UBE2N promotes cell viability and glycolysis by promoting Axin1 ubiquitination in prostate cancer cells.

BACKGROUND: Ubiquitin-conjugating enzyme E2 N (UBE2N) is recognized in the progression of some cancers; however, little research has been conducted to describe its role in prostate cancer. The purpose of this paper is to explore the function and mechanism of UBE2N in prostate cancer cells. METHODS: UBE2N expression was detected in Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data, prostate cancer tissue microarrays, and prostate cancer cell lines, respectively. UBE2N knockdown or overexpression was used to analyze its role in cell viability and glycolysis of prostate cancer cells and tumor growth. XAV939 or Axin1 overexpression was co-treated with UBE2N overexpression to detect the involvement of the Wnt/ß-catenin signaling and Axin1 in the UBE2N function. UBE2N interacting with Axin1 was analyzed by co-immunoprecipitation assay. RESULTS: UBE2N was upregulated in prostate cancer and the UBE2N-high expression correlated with the poor prognosis of prostate cancer. UBE2N knockdown inhibited cell viability and glycolysis in prostate cancer cells and restricted tumor formation in tumor-bearing mice. Wnt/ß-catenin inhibition and Axin1 overexpression reversed the promoting viability and glycolysis function of UBE2N. UBE2N promoted Axin1 ubiquitination and decreased Axin1 protein level.

A small molecule compound 759 inhibits the wnt/beta-catenin signaling pathway via increasing the Axin protein stability.

Wnt/ß-catenin signaling plays important role in cancers. Compound 759 is one of the compounds previously screened to identify inhibitors of the Wnt/ß-catenin pathway in A549 cells [Lee et al. in Bioorg Med Chem Lett 20:5900-5904, 2010]. However, the mechanism by which Compound 759 induces the inhibition of the Wnt/ß-catenin pathway remains unknown. In our study, we employed various assays to comprehensively evaluate the effects of Compound 759 on lung cancer cells. Our results demonstrated that Compound 759 significantly suppressed cell proliferation and Wnt3a-induced Topflash activity and arrested the cell cycle at the G1 stage. Changes in Wnt/ß-catenin signaling-related protein expression, gene activity, and protein stability including Axin, and p21, were achieved through western blot and qRT-PCR analysis. Compound 759 treatment upregulated the mRNA level of p21 and increased Axin protein levels without altering the mRNA expression in A549 cells. Co-treatment of Wnt3a and varying doses of Compound 759 dose-dependently increased the amounts of Axin1 in the cytosol and inhibited ß-catenin translocation into the nucleus. Moreover, Compound 759 reduced tumor size and weight in the A549 cell-induced tumor growth in the in vivo tumor xenograft mouse model. Our findings indicate that Compound 759 exhibits potential anti-cancer activity by inhibiting the Wnt/ß-catenin signaling pathway through the increase of Axin1 protein stability.

Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate ß-Catenin Signaling.

AXIN1 is a major component of the ß-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to ß-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3ß, ß-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper ß-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected ß-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on ß-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the ß-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect ß-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other ß-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. SIGNIFICANCE: Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its ß-catenin regulatory function, whereas the majority are passenger mutations.

AXIN1/MYC Axis Mediated the Osimertinib Resistance in EGFR Mutant Non-Small Cell Lung Cancer Cells.

Osimertinib, a promising and approved third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is a standard strategy for EGFR-mutant non-small cell lung cancer (NSCLC) patients. However, developed resistance is unavoidable, which reduces its long-term effectiveness. In this study, RNA sequencing was performed to analyze differentially expressed genes (DEGs). The PrognoScan database and Gene Expression Profiling Interactive Analysis (GEPIA) were used to identify the key genes for clinical prognosis and gene correlation respectively. Protein expression was determined by western blot analysis. Cell viability assay and Ki67 staining were used to evaluate the effect of osimertinib on tumor cells. Finally, we screened out two hub genes, myelocytomatosis oncogene (Myc) and axis inhibition protein 1 (Axin1), upregulated in three osimertinib-resistant cell lines through RNA sequencing and bioinformatics analysis. Next, cell experiment confirmed that expression of C-MYC and AXIN1 were elevated in different EGFR mutant NSCLC cell lines with acquired resistance to osimertinib, compared with their corresponding parental cell lines. Furthermore, we demonstrated that AXIN1 upregulated the expression of C-MYC and mediated the acquired resistance of EGFR mutant NSCLC cells to osimertinib in vitro. In conclusion, AXIN1 affected the sensitivity of EGFR mutant NSCLC to osimertinib via regulating C-MYC expression in vitro. Targeting AXIN1/MYC signaling may be a potential new strategy for overcoming acquired resistance to osimertinib.

APC mutations disrupt ß-catenin destruction complex condensates organized by Axin phase separation.

The Wnt/ß-catenin pathway is critical to maintaining cell fate decisions. Recent study showed that liquid-liquid-phase separation (LLPS) of Axin organized the ß-catenin destruction complex condensates in a normal cellular state. Mutations inactivating the APC gene are found in approximately 80% of all human colorectal cancer (CRC). However, the molecular mechanism of the formation of ß-catenin destruction complex condensates organized by Axin phase separation and how APC mutations impact the condensates are still unclear. Here, we report that the ß-catenin destruction complex, which is constructed by Axin, was assembled condensates via a phase separation process in CRC cells. The key role of wild-type APC is to stabilize destruction complex condensates. Surprisingly, truncated APC did not affect the formation of condensates, and GSK 3ß and CK1α were unsuccessfully recruited, preventing ß-catenin phosphorylation and resulting in accumulation in the cytoplasm of CRCs. Besides, we propose that the phase separation ability of Axin participates in the nucleus translocation of ß-catenin and be incorporated and concentrated into transcriptional condensates, affecting the transcriptional activity of Wnt signaling pathway.

The scaffold protein AXIN1: gene ontology, signal network, and physiological function.

AXIN1, has been initially identified as a prominent antagonist within the WNT/ß-catenin signaling pathway, and subsequently unveiled its integral involvement across a diverse spectrum of signaling cascades. These encompass the WNT/ß-catenin, Hippo, TGFß, AMPK, mTOR, MAPK, and antioxidant signaling pathways. The versatile engagement of AXIN1 underscores its pivotal role in the modulation of developmental biological signaling, maintenance of metabolic homeostasis, and coordination of cellular stress responses. The multifaceted functionalities of AXIN1 render it as a compelling candidate for targeted intervention in the realms of degenerative pathologies, systemic metabolic disorders, cancer therapeutics, and anti-aging strategies. This review provides an intricate exploration of the mechanisms governing mammalian AXIN1 gene expression and protein turnover since its initial discovery, while also elucidating its significance in the regulation of signaling pathways, tissue development, and carcinogenesis. Furthermore, we have introduced the innovative concept of the AXIN1-Associated Phosphokinase Complex (AAPC), where the scaffold protein AXIN1 assumes a pivotal role in orchestrating site-specific phosphorylation modifications through interactions with various phosphokinases and their respective substrates.

Mannose attenuates intestinal epithelial tight junction damage in experimental colitis mice by activating the AXIN-AMPK pathway.

Mannose is a unique natural sugar that can be found in a variety of fruits and vegetables. During the past decades, mannose has been reported to be effective in promoting immune tolerance and suppressing inflammatory diseases. Metabolic dysfunction and altered inflammation have clear implications for the development and progression of inflammatory diseases. Herein, we intended to reveal the molecular mechanism of mannose in protecting against intestinal epithelial damage in experimental colitis. We showed that mannose treatment significantly attenuated dextran sodium sulfate (DSS)-induced intestinal barrier damage. The AMPK pathway was responsible for the mannose-mediated protective effect in DSS-induced intestinal epithelial damage. Mechanistically, mannose promoted the axis inhibition protein (AXIN)-based AMPK activation, thereby preventing mitochondrial dysfunction and tight junction disruption in response to the DSS challenge. Cumulatively, the results indicate the use of mannose as a novel approach to treat IBD and other diseases involving tight junction dysfunction. The therapeutic effect of mannose is related to its regulatory function in AMPK pathway activation.

Axin2 depletion in macrophages alleviated senescence and increased immune response after myocardial infarction.

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.

Hypoxia mediates immune escape of pancreatic cancer cells by affecting miR-1275/AXIN2 in natural killer cells.

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.

Tripartite motif 31 drives gastric cancer cell proliferation and invasion through activating the Wnt/ß-catenin pathway by regulating Axin1 protein stability.

Mounting evidence has proposed the importance of the Wnt/ß-catenin pathway and tripartite motif 31 (TRIM31) in certain malignancies. Our research aimed to clarify the correlation between aberrant TRIM31 expression and the Wnt/ß-catenin pathway during gastric cancer (GC) oncogenesis and development. TRIM31 was drastically elevated in GC tissues and was closely associated with aggressive clinical outcomes and poor prognosis. Moreover, TRIM31 downregulation attenuated GC cell proliferation and invasion in vitro. Mechanistically, TRIM31 could bind and ubiquitinate Axin1 protein, thereby facilitating the activation of the Wnt/ß-catenin pathway. Additionally, Axin1 knockdown partially abrogated the inhibitory effects on the proliferative, invasive and migratory abilities of GC cells induced by TRIM31 silencing. Furthermore, TRIM31 was negatively correlated with Axin1 protein expression in GC tissues. In summary, we revealed a new TRIM31-Axin1-Wnt/ß-catenin axis that contributed greatly to the progression of GC, and targeting this regulatory axis may represent an effective treatment for GC patients.

RHAMM/hyaluronan inhibit ß-catenin degradation, enhance downstream signaling, and facilitate fibrosarcoma cell growth.

Increased hyaluronan deposition (HA) in various cancer tissues, including sarcomas, correlates with disease progression. The receptor for hyaluronic acid-mediated motility (RHAMM) expression is elevated in most human cancers. ß-catenin is a critical downstream mediator of the Wnt signaling pathways, facilitating carcinogenic events characterized by deregulated cell proliferation. We previously showed that low molecular weight (LMW) HA/RHAMM/ß-catenin signaling axis increases HT1080 fibrosarcoma cell growth. Here, focusing on mechanistic aspects and utilizing immunofluorescence and immunoprecipitation, we demonstrate that LMW HA treatment enhanced RHAMM intracellular localization (p ≤ 0.001) and RHAMM/ß-catenin colocalization in HT1080 fibrosarcoma cells (p ≤ 0.05). Downregulating endogenous HA attenuated the association of RHAMM/ß-catenin in HT1080 fibrosarcoma cells (p ≤ 0.0.01). Notably, Axin-2, the key ß-catenin degradation complex component, and RHAMM were demonstrated to form a complex primarily to cell membranes, enhanced by LMW HA (p ≤ 0.01). In contrast, LMW HA attenuated the association of ß-catenin and Axin-2 (p ≤ 0.05). The utilization of FH535, a Wnt signaling inhibitor, showed that LMW HA partially rescued the Wnt-dependent growth of HT1080 cells and restored the expression of Wnt/ß-catenin mediators, cyclin-D1 and c-myc (p ≤ 0.05). B6FS fibrosarcoma cells with different HA metabolism do not respond to the LMW HA growth stimulus (p = NS). The present study identifies a novel LMW HA/RHAMM mechanism in a fibrosarcoma model. LMW HA regulates intracellular RHAMM expression, which acts as a scaffold protein binding ß-catenin and Axin-2 at different cellular compartments to increase ß-catenin expression, transcriptional activity, and fibrosarcoma growth.

Phosphorylation of axin within biomolecular condensates counteracts its tankyrase-mediated degradation.

Axin (also known as AXIN1) is a central negative regulator of the proto-oncogenic Wnt/ß-catenin signaling pathway, as axin condensates provide a scaffold for the assembly of a multiprotein complex degrading ß-catenin. Axin, in turn, is degraded through tankyrase. Consequently, tankyrase small-molecule inhibitors block Wnt signaling by stabilizing axin, revealing potential for cancer therapy. Here, we discovered that axin is phosphorylated by casein kinase 1 alpha 1 (CSNK1A1, also known as CK1α) at an N-terminal casein kinase 1 consensus motif, and that this phosphorylation is antagonized by the catalytic subunit alpha of protein phosphatase 1 (PPP1CA, hereafter referred to as PP1). Axin condensates promoted phosphorylation by enriching CK1α over PP1. Importantly, the phosphorylation took place within the tankyrase-binding site, electrostatically and/or sterically hindering axin-tankyrase interaction, and counteracting tankyrase-mediated degradation of axin. Thus, the presented data propose a novel mechanism regulating axin stability, with implications for Wnt signaling, cancer therapy and self-organization of biomolecular condensates.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia.

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the ß-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

Pancreatic stellate cell-derived exosomal tRF-19-PNR8YPJZ promotes proliferation and mobility of pancreatic cancer through AXIN2.

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.

Pharmacological targeting of Axin2 suppresses cell growth and metastasis in colorectal cancer.

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.

Axin2-CreERT2 mediated knockout of bone morphogenetic protein receptor type 1A caused abnormal secondary dentine and altered cell fate of Axin2-expressing odontogenic cells.

AIM: Inducing odontogenic differentiation and tubular dentine formation is extremely important in dentine repair and tooth regeneration. Bone morphogenic proteins (BMPs) signalling plays a critical role in dentine development and tertiary dentine formation, whilst how BMPR1A-mediated signalling affects odontoblastic differentiation of Axin2-expressing (Axin2+ ) odontogenic cells and tubular dentine formation remains largely unknown. This study aims to reveal the cellular and molecular mechanisms involved in the formation of secondary dentine. METHODOLOGY: Axin2lacZ/+ mice harvested at post-natal 21 (P21) were used to map Axin2+ mesenchymal cells. Axin2CreERT2/+ ; R26RtdTomato/+ mice and Axin2CreERT2/+ ; R26RDTA/+ ; R26RtdTomato/+ mice were generated to observe the tempo-spatial distribution pattern of Axin2-lineage cells and the effect of ablation of Axin2+ cells on dentinogenesis, respectively. A loss-of-function model was established with Axin2CreERT2/+ ; Bmpr1afl/fl ; R26RtdTomato/+ (cKO) mice to study the role of BMP signalling in regulating Axin2+ cells. Micro-computed tomography, histologic and immunostainings, and other approaches were used to examine biological functions, including dentine formation, mineralization and cell differentiation in cKO mice. RESULTS: The results showed rich expression of Axin2 in odontoblasts at P21. Lineage tracing assay confirmed the wide distribution of Axin2 lineage cells in odontoblast layer and dental pulp during secondary dentine formation (P23 to P56), suggesting that Axin2+ cells are important cell source of primary odontoblasts. Ablation of Axin2+ cells (DTA mice) significantly impaired secondary dentine formation characterized with notably reduced dentine thickness (Mean of control: 54.11 µm, Mean of DTA: 27.79 µm, p = .0101). Furthermore, malformed osteo-dentine replaced the tubular secondary dentine in the absence of Bmpr1a with irregular cell morphology, abnormal cellular process formation and lack of cell-cell tight conjunction. Remarkably increased expression of osteogenic markers like Runx2 and DMP1 was detected, whilst DSP expression was observed in a dispersed manner, indicating an impaired odontogenic cell fate and failure in producing tubular dentine in cKO mice. CONCLUSIONS: Axin2+ cells are a critical population of primary odontoblasts which contribute to tubular secondary dentine formation, and BMP signalling pathway plays a vital role in maintaining the odontogenic fate of Axin2+ cells.

Structural Basis of the Interaction between Human Axin2 and SIAH1 in the Wnt/ß-Catenin Signaling Pathway.

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.

BMSC-derived extracellular vesicles promoted osteogenesis via Axin2 inhibition by delivering MiR-16-5p.

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.

Axin2 coupled excessive Wnt-glycolysis signaling mediates social defect in autism spectrum disorders.

Social dysfunction is the core syndrome of autism spectrum disorder (ASD) and lacks effective medicine. Although numerous risk genes and relevant environmental factors have been identified, the convergent molecular mechanism underlying ASD-associated social dysfunction remains largely elusive. Here, we report aberrant activation of canonical Wnt signaling and increased glycolysis in the anterior cingulate cortex (ACC, a key brain region of social function) of two ASD mouse models (Shank3-/- and valproic acid-treated mice) and their corresponding human neurons. Overexpressing ß-catenin in the ACC of wild-type mice induces both glycolysis and social deficits. Suppressing glycolysis in ASD mice partially rescued synaptic and social phenotype. Axin2, a key inhibitory molecule in Wnt signaling, interacts with the glycolytic enzyme enolase 1 (ENO1) in ASD neurons. Surprisingly, an Axin2 stabilizer, XAV939, effectively blocked Axin2/ENO1 interaction, switched glycolysis/oxidative phosphorylation balance, promoted synaptic maturation, and rescued social function. These data revealed excessive neuronal Wnt-glycolysis signaling as an important underlying mechanism for ASD synaptic deficiency, indicating Axin2 as a potential therapeutic target for social dysfunction.

SUMOylation of RNF146 results in Axin degradation and activation of Wnt/ß-catenin signaling to promote the progression of hepatocellular carcinoma.

Aberrant SUMOylation contributes to the progression of hepatocellular carcinoma (HCC), yet the molecular mechanisms have not been well elucidated. RING-type E3 ubiquitin ligase RNF146 is a key regulator of the Wnt/ß-catenin signaling pathway, which is frequently hyperactivated in HCC. Here, it is identified that RNF146 can be modified by SUMO3. By mutating all lysines in RNF146, we found that K19, K61, K174 and K175 are the major sites for SUMOylation. UBC9/PIAS3/MMS21 and SENP1/2/6 mediated the conjugation and deconjugation of SUMO3, respectively. Furthermore, SUMOylation of RNF146 promoted its nuclear localization, while deSUMOylation induced its cytoplasmic localization. Importantly, SUMOylation promotes the association of RNF146 with Axin to accelerate the ubiquitination and degradation of Axin. Intriguingly, only UBC9/PIAS3 and SENP1 can act at K19/K175 in RNF146 and affect its role in regulating the stability of Axin. In addition, inhibiting RNF146 SUMOylation suppressed the progression of HCC both in vitro and in vivo. And, patients with higher expression of RNF146 and UBC9 have the worst prognosis. Taken together, we conclude that RNF146 SUMOylation at K19/K175 promotes its association with Axin and accelerates Axin degradation, thereby enhancing ß-catenin signaling and contributing to cancer progression. Our findings reveal that RNF146 SUMOylation is a potential therapeutic target in HCC.