Introduction

Oncology Reports

1021-335X 1791-2431

D.A. Spandidos

10.3892/or.2023.8613

OR-50-4-08613

Review

Targeting the deubiquitinase USP2 for malignant tumor therapy (Review)

Zhang

Shilong

1 * Guo

1 * Zhang

Shenjie

1 * Wang

Zhi

1 Zhang

Yewei

1 Zuo

Shi

1 2

1Department of Hepatobiliary Surgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China 2Precision Medicine Research Institute of Guizhou, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China

Correspondence to: Dr Shi Zuo, Department of Hepatobiliary Surgery, The Affiliated Hospital of Guizhou Medical University, 28 Guiyi Street, Guiyang, Guizhou 550004, P.R. China, E-mail: drzuoshi@gmc.edu.cn *

Contributed equally

10 2023

07 08 2023

50 4

176

14032023 24072023

2023

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

The ubiquitin-proteasome system is a major degradation pathway for >80% of proteins in vivo. Deubiquitylases, which remove ubiquitinated tags to stabilize substrate proteins, are important components involved in regulating the degradation of ubiquitinated proteins. In addition, they serve multiple roles in tumor development by participating in physiological processes such as protein metabolism, cell cycle regulation, DNA damage repair and gene transcription. The present review systematically summarized the role of ubiquitin-specific protease 2 (USP2) in malignant tumors and the specific molecular mechanisms underlying the involvement of USP2 in tumor-associated pathways. USP2 reverses ubiquitin-mediated degradation of proteins and is involved in aberrant proliferation, migration, invasion, apoptosis and drug resistance of tumors. Additionally, the present review summarized studies reporting on the use of USP2 as a therapeutic target for malignancies such as breast, liver, ovarian, colorectal, bladder and prostate cancers and glioblastoma and highlights the current status of pharmacological research on USP2. The clinical significance of USP2 as a therapeutic target for malignant tumors warrants further investigation.

ubiquitin-proteasome system deubiquitination ubiquitin-specific protease 2 malignant tumors targeted therapy

The National Natural Science Foundation of China

82260535

The 2022 National Foundation incubation Program of the Affiliated Hospital of Guizhou Medical University

gyfynsfc-2022-7

The present study was funded by The National Natural Science Foundation of China (grant no. 82260535) and The 2022 National Foundation incubation Program of the Affiliated Hospital of Guizhou Medical University (grant no. gyfynsfc-2022-7).

1. Introduction

Proteins, the material basis of life, are essential components of all cells, tissues and organs in the body. Intracellular proteins are predominantly degraded through the lysosomal pathway, cysteine-containing aspartate protease pathway and ubiquitin-proteasome pathway (1–4). The ubiquitin-proteasome system is the primary protein degradation pathway in vivo. More than 80% of proteins in the body are degraded through this pathway, which is involved in various metabolic processes in the body (4). The ubiquitin-proteasome pathway can degrade the cell cycle protein cyclin (5,6), spindle-related proteins (7), cell surface receptors such as epidermal growth factor (8), transcription factors (9), the tumor inhibitory factor p53 and oncogenic products (10). In addition, abnormal intracellular proteins are degraded by the ubiquitin-proteasome pathway under stress conditions. Ubiquitin, ubiquitin-activating (E1) enzymes, ubiquitin-binding (E2) enzymes, ubiquitin ligases (E3), protein hydrolases and deubiquitinating enzymes are the main components of the ubiquitin-proteasome pathway (9,11). In the presence of ATP, the glycine (Gly) residue at the C-terminus of ubiquitin forms a high-energy lipid bond with the sulfur group (SH) of the cysteine residue of an E1 enzyme, and the activated ubiquitin is subsequently transferred to an E2 enzyme. In the presence of an E3 ubiquitin ligase, ubiquitin is transferred from the E2 enzyme to the substrate protein, forming an isopeptide bond with the ε-NH2 group of the Lys residue of the substrate protein. Subsequently, the C-terminus of the next ubiquitin molecule is connected to the Lys48 residue of the previous ubiquitin molecule, thus completing polyubiquitination. The ubiquitinated substrate proteins are recognized by cap-shaped regulatory particles of the 19S proteasome and transported into the cylindrical core of 20S, where they are hydrolyzed into oligopeptides and amino acids by various enzymes and are eventually released from the proteasome, thereby completing ubiquitin-mediated degradation (12–14). Ubiquitin molecules involved in ubiquitination are dissociated from substrate proteins and can be reused in the cytoplasm (Fig. 1). E3 ubiquitin ligases, which serve a key role in ubiquitin-mediated degradation of proteins, specifically mark the substrate proteins and attach ubiquitin to them for degradation (9,15). Deubiquitination is an important mechanism for maintaining intracellular protein stability and is closely associated with the development of cancer. Deubiquitinating enzymes (DUBs) hydrolyze the isopeptide bonds in ubiquitinated substrate proteins, thereby dissociating the ubiquitinated molecules from the substrate proteins and inhibiting ubiquitin-mediated protein degradation. A flowchart demonstrating the mechanism of the ubiquitin-proteasome pathway is shown in Fig. 1 (16–18). DUBs are a large family of proteasomes. It is known that ~100 DUBs are encoded by the human genome, which can be classified as ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor-related proteases (OTUs), Machado-Joseph disease (MJD) deubiquitinases and metalloproteases (19–22). Except for the metalloproteinase family, all other deubiquitinases are cysteine proteases; of which, USPs are the most structurally diverse class of deubiquitinases with the largest known membership. USPs inhibit protein degradation by removing ubiquitin from substrate proteins through interaction with a catalytic triplet of residues (cysteine, histidine and aspartate) (23). USPs are involved in regulation of apoptosis (23), protein transport (24), regulation of the cell cycle (25), DNA damage repair (26), chromatin remodeling and protein signaling (27,28). In addition to inhibiting the degradation of ubiquitinated substrate proteins, USPs can regulate related signaling pathways by affecting protein activity. For example, in the TGF-β signaling pathway, USP15 and CYLD lysine 63 deubiquitinase (CYLD) affect the stability of the mothers against decapentaplegic (SMAD) protein in Drosophila by antagonizing SMAD protein-specific E3 ligase 2, which in turn negatively regulates the activation of the TGF-β pathway (29). USP is involved in the regulation of multiple cancer-related pathways, including p53, Wnt/β-catenin, TGF-β and protein kinase B (Akt) pathways. For example, the overexpression of USP2a stabilizes murine double minute 2 (MDM2) through direct deubiquitination, thereby enhancing the degradation of the tumor suppressor protein p53. The downregulation of p53 eventually leads to tumor progression (30). Overexpression of USP10 directly deubiquitinates and stabilizes Krüppel-like factor 4 protein, which directly binds to the promoter region of tissue inhibitor of metalloproteinase-3 (a tumor suppressor gene) to promote its transcription and exert positive anti-tumor effects (31).

USP2, the second member of the USP family, was discovered in chicken muscle in 1997 and was originally defined as UBP41 (32). It is localized on the long arm of human chromosome 11 (11q23.3) and is highly conserved in eukaryotes. According to the Uniprot Protein Data Bank (Accession number: 075604), the mRNA precursor of USP2 can generate four mRNA isoforms, namely, USP2-1, USP2-2, USP2-3 and USP2-4, owing to different splicing patterns. USP2-1 (USP2a/USP2-69) contains 605 amino acids and has a molecular weight of 68,072 Da. USP2-2 (USP2b) contains 353 amino acids and has a molecular weight of 40,638 Da, with 1–252 amino acid residues missing and 253–258 amino acid residues mutated from PGRDGM to MLNKAK (when compared with USP2a). USP2-3 (USP2c) contains 362 amino acids and has a molecular weight of 41,682 Da. USP2-4 contains 396 amino acids and has a molecular weight of 45,241 Da (www.uniprot.org/UniProtKB/O75604/entry). The isomers of USP2 have similar structures, both consisting of an N-terminal structural domain of variable length and a C-terminal structural domain with 347 amino acids, where the C-terminus has the characteristic catalytic triad of the USP family (Cys, His, and Asp/Asn) (33). Specifically, USP2a has the largest N-terminal domain with 258 amino acids, while that of USP2b, USP2c and USP2-4 has 6, 15 and 49 amino acids, respectively (34–37). The basic structure of USP2 is summarized in Fig. 2. Factors involved in isomeric splicing of USP2 under physiological conditions mainly include changes in the circadian rhythm, nutritional status and androgen levels (38). The circadian rhythm and nutritional status primarily affect the changes in USP2b. Pouly et al (39) reported that USP2b expression continuously increases in mouse kidney tissues during the light phase, reaching the highest value at 12 noon, and then gradually decreases from noon to midnight. Starvation leads to an increase in the mRNA expression of USP2b in mice but does not affect the expression of USP2a (40). Although androgens can promote alternative splicing of the USP2a gene, they do not affect USP2b and other isomers (41). In addition, multiple cytokines and signaling pathways are involved in USP2 splicing. For example, activation of protein kinase C (PKC) signaling can promote alternative splicing of USP2b and inhibit alternative splicing of USP2a in macrophages (42). The cytokine interleukin-1β (IL-1β) can promote alternative splicing of USP2a in mesenchymal stem cells (MSCs) (43). TNF-α can downregulate the mRNA and protein expression of USP2c in the liver while promoting the alternative splicing of USP2a (44). TGF-β1 and platelet-derived growth factor-BB (PDGF-BB) can both promote alternative splicing of USP2a; however, the effects of PDGF-BB are most pronounced (45,46). All isomers have the same structure at the C-terminus, including catalytically active Cys and His residues, whereas the N-terminus can interact with different proteins and perform different physiological functions (33,47). For example, when the N-terminus of USP2 binds to receptor interacting protein-1 (RIP1) protein, it removes the ubiquitinated molecules on RIP1 protein and increases RIP1 expression, which in turn promotes apoptosis (48). The N-terminus binds to and stabilizes the cyclin D1 (CCND1) protein to promote cell cycle progression from the G₁ to the S phase (49). The present review focused on the role and function of USP2 in cancer-related signaling pathways and its potential application value in cancer therapy.

2. Role of USP2 in the biological behavior of malignant tumors

Initially, USP2 was thought to be expressed in only human testicular tissue (50); however, with the continuous advancement of detection techniques, USP2 expression has been observed in various cells, including macrophages, and tissues and organs, including the heart, liver, kidney, breast, brain and skeletal muscle (47,51–56). Previous studies have demonstrated that USP2 promotes tumor cell proliferation by stabilizing proteins such as CCND1 and CCNA1. In addition, USP2 promotes epithelial-mesenchymal transition (EMT) and affects the sensitivity of tumor cells to chemotherapeutic drugs by stabilizing β-catenin protein through deubiquitination (55,57). This section discusses the specific molecular mechanisms through which USP2 participates in biological processes and highlights the significance of USP2 as a therapeutic target for tumors.

<sec> <title>USP2 enhances cell cycle and mitosis and promotes abnormal proliferation of tumor cells

Normal cell division, proliferation, differentiation and ageing maintain the self-stability of the body. Cell cycle disturbances can lead to abnormal cell proliferation, which is a common feature of tumor cells (57). The role of USP2 in cell cycle regulation has been well demonstrated (59,60). CCND1 is abnormally overexpressed in various tumor cells. Shan et al (61) screened 76 DUBs in vitro to assess their catalytic ability to target CCND1. They identified USP2 as a specific DUB of CCND1, which can directly interact with CCND1, reduce the polymeric ubiquitination-dependent degradation of CCND1 and promote tumor cell proliferation. In the human embryonic kidney cell line 293T, USP2a has been shown to deubiquitinate CCND1, thereby facilitating cell cycle progression from the G₁ to the S phase (38). A study demonstrated that the protein expression of CCND1 is significantly higher in human breast cancer MCF-7 cells and prostate cancer PC3 cells than in normal cells (38). USP2 knockdown attenuates CCND1 deubiquitination and stability, promotes ubiquitin-mediated degradation, reduces CCND1 expression, inhibits cell progression from the G₁ to the S phase and suppresses cell proliferation (38). In addition, USP2a is a downstream target of lithocholic acid (LCA) hydroxyamide (LCAHA), and LCAHA can destabilize CCND1 by inhibiting the expression of the deubiquitinating enzyme USP2a (62). It induces G₀/G₁ phase arrest in colon cancer cells (HCT116), thus exerting an active anti-tumor effect (62). Leptin and adiponectin are two hormones secreted by adipose tissue that have contradictory effects on USP2 expression in tumor cells. Leptin targets USP2 to upregulate the protein expression of CCND1 to promote cell cycle progression and tumorigenesis(63). Adiponectin targets USP2 to promote ubiquitin-mediated degradation of CCND1 protein, resulting in cell cycle arrest and inhibition of tumor progression (63). As the intracellular overexpression of CCND1 is a decisive factor in the development of some tumors, CCND1 has been used to assess the potential of USP2 inhibitors as important indicators of the efficacy of antineoplastic drugs (49,64). USP2 can directly recognize and deubiquitinate CCND1, thereby preventing its degradation, stabilizing its expression and promoting tumor development. Similarly, USP2a can inhibit CCNA1 protein degradation through deubiquitination, stabilize CCNA1 protein expression and promote the progression of bladder cancer T24 cells from G₁ to S phase, which in turn promotes bladder cancer T24 cell proliferation (65).

The oncoprotein c-Myc serves a key role in the development, progression and maintenance of cancer, particularly influencing the proliferation of tumor cells (66,67). Inhibition of c-Myc expression promotes the senescence of different types of tumor cells, whereas its overexpression inhibits the senescence of melanoma cells and exerts pro-tumor effects (68,69). A recent study demonstrated that c-Myc upregulates USP2-AS1 expression by promoting the transcription of lncRNA USP2-AS1. USP2-AS1 stabilizes E2F1 mRNA and increases E2F1 expression by interacting with the RNA-binding protein G3BP1, which in turn attenuates the senescence of HCT116 and A549 cells and serves a pro-cancer function of c-Myc (70).

Aurora-A, a serine/threonine protein kinase, is a mitotic regulator essential for the replication, maturation and segregation of centrosomes and the subsequent spindle assembly. Overexpression of Aurora-A inhibits Hec1 phosphorylation at serine 55 (Hec1-S55) during metaphase and destabilizes the kinetochore-microtubule attachment, which in turn induces tumor development (71,72). Shi et al (73) reported that USP2a reverses ubiquitin-mediated degradation of Aurora-A and promotes mitosis in pancreatic cancer MIA PaCa-2 cells. They used small interfering RNAs (siRNAs) to knock down USP2a in MIA PaCa-2 cells, which enhanced ubiquitin-mediated degradation of Aurora-A and significantly inhibited the proliferation of tumor cells. Therefore, USP2a may promote tumor cell proliferation by stabilizing the Aurora-A protein, and targeting USP2a may represent an effective strategy for inhibiting the abnormal proliferation of tumor cells.

USP2 promotes EMT and enhances the migratory and invasive capabilities of tumor cells

EMT is an important biological process in which epithelial cells acquire the ability to migrate and invade (74). TGF-β signaling can induce the transcription of related genes, promote EMT and enhance the migratory and invasive capabilities of tumor cells (75,76). TGF-β binds to two types of transmembrane serine/threonine kinase receptor heterologous complexes to initiate cellular responses (77). Receptor kinases activate the intracellular signaling protein SMAD to form heterologous protein complexes that are transferred to the nucleus, where they regulate the transcription of EMT-related genes, such as Snail, Slug (zinc-finger proteins), Twist, N-cadherin and E-cadherin (78). USP2a promotes the migratory and invasive capabilities of non-small cell lung cancer A549 cells by removing the K33-linked polyubiquitin chain from the TGF-β receptor, thereby promoting the binding of receptor-regulated SMAD (R-SMAD) to the TGF-β receptor and upregulating the expression of Snail (79). A study demonstrated that knockdown of USP2a or treatment with the USP2a-specific inhibitor ML364 (10 µM) effectively inhibits the migratory and invasive capabilities of tumor cells. In addition, ML364 significantly prolongs the survival of nude mice injected with hepatocellular carcinoma (HCC) Hep3B cells (via the tail vein) and attenuated lung metastasis (79). Therefore, USP2a may serve as a potential therapeutic target for cancer.

Wnt/β-catenin signaling serves a key role in EMT in tumor cells (80–82). Wnt is a secreted glycoprotein that interacts with specific receptors on the cell surface through autocrine and paracrine mechanisms and induces the accumulation of β-catenin through phosphorylation and dephosphorylation of downstream proteins. β-catenin serves an important role in cell adhesion by interacting with E-cadherin at cell junctions and participating in the formation of adhesion bonds. Free β-catenin can enter the nucleus and interact with T-cell Factor/Lymphoid Enhancing Factor DNA-binding proteins to increase the transcription of EMT-related genes (83). In a previous study, a total of 68 DUBs potentially related to β-catenin were analyzed via immunoprecipitation and GST pull-down assays. USP2a was identified as a DUB that directly interacts with β-catenin and positively regulates its levels and activity. Further experiments revealed that USP2a removes the ubiquitin molecules on β-catenin and prevents its degradation, which in turn enhances the activity of the Wnt/β-catenin pathway and promotes the EMT of tumor cells. In addition, knockdown of USP2a or treatment with ML364 downregulated the protein expression of β-catenin in human breast cancer BT549 cells and inhibited the migratory and invasive capabilities of tumor cells (84).

USP2 inhibits p53-mediated cell death

The tumor suppressor gene p53 is a downstream target of USP2. It repairs damaged DNA, induces apoptosis and regulates the cell cycle, thereby preventing carcinogenesis (85,86). Loss of p53 gene is another major cause of tumorigenesis, and >50% of patients with malignant tumors may have p53 gene mutations (87). MDM2 is an E3 ubiquitin ligase that promotes the degradation of p53 protein through the ubiquitin-proteasome pathway (88). Although USP2 cannot directly affect the ubiquitin-mediated degradation of p53, it can inhibit the ubiquitin-mediated degradation of MDM2 protein by specifically recognizing and hydrolyzing the isopeptide bond in MDM2. This inhibition increases protein stability and indirectly inhibits p53 expression in prostate cancer and cutaneous T-cell lymphoma (30,89,90). MDM4 is an important regulator of p53 upstream and is similar to MDM2 in terms of structure and function. Its high expression inactivates p53 and induces tumor development. USP2a can directly stabilize the protein expression of MDM4 in glioma cells through deubiquitination and promote the ubiquitin-mediated degradation of p53. In glioma cells with USP2a knockdown, MDM4 expression is downregulated and p53 protein is transported to mitochondria, promoting cytochrome c-induced apoptosis (91). A study demonstrated that the expression of p53 is downregulated in hepatoma HepG2 cells and breast cancer MCF-7 cells. The content of p53 in these cells is significantly higher following leptin treatment than prior to treatment. However, USP2 knockdown inhibits the leptin-induced increase in intracellular p53 levels, indicating that the tumor-suppressing effects of leptin rely on the deubiquitinating effects of USP2 on p53 protein (92). Therefore, USP2 may serve as an effective therapeutic target for malignancies characterized by the loss of function of p53 gene.

USP2 reduces sensitivity to chemotherapeutic drugs

Drug resistance and metastasis are the major causes of death among patients with cancer. Developing effective strategies for reversing drug resistance and elucidating mechanisms underlying drug resistance constitute the primary focus of modern medical research. Clinical conventional chemotherapeutic drugs mainly exert their cytotoxic effects by inducing apoptosis through the mitochondrial and endoplasmic reticulum stress-mediated autophagic pathways (93–95). The anti-apoptotic regulator cFILP serves an important role in death receptor signaling, and its overexpression is one of the primary mechanisms through which tumor cells acquire drug resistance (96,97). On the one hand, cFILP competes with the precursor caspase-8 to bind to Fas-associated death domain-containing protein (FADD), which inhibits apoptosis and promotes drug resistance in tumor cells (98). On the other hand, cFILP interacts with Akt and enhances the anti-apoptotic function of Akt by regulating the activity of glycogen synthase kinase-3 β (GSK3β) to promote drug resistance in tumor cells (99,100). Previous studies have reported that USP2 stabilizes the protein expression of cFILP and promotes the proliferation of HCC Huf7 cells through deubiquitination. Inhibition of USP2 can reduce cFILP expression in sorafenib-resistant Huf7-SR cells, promote apoptosis and increase sorafenib sensitivity (98). Additionally, USP2 negatively regulates the expression of miRNA-1915-3P in oxaliplatin-resistant colorectal cancer (CRC) cells; inhibits apoptosis and promotes the proliferative, migratory and invasive capabilities of tumor cells. Knockdown of USP2 promotes apoptosis and increases the sensitivity of CRC cells to oxaliplatin (101). In addition, knockdown of USP2 or treatment with ML363 enhances the sensitivity of triple-negative breast cancer cells to doxorubicin (102).

3. Targeting USP2 for cancer therapy

In molecularly targeted therapy, specific oncogenes or gene fragments can be targeted and corresponding targeted drugs can be developed to act at the cellular level. When these targeted drugs enter the human body, they specifically target the cancer-inducing sites, leading to the specific elimination of tumor cells without damaging normal cells (103–105). Therefore, molecularly targeted therapy is considered an effective therapeutic strategy for cancer in modern medicine and is a major focus of cancer research. USP7, a member of the DUB family, can promote tumor development by stabilizing the E3 ubiquitin ligase MDM2, promoting p53 degradation and reducing the expression of downstream proteins of p53 (106,107). Elevated USP7 expression is closely associated with the development of several cancers and USP7 is an important target for the treatment of prostate cancer (108), malignant melanoma (109), ovarian cancer (110), multiple myeloma (111) and CRC (112). In addition to USP7, other USPs such as CYLD, USP1, USP6, USP8, USP9X, USP11, USP15 and USP28, are considered potential therapeutic targets for various cancers (23). Studies have demonstrated that USP2, a multifunctional cysteine protease, is a key regulator of ubiquitin-mediated degradation of fatty acid synthase (FAS), MDM2, MDM4, epidermal growth factor receptor (EGFR), the cell cycle proteins A1 and D1 and other oncogenic proteins, and is closely associated with the development of a number of tumors (59,91,106,113). Therefore, targeting USP2 is a promising strategy for tumor treatment. This section discusses the current research status of USP2 in cancer therapy and summarizes the targets and related molecular mechanisms of USP2 (Table I and Fig. 3).

<sec> <title>Targeting USP2 for breast cancer

USP2 expression is low in invasive ductal carcinoma (114) but high in estrogen receptor-positive, progesterone receptor-positive and triple-negative breast cancers and distant metastatic sites. High expression of USP2 is significantly associated with a poor prognosis in breast cancer (57). USP2 promotes distant metastasis and invasion in triple-negative breast cancer. Its overexpression upregulates MMP2 to promote the migratory and invasive capabilities of breast cancer cells, whereas its silencing significantly attenuates these capabilities (57). Therefore, USP2 may be used as a prognostic biomarker and therapeutic target for triple-negative breast cancer.

The Twist protein is a highly conserved basic helix-loop-helix transcription factor that is repressed in normal tissue cells but overexpressed in triple-negative breast cancer and various metastatic tumors (115,116). It serves a key role in the self-renewal and EMT of tumor stem cells (117,118). USP2 is associated with the upregulation of Twist protein in clinical tumor specimens. Inhibition of USP2 expression promotes the ubiquitin-mediated degradation of Twist, thereby inhibiting tumor stem cell properties in vitro and tumorigenicity in vivo (102). The USP2 inhibitor ML364 inhibits tumor growth and enhances the sensitivity to Adriamycin (102). In addition, the molecular chaperone function of heat shock protein 90 (HSP90) serves a critical role in maintaining the stability of various intracellular proteins and is closely associated with the development of several tumors (119,120). Clinical trials have demonstrated the anticancer effects of multiple HSP90 inhibitors, both as monotherapy and combination therapy with ErbB2-targeting agents (121,122). A preliminary clinical trial of tanespimycin (17-AAG) provides additional evidence for the use of HSP90 inhibitors in the treatment of ErbB2-positive breast cancer (123). In a recent study, HSP90 inhibitors were found to promote the ubiquitin-mediated degradation of ErbB2; however, these effects were reversed by USP2. Additionally, ML364 not only enhanced the degradation of ErbB2 by HSP90 inhibitors but also inhibited the growth of ErbB2-positive breast cancer cells and transplanted tumors in mice in vivo (56). Therefore, USP2 may serve as a prognostic biomarker and therapeutic target for breast cancer.

Targeting USP2 for hepatocellular carcinoma

USP2 exerts pro-carcinogenic effects in malignant tumors such as breast and lung cancers; however, its role in liver cancer remains unclear. The expression of different isoforms of USP2 in the liver is controversial. In one study, USP2c was identified as the major isoform of USP2 protein in the liver (~89% of total USP2), whereas USP2b protein was not detected in the liver (44). However, other studies have reported that USP2b is the major isoform of USP2 in the liver (33,41). Nadolny et al (55) used an isoform-specific probe technique to detect USP2 in normal human and mouse liver tissues and identified USP2b as the major isoform of USP2 in the liver. The mRNA and protein expression of USP2 is significantly lower in clinical primary HCC tumor tissues than in para-carcinoma and healthy liver tissues, and the expression of USP2b is consistent with the total USP2 expression (55). Furthermore, USP2b can exert both pro- and anti-cancer effects. On the one hand, overexpression of USP2b promotes bile acid-induced apoptosis and necrosis of tumor cells to exert anti-tumor effects; on the other hand, overexpression of USP2b promotes tumor cell proliferation, colony formation and wound healing to exert pro-cancer effects. Therefore, the ability of USP2b to act as a tumor suppressor or initiator depends on the cell state and the specific underlying molecular mechanisms warrant further investigation.

Antisense RNAs refer to RNA molecules that are complementary to mRNAs. They inhibit the translation of mRNAs and block gene function by specifically and complementarily binding to mRNAs (124). Given the medicinal value of antisense RNAs, their role in cell growth and differentiation needs to be intensively investigated (125). The expression of ubiquitin-specific peptidase 2 antisense RNA 1 (lncRNA USP2-AS1), a USP2-specific antisense RNA, is significantly higher in HCC tissues than in paraneoplastic tissues. The high expression of USP2-AS1 is significantly associated with a poorer prognosis. Knockdown of USP2-AS1 promotes the ubiquitin-mediated degradation of Y-box binding protein 1-mediated hypoxia-inducible factor 1α; inhibits the proliferative, migratory and invasive abilities of HCC cells and reduces the tumorigenicity of HCC cells in mice (126).

cFILP is an important regulator of death receptor signaling that inhibits tumor cell apoptosis by competing with caspase-8 to bind to the junction protein FADD (127). cFILP overexpression is one of the major causes of resistance to death receptor-mediated apoptosis and chemotherapy (128). Liu et al (98) reported that cFILP expression is significantly elevated in sorafenib-resistant HCC cells, and overexpression of USP2 can promote ubiquitin-mediated degradation of cFILP protein by stabilizing the E3 ubiquitin ligase ITCH to enhance the sensitivity of HCC cells to sorafenib. Therefore, USP2 and cFILP may serve as potential targets for reversing sorafenib resistance in HCC cells.

Targeting USP2 for ovarian cancer

Yang et al (129) examined tumor specimens from 40 patients with ovarian plasmacytoid cystic adenocarcinoma and found that the expression of USP2, USP14 and UBE4A (an ubiquitin-related factor) is significantly higher in ovarian cancer tissues than in peri-cancerous tissues and normal ovarian tissues. These findings suggest that the ubiquitin-proteasome pathway is involved in the development of ovarian cancer. USP2-AS1, an antisense RNA of USP2, is significantly upregulated in ovarian cancer tissues, and its knockdown can inhibit the proliferative, migratory and invasive abilities of ovarian cancer cells. USP2-AS1 can compete for endogenous RNAs to regulate the expression of downstream genes by sponging miRNAs (126). Guo et al (130) investigated the specific molecular mechanisms through which USP2-AS1 promotes ovarian cancer progression. USP2-AS1 and miRNA-520d-3P were found to bind to each other, and silencing of miRNA-520d-3P reversed the USP2-AS1-induced proliferative, migratory and invasive abilities of ovarian cancer cells. The specific mechanism is related to the involvement of USP2-AS1 in the ubiquitinated degradation of miRNA-520d-3P downstream gene KIAA1522. Therefore, USP2 and USP2-AS1 may serve as potential targets for the treatment of ovarian cancer.

Targeting USP2 for colorectal cancer

USP2, a specific deubiquitinating enzyme of CCND1, is highly expressed in colon cancer cells (HCT116). Treatment of HCT116 cells with ML364 can promote the ubiquitin-mediated degradation of CCND1, inhibit tumor cell proliferation and induce the arrest of tumor cells in the G₁ phase (49). In addition, the expression of USP2-AS1 is significantly elevated in clinical colon adenocarcinoma tumor tissues and is positively correlated with tumor size, grade and TNM stage. Knockdown of USP2-AS1 can promote phosphorylation and ubiquitin-mediated degradation of Yes-associated protein 1 (YAP1) to inhibit the activation of the Hippo/YAP1 signaling pathway, which in turn inhibits the proliferative, migratory and invasive abilities of tumor cells and reduces the tumorigenicity and distant metastatic ability of cancer cells in mice (131).

Oxaliplatin is widely used as the first-line chemotherapeutic agent for the treatment of advanced CRC in clinical settings. The expression of miRNA-1915-3P is reduced in oxaliplatin-resistant CRC cells and overexpression of miRNA-1915-3P downregulates the oncogenes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and USP2, thus inhibiting tumor proliferation, metastasis and invasion (101). USP2 is a negative regulator of miRNA-1915-3P. Overexpression of USP2 restores the proliferative capacity of tumor cells, whereas its knockdown inhibits tumor cell proliferation (101). Therefore, small-molecule inhibitors of USP2 may be used to induce oxaliplatin sensitivity in advanced CRC.

Targeting USP2 for glioblastoma

Glioblastoma (GBM) is an astrocytic tumor characterized by rapid growth, high malignancy and high mortality rates (132). As a cancer-promoting factor, TGF-β serves an important role in the development of GBM (133,134). SMAD7, a key negative regulator of TGF-β signaling, exerts its inhibitory effects on TGF-β by blocking receptor activity and inducing receptor degradation (135). USP2 expression is significantly lower in GBM tissues than in normal human brain tissues and the prognosis of patients with lower USP2 expression is worse. Overexpression of USP2 can break the isopeptide bond between ubiquitin and the Lys27 and Lys48 residues of SMAD7 protein, reduce the recruitment of SMAD7 protein to the E3 ubiquitin ligase HERC3 and inhibit ubiquitin-mediated degradation of SMAD7, thereby inhibiting the activation of the TGF-β signaling pathway and the progression of GBM (54). Abnormal DNA methylation transferase 3A (DNMT3A)-mediated methylation of USP2 is the main cause of low expression of USP2 in GBM tissues, and the DNMT3A inhibitor GSI-1027 can induce USP2 expression to exert anti-tumor effects against GBM (54).

Previous studies have reported that MDM4 can promote endogenous apoptosis by regulating the expression of oncogene p53 and that USP2a can interact with MDM4 to inhibit its ubiquitin-mediated degradation (91,136,137). The expression of MDM4 and USP2a is significantly lower in GBM tissues than in normal brain tissues and is positively associated with the prognosis of GBM; that is, the higher the expression, the more improved the prognosis. Knockdown of USP2a promotes UV irradiation-induced cytochrome c release, p53 protein expression and apoptosis in U87MG glioma cells, whereas simultaneous upregulation of MDM4 can reverse these effects (91). Therefore, USP2 and MDM4 may serve as effective targets for the treatment of GBM.

Targeting USP2 for bladder cancer

Bladder cancer is the most common life-threatening tumor of the urinary system. Tight junction protein 1 (TJP1) interacts with TWIST1 to enhance the invasive ability of tumor cells and promotes bladder cancer progression by affecting vascular remodeling (53). TJP1 expression is significantly higher in clinical bladder cancer tissues compared with healthy bladder tissues and is associated with tumor angiogenesis and overall survival of patients (138). In vitro studies have demonstrated that overexpression of TJP1 promotes the expression of TWIST1 and chemokine C-C motif ligand 2 (CCL2) in tumor cells, stimulating tumor cells to recruit more macrophages, which secrete VEGF under CCL2 stimulation and enhance tumor angiogenesis. Knockdown of TJP1 inhibits, and overexpression of TWIST1 promotes, vascular remodeling in bladder cancer. TJP1 promotes vascular remodeling by reversing ubiquitin-mediated degradation of TWIST1 by recruiting USP2, whereas knockdown of USP2 promotes ubiquitin-mediated degradation of TWIST1, reduces tumor angiogenesis and exerts positive anti-tumor effects (53). In addition, USP2a gene is highly expressed in bladder cancer cells and there is a physical interaction between USP2a and CCNA1. USP2a inhibits ubiquitination degradation of CCNA1 protein through deubiquitylation, which in turn increases CCNA1 protein expression and exerts a positive pro-oncogenic effect. Therefore, USP2 and TJP1 may serve as effective therapeutic targets for bladder cancer.

Targeting USP2 for prostate cancer

The positive regulation of FAS by USP2 serves a key role in influencing the malignant behavior of prostate cancer (139–141). USP2 is significantly upregulated in prostate cancer tissues; however, it is either not expressed or is downregulated in healthy prostate tissues. The protein expression of USP2a is directly associated with the malignant behavior of prostate cancer. Overexpression of USP2a promotes the proliferation of LNCaP cells. Following USP2a knockdown, ubiquitin-mediated degradation of FAS is enhanced and the protein expression of FAS is significantly downregulated, resulting in apoptosis of tumor cells, which can be reversed by the proteasome inhibitor MG-132 (142). Previous studies have reported that acid ceramidase (ACD), which metabolizes ceramide to sphingomyelin, is upregulated in prostate cancer (143). Mizutani et al (144) reported that treatment with the androgen receptor antagonist bicalutamide (Casodex) decreased the protein expression of adrenocortical dysplasia homologue (ACD) in LNCaP cells, whereas treatment with MG132 restored the activity of ACD protein. These findings suggest that the ubiquitin-proteasome pathway is involved in the modification of ACD protein. The oncogenic role of USP2 in prostate cancer has been demonstrated in previous studies (139,144). The protein expression of ACD is promoted upon USP2 overexpression and inhibited upon USP2 silencing in LNCaP cells. However, silencing or overexpression of SKP2, an E3 ubiquitin ligase, does not alter the activity of ACD protein in LNCaP cells, suggesting that ACD activity is affected by deubiquitination of USP2, independent of SKP2 (144). Therefore, USP2, FAS and ACD may influence the malignant behavior of prostate cancer and serve as potential therapeutic targets.

Targeting USP2 for cutaneous T-cell lymphoma

Cutaneous T-cell lymphoma (CTCL) is caused by the clonal proliferation of T lymphocytes originating in the skin and is a type of extranodal non-Hodgkin lymphoma. Psoralen with ultraviolet A (PUVA) phototherapy is a common treatment strategy for CTCL in clinical settings (145,146). A study demonstrated that USP2 is expressed in both quiescent and activated T lymphocytes, and its expression is significantly reduced in advanced CTCL. Treatment of MyLa2000 cells with PUVA or the p53 agonist nutlin3a significantly increases the protein expression of USP2 and p53 and promoted apoptosis (90). Silencing of USP2, which acts as a tumor suppressor, reduces the protein expression of MDM2 and enhances the transcriptional activity of p53, thereby promoting apoptosis and enhancing the sensitivity of MyLa2000 cells to PUVA and nutlin3a. In addition, p53 induced USP2 expression and stabilized MDM2 protein via deubiquitination, which in turn inhibited the pro-apoptotic activity of p53, forming a negative feedback loop (90). Therefore, small-molecule inhibitors of USP2 may serve as sensitizing agents in CTCL.

Targeting USP2 for gastric cancer

E2F transcription factor 4 (E2F4), a key factor regulating cell cycle progression, binds to DNA to promote the progression of cells from the G₀ to the G₁ and S phases and is involved in tumor progression (147). E2F4 can directly regulate the transcription of ATG2A and ULK2 proteins, leading to the autophagic degradation of metallothionein; it can maintain zinc homeostasis in tumor cells and promote the proliferative, migratory and invasive abilities of gastric cancer cells (59). High expression of USP2 and E2F4 in gastric cancer tissues is associated with a poor prognosis. Emetine, an autophagy inhibitor, can block the interaction between USP2 and E2F4 and promote E2F4 degradation for an oncogenic effect, which can be reversed upon USP2 overexpression (59). Therefore, USP2 and E2F4 may serve as potential biomarkers for maintaining zinc homeostasis in the treatment of gastric cancer.

Targeting USP2 for lung cancer

Previous studies have demonstrated the involvement of multiple USPs in the tumorigenesis and chemotherapy resistance of lung cancer (148,149). USPs may serve as therapeutic targets for lung cancer. For instance, inhibition of USP1 and USP51 can increase cisplatin sensitivity in lung cancer (150,151); inhibition of USP5 and USP28 can promote apoptosis of tumor cells (152,153) and promotion of USP52 and USP7 can inhibit the proliferative, migratory and invasive abilities of lung cancer cells (107,154). However, the role of USP2 in lung cancer remains elusive. Zhang et al (155) reported that USP2 expression is upregulated in the lung cancer cell lines H1229 and H1270. Knockdown of USP2 promotes ubiquitin-mediated degradation of SKP2 and inhibits the growth of tumor cells. Mechanistically, USP2 interacts with SKP2 and stabilizes its expression to promote lung cancer progression. Therefore, USP2 and SKP2 may serve as potential therapeutic targets for lung cancer.

Targeting USP2 for renal clear cell carcinoma

Renal clear cell carcinoma is a common malignant tumor of the urinary system. The proliferation and apoptosis of cancer cells cannot be achieved without the participation of USPs (156,157). The clinical significance of USP2 in renal clear cell carcinoma has been demonstrated (158). The mRNA and protein expression of USP2 is significantly lower in cancer tissues than in para-cancerous and healthy kidney tissues. Studies have verified the low protein expression of USP2 in most cancer tissues via immunohistochemical analysis (56,159,160). Overexpression of USP2 inhibits the proliferative, migratory and invasive abilities of kidney cancer cells (A498 and CAKi-1) (158). In addition, the abnormal expression of USP2 is closely related to the clinical stage, pathological grade and prognosis of patients with renal cancer, and USP2 has been identified as an independent risk factor for renal clear cell carcinoma (158). Therefore, USP2 is a potential target for the diagnosis and treatment of renal clear cell carcinoma.

Targeting USP2 for the treatment of hematological tumors

The role of USP2 in stabilizing CCND1 during cell proliferation is well established. Davis et al (49) were the first to reveal the epigenetic regulation mechanism of USP2 in a nested cell lymphoma model. They found that USP2 expression is significantly reduced, the ubiquitin-mediated degradation of CCND1 protein is significantly enhanced, the cell cycle is arrested in the G₁ phase and the proliferative capacity of tumor cells is significantly reduced after Mino cells were treated with the USP2-specific small-molecule inhibitor ML364. However, CCND1 degradation was reversed and the proliferative capacity of cells was restored after the cells were treated with the proteasome inhibitor MG132. 6-thioguanine (6-TG), an anti-tumor agent clinically used in the treatment of acute leukemia and chronic granulocytic leukemia, is a potent inhibitor of USP2 (161,162). 6-TG forms covalent bonds with the Cys276 residue of USP2 to inhibit USP2 in a non-competitive and slow-binding manner. Therefore, it can be used in the clinical treatment of tumors characterized by USP2 upregulation (139). Lin et al (163) reported that disulfiram, a clinical therapeutic agent for alcohol dependence, competitively inhibited the protein activity of both USP2 and USP21. Altogether, the combination of 6-TG and disulfiram may be used for the clinical treatment of USP2-associated tumors.

Lysine (K)-specific methyltransferase 2A (KMT2A) serves an important role in embryonic development and the hematopoietic system. The translocation of the KMT2A gene produces a KMT2A fusion protein that directly binds to DNA and upregulates gene transcription, leading to the development of acute myeloid leukemia (AML) in infants and children (164,165). USP2 serves as a chaperone gene for KMT2A and the poor clinical prognosis of children with KMT2A-USP2-positive AML has been associated with the aberrant expression of USP2 (166–168). In a prospective study, Meyer et al (169) reported that a very small number of patients with acute leukemia have rearranged USP2 and USP8 genes and that the conserved region of the deubiquitinating enzyme ‘UCH-domain’ fuses to an extended 5´-MLL portion, which formed the fusion proteins MLL-USP2 and MLL-USP8. Deubiquitination of USP2 stabilizes MDM protein and indirectly enhances the degradation of p53, which may be an important mechanism affecting the development of MLL-USP2 type leukemia. Therefore, USP2 may serve as a potential therapeutic target for AML.

4. Pharmacological studies of USP2

With the rapid development of structural biology and small-molecule drugs, targeted therapy has emerged as the most promising strategy in clinical tumor treatment. Recent studies have revealed the role of DUBs in life activities and identified DUBs as potential therapeutic targets for tumors (170). USP2 is closely associated with the development of various tumors, such as breast cancer, liver cancer, CRC, GBM and hematological tumors (49,53,54,56,126). At present, ML364 is the most common small-molecule inhibitor used in clinical trials; other inhibitors include Q29, STD1T and LCAHA (23). The chemical structures of these small-molecule inhibitors are shown in Fig. 4, and key information regarding their mechanism of action and targets is summarized in Table II. The use of USP2 as a target for tumor treatment has received increasing attention from researchers. Although several USP2-targeted agents have shown positive anticancer effects in different cancers, the identified targeting agents are undergoing preclinical investigation at present. Therefore, these agents should be evaluated via complex and comprehensive techniques to provide a theoretical basis for their clinical application.

<sec> <title>ML364

ML364 is the most commonly used specific small-molecule inhibitor of USP2 (IC₅₀=1.1 µM; Fig. 4A). It directly binds to USP2, induces ubiquitin-mediated degradation of cyclin D1, leads to cell cycle arrest in the G₀/G₁ phase, inhibits the proliferation of HCT116 and Mino cells and exerts positive antitumor effects (49). In addition, ML364 enhances ubiquitin-mediated degradation of ErbB2 by HSP90 inhibitors through inhibition of USP2 in the treatment of ErbB2-positive breast cancer (56).

Beta-lapachone

Beta-lapachone (Q29) is a natural naphthoquinone compound (Fig. 4B) that was used in several phase II clinical trials in the early 21st century for the treatment of pancreatic cancer, head and neck tumors and smooth muscle sarcoma (171). In subsequent studies, Q29 was found to exert positive antitumor effects by selectively and irreversibly inhibiting the oxidation of cysteine residues of USP2 and promoting the production of reactive oxygen species, thereby interfering with cell cycle progression and inducing apoptosis in tumor cells (172).

LCAHA

LCA, a secondary bile acid, serves an important role in lipid metabolism, and several derivatives of LCA have anticancer activity (173,174). The most active, LCAHA (Fig. 4C; IC₅₀=5.8 µM), can directly inhibit the biological activity of USP2a, induce G₀/G₁-phase arrest in HCT116 cells and ubiquitously degrade cell cycle protein D1, thereby exerting positive anticancer effects (62).

STD1T

Tomala et al (64) used saturation transfer difference nuclear magnetic resonance spectroscopy to screen USP2 protein and found that STD1 could directly bind to USP2a and inhibit its activity. As a derivative of STD1, STD1T (IC₅₀=3.3 µM) has a stronger inhibitory effect on USP2a (Fig. 4D) and can significantly reduce the expression of CCND1 in HCT116 and MCF-7 cells at a concentration of 20 µM, thus exerting a positive oncogenic effect.

6-TG

6-TG is a clinical agent for the treatment of AML and chronic granulocytic leukemia (161,162) (Fig. 4E). Chuang et al (139) used enzyme kinetic and X-ray crystallographic data to verify that 6-TG is a small-molecule inhibitor of USP2 that forms covalent bonds with the Cys276 residue of USP2 to inhibit its expression. This finding provides a rationale for the clinical use of 6-TG in the treatment of tumors with USP2 upregulation.

PR619

Altun et al (175) used high-throughput screening and structural optimization to identify PR619 (Fig. 4F) as a broad-spectrum inhibitor of USP2, USP4, USP5, USP7, USP8, USP15, USP20, USP28 and USP47. PR619 can inhibit USP2 in HCT116 cells and induce tumor cell death.

RA-9

Chalcones, members of the flavonoid family, can regulate the malignant behavior of tumors, such as tumor accretion, invasion and metastasis, by targeting the ubiquitin-proteasome system (176,177). Issaenko et al (178) found that the chalcone derivative RA-9 (Fig. 4G) inhibits the activity of USP2, USP5 and USP8; downregulates the expression of CCND1 in breast, ovarian and cervical cancer cells, upregulates the expression of oncogenes p53, p27 and p16 to promote apoptosis and exerts positive anticancer effects.

NSC632839

Aleo et al (179) were the first to identify NSC632839 (Fig. 4H) as a DUB inhibitor that can induce apoptosis by stabilizing the second mitochondria-derived activator of caspases. Nicholson et al (180) verified the inhibitory effects of NSC632839 and found that it inhibits both USP2 and USP7 at the half-maximal effective concentration (EC₅₀) of 45±4 and 37±1 µM, respectively.

Compound 14

Vamisetti et al (181) used a unique fluorescence quenching assay and found that Compound 14 (Fig. 4I) inhibited the protein activity of USP2 and USP7 via a non-competitive mechanism (IC₅₀=250 nM). Additionally, the fluorine atom in Compound 14 could reverse the selectivity between USP2 and USP7. Therefore, Compound 14 was identified as a reversible inhibitor of USP2 and USP7.

5. Concluding remarks and potential future directions

USP2 is closely associated with the development of several types of malignant tumors. USP2a, which has been more intensively investigated, is upregulated in prostate, gastric and lung cancers and downregulated in bladder cancer, renal clear cell carcinoma and GBM. The expression of USP2 differs among different types of breast cancers. It is low in invasive ductal carcinoma but high in estrogen receptor-positive and triple-negative breast cancers. USP2 regulates the stability of key tumor-associated proteins such as CCND1, CCNA1, MDM2, MDM4 and FAS. Therefore, targeting USP2 represents an effective strategy for the treatment of related malignancies. ML364, a small-molecule inhibitor of USP2, can cause cell cycle arrest, promote the expression of p53 and exert anti-tumor effects in vitro. In addition, other inhibitors of USP2, such as β-lapachone (a naphthoquinone), chalcone (a flavonoid), 6-TG and stigmasterol acid and its derivatives exert anti-tumor effects in different cancers.

Ubiquitination, one of the post-translational modifications, serves an important role in the development and malignant behavior of several cancers and influences protein expression and signal transduction. DUBs can regulate the stability of substrate proteins by removing ubiquitin tags, thereby regulating the cascade responses of the cell cycle, DNA damage repair, invasion, metastasis and other signaling pathways. Targeting DUBs represents an effective strategy for the treatment of cancer. Some USP-targeted drugs are undergoing investigation in phase II clinical trials. USP2 primarily influences the expression of CCND1, MDM2, p53 and other proteins by regulating ubiquitin-mediated protein degradation, which in turn affects tumor development. However, the following questions remain to be addressed: i) How do transcription factors recognize USP2 and regulate its transcription? ii) In addition to affecting the cell cycle and cell death, does USP2 regulate other biological processes? iii) What are the specific molecular mechanisms through which USP2 regulates the expression of related factors?

The function of USP2 may be related to the cell or tissue type, and deletion/overexpression of USP2 in specific cells/tissues may have different effects on biological processes in tumors. To date, studies on USP2 in tumors have mainly focused on two isomers, namely, USP2a and USP2b. Studies on other isomers in tumors are lacking. An in-depth investigation of the biological effects and specific molecular mechanisms of USP2 in different malignancies may provide a theoretical basis for the development of safe and effective targeted drugs. In conclusion, USP2 may serve as a potential therapeutic target for cancer, and the clinical significance of USP2 in developing targeted drugs should be comprehensively evaluated.

Acknowledgements

Not applicable.

Availability of data and materials

Data sharing is not applicable to this article, as no data sets were generated or analyzed during the current study.

Authors' contributions

SLZ, YG, and SJZ contributed to the review of data collection, manuscript writing and revision. SZ was involved in the consultation process and article revision. YWZ and ZW participated in the collection and arrangement of materials.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figure 1.

Ubiquitin-proteasome system. In the presence of ATP, the glycine residue at the C-terminus of ubiquitin forms a high-energy lipid bond with the SH of the cysteine residue of an E1 enzyme, and the activated ubiquitin is subsequently transferred to an E2 enzyme. In the presence of an E3 ubiquitin ligase, ubiquitin is transferred from the E2 enzyme to the substrate protein, forming an isopeptide bond with the ε-NH2 group of the Lys residue of the substrate protein. Subsequently, the C-terminus of the next ubiquitin molecule is connected to the Lys48 residue of the previous ubiquitin molecule, thus completing polyubiquitination. The ubiquitinated substrate proteins are recognized by the cap-shaped regulatory particles of the 19S proteasome and transported into the cylindrical core of 20S, where they are hydrolyzed into oligopeptides and amino acids by various enzymes and are eventually released from the proteasome, thereby completing degradation. However, deubiquitinating enzymes can reverse ubiquitination by hydrolyzing the isopeptide bonds in ubiquitinated substrate proteins and dissociating ubiquitin molecules from the substrate proteins. SH, sulfur group.

Figure 2.

Structure of the deubiquitinating enzyme USP2. USP2, ubiquitin-specific protease 2.

Figure 3.

Mechanisms through which USP2 participates in cancer-related pathways. Upper right quadrant: USP2 stabilizes the expression of CCND1, CCNA1 and Aurora-A proteins through deubiquitination and promotes cell mitosis and cell cycle progression, which in turn promotes abnormal proliferation of tumor cells. Upper left quadrant: USP2 inhibits the ubiquitin-mediated degradation of β-catenin, SMAD7 and MMP2 proteins through deubiquitination and promotes epithelial-mesenchymal transition, which in turn enhances the migratory and invasive abilities of tumor cells. Lower left quadrant: USP2 directly interacts with and stabilized MDM2/4, promoting the ubiquitin-mediated degradation of the substrate protein p53 and inhibiting p53-dependent cell death. Lower right quadrant: USP2 can stabilize cFILP protein through deubiquitination, regulate GSK3β activity to enhance the anti-apoptotic function of Akt and induce chemotherapy resistance in tumor cells. USP2, ubiquitin-specific protease 2.

Figure 4.

Chemical structural formula of inhibitors. Structural formula of (A) ML364, (B) β-lapachone, (C) LCAHA, (D) STD1T, (E) 6-TG, (F) PR619, (G) RA-9, (H) NSC632839 and (I) compound 14. LCAHA, lithocholic acid hydroxyamide; 6-TG, 6-thioguanine.

Table I.

Reported targets of USP2.

First author, year	USP2 target	Tumor	Mode of action of USP2	Pathway involved	(Refs.)
Magiera et al, 2017	CCND1	Colorectal, breast and	Deubiquitination stabilizes CCND1	Cell cycle	(62,63)
Nepal et al, 2015		hepatoma cancer	protein and promotes cell cycle progression
Kim et al, 2012	CCNA1	Bladder cancer	Deubiquitination stabilizes CCNA1 protein and promotes cell cycle progression	Cell cycle	(65)
Shi et al, 2011	Aurora-A	Pancreatic cancer	Deubiquitination stabilizes Aurora-A protein and promotes cell mitosis	Cell mitosis	(73)
Kim et al, 2018	β-catenin	Breast cancer	Deubiquitination stabilizes β-catenin protein and promotes the migratory and invasive abilities of cells	Wnt/β-catenin pathway	(84)
Bonacci et al, 2020	MDM2/4	Testicular Cancer/	Direct stabilization of MDM2/4	p53 pathway	(30,89,91)
Stevenson et al, 2007		glioblastoma	activity and promotion of ubiquitin-
Wang et al, 2014			mediated degradation of p53, the downstream target of MDM2/4
Liu et al, 2022	TWIST1	Bladder cancer	Deubiquitination stabilizes TWIST1 protein and promotes vascular remodeling	Epithelialmesenchymal transition	(53)
Tu et al, 2022	SMAD7	Glioblastoma	Deubiquitination stabilizes SMAD7 protein, reduces SMAD7 recruitment of the E3 ligase HERC3 and inhibits the TGF-β signaling pathway	TGF-β signaling pathway	(54)
Qu et al, 2015	MMP2	Breast cancer	Deubiquitination stabilizes MMP2 protein and promotes the migratory and invasive abilities of cells	Epithelialmesenchymal transition	(57)
Xiao et al, 2022	E2F4	Gastric cancer	Deubiquitination stabilizes E2F4 protein and promotes cell proliferation	Cell proliferation	(59)
Liu et al, 2018	cFILP	T-cell lymphoma	Deubiquitination stabilizes cFILP protein and promotes drug resistance in tumor cells	Endoplasmic reticulum stress	(98)
Liu et al, 2018	ITCH	T-cell lymphoma	Direct stabilization of ITCH protein and degradation of the downstream target cFILP	Endoplasmic reticulum stress	(98)
Graner et al, 2004	FAS	Prostate cancer	Deubiquitination stabilizes FAS expression and inhibits cell apoptosis	Cell apoptosis	(142)
Zhang et al, 2021	Skp2	Lung cancer	Direct stabilization of Skp2 expression and promotion of substrate protein degradation	Cell cycle	(155)

USP2, ubiquitin-specific protease 2; CCND1, cyclin D1; MDM2, murine double minute 2; SMAD, mothers against decapentaplegic; E2F4, E2F transcription factor 4; FAS, fatty acid synthase.

Table II.

USP2 inhibitors and their pharmacological mechanisms of action.

First author, year	Inhibitor	Clinical trials	ClinicalTrials.gov ID	USP targets	Tumor	Mechanism	(Refs.)
Davis et al, 2016	ML364	/	/	USP2	Breast cancer/colorectal cancer/	It inhibits USP2 deubiquitination;	(49,56)
Zhang et al, 2020					mantle cell lymphoma	promotes the degradation of CCND1 and β-catenin; blocks cell cycle progression and inhibits the proliferative, migratory and invasive abilities of tumor cells
Savage et al, 2008	β- lapachone	I/II	NCT00075933 NCT00102700 NCT00524524	USP2	/	It selectively and irreversibly inhibits the oxidation of USP2 cysteine residues, interferes with cell cycle progression and promotes apoptosis	(171)
Nguyen et al, 2021	LCAHA	/	/	USP2a	Colorectal cancer	It promotes ubiquitin-mediated degradation of CCND1 protein and induces G₀/G₁-phase progression of tumor cells	(173)
Tomala et al, 2018	STD1T	/	/	USP2a	Colorectal cancer/breast cancer	It binds directly to USP2a, inhibits CCND1 protein expression and blocks cell cycle progression	(64)
Chuang et al, 2018	6-TG	I/II	NCT00587873 NCT00504660 NCT00588536	USP2	/	It interacts with the Cys276 residue of USP2 in a non-competitive and slow-binding manner to inhibit USP2 through covalent bonding	(139)
Altun et al, 2011	PR619	/	/	USP2/4/5/7/8/15/20/28/47	Colorectal cancer	It induces tumor cell death; however, the exact mechanism remains unclear	(175)
Issaenko et al, 2012	RA-9	/	/	USP2/5/8	Breast cancer/ovarian cancer	It promotes ubiquitin-mediated degradation of CCND1 protein and upregulates the expression of P53, P27 and other tumor suppressor genes, thereby promoting P53-dependent cell death	(178)
Nicholson et al, 2008	NSC632839	/	/	USP2/7	/	It inhibits USP2 activity and stabilizes Smac protein expression, thereby inducing cell apoptosis	(180)
Vamisetti et al, 2019	Compound 14	/	/	USP2/7	/	Non-competitive mechanisms inhibit USP2 protein activity	(181)

USP2, ubiquitin-specific protease 2; LCAHA, lithocholic acid hydroxyamide; 6-TG, 6-thioguanine.