The execution of apoptosis is countered by the action of anti-apoptotic proteins (58). Since certain substrates of USP9X, such as MCL-1 and XIAP, are key factors in cellular apoptosis signaling pathways that drive cell apoptosis, targeting these substrates via USP9X might lead to the regulation of cell apoptosis (41–45). USP9X displays both pro- and anti-apoptotic functions, mediated by the deubiquitination of critical components of the apoptotic signaling networks (53,59). Previous studies have reported that the expression of stress-sensing, pro-apoptotic kinases is regulated by USP9X to initiate the apoptotic JNK signaling cascade (59,60). The pro-apoptotic kinase, apoptosis signal-regulating kinase 1 (ASK-1), is activated under conditions of oxidative stress, leading to the selective activation of the JNK signaling pathways (59). USP9X also interacts with ASK-1, protecting it from proteasomal degradation to mediate oxidative stress-induced cell death (59). USP9X also activates and stabilizes the dual leucine zipper kinase in response to extracellular and intracellular stress in neurons, thereby enabling the activation of pro-apoptotic JNK signaling (60).

By contrast, USP9X enhances the activities of a large spectrum of anti-apoptotic factors for cell survival. In the classical mitochondrial apoptosis pathway, the pro-survival B-cell lymphoma-2 family proteins, including MCL-1, preserve mitochondrial integrity and indirectly inhibit the activation of caspase-3 and −7, ultimately limiting the rate of cell apoptosis (17). USP9X stabilizes MCL-1 through its interaction with the protein and the removal of the Lys48-linked polyubiquitin chains that mark a protein for proteasomal degradation (53). A recent study reported that the RNA helicase Asp-Glu-Ala-Asp-box polypeptide 3 interacted with the N-terminus of USP9X and participated in deubiquitination of MCL-1 (61). Therefore, human tumors with a high level of MCL-1 expression may be accompanied by the overexpression of USP9X (53,62). Upregulated expression of USP9X promotes tumorigenicity and cell survival through stabilizing cell death regulators, including X-linked inhibitor of apoptosis protein (XIAP), and inhibiting the induction of apoptosis by specifically stabilizing MCL-1 (53). WP1130 is a small molecule that directly decreases the DUB activity of USP9X, which leads to the downregulation of MCL-1, ultimately facilitating apoptosis (63,64). A previous study reported that apoptosis was induced by the inhibition of USP9X at least partly through oxidative stress, which activated DNA damage responses and stress-associated mitogen-activated protein kinase (MAPK) signaling pathways (43). Therefore, USP9X, when co-expressed with multiple apoptosis-associated proteins, exerts an anti-apoptotic role in cancers, such as oral cancer, prostate cancer, chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) (65–67). Overexpression or depletion of USP9X is therefore an important factor in cell apoptosis and survival.

Regulation of mitosis safeguards cellular integrity and its failure contributes to the progression, maintenance and drug resistance of cancer (68–70). In the cell cycle, the mitotic checkpoint complex (MCC) senses the orientation of sister chromatids on the mitotic spindle and restricts the activity of anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase to initiate mitotic exit, consequently ensuring that chromosome segregation and anaphase are able to occur (71). In this way, the spindle assembly checkpoint is strengthened and chromosomal stability is made more secure through restricting APC/C-mediated MCC turnover (71). By contrast, downregulation of USP9X contributes to a reduction in the efficacy of the spindle assembly checkpoint, an increase in chromosome segregation defects and chromosomal instability, which leads to the subsequent promotion of cancer (71). Activating the mitotic phosphorylation of USP9X promotes cell survival through counteracting mitotic ubiquitination and the ensuing proteasomal degradation of Wilms' tumor protein 1, the latter of which modulates the transcription and secretion of CXC motif chemokine ligand 8/interleukin-8 in mitosis (72). In addition, the dephosphorylation of USP9X mediated by cell division cycle 14B (CDC14B) has previously been shown to promote mitotic apoptosis (72). USP9X is an integral component of the centrosome, where it functions to stabilize certain centrosome proteins, centrosomal protein 55 and pericentriolar material 1, thereby promoting centrosome biogenesis (73). Activation of USP9X enhances centrosome amplification and chromosome instability, whereas inactivation of USP9X leads to an impairment of centrosome duplication (40). It has been previously reported that USP9X may stabilize cell cycle-associated proteins to control ribosomal stalling (74), regulate centrosome duplication (40), and antagonize mitotic cell death and chemoresistance (41). Proteins, such as USP9X, that coordinate the normal functioning of the cell cycle may also trigger inappropriate cell divisions, thereby taking on dysfunctional roles in certain pathological disorders, including X-linked intellectual disability (16,18) and malignancies (40).

USP9X also serves pivotal roles in cell migration and invasion. Doublecortin (DCX) is a microtubule-associated protein involved in vesicle transport and microtubule dynamics (75). The C-terminus of USP9X binds DCX, which acts as a regulator of neuronal cell migration (76). Loss of USP9X has been shown not only to reduce axon growth, but also to cause a reduction in neuronal cell migration both in vivo and in vitro via USP9X-mediated disruption of the neuronal cytoskeleton (18). Accordingly, USP9X is required for normal neuronal cell migration. In addition, USP9X-mediated deubiquitination of integrin a5b1 serves a role in a5b1-dependent cell migration (77). A previous study reported that USP9X promoted TGF-β-dependent cancer progression and metastasis through interaction with SMAD4 in the TGF-β signaling pathway, thereby inhibiting the E3 ubiquitin-protein ligase TIF1γ-mediated ubiquitination of SMAD4 (46). SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) was originally identified to block the TGF/bone morphogenetic protein (BMP) signaling pathway by specifically degrading SMAD1 and SMAD5, as well as TGF/BMP receptors (78,79). The negative regulation of USP9X destabilizes SMURF1 and inhibits SMURF1-dependent cell migration in breast cancer cells (80). USP9X has also been shown to activate the prostaglandin E synthase (PTGES)/prostaglandin E2 (PGE2) pathway, thereby promoting metastatic features of non-small cell lung cancer (NSCLC) cells through the deubiquitination and stabilization of PTGES, which functions as a key enzyme for the process of PGE2 synthesis in the arachidonic acid pathway (81). It has previously been demonstrated that USP9X is able to disrupt neuronal cell migration and growth, which is associated with X-linked intellectual disability; therefore, USP9X may serve roles in both neurodevelopment and the modulation of neural apoptosis (18). Taken together, these findings demonstrate the importance of UPS9X in regulating cell migration and invasion.

USP9X serves important roles in DNA damage repair (82). Genomic instability is a cancer hallmark, often discovered at early stages of the tumorigenesis process (83). The repair of DNA double-strand breaks may trigger the modification of proteins via polyubiquitin or monoubiquitin around the damage sites (84). During DNA replication, the ataxia telangiectasia-mutated and Rad3-related kinase/checkpoint kinase 1 pathway is activated to coordinate both fork-repair processes and checkpoint responses. In a previous study, USP9X was found to enhance the maintenance of DNA damage checkpoint responses and improve DNA replication fork stability by modulating the checkpoint adaptor and DNA replication factor claspin during S phase, thereby preventing DNA damage accumulation or the DNA replication blockade (85). In addition, USP9X depletion was shown to block the progression of DNA replication forks, which increased sensitivity to ionizing radiation, indirectly impairing genome integrity and leading to excessive endogenous DNA damage (85). Loss of USP9X also affects the radiosensitivity of, and cell survival in, glioblastoma through a number of MCL-independent and -dependent mechanisms (86). The potential involvement of USP9X during radiotherapy remains unknown; however, a previous study reported that it may promote lung cancer radioresistance via epigenetically inducing TGF-β2 transcription, which serves to increase the survival of lung cancer cells (26). Therefore, USP9X serves an important role in the stability of the genome during DNA replication. The positive regulation of the DNA damage repair process may also indicate that USP9X is a potential tumor promoter in cancer, also suggesting its candidacy as a potential clinical target in tumor therapy.

USP9X expression level has been shown to differ in tissues and cell lines (Fig. 4A). Furthermore, increased expression levels of USP9X are found in certain cancer types, including breast and lung cancer, and glioblastoma (Fig. 4B; Table I), according to the data from the ProteomicsDB database (87). The therapeutic potential of USP9X in different types of cancer has been demonstrated through considering its deubiquitination capabilities on multiple regulated substrates and critical signaling pathways (88,89). As for the survival and prognosis value of USP9X, high expression of USP9X in adrenocortical, and bladder urothelial carcinoma tissues is associated with lower disease-free survival rates of patients, although it is associated with higher overall survival (OS) rates in cholangiocarcinoma (CHOL; Fig. 5), according to the data from GEPIA (http://gepia.cancer pku.cn/) (90).

The available evidence demonstrates that the USP9X-induced aggressive and metastatic phenotypes of cancer are regulated by the abnormal expression of non-coding RNAs (91–97) (Table II). The long non-coding RNA LINC01433 has been shown to enhance the interaction between USP9X and Yes-associated protein (YAP), thereby stabilizing YAP and leading to the promotion of tumor progression and chemotherapy resistance in gastric cancer (GC) cells (91). In addition, the circular RNA hsa_circ_0008434 is highly expressed in GC and serves the role of upregulating the expression of USP9X, thereby promoting the malignant phenotypes of GC cells by serving as a microRNA (miR) sponge for miR-6838-5p (93). Moreover, downregulation of the cellular FADD-like interleukin-1β-converting enzyme-inhibitory protein, which is induced by WP1130, a type of selective USP9X inhibitor that decreases the activity of USP9X, was shown to be regulated by miR-708 through the inhibition of USP9X, which induces the apoptosis of CaSki cells (98). In addition, miR-132 was previously shown to target USP9X-induced EMT, which led to inhibition of the migration and invasive capabilities of NSCLC cells (97).

USP9X has been shown to exert opposing effects in hematological malignancies. It has been reported that USP9X can promote tumor cell survival in human diffuse large B-cell lymphoma, multiple myeloma and follicular lymphoma through deubiquitinating MCL-1, which protects it from degradation, resulting in poor clinical outcomes (38,45,99,100). In addition, a high level of USP9X in patients with aggressive B-cell lymphoma has been shown to be associated with excessive B-cell proliferation, resulting in an adverse prognosis and resistance to treatment therapies through the deubiquitination and stabilization of XIAP, independent of MCL-1 (41). A BCR-ABL-positive diagnosis is a typical characteristic of patients with CML. The WP1130-mediated inhibition of USP9X is associated with a reduction in MCL-1 levels, followed by blockade of BCR-ABL kinase signaling, which leads to the rapid onset of CML cell apoptosis (21). However, USP9X is not involved in BCR-ABL ubiquitination or cellular localization (101). Moreover, USP9X silencing was shown to lead to lymphoma growth suppression, leading to decreased chemotherapy resistance in B-cell lymphoma (63). Internal tandem duplications of FMS-like tyrosine kinase 3 (FLT3-ITD) occur frequently in AML, and is associated with poor outcomes in patients with AML. USP9X interacts with FLT3-ITD to inhibit its Lys63-linked polyubiquitination (43,102). Furthermore, FLT3-ITD reversely induces the ubiquitination and tyrosine-phosphorylation of USP9X, thereby promoting its proteasomal degradation (102). USP9X also stabilizes RNA m6A demethylase ALKBH5 by removing the K48-linked polyubiquitin chain at K57 and promotes AML cell survival (44). Taken together, these findings identify USP9X as an oncogenic gene that promotes the development of hematological malignancies, and the therapeutic targeting of USP9X may lead to preferential inhibition of certain types of leukemia.

USP9X also exerts negative effects in hematological malignancies by acting as a tumor suppressor gene. USP9X has been identified as a novel leukemia susceptibility gene associated with B-cell acute lymphoblastic leukemia (B-ALL) and multiple neurodevelopmental and congenital abnormalities (103). Low USP9X expression is associated with decreased survival in patients with high-risk B-ALL (103). Genetic or pharmacological inhibition of USP9X can restrict JAK signaling to enhance the survival of cytokine receptor-like factor 2-positive B-ALL in patients with Down syndrome (104), which suggests that USP9X may exert differential effects in different types of leukemia.

The underlying mechanisms of USP9X in breast cancer are complex. YAP is an important oncogene that drives cancer progression, and dysregulation of the Hippo/YAP1 signaling pathway is involved in breast cancer development (105). YAP1 is deubiquitinated and stabilized by USP9X, thereby promoting breast cancer cell survival and resulting in chemotherapy resistance (47). USP9X stabilizes Snail1, a key factor regulating the EMT process, contributing to metastasis and chemoresistance in triple-negative breast cancer (48). Furthermore, downregulation of USP9X renders estrogen receptor α-positive breast cancer resistant to tamoxifen, leading to a poor outcome for patients following adjuvant tamoxifen treatment (89). USP9X can modulate centrosome biogenesis and enhance breast carcinogenesis by deubiquitinating and stabilizing the centriolar satellite protein, CEP131 (40). In a previous study, the arginine methylation of USP9X was shown to improve its interaction with Tudor domain-containing protein 3 (TDRD3), which led to a subsequent enhancement of its anti-apoptotic activities in breast cancer cells (35). The aforementioned study also reported that downregulation of TDRD3 improved the sensitivity of chemotherapeutic drug-induced apoptosis in breast cancer cells, which is likely due to its regulation of USP9X DUB activity on the anti-apoptotic protein MCL-1 and stress granule localization (35).

Obesity may be a risk factor for the development of breast cancer, as it is associated with reduced survival and increases the risk of distant metastasis for female patients diagnosed with breast cancer (106). Plasma free fatty acids further facilitate this biological progression of breast cancer in obese patients (46). USP9X can also be recruited by the transcription factor Nanog to stabilize the hypoxia-inducible factor-1α protein, which leads to an enhancement of self-renewal in breast cancer stem cells (49). Moreover, USP9X selectively promotes activation of the Notch developmental signaling pathway in triple-negative breast cancer, and small molecule EOAI3402143 (G9)-mediated USP9X inhibition specifically inhibits the Notch pathway, which leads to a remodeling of the tumor immune landscape and suppresses tumor growth (107). Taken together, these studies suggest that a diverse range of USP9X-associated mechanisms are involved in breast cancer.

Histone lysine demethylase 4C (KDM4C), a substrate of USP9X, is associated with poor clinical outcomes in patients with lung cancer (50). A recent study reported that USP9X activated the TGF-β/SMAD signaling pathway, thereby inducing radioresistance by deubiquitinating and stabilizing KDM4C in lung cancer (50). USP9X is also positively correlated with the dual-specificity protein kinase known as threonine tyrosine kinase, which modulates cell proliferation, migration and tumorigenesis in NSCLC (51). Furthermore, the inhibition of USP9X has been shown to contribute towards MCL-1-mediated proteasomal degradation, radiosensitivity and apoptosis in NSCLC cells (52). Moreover, the chemotherapeutic agent pemetrexed has been shown to induce apoptosis by increasing the expression of the pro-apoptotic protein Noxa, thereby activating the Noxa-USP9X-MCL-1 axis, which demonstrates that USP9X serves a critical role in human lung cancer cells (108). Collectively, these studies have shown that USP9X exerts a range of different functions in lung cancer.

Pancreatic cancer is a lethal malignancy, and effective targeted therapies are urgently required to combat its high morbidity rates (2.6%) and mortality (4.7%) rates worldwide in 2020 (109). To date, USP9X has been reported to show tumor-suppressing potential in pancreatic cancer (110–112). USP9X suppressed tumorigenesis in a V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mouse model of pancreatic ductal adenocarcinoma (PDAC) and its downregulation enhanced malignant transformation, protecting PDAC cells from anoikis (110). However, these properties of USP9X in pancreatic cancer predominantly depend on its intrinsic deubiquitinase activity. USP9X mediates acute MCL-1 stabilization and protection from apoptosis in response to MAPK suppression caused by MEK inhibitors (113), which may provide a promising therapeutic strategy for pancreatic cancer.

The differential expression or genetic alterations of USP9X have been identified in multiple types of human cancer, which suggests that it potentially exerts different roles in tumor progression. Higher expression levels of USP9X are observed in oral squamous cell carcinoma (OSCC) cells, where it drives oral tumorigenesis by deubiquitinating and stabilizing the anti-apoptotic protein MCL-1, which was shown to correlate with poor outcomes in patients with OSCC (53). In addition, aldehyde dehydrogenase 1 family member A3 (ALDH1A3) functions as a key enzyme for maintaining the self-renewal and mesenchymal (MES) features of glioblastoma stem cells (54). Depletion of USP9X leads to a marked downregulation of ALDH1A3, which leads to a loss of the tumorigenic and self-renewal capabilities of MES glioblastoma stem cells. By contrast, a high expression level of USP9X is indicative of potent tumorigenic capability in MES glioblastoma stem cells with enrichment of ALDH1A3 (114). A previous study reported that USP9X-mediated deubiquitination and stabilization of Ets-1 promoted the expression of the N-RAS oncogene and carcinogenesis in melanoma (56).

Although USP9X has been reported to exert oncogenic functions in several different types of cancer, a number of previous studies have also reported the tumor-suppressive properties of USP9X in carcinogenesis. In the murine intestine, USP9X is necessary for tissue homeostasis and regeneration following acute colitis (55); it regulates the function and protein expression levels of the tumor suppressor FBW7, which consequently protects it from proteasomal degradation (55). Therefore, the restricted level of c-Myc that is regulated by USP9X via stabilization of FBW7 reduces the risk of colitis-mediated colorectal cancer (CRC) in mice, whereas the silencing of USP9X is associated with a poor prognosis in human CRC (55). USP9X expression is downregulated in CRC and CHOL (89). Moreover, USP9X is involved in the activation of apoptosis in CHOL, which also suppresses tumor cell proliferation (57). USP9X exerts its tumor-suppressive functions through deubiquitinating Egl-9 family hypoxia inducible factor 3, thereby activating the apoptosis signaling pathway components, kinesin KIF1Bβ and cleaved caspase-3 (57). USP9X also targets and regulates the stability of angiomotin to indirectly inhibit YAP/transcriptional coactivator with PDZ-binding motif activity, and a low level of USP9X is correlated with poor clinical outcome in renal clear cell carcinoma (115). Taken together, these findings suggest that there may be potential for USP9X to be used as a therapeutic target for the treatment of certain types of cancer.