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Hepatoblastoma, the most prevalent pediatric liver cancer, almost always carries a WNT-activating CTNNB1 mutation, yet exhibits notable molecular heterogeneity. To characterize this heterogeneity and identify novel targeted therapies, we perform comprehensive analysis of hepatoblastomas and tumor-derived organoids using single-cell RNA-seq/ATAC-seq, spatial transcriptomics, and high-throughput drug profiling. We identify two distinct tumor epithelial signatures: hepatic 'fetal' and WNT-high 'embryonal', displaying divergent WNT signaling patterns. The fetal group is enriched for liver-specific WNT targets, while the embryonal group is enriched in canonical WNT target genes. Gene regulatory network analysis reveals enrichment of regulons related to hepatic functions such as bile acid, lipid and xenobiotic metabolism in the fetal subtype but not in the embryonal subtype. In addition, the dichotomous expression pattern of the transcription factors HNF4A and LEF1 allows for a clear distinction between the fetal and embryonal tumor cells. We also perform high-throughput drug screening using patient-derived tumor organoids and identify sensitivity to HDAC inhibitors. Intriguingly, embryonal and fetal tumor organoids are sensitive to FGFR and EGFR inhibitors, respectively, indicating a dependency on EGF/FGF signaling in hepatoblastoma tumorigenesis. In summary, our data uncover the molecular and drug sensitivity landscapes of hepatoblastoma and pave the way for the development of targeted therapies., Competing Interests: Competing interests: H.C. is currently head of pharma Research Early Development (pRED) at Roche and is an inventor on several patents related to organoid technology. His full disclosure is given at www.uu.nl/staff/JCClevers . The remaining authors declare no competing interests., (© 2024. The Author(s).)

Wnt/β-Catenin pathway plays an important role in the occurrence and progression of malignant tumors, especially PD-L1-mediated tumor immune evasion. However, the role of TCF/LEF, an important member of the Wnt/β-catenin pathway, in the tumor immunosuppressive microenvironment of lung adenocarcinoma (LUAD) remains unknown. LUAD tissue-coding RNA expression data from The Cancer Genome Atlas and TIMER databases were used to analyze the expression of TCF/LEF transcription factors and their correlation with various immune cell infiltration. Immunohistochemistry and immunofluorescence were used to detect tissue protein staining in 105 patients with LUAD. LEF1, TCF7, TCF7L1 and TCF7L2 were all aberrantly expressed in the tumor tissues of LUAD patients with the data from The Cancer Genome Atlas (TCGA) database, tumor immune estimation resource (TIMER) database and results of immunohistochemistry, but only LEF1 expression was associated with 5-year overall survival in LUAD patients. LEF1 protein expression was associated with advanced tumor node metastasis (TNM) stage, lymphatic metastasis and local invasion in 105 cases LUAD patients. At the same time, LEF1 mRNA expression was also associated with immunosuppressive microenvironment in LUAD patients with the data from TCGA database and TIMER database. Results of immunohistochemistry and immunofluorescence in tumor tissues of 105 cases LUAD patients showed that there was a positively correlation between LEF1 protein expression and the infiltration of M2 macrophages and Treg cells. LEF1 was highly expressed in tumor tissues of LUAD patients, and highly expressed LEF1 was associated with the immunosuppressive microenvironment of LUAD patients., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The homologous proteins identified as cellular retinoic acid-binding proteins I and II ( CRABP-I and CRABP-II ) belong to a subset of intracellular proteins characterized by their robust affinity for retinoic acid, which plays an indispensable role in the development of hair follicle, including differentiation, proliferation, and apoptosis in keratinocytes. Previous research on Hu sheep hair follicles revealed the specific expression CRABP1 in dermal papilla cells (DPCs), suggesting that CRABP1 has a potential role in regulating the DPC population. Therefore, the main purpose of this study is to expose the performance of the CRABP1 genes in the development and proliferation of DPCs., Methods: Initially, overexpression and inhibition of CRABP1 in the DPCs were conducted through overexpression vector and siRNA. CCK-8, EDU, and RT-PCR cell cycle assays and immunostaining were performed to evaluate the proliferation and cell cycle of dermal papilla cells (DPCs). Although, the influence of CRABP1 upon β-catenin in dermal papilla cells (DPCs) was found using immunofluorescence labeling. Finally, RT-PCR was conducted to assess the impact of CRABP1 on the expression levels of CTNNB1, TCF4, and LEF1 in DPCs involved in the Wnt/β-catenin signaling pathway., Results: The results showed that CRABP1 overexpression promotes the growth rates of DPCs and significantly enhances the proportion of S-phase cells compared with the control group ( p < 0.05). The results were the opposite when CRABP1 was a knockdown. In contrast, there was a significant decline in the mRNA expression levels of CTNNβ1 , LEF1 ( p < 0.05), and TCF4 ( p < 0.01) by CRABP1 knockdown., Conclusions: This study found that CRABP1 influences the expression of important genes within the Wnt/β-catenin signaling pathway and promotes DPC proliferation. This investigation provides a theoretical framework to explain the mechanisms that control hair follicle morphogenesis and development.

The Wnt/β-catenin signaling pathway plays numerous essential roles in animal development and tissue/stem cell maintenance. The activation of genes regulated by Wnt/β-catenin signaling requires the nuclear accumulation of β-catenin, a transcriptional co-activator. β-catenin is recruited to many Wnt-regulated enhancers through direct binding to T-cell factor/lymphoid enhancer factor (TCF/LEF) family transcription factors. β-catenin has previously been reported to form phase-separated biomolecular condensates (BMCs), which was implicated as a component of β-catenin's mechanism of action. This function required aromatic amino acid residues in the intrinsically disordered regions (IDRs) at the N- and C-termini of the protein. In this report, we further explore a role for β-catenin BMCs in Wnt target gene regulation. We find that β-catenin BMCs are miscible with LEF1 BMCs in vitro and in cultured cells. We characterized a panel of β-catenin mutants with different combinations of aromatic residue mutations in human cell culture and Drosophila melanogaster. Our data support a model in which aromatic residues across both IDRs contribute to BMC formation and signaling activity. Although different Wnt targets have different sensitivities to loss of β-catenin's aromatic residues, the activation of every target examined was compromised by aromatic substitution. These mutants are not defective in nuclear import or co-immunoprecipitation with several β-catenin binding partners. In addition, residues in the N-terminal IDR with no previously known role in signaling are clearly required for the activation of various Wnt readouts. Consistent with this, deletion of the N-terminal IDR results in a loss of signaling activity, which can be rescued by the addition of heterologous IDRs enriched in aromatic residues. Overall, our work supports a model in which the ability of β-catenin to form biomolecular condensates in the nucleus is tightly linked to its function as a transcriptional co-regulator., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Stewart et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Androgenetic alopecia (AGA) is a prevalent hair loss disorder with psychological repercussions. Traditional treatments have limitations, leading to the exploration of regenerative therapies such as exosomes derived from adipose tissue stem cells (ASC-Exosomes)., Methods: First, using human hair follicle (HF) dermal papilla cells (hDPCs) treated with ASC-Exosomes, ALP, VCAN, β-catenin, and LEF-1 levels with RT-PCR and p-GSK3β, GSK3β, β-catenin, ALP, and β-actin levels with western blot analysis were assessed. Hair shaft elongation test and assay for ALP, Ki-67, and β-catenin were done using human HF organ culture. Patients with AGA had ASC-Exosomes treatment and were evaluated for hair counts, photographic assessments, subjective satisfaction, and safety profiles., Results: ASC-Exosomes impact hDPCs, increasing proliferation and the upregulation of hair growth-related genes, including ALP, VCAN, β-catenin, and LEF-1. The Wnt/β-catenin pathway was activated, indicating their role in promoting hair growth. ASC-Exosomes also promoted hair shaft elongation and ALP activity, suggesting a potential for hair regeneration. Thirty participants with AGA enrolled and treated over 24 weeks. The subjects experienced a significant increase in total hair density, improved global photographic assessments, and reported higher subjective satisfaction without severe adverse reactions., Conclusion: This research contributes to the growing body of evidence supporting the use of exosomes in hair loss treatment, offering a safe and effective alternative for individuals with AGA., (© 2024 the International Society of Dermatology.)

Endometrial stromal tumors (ESTs) are uncommon uterine mesenchymal lesions. Nuclear expression of β-catenin, an indication of activated Wnt/β-catenin signaling pathway, was described in 50% to 92% of low-grade ESTs, including endometrial stromal nodule and low-grade endometrial stromal sarcoma. Activation of the Wnt/β-catenin signaling pathway leads to the translocation of β-catenin into the nucleus and interaction with the T-cell factor/lymphoid enhancer-binding factor-1 (LEF1) family of transcription factors to regulate cell proliferation, differentiation, migration, and survival. Immunohistochemical analysis of β-catenin and LEF1 was performed in 2 endometrial stromal nodules and 20 low-grade endometrial stromal sarcomas and demonstrated 90.9% and 81.8% positive rates for β-catenin and LEF1, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of β-catenin and LEF1 were 90.9% versus 81.8%, 81.0% versus 85.7%, 83.3% versus 85.7%, 89.5% versus 81.8%, respectively, in the diagnosis of low-grade ESTs. There is no statistical significance of the performance of β-catenin and LEF1 in all ESTs ( P = 0.664) or in primary or metastatic/recurrent settings ( P = 0.515 and 0.999, respectively). Only 3 smooth muscle tumors showed focal and weak positivity for LEF1. Our results indicate LEF1 can be a useful marker in aiding a diagnosis of low-grade EST and differentiating from smooth muscle tumors alone or in combination with β-catenin., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 by the International Society of Gynecological Pathologists.)

Recent studies have highlighted the potential of Mesenchymal Stem Cells (MSCs) as an alternative treatment for Alopecia Areata (AA) due to their immunosuppressive properties. While MSCs have shown promise in cell experiments, their effectiveness in vivo remains uncertain. This study aims to validate local administration of MSC therapy's efficacy in AA treatment through animal experiments. AA was induced through Interferon-gamma (IFN-γ) administration in mice, and MSC treatment (MSCT)'s effects were assessed visually and through tissue analysis. The MSC-treated group showed more hair regrowth compared to the control (CTL) group. MSCT notably reduced local inflammatory cytokines (JAK1, JAK2, STAT1, STAT3, IFN-γR, IL-1β, IL-16, IL-17α, and IL-18) in AA-induced mice's skin, but systemic cytokine levels remained unchanged. Furthermore, MSC treatment normalized the expression of Wnt/β-catenin signaling pathway genes (LEF1 and β-catenin) and growth factors (FGF7 and FGF2), which are crucial for hair cycle regulation. This study lays the groundwork for further exploring MSCs as a potential treatment for AA, but more research is needed to fully understand their therapeutic potential.

Hair loss affects men and women of all ages. Myokines, which are mainly secreted by skeletal muscles during exercise, have numerous health benefits. VEGF, IGF-1, FGF and irisin are reprehensive myokines. Although VEGF, IGF-1 and FGF are positively associated with hair growth, few studies have researched the effects of irisin on hair growth. Here, we investigated whether irisin promotes hair growth using in vitro, ex vivo and in vivo patch assays, as well as mouse models. We show that irisin increases proliferation, alkaline phosphatase (ALP) activity and mitochondrial membrane potential in human dermal papilla cells (hDPCs). Irisin activated the Wnt/β-catenin signalling pathway, thereby upregulating Wnt5a, Wnt10b and LEF-1, which play an important role in hair growth. Moreover, irisin enhanced human hair shaft elongation. In vivo, patch assays revealed that irisin promotes the generation of new hair follicles, accelerates entry into the anagen phase, and significantly increases hair growth in C57BL/6 mice. However, XAV939, a Wnt/β-catenin signalling inhibitor, suppressed the irisin-mediated increase in hair shaft and hair growth. These results indicate that irisin increases hair growth via the Wnt/β-catenin pathway and highlight its therapeutic potential in hair loss treatment., (© 2024 The Author(s). Experimental Dermatology published by John Wiley & Sons Ltd.)

Hair follicle (HF) regeneration during wound healing continues to present a significant clinical challenge. Dermal papilla cell-derived exosomes (DPC-Exos) hold immense potential for inducing HF neogenesis. However, the accurate role and underlying mechanisms of DPC-Exos in HF regeneration in wound healing remain to be fully explained. This study, represents the first analysis into the effects of DPC-Exos on fibroblasts during wound healing. Our findings demonstrated that DPC-Exos could stimulate the proliferation and migration of fibroblasts, more importantly, enhance the hair-inducing capacity of fibroblasts. Fibroblasts treated with DPC-Exos were capable of inducing HF neogenesis in nude mice when combined with neonatal mice epidermal cells. In addition, DPC-Exos accelerated wound re-epithelialization and promoted HF regeneration during the healing process. Treatment with DPC-Exos led to increased expression levels of the Wnt pathway transcription factors β-catenin and Lef1 in both fibroblasts and the dermis of skin wounds. Specifically, the application of a Wnt pathway inhibitor reduced the effects of DPC-Exos on fibroblasts and wound healing. Accordingly, these results offer evidence that DPC-Exos promote HF regeneration during wound healing by enhancing the hair-inducing capacity of fibroblasts and activating the Wnt/β-catenin signaling pathway. This suggests that DPC-Exos may represent a promising therapeutic strategy for achieving regenerative wound healing., (© 2024. The Author(s).)

With the wide application of bromuconazole (BRO), a kind of triazole fungicide, the environmental problems caused by BRO have been paid more and more attention. In this study, adult male zebrafish were exposed to environmental related concentration and the maximum non-lethal concentration for zebrafish larvae (0,50 ng/L and 7.5 mg/L) for 7 days, respectively. Zebrafish exposed to BRO exhibited a significant reduction in body length and an increase in fatness index, indicating adverse physiological changes. Notably, the exposed zebrafish showed enlarged heart ventricular volumes and thinner heart walls. Transcriptome analysis of heart samples showed that BRO exposure mainly affected pathways related to cardiac energy metabolism. In addition, the amount of ATP in the heart tissue was correspondingly reduced, and the expression levels of genes related to controlling ion balance and myosin synthesis in the heart were also altered. The study extended its findings to the rat cardiomyocytes (H9C2), where similar cardiotoxic effects including changes in transcription of genes related to energy metabolism and heart function were also observed, suggesting a potential universal mechanism of BRO-induced cardiotoxicity. In a doxorubicin (DOX) induced larval zebrafish heart failure model, the expression of lymphoid enhancer-binding factor 1(LEF1), a key gene in the Wnt/β-catenin signaling pathway, was significantly increased in larval zebrafish and adult fish heart tissues and cardiomyocytes, suggesting that LEF1 might play an important role in BRO-induced cardiotoxicity. Taken together, BRO exposure could interfere with cardiac function and metabolic capacity by abnormal activation the expression of LEF1. The study emphasized the urgent need for monitoring and regulating BRO due to its harmful effects on the hearts of aquatic organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias., (© 2024. The Author(s).)

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice ( Osr2-cre KI ; Rosa26R-Fgf8 ) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-cre KI ; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-cre KI ; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-cre KI ; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin , Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-cre KI ; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-cre KI ; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.

Background: The objective of this study is to investigate the mechanism by which low-level laser stimulation promotes the proliferation of intraepithelial hair follicle stem cells (HFSCs) in wounds. This research aims to expand the applications of laser treatment, enhance wound repair methods, and establish a theoretical and experimental foundation for achieving accelerated wound healing., Methods: The experimental approach involved irradiating a cell model with low-level laser to assess the proliferation of HFSCs and examine alterations in the expression of proteins related to the Wnt/β-catenin signaling pathway. A mouse back wound model was established to investigate the effects of low-level laser irradiation on wound healing rate, wound microenvironment, and the proliferation of HFSCs in relation to the Wnt/β-catenin signaling pathway., Results: The research findings indicate that low-level laser light effectively activates the Wnt signaling pathway, leading to the increased accumulation of core protein β-catenin and the upregulation of key downstream gene Lef 1. Consequently, this regulatory mechanism facilitates various downstream biological effects, including the notable promotion of HFSC proliferation and differentiation into skin appendages and epithelial tissues. As a result, the process of wound healing is significantly accelerated., Conclusion: Low levels of laser activates the Wnt signalling pathway, promotes the regeneration of hair follicle stem cells and accelerates wound healing., (© 2024 The Author(s). Skin Research and Technology published by John Wiley & Sons Ltd.)

The Wnt/β-catenin pathway plays a critical role in cell fate specification, morphogenesis, and stem cell activation across diverse tissues, including the skin. In mammals, the embryonic surface epithelium gives rise to the epidermis as well as the associated appendages including hair follicles and mammary glands, both of which depend on epithelial Wnt/β-catenin activity for initiation of their development. Later on, Wnts are thought to enhance mammary gland growth and branching, whereas in hair follicles, they are essential for hair shaft formation. In this study, we report a strong downregulation of epithelial Wnt/β-catenin activity as the mammary bud progresses to branching. We show that forced activation of epithelial β-catenin severely compromises embryonic mammary gland branching. However, the phenotype of conditional Lef1-deficient embryos implies that a low level of Wnt/β-catenin activity is necessary for mammary cell survival. Transcriptomic profiling suggests that sustained high β-catenin activity leads to maintenance of mammary bud gene signature at the expense of outgrowth/branching gene signature. In addition, it leads to upregulation of epidermal differentiation genes. Strikingly, we find a partial switch to hair follicle fate early on upon stabilization of β-catenin, suggesting that the level of epithelial Wnt/β-catenin signaling activity may contribute to the choice between skin appendage identities., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (T H 2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed T H 2-multipotent progenitors (T H 2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (T reg ) cells and follicular helper T (T FH ) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between T H 2-MPP, T H 2 effectors, T reg cells and T FH cells. T H 2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify T H 2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells., (© 2024. Springer Nature America, Inc.)

Acquired resistance is a significant hindrance to clinical application of lenvatinib in unresectable hepatocellular carcinoma (HCC). Further in-depth investigation of resistance mechanisms can help to develop additional therapeutic strategies to overcome or delay resistance. In our study, two lenvatinib-resistant (LR) HCC cell lines were established by treatment with gradient increasing concentration of lenvatinib, named Hep3B-LR and HepG2-LR. Interestingly, continuous lenvatinib treatment reinforced epithelial-mesenchymal transition (EMT), cell migration, and cell invasion. Gene set enrichment analysis (GSEA) enrichment analysis of RNA-sequencing from Hep3B-LR and corresponding parental cells revealed that activation of Wnt signaling pathway was involved in this adaptive process. Active β-catenin and its downstream target lymphoid enhancer binding factor 1 (LEF1) were significantly elevated in LR HCC cells, which promoted lenvatinib resistance through mediating EMT-related genes. Data analysis based on Gene Expression Omnibus (GEO) and the Cancer Genome Atlas Program (TCGA) databases suggests that LEF1, as a key regulator of EMT, was a novel molecular target linked to lenvatinib resistance and poor prognosis in HCC. Using a small-molecule specific inhibitor ICG001 and knocking down LEF1 showed that targeting LEF1 restored the sensitivity of LR HCC cells to lenvatinib. Our results uncover upregulation of LEF1 confers lenvatinib resistance by facilitating EMT, cell migration, and invasion of LR HCC cells, indicating that LEF1 is a novel therapeutic target for overcoming acquired lenvatinib resistance., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Forkhead box P3 (Foxp3)-expressing regulatory T (Treg) cells play essential roles in immune homeostasis but also contribute to establish a favorable environment for tumor growth by suppressing anti-tumor immune responses. It is thus necessary to specifically target tumor-infiltrating Treg cells to minimize effects on immune homeostasis in cancer immunotherapy. However, molecular features that distinguish tumor-infiltrating Treg cells from those in secondary lymphoid organs remain unknown. Here we characterize distinct features of tumor-infiltrating Treg cells by global analyses of the transcriptome and chromatin landscape. They exhibited activated phenotypes with enhanced Foxp3-dependent transcriptional regulation, yet being distinct from activated Treg cells in secondary lymphoid organs. Such differences may be attributed to the extensive clonal expansion of tumor-infiltrating Treg cells. Moreover, we found that TCF7 and LEF1 were specifically downregulated in tumor-infiltrating Treg cells both in mice and humans. These factors and Foxp3 co-occupied Treg suppressive function-related gene loci in secondary lymphoid organ Treg cells, whereas the absence of TCF7 and LEF1 accompanied altered gene expression and chromatin status at these gene loci in tumor-infiltrating Treg cells. Functionally, overexpression of TCF7 and LEF1 in Treg cells inhibited the enhancement of Treg suppressive function upon activation. Our results thus show the downregulation of TCF7 and LEF1 as markers of highly suppressive Treg cells in tumors and suggest that their absence controls the augmentation of Treg suppressive function in tumors. These molecules may be potential targets for novel cancer immunotherapy with minimum effects on immune homeostasis., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Competing Interests: The authors declare no conflict of interest.

Backgrounds: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovial hyperplasia. Maintaining a balance between the proliferation and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs) is crucial for preventing the erosion of bone and cartilage and, ultimately, mitigating the progression of RA. We found that the lncRNA LEF1-AS1 was expressed at low levels in the RASFs and inhibited their abnormal proliferation by targeting PIK3R2 protein and regulating the PI3K/AKT signal pathway through its interaction with miR-30-5p. In this study, we fabricated a nano-drug delivery system for LEF1-AS1 using Zn-Adenine nanoparticles (NPs) as a novel therapeutic strategy against RA., Methods: The expression levels of LEF1-AS1, miR-30-5p, PIK3R2, p-PI3K, and p-AKT were detected in the primary RASFs and a human fibroblast-like synovial cell line (HFLS). Zn-Adenine nanoparticles (NPs) were functionalized with anti-CD305 antibody to construct (Zn-Adenine)@Ab. These NPs were then loaded with LEF1-AS1 to form (Zn-Adenine)@Ab@lncRNA LEF1-AS1. Finally, the (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs were locally injected into a rat model with collagen-induced arthritis (CIA). The arthritic injuries in each group were evaluated by HE staining and other methods., Results: LEF1-AS1 was expressed at low levels in the primary RASFs. High expression levels of LEF1-AS1 were detected in the HFLS cells, which corresponded to a significant downregulation of miR-30-5p. In addition, the expression level of PIK3R2 was significantly increased, and that of p-PI3K and p-AKT were significantly downregulated in these cells. The (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly inhibited the proliferation of RASFs and decreased the production of inflammatory cytokines (IL-1β, IL-6, TNF-α). Intra-articular injection (IAI) of (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly alleviated cartilage destruction and joint injury in the CIA-modeled rats., Conclusions: LEF1-AS1 interacts with miR-30-5p to inhibit the abnormal proliferation of RASFs by regulating the PI3K/AKT signal pathway. The (Zn-Adenine)@Ab NPs achieved targeted delivery of the loaded LEF1-AS1 into the RASFs, which improved the cellular internalization rate and therapeutic effects. Thus, LEF1-AS1 is a potential target for the treatment of RA., (© 2023. The Author(s).)

The study of aging and its mechanisms, such as cellular senescence, has provided valuable insights into age-related pathologies, thus contributing to their prevention and treatment. The current abundance of high-throughput data combined with the surge of robust analysis algorithms has facilitated novel ways of identifying underlying pathways that may drive these pathologies. For the purpose of identifying key regulators of lung aging, we performed comparative analyses of transcriptional profiles of aged versus young human subjects and mice, focusing on the common age-related changes in the transcriptional regulation in lung macrophages, T cells, and B immune cells. Importantly, we validated our findings in cell culture assays and human lung samples. Our analysis identified lymphoid enhancer binding factor 1 (LEF1) as an important age-associated regulator of gene expression in all three cell types across different tissues and species. Follow-up experiments showed that the differential expression of long and short LEF1 isoforms is a key regulatory mechanism of cellular senescence. Further examination of lung tissue from patients with idiopathic pulmonary fibrosis, an age-related disease with strong ties to cellular senescence, revealed a stark dysregulation of LEF1. Collectively, our results suggest that LEF1 is a key factor of aging, and its differential regulation is associated with human and murine cellular senescence., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Background: Lymphocyte enhancer-binding factor-1 (LEF1) is responsible for melanocyte proliferation, migration and differentiation and its downregulation may result in depigmentation in vitiligo. Narrowband UVB (NB-UVB) phototherapy is known to enhance melanocyte migration from hair follicles to lesional epidermis; hence, it may have a role in the upregulation of LEF1., Objectives: We intended to assess the expression of LEF1 both before and after NB-UVB therapy and correlate it with the extent of re-pigmentation., Materials and Methods: In this prospective cohort study, 30 patients of unstable non-segmental vitiligo were administered NB-UVB phototherapy for 24 weeks. Skin biopsies were obtained from acral and non-acral sites in all patients, both prior to initiation and after completion of phototherapy and LEF1 expression was measured., Results: Amongst the 16 patients who completed the study, at 24 weeks, all patients achieved > 50% re-pigmentation. However, > 75% re-pigmentation was achieved in only 11.1% of acral patches, whereas it was achieved in a significantly higher number of non-acral patches (66.6%) (p = 0.05). A significant increase was observed in the mean fluorescent intensity of the LEF1 gene in both acral as well as non-acral areas at 24 weeks as compared to baseline (p = 0.0078), However, no difference was observed between acral and non-acral lesions in the LEF1 expression at 24 weeks or the change in LEF1 expression from baseline., Conclusion: LEF1 expression modulates the re-pigmentation of vitiligo lesions after treatment with NBUVB phototherapy., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The role of programmed cell death 1 (PD1) in cancer immune evasion is of considerable importance, prompting the development of monoclonal antibodies that specifically target PD-1 to enhance the immune system for cancer therapy. Nevertheless, the efficacy of PD1/programmed cell death-Ligand 1 (PD-L1) blocking antibodies is limited to certain patients or tumor types. Although researchers have demonstrated the influence of PD-1 on the positive selection of T cells, its effect on the T-cell repertoire remains uncertain. Lymphoid enhancer binding factor 1 (LEF1) has been known to play a critical role as a transcription factor in the development and maturation of T cells. Despite the greater focus on the study of its homologous protein, T cell factor 1 (TCF1), we discovered that LEF1 had a positive regulatory effect on the transcription of PD1 in mature T cells, including CD4+ T cells, CD8+ T cells, and Treg cells. This finding was observed in LEF1 knockout and LEF1-stimulated mice models. Additionally, we confirmed the direct regulation of PD1 by LEF1 in tumor-infiltrating lymphocytes through tumor-implantation experiments. The direct regulation of PD1 by LEF1 was further validated in the LEF1 knockout cell line. The results of our study provide novel perspectives on the regulation of PD1 in immune responses and investigate potential approaches for clinical anti-PD1 therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is the most prevalent cancer of the mouth, characterised by rapid progression and poor prognosis. Hence, an urgent need exists for the development of predictive targets for early diagnosis, prognosis determination, and clinical therapy. Dysregulation of lymphoid enhancer-binding factor 1 (LEF1), an important transcription factor involved in the Wnt-β-catenin pathway, contributes to the poor prognosis of OSCC. Herein, we aimed to explore the correlation between LEF1 and histone lysine demethylase 4 A (KDM4A). Results show that the KDM4A complex is recruited by LEF1 and specifically binds the LATS2 promoter region, thereby inhibiting its expression, and consequently promoting cell proliferation and impeding apoptosis in OSCC. We also established NOD/SCID mouse xenograft models using CAL-27 cells to conduct an in vivo analysis of the roles of LEF1 and KDM4A in tumour growth, and our findings show that cells stably suppressing LEF1 or KDM4A have markedly decreased tumour-initiating capacity. Overall, the results of this study demonstrate that LEF1 plays a pivotal role in OSCC development and has potential to serve as a target for early diagnosis and treatment of OSCC., (© 2023. The Author(s).)

The Wnt signaling pathway plays a critical role in regulating normal cellular processes, including proliferation, differentiation, and apoptosis. Dysregulation of Wnt signaling has been implicated in various human diseases, including cancer. β-catenin and LEF1 are key mediators of Wnt signaling, and their dysregulation is a hallmark of many cancer types. In this study, we aimed to identify the deubiquitinases (DUBs) that regulate the Wnt signaling pathway through the essential component LEF1. Screening candidate DUBs from the human DUB library, we discovered that OTUD7B interacts with LEF1 and activates Wnt signaling. OTUD7B and LEF1 interact with each other through the UBA and HMG domains, respectively. Furthermore, OTUD7B promotes the nuclear localization of LEF1, leading to an increased interaction with β-catenin in the nucleus while not noticeably affecting ubiquitination on LEF1. Using qPCR array analysis, we found that OTUD7B overexpression leads to an upregulation of 75% of the tested Wnt target genes compared to the control. These findings suggest that OTUD7B may serve as a potential therapeutic target in human diseases, including cancers where Wnt signaling is frequently dysregulated.

Despite the improvement of current classical treatment, the prognosis of esophageal squamous cell carcinoma (ESCC) remains poor. Immunotherapy, as a new treatment method, has revolutionized the therapy of various cancer types and created more attractive for ESCC. Cancer-testis genes (CTGs), because of its characteristic expression and immunomodulation property, are considered as the ideal targets for tumor immunotherapy. However, the ESCC-specific CTGs, especially long non-coding RNA (lncRNA), has not been elucidated. In the present study, a systematic strategy was adopted to screen ESCC-specific cancer-testis lncRNA (CT-lncRNA). Collectively, 447 genes were recognized as ESCC-specific CT-lncRNAs, in particularly LEF1-AS1 showed the most aberrantly expression and clinically associated with poor outcome. Functional assays revealed that H3K27 acetylation in LEF1-AS1 promoter might give rise to the activation of LEF1-AS1 during ESCC tumorigenesis. The activated LEF1-AS1 was predominantly localized in the cytoplasm implicated in regulation of apoptosis and proliferation capacities of ESCC cells in vitro and in vivo. Further mechanistic studies unveiled that LEF1-AS1 participated in ESCC by interacting with RNA binding protein PDCD5 through weakened its nuclear translocation binding to TP53, leading to p53 degradation and disruption the transcription of downstream genes. Taken together, our findings suggest that LEF1-AS1 acts as a CT-lncRNA and might be an ideal immunotherapeutic target for clinical intervention for ESCC., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: High-intensity chemotherapy regimens are often used in adult T-cell lymphoblastic lymphoma (T-LBL) patients. Nevertheless, the response rate remains unsatisfactory due to emergence of chemoresistance. Growing evidence has shown that long non-coding RNAs (lncRNAs) are involved in tumor progression and chemoresistance. Herein, we investigated the potential role of lncRNAs in T-LBLs., Methods: RNAseq was used to screen and identify candidate lncRNAs associated with T-LBL progression and chemoresistance. Luciferase reporter assay was used to examine the binding of miR-371b-5p to the 3'UTR of Smad2 and LEF1, and the binding of TCF-4/LEF1 to the promoter of LINC00183. Chromatin immunoprecipitation assay was undertaken to analyze the connection between LEF1 and the LINC00183 promoter region. RNA immunoprecipitation assays were used to explore the mechanism whereby LINC00183 regulated miR-371b-5p. MTT and flow cytometry assays were used to measure apoptosis of T-LBL cells., Results: LINC00183 was upregulated in T-LBL progression and chemoresistant tissues in both the Sun Yat-sen University Cancer Center dataset and the First Affiliated Hospital of Anhui Medical University dataset. High expression of LINC00183 was correlated with poorer overall survival and progression-free survival of T-LBL patients compared to those with low expression of LINC00183. Furthermore, miR-371b-5p was negatively regulated by LINC00183. In vivo and in vitro assays showed that LINC00183-mediated T-LBL chemoresistance depended on miR-371b-5p expression. The direct binding of miR-371b-5p to Smad2 and LEF1 was verified by luciferase assays. It was shown that TCF4/LEF1 could bind to the LINC00183 promoter site and increase its transcript level. Downregulation of miR-371b-5p led to increased expression of Smad2/LEF1, and in turn increased LINC00183 expression. Additionally, phospho-Smad2 promotes nuclear translocation of β-catenin, LINC00183 downregulation decreased chemoresistance induced by β-catenin and TGF-β1 in T-LBL cells., Conclusion: We unraveled a β-catenin-LINC00183-miR-371b-5p-Smad2/LEF1 feedback loop that promotes T-LBL progression and chemoresistance, indicating that LINC00183 may serve as a potential therapeutic target in T-LBLs., (© 2023. The Author(s).)

Objectives: This study aimed to investigate the LEF-1-mediated Wnt/β-catenin pathway for its biological functions and prognostic value in colon cancer (CC). Furthermore, the potential molecular mechanism of β-sitosterol in CC was investigated in vitro., Methods: Clinical information and gene expression profiles from CC patients were obtained based on Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. In addition, we applied R software "Limma" package for the differential analysis of LEF-1 between cancer and para-carcinoma tissue samples. Kaplan-Meier (KM) survival analysis was adopted for analyzing whether LEF-1 was of prognostic significance. Moreover, gene set enrichment analysis (GSEA) was adopted for pathway enrichment analysis and visualization. In addition, CCK8, plate cloning, scratch and high-content screening (HCS) imaging assays were performed to examine the therapeutic efficacy of β-sitosterol in human CC HCT116 cells. siRNA technology was employed to knock down LEF1 expression in HCT116 cells. qRT-PCR and Western-blot (WB) analysis were carried out to analyze the HCT-116 mRNA and protein expression levels, respectively., Results: LEF-1 was up-regulated within CC and acted as an oncogenic gene. LEF-1 up-regulation predicted the dismal prognostic outcome and activated the Wnt/β-catenin pathway. β-sitosterol effectively suppressed HCT116 cells proliferation and invasion. For the mechanism underlying β-sitosterol, β-sitosterol was found to significantly down-regulate LEF-1 gene and protein expression and disrupt Wnt/β-catenin pathway transmission in HCT116 cells. After suppressing LEF-1 expression, its downstream targets including C-myc, Survivin and CCND1 were also down-regulated., Conclusion: According to our results, LEF-1 down-regulation can effectively block Wnt/β-catenin pathway, inhibit CC cell growth and migration. Collectively, β-sitosterol can be used to treat CC, which can provide anti-tumor activity by targeting LEF-1., Competing Interests: Declaration of Competing Interest The authors declared no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Transcription factor lymphoid enhancer-binding factor 1 (LEF1) is a downstream mediator of the Wnt/β-catenin signaling pathway. It is expressed in dermal papilla and surrounding cells in the hair follicle, promoting cell proliferation, and differentiation., Results: Here, we report that LEF1 is also expressed all through the hair cycle in the terminal Schwann cells (TSCs), a component of the lanceolate complex located at the isthmus. The timing of LEF1 appearance at the isthmus coincides with that of hair follicle innervation. LEF1 is not found at the isthmus in the aberrant hair follicles in nude mice. Instead, LEF1 in TSCs is found in the de novo hair follicles reconstituted on nude mice by stem cells chamber graft assay. Cutaneous denervation experiment demonstrates that the LEF1 expression in TSCs is independent of nerve endings. At last, LEF1 expression in the interfollicular epidermis during the early stage of skin development is significantly suppressed in transgenic mice with T-cell factor 3 (TCF3) overexpression., Conclusion: We reveal the expression dynamics of LEF1 in skin during development and hair cycle. LEF1 expression in TSCs indicates that the LEF1/Wnt signal might help to establish a niche at the isthmus region for the lanceolate complex, the bulge stem cells and other neighboring cells., (© 2022 American Association of Anatomists.)

Follicular atresia is a natural physiological phenomenon in poultry reproduction. It is well known that follicular atresia is caused by both autophagy and apoptosis of granulosa cells. In current experiment, we evaluated the function of miR-34a-5p on autophagy and apoptosis in chicken follicular atresia. First, the follicular atresia model of chicken was successfully constructed by subcutaneous injection of tamoxifen (TMX), and found the expression of miR-34a-5p in the atresia follicles obviously increased. Then, we confirmed that miR-34a-5p accelerates autophagy and apoptosis of chicken granulose cells in vitro, and miR-34a-5p could induce apoptosis by mediating autophagy. Mechanistically, lymphoid enhancer binding factor 1 (LEF1) was deemed as a target gene for miR-34a-5p. On the contrary, LEF1 overexpression attenuated the autophagy and apoptosis of chicken granular cells. In addition, it was confirmed that the miR-34a-5p/LEF1 axis plays a regulatory role in chicken granulosa cells by mediating the Hippo-YAP signaling pathway. Taken together, this study demonstrated that miR-34a-5p contributes to autophagy and apoptosis of chicken follicular granulosa cells by targeting LEF1 to mediate the Hippo-YAP signaling pathway., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Unrestrained transcriptional activity of β-CATENIN and its binding partner TCF7L2 frequently underlies colorectal tumor initiation and is considered an obligatory oncogenic driver throughout intestinal carcinogenesis. Yet, the TCF7L2 gene carries inactivating mutations in about 10% of colorectal tumors and is non-essential in colorectal cancer (CRC) cell lines. To determine whether CRC cells acquire TCF7L2-independence through cancer-specific compensation by other T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family members, or rather lose addiction to β-CATENIN/TCF7L2-driven gene expression altogether, we generated multiple CRC cell lines entirely negative for TCF/LEF or β-CATENIN expression. Survival of these cells and the ability to propagate them demonstrate their complete β-CATENIN- and TCF/LEF-independence. Nonetheless, one β-CATENIN-deficient cell line eventually became senescent, and absence of TCF/LEF proteins and β-CATENIN consistently impaired CRC cell proliferation, reminiscent of mitogenic effects of WNT/β-CATENIN signaling in the healthy intestine. Despite this common phenotype, β-CATENIN-deficient cells exhibited highly cell-line-specific gene expression changes with little overlap between β-CATENIN- and TCF7L2-dependent transcriptomes. Apparently, β-CATENIN and TCF7L2 independently control sizeable fractions of their target genes. The observed divergence of β-CATENIN and TCF7L2 transcriptional programs, and the finding that neither β-CATENIN nor TCF/LEF activity is strictly required for CRC cell survival has important implications when evaluating these factors as potential drug targets., (© 2023. The Author(s).)

Aims: Colorectal liver metastasis (CRLM) is the leading death-causing among colorectal cancer (CRC) patients. Recently, a novel tumor-related microRNA, miR-621, has been identified as a tumor suppressor in diverse tumor types, but its role in CRLM remains unclear and requires further investigation., Main Methods: To elucidate novel regulators of CRLM progression, we used a well-established CRLM animal model. After serially transplanting human colon carcinoma cell lines Caco-2 into the liver, we obtained liver metastatic variants that exhibited a strong ability for invasion and metastasis. High-throughput sequencing was conducted on these newly established cell lines. After comparison and prediction between the two cell lines: parental Caco-2 (hereafter referred to as F0) and F3, miR-621 was identified as a candidate regulator for lymphoid enhancer-binding factor 1 (LEF1) expression. Further validation was achieved with dual-luciferase reporter assay., Key Findings: The gain- and loss-of-function validation showed that miR-621 inhibits cell viability, cell cycle progression, colony formation, and proliferation in vitro. Meanwhile, miR-621 could reverse EMT malignant phenotype. LEF1, an important downstream mediator of activated Wnt/β-catenin signaling pathway, was validated as the direct functional target of miR-621. miR-621 interacts directly with the LEF1 3'-UTR and post-transcriptionally suppresses LEF1 expression. Moreover, LEF1 overexpression reversed the effect of miR-621. LEF1 silencing counteracted miR-621 down-regulation-induced effects. Further in vivo experiments revealed that miR-621 over-expression suppressed CRLM, but LEF1 abrogated the inhibitory effect of miR-621., Significance: MiR-621 is a vital tumor suppressor in CRC and could be a promising anti-cancer therapeutic target., Competing Interests: Declaration of competing interest All authors declare that there is no conflict of interest., (Copyright © 2022. Published by Elsevier Inc.)

Purpose: Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is not a uniform entity but consists of various disease subtypes associated with Epstein-Barr virus (EBV) infection. Lymphoid enhancer binding factor-1 (LEF-1), a member of the T-cell factor/LEF family of transcription factors, plays a significant role in NK-cell and T-cell development. We aimed to explore the expression of LEF-1 in ENKTL and evaluate the applicability of LEF-1 in the diagnosis of ENKTL., Methods: The expression of LEF-1 was investigated in tissue samples harvested from patients with ENKTL by immunohistochemistry., Results: LEF-1 staining was not observed in 85 of 88 ENKTL cases (97%). Eighty-six of the 88 cases (98%) were positive for CD56, whereas all the tested cases were negative for both CD16 and CD27. Of the cytotoxic T-cell-like features studied, 81 cases (92%) were negative for CD8, 85 of 88 cases (97%) were immunoreactive for the recombinant linker for activation of T cells (LAT), all (100%) were immunoreactive for TIA-1, 85 (97%) were immunoreactive for granzyme B and 65 (74%) were immunoreactive for perforin., Conclusion: Loss of LEF-1 expression is a highly specific diagnostic indicator of ENKTL., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Synergies of transcription factors, chromatin modifiers and their target genes are vital for cell fate determination in human cancer. Although the importance of numerous epigenetic machinery for regulating gliomagenesis has been previously recognized, how chromatin modifiers collaborate with specific transcription factors remains largely elusive. Herein we report that Pontin chromatin remodelling factor acts as a coactivator for LEF1 to activate TGFβ/SMAD signalling, thereby contributing to gliomagenesis. Pontin is highly expressed in gliomas, and its overexpression paralleled the grade elevation and poor prognosis of patients. Functional studies verified its oncogenic roles in GBM cells by facilitating cell proliferation, survival and invasion both in vitro and in vivo. RNA sequencing results revealed that Pontin regulated multiple target genes involved in TGFβ/SMAD signalling. Intriguingly, we found that Pontin amplified TGFβR2 gene transcription by recruiting LEF1, thereby activating TGFβ/SMAD signalling and facilitating gliomagenesis. Furthermore, higher TGFβR2 expression conferred worse patient outcomes in glioma. To conclude, our study revealed that the Pontin-LEF1 module plays a crucial role in driving TGFβR2 gene transcription, which could be exploited to target TGFβ/SMAD signalling for anti-glioma therapy., (© 2022. The Author(s).)

Purpose: Bronchopulmonary dysplasia (BPD) is a long-term respiratory condition. More than a quarter of extremely premature newborns are harmed by BPD. At present, there are no apparent effective drugs or treatments for the condition. In this study, we aimed to investigate the functional role and mechanism of lymphoid enhancer-binding factor 1 ( Lef1 ) in BPD in vitro ., Materials and Methods: Blood samples from BPD patients and healthy volunteers were gathered, and an in vitro model of BPD was developed in alveolar epithelial cells (AECs) MLE-12 induced by hyperoxia. Then expression of krüppel-like factor 4 ( KLF4 / Klf4 ) and LEF1 / Lef1 were evaluated. After Lef1 overexpressing plasmid and the vector were transfected into hyperoxia-induced MLE-12 cells, cell proliferation assays were carried out. Cell apoptosis was investigated by a flow cytometry assay, and apoptosis related proteins Bcl-2, cleaved-caspase 3 and 9 were analyzed by a western blot assay. The binding between Klf4 and Lef1 promoter predicted on the JASPAR website was verified using luciferase and ChIP assays. For further study of the mechanism of Klf4 and Lef1 in BPD, gain-of-function experiments were performed., Results: The mRNA levels of KLF4 / Klf4 and LEF1 / Lef1 were diminished in clinical BPD serum samples and hyperoxia-induced MLE-12 cells. Overexpression of Lef1 stimulated AEC proliferation and suppressed AEC apoptosis induced by hyperoxia. Mechanically, Klf4 bound to Lef1 's promoter region and aids transcription. Moreover, the results of gain-of-function experiments supported that Klf4 could impede AEC damage induced by hyperoxia via stimulating Lef1 ., Conclusion: Klf4 and Lef1 expression levels were declined in hyperoxia-induced AECs, and Lef1 could be transcriptionally activated by Klf4 and protect against hyperoxia-induced AEC injury in BPD. As a result, Lef1 might become a prospective therapeutic target for BPD.

Pulmonary hypertension (PH) is mainly characterized by abnormal pulmonary vascular hyperplasia and vascular remodeling, but its mechanism is complicated and currently unclear. Cysteine and glycine-rich protein 2 (Csrp2) has been reported to promote cell proliferation and migration, and affect cell cycle progression. As a new invasive actin-binding factor, Csrp2 increased the invasion and even metastasis of some cancer cells. It was associated with tumor recurrence and chemotherapy resistance. However, the role of Csrp2 in PH remains unknown. We found that Csrp2 expression was increased both in pulmonary arteries (PAs) and smooth muscle cells (PASMCs) in PH. Csrp2 enhanced PASMC proliferation and phenotypic transition. The Wnt3α-β-catenin/lymphoid enhancer-binding factor 1 (LEF1) pathway is involved in cell proliferation and phenotypic transition regulated by Csrp2 expression. These results suggest that hypoxia downregulates YinYang-1 (YY1) and then increases Csrp2 expression. Increased Csrp2 promotes PASMC proliferation and phenotypic transition by activating the Wnt3α-β-catenin/LEF1 pathways, which leads to pulmonary vascular remodeling and even provides a new theoretical basis for studying the pathogenesis and therapeutic targets of PH., (© 2022 Wiley Periodicals LLC.)

The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2-transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: COL10A1 is a secreted, short-chain collagen found in several types of cancer. Studies have shown that COL10A1 aberrant expression is considered an oncogenic factor. However, its underlying mechanisms and regulation of gastric cancer remain undefined., Methods: The data on the expression of COL10A1, clinicopathological characteristics, and outcome of patients with GC were obtained from The Cancer Genome Atlas. The ALGGEN-PROMO database defined the related transcription factors. Quantitative real-time reverse transcription-polymerase chain reaction and western blotting analysis were used to identify the differential expression levels of COL10A1 and related transcription factors., Results: We found that high COL10A1 expression is an independent risk factor for gastric cancer. Upregulation of LEF1 and Wnt2 was also observed in gastric cancer, suggesting a potential correlation between LEF1/COL10A1 regulation in the Wnt2 signaling pathway., Conclusion: High COL10A1 expression may contribute to poor outcomes via upregulation of LEF1 and Wnt2 in gastric cancer., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Low expression of CTBP2 and CASP8AP2 correlated with poor outcome and predicted risk of relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to investigate the molecular mechanism by which CASP8AP2 regulates LEF1 expression by interacting with CtBP2 and ZEB2 in Acute lymphoblastic lymphoma (ALL). There was an interaction between CASP8AP2, ZEB2, and CtBP2, and then the interaction between CtBP2 and ZEB2 was observed after downregulating the expression of CASP8AP2. The wild type (containing the ZEB2 binding site) or mutant (containing a mutant binding site) LEF1 gene promoter sequence was inserted into the pGL3-basic plasmid, and a dual-luciferase reporter gene detection system was used to observe how CASP8AP2, ZEB2, and CtBP2 regulate the transcription of the LEF1 gene. We conclude that CASP8AP2, CtBP2, and ZEB2 can all bind to the LEF1 gene promoter region and reduce the luciferase activity of the LEF1 promoter. Meanwhile, the interaction of ZEB2 and the LEF1 promoter was significantly weakened after downregulation of CASP8AP2. Knockdown of CASP8AP2 in the 697 cell lines resulted in the significant upregulation of the mRNA expression levels of the stemness-related genes CD44 , JAG1 , and SALL4 . In conclusion, CASP8AP2 is vital for the interaction between CtBP2 and ZEB2, inhibiting LEF1 and stemness-related genes expression ALL.Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2022.2033369 .

Cartilaginous airways of larger mammals and the mouse trachea contain at least 3 well-established stem cell compartments, including basal cells of the surface airway epithelium (SAE) and ductal and myoepithelial cells of the submucosal glands (SMG). Here we demonstrate that glandular Sox9-expressing progenitors capable of SAE repair decline with age in mice. Notably, Sox9-lineage glandular progenitors produced basal and ciliated cells in the SAE, but failed to produce secretory cells. Lef1 was required for glandular Sox9 lineage contribution to SAE repair, and its deletion significantly reduced proliferation following injury. By contrast, in vivo deletion of Sox9 enhanced proliferation of progenitors in both the SAE and SMG shortly following injury, but these progenitors failed to proliferate in vitro in the absence of Sox9, similar to that previously shown for Lef1 deletion. In cystic fibrosis ferret airways, Sox9 expression inversely correlated with Ki67 proliferative marker expression in SMG and the SAE. Using in vitro and ex vivo models, we demonstrate that Sox9 is extinguished as glandular progenitors exit ducts and proliferate on the airway surface and that Sox9 is required for migration and proper differentiation of SMG, but not surface airway, progenitors. We propose a model whereby Wnt/Lef1 and Sox9 signals differentially regulate the proliferative and migratory behavior of glandular progenitors, respectively., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: LEF1 encodes a transcription factor acting downstream of the WNT-β-catenin signaling pathway. It was recently suspected as a candidate for ectodermal dysplasia in 2 individuals carrying 4q35 microdeletions. We report on 12 individuals harboring LEF1 variants., Methods: High-throughput sequencing was employed to delineate the genetic underpinnings of the disease. Cellular consequences were characterized by immunofluorescence, immunoblotting, pulldown assays, and/or RNA sequencing., Results: Monoallelic variants in LEF1 were detected in 11 affected individuals from 4 unrelated families, and a biallelic variant was detected in an affected individual from a consanguineous family. The phenotypic spectrum includes various limb malformations, such as radial ray defects, polydactyly or split hand/foot, and ectodermal dysplasia. Depending on the type and location of LEF1 variants, the inheritance of this novel Mendelian condition can be either autosomal dominant or recessive. Our functional data indicate that 2 molecular mechanisms are at play: haploinsufficiency or loss of DNA binding are responsible for a mild to moderate phenotype, whereas loss of β-catenin binding caused by biallelic variants is associated with a severe phenotype. Transcriptomic studies reveal an alteration of WNT signaling., Conclusion: Our findings establish mono- and biallelic variants in LEF1 as a cause for a novel syndrome comprising limb malformations and ectodermal dysplasia., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: Fucosylation alteration is involved in several steps of human cancer pathogenesis. Dysregulated long non-coding RNA (lncRNA) often leads to malignancy in colorectal cancer (CRC)., Methods: Differential levels of LEF1-AS1, LEF1 and FUT8 are analyzed by qRT-PCR and western blot. Chip, RIP, EMSA and luciferase reporter assay confirm the direct interaction among LEF1-AS1, MLL1, H3K4me3, LEF1 and FUT8. Functionally, CRC cell proliferation, migration and invasion are analyzed by CCK8 assay, colony formation assay, transwell assay and flow cytometry. The xenografts nude mice models, lung metastasis and liver metastasis are established to determine the effect of LEF1-AS1/LEF1/FUT8 axis on CRC progression in vivo., Results: Here, we identify that LEF1-AS1 and LEF1 are higher in CRC tissues than that in adjacent tissues, as well as upregulated in CRC cell lines than that in normal colorectal cells. Altered levels of LEF1-AS1 modulate LEF1 expression, while altered LEF1 could not regulate LEF1-AS1. LEF1-AS1 recruits MLL1 to the promoter region of LEF1, induces H3K4me3 methylation modification and mediates LEF1 transcription. Furthermore, α1-6 fucosyltransferase FUT8 is overexpressed in CRC tissues and positively correlated to LEF1. FUT8 is a direct target of transcription factor LEF1, which regulates FUT8 level. Altered FUT8 also regulates the core fucosylation of CRC cells, and LEF1-AS1 mediates FUT8 level through activation of Wnt/β-catenin/LEF1 pathway, thereby resulting in β-catenin nuclear translocation. In addition, LEF1-AS1 mediates the proliferation, migration and invasion of CRC cells in vitro. LEF1-AS1 silence hinders the tumorigenesis, liver and lung metastasis of SW620 cells in vivo, while overexpressed FUT8 abolishes the suppressive impact of LEF1-AS1 repression on the biological behavior of SW620 cells., Conclusion: Our studies uncovered a novel mechanism for constitutive LEF1-AS1/LEF1/FUT8 axis in CRC progression by regulating α1, 6-fucosylation via Wnt/β-catenin pathway, and consequently, as a potential therapeutic target in CRC., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cartilage mineralization is a tightly controlled process, imperative for skeletal growth and fracture repair. However, in osteoarthritis (OA), cartilage mineralization may impact the joint range of motion, inflict pain, and increase chances for joint effusion. Here we attempt to understand the link between inflammation and cartilage mineralization by targeting Sirtuin 1 (SIRT1) and lymphoid enhancer binding factor 1 (LEF1), both reported to have contrasting effects on cartilage. We find that inflammatory-dependent cleavage of SIRT1 or its cartilage-specific genetic ablation, directly enhanced LEF1 expression accompanied by a catabolic response. Applying a posttraumatic OA (PTOA) model to cartilage-specific Sirt1 nulls displayed severe OA, which was accompanied by synovitis, meniscal mineralization, and osteophyte formation of the lateral joint compartment. Alternatively, cartilage-specific Lef1 nulls presented reduced lateral mineralization, OA severity, and local pain. Differential gene expression analysis revealed that Lef1 ablation reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Toll-like receptor (Tlr) pathways, while enhancing SRY-Box transcription factor 9 (Sox9) and cartilaginous extracellular matrix genes. The results support a link between inflammation and Lef1-dependent cartilage mineralization, mediated by the inactivation of Sirt1. By ablating Lef1 in a PTOA model, the structural and pain-related phenotypes of OA were reduced, in part, by preventing cartilage mineralization of the lateral joint compartment, partially manifested by meniscal tissue mineralization. Overall, these data provide a molecular axis to link between inflammation and cartilage in a PTOA model.

Hutchinson-Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in the human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. Here, we studied the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, we investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected β-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. A chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to the early-stage epithelial developmental markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS.

T lymphocyte acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease affecting T cells at multiple stages of their development and is characterized by frequent genomic alterations. The transcription factor LEF1 is inactivated through mutation in a subset of T-ALL cases but elevated LEF1 expression and activating mutations have also been identified in this disease. Here we show, in a murine model of T-ALL arising due to E2a inactivation, that the developmental timing of Lef1 mutation impacts its ability to function as a cooperative tumor suppressor or oncogene. T cell transformation in the presence of LEF1 allows leukemic cells to become addicted to its presence. In contrast, deletion prior to transformation both accelerates leukemogenesis and results in leukemic cells with altered expression of genes controlling receptor-signaling pathways. Our data demonstrate that the developmental timing of Lef1 mutations impact its apparent oncogenic or tumor suppressive characteristics and demonstrate the utility of mouse models for understanding the cooperation and consequence of mutational order in leukemogenesis., Competing Interests: BK is on the Scientific Advisory Board for Century Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer PN declared a shared affiliation, with no collaboration, with the author MS to the handling editor at the time of review., (Copyright © 2022 Carr, McGregor, Dias, Verykokakis, Le Beau, Xue, Sigvardsson, Bartom and Kee.)

Nuclear pore complex (NPC) embedded in the nuclear envelope, is the only channel for macromolecule nucleocytoplasmic transportation and has important biological functions. However, the deregulation of specific nucleoporins (Nups) and NPC-Nup-based mechanisms and their function in tumour progression remain poorly understood. Here, we aimed to identify the Nups that contribute to HCC progression and metastasis in 729 primary hepatocellular carcinoma (HCC) cases using molecular, cytological, and biochemical techniques. Our results revealed elevated Nup93 expression in HCC tissues, especially in cases with metastasis, and was linked to worse prognosis. Furthermore, Nup93 knockdown suppressed HCC cell metastasis and proliferation, while Nup93 overexpression promoted these activities. We observed that Nup93 promotes HCC metastasis and proliferation by regulating β-catenin translocation. In addition, we found that Nup93 interacted with β-catenin directly, independent of importin. Furthermore, LEF1 and β-catenin facilitated the Nup93-mediated metastasis and proliferation in HCC via a positive feedback loop. Thus, our findings provide novel insights into the mechanisms underlying the Nup93-induced promotion of HCC metastasis and suggest potential therapeutic targets in the LEF1-Nup93-β-catenin pathway for HCC therapeutics., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Hair follicle stem cells (HFSCs) play a significant role in hair development. miR-1 has been reported as an important regulatory factor that affects hair follicle growth and development, but its regulatory mechanism on HFSC development remains unknown. In this study, the molecular mechanism of miR-1 in regulating HFSC proliferation and differentiation was investigated. High-throughput RNA-seq and integrated analysis were performed to identify differentially transcribed mRNAs and microRNAs (miRNAs) in HFSCs co-cultured with dermal papilla cells (named dHFSCs) and control HFSCs. We then determined the molecular function of miR-1 in HFSCs. Compared with HFSCs, 13 differentially transcribed miRNAs were identified in dHFSCs. The in vitro results indicated that the overtranscription of miR-1 inhibited HFSC proliferation, but enhanced HFSC differentiation by targeting IGF1R and LEF1 genes. This study provides new insights into the molecular mechanisms of HFSC development. Approval ID (2014ZX08008-002).

Induction of new hair follicles (HFs) may be an ultimate treatment goal for alopecia; however, functional cells with HF inductivity must be expanded in bulk for clinical use. In vitro culture conditions are completely different from the in vivo microenvironment. Although fetal and postnatal dermal cells (DCs) have the potential to induce HFs, they rapidly lose this HF inductivity during culture, accompanied by a drastic change in gene expression. This suggests that epigenetic regulation may be involved. Of the various histone deacetylases (HDACs), Class I HDACs are noteworthy because they are ubiquitously expressed and have the strongest deacetylase activity. This study revealed that DCs from postnatal mice rapidly lose HF inductivity and that this reduction is accompanied by a significant decrease in histone H3 acetylation. However, MS-275, an inhibitor of class I HDACs, preserves HF inductivity in DCs during culture, increasing alkaline phosphatase activity and upregulating HF inductive genes such as BMP4, HEY1, and WIF1. In addition, the inhibition of class I HDACs activates the Wnt signaling pathway, the most well-described molecular pathway in HF development, via increased histone H3 acetylation within the promoter region of the Wnt transcription factor LEF1. Our results suggest that class I HDACs could be a potential target for the neogenesis of HFs., (© 2021. The Author(s).)

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

Melanoma is considered as the most common malignancy among skin cancers. The roles of many long non-coding RNAs (lncRNAs) have been clearly identified in multiple tumors. Nevertheless, lncRNA MSC antisense RNA 1 (MSC-AS1) has not been deeply investigated melanoma. In the present study, RT-qPCR and western blot analyses were used to measure the expression of RNAs and proteins. Functional and in vivo assays were implemented to detect the function of genes in melanoma. RNA pull-down, RIP and luciferase reporter assays were applied for determining interactions between RNA and protein molecules. It was observed that MSC-AS1 and lymphoid enhancer-binding factor 1 (LEF1) were remarkably up-regulated while microRNA-302a-3p (miR-302a-3p) down-regulated in melanoma cell lines. The silencing of MSC-AS1 hindered cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro and tumor growth in vivo. Furthermore, MSC-AS1 regulated LEF1 expression through sponging miR-302a-3p and recruiting insulin like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Eventually, LEF1 overexpression rescued cell progression impaired by MSC-AS1 knock-down. In summary, our research identified the MSC-AS1/miR-302a-3p/IGF2BP2/LEF1 axis in melanoma development, which indicated that MSC-AS1 is a potential biomarker in the treatment of melanoma., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)