The role of ubiquitination and deubiquitination in tumor invasion and metastasis

1Protein 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. 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).

2Ubiquitination 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. 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. 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. 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 (Fig. 1).

3The Ub cascade and DUBs synergistically regulate protein turnover and function in numerous signaling pathways to maintain cellular invasion and metastasis.

4Tumor 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,. The epithelial-mesenchymal transition (EMT) is an equally crucial determinant during the metastatic cascade.

5Ubiquitination 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.

6FBXW2 is a substrate recognition receptor in the SKP1-Cullin1-F-box protein (SCF) E3 Ub ligase complex. FBXW2 suppresses proliferation and invasion of lung cancer cells by targeting S phase kinase-associated protein 2 (SKP2) and β-catenin (Fig. 2). FBXW2 is downregulated and negatively correlated with β-catenin in lymph-node metastasis,. Meanwhile, FBXW2 is a novel substrate of β-transducin repeat-containing protein 1 (β-TrCP1). Following growth factor stimulation, β-TrCP1 targets FBXW2 for ubiquitination degradation. The accumulation of SKP2 subsequently leads to the degradation of tumor suppressors and apoptosis-inducing substrates, such as p21, p27, p130, and FOXO1, to promote tumor cell proliferation, growth, and survival. The β-TrCP-FBXW2-SKP2 signaling cascade forms the oncogene (β-TrCP1)-tumor suppressor gene (FBXW2)-oncogene (SKP2) axis that regulates the growth and survival of lung cancer cells via targeting each other for degradation, which is a process of crucial crosstalk among F-box proteins. In hepatocellular carcinoma (HCC), FBXW2 targets transforming growth factor-β-activated kinase 1 (TAK1) for K48-linked polyubiquitination and degradation to inhibit cancer progression (Fig. 2). Clinically, FBXW2 levels could be a significant indicator of the prognosis and survival of patients with cancer.

7FBW7 is a substrate recognition receptor of the SCFFBW7 E3 Ub ligase complex. It functions as a tumor suppressor and mediates ubiquitination-induced degradation of various oncogenic proteins, including c-MYC, NOTCH, c-JUN, and cyclin E. In gastric cancer, transcription activator Brg1 (Brg1) binds to the promoter of Snail, which subsequently promotes EMT and metastasis. Meanwhile, Brg1 is a Ub substrate of the SCFFBW7 E3 ligase complex. Casein kinase 1 (CK1)δ-mediated phosphorylation at Ser31/Ser35 sites of Brg1 strengthens FBW7-binding capacity, thus accelerating the ubiquitination of Brg1 (Fig. 2). Methylation induces epigenetic silencing of the FBW7 gene. Decitabine (DAC) epigenetically activates FBW7 expression via its demethylation. DAC-activated FBW7 promotes myeloid leukemia cell differentiation protein Mcl-1 ubiquitination and degradation to suppress lung cancer growth. YTH domain-containing family protein 2 (YTHDF2) is the N6-methyladenosine (m6A) reader protein and promotes the decay of the m6A-modified mRNAs. FBW7 can degrade YTHDF2 by ubiquitination and suppress the propagation of ovarian cancer (Fig. 2).

8UBR7 belongs to the Ub-protein ligase E3 component N-recognin (UBR) family and has a unique plant homeodomain (PHD) finger. PHD fingers are central “readers” of histone post-translational modifications. UBR7-PHD finger monoubiquitinates histone H2B in triple-negative breast tumors at lysine 120 (H2BK120Ub). H2BK120Ub enhances cadherin-4 (CDH4) transcription activity and expression level. CDH4 overexpression notably suppresses EMT and reduces cellular proliferation, migration, and invasion (Fig. 2). Meanwhile, CDH4 can regulate the Wnt/β-catenin signaling pathway. It alters the nuclear localization of β-catenin to the cytoplasm, which downregulates β-catenin target genes, including AXIN2, G1/S-specific cyclin-D1, C-MYC, COX2, and MMP7, to suppress tumor metastasis,.

9Notably, as an E3 Ub ligase, Parkin can degrade substrate proteins associated with Parkinson's Disease (PD). Meanwhile, Parkin acts as a tumor suppressor, and its expression is downregulated in various tumors. 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,. 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. In intrahepatic cholangiocarcinoma (ICC), Parkin targets pyruvate kinase PKM2 for ubiquitination degradation to suppress migration and proliferation (Fig. 2).

10UBE3C belongs to the HECT family of E3 Ub ligases. It functions as a tumor promoter and is aberrantly expressed in breast cancer, hepatocellular carcinoma, and renal cell carcinoma. In non-small cell lung cancer (NSCLC) tissues, UBE3C maintains cancer stemness by ubiquitinating and promoting neuroblast differentiation-associated protein AHNAK (AHNAK) degradation. AHNAK is a cofactor that assists P53 binding to stemness-related gene promoters. UBE3C-mediated degradation of AHNAK abrogates P53-AHNAK complex-mediated inhibition of gene expression, which enhances lung cancer cell stemness and NSCLC growth and metastasis,. UBE3C targets AXIN1 for ubiquitination degradation to activate β-catenin signaling in gastric cancer (Fig. 2).

11Tumor metastasis involves the reorganization of the cytoskeleton, whose activities are controlled by GTPases. When bound to guanosine diphosphate (GDP), GTPases are inactivated, which is regulated by GTPase-activating protein (GAP). 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 (Fig. 2). The TRIM65-ARHGAP35-Rho A axis enhances cancer cell migration by modulating the actin cytoskeleton.

12FBXO22, one of the F-box-only proteins, is the substrate-recognizing subunit of the SCF E3 Ub ligase complex. FBXO22 ubiquitinates nuclear PTEN at lysine 221 (K221). Nuclear PTEN exerts potent tumor inhibition capacity. In CRC tissues, FBXO22 overexpression contributes to the downregulation of nuclear PTEN to promote tumorigenesis, which is reversed by the mutation of K221. FBXO22 is upregulated and negatively correlated with p21 in HCC. FBXO22 functions as an oncogene by mediating the ubiquitination and degradation of p21 to promote HCC pathogenesis and progression. FBXO22 mediates Lys-63-linked liver kinase B1 (LKB1) polyubiquitination and inhibits LKB1-AMPK-mTOR signaling in lung adenocarcinoma. FBXO22 promotes melanoma angiogenesis and migration of tumor cells via upregulating HIF-1α and vascular endothelial growth factor A (VEGFA) (Fig. 2).

13DUBs 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. 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.

14BAP1 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. In ICC, BAP1 inhibits ERK1/2 and JNK/c-Jun pathways. 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. The loss of cystine transport mediated by SLC7A11 induces ferroptosis to inhibit tumor development and metastasis (Fig. 3). In summary, as a major tumor suppressor, mutated BAP1 is associated with numerous human malignancies, which is defined as “BAP1 cancer syndrome”.

15In the TGF-β signaling pathway, SMAD7 recruits E3 ligase SMURF2 to TGF-β type I receptor (TβRI). However, E3 Ub-protein ligases RNF12 and Itchy homolog can degrade SMAD7 to antagonize its suppression,. In breast cancer, OTUD1 can selectively eliminate the K48-linked Ub chain from SMAD7 to stabilize it (Fig. 3). Moreover, OTUD1 can cleave the K33-linked Ub chain on the lysine 220 site and unveil the PY motif of SMAD7. The exposed SMAD7 PY motif subsequently binds the WW domain of SMURF2 to degrade TβRI via ubiquitination,. In summary, OTUD1 suppresses the TGF-β-induced metastasis by exerting dual effects on SMAD7.

16The EGFR/PI3K/AKT pathway is aberrantly activated in various cancers. PTEN can negatively regulate AKT kinase activity. In addition, another molecular regulatory mechanism exists. Nuclear USP43 is physically associated with the nucleosome remodeling and deacetylase (NuRD) complex. The USP43-NuRD complex coordinately catalyzes H2BK120 deubiquitination to suppress downstream EGFR (Fig. 3). Simultaneously, activated AKT can phosphorylate cytoplasmic USP43 on Ser29, phosphorylated USP43 is detained in the cytoplasm, reduced nuclear USP43 and accumulated EGFR potentiate the EGFR/PI3K/AKT pathway to promote breast cell proliferation and invasion. USP43 is a hub of the USP43-NuRD complex, the reciprocally inhibitory loop between the USP43-NuRD complex and the EGFR/PI3K/AKT pathway synergistically modulates breast carcinogenesis. The ratio of nuclear/cytoplasmic USP43 is a worthy prognostic indicator.

17USP7 can coordinate with histone-lysine N-methyltransferase EZH2 (EZH2)-catalyzed methylation to remove ubiquitination and enhance FOXA1 protein stability, promoting prostate cancer growth. Moreover, USP7 can stabilize EZH2 via deubiquitination (Fig. 3). USP7 is upregulated in M2 macrophages. USP7 inhibition can induce the polarization of tumor-associated macrophages from M2 into M1 by activating the P38 MAPK pathway and upregulating the expression of programmed cell death 1 ligand 1 (PD-L1) in the tumor microenvironment. Therefore, USP7 blockade combined with anti-PD-1 immunotherapy exert an inhibitory effect on tumors in lung cancer.

18Transcriptional coactivator YAP1 (YAP) and tafazzin (TAZ) are generally downregulated by the canonical Hippo pathway. 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,. 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. 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,. Meanwhile, activated EGF-RAS signaling strengthens OTUB2 SUMOylation and elevates YAP/TAZ protein levels to promote cancer stemness and metastasis,. 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.

19Snail1 is a critical EMT-driving transcription factor and confers tumor metastatic and cancer stem cell-like properties. The E3 ligases β-TrCP1 and F-box/LRR-repeat protein 14 (FBXL14) can degrade Snail1 via ubiquitination. In breast cancer, Dub3 accounts for Snail1 stabilization, and inflammatory cytokine IL-6 can increase the expression of Dub3. Meanwhile, Dub3 also inhibits the activity of β-TrCP1 and FBXL14 to block Snail1 ubiquitination (Fig. 3). Overall, Dub3 senses inflammatory stimulation and converts it into Snail1 stabilization.

20Although 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. 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, selectively. In tumor xenografts, small-molecule PROTACs can significantly attenuate tumor progression.

21This review provides a glimpse into the importance and extensiveness of ubiquitination component-mediated tumor invasion and metastasis, which represents a worthy research prospect.