1 protein is produced via two IRESs in it's 5′ UTR. IRES-driven translation of LEF-1 was highly sensitive to Bcr-Abl as treatment with imatinib reduced IRES activity 5 fold. Transfection of CML cells with dicistronic mRNAs suggests that Bcr-Abl stimulates LEF-1 protein production through steps in the nucleus and cytoplasm. We propose that, in addition to its strong effects on cap-dependent translation in CML, Bcr-Abl is an important regulator of alternative translation pathways. mRNAs that are translated via IRES-dependent mechanisms are particularly relevant to cancer since they encode proteins that regulate cell proliferation and survival. Together, these observations underscore the important role which dysregulated translation plays in transformation, and suggest novel approaches with which to counteract the transforming properties of Bcr-Abl.

LEF-1 Transcription Factor Regulates Proliferation and Differentiation of Myeloid Progenitors in Healthy Individuals and in Patients with Severe Congenital Neutropenia (CN).

Wnt signaling pathway plays a key role in the signal transmission during cell growth, proliferation, differentiation and survival, especially in rapidly self-renewing tissues, such as bone marrow, skin, and intestinal mucosa. Regulation of target gene expression by Wnt signaling requires the presence of lymphoid enhancer factor 1 (LEF-1) and/or T-cell factors (TCFs) within the nucleus. Previously we reported 20 times lower or even absent expression of LEF-1 mRNA and protein in CD33+ myeloid progenitors from patients with severe congenital neutropenia (CN), as compared to healthy controls. Abolished LEF-1 expression in CD33+ progenitors from CN patients was accompanied by low levels of LEF-1 target genes, such as cyclin D1, c-myc, ELA2, and survivin.In the present study we aimed to characterize the role of LEF-1 transcription factor in myeloid cells from healthy individuals and from CN patients. Expression profile of LEF-1 mRNA during myeloid differentiation was measured by laser-assisted single-cell picking and real-time quantitative RT-PCR of different myeloid precursors (myeloblasts, promyelocytes, myelocytes/metamyelocytes, and mature granulocytes) from bone marrow smears of healthy controls and CN patients. LEF-1 mRNA was predominantly expressed in promyelocytes and myelocytes/metamyelocytes from healthy donors and was dramatically down-regulated in these cell types from CN patients. To investigate the functional consequence of LEF-1 downregulation, we specifically inhibitied LEF-1 expression in HL-60 promyelocytic cells and CD34+ cells from healthy individuals by lentiviral transduction of anti-LEF-1 shRNA. Interestingly, inhibition of LEF-1 resulted in down-regulation of LEF-1 target genes (cyclin D1, survivin, and c-myc), hematopoietic transcription factors C/EBPα and C/EBPε , and was accompanied by dramatically reduced proliferation and increased apoptosis in both HL-60 cells and CD34+ progenitors. Moreover, lentiviral transduction using vector containing LEF-1-GFP reporter into CD34+ cells from one CN patient led to normalization of expression of LEF-1 target genes (c-myc, survivin, cyclin D1), increased expression of C/EBPα , C/EBPε , and partial restoration of myelopoiesis.To investigate the cause of reduced LEF-1 expression in myeloid progenitors from CN patients, we sequenced the LEF-1 gene. We could not find any mutations in 15 CN patients studied. Therefore, regulatory mechanisms of LEF-1 expression, which are different from healthy controls, exist in CN. Interestingly, noggin, one of the activators of LEF-1 expression is significantly downregulated in CN patients. When exposed to noggin-containing medium, LEF-1 and LEF-1 target genes were induced in CD33+ cells from one CN patient, although the effect of noggin on myelopoiesis have to be analysed.Taken together, LEF-1 transcription factor regulate proliferation and differentiation of myeloid progenitors and is involved in the pathomechanism of CN.

Alterations of Lymphoid Enhancer Factor-1 Isoform Expression in Solid Tumors and Acute Leukemias

Two major transcripts of lymphoid enhancer factor-1 (LEF-1) have been described. The long isoform with b-catenin binding domain functions as a transcriptional enhancer factor. The short isoform derives from an intronic promoter and exhibits dominant negative activity. Recently, alterations of LEF-1 isoforms distribution have been described in colon cancer. In the current study we employed a quantitative real-time reverse transcription PCR method (TaqMan) to analyze expression of LEF-1 isoforms in a large cohort of human tumor (n = 304) and tumor-free control samples (n = 56). The highest expression level of LEF-1 was found in carcinoma samples whereas brain cancer samples expressed little. Expression of LEF-1 was different in distinct cancer types. For example, the mRNA level of LEF-1 was lower in testicular tumor samples compared with tumor-free control samples. Besides epithelial cancers, significant LEF-1 expression was also found in hematopoietic cells. In hematological malignancies, overall LEF-1 level was higher in lymphocytic leukemias compared with myeloid leukemias and normal hematopoiesis. However, acute myeloid leukemia and acute lymphocytic leukemia showed a significantly increased fraction of the oncogenic LEF-1 compared with chronic lymphocytic leukemia and chronic myeloid leukemia. Taken together, these data suggest that LEF-1 is abundantly expressed in human tumors and the ratio of the oncogenic and the dominant negative short isoform altered not only in carcinomas but also in leukemia.

Alterations of Lymphoid Enhancer Factor-1 Isoform Expression in Solid Tumors and Acute Leukemias

<title>Abstract</title> Two major transcripts of lymphoid enhancer factor-1 (LEF-1) have been described. The long isoform with β-catenin binding domain functions as a transcriptional enhancer factor. The short isoform derives from an intronic promoter and exhibits dominant negative activity. Recently, alterations of LEF-1 isoforms distribution have been described in colon cancer. In the current study we employed a quantitative real-time reverse transcription PCR method (TaqMan) to analyze expression of LEF-1 isoforms in a large cohort of human tumor (<italic>n</italic>=304) and tumor-free control samples (<italic>n</italic>=56). The highest expression level of LEF-1 was found in carcinoma samples whereas brain cancer samples expressed little. Expression of LEF-1 was different in distinct cancer types. For example, the mRNA level of LEF-1 was lower in testicular tumor samples compared with tumor-free control samples. Besides epithelial cancers, significant LEF-1 expression was also found in hematopoietic cells. In ematological malignancies, overall LEF-1 level was higher in lymphocytic leukemias compared with myeloid leukemias and normal hematopoiesis. However, acute myeloid leukemia and acute lymphocytic leukemia showed a significantly increased fraction of the oncogenic LEF-1 compared with chronic lymphocytic leukemia and chronic myeloid leukemia. Taken together, these data suggest that LEF-1 is abundantly expressed in human tumors and the ratio of the oncogenic and the dominant negative short isoform altered not only in carcinomas but also in leukemia. <b>FullText for HTML: </b><a target="_blank" class="col_4A90E2" href="https://doi.org/10.1093/abbs/37.3.173">https://doi.org/10.1093/abbs/37.3.173</a>

Defective Expression of LEF-1 Transcription Factor mRNA and - Protein in Patients with Severe Congenital Neutropenia (Kostmann'S Syndrome).

Severe congenital neutropenia (SCN) is characterized by “maturation arrest” of myeloid progenitor cells at the promyelocytic/myelocytic stage with the absence or only few mature neutrophils in the bone marrow and peripheral blood. Significant progress in the treatment of SCN patients has been achieved in the last 15 years by administration of granulocyte colony-stimulating factor (G-CSF), which significantly increases the number of neutrophils leading to an improvement of the quality of life. To date, the pathophysiology and underlying genetic defect in patients with congenital neutropenia is still under investigation. Wnt signalling pathway orchestrates a number of cellular programs such as proliferation, differentiation and cell fate determination in many tissues. In the present study we investigated the mRNA and protein expression patterns of Wnt signalling peptides, such as the High Mobility Group (HMG) box containing transcription factors such as lymphoid enhancer factor-1 (LEF-1) and T cell factors (TCFs), as well as β-catenin in CD33+ bone marrow myeloid progenitor cells from SCN patients (n = 6) in comparison to those of patients with cyclic neutropenia (n = 4) and G-CSF-treated healthy controls (n = 3). All SCN and cyclic neutropenia patients are under G-CSF therapy. mRNA expression of genes of interest was measured by quantitative real-time PCR. Protein expression was assessed by immunofluorescence staining, visualized and recorded by confocal microscopy. We found that CD33+ cells from patients with SCN exhibited 20 times lower or even absent expression of LEF-1 mRNA and protein, as compared to healthy G-CSF treated controls (mRNA expression ratio: SCN patients 0.83 ± 0.38 AU vs. healthy controls: 15.1 ± 0.4 AU; p < 0.0001). Intriguingly, LEF-1 mRNA expression levels on CD33+ cells from cyclic neutropenia patients were comparable to those of healthy controls. Immunostaining with anti-LEF-1 polyclonal antibody (kindly provided by Dr. R. Grosschedl) and confocal microscopy analysis revealed that LEF-1 protein was detectable at the expected level in CD33+ cells from healthy G-CSF treated controls. In patients with cyclic neutropenia LEF-1 protein expression in myeloid progenitor cells was comparable to healthy individuals. In contrast, in CD33+ cells from patients with SCN, LEF-1 protein was not detectable. mRNA expression of other TCFs: TCF-1, TCF-3, TCF-4 in SCN was not significantly different from healthy individuals. However, the expression level of LEF-1 binding partner in the Wnt pathway, β-catenin, was increased in SCN patients (SCN patients: 224.7 ± 42.4 AU vs. healthy controls: 107.2 ± 7.3 AU, p = 0.052). The defect in LEF-1 expression in SCN patients was further substantiated by the fact that mRNA expression of LEF-1 target genes such as c-myc, cyclin D1, survivin and neutrophil elastase were also significantly downregulated. In conclusion, our results suggest that defective LEF-1 expression might have an impact on the pathogenesis of SCN. In addition, it may help to distinguish SCN from cyclic neutropenia patients.

Lymphoid Enhancer Factor-1 Links Two Hereditary Leukemia Syndromes through Core-binding Factor α Regulation of ELA2

Two hereditary human leukemia syndromes are severe congenital neutropenia (SCN), caused by mutations in the gene ELA2, encoding the protease neutrophil elastase, and familial platelet disorder with acute myelogenous leukemia (AML), caused by mutations in the gene AML1, encoding the transcription factor core-binding factor alpha (CBFalpha). In mice, CBFalpha regulates the expression of ELA2, suggesting a common link for both diseases. However, gene-targeted mouse models have failed to reproduce either human disease, thus prohibiting further in vivo studies in mice. Here we investigate CBFalpha regulation of the human ELA2 promoter, taking advantage of bone marrow obtained from patients with either illness. In particular, we have identified novel ELA2 promoter substitutions (-199 C to A) within a potential motif for lymphoid enhancer factor-1 (LEF-1), a transcriptional mediator of Wnt/beta-catenin signaling, in SCN patients. The LEF-1 motif lies adjacent to a potential CBFalpha binding site that is in a different position in human compared with mouse ELA2. We find that LEF-1 and CBFalpha co-activate ELA2 expression. In vitro, the high mobility group domain of LEF-1 interacts with the runt DNA binding and proline-, serine-, threonine-rich activation domains of CBFalpha. ELA2 transcript levels are up-regulated in bone marrow of an SCN patient with the -199 C to A substitution. Conversely, a mutation of the CBFalpha activation domain, found in a patient with familial platelet disorder with AML, fails to stimulate the ELA2 promoter in vitro, and bone marrow correspondingly demonstrates reduced ELA2 transcript. Observations in these complementary patients indicate that LEF-1 cooperates with CBFalpha to activate ELA2 in vivo and also suggest the possibility that up-regulating promoter mutations can contribute to SCN. Two hereditary AML predisposition syndromes may therefore intersect via LEF-1, potentially linking them to more generalized cancer mechanisms.

A New β-Catenin-dependent Activation Domain in T Cell Factor

Transcription of the lymphoid enhancer factor-1 (LEF1) gene is aberrantly activated in sporadic colon cancer, whereas this gene is not expressed in the normal adult colon. We have shown previously that promoter 1 of the LEF1 gene is activated by T cell factor (TCF)-beta-catenin complexes in transient transfection assays, suggesting that LEF1 is a target of the Wnt pathway in colon cancer. To further explore the link between LEF1 expression and the Wnt pathway, we studied two response elements in the promoter. Surprisingly we found that the LEF1 promoter is selectively activated by specific isoforms of the LEF/TCF transcription factor family that contain an alternative C-terminal "E" tail. These isoforms, TCF-1E and TCF-4E, activate the promoter in a beta-catenin-dependent manner. We show that a complete E-tail domain is necessary for full activity and delimits residues within two highly conserved peptide motifs within the tail that are required (KKCRARFG; WCXXCRRKKKC). These peptide motifs are not only conserved among the TCF family members but are also found in two newly identified DNA-binding proteins named papillomavirus binding factor and GLUT4 enhancer factor. This study thus identifies a new and unique set of motifs used by the Wnt pathway for target gene regulation.

Loss of Expression of the WNT/β-Catenin-Signaling Pathway Transcription Factors Lymphoid Enhancer Factor-1 (LEF-1) and T Cell Factor-1 (TCF-1) in a Subset of Peripheral T Cell Lymphomas

T cell factor-1 (TCF-1) and lymphoid enhancer factor-1 (LEF-1), members of the TCF/LEF family of transcription factors, play a significant role in T cell development and are expressed in thymocytes and peripheral CD3+ T cells. Previously, precursor T lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL/LyL) was found to express TCF-1, and we find that 9 of 10 cases of T-ALL/LyL express LEF-1 as well as TCF-1, exhibiting uniform nuclear immunostaining for both transcription factors. In addition, a significant subset of cases of peripheral T cell lymphoma (PTCL), 39 of 81 cases (48%), are immunoreactive for LEF-1 and/or TCF-1, with 36 of 38 cases immunoreactive for both, indicating that these transcription factors are coordinately expressed in PTCL. The vast majority of LEF-1+ and/or TCF-1+ PTCL (34 of 39 or 87%) exhibit a composite Th1 T-cell-like immunophenotype, based on expression of Th1 T cell-associated, but not Th2 T cell-associated, chemokine receptors and activation markers. Of the Th1-like PTCL studied, 33 of 42 (79%) were immunoreactive for LEF-1 and 32 of 42 (76%) were immunoreactive for TCF-1, including most cases of angioimmunoblastic lymphoma and all cases of lymphoepithelioid lymphoma. Surprisingly, none of the 21 cases of Th2-like PTCL studied, all cases of anaplastic large cell lymphoma, were immunoreactive for LEF-1 or TCF-1 (P < 0.0001), suggesting that LEF-1 and TCF-1 transcription factor expression may be lost in Th2 T cells or Th2-like PTCL. LEF-1 and TCF-1 immunostaining can serve to identify specific subtypes of PTCL, and lends support to a bipartite model of PTCL development, based on expression of activation markers.