CTNNB1-mutated hepatocellular carcinomas are characterized by a distinctive morphology and activation of the Wnt pathway. AXIN1 also plays a key role in the Wnt pathway, but the morphology of AXIN1-mutated tumors has not been examined. In addition, there are ongoing questions on the ability of AXIN1 mutations to activate the Wnt pathway in hepatocellular carcinoma. AXIN1 mutated tumors (N=18) were studied, along with control groups: CTNNB1 (N=17), APC (6), or "Other" genes in the Wnt pathway (5). Wnt pathway activation was studied by immunostains for beta-catenin and glutamine synthetase. Findings were supplemented by gene expression analysis using TCGA data. On histologic examination, the classic morphology associated with beta-catenin mutations was found in all 4 groups: 8/18 AXIN1 (44%), 10/17 CTNNB1 (59%), 4/6 APC (67%), and 1/5 Other (20%). By immunohistochemistry, Wnt pathway activation was found in 11/18 AXIN1 (61%), 15/17 CTTNB1 (88%), 6/6 APC (100%), and 5/5 (100%) of Other. In AXIN1-mutated tumors, the Wnt pathway was weakly activated. Glutamine synthetase stains also highlighted a new "progressed pattern" associated with distinct subnodules of staining. Tertiary lymphoid structures were uncommon except for cases with CTTNNB1 mutations plus additional mutations in the Wnt pathway. In summary, the classic morphology associated with CTNNB1 mutations is found in hepatocellular carcinomas with mutations in AXIN1, APC, and other Wnt genes. AXIN1 mutated tumors have Wnt activation that is detectable but at lower levels than CTNNB1 mutated tumors. As tumors progress, their level of Wnt activation can change.

The functions of circRNAs in hepatocellular carcinoma (HCC) till needs to be further elucidated. We assessed the biological functions of circGDI2 in vitro and in vivo by gain or loss of function experiments. Then, fuorescence in situ hybridization (FISH), immunofluorescence (IF), RNA pull-down, mass spectrometry, and RNA immunoprecipitation (RIP) were applied to explore the interaction between circGDI2 and heterogeneous nuclear ribonucleoprotein C (HNRNPC). Finally, in vitro and in vivo experiments were performed to explore the influence of circGDI2 on the anti-tumor activity of LGK-974, a porcupine O-acyltransferase (PORCN) inhibitor. CircGDI2 was significantly overexpressed in HBV-related HCC, and its high expression was significantly associated with the growth and invasion characteristics of HCC. Functional experiments indicated that circGDI2 promoted the proliferation and metastasis of HCC cells both in vitro and in vivo. Mechanistic investigations revealed that circGDI2 physically binds to HNRNPC, facilitating its interaction with mPORCN, which stabilizes mRNA and promotes PORCN expression, thereby activating the Wnt signaling pathway and driving tumor proliferation and metastasis. Additionally, we found that the PORCN inhibitor LGK-974 effectively suppressed the proliferation and metastasis of HCC cells both in vitro and in vivo, and a series of experiments demonstrated that knocking down circGDI2 could enhance the antitumor effect of LGK-974, thereby maximizing the inhibition of HCC. CircGDI2 played a crucial role in the progression of HCC by interacting with HNRNPC to promote the Wnt signaling pathway. Meanwhile, LGK-974 can effectively inhibit HCC and targeting circGDI2 can enhance the antitumor effect of LGK-974.

Trophoblast and amniotic lineages, representing key extra-embryonic tissues, can be differentiated from human pluripotent stem cells (hPSCs) under chemically defined conditions. However, the regulatory mechanisms coordinating the fate decision between these lineages during PSC differentiation remain incompletely understood. Leveraging CRISPR/Cas9-mediated loss-of-function screening in lineage-reporter PSCs, we identified the transcription factor HAND1 as a critical determinant controlling the bifurcation of trophoblast and amniotic lineages. Genetic ablation ofHAND1effectively abrogated the amniotic differentiation capacity of PSCs while concomitantly enhancing their trophoblast differentiation potential. Conversely, ectopic HAND1 overexpression impaired trophoblast differentiation. Notably, forced HAND1 expression in human trophoblast stem cells (TSCs) induced transcriptional reprogramming toward an amniotic fate, indicating its lineage-instructive capability. Mechanistic analyses demonstrated that HAND1 interacts with the TCFs and Wnt signaling effectors β-catenin to form a transcriptional complex that antagonistically modulates the balance between trophoblast- and amnion-associated gene regulatory networks. Collectively, our findings establish HAND1 as a master regulator orchestrating the amniotic versus trophoblast lineage choice during human PSC differentiation, thereby illuminating fundamental regulatory mechanism underlying extra-embryonic lineage specification.

Radiation proctitis (RP) is a frequent complication of pelvic radiotherapy that compromises treatment delivery and patient quality of life, yet the factors shaping injury severity remain incompletely defined. We prospectively profiled pretreatment fecal microbiomes and metabolomes from 55 patients and stratified them by outcome into mild versus severe RP. Baseline microbial composition showed Bacteroidales enriched in severe RP and Firmicutes enriched in mild cases. Multi-omics integration highlighted nicotinate/nicotinamide pathways; severe RP was characterized by concomitant reductions in both fecal and tissue NAD⁺ levels, along with an enrichment of microbial nicotinate/nicotinamide metabolism genes, primarily contributed by Bacteroides ovatus, B. xylanisolvens, and B. fragilis. In mice, fecal microbiota transplantation from severe-RP donors exacerbated radiation-induced colorectal injury and decreased colorectal NAD⁺, supporting a causal role for the microbiota. Gavage with Bacteroides similarly worsened pathology and lowered NAD⁺, whereas nicotinamide mononucleotide (NMN) supplementation attenuated the injury. Mechanistically, Bacteroides gavage reduced mitochondrial membrane potential, decreased the Lgr5⁺ stem-cell proportion and proliferative indices, associated with Wnt pathway modulation. NMN reversed these effects in parallel with NAD⁺ restoration. Together, these results identify a microbiota‒metabolite association wherein Bacteroidales enrichment is associated with NAD⁺ depletion, reduced mucosal proliferative capacity, and exacerbated radiation-induced colorectal injury. The work deepens insight into RP pathogenesis and suggests a potential basis for microbiome- and metabolite-targeted approaches to attenuate severe RP.

Gastric cancer (GC) is a leading cause of cancer-related deaths and has high recurrence rate. Although fibronectin domain-containing protein 1 (FNDC1) is implicated in GC progression, its molecular mechanisms remain unclear. Multi-omics analyses (TCGA, GEO datasets) were used to assess FNDC1 expression and clinical correlation. Invitro (cell proliferation, invasion, EMT markers) and invivo (xenograft) experiments, combined with molecular assays (Co-IP, WB, ChIP), explored FNDC1's function and mechanism. FNDC1 was significantly upregulated in GC, correlating with advanced clinicopathological features and poor prognosis. Knockdown of FNDC1 suppressed GC cell proliferation, invasion, and metastasis by inhibiting EMT and Wnt/β-catenin signaling. Mechanistically, FNDC1 competitively bound the WD5 domain (residues 224-254) of Gβ2, disrupting Gβγ-Dvl1 interaction. This prevented Dvl1 degradation, promoted Axin1 ubiquitination, and destabilized the β-catenin-destruction complex (GSK3 β-APC-Axin1), leading to β-catenin accumulation and Wnt pathway activation. FNDC1 drives GC malignancy by targeting the Gβ2-Dvl1 axis to activate Wnt/β-catenin signaling, suggesting FNDC1 as a novel prognostic biomarker and therapeutic target.

The pathogenesis of type 2 diabetes is characterized by insulin resistance and a progressive decline in β-cell function. A key driver of this dysfunction is the loss of β-cell identity, which reduces functional β-cell mass and leads to inadequate insulin secretion. Periodontal pathogens have been implicated in promoting insulin resistance; however, their role in the transformation of β-cell identity remains poorly understood. This study aims to investigate the impact of periodontal pathogen Fusobacterium nucleatum (F. nucleatum) on β-cell identity maintenance and the underlying molecular mechanisms. Single-cell RNA sequencing (scRNA-seq) data from human pancreatic islets of nondiabetic (ND), prediabetic (Pre-T2D), and type 2 diabetes (T2D) donors were analyzed to assess changes in β-cell proportion, differentiation trajectory, and associated molecular pathways. The Single-cell Analysis of Host-Microbiome Interactions (SAHMI) method was used to detect F. nucleatum sequences in pancreatic islets. Pearson correlation analysis identified key genes associated with the action of F. nucleatum, followed by in vitro validation using a co-culture model of F. nucleatum and MIN6 cells to elucidate the underlying mechanisms. scRNA-seq analysis revealed a reduced proportion of β-cells and decreased expression of key β-cell identity-maintenance genes in the T2D group. The expression levels of transdifferentiation markers and β-cell disallowed genes were elevated, alongside a trend toward α-cell transdifferentiation. The NF-κB signaling pathway was significantly activated in the T2D group, accompanied by a significant increase in the SPP1 inflammatory signal, while the WNT pathway was markedly diminished. Integrated Pearson correlation and in vitro analyses identified the cell cycle regulator CDKN1C as a central mediator through which F. nucleatum promotes β-cell identity loss. Mechanistically, F. nucleatum activated the NF-κB pathway, leading to downregulation of CDKN1C expression and thereby promoting loss of β-cell identity, which played an important role in the progression of diabetes associated with periodontitis. This study demonstrates that β-cells in T2D primarily undergo transdifferentiation towards α-cells, and the periodontal pathogen F. nucleatum promotes β-cell identity loss via NF-κB-mediated downregulation of CDKN1C.

Identification of an Anoikis-Related Gene Signature to Predict the Prognosis in Patients With Oral Squamous Cell Carcinoma

MicroRNAs (miRNAs) are key regulators of bone regeneration. We investigated the role of miR-21 in humeral fracture (HF) healing, focusing on its interaction with the SMAD7/Wnt/β-catenin signaling axis. Serum and tissue levels of miR-21 were analyzed in HF patients and healthy controls using qRT-PCR. A rat HF model was established, and agomiR-21 was administered to evaluate its effect on fracture healing. Histological analysis using hematoxylin and eosin (H&E) staining was performed to assess bone tissue morphology and fracture healing. In vitro, MC3T3-E1 osteoblasts were transfected with miR-21 mimics to assess cell viability (CCK-8), proliferation (EdU), apoptosis (flow cytometry), migration (Transwell assay), and osteogenic differentiation (Western blot). The downstream target of miR-21 was identified using bioinformatics and validated via luciferase reporter and RNA immunoprecipitation assays. Rescue experiments involving SMAD7 overexpression and Wnt/β-catenin inhibitors (DKK1) were conducted to explore the mechanism of action. Serum analysis revealed reduced β-catenin and phosphorylated GSK3β (p-GSK3β) levels in humeral fracture (HF) patients compared to healthy controls, indicating suppressed Wnt/β-catenin signaling. In MC3T3-E1 cells, miR-21 overexpression enhanced osteogenic markers and increased total β-catenin, active β-catenin, and p-GSK3β levels, confirming activation of the Wnt/β-catenin pathway. Dual-luciferase and expression analyses demonstrated that miR-21 directly targets SMAD7, a known Wnt pathway inhibitor. Co-transfection with SMAD7 reversed the effects of miR-21 on β-catenin activation, ALP activity, and osteogenic gene expression. Additionally, miR-21 knockdown impaired osteogenesis, further supporting its regulatory role. Importantly, in vivo, agomiR-21 treatment enhanced osteoblast activity and significantly promoted bone healing in a rat fracture model, reinforcing the therapeutic potential of miR-21-mediated SMAD7 suppression and Wnt/β-catenin pathway activation in bone regeneration. miR-21 promotes bone regeneration after humeral fracture by enhancing osteoblast function and activating Wnt/β-catenin signaling through direct inhibition of SMAD7.

Diabetic wounds are a critical clinical challenge due to chronic inflammation, impaired tissue regeneration, and bacterial infection. The absence of multifunctional therapeutic systems that can address these pathological barriers simultaneously is a major obstacle in managing diabetic wounds. In this study, we developed a versatile photothermal-responsive hydrogel (HPCT) by incorporating Ti3C2 MXene nanosheets and Cur-Cu nanoparticles (NPs) into a dual-network polymer matrix. This hydrogel exhibits robust physicochemical stability, efficient photothermal conversion, and on-demand release properties. Due to the synergistic effects of its components, HPCT effectively scavenges ROS/RNS, reprograms macrophages, and promotes fibroblast and endothelial proliferation and migration. Additionally, it exhibits potent antibacterial activity. Transcriptomic analysis reveals that the bioactive material activates the PI3K-AKT and Wnt signaling pathways, providing a mechanistic basis for its regenerative potential. In vivo, HPCT significantly accelerates wound closure in diabetic mice by reducing inflammation and oxidative stress, enhancing angiogenesis, and improving collagen deposition while maintaining good biocompatibility. Overall, this multifunctional hydrogel is a promising therapeutic strategy for treating diabetic and other chronic wounds.

Colitis-associated carcinoma (CAC) represents ~1% of colorectal carcinomas and has important differences from sporadic colorectal carcinoma (sCRC). The precursors and carcinomas that arise in the setting of IBD are uniquely challenging to visualize by endoscopy and diagnose via histology, and the rising prevalence of IBD amplifies the challenges of surveillance to informed management. Although in broad strokes, CAC and sCRC share molecular features (~85% chromosomal instability pathway 15% microsatellite instability high (MSI-H)), CAC has a distinct distribution of molecular abnormalities, including lower frequencies of APC and KRAS mutations, greater prevalence of IDH1R132H, and more frequent copy number alterations (e.g., MYC amplifications), and functional data indicate that most CACs show far less dependence on Wnt signaling than sCRC, suggesting a distinct pathogenesis from the earliest stages. Although there are significant gaps in our knowledge of the pathogenesis of CAC, our understanding is growing. This review summarizes how chronic colitis reshapes epithelial homeostasis and somatic evolution, resulting in the distinctive pathogenesis of CAC, and highlights knowledge gaps that could be addressed by applying multimodal technologies to well-annotated clinical material. The review is structured in two sections, the first introducing the IBDs and the homeostatic mechanisms that preserve integrity and prevent colorectal neoplasia. The second section compares failure modes in sporadic and colitic settings and describes the differences in the resulting neoplasms.

Immunomodulatory-osteogenic dual-functional nanofibers augment screw anchorage in osteoporotic bone

As the use of immune checkpoint inhibitors for the treatment of cancer has expanded, convincing data have emerged correlating active WNT signaling with resistance to immunotherapies. To identify mechanisms through which WNT signaling limits anti-tumor immunity, we examined the response to WNT inhibition in a variety of human cancer cell lines that harbor distinct WNT pathway mutations. Our data show that inhibition of WNT signaling leads to activation of the TBK1/IRF3 dsRNA-sensing pathway and expression of interferon-stimulated genes (ISGs), independently of IFN/JAK/STAT signaling. Mechanistically, we show that WNT inhibition leads to increased chromatin accessibility at genomic loci harboring endogenous retroviruses (ERVs), resulting in ERV re-expression and activation of the dsRNA response. Increased ISG expression following WNT inhibition does not involve decreased MAP kinase signaling and therefore differs from reports documenting ISG induction in response to inhibition of other oncogenic pathways. Given the variety of tumor cell lines and WNT pathway mutations examined, these data suggest a mechanism by which WNT may drive immune evasion and several therapeutic avenues to reverse it, including tumor-targeted type 1 interferon stimulation and/or epigenetic therapies.

ABSTRACT Endometrial polyps (EPs) are common uterine lesions associated with abnormal uterine bleeding and infertility, yet their pathogenesis remains poorly defined. Here, we performed single‐cell RNA sequencing of normal endometrium, para‐polyp, and polyp tissues, identifying distinct cellular compositions and transcriptional programs. EPs showed enhanced estrogen signaling and increased epithelial proliferation, accompanied by decreased expression of cytokines and reduced T cell cytotoxicity. Notably, we observed epithelial subpopulations with elevated copy number variations and transcription factors associated with hyperplasia. Cell–cell communication analyzes revealed aberrant stromal‐epithelial crosstalk, characterized by upregulated WNT, IGF, and VEGF signaling originating from stromal cells. Spatial transcriptomic analyzes further demonstrated enhanced WNT signaling between stromal and epithelial compartments in endometrial cancer. In vitro glandular organoid models showed that epithelial transcriptional alterations contribute to polyp formation. These findings highlight a critical role of stromal‐epithelial interactions in EP development and suggest potential therapeutic targets.

Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC, and silenced BASP1, we generated stable cell lines overexpressing BASP1, either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR-based system. BASP1 encodes a neuronal signaling protein and transcriptional corepressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.

Colorectal cancer (CRC) is a multifactorial malignancy driven by dysregulated inflammatory signaling, aberrant cell proliferation, defective apoptosis, loss of tumor suppressor functions, and aberrant activation of oncogenic signaling pathways. The identification of multi-target agents capable of simultaneously modulating these interconnected pathways represents a promising therapeutic strategy. In the present study, the multi-target therapeutic potential of mangiferin, a bioactive C-glucosyl xanthone, was systematically evaluated against key CRC-associated molecular targets using structure-based molecular docking. Molecular docking was performed using AutoDock Vina against proteins involved in inflammation (TNF-α and COX-2), proliferation and angiogenesis (K-Ras, BRAF, β-catenin, VEGFR-2, and Cyclin D1), apoptosis (Bcl-2, BAX, and Caspase-3), tumor suppression (p53 and APC), and the PI3K/Akt/mTOR signaling pathway (PI3K-α, AKT, and mTOR). Protein structures were retrieved from the RCSB Protein Data Bank and docked following binding-site prediction using CASTp. Mangiferin exhibited strong to moderate binding affinities toward all selected targets, with docking scores ranging from −10.5 to −7.0 kcal/mol. The strongest binding affinity was observed with TNF-α (−10.5 kcal/mol), followed by AKT (−10.3 kcal/mol), COX-2 (−9.8 kcal/mol), BRAF (−9.4 kcal/mol), and PI3K-α (−9.4 kcal/mol), indicating effective targeting of inflammatory mediators and oncogenic survival signaling pathways. Moderate binding affinities were observed for VEGFR-2 and p53 (−8.5 kcal/mol), K-Ras and APC (−8.4 kcal/mol), and Bcl-2 (−8.2 kcal/mol), suggesting potential inhibition of angiogenesis, cell proliferation, and anti-apoptotic signaling. Additionally, mangiferin demonstrated favorable interactions with β-catenin (−7.7 kcal/mol), caspase-3 (−7.6 kcal/mol), mTOR (−7.5 kcal/mol), BAX (−7.1 kcal/mol), and Cyclin D1 (−7.0 kcal/mol), highlighting its capacity to modulate Wnt signaling, apoptosis, cell-cycle regulation, and PI3K/AKT/mTOR pathway components. Overall, mangiferin–protein complexes were stabilized through a combination of hydrogen bonding and hydrophobic π-interactions, supporting mangiferin as a promising multi-target phytochemical with therapeutic relevance in colorectal cancer.

INAFM2, the human homolog of the Drosophila inaF, is a predicted membrane protein with no known function in vertebrates. Through an in vivo genome-wide transcriptional activation screen, we uncovered INAFM2 as a potent driver of metastasis, leading us to propose naming the vertebrate gene and its protein product ROME (regulator of metastasis). We discovered ROME’s subcellular localization, posttranslational modifications, and transcriptional profiles related to its expression. ROME negatively regulates the canonical Wnt pathway by directly binding to β-catenin. Blocking rome expression in zebrafish embryos results in severe developmental defects and early mortality, which can be reversed by inhibiting the canonical Wnt pathway. Notably, we demonstrate that ROME expression regulates human cancer cell motility and invasion in vitro and metastasis in vivo in both zebrafish and immunodeficient mice via tail vein and orthotopic injection models. ROME-mediated increase in cancer cell intravasation is dependent on its direct interaction with vimentin. Furthermore, we show that elevated ROME expression correlates with poorer patient survival in multiple human cancers. Taken together, this is the first report of the vertebrate ROME gene producing a biologically active plasma membrane glycoprotein that is critical for normal development and metastasis.Significance:This is the first report of a detailed characterization of the molecular features of ROME (INAFM2) protein in mammalian cells and its biochemical and biological functions related to vertebrate development and cancer metastasis.

Androgenetic alopecia(AGA) is the most common form of hair loss worldwide. By middle age, approximately two-thirds of men and 40% of women are affected. The pathophysiology of AGA involves complex interactions among genetic predisposition, androgen sensitivity, and dysregulation of the hair follicles (HFs) cycle. Among the key regulatory pathways, the Wnt/β-catenin signaling pathway plays a central role in maintaining homeostasis of hair follicle stem cell (HFSCs) and dermal papilla cells (DPCs), as well as in promoting the transition to the anagen phase. Endogenous Wnt inhibitors, such as dickkopfs (DKKs) and secreted frizzled-related proteins (sFRPs), are upregulated under the influence of dihydrotestosterone and can modulate canonical Wnt signaling in DPCs and HFSCs via paracrine mechanisms, potentially contributing to the development of AGA. Targeted inhibition of these endogenous negative regulators may therefore represent a promising therapeutic strategy for AGA. This review summarizes current insights into the role of Wnt signaling in hair follicle biology, the characteristics and functions of DKKs and sFRPs in AGA, and their potential as therapeutic targets.

CYLD cutaneous syndrome (CCS) skin tumours develop from puberty onwards, can number in the hundreds and progressively grow over time. CCS patients lack medical therapies and require repeated surgery to control tumour burden. CYLD loss of heterozygosity (LOH) drives tumour growth, and CCS tumours have previously been shown to demonstrate increased canonical NF-κB and Wnt signalling. Here, we demonstrate evidence of non-canonical NF-κB signalling in CCS tumour keratinocytes, with increased p100 to p52 processing and RelB protein expression compared to normal skin. Utilizing complementary transcriptomics and proteomics on patient derived CCS tumour cell fractions, we identify IκB kinase alpha (IKKα) as a candidate target in the non-canonical NF-κB signalling pathway. A novel, highly selective, IKKα inhibitor (SU1644) used in patient derived CCS tumour spheroid cultures demonstrated that IKKα inhibition reduced tumour spheroid viability. These data provide the pre-clinical rationale for the assessment of topical IKKα inhibitors as a novel preventative treatment for CCS.

Hair and nails, though structurally different, both rely on Wnt/β-catenin signaling for regeneration. To identify small molecules that enhance this pathway, we screened 5,170 FDA-approved and natural compounds using HEK293 cells with a TCF/LEF luciferase reporter. Nine compounds significantly activated Wnt signaling, including antivirals (imidocarb, proflavine, aminoacridine), anticancer agents (entinostat, enzastaurin, abemaciclib), and GSK-3β inhibitors (BIO, CP21R7). Aminoacridine and proflavine markedly upregulated RSPO3 and RSPO4 while suppressing WIF1. In mice, aminoacridine-especially combined with minoxidil-showed the strongest hair regrowth, while proflavine and imidocarb enhanced nail elongation. These results reveal new therapeutic candidates for hair and nail regeneration.

Wnt signaling is a key driver of colorectal cancer (CRC) progression, yet directly inhibiting it remains a major challenge. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression, thereby modulating oncogenic pathways. However, the role of miR-27a-5p and its underlying mechanisms in CRC remains largely unknown. Bioinformatics analyses and paired clinical CRC specimens were used to evaluate miR-27a-5p expression levels and their association with prognosis. CCK-8, colony formation, wound healing, Transwell invasion, and epithelial–mesenchymal transition (EMT) marker analysis were performed to assess the effects of miR-27a-5p on the malignancy of CRC cells. The potential underlying mechanisms were investigated using dual-luciferase reporter assays, RNA-seq, HPLC-UV, immunoprecipitation/co-immunoprecipitation and immunofluorescence. Xenograft models were used to evaluate the in vivo role of miR-27a-5p in CRC. miR-27a-5p was downregulated in CRC, and its low expression correlated with poorer prognosis. miR-27a-5p directly targeted GFPT2, the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP), thereby decreasing intracellular uridine 5′-diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc) levels and global protein O-linked β-N-acetylglucosaminylation (O-GlcNAcylation), which in turn reduced β-catenin O-GlcNAcylation, inhibited its nuclear accumulation, and suppressed its transcriptional activity, leading to attenuation of Wnt signaling. Restoring miR-27a-5p expression in CRC cells suppressed proliferation, migration, invasion, and EMT, whereas GFPT2 overexpression or glucosamine supplementation partially reversed the inhibited malignant behaviors. Conversely, β-catenin knockdown attenuated the malignant phenotypes and expression of EMT/Wnt targets induced by miR-27a-5p inhibition, supporting a β-catenin-dependent mechanism. In mouse xenografts, treatment with the O-GlcNAc transferase (OGT) inhibitor OSMI-1 attenuated the accelerated tumor growth driven by miR-27a-5p inhibition, supporting an O-GlcNAcylation-dependent mechanism in vivo. These findings reveal a novel miR-27a-5p–GFPT2–HBP axis that links metabolic reprogramming to Wnt signaling in CRC by suppressing β-catenin activity through the reduction of UDP-GlcNAc-dependent O-GlcNAcylation, thereby restraining CRC progression. This suggests that targeting this axis could attenuate Wnt signaling and slow CRC progression.