The role of ubiquitination and deubiquitination in tumor invasion and metastasis

Ubiquitination is vital for multiple cellular processes via dynamic modulation of proteins related to cell growth, proliferation, and survival. Of the ubiquitination system components, E3 ubiquitin ligases and deubiquitinases have the most prominent roles in modulating tumor metastasis. This review will briefly summarize the observations and underlying mechanisms of multiple E3 ubiquitin ligases and deubiquitinases to regulate tumor metastasis. Further, we will discuss the relationship and importance between ubiquitination components and tumor progression.

Protein ubiquitination is a dynamic, multifaceted post-translational modification involved in multiple cellular processes. Ubiquitin (Ub), a 76-amino acid protein, features seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), which can each be ubiquitinated to form distinctive forms of polyubiquitin chains. Different polyubiquitin chains mediate distinct signaling pathways to determine the fate of substrate proteins [1]. For example, K48/K11-linked chains are responsible for targeting substrate proteins for proteasomal degradation, while other chains perform non-degradative roles in controlling protein interactions, cellular localization, and signaling transduction (Fig. 1).
Ubiquitination is catalyzed by a three-enzyme cascade composed of the E1 Ub-activating enzyme, the E2 Ub-conjugating enzyme, and the E3 Ub ligase. E1 recruits and activates Ub by utilizing the energy of ATP. Activated Ub is transferred to E2, which can transfer Ub to the target substrate. E3 Ub ligase selectively recognizes a substrate protein by forming an iso-peptide bond between the COOH-terminal glycine of Ub and a lysine residue of the substrate. In addition, E3 Ub ligase recruits the E2-Ub complex and catalyzes the transfer of Ub to the substrate from E2 [2]. Different subtypes of E3s (the RING type E3s, the HECT type E3s, and the RBR type E3s) are the most critical component of the ubiquitination cascade for the substrate recognition capacity [2]. Meanwhile, ubiquitination is a dynamic and reversible process. Deubiquitinases (DUBs) can act as an "eraser" that reverses Ub signals. Most DUBs remove Ub moieties from proteins to prevent substrate proteins from degradation. However, some proteasome-related DUBs, including USP14, UCHL5, PSMD14, and PSMD7, are localized in 19S particles of the proteasome [3]. The roles of these DUBs are to deubiquitinate the substrates and facilitate their degradation in 20S particles of the proteasome. Alternatively, DUBs can alter signals by non-degradation ubiquitination [4] (Fig. 1).

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The Ub cascade and DUBs synergistically regulate protein turnover and function in numerous signaling pathways to maintain cellular invasion and metastasis.

A novel insight into tumor metastasis
Tumor metastasis remains the primary cause of cancer-associated mortality. Metastasis involves tumor cell motility, intravasation into the adjacent tissues, circulation, and extravasation to distant organs. Simultaneously, the process is caused by genome instability where cancer cells can reprogram tumor metabolism, resist cell death, avoid immune destruction, and constitute the tumor microenvironment [5,6]. The epithelial-mesenchymal transition (EMT) is an equally crucial determinant during the metastatic cascade [7].
Ubiquitination and deubiquitination broadly participate in various processes involved in protein modification and regulation. Aberrant dysregulation induces tumorigenesis. This review is primarily focused on recent novel observations and underlying mechanisms concerning E3 ligases and DUBs in order to contribute to further elucidating the role of ubiquitination and deubiquitination in tumor invasion and metastasis.

Parkin
Notably, as an E3 Ub ligase, Parkin can degrade substrate proteins associated with Parkinson's Disease (PD) [19]. Meanwhile, Parkin acts as a tumor suppressor, and its expression is downregulated in various tumors [20]. Parkin is an E3 Ub ligase for hypoxia-inducible factor 1α (HIF-1α) and can ubiquitinate HIF-1α at lysine 477 (K477), inhibit HIF-1α transcriptional activity, and induce its degradation to suppress breast cancer cells invasion and metastasis [21,22]. Parkin regulates HIF-1α in a Von Hippel-Lindau-independent manner, unveiling an additional layer of regulation for HIF-1α in cells. Phosphoglycerate dehydrogenase (PHGDH) is the first rate-limiting enzyme of serine synthesis. PHGDH overexpression activates serine synthesis to promote cancer progression. Parkin expression is inversely correlated with PHGDH expression in breast and lung cancer. Parkin interacts with PHGDH and ubiquitinates PHGDH at lysine 330, leading to PHGDH degradation to suppress serine synthesis [23]. In intrahepatic cholangiocarcinoma (ICC), Parkin targets pyruvate kinase PKM2 for ubiquitination degradation to suppress migration and proliferation [24] (Fig. 2). Ubiquitination is catalyzed by a three-enzyme cascade composed of the E1 Ub-activating enzyme, the E2 Ub-conjugating enzyme, and the E3 Ub ligase. The E3 ligase selectively recognizes substrate proteins by forming an iso-peptide bond and recruits the Ub-E2 complex to catalyze the transfer of Ub to the substrate from E2. Elongation and distinct polyubiquitin chains are involved in protein degradation, signal transduction, and transcriptional activity. Deubiquitinases remove Ub moieties from substrate proteins with high specificity and reverse Ub signals to maintain cellular dynamic ubiquitination. FBXW2 targets SKP2, β-catenin, and TAK1, FBW7 targets Brg1, Mcl-1, and YTHDF2, and Parkin targets HIF-1α, PHGDH, and PKM2 for ubiquitination degradation to suppress tumor proliferation and metastasis, respectively. UBR7 monoubiquitinates histone H2B to suppress EMT and nuclear β-catenin. UBE3C targets AHNAK and AXIN1 for ubiquitination-induced degradation. TRIM65 ubiquitinates ARHGAP35, FBXO22 ubiquitinates nuclear PTEN and p21 to enhance cancer cell migration, respectively. FBXO22 mediates Lys-63-linked LKB1 ubiquitination. FBXO22 upregulates HIF-1α and VEGFA to promote tumor proliferation and metastasis.

Tripartite motif-containing protein 65 (TRIM65)
Tumor metastasis involves the reorganization of the cytoskeleton, whose activities are controlled by GTPases [31]. When bound to guanosine diphosphate (GDP), GTPases are inactivated, which is regulated by GTPase-activating protein (GAP) [32]. Rho A belongs to the Rho family of GTPases and regulates the cytoskeleton. Rho GTPase-activating protein 35 (ARHGAP35), a Rho GAP, regulates polarized cell migration and inhibits Rho GTPase. In colorectal cancer (CRC), E3 Ub ligase TRIM65 ubiquitinates and degrades ARHGAP35, which leads to subsequent elevated Rho GTPase activity and cytoskeleton remodeling [33] ( Fig.  2). The TRIM65-ARHGAP35-Rho A axis enhances cancer cell migration by modulating the actin cytoskeleton.

DUBs
DUBs can reverse ubiquitination by cleaving the isopeptide bond between the Ub and the substrate. Currently, over 100 DUBs have been identified and can be divided into six subclasses: i) Ub-specific proteases (USPs); ii) ovarian tumor proteases (OTUs); iii) Ub C-terminal hydrolases (UCHs); iv) Machado-Joseph disease proteases; v) JAB1/MPN/ Mov34 metalloenzymes; and vi) monocyte chemotactic protein-induced protein [39]. DUBs act as tumor suppressors or oncogenes and play essential roles in regulating various types of tumors (Table 1). DUBs have emerged as promising therapeutic targets in cancer. USP1 induces platinum resistance, cancer cell stemness, and metastatic dissemination in ovarian cancer. [68] USP1 deubiquitinates KPNA2 and enhances pro-metastatic genes expression.
The intervention of USP1 via pimozide or ML323 suppresses metastasis. [69] USP1 deubiquitinates and increases TAZ protein stability. Loss of USP1 reduces TAZ to inhibit cell proliferation and migration, and USP1 is a potential therapeutic target in triple-negative breast cancer (TNBC). [70] USP1 deubiquitinates and stabilizes ribosomal protein S16 (RPS16).
USP3 promotes TGF-β1-induced EMT and cell migration in gastric cancer.
USP5 is overexpressed in non-small cell lung cancer to promote EMT, invasion, and metastasis. [78] USP6 Oncogene USP6 promotes invasion and metastasis, and acts as an efficient prognostic biomarker.
USP6 is highly overexpressed in colon cancer. [79] USP7 (HAUSP) Oncogene USP7 promotes the circulation of tumor cells (CTCs) to reside in the bone marrow.
Under hypoxia, E3 ligase HectH9 is required for K63-polyubiquitinated HAUSP to promote EMT and metastasis in lung cancer. [81] USP7 promotes proliferation and invasion. Overexpressed USP7 represents a worse overall survival and acts as an independent prognostic indicator in epithelial ovarian cancer (EOC) and oral squamous cell carcinoma (OSCC). [82,83] USP7 overexpression activates the PI3K/AKT signaling pathway.
USP8 suppresses apoptosis and promotes proliferation, invasion, and metastasis in cholangiocarcinoma. [86] TSG (Tumor  suppressor  gene) USP8 is a protective factor and prognosticates better clinical outcomes.
Plasma-free fatty acids (FFA) promote the SMAD4-USP9X interaction via ERK to elicit TGF-β-induced metastasis for obese breast cancer patients.
USP10 is highly expressed and stabilizes NLRP7 to promote cell proliferation and metastasis in colorectal cancer. [91]
USP11 enhances TGFβ-induced EMT to promote breast cancer metastasis. USP11 is an independent prognostic predictor.
USP11 is overexpressed and promotes migration and metastasis in hepatocellular carcinoma. TSG USP11 deubiquitinates ARID1A and prevents its degradation to inhibit SDC2 activation.
High USP15 expression indicates a worse prognosis, and USP15 could be a therapeutic target in hepatocellular carcinoma. [101] USP15 promotes β-catenin nuclear translocation and activates the Wnt/β-catenin pathway.
USP15 is upregulated and promotes EMT, cell proliferation, and metastasis.
Expression of USP28 is elevated in colon and breast carcinomas.

USP29
Oncogene USP29 interacts simultaneously with Snail and SCP1 to stabilize Snail via deubiquitination and dephosphorylation.
USP33 is overexpressed and is a prognostic biomarker and therapeutic target in hepatocellular carcinoma. [118,119] TSG USP33 can deubiquitinate and stabilize Robo1 to inhibit EMT and cell migration in a Slit-Robo pathway-dependent manner.
USP33 expression is downregulated and it is an independent prognostic marker in colorectal cancer and gastric cancer. The expression of USP47 is elevated, and silencing USP47 can promote Snail degradation and attenuate EMT in colorectal cancer. USP47 abrogates the SMURF2-mediated ubiquitination of special AT-rich sequence-binding protein-1 (SATB1) to promote colon cacer cell proliferation and metastasis.

USP48
Oncogene USP48 promotes migration and invasion. Ablation of USP48 increases the responsiveness to carboplatin treatment in ovarian cancer. [131]
The overexpressed p-USP51 is correlated to a poor prognosis for breast cancer patients, and the CDK4/6-USP51-ZEB1 axis could be a viable therapeutic target. [132][133][134] USP51 increases FAT4 protein level and is imperative for FAT4's function.

USP51 suppression contributes to the inhibition of FAT4 and promotes proliferation and invasion of endometrial cancer (EC). USP54
Oncogene USP54 is of pro-tumorigenic properties. USP54 is upregulated in colorectal carcinoma and is a promising therapeutic target. [135] OTUB1 Oncogene OTUB1 stabilizes Snail to promote metastasis. OTUB1 is highly expressed in esophageal squamous cell carcinoma (ESCC), and higher expression of OTUB1 predicts poor prognosis. [136,137] OTUB1 induces EMT to promote metastasis. OTUB1 is overexpressed and related to poor survival and serves as an independent prognostic factor in colorectal cancer (CRC).
OTUB2 and U2AF2 are highly expressed and associated with metastasis and poor survival. OTUB2 may serve as a potential prognostic indicator and therapeutic target in NSCLC. [138,139] EGF/KRAS-induced SUMOylation of OTUB2 can deubiquitinate and activate YAP/TAZ.
CHIP knockdown increases lung cancer cell invasion in an OTUD3 and GRP78-dependent manner. [140] TSG OTUD3 deubiquitinates and stabilizes PTEN. Reduction of OTUD3 causes decreased PTEN abundance and correlates with breast cancer progression. [141] OUTD6B TSG OTUD6B couples pVHL to form the CBC VHL complex to decrease its ubiquitination degradation, thereby attenuating HIF-1α.
OTUD6B is positively correlated with pVHL, but negatively with HIF-1α and vascular endothelial growth factor in hepatocellular carcinoma. [142] OUTD7B TSG OTUD7B promotes proliferation and metastasis via the Akt/VEGF signal pathway.
OTUD7B is highly expressed in lung squamous carcinoma and adenocarcinoma, and correlates with a worse prognosis, and may be an independent predictive indicator. [143]
CYLD mutant enhances squamous cell carcinoma growth and migration in an AP1-dependent manner. Snail1 inhibits CYLD to promote BCL-3 nuclear translocation, activating cycling D1 and N-cadherin.

Upregulation of CYLD expression can repress proliferation and invasion in melanoma. UCHL1
Oncogene UCHL1 promotes EMT. UCHL1 is overexpressed, and knockdown can induce MET in metastatic prostate cancer.
ERK inhibitor U0126 can block multidrug resistance and invasion in UCHL1-overexpressed breast cancer cells. UCHL1 compromises VHL-mediated ubiquitination of HIF-1α to promote metastasis. UCHL1 is overexpressed in breast and lung cancer. It may be a prognostic marker and therapeutic target. UCHL1 expression is positively associated with renal cell cancer's (RCC) metastatic phenotype.
UCHL1 might serve as a potential diagnostic and prognosis biomarker for RCC patients. UCHL1 deubiquitinates TGFβ type I receptor and SMAD2. The UCHL1 inhibitor 6RK73 suppresses TGFβ-induced metastasis, and UCHL1 could potentially target triple-negative breast cancer (TNBC) treatment.
COPS5 relates to HK2 overexpression, and Curcumin can inhibit CSN5 activity to decrease HK2, and to repress glycolysis and metastasis in hepatocellular carcinoma. [158,159] COPS5 deubiquitinates and stabilizes ZEB1. COPS5 expression is elevated and its knockdown can suppress EMT and metastasis. COPS6 Oncogene COPS6 increases CHIP self-ubiquitination to elevate EGFR stability.
COPS6 is overexpressed and the CSN6-CHIP-EGFR axis could be a therapeutic target in glioblastoma. [160,161] COPS6 inhibits the autophagy of CathepsinL (CTSL) via the mTOR pathway.
COPS6 and CTSL are overexpressed and indicate aggressive cervical cancer. ATXN3 Oncogene ATXN3 deubiquitinates KLF4. High ATXN3 and KLF4 expression are associated with a poor prognosis in breast cancer patients. [162]

DUBs inhibiting metastasis
Ub carboxyl-terminal hydrolase BAP1 (BAP1) BAP1 belongs to the UCH domain-containing proteins, and it can physically bind to and deubiquitinate PTEN to stabilize PTEN protein. Downregulated BAP1 leads to the decrease of PTEN protein levels and the activation of the Akt signaling pathway, therefore promoting malignant transformation and metastasis in prostate cancer. Clinically, low BAP1 expression is positively correlated with aggressive prostate tumor proliferation and lymphatic metastasis [40]. In ICC, BAP1 inhibits ERK1/2 and JNK/c-Jun pathways [41]. Moreover, BAP1 mediates the metabolic regulation of ferroptosis and tumor suppression. BAP1 reduces histone 2A ubiquitination (H2AUb) on the cystine/glutamate transporter (SLC7A11) promoter and represses SLC7A11 expression in a deubiquitination-dependent manner [42]. The loss of cystine transport mediated by SLC7A11 induces ferroptosis to inhibit tumor development and metastasis [43] (Fig. 3). In summary, as a major tumor suppressor, mutated BAP1 is associated with numerous human malignancies, which is defined as "BAP1 cancer syndrome" [44].

SUMOylated-Ub thioesterase OTUB2 (OTUB2)
Transcriptional coactivator YAP1 (YAP) and tafazzin (TAZ) are generally downregulated by the canonical Hippo pathway [56]. However, YAP and TAZ are hyperactivated and induce tumor proliferation and metastasis, while the Hippo pathway is still active in multiple malignancies, including breast cancer [57,58]. DUB OTUB2 mediates the activation of YAP and TAZ in a Hippo-independent manner (Fig. 3). Mechanistically, OTUB2 is poly-SUMOylated at lysine 233 (K233), SUMOylated-OTUB2 can subsequently bind YAP/TAZ through SUMO-interacting motif in YAP and TAZ [58]. OTUB2 deubiquitinates and activates YAP and TAZ, and accumulated YAP and TAZ translocate into the nucleus in which they interact with TEA domain family transcription factors and transcriptionally activate genes to potentiate cell proliferation and metastasis [59,60]. Meanwhile, activated EGF-RAS signaling strengthens OTUB2 SUMOylation and elevates YAP/TAZ protein levels to promote cancer stemness and metastasis [58,61]. In summary, the novel SUMOylated-OTUB2-mediated regulatory mechanism expands the complexity of YAP/TAZ beyond the Hippo pathway. OTUB2 may be a potential drug target to suppress cancer progression for patients harboring RAS mutations.

Conclusion
Although this brief review only scratches the surface of ubiquitination and deubiquitination in cancer, it highlights the significance of E3s and DUBs in a range of processes involved in tumor progression. Ubiquitination components are potential therapeutic targets for cancer treatment [65]. However, several issues remain obstacles for targeted therapy. DUBs share similar structural characteristics among family members, and ubiquitination involves substantial conformational changes. We still endeavor to deal with the challenges ahead, such as defining novel E3s, DUBs, and targeted substrates, investigating whether there exists unknown crosstalk among distinct E3s or DUBs, and decoding the unknown pathways linking ubiquitination with other cellular physiological mechanisms. Several valuable E3s or DUBs are promising clinical prognostic indexes and drug targets. Proteolysis Targeting Chimeras (PROTACs) exploit the intracellular Ub-proteasome system to degrade target proteins [66], selectively. In tumor xenografts, small-molecule PROTACs can significantly attenuate tumor progression [67].
This review provides a glimpse into the importance and extensiveness of ubiquitination component-mediated tumor invasion and metastasis, which represents a worthy research prospect.