肺癌是全球死亡率最高的癌症，越来越多的证据表明，肺癌干细胞（CSCs）与肺癌预后不良、复发和转移相关。因此，迫切需要鉴定针对肺癌CSCs的新生物标志物和治疗靶点。我们使用The Cancer Genome Atlas（TCGA）肺腺癌（LUAD）亚群的509名患者的全基因组RNA测序数据，计算了1554个Reactome基因集的单样本基因集富集分析（ssGSEA），以鉴定与mRNA表达为基础的干细胞指数（mRNAsi）相关的途径。通过体外球形形成测定、迁移测定、侵袭测定和体内异种移植动物模型，检验了泛素特异性蛋白酶5（USP5）对CSC类特性和转移的表型效应。我们进行了环己亚胺甲酰蚋（Cycloheximide）追踪测定、共免疫沉淀测定和去泛素化测定，以确认USP5对β-连环蛋白（β-catenin）去泛素化的影响。我们证明了USP5表达与干细胞相关特征和肺癌样本不良预后呈正相关。沉默内源性USP5减少了CSC类特性、上皮-间质转化（EMT）和体内外的转移。此外，USP5与β-连环蛋白相互作用，导致去泛素化、β-连环蛋白的稳定和Wnt/β-连环蛋白途径的激活。因此，USP5的表达与人类肺癌中Wnt/TCF通路标志物的富集得分呈正相关。沉默β-连环蛋白表达抑制USP5增强的球形形成。用小分子化合物WP1130靶向USP5可以促进β-连环蛋白的降解，对球形形成、迁移和侵袭具有很强的抑制作用。最后，我们在TCGA-LUAD和Rousseaux_2013数据集中确定了一个贫预后的肿瘤亚组，其特征为高水平的USP5、Wnt信号评分和干细胞评分。这些发现揭示了USP5增强的Wnt/β-连环蛋白信号通路在促进肺癌干细胞特性和转移中的临床证据，暗示针对USP5可能对改善肺癌治疗产生有益效果。© 2023. BioMed Central Ltd., part of Springer Nature.

Lung cancer has the highest mortality rate in the world, and mounting evidence suggests that cancer stem cells (CSCs) are associated with poor prognosis, recurrence, and metastasis of lung cancer. It is urgent to identify new biomarkers and therapeutic targets for targeting lung CSCs.We computed the single-sample gene set enrichment analysis (ssGSEA) of 1554 Reactome gene sets to identify the mRNA expression-based stemness index (mRNAsi)-associated pathways using the genome-wide RNA sequencing data of 509 patients from The Cancer Genome Atlas (TCGA) cohort of lung adenocarcinoma (LUAD). Phenotypic effects of ubiquitin-specific peptidase 5 (USP5) on the CSC-like properties and metastasis were examined by in vitro sphere formation assay, migration assay, invasion assay, and in vivo xenografted animal models. Cycloheximide chase assay, co-immunoprecipitation assay, and deubiquitination assay were performed to confirm the effect of USP5 on the deubiquitination of β-catenin.We demonstrated that USP5 expression were positively correlated with the stemness-associated signatures and poor outcomes in lung cancer specimens. Silencing of endogenous USP5 reduced CSC-like characteristics, epithelial-mesenchymal transition (EMT), and metastasis in vitro and in vivo. Furthermore, USP5 interacted with β-catenin, which resulted in deubiquitination, stabilization of β-catenin, and activation of Wnt/β-catenin pathway. Accordingly, expression of USP5 was positively correlated with the enrichment score of the Wnt/TCF pathway signature in human lung cancer. Silencing of β-catenin expression suppressed USP5-enhancing sphere formation. Targeting USP5 with the small molecule WP1130 promoted the degradation of β-catenin, and showed great inhibitory effects on sphere formation, migration, and invasion. Finally, we identified a poor-prognosis subset of tumors characterized by high levels of USP5, Wnt signaling score, and Stemness score in both TCGA-LUAD and Rousseaux_2013 datasets.These findings reveal a clinical evidence for USP5-enhanced Wnt/β-catenin signaling in promoting lung cancer stemness and metastasis, implying that targeting USP5 could provide beneficial effects to improve lung cancer therapeutics.© 2023. BioMed Central Ltd., part of Springer Nature.