LEF1 Overexpression Research Articles

TCF7 and LEF-1 downregulation in sepsis promotes immune suppression by inhibiting CD4+ T cell proliferation

BackgroundPrevious studies have shown that sepsis is implicated in a reduction in the number and function of CD4+ T cells. TCF7 and LEF-1 facilitate early T cell development and lineage selection of CD4+ T cells. However, the function and mechanism of TCF7 and LEF-1 in sepsis are uncharacterized. This study intended to delineate effect of TCF7 and LEF-1 on sepsis and the impact on proliferation of CD4+ T cells in sepsis. MethodsA mouse sepsis model was constructed by cecal ligation and puncture (CLP) method. Expression of TCF7 and LEF-1 in sepsis was investigated using bioinformatics analysis and molecular experiments. We then constructed TCF7 and LEF-1 overexpression cell lines to investigate their effects on proliferation, apoptosis, effector activation, and immunosuppressive molecules of CD4+ T cells in sepsis. ResultsTCF7 and LEF-1 were downregulated in sepsis. As the duration of sepsis induction increased, the levels of TCF7 and LEF-1 gradually decreased, as did the number of CD4+ T cells. Cell experiments showed that overexpression of TCF7 and LEF-1 enhanced proliferation and effector activation of CD4+ T cells, reduced apoptosis, decreased PD-1 and LAG3 expression, and promoted immune response in sepsis. ConclusionIn conclusion, this study confirmed that downregulation of TCF7 and LEF-1 expression in sepsis inhibited proliferation of CD4+ T cells, leading to immune suppression. This finding suggested that TCF7 and LEF-1 were potential biological targets for sepsis and indicated that immunotherapy aimed at improving CD4+ T cell proliferation may be a new strategy for immune therapy in sepsis patients.

Striking Association of Lymphoid Enhancing Factor (LEF1) Overexpression and DUSP22 rearrangements in Anaplastic Large Cell Lymphoma

Introduction: Anaplastic large cell lymphomas (ALCLs) are CD30-positive T-cell lymphomas classified into ALK-positive and ALK-negative. The ALK-negative ALCLs are a genetically heterogeneous group that includes DUSP22-rearranged, TP63-rearranged and triple-negative cases. The DUSP22-rearranged ALCLs characteristically grow in a diffuse sheet-like pattern and are composed of intermediate-sized monomorphous hallmark cells, admixed with smaller cells with prominent nuclear indentations and occasional central nuclear pseudo inclusions (King RL et al, Am J Surg Pathol. 2016). Further, DUSP22-rearranged ALCL is a molecularly distinct subset of ALCL with a unique immunogenic phenotype that portends a favorable prognosis (Luchtel RA et al. Blood 2018). Lymphoid enhancer binding factor (LEF1) is a crucial nuclear mediator of the Wnt/ β-catenin pathway and a transcription factor involved in T-cell maturation. However, limited data exists on the association of LEF1 in T-cell lymphomas, including ALCL. The aims of this study were to explore LEF1 expression by immunohistochemistry in different subsets of ALCLs and to analyze the association of Wnt/ β-catenin pathway in this cohort. Methods: After approval by the institutional review board, we screened pathology reports from January 1, 1995 to December 31, 2019 and identified patients with a diagnosis of ALCL using our institutional database. Details on fluorescence in situ hybridization studies in ALK-negative cases were documented. Immunostaining for LEF1 and β catenin was performed on all cases where formalin fixed paraffin embedded tissue was available. The presence or absence of β catenin nuclear staining was recorded. LEF1 nuclear staining in the neoplastic ALCL cells was graded as negative (0), weak (1+), intermediate (2+) or strong (3+) and the percentage of LEF1-positive neoplastic cells was detailed (Figure 1). LEF1 scoring was performed by manual counting of the stained nuclei out of all tumor nuclei by two pathologists (A.R. and M.S.) and the grading was reviewed by another pathologist (P.J.K.). Results: We evaluated 45 ALCL cases, including 41 (91.1%) ALK-negative and 4 (8.9%) ALK-positive ALCLs. Among the ALK-negative ALCLs, there were16 DUSP22-rearranged, and 25 non-DUSP22-rearanged (7 TP63-rearranged and 18 triple-negative) cases. Fifteen (94%) of 16 DUSP22-rearranged ALCLs showed strong expression of LEF1 in >75% tumor cells (Figure 2). In contrast, this strong and uniform LEF1 overexpression was only detected in 1 of 29 (3%) of the remaining ALCL cases (P <.0001) (Table 1). We previously characterized a distinctive molecular signature in ALK-negative ALCLs with DUSP22-rearrangements using gene expression microarray analysis on frozen ALCL tissue samples (Luchtel RA et al. Blood 2018). On examining LEF1 gene expression in this previously published dataset, DUSP22 -rearranged cases had significantly higher LEF1 expression than any other genetic subtype (triple-negative, TP63-rearranged, or ALK-positive). Overall, mean LEF1 expression was 6.3-fold higher in ALCLs with DUSP22 rearrangements than in ALCLs without DUSP22-rearrangements (P=0.0001). LEF1 is a coactivator of the Wnt/β-catenin pathway; therefore, it is reasonable to hypothesize the overexpression of LEF1 may promote Wnt/β-catenin signaling. However, all LEF1-positive cases were negative for β-catenin by immunohistochemistry, suggesting that LEF1 overexpression is independent of the Wnt/β-catenin pathway signaling in these cases. Conclusions: There is a striking association between high LEF1 expression and DUSP22-rearrangements in ALCLs. The strong and uniform LEF1 expression pattern has a high positive predictive value (94%) and high negative predictive value (96%) for DUSP22- rearrangement in ALK-negative ALCLs (Table 2). The combination of characteristic morphologic and molecular features of DUSP22-rearranged cases with the unique LEF1 expression further emphasizes that DUSP22-rearranged ALCL represents a distinct clinicopathologic subset of ALCL. Disclosures No relevant conflicts of interest to declare.

Striking Association of Lymphoid Enhancing Factor (LEF1) Overexpression and DUSP22 Rearrangements in Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphomas (ALCLs) are broadly classified into ALK-positive and ALK-negative. ALK-negative ALCL is composed of DUSP22-rearranged, TP63-rearranged, and triple-negative cases. While lymphoid enhancer-binding factor (LEF1) plays a crucial role in T-cell maturation, limited data exist on its expression in T-cell lymphomas, including ALCL. We characterized the expression of LEF1 in ALCL by immunohistochemistry. LEF1 nuclear expression in the neoplastic cells was graded as negative (0), weak (1+), intermediate (2+), or strong (3+), with the percentage of LEF1-positive neoplastic cells recorded. A total of 45 ALCL cases were evaluated, of which 16 were DUSP22-rearranged. About 93.8% (15/16) DUSP22-rearranged cases showed strong expression of LEF1 in >75% tumor cells, compared with 3.4% (1/29) non-DUSP22-rearranged ALCL (P<0.0001). The striking association of LEF1 protein overexpression with DUPS22 rearrangement in ALCL was further confirmed by a gene expression profiling study which revealed significantly higher LEF1 expression in DUSP22-rearranged ALCL compared with other ALCL subtypes (P=0.0001). Although LEF1 is a nuclear mediator of the Wnt/β-catenin pathway, CTNNB1 RNA and protein levels were not overexpressed in LEF1-positive cases, suggesting the LEF1 overexpression in ALCL may not be involved in the Wnt/β-catenin pathway. The strong and uniform LEF1 expression pattern has a high positive predictive value (93.8%) and high negative predictive value (96%) for DUSP22 rearrangement in ALK-negative ALCL. The combination of characteristic morphologic and molecular features of DUSP22-rearranged cases with the high LEF1 expression further emphasizes that DUSP22-rearranged ALCL represents a distinct clinicopathologic subset of ALCL.

Transplantation of hMSCs Genome Edited with LEF1 Improves Cardio-Protective Effects in Myocardial Infarction

Stem cell-based therapy is one of the most attractive approaches to ischemic heart diseases, such as myocardial infarction (MI). We evaluated the cardio-protective effects of the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) stably expressing lymphoid enhancer-binding factor 1 (LEF1; LEF1/hUCB-MSCs) in a rat model of MI. LEF1 overexpression in hUCB-MSCs promoted cell-proliferation and anti-apoptotic effects in hypoxic conditions. For the application of its therapeutic effects in vivo, the LEF1 gene was introduced into an adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor site on chromosome 19 by CRISPR/Cas9-mediated gene integration in hUCB-MSCs. Transplantation of LEF1/hUCB-MSCs onto the infarction region in the rat model significantly improved overall survival. The cardio-protective effect of LEF1/hUCB-MSCs was proven by echocardiogram parameters, including greatly improved left-ventricle ejection fraction (EF) and fractional shortening (FS). Moreover, histology and immunohistochemistry successfully presented reduced MI region and fibrosis by LEF1/hUCB-MSCs. We found that these overall positive effects of LEF1/hUCB-MSCs are attributed by increased proliferation and survival of stem cells in oxidative stress conditions and by the secretion of various growth factors by LEF1. In conclusion, this study suggests that the stem cell-based therapy, conjugated with genome editing of transcription factor LEF1, which promotes cell survival, could be an effective therapeutic strategy for cardiovascular disease.

968 Enhancing the efficiency of engineered hair follicles with master regulators and extrinsic factors

Each year, more than one million patients are hospitalized in the U.S. for significant skin loss due to thermal and pressure injuries, chronic diabetic ulcers or genetic skin diseases. The ability to generate engineered human skin constructs (HSCs) has provided a promising therapy for these patients. However, it remained an unsolved challenge to incorporate hair follicles (HFs) into HSCs in order to improve wound healing and skin function. We initially employed a 3D-printing strategy to induce human HF formation within HSCs through guiding physiological 3D organization of cells in the HF microenvironment. Grafting our HF-bearing HSCs onto immunodeficient mice after their vascularization in vitro resulted in efficient growth of human HFs. However, when HSCs were maintained in vitro instead of grafting, the efficiency of HF induction was as low as 19%, due in part to incomplete restoration of DPC hair inductive gene signature achieved solely through 3D spheroid formation. To further enhance hair inductivity, we leveraged two master regulator (MR) genes, Lef1 and Fli1, which we previously identified as the key regulators of the intact DPC gene signature, as well as extrinsic factors targeting Wnt-signaling. Using RNA-sequencing, we confirmed that Lef1 overexpression and 3D-spheroid culture synergistically restored the intact DPC gene signature. Lef1 overexpression in DPCs markedly increased the efficiency of HF induction from 19% to 70%. Treatment of HSCs with recombinant Wnt10b and Wnt-activator small molecule, CHIR99021, also increased the efficiency to 50%, albeit lower than that was achieved with Lef1 overexpression. Overall, this data suggested that overexpressing Lef1 in DPCs or extrinsically targeting Wnt-signaling in HSCs can be an effective strategy to promote the generation of engineered human HFs from cultured cells.

LEF-1 drives aberrant β-catenin nuclear localization in myeloid leukemia cells.

Canonical Wnt/β-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear β-catenin even where cytosolic β-catenin is abundant. Control of the subcellular localization of β-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of β-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the β-catenin interactome in myeloid leukemia cells and identified putative novel β-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear β-catenin) versus Wnt-unresponsive cells (low nuclear β-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of β-catenin. The relative levels of nuclear LEF-1 and β-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed β-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and β-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first β-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear β- catenin level in human myeloid leukemia.

Regulatory role ofLEF-1in the proliferation of Arbas White Cashmere goat dermal papilla cells

Cashmere, which has high economic value, is made from the secondary hair follicles of cashmere goat skin. Dermal papilla cells (DPCs) are considered the center for regulation of hair growth, which is closely related to hair follicle growth. We constructed LEF-1 overexpression and interference experimental groups of goat DPCs to investigate LEF-1 regulation of DPCs proliferation by Wnt signaling, and provide a theoretical basis for improving cashmere yield. In primary DPCs, LEF-1, β-catenin, C-myc, and cyclin D1 expression in the LEF-1 overexpression group was 9.25-, 1.27-, 1.74-, and 1.63-fold, respectively, that of the control. LEF-1, β-catenin, C-myc, and cyclin D1 expression in the LEF-1 interference group was 0.20-, 0.75-, 0.38-, and 0.39-fold, respectively, that of the control. In secondary DPCs, LEF-1, β-catenin, C-myc, and cyclin D1 expression in the LEF-1 overexpression group was 10.53-, 1.48-, 1.64-, and 1.39-fold, respectively, that of the control. LEF-1, β-catenin, C-myc, and cyclin D1 expression in the LEF-1 interference group was 0.21-, 0.71-, 0.40-, and 0.36-fold, respectively, that of the control. Primary and secondary DPCs proliferation rates changed with LEF-1 expression. Therefore, the LEF-1 regulation pattern of cell proliferation through Wnt signaling is similar in both DPCs.

A Reciprocal Interaction between \u03b2-Catenin and Osterix in Cementogenesis

Although accumulating evidence indicates that both β-catenin and osterix (Osx) are essential for bone and tooth development, few studies have investigated the interaction of these two key proteins in the context of cementogenesis. In this study, we used transgenic mice with constitutively active β-catenin and inactive Osx in the dental mesenchyme to address this question. We found that cementoblasts with constitutively active β-catenin require Osx to produce excessive cellular cementum, and that ablation of Osx prevents this abnormal accumulation. Importantly, cementoblasts transduced with retrovirus expressing constitutively active β-catenin exhibited upregulation of Osx expression through direct binding to the promoter region of Osx. Osx regulates Lef1 expression and consequently could regulate T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding activity in Wnt/β-catenin signaling. However, the loss of Tcf/Lef binding activity by Osx ablation was not rescued by transduction of retrovirus expressing constitutively active β-catenin or ectopic Lef1 overexpression. These results suggest that the Tcf/Lef binding activity of Wnt/β-catenin signaling is Osx-dependent during cementogenesis. Moreover, Osx differentially regulates the expression of various Tcf family members, suggesting that Osx regulates cementogenesis by utilizing various Tcf/Lef-dependent mechanisms. This is the first report to show that downstream Osx signaling through Tcf/Lefs is critical for cementogenesis.

LEF-1 Regulates Tyrosinase Gene Transcription In Vitro.

TYR, DCT and MITF are three important genes involved in maintaining the mature phenotype and producing melanin; they therefore participate in neural crest cell development into melanocytes. Previous studies have revealed that the Wnt signaling factor lymphoid enhancer-binding factor (LEF-1) can enhance DCT and MITF gene expression. However, whether LEF-1 also affects TYR gene expression remains unclear. In the present study, we found that LEF-1 regulated TYR transcription in vitro. LEF-1 overexpression increased TYR gene promoter activity, whereas LEF-1 knockdown by RNA interference significantly decreased TYR expression. Moreover, the core GTTTGAT sequence (-56 to -50) within the TYR promoter is essential for the effect of LEF-1 on TYR expression, and chromatin immunoprecipitation (ChIP) assay indicated that endogenous LEF-1 interacts with the TYR promoter. In addition, we observed a synergistic transactivation of the TYR promoter by LEF-1 and MITF. These data suggest that Wnt signaling plays an important role in regulating melanocyte development and differentiation.

Lef1 Contributes to the Differentiation of Bulge Stem Cells by Nuclear Translocation and Cross-Talk with the Notch Signaling Pathway

Lymphoid enhancer binding factor-1 (Lef1) is an essential regulatory protein in the Wnt signal pathway, which controls cell growth and differentiation. Investigators in the field of skin biology have confirmed that multipotent bulge stem cells (BSCs) are responsible for hair follicle development and regeneration. However, the role of Lef1 remains poorly understood. In this study, we investigated the pattern of Lef1 expression at different stages of the hair growth cycle. Lef1 was strongly expressed during anagen but attenuated in both catagen- and telogen-phase hair follicles in vivo. When stem cells were induced to differentiate toward a hair fate in a co-culture system, Lef1 was notably up-regulated and accumulated in the nucleus, appearing to activate the target protein c-myc and jagged1. Simultaneously, the Wnt and Notch signaling pathways were co-activated, as confirmed by the increased expression of β-catenin and notch1. Plasmids expressing Lef1 and ΔNLef1, a construct in which the β-catenin-binding domain of Lef1 was deleted, were used to evaluate the effects of Lef1 on stem cell differentiation. Lef1 overexpression promoted bulge stem cell differentiation toward a hair fate, which was accompanied by the subsequent migration of β-catenin into the nucleus, whereas no changes were observed in the control group. Taken together, our results demonstrate that Lef1 plays an important role in bulge stem cell differentiation, promoting β-catenin translocation into the nucleus, activating downstream signaling molecules, eventually causing hair follicle bulge stem cells to adopt the hair fate.

Abstract 5289: In human endothelial cell line Wnt-beta-catenin pathway controls transcription of lymphoid enhancer-binding factor 1, a representative of vertebrate-specific Lef1/Tcf1 sub-family enhancing matrix metalloproteinase-2 transcription and endothelial cell invasion

Abstract Background: Wnt signaling is activated in many types of cancer and normal physiological processes. Various Wnt-related secreted factors may influence angiogenesis both in the tumor microenvironment and in normal tissues by direct action on endothelial cells. The mechanism of this Wnt action in angiogenesis is not well defined. Gradl D, König A, and Wedlich D (2002) show that members of Tcf1/Lef1 sub-family generate stronger Wnt throughput and Xenopus’ secondary axis induction per protein unit than members of Tcf3/Tcf4 sub-family. This stronger activity might require different, may be, stronger chemotherapy tools for angiogenesis control than the tools necessary to prevent colon cancer where Tcf4 signaling is prominent. We hypothesize that endothelial cells are responsive to Wnt signals and that Lef1, a member of the vertebrate-specific Wnt/beta-catenin throughput-inducing transcription factors’ sub-family Lef1/Tcf1, mediates this responsiveness and promotes endothelial cell invasion. Methods: A human endothelial cell line, EAhy926 was exposed to Wnt3a or directly transfected with Lef1. Readouts included assessment of nuclear β-catenin, Wnt throughput with a SuperTOPflash reporter assay, induction of Lef1 transcription, induction of matrix metalloproteinase (MMP)-2 transcription, cell proliferation and cell invasion through a matrix in vitro. The effects on MMP2 were also evaluated in the presence of Lef1 silencing siRNA. Results: Wnt3a increased nuclear β-catenin staining identified by DNA staining of nucleus with DAPI and up-regulated Wnt/β-catenin throughput without changing the activity of negative control reporter with mutated Lef/Tcf binding sites (FOP). Wnt3a increased Lef1 transcription and activity of the Lef1 promoter. Both Wnt3a treatment and Lef1 overexpression induced MMP2 transcription but this effect was completely abrogated in the presence of Lef1 siRNA. Inhibition of Lef1 also reduced basal MMP2 levels suggesting that Lef1 regulates MMP2 expression even in the absence of exogenous Wnt pathway activation. Lef1 slightly enhanced proliferation of EAhy926 cells after prolonged periods (96 and 120 hours post-transfection) and increased invasion by more than two-fold. Conclusions: EAhy926 cell line activates canonical Wnt signaling in response to Wnt3a ligand. A representative of vertebrate-specific Lef1/Tcf1 sub-family Lef1 specifically regulates MMP2 transcription in these cells and promotes endothelial cell invasion. This cell line provides a convenient alternative to primary human umbilical vein endothelial cells (HUVEC) in the study of angiogenesis and the role of Wnt signaling on endothelial cell function. 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 5289. doi:1538-7445.AM2012-5289

Lymphoid enhancing factor 1 (Lef-1) overexpression in epithelial ovarian, fallopian tube and peritoneal cancers and associations with clinical factors

Lef-1 mRNA levels were statistically elevated in cases of ovarian, fallopian tube or peritoneal cancer when compared to non-cancerous controls. Among cancer cases, levels of Lef-1 were statistically different between stage and histology. Lef-1 overexpression may be predictive of poor overall survival. These findings suggest that Lef-1 overexpression may contribute to ovarian, fallopian tube and peritoneal carcinogenesis, and that further investigation is warranted.

Lymphoid enhancing factor 1 (Lef-1) is over expressed in benign endometria as body mass index increases

Lymphoid enhancing factor 1 (Lef-1) is over expressed in benign endometria as body mass index increases

Lymphoid Enhancing Factor 1 (Lef-1) overexpression in epithelial ovarian, fallopian tube and peritoneal cancer and associations with clinical factors

Lef-1 mRNA levels were statistically elevated in cases of ovarian, fallopian tube or peritoneal cancer when compared to non-cancerous controls. Among cancer cases, levels of Lef-1 were statistically different between stage and histology. Lef-1 overexpression may be predictive of poor overall survival. These findings suggest that Lef-1 overexpression may contribute to ovarian, fallopian tube and peritoneal carcinogenesis, and that further investigation is warranted.

Lymphoid enhancer-binding factor 1, a representative of vertebrate-specific Lef1/Tcf1 sub-family, is a Wnt-beta-catenin pathway target gene in human endothelial cells which regulates matrix metalloproteinase-2 expression and promotes endothelial cell invasion

BackgroundWnt signaling is activated in many types of cancer and normal physiological processes. Various Wnt-related secreted factors may influence angiogenesis both in the tumor microenvironment and in normal tissues by direct action on endothelial cells. The mechanism of this Wnt action in angiogenesis is not well defined. We hypothesize that endothelial cells are responsive to Wnt signals and that Lef1, a member of the vertebrate-specific Wnt/beta-catenin throughput-inducing transcription factors' sub-family Lef1/Tcf1, mediates this responsiveness and promotes endothelial cell invasion.MethodsA human endothelial cell line, EAhy926 was exposed to Wnt3a or directly transfected with Lef1. Readouts included assessment of nuclear beta-catenin, Wnt throughput with a SuperTOPflash reporter assay, induction of Lef1 transcription, induction of matrix metalloproteinase (MMP)-2 transcription, cell proliferation and cell invasion through a matrix in vitro. The effects on MMP2 were also evaluated in the presence of Lef1 silencing siRNA.ResultsWnt3a increased nuclear beta-catenin and up-regulated Wnt/beta-catenin throughput. Wnt3a increased Lef1 transcription and activity of the Lef1 promoter. Both Wnt3a treatment and Lef1 overexpression induced MMP2 transcription but this effect was completely abrogated in the presence of Lef1 siRNA. Inhibition of Lef1 also reduced basal MMP2 levels suggesting that Lef1 regulates MMP2 expression even in the absence of exogenous Wnt pathway activation. Lef1 slightly increased proliferation of EAhy926 cells and increased invasion by more than two-fold.ConclusionsEAhy926 cells activate canonical Wnt signaling in response to Wnt3a ligand. The Wnt target Lef1 specifically regulates MMP2 expression in these cells and promotes endothelial cell invasion. The EAhy926 cell line provides a convenient alternative to primary human umbilical vein endothelial cells (HUVEC) in the study of angiogenesis and the role of Wnt signaling on endothelial cell function.

Abstract LB-367: Wnt-dependent transcription factor LEF-1 controls endothelial cell invasion through changes of MMP-2 expression

Abstract Background: Wnt signaling is involved in many events including embryogenesis, stem cell regulation, morphogenesis, cell fate determination, and oncogenesis. The role of Wnt pathway in angiogenesis is proposed, however the mechanism is still unclear. Most cancers express elevated levels of different Wnts when compared to normal cells. LEF-1 is a participant in and a target gene of the Wnt/ -catenin pathway. LEF-1 increases the in vitro invasiveness of cancer cells. Hypothesis: We suggest that LEF-1 controls the invasion also in endothelial cells. We hypothesize further that cancer affects local tumor angiogenesis via Wnt pathway-dependent up-regulation of LEF-1 in endothelial cells (EC). Methods: To test this hypothesis we construct a model system: endothelial cell line EaHy926 treated with Wnt3a (mostly known as a canonical Wnt signal) conditioned medium or grown in co-culture with L-cells producing Wnt3a. Results: Wnt3a increases nuclear ß-catenin in human endothelial cell line, and up-regulates LEF/TCF-dependent promoter activity. Wnt3a augments its target gene's LEF-1 promoter activity and LEF-1 mRNA concentration, demonstrating involvement of some transcription factors with E-tails. Both Wnt3a treatment and LEF-1 overexpression elevate MMP-2 mRNA level. Elimination of Wnt-dependent induction of MMP-2 gene expression by LEF-1 siRNA confirms the LEF-1 role in the MMP-2 control mechanism. Loosening up the extracellular matrix MMPs allows the cells to move around. These enzymes are integral part of invasion process. Indeed, LEF-1 up-regulates endothelial cells invasiveness in Matrigel matrix. LEF-1 increases also EC proliferation, in agreement with published data for parental primary endothelial culture HUVEC. Conclusions: In endothelial cells canonical (ß-catenin) Wnt signaling enhances invasiveness of EC via LEF-1 - dependent regulation of MMP-2 expression. There is a number of similarities between primary cells HUVEC, studied by others, and cell line EAhy926 in their response to Wnt3a. This indicates that EAhy926 cell line conserves many components of angiogenesis control by Wnt/β-catenin pathway and could serve as a simplified model to study this regulation, replacing primary endothelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-367.

Lymphoid Enhancer Binding Factor 1 Regulates Transcription through Gene Looping

Efficient transcription depends upon efficient physical and functional interactions between transcriptosome complexes and DNA. We have previously shown that IL-1beta-induced lymphoid enhancer binding factor 1 (Lef1) regulates the transcription of its target genes COX2 and MMP13 in mouse chondrocytes by binding to the Lef1 binding sites located in the 3' region. In this study, we investigated how the 3' region-bound Lef1 regulates expression of target genes. IL-1beta stimulation induced gene looping in COX2 and MMP13 genomic loci, which is mediated by the physical interaction of Lef1 with its binding partners, including beta-catenin, AP-1, and NF-kappaB. As shown by chromosome conformation capture (3C) assay, the 5' and 3' genomic regions of these genes were juxtaposed in an IL-1beta-stimulation dependent manner. Lef1 played a pivotal role in this gene looping; Lef1 knockdown decreased the incidence of gene looping, while Lef1 overexpression induced it. Physical interactions between the 3' region-bound Lef1 and promoter-bound transcription factors AP-1 or NF-kappaB in COX2 and MMP13, respectively, were increased upon stimulation, leading to synergistic up-regulation of gene expression. Knockdown of RelA or c-Jun decreased the formation of gene loop and down-regulated cyclooxygenase 2 (COX2) or matrix metalloproteinase 13 (MMP13) transcription levels. However, overexpression of RelA or c-Jun along with Lef1 increased the looping and their expression levels. Our results indicate a novel function of Lef1, as a mediator of gene looping between 5' and 3' regions. Gene looping may serve to delineate the transcription unit in the inducible gene transcription of mammalian cells.

LEF1 in androgen-independent prostate cancer: regulation of androgen receptor expression, prostate cancer growth, and invasion.

A major obstacle in treating prostate cancer is the development of androgen-independent disease. In this study, we examined LEF1 expression in androgen-independent cancer as well as its regulation of androgen receptor (AR) expression, prostate cancer growth, and invasion in androgen-independent prostate cancer cells. Affymetrix microarray analysis of LNCaP and LNCaP-AI (androgen-independent variant LNCaP) cells revealed 100-fold increases in LEF1 expression in LNCaP-AI cells. We showed that LEF1 overexpression in LNCaP cells resulted in increased AR expression and consequently enhanced growth and invasion ability, whereas LEF1 knockdown in LNCaP-AI cells decreased AR expression and, subsequently, growth and invasion capacity. Chromatin immunoprecipitation, gel shift, and luciferase assays confirmed LEF1 occupancy and regulation of the AR promoter. Thus, we identified LEF1 as a potential marker for androgen-independent disease and as a key regulator of AR expression and prostate cancer growth and invasion. LEF1 is highly expressed in androgen-independent prostate cancer, potentially serving as a marker for androgen-independent disease.

Identification of prognostically-relevant signature genes differentially expressed between primary colorectal carcinoma and hepatic metastasis

4116 Background: Colorectal cancer (CRC) is the third most common cancer in the US. Hepatic metastasis (HM), the most common distant metastasis from CRC and occurring in about 60% of CRC patients during the course of their treatment, is a significant clinical problem. Although several clinical prognostic factors for HM from CRC have been identified, little is known about its molecular aspect. Methods: Under IRB approved protocols, we profiled gene expression in a set of 30 specimens from primary CRC and 31 unmatched specimens of HM from CRC. To survey the molecular variations between the two groups, we used cDNA microarrays containing ∼22,000 human genes, and identified differentially expressed genes using the Significance Analysis of Microarrays (SAM) method. To further characterize the clinical relevance of differentially expressed genes, we used immunohistochemistry (IHC) on tissue microarrays of tumor tissues obtained from an independent dataset of 154 patients with primary CRC. Results: Supervised analysis using SAM identified &gt;200 genes with significantly higher expression in HM from CRC compared to primary CRC (and also compared to normal liver), with a false discovery rate (FDR) of &lt;1%. IHC staining of two highly ranked differentially expressed signature genes, SPP1 (osteopontin) and LEF1 (lymphoid enhancer factor-1; a transcriptional mediator of Wnt pathway activation), showed protein overexpression (OE) in 60% and 44% of CRC cases, respectively; with a significant correlation with liver involvement in LEF1 expression (chi-square p=0.04) vs. SPP1 OE (p=0.14). Kaplan Meier analysis revealed significantly worsening survival in patients with LEF1 OE (p&lt;0.01), but not SPP1 (p=0.14). Multivariate analysis identified several relevant prognostic markers: stage (p&lt;0.001), nodal status (p &lt;0.01) and LEF1 OE (p=0.02). Conclusions: Among signature genes differentially expressed between CRC and HM, we demonstrate LEF1, not SPP1, OE to be a poor prognostic factor for survival. Further studies are needed to characterize other signature genes, and to refine our understanding of the role of altered LEF1 expression in CRC tumorigenesis. No significant financial relationships to disclose.

Overexpression of beta-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators.

beta-Catenin promotes epithelial architecture by forming cell surface complexes with E-cadherin and also interacts with TCF/LEF-1 in the nucleus to control gene expression. By DNA transfection, we overexpressed beta-catenin and/or LEF-1 in NIH 3T3 fibroblasts, corneal fibroblasts, corneal epithelia, uveal melanoma cells, and several carcinoma cell lines. In all cases (with or without LEF-1), the abundant exogenous beta-catenin localizes to the nucleus and forms distinct nuclear aggregates that are not associated with DNA. Surprisingly, we found that with time (5-8 d after transfection) cells overexpressing beta-catenin all undergo apoptosis. LEF-1 does not need to be present. Moreover, LEF-1 overexpression in the absence of exogenous beta-catenin does not induce apoptosis, even though some endogenous beta-catenin moves with the exogenous LEF-1 into the nucleus. TOPFLASH/FOPFLASH reporter assays showed that full-length beta-catenin is able to induce LEF-1-dependent transactivation, whereas Arm beta-catenin totally abolishes the transactivating function. However, Arm beta-catenin, containing deletions of known LEF-1-transactivating domains, has the same apoptotic effects as full-length beta-catenin. Overexpressed beta-catenin also induces apoptosis in cells transfected with nuclear localization signal-deleted LEF-1 that localizes only in the cytoplasm. Thus, the apoptotic effects of overexpressed exogenous beta-catenin do not rely on its transactivating function with nuclear LEF-1. Overexpressed delta-catenin, containing 10 Arm repeats, induces only minor apoptosis, suggesting that the major apoptotic effect may be due to domains specific to beta-catenin as well as to Arm repeats. The absence of p53, Rb, cyclin D1, or E2F1 does not affect the apoptotic effect of overexpressed beta-catenin, but Bcl-x(L) reduces it. We hypothesize that in vivo apoptosis of cells overexpressing beta-catenin might be a physiological mechanism to eliminate them from the population.