Single-cell response to Wnt signaling activation reveals uncoupling of Wnt target gene expression.

Wnt signaling drives nuclear translocation of ß-catenin and its subsequent association with the DNA-bound TCF/LEF transcription factors, which dictate target gene specificity by recognizing Wnt responsive elements across the genome. ß-Catenin target genes are therefore thought to be collectively activated upon Wnt pathway stimulation. However, this appears in contrast with the non-overlapping patterns of Wnt target gene expression in several contexts, including early mammalian embryogenesis. Here we followed Wnt target gene expression in human embryonic stem cells after Wnt pathway stimulation at a single-cell resolution. Cells changed gene expression program over time consistent with three key developmental events: i) loss of pluripotency, ii) induction of Wnt target genes, and iii) mesoderm specification. Contrary to our expectation, not all cells displayed equal amplitude of Wnt target gene activation; rather, they distributed in a continuum from strong to weak responders when ranked based on the expression of the target AXIN2. Moreover, high AXIN2 did not always correspond to elevated expression of other Wnt targets, which were activated in different proportions in individual cells. The uncoupling of Wnt target gene expression was also identified in single cell transcriptomics profiling of other Wnt-responding cell types, including HEK293T, murine developing forelimbs, and human colorectal cancer. Our finding underlines the necessity to identify additional mechanisms that explain the heterogeneity of the Wnt/ß-catenin-mediated transcriptional outputs in single cells.

In silico optimization of peptides that inhibit Wnt/ß-catenin signaling.

The Wnt/ß-catenin signaling pathway causes transcriptional activation through the interaction between ß-catenin and T cell-specific transcription factor (TCF) and regulates a wide variety of cellular responses, including proliferation, differentiation and cell motility. Excessive transcriptional activation of the Wnt/ß-catenin pathway is implicated in developing or exacerbating various cancers. We have recently reported that liver receptor homolog-1 (LRH-1)-derived peptides inhibit the ß-catenin/TCF interaction. In addition, we developed a cell-penetrating peptide (CPP)-conjugated LRH-1-derived peptide that inhibits the growth of colon cancer cells and specifically inhibits the Wnt/ß-catenin pathway. Nonetheless, the inhibitory activity of the CPP-conjugated LRH-1-derived peptide was unsatisfactory (ca. 20 µM), and improving the bioactivity of peptide inhibitors is required for their in vivo applications. In this study, we optimized the LRH-1-derived peptide using in silico design to enhance its activity further. The newly designed peptides showed binding affinity toward ß-catenin comparable to the parent peptide. In addition, the CPP-conjugated stapled peptide, Penetratin-st6, showed excellent inhibition (ca. 5 µM). Thus, the combination of in silico design by MOE and MD calculations has revealed that logical molecular design of PPI inhibitory peptides targeting ß-catenin is possible. This method can be also applied to the rational design of peptide-based inhibitors targeting other proteins.

SOX9 and TCF transcription factors associate to mediate Wnt/ß-catenin target gene activation in colorectal cancer.

Activation of the Wnt/ß-catenin pathway regulates gene expression by promoting the formation of a ß-catenin-T-cell factor (TCF) complex on target enhancers. In addition to TCFs, other transcription factors interact with the Wnt/ß-catenin pathway at different levels to produce tissue-specific patterns of Wnt target gene expression. The transcription factor SOX9 potently represses many Wnt target genes by downregulating ß-catenin protein levels. Here, we find using colony formation and cell growth assays that SOX9 surprisingly promotes the proliferation of Wnt-driven colorectal cancer (CRC) cells. In contrast to how it indirectly represses Wnt targets, SOX9 directly co-occupies and activates multiple Wnt-responsive enhancers in CRC cells. Our examination of the binding site grammar of these enhancers shows the presence of TCF and SOX9 binding sites that are necessary for transcriptional activation. In addition, we identify a physical interaction between the DNA-binding domains of TCFs and SOX9 and show that TCF-SOX9 interactions are important for target gene regulation and CRC cell growth. Our work demonstrates a highly context-dependent effect of SOX9 on Wnt targets, with the presence or absence of SOX9-binding sites on Wnt-regulated enhancers determining whether they are directly activated or indirectly repressed by SOX9.

Development of a penetratin-conjugated stapled peptide that inhibits Wnt/ß-catenin signaling.

Wnt/ß-catenin pathway triggers the formation of a complex between ß-catenin and T cell-specific transcription factor (TCF), which induces transcriptional activation. Excessive transcriptional activation of this pathway is associated with the development, cause, and deterioration of various cancers. Therefore, the Wnt/ß-catenin pathway is an attractive drug target for cancer therapeutics and small molecule- and peptide-based protein-protein interaction (PPI) inhibitors have been developed. However, peptide-based PPI inhibitors generally have low cell-membrane permeability because of their large molecular size. To improve cell-membrane permeability, conjugating cell-penetrating peptides (CPPs) to PPI-inhibiting peptides is a useful method for developing intracellularly targeted PPI inhibitors. In this study, we focused on the interaction between ß-catenin and liver receptor homologue-1 (LRH-1) and designed and synthesized a series of LRH-1-derived peptides to develop inhibitors against Wnt/ß-catenin signaling. The results showed that a penetratin-conjugated LRH-1-derived peptide (Penetratin-st7) predominantly inhibited DLD-1 cell growth at 20 µM treatment via inhibition of the Wnt signaling pathway. This result suggests that Penetratin-st7 is one of promising PPI inhibitors between TCF and ß-catenin.

Synthesis of Unsymmetrical Squaramides as Allosteric GSK-3ß Inhibitors Promoting ß-Catenin-Mediated Transcription of TCF/LEF in Retinal Pigment Epithelial Cells.

The glycogen synthase kinase 3ß (GSK-3ß) is a ubiquitous enzyme that is a validated target for the development of potential therapeutics useful in several diseases including retinal degeneration. Aiming at developing an innovative class of allosteric inhibitors of GSK-3ß potentially useful for retinal degeneration, we explored the class of squaramides. The developed compounds (6 a-l) were obtained through a nontoxic one-pot synthetic protocol, which employs low-cost goods and avoids any purification step. Ethanol was used as the reaction solvent, simultaneously allowing the pure reaction products' recovery (by precipitation). Out of this set of squaramides, 6 j stood out, from computational and enzymatic converging data, as an ATP non-competitive inhibitor of GSK-3ß of micromolar potency. When engaged in cellular studies using retinal pigment epithelial cells (ARPE-19) transfected with a luciferase reporter gene under the control of T-cell factor/lymphoid enhancer factor (TCF/LEF) binding sites, 6 j was able to dose-dependently induce ß-catenin nuclear accumulation, as shown by the increased luciferase activity at a concentration of 2.5 µM.

High glucose impairs osteogenic differentiation of embryonic stem cells via early diversion of beta-catenin from Forkhead box O to T cell factor interaction.

BACKGROUND: Diabetes, which is characterized by an increase in blood glucose concentration, is accompanied by low bone turnover, increased fracture risk, and the formation of embryonic skeletal malformations. Yet, there are few studies elucidating the underlying alterations in signaling pathways leading to these osteogenic defects. We hypothesized here that bone formation deficiencies in a high glucose environment result from altered activity of beta-catenin (CTNNB1), a key contributor to osteogenic differentiation, dysregulation of which has also been implicated in the development of diabetes. METHODS: To test this hypothesis, we used a previously established embryonic stem cell (ESC) model of differentiation that mimics the diabetic environment of the developing embryo. We differentiated murine ESCs within osteogenic-inducing media containing either high (diabetic) or low (physiological) levels of D-glucose and performed time course analyses to study the influence of high glucose on early and late bone cell differentiation. RESULTS: Endpoint measures for osteogenic differentiation were reduced in a glucose-dependent manner and expression of precursor-specific markers altered at multiple time points. Furthermore, transcriptional activity of the lymphoid enhancer factor (LEF)/T cell factor (TCF) transcription factors during precursor formation stages was significantly elevated while levels of CTNNB1 complexed with Forkhead box O 3a (FOXO3a) declined. Modulation of AKT, a known upstream regulator of both LEF/TCF and FOXO3a, as well as CTNNB1 rescued some of the reductions in osteogenic output seen in the high glucose condition. CONCLUSIONS: Within our in vitro model, we found a clear involvement of LEF/TCF and FOXO3a signaling pathways in the regulation of osteogenic differentiation, which may account for the skeletal deficiencies found in newborns of diabetic mothers.

ICAT promotes colorectal cancer metastasis via binding to JUP and activating the NF-κB signaling pathway.

BACKGROUND: The inhibitor of ß-catenin and T-cell factor (ICAT) is a direct negative regulator of the canonical Wnt signaling pathway, which is an attractive therapeutic target for colorectal cancer (CRC). Accumulating evidence suggests that ICAT interacts with other proteins to exert additional functions, which are not yet fully elucidated. METHODS: The overexpression of ICAT of CRC cells was conducted by lentivirus infection and plasmids transfection and verified by quantitative real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and Western blotting. The effect of ICAT on the mobility of CRC cells was assessed by wound healing assay and transwell assay in vitro and lung metastasis in vivo. New candidate ICAT-interacting proteins were explored and verified using the STRING database, silver staining, co-immunoprecipitation mass spectrometry analysis (Co-IP/MS), and immunofluorescence (IF) staining analysis. RESULT: Inhibitor of ß-catenin and T-cell factor overexpression promoted in vitro cell migration and invasion and tumor metastasis in vivo. Co-IP/MS analysis and STRING database analyses revealed that junction plakoglobin (JUP), a homolog of ß-catenin, was involved in a novel protein interaction with ICAT. Furthermore, JUP downregulation impaired ICAT-induced migration and invasion of CRC cells. In addition, ICAT overexpression activated the NF-κB signaling pathway, which led to enhanced CRC cell migration and invasion. CONCLUSION: Inhibitor of ß-catenin and T-cell factor promoted CRC cell migration and invasion by interacting with JUP and the NF-κB signaling pathway. Thus, ICAT could be considered a protein diagnostic biomarker for predicting the metastatic ability of CRC.

Targeting the interaction of ß-catenin and TCF/LEF transcription factors to inhibit oncogenic Wnt signaling.

The Wnt/ß-catenin signaling pathway is crucially involved in embryonic development, stem cell maintenance and tissue renewal. Hyperactivation of this pathway is associated with the development and progression of various types of cancers. The transcriptional coactivator ß-catenin represents a pivotal component of the pathway and its interaction with transcription factors of the TCF/LEF family is central to pathway activation. Inhibition of this crucial protein-protein interaction via direct targeting of ß-catenin is considered a promising strategy for the inactivation of oncogenic Wnt signaling. This review summarizes advances in the development of Wnt antagonists that have been shown to directly bind ß-catenin.

Regulation of c-Fos gene transcription by stimulus-responsive protein kinases.

The basic region leucin zipper (bZIP) protein c-Fos constitutes together with other bZIP proteins the AP-1 transcription factor complex. Expression of the c-Fos gene is regulated by numerous extracellular signaling molecules including mitogens, metabolites, and ligands for receptor tyrosine kinases, G protein-coupled receptors, and cytokine receptors. Here, we analyzed the effects of the stimulus-responsive MAP kinases ERK1/2 (extracellular signal-regulated protein kinase), JNK (c-Jun N-terminal protein kinase) and p38 protein kinase on transcription of the c-Fos gene. We used chromatin-integrated c-Fos promoter-luciferase reporter genes containing inactivating point mutations of DNA binding sites for distinct transcription factors. ERK1/2, JNK, and p38 protein kinases were specifically activated following expression of either a mutant of B-Raf, a truncated version of mitogen-activated/extracellular signal responsive kinase kinase kinase-1 (MEKK1), or a mutant of MAP kinase kinase-6 (MKK6), respectively. The results show that the DNA binding sites for serum response factor (SRF) and for the ternary complex factor (TCF) are of major importance for stimulating c-Fos promoter activity by MAP kinases. ERK1/2 and p38-induced stimulation of the c-Fos promoter additionally required the DNA binding site for the transcription factor AP-1. Mutation of the DNA binding site for STAT had no or only a small effect on c-Fos promoter activity. We conclude that MAP kinases do not activate distinct transcription factors involving distinct genetic elements. Rather, these kinases mainly target SRF and TCF proteins, leading to an activation of transcription of the c-Fos gene via the serum response element.

TPL-2 Inhibits IFN-ß Expression via an ERK1/2-TCF-FOS Axis in TLR4-Stimulated Macrophages.

TPL-2 kinase plays an important role in innate immunity, activating ERK1/2 MAPKs in myeloid cells following TLR stimulation. We investigated how TPL-2 controls transcription in TLR4-stimulated mouse macrophages. TPL-2 activation of ERK1/2 regulated expression of genes encoding transcription factors, cytokines, chemokines, and signaling regulators. Bioinformatics analysis of gene clusters most rapidly induced by TPL-2 suggested that their transcription was mediated by the ternary complex factor (TCF) and FOS transcription factor families. Consistently, TPL-2 induced ERK1/2 phosphorylation of the ELK1 TCF and the expression of TCF target genes. Furthermore, transcriptomic analysis of TCF-deficient macrophages demonstrated that TCFs mediate approximately half of the transcriptional output of TPL-2 signaling, partially via induced expression of secondary transcription factors. TPL-2 signaling and TCFs were required for maximal TLR4-induced FOS expression. Comparative analysis of the transcriptome of TLR4-stimulated Fos -/- macrophages indicated that TPL-2 regulated a significant fraction of genes by controlling FOS expression levels. A key function of this ERK1/2-TCF-FOS pathway was to mediate TPL-2 suppression of type I IFN signaling, which is essential for host resistance against intracellular bacterial infection.

A ß-Catenin-TCF-Sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer.

BACKGROUND & AIMS: Sporadic colorectal cancers arise from initiating mutations in APC, producing oncogenic ß-catenin/TCF-dependent transcriptional reprogramming. Similarly, the tumor suppressor axis regulated by the intestinal epithelial receptor GUCY2C is among the earliest pathways silenced in tumorigenesis. Retention of the receptor, but loss of its paracrine ligands, guanylin and uroguanylin, is an evolutionarily conserved feature of colorectal tumors, arising in the earliest dysplastic lesions. Here, we examined a mechanism of GUCY2C ligand transcriptional silencing by ß-catenin/TCF signaling. METHODS: We performed RNA sequencing analysis of 4 unique conditional human colon cancer cell models of ß-catenin/TCF signaling to map the core Wnt-transcriptional program. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, chromatin immunoprecipitation sequencing, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) knockout, and CRISPR epigenome editing, which were cross-validated with human tissue chromatin immunoprecipitation sequencing datasets, to identify functional gene enhancers mediating GUCY2C ligand loss. RESULTS: RNA sequencing analyses reveal the GUCY2C hormones as 2 of the most sensitive targets of ß-catenin/TCF signaling, reflecting transcriptional repression. The GUCY2C hormones share an insulated genomic locus containing a novel locus control region upstream of the guanylin promoter that mediates the coordinated silencing of both genes. Targeting this region with CRISPR epigenome editing reconstituted GUCY2C ligand expression, overcoming gene inactivation by mutant ß-catenin/TCF signaling. CONCLUSIONS: These studies reveal DNA elements regulating corepression of GUCY2C ligand transcription by ß-catenin/TCF signaling, reflecting a novel pathophysiological step in tumorigenesis. They offer unique genomic strategies that could reestablish hormone expression in the context of canonical oncogenic mutations to reconstitute the GUCY2C axis and oppose transformation.

T cell-derived tumor necrosis factor induces cytotoxicity by activating RIPK1-dependent target cell death.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1's death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.

lncRNA OXCT1-AS1 Promotes Metastasis in Non-Small-Cell Lung Cancer by Stabilizing LEF1, In Vitro and In Vivo.

Long noncoding RNAs (lncRNAs) play nonnegligible roles in the metastasis of non-small-cell lung cancer (NSCLC). This study is aimed at investigating the biological role of lncRNA OXCT1-AS1 in NSCLC metastasis and the underlying regulatory mechanisms. The expression profiles of lncRNA OXCT1-AS1 in different NSCLC cell lines were examined. Then, the biological function of lncRNA OXCT1-AS1 in NSCLC metastasis was explored by loss-of-function assays in vitro and in vivo. Further, the protective effect of lncRNA OXCT1-AS1 on lymphoid enhancer factor 1 (LEF1) was examined using RNA pull-down and RNA immunoprecipitation assays. Additionally, the role of LEF1 in NSCLC metastasis was investigated. Results indicated that lncRNA OXCT1-AS1 expression was significantly increased in NSCLC cell lines. Functional analysis revealed that knockdown of lncRNA OXCT1-AS1 impaired invasion and migration in vitro. Additionally, the ability of lncRNA OXCT1-AS1 to promote NSCLC metastasis was also confirmed in vivo. Mechanistically, through direct interaction, lncRNA OXCT1-AS1 maintained LEF1 stability by blocking NARF-mediated ubiquitination. Furthermore, LEF1 knockdown impaired invasion and migration of NSCLC in vitro and in vivo. Collectively, these data highlight the ability of lncRNA OXCT1-AS1 to promote NSCLC metastasis by stabilizing LEF1 and suggest that lncRNA OXCT1-AS1 represents a novel therapeutic target in NSCLC.

Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif.

Transcriptional regulation by Wnt signalling is primarily thought to be accomplished by a complex of ß-catenin and TCF family transcription factors (TFs). Although numerous studies have suggested that additional TFs play roles in regulating Wnt target genes, their mechanisms of action have not been investigated in detail. We characterised a Wnt-responsive element (WRE) downstream of the Wnt target gene Axin2 and found that TCFs and Caudal type homeobox (CDX) proteins were required for its activation. Using a new separation-of-function TCF mutant, we found that WRE activity requires the formation of a TCF/CDX complex. Our systematic mutagenesis of this enhancer identified other sequences essential for activation by Wnt signalling, including several copies of a novel CAG DNA motif. Computational and experimental evidence indicates that the TCF/CDX/CAG mode of regulation is prevalent in multiple WREs. Put together, our results demonstrate the complex nature of cis- and trans- interactions required for signal-dependent enhancer activity.

Linderapyrone: A Wnt signal inhibitor isolated from Lindera umbellata.

Linderapyrone, a Wnt signal inhibitor was isolated from the methanolic extract of the stems and twigs of Lindera umbellata together with epi-(-)-linderol A. Linderapyrone inhibited TCF/ß-catenin transcriptional activity that was evaluated using cell-based TOPFlash luciferase assay system. To evaluate the structure-activity relationship and mechanism, we synthesized linderapyrone and its derivatives from piperitone. As the results of further bioassay for synthesized compounds, we found both of pyrone and monoterpene moieties were necessary for inhibitory effect. cDNA microarray analysis in a linderapyrone derivative treated human colorectal cancer cells showed that this compound downregulates Wnt signaling pathway. Moreover, we successes to synthesize the derivative of linderapyrone that has stronger inhibitory effect than linderapyrone and ICG-001 (positive control).

Genome-scale CRISPR-Cas9 screen of Wnt/ß-catenin signaling identifies therapeutic targets for colorectal cancer.

Aberrant activation of Wnt/ß-catenin pathway is a key driver of colorectal cancer (CRC) growth and of great therapeutic importance. In this study, we performed comprehensive CRISPR screens to interrogate the regulatory network of Wnt/ß-catenin signaling in CRC cells. We found marked discrepancies between the artificial TOP reporter activity and ß-catenin-mediated endogenous transcription and redundant roles of T cell factor/lymphoid enhancer factor transcription factors in transducing ß-catenin signaling. Compiled functional genomic screens and network analysis revealed unique epigenetic regulators of ß-catenin transcriptional output, including the histone lysine methyltransferase 2A oncoprotein (KMT2A/Mll1). Using an integrative epigenomic and transcriptional profiling approach, we show that KMT2A loss diminishes the binding of ß-catenin to consensus DNA motifs and the transcription of ß-catenin targets in CRC. These results suggest that KMT2A may be a promising target for CRCs and highlight the broader potential for exploiting epigenetic modulation as a therapeutic strategy for ß-catenin-driven malignancies.

ADCK1 activates the ß-catenin/TCF signaling pathway to promote the growth and migration of colon cancer cells.

As a result of mutations in the upstream components of the Wnt/ß-catenin signaling pathway, this cascade is abnormally activated in colon cancer. Hence, identifying the activation mechanism of this pathway is an urgent need for the treatment of colon cancer. Here, we found an increase in ADCK1 (AarF domain-containing kinase 1) expression in clinical specimens of colon cancer and animal models. Upregulation of ADCK1 expression promoted the colony formation and infiltration of cancer cells. Downregulation of ADCK1 expression inhibited the colony formation and infiltration of cancer cells, in vivo tumorigenesis, migration, and organoid formation. Molecular mechanistic studies demonstrated that ADCK1 interacted with TCF4 (T-cell factor 4) to activate the ß-catenin/TCF signaling pathway. In conclusion, our research revealed the functions of ADCK1 in the development of colon cancer and provided potential therapeutic targets.

Small-Molecule Inhibitors Targeting the Canonical WNT Signaling Pathway for the Treatment of Cancer.

Canonical WNT signaling is an important developmental pathway that has attracted increased attention for anticancer drug discovery. From the production and secretion of WNT ligands, their binding to membrane receptors, and the ß-catenin destruction complex to the expansive ß-catenin transcriptional complex, multiple components have been investigated as drug targets to modulate WNT signaling. Significant progress in developing WNT inhibitors such as porcupine inhibitors, tankyrase inhibitors, ß-catenin/coactivators, protein-protein interaction inhibitors, casein kinase modulators, DVL inhibitors, and dCTPP1 inhibitors has been made, with several candidates (e.g., LGK-974, PRI-724, and ETC-159) in human clinical trials. Herein we summarize recent progress in the drug discovery and development of small-molecule inhibitors targeting the canonical WNT pathway, focusing on their specific target proteins, in vitro and in vivo activities, physicochemical properties, and therapeutic potential. The relevant opportunities and challenges toward maintaining the balance between efficacy and toxicity in effectively targeting this pathway are also highlighted.

Emodin Suppresses the Migration and Invasion of Melanoma Cells.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), as an active ingredient in rhubarb roots and rhizomes, has been reported to possess various pharmacological properties including anti-tumor effects. Recent studies have confirmed that emodin inhibited cell proliferation and induced apoptosis of cancer cells. However, the inhibitory effect of emodin on the migration and invasion of melanoma cells and its underlying mechanism are still unclear. In the study, we observed the impercipient effects of emodin in B16F10 and A375 melanoma cells with strong metastatic abilities, focusing on the functions and mechanisms of migration and invasion of B16F10 and A375 melanoma cells. Cell counting kit-8 (CCK-8), colony formation test and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining tests confirmed that emodin possessed anti-proliferative and pro-apoptotic activities in B16F10 and A375 cells. The inhibitory effects on the migration and invasion of B16F10 and A375 cells were proved by wound healing assay and Transwell methods. Moreover, immunofluorescence assay approved the decrease in protein expression of matrix metalloproteinas (MMP)-2/-9 by emodin, and Western blot analyses revealed that emodin could increase the Bax/Bcl-2 ratio and inhibit the MMP-2/-9 protein expression and Wnt/ß-catenin pathway in a dose-depended manner. BML-284, as an agonist of Wnt/ß-catenin signaling pathway, reversed the effects of emodin on cell growth, migration and invasion in B16F10 cells. These findings may suggest that emodin treatment can be a promising therapeutic strategy for melanoma with highly metastatic abilities.

HIC1 Represses Atoh1 Transcription and Hair Cell Differentiation in the Cochlea.

Across species, expression of the basic helix-loop-helix transcription factor ATOH1 promotes differentiation of cochlear supporting cells to sensory hair cells required for hearing. In mammals, this process is limited to development, whereas nonmammalian vertebrates can also regenerate hair cells after injury. The mechanistic basis for this difference is not fully understood. Hypermethylated in cancer 1 (HIC1) is a transcriptional repressor known to inhibit Atoh1 in the cerebellum. We therefore investigated its potential role in cochlear hair cell differentiation. We find that Hic1 is expressed throughout the postnatal murine cochlear sensory epithelium. In cochlear organoids, Hic1 knockdown induces Atoh1 expression and promotes hair cell differentiation, while Hic1 overexpression hinders differentiation. Wild-type HIC1, but not the DNA-binding mutant C521S, suppresses activity of the Atoh1 autoregulatory enhancer and blocks its responsiveness to ß-catenin activation. Our findings reveal the importance of HIC1 repression of Atoh1 in the cochlea, which may be targeted to promote hair cell regeneration.