Spotlights on ubiquitin-specific proteases in lung cancer: from multifaceted pathophysiological mechanisms to potential therapeutic targets

USP7

Ubiquitin-Specific Peptidase 7 (USP7) comprises two domains: an N-terminal TRAF domain and a C-terminal ubiquitin-like domain, the TRAF domain of USP7 binds to the [P/A/E]-X-X-S motif in multiple substrates, such as p53, MDM2, MDM4, pyruvate kinase M2, Epstein-Barr nuclear antigen 1, microchromosome maintenance complex-associated protein, F-box protein 38, tripeptidyl peptidase 1, and ubiquitin-conjugating enzymes E2 and E1,it plays a crucial role in maintaining normal protein function and preventing disease, participating in the construction and regulation of protein interaction networks, and sustaining cellular genomic stability and survival (Carreira et al., 2023; Choi et al., 2020; Honda, Okumura & Chihara, 2023; Jagannathan et al., 2014; Park & Baek, 2023; Penev et al., 2022; Qi et al., 2020).

Research has revealed that USP7 stabilises the oestrogen receptor β (ERβ) through deubiquitination, inhibits the SUMOylation of peroxiredoxin-3 (PRDX3), and mitigates reactive oxygen species (ROS) accumulation, this mechanism promotes the development of osimertinib resistance in non-small cell lung cancer (NSCLC), offering novel insights for overcoming osimertinib resistance challenges (Meng et al., 2024). He et al. (2023) indicate that USP7, acting as a deubiquitinating enzyme for c-Abl, induces the accumulation of c-Abl in the cytoplasm, promotes its phosphorylation, and stabilises hexokinase 2 (HK2), this process drives glycolysis in non-small cell lung cancer tumour cells, ultimately facilitating tumour cell proliferation and metastasis. Huang et al. (2021) demonstrated that USP7 promotes the autoregulation of SMAD3 through its deubiquitinating enzyme activity, thereby inhibiting the proliferation of p53-deficient lung cancer cells. Furthermore, studies have indicated that USP7 stabilises KRAS through deubiquitination, thereby stimulating the proliferation of NSCLC cells, moreover, when USP7 inhibitors are combined with KRAS inhibitors, they exhibit a synergistic inhibitory effect on NSCLC cells resistant to KRAS inhibitors (Huang et al., 2024). Further research indicates that USP7, by binding to the PVDS motif of Raf-1, can deubiquitinate multiple polyubiquitin chains on Raf-1, thereby reducing the extent of its threonine phosphorylation, this process inhibits the activity of the ERK1/2 signalling pathway, consequently arresting the proliferation of lung adenocarcinoma cells (Park, Hwang & Baek, 2022).

USP9X

Ubiquitin-Specific Peptidase 9, X-linked (USP9X) is an X-linked USP protein associated with embryonic and neural development (Kugler & Lasko, 2009). It possesses unique functional characteristics, capable of both promoting and inhibiting apoptosis, with these functions mediated by its deubiquitination effects on key components of the apoptotic signalling network (Park & Baek, 2022; Sulkshane et al., 2021). Previous studies have demonstrated that USP9X modulates the expression of stress-responsive and pro-apoptotic kinases, thereby initiating the JNK apoptotic signalling cascade (Niu et al., 2022; Park & Baek, 2022). In cancer cells, USP9X also exerts regulatory control over numerous tumour functions, encompassing cell adhesion, cell polarity, cell death, and inflammatory processes (Gao et al., 2024; Moon et al., 2021).

Research indicates that USP9X serves as a crucial interacting partner for histone demethylase 4C (KDM4C), capable of deubiquitinating KDM4C and maintaining its stability, this subsequently reduces the level of histone H3 lysine 9 trimethylation (H3K9me3) at the transforming growth factor-β2 (TGF-β2) promoter, ultimately promoting radiotherapy resistance in lung cancer cells by activating the SMAD/ATM/Chk2 signalling pathway (Jie et al., 2021). Moreover, REV1 plays a pivotal role in the development of radiotherapy resistance in lung cancer, its abnormally high expression is triggered by deubiquitination mediated by USP9X,specifically, acting as a scaffold protein, REV1 promotes the binding of Rad18 to CTH and accelerates CTH degradation,this induces abnormalities in the tumour metabolic microenvironment, ultimately leading to the emergence of radiotherapy resistance (Chen et al., 2024).

USP10

Ubiquitin-Specific Peptidase 10 (USP10), as a cysteine protease, primarily mediates the thiol-dependent hydrolysis of ester, thioester, or peptide bonds formed by the carboxyl-terminal glycine residue of ubiquitin, through this hydrolysis process, the Ub moiety is removed from the target protein (Meyer et al., 2020). Among numerous targets, the tumour protein p53, cystic fibrosis transmembrane conductance regulator, adenosine monophosphate (AMP)-activated protein kinase alpha, silencer protein 6, and nuclear factor κB essential regulator constitute key targets of USP10, these targets are profoundly involved in vital cellular processes, which fully accounts for USP10’s pivotal role in cellular metabolism, signal transduction, and tumourigenesis (Tao et al., 2022; Wang et al., 2023c).

He et al. (2021b) demonstrated that USP10 activates phosphatase and tensin homolog (PTEN) by removing the K63-linked polyubiquitin chain from PTEN, thereby inhibiting the AKT/mTOR signalling pathway and ultimately suppressing the proliferation of NSCLC tumour cells. Additionally, studies have reported that USP10, acting as a deubiquitinating enzyme for histone deacetylase 6 (HDAC6), regulates the stability of HDAC6,when USP10 is knocked down or inhibited, cells harbouring mutant or loss-of-function p53 become sensitive to cisplatin (Zhang et al., 2021). Moreover, USP10 promotes the proliferation, migration, and invasion of NSCLC cells by deubiquitinating and thereby stabilising the eukaryotic initiation factor 4G1 (EIF4G1), leading to increased EIF4G1 levels (Li et al., 2024a).

USP14

Ubiquitin-Specific Peptidase 14 (USP14), comprising 494 amino acids and two distinct domains, features a ubiquitin-like domain at its N-terminus that regulates proteasome activity, whilst its C-terminal USP domain governs the deubiquitinating enzyme activity of USP14 (Boughton et al., 2021). Research indicates that USP14 activity is subject to stringent regulation, with its regulatory mechanisms encompassing association with the proteasome and various post-translational modifications, such as phosphorylation (Wang et al., 2021a; Wang, Zhang & Wu, 2023b). Once USP14 becomes dysregulated, it may trigger a series of pathological abnormalities, such as cancer, neurodegenerative diseases, autophagy, immune responses, and viral infections (Wang et al., 2021a).

Research has revealed that following interaction between ceramide synthase 1 (CERS1) and USP14, the PI3K/AKT/mTOR signalling pathway is downregulated, thereby inhibiting the proliferation, migration, invasion, and brain metastasis capabilities of NSCLC cells (Yan et al., 2021). Additionally, research indicates that in lung adenocarcinoma, overexpression of USP14 promotes the accumulation of β-catenin (β-catenin 17), thereby driving tumour cell proliferation, furthermore, USP14 regulates lung tumourigenesis via apoptotic and autophagic pathways and is critically important for NSCLC migration, this process is achieved by deubiquitinating actin cross-linking family protein 7 (Acf7) and maintaining its stability (Lee et al., 2023). Moreover, in cisplatin-resistant lung cancer, USP14 exhibits high expression levels and correlates with poor prognosis. Reducing USP14 expression enhances cisplatin’s antitumour efficacy against lung cancer cells, whilst aptamers—siRNA nano-zinc carriers represent an innovative drug delivery system for targeted therapy, they effectively suppress resistance in cisplatin-resistant lung cancer cells and demonstrate antitumour efficacy in vivo (Zhao et al., 2023).

USP22

Ubiquitin-Specific Peptidase 22 (USP22) functions as a subunit of the human Spt-Ada-Gcn5—acetyltransferase (SAGA) complex, plays a pivotal role in histone modification, it facilitates histone acetylation via GCN5 whilst simultaneously deubiquitinating histones H2B and H2A through its own activity, and is deeply involved in regulating gene transcription (Grasser, Rubio & Barneche, 2021; Zhou et al., 2021). In normal tissues, USP22 actively participates in the growth, development, and phenotypic conversion processes of T cells and B cells, in the context of cancer, USP22 may induce alterations in the immune microenvironment and has been identified as a significant biomarker for cancer mortality, being closely associated with cancer recurrence, metastasis, and patient survival outcomes (Guo et al., 2022).

Research indicates that USP22 expression is promoted by Activator Protein 2 (AP2) and c-Myc, with AP2 acting as a key transcription factor driving USP22 expression, this may partially occur through upregulating USP22’s transcriptional levels, thereby enhancing the malignancy of NSCLC (Sun et al., 2022). USP22 not only enhances EGFR signalling activity but also promotes resistance to EGFR-TKIs in lung cancer patients, furthermore, USP22 drives the acquisition of stem cell-like characteristics in NSCLC cells by regulating BMI1 signalling (Thai et al., 2021). Further research has demonstrated that, through the establishment of a mouse xenograft model, USP22 overexpression induces gefitinib resistance, moreover, in vitro experiments show that deubiquitination of the mouse homologue of the double microbody 2 gene (MDM2) inhibits ferroptosis in NSCLC cells (Lu, Li & Xu, 2024a).

USP35

Ubiquitin Specific Peptidase 35 (USP35) belongs to the peptidase C19 family and plays a crucial role in the execution of multiple cellular functions, recent research evidence indicates that USP35 is deeply involved in regulating the cell cycle, determining cell fate, and driving cancer progression, specifically, USP35 effectively modulates the mitotic process by deubiquitinating Aurora B kinase, thereby preventing its degradation by the APC/CDH1 complex (Park et al., 2023). According to relevant reports, USP35 can also regulate the abundance of mitochondrial fusion protein 2 (MFN2) and PARK2-mediated mitochondrial autophagy (Qin et al., 2022). Moreover, USP35 inhibits NFκB activation by stabilising ABIN-2, thereby impeding tumour growth (Zou et al., 2024).

Research indicates that knocking down USP35 induces a series of effects: not only does it inhibit the growth, colony formation, and tumour invasion of lung cancer cells, but it also accelerates ferroptosis-mediated lipid peroxidation and intracellular iron overload, this leads to reduced glutathione (GSH) levels and decreased glutathione peroxidase 4 (GPX4) activity, while simultaneously enhancing the sensitivity of lung cancer cells to cisplatin and paclitaxel (Tang et al., 2021). Furthermore, the stability of USP35 is regulated or maintained by fusin (FUS), USP35 interacts with vascular endothelial growth factor A (VEGFA), and FUS-stabilised USP35 promotes the growth, invasion, and angiogenesis of NSCLC cells by deubiquitinating VEGFA (Li et al., 2024c). Liu et al. (2022) demonstrated that USP35 directly associates with the apoptosis-inhibiting protein BIRC3, modifying it via its deubiquitinating enzyme activity to prevent proteasomal degradation, by stabilising BIRC3, USP35 inhibits cisplatin-induced apoptosis in NSCLC cells, ultimately conferring resistance to cisplatin. Wang et al. (2022) indicate that in NSCLC, USP35 alleviates endoplasmic reticulum stress-induced apoptosis by stabilising ribosomal binding protein 1.

USP38

Recombinant Ubiquitin-Specific Peptidase 38 (USP38) was initially identified as a negative regulator of type I interferon signalling, acting by modulating the ubiquitination of TANK-binding kinase 1 (He et al., 2021a). Simultaneously, it enhances its own stability through automated deubiquitination (Hu et al., 2024). Research indicates that USP38 possesses the capacity to recognise multiple types of ubiquitin modifications, encompassing K11 (Wang et al., 2024), K27 (Yi et al., 2022), K33 (Zhang et al., 2024), K48 (Zhang et al., 2024) and K63 (Zhan et al., 2020). Moreover, USP38 exerts crucial regulatory functions across multiple pathways, including inflammatory responses, antiviral defence, cell proliferation, migration, invasion, DNA damage repair, and chemotherapy resistance (Huang et al., 2022; Wang et al., 2023a; Wang et al., 2021b; Yi et al., 2022; Zheng et al., 2022).

Previous studies have demonstrated that USP38 promotes the proliferation of lung cancer cells by stabilising the c-myc protein (Xu et al., 2021). Zhang et al. (2024) noted that in LUAD cells, USP38 participates in maintaining the stability of the transcription factor KLF5 through its deubiquitinating activity, thereby promoting tumour cell proliferation, specifically, USP38 knockdown increases KLF5 ubiquitination levels, whereas USP38 overexpression reduces KLF5 ubiquitination, this occurs because USP38 removes K33- and K48-linked ubiquitin chains from KLF5, thereby inhibiting proteasomal degradation of the protein and ultimately increasing KLF5 expression, consequently, the USP38/KLF5 axis holds promise as a potential therapeutic target for lung adenocarcinoma.

USP39

Ubiquitin-Specific Peptidase 39 (USP39) functions as a pivotal splicing factor, playing a crucial role in cellular activities, it is essential for maintaining the integrity of the mitotic spindle checkpoint and regulates the mRNA levels of Aurora B (Francois et al., 2022). Moreover, within NSCLC tissues, USP39 further stabilises the pyruvate dehydrogenase E1α subunit within the pyruvate dehydrogenase complex through deubiquitination, this action regulates the conversion of pyruvate into the tricarboxylic acid (TCA) cycle, ultimately influencing mitochondrial respiration, cellular proliferation, and tumour growth (Becirovic et al., 2024).

Mechanism of action and selectivity profile of USPs inhibitors

USPs inhibitors competitively or non-competitively block interactions with ubiquitin chains or ubiquitinated substrates by binding with high affinity to the catalytic active site of the target enzyme (Kategaya et al., 2017). This inhibitory effect prevents the efficient removal of ubiquitin tags from substrate proteins, leading to persistently elevated ubiquitin levels. Its downstream effects manifest primarily in two ways: firstly, for proteins primarily degraded via the proteasome pathway, sustained ubiquitination promotes their recognition and degradation by the 26S proteasome, thereby reducing the intracellular abundance of oncogenic proteins; Secondly, it disrupts numerous physiological processes dependent on reversible ubiquitination, such as DNA damage repair, cellular metabolism, and immune signalling pathways,this ultimately induces cell cycle arrest, apoptosis, or enhances the efficacy of chemotherapy/immunotherapy (Mani & Gelmann, 2005). Given the high structural similarity among catalytic domains within the USP family, achieving high selectivity represents a core challenge in inhibitor development. Current design strategies primarily leverage relatively variable regions surrounding the active site or unique allosteric pockets, alongside differences in spatial distribution among family members within specific organelles or protein complexes, this approach confers selectivity for particular USPs, enabling precise regulation of specific pathways while minimising off-target toxicity (Kazi et al., 2025).

USP7 inhibitors

Several USP7 inhibitors have been developed to date, including representative compounds such as P5091 (as shown in Fig. 3B), HBX19818 (as shown in Fig. 3C), and GNE-6776 (as shown in Fig. 3D), P5091 demonstrated significant immunomodulatory effects in lung cancer models, reducing IL-10 levels while elevating IFN-γ and TNF-α levels, and enhancing the cytotoxicity of both CD4+ and CD8+ T cells (Dai et al., 2020). HBX19818 inhibits the deubiquitination of polyubiquitinated p53 by USP7, providing experimental evidence for its role in stabilising the p53 protein and exerting tumour-suppressing effects (Reverdy et al., 2012). Furthermore, our team demonstrated that USP7 inhibitors HBX41108 and GNE-6776, either alone or in combination with AMG510 (as shown in Fig. 3E), more effectively suppressed the proliferation of AMGR cells, this approach may represent an effective therapeutic strategy for inhibiting NSCLC cell proliferation and overcoming AMG510 resistance, particularly in KRAS G12C-mutated NSCLC (Huang et al., 2024). Concurrently, the new generation of USP7 inhibitors demonstrates enhanced potency and selectivity. The inhibitors FT671 (as shown in Fig. 3F) and FT827 (as shown in Fig. 3G) exhibit high-affinity and high-specificity inhibition of USP7 in vitro and in human cells. Co-crystallisation structures reveal that both compounds target a dynamic pocket near the catalytic centre of the USP7 autoinhibitory apolipoprotein structure. In vivo, FT671 destabilises USP7 substrates including MDM2, elevates p53 levels, drives transcription of p53 target genes, induces expression of the tumour suppressor gene p21, and ultimately inhibits tumour growth in mice (Schauer et al., 2020; Turnbull et al., 2017).

USP14 inhibitors

IU1 (as shown in Fig. 3H), as the first reported selective inhibitor of USP14, binds to USP14, inducing a conformational change in the BL2 loop, this inhibits its deubiquitinating activity, enhances proteasomal degradation, suppresses lung cancer cell proliferation, and induces apoptosis (Srinivasan et al., 2023). Furthermore, USP14 acts as a regulator of the primary double-strand break repair pathway in non-small cell lung cancer (NSCLC). Ionizing radiation treatment increases USP14 levels and double-strand break recruitment in NSCLC cell lines, pharmacological inhibition of USP14 using IU1 enhances radiosensitivity in NSCLC cell lines (Sharma & Almasan, 2020). b-AP15 (as shown in Fig. 3I) recognises the 19S proteasome, inhibiting the activity of the deubiquitinating enzymes USP14 and UCHL5 associated with the 19S regulatory particle, this leads to the accumulation of polyubiquitin chains, thereby suppressing the proteasome’s degradation function, ultimately, it induces cell death by activating the cathepsin-D-dependent lysosomal apoptosis pathway (Chitta et al., 2015).

Other USP member inhibitors

WP1130 (as shown in Fig. 3J) is a commonly used USP9X inhibitor that efficiently degrades ALDH1A3 and exerts a marked inhibitory effect on the growth of orthotopic xenografts derived from glioblastoma stem cells (Chen et al., 2019). Moreover, Gambogic acid (NGA) (as shown in Fig. 3K), an active component of garlic, also acts as a USP9X inhibitor, It significantly suppresses the proliferation of osteosarcoma cells through ubiquitin-proteasome-mediated degradation of SOX2 in both in vitro and in vivo experiments, whilst exhibiting minimal toxic side effects (Chen et al., 2020). ML323 (as shown in Fig. 3L) is a novel USP1/UAF1 deubiquitinating enzyme complex inhibitor exhibiting reversible nanomolar-level inhibitory activity and outstanding selectivity towards USP1/UAF1, furthermore, ML323 potentiates the cytotoxicity of cisplatin and increases endogenous monoubiquitination levels of PCNA and FANCD2 (Dexheimer et al., 2010). The secoiridoid glycoside gentiopicroside (as shown in Fig. 3M), acting as a specific inhibitor of USP22, attenuates the immunosuppressive function of Tregs by downregulating Foxp3 expression, concurrently, it suppresses tumour PD-L1 expression, thereby enhancing anti-tumour immunity and inhibiting tumour growth in mice with lung adenocarcinoma (Lu et al., 2024b).

Based on existing research, developing selective inhibitors targeting specific deubiquitinating enzymes represents another precision strategy. These enzymes exhibit preferences for particular ubiquitin chain types; for instance, members such as USP35 and USP38 tend to remove (Lysine residue at position 48 of the ubiquitin molecule)—linked ubiquitin chains from substrate proteins, thereby stabilising oncogenic proteins like BIRC3 and KLF5 and inducing drug resistance, conversely, USP10 and USP38 also participate in editing K63-linked ubiquitin chains, regulating the PTEN/AKT/mTOR pathway and DNA repair processes respectively (Gao et al., 2023; Hu et al., 2017). Furthermore, USPs participate in regulating key signalling pathways such as p53 and NF-κB, as illustrated in Table 2. Inhibiting USPs may disrupt these pathways, potentially impeding the proliferation and survival of lung cancer cells (Jiang et al., 2025; Li et al., 2024b; Pan & Deng, 2025; Wei et al., 2025). USP inhibitors may also induce apoptosis by elevating cellular stress levels and potentially reverse chemotherapy resistance in lung cancer cells. As illustrated in Fig. 4, this offers novel therapeutic avenues for intervening in tumour proliferation, metastasis, and treatment resistance (Chen, Liu & Zhou, 2021).