Overexpression of USP8 inhibits inflammation and ferroptosis in chronic obstructive pulmonary disease by regulating the OTUB1/SLC7A11 signaling pathway.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a familiar disease, and owns high morbidity and mortality, which critically damages the health of patients. Ubiquitin-specific peptidase 8 (USP8) is a pivotal protein to join in the regulation of some diseases. In a previous report, it was determined that USP8 expression is down-regulated in LPS-treated BEAS-2B cells, and USP8 restrains inflammatory response and accelerates cell viability. However, the regulatory roles of USP8 on ferroptosis in COPD are rarely reported, and the associated molecular mechanisms keep vague. OBJECTIVE: To investigate the regulatory functions of USP8 in COPD progression. MATERIAL AND METHODS: The lung functions were measured through the Buxco Fine Pointe Series Whole Body Plethysmography (WBP). The Fe level was tested through the Fe assay kit. The protein expressions were assessed through western blot. The levels of tumor necrosis -factor-α, interleukin 6, and interleukin 8 were evaluated through enzyme-linked immunosorbent serologic assay. Cell viability was tested through CCK-8 assay. RESULTS: In this work, it was discovered that overexpression of USP8 improved lung function in COPD mice. In addition, overexpression of USP8 repressed ferroptosis by regulating glutathione peroxidase 4 and acyl-CoA synthetase long-chain family 4 expressions in COPD mice. Overexpression of USP8 suppressed inflammation in COPD mice. Furthermore, overexpression of USP8 suppressed ferroptosis in COPD cell model. At last, it was verified that overexpression of USP8 accelerated ubiquitin aldehyde-binding protein 1 (OTUB1)/solute carrier family 7 member 11 (SLC7A11) pathway. CONCLUSION: This study manifested that overexpression of USP8 restrained inflammation and ferroptosis in COPD by regulating the OTUB1/SLC7A11 signaling pathway. This discovery hinted that USP8 could be a potential target for COPD treatment.

Deubiquitylase OTUD3 regulates integrated stress response to suppress progression and sorafenib resistance of liver cancer.

The integrated stress response (ISR) is activated in response to intrinsic and extrinsic stimuli, playing a role in tumor progression and drug resistance. The regulatory role and mechanism of ISR in liver cancer, however, remain largely unexplored. Here, we demonstrate that OTU domain-containing protein 3 (OTUD3) is a deubiquitylase of eukaryotic initiation factor 2α (eIF2α), antagonizing ISR and suppressing liver cancer. OTUD3 decreases interactions between eIF2α and the kinase EIF2ΑK3 by removing K27-linked polyubiquitylation on eIF2α. OTUD3 deficiency in mice leads to enhanced ISR and accelerated progression of N-nitrosodiethylamine-induced hepatocellular carcinoma. Additionally, decreased OTUD3 expression associated with elevated eIF2α phosphorylation correlates with the progression of human liver cancer. Moreover, ISR activation due to decreased OTUD3 expression renders liver cancer cells resistant to sorafenib, while the combined use of the ISR inhibitor ISRIB significantly improves their sensitivity to sorafenib. Collectively, these findings illuminate the regulatory mechanism of ISR in liver cancer and provide a potential strategy to counteract sorafenib resistance.

FUS-stabilized USP35 promotes growth, invasion and angiogenesis in NSCLC through deubiquitinating VEGFA.

Vascular endothelial growth factor A (VEGFA) is an important regulator for non-small cell lung cancer (NSCLC). Our study aimed to reveal its upstream pathway to provide new ideas for developing the therapeutic targets of NSCLC. The mRNA and protein levels of VEGFA, ubiquitin-specific peptidase 35 (USP35), and FUS were determined by quantitative real-time PCR and Western blot. Cell proliferation, apoptosis, invasion and angiogenesis were detected using CCK8 assay, EdU assay, flow cytometry, transwell assay and tube formation assay. The interaction between USP35 and VEGFA was assessed by Co-IP assay and ubiquitination assay. Animal experiments were performed to assess USP35 and VEGFA roles in vivo. VEGFA had elevated expression in NSCLC tissues and cells. Interferences of VEGFA inhibited NSCLC cell proliferation, invasion, angiogenesis, and increased apoptosis. USP35 could stabilize VEGFA protein level by deubiquitination, and USP35 knockdown suppressed NSCLC cell growth, invasion and angiogenesis via reducing VEGFA expression. FUS interacted with USP35 to promote its mRNA stability, thereby positively regulating VEGFA expression. Also, USP35 silencing could reduce NSCLC tumorigenesis by downregulating VEGFA. FUS-stabilized USP35 facilitated NSCLC cell growth, invasion and angiogenesis through deubiquitinating VEGFA, providing a novel idea for NSCLC treatment.

MRPL35 Induces Proliferation, Invasion, and Glutamine Metabolism in NSCLC Cells by Upregulating SLC7A5 Expression.

BACKGROUND: Mitochondrial ribosomal protein L35 (MRPL35) has been reported to contribute to the growth of non-small cell lung cancer (NSCLC) cells. However, the functions and mechanisms of MRPL35 on glutamine metabolism in NSCLC remain unclear. METHODS: The detection of mRNA and protein of MRPL35, ubiquitin-specific protease 39 (USP39), and solute carrier family 7 member 5 (SLC7A5) was conducted using qRT-PCR and western blotting. Cell proliferation, apoptosis, and invasion were evaluated using the MTT assay, EdU assay, flow cytometry, and transwell assay, respectively. Glutamine metabolism was analyzed by detecting glutamine consumption, α-ketoglutarate level, and glutamate production. Cellular ubiquitination analyzed the deubiquitination effect of USP39 on MRPL35. An animal experiment was conducted for in vivo analysis. RESULTS: MRPL35 was highly expressed in NSCLC tissues and cell lines, and high MRPL35 expression predicted poor outcome in NSCLC patients. In vitro analyses suggested that MRPL35 knockdown suppressed NSCLC cell proliferation, invasion, and glutamine metabolism. Moreover, MRPL35 silencing hindered tumor growth in vivo. Mechanistically, USP39 stabilized MRPL35 expression by deubiquitination and then promoted NSCLC cell proliferation, invasion, and glutamine metabolism. In addition, MRPL35 positively affected SLC7A5 expression in NSCLC cells in vitro and in vivo. Moreover, the anticancer effects of MRPL35 silencing could be rescued by SLC7A5 overexpression in NSCLC cells. CONCLUSION: MRPL35 expression was stabilized by USP39-induced deubiquitination in NSCLC cells, and knockdown of MRPL35 suppressed NSCLC cell proliferation, invasion, and glutamine metabolism in vitro and impeded tumor growth in vivo by upregulating SLC7A5, providing a promising therapeutic target for NSCLC.

Endothelial Dickkopf-1 Promotes Smooth Muscle Cell-derived Foam Cell Formation via USP53-mediated Deubiquitination of SR-A During Atherosclerosis.

Background: Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of endothelial DKK1 in modulating adjacent smooth muscle cells (SMCs) in atherosclerosis remains unclear. This study investigated the role of EC-secreted DKK1 in SMC-derived foam cell formation under shear stress, in vitro and in vivo. Methods: Parallel-plate co-culture flow system was used to explore the cellular communication between ECs and SMCs under shear stress in vitro. Endothelium-specific knockout of DKK1 (DKK1ECKO/APOE-/-) and endothelium-specific overexpression of DKK1 (DKK1ECTg) mice were constructed to investigate the role of endothelial DKK1 in atherosclerosis and SMC-derived foam cell formation in vivo. RNA sequencing (RNA-seq) was used to identify the downstream targets of DKK1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot, coimmunoprecipitation (Co-IP) assays and chromatin immunoprecipitation (ChIP) experiments were conducted to explore the underlying regulatory mechanisms. Results: DKK1 is transcriptionally upregulated in ECs under conditions of low shear stress, but not in co-cultured SMCs. However, DKK1 protein in co-cultured SMCs is increased via uptake of low shear stress-induced endothelial DKK1, thereby promoting lipid uptake and foam cell formation in co-cultured SMCs via the post-translational upregulation of scavenger receptor-A (SR-A) verified in parallel-plate co-culture flow system, DKK1ECKO and DKK1ECTg mice. RNA sequencing revealed that DKK1-induced SR-A upregulation in SMCs is dependent on Ubiquitin-specific Protease 53 (USP53), which bound to SR-A via its USP domain and cysteine at position 41, exerting deubiquitination to maintain the stability of the SR-A protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby mediating the effect of DKK1 on lipid uptake in SMCs. Moreover, DKK1 regulates the transcription of USP53 by facilitating the binding of transcription factor CREB to the USP53 promoter. SMC-specific overexpression of USP53 via adeno-associated virus serotype 2 vectors in DKK1ECKO/APOE-/- mice reversed the alleviation of atherosclerotic plaque burden, SR-A expression and lipid accumulation in SMCs within plaques resulting from DKK1 deficiency. Conclusions: Our findings demonstrate that, endothelial DKK1, induced by pathological low shear stress, acts as an intercellular mediator, promoted the foam cell formation of SMCs. These results suggest that targeted intervention with endothelial DKK1 may confer beneficial effects on atherosclerosis.

Effect of ubiquitin-specific proteinase 43 on ovarian serous adenocarcinoma and its clinical significance.

BACKGROUND: Ovarian cancer stands as a highly aggressive malignancy. The core aim of this investigation is to uncover genes pivotal to the progression and prognosis of ovarian cancer, while delving deep into the intricate mechanisms that govern their impact. METHODS: The study entailed the retrieval of RNA-seq data and survival data from the XENA database. Outliers were meticulously excluded in accordance with TCGA guidelines and through principal components analysis. The R package 'deseq2' was harnessed to extract differentially expressed genes. WGCNA was employed to prioritise these genes, and Cox regression analysis and survival analysis based on disease-specific time were conducted to identify significant genes. Immunohistochemistry validation was undertaken to confirm the distinct expression of USP43. Furthermore, the influence of USP43 on the biological functions of ovarian cancer cells was explored using techniques such as RNA interference, western blotting, scratch assays, and matrigel invasion assays. The examination of immune infiltration was facilitated via CIBERSORT. RESULTS: The study unearthed 5195 differentially expressed genes between ovarian cancer and normal tissue, comprising 3416 up-regulated and 1779 down-regulated genes. WGCNA pinpointed 204 genes most intimately tied to tumorigenesis. The previously undisclosed gene USP43 exhibited heightened expression in tumour tissues and exhibited associations with overall survival and disease-specific survival. USP43 emerged as a driver of cell migration (43.27 ± 3.91% vs 19.69 ± 1.94%) and invasion ability (314 ± 32 vs 131 ± 12) through the mechanism of epithelial mesenchymal transition, potentially mediated by the KRAS pathway. USP43 was also identified as a booster of CD4+ T memory resting cell infiltration, while concurrently reducing M1 macrophages within cancer, thereby fostering a milieu with relatively immune suppressive traits. Interestingly, USP43 demonstrated connections with epigenetically regulated-mRNAsi, although not with mRNAsi. CONCLUSION: This study underscores the role of USP43 in facilitating tumour migration and invasion. It postulates USP43 as a novel therapeutic target for ovarian cancer treatment.

Ovarian cancer is the most deadly tumour among all gynecological tumours. Thus we tried to explore the relevant mechanism of ovarian cancer because its occurrence and development mechanism has not been fully elucidated. We used bioinformatics methods to perform differential gene analysis on ovarian cancer tissues and normal tissues, and used methods such as WGCNA and COX regression analysis to find the gene USP43 related to tumour development and prognosis. USP43 is a gene that has not been studied in ovarian cancer before. Through RNA interference technology, we found that it can promote the migration and invasion ability of ovarian cancer and promote epithelial-mesenchymal transition of ovarian cancer cells. In addition, this gene has also been proven to be related to tumour immunity and tumour stemness. These results indicate that USP43 can promote the tumorigenesis of ovarian cancer and can be used as a drug target.

USP38 exacerbates pressure overload-induced left ventricular electrical remodeling.

BACKGROUND: Ubiquitin-specific protease 38 (USP38), belonging to the USP family, is recognized for its role in controlling protein degradation and diverse biological processes. Ventricular arrhythmias (VAs) following heart failure (HF) are closely linked to ventricular electrical remodeling, yet the specific mechanisms underlying VAs in HF remain inadequately explored. In this study, we examined the impact of USP38 on VAs in pressure overload-induced HF. METHODS: Cardiac-specific USP38 knockout mice, cardiac-specific USP38 transgenic mice and their matched control littermates developed HF induced by aortic banding (AB) surgery. After subjecting the mice to AB surgery for a duration of four weeks, comprehensive investigations were conducted, including pathological analysis and electrophysiological assessments, along with molecular analyses. RESULTS: We observed increased USP38 expression in the left ventricle of mice with HF. Electrocardiogram showed that the USP38 knockout shortened the QRS interval and QTc, while USP38 overexpression prolonged these parameters. USP38 knockout decreased the susceptibility of VAs by shortening action potential duration (APD) and prolonging effective refractory period (ERP). In addition, USP38 knockout increased ion channel and Cx43 expression in ventricle. On the contrary, the increased susceptibility of VAs and the decreased expression of ventricular ion channels and Cx43 were observed with USP38 overexpression. In both in vivo and in vitro experiments, USP38 knockout inhibited TBK1/AKT/CAMKII signaling, whereas USP38 overexpression activated this pathway. CONCLUSION: Our data indicates that USP38 increases susceptibility to VAs after HF through TBK1/AKT/CAMKII signaling pathway, Consequently, USP38 may emerge as a promising therapeutic target for managing VAs following HF.

RUNX1 regulates MCM2/CDC20 to promote COAD progression modified by deubiquitination of USP31.

Colon adenocarcinoma (COAD) is the second leading cause of cancer death, and there is still a lack of diagnostic biomarkers and therapeutic targets. In this study, bioinformatics analysis of the TCGA database was used to obtain RUNX1, a gene with prognostic value in COAD. RUNX1 plays an important role in many malignancies, and its molecular regulatory mechanisms in COAD remain to be fully understood. To explore the physiological role of RUNX1, we performed functional analyses, such as CCK-8, colony formation and migration assays. In addition, we investigated the underlying mechanisms using transcriptome sequencing and chromatin immunoprecipitation assays. RUNX1 is highly expressed in COAD patients and significantly correlates with survival. Silencing of RUNX1 significantly slowed down the proliferation and migratory capacity of COAD cells. Furthermore, we demonstrate that CDC20 and MCM2 may be target genes of RUNX1, and that RUNX1 may be physically linked to the deubiquitinating enzyme USP31, which mediates the upregulation of RUNX1 protein to promote transcriptional function. Our results may provide new insights into the mechanism of action of RUNX1 in COAD and reveal potential therapeutic targets for this disease.

In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer.

Functionally characterizing the genetic alterations that drive pancreatic cancer is a prerequisite for precision medicine. Here, we perform somatic CRISPR/Cas9 mutagenesis screens to assess the transforming potential of 125 recurrently mutated pancreatic cancer genes, which revealed USP15 and SCAF1 as pancreatic tumor suppressors. Mechanistically, we find that USP15 functions in a haploinsufficient manner and that loss of USP15 or SCAF1 leads to reduced inflammatory TNFα, TGF-ß and IL6 responses and increased sensitivity to PARP inhibition and Gemcitabine. Furthermore, we find that loss of SCAF1 leads to the formation of a truncated, inactive USP15 isoform at the expense of full-length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 alterations are observed in 31% of pancreatic cancer patients. Our results highlight the utility of in vivo CRISPR screens to integrate human cancer genomics and mouse modeling for the discovery of cancer driver genes with potential prognostic and therapeutic implications.

USP39 Promotes the Viability and Migration of Head and Neck Squamous Cell Carcinoma Cell by Regulating STAT1.

Objective: Ubiquitin-specific peptidase 39 (USP39) plays a carcinogenic role in many cancers, but little research has been conducted examining whether it is involved in head and neck squamous cell carcinoma (HNSCC). Therefore, this study explored the functional role of USP39 in HNSCC. Method: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins (DEPs) between the HNSCC tumor and adjacent healthy tissues. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to assess the functional enrichment of DEPs. Immunohistochemistry was used to detect protein expression. The viability and migration of two HNSCC cell lines, namely CAL27 and SCC25, were detected using the cell counting kit-8 assay and a wound healing assay, respectively. Quantitative real-time PCR was used to detect the expression level of signal transducer and activator of transcription 1 (STAT1) mRNA. Results: LC-MS/MS results identified 590 DEPs between HNSCC and adjacent tissues collected from 4 patients. Through GO and KEGG pathway analyses, 34 different proteins were found to be enriched in the spliceosome pathway. The expression levels of USP39 and STAT1 were significantly higher in HNSCC tumor tissue than in adjacent healthy tissue as assessed by LC-MS/MS analysis, and the increased expression of USP39 and STAT1 protein was confirmed by immunohistochemistry in clinical samples collected from 7 additional patients with HNSCC. Knockdown of USP39 or STAT1 inhibited the viability and migration of CAL27 and SCC25 cells. In addition, USP39 knockdown inhibited the expression of STAT1 mRNA in these cells. Conclusion: Our findings indicated that USP39 knockdown may inhibit HNSCC viability and migration by suppressing STAT1 expression. The results of this study suggest that USP39 may be a potential new target for HNSCC clinical therapy or a new biomarker for HNSCC.

Ubiquitin-specific protease 54 regulates GLUT1-mediated aerobic glycolysis to inhibit lung adenocarcinoma progression by modifying p53 degradation.

Lung adenocarcinoma (LUAD) is one of the most prevalent types of cancer. Ubiquitination is crucial in modulating cell proliferation and aerobic glycolysis in cancer. The frequency of TP53 mutations in LUAD is approximately 50%. Currently, therapeutic targets for wild-type (WT) p53-expressing LUAD are limited. In the present study, we systemically explored the expression of ubiquitin-specific protease genes using public datasets. Then, we focused on ubiquitin-specific protease 54 (USP54), and explored its prognostic significance in LUAD patients using public datasets, analyses, and an independent cohort from our center. We found that the expression of USP54 was lower in LUAD tissues compared with that in the paracancerous tissues. Low USP54 expression levels were linked to a malignant phenotype and worse survival in patients with LUAD. The results of functional experiments revealed that up-regulation of USP54 suppressed LUAD cell proliferation in vivo and in vitro. USP54 directly interacted with p53 protein and the levels of ubiquitinated p53 were inversely related to USP54 levels, consistent with a role of USP54 in deubiquitinating p53 in p53-WT LUAD cells. Moreover, up-regulation of the USP54 expression inhibited aerobic glycolysis in LUAD cells. Importantly, we confirmed that USP54 inhibited aerobic glycolysis and the growth of tumor cells by a p53-mediated decrease in glucose transporter 1 (GLUT1) expression in p53-WT LUAD cells. Altogether, we determined a novel mechanism of survival in the p53-WT LUAD cells to endure the malnourished tumor microenvironment and provided insights into the role of USP54 in the adaptation of p53-WT LUAD cells to metabolic stress.

Ubiquitin-specific peptidase 15 regulates the TFAP4/PCGF1 axis facilitating liver metastasis of colorectal cancer and cell stemness.

The tumor recurrence and metastasis of colorectal cancer (CRC) are responsible for most of CRC-linked mortalities. It is an urgent need to deeply investigate the pathogenesis of CRC metastasis and look for novel targets for its treatment. The current study aimed to investigate the effects of ubiquitin-specific peptidase 15 (USP-15) on the CRC progression. In vivo, a mouse model of liver metastasis of CRC tumor was established to investigate the role of USP-15. In vitro, the migrated and invasive abilities of CRC cells were assessed by transwell assay. Cell stemness was evaluated by using sphere formation assay. The underlying mechanism was further explored by employing the co-immunoprecipitation, dual luciferase reporter assay, oligonucleotide pull-down assay, and chromatin immunoprecipitation assay. The results showed that USP-15 was upregulated in CRC patients with liver metastasis and high metastatic potential cell lines of CRC. Loss of USP-15 repressed the epithelial-to-mesenchymal transition (EMT), migration, invasion, and stemness properties of CRC cells in vitro. Downregulation of USP-15 reduced the liver metastasis of mice in vivo. USP-15 upregulation obtained the contrary effects. Subsequently, USP-15 deubiquitinated transcription factor AP-4 (TFAP4) and enhanced its protein stability. TFAP4 could transcriptionally activated polycomb group ring finger 1 (PCGF1). The pro-cancer effects of USP-15 were rescue by the knockdown of TFAP4 or PCGF1. In conclusions: USP-15 facilitated the liver metastasis by the enhancement of cell stemness and EMT in CRC, which was at least partly mediated by the deubiquitination of TFAP4 upon the upregulation of PCGF1.

E2F1-regulated USP5 contributes to the tumorigenic capacity of glioma stem cells through the maintenance of OCT4 stability.

Mesenchymal glioma stem cells (MES GSCs) are a subpopulation of cells in glioblastoma (GBM) that contribute to a worse prognosis owing to their highly aggressive nature and resistance to radiation therapy. Here, OCT4 is characterized as a critical factor in sustaining the stemness phenotype of MES GSC. We find that OCT4 is expressed intensively in MES GSC and is intimately associated with poor prognosis, moreover, OCT4 depletion leads to diminished invasive capacity and impairment of the stem phenotype in MES GSC. Subsequently, we demonstrated that USP5 is a deubiquitinating enzyme which directly interacts with OCT4 and preserves OCT4 stability through its deubiquitination. USP5 was additionally proven to be aberrantly over-expressed in MES GSCs, and its depletion resulted in a noticeable diminution of OCT4 and consequently a reduced self-renewal and tumorigenic capacity of MES GSCs, which can be substantially restored by ectopic expression of OCT4. In addition, we detected the dominant molecule that regulates USP5 transcription, E2F1, with dual luciferase reporter gene analysis. In combination, targeting the E2F1-USP5-OCT4 axis is a potentially emerging strategy for the therapy of GBM.

OTUD1 enhances gastric cancer aggressiveness by deubiquitinating EBV-encoded protein BALF1 to stabilize the apoptosis inhibitor Bcl-2.

The Epstein-Barr virus (EBV) is implicated in several cancers, including EBV-associated gastric cancer (EBVaGC). This study focuses on EBV-encoded BALF1 (BamH1 A fragment leftward reading frame 1), a key apoptosis regulator in EBV-related cancers, whose specific impact on EBVaGC was previously unknown. Our findings indicate that BALF1 overexpression in gastric cancer cells significantly enhances their proliferation, migration, and resistance to chemotherapy-induced apoptosis, confirming BALF1's oncogenic potential. A novel discovery is that BALF1 undergoes degradation via the ubiquitin-proteasome pathway. Through analysis of 69 deubiquitinating enzymes (DUBs), ovarian tumor protease (OTU) domain-containing protein 1 (OTUD1) emerged as a vital regulator for maintaining BALF1 protein stability. Furthermore, BALF1 was found to play a role in regulating the stability of the B-cell lymphoma-2 (Bcl-2) protein, increasing its levels through deubiquitination. This mechanism reveals BALF1's multifaceted oncogenic role in gastric cancer, as it contributes both directly and indirectly to cancer progression, particularly by stabilizing Bcl-2, known for its anti-apoptotic characteristics. These insights significantly deepen our understanding of EBV's involvement in the pathogenesis of gastric cancer. The elucidation of OTUD1's role in BALF1 regulation and its influence on Bcl-2 stabilization provide new avenues for therapeutic intervention in EBVaGC, bridging the gap between viral oncogenesis and cellular protein regulation and offering a more holistic view of gastric cancer development under the influence of EBV.

USP15 facilitates the progression of bladder cancer by amplifying the activation of the NF-κB signaling pathway.

USP15, a pivotal member of the deubiquitinase family, plays a crucial role in orchestrating numerous vital biological processes, including the regulation of NF-κB signaling pathway and deubiquitination of proto-oncogenes. In various cancers, USP15 has been validated to exhibit up-regulated expression, impacting the initiation and progression of cancer. However, its precise mechanism in bladder cancer remains elusive. Our study shed light on the significant overexpression of USP15 in bladder cancer cells compared to normal bladder cells, correlating with a poorer prognosis for bladder cancer patients. Strikingly, attenuation of USP15 expression greatly attenuated the proliferation, migration, and invasion of bladder cancer cells. Moreover, upregulation of USP15 was found to drive cancer progression through the activation of the NF-κB signaling pathway. Notably, USP15 directly deubiquitinates BRCC3, heightening its expression level, and subsequent overexpression of BRCC3 counteracted the antitumoral efficacy of USP15 downregulation. Overall, our findings elucidated the carcinogenic effects of USP15 in bladder cancer, primarily mediated by the excessive activation of the NF-κB signaling pathway, thereby promoting tumor development. These results underscore the potential of USP15 as a promising therapeutic target for bladder cancer in the future.

Deubiquitinating enzyme OTUD4 stabilizes RBM47 to induce ATF3 transcription: a novel mechanism underlying the restrained malignant properties of ccRCC cells.

Dysregulation of deubiquitination contributes to various diseases, including cancer, and aberrant expression of deubiquitinating enzymes is involved in carcinoma progression. As a member of the ovarian tumor (OTU) deubiquitinases, OTUD4 is considered a tumor suppressor in many kinds of malignancies. The biological characteristics and mechanisms of OTUD4 in clear cell renal cell carcinoma (ccRCC) remain unclear. The downregulation of OTUD4 in ccRCC was confirmed based on the TCGA database and a validation cohort of 30-paired ccRCC and para-carcinoma samples. Moreover, OTUD4 expression was detected by immunohistochemistry in 50 cases of ccRCC tissues, and patients with lower levels of OTUD4 showed larger tumor size (p = 0.015). TCGA data revealed that patients with high expression of OTUD4 had a longer overall survival rate. In vitro and in vivo studies revealed that downregulation of OTUD4 was essential for tumor cell growth and metastasis in ccRCC, and OTUD4 overexpression inhibited these malignant phenotypes. We further found that OTUD4 sensitized ccRCC cells to Erastin-induced ferroptosis, and ferrostain-1 inhibited OTUD4-induced ferroptotic cell death. Mechanistic studies indicated that OTUD4 functioned as an anti-proliferative and anti-metastasic factor through the regulation of RNA-binding protein 47 (RBM47)-mediated activating transcription factor 3 (ATF3). OTUD4 directly interacted with RBM47 and promoted its stability via deubiquitination events. RBM47 was critical in ccRCC progression by regulating ATF3 mRNA stability, thereby promoting ATF3-mediated ferroptosis. RBM47 interference abolished the suppressive role of OTUD4 overexpression in ccRCC. Our findings provide mechanistic insight into OTUD4 of ccRCC progression and indicate a novel critical pathway OTUD4/RBM47/ATF3 may serve as a potential therapeutic pathway for ccRCC.

USP46 promotes the interferon antiviral signaling in black carp by deubiquitinating TBK1.

Ubiquitin-specific peptidase 46 (USP46) functions as a deubiquitinating enzyme, facilitating the removal of ubiquitin molecules attached to substrate proteins and playing a critical role in cancer and neurodegenerative diseases. However, its function in innate antiviral immunity is unknown. In this study we cloned and identified bcUSP46, a homolog of USP46 from black carp. We discovered that overexpression of bcUSP46 enhanced the transcription of interferon (IFN) promoters and increased the expression of IFN, PKR, and Mx1. In addition, bcUSP46 knockdown significantly inhibited the expression of ISG genes, as well as the antiviral activity of the host cells. Interestingly, when bcUSP46 was co-expressed with the RLR factors, it significantly enhanced the activity of the IFN promoter mediated by these factors, especially TANK-binding kinase 1 (TBK1). The subsequent co-immunoprecipitation (co-IP) and immunofluorescence (IF) assay confirmed the association between bcUSP46 and bcTBK1. Noteworthily, co-expression of bcUSP46 with bcTBK1 led to an elevation of bcTBK1 protein level. Further analysis revealed that bcUSP46 stabilized bcTBK1 by eliminating the K48-linked ubiquitination of bcTBK1. Overall, our findings highlight the unique role of USP46 in modulating TBK1/IFN signaling and enrich our knowledge of the function of deubiquitination in regulating innate immunity in vertebrates.

USP3 promotes cisplatin resistance in non-small cell lung cancer cells by suppressing ACOT7-regulated ferroptosis.

This study aims to investigate the role and mechanism of ubiquitin-specific protease 3 (USP3) in cisplatin (DDP) in non-small cell lung cancer (NSCLC). USP3 expression in NSCLC cells was detected using reverse transcription quantitative PCR and Western blot. DDP-resistant cells were constructed and cell counting kit-8 assay determined the IC 50 of cells to DDP. USP3 expression was silenced in DDP-resistant cells, followed by detection of cell proliferation by clone formation assay, iron ion contents, ROS, MDA, and GSH levels by kits, GPX4 and ACSL4 protein expressions by Western blot. The binding between USP3 and ACOT7 was analyzed using Co-IP, and the ubiquitination level of ACOT7 was measured. USP3 and ACOT7 were highly expressed in NSCLC cells and further increased in drug-resistant cells. USP3 silencing reduced the IC 50 of cells to DDP and diminished the number of cell clones. Moreover, USP3 silencing suppressed GSH and GPX4 levels, upregulated iron ion contents, ROS, MDA, and ACSL4 levels, and facilitated ferroptosis. Mechanistically, USP3 upregulated ACOT7 protein expression through deubiquitination. ACOT7 overexpression alleviated the promoting effect of USP7 silencing on ferroptosis in NSCLC cells and enhanced DDP resistance. To conclude, USP3 upregulated ACOT7 protein expression through deubiquitination, thereby repressing ferroptosis in NSCLC cells and enhancing DDP resistance.

OTUD4 promotes the progression of glioblastoma by deubiquitinating CDK1 and activating MAPK signaling pathway.

Glioblastoma, IDH-Wild type (GBM, CNS WHO Grade 4) is a highly heterogeneous and aggressive primary malignant brain tumor with high morbidity, high mortality, and poor patient prognosis. The global burden of GBM is increasing notably due to limited treatment options, drug delivery problems, and the lack of characteristic molecular targets. OTU deubiquitinase 4 (OTUD4) is a potential predictive factor for several cancers such as breast cancer, liver cancer, and lung cancer. However, its function in GBM remains unknown. In this study, we found that high expression of OTUD4 is positively associated with poor prognosis in GBM patients. Moreover, we provided in vitro and in vivo evidence that OTUD4 promotes the proliferation and invasion of GBM cells. Mechanism studies showed that, on the one hand, OTUD4 directly interacts with cyclin-dependent kinase 1 (CDK1) and stabilizes CDK1 by removing its K11, K29, and K33-linked polyubiquitination. On the other hand, OTUD4 binds to fibroblast growth factor receptor 1 (FGFR1) and reduces FGFR1's K6 and K27-linked polyubiquitination, thereby indirectly stabilizing CDK1, ultimately influencing the activation of the downstream MAPK signaling pathway. Collectively, our results revealed that OTUD4 promotes GBM progression via OTUD4-CDK1-MAPK axis, and may be a prospective therapeutic target for GBM treatment.

Deubiquitinase USP4 suppresses antitumor immunity by inhibiting IRF3 activation and tumor cell-intrinsic interferon response in colorectal cancer.

Despite the approval of immune checkpoint blockade (ICB) therapy for various tumor types, its effectiveness is limited to only approximately 15% of patients with microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) colorectal cancer (CRC). Approximately 80%-85% of CRC patients have a microsatellite stability (MSS) phenotype, which features a rare T-cell infiltration. Thus, elucidating the mechanisms underlying resistance to ICB in patients with MSS CRC is imperative. In this study, we demonstrate that ubiquitin-specific peptidase 4 (USP4) is upregulated in MSS CRC tumors and negatively regulates the immune response against tumors in CRC. Additionally, USP4 represses the cellular interferon (IFN) response and antigen presentation and impairs PRR signaling-mediated cell death. Mechanistically, USP4 impedes the nuclear localization of interferon regulator Factor 3 (IRF3) by deubiquitinating the K63-polyubiquitin chain of TRAF6 and IRF3. Knockdown of USP4 enhances the infiltration of T cells in CRC tumors and overcomes ICB resistance in an MC38 syngeneic mouse model. Moreover, published datasets revealed that patients showing higher USP4 expression exhibited decreased responsiveness to anti-PD-L1 therapy. These findings highlight an essential role of USP4 in the suppression of antitumor immunity in CRC.