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Background Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world, with a high likelihood of metastasis and a dismal prognosis. The reprogramming of glucose metabolism is critical in the development of HCC. The Warburg effect has recently been confirmed to occur in a variety of cancers, including HCC. However, little is known about the molecular biological mechanisms underlying the Warburg effect in HCC cells. In this study, we sought to better understand how methyltransferase 5, N6‐adenosine (METTL5) controls the development of HCC and the Warburg effect. Methods In the current study, quantitative real‐time polymerase chain reaction and Western blotting were used to detect the expression of METTL5 in HCC tissues and cell lines. Several different cell models and animal models were established to determine the role of METTL5 in glucose metabolism reprogramming and the underlying molecular mechanism of HCC. Glutathione‐S‐transferase pulldown, coimmunoprecipitation, RNA sequencing, non‐targeted metabolomics, polysome profiling, and luciferase reporter assays were performed to investigate the molecular mechanisms of METTL5 in HCC cells. Results We discovered that METTL5 drove glucose metabolic reprogramming to promote the proliferation and metastasis of HCC. Mechanistically, upregulation of METTL5 promoted c‐Myc stability and thus activated its downstream glycolytic genes lactate dehydrogenase A (LDHA), enolase 1 (ENO1), triosephosphate isomerase 1 (TPI1), solute carrier family 2 member 1 (SLC2A1), and pyruvate kinase M2 (PKM2). The c‐Box and ubiquitin binding domain (UBA) regions of ubiquitin specific peptidase 5 (USP5) binded to c‐Myc protein and inhibited K48‐linked polyubiquitination of c‐Myc. Further study revealed that METTL5 controled the USP5 translation process, which in turn regulated the ubiquitination of c‐Myc. Furthermore, we identified cAMP responsive element binding protein 1 (CREB1)/P300 as a critical transcriptional regulator of METTL5 that promoted the transcription of METTL5 in HCC. In patient‐derived tumor xenograft (PDX) models, adenovirus‐mediated knockout of METTL5 had a good antitumor effect and prolonged the survival of PDX‐bearing mice. Conclusions These findings point to a novel mechanism by which CREB1/P300‐METTL5‐USP5‐c‐Myc controls abnormal glucose metabolism and promotes tumor growth, suggesting that METTL5 is a potential therapeutic target and prognostic biomarker for HCC.

The role of SLUG in epithelial-mesenchymal transition during tumor progression has been thoroughly studied, but its precise regulation remains poorly explored. The affinity purification, mass spectrometry and CO-IP were performed to identify the interaction between SLUG and ubiquitin-specific protease 5 (USP5). Cycloheximide chase assays and deubiquitination assays confirmed that the effect of USP5 on the deubiquitin of SLUG. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional regulation of E-cadherin by SLUG effected by USP5. EMT related markers was detected by western blotting and immunofluorescence. Molecular docking, SPR sensor (biacore) and co-location were detected to prove Formononetin targets USP5. Bioinformatics analysis was used to study the relation of USP5 and SLUG to malignancy degree of HCC. Cell migration, invasion in HCC cells and xenografts model in nude mouse were conducted to detect the promotion of USP5 and the inhibition of Formononetin on EMT. USP5 interacts with and stabilizes SLUG to regulate its abundance through USP5 deubiquitination activities in epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC). USP5 is highly expressed and positively correlated with SLUG expression in HCC with high malignancy. Knockdown of USP5 inhibits SLUG deubiquitination and inhibits HCC cells proliferation, metastasis, and invasion, while overexpression of USP5 promotes SLUG stability and EMT in vitro and in vivo. Through virtual screening, we found that Formononetin exhibits excellent binding to USP5. Moreover, Formononetin inhibits deubiquitinating activities of USP5 to SLUG and consequently impedes the EMT and malignant progression of HCC. Our findings reveal that USP5 serve as a potential target for tumor intervention and provide a preliminary antitumor therapy for inhibit EMT by targeting USP5 or its interaction with SLUG in HCC.

Background Triple-negative breast cancer (TNBC) remains the most challenging subtype of breast cancer and lacks definite treatment targets. Aerobic glycolysis is a hallmark of metabolic reprogramming that contributes to cancer progression. PFKP is a rate-limiting enzyme involved in aerobic glycolysis, which is overexpressed in various types of cancers. However, the underlying mechanisms and roles of the posttranslational modification of PFKP in TNBC remain unknown. Methods To explore whether PFKP protein has a potential role in the progression of TNBC, protein levels of PFKP in TNBC and normal breast tissues were examined by CPTAC database analysis, immunohistochemistry staining (IHC), and western blotting assay. Further CCK-8 assay, colony formation assay, EDU incorporation assay, and tumor xenograft experiments were used to detect the effect of PFKP on TNBC progression. To clarify the role of the USP5-PFKP pathway in TNBC progression, ubiquitin assay, co-immunoprecipitation (Co-IP), mass spectrometry-based protein identification, western blotting assay, immunofluorescence microscopy, in vitro binding assay, and glycolysis assay were conducted. Results Herein, we showed that PFKP protein was highly expressed in TNBC, which was associated with TNBC progression and poor prognosis of patients. In addition, we demonstrated that PFKP depletion significantly inhibited the TNBC progression in vitro and in vivo. Importantly, we identified that PFKP was a bona fide target of deubiquitinase USP5, and the USP5-mediated deubiquitination and stabilization of PFKP were essential for cancer cell aerobic glycolysis and TNBC progression. Moreover, we found a strong positive correlation between the expression of USP5 and PFKP in TNBC samples. Notably, the high expression of USP5 and PFKP was significantly correlated with poor clinical outcomes. Conclusions Our study established the USP5-PFKP axis as an important regulatory mechanism of TNBC progression and provided a rationale for future therapeutic interventions in the treatment of TNBC.

Glioblastoma multiforme (GBM) is one of the most malignant primary tumors in humans. Despite standard therapeutic strategy with tumor resection combined with radiochemotherapy, the prognosis remains disappointed. Recently, deubiquitinating enzymes (DUBs) has been reported as potential cancer therapy targets due to their multifunctions involved in the regulation of tumorigenesis, cell cycle, apoptosis, and autophagy. In this study, we found that knockdown of ubiquitin specific protease (USP5), a family member of DUB, could significantly suppress GBM cell line U251 and DBTRG-05MG proliferation and colony formation by inducing cell cycle G1/S arrest, which was correlated with downregulation of CyclinD1 protein level. CyclinD1 had been reported to play a critical role in the tumorigenesis and development of GBM via regulating cell cycle transition. Overexpression of USP5 could significantly extend the half-life of CyclinD1, while knockdown of USP5 decreased the protein level of CyclinD1, which could be restored by proteasome inhibitor MG-132. Indeed, USP5 was found to directly interact with CyclinD1, and decrease its K48-linked polyubiquitination level. Furthermore, knockdown of USP5 in U251 cells remarkably inhibited tumor growth in vivo . Taken together, these findings demonstrate that USP5 plays a critical role in tumorigenesis and progression of GBM by stabilizing CyclinD1 protein. Targeting USP5 could be a potential therapeutic strategy for GBM.

Background 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. Methods 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. Results 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. Conclusions 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.

Increasing studies have reported that dysregulated lipid metabolism is an independent risk factor for breast cancer (BC); it would be, therefore, enlightening to investigate the relationship between metabolic reprogramming and the tumor microenvironment in the future. Ferroptosis, a novel form of programmed cell death, is characterized by glutathione (GSH) depletion and inactivation of glutathione peroxidase 4 (GPX4), the central regulator of the antioxidant system. While the close association between fatty acid metabolism and ferroptosis has been studied in various diseases, the interplay between the key fatty acid metabolic enzyme fatty acid synthase (FASN) and ferroptosis in BC remains unexplored. At the beginning of the current study, we demonstrated that FASN expression positively correlates with an immune-cold tumor microenvironment in BC. Subsequent findings revealed that FASN knockdown promotes GPX4 degradation-induced ferroptosis, thereby enhancing the efficacy of anti-programmed cell death protein 1 (PD-1) immunotherapy. Co-immunoprecipitation coupled with mass spectrometry (IP/MS) and co-IP experiments demonstrated that ubiquitin specific protease 5 (USP5) stabilizes GPX4 by binding to and deubiquitinating it. Furthermore, knockdown of FASN inhibited the palmitoylation of USP5, reducing its interaction with GPX4 and consequently increasing GPX4 ubiquitination and degradation. Our results demonstrate that FASN suppresses ferroptosis in BC by stabilizing GPX4 via USP5-mediated mechanisms, highlighting FASN inhibition as a potential therapeutic approach to enhance immunotherapy response.

Background Cav3.2 channels facilitate nociceptive transmission and are upregulated in DRG neurons in response to nerve injury or peripheral inflammation. We reported that this enhancement of Cav3.2 currents in afferent neurons is mediated by deubiquitination of the channels by the deubiquitinase USP5, and that disrupting USP5/Cav3.2 channel interactions protected from inflammatory and neuropathic pain. Results Here we describe the development of a small molecule screening assay for USP5- Cav3.2 disruptors, and report on two hits of a ~5000 compound screen - suramin and the flavonoid gossypetin. In mouse models of inflammatory pain and neuropathic pain, both suramin and gossypetin produced dose-dependent and long-lasting mechanical anti-hyperalgesia that was abolished or greatly attenuated in Cav3.2 null mice. Suramin and Cav3.2/USP5 Tat-disruptor peptides were also tested in models of diabetic neuropathy and visceral pain, and provided remarkable protection. Conclusions Overall, our findings provide proof of concept for a new class of analgesics that target T-type channel deubiquitination.

Objective Targeting deubiquitinases (DUBs) has emerged as a promising avenue for anticancer drug development. However, the effect and mechanism of pan-DUB inhibitor EOAI on non-small cell lung cancer (NSCLC) remains to be studied. Materials and methods The expression of ubiquitin-specific peptidase 5 (USP5) in NSCLC was evaluated by immunohistochemistry. The effect of the USP5 inhibitor, EOAI, on NSCLC cell growth and cell cycle was evaluated by CCK-8 and PI staining. Apoptosis was detected by Annexin V-FITC/PI double staining. Autophagy was examined by LC3 immunofluorescence. Comet assay and γ-H2AX immunofluorescence staining were used to detect DNA damage, and Western blotting was used to detect the expression of apoptosis, cycle, autophagy and DNA damage-related proteins. In vivo experiments demonstrated the effect of EOAI on NSCLC. Results We also found that USP5 was significantly upregulated in NSCLC tissues in this study. In addition, we show that EOAI can cause DNA damage in NSCLC cells while modulating the transcriptional activity of P53, thereby inducing cell cycle arrest in NSCLC cells, autophagy and apoptosis. In vivo experiments have shown that EOAI can inhibit tumors and synergistically enhance the anti-tumor effect of cisplatin. Conclusion USP5-mediated epigenetic regulation of oncogenes promotes the occurrence of NSCLC, which provides ideas for developing potential targeted therapy.

Background Glycolysis, a classic characteristic of cancer cells, can drive cancer progression by generating lactate, which play as a key immunosuppressive mediator. Currently, ubiquitin-specific proteases 5 (USP5) has been demonstrated to facilitate tumor cell survival in multiple myeloma (MM), whereas whether USP5 was involved in glycolysis-lactate production pathway and immunosuppressive microenvironment formation in MM remain unknown. Methods The gene and protein expression characteristics were assessed via qRT-PCR and western blot. MM cell survival was determined by CCK-8 and flow cytometry analysis. Glycolysis was evaluated by examining glucose uptake, lactate production and ATP level via corresponding kits. Tumor-associated macrophages polarization was tested via measurement of M1/M2-like macrophage markers using qRT-PCR and flow cytometry methods. Dual-luciferase reporter, chromatin immunoprecipitation and co-immunoprecipitation assays were conducted to verified molecular relationship. Xenograft model was used for verified cellular findings. Results USP5 was abnormally overexpressed in MM patients and cell lines. Knockdown of USP5 could restrain MM cell survival and glycolysis activity, thus reducing lactate-mediated immunosuppressive M2-like macrophage polarization in vitro and in vivo, whereas overexpression of USP5 play opposite impacts. Mechanistically, USP5 could downregulate the ubiquitination modification of STAT to stabilize STAT2 protein, thus activating PFKFB4 transcription. Moreover, STAT2 could overturn the regulatory role of USP5 on MM cell survival, glycolysis and lactate-mediated immunosuppressive M2-like macrophage polarization. Conclusion These findings elucidated that USP5 served as a regulator of glycolysis-lactate to stimulate M2-like macrophage formation by regulating STAT2-PFKFB4 signaling, which supported that USP5 could be a viable therapeutic target of MM treatment.