**7. DUBs and Metastasis**

Metastasis, which is the ability of cancer cells spread to di fferent tissues or organs, is regulated by many mechanisms. It is a series of biological processes including various invasion-metastasis cascades. Multiple reports have suggested the role of DUBs in controlling these mechanisms. The epithelial–mesenchymal transition (EMT) represents one of the most important invasive events in cancer metastasis. It refers to a change of a subset of adhesion molecules in cells: adopting a migratory and invasive behavior [135]. Numerous DUBs are involved in cancer cell invasiveness through the regulation of di fferent EMT transcription factors (Figure 4).

**Figure 4.** Roles of DUBs in epithelial–mesenchymal transition (EMT) in cancer metastasis. Epithelial cells are held together by numerous proteins, including tight junctions, adherens junctions, and desmosomes. These cells express molecules that are associated with the epithelial state, such as E-cadherin in epithelial state, and N-cadherin in mesenchymal state. Induction of EMT induces different EMT-inducing transcription factors (EMT-TFs) such as SNAIL, SLUG, and TWIST. These factors can then inhibit the epithelial state-related genes, such as E-cadherin, and activate the mesenchymal state related genes, such as N-cadherin. Various DUBs have been shown to interact with different EMT regulators. EMT is a reversible process, and mesenchymal cells can revert to the epithelial state by undergoing mesenchymal–epithelial transition (MET). A detailed description can be found in the main text.

SNAIL is a key regulator of EMT and plays an important role in tumor progression and metastasis. A group of DUBs, including OTUB1, DUB3, and USP3, are reported to stabilize Snail through preventing its ubiquitination and proteasomal degradation. OTUB1 promotes metastasis of esophageal squamous cell carcinoma through the stabilization of Snail [92]. DUB3 is found to be overexpressed in breast cancer, and depletion of DUB3 leads to Snail1 destabilization, which suppresses EMT, tumor invasiveness, and metastasis [90]. In addition, DUB3 also interacts with SLUG and TWIST and prevents their degradation, thereby promoting migration, invasion, and cancer stem cell-like properties in breast cancer cells [91]. Moreover, USP3 is significantly upregulated in glioblastomas and gastric cancer (GC). Clinicopathological data demonstrate that USP3 correlates with a shorter overall and relapse-free survival in glioblastomas [136]. It has also been reported that USP3 interacts with and stabilizes SUZ12 via deubiquitination. Expression of SUZ12 is negatively correlated with E-cadherin, which promotes migration and EMT in GC cells [95]. SMAD4 has been found to regulate EMT. USP17 is upregulated in osteosarcoma tissues and stabilizes SMAD4 through its DUB activity, leading to enhanced osteosarcoma cell invasion [94].

In addition to EMT mediators, DUBs target other molecules involved in cancer invasiveness. High expression of 14-3-3 γ is found in various cancers, such as breast cancer and NSCLC [137,138]. Overexpression of 14-3-3 γ promotes cell migration and invasion and correlates with the invasiveness of cancer cells. USP37 regulates the stability of 14-3-3 γ through its DUB activity [97]. Another DUB, 26S proteasome non-ATPase regulatory subunit 14 (PSMD14), is a posttranslational regulator of growth factor receptor bound protein 2 (GRB2). PSMD14 is significantly upregulated in HCC tissues, and it inhibits the degradation of GRB2 via deubiquitination. Overexpression of PSMD14 correlates with vascular invasion, tumor recurrence, and poor tumor-free and overall survival in patients with HCC [93]. The small GTPase Ras-related protein RAB7 is an early-induced melanoma driver and endocytosis protein that favors tumor invasion [139]. It is suggested to play roles in modulating endosomal maturation and autophagosome resolution in various cell types [140,141]. It was recently shown to be regulated by USP32 [96].

### **8. DUBs as Therapeutic Targets for Cancer Treatment**

As mentioned above, DUBs have been shown to deubiquitinate many targets involved in di fferent characteristics of cancer (Table 2), suggesting that DUBs may be potential therapeutic targets in cancer treatment. Indeed, many studies have been conducted to examine the potential of DUBs in cancer therapeutics. As DUBs are part of the proteasome system, proteasome inhibitors target them, which has shown promising successes for cancer treatment. Several examples are given below. Bortezomib, the first proteasome inhibitor, has entered clinical practice to treat relapsed multiple myeloma and showed outstanding antimyeloma activity [142,143]. In addition, combination of bortezomib and epirubicin significantly increases the sensitivity of colorectal carcinoma cells to apoptosis [144]. Due to the resistance to bortezomib, next-generation proteasome inhibitors carfilzomib and ixazomib have been approved. Carfilzomib irreversibly binds to the β-5 subunit of the proteasome [145]. A preclinical study has demonstrated that carfilzomib increased e fficacy against bortezomib-resistant multiple myeloma [146]. In the Phase 2 and Phase 3 clinical trials, single-agent carfilzomib provided durable anticancer activity in patients with relapsed and/or refractory multiple myeloma [147]. Ixazomib, the first oral proteasome inhibitor to enter the clinic, is now commonly used for multiple myeloma treatment. It is an e fficacious and long-term therapy for patients with advanced stage multiple myeloma [148]. In a double-blind Phase 3 trial, the use of ixazomib significantly improved progression-free survival in patients with relapsed and/or refractory multiple myeloma [149].

In addition to proteasome inhibitors, numerous DUB therapeutic targets have been developed. One excellent and classical example is USP7. Activating p53 by inhibiting MDM2 is a major direction of cancer treatment [150,151]. Nutlin-3 from Roche and RITA (2,5-bis(5-hydroxymethyl-2-thienyl)furan (NSC652287)) from the National Cancer Institute have been developed for interfering with the MDM2/p53 interaction to induce p53 and therefore cell death in human tumor cells [152–154]. They represent an important class of small molecules that has significant antitumor e ffects without obvious toxicity in mice [153,155], which further suggests that promoting MDM2 degradation will provide a therapeutic benefit when treating p53-related cancers. Additionally, USP7 silencing promotes the degradation of MDM2 and thus abrogates p53 degradation. Targeting DUBs might provide a new direction for cancer treatment, as it has the advantage of a simpler mechanism than targeting UBQ ligases or the 26S proteasome [150,156]. A small molecule lead-like inhibitor of USP7, HBX41108, which stabilizes and activates p53, was identified using high-throughput screening [156]. This inhibitor symbolizes a milestone in DUB drug development and sheds light on new potential cancer therapies using DUB inhibitors.

In addition, many cancer studies have focused on the apoptotic role of DUBs and exploited this role for chemotherapy. A drug screening study demonstrated that the small molecule DUB inhibitor b-AP15 inhibits two DUBs, USP14 and UCHL5. Treatment with b-AP15 results in apoptosis of human Waldenström macroglobulinemia (WM) cell lines and primary WM tumor cells [157]. In another chemotherapeutic study, pharmacological targeting of USP14 with the FDA-approved small-molecule inhibitor VLX1570 decreased viability in endometrial cancer cells through cell cycle arrest and caspase 3-mediated apoptosis [158]. The oncogenic transcription factor pre-B cell leukemia homeobox-1 (PBX1) promotes advanced PCa cell proliferation. USP9X interacts with and stabilizes the PBX1 protein by attenuating its Lys48-linked polyubiquitination. The USP9X inhibitor WP1130 markedly induces PBX1 degradation and promotes PCa cell apoptosis [159]. The selected DUB inhibitors that target on cancer cells are summarized in Table 3. To conclude, DUBs play multiple roles in cellular functions. The aberrant expression and regulation of these enzymes have been shown to contribute to promote tumorigenesis, making them promising therapeutic targets for cancer therapy.


**Table 3.** Summary of known DUB inhibitors that are targeted in cancer cells.

**Author Contributions:** Conceptualization, J.C. and W.K.F.T.; writing the review and editing, K.P.L., J.C. and W.K.F.T. All authors have read and agreed to the published version of the manuscript.

**Funding:**This work is supported by the funding of National Natural Science Foundation of China (number 81660610), and the National Natural Science Foundation of Guangxi Province (number 2019GXNSFFA245001, 2017GXNSFDA198019, 2018GXNSFAA281159) for Jian Chen. The pathology works in WKF Tse Laboratory were partially supported by the Japan Society for the Promotion of Science Bilateral Open Partnership Joint Research Project AJ179064, the joint research program of the Institute for Molecular and Cellular Regulation, Gunma University, Japan (18003); and the National Institute of Basic Biology Collaborative Research Program, Japan (19-307).

**Acknowledgments:** K.P.L. is supported by Hong Kong SAR, Macao SAR, and Taiwan Province Talent Young Scientist Program of Guangxi.

**Conflicts of Interest:** The authors declare no conflict of interest.
