Gallbladder cancer (GBC) is an extremely malignant tumor with a high mortality rate. Little is known about its invasion and metastasis mechanism so far. To identify the driver genes in GBC metastasis, we performed a mRNA microarray of metastatic GBC and paired non-tumor samples, and found PLEK2 was markedly upregulated in GBC tissues. Next, the expression of PLEK2 in GBC were examined in a larger cohort of patients by qRT-PCR, western blot and IHC staining. The clinicopathologic correlation of PLEK2 was determined by statistical analyses. The biological involvement of PLEK2 in GBC metastasis and the underlying mechanisms were investigated. In this study, we found that PLEK2 had higher expression in GBC tumor tissues compared to non-cancerous adjacent tissues and cholecystolithiasis tissues. The clinicopathologic analyses showed PLEK2 expression was positively correlated with tumor TNM stage, distant metastasis and PLEK2 was an independent predictor of overall survival (OS) in GBC patients. The cellular function assays showed PLEK2 promoted GBC cells migration, invasion and liver metastasis in mouse model via the regulation of epithelial-mesenchymal transition (EMT) process. Our mass spectrum and co-immunoprecipitation (co-IP) assays demonstrated that PLEK2 could interact with the kinase domain of EGFR and suppress EGFR ubiquitination mediated by c-CBL, leading to constitutive activation of EGFR signaling. Furthermore, RNA-sequencing and qRT-PCR results demonstrated chemokine (C-C motif) ligand 2 (CCL2), a target gene downstream of PLEK2/EGFR signaling, mediated the motility-promoting function of PLEK2. On the basis of these collective data, we propose that PLEK2 promotes the invasion and metastasis of GBC by EGFR/CCL2 pathway and PLEK2 can serve as a potential therapeutic target for GBC treatment.

Background: 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. Methods: 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. Results: 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. Conclusion: 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.