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Abstract

Ubiquitin-specific protease 5 (USP5), a member of the ubiquitin-specific proteases (USPs) family, functions by specifically removing ubiquitin chains from target proteins for stabilization and degrading unbound polyubiquitin chains to maintain a steady-state monoubiquitin pool. Ubiquitin-specific protease 5 regulates various cellular activities, including DNA double-strand break repair, transmission of neuropathic and inflammatory pain signals, immune response, and tumor cell proliferation. Furthermore, USP5 is involved in the development of multiple tumors such as liver, lung, pancreatic, and breast cancers as well as melanoma. Downstream regulatory mechanisms associated with USP5 are complex and diverse. Ubiquitin-specific protease 5 has been revealed as an emerging target for tumor treatment. This study has introduced some molecules upstream to control the expression of USP5 at the levels of transcription, translation, and post-translation. Furthermore, the study incorporated inhibitors known to be associated with USP5, including partially selective deubiquitinase (DUB) inhibitors such as WP1130, EOAI3402143, vialinin A, and chalcone derivatives. It also included the ubiquitin-activating enzyme E1 inhibitor, PYR-41. These small molecule inhibitors impact the occurrence and development of various tumors. Therefore, this article comprehensively reviews the pivotal role of USP5 in different signaling pathways during tumor progression and resumes the progress made in developing USP5 inhibitors, providing a theoretical foundation for their clinical translation.

Keywords

Ubiquitin-specific protease 5 ubiquitination deubiquitination tumor inhibitor

Introduction

Ubiquitination, a crucial post-translational protein modification in tumor cells, regulates various physiological functions and pathological processes such as cell cycle progression, signal transduction, and carcinogenesis.^1,2 Ubiquitination primarily involves three classes of enzymes: ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3.³ The assembly of ubiquitin into mixed chains, including seven lysine (Lys) residues and N-terminal methionine (Met1) determines substrate protein fate, including degradation, specific interactions, transport, and activity regulation. Ubiquitination, along with its counterpart process—deubiquitination—establishes a dynamic equilibrium, maintaining cellular homeostasis by governing protein degradation and stability.⁴ Approximately 100 deubiquitinating enzymes in the human genome fall into seven homologous families based on differences in amino acid sequence and structure: ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), Otubain proteases (OTUs), Machado-Joseph disease proteases (MJDs), MINDYs protease family, ZUP1 protease family, and AB1/MPN/Mov34 metalloenzymes (JAMMs).^5-9 Except for JAMMs, which are metalloproteases, all other deubiquitinases belong to the cysteine protease family.¹⁰

The functions of deubiquitinases in the human body can be categorized into three groups: removing monoubiquitin from substrate proteins, eliminating polyubiquitin chains from post-translationally modified protein substrates, resulting in a reversal of ubiquitin signaling, and altering ubiquitin modification by trimming ubiquitin chains.¹¹ Ubiquitin-specific protease 5 (USP5), also known as isopeptidase T (ISOT), is a cysteine deubiquitinating enzyme in the USP family.¹²

Increasing evidence indicates the involvement of USP5 in various cellular processes such as DNA repair,¹³ stress response,¹⁴ and inflammation.¹⁵ Moreover, it is closely associated with tumor occurrence and development,¹⁶ promoting the biological characteristics of multiple tumors, including drug resistance. This review focuses on elucidating the structure and function of USP5 in cancer, exploring its targeted inhibitors, and discussing their therapeutic potential.

Landscape of USP5

Ubiquitin-specific protease 5 was initially identified in 1995¹² and subsequently recognized as a functional homolog of USP14 in 1997.¹⁷ This gene generates three isoforms of USP5: ISOT-S (initially referred to as ISOT, later renamed USP5), ISOT-L, and ISOT-3. The ISOT-S and ISOT-L exhibit a different splicing pattern of the 15th exon. The ISOT-L, unlike ISOT-S, presents a deletion of 23 amino acids in its ubiquitin-binding domain.¹⁸ Ubiquitin-specific protease 5 is highly conserved between humans and mice, with a similarity of 98.7%. In humans, it has a molecular weight of 93.3 kDa.¹⁹ Its encoding gene is located on chromosome 12p13, near the human CD4 gene.²⁰

Unlike most deubiquitinases that exhibit substrate-dependent enzymatic activity, USP5 uniquely removes ubiquitin molecules directly from free polyubiquitin chains, stabilizing the free ubiquitin pool. Extensive studies confirm the ability of USP5 to cleave five types of linkages: K6, K29, K48, K63, and linear linkages. Notably, USP5 exhibits a higher affinity for K48-linked polyubiquitin chains and a lower affinity for linear ubiquitin chains and those linked to K63.^19,21

Ubiquitin-specific protease 5 is a multi-domain enzyme with 835 residues.²² Ubiquitin-specific protease 5 contains at least four ubiquitin binding sites^4,22,23 (S1’, S1, S2, and S3). The S1’ site binds to the zinc finger ubiquitin-specific protease (ZnF-UBP) domain (residues 173-283), the S1 site binds to ubiquitin—specific processing protease (USP), the S2 and S3 sites bind to two ubiquitin-associated (UBA) domains (residues 634-684 and 698-749)²⁴ (see Figure 1). The ZnF-UBP domain is involved in substrate affinity and specificity.²² The ZnF-UBP domain recognizes ubiquitin, binding to its di-glycine motifs and facilitating proximal cleavage. The USP domain does not directly bind to ubiquitin but enhances the catalytic activity of USP5 through conformational effects. The UBA domains interact with distal ubiquitin via the S2 and S3 sites. These domains, connected through flexible peptide loops, enable USP5 to cleave various types of polyubiquitin chains.^24,26,27

Figure 1.

Schematic representation of the USP5 domains.

The cBioPortal online platform ((https://www.cbioportal.org/), utilizing The Cancer Genome Atlas (TCGA) data, demonstrated the genetic mutations of USP5 across various types of cancer. The top five most frequent alterations in the USP5 gene were found in uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS), ovarian serous cystadenocarcinoma (OV), skin cutaneous melanoma (SKCM), and testicular germ cell tumors (TGCT). The most common forms of genetic change detected in the USP5 gene were mutation, amplification, and deep deletion. In UCEC, USP5 had the highest frequency of USP5 gene mutation (6.05%), and in UCS, USP5 had the highest frequency of gene amplification (7.02%). The specific mutations can be shown in Figure 2.

Figure 2.

USP5 genetic alteration in TCGA PanCancer Atlas (https://www.cbioportal.org/).

Multiple molecules located upstream directly or indirectly control the expression of USP5. The E2F1 acts as a transcription factor that positively regulates the expression of USP5 in mesenchymal glioma stem cells (MES GSCs) at the transcriptional level, as determined by dual luciferase reporter gene analysis.²⁸ Ubiquitin-specific protease 5 is targeted and negatively regulated by miR-1256.²⁹ MiR-23a-3p specifically acts on USP5, leading to decreased expression and perhaps enhancing the relief of inflammatory pain via modulating the HDAC2/NRF2 pathway.³⁰ At the translational level, METTL5 promotes USP5 translation in an m6A-dependent manner.³¹ In addition, post-translational modifications, including ubiquitination and SUMOylation, also have an impact on USP5. Smurf1, a HECT-type E3 ligase, facilitates the ubiquitination of USP5 and suppresses the production of Tumor Necrosis Factor-alpha (TNF-α).³² The interaction between USP5 and Cav3.2 calcium channels can be influenced by the SUMOylation state of USP5. When the SUMOylation of USP5 is decreased, there is an increase in the interactions between USP5 and Cav3.2 calcium channels.³³ The KRas indirectly regulates USP5 activity by upregulating cellular ROS levels, leading to the formation of USP5 homodimers, enhanced USP5 enzymatic activity, and the accumulation of USP5 protein.³⁴ Long non-coding RNAs (LncRNAs) interact with specific proteins to regulate downstream effectors. Ubiquitin-specific protease 5 is the specific RNA-binding protein (RBP) for LOC85009. It inhibits USF1 ubiquitination, while LOC85009 sequesters USP5, causing the USF1 protein to destabilize.³⁵ The LNC01468 had a high affinity for USP5, attracting it to deubiquitylate PAI1 and inhibit its degradation.³⁶ The LncRNA MAFG-AS1 functions to stabilize Hu antigen R by recruiting USP5, thereby preventing the degradation process.³⁷

Underlying Mechanism Associated to USP5 in Human Cancer

Based on the TCGA Pan-Cancer data from the UCSC database (https://xenabrowser.net/), aberrant mRNA expression was observed in 20 tumors, with 14 demonstrating upregulation and 6 showing downregulation compared with normal tissues (see Figure 3). The role of USP5 in tumors is underscored by a study by Meyers et al.³⁸ In a whole-genome CRISPR-Cas9 knockout screening of 804 tumor cell lines across 28 cancer types, USP36 and USP5 emerged as the most crucial tumor-related genes within the USPs family. Ubiquitin-specific protease 5 regulates the stability of downstream proteins such as p53,²¹ FoxM1,³⁹ β-catenin,⁴⁰ HDAC2,³⁰ and TUFM⁴¹ (see Figure 4), promoting or inhibiting effects on cancer development across various tumors. Research indicates that USP5 promotes the occurrence and progression of liver, lung, breast, ovarian, and colorectal cancers^41-48 (see Figure 3), while simultaneously exhibiting inhibitory effects on melanoma tumor growth.^14,49

Figure 3.

USP5 is aberrantly expressed in different tumors.

Figure 4.

The role of USP5 in tumors (by Figdraw2.0).

The role of USP5 in the proliferation of cancer

Lung cancer cells exhibit rapid growth and high proliferation rates, which are closely related to short cell cycle intervals.⁵⁰ D-type cyclins, including cyclin D1 (CCND1), cyclin D2 (CCND2), and cyclin D3 (CCND3), crucially interact with cyclin-dependent kinases 4/6 (CDK4/6) in lung cancer, activating them.^51-53 In humans, these cyclins are encoded by the cyclin D geneIn lung cancer, elevated levels of CCND1 promote carcinogenesis, cell cycle progression, metastasis, and drug resistance.⁵⁴ The USP5 binds to CCND1, reducing its polyubiquitination, stabilizing it, and prolonging its half-life. This promotes non-small cell lung cancer (NSCLC) cell proliferation and colony formation.⁵⁵ Thus, inhibiting USP5 expression can lead to CCND1 degradation in NSCLC cells and cell cycle arrest. In lung cancer cells, epithelial–mesenchymal transition (EMT) causes loss of intercellular adhesion and cellular polarity, increases the expression of mesenchymal markers and migration ability, induces resistance to apoptosis, and promotes the production of extracellular matrix components. Ubiquitin-specific protease 5 induces EMT through the glycogen synthase kinase 3-mediated activation of the Wingless/integrated/β-catenin pathway.⁴⁰ Hepatocellular carcinoma (HCC) is the most prevalent malignant liver tumor. p53 is a classic regulator of cell proliferation and has been associated with carcinogenesis,⁵⁶ modulating cell cycle arrest and apoptosis. Both p53 and its inhibitor Mdm2 undergo ubiquitination and proteasomal degradation.⁵⁷ In general, deubiquitinases regulate the stability of p53 by removing ubiquitin from substrate proteins or stabilizing E3 ligases. Ubiquitin-specific protease 5 removes single ubiquitin chains from unanchored substrates with polyubiquitin chains at their proximal ends. Inhibiting USP5 expression allows polyubiquitin chains to competitively bind to proteasomes, preventing p53 degradation.²¹ In addition, inhibition of USP5 activates the p14 ARF/p53 signaling pathway in HCC cells, indicating USP5 as a potential therapeutic target for HCC treatment.⁴⁵

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive human malignant tumor, with a 5-year overall survival rate of only 6%.⁵⁸ FoxM1 belongs to the Forkhead transcription factor superfamily, promoting pancreatic cancer cell proliferation and progression.³⁹ In addition, USP5 expression is significantly upregulated within pancreatic cancer cell lines, enhancing FoxM1 stability and extending its half-life.⁴⁸ Treatment with MG-132, a proteasome inhibitor, restores FoxM1 expression levels. Furthermore, inhibiting USP5 expression accelerates the FoxM1 degradation rate, ultimately slowing cellular proliferation in pancreatic cancer cell lines and growth in xenograft tumor mouse models.

Hypoxia-inducible factor 2α (HIF2α) is pivotal in breast tumor growth and metastasis.⁵⁹ Ubiquitin-specific protease 5 interacts with HIF2α within breast tumors, stabilizing and preventing its ubiquitin-proteasome degradation. Consequently, this stabilization promotes the transcription of HIF2α target genes such as SLC2A1, PLOD2, P4HA1, and VEGFA.⁴⁶ Suppressing USP5 expression attenuates breast cancer cell proliferation, colony formation. Targeting USP5, by enhancing the stability of HIF2α may serve as a promising therapeutic strategy against breast cancer.

Epithelial ovarian cancer (EOC) is a prevalent gynecological malignancy with a typically unfavorable prognosis.⁴³ The USP5 expression is upregulated in ovarian cancer, exhibiting a negative correlation with the prognosis of patients with serous ovarian carcinoma. An isoform of histone deacetylase 2 (HDAC2), serves as a cellular adaptive factor governing cell cycle progression in vitro and metastasis in vivo.⁶⁰ Suppressing USP5 expression reduces post-translational HDAC2 levels, enhancing the transcriptional activity of p27,⁶¹ a crucial cell cycle inhibitor. This induces G0/G1 phase arrest in ovarian cancer cells and inhibits xenograft growth in nude mice. These findings suggest that USP5 promotes ovarian cancer and may be involved in its regulatory mechanisms.

The role of USP5 in the migration of cancer cells

Hepatocellular carcinoma is closely linked to EMT.^62,63 Epithelial–mesenchymal transition enables tumor cells to acquire an invasive mesenchymal phenotype, enhancing their migratory capacity. Consequently, inhibiting EMT represents a crucial strategy for suppressing tumor metastasis. In HCC cells, high USP5 expression levels are significantly associated with tumor malignancy and pathological grading.⁴⁷ The zinc finger transcription factor SLUG inhibits E-cadherin transcription by binding to E-box elements in the proximal promoter region during the EMT.^64-67 Aberrant SLUG expression, observed in various cancers, regulates tumor cell invasion and metastasis.⁶⁸ In liver cancer cells, the overexpression of SLUG indicates the presence of a stabilizing factor.⁴⁷ Through pull-down and mass spectrometry analysis, an interaction between USP5 and SLUG has been identified in liver cancer cells, inhibiting SLUG ubiquitination and stabilizing it. Knocking down USP5 increases the degradation of SLUG in HCC, suppressing EMT and liver cancer cell metastasis. Furthermore, inhibiting USP5 expression causes cell cycle arrest at the G1 phase, inhibiting tumor growth in liver cancer cells.⁴⁵

As an oncogene, WT1 plays a pivotal role in regulating downstream proteins including cyclin D1, B-cell lymphoma 2 (Bcl-2), and E-cadherin, enhancing pancreatic cancer cell proliferation, inhibiting cell apoptosis, and promoting cancer cell metastasis.^69-71 Overexpression of USP5 was shown to deubiquitinate WT1 protein and stabilize its expression in vivo. This stabilization reduces E-cadherin expression while promoting pancreatic cancer metastasis.⁷² Moreover, USP5 also activates the STAT3 signaling pathway, facilitating the occurrence and progression of pancreatic cancer.⁴² Thus, targeting USP5 emerges as a potential therapeutic strategy for treating pancreatic cancer.

The role of USP5 in patients’ prognosis

The rate of recurrence and clinical staging determines the prognosis of cancer. Elevated levels of USP5 expression have been identified as a negative prognostic indicator for pancreatic cancer, leading to a reduced overall survival time in patients.⁴⁸ The study revealed that USP5 interacts with c-Maf, protecting it from degradation by decreasing ubiquitination. This interaction eventually results in the extended life of multiple myeloma cells and accelerates the course of the disease.⁷³ Moreover, it has been demonstrated that USP5 promotes bladder urothelial carcinoma growth, which is associated with a poor prognosis for patients.³⁷ Ubiquitin-specific protease 5 also exhibits high expression in NSCLC and is correlated with overall survival and metastasis.⁷⁴ The LncRNA LINC01468 plays a role in the progression of lung adenocarcinoma (LUAD). It interacts with USP5 to inhibit ubiquitination-mediated degradation of the plasminogen activator inhibitor-1 (PAI1) protein, thereby stabilizing its expression in lung cancer. This suggests that USP5 may provide new insights into the mechanisms driving the progression of lung cancer.³⁶

The role of USP5 in immunity of cancer

Programmed cell death ligand 1 (PD-L1; CD274) is a widely recognized immune inhibitory molecule that enables cancer cells to evade immune surveillance and serves as a key target for cancer immunotherapy.^75,76 T-cell immunity selectively recognizes and eliminates pathogens and abnormal cells, including cancer cells.⁷⁵ The interaction between PD-L1 and PD-1 leads to the dephosphorylation of the T-cell receptor.⁷⁷ PD-L1 is a crucial immune checkpoint protein that hinders the ability of T-cells to eliminate cancer cells by diminishing their proliferation and activity. Elevated levels of PD-L1 protein expression have been detected in many cancer types, resulting in the facilitation of immune evasion in cancer cells.⁷⁸ Antibodies that specifically target PD-L1 have significantly transformed the approach to treating advanced-stage malignancies, including melanoma, lung cancer, breast cancer, kidney cancer, and other types of cancer.⁷⁹ Ubiquitin-specific protease 5 directly interacts with PD-L1 and removes its polyubiquitin chains, increasing its stability, preventing its degradation, and increasing its protein level.⁴⁴ In NSCLC tissues, the expression of USP5 is elevated and positively correlates with the level of PD-L1, a correlation closely associated with poor prognosis. Knockdown of USP5 delayed tumor growth in a mouse model of lung cancer. Notably, two isoforms of USP5 have been identified in NSCLC, with the shorter form playing a more significant role in regulating the stability of PD-L1, promoting tumor immune evasion, and exerting tumor regulatory effects.

The PD-1 functions as a receptor in colorectal cancer, suppressing T-cells that contribute to the promotion of immune evasion in cancer. Ubiquitin-specific protease 5 removes ubiquitin molecules from PD-1, leading to its stabilization. The ERK phosphorylates PD-1, which enhances the interaction between USP5 and PD-1. The elimination of USP5 in T-cells under specific conditions boosts the production of effector cytokines and hinders the formation of colorectal tumors.⁸⁰

The role of USP5 in the resistance of cancer to therapy

Doxorubicin is a common treatment for colorectal tumors,^81,82 inhibiting DNA replication and damaging DNA to exert an anti-tumor effect. It significantly inhibits the luciferase activity driven by the USP5 promoter and reduces the transcription level of USP5 in colorectal tumor cells.⁴¹ Tu translation elongation factor, mitochondrial (TUFM), a widely expressed mitochondrial protein,⁸³ plays a crucial role in promoting colorectal adenoma progression to cancerous tissue.⁸⁴ Mass spectrometry reveals that USP5 deubiquitinates TUFM at the K48 site, maintaining its stability. This process is regulated by its upstream transcription factor early B-cell factor-1 (EBF1). Furthermore, EBF1 overexpression leads to elevated levels of both USP5 and TUFM while reducing the sensitivity of colorectal cancer cells to doxorubicin. Henceforth, targeting the EBF1/USP5/TUFM axis could be an effective strategy for mitigating resistance in colorectal cancer. In addition, USP5 functions as the deubiquitinase for PD-1. The combined administration of the USP5 inhibitor EOAI3402143 with either anti-CTLA-4 or trametinib has a synergistic effect in inhibiting the growth of mice colorectal cancers.⁸⁰

Small Molecule Inhibitors of USP5

As a deubiquitinase, USP5 cleaves ubiquitin from substrate proteins and unanchored polyubiquitin chains at specific sites, including Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63. Due to its abnormal activation or expression in various malignant tumors or tumor microenvironments and its role in regulating the stability of downstream oncogenic proteins, targeting USP5 inhibition emerges as a promising strategy for cancer therapy.⁸⁵ An α, β-unsaturated ketone with a sterically accessible β-carbon is a crucial molecular factor in conferring partial selectivity as a DUB inhibitor and determining its inhibitory activity.⁸⁶ When the thiol group of cysteine in DUB attacks the α, β-unsaturated carbonyl group of the DUB inhibitor by a nucleophilic addition reaction, it forms a covalent complex. This complex leads to an increase in the accumulation of polyubiquitin chains and a reduction in the degradation rate. The monomeric ubiquitin pool decreases, and individual DUB activity overall decreases, thereby affecting the cellular level and activity of cancer proteins regulated by DUB.⁸⁷ Ubiquitin-specific protease 5 inhibitors with a similar structure include WP1130,⁸⁷ EOAI3402143,⁸⁸ PYR-41,⁸⁹ and chalcone-based derivatives,⁹⁰ which are discussed below and summarized in Table 1. The information related to USP5 inhibitors, including chemical structures, PubChem CID, molecular formula and IC50 values, was presented in Figure 5. Consequently, USP5 represents an ideal candidate for anticancer treatment. Developing inhibitors specifically targeting USP5 could represent a novel effective approach to tumor management.

Table 1.

Small molecule inhibitors of USP5 and related diseases.

Compounds	Reported targets	Diseases
WP1130 (Degrasyn)	USP5, UCH37, UCH-L1, USP9X, USP14	Renal cancer, lung cancer, hepatocellular cancer, chronic myelogenous leukemia,⁹¹ pancreatic ductal adenocarcinoma,⁶⁹ melanoma⁹²
EOAI3402143 (G9)	USP9X, USP24, USP5	Melanoma,⁹³ non-small cell lung cancer⁵⁵
PYR-41	USP5, UBE1	Mantle cell lymphoma cancer⁹⁴
Vialinin A	USP5	Colonic cancer, hepatic carcinoma⁹⁵
Chalcone derivatives (AM146, RA-9, RA-14)	UCH-L1, UCH-L3, USP2, USP5, USP8	Breast cancer, ovarian cancer and cervical cancer⁹⁰

UBE, ubiquitin-activating enzyme; UCH, ubiquitin C-terminal hydrolase; USP, ubiquitin-specific protease.

Figure 5.

Chemical structures of USP5 inhibitors.

WP1130 (degrasyn)

WP1130, a second-generation tyrphostin derivative (degrasyn), was discovered through screening for AG490-like molecules that inhibit the activation of Stat molecules by interleukin 6 (IL-6) and IL-3.⁸⁷ WP1130 has superior effectiveness in inducing apoptosis in both myeloid and lymphoid tumor cells, with an IC50 range of around 0.5 to 2.5 μM.⁹¹ Mantle cell lymphoma Z138 cells displayed the most apoptotic sensitivity to WP1130, with an IC50 of approximately 1 μM.⁹⁶ Unlike MG132, which directly inhibits the proteasome, WP1130 rapidly induces the accumulation of polyubiquitinated target proteins, leading to apoptosis in tumor cells.⁹⁷ It selectively inhibits deubiquitinases responsible for the removal of K48- and K63-specific ubiquitin chains, including USP5, UCH37, UCH-L1, USP9X,⁹⁸ and USP14.⁸⁷ The molecular structure of WP1130 contains a cross-conjugated α, β-unsaturated dienone with two sterically accessible electrophilic β-carbons.⁸⁶ The α, β-unsaturated carbonyl group in WP1130 can create covalent adducts with the cystine in the active site of USP5, which can hinder its deubiquitinating activity.⁸⁷ Consequently, inhibition of USP5 leads to an accumulation of unanchored polyubiquitin chains within cells and a reduction in monomeric ubiquitin, increasing p53 levels in a dose-dependent manner and suppressing tumor formation.²¹

WP1130 effectively inhibits tumor development in mouse models with xenograft melanoma and chronic myeloid leukemia tumor cells.⁹¹ Furthermore, derivatives of WP1130 with reduced activity, such as WP1066 and WP1034, exhibit anti-proliferative and pro-apoptotic effects in diseases such as chronic myelogenous leukemia,⁹¹ glioblastoma multiforme,⁹⁹ and myeloproliferative disorders.¹⁰⁰ It hinders the spread of PDAC by blocking the USP5-induced production of WT1 and E-cadherin, which are responsible for metastasis. Moreover, degrasyn hampers the occurrence and advancement of melanoma and leukemia by degrading c-Myc and BCR-ABL via ubiquitin-mediated pathways.^92,101

EOAI3402143(G9)

EOAI3402143(G9) was selected as a small molecule inhibitor from more than 220 compounds that retained the inhibitory activity of WP1130 against Usp9x and improved its solubility. G9 was better than WP1130 in USP9x catalytic activity, water-soluble activity, and cell USP9x inhibitory activity.⁸⁸ G9 inhibits USP9x, USP24, and USP5. The IC50 of G9 is reported to be 1 μM.⁹³ G9 inhibits the function of Usp9x and Usp24 by creating a covalent, gradually reversible connection with cysteine residues.⁸⁸ However, the mechanism by which G9 inhibits USP5 has yet to be identified. Moreover, clinical trials for G9 have not yet been conducted.

G9 suppresses USP9X, leading to aggresomal translocation, ultimately triggering apoptosis in FLT3-ITD-positive acute myeloid leukemia (AML) cells¹⁰² and Ruxolitinib-resistant JAK2-V617F-positive leukemic cells.¹⁰³ G9 exhibited a dose-dependent inhibition of Usp9x and Usp24 activity. It also induced apoptosis in tumor cells and led to either full regression or suppression of myeloma tumors.⁸⁸ When G9 is utilized, similar effects are observed as when USP5 is knocked down, leading to the induction of FAS and sensitization to apoptosis. This ultimately results in significant inhibition of melanoma tumor growth.⁹³ G9 inhibits the function of USP5, resulting in reduced production of the CCND1 protein and eventually suppressing the proliferation of NSCLC cells in vitro and limiting the formation of tumors in vivo.⁵⁵

PYR-41

Ubiquitin-activating enzyme 1 (UBE1) plays a pivotal role in the regulation of the ubiquitination cycle. Its inhibitor PYR-41 is an effective blocker of UBE1-mediated ubiquitination reactions, exhibiting significant inhibitory effects on USP5.⁹⁴ The IC50 of PYR-41 is reported to be less than 10 μM.⁸⁹ PYR-41 possesses an α, β-unsaturated enone functional group that has a readily available β-carbon. The α, β-unsaturated enones exhibit a rapid reaction with sulfhydryl groups via a Michael addition reaction. The suppression of both UBE1 and USP5 was achieved by PYR-41-induced covalent protein cross-linking, which was accompanied by the inhibition of the enzymatic activity of the target proteins.⁹⁴ Elevated levels of PYR-41 in lymphoma tumor cells downregulate the expression of USP5 by inducing protein crosslinking into W structures instead of degradation pathways. This selective protein crosslinking may impact signal transduction and ubiquitin cycling, modulating protein expression. PYR-41-related compounds have demonstrated promising anti-tumor activity in animal models of prostate¹⁰⁴ and breast cancers,¹⁰⁵ among others, providing potential alternative strategies for cancer treatment.

Vialinin A

Vialinin A, a small molecule compound extracted and isolated from the Chinese fungus Ganoderma lucidum, possesses antioxidant properties.¹⁰⁶ Vialinin A displayed competitive binding with Ub at the S1 and/or S1′ sites of USP5, with an IC50 value of 5.9 μM. It exhibited the highest efficacy in the competition. However, further research is needed to investigate the mechanism and binding characteristics of vialinin A with USP5.¹⁰⁷ Vialinin A effectively regulates the production of TNF-α and prevents the progression of rat eosinophilic leukemia.¹⁰⁸ Furthermore, vialinin A dose-dependently inhibits vascular endothelial growth factor expression, antagonizing human umbilical vein endothelial cell-mediated neovascularization.¹⁰⁹ Ultimately, it exerts anticancer effects through its antioxidant and anti-angiogenic actions. Toshio Norikura et al⁹⁵ were the first to discover that vialinin A reduces the proliferative activity of human colon tumor and liver cancer cells.

Chalcones derivatives

Chalcones serve as precursors for flavonoids and isoflavonoids, which are common chemical structures found in many naturally occurring substances.¹¹⁰ A multitude of chalcone derivatives were synthesized due to their simple synthesis procedure. The chalcone-based derivatives, such as RA-14, AM146, and RA-9, from the “RA” and “AM” series of compounds exhibit partially selective inhibitory activity against DUB. These small molecules possess a reactive α, β-unsaturated carbonyl group that may undergo a Michael addition reaction with the sulfhydryl of cysteines at the active sites in DUB, resulting in the formation of covalent compounds. This leads to the rapid accumulation of polyubiquitinated proteins and a reduction in the available free ubiquitin. These chalcone derivatives effectively inhibit the activity of DUB, such as USP5, UCH-L3, USP2, UCH-L1, and USP8, leading to irreversible cell cycle arrest in breast, ovarian, and cervical cancer cells (IC50 = 1.5-12.5 µM), as well as inhibiting their proliferation and initiating apoptosis.⁹⁰

Discussion

With the advancement of research, the understanding of USP5 has been progressively elucidated. Ubiquitin-specific protease 5 plays a crucial role in the onset and progression of malignancies and has become a prospective target for therapy. However, there are inherent difficulties that must be resolved in the development of USP5 inhibitors. First, these inhibitors do not specifically inhibit the expression of USP5, complicating their effects on different types of tumors.²¹,^111-113 Second, since USP5 is involved in various in vivo physiological activities such as DNA repair¹¹⁴ and normal immune responses,¹¹⁵ its inhibitors may cause certain side effects during tumor treatment. Therefore, it is crucial to comprehensively understand the role of USP5 in different tumors, explore specific regulatory mechanisms, and assess the effects of USP5 inhibitors through animal experiments.

Targeted gene therapy is a promising way of cancer therapy. A galactose-decorated lipopolyplex (Gal-SLP) is developed to co-delivery sorafenib and USP22 shRNA for synergetic hepatocellular carcinoma (HCC) therapy.¹¹⁶ DeUBiquitinase-TArgeting Chimeras (DUBTACs) has been revealed as a promising approach against cancer. It facilitates the recruitment of a deubiquitinase to prevent the degradation of the target protein by the proteasome, thereby stabilizing the target protein.¹¹⁷ Deubiquitinating enzymes that are specifically targeted can be found in two different kinds of binding: covalently bound and non-covalently bound. The ligand EN523 forms a covalent bond with OTUB1 by selectively interacting with the non-catalytic allosteric cysteine C23 in OTUB1. This interaction synergistically combines with lumacaftor to stabilize the ΔF508-CFTR protein and increase chloride channel conductance in human cystic fibrosis bronchial epithelial cells.¹¹⁸ USP7 inhibitors interact with USP7 as the ligand in a non-covalent manner, resulting in the formation of DUBTAC. DUBTAC, a compound that binds to the CFTR ligand Lumacaftor, effectively stabilizes the ΔF508-CFTR mutant protein in human cystic fibrosis bronchial epithelial cells (CFBE41o-4.7 ΔF508-CFTR). Its efficiency is equivalent to that of OTUB1-based CFTR DUBTAC NJH-2-057.¹¹⁹ USP5 plays a significant role in the development of the majority of malignancies, indicating that it might be a reliable target for treating tumors. Thus, further investigation of USP5 in DUBTAC is needed.

Conclusions

In conclusion, this review provides a comprehensive examination of USP5, shedding light on its functional domains and diverse roles in various cancers. Ubiquitin-specific protease 5 influences processes like proliferation, apoptosis, autophagy, and migration of tumor cells. In the past decade, research on deubiquitinases has advanced, presenting them as promising targets for tumor treatment. The current investigation of small-molecule inhibitors, such as WP1130, PYR-41, and vialinin A, has revealed the possibility of targeting USP5 and shown interesting opportunities for treating tumors.

Footnotes

Acknowledgements

We thank Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University and the Health Commission of Hubei Province (WJ2023M175) for support and encouragement. And we also thank the drawing tools provided by Figdraw ().

Author Contributions

QH and YJ conceived and designed this topic, JW and SF drafted the article. All authors read and approved the final article.

Declaration of conflicting interests:

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding:

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Health Commission of Hubei Province (WJ2023M175).

Availability of Data and Materials

Main data are shown in this article, and additional data about this study could be obtained from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate

Not applicable.

ORCID iDs

Shizhen Fang

Qingquan Hua

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Unraveling the Mechanism of Action of Ubiquitin-Specific Protease 5 and Its Inhibitors in Tumors

Abstract

Keywords

Introduction

Landscape of USP5

Underlying Mechanism Associated to USP5 in Human Cancer

The role of USP5 in the proliferation of cancer

The role of USP5 in the migration of cancer cells

The role of USP5 in patients’ prognosis

The role of USP5 in immunity of cancer

The role of USP5 in the resistance of cancer to therapy

Small Molecule Inhibitors of USP5

WP1130 (degrasyn)

EOAI3402143(G9)

PYR-41

Vialinin A

Chalcones derivatives

Discussion

Conclusions

Footnotes

Acknowledgements

Author Contributions

Declaration of conflicting interests:

Funding:

Availability of Data and Materials

Ethics Approval and Consent to Participate

ORCID iDs

References