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USP7 promotes deubiquitination and stabilization of Axin. a, b Knockout of USP7 decreases the protein level, but not the mRNA level, of AXIN1 in HEK293T cells (a) and YAPC cells (b). Error bars denote the SD between four replicates. c Proteasome inhibitor Bortezomib and E1 inhibitor MLN7243, but not lysosome inhibitor Bafilomycin A1, inhibit the effect of USP7 knockout on the expression of AXIN1 protein in HEK293T cells. HEK293T control and USP7 gRNA bearing cells were treated with DMSO, 5 µM Bortezomib, 5 µM MLN7243 or 0.1 µM Bafilomycin A1 overnight. d Knockout of USP7 promotes Wnt3a-induced AXIN1 degradation and accumulation of active β-catenin in HEK293T cells. HEK293T control and USP7 gRNA bearing cells were incubated with control medium or 20% Wnt3a CM overnight. e Knockout of USP7 decreases the protein stability of AXIN1 upon Wnt3a treatment in HEK293T cells. Cells were treated with 100 µg/ml cycloheximide (CHX) to block de novo protein synthesis and simultaneously stimulated with 20% Wnt3a CM to induce AXIN1 degradation. The intensity of AXIN1 and Tubulin bands was quantified by ImageJ, and quantification of normalized AXIN1 band intensity is shown in the bottom panel. f Overexpression of wild-type USP7, but not the C223A mutant, stabilizes co-expressed Axin1 protein. GFP serves as a control for transfection efficiency. g Overexpression of wild-type USP7, but not the C233A mutant, decreases poly-ubiquitination of co-expressed Axin1. h Knockout of USP7 enhances poly-ubiquitination of endogenous AXIN1 in YAPC cells. Control and USP7 gRNA bearing YAPC cells were incubated with 10 µM MG132 overnight and harvested for TUBE assay. i Recombinant USP7 protein (rUSP7) efficiently erases poly-ubiquitination of Axin1 but not β-catenin in an in vitro deubiquitination assay. j Wnt3a-induced poly-ubiquitination of endogenous AXIN1 is sustained upon USP7 knockout in HEK293T cells. HEK293T control and USP7 gRNA bearing cells were treated with 30% Wnt3a CM and 20 µM MG132 and harvested at different time points for TUBE assay., Source data for Fig. 3a, b, c, d, e, f, g, h, i, and j are provided as Source Data file
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Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, t...
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... S3). Given that USP7 is a specific deubiquitinase that stabilizes protein expression in a posttranslational manner, 34 we hypothesized that the histone lactylation (Pan-Kla) promotes CMC1 expression via USP7 in a post-translational manner rather than its direct modification on the Cmc1 promoter. ...
... To further confirm that USP7 deubiquitinates CMC1, we performed the in-vivo de-ubiquitination assay in 293T cell line as previously described. 34 In the condition of USP7 overexpression, the poly-ubiquitination of exogenous CMC1 significantly decreased ( Figure 5(h)). Finally, we utilized a specific USP7 inhibitor, P5091, 40 to test the efficacy of targeting CMC1 by inhibiting USP7 activity in the presence of lactate addition. ...
T cell immunity is critical for human defensive immune response. Exploring the key molecules during the process provides new targets for T cell-based immunotherapies. CMC1 is a mitochondrial electron transport chain (ETC) complex IV chaperon protein. By establishing in-vitro cell culture system and Cmc1 gene knock out mice, we evaluated the role of CMC1 in T cell activation and differentiation. The B16-OVA tumor model was used to test the possibility of targeting CMC1 for improving T cell anti-tumor immunity. We identified CMC1 as a positive regulator in CD8⁺T cells activation and terminal differentiation. Meanwhile, we found that CMC1 increasingly expressed in exhausted T (Tex) cells. Genetic lost of Cmc1 inhibits the development of CD8⁺T cell exhaustion in mice. Instead, deletion of Cmc1 in T cells prompts cells to differentiate into metabolically and functionally quiescent cells with increased memory-like features and tolerance to cell death upon repetitive or prolonged T cell receptor (TCR) stimulation. Further, the in-vitro mechanistic study revealed that environmental lactate enhances CMC1 expression by inducing USP7, mediated stabilization and de-ubiquitination of CMC1 protein, in which a mechanism we propose here that the lactate-enriched tumor microenvironment (TME) drives CD8⁺TILs dysfunction through CMC1 regulatory effects on T cells. Taken together, our study unraveled the novel role of CMC1 as a T cell regulator and its possibility to be utilized for anti-tumor immunotherapy.
... Dvl is ubiquitinated by NEDL1, KLHL12, NEDD4L, Huwe1, Inversin, and ITCH and deubiquitinated by CYLD, USP9X, and USP14 (Miyazaki et al. 2004;Angers et al. 2006;Wei et al. 2012;Ding et al. 2013;Jung et al. 2013;de Groot et al. 2014;Nielsen et al. 2019). A component of the destruction complex, Axin, is ubiquitinated by RNF146, Smurf1/2, and Siah1 and deubiquitinated by USP7, USP34, and USP44 (Kim and Jho 2010;Lui et al. 2011;Zhang et al. 2011;Fei et al. 2013;Ji et al. 2017;Ji et al. 2019;Huang et al. 2020). APC is ubiquitinated by HectD1 and RNF61 and deubiquitinated by Trabid and USP15 (Tran et al. 2008;Huang et al. 2009;Tran et al. 2013;Lee et al. 2018). ...
Wnt signaling plays a crucial role in embryonic development and homeostasis maintenance. Delicate and sensitive fine-tuning of Wnt signaling based on the proper timings and positions is required to balance cell proliferation and differentiation and maintain individual health. Therefore, homeostasis is broken by tissue hypoplasia or tumor formation once Wnt signal dysregulation disturbs the balance of cell proliferation. The well-known regulatory mechanism of Wnt signaling is the molecular reaction associated with the cytoplasmic accumulation of effector β-catenin. In addition to β-catenin, most Wnt effector proteins are also regulated by ubiquitin-dependent modification, both qualitatively and quantitatively. This review will explain the regulation of the whole Wnt signal in four regulatory phases, as well as the different ubiquitin ligases and the function of deubiquitinating enzymes in each phase. Along with the recent results, the mechanism by which RNF43 negatively regulates the surface expression of Wnt receptors, which has recently been well understood, will be detailed. Many RNF43 mutations have been identified in pancreatic and gastrointestinal cancers and examined for their functional alteration in Wnt signaling. Several mutations facilitate or activate the Wnt signal, reversing the RNF43 tumor suppressor function into an oncogene. RNF43 may simultaneously play different roles in classical multistep tumorigenesis, as both wild-type and mutant RNF43 suppress the p53 pathway. We hope that the knowledge obtained from further research in RNF43 will be applied to cancer treatment in the future despite the fully unclear function of RNF43.
... 51 Ubiquitination has widely reported to regulate the level of Axin, such as RNF146 52 and Smurf1. 53 And USP proteins such as USP7 54 and USP34 55 would stabilize the protein level of Axin to inhibit Wnt signaling. Here, in this study, we found that BIRC6, acting as an E3 ligase, interacted to regulate the ubiquitination level of Axin in RCC cells. ...
The mechanism underlying the development of renal cell carcinoma (RCC) remains unclear, and effective prevention and therapeutic measures are lacking. BIRC6, a protein inhibitor of apoptosis, has attracted great interest. Our data indicated that overexpression of BIRC6 elevated cell growth, colony formation, migration, and invasion of cultured RCC cells, while siRNA knockdown of BIRC6 suppressed these processes. Additionally, BIRC6 was highly expressed in RCC clinical samples along with a downregulated level of Axin. Immunoprecipitation assays found that BIRC6 interacted with Axin and the two proteins colocalized within the cytoplasm of RCC cells. Overexpression of BIRC6 promoted the ubiquitination modification of Axin, while genetic knockdown of BIRC6 suppressed it. Furthermore, overexpression of BIRC6 significantly promoted the turnover of Axin, suggesting BIRC6’s inhibitory effect on Axin protein stability. BIRC6 was also upregulated in cancer stem-like cells of RCC and increased the drug resistance of RCC cells against sunitinib. Western blotting assays showed that the overexpression of BIRC6 upregulated CXCR4 protein expression and activated the β-catenin pathway. Two cell lines were then constructed with BIRC6 overexpressed by lentiviruses. Pharmacological administration of a Wnt/β-catenin inhibitor, XAV-939, or genetic knockdown of β-catenin inhibited cell growth, tumor sphere formation, colony formation, migration, and invasion of BIRC6-overexpressed cells. In vivo administration of XAV-939 markedly suppressed the tumorigenesis of BIRC6-overexpressed RCC cells in nude mice. In conclusion, we propose that BIRC6 activates the β-catenin signaling pathway via mediating the ubiquitination and degradation of Axin, promoting the growth, stemness, and drug resistance of RCC cells. This project aims to elucidate the role of BIRC6 as a potential therapeutic target and provide new insights into the clinical treatment of RCC.
... These stabilizing influences have been documented through various mechanisms. Reports have indicated that deubiquitinating enzymes, including USP7 [69], USP44 [70], USP49 [71], and UCHL5 [72], can reduce the ubiquitination of AXIN1, thereby hampering WNT/β-catenin signaling. DAB2 contributes to an increase in the protein level of AXIN1 by impeding the dephosphorylation of PP1 on AXIN1 [73,74]. ...
AXIN1, has been initially identified as a prominent antagonist within the WNT/β-catenin signaling pathway, and subsequently unveiled its integral involvement across a diverse spectrum of signaling cascades. These encompass the WNT/β-catenin, Hippo, TGFβ, AMPK, mTOR, MAPK, and antioxidant signaling pathways. The versatile engagement of AXIN1 underscores its pivotal role in the modulation of developmental biological signaling, maintenance of metabolic homeostasis, and coordination of cellular stress responses. The multifaceted functionalities of AXIN1 render it as a compelling candidate for targeted intervention in the realms of degenerative pathologies, systemic metabolic disorders, cancer therapeutics, and anti-aging strategies. This review provides an intricate exploration of the mechanisms governing mammalian AXIN1 gene expression and protein turnover since its initial discovery, while also elucidating its significance in the regulation of signaling pathways, tissue development, and carcinogenesis. Furthermore, we have introduced the innovative concept of the AXIN1-Associated Phosphokinase Complex (AAPC), where the scaffold protein AXIN1 assumes a pivotal role in orchestrating site-specific phosphorylation modifications through interactions with various phosphokinases and their respective substrates.
... Noncanonical Wnt signaling operates without involving β-catenin and is activated by Wnt ligands, which bind to a receptor complex consisting of frizzled, receptor tyrosine kinase-like orphan receptor 1/2, or receptor tyrosine kinase. The canonical pathway is based on the stability of the transcription cofactor, β-catenin [100]. Wnt signaling is activated when Wnt ligands bind to frizzled receptors along with lipoprotein receptor-related protein 5/6 (LRP5/6) [101]. ...
Over the past decade, the role of the 14–3-3 protein has received increasing interest. Seven subtypes of 14–3-3 proteins exhibit high homology; however, each subtype maintains its specificity. The 14–3-3ε protein is involved in various physiological processes, including signal transduction, cell proliferation, apoptosis, autophagy, cell cycle regulation, repolarization of cardiac action, cardiac development, intracellular electrolyte homeostasis, neurodevelopment, and innate immunity. It also plays a significant role in the development and progression of various diseases, such as cardiovascular diseases, inflammatory diseases, neurodegenerative disorders, and cancer. These immense and various involvements of 14–3-3ε in diverse processes makes it a promising target for drug development. Although extensive research has been conducted on 14–3-3 dimers, studies on 14–3-3 monomers are limited. This review aimed to provide an overview of recent reports on the molecular mechanisms involved in the regulation of binding partners by 14–3-3ε, focusing on issues that could help advance the frontiers of this field.
... USP7 can negatively regulate Wnt signaling by directly interacting with Axin through its tumor necrosis factor (TNF) receptor associated factor (TRAF) structural domain, promoting the deubiquitination and stabilization of Axin. [3] The homonymous protein encoded by the enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that acts as an episodic silencing factor during tumorigenesis and progression. Recently, EZH2 was shown to play an important role in parathyroid development by regulating the expression of T-box transcription factor 1 (TBX1) and glial cells missing transcription factor 2 (GCM2). ...
... Therefore, there is an urgent need to evaluate the impact of proteasome expression and activity in cancer as an enzymatic unit, rather than the evaluation of the role of separated subunits. Proteasome expression in cancer is associated with oncogenic signaling pathways, such as the PI3K/Akt/mTOR, NF-κB, Cyclin D1 signaling, interferon signaling and Wnt-β catenin pathways [26][27][28][29][30]. ...
Proteasomes are involved in the maintenance of cellular protein homeostasis and the control of numerous cellular pathways. Single proteasome genes or subunits have been identified as important players in cancer development and progression without considering the proteasome as a multi-subunit protease. We here conduct a comprehensive pan-cancer analysis encompassing transcriptional, epigenetic, mutational landscapes, pathway enrichments, and survival outcomes linked to the 20S proteasome core complex. The impact of proteasome gene expression on patient survival exhibits a cancer-type dependent pattern. Escalated proteasome expression associates with elevated activation of oncogenic pathways, such as DNA repair, MYC- controlled gene networks, MTORC1 signaling, oxidative phosphorylation, as well as metabolic pathways including glycolysis and fatty acid metabolism. Vice versa, potential loss of function variants correlates with improved survival. The TCGA-derived outcomes are further supported by gene expression analysis of THP-1 cells. Our study reframes these subunits as an integrated functional ensemble, rather than separated subunits.
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... Almac4 is a highly potent and selective small-molecule inhibitor of USP7 that has shown significant antitumor activity in preclinical models of cancer [15]. Almac4 is a second generation USP7 inhibitor, along with GNE-6776, XL188, XL177A, FT671, and FT827, and these second generation inhibitors are thought to have fewer off-target activities compared to earlier generation USP7 inhibitors [16][17][18]. Specific pharmacologic inhibition of USP7 would allow us to study the direct effects of USP7 on its various targets and understand the functional role of USP7 in NB. We therefore hypothesized that USP7 inhibition with Almac4 would be effective against NB cells via modulation of ubiquitination of key proteins important in NB pathogenesis. ...
Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.
... AXIN1 has a strong influence on Wnt/β-catenin signaling and is the central scaffolding protein responsible for the formation of the β-catenin destruction complex [157]. ...
The major challenges in Osteosarcoma (OS) therapy are its heterogeneity and drug resistance. The development of new therapeutic approaches to overcome the major growth mechanisms of OS is urgently needed. The search for specific molecular targets and promising innovative approaches in OS therapy, including drug delivery methods, is an urgent problem. Modern regenerative medicine focuses on harnessing the potential of mesenchymal stem cells (MSCs) because they have low immunogenicity. MSCs are important cells that have received considerable attention in cancer research. Currently, new cell-based methods for using MSCs in medicine are being actively investigated and tested, especially as carriers for chemotherapeutics, nanoparticles, and photosensitizers. However, despite the inexhaustible regenerative potential and known anticancer properties of MSCs, they may trigger the development and progression of bone tumors. A better understanding of the complex cellular and molecular mechanisms of OS pathogenesis is essential to identify novel molecular effectors involved in oncogenesis. The current review focuses on signaling pathways and miRNAs involved in the development of OS and describes the role of MSCs in oncogenesis and their potential for antitumor cell-based therapy.
... Further experiments showed that defective differentiation and mineralization occur in Otub1-null osteoblasts while their proliferation remained unchanged. Although several studies have revealed the ubiquitin-specific proteases (USPs) family regulate the function of osteoblasts or osteoclasts, [40][41][42][43] our results suggest that OTUB1 is a positive regulator of osteogenesis, which is a previously unidentified important physiological function in the OTU deubiquitinase family. ...
Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Dysregulation of this process leads to multiple diseases, including osteoporosis. However, the underlying molecular mechanisms are not fully understood. Here, we show that the global and conditional osteoblast knockout of a deubiquitinase Otub1 result in low bone mass and poor bone strength due to defects in osteogenic differentiation and mineralization. Mechanistically, the stability of FGFR2, a crucial regulator of osteogenesis, is maintained by OTUB1. OTUB1 attenuates the E3 ligase SMURF1-mediated FGFR2 ubiquitination by inhibiting SMURF1’s E2 binding. In the absence of OTUB1, FGFR2 is ubiquitinated excessively by SMURF1, followed by lysosomal degradation. Consistently, adeno-associated virus serotype 9 (AAV9)-delivered FGFR2 in knee joints rescued the bone mass loss in osteoblast-specific Otub1 -deleted mice. Moreover, Otub1 mRNA level was significantly downregulated in bones from osteoporotic mice, and restoring OTUB1 levels through an AAV9-delivered system in ovariectomy-induced osteoporotic mice attenuated osteopenia. Taken together, our results suggest that OTUB1 positively regulates osteogenic differentiation and mineralization in bone homeostasis by controlling FGFR2 stability, which provides an optical therapeutic strategy to alleviate osteoporosis.
