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Research Progress for Targeting Deubiquitinases in Gastric Cancers

Simple Summary The deubiquitinase-mediated cleavage of ubiquitin chains from substrate proteins plays a crucial role in regulating protein degradation, activities, interactions, and localization. The dysregulation of multiple deubiquitinases has been implicated in various human diseases, especially cancer. More importantly, many small molecules targeting oncogenic deubiquitinases have been discovered, some of which have exhibited promising anti-cancer effects and entered clinical trials. Gastric cancer remains one of the most common and fatal malignancies. In this review, we aim to summarize the multifaceted roles of deubiquitinases in gastric tumorigenesis. We also present the upstream regulation of specific deubiquitinases and the research progress of several deubiquitinase-associated small molecules for gastric cancer therapy. Together, this review will improve our understanding of the biological role of deubiquitinases as well as the therapeutic potential of targeting deubiquitinases in gastric cancer. Abstract Gastric cancers (GCs) are malignant tumors with a high incidence that threaten global public health. Despite advances in GC diagnosis and treatment, the prognosis remains poor. Therefore, the mechanisms underlying GC progression need to be identified to develop prognostic biomarkers and therapeutic targets. Ubiquitination, a post-translational modification that regulates the stability, activity, localization, and interactions of target proteins, can be reversed by deubiquitinases (DUBs), which can remove ubiquitin monomers or polymers from modified proteins. The dysfunction of DUBs has been closely linked to tumorigenesis in various cancer types, and targeting certain DUBs may provide a potential option for cancer therapy. Multiple DUBs have been demonstrated to function as oncogenes or tumor suppressors in GC. In this review, we summarize the DUBs involved in GC and their associated upstream regulation and downstream mechanisms and present the benefits of targeting DUBs for GC treatment, which could provide new insights for GC diagnosis and therapy.


Introduction
Gastric cancer (GC) is the fifth most common cancer and the fourth leading cause of cancer-related mortality worldwide. Based on estimates from the GLOBOCAN database, more than 1 million new GC cases and an estimated 769,000 GC deaths occurred worldwide in 2020 [1]. As with other cancer types, GC development is a multistage process involving genetic and epigenetic alterations [2,3]. In addition to host factors, other etiological factors, including a high-salt diet, tobacco use, and infectious agents, such as Helicobacter pylori etiological factors, including a high-salt diet, tobacco use, and infectious agents, such as Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV), also play a significant role in the initiation and progression of GC [4]. Despite advances in understanding the mechanisms underlying GC and improved therapies, almost one-third of patients with GC are diagnosed at a late stage, with a 5-year survival rate below 20% [3,5,6]. Therefore, there is an urgent need to develop novel biomarkers and therapeutic targets for treating GC.
Deubiquitinases (DUBs) are isopeptidases that can cleave a single ubiquitin or entire ubiquitin chains from target proteins, thereby counteracting protein ubiquitylation, a post-translational modification fundamental for the regulation of protein stability, activity, subcellular localization, and interactions [7,8]. DUBs are involved in many physiological processes, such as apoptosis, autophagy, and the cell cycle, and pathological processes, such as neurodegenerative diseases and cancers [9][10][11][12][13][14]. Hence, DUBs have attracted attention as therapeutic targets, and DUB inhibitors have been developed, with some now in preclinical development or clinical trials [15]. To date, approximately 100 DUBs have been reported and are classified into seven subfamilies based on their sequence and structural similarities as follows: ubiquitin-specific proteases (USPs), ubiquitin carboxy-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Jab1/MPN domain-associated metalloenzymes (JAMMs), Machado-Joseph disease proteases (MJDs), the monocyte chemotactic protein-induced protease family (MINDYs), and Zn-finger and UFSP domain proteins (ZUFSPs) (Figure 1) [16]. During the past decades, many studies have demonstrated that multiple DUBs are implicated in the development of GC. In this review, we first summarize and discuss the DUBs related to GC, with a summary of their mechanisms of action and regulation (Table  1). In addition, we document the potential of pharmacological interventions that target DUBs (Table 2). Taken together, the present review aims to provide a better understanding of the molecular mechanisms underlying GC development associated with DUBs and how they can be targeted for GC treatment. During the past decades, many studies have demonstrated that multiple DUBs are implicated in the development of GC. In this review, we first summarize and discuss the DUBs related to GC, with a summary of their mechanisms of action and regulation (Table 1). In addition, we document the potential of pharmacological interventions that target DUBs (Table 2). Taken together, the present review aims to provide a better understanding of the molecular mechanisms underlying GC development associated with DUBs and how they can be targeted for GC treatment.

USP10
None reported None reported USP10 was an independent prognostic marker for patients with GC. [31] 3-Deazaneplanocin A treatment upregulates USP10 expression by reducing EZH2 binding on its promoter p53 Stabilization of p53 by USP10 seemed to be correlated with the sensitivity of GC cells to 3-Deazaneplanocin A. [32] CircCOL1A2 upregulates USP10 expression by sponging miR-1286 RFC2 CircCOL1A2 sponges miR-1286 to promote GC cell migration and invasion by increasing USP10 level to stabilize RFC2.

USP22
None reported None reported Coordinate expression of USP22 and BMI1 correlated with GC progression and treatment failure. [45] None reported None reported High expression of USP22 correlated with GC progression and has synergistic effects with c-Myc. [46] None reported BMI1 USP22 contributed to gastric CSC stemness maintenance and GC progression by stabilizing BMI1. [47] None reported None reported USP22 siRNA-loaded nanoliposomes decorated with CD44 antibodies selectively target and eliminate CD44 + GC stem cells. [48] None reported None reported Positive co-expression of USP22 and HSP90 might be more effective in predicting prognosis of GC. [49] None miR-500 inhibited CYLD expression via targeting its 3 -UTR None reported Upregulated miR-500 promoted GC cell proliferation and cisplatin resistance by repressing CYLD, OTUD7B, and TAX1BP1 and activating NF-κB signaling. [69] miR-130b inhibited CYLD expression via targeting its 3 -UTR None reported Upregulated miR-130b promoted GC cell proliferation and inhibited apoptosis by repressing CYLD. [70] miR-20a negatively regulated CYLD expression by targeting its 3 UTR None reported Upregulated miR-20a augmented the resistance of GC cells to cisplatin by inhibiting CYLD and activating NF-κB signaling and its downstream targets. [71] miR-425-5p negatively regulated CYLD expression by targeting its 3 UTR None reported Upregulated miR-425-5p may promote GC cell migration and invasion by repressing CYLD. [72] CYLD expression was inversely correlated with hypermethylation of its promoter, which could be induced by some infectious agents

None reported
Decreased CYLD level may be associated with gender, patient's age, high grade, and no lymph-node metastasis in GC patients. [73] LncRNA CRAL functions as a ceRNA to upregulate CYLD expression via absorbing miR-505 None reported LncRNA CRAL improved cisplatin resistance via the miR-505/CYLD/AKT axis in GC cells. [74] miR-454 inhibited CYLD expression via targeting its 3 -UTR None reported miR-454 supported survival and induced oxaliplatin resistance in GC cells by repressing CYLD. [75] Exosomal miR-588 from M2 polarized macrophages inhibited CYLD expression via targeting its 3 -UTR None reported Exosomal miR-588 contributed to cisplatin GC cell resistance by repressing CYLD. [76] ZNF333 decreased CYLD level by binding to its promoter None reported ALKBH5 promoted the process of bile-acid-induced gastric intestinal metaplasia by ZNF333/CYLD/CDX2 pathway.

CSN5
None reported RUNX3 CSN5 facilitates nuclear export and degradation of RUNX3. [101] None reported None reported CSN5 knockdown inhibited proliferation and promoted apoptosis of GC cells by regulating p53-related apoptotic pathways. [102] None reported p14ARF CSN5 potentiated GC progression by decreasing p14ARF expression through non-ubiquitin pathway. [103] The activity of CSN5 was enhanced by IKKβ, and IKKβ expression was inhibited by DAPK1 PD-L1 Overexpression of DAPK1 enhanced NK cell killing and suppressed tumor immune evasion in GC cells by inhibiting the IKKβ/CSN5/PD-L1 axis. [104]

MJDs
Ataxin-3 None reported None reported Decreased Ataxin-3 expression correlated with clinicopathologic features of GC. [106]  None reported p14ARF CSN5 potentiated GC progression by decreasing p14ARF expression through nonubiquitin pathway. [103] The activity of CSN5 was enhanced by IKKβ, and IKKβ expression was inhibited by DAPK1

MJDs
Ataxin-3 None reported None reported Decreased Ataxin-3 expression correlated with clinicopathologic features of GC.
[106] None reported p14ARF CSN5 potentiated GC progression by decreasing p14ARF expression through nonubiquitin pathway. [103] The activity of CSN5 was enhanced by IKKβ, and IKKβ expression was inhibited by DAPK1

MJDs
Ataxin-3 None reported None reported Decreased Ataxin-3 expression correlated with clinicopathologic features of GC.

USPs and GC
USPs constitute the largest family of human DUBs, with 56 members. As shown in Figure 2, at least 25 USPs are linked to GC.    [113] IC50, the half maximal inhibitory concentration.

USPs and GC
USPs constitute the largest family of human DUBs, with 56 members. As shown in Figure 2, at least 25 USPs are linked to GC. Figure 2. USP-related upstream regulation and downstream mechanisms in GC. USPs marked in red and green represent oncoproteins and tumor suppressors, respectively, while purple-labeled USPs play a dual role. The text in the outer and inner circles of the USPs describes the upstream regulatory events and downstream substrates or signaling pathways, respectively.

USPs and GC
USPs constitute the largest family of human DUBs, with 56 members. As shown in Figure 2, at least 25 USPs are linked to GC.  [113] IC50, the half maximal inhibitory concentration.

USPs and GC
USPs constitute the largest family of human DUBs, with 56 members. As shown in Figure 2, at least 25 USPs are linked to GC.

USP3
Studies have highlighted the role of USP3 in GC progression. USP3 is overexpressed in GC cells and tissues and can serve as a prognostic marker for patients [20,21]. USP3 facilitates GC cell growth and metastasis in vitro and in vivo by modulating cell cycle

USP3
Studies have highlighted the role of USP3 in GC progression. USP3 is overexpressed in GC cells and tissues and can serve as a prognostic marker for patients [20,21]. USP3 facilitates GC cell growth and metastasis in vitro and in vivo by modulating cell cycle control and epithelial-mesenchymal transition (EMT)-related molecules [20]. Wu et al. identified differentially expressed proteins in BGC-823 cells stably expressing USP3 [21]. Among them, SUZ12, a scaffolding component of the PRC2 complex, and COL9A3/COL6A5, collagen family members, were deubiquitinated and stabilized by USP3, which accounts for its role in promoting invasion and migration [21,22]. In addition to SUZ12, the core PRC2 complex also comprises the histone methyltransferase EZH2 and the scaffolding component EED. As an epigenetic regulator complex, PRC2 trimethylates lysine 27 on histone H3 tails to modulate its several thousand target genes, including various genes involved in cancer proliferation, migration, and invasion, such as E-cadherin and p14ARF [114]. However, it is unknown whether these three proteins are involved in the USP3-directed proliferation of GC cells, and future studies should examine their oncogenic roles in GC.
Additionally, non-coding RNAs, including circular RNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs), play critical roles in modulating GC [115][116][117][118]. Hsa_circ_0017639, a circular RNA, is increased in GC cell lines and promotes proliferation and migration by increasing USP3 expression by sponging miR-224-5p [23]. In addition, Jin et al. demonstrated that exosomal lncRNA SND1-IT1 secreted from GC cells not only recruited DDX54, a DEAD-box RNA helicase that binds to specific RNAs, to enhance USP3 mRNA stability, but also bound to miR-1245b-5p to upregulate USP3 expression, thus leading to SNAIL1 stabilization and inducing the malignant transformation of gastric mucosa cells [24]. Taken together, these studies suggest that targeting USP3 may be a potential treatment for GC.

USP7 and USP11
USP7, also called herpes-virus-associated ubiquitin-specific protease, deubiquitinates many substrate proteins involved in the cell cycle, DNA damage responses, and immune responses [119,120]. Accordingly, the aberrant expression and activity of USP7 have been found in GC. Wang et al. showed that USP7 is highly expressed in GC and increases the expression of programmed cell death protein 1 (PD-L1), a pivotal immune checkpoint molecule that decreases T-cell immune responses [26]. Furthermore, USP7 knockdown or inhibition with its inhibitor Almac4, developed by Gavory et al., conferred GC cell sensitivity to T-cell cytotoxicity, reduced proliferation, and induced cell cycle arrest by stabilizing p53, which interacts with USP7 [26,107,121]. Similarly, the USP7 inhibitor C9, a quinazolin-4(3H)-one derivative, also suppressed GC cell proliferation by upregulating p53 and its downstream target p21 [108].
To better understand the USP7 binding network in tumor cells, Anna et al. performed affinity purification coupled with mass spectrometry to identify interactions in GC cells overexpressing USP7. In addition to several reported binding proteins (such as USP11 and TRIP12), this study also identified DHX40 and DDX24, two DEAD/DEAH-box RNA helicases, as novel targets of USP7, providing preliminary evidence that USP7 may regulate RNA metabolism [25]. Furthermore, these interactions were confirmed with nasopharyngeal carcinoma cells. However, the role of these USP7 substrates in GC carcinogenesis were not investigated [25]. Interestingly, a recent study suggested that USP11 might play an oncogenic role in GC by affecting RhoA and Ras-mediated signaling pathways [34]. Moreover, a previous study indicated that H. pylori decreases the expression and activity of USP7 in infected GC cells. However, the exact function of H. pylori in regulating USP7 expression remains unclear [122].
In addition, Zhang et al. found that USP7 stabilizes hnRNPA1 in cancer-associated fibroblasts (CAFs) to contribute to the packaging and release of exosomes containing miR-522. Paclitaxel and cisplatin also promoted miR-522 secretion by activating the USP7/hnRNPA1 axis. The lipoxygenase ALOX15 is important in mediating the lipid peroxidation that drives ferroptosis. Further, exosomal miR-522 repressed ALOX15 expression and lipid peroxide accumulation, inhibited ferroptosis, and ultimately lead to acquired chemoresistance in GC cells [27].

USP9X, USP36, and USP49
USP9X has been shown to function as either an oncogene or tumor suppressor, depending on the type of cancer [123]. Increased USP9X expression is associated with a poor prognosis in patients with GC, suggesting an oncogenic role [28]. Consistent with this evidence, another study showed that silencing USP9X represses the migration, invasion, and colony formation ability of GC cells. Moreover, hsa_circ_0008434, a miRNA sponge for miR-6838-5p, increased USP9X expression and promoted GC progression [29].
Hippo signaling has been implicated in regulating cell growth, metastasis, and chemoresistance in GC [124]. As the core downstream effectors, YAP and TAZ activity is controlled by a conserved kinase cassette. In mammals, once Hippo signaling is activated, MST1/2 kinases phosphorylate and activate LATS1/2 kinases, which further phosphorylate YAP/TAZ for cytoplasmic sequestration or degradation. Inhibiting the Hippo pathway leads to the nuclear translocation of YAP/TAZ, which regulate target gene expression by binding to TEAD coactivators [125,126]. Additionally, several DUBs activate or repress Hippo signaling through different mechanisms [127]. For instance, USP9X promotes breast cancer cell survival and attenuates cell sensitivity to chemotherapy by deubiquitinating and stabilizing YAP [128]. Zhang et al. demonstrated that LINC01433, a lncRNA positively related to GC progression, increased YAP stability by enhancing its interaction with USP9X and decreased YAP phosphorylation by weakening its association with LATS1. Because YAP binds to the LINC01433 promoter and activates its transcription, this positive feedback loop could be a therapeutic target for GC treatment [30].
Two studies have suggested that DUB1 and USP49 also contribute to GC development by regulating the Hippo signaling pathway [58,67]. DUB1 is a short form of USP36, but its role in tumorigenesis remains largely unknown [129,130]. However, it has a role in GC progression. Wang et al. found that DUB1 is capable of interacting with, deubiquitinating, and stabilizing the Hippo signaling effector TAZ [58]. USP49 silencing decreases cell proliferation, migration, and invasion and enhances GC cell sensitivity to chemotherapy; however, this effect can be reversed by YAP1 overexpression. Because USP49 is a target gene modulated by YAP1/TEAD4, this result indicates that USP49 and YAP1 form a positive feedback loop to support the malignant progression of GC [67].

USP10
USP10 plays a dual role in different human cancer types [131]. Zeng et al. showed that USP10 expression was lower in GC cell lines and clinical samples than in their noncancerous counterparts. More importantly, decreased USP10 expression indicates several highly malignant clinicopathological features and poor survival in patients with GC, suggesting that USP10 may be a prognostic biomarker [31]. Additionally, the calcium-binding protein S100A12 was found to be a GC prognostic marker, and its levels correlated with USP10 [132,133]. Given that USP10 and S100A12 are both located in the cytoplasm, USP10 may regulate the stability of S100A12 via deubiquitylation [132]. Therefore, future studies should investigate the role of USP10 in GC proliferation and metastasis. Moreover, the USP10-mediated stabilization of p53 likely contributes to the sensitivity of GC cells to 3-deazaneplanocin A, a histone methylation inhibitor that depletes EZH2, the enzymatic component of the PRC2 complex that catalyzes H3K27me3 [32]. As p53 turnover is regulated by a variety of DUBs [134], whether these DUBs influence chemotherapy resistance in GC cells warrants further study. While these results indicate that USP10 functions as a Cancers 2022, 14, 5831 9 of 23 tumor suppressor, one study showed that USP10 promoted GC cell migration and invasion by stabilizing replication factor C subunit 2 [33]. Collectively, the function of USP10 in GC remains unclear and requires further studies.

USP13, USP29, and USP37
As a master transcriptional factor that induces EMT, Snail is frequently overexpressed in tumors and drives tumor progression, cell survival, metastasis, and stem cell properties [135]. E3 ligases and DUBs are involved in the phosphorylation-dependent ubiquitination and proteasomal degradation of Snail [135]. For instance, GSK-3β phosphorylates Snail at six Ser sites. Snail phosphorylation at four Ser sites (Ser-107, 111, 115, and 119) causes cytoplasmic localization, whereas phosphorylation at two Ser sites (Ser-96 and 100) promotes ubiquitination and degradation by β-TrCP [136]. USP13, USP29, and USP37 were reported to promote the metastasis of GC by deubiquitinating and stabilizing Snail [35,36,54,59]. In addition, the upstream events of USP29 and USP37 are relevant for Snail regulation. Specifically, USP29, induced by TGFβ, TNFα, and hypoxia, increases the interaction between Snail and phosphatase SCP1, resulting in the dephosphorylation and deubiquitination of Snail, preventing its degradation [54]. USP37, which is transcriptionally activated by PLAG1-like zinc finger 2, stabilizes Snail in a GSK-3β phosphorylation-dependent manner [59]. Overall, these studies demonstrate the complexity of Snail regulation and provide potential therapeutic targets for the treatment of GC.

USP14
USP14 is a proteasome-associated DUB that plays a dual role in regulating protein degradation. Although it cleaves ubiquitin, it also promotes protein degradation by activating the proteasome [137,138]. USP14 has been reported to be an oncogene in GC [139]. USP14 levels are elevated in GC and may be an independent disease-free survival marker in patients [38,39]. In one study, USP14 was found to promote GC cell proliferation, invasion, and migration by stabilizing the EMT protein vimentin. Furthermore, the authors showed that miR-320a acts as a tumor suppressor by reducing vimentin expression and preventing USP14 from stabilizing this protein [37]. However, Fu et al. showed that USP14 depletion did not lead to cell death but sensitized GC cells to cisplatin-induced apoptosis [38]. As one chemical modification commonly found in eukaryotic mRNAs, N 6 -methyladenosine (m 6 A) plays a crucial role in modulating mRNA processing; studies have indicated that dysregulated m 6 A regulators are involved in cancer progression [140]. The m 6 A reader YTH N 6 -methyladenosine RNA binding protein 1 (YTHDF1) promotes gastric carcinogenesis by modulating the translation of FDZ7, a key Wnt receptor [141]. YTHDF1 also increased USP14 levels in an m 6 A-dependent manner, and USP14 overexpression reversed the tumor-suppressive effects elicited by YTHDF1 silencing [39]. Additionally, the USP14 inhibitor IU1 restricted GC cell viability and metastasis triggered by YTHDF1 overexpression [39,109]. Together, these findings provide novel insights into the USP14-related mechanisms underlying malignancy in GC.

USP15
The role of USP15 in GC is less defined and contradictory. Zheng et al. suggested that the ectopic expression of USP15 could suppress GC cell growth, migration, and invasion through the deubiquitination of IκBα by the COP9 signalosome (CSN) complex, thereby hindering NF-κB activity [40,142]. In contrast, USP15 knockdown attenuated the activity of Wnt/β-catenin signaling and inhibited GC progression both in vitro and in vivo, but the mechanism through which USP15 regulates this pathway remains unclear [41]. As reviewed by Das et al., USP15 can either activate or suppress NF-κB and Wnt/β-catenin signaling depending on its effects on different proteins in various cellular contexts [143]. Furthermore, Huangfu et al. suggested that USP15 may assist in GC development by acting as a target that is regulated by the LINC00205/miR-26a axis [42]. In short, the effects of USP15 on specific signaling pathways, such as NF-κB and Wnt/β-catenin, and on tumorigenesis and metastasis in GC require further study.

USP22
USP22 has been identified as producing cancer-stem-cell-like qualities, including aggressive growth, metastasis, and therapy resistance [144]. Unlike other DUBs, the USP22 zinc-finger ubiquitin-binding domain does not directly bind to ubiquitin and instead is recruited and activated by the SAGA complex, forming a subcomplex (DUB module) with other proteins [145,146]. By regulating a broad range of histone and non-histone protein substrates, USP22 is associated with oncogenesis in GC [145]. Several studies have suggested that USP22 expression is upregulated in GC tissues and cells [45][46][47][49][50][51][52]. In gastric tumors, USP22 expression was found to be positively correlated with the expression of the three well-known oncoproteins BMI1, c-Myc, and HSP90, which better predicted GC progression and prognosis than other methods [45,46,49]. Notably, USP22 stabilizes BMI1 and c-Myc, but not HSP90 [47,49,50]. USP22 maintained GC cell stemness by stabilizing BMI1 and promoted proliferation and metastasis by activating the FOXO1 and YAP signaling pathway [47,50]. However, these studies did not provide evidence that USP22 can stabilize BMI1 or c-Myc through deubiquitination, but this role was later identified in glioma and breast cancer cells [147,148]. As a guanine nucleotide exchange factor, son of sevenless 1 (SOS1) catalyzes GTP-bound RAS formation, whose activation elicits oncogenic signaling pathways [149]. Lim et al. proposed that USP22 upregulates SOS1 expression in GC, thus leading to the activation of RAS/ERK and RAS/PI3K/AKT pathways [52]. USP22 can also be modulated by the POU2F1/miR-4490 axis to further increase GC proliferation and metastasis [51].
Although the aforementioned studies indicate that USP22 is a GC oncogene, a metaanalysis noted contradictions between USP22 expression, tumor size, differentiation state, tumor stage, and clinical outcomes in patients with gastric tumors. However, these inconsistencies may be due to the small sample size, suggesting that the role of USP22 should be analyzed in larger randomized controlled clinical trials [150]. Moreover, there are discrepancies in USP22 s effects on malignant behaviors. Ma et al. reported that USP22 only affects cell proliferation, whereas other authors have shown that USP22 affects GC cell proliferation, cell migration, and apoptosis [47,[50][51][52]. Such controversial conclusions may be explained by differences in the cell context, including variations in USP22 levels among cell lines. Taken together, the role and downstream effectors of USP22 in GC warrant further investigation.

USP28
USP28 has been validated as an oncoprotein and therapeutic target in various cancer types, and a list of oncogenic substrates of USP28 have been reported, such as JUN, NOTCH1, CCNE, and LSD1 [151,152]. For instance, USP28 was identified as a DUB of LSD1 and conferred stem-cell-like characteristics in breast cancer [153]. Likewise, Zhao et al. found that USP28 was highly expressed in GC and was conducive to proliferation and metastasis, mainly due to its ability to increase LSD1 levels [53]. The same group subsequently designed and synthesized new [1][2][3] triazolo [4,5-d] pyrimidine derivatives as potent USP28 inhibitors. Among them, compound 19 potently inhibited USP28 activity with an IC 50 of 1.10 ± 0.02 µM and induced its degradation at higher doses, thereby exhibiting cytotoxic effects against GC cells [110]. In addition to LSD1, compound 19 also promotes c-Myc degradation, another substrate of USP28 [110]. Hu et al. showed that two GC cell lines, MKN-45 and SGC-7901, were sensitive to lanatoside C, an FDA-approved cardiac glycoside derived from Digitalis lanata. They also showed that lanatoside C partially promoted c-Myc degradation by reducing its interactions with USP28 [111]. These studies suggest that c-Myc is a potential downstream effector responsible for the oncogenic role of USP28 in GC.

USP32
USP32 was reported to be one protein that contributed to the chimeric Tre2 (USP6) oncogene [154]. The pro-cancer effects of USP32 have been observed in breast cancer and glioblastoma [155,156]. Additionally, USP32 promotes GC cell growth, metastasis, and chemoresistance by upregulating SMAD2, an important protein in the TGF-β signaling pathway [55]. However, it is unclear whether USP32 regulates the ubiquitination level of SMAD2.

USP33
USP33 expression is reduced in GC tissues and cell lines, which correlates with poor patient survival [56,57]. Additionally, USP33 overexpression suppresses the proliferation, migration, and invasion of gastric adenocarcinoma cells [56]. In addition, Xia et al. demonstrated that USP33 deubiquitinates and stabilizes ROBO1, which is required for the SLIT2-mediated inhibition of EMT and GC cell metastasis [57].

USP39
Although USP39 was initially identified as a DUB without ubiquitin hydrolysis activity, other studies have suggested that it plays a role in RNA splicing and oncogenesis in multiple malignant tumors [157,158]. Intriguingly, USP39 stabilizes SP1, ZEB1, and FOXM1 through its deubiquitination activity, thus supporting the progression of hepatocellular carcinoma and breast cancer [159][160][161]. Silencing USP39 expression hinders MGC-803 GC cell proliferation and induces G2/M arrest and PARP cleavage [60]. In line with these results, USP39 knockdown also decreased the proliferation of MGC-803 and HGC-27 cells. Moreover, miR-133a can suppress USP39 expression by directly targeting its 3 UTR, thereby acting as a tumor suppressor in GC [61]. However, the mechanisms by which USP39 depletion inhibits GC proliferation require further investigation.

USP44
Aneuploidy is a common characteristic of tumor cells [162]. Several studies have demonstrated that USP44 dysregulation results in aneuploidy, which has also been observed in GC tissues [63,163]. Furthermore, the combination of elevated USP44 expression and DNA aneuploidy provides useful prognostic information [63]. In addition, USP44 is regulated by the circFOXO3/miR-143-3p axis in GC, in which circFOXO3 functions as a miR-143-3p sponge to promote USP44-directed malignancy in gastric carcinoma [64].

CYLD
CYLD was initially described in cylindromatosis and is currently considered a tumor suppressor that negatively regulates multiple signaling pathways, such as NF-κB, AKT, and Wnt [167][168][169]. Ghadami et al. showed that CYLD expression was decreased in GC tissues owing to the hypermethylation of its promoter. They also revealed a direct cor-relation between H. pylori, EBV, and CMV infections and CYLD hypermethylation and downregulated protein expression, suggesting that infectious-agent-induced CYLD hypermethylation may be a significant mechanism of GC development [73]. Several studies have shown that CYLD can also be regulated by non-coding RNAs and serve as a downstream effector by modulating the progression and chemoresistance of GC. For example, miR-130b, miR-425-5p, miR-454, and exosomal miR-588 from M2-polarized macrophages negatively regulated CYLD expression by targeting its 3 -UTR, thus promoting GC cell proliferation, migration, invasion, and treatment resistance [70,72,75,76]. In addition, the activation of NF-κB by miR-362, miR-500, and miR-20a plays a significant role in the survival and cisplatin resistance of GC cells [68,69,71]. Notably, miR-500 also activates NF-κB signaling by directly repressing two other negative regulators of NF-κB, OTUD7B, and TAX1BP1 [69]. miR-505 also targets the 3 -UTR of CYLD mRNA. However, miR-505 can be sponged by CRAL and increase CYLD levels, which decreases AKT signaling and increases treatment susceptibility in GC cells [74]. Furthermore, the ALKBH5/ZNF333/CYLD axis participates in the development of gastric intestinal metaplasia, a precursor of GC. In bile-acid-induced gastric intestinal metaplasia, ZNF333 activates NF-κB signaling by repressing CYLD expression [77]. Taken together, these studies suggest that targeting proteins and miRNA that reduce CYLD levels may be a useful therapeutic strategy for GC.

Other USPs Involved in GC
In addition to the USPs mentioned previously herein, there are several other GCassociated USPs, including USP1, USP2, USP20, USP21, and USP42. USP1 levels were increased in GC cell lines and clinical samples, and its overexpression confers poor patient survival rates. USP1 knockdown inhibits their proliferation, migration, and invasion [17,18]. Mechanistically, USP1 promotes metastasis by stabilizing the inhibitor of DNA binding-2 protein [18]. USP2 was recently reported to harbor oncogenic properties by promoting E2F4-mediated cytoprotective autophagy and zinc homeostasis. By blocking the USP2-E2F4 interaction, emetine inhibited autophagy and GC aggressiveness, suggesting the therapeutic potential of targeting the USP2-E2F4 axis [19].
USP20 promotes the tumorigenesis of several cancer types, such as breast and cervical cancer; however, it exhibits inhibitory effects on GC [16]. Past reports have indicated that USP20 overexpression inhibited cell proliferation and delayed the cell cycle transition from the G1 to S phase by stabilizing Claspin [43,170]. Additionally, USP21 elevates MAPK1 expression through the transcription factor GATA3, thereby contributing to tumor growth and stemness in GC [44]. Hou et al. showed that higher USP42 expression was associated with poor prognosis in patients with GC. In vitro and in vivo studies showed that USP42 knockdown inhibited GC cell survival and invasion, suggesting that USP42 may be a therapeutic target [62].

UCHs and GC
All members of this family have been reported to be associated with GC ( Figure 3). UCHL1, also called PGP9.5, controls intracellular ubiquitin levels and is related to tumorigenesis in various cancer types [171]. However, UCHL1 has a paradoxical role in GC. UCHL1 promoter hypermethylation is a common event in several types of primary digestive tumors, including GC [78][79][80][81][82]. Moreover, UCHL1 hypermethylation is more frequent in advanced-stage GC [78,81]. In addition, this hypermethylation is associated with poor prognosis [82]. Furthermore, galangin cytotoxicity in SNU-484 cells may occur through increasing UCHL1 levels [112]. These studies indicate that UCHL1 may serve as a tumor suppressor and a diagnostic marker for GC. However, two other studies reported higher UCHL1 expression in liver metastases from GC and gastric cardiac adenocarcinoma, likely because UCHL1 overexpression increases the proliferation, migration, and invasion capabilities of GC cells [83,84]. Lastly, the pro-cancer effect of UCHL1 is mechanistically correlated with activated Akt and ERK1/2 pathways [83]. Thus, the role of UCHL1 in GC requires further investigation. Similar to USP14 and PSMD14, UCHL5 is also a proteasome-associated DUB. Highe UCHL5 expression is a predictor of increased survival in a subgroup of patients with early-stage GC [86]. However, another study suggested that UCHL5 stabilizing NFRKB a chromatin-remodeling protein, may promote GC cell proliferation and metastasis [87] Therefore, the biological function of UCHL5 requires more investigation.
One recent study reported that UCHL3 promotes GC cell migration and invasion by upregulating IGF2 [85]. In addition, Yan et al. found that BAP1 levels are decreased in GC which was linked to advanced tumor features and unfavorable survival, suggesting it role as a tumor suppressor [88]. Moreover, another study suggested that BAP1 upregula tion is essential for ferroptosis induced by 3,3′-diindolylmethane in BGC-823 cells, sug gesting that BAP1-induced ferroptosis could be one of the potential mechanisms by which it suppresses GC progression [113].

OTUs and GC
Three members of this family, including OTUB1, OTUB2, and A20, have been linked to GC (Figure 4). Weng et al. found that patients with GC with high OTUB1 expression were associated with several advanced clinical features, such as invasion depth, lymph node status, and nerve invasion, and these patients had lower disease-specific surviva rates. OTUB1 was found to be active in GC cell invasion and migration [89], but the un derlying mechanism is unknown. Similar to OTUB1, OTUB2 was also found to be overex pressed in GC tissues and cell lines and predicted a poor prognosis [90,91]. Silencing OTUB2 inhibited GC cell growth, metastasis, and sphere formation. Mechanistically OTUB2 acts as a potential driver oncogene in GC by deubiquitinating and stabilizing th demethylase KDM1A, and epithelial keratin KRT80 [90,91]. As previously reported KDM1A and KRT80 contributed to GC progression by regulating KLF2 expression and the PI3K/Akt pathway, respectively [172,173]. Similar to USP14 and PSMD14, UCHL5 is also a proteasome-associated DUB. Higher UCHL5 expression is a predictor of increased survival in a subgroup of patients with early-stage GC [86]. However, another study suggested that UCHL5 stabilizing NFRKB, a chromatin-remodeling protein, may promote GC cell proliferation and metastasis [87]. Therefore, the biological function of UCHL5 requires more investigation.
One recent study reported that UCHL3 promotes GC cell migration and invasion by upregulating IGF2 [85]. In addition, Yan et al. found that BAP1 levels are decreased in GC, which was linked to advanced tumor features and unfavorable survival, suggesting its role as a tumor suppressor [88]. Moreover, another study suggested that BAP1 upregulation is essential for ferroptosis induced by 3,3 -diindolylmethane in BGC-823 cells, suggesting that BAP1-induced ferroptosis could be one of the potential mechanisms by which it suppresses GC progression [113].

OTUs and GC
Three members of this family, including OTUB1, OTUB2, and A20, have been linked to GC (Figure 4). Weng et al. found that patients with GC with high OTUB1 expression were associated with several advanced clinical features, such as invasion depth, lymph node status, and nerve invasion, and these patients had lower disease-specific survival rates. OTUB1 was found to be active in GC cell invasion and migration [89], but the underlying mechanism is unknown. Similar to OTUB1, OTUB2 was also found to be overexpressed in GC tissues and cell lines and predicted a poor prognosis [90,91]. Silencing OTUB2 inhibited GC cell growth, metastasis, and sphere formation. Mechanistically, OTUB2 acts as a potential driver oncogene in GC by deubiquitinating and stabilizing the demethylase KDM1A, and epithelial keratin KRT80 [90,91]. As previously reported, KDM1A and KRT80 contributed to GC progression by regulating KLF2 expression and the PI3K/Akt pathway, respectively [172,173]. A20, also known as TNFAIP3, is a ubiquitin-editing enzyme with both DUB and E3 ligase activities [174]. A20 expression is increased in GC tissues and cell lines, which is associated with poor clinical outcomes [92,93]. In vitro studies have suggested that A20 downregulation suppresses the proliferation, migration, and invasion of MGC-803 GC cells [93]. In addition, A20 was found to be the target of miR-200a in GC cells (MGC-803 and SGC7901) resistant to TRAIL-induced apoptosis. Overexpressing miR-200a or depleting A20 enhanced apoptosis by reducing RIP1 polyubiquitination and promoting caspase-8 cleavage [94]. These results were consistent with a previous study showing that A20 mediates the polyubiquitination of RIP1, which in turn binds to and inhibits caspase-8 activation [175]. Later studies suggested that RIP1 is involved in multiple cellular signaling pathways and processes, such as NF-κB activation and apoptosis, and promotes GC growth and invasion [176]. Moreover, the DUB activity of A20 counteracted the ubiquitination of procaspase-8, thereby restricting caspase-8 activity and apoptosis [96]. Consistently, NF-κB activation induced A20 upregulation during H. pylori infection, mainly because USP48 stabilizes nuclear RelA and promotes its transcriptional activity [95][96][97][98]. Together, USP48 and A20 promoted GC cell survival during H. pylori infection and suggested an oncogenic role for A20. Conversely, one study reported that H. pylori infection increased miR-29a-3p, which promoted the migration of gastric epithelial cells by reducing A20 expression, indicating that A20 silencing may induce EMT to promote GC progression [99]. Thus, the role of A20 in GC might be context-dependent and requires further investigation.  A20, also known as TNFAIP3, is a ubiquitin-editing enzyme with both DUB and E3 ligase activities [174]. A20 expression is increased in GC tissues and cell lines, which is associated with poor clinical outcomes [92,93]. In vitro studies have suggested that A20 downregulation suppresses the proliferation, migration, and invasion of MGC-803 GC cells [93]. In addition, A20 was found to be the target of miR-200a in GC cells (MGC-803 and SGC7901) resistant to TRAIL-induced apoptosis. Overexpressing miR-200a or depleting A20 enhanced apoptosis by reducing RIP1 polyubiquitination and promoting caspase-8 cleavage [94]. These results were consistent with a previous study showing that A20 mediates the polyubiquitination of RIP1, which in turn binds to and inhibits caspase-8 activation [175]. Later studies suggested that RIP1 is involved in multiple cellular signaling pathways and processes, such as NF-κB activation and apoptosis, and promotes GC growth and invasion [176]. Moreover, the DUB activity of A20 counteracted the ubiquitination of procaspase-8, thereby restricting caspase-8 activity and apoptosis [96]. Consistently, NF-κB activation induced A20 upregulation during H. pylori infection, mainly because USP48 stabilizes nuclear RelA and promotes its transcriptional activity [95][96][97][98]. Together, USP48 and A20 promoted GC cell survival during H. pylori infection and suggested an oncogenic role for A20. Conversely, one study reported that H. pylori infection increased miR-29a-3p, which promoted the migration of gastric epithelial cells by reducing A20 expression, indicating that A20 silencing may induce EMT to promote GC progression [99]. Thus, the role of A20 in GC might be context-dependent and requires further investigation.

JAMMs and GC
Three members of this family, PSMD14, CSN5, and BRCC3, have been found to be associated with GC ( Figure 5). PSMD14, also known as Rpn11 and POH1, is a subunit of the proteasomal 19S regulatory particle, which functions as a DUB [177]. PSMD14 over-

JAMMs and GC
Three members of this family, PSMD14, CSN5, and BRCC3, have been found to be associated with GC ( Figure 5). PSMD14, also known as Rpn11 and POH1, is a subunit of the proteasomal 19S regulatory particle, which functions as a DUB [177]. PSMD14 overexpression has been reported to be tumorigenic and promote cancer progression through multiple mechanisms [177][178][179][180][181], such as stabilizing the alternative splicing factor PTBP1 to promote GC tumorigenesis [100].
CSN5, the catalytic subunit of the COP9 signalosome, also called COPS5 or JAB1, may play a role in GC [182]. CSN5 overexpression contributes to GC by modulating the stability or expression of several tumorigenic proteins. Silencing CSN5 suppresses GC cell growth and induces apoptosis by regulating P53 and BAX expression [102]. Additionally, CSN5 induces non-ubiquitin proteasomal degradation of the tumor suppressor p14ARF [103]. CSN5 also facilitates the nuclear export and degradation of the tumor suppressor RUNX3 [101]. Previous studies suggested that TNFα and CCL5 increase CSN5, stabilize PD-L1, and facilitate the immune escape of breast cancer cells and colorectal cancer cells [183,184]. CSN5 also stabilizes PD-L1 in GC cells. Furthermore, CSN5 activity in GC cells is regulated by the DAPK1/IKKβ axis [104]. Collectively, these studies indicate that CSN5 could be a novel therapeutic target in GC.
Lastly, Hu et al. reported that BRCC3 is upregulated in GC and is regulated by the lncRNA TMPO-AS1/miR-126-5p axis. TMPO-AS1 sponges miR-126-5p to upregulate BRCC3 expression, thereby activating the PI3K/Akt/mTOR pathway, which leads to malignancy [105]. CSN5, the catalytic subunit of the COP9 signalosome, also called COPS5 or JAB1, may play a role in GC [182]. CSN5 overexpression contributes to GC by modulating the stability or expression of several tumorigenic proteins. Silencing CSN5 suppresses GC cell growth and induces apoptosis by regulating P53 and BAX expression [102]. Additionally, CSN5 induces non-ubiquitin proteasomal degradation of the tumor suppressor p14ARF [103]. CSN5 also facilitates the nuclear export and degradation of the tumor suppressor RUNX3 [101]. Previous studies suggested that TNFα and CCL5 increase CSN5, stabilize PD-L1, and facilitate the immune escape of breast cancer cells and colorectal cancer cells [183,184]. CSN5 also stabilizes PD-L1 in GC cells. Furthermore, CSN5 activity in GC cells is regulated by the DAPK1/IKKβ axis [104]. Collectively, these studies indicate that CSN5 could be a novel therapeutic target in GC.

MJDs and GC
In the MJD family, only Ataxin-3 has been reported to be associated with GC tumorigenesis. Ataxin-3 levels were found to be decreased in GC tissues and cells, which correlated with clinicopathological characteristics, including tumor size, Lauren classification, histologic differentiation, and p53 mutation status [106]. However, the molecular mechanisms underlying this process have not yet been elucidated.

Conclusions and Perspectives
In the past few years, DUBs and tumorigenesis have been linked. Here, we summarize the regulatory roles of DUBs in the occurrence and development of GC. As shown in Figures 2-5, most DUBs promote GC progression, whereas several DUBs play an inhibitory role or exert context-dependent effects. Moreover, the regulatory mechanisms of DUBs are complicated and involve multiple targets and signaling pathways, yet some regulatory mechanisms have not been discovered (Table 1). Although the USP subfamily has received the most attention in GC research, other subfamilies, namely, OTUs, UCHs, JAMMs, and MJDs, have received attention as well. However, there have been no reports on MINDYs and ZUFSPs to date. Therefore, we speculate that USPs may be the most promising biomarkers for GC diagnosis and treatment, and we believe that the function of other DUB subfamilies requires further in-depth exploration.
In addition, DUB regulation is complex. Although the regulatory mechanisms of

MJDs and GC
In the MJD family, only Ataxin-3 has been reported to be associated with GC tumorigenesis. Ataxin-3 levels were found to be decreased in GC tissues and cells, which correlated with clinicopathological characteristics, including tumor size, Lauren classification, histologic differentiation, and p53 mutation status [106]. However, the molecular mechanisms underlying this process have not yet been elucidated.

Conclusions and Perspectives
In the past few years, DUBs and tumorigenesis have been linked. Here, we summarize the regulatory roles of DUBs in the occurrence and development of GC. As shown in Figures 2-5, most DUBs promote GC progression, whereas several DUBs play an inhibitory role or exert context-dependent effects. Moreover, the regulatory mechanisms of DUBs are complicated and involve multiple targets and signaling pathways, yet some regulatory mechanisms have not been discovered (Table 1). Although the USP subfamily has received the most attention in GC research, other subfamilies, namely, OTUs, UCHs, JAMMs, and MJDs, have received attention as well. However, there have been no reports on MINDYs and ZUFSPs to date. Therefore, we speculate that USPs may be the most promising biomarkers for GC diagnosis and treatment, and we believe that the function of other DUB subfamilies requires further in-depth exploration.
In addition, DUB regulation is complex. Although the regulatory mechanisms of some GC-related DUBs remain to be discovered, non-coding RNAs have been reported to modulate the expression of DUBs, such as USP3 and CYLD, in GC (Table 1). Intriguingly, three studies discovered that non-coding RNAs are also key mediators of interactions between GC cells and other cell types, including gastric mucosa cells, CAFs, and M2-polarized macrophages, and that USP3, USP7, and CYLD are linked to this process [24,27,76]. Furthermore, several infectious agents, cytokines, and antitumor agents also affect the expression of several DUBs, such as USP3, USP7, USP10, USP29, CYLD, and A20 [21,27,32,54,73,97,98,122] (Table 1), which implicates unknown pro-cancer effects under certain circumstances.
Finally, research on the therapeutic potential of DUB inhibitors for GC therapy is limited despite the efforts made in their development. Some inhibitors show promising therapeutic effects in various cancer types [15,185]. For instance, pharmacological inhibition of USP28, USP1, and USP7 with their inhibitors efficiently hindered in vitro and in vivo tumor growth or metastasis in squamous cell carcinoma, breast cancer, and colon cancer, respectively [186][187][188]. However, only a few studies have demonstrated the antitumor effects of USP7, USP14, and USP28 inhibitors on GC cells. Moreover, compounds that upregulate DUB tumor suppressors also exhibited activity against GC cells, providing another direction for drug development (Table 2). In conclusion, the investigation of DUBs in the pathogenesis and treatment of GC requires more work, which may provide clues for GC treatment in the future.