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BACKGROUND: Hypodontia is the most prevalent dental anomaly in humans, and is primarily attributed to genetic factors. Although genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNP) associated with hypodontia, genetic risk assessment remains challenging due to population-specific SNP variants. Therefore, we aimed to conducted a genetic analysis and developed a machine-learning-based predictive model to examine the association between previously reported SNPs and hypodontia in the Saudi Arabian population. Our case-control study included 106 participants (aged 8-50 years; 64 females and 42 males), comprising 54 hypodontia cases and 52 controls. We utilized TaqManTM Real-Time Polymerase Chain Reaction and allelic genotyping to analyze three selected SNPs (AXIN2: rs2240308, PAX9: rs61754301, and MSX1: rs12532) in unstimulated whole saliva samples. The chi-square test, multinomial logistic regression, and machine-learning techniques were used to assess genetic risk by using odds ratios (ORs) for multiple target variables. RESULTS: Multivariate logistic regression indicated a significant association between homozygous AXIN2 rs2240308 and the hypodontia phenotype (ORs [95% confidence interval] 2.893 [1.28-6.53]). Machine-learning algorithms revealed that the AXIN2 homozygous (A/A) genotype is a genetic risk factor for hypodontia of teeth #12, #22, and #35, whereas the AXIN2 homozygous (G/G) genotype increases the risk for hypodontia of teeth #22, #35, and #45. The PAX9 homozygous (C/C) genotype is associated with an increased risk for hypodontia of teeth #22 and #35. CONCLUSIONS: Our study confirms a link between AXIN2 and hypodontia in Saudi orthodontic patients and suggests that combining machine-learning models with SNP analysis of saliva samples can effectively identify individuals with non-syndromic hypodontia.
Subject(s)
Anodontia , Axin Protein , Machine Learning , Polymorphism, Single Nucleotide , Humans , Axin Protein/genetics , Anodontia/genetics , Case-Control Studies , Female , Male , Adolescent , Child , Adult , PAX9 Transcription Factor/genetics , Middle Aged , Saudi Arabia , Genetic Testing/methods , MSX1 Transcription Factor/genetics , Genotype , Young Adult , PhenotypeABSTRACT
AXIN1 and AXIN2 are homologous proteins that inhibit the Wnt/ß-catenin signaling pathway, which is frequently hyperactive in colorectal cancer. Stabilization of AXIN1 and AXIN2 by inhibiting their degradation through tankyrase (TNKS) allows the attenuation of Wnt signaling in cancer, attracting interest for potential targeted therapy. Here, we found that knockout or knockdown of AXIN2 in colorectal cancer cells increased the protein stability of AXIN1. The increase in AXIN1 overcompensated for the loss of AXIN2 with respect to protein levels; however, functionally it did not because loss of AXIN2 activated the pathway. Moreover, AXIN2 was highly essential in the context of TNKS inhibition because TNKS-targeting small-molecule inhibitors completely failed to inhibit Wnt signaling and to stabilize AXIN1 in AXIN2 knockout cells. The increased AXIN1 protein stability and the impaired stabilization by TNKS inhibitors indicated disrupted TNKS-AXIN1 regulation in AXIN2 knockout cells. Concordantly, mechanistic studies revealed that co-expression of AXIN2 recruited TNKS to AXIN1 and stimulated TNKS-mediated degradation of transiently expressed AXIN1 wild-type and AXIN1 mutants with impaired TNKS binding. Taken together, our data suggest that AXIN2 promotes degradation of AXIN1 through TNKS in colorectal cancer cells by directly linking the two proteins, and these findings may be relevant for TNKS inhibition-based colorectal cancer therapies.
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OBJECTIVES: The prevalence of microsatellite instability (MSI) subtype among all colon cancers in India is about 30â¯%, approximately two times more than that of western population suggesting different molecular pathogeneses. METHODS: A NanoString analysis-based Pan cancer differential expression (DE) profile was determined in a primary cohort of early-stage CRC (tumor=10, normal=7), and correlated against MSI status. Using RT-PCR, tumor-specific DE genes were validated in another cohort of MSI-high CRC (n=15). RESULTS: Among the most differentially expressed genes, AXIN2, ETV4, and RNF43 were tumor cell-specific signals, while a set of genes including COL11A1, COMP, INHBA, SPP1, MMP3, TLR2, and others were immune cell-specific signals, that had a differential expression between MSI and MSS groups. When overlapped with The Cancer Genome Atlas (TCGA) studies using the Tumor immune estimation resource tool (TIMER), and protein-protein interaction analysis by STRING.db, these genes were segregated to representative tumor cells and immune cells. On validation, the tumor-specific gene signals were inversely associated with TLR4 expression. CONCLUSIONS: The differential expression distribution of AXIN2, ETV4, and RNF43 among tumor and immune cells, suggests more than one pathological subset in the MSI-H subgroup of early-stage CRC in the Indian population.
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Here, we investigated the role of the canonical Wnt signaling pathway transcriptional regulators at the neuromuscular junction. Upon applying a denervation paradigm, the transcription levels of Ctnnb1, Tcf7l1, Tle1, Tle2, Tle3, and Tle4 were significantly downregulated. A significant decrease in canonical Wnt signaling activity was observed using the denervation paradigm in Axin2-lacZ reporter mice. Alterations in the transcriptional profile of the myogenic lineage in response to agrin (AGRN) suggested that TLE3 and TLE4, family members of groucho transducin-like enhancer of split 3 (TLE3), transcriptional repressors known to antagonize T cell factor/lymphoid enhancer factor (TCF)-mediated target gene activation, could be important regulators of canonical Wnt signaling activity at the postsynapse. Knockouts of these genes using CRISPR/Cas9 gene editing in primary skeletal muscle stem cells, called satellite cells, led to decreased AGRN-dependent acetylcholine receptor (CHRN) clustering and reduced synaptic gene transcription upon differentiation of these cells. Overall, our findings demonstrate that TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and CHRN clustering at the neuromuscular junction.
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The Wnt pathway plays critical roles in neurogenesis. The expression of Axin2 is induced by Wnt/ß-catenin signaling, making this gene a reliable indicator of canonical Wnt activity. We employed pulse-chase genetic lineage tracing with the Axin2-CreERT2 allele to follow the fate of Axin2+ lineage in the adult hippocampal formation. We found Axin2 expressed in astrocytes, neurons and endothelial cells, as well as in the choroid plexus epithelia. Simultaneously with the induction of Axin2 fate mapping by tamoxifen, we marked the dividing cells with 5-ethynyl-2'-deoxyuridine (EdU). Tamoxifen induction led to a significant increase in labeled dentate gyrus granule cells three months later. However, none of these neurons showed any EdU signal. Conversely, six months after the pulse-chase labeling with tamoxifen/EdU, we identified granule neurons that were positive for both EdU and tdTomato lineage tracer in each animal. Our data indicates that Axin2 is expressed at multiple stages of adult granule neuron differentiation. Furthermore, these findings suggest that the integration process of adult-born neurons from specific cell lineages may require more time than previously thought.
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Bone tissue provides structural support for our bodies, with the inner bone marrow (BM) acting as a hematopoietic organ. Within the BM tissue, two types of stem cells play crucial roles: mesenchymal stem cells (MSCs) (or skeletal stem cells) and hematopoietic stem cells (HSCs). These stem cells are intricately connected, where BM-MSCs give rise to bone-forming osteoblasts and serve as essential components in the BM microenvironment for sustaining HSCs. Despite the mid-20th century proposal of BM-MSCs, their in vivo identification remained elusive owing to a lack of tools for analyzing stemness, specifically self-renewal and multipotency. To address this challenge, Cre/loxP-based cell lineage tracing analyses are being employed. This technology facilitated the in vivo labeling of specific cells, enabling the tracking of their lineage, determining their stemness, and providing a deeper understanding of the in vivo dynamics governing stem cell populations responsible for maintaining hard tissues. This review delves into cell lineage tracing studies conducted using commonly employed genetically modified mice expressing Cre under the influence of LepR, Gli1, and Axin2 genes. These studies focus on research fields spanning long bones and oral/maxillofacial hard tissues, offering insights into the in vivo dynamics of stem cell populations crucial for hard tissue homeostasis.
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OBJECTIVE AND DESIGN: This study aimed to investigate Axin2 effects on myocardial infarction (MI) using a macrophage Axin2 conditional knockout (cKO) mouse model, RAW264.7 cell line, and human subepicardial tissues from patients with coronary artery bypass graft (CABG). MATERIAL OR SUBJECTS: Axin2 cKO mice showed decreased cardiac function, reduced edema, increased lymphangiogenesis, and improved repair in MI Few studies border zones. Hypoxic macrophages with Axin2 depletion exhibited decreased senescence, elevated IL6 expression, and increased LYVE1 transcription. Senescent macrophages decreased in patients with CABG and low Axin2 expression. TREATMENT: Treatment options included in this study were MI induction in Axin2 cKO mice, in vitro experiments with RAW264.7 cells, and analysis of human subepicardial tissues. METHODS: Assays included MI induction, in vitro experiments, and tissue analysis with statistical tests applied. RESULTS: Axin2 cKO improved cardiac function, reduced edema, enhanced lymphangiogenesis, and decreased senescence. Hypoxic macrophages with Axin2 depletion showed reduced senescence, increased IL6 expression, and elevated LYVE1 transcription. Senescent macrophages decreased in patients with CABG and low Axin2 expression. CONCLUSION: Targeting Axin2 emerges as a novel therapeutic strategy for regulating cardiac lymphatics and mitigating cell senescence post-MI, evidenced by improved outcomes in Axin2-deficient conditions.
Subject(s)
Interleukin-6 , Myocardial Infarction , Humans , Mice , Animals , Interleukin-6/metabolism , Myocardial Infarction/genetics , Macrophages , Immunity , Edema/metabolism , Mice, Inbred C57BL , Myocardium , Axin Protein/genetics , Axin Protein/metabolismABSTRACT
OBJECTIVES: To assess the effect of Tideglusib and CHIR99021 small molecules on the odontogenic differentiation potential of human dental pulp stem cells (hDPSCs) via Wnt/ß-catenin pathway activation. METHODOLOGY: hDPSCs were isolated from impacted third molars indicated for extraction and were characterized by flow cytometry. hDPSCs were then induced to differentiate into odontogenic lineage in the presence of Tideglusib and CHIR99021. Odontogenic differentiation was evaluated using Alizarin Red stain and RT-PCR for expression of odontogenic specific differentiation markers: DSPP, DMP1, ALP, OPN, and RUNX2 in relation to undifferentiated cells. RT-PCR was also conducted to assess the expression of Wnt/ß-catenin pathway activation marker (AXIN2). One-way ANOVA Kruskal-Wallis test was used for statistical analysis. RESULTS: Wnt/ß-catenin pathway was successfully activated by Tideglusib and CHIR99021 in hDPSCs where AXIN2 was significantly upregulated. Successful odontogenic differentiation was confirmed by Alizarin Red staining of calcified nodules. RT-PCR for odontogenic differentiation markers DSPP, DMP1, and RUNX expression by hDPSCs induced by CHIR99021 was higher than that expressed by hDPSCs induced by Tideglusib, whereas expression of OPN and ALP was higher in Tideglusib-induced cells than in CHIR99021-induced cells. CONCLUSIONS: Both small molecules successfully induced odontogenic differentiation of hDPSCs through Wnt/ß-catenin pathway activation. CLINICAL RELEVANCE: These findings suggest that Tideglusib and CHIR99021 can be applied clinically in pulp regeneration to improve strategies for vital pulp regeneration and to promote dentine repair.
Subject(s)
Dental Pulp , beta Catenin , Humans , Regeneration , Antigens, Differentiation , Stem CellsABSTRACT
BACKGROUND: The Wnt signaling pathway regulates crucial cellular processes, including stem cell development and tissue repair. Dysregulation of this pathway, particularly ß-catenin stabilization, is linked to colorectal carcinoma and other tumors. Axin2, a critical component in the pathway, plays a role in ß-catenin regulation. This study examines Axin2 expression in normal gastric mucosa and various gastric pathologies. METHODS: Formalin-fixed and paraffin-embedded tissue samples from normal stomach, gastritis, intestinal metaplasia (IM), and gastric carcinoma were collected. Axin2 and ß-catenin expression were evaluated using RNA in situ hybridization and immunohistochemistry, respectively. Histo-scores (H-scores) were calculated to quantify expression levels of Axin2. Associations between Axin2 expression and clinicopathological variables were examined. RESULTS: Axin2 expression was examined in normal stomach, gastritis, and IM tissues. Axin2 expression was mainly observed in the surface and isthmus areas in the normal stomach and gastritis, whereas Axin2 expression was markedly higher at the bases of IM. Axin2 H-scores were significantly elevated in IM (mean ± standard deviation [SD], 87.0 ± 38.9) compared to normal (mean ± SD, 18.0 ± 4.5) and gastritis tissues (mean ± SD, 33.0 ± 18.6). In total, 30% of gastric carcinomas showed higher Axin2 expression. Axin2 expression did not have significant associations with age, sex, Lauren classification, histological differentiation, invasion depth, and lymph node metastasis. However, a strong positive correlation was observed between Axin2 and nuclear ß-catenin in gastric carcinomas (p < .001). CONCLUSIONS: Axin2 expression was significantly increased in IM compared to normal and gastritis cases. In addition, Axin2 showed a strong positive association with nuclear ß-catenin expression in gastric carcinomas, demonstrating a close relationship with abnormal Wnt/ß-catenin signaling pathway.
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Given the increasing incidence of pancreatic cancer and the low survival rate, the exploration of the complex tumor microenvironment and the development of novel treatment options is urgent. NK cells, known for their cytotoxic abilities and modulation of other immune cells, are vital in recognizing and killing cancer cells. However, hypoxic conditions in the tumor microenvironment have been found to impair NK cell functionality and contribute to tumor immune escape. Therefore, we aimed to uncover the mechanism through which hypoxia mediates the immune escape of pancreatic cancer cells, focusing on the influence of miR-1275/AXIN2 on NK cells. Using a combination of GEO dataset screening, Tumor Immune Estimation Resource 2.0 immunoscore screening, and the Cancer Genome Atlas data, we identified a correlation between miR-1275 and NK cells. The down-regulation of miR-1275 was associated with decreased NK cell activity and survival in patients with pancreatic cancer. Pathway analysis further linked miR-1275 expression with the hypoxic HIF1A pathway. In vitro experiments were conducted using the NK-92 cell, revealing that hypoxia significantly reduced miR-1275 expression and correspondingly decreased the cell-killing ability of NK cells. Upregulation of miR-1275 increased perforin, IFN-γ and TNF-α expression levels and enhanced NK cell cytotoxicity. Additionally, miR-1275 was found to bind to and inhibit AXIN2 expression, which when overexpressed, partially alleviated the promotive effect of upregulated miR-1275 on NK-92 cell killing ability. In conclusion, this research underscores the critical role of the miR-1275/AXIN2 axis in hypoxia-mediated immune escape in pancreatic cancer, thus opening new potential avenues for treatment strategies.
Subject(s)
MicroRNAs , Pancreatic Neoplasms , Humans , Killer Cells, Natural , Hypoxia/genetics , Hypoxia/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Tumor Microenvironment/genetics , Axin Protein/metabolismABSTRACT
Parkinson disease (PD) is a major neurodegenerative disease that greatly undermines people's health and for which effective therapeutic strategies are currently limited. This study dissected the effects of expression changes of AXIN2, a modulator of the Wnt/beta-catenin signaling pathway, the transcription factor CREB1, and of the microRNA miR-15b-5p on apoptosis and the inflammatory response in a PD mouse model in vivo and in a cellular PD model in vitro. The analyses demonstrated low CREB1 and miR-15b-5p expression and high AXIN2 expression in both models. miR-15b-5p overexpression or AXIN2 knockdown alleviated the inflammatory response indicated by decreased levels of TNF-α, IL-6, and IL-1ß and apoptosis indicated by decreased levels of cleaved caspase-3 and Bax and elevated Bcl-2. Protection by miR-15b-5p upregulation was counteracted by the simultaneous overexpression of AXIN2. miR-15b-5p targeted AXIN2. CREB1 promoted miR-15b-5p expression, which activated the Wnt/ß-catenin pathway by inhibiting AXIN2. Collectively, the data indicate that transcriptional expression of miR-15b-5p can be promoted by CREB1 to inhibit AXIN2 and activate Wnt/ß-catenin, thereby reducing the inflammatory response and apoptosis in these PD models. These data suggest the CREB1/miR-15b-5p/AXIN2 axis is a potential therapeutic target in PD patients.
Subject(s)
MicroRNAs , Neurodegenerative Diseases , Parkinson Disease , Animals , Mice , Humans , beta Catenin , Parkinson Disease/genetics , Apoptosis , Inflammation , MicroRNAs/genetics , Cyclic AMP Response Element-Binding Protein , Axin Protein/geneticsABSTRACT
INTRODUCTION: The dysregulation of the c-Jun NH2-terminal kinase (JNK) pathway has been increasingly reported in human malignancies. Aberrant expression of the JNK pathway has also been implicated in the progression of Esophageal Squamous Cell Carcinoma (ESCC). However, the specific role and regulatory mechanisms of JNK2 in ESCC have not been extensively investigated. METHODS: In this study, we examined JNK2 expression in patient samples and performed experiments involving the knockdown and inhibition of the JNK2 in ESCC cell lines. RESULTS: Higher JNK2 expression was observed in tumor tissues compared to adjacent tissues. JNK2 overexpression was associated with advanced disease stages and poor prognosis. Furthermore, knockdown or inhibition of JNK2 in ESCC cell lines resulted in a decrease in cell proliferation and migration. CONCLUSION: Additionally, a significant decrease in the expression of ß-catenin and vimentin, along with an increase in the expression of Axin2, was observed upon downregulation of JNK2. Our study provides insight into the role of JNK2 in ESCC and its potential regulatory mechanism, offering a potential therapeutic strategy for ESCC patients with aberrant JNK2 expression.
Subject(s)
Carcinoma, Squamous Cell , Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Humans , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Down-Regulation , Esophageal Neoplasms/genetics , Esophageal Neoplasms/metabolism , Esophageal Squamous Cell Carcinoma/genetics , Gene Expression Regulation, Neoplastic , Janus Kinase 2ABSTRACT
Classically, Axin1 is considered a regulator of Wnt/ß-catenin signaling. However, Axin1's roles in host-microbial interactions have been unknown. Our recent study has demonstrated that deletion of intestinal epithelial Axin1 in epithelial cells and Paneth cells protects the host against colitis by enhancing Akkermansia muciniphila. Loss of intestinal epithelial or Paneth cell Axin1 results in increased Wnt/ß-catenin signaling, proliferation, and cell migration. This is associated with morphologically altered goblet and Paneth cells, including increased Muc2 and decreased lysozyme. Axin1 deletion specifically enriched Akkermansia muciniphila. Akkermansia muciniphila in Axin1 knockout mice is the driver of protection against DSS-induced inflammation. Here, we feature several significant conceptual changes, such as differences between Axin1 and Axin2, Axin1 in innate immunity and microbial homeostasis, and Axin1 reduction of Akkermansia muciniphila. We discuss an important trend in the field related to Paneth cells and tissue-specific Axin1 manipulation of microbiome in health and inflammation.
Subject(s)
Axin Protein , Colitis , Gastrointestinal Microbiome , Microbiota , Animals , Mice , Axin Protein/genetics , beta Catenin , Colitis/chemically induced , Colitis/genetics , Inflammation , Paneth CellsABSTRACT
In recent research, there has been a growing awareness of the role of genetic factors in the positioning and eruption of teeth in the maxilla and mandible. This study aimed to evaluate the potential of specific polymorphic markers of single nucleotide polymorphisms (SNPs) located within the PAX9, MSX1, AXIN2, and IRF6 genes to determine the predisposition to tooth impaction. The study participants were divided into two groups: the first group consisted of individuals with at least one impacted secondary tooth. In contrast, the second group (control group) had no impacted teeth in their jaws. To analyze the genes, real-time PCR (polymerase chain reaction) and TaqMan probes were utilized to detect the selected polymorphisms. The findings suggest that disruptions in the structure and function of the mentioned genetic factors such as polymorphic and haplotype variants of PAX9, MSX1, AXIN2, and IRF6 genes, which play a direct role in tooth and periodontal tissue development, might be significant factors in tooth impaction in individuals with genetic variations. Therefore, it is reasonable to hypothesize that tooth impaction may be influenced, at least in part, by the presence of specific genetic markers, including different allelic variants of the PAX9, AXIN2, and IRF6 genes, and especially MSX1.
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BACKGROUND: WNT signaling is pivotal in embryogenesis and tissue homeostasis. Aberrant WNT signaling, due to mutations in components of this pathway, contributes to the development and progression of human cancers, including colorectal cancer. AXIN2, encoded by the AXIN2 gene, is a key negative regulator and target of the canonical WNT signaling pathway. Germline mutations in AXIN2 are associated with absence of permanent teeth (hypo- and oligodontia) and predisposition to gastrointestinal polyps and cancer. The limited number of patients makes an accurate genotype-phenotype analysis currently challenging. CASE PRESENTATION: We present the case of a 55-year-old male with colorectal polyposis and hypodontia. Genetic testing confirmed a novel frameshift germline mutation in exon 8 of the AXIN2 gene. In addition, we provide an updated overview of germline AXIN2 mutations reported in literature. CONCLUSIONS: Although the number of missing teeth is less severe in our patient than in some previously reported cases, our findings provide additional evidence that missing teeth and gastrointestinal neoplasia are associated with rare pathogenic AXIN2 germline mutations.
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The pancreatic stellate cells (PSCs) play an important role in the development of pancreatic cancer (PC) through mechanisms that remain unclear. Exosomes secreted from PSCs act as mediators for communication in PC. This study aimed to explore the role of PSC-derived exosomal small RNAs derived from tRNAs (tDRs) in PC cells. Exosomes from PSCs were extracted and used to detect their effects on PC cell proliferation, migration and invasion. Exosomal tDRs profiling was performed to identify PSC-derived exosomal tDRs. ISH and qRT-PCR were used to examine the tRF-19-PNR8YPJZ levels and clinical value in clinical samples. The biological function of exosomal tRF-19-PNR8YPJZ was determined using the CCK-8, clone formation, wound healing and transwell assays, subcutaneous tumour formation and lung metastatic models. The relationship between the selected exosomal tRF-19-PNR8YPJZ and AXIN2 was determined by RNA sequencing, luciferase reporter assay. PSC-derived exosomes promoted the proliferation, migration, and invasion of PC cells. Novel and abundant tDRs are found to be differentially expressed in PANC-1 cells after treatment with PSC-derived exosomes, such as tRF-19-PNR8YPJZ. PC tissue samples showed markedly higher levels of tRF-19-PNR8YPJZ than normal controls. Patients with PC exhibiting high tRF-19-PNR8YPJZ expression had a highly lymph node invasion, metastasis, perineural invasion, advanced clinical stage and poor overall survival. Exosomal tRF-19-PNR8YPJZ from PSCs targeted AXIN2 in PC cells and decreased its expression, thus activating the Wnt pathway and promoting proliferation and metastasis. Exosomal tRF-19-PNR8YPJZ from PSCs promoted proliferation and metastasis in PC cells via AXIN2.
Subject(s)
Exosomes , MicroRNAs , Pancreatic Neoplasms , Humans , Pancreatic Stellate Cells/metabolism , Cell Line, Tumor , Cell Movement/genetics , Pancreatic Neoplasms/pathology , Exosomes/metabolism , Cell Proliferation/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Axin Protein/genetics , Axin Protein/metabolism , Pancreatic NeoplasmsABSTRACT
BACKGROUND AND PURPOSE: The scaffold molecule Axin2 is constitutively activated in colorectal cancer (CRC) and functions as a potent promoter of CRC behaviour. Pharmacological targeting of Axin2 may therefore exert a therapeutic effect in patients with CRC. Here, we discovered a potent small-molecule inhibitor of Axin2, based on the mechanism by which Axin2 is regulated post-translationally, and investigated its antitumour effects. EXPERIMENTAL APPROACH: Compound discovery and its inhibitory action on Axin2 protein were revealed by microscale thermophoresis, in vitro kinase assay, quantitative kinetic assay, immunoblotting/immunoprecipitation, RT-qPCR and cycloheximide pulse-chase assay. Compound antitumour effects and the underlying mechanisms were evaluated in multiple cell-based assays and mouse models. KEY RESULTS: We discovered that glycogen synthase kinase 3ß (GSK3ß) phosphorylates Axin2 at two consensus motifs and coupled Axin2 phosphorylation to its ubiquitination (mediated by the E3 ligase ß-Trcp2) and proteasomal degradation. The binding of Axin2 to GSK3ß in CRC cells is faint, which enables most of the Axin2 protein to maintain an unphosphorylated status and thereby permits the cells to preserve high levels of Axin2. Importantly, we identified a small-molecule compound CW85319 that enhances Axin2's interaction with GSK3ß via forming a high affinity for Axin2. Treatment of CRC cells with CW85319 enhanced Axin2 binding with GSK3ß, thereby promoting Axin2 phosphorylation, subsequent ubiquitination, and degradation. Furthermore, we demonstrated that CW85319 efficiently suppressed Axin2-driven CRC growth and metastasis, without eliciting side toxicity. CONCLUSIONS AND IMPLICATIONS: These findings suggest that pharmacological targeting of Axin2 by CW85319 may provide therapeutic benefits against certain human cancers, especially CRC.
Subject(s)
Colorectal Neoplasms , Mice , Animals , Humans , Cell Line, Tumor , Glycogen Synthase Kinase 3 beta , Disease Models, Animal , Immunoblotting , Colorectal Neoplasms/metabolism , Axin Protein/metabolismABSTRACT
Interleukin (IL)-35, a member of the IL-12 family, functions as an immunosuppressive cytokine that plays a crucial role in the regulation of immune-related disorders and inflammatory diseases. Adipose tissue, which is now recognized as an immune organ, is regulated by immunocytes through various signaling pathways, including the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) pathway and the Wnt/ß-actin pathway. However, there is limited research regarding the effects of IL-35 on adipogenesis. Our current findings indicated that IL-35 impedes the proliferation and promotes the cytotoxicity of 3T3-L1 preadipocytes. Furthermore, IL-35 inhibited the adipogenic differentiation, as well as suppressed triglyceride and lipid accumulation. Additionally, the expression of PPARγ and C/EBPα, two key regulators of adipogenesis, were both down-regulated with IL-35 treatment. In order to explicate the mechanisms underlying the effects of IL-35, we conducted an investigation into the expression of Axin2, an intracellular inhibitor of Wnt/ß-catenin signaling, in 3T3-L1 preadipocyte cells. Gene silencing of Axin2 through small interfering RNAs (siRNAs) enhanced PPARγ and C/EBPα expression while decreasing nuclear ß-catenin levels in the presence of IL-35. Furthermore, in IL-35-treated cells, Axin2 knockdown boosted adipogenic differentiation (as measured by increased Oil Red O staining). These findings imply that IL-35 regulates Axin2 expression and thereby plays an important role in adipocyte development.
Subject(s)
Adipogenesis , PPAR gamma , Mice , Animals , PPAR gamma/metabolism , beta Catenin/metabolism , Wnt Signaling Pathway , Cell Differentiation , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , CCAAT-Enhancer-Binding Protein-alpha/pharmacology , RNA, Small Interfering/pharmacology , Interleukins/pharmacology , 3T3-L1 Cells , Axin Protein/pharmacologyABSTRACT
Osteoporosis (OP) is a systemic bone disease caused by an imbalance in osteogenesis and osteoclastic resorption. Extracellular vesicles (EVs)-encapsulated miRNAs from bone mesenchymal stem cells (BMSCs) have been reported to participate in osteogenesis. MiR-16-5p is one of the miRNAs that regulates osteogenic differentiation; however, studies have shown that its role in osteogenesis is controversial. Thus, this study aims to investigate the role of miR-16-5p from BMSC-derived extracellular vesicles (EVs) in osteogenic differentiation and uncover the underlying mechanisms. In this study, we used an ovariectomized (OVX) mouse model and an H2O2-treated BMSCs model to investigate the effects of BMSC-derived EVs and EV-encapsulated miR-16-5p on OP and the underlying mechanisms. Our results proved that the miR-16-5p level was significantly decreased in H2O2-treated BMSCs, bone tissues of OVX mice, and lumbar lamina tissues from osteoporotic women. EVs-encapsulated miR-16-5p from BMSCs could promote osteogenic differentiation. Moreover, the miR-16-5p mimics promoted osteogenic differentiation of H2O2-treated BMSCs, and the effects exerted by miR-16-5p were mediated by targeting Axin2, a scaffolding protein of GSK3ß that negatively regulates the Wnt/ß-catenin signaling pathway. This study provides evidence that EVs-encapsulated miR-16-5p from BMSCs could promote osteogenic differentiation by repressing Axin2.
Subject(s)
Extracellular Vesicles , MicroRNAs , Osteoporosis , Female , Mice , Animals , Osteogenesis , Hydrogen Peroxide/metabolism , MicroRNAs/metabolism , Bone and Bones/metabolism , Cell Differentiation , Osteoporosis/genetics , Osteoporosis/metabolism , Extracellular Vesicles/metabolism , Axin Protein/genetics , Axin Protein/metabolism , Axin Protein/pharmacologyABSTRACT
The scaffolding protein Axin is an important regulator of the Wnt signaling pathway, and its dysfunction is closely related to carcinogenesis. Axin could affect the assembly and dissociation of the ß-catenin destruction complex. It can be regulated by phosphorylation, poly-ADP-ribosylation, and ubiquitination. The E3 ubiquitin ligase SIAH1 participates in the Wnt pathway by targeting various components for degradation. SIAH1 is also implicated in the regulation of Axin2 degradation, but the specific mechanism remains unclear. Here, we verified that the Axin2-GSK3 binding domain (GBD) was sufficient for SIAH1 binding by the GST pull-down assay. Our crystal structure of the Axin2/SIAH1 complex at 2.53 Å resolution reveals that one Axin2 molecule binds to one SIAH1 molecule via its GBD. These interactions critically depend on a highly conserved peptide 361EMTPVEPA368 within the Axin2-GBD, which forms a loop and binds to a deep groove formed by ß1, ß2, and ß3 of SIAH1 by the N-terminal hydrophilic amino acids Arg361 and Thr363 and the C-terminal VxP motif. The novel binding mode indicates a promising drug-binding site for regulating Wnt/ß-catenin signaling.